A Microfluidic Device for Kinetic Optimization of Protein Crystallization and In Situ Structure Determination

The unprecedented economies of scale and unique mass transport properties of microfluidic devices made them viable nano-volume protein crystallization screening platforms. However, realizing the full potential of microfluidic crystallization requires complementary technologies for crystal optimization and harvesting. In this paper, we report a microfluidic device which provides a link between chip-based nanoliter volume crystallization screening and structure analysis through “kinetic optimization” of crystallization reactions and in situ structure determination. Kinetic optimization through systematic variation of reactor geometry and actuation of micromechanical valves is used to screen a large ensemble of kinetic trajectories that are not practical with conventional techniques. Using this device, we demonstrate control over crystal quality, reliable scale-up from nanoliter volume reactions, facile harvesting and cryoprotectant screening, and protein structure determination at atomic resolution from data collected in-chip

Prevalence of healthcare-associated infections, estimated incidence and composite antimicrobial resistance index in acute care hospitals and long-term care facilities: results from two European point prevalence surveys, 2016 to 2017

Point prevalence surveys of healthcare-associated infections (HAI) and antimicrobial use in the European Union and European Economic Area (EU/EEA) from 2016 to 2017 included 310,755 patients from 1,209 acute care hospitals (ACH) in 28 countries and 117,138 residents from 2,221 long-term care facilities (LTCF) in 23 countries. After national validation, we estimated that 6.5% (cumulative 95% confidence interval (cCI): 5.4-7.8%) patients in ACH and 3.9% (95% cCI: 2.4-6.0%) residents in LTCF had at least one HAI (country-weighted prevalence). On any given day, 98,166 patients (95% cCI: 81,022-117,484) in ACH and 129,940 (95% cCI: 79,570-197,625) residents in LTCF had an HAI. HAI episodes per year were estimated at 8.9 million (95% cCI: 4.6-15.6 million), including 4.5 million (95% cCI: 2.6-7.6 million) in ACH and 4.4 million (95% cCI: 2.0-8.0 million) in LTCF; 3.8 million (95% cCI: 3.1-4.5 million) patients acquired an HAI each year in ACH. Antimicrobial resistance (AMR) to selected AMR markers was 31.6% in ACH and 28.0% in LTCF. Our study confirmed a high annual number of HAI in healthcare facilities in the EU/EEA and indicated that AMR in HAI in LTCF may have reached the same level as in ACH

A Microfluidic Device for Kinetic Optimization of Protein Crystallization and In Situ Structure Determination

The unprecedented economies of scale and unique mass transport properties of microfluidic devices made them viable nano-volume protein crystallization screening platforms. However, realizing the full potential of microfluidic crystallization requires complementary technologies for crystal optimization and harvesting. In this paper, we report a microfluidic device which provides a link between chip-based nanoliter volume crystallization screening and structure analysis through “kinetic optimization” of crystallization reactions and in situ structure determination. Kinetic optimization through systematic variation of reactor geometry and actuation of micromechanical valves is used to screen a large ensemble of kinetic trajectories that are not practical with conventional techniques. Using this device, we demonstrate control over crystal quality, reliable scale-up from nanoliter volume reactions, facile harvesting and cryoprotectant screening, and protein structure determination at atomic resolution from data collected in-chip

Global increases in both common and rare copy number load associated with autism.

Children with autism have an elevated frequency of large, rare copy number variants (CNVs). However, the global load of deletions or duplications, per se, and their size, location and relationship to clinical manifestations of autism have not been documented. We examined CNV data from 516 individuals with autism or typical development from the population-based Childhood Autism Risks from Genetics and Environment (CHARGE) study. We interrogated 120 regions flanked by segmental duplications (genomic hotspots) for events >50 kbp and the entire genomic backbone for variants >300 kbp using a custom targeted DNA microarray. This analysis was complemented by a separate study of five highly dynamic hotspots associated with autism or developmental delay syndromes, using a finely tiled array platform (>1 kbp) in 142 children matched for gender and ethnicity. In both studies, a significant increase in the number of base pairs of duplication, but not deletion, was associated with autism. Significantly elevated levels of CNV load remained after the removal of rare and likely pathogenic events. Further, the entire CNV load detected with the finely tiled array was contributed by common variants. The impact of this variation was assessed by examining the correlation of clinical outcomes with CNV load. The level of personal and social skills, measured by Vineland Adaptive Behavior Scales, negatively correlated (Spearman's r = -0.13, P = 0.034) with the duplication CNV load for the affected children; the strongest association was found for communication (P = 0.048) and socialization (P = 0.022) scores. We propose that CNV load, predominantly increased genomic base pairs of duplication, predisposes to autism

Long-Term Monitoring of Bacteria Undergoing Programmed Population Control in a Microchemostat

Using an active approach to preventing biofilm formation, we implemented a microfluidic bioreactor that enables long-term culture and monitoring of extremely small populations of bacteria with single-cell resolution. We used this device to observe the dynamics of Escherichia coli carrying a synthetic “population control” circuit that regulates cell density through a feedback mechanism based on quorum sensing. The microfluidic bioreactor enabled long-term monitoring of unnatural behavior programmed by the synthetic circuit, which included sustained oscillations in cell density and associated morphological changes, over hundreds of hours

Identification and evolution of a plant cell wall specific glycoprotein glycosyl transferase, ExAD

Extensins are plant cell wall glycoproteins that act as scaffolds for the deposition of the main wall carbohydrate polymers, which are interlocked into the supramolecular wall structure through intra- and inter-molecular iso-di-tyrosine crosslinks within the extensin backbone. In the conserved canonical extensin repeat, Ser-Hyp(4), serine and the consecutive C4-hydroxyprolines (Hyps) are substituted with an α-galactose and 1–5 β- or α-linked arabinofuranoses (Arafs), respectively. These modifications are required for correct extended structure and function of the extensin network. Here, we identified a single Arabidopsis thaliana gene, At3g57630, in clade E of the inverting Glycosyltransferase family GT47 as a candidate for the transfer of Araf to Hyp-arabinofuranotriose (Hyp-β1,4Araf-β1,2Araf-β1,2Araf) side chains in an α-linkage, to yield Hyp-Araf(4) which is exclusively found in extensins. T-DNA knock-out mutants of At3g57630 showed a truncated root hair phenotype, as seen for mutants of all hitherto characterized extensin glycosylation enzymes; both root hair and glycan phenotypes were restored upon reintroduction of At3g57630. At3g57630 was named Extensin Arabinose Deficient transferase, ExAD, accordingly. The occurrence of ExAD orthologs within the Viridiplantae along with its’ product, Hyp-Araf(4), point to ExAD being an evolutionary hallmark of terrestrial plants and charophyte green algae

Fifteen new risk loci for coronary artery disease highlight arterial-wall-specific mechanisms

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Although 58 genomic regions have been associated with CAD thus far, most of the heritability is unexplained, indicating that additional susceptibility loci await identification. An efficient discovery strategy may be larger-scale evaluation of promising associations suggested by genome-wide association studies (GWAS). Hence, we genotyped 56,309 participants using a targeted gene array derived from earlier GWAS results and performed meta-analysis of results with 194,427 participants previously genotyped, totaling 88,192 CAD cases and 162,544 controls. We identified 25 new SNP-CAD associations (P < 5 × 10(-8), in fixed-effects meta-analysis) from 15 genomic regions, including SNPs in or near genes involved in cellular adhesion, leukocyte migration and atherosclerosis (PECAM1, rs1867624), coagulation and inflammation (PROCR, rs867186 (p.Ser219Gly)) and vascular smooth muscle cell differentiation (LMOD1, rs2820315). Correlation of these regions with cell-type-specific gene expression and plasma protein levels sheds light on potential disease mechanisms

Goal-directed haemodynamic therapy during general anaesthesia for noncardiac surgery:a systematic review and meta-analysis

BACKGROUND: During general anaesthesia for noncardiac surgery, there remain knowledge gaps regarding the effect of goal-directed haemodynamic therapy on patient-centred outcomes. METHODS: Included clinical trials investigated goal-directed haemodynamic therapy during general anaesthesia in adults undergoing noncardiac surgery and reported at least one patient-centred postoperative outcome. PubMed and Embase were searched for relevant articles on March 8, 2021. Two investigators performed abstract screening, full-text review, data extraction, and bias assessment. The primary outcomes were mortality and hospital length of stay, whereas 15 postoperative complications were included based on availability. From a main pool of comparable trials, meta-analyses were performed on trials with homogenous outcome definitions. Certainty of evidence was evaluated using Grading of Recommendations, Assessment, Development, and Evaluations (GRADE). RESULTS: The main pool consisted of 76 trials with intermediate risk of bias for most outcomes. Overall, goal-directed haemodynamic therapy might reduce mortality (odds ratio=0.84; 95% confidence interval [CI], 0.64 to 1.09) and shorten length of stay (mean difference=–0.72 days; 95% CI, –1.10 to –0.35) but with low certainty in the evidence. For both outcomes, larger effects favouring goal-directed haemodynamic therapy were seen in abdominal surgery, very high-risk surgery, and using targets based on preload variation by the respiratory cycle. However, formal tests for subgroup differences were not statistically significant. Goal-directed haemodynamic therapy decreased risk of several postoperative outcomes, but only infectious outcomes and anastomotic leakage reached moderate certainty of evidence. CONCLUSIONS: Goal-directed haemodynamic therapy during general anaesthesia might decrease mortality, hospital length of stay, and several postoperative complications. Only infectious postoperative complications and anastomotic leakage reached moderate certainty in the evidence

BRCA2 polymorphic stop codon K3326X and the risk of breast, prostate, and ovarian cancers

Background: The K3326X variant in BRCA2 (BRCA2*c.9976A>T; p.Lys3326*; rs11571833) has been found to be associated with small increased risks of breast cancer. However, it is not clear to what extent linkage disequilibrium with fully pathogenic mutations might account for this association. There is scant information about the effect of K3326X in other hormone-related cancers. Methods: Using weighted logistic regression, we analyzed data from the large iCOGS study including 76 637 cancer case patients and 83 796 control patients to estimate odds ratios (ORw) and 95% confidence intervals (CIs) for K3326X variant carriers in relation to breast, ovarian, and prostate cancer risks, with weights defined as probability of not having a pathogenic BRCA2 variant. Using Cox proportional hazards modeling, we also examined the associations of K3326X with breast and ovarian cancer risks among 7183 BRCA1 variant carriers. All statistical tests were two-sided. Results: The K3326X variant was associated with breast (ORw = 1.28, 95% CI = 1.17 to 1.40, P = 5.9x10- 6) and invasive ovarian cancer (ORw = 1.26, 95% CI = 1.10 to 1.43, P = 3.8x10-3). These associations were stronger for serous ovarian cancer and for estrogen receptor–negative breast cancer (ORw = 1.46, 95% CI = 1.2 to 1.70, P = 3.4x10-5 and ORw = 1.50, 95% CI = 1.28 to 1.76, P = 4.1x10-5, respectively). For BRCA1 mutation carriers, there was a statistically significant inverse association of the K3326X variant with risk of ovarian cancer (HR = 0.43, 95% CI = 0.22 to 0.84, P = .013) but no association with breast cancer. No association with prostate cancer was observed. Conclusions: Our study provides evidence that the K3326X variant is associated with risk of developing breast and ovarian cancers independent of other pathogenic variants in BRCA2. Further studies are needed to determine the biological mechanism of action responsible for these associations