A metabolic-dysfunction associated steatotic liver acinus biomimetic induces pancreatic islet dysfunction in a coupled microphysiology system.

Preclinical and clinical studies suggest that lipid-induced hepatic insulin resistance is a primary defect that predisposes to dysfunction in pancreatic islets, implicating a perturbed liver-pancreas axis underlying the comorbidity of T2DM and MASLD. To investigate this hypothesis, we developed a human biomimetic microphysiological system (MPS) coupling our vascularized liver acinus MPS (vLAMPS) with primary islets on a chip (PANIS) enabling MASLD progression and islet dysfunction to be quantitatively assessed. The modular design of this system (vLAMPS-PANIS) allows intra-organ and inter-organ dysregulation to be deconvoluted. When compared to normal fasting (NF) conditions, under early metabolic syndrome (EMS) conditions, the standalone vLAMPS exhibited characteristics of early stage MASLD, while no significant differences were observed in the standalone PANIS. In contrast, with EMS, the coupled vLAMPS-PANIS exhibited a perturbed islet-specific secretome and a significantly dysregulated glucose stimulated insulin secretion (GSIS) response implicating direct signaling from the dysregulated liver acinus to the islets. Correlations between several pairs of a vLAMPS-derived and a PANIS-derived secreted factors were significantly altered under EMS, as compared to NF conditions, mechanistically connecting MASLD and T2DM associated hepatic factors with islet-derived GLP-1 synthesis and regulation. Since vLAMPS-PANIS is compatible with patient-specific iPSCs, this platform represents an important step towards addressing patient heterogeneity, identifying complex disease mechanisms, and advancing precision medicine.

Assessment of in vitro colistin susceptibility of carbapenem-resistant clinical Gram-negative bacterial isolates using four commercially available systems & Whole-genome sequencing: A diagnostic accuracy study.

AIM: To analyze the diagnostic utility of commercially available platforms and Whole-genome sequencing (WGS) for accurate determination of colistin susceptibility test results. MATERIAL & METHODS: An exploratory diagnostic accuracy study was conducted in which sixty carbapenem-resistant Gram-negative bacteria were subjected to identification and AST using MALDI-TOF MS & MicroScan walkaway 96 Plus. Additional AST was performed using the BD Phoenix system and Mikrolatest colistin kit. The test isolates were subjected to Vitek-2 and WGS at CRL, Bengaluru. RESULTS: There was no statistically significant agreement between the colistin susceptibility results obtained by WGS, with those of commercial phenotypic platforms. The MicroScan 96 Plus had the highest sensitivity (31 %) & NPV (77 %), and the BD Phoenix system had the highest specificity (97 %) and PPV (50 %), respectively, for determining colistin resistance. CONCLUSION: The utility of WGS as a tool in AMR surveillance and validation of phenotypic AST methods should be explored further.

A rare case of colonic diverticulum in a Bitch.

Abstract. Background: Colonic diverticulum is one of the rare findings in dogs characterized by an out-pouching of mucosal and submucosal layers through the defect in muscularis layer of the colon. Case description: A five years old intact female Labrador was presented with an anamnesis of dyschezia and tenesmus. Findings/treatment and outcome: Rectal examination was normal, and the survey radiograph showed an almost crescent shaped abnormal dilatation (10.52 cm × 6.21 cm) with gas and increased radiopaque material, dorsal to the urinary bladder and ventral to the descending colon suggesting fecal stasis. Ultrasonographic examination revealed gas-filled out-pouching with hyperechoic colon wall and acoustic shadowing. Exploratory celiotomy confirmed the diagnosis of colonic diverticulum, and diverticulectomy was performed. All four layers of the colonic wall were detected histopathologically in the biopsy sample and excluded neoplasia. The dog recovered uneventfully with no post-operative complications. Conclusion: This surgery produced an excellent resolution of clinical signs. To our knowledge, this is one of the few cases of colonic diverticulum reported in dogs.

Novel multidrug-resistant sublineages of Staphylococcus aureus clonal complex 22 discovered in India.

Staphylococcus aureus is a major pathogen in India causing community and nosocomial infections, but little is known about its molecular epidemiology and mechanisms of resistance in hospital settings. Here, we use whole-genome sequencing (WGS) to characterize 478 S. aureus clinical isolates (393 methicillin-resistant Staphylococcus aureus (MRSA) and 85 methicilin-sensitive Staphylococcus aureus (MSSA) collected from 17 sentinel sites across India between 2014 and 2019. Sequencing results confirmed that sequence type 22 (ST22) (142 isolates, 29.7%), ST239 (74 isolates, 15.48%), and ST772 (67 isolates, 14%) were the most common clones. An in-depth analysis of 175 clonal complex (CC) 22 Indian isolates identified two novel ST22 MRSA lineages, both Panton-Valentine leukocidin+, both resistant to fluoroquinolones and aminoglycosides, and one harboring the the gene for toxic shock syndrome toxin 1 (tst). A temporal analysis of 1797 CC22 global isolates from 14 different studies showed that the two Indian ST22 lineages shared a common ancestor in 1984 (95% highest posterior density [HPD]: 1982-1986), as well as evidence of transmission to other parts of the world. Moreover, the study also gives a comprehensive view of ST2371, a sublineage of CC22, as a new emerging lineage in India and describes it in relationship with the other Indian ST22 isolates. In addition, the retrospective identification of a putative outbreak of multidrug-resistant (MDR) ST239 from a single hospital in Bangalore that persisted over a period of 3 years highlights the need for the implementation of routine surveillance and simple infection prevention and control measures to reduce these outbreaks. To our knowledge, this is the first WGS study that characterized CC22 in India and showed that the Indian clones are distinct from the EMRSA-15 clone. Thus, with the improved resolution afforded by WGS, this study substantially contributed to our understanding of the global population of MRSA. IMPORTANCE The study conducted in India between 2014 and 2019 presents novel insights into the prevalence of MRSA in the region. Previous studies have characterized two dominant clones of MRSA in India, ST772 and ST239, using whole-genome sequencing. However, this study is the first to describe the third dominant clone, ST22, using the same approach. The ST22 Indian isolates were analyzed in-depth, leading to the discovery of two new sublineages of hospital-acquired Staphylococcus aureus in India, both carrying antimicrobial resistance genes and mutations, which limit treatment options for patients. One of the newly characterized sublineages, second Indian cluster, carries the tsst-1 virulence gene, increasing the risk of severe infections. The geographic spread of the two novel lineages, both within India and internationally, could pose a global public health threat. The study also sheds light on ST2371 in India, a single-locus variant of ST22. The identification of a putative outbreak of MDR ST239 in a single hospital in Bangalore emphasizes the need for routine surveillance and simple infection prevention and control measures to reduce these outbreaks. Overall, this study significantly contributes to our understanding of the global population of MRSA, thanks to the improved resolution afforded by WGS.

An insight into whole genome sequencing data of methicillin-resistant Staphylococcus aureus circulating in a teaching hospital in North India.

PURPOSE: To provide an insight into the Whole-genome sequencing (WGS) data of MRSA strains circulating in a teaching hospital in north India. METHODS: An exploratory study was conducted in which fifty non-repetitive MRSA isolates obtained from pus samples of inpatients from July 2018 to February 2019 were subjected to preliminary identification (ID) and antibiotic susceptibility testing (AST) at our centre. These isolates were later sent to Central Research Laboratory, India for further testing using the VITEK-2 compact system followed by WGS. Only eighteen isolates were eventually considered for final analysis and the rest (n â€‹= â€‹32) were excluded due to various technical reasons. RESULTS: The WGS confirmed MRSA isolates predominantly belonged to CC22 (56.25%) and CC30 (31.25%). The CC22 MRSA strains carried SCCmec types IVa (77.8%) & IVc (22.2%) and belonged to spa types t005 (44.4%), t4584 (22.2%), t11808 (11.1%), t1328 (11.1%) and t309 (11.1%), respectively. The MRSA isolates of CC30 carried SCCmec types IVa (60%), IVg (20%) & V (20%) and belonged to spa types t021 (80%) & t2575 (20%), respectively. One MRSA isolate carried a novel SCCmec type V. The luk-PV and tsst-1 genes were present in 93.75% and 33.33% of MRSA isolates, respectively. The concordance between the phenotypic and genotypic AST results was 100% for Beta-lactams, Fluoroquinolones, Tetracyclines & Lipoglycopeptides, respectively. CONCLUSIONS: Through this study, we intend to embark upon a relatively newer avenue of clinical-genomic surveillance of nosocomial bacterial isolates like MRSA, which would help us improve the existing infection control and antibiotic stewardship practices in our hospital.

Estimation of baseline IgG antibody levels to 23 pneumococcal vaccine-type capsular polysaccharides in healthy vaccine naïve Indian adults.

Since immunological responses to pneumococcal vaccines are assessed by a fold-increase in antibody levels relative to pre-immunization levels, it is therefore critical to determine baseline antibody levels to establish putative threshold as a measure of normal response. Herein, for the first time, we measured baseline IgG antibody levels in 108 healthy unvaccinated Indian adults using WHO-recommended ELISA. Median baseline IgG concentration ranged between 0.54 µg/mL to 12.35 µg/mL. Highest levels of baseline capsule polysaccharide (cPS)-specific IgG were found against types 14, 19A, and 33F. Whereas, lowest baseline IgG levels were observed against types 3, 4, and 5. Overall, ∼79% of study population had median baseline IgG levels ≥1.3 µg/mL against 74% of cPS's. Substantial baseline antibody levels in unvaccinated adults were observed. The study would be critical in bridging gaps in baseline immunogenicity data and may offer a valuable foundation for evaluating immune response of Indian adults to pneumococcal vaccination.

Dexmedetomidine as an Adjuvant to 0.25% Bupivacaine in Ultrasound Guided Femoral Nerve Block for Preoperative Positioning and Postoperative Analgesia in Patients Undergoing Elective Surgery for Fracture Shaft of Femur.

Background: Femur fracture causes excruciating pain and surgical repair is recommended. To obtain satisfactory patient co-operation in the perioperative period, various analgesics have been used. Femoral nerve block (FNB) provides an excellent alternative for analgesia in the perioperative period. Dexmedetomidine of up to 2 µg.kg-1 has been used in FNB as adjuvants in lower limb surgeries. Aims: The aim was to study the effect of addition of Dexmedetomidine to Bupivacaine in FNB on the comfort of positioning for subarachnoid block (SAB) and postoperative analgesia. Materials and Methods: Prospective, randomized, double-blind design was followed. Seventy American Society of Anesthesiologist I and II patients aged 18-70 years of either gender were randomly allocated into Group B (20 mL 0.25% Bupivacaine + 2 mL Normal Saline) and Group BD (received 20 mL 0.25% Bupivacaine + Dexmedetomidine 2 µg.kg-1 diluted to 2 mL) for FNB. Numerical rating scale (NRS) was recorded before and after FNB and comfort of positioning graded. After 10 min, subarachnoid block (SAB) was administered. NRS was recorded postoperatively until 24 h. Results: The comfort of positioning improved in both the groups after FNB but was statistically not significant when compared among the groups (P = 0.7). Duration of postoperative analgesia was significantly higher in the Group BD (741 min ± 97 min) compared to the Group B (440 min ± 45 min) (P = 0.001) and was statistically significant. Conclusion: FNB improved the comfort of positioning for SAB, but the addition of Dexmedetomidine did not have any added advantages with respect to comfort of positioning. However, the addition of Dexmedetomidine significantly increased the duration of postoperative analgesia with minimal hemodynamic changes.

Emergence of a multidrug-resistant and virulent Streptococcus pneumoniae lineage mediates serotype replacement after PCV13: an international whole-genome sequencing study.

BACKGROUND: Serotype 24F is one of the emerging pneumococcal serotypes after the introduction of pneumococcal conjugate vaccine (PCV). We aimed to identify lineages driving the increase of serotype 24F in France and place these findings into a global context. METHODS: Whole-genome sequencing was performed on a collection of serotype 24F pneumococci from asymptomatic colonisation (n=229) and invasive disease (n=190) isolates among individuals younger than 18 years in France, from 2003 to 2018. To provide a global context, we included an additional collection of 24F isolates in the Global Pneumococcal Sequencing (GPS) project database for analysis. A Global Pneumococcal Sequence Cluster (GPSC) and a clonal complex (CC) were assigned to each genome. Phylogenetic, evolutionary, and spatiotemporal analysis were conducted using the same 24F collection and supplemented with a global collection of genomes belonging to the lineage of interest from the GPS project database (n=25 590). FINDINGS: Serotype 24F was identified in numerous countries mainly due to the clonal spread of three lineages: GPSC10 (CC230), GPSC16 (CC156), and GPSC206 (CC7701). GPSC10 was the only multidrug-resistant lineage. GPSC10 drove the increase in 24F in France and had high invasive disease potential. The international dataset of GPSC10 (n=888) revealed that this lineage expressed 16 other serotypes, with only six included in 13-valent PCV (PCV13). All serotype 24F isolates were clustered in a single clade within the GPSC10 phylogeny and long-range transmissions were detected from Europe to other continents. Spatiotemporal analysis showed GPSC10-24F took 3-5 years to spread across France and a rapid change of serotype composition from PCV13 serotype 19A to 24F during the introduction of PCV13 was observed in neighbouring country Spain. INTERPRETATION: Our work reveals that GPSC10 alone is a challenge for serotype-based vaccine strategy. More systematic investigation to identify lineages like GPSC10 will better inform and improve next-generation preventive strategies against pneumococcal diseases. FUNDING: Bill & Melinda Gates Foundation, Wellcome Sanger Institute, and the US Centers for Disease Control and Prevention.

Molecular characterisation of carbapenem-resistant Klebsiella pneumoniae clinical isolates: preliminary experience from a tertiary care teaching hospital in the Himalayas.

BACKGROUND: There is a lack of whole-genome sequencing (WGS) data on multidrug-resistant (MDR) bacteria from the Uttarakhand region of India. The aim of this study was to generate WGS data of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates recovered from patients in Uttarakhand's tertiary care centre. METHODS: A cross-sectional study included 29 MDR K. pneumoniae test isolates obtained from various clinical samples submitted to the bacteriology laboratory for culture and sensitivity testing from July 2018 to August 2019. After preliminary identification and antibiotic susceptibility testing, these isolates were subjected to WGS. RESULTS: A total of 27 of 29 isolates were CRKP. ST14 was the most common sequence type (n=8 [29.6%]). Carbapenem resistance was mainly encoded by OXA-48-like genes (21/27 [77.8%]). All isolates had a varied arsenal of resistance genes to different antibiotic classes. KL2 (9/27 [33.3%]) and KL51 (8/27 [29.6%]) were dominant K loci types. O1 and O2 together accounted for 88.9% (n=27) of CRKP isolates. Genes encoding yersiniabactin (ybt) and aerobactin (iuc) were identified in 88.9% (24/27) and 29.6% (8/27) of isolates. The predominant plasmid replicons present were ColKP3 (55.5%), IncFII(K) (51.8%) and IncFIB(pQil) (44.4%). CONCLUSIONS: This study emphasises the need for continued genomic surveillance of MDR bacteria that could be instrumental in developing treatment guidelines based on integrating phenotypic and molecular methods.

Implementing Whole-Genome Sequencing for Ongoing Surveillance of Antimicrobial Resistance: Exemplifying Insights Into Klebsiella pneumoniae.

In this Supplement, we detail outputs of the National Institute for Health Research Global Health Research Unit on Genomic Surveillance of Antimicrobial Resistance project, covering practical implementation of whole-genome sequencing across our consortium, which consists of laboratories in Colombia, India, Nigeria, and the Philippines.

High-Resolution Genomic Profiling of Carbapenem-Resistant Klebsiella pneumoniae Isolates: A Multicentric Retrospective Indian Study.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a threat to public health in India because of its high dissemination, mortality, and limited treatment options. Its genomic variability is reflected in the diversity of sequence types, virulence factors, and antimicrobial resistance (AMR) mechanisms. This study aims to characterize the clonal relationships and genetic mechanisms of resistance and virulence in CRKP isolates in India. MATERIALS AND METHODS: We characterized 344 retrospective K. pneumoniae clinical isolates collected from 8 centers across India collected in 2013-2019. Susceptibility to antibiotics was tested with VITEK 2. Capsular types, multilocus sequence type, virulence genes, AMR determinants, plasmid replicon types, and a single-nucleotide polymorphism phylogeny were inferred from their whole genome sequences. RESULTS: Phylogenetic analysis of the 325 Klebsiella isolates that passed quality control revealed 3 groups: K. pneumoniae sensu stricto (n = 307), K. quasipneumoniae (n = 17), and K. variicola (n = 1). Sequencing and capsular diversity analysis of the 307 K. pneumoniae sensu stricto isolates revealed 28 sequence types, 26 K-locus types, and 11 O-locus types, with ST231, KL51, and O1V2 being predominant. blaOXA-48-like and blaNDM-1/5 were present in 73.2% and 24.4% of isolates, respectively. The major plasmid replicon types associated with carbapenase genes were IncF (51.0%) and Col group (35.0%). CONCLUSION: Our study documents for the first time the genetic diversity of K and O antigens circulating in India. The results demonstrate the practical applicability of genomic surveillance and its utility in tracking the population dynamics of CRKP. It alerts us to the urgency for longitudinal surveillance of these transmissible lineages.

Good Financial Grant Practice: A Tool for Developing and Demonstrating Institutional Financial and Grant Management Capacity in Global Health.

The administration and governance of grant funding across global health organizations presents enormous challenges. Meeting these challenges is crucial to ensuring that funds are used in the most effective way to improve health outcomes, in line with the United Nations' Sustainable Development Goal 3, "Ensure healthy lives and promote well-being for all at all ages." The Good Financial Grant Practice (GFGP) Standard (ARS 1651) is the world's first and, currently, only international standard for the financial governance and management of grant funding. Through consensus building and global harmonization between both low- and middle-income and high-income country players, the GFGP Standard has achieved a leveling impact: GFGP applies equally to, and can be implemented by, all types of organization, regardless of location, size, or whether they predominantly give or receive funding. GFGP can be used as a tool for addressing some of the challenges of the current funding model. Here, we describe our experiences and lessons learned from implementing GFGP across 4 diverse research institutions in India, Nigeria, Colombia, and the Philippines as part of our National Institute for Health Research Global Health Research Unit on Genomic Surveillance of Antimicrobial Resistance.

Rapid Genomic Characterization and Global Surveillance of Klebsiella Using Pathogenwatch.

BACKGROUND: Klebsiella species, including the notable pathogen K. pneumoniae, are increasingly associated with antimicrobial resistance (AMR). Genome-based surveillance can inform interventions aimed at controlling AMR. However, its widespread implementation requires tools to streamline bioinformatic analyses and public health reporting. METHODS: We developed the web application Pathogenwatch, which implements analytics tailored to Klebsiella species for integration and visualization of genomic and epidemiological data. We populated Pathogenwatch with 16 537 public Klebsiella genomes to enable contextualization of user genomes. We demonstrated its features with 1636 genomes from 4 low- and middle-income countries (LMICs) participating in the NIHR Global Health Research Unit (GHRU) on AMR. RESULTS: Using Pathogenwatch, we found that GHRU genomes were dominated by a small number of epidemic drug-resistant clones of K. pneumoniae. However, differences in their distribution were observed (eg, ST258/512 dominated in Colombia, ST231 in India, ST307 in Nigeria, ST147 in the Philippines). Phylogenetic analyses including public genomes for contextualization enabled retrospective monitoring of their spread. In particular, we identified hospital outbreaks, detected introductions from abroad, and uncovered clonal expansions associated with resistance and virulence genes. Assessment of loci encoding O-antigens and capsule in K. pneumoniae, which represent possible vaccine candidates, showed that 3 O-types (O1-O3) represented 88.9% of all genomes, whereas capsule types were much more diverse. CONCLUSIONS: Pathogenwatch provides a free, accessible platform for real-time analysis of Klebsiella genomes to aid surveillance at local, national, and global levels. We have improved representation of genomes from GHRU participant countries, further facilitating ongoing surveillance.

Streptococcus pneumoniae genomic datasets from an Indian population describing pre-vaccine evolutionary epidemiology using a whole genome sequencing approach.

Globally, India has a high burden of pneumococcal disease, and pneumococcal conjugate vaccine (PCV) has been rolled out in different phases across the country since May 2017 in the national infant immunization programme (NIP). To provide a baseline for assessing the impact of the vaccine on circulating pneumococci in India, genetic characterization of pneumococcal isolates detected prior to introduction of PCV would be helpful. Here we present a population genomic study of 480 Streptococcus pneumoniae isolates collected across India and from all age groups before vaccine introduction (2009-2017), including 294 isolates from pneumococcal disease and 186 collected through nasopharyngeal surveys. Population genetic structure, serotype and antimicrobial susceptibility profile were characterized and predicted from whole-genome sequencing data. Our findings revealed high levels of genetic diversity represented by 110 Global Pneumococcal Sequence Clusters (GPSCs) and 54 serotypes. Serotype 19F and GPSC1 (CC320) was the most common serotype and pneumococcal lineage, respectively. Coverage of PCV13 (Pfizer) and 10-valent Pneumosil (Serum Institute of India) serotypes in age groups of ≤2 and 3-5 years were 63-75 % and 60-69 %, respectively. Coverage of PPV23 (Merck) serotypes in age groups of ≥50 years was 62 % (98/158). Among the top five lineages causing disease, GPSC10 (CC230), which ranked second, is the only lineage that expressed both PCV13 (serotypes 3, 6A, 14, 19A and 19F) and non-PCV13 (7B, 13, 10A, 11A, 13, 15B/C, 22F, 24F) serotypes. It exhibited multidrug resistance and was the largest contributor (17 %, 18/103) of NVTs in the disease-causing population. Overall, 42 % (202/480) of isolates were penicillin-resistant (minimum inhibitory concentration ≥0.12 µg ml-1) and 45 % (217/480) were multidrug-resistant. Nine GPSCs (GPSC1, 6, 9, 10, 13, 16, 43, 91, 376) were penicillin-resistant and among them six were multidrug-resistant. Pneumococci expressing PCV13 serotypes had a higher prevalence of antibiotic resistance. Sequencing of pneumococcal genomes has significantly improved our understanding of the biology of these bacteria. This study, describing the pneumococcal disease and carriage epidemiology pre-PCV introduction, demonstrates that 60-75 % of pneumococcal serotypes in children ≤5 years are covered by PCV13 and Pneumosil. Vaccination against pneumococci is very likely to reduce antibiotic resistance. A multidrug-resistant pneumococcal lineage, GPSC10 (CC230), is a high-risk clone that could mediate serotype replacement.

COMPARISON OF PERFORMANCE CHARACTERISTICS BETWEEN LATERAL FLOW, ELISA AND ELECTROCHEMILUMINESCENCE IMMUNOASSAYS FOR THE DETECTION OF SARS-COV-2 ANTIBODIES AMONG HEALTHCARE WORKERS

BackgroundMedical professionals and researchers have been urging the need for wide and rapid testing of citizens in order to plan measures that can contain the spread of the virus. Antibody tests play an important role throughout the patient care pathway and are vital for the management and surveillance of the virus. Although RT-PCR is considered as the gold standard, serological tests based on antibodies are helpful for on-time detection. We performed one to one assessment of point-of-care lateral flow assay (POCTs), enzyme immunoassay (EIAs), electrochemiluminescence immunoassay (CLIA), to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG antibody. Materials and Methods611 healthcare workers were recruited between November and December 2020 at Central Research Laboratory, KIMS. Collected serum samples were analysed according to manufacturers protocol. The Standard Q IgG/IgM combo assay, Anti-SARS CoV-2 Human IgG ELISA, and the Elecsys(R) to measure the IgG titer of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ResultsThe kits displayed a sensitivity of 61.2%,79.5%, 91.8% and specificity of 61.7%,64.1%,80.2% for the Standard Q IgG/IgM combo assay, Anti-SARS CoV-2 Human IgG ELISA, and the Elecsys(R) in order. ConclusionOur results indicate high sensitivity and specificity for the Elecsys(R) assay compared to Anti-SARS CoV-2 Human IgG ELISA, the Standard Q IgG/IgM combo assay.

Critical comparison of image analysis workflows for quantitative cell morphological evaluation in assessing cell response to biomaterials.

Quantitative image analysis is an important tool in understanding cell fate processes through the study of cell morphological changes in terms of size, shape, number, and orientation. In this context, this work explores systematically the main challenges involved in the quantitative analysis of fluorescence microscopy images and also proposes a new protocol while comparing its outcome with the widely used ImageJ analysis. It is important to mention that fluorescence microscopy is by far most widely used in biocompatibility analysis (observing cell fate changes) of implantable biomaterials. In this study, we employed two different image analyses toolsets: (a) the conventionally employed ImageJ software, and (b) a recently developed automated digital image analyses framework, called ImageMKS. While ImageJ offers a powerful toolset for image analyses, it requires sophisticated user expertise to design and iteratively refine the analyses workflow. This workflow primarily comprises a sequence of image transformations that typically involve de-noising and labeling of features. On the other hand, ImageMKS automates the image analyses protocol to a large extent, and thereby mitigates the influence of the user bias on the final results. This aspect is addressed using a case study of C2C12 mouse myoblast cells grown on poly(vinylidene difluoride) (PVDF) based polymeric substrates. In particular, we used a number of fluorescence microscopy images of these mouse myoblasts grown on PVDF-based nanobiocomposites under the influence of electric field. In addition to the MKS workflows requiring much less user time because of their automation, it was observed that ImageMKS workflows consistently produced more reliable results that correlated better with the previously reported experimental studies.

Engineering Biophysical Cues for Controlled 3D Differentiation of Endoderm Derivatives.

Biophysical cues synergize with biochemical cues to drive differentiation of pluripotent stem cells through specific phenotypic trajectory. Tools to manipulate the cell biophysical environment and identify the influence of specific environment perturbation in the presence of combinatorial inputs will be critical to control the development trajectory. Here we describe the procedure to perturb biophysical environment of pluripotent stem cells while maintaining them in 3D culture configuration. We also discuss a high-throughput platform for combinatorial perturbation of the cell microenvironment, and detail a statistical procedure to extract dominant environmental influences.

Engineered peptide modified hydrogel platform for propagation of human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) have enormous potential to alleviate cell needs for regenerative medicine, however these cells require expansion in cell colonies to maintain cell-cell contact, thus limiting the scalability needed to meet the demands of cell therapy. While the use of a Rho-associated protein kinase (ROCK) inhibitor will allow for culture of single cell hPSCs, typically only 50% of cells are recovered after dissociation. When hPSCs lose cell-cell contact through E-cadherin, dissociation induced apoptosis occurs. In this study, we hypothesized that the extracellular E-cadherin domain of hPSCs will bind to synthetic E-cadherin peptides presented on a hydrogel substrate, mimicking the required cell-cell contact and thereby retaining single-cell viability and clonogenicity. Hence, the objective of this study was to functionalize alginate hydrogels with synthetic peptides mimicking E-cadherin and evaluate peptide performance in promoting cell attachment, viability, maintaining pluripotency, and differentiation potential. We observed that alginate conjugated with synthetic E-cadherin peptides not only supported initial cell attachment with high viability, but also supported hPSC propagation and high fold expansion. hPSCs propagated on the peptide modified substrates maintained the hPSC characteristic pluripotency and differentiation potential, characterized by both spontaneous and directed differentiation. STATEMENT OF SIGNIFICANCE: Human pluripotent stem cells (hPSCs) have enormous potential to alleviate cell needs for regenerative medicine and cell therapy. However, scalable culture of hPSCs is challenged by its need for maintenance of cell-cell contact, dissociation of which triggers the apoptotic pathway. Hence hPSCs are commonly maintained as colonies over Matrigel coated culture plates. Furthermore, use of xenogenic and undefined Matrigel compromises the translational potential of hPSCs. In this work we have developed a completely defined substrate to enable adherent culture of hPSCs as single cells. This substrate prevents apoptosis of the single cells and allows significant fold expansion of hPSCs while maintaining pluripotency and differentiation potential. The developed substrate is expected to be a cost-effective and translatable alternative to Matrigel.

Visualizing variation within Global Pneumococcal Sequence Clusters (GPSCs) and country population snapshots to contextualize pneumococcal isolates.

Knowledge of pneumococcal lineages, their geographic distribution and antibiotic resistance patterns, can give insights into global pneumococcal disease. We provide interactive bioinformatic outputs to explore such topics, aiming to increase dissemination of genomic insights to the wider community, without the need for specialist training. We prepared 12 country-specific phylogenetic snapshots, and international phylogenetic snapshots of 73 common Global Pneumococcal Sequence Clusters (GPSCs) previously defined using PopPUNK, and present them in Microreact. Gene presence and absence defined using Roary, and recombination profiles derived from Gubbins are presented in Phandango for each GPSC. Temporal phylogenetic signal was assessed for each GPSC using BactDating. We provide examples of how such resources can be used. In our example use of a country-specific phylogenetic snapshot we determined that serotype 14 was observed in nine unrelated genetic backgrounds in South Africa. The international phylogenetic snapshot of GPSC9, in which most serotype 14 isolates from South Africa were observed, highlights that there were three independent sub-clusters represented by South African serotype 14 isolates. We estimated from the GPSC9-dated tree that the sub-clusters were each established in South Africa during the 1980s. We show how recombination plots allowed the identification of a 20 kb recombination spanning the capsular polysaccharide locus within GPSC97. This was consistent with a switch from serotype 6A to 19A estimated to have occured in the 1990s from the GPSC97-dated tree. Plots of gene presence/absence of resistance genes (tet, erm, cat) across the GPSC23 phylogeny were consistent with acquisition of a composite transposon. We estimated from the GPSC23-dated tree that the acquisition occurred between 1953 and 1975. Finally, we demonstrate the assignment of GPSC31 to 17 externally generated pneumococcal serotype 1 assemblies from Utah via Pathogenwatch. Most of the Utah isolates clustered within GPSC31 in a USA-specific clade with the most recent common ancestor estimated between 1958 and 1981. The resources we have provided can be used to explore to data, test hypothesis and generate new hypotheses. The accessible assignment of GPSCs allows others to contextualize their own collections beyond the data presented here.

A mosaic tetracycline resistance gene tet(S/M) detected in an MDR pneumococcal CC230 lineage that underwent capsular switching in South Africa.

OBJECTIVES: We reported tet(S/M) in Streptococcus pneumoniae and investigated its temporal spread in relation to nationwide clinical interventions. METHODS: We whole-genome sequenced 12 254 pneumococcal isolates from 29 countries on an Illumina HiSeq sequencer. Serotype, multilocus ST and antibiotic resistance were inferred from genomes. An SNP tree was built using Gubbins. Temporal spread was reconstructed using a birth-death model. RESULTS: We identified tet(S/M) in 131 pneumococcal isolates and none carried other known tet genes. Tetracycline susceptibility testing results were available for 121 tet(S/M)-positive isolates and all were resistant. A majority (74%) of tet(S/M)-positive isolates were from South Africa and caused invasive diseases among young children (59% HIV positive, where HIV status was available). All but two tet(S/M)-positive isolates belonged to clonal complex (CC) 230. A global phylogeny of CC230 (n=389) revealed that tet(S/M)-positive isolates formed a sublineage predicted to exhibit resistance to penicillin, co-trimoxazole, erythromycin and tetracycline. The birth-death model detected an unrecognized outbreak of this sublineage in South Africa between 2000 and 2004 with expected secondary infections (effective reproductive number, R) of ∼2.5. R declined to ∼1.0 in 2005 and <1.0 in 2012. The declining epidemic could be related to improved access to ART in 2004 and introduction of pneumococcal conjugate vaccine (PCV) in 2009. Capsular switching from vaccine serotype 14 to non-vaccine serotype 23A was observed within the sublineage. CONCLUSIONS: The prevalence of tet(S/M) in pneumococci was low and its dissemination was due to an unrecognized outbreak of CC230 in South Africa. Capsular switching in this MDR sublineage highlighted its potential to continue to cause disease in the post-PCV13 era.