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  • 1.
    Abedi, Mohammad R.
    et al.
    Region Örebro län. Department of Laboratory Medicine, Section for Transfusion Medicine.
    Doverud, Ann-Charlotte
    Department of Laboratory Medicine, Section for Transfusion Medicine, Örebro University Hospital. Örebro, Sweden.
    Preparation and Pathogen Inactivation of Double Dose Buffy Coat Platelet Products using the INTERCEPT Blood System2012Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 70, artikel-id UNSP e4414Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Blood centers are faced with many challenges including maximizing production yield from the blood product donations they receive as well as ensuring the highest possible level of safety for transfusion patients, including protection from transfusion transmitted diseases. This must be accomplished in a fiscally responsible manner which minimizes operating expenses including consumables, equipment, waste, and personnel costs, among others.

    Several methods are available to produce platelet concentrates for transfusion. One of the most common is the buffy coat method in which a single therapeutic platelet unit (>= 2.0 x10(11) platelets per unit or per local regulations) is prepared by pooling the buffy coat layer from up to six whole blood donations. A procedure for producing "double dose" whole blood derived platelets has only recently been developed.

    Presented here is a novel method for preparing double dose whole blood derived platelet concentrates from pools of 7 buffy coats and subsequently treating the double dose units with the INTERCEPT Blood System for pathogen inactivation. INTERCEPT was developed to inactivate viruses, bacteria, parasites, and contaminating donor white cells which may be present in donated blood. Pairing INTERCEPT with the double dose buffy coat method by utilizing the INTERCEPT Processing Set with Dual Storage Containers (the "DS set"), allows blood centers to treat each of their double dose units in a single pathogen inactivation processing set, thereby maximizing patient safety while minimizing costs. The double dose buffy coat method requires fewer buffy coats and reduces the use of consumables by up to 50% (e.g. pooling sets, filter sets, platelet additive solution, and sterile connection wafers) compared to preparation and treatment of single dose buffy coat platelet units. Other cost savings include less waste, less equipment maintenance, lower power requirements, reduced personnel time, and lower collection cost compared to the apheresis technique.

  • 2. Afdile, Mamdooh
    et al.
    Jääskeläinen, Iiro P.
    Post-Movie Subliminal Measurement (PMSM), for Investigating Implicit Social Bias2020Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 156Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    This protocol describes the use of movies to investigate brain mechanisms underlying implicit social biases during functional magnetic resonance imaging. When the face of a protagonist is presented after a movie subliminally, it evokes an implicit response based on knowledge of the protagonist gained during the movie.

  • 3.
    Agathangelidis, Andreas
    et al.
    Ctr Res & Technol Hellas, Inst Appl Biosci, Thessaloniki, Greece.
    Sutton, Lesley Ann
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi. Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.
    Hadzidimitriou, Anastasia
    Ctr Res & Technol Hellas, Inst Appl Biosci, Thessaloniki, Greece.
    Tresoldi, Cristina
    IRCCS San Raffaele Sci Inst, Div Immunol Transplantat & Infect, Milan, Italy.
    Langerak, Anton W.
    Erasmus Univ, Med Ctr, Lab Med Immunol, Dept Immunol, Rotterdam, Netherlands.
    Belessi, Chrysoula
    Nikea Gen Hosp, Hematol Dept, Piraeus, Greece.
    Davi, Frederic
    Hop La Pitie Salpetriere, AP HP, Dept Hematol, Paris, France;UPMC Univ Paris 06, UMRS 1138, Paris, France.
    Rosenquist, Richard
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi. Karolinska Inst, Dept Mol Med & Surg, Stockholm, Sweden.
    Stamatopoulos, Kostas
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi. Ctr Res & Technol Hellas, Inst Appl Biosci, Thessaloniki, Greece.
    Ghia, Paolo
    IRCCS Ist Scientifico San Raffaele, Div Expt Oncol, Milan, Italy;Univ Vita Salute San Raffaele, Milan, Italy.
    Immunoglobulin Gene Sequence Analysis In Chronic Lymphocytic Leukemia: From Patient Material To Sequence Interpretation2018Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 141, artikel-id e57787Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    During B cell maturation, the complex process of immunoglobulin (IG) gene V(D)J recombination coupled with somatic hypermutation (SHM) gives rise to a unique DNA sequence within each individual B cell. Since B cell malignancies result from the clonal expansion of a single cell, IG genes represent a unique molecular signature common to all the malignant cells within an individual patient; thus, IG gene rearrangements can be used as clonal markers. In addition to serving as an important clonal identifier, the IG gene sequence can act as a 'molecular timeline' since it is associated with specific developmental stages and hence reflects the history of the B cell involved in the neoplastic transformation. Moreover, for certain malignancies, in particular chronic lymphocytic leukemia (CLL), the IG gene sequence holds prognostic and potentially predictive capabilities. That said, extrapolating meaningful conclusions from IG gene sequence analysis would be impossible if robust methods and tools were not available to aid in their analysis. This article, drawing on the vast experience of the European Research Initiative on CLL (ERIC), details the technical aspects and essential requirements necessary to ensure reliable and reproducible IG gene sequence analysis in CLL, a test that is now recommended for all CLL patients prior to treatment. More specifically, the various analytical stages are described ranging from the identification of the clonotypic IG gene rearrangement and the determination of the nucleotide sequence to the accurate clinical interpretation of the IG gene sequence data.

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  • 4.
    Aibara, Shintaro
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Andréll, Juni
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Singh, Vivek
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Amunts, Alexey
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Rapid Isolation of the Mitoribosome from HEK Cells2018Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 140, artikel-id e57877Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondria! genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart. Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only similar to 10(10) cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

  • 5.
    Andersson, Eva A
    et al.
    Gymnastik- och idrottshögskolan, GIH, Institutionen för idrotts- och hälsovetenskap, Åstrandlaboratoriet. Karolinska institutet.
    Frank, Per
    Gymnastik- och idrottshögskolan, GIH, Institutionen för idrotts- och hälsovetenskap, Åstrandlaboratoriet. Karolinska institutet.
    Pontén, Marjan
    Gymnastik- och idrottshögskolan, GIH, Institutionen för idrotts- och hälsovetenskap, Åstrandlaboratoriet.
    Ekblom, Björn
    Gymnastik- och idrottshögskolan, GIH, Institutionen för idrotts- och hälsovetenskap, Åstrandlaboratoriet, Björn Ekbloms forskningsgrupp.
    Ekblom, Maria
    Gymnastik- och idrottshögskolan, GIH, Institutionen för idrotts- och hälsovetenskap, Åstrandlaboratoriet. Karolinska institutet.
    Moberg, Marcus
    Gymnastik- och idrottshögskolan, GIH, Institutionen för idrotts- och hälsovetenskap, Åstrandlaboratoriet.
    Sahlin, Kent
    Gymnastik- och idrottshögskolan, GIH, Institutionen för idrotts- och hälsovetenskap, Åstrandlaboratoriet.
    Improving Strength, Power, Muscle Aerobic Capacity, and Glucose Tolerance through Short-term Progressive Strength Training Among Elderly People.2017Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 125Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    This protocol describes the simultaneous use of a broad span of methods to examine muscle aerobic capacity, glucose tolerance, strength, and power in elderly people performing short-term resistance training (RET). Supervised progressive resistance training for 1 h three times a week over 8 weeks was performed by RET participants (71±1 years, range 65-80). Compared to a control group without training, the RET showed improvements on the measures used to indicate strength, power, glucose tolerance, and several parameters of muscle aerobic capacity. Strength training was performed in a gym with only robust fitness equipment. An isokinetic dynamometer for knee extensor strength permitted the measurement of concentric, eccentric, and static strength, which increased for the RET group (8-12% post- versus pre-test). The power (rate of force development, RFD) at the initial 0-30 ms also showed an increase for the RET group (52%). A glucose tolerance test with frequent blood glucose measurements showed improvements only for the RET group in terms of blood glucose values after 2 h (14%) and the area under the curve (21%). The blood lipid profile also improved (8%). From muscle biopsy samples prepared using histochemistry, the amount of fiber type IIa increased, and a trend towards a decrease in IIx in the RET group reflected a change to a more oxidative profile in terms of fiber composition. Western blot (to determine the protein content related to the signaling for muscle protein synthesis) showed a rise of 69% in both Akt and mTOR in the RET group; this also showed an increase in mitochondrial proteins for OXPHOS complex II and citrate synthase (both ~30%) and for complex IV (90%), in only the RET group. We demonstrate that this type of progressive resistance training offers various improvements (e.g., strength, power, aerobic capacity, glucose tolerance, and plasma lipid profile).

  • 6.
    Aresh, Bejan
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Genetisk utvecklingsbiologi.
    Peuckert, Christiane
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för organismbiologi, Evolution och utvecklingsbiologi.
    Dissection and Culture of Mouse Embryonic Kidney2017Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 123, artikel-id e55715Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant. One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

  • 7.
    Assadian, Farzaneh
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Sandström, Karl
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för kirurgiska vetenskaper, Öron-, näs- och halssjukdomar.
    Laurell, Göran
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för kirurgiska vetenskaper, Öron-, näs- och halssjukdomar.
    Svensson, Catharina
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Akusjärvi, Göran
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Punga, Tanel
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils2015Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, Vol. 105, artikel-id e53374Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

  • 8.
    Augier, Eric
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Centrum för social och affektiv neurovetenskap. Linköpings universitet, Medicinska fakulteten.
    Dulman, Russell S.
    National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, USA.
    Singley, Erick
    National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, USA.
    Heilig, Markus
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Centrum för social och affektiv neurovetenskap. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Närsjukvården i centrala Östergötland, Psykiatriska kliniken.
    A Method for Evaluating the Reinforcing Properties of Ethanol in Rats without Water Deprivation, Saccharin Fading or Extended Access Training2017Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 119, artikel-id e53305Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods require saccharin/sucrose fading, water deprivation and/or extended training to initiate operant responding in rats. This paper describes a novel and efficient method to quickly initiate operant responding for ethanol that is convenient for experimenters and does not require water deprivation or saccharin/sucrose fading, thus eliminating the potential confound of using sweeteners in ethanol operant self-administration studies. With this method, Wistar rats typically acquire and maintain self-administration of a 20% ethanol solution in less than two weeks of training. Furthermore, blood ethanol concentrations and rewards are positively correlated for a 30 min self-administration session. Moreover, naltrexone, an FDA-approved medication for alcohol dependence that has been shown to suppress ethanol self-administration in rodents, dose-dependently decreases alcohol intake and motivation to consume alcohol for rats self-administering 20% ethanol, thus validating the use of this new method to study the reinforcing properties of alcohol in rats.

  • 9.
    B. Kumar, Ramakrishnan
    et al.
    Karolinska institutet, Sverige.
    Zhu, Lin
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik.
    Hebert, Hans
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik. Karolinska institutet, Sverige.
    Jegerschöld, Caroline
    Karolinska institutet, Sverige.
    Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy2017Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 121, artikel-id e55148Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

  • 10.
    Bagheri, Maryam
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Rezakhani, Arjang
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Roghani, Mehrdad
    Neurophysiology Research Center, Shahed University, Iran.
    Joghataei, Mohammad T.
    Iran University of Medical Science, Iran.
    Mohseni, Simin
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes2015Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 106, s. e53113-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 mu m(2)). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta(1-40) (A beta(1-40)) with or without a therapeutic intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

  • 11. Baharom, Faezzah
    et al.
    Rankin, Gregory
    Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Medicin.
    Scholz, Saskia
    Pourazar, Jamshid
    Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Medicin.
    Ahlm, Clas
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Infektionssjukdomar.
    Blomberg, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Medicin.
    Smed-Sörensen, Anna
    Human lung dendritic cells: spatial distribution and phenotypic identification in endobronchial biopsies using immunohistochemistry and flow cytometry2017Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 119, artikel-id e55222Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In order to maintain tolerance or to induce an immune response, the immune system must appropriately handle inhaled antigens. Lung dendritic cells (DCs) are essential in maintaining a delicate balance to initiate immunity when required without causing collateral damage to the lungs due to an exaggerated inflammatory response. While there is a detailed understanding of the phenotype and function of immune cells such as DCs in human blood, the knowledge of these cells in less accessible tissues, such as the lungs, is much more limited, since studies of human lung tissue samples, especially from healthy individuals, are scarce. This work presents a strategy to generate detailed spatial and phenotypic characterization of lung tissue resident DCs in healthy humans that undergo a bronchoscopy for the sampling of endobronchial biopsies. Several small biopsies can be collected from each individual and can be subsequently embedded for ultrafine sectioning or enzymatically digested for advanced flow cytometric analysis. The outlined protocols have been optimized to yield maximum information from small tissue samples that, under steady-state conditions, contain only a low frequency of DCs. While the present work focuses on DCs, the methods described can directly be expanded to include other (immune) cells of interest found in mucosal lung tissue. Furthermore, the protocols are also directly applicable to samples obtained from patients suffering from pulmonary diseases where bronchoscopy is part of establishing the diagnosis, such as chronic obstructive pulmonary disease (COPD), sarcoidosis, or lung cancer.

  • 12.
    Balian, Alien
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär ytfysik och nanovetenskap. Linköpings universitet, Tekniska fakulteten. Wallenberg Centre for Molecular Medicine (WCMM), Sweden.
    Garcia Gonzalez, Javier
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär ytfysik och nanovetenskap. Linköpings universitet, Tekniska fakulteten. Wallenberg Centre for Molecular Medicine (WCMM), Sweden.
    Bastida, Nora
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär ytfysik och nanovetenskap. Linköpings universitet, Tekniska fakulteten.
    Akhtar, Khadija-Tul Kubra
    Linköpings universitet, Institutionen för fysik, kemi och biologi. Linköpings universitet, Tekniska fakulteten.
    Borsa, Baris Ata
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär ytfysik och nanovetenskap. Linköpings universitet, Tekniska fakulteten. Wallenberg Centre for Molecular Medicine (WCMM), Sweden.
    Hernandez, Frank
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär ytfysik och nanovetenskap. Linköpings universitet, Tekniska fakulteten. Wallenberg Centre for Molecular Medicine (WCMM), Sweden.
    Kinetic Screening of Nuclease Activity using Nucleic Acid Probes2019Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 153, artikel-id e60005Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable. Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

  • 13. Bello, M. A.
    et al.
    Ruiz-León, Y.
    Sandoval-Sierra, J. V.
    Rezinciuc, Svetlana
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Diéguez-Uribeondo, J.
    Scanning electron microscopy (SEM) protocols for problematic plant, oomycete, and fungal samples2017Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, Vol. 2017, nr 120, artikel-id e55031Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricalesas examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM. Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells. The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.

  • 14.
    Berthold, Anne
    et al.
    Department of Biology, Mt. Allison University, Canada.
    Faucillion, Marie-Line
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Nilsson, Ingela
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Golovchenko, Maryna
    Biology Centre Czech Academy of Sciences, Institute of Parasitology, Czech Republic.
    Lloyd, Vett
    Department of Biology, Mt. Allison University, Canada.
    Bergström, Sven
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Rudenko, Natalie
    Biology Centre Czech Academy of Sciences, Institute of Parasitology, Czech Republic.
    Cultivation Methods of Spirochetes from Borrelia burgdorferi Sensu Lato Complex and Relapsing Fever Borrelia2022Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, Vol. 2022, nr 189, artikel-id e64431Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Borrelia consists of three groups of species, those of the Lyme borreliosis (LB) group, also known as B. burgdorferi sensu lato (s.l.) and recently reclassified into Borreliella, the relapsing fever (RF) group Borrelia, and a third reptile-associated group of spirochetes. Culture-based methods remain the gold standard for the laboratory detection of bacterial infections for both research and clinical work, as the culture of pathogens from bodily fluids or tissues directly detects replicating pathogens and provides source material for research. Borrelia and Borreliella spirochetes are fastidious and slow growing, and thus are not commonly cultured for clinical purposes; however, culture is necessary for research. This protocol demonstrates the methodology and recipes required to successfully culture LB and RF spirochetes, including all recognized species from B. burgdorferi s.l. complex including B. afzelii, B. americana, B. andersonii, B. bavariensis, B. bissettii/bissettiae, B. burgdorferi sensu stricto (s.s.), B. californiensis, B. carolinensis, B. chilensis, B. finlandensis, B. garinii, B. japonica, B. kurtenbachii, B. lanei, B. lusitaniae, B. maritima, B. mayonii, B. spielmanii, B. tanukii, B. turdi, B. sinica, B. valaisiana, B. yangtzensis, and RFspirochetes, B. anserina, B. coriaceae, B. crocidurae, B. duttonii, B. hermsii, B. hispanica, B. persica, B. recurrentis, and B. miyamotoi. The basic medium for growing LB and RF spirochetes is the Barbour-Stoenner-Kelly (BSK-II or BSK-H) medium, which reliably supports the growth of spirochetes in established cultures. To be able to grow newly isolated Borrelia isolates from tick-or host-derived samples where the initial spirochete number is low in the inoculum, modified Kelly-Pettenkofer (MKP) medium is preferred. This medium also supports the growth of B. miyamotoi. The success of the cultivation of RF spirochetes also depends critically on the quality of ingredients.

  • 15.
    Bimpisidis, Zisis
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för organismbiologi, Jämförande fysiologi.
    König, Niclas
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för organismbiologi, Jämförande fysiologi.
    Wallén-Mackenzie, Åsa
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för organismbiologi, Jämförande fysiologi.
    Two Different Real-Time Place Preference Paradigms Using Optogenetics within the Ventral Tegmental Area of the Mouse2020Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 156, artikel-id e60867Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Understanding how neuronal activation leads to specific behavioral output is fundamental for modern neuroscience. Combining optogenetics in rodents with behavioral testing in validated paradigms allows the measurement of behavioral consequences upon stimulation of distinct neurons in real-time with high spatial and temporal selectivity, and thus the establishment of causal relationships between neuronal activation and behavior. Here, we describe a step-by-step protocol fora real-time place preference (RT-PP) paradigm, a modified version of the classical conditioned place preference (CPP) test. The RT-PP is performed in a three-compartment apparatus and can be utilized to answer if optogenetic stimulation of a specific neuronal population is rewarding or aversive. We also describe an alternative version of the RT-PP protocol, the socalled neutral compartment preference (NCP) protocol, which can be used to confirm aversion. The two approaches are based on extensions of classical methodology originating from behavioral pharmacology and recent implementation of optogenetics within the neuroscience field. Apart from measuring place preference in real time, these setups can also give information regarding conditioned behavior. We provide easyto-follow step-by-step protocols alongside examples of our own data and discuss important aspects to consider when applying these types of experiments.

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  • 16.
    Bjerling, Pernilla
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Olsson, Ida
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab. Swedish Univ Agr Sci, Dept Microbiol, S-90183 Umea, Sweden.
    Meng, Xi'nan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast2012Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 59, s. e3454-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular (1). Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome(2). The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP (3). Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour (4). In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast (5). In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) (6). In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM (7). The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

  • 17.
    Braian, Clara
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Medicinska fakulteten.
    Svensson, Mattias
    Karolinska Institute, Sweden.
    Brighenti, Susanna
    Karolinska Institute, Sweden.
    Lerm, Maria
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Medicinska fakulteten.
    Parasa, Venkata R.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Medicinska fakulteten. Karolinska Institute, Sweden.
    A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection2015Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 104, s. 1-9, artikel-id e53084Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

  • 18.
    Calitz, Carlemi
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Pavlovic, Natasa
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Rosenquist, Jenny
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - Ångström, Polymerkemi.
    Zagami, Claudia
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Samanta, Ayan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - Ångström, Polymerkemi.
    Heindryckx, Femke
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    A Biomimetic Model for Liver Cancer to Study Tumor-Stroma Interactions in a 3D Environment with Tunable Bio-Physical Properties2020Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 162, artikel-id e61606Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a fibrotic environment actively supporting and driving hepatocarcinogenesis. Insight into hepatocarcinogenesis in terms of the interplay between the tumor stroma microenvironment and tumor cells is thus of considerable importance. Three-dimensional (3D) cell culture models are proposed as the missing link between current in vitro 2D cell culture models and in vivo animal models. Our aim was to design a novel 3D biomimetic HCC model with accompanying fibrotic stromal compartment and vasculature. Physiologically relevant hydrogels such as collagen and fibrinogen were incorporated to mimic the bio-physical properties of the tumor ECM. In this model LX2 and HepG2 cells embedded in a hydrogel matrix were seeded onto the inverted transmembrane insert. HUVEC cells were then seeded onto the opposite side of the membrane. Three formulations consisting of ECM-hydrogels embedded with cells were prepared and the bio-physical properties were determined by rheology. Cell viability was determined by a cell viability assay over 21 days. The effect of the chemotherapeutic drug doxorubicin was evaluated in both 2D co-culture and our 3D model for a period of 72h. Rheology results show that bio-physical properties of a fibrotic, cirrhotic and HCC liver can be successfully mimicked. Overall, results indicate that this 3D model is more representative of the in vivo situation compared to traditional 2D cultures. Our 3D tumor model showed a decreased response to chemotherapeutics, mimicking drug resistance typically seen in HCC patients.

  • 19.
    Calogiuri, Tullia
    et al.
    Wageningen Univ & Res, Soil Biol Grp, Wageningen, Netherlands.;Wageningen Univ & Res, Soil Chem & Chem Soil Qual, Wageningen, Netherlands..
    Hagens, Mathilde
    Wageningen Univ & Res, Soil Chem & Chem Soil Qual, Wageningen, Netherlands..
    Van Groenigen, Jan Willem
    Wageningen Univ & Res, Soil Biol Grp, Wageningen, Netherlands..
    Corbett, Thomas
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Geovetenskapliga sektionen, Institutionen för geovetenskaper.
    Hartmann, Jens
    Univ Hamburg, Inst Geol, Ctr Earth Syst Res & Sustainabil, Hamburg, Germany..
    Hendriksen, Rick
    Wageningen Univ & Res, Tupola, Wageningen, Netherlands..
    Janssens, Iris
    Univ Antwerp, IMEC, Dept Comp Sci, IDLab, Antwerp, Belgium..
    Janssens, Ivan A.
    Univ Antwerp, Biol Dept, Plants & Ecosyst PLECO, Antwerp, Belgium..
    Dominguez, Guillermo Ledesma
    Univ Antwerp, Biol Dept, Plants & Ecosyst PLECO, Antwerp, Belgium..
    Loescher, Grant
    Univ Hamburg, Inst Geol, Ctr Earth Syst Res & Sustainabil, Hamburg, Germany..
    Mortier, Steven
    Univ Antwerp, IMEC, Dept Comp Sci, IDLab, Antwerp, Belgium..
    Neubeck, Anna
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Geovetenskapliga sektionen, Institutionen för geovetenskaper, Paleobiologi.
    Niron, Harun
    Univ Antwerp, Biol Dept, Plants & Ecosyst PLECO, Antwerp, Belgium..
    Poetra, Reinaldy P.
    Univ Hamburg, Inst Geol, Ctr Earth Syst Res & Sustainabil, Hamburg, Germany..
    Rieder, Lukas
    Univ Hamburg, Inst Geol, Ctr Earth Syst Res & Sustainabil, Hamburg, Germany..
    Struyf, Eric
    Univ Antwerp, Biol Dept, Plants & Ecosyst PLECO, Antwerp, Belgium..
    Van Tendeloo, Michiel
    Univ Antwerp, Res Grp Sustainable Energy Air & Water Technol, Antwerp, Belgium..
    De Schepper, Tom
    Univ Antwerp, IMEC, Dept Comp Sci, IDLab, Antwerp, Belgium..
    Verdonck, Tim
    Univ Antwerp, IMEC, Dept Math, Antwerp, Belgium..
    Vlaeminck, Siegfried E.
    Univ Antwerp, Res Grp Sustainable Energy Air & Water Technol, Antwerp, Belgium..
    Vicca, Sara
    Univ Antwerp, Biol Dept, Plants & Ecosyst PLECO, Antwerp, Belgium..
    Vidal, Alix
    Wageningen Univ & Res, Soil Biol Grp, Wageningen, Netherlands..
    Design and Construction of an Experimental Setup to Enhance Mineral Weathering through the Activity of Soil Organisms2023Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 201, artikel-id e65563Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Enhanced weathering (EW) is an emerging carbon dioxide (CO2) removal technology that can contribute to climate change mitigation. This technology relies on accelerating the natural process of mineral weathering in soils by manipulating the abiotic variables that govern this process, in particular mineral grain size and exposure to acids dissolved in water. EW mainly aims at reducing atmospheric CO2 concentrations by enhancing inorganic carbon sequestration. Until now, knowledge of EW has been mainly gained through experiments that focused on the abiotic variables known for stimulating mineral weathering, thereby neglecting the potential influence of biotic components. While bacteria, fungi, and earthworms are known to increase mineral weathering rates, the use of soil organisms in the context of EW remains underexplored. This protocol describes the design and construction of an experimental setup developed to enhance mineral weathering rates through soil organisms while concurrently controlling abiotic conditions. The setup is designed to maximize weathering rates while maintaining soil organisms' activity. It consists of a large number of columns filled with rock powder and organic material, located in a climate chamber and with water applied via a downflow irrigation system. Columns are placed above a fridge containing jerrycans to collect the leachate. Representative results demonstrate that this setup is suitable to ensure the activity of soil organisms and quantify their effect on inorganic carbon sequestration. Challenges remain in minimizing leachate losses, ensuring homogeneous ventilation through the climate chamber, and avoiding flooding of the columns. With this setup, an innovative and promising approach is proposed to enhance mineral weathering rates through the activity of soil biota and disentangle the effect of biotic and abiotic factors as drivers of EW.

  • 20.
    Castells-Nobau, Anna
    et al.
    Radboud University of Nijmegen, Netherlands.
    Nijhof, Bonnie
    Radboud University of Nijmegen, Netherlands.
    Eidhof, Ilse
    Radboud University of Nijmegen, Netherlands.
    Wolf, Louis
    Radboud University of Nijmegen, Netherlands.
    Scheffer-de Gooyert, Jolanda M.
    Radboud University of Nijmegen, Netherlands.
    Monedero Cobeta, Ignacio
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Medicinska fakulteten. University of Autonoma Madrid, Spain.
    Torroja, Laura
    University of Autonoma Madrid, Spain.
    van der Laak, Jeroen A. W. M.
    Radboud University of Nijmegen, Netherlands; Radboud University of Nijmegen, Netherlands.
    Schenck, Annette
    Radboud University of Nijmegen, Netherlands.
    Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology2017Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 123, artikel-id e55395Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms Drosophila NMJ Morphometrics and Drosophila NMJ Bouton Morphometrics, available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

  • 21. Desmarais, Samantha M.
    et al.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    de Pedro, Miguel A.
    Huang, Kerwyn Casey
    Isolation and preparation of bacterial cell walls for compositional analysis by Ultra Performance Liquid Chromatography2014Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 83, s. UNSP e51183-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan's central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

  • 22.
    Dias, Jorge
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Svedberg, Gustav
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Nystrand, M.
    Svahn Andersson, Helene
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Gantelius, Jesper
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Rapid nanoprobe signal enhancement by in situ gold nanoparticle synthesis2018Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, Vol. 2018, nr 133, artikel-id e57297Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good's buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera. 

  • 23.
    Eriksson, Anna U.
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Svensson, Christoffer
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Hörnblad, Andreas
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Cheddad, Abbas
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Kostromina, Elena
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Eriksson, Maria
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Norlin, Nils
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Pileggi, Antonello
    Cell Transplants Center, Diabetes Research Institute, University of Miami.
    Sharpe, James
    EMBL-CRG Systems Biology Program, Centre for Genomic Regulation, Catalan Institute of Research and Advance Studies.
    Georgsson, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för datavetenskap.
    Alanentalo, Tomas
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Ahlgren, Ulf
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Near infrared optical projection tomography for assessments of beta-cell mass distribution in diabetes research2013Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, Vol. 71, artikel-id e50238Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen's (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR)2; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction2 and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations3. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.

    Ladda ner fulltext (pdf)
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  • 24.
    Fard, Shahrzad Shirazi
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Medicinsk utvecklingsbiologi.
    Blixt, Maria
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Medicinsk utvecklingsbiologi.
    Hallböök, Finn
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Medicinsk utvecklingsbiologi.
    Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments2015Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 103, artikel-id e53202Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental manipulations in ovo/in vivo makes the chicken embryonic retina a versatile and very efficient experimental model. Although the chicken retina is easy to target in ovo by intraocular injections or electroporation, the effective and exact concentration of the reagents within the retina may be difficult to fully control. This may be due to variations of the exact injection site, leakage from the eye or uneven diffusion of the substances. Furthermore, the frequency of malformations and mortality after invasive manipulations such as electroporation is rather high. This protocol describes an ex ovo technique for culturing whole retinal explants from chicken embryos and provides a method for controlled exposure of the retina to reagents. The protocol describes how to dissect, experimentally manipulate, and culture whole retinal explants from chicken embryos. The explants can be cultured for approximately 24 hr and be subjected to different manipulations such as electroporation. The major advantages are that the experiment is not dependent on the survival of the embryo and that the concentration of the introduced reagent can be varied and controlled in order to determine and optimize the effective concentration. Furthermore, the technique is rapid, cheap and together with its high experimental success rate, it ensures reproducible results. It should be emphasized that it serves as an excellent complement to experiments performed in ovo.

  • 25.
    Galichanin, Konstantin
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Oftalmiatrik. Karolinska Inst, St Eriks Eye Hosp, S-10401 Stockholm, Sweden.
    Talebizadeh, Nooshin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Oftalmiatrik.
    Söderberg, Per
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Oftalmiatrik.
    Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract2012Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 69, artikel-id e4016Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cataract is the leading cause of blindness in the world (1). The World Health Organization defines cataract as a clouding of the lens of the eye which impedes the transfer of light. Cataract is a multi-factorial disease associated with diabetes, smoking, ultraviolet radiation (UVR), alcohol, ionizing radiation, steroids and hypertension. There is strong experimental (2-4) and epidemiological evidence (5,6) that UVR causes cataract. We developed an animal model for UVR B induced cataract in both anesthetized (7) and non-anesthetized animals (8). The only cure for cataract is surgery but this treatment is not accessible to all. It has been estimated that a delay of onset of cataract for 10 years could reduce the need for cataract surgery by 50% (9). To delay the incidence of cataract, it is needed to understand the mechanisms of cataract formation and find effective prevention strategies. Among the mechanisms for cataract development, apoptosis plays a crucial role in initiation of cataract in humans and animals (10). Our focus has recently been apoptosis in the lens as the mechanism for cataract development (8,11,12). It is anticipated that a better understanding of the effect of UVR on the apoptosis pathway will provide possibilities for discovery of new pharmaceuticals to prevent cataract. In this article, we describe how cataract can be experimentally induced by in vivo exposure to UVR-B. Further RT-PCR and immunohistochemistry are presented as tools to study molecular mechanisms of UVR-B induced cataract.

  • 26. Gallstedt, Mikael
    et al.
    Pettersson, Henrik
    Johansson, Therese
    Newson, William R.
    Johansson, Eva
    Hedenqvist, Mikael S.
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknologi.
    Film Extrusion of Crambe abyssinica/Wheat Gluten Blends2017Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 119, artikel-id e54770Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Crambe abyssinica is a plant with potential for use in industrial (non-food) plant oil production. The side stream from this oil production is a high-protein crambe meal that has limited value, as it is not fit for food or feed use. However, it contains proteins that could potentially make it a suitable raw material for higher-value products. The purpose of this study was to find methods of making this side stream into extruded films, showing that products with a higher value can be produced. The study mainly considered the development of material compositions and methods of preparing and extruding the material. Wheat gluten was added as a supportive protein matrix material, together with glycerol as a plasticizer and urea as a denaturant. The extrudate was evaluated with respect to mechanical (tensile testing) and oxygen barrier properties, and the extrudate structure was revealed visually and by scanning electron microscopy. A denser, more homogeneous material had a lower oxygen transmission rate, higher strength, and higher extensibility. The most homogeneous films were made at an extruder die temperature of 125-130 degrees C. It is shown here that a film can be extruded with promising mechanical and oxygen barrier properties, the latter especially after a final compression molding step.

  • 27.
    Garcia-Hernandez, Vicky
    et al.
    Univ Michigan, MI 48109 USA.
    Neumann, Philipp-Alexander
    Tech Univ Munich, Germany.
    Koch, Stefan
    Linköpings universitet, Institutionen för biomedicinska och kliniska vetenskaper, Avdelningen för molekylär medicin och virologi. Linköpings universitet, Medicinska fakulteten.
    Lyons, Renae
    Univ Michigan, MI 48109 USA.
    Nusrat, Asma
    Univ Michigan, MI 48109 USA.
    Parkos, Charles A.
    Univ Michigan, MI 48109 USA.
    Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model2021Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 168, artikel-id e62135Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. However, robust standards for objective and reproducible quantification of disease severity remain to be defined. Most colitis analysis methods rely on limited histological scoring of small segments of intestine, leading to partial or biased analyses. Here, we combine high-resolution image acquisition and longitudinal analysis of the entire colon to quantify intestinal injury and ulceration in the dextran sodium sulfate (DSS) induced model of murine colitis. This protocol allows for the generation of objective and reproducible results without extensive user training. Here, we provide comprehensive details on sample preparation and image analysis using examples of data from DSS induced colitis. This method can be easily adapted to other models of murine colitis that have significant inflammation associated with mucosal injury. We demonstrate that the fraction of inflamed/injured and eroded/ulcerated mucosa relative to the complete length of the colon closely parallels clinical findings such as weight loss amid DSS-induced disease progression. This histological protocol provides a reliable time and cost-effective aid to standardize analyses of disease activity in an unbiased way in DSS colitis experiments.

  • 28.
    Gällstedt, Mikael
    et al.
    SIG Combibloc, Switzerland.
    Pettersson, Henrik
    RISE - Research Institutes of Sweden (2017-2019), Bioekonomi. RISE., Innventia.
    Johansson, Therese
    RISE - Research Institutes of Sweden (2017-2019), Bioekonomi. RISE., Innventia.
    Newson, William R.
    SLU Swedish University of Agricultural Sciences, Sweden.
    Johansson, Eva
    SLU Swedish University of Agricultural Sciences, Sweden.
    Hedenqvist, Mikael S.
    KTH Royal Institute of Technology, Sweden.
    Film extrusion of Crambe abyssinica/wheat gluten blends2017Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, Journal of Visualized Experiments, Vol. 2017, nr 119, artikel-id e54770Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Crambe abyssinica is a plant with potential for use in industrial (non-food) plant oil production. The side stream from this oil production is a high-protein crambe meal that has limited value, as it is not fit for food or feed use. However, it contains proteins that could potentially make it a suitable raw material for higher-value products. The purpose of this study was to find methods of making this side stream into extruded films, showing that products with a higher value can be produced. The study mainly considered the development of material compositions and methods of preparing and extruding the material. Wheat gluten was added as a supportive protein matrix material, together with glycerol as a plasticizer and urea as a denaturant. The extrudate was evaluated with respect to mechanical (tensile testing) and oxygen barrier properties, and the extrudate structure was revealed visually and by scanning electron microscopy. A denser, more homogeneous material had a lower oxygen transmission rate, higher strength, and higher extensibility. The most homogeneous films were made at an extruder die temperature of 125-130 °C. It is shown here that a film can be extruded with promising mechanical and oxygen barrier properties, the latter especially after a final compression molding step.

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  • 29.
    Hanrieder, Jörg
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap. Chalmers, Dept Chem & Biol Engn, Gothenburg, Sweden.
    Ljungdahl, Anna
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    Andersson, Malin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease2012Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, Vol. 14, nr 60, artikel-id e3445Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples(2) (Fig.1). A 12 μm thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity(2). Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson's disease (PD). PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age(3,4). The most common symptomatic treatment is based on dopamine replacement using L-DOPA(5). However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)(1,3,6). One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA(7). The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control(7,8). However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson's disease and L-DOPA induced dyskinesia. MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin's opioid-receptor binding capacity(9). This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.

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    Jove
  • 30.
    Hauptmann, Giselbert
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Söll, Iris
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Krautz, Robert
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Theopold, Ulrich
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos2016Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 107, artikel-id e53830Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

  • 31.
    Henricson, Joakim
    et al.
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för läkemedelsforskning. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Hjärt- och Medicincentrum, Hudkliniken i Östergötland.
    Toll John, Rani
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för läkemedelsforskning. Linköpings universitet, Medicinska fakulteten.
    Anderson, Chris
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Hjärt- och Medicincentrum, Hudkliniken i Östergötland.
    Björk Wilhelms, Daniel
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för läkemedelsforskning. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Närsjukvården i centrala Östergötland, Akutkliniken.
    Diffuse Reflectance Spectroscopy: Getting the Capillary Refill Test Under Ones Thumb2017Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 130, artikel-id e56737Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The capillary refill test was introduced in 1947 to help estimate circulatory status in critically ill patients. Guidelines commonly state that refill should occur within 2 s after releasing 5 s of firm pressure (e.g., by the physicians finger) in the normal healthy supine patient. A slower refill time indicates poor skin perfusion, which can be caused by conditions including sepsis, blood loss, hypoperfusion, and hypothermia. Since its introduction, the clinical usefulness of the test has been debated. Advocates point out its feasibility and simplicity and claim that it can indicate changes in vascular status earlier than changes in vital signs such as heart rate. Critics, on the other hand, stress that the lack of standardization in how the test is performed and the highly subjective nature of the naked eye assessment, as well as the tests susceptibility to ambient factors, markedly lowers the clinical value. The aim of the present work is to describe in detail the course of the refill event and to suggest potentially more objective and exact endpoint values for the capillary refill test using diffuse polarization spectroscopy.

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  • 32.
    Israel Nazarious, Miracle
    et al.
    Luleå tekniska universitet, Institutionen för system- och rymdteknik, Rymdteknik. School of Geosciences, University of Aberdeen.
    Vakkada Ramachandran, Abhilash
    Luleå tekniska universitet, Institutionen för system- och rymdteknik, Rymdteknik.
    Zorzano, Maria-Paz
    Centro de Astrobiología (INTA-CSIC); School of Geosciences, University of Aberdeen.
    Martin-Torres, Javier
    Instituto Andaluz de Ciencias de la Tierra (UGR-CSIC); School of Geosciences, University of Aberdeen.
    Measuring Electrical Conductivity to Study the Formation of Brines Under Martian Conditions2021Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, Vol. 173, artikel-id e61217Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    This paper describes a protocol to design experiments to study the formation of brines under Martian conditions and monitor the process with electrical conductivity measurements. We used the Engineering Qualification Model (EQM) of Habitability: Brines, Irradiation, and Temperature (HABIT)/ExoMars 2022 instrument for the experiment setup but we provide a brief account of constructing a simple and inexpensive electrical conductivity measurement setup. The protocol serves to calibrate the electrical conductivity measurements of the salt deliquescence into brine in a simulated Martian environment. The Martian conditions of temperature (-70 °C to 20 °C), relative humidity (0% to 100%) and pressure (7 - 8 mbar) with carbon-dioxide atmosphere were simulated in the SpaceQ Mars simulation chamber, a facility at the Luleå University of Technology, Sweden. The hydrate form of the known amount of salt accommodated between a pair of electrodes and thus the electrical conductivity measured depends predominantly on its water content and the temperature and relative humidity of the system. Electrical conductivity measurements were carried out at 1 Hz while exposing salts to a continuously increasing relative humidity (to force transitioning through various hydrates) at different Martian temperatures. For demonstration, a day-night cycle at Oxia Planum, Mars (the landing site of ExoMars 2022 mission) was recreated.

  • 33. Jagadeesan, Srikanth
    et al.
    Workman, Michael J.
    Herland, Anna
    KTH, Skolan för elektroteknik och datavetenskap (EECS), Intelligenta system, Mikro- och nanosystemteknik.
    Svendsen, Clive N.
    Vatine, Gad D.
    Generation of a Human iPSC-Based Blood-Brain Barrier Chip2020Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 157, artikel-id e60925Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB's role in health and disease. We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

  • 34.
    Jin, Zhe
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Fysiologi.
    Jin, Yang
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Fysiologi.
    Birnir, Bryndis
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Fysiologi.
    GABA-activated single-channel and tonic currents in rat brain slices2011Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 53Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The GABA(A) channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, respectively. The GABA(A) channel is a pentameric GABA-gated chloride channel. The channel subunits are grouped into 8 families (α1-6, β1-3, γ1-3, δ, ε, θ, π and ρ). Two alphas, two betas and one 3(rd) subunit form the functional channel. By combining studies of sub-type specific GABA-activated single-channel molecules with studies including all populations of GABA(A) channels in the neuron it becomes possible to understand the basic mechanism of neuronal inhibition and how it is modulated by pharmacological agents. We use the patch-clamp technique to study the functional properties of the GABA(A) channels in alive neurons in hippocampal brain slices and record the single-channel and whole-cell currents. We further examine how the channels are affected by different GABA concentrations, other drugs and intra and extracellular factors. For detailed theoretical and practical description of the patch-clamp method please see The Single-Channel Recordings edited by B Sakman and E Neher.

  • 35.
    Kampf, Caroline
    et al.
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylär och morfologisk patologi.
    Olsson, Ingmarie
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylär och morfologisk patologi.
    Ryberg, Urban
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylär och morfologisk patologi.
    Sjöstedt, Evelina
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylär och morfologisk patologi.
    Pontén, Fredrik
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylär och morfologisk patologi.
    Production of tissue microarrays, immunohistochemistry staining and digitalization within the human protein atlas2012Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 63, artikel-id e3620Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The tissue microarray (TMA) technology provides the means for high-throughput analysis of multiple tissues and cells. The technique is used within the Human Protein Atlas project for global analysis of protein expression patterns in normal human tissues, cancer and cell lines. Here we present the assembly of 1 mm cores, retrieved from microscopically selected representative tissues, into a single recipient TMA block. The number and size of cores in a TMA block can be varied from approximately forty 2 mm cores to hundreds of 0.6 mm cores. The advantage of using TMA technology is that large amount of data can rapidly be obtained using a single immunostaining protocol to avoid experimental variability. Importantly, only limited amount of scarce tissue is needed, which allows for the analysis of large patient cohorts (1 2). Approximately 250 consecutive sections (4 μm thick) can be cut from a TMA block and used for immunohistochemical staining to determine specific protein expression patterns for 250 different antibodies. In the Human Protein Atlas project, antibodies are generated towards all human proteins and used to acquire corresponding protein profiles in both normal human tissues from 144 individuals and cancer tissues from 216 different patients, representing the 20 most common forms of human cancer. Immunohistochemically stained TMA sections on glass slides are scanned to create high-resolution images from which pathologists can interpret and annotate the outcome of immunohistochemistry. Images together with corresponding pathology-based annotation data are made publically available for the research community through the Human Protein Atlas portal (www.proteinatlas.org) (Figure 1) (3 4). The Human Protein Atlas provides a map showing the distribution and relative abundance of proteins in the human body. The current version contains over 11 million images with protein expression data for 12.238 unique proteins, corresponding to more than 61% of all proteins encoded by the human genome.

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  • 36.
    Karlgren, Anna
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för evolution, genomik och systematik, Evolutionär funktionsgenomik.
    Carlsson, Jenny
    Gyllenstrand, Niclas
    Lagercrantz, Ulf
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för evolution, genomik och systematik, Evolutionär funktionsgenomik.
    Sundström, Jens
    Non-radioactive in situ hybridization protocol applicable for Norway spruce and a range of plant species.2009Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 26Artikel i tidskrift (Refereegranskat)
  • 37. Katchy, Anne
    et al.
    Williams, Cecilia
    Profiling of estrogen-regulated microRNAs in breast cancer cells.2014Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 84, artikel-id e51285Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ERα, and ERβ. ERα is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ERα's regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ERα-positive breast cancer cell lines were compared before and after estrogen-activation using both the µParaflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations, and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

  • 38.
    Kraus, Robert H. S.
    et al.
    Wageningen University, The Netherlands.
    van Hooft, Pim
    Wageningen University, The Netherlands.
    Waldenström, Jonas
    Linnéuniversitetet, Fakultetsnämnden för naturvetenskap och teknik, Institutionen för naturvetenskap, NV.
    Latorre-Margalef, Neus
    Linnéuniversitetet, Fakultetsnämnden för naturvetenskap och teknik, Institutionen för naturvetenskap, NV.
    Ydenberg, Ronald C.
    Wageningen University, The Netherlands ; Simon Fraser University, Canada.
    Prins, Herbert H. T.
    Wageningen University, The Netherlands.
    Avian influenza surveillance with FTA cards: field methods, biosafety, and transportation issues solved2011Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 54, artikel-id 2832Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Avian Influenza Viruses (AIVs) infect many mammals, including humans(1). These AIVs are diverse in their natural hosts, harboring almost all possible viral subtypes(2). Human pandemics of flu originally stem from AIVs(3). Many fatal human cases during the H5N1 outbreaks in recent years were reported. Lately, a new AIV related strain swept through the human population, causing the 'swine flu epidemic'(4). Although human trading and transportation activity seems to be responsible for the spread of highly pathogenic strains(5), dispersal can also partly be attributed to wild birds(6, 7). However, the actual reservoir of all AIV strains is wild birds. In reaction to this and in face of severe commercial losses in the poultry industry, large surveillance programs have been implemented globally to collect information on the ecology of AIVs, and to install early warning systems to detect certain highly pathogenic strains(8-12). Traditional virological methods require viruses to be intact and cultivated before analysis. This necessitates strict cold chains with deep freezers and heavy biosafety procedures to be in place during transport. Long-term surveillance is therefore usually restricted to a few field stations close to well equipped laboratories. Remote areas cannot be sampled unless logistically cumbersome procedures are implemented. These problems have been recognised(13, 14) and the use of alternative storage and transport strategies investigated (alcohols or guanidine)(15-17). Recently, Kraus et al.(18) introduced a method to collect, store and transport AIV samples, based on a special filter paper. FTA cards(19) preserve RNA on a dry storage basis(20) and render pathogens inactive upon contact(21). This study showed that FTA cards can be used to detect AIV RNA in reverse-transcription PCR and that the resulting cDNA could be sequenced and virus genes and determined. In the study of Kraus et al.(18) a laboratory isolate of AIV was used, and samples were handled individually. In the extension presented here, faecal samples from wild birds from the duck trap at the Ottenby Bird Observatory (SE Sweden) were tested directly to illustrate the usefulness of the methods under field conditions. Catching of ducks and sample collection by cloacal swabs is demonstrated. The current protocol includes up-scaling of the work flow from single tube handling to a 96-well design. Although less sensitive than the traditional methods, the method of FTA cards provides an excellent supplement to large surveillance schemes. It allows collection and analysis of samples from anywhere in the world, without the need to maintaining a cool chain or safety regulations with respect to shipping of hazardous reagents, such as alcohol or guanidine.

  • 39.
    Kürten, Charlotte
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Syren, Per-Olof
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes2016Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 107, artikel-id e53168Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.

  • 40.
    Luo, Zhengkang
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Thorvaldson, Lina
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Blixt, Martin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Singh, Kailash
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk cellbiologi.
    Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry2019Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 144, artikel-id e58848Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

  • 41. Nicholas, Sarah
    et al.
    Thyselius, Malin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Fysiologi.
    Holden, Marissa
    Nordström, Karin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap, Fysiologi.
    Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies.2018Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 135, artikel-id e57711Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    With an estimated 6000 species worldwide, hoverflies are ecologically important as alternative pollinators to domesticated honeybees. However, they are also a useful scientific model to study motion vision and flight dynamics in a controlled laboratory setting. As the larvae develop in organically polluted water, they are useful models for investigating investment in microbial immunity. While large scale commercial breeding for agriculture already occurs, there are no standardized protocols for maintaining captive populations for scientific studies. This is important as commercial captive breeding programs focusing on mass output during peak pollination periods may fail to provide a population that is consistent, stable and robust throughout the year, as is often needed for other research purposes. Therefore, a method to establish, maintain and refresh a captive research population is required. Here, we describe the utilization of an artificial hibernation cycle, in addition to the nutritional and housing requirements, for long term maintenance of Eristalis tenax. Using these methods, we have significantly increased the health and longevity of captive populations of E. tenax compared to previous reports. We furthermore discuss small scale rearing methods and options for optimizing yields and manipulating population demographics.

  • 42.
    Nilvebrant, Johan
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Alm, Tove
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Orthogonal protein purification facilitated by a small bispecific affinity tag2012Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 59, s. 1-5Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization. To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used. The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions. The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies. Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding, were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A, a small, bispecific molecule with affinity for both HSA and the novel target was identified. The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix. This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination.

  • 43.
    Nordling, Sofia
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Nilsson, Bo
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Magnusson, Peetra
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    A novel in vitro model for studying the interactions between human whole blood and endothelium2014Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, Vol. 21, nr 93, artikel-id e52112Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel. We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNFα) and found the presence of CD16(+) cells to be significantly higher on TNFα stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

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  • 44.
    Nyström, Sofie
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Bäck, Marcus
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Nilsson, Peter
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Hammarström, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging2017Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 128, artikel-id e56279Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimers and Parkinsons disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive beta-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

  • 45.
    Otterbring, Tobias
    et al.
    Karlstads universitet, Fakulteten för humaniora och samhällsvetenskap (from 2013), Centrum för tjänsteforskning (from 2013). Karlstads universitet, Fakulteten för humaniora och samhällsvetenskap (from 2013), Institutionen för sociala och psykologiska studier (from 2013). Aarhus University, Denmark.
    Wästlund, Erik
    Karlstads universitet, Fakulteten för humaniora och samhällsvetenskap (from 2013), Centrum för tjänsteforskning (from 2013). Karlstads universitet, Fakulteten för humaniora och samhällsvetenskap (from 2013), Institutionen för sociala och psykologiska studier (from 2013).
    Shams, Poja
    Karlstads universitet, Fakulteten för humaniora och samhällsvetenskap (from 2013), Centrum för tjänsteforskning (from 2013). Karlstads universitet, Fakulteten för humaniora och samhällsvetenskap (from 2013), Handelshögskolan (from 2013).
    Spotlighting Customers' Visual Attention at the Stock, Shelf and Store Levels with the 3S Model2019Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 147, s. 1-6, artikel-id e58846Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Several models of the in-store search process exist in the fields of retailing, marketing, and consumer-based research. The present article presents a new conceptualization of this search process, which captures customers' visual attention at three distinct levels of analysis: Stock, Shelf, and Store. We refer to this conceptualization as the 3S Model and illustrate its usefulness through three eye-tracking studies, one from each level of analysis. Our experimental examples, which range from manipulating certain stimuli on a single product (e.g., the placement of textual and pictorial packaging elements) to manipulating the entire shopping trip for customers during their stay in a store (e.g., through more or less specific shopping tasks), highlight the broad applicability of this alternative approach for understanding customers' in-store search behavior. Thus, our model can be seen as a helpful tool for researchers interested in how to conduct experimental eye-tracking studies that shed light on the perceptual processes preceding product choices and purchase decisions. The 3S Model is equally suitable in controlled lab conditions and under ecologically valid settings in the real retail environment. Furthermore, it can be used from the micro level, with a focus on the meaningful metrics on a particular product, through the intermediate level, with the emphasis on the area surrounding products in shelves and other in-store spaces, all the way to the macro level, examining customers' navigational paths throughout a store as a function of their shopping tasks, cognitive capacity, or ability to acquire in-store information.

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  • 46.
    Padhan, Narendra
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method2018Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 139, artikel-id e56794Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pl), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

  • 47.
    Panaliappan, Tamilarasan K.
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Slekiene, Lina
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM). Department of Histology and Embryology, Medical Academy, Lithuanian University of Health Sciences, Lithuania.
    Gunhaga, Lena
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    CAM-Delam Assay to Score Metastatic Properties by Quantifying Delamination and Invasion Capacity of Cancer Cells2022Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, Vol. 2022, nr 184, artikel-id e64025Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The major cause of cancer-related deaths is metastasis formation (i.e., when cancer cells spread from the primary tumor to distant organs and form secondary tumors). Delamination, defined as the degradation of the basal lamina and basement membrane, is the initial process that facilitates the transmigration and spread of cancer cells to other tissues and organs. Scoring the delamination capacity of cancer cells would indicate the metastatic potential of these cells. We have developed a standardized method, the ex ovo CAM-Delam assay, to visualize and quantify the ability of cancer cells to delaminate and invade, thereby being able to assess metastatic aggressiveness. Briefly, the CAM-Delam method includes seeding cancer cells in silicone rings on the chick chorioallantoic membrane (CAM) at embryonic day 10, followed by incubation from hours to a few days. The CAM-Delam assay includes the use of an internal humidified chamber during chick embryo incubation. This novel approach increased embryo survival from 10%-50% to 80%-90%, which resolved previous technical problems with low embryo survival rates in different CAM assays. Next, the CAM samples with associated cancer cell clusters were isolated, fixed, and frozen. Finally, cryostat-sectioned samples were visualized and analyzed for basement membrane damage and cancer cell invasion using immunohistochemistry. By evaluating various known metastatic and non-metastatic cancer cell lines designed to express green fluorescent protein (GFP), the CAM-Delam quantitative results showed that the delamination capacity patterns reflect metastatic aggressiveness and can be scored into four categories. Future use of this assay, apart from quantifying delamination capacity as an indication of metastatic aggressiveness, is to unravel the molecular mechanisms that control delamination, invasion, the formation of micrometastases, and changes in the tumor microenvironment.

  • 48.
    Qi, Xiaoying
    et al.
    Binzhou Med Univ, Med & Pharm Res Ctr, Yantai, Peoples R China.
    Zhang, Yunyun
    Yantai Zestern Biotech Co LTD, Yantai, Peoples R China.
    Zhang, Yuan
    Binzhou Med Univ, Med & Pharm Res Ctr, Yantai, Peoples R China.
    Ni, Tianhui
    Binzhou Med Univ, Precis Med Res Ctr, Yantai, Shandong, Peoples R China.
    Zhang, Wenfeng
    Yantai Zestern Biotech Co LTD, Yantai, Peoples R China.
    Yang, Chunhua
    Binzhou Med Univ, Med & Pharm Res Ctr, Yantai, Peoples R China.
    Mi, Jia
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Analytisk kemi. Binzhou Med Univ, Med & Pharm Res Ctr, Yantai, Peoples R China.
    Zhang, Jiandi
    Yantai Zestern Biotech Co LTD, Yantai, Peoples R China;Binzhou Med Univ, Precis Med Res Ctr, Yantai, Shandong, Peoples R China.
    Tian, Geng
    Binzhou Med Univ, Med & Pharm Res Ctr, Yantai, Peoples R China.
    High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)2018Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 138, artikel-id e56885Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and tissue level significantly hampers the progress in proteomic research. Results derived from currently available immunoblot techniques are also relative, preventing any efforts to combine independent studies with a large-scale analysis of protein samples. In this study, we demonstrate the process of quantitative dot blot analysis (QDB) to achieve absolute quantification in a high throughput format. Using a commercially available protein standard, we are able to determine the absolute content of capping actin protein, gelsolin-like (CAPG) in protein samples prepared from three different mouse tissues (kidney, spleen, and prostate) together with a detailed explanation of the experimental details. We propose the QDB analysis as a convenient, quantitative, high throughput immunoblot method of absolute quantification of individual proteins at the cellular and tissue level. This method will substantially aid biomarker validation and pathway verification in various areas of biological and biomedical research.

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  • 49.
    Ree, Anbjorn
    et al.
    Univ Oslo, Norway.
    Morrison, India
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Centrum för social och affektiv neurovetenskap. Linköpings universitet, Medicinska fakulteten.
    Olausson, Håkan
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Centrum för social och affektiv neurovetenskap. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Neurofysiologiska kliniken US.
    Sailer, Uta
    Univ Oslo, Norway.
    Heilig, Markus
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Centrum för social och affektiv neurovetenskap. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Närsjukvården i centrala Östergötland, Psykiatriska kliniken inkl beroendekliniken.
    Mayo, Leah
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Centrum för social och affektiv neurovetenskap. Linköpings universitet, Medicinska fakulteten.
    Using Facial Electromyography to Assess Facial Muscle Reactions to Experienced and Observed Affective Touch in Humans2019Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 145, artikel-id e59228Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Affective

  • 50.
    Rubino, Stefano
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper, Tillämpad materialvetenskap.
    Melin, Petter
    Spellward, Paul
    Leifer, Klaus
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper, Tillämpad materialvetenskap.
    Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam2014Ingår i: Journal of Visualized Experiments, E-ISSN 1940-087X, nr 89, artikel-id e51463Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms or solutions. We make use of a custom built cryo-transfer station and a modified cryo-SEM preparation chamber2. The spores are taken from a culture, plunge-frozen in a liquid nitrogen slush and observed in the cryo-SEM to select a region of interest. A thin lamella is then extracted using the FIB, attached to a TEM grid and subsequently thinned to electron transparency. The grid is transferred to a cryo-TEM holder and into a TEM for high resolution studies. Thanks to the introduction of a cooled nanomanipulator tip and a cryo-transfer station, this protocol is a straightforward adaptation to cryogenic temperature of the routinely used FIB preparation of TEM samples. As such it has the advantages of requiring a small amount of modifications to existing instruments, setups and procedures; it is easy to implement; it has a broad range of applications, in principle the same as for cryo-TEM sample preparation. One limitation is that it requires skillful handling of the specimens at critical steps to avoid or minimize contaminations.

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