Evaluation of methods for whole genome sequencing of Enterococcus faecium in a diagnostic laboratory

Enterococcus faecium is an important nosocomial pathogen associated with hospital transmission and outbreaks. Based on growing evidence that bacterial whole genome sequencing enhances hospital outbreak investigation of other bacterial species, our aim was to develop and evaluate methods for low volume clinical sequencing of E. faecium. Using a test panel of 22 E. faecium isolates associated previously with hospital transmission, we developed laboratory protocols for DNA extraction and library preparation, which in combination with the Illumina MiniSeq can generate sequence data within 24 hours. The final laboratory protocol took 3.5 hours and showed 98% reproducibility in producing sufficient DNA for sequencing. Repeatability and reproducibility assays based on the laboratory protocol and sequencing demonstrated 100% accuracy in assigning species, sequence type (ST) and (when present) detecting vanA or vanB, with all isolates passing the quality control metrics. Minor variation was detected in base calling of the same isolate genome when tested repeatedly due to variations in mapping and base calling, but application of a SNP cut-off (<15 SNPs) to assign isolates to outbreak clusters showed 100% reproducibility. An evaluation of contamination showed that controls and test E. faecium sequence files contained <0.34% and <2.12% of fragments matching another species, respectively. Deliberate contamination experiments confirmed that this was insufficient to impact on data interpretation. Further work is required to develop informatic tools prior to implementation into clinical practice.

Towards Biomanufacturing of Cell-Derived Matrices

Cell-derived matrices (CDM) are the decellularised extracellular matrices (ECM) of tissues obtained by the laboratory culture process. CDM is developed to mimic, to a certain extent, the properties of the needed natural tissue and thus to obviate the use of animals. The composition of CDM can be tailored for intended applications by carefully optimising the cell sources, culturing conditions and decellularising methods. This unique advantage has inspired the increasing use of CDM for biomedical research, ranging from stem cell niches to disease modelling and regenerative medicine. However, while much effort is spent on extracting different types of CDM and exploring their utilisation, little is spent on the scale-up aspect of CDM production. The ability to scale up CDM production is essential, as the materials are due for clinical trials and regulatory approval, and in fact, this ability to scale up should be an important factor from the early stages. In this review, we first introduce the current CDM production and characterisation methods. We then describe the existing scale-up technologies for cell culture and highlight the key considerations in scaling-up CDM manufacturing. Finally, we discuss the considerations and challenges faced while converting a laboratory protocol into a full industrial process. Scaling-up CDM manufacturing is a challenging task since it may be hindered by technologies that are not yet available. The early identification of these gaps will not only quicken CDM based product development but also help drive the advancement in scale-up cell culture and ECM extraction.

Validation of manual blood culture bottles v2

Use of equipment-free, “manual” blood cultures is still widespread in low-resource settings, as requirements for implementation of automated systems are often not met. Quality of manual blood culture bottles currently on the market, however, is usually unknown. An acceptable quality in terms of yield and speed of growth can be ensured by evaluating the bottles using simulated blood cultures. In these experiments, bottles from different systems are inoculated in parallel with blood and a known quantity of bacteria. Based on literature review and personal experiences, we propose a short and practical protocol for an efficient evaluation of manual blood culture bottles, aimed at research or reference laboratories in low-resource settings. This laboratory protocol was used in a study for Médecins Sans Frontières' Mini-Lab project, which aims to bring clinical bacteriology to low-resource settings. Three bottle types were evaluated in this study; two "manual" blood culture bottles and one automated system.

Chelex DNA extraction Protocol v1

Laboratory protocol for DNA extraction from animal tissue with Chelex.

Vertical Discrepancy in Height of Morse Cone Abutments Submitted to Different Torque Forces

The present study aimed to evaluate the influence of manual torque (10 Ncm) versus clinical torque (30 Ncm), which is recommended by the manufacturer, on the total length of morse cone implant abutments. Twenty specimens were prepared and distributed into two groups: group 1 with ten analogs for morse cone type implant, and group 2 with ten morse type implants, size 4.3 × 15 cm. In each group, the distance between the implant platform to the top of the prosthetic abutment (abutment height) was measured and subjected to a torque of 10 Ncm. Then, the 30 Ncm torque was applied to the same abutment, and abutment height was measured. The distance between the top of the abutment and the implant/analog base was measured. In order to verify the clinical reproducibility of the experiment, comparisons between the abutment height of the analog at 10 Ncm and the implant at 30 Ncm were performed, showing a greater discrepancy in torque for the 10 Ncm analog (p < 0.05). In order to verify if the change in the laboratory protocol from 10 to 30 Ncm could minimize the differences in the height of the prosthetic abutments, the abutment height in groups 1 and 2 was compared with 30 Ncm, and no significant difference was observed (p > 0.05). The data indicated that the manual torque and the torque recommended by the manufacturer influence the total length of the prosthetic abutments of morse cone implants.

Highly contiguous assemblies of 101 drosophilid genomes

Over 100 years of studies in Drosophila melanogaster and related species in the genus Drosophila have facilitated key discoveries in genetics, genomics, and evolution. While high-quality genome assemblies exist for several species in this group, they only encompass a small fraction of the genus. Recent advances in long-read sequencing allow high-quality genome assemblies for tens or even hundreds of species to be efficiently generated. Here, we utilize Oxford Nanopore sequencing to build an open community resource of genome assemblies for 101 lines of 93 drosophilid species encompassing 14 species groups and 35 sub-groups. The genomes are highly contiguous and complete, with an average contig N50 of 10.5 Mb and greater than 97% BUSCO completeness in 97/101 assemblies. We show that Nanopore-based assemblies are highly accurate in coding regions, particularly with respect to coding insertions and deletions. These assemblies, along with a detailed laboratory protocol and assembly pipelines, are released as a public resource and will serve as a starting point for addressing broad questions of genetics, ecology, and evolution at the scale of hundreds of species.

Metabolic Reprogramming of GMP Grade Cord Tissue Derived Mesenchymal Stem Cells Enhances Their Suppressive Potential in GVHD

Acute graft-vs.-host (GVHD) disease remains a common complication of allogeneic stem cell transplantation with very poor outcomes once the disease becomes steroid refractory. Mesenchymal stem cells (MSCs) represent a promising therapeutic approach for the treatment of GVHD, but so far this strategy has had equivocal clinical efficacy. Therapies using MSCs require optimization taking advantage of the plasticity of these cells in response to different microenvironments. In this study, we aimed to optimize cord blood tissue derived MSCs (CBti MSCs) by priming them using a regimen of inflammatory cytokines. This approach led to their metabolic reprogramming with enhancement of their glycolytic capacity. Metabolically reprogrammed CBti MSCs displayed a boosted immunosuppressive potential, with superior immunomodulatory and homing properties, even after cryopreservation and thawing. Mechanistically, primed CBti MSCs significantly interfered with glycolytic switching and mTOR signaling in T cells, suppressing T cell proliferation and ensuing polarizing toward T regulatory cells. Based on these data, we generated a Good Manufacturing Process (GMP) Laboratory protocol for the production and cryopreservation of primed CBti MSCs for clinical use. Following thawing, these cryopreserved GMP-compliant primed CBti MSCs significantly improved outcomes in a xenogenic mouse model of GVHD. Our data support the concept that metabolic profiling of MSCs can be used as a surrogate for their suppressive potential in conjunction with conventional functional methods to support their therapeutic use in GVHD or other autoimmune disorders.

124 Skeletal muscle RNA and insulin sensitivity during insufficient sleep

Introduction Insufficient sleep is associated with a down-regulation of genes involved in glycolysis, in conjunction with an upregulation of genes involved in lipid metabolism in skeletal muscle. However, whether changes in RNA are associated with impairments in insulin sensitivity is unclear. We therefore tested the hypothesis that insufficient sleep will induce alterations in skeletal muscle RNA that correlate with changes in insulin sensitivity. Methods As part of an ongoing study, sixteen sedentary, healthy, lean adults (24.9±3.4y; 22.6±1.7kg/m2; 6F; mean±SD) participated in a controlled 6-day in-laboratory protocol with 9h in bed (habitual sleep) followed by 4 nights of 5h in bed (insufficient sleep), achieved by delaying bedtime by 4 hours. For one week prior to the study, participants maintained a 9h sleep schedule based on their habitual bed and wake times. Participants consumed energy-balanced diets 3 days prior to and throughout the laboratory protocol. Whole body insulin sensitivity was assessed using glucose infusion rate from a hyperinsulinemic euglycemic clamp before and after 4 nights of insufficient sleep. Skeletal muscle biopsies of the vastus lateralis were taken immediately before each clamp. In a subset of subjects (n=12), RNA sequencing was performed (Novogene Co., Ltd). Generalized linear model likelihood ratio tests were completed using the DESeq2/EdgeR R packages with a false discovery rate (FDR) cut-off of 5%. P-values were adjusted for multiple comparisons using the Benjamini-Hochberg method and a corrected p-value of 0.05 and log2 fold-change of 0 were set as the threshold for statistical significance. Results Insulin sensitivity was impaired by 6% following insufficient sleep (10.1±1.4 vs 9.1±1.1mg/kg/min, p&lt;0.05, mean±SEM). Preliminary results from skeletal muscle RNAseq analyses suggest approximately 25 genes were down-regulated and 60 genes were up-regulated. Down-regulated genes were involved in insulin-like growth factor binding and signal transduction (p=8.4e-11), while up-regulated genes were involved in glycolysis and ATP binding (p=1.1e-9). While there were trends for associations between changes in gene expression and insulin sensitivity, these relationships did not reach statistical significance. Conclusion Preliminary findings suggest insufficient sleep alters skeletal muscle RNA. Changes in these aforementioned pathways may contribute to metabolic dysregulation during insufficient sleep. Support (if any) NIH K01DK110138, R03 DK118309, UL1 TR002535, and GCRC RR-00036

The Use of Antifactor Xa Assays in a Comprehensive Pediatric Extracorporeal Membrane Oxygenation Anticoagulation Protocol is Associated with Increased Survival and Significant Blood Product Cost-Savings

We augmented our standard extracorporeal membrane oxygenation laboratory protocol to include antifactor Xa assays, thromboelastography, and antithrombin measurements. We performed a retrospective chart review to determine outcomes for patients placed on extracorporeal membrane oxygenation (ECMO) prior to and after the initiation of our anticoagulation laboratory protocol. A total of 663 consecutive ECMO runs were evaluated from January 1, 2007 to June 30, 2018. Of these patients, 252 were on ECMO prior to initiation of the anticoagulation laboratory protocol on September 1, 2011, and 411 patients were on ECMO after initiation of the protocol. There were no major changes to our extracorporeal membrane oxygenation circuit or changes to our transfusion threshold during this continuous study period. Transfusion utilization data revealed statistically significant decreases in almost all blood components, and a savings in blood component inflation-adjusted acquisition costs of 31% bringing total blood product cost-savings to $309,905 per year. In addition, there was an increase in survival to hospital discharge from 45 to 56% associated with the initiation of the protocol (p = 0.004). Our data indicate that implementation of a standardized ECMO anticoagulation protocol, which titrates unfractionated heparin infusions based on antifactor Xa assays, is associated with reduced blood product utilization, significant blood product cost savings, and increased patient survival. Future prospective evaluation is needed to establish an antifactor Xa assay-driven ECMO anticoagulation strategy as both clinically superior and cost-effective.