Search

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network. By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules. The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

Chlamydia abortus, the aetiological agent of enzootic abortion of ewes, is a major cause of reproductive loss in small ruminants worldwide, accounting for significant economic losses to the farming industry. Disease can be managed through the use of commercial inactivated or live whole organism-based vaccines, although both have limitations particularly in terms of efficacy, safety and disease-associated outbreaks. Here we report a comparison of two experimental vaccines (chlamydial outer membrane complex (COMC) and octyl glucoside (OG)-COMC) based on detergent extracted outer membrane preparations of C. abortus and delivered as prime-boost immunisations, with the commercial live vaccine Cevac® Chlamydia in a pregnant sheep challenge model. No abortions occurred in either experimental vaccine group, while a single abortion occurred in the commercial vaccine group. Bacterial shedding, as a measure of potential risk of transmission of infection to naïve animals, was lowest in the COMC vaccinated group, with reductions of 87.5%, 86.4% and 74% observed for the COMC, OG-COMC and live commercial vaccine groups, respectively, compared to the unvaccinated challenge control group. The results show that the COMC vaccine performed the best and is a safer efficacious alternative to the commercial vaccines. However, to improve commercial viability, future studies should optimise the antigen dose and number of inoculations required.

Exercise mobilizes angiogenic cells, which stimulate vascular repair. However, limited research suggests exercise-induced increase of endothelial progenitor cell (EPCs) is completely lacking in type 1 diabetes (T1D). Clarification, along with investigating how T1D influences exercise-induced increases of other angiogenic cells (hematopoietic progenitor cells; HPCs) and cell surface expression of chemokine receptor 4 (CXCR4) and 7 (CXCR7), is needed. Thirty T1D patients and 30 matched non-diabetes controls completed 45 min of incline walking. Circulating HPCs (CD34+, CD34+CD45dim) and EPCs (CD34+VEGFR2+, CD34+CD45dimVEGFR2+), and subsequent expression of CXCR4 and CXCR7, were enumerated by flow cytometry at rest and post-exercise. Counts of HPCs, EPCs and expression of CXCR4 and CXCR7 were significantly lower at rest in the T1D group. In both groups, exercise increased circulating angiogenic cells. However, increases was largely attenuated in the T1D group, up to 55% lower, with CD34+ (331 ± 437 Δcells/mL vs. 734 ± 876 Δcells/mL p = 0.048), CD34+VEGFR2+ (171 ± 342 Δcells/mL vs. 303 ± 267 Δcells/mL, p = 0.006) and CD34+VEGFR2+CXCR4+ (126 ± 242 Δcells/mL vs. 218 ± 217 Δcells/mL, p = 0.040) significantly lower. Exercise-induced increases of angiogenic cells is possible in T1D patients, albeit attenuated compared to controls. Decreased mobilization likely results in reduced migration to, and repair of, vascular damage, potentially limiting the cardiovascular benefits of exercise.Trial registration: ISRCTN63739203.

Aims and ObjectivesThe aim of this study was to assess, for the first time in a hard-to-reach population, the risk factors for leg ulceration among PWID, with the objective of making improvements to prevention and care. Background An estimated 4.8 million people globally inject drugs with potential for injecting related harm. Skin and vein damage associated with drug injecting is increasing. Leg ulceration is a chronic condition which in the UK has a prevalence of 15% amongst people who have injected drugs (PWID) compared to 1% in the general population. Glasgow has the highest rate of problematic drug use in Scotland with approximately 13,900 individuals, about 50% of whom are thought to inject. However, the reasons for high prevalence of leg ulceration among PWID are unknown. To support improvements in prevention and care, the dearth of evidence around risk factors for leg ulceration in PWID needs to be addressed.Design A cross-sectional survey of 200 current and former injectors recruited from drugs services in Glasgow, Scotland, to measure skin problems, leg ulceration and injecting habits is reported following STROBE guidelines. Logistic regression modelling examined whether demographics and injecting habits predicted leg ulceration. Results: The likelihood of leg ulceration was increased for those who injected in the groin and the leg. Additionally, injecting in the groin and leg were associated with having a DVT.Conclusion The primary risk factors for leg ulceration in PWID are injecting in the groin and the legs and these are clinically linked to deep vein thrombosis. Injecting into the femoral vein is increasingly common practice for PWID and healthcare practitioners should advise injectors of the increased risk of leg ulceration and DVT and discourage injecting into these areas.

Bacterial transmission from contaminated surfaces via hand contact plays a critical role in disease spread. However, the fomite‐to‐finger transfer efficiency of microorganisms during multiple sequential surface contacts with and without gloves has not been formerly investigated. We measured the quantity of Escherichia coli on fingertips of participants after 1‐8 sequential contacts with inoculated plastic coupons with and without nitrile gloves. A Bayesian approach was used to develop a mechanistic model of pathogen accretion to examine finger loading as a function of the difference between E coli on surfaces and fingers. We used the model to determine the coefficient of transfer efficiency (λ ), and influence of swabbing efficiency and finger area. Results showed that λ for bare skin was higher (49%, 95% CI = 32%‐72%) than for gloved hands (30%, CI = 17%‐49%). Microbial load tended toward a dynamic equilibrium after four and six contacts for gloved hands and bare skin, respectively. Individual differences between volunteers’ hands had a negligible effect compared with use of gloves (P < .01). Gloves reduced loading by 4.7% (CI = −12%‐21%) over bare skin contacts, while 20% of participants accrued more microorganisms on gloved hands. This was due to poor fitting, which created a larger finger surface area than bare hands.

Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since its first use as a vaccine, VACV has been developed as a vector for therapeutic vaccines and as an oncolytic virus. These applications take advantage of VACV's easily manipulated genome and broad host range as an outstanding platform to generate recombinant viruses with a variety of therapeutic applications. Several methods have been developed to generate recombinant VACV, including marker selection methods and transient dominant selection. Here, we present a refinement of a host range selection method coupled with visual identification of recombinant viruses. Our method takes advantage of selective pressure generated by the host antiviral protein kinase R (PKR) coupled with a fluorescent fusion gene expressing mCherry-tagged E3L, one of two VACV PKR antagonists. The cassette, including the gene of interest and the mCherry-E3L fusion is flanked by sequences derived from the VACV genome. Between the gene of interest and mCherry-E3L is a smaller region that is identical to the first ~150 nucleotides of the 3' arm, to promote homologous recombination and loss of the mCherry-E3L gene after selection. We demonstrate that this method permits efficient, seamless generation of rVACV in a variety of cell types without requiring drug selection or extensive screening for mutant viruses.

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been successfully used for identification of protein-protein interactions (PPIs) in mammalian cells. However, requirements of toxic hydrogen peroxide (H(2)O(2)) in APEX-based PL, longer incubation time with biotin (16–24 h), and higher incubation temperature (37 °C) in BioID-based PL severely limit their applications in plants. The recently described TurboID-based PL addresses many limitations of BioID and APEX. TurboID allows rapid proximity labeling of proteins in just 10 min under room temperature (RT) conditions. Although the utility of TurboID has been demonstrated in animal models, we recently showed that TurboID-based PL performs better in plants compared to BioID for labeling of proteins that are proximal to a protein of interest. Provided here is a step-by-step protocol for the identification of protein interaction partners using the N-terminal Toll/interleukin-1 receptor (TIR) domain of the nucleotide-binding leucine-rich repeat (NLR) protein family as a model. The method describes vector construction, agroinfiltration of protein expression constructs, biotin treatment, protein extraction and desalting, quantification, and enrichment of the biotinylated proteins by affinity purification. The protocol described here can be easily adapted to study other proteins of interest in Nicotiana and other plant species.

Staphylococcus aureus is an important bacterial pathogen. This study utilized known staphylococcal epidemiology to track S. aureus between patients, surfaces, staff hands and air in a ten-bed intensive care unit (ICU). Methods: Patients, air and surfaces were screened for total colony counts and S. aureus using dipslides, settle plates and an MAS-100 slit-sampler once a month for 10 months. Data were modelled against proposed standards for air and surfaces, and ICU-acquired staphylococcal infection. Whole-cell genomic typing (WGS) demonstrated possible transmission pathways between reservoirs. Results: Frequently touched sites were more likely to be contaminated (>12 cfu/cm2; p = 0.08). Overall, 235 of 500 (47%) sites failed the surface standard (≤2.5 cfu/cm2); 20 of 40 (50%) passive air samples failed the “Index of Microbial Air” standard (2 cfu/9 cm plate/h), and 15/40 (37.5%) air samples failed the air standard (3). Settle plate data were closer to surface counts than automated air data; the surface count most likely to reflect pass/fail rates for air was 5 cfu/cm2. Surface counts/bed were associated with staphylococcal infection rates (p = 0.012). Of 34 pairs of indistinguishable S. aureus, 20 (59%) showed autogenous transmission, with another four (12%) occurring between patients. Four (12%) pairs linked patients with hand-touch sites and six (18%) linked airborne S. aureus, staff hands and hand-touch sites. Conclusion: Most ICU-acquired S. aureus infection is autogenous, while staff hands and air were rarely implicated in onward transmission. Settle plates could potentially be used for routine environmental screening. ICU staphylococcal infection is best served by admission screening, systematic cleaning and hand hygiene.

Detrusor smooth muscle (DSM) cells present within the urinary bladder wall ultimately facilitate urine storage and voiding. Preparation of the viable, fresh, and isolated DSM cells presents an important technical challenge whose achievement provides optimal cells for subsequent functional and molecular studies. The method developed and elaborated herein, successfully used by our group for over a decade, describes dissection of human urinary bladder specimens obtained from open bladder surgeries followed by an enzymatic two-step treatment of DSM pieces and mechanical trituration to obtain freshly isolated DSM cells. The initial step involves dissection to separate the DSM layer (also known as muscularis propria) from mucosa (urothelium, lamina propria, and muscularis mucosa) and the adjacent connective, vascular, and adipose tissues present. The DSM is then cut into pieces (2–3 mm x 4–6 mm) in nominal Ca2+-containing dissection/digestion solution (DS). DSM pieces are next transferred to and sequentially treated separately with DS containing papain and collagenase at ~37 °C for 30–45 min per step. Following washes with DS containing enzyme-free bovine serum and trituration with a fire-polished pipette, the pieces release single DSM cells. Freshly isolated DSM cells are ideally suited for patch-clamp electrophysiological and pharmacological characterizations of ion channels. Specifically, we show that the TRPM4 channel blocker 9-phenanthrol reduces voltage-step evoked cation currents recorded with the amphotericin-B perforated patch-clamp approach. DSM cells can also be studied by other techniques such as single cell RT-PCR, microarray analysis, immunocytochemistry, in situ proximity ligation assay, and Ca2+ imaging. The main advantage of utilizing single DSM cells is that the observations made relate directly to single cell characteristics revealed. Studies of freshly isolated human DSM cells have provided important insights characterizing the properties of various ion channels including cation-permeable in the urinary bladder and will continue as a gold standard in elucidating DSM cellular properties and regulatory mechanisms.

The lysosomal endoprotease legumain (asparaginyl endoprotease) has been proposed as a putative biomarker in prostate tumours, in which the enzyme is markedly overexpressed. Overexpression, coupled with highly selective specificity for cleavage of substrates at the C-terminus of asparagine (Asn) residues, make legumain an attractive biochemical target for potential diagnosis, prognosis and treatment. We report the design, synthesis, characterisation and preliminary evaluation of a new rhodamine-B (Rho-B)-labelled legumain peptide substrate probe5[Rho-Pro-Ala-Asn-PEG-AQ(4-OH)] and its selective targeting to lysosomes in PC3 prostate cancer cells. Probe5was efficiently activated by recombinant human legumain to afford the high quantum yield reporter fluorophore tripeptide4b(Rho-Pro-Ala-Asn-OH) with concomitant release of intense fluorescence. Furthermore, probe5was activated upon incubation with homogenates derived from fresh-frozen tissue material of prostatectomy specimens. Probe5represents a new viable biochemical tool for probing the activity of legumain with the potential to be used in ex vivo diagnostics in the cancer pathology laboratory.

The complexity of data generated by mass cytometry has necessitated new tools to rapidly visualize analytic outcomes. Clustering methods like Cytosplore or FlowSOM are used for the visualization and identification of cell clusters. For downstream analysis, a newly developed R package, Cytofast, can generate a rapid visualization of results from clustering methods. Cytofast takes into account the phenotypic characterization of cell clusters, calculates the cell cluster abundance, then quantitatively compares groups. This protocol explains the applications of Cytofast to the use of mass cytometry data based on modulation of the immune system in the tumor microenvironment (i.e., the natural killer [NK] cell response) upon tumor challenge followed by immunotherapy (PD-L1 blockade). Demonstration of the usefulness of Cytofast with FlowSOM and Cytosplore is shown. Cytofast rapidly generates visual representations of group-related immune cell clusters and correlations with immune system composition. Differences are observed in the clustering analysis, but separation between groups are visible with both clustering methods. Cytofast visually shows the patterns induced by PD-L1 treatment that include a higher abundance of activated NK cell subsets, expressing a higher intensity of activation markers (i.e., CD54 or CD11c).

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. Meningococcus is a transient colonizer of human nasopharynx of approximately 10% of healthy subject. In particular circumstances, it acquires an invasive ability to penetrate the mucosal barrier and invades the bloodstream causing septicaemia. In the latest case, fulminating sepsis could arise even without the consequent development of meningitis. Conversely, bacteria could poorly multiply in the bloodstream, cross the blood brain barrier, reach the central nervous system, leading to fulminant meningitis. The murine models of bacterial meningitis represent a useful tool to investigate the host-pathogen interactions and to analyze the pathogenetic mechanisms responsible for this lethal disease. Although, several experimental model systems have been evaluated over the last decades, none of these were able to reproduce the characteristic pathological events of meningococcal disease. In this experimental protocol, we describe a detailed procedure for the induction of meningococcal meningitis in a mouse model based on the intracisternal inoculation of bacteria. The peculiar signs of human meningitis were recorded in the murine host through the assessment of clinical parameters (e.g., temperature, body weight), evaluation of survival rate, microbiological analysis and histological examination of brain injury. When using intracisternal (i.cist.) inoculum, meningococci complete delivery directly into cisterna magna, leading to a very efficient meningococcal replication in the brain tissue. A 1,000-fold increase of viable count of bacteria is observed in about 18 h. Moreover, meningococci are also found in the spleen, and liver of infected mice, suggesting that the liver may represent a target organ for meningococcal replication.

Background The purpose of this paper is to highlight the range of cleaning practices and processes in 11 Australian hospitals and to discuss the challenges this variation poses to the implementation of clinical trials or changes to hospital cleaning practices. Methods A cross-sectional study design was used to determine cleaning practices and processes in hospitals participating in the ‘Researching Effective Approaches to Cleaning in Hospitals' (REACH) study. A standardised template and approach was used to collect information. Data collection activities included structured on-site discussions, a review of hospital practices and a document review of policy and procedural documents related to cleaning. Results Variations in the auditing process used to evaluate environmental cleanliness, cleaning practices, product use, training and communication pathways available to cleaning staff were identified. There was also variation in workforce structure and responsibilities for cleaning. Conclusion This paper is the first to describe the differences in cleaning practices between Australian hospitals. The variations identified present a number of challenges for the conduct of research and have important implications for both monitoring of and standards for cleanliness. These challenges include implementing a practice change or cleaning study where hospitals have different processes, practices and structures.

Biotin is an attractive post-translational modification of proteins that provides a powerful tag for the isolation and detection of protein. Enzymatic biotinylation by the E. coli biotin-protein ligase BirA is highly specific and allows for the biotinylation of target proteins in their native environment; however, the current usage of BirA mediated biotinylation requires the presence of a synthetic acceptor peptide (AP) in the target protein. Therefore, its application is limited to proteins that have been engineered to contain the AP. The purpose of the present protocol is to use the bacterial display of a peptide derived from an unmodified target protein to select for BirA variants that biotinylates the peptide. The system is based on a single plasmid that allows for the co-expression of BirA variants along with a scaffold for the peptide display on the bacterial surface. The protocol describes a detailed procedure for the incorporation of the target peptide into the display scaffold, creation of the BirA library, selection of active BirA variants and initial characterization of the isolated BirA variants. The method provides a highly effective selection system for the isolation of novel BirA variants that can be used for the further directed evolution of biotin-protein ligases that biotinylate a native protein in complex solutions.

Background: Staphylococcus aureus remains an important bacterial pathogen worldwide. This study utilized known staphylococcal epidemiology to track S. aureus between different ecological reservoirs in one ten-bed intensive care unit (ICU).Methods: Selected hand-touch surfaces, staff hands and air were systematically screened ten times during ten months, with patients screened throughout the study. S. aureus isolates were subjected to spa typing and epidemiological analyses, followed by wholegenome sequencing (WGS) to provide single nucleotide polymorphism (SNP) data.Results: Multiple transmission pathways between patients and reservoirs were investigated. There were 34 transmission events, of which 29 were highly related (

BackgroundNow that cleaning and decontamination are recognised as integral to infection control, it is timely to examine the process in more detail. This is because cleaning practices vary widely within healthcare districts and it is likely that both time and energy are needlessly wasted with ill-defined duties. Furthermore, inadequate cleaning will not reduce the infection risk but may even enhance it. The process would benefit from a systematic appraisal, with each component placed within an evidence-based and ordered protocol.MethodsA literary search was performed on ‘hospital cleaning’, with focus on manual aspects of cleaning, pathogen reservoirs and transmission, hand-hygiene, staff responsibilities and patient comfort.ResultsThere were no articles providing an evidence-based practical approach to systematic cleaning in hospitals. This review therefore proposes a simple four-step guide for daily cleaning of the occupied bed space. STEP 1 (Look) describes a visual assessment of the area to be cleaned; STEP 2 (Plan) argues why the bed space needs preparation before cleaning; STEP 3 (Clean) covers surface cleaning/decontamination; and STEP 4 (Dry) is the final stage whereby surfaces are allowed to dry.ConclusionGiven the lack of articles providing practical cleaning guidance, this review proposes a four-step protocol based on evidence if available, or justified where not. Each step is presented, discussed and risk-assessed. It is likely that a systematic cleaning process would reduce the risk of healthcare-acquired infection for everyone, including outbreaks, in addition to heightened confidence in overall quality of care.

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

Background: Breast cancer diagnosis may be a teachable moment for lifestyle behaviour change and to prevent adjuvant therapy associated weight gain. We assessed the acceptability and effectiveness of two weight control programmes initiated soon after breast cancer diagnosis to reduce weight amongst overweight or obese women and prevent gains in normal-weight women.Methods: Overweight or obese (n = 243) and normal weight (n = 166) women were randomised to a three-month unsupervised home (home), a supervised community weight control programme (community) or to standard written advice (control). Primary end points were change in weight and body fat at 12 months. Secondary end points included change in insulin, cardiovascular risk markers, quality of life and cost-effectiveness of the programmes.Results: Forty-three percent of eligible women were recruited. Both programmes reduced weight and body fat: home vs. control mean (95% CI); weight −2.3 (−3.5, −1.0) kg, body fat −1.6 (−2.6, −0.7) kg, community vs. control; weight −2.4 (−3.6, −1.1) kg, body fat −1.4 (−2.4, −0.5) kg (all p Conclusions: The programmes were equally effective for weight control, but the community programme had additional benefits.

Background Chlamydia trachomatis (C. trachomatis) is the most common bacterial sexually transmitted infection in the UK. Recent studies suggest that in addition to the genital tract, C. trachomatis is found in the throat and rectum, suggesting the number of infections is under-reported. There is an urgent need to study the impact of extending diagnosis to include extra-genital samples; however, there is a lack of evidence on the acceptability of asking young women to provide these samples. Method A mixed methods single group feasibility study explored the acceptability of combined genital and extra-genital testing in young women aged 16–25 years consecutively attending a sexual health centre in Edinburgh, Scotland. Young women were asked to complete a self- administered anonymous questionnaire whether they would be willing to give self-taken throat and ano-rectal samples. Interviews with women (n = 20) willing to self-sample were conducted before and after self-sampling, and these explored the underlying reasons behind their decision, and feelings about the tests. Results Of 500 women recruited to the study, 422 (84.4%) women provided sufficient data for analysis. From completed questionnaires, 86.3% of respondents reported willingness to self-sample from the throat. Willingness of ano-rectal self-sampling was lower (59.1%), particularly in women under 20 (

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting them for degradation. This system is exploited in many non-plant eukaryotes, such that a target protein, tagged with the AID motif, is degraded upon auxin addition. The level of TIR1 expression is critical; excessive expression leads to degradation of the AID-tagged protein even in the absence of auxin, whereas low expression leads to slow depletion. A β-estradiol-inducible AID system was created, with expression of TIR1 under the control of a β-estradiol inducible promoter. The level of TIR1 is tunable by changing the time of incubation with β-estradiol before auxin addition. This protocol describes how to rapidly deplete a target protein using the AID system. The appropriate β-estradiol incubation time depends on the abundance of the target protein. Therefore, efficient depletion depends on optimal timing that also minimizes auxin-independent depletion.

The cytoskeletal protein vimentin plays a key role in positioning of organelles within the cytosol and has been linked to the regulation of numerous cellular processes including autophagy, however, how vimentin regulates autophagy remains relatively unexplored. Here we report that inhibition of vimentin using the steroidal lactone Withaferin A (WFA) causes vimentin to aggregate, and this is associated with the relocalisation of organelles including autophagosomes and lysosomes from the cytosol to a juxtanuclear location. Vimentin inhibition causes autophagosomes to accumulate, and we demonstrate this results from modulation of mechanistic target of rapamycin (mTORC1) activity, and disruption of autophagosome-lysosome fusion. We suggest that vimentin plays a physiological role in autophagosome and lysosome positioning, thus identifying vimentin as a key factor in the regulation of mTORC1 and autophagy.

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Inflammatory Bowel Disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis, is characterised by chronic inflammation of the gastrointestinal tract. Aetiology involves a combination of genetic and environmental factors resulting in abnormal immune responses to intestinal microbiota. Genetic studies have strongly linked genes involved in autophagy to CD, and genes involved in the unfolded protein response (UPR) to IBD. The UPR is triggered in response to accumulation of misfolded proteins in the endoplasmic reticulum (ER) and autophagy plays a key role to relieve ER-stress and restore homeostasis. This review summarises the known interactions between autophagy and the UPR and discusses the impact of these converging pathways on IBD pathogenesis. With a paucity of effective long-term treatments for IBD, targeting of synergistic pathways may provide novel and more effective therapeutic options.

BackgroundGenetic studies have strongly linked autophagy to Crohn’s disease (CD), and stimulating autophagy in CD patients may be therapeutically beneficial. The aim of this study was to evaluate the effect of current inflammatory bowel disease (IBD) drugs on autophagy and investigate molecular mechanisms of action and functional outcomes in relation to this cellular process.MethodsAutophagy marker LC3 was evaluated by confocal fluorescence microscopy and flow cytometry. Drug mechanism of action was investigated by polymerase chain reaction (PCR) array with changes in signaling pathways examined by immunoblot and quantitative reverse transcription PCR (RT-qPCR). Clearance of adherent-invasive Escherichia coli (AIEC) and levels of pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) were evaluated by gentamicin protection assays and RT-qPCR, respectively. The marker LC3 was analyzed in peripheral blood mononuclear cells (PBMCs) from pediatric patients by flow cytometry.ResultsAzathioprine induces autophagy via mechanisms involving modulation of mechanistic target of rapamycin (mTORC1) signaling and stimulation of the unfolded protein response (UPR) sensor PERK. Induction of autophagy with azathioprine correlated with the enhanced clearance of AIEC and dampened AIEC-induced increases in TNFα. Azathioprine induced significant increase in autophagosome bound LC3-II in PBMC populations ex vivo, supporting in vitro findings. In patients, the CD-associated ATG16L1 T300A single-nucleotide polymorphism did not attenuate azathioprine induction of autophagy.ConclusionsModulation of autophagy via mTORC1 and the UPR may contribute to the therapeutic efficacy of azathioprine in IBD.

Purpose: Dietary nitrate (NO3-) has repeatedly been shown to improve endurance and intermittent, high-intensity events in temperate conditions. However, the ergogenic effects of dietary NO3- on intermittent exercise performance in hot conditions has yet to be investigated. Methods: In a randomised, counterbalanced, double-blind crossover study, twelve 5recreationally trained males ingested a nitrate-rich beetroot juice shot (BRJ) (6.2 mmol NO3-) or a nitrate-depleted placebo (PLA) (0.05). There was a reduced peak (BRJ: 659±100W vs. PLA: 693±139W; p=0.056) and mean power (BRJ: 543±29W vs PLA: 575±38W; p=0.081) following BRJ compared to PLA in the hot and humid condition, but this was not statistically significant. There was no effect of supplement on total work done irrespective of environmental 69 condition. However, ~75% of participants experienced performance decreases following BRJ 70 in the hot and humid environment. No differences were observed between trials for tympanic 71 temperature measured at the conclusion of the exercise trial. Conclusion: In conclusion, an acute dose of inorganic dietary NO3- does not improve repeated sprint performance in either temperate, or hot and humid conditions.

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world’s population is believed to be infected with Mycobacterium tuberculosis. Despite decades of research, many of the mechanisms behind the success of M. tuberculosis as a pathogenic organism remain to be investigated, and the development of safer, more effective antimycobacterial drugs are urgently needed to tackle the rise and spread of drug resistant tuberculosis. However, the progression of tuberculosis research is bottlenecked by traditional mammalian infection models that are expensive, time consuming, and ethically challenging. Previously we established the larvae of the insect Galleria mellonella (greater wax moth) as a novel, reproducible, low cost, high-throughput and ethically acceptable infection model for members of the M. tuberculosis complex. Here we describe the maintenance, preparation, and infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Using this infection model, mycobacterial dose dependent virulence can be observed, and a rapid readout of in vivo mycobacterial burden using bioluminescence measurements is easily achievable and reproducible. Although limitations exist, such as the lack of a fully annotated genome for transcriptomic analysis, ontological analysis against genetically similar insects can be carried out. As a low cost, rapid, and ethically acceptable model for tuberculosis, G. mellonella can be used as a pre-screen to determine drug efficacy and toxicity, and to determine comparative mycobacterial virulence prior to the use of conventional mammalian models. The use of the G. mellonella-mycobacteria model will lead to a reduction in the substantial number of animals currently used in tuberculosis research.

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2-D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3-D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

10 páginas, 4 figuras., Successful mammalian pregnancies are a result of complex physiological, endocrinological, and immunological processes that combine to create an environment where the mother is tolerant to the semi-allogeneic fetus. Our knowledge of the mechanisms that contribute to maternal tolerance is derived mainly from human and murine studies of haemochorial placentation. However, as this is the most invasive type of placentation it cannot be assumed that identical mechanisms apply to the less invasive epitheliochorial placentation found in other species such as ruminants. Here, we examine three features associated with reproductive immune regulation in a transformed ovine trophoblast cell line and ex-vivo ovine reproductive tissues collected at term, namely: major histocompatibility complex (MHC) expression, Indoleamine 2,3 dioxygenase-1 (IDO-1) expression, and Natural Killer (NK) cell infiltration. High levels of MHC class I protein expression were detected at the surface of the trophoblast cell line using a pan-MHC class I specific monoclonal antibody. The majority of MHC class I transcripts isolated from the cell line clustered with classical MHC alleles. Transcriptional analysis of placental tissues identified only classical MHC class I transcripts. We found no evidence of constitutive transcription of IDO-1 in either the trophoblast cell line or placental tissues. Ex-vivo tissues collected from the materno-fetal interface were negative for cells expressing NKp46/NCR1. Collectively, these observations suggest that the relatively non-invasive synepitheliochorial placentation found in sheep has a more limited requirement for local immunoregulation compared to the more invasive haemochorial placentation of primates and rodents., SW, NW, CM, KB, JP-A, and GE were funded by the Scottish Government Rural and Environment Science and Analytical Services (RESAS) Strategic Research Programme (SRP). SW, CM, and GE also received funding from The route to identification of immunological correlates of protection in ruminants Industrial Partnership Award with Bio-Rad Laboratories (grant numbers BB/I019863/1; BB/I020519/1). KB and GE also receive funding the European Union’s Horizon 2020 research and innovation programme under grant agreement N◦731014 (VetBioNet).

Summary Background A previous study among neonatal intensive care unit (NICU) nurses showed that the antibacterial efficacy of alcohol-based handrubs (ABHR) can be achieved in 15 s instead of 30 s with a significant increase in the frequency of hand antisepsis. This study aimed to examine 15-s vs 30-s antisepsis performance by measuring microbial load on fingertips and compliance among nurses in a low-risk gynaecological ward. Methods An independent trained observer monitored the frequency and compliance with hand antisepsis during shifts in a crossover design. Fingertips including thumbs were rinsed in soy broth before hand rubbing at the beginning of a shift and then hourly to determine the bacterial load. Performance activity was assigned to the contamination class of the Fulkerson scale. Immediately before the lunch break, volunteers cleaned their hands for a randomly determined application time of 15 or 30 s. Results Examination of bacterial load on fingertips revealed no difference between 15 vs 30 s application time. Controlled hand antisepsis before the lunch break also showed no difference in efficacy for either test series. Participants rubbing for 15 s were more likely to perform hand antisepsis compared with those rubbing for 30 s (P=0.2). The compliance increased from 54.7% to 69.5% in the 15-s trial. Discussion Shortening the duration for hand antisepsis did not decrease efficacy. Shortening the application time to 15 s should be considered within the critical components of a successful multimodal intervention strategy to improve hand-hygiene compliance in clinical practice.

© 2019 Journal of Visualized Experiments., The association of Zika virus (ZIKV) infection with neurological complications during the recent worldwide outbreak and the lack of approved vaccines and/or antivirals have underscored the urgent need to develop ZIKV reverse genetic systems to facilitate the study of ZIKV biology and the development of therapeutic and/or prophylactic approaches. However, like with other flaviviruses, the generation of ZIKV full-length infectious cDNA clones has been hampered due to the toxicity of viral sequences during its amplification in bacteria. To overcome this problem, we have developed a nontraditional approach based on the use of bacterial artificial chromosomes (BACs). Using this approach, the full-length cDNA copy of the ZIKV strain Rio Grande do Norte Natal (ZIKV-RGN) is generated from four synthetic DNA fragments and assembled into the single-copy pBeloBAC11 plasmid under the control of the human cytomegalovirus (CMV) immediate-early promoter. The assembled BAC cDNA clone is stable during propagation in bacteria, and infectious recombinant (r)ZIKV is recovered in Vero cells after transfection of the BAC cDNA clone. The protocol described here provides a powerful technique for the generation of infectious clones of flaviviruses, including ZIKV, and other positive-strand RNA viruses, particularly those with large genomes that have stability problems during bacterial propagation., This work was supported in part by grants from the Spanish Ministry of Economy and Competitiveness (MINECO, grant number BFU2016-79127-R) to F.A.T. and the National Institutes of Health (NIH, grant number 1R21AI120500) to L.M.S. and F.A.T.

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling. ispartof: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS issue:143 ispartof: location:United States status: published

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90° angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS., This work was supported by grants from the Spanish Ministerio de Economía y Competitividad (MINECO), Plan Nacional de Investigación Científica (SAF2016-77561-R to M.I., which was in part granted with FEDER-EC funding).

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Populational analyses of the morphological and functional alteration of endocytic proteins are challenging due to the demand of image capture at a single cell level and statistical image analysis at a populational level. To overcome this difficulty, we used imaging flow cytometry and transcriptomic profiling (RNA-seq) to determine altered subcellular localization of the cluster of differentiation 1d protein (CD1d) associated with impaired endocytic gene expression in human dendritic cells (DCs), which were exposed to the common lipophilic air pollutant benzo[a]pyrene. The colocalization of CD1d and endocytic marker Lamp1 proteins from thousands of cell images captured with imaging flow cytometry was analyzed using IDEAS and ImageJ-Fiji programs. Numerous cellular images with co-stained CD1d and Lamp1 proteins were visualized after gating on CD1d(+)Lamp1(+) DCs using IDEAS. The enhanced CD1d and Lamp1 colocalization upon BaP exposure was further demonstrated using thresholded scatterplots, tested with Mander's coefficients for co-localized intensity, and plotted based on the percentage of co-localized areas using ImageJ-Fiji. Our data provide an advantageous instrumental and bioinformatic approach to measure protein colocalization at both single and populational cellular levels, supporting an impaired functional outcome of transcriptomic alteration in pollutant-exposed human DCs.

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fcγ receptor (FcγR)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to FcγRs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fcγ receptor- or complement receptor-mediated phagocytosis.

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barre syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.