Structural variants drive context-dependent oncogene activation in cancer.

Higher-order chromatin structure is important for the regulation of genes by distal regulatory sequences1,2. Structural variants (SVs) that alter three-dimensional (3D) genome organization can lead to enhancer-promoter rewiring and human disease, particularly in the context of cancer3. However, only a small minority of SVs are associated with altered gene expression4,5, and it remains unclear why certain SVs lead to changes in distal gene expression and others do not. To address these questions, we used a combination of genomic profiling and genome engineering to identify sites of recurrent changes in 3D genome structure in cancer and determine the effects of specific rearrangements on oncogene activation. By analysing Hi-C data from 92 cancer cell lines and patient samples, we identified loci affected by recurrent alterations to 3D genome structure, including oncogenes such as MYC, TERT and CCND1. By using CRISPR-Cas9 genome engineering to generate de novo SVs, we show that oncogene activity can be predicted by using 'activity-by-contact' models that consider partner region chromatin contacts and enhancer activity. However, activity-by-contact models are only predictive of specific subsets of genes in the genome, suggesting that different classes of genes engage in distinct modes of regulation by distal regulatory elements. These results indicate that SVs that alter 3D genome organization are widespread in cancer genomes and begin to illustrate predictive rules for the consequences of SVs on oncogene activation.

Azithromycin to Reduce Mortality - An Adaptive Cluster-Randomized Trial.

BACKGROUND: Twice-yearly mass distribution of azithromycin to children is a promising intervention to reduce childhood mortality in sub-Saharan Africa. The World Health Organization recommended restricting distribution to infants 1 to 11 months of age to mitigate antimicrobial resistance, although this more limited treatment had not yet been tested. METHODS: We randomly assigned rural communities in Niger to four twice-yearly distributions of azithromycin for children 1 to 59 months of age (child azithromycin group), four twice-yearly distributions of azithromycin for infants 1 to 11 months of age and placebo for children 12 to 59 months of age (infant azithromycin group), or placebo for children 1 to 59 months of age. Census workers who were not aware of the group assignments monitored mortality twice yearly over the course of 2 years. We assessed three primary community-level mortality outcomes (deaths per 1000 person-years), each examining a different age group and pairwise group comparison. RESULTS: A total of 1273 communities were randomly assigned to the child azithromycin group (1229 were included in the analysis), 773 to the infant azithromycin group (751 included in the analysis), and 954 to the placebo group (929 included in the analysis). Among 382,586 children, 419,440 person-years and 5503 deaths were recorded. Lower mortality among children 1 to 59 months of age was observed in the child azithromycin group (11.9 deaths per 1000 person-years; 95% confidence interval [CI], 11.3 to 12.6) than in the placebo group (13.9 deaths per 1000 person-years; 95% CI, 13.0 to 14.8) (representing 14% lower mortality with azithromycin; 95% CI, 7 to 22; P<0.001). Mortality among infants 1 to 11 months of age was not significantly lower in the infant azithromycin group (22.3 deaths per 1000 person-years; 95% CI, 20.0 to 24.7) than in the placebo group (23.9 deaths per 1000 person-years; 95% CI, 21.6 to 26.2) (representing 6% lower mortality with azithromycin; 95% CI, -8 to 19). Five serious adverse events were reported: three in the placebo group, one in the infant azithromycin group, and one in the child azithromycin group. CONCLUSIONS: Azithromycin distributions to children 1 to 59 months of age significantly reduced mortality and was more effective than treatment of infants 1 to 11 months of age. Antimicrobial resistance must be monitored. (Funded by the Bill and Melinda Gates Foundation; AVENIR ClinicalTrials.gov number, NCT04224987.).

A calcineurin-Hoxb13 axis regulates growth mode of mammalian cardiomyocytes.

A major factor in the progression to heart failure in humans is the inability of the adult heart to repair itself after injury. We recently demonstrated that the early postnatal mammalian heart is capable of regeneration following injury through proliferation of preexisting cardiomyocytes1,2 and that Meis1, a three amino acid loop extension (TALE) family homeodomain transcription factor, translocates to cardiomyocyte nuclei shortly after birth and mediates postnatal cell cycle arrest3. Here we report that Hoxb13 acts as a cofactor of Meis1 in postnatal cardiomyocytes. Cardiomyocyte-specific deletion of Hoxb13 can extend the postnatal window of cardiomyocyte proliferation and reactivate the cardiomyocyte cell cycle in the adult heart. Moreover, adult Meis1-Hoxb13 double-knockout hearts display widespread cardiomyocyte mitosis, sarcomere disassembly and improved left ventricular systolic function following myocardial infarction, as demonstrated by echocardiography and magnetic resonance imaging. Chromatin immunoprecipitation with sequencing demonstrates that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and cell cycle. Finally, we show that the calcium-activated protein phosphatase calcineurin dephosphorylates Hoxb13 at serine-204, resulting in its nuclear localization and cell cycle arrest. These results demonstrate that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and proliferation and provide mechanistic insights into the link between hyperplastic and hypertrophic growth of cardiomyocytes.

IL-33 potentiates histaminergic itch.

BACKGROUND: Itch is a common symptom that can greatly diminish quality of life. Histamine is a potent endogenous pruritogen, and while antihistamines are often the first-line treatment for itch, in conditions like chronic spontaneous urticaria (CSU), many patients remain symptomatic while receiving maximal doses. Mechanisms that drive resistance to antihistamines are poorly defined. OBJECTIVES: Signaling of the alarmin cytokine IL-33 in sensory neurons is postulated to drive chronic itch by inducing neuronal sensitization to pruritogens. Thus, we sought to determine if IL-33 can augment histamine-induced (histaminergic) itch. METHODS: Itch behavior was assessed in response to histamine after IL-33 or saline administration. Various stimuli and conditional and global knockout mice were utilized to dissect cellular mechanisms. Multiple existing transcriptomic data sets were evaluated, including single-cell RNA sequencing of human and mouse skin, microarrays of isolated mouse mast cells at steady state and after stimulation with IL-33, and microarrays of skin biopsy samples from subjects with CSU and healthy controls. RESULTS: IL-33 amplifies histaminergic itch independent of IL-33 signaling in sensory neurons. Mast cells are the top expressors of the IL-33 receptor in both human and mouse skin. When stimulated by IL-33, mouse mast cells significantly increase IL-13 levels. Enhancement of histaminergic itch by IL-33 relies on a mast cell- and IL-13-dependent mechanism. IL-33 receptor expression is increased in lesional skin of subjects with CSU compared to healthy controls. CONCLUSIONS: Our findings suggest that IL-33 signaling may be a key driver of histaminergic itch in mast cell-associated pruritic conditions such as CSU.

The State of Patient-Reported Outcome Measures in Rheumatology.

OBJECTIVE: We sought to evaluate the quality and timeliness of patient-reported outcome (PRO) measure reporting, which have not been previously studied. METHODS: Clinical trials that informed new US Food and Drug Administration (FDA) approvals for the first rheumatological indication between 1995 and 2021 were identified. Data were recorded to determine whether collected PROs were published, met minimum clinically important difference (MCID) or statistical significance (P < 0.05) thresholds, and were consistent with Consolidated Standards of Reporting Trials (CONSORT)-PRO standards. Hazard ratios and Kaplan-Meier estimate were used to assess the time from FDA approval to PRO publication. RESULTS: Thirty-one FDA approvals corresponded with 110 pivotal trials and 262 reported PROs. Of the 90 included studies, 1 (1.1%) met all 5 recommended items, 10 (11.1%) met 4 items, 17 (18.9%) met 3 items, 21 (23.3%) met 2 items, 26 (28.9%) met 1 item, and 15 (16.7%) met none of the reporting standards. Most PROs met MCID thresholds (149/262; 56.9%) and were statistically significant (223/262; 85.1%). Of our subset analysis, one-third of PROs were not published upfront (70/212; 33%) and 1 of 9 (22/212; 10.4%) remained unpublished ≥ 4 years after initial trial reporting. Publication rates were highest for the Health Assessment Questionnaire-Disability Index (97.4%) and lowest for the 36-item Short Form Health Survey (81.8%). Less than half of these published PROs met MCID and statistical significance thresholds (94/212; 44.3%). CONCLUSION: One in 9 PROs remained unpublished for ≥ 4 years after initial trial reporting, and compliance with CONSORT-PRO reporting guidelines was poor. Efforts should be made to ensure PROs are adequately reported and expeditiously published.

From Squaric Acid Amides (SQAs) to Quinoxaline-Based SQAs─Evolution of a Redox-Active Cathode Material for Organic Polymer Batteries.

The search for new redox-active organic materials (ROMs) is essential for the development of sustainable energy-storage solutions. In this study, we present a new class of cyclobuta[b]quinoxaline-1,2-diones or squaric acid quinoxalines (SQXs) as highly promising candidates for ROMs featuring exceptional stability and high redox potentials. While simple 1,2- and 1,3-squaric acid amides (SQAs), initially reported by Hünig and coworkers decades ago, turned out to exhibit low stability in their radical cation oxidation states, we demonstrate that embedding the nitrogen atoms into a quinoxaline heterocycle leads to robust two-electron SQX redox systems. A series of SQX compounds, as well as their corresponding radical cations, were prepared and fully characterized, including EPR spectroscopy, UV-vis spectroscopy, and X-ray diffraction. Based on the promising electrochemical properties and high stability of the new ROM, we developed SQX-functionalized polymers and investigated their physical and electrochemical properties for energy-storage applications. These polymers showed remarkable thermal stability well above 200 °C with reversible redox properties and potentials of about 3.6 V vs Li+/Li. By testing the galvanostatic cycling performance in half-cells with lithium-metal counter electrodes, a styrene-based polymer with SQX redox side groups showed stable cycling for single-electron oxidation for more than 100 cycles. These findings render this new class of redox-active polymers as highly promising materials for future energy-storage applications.

A Cross-sectional Study of Patients With Extrapulmonary Tuberculosis and Normal Chest Radiographs - What Characteristics Were Associated With Sputum Culture Positivity?

BACKGROUND: Recognizing pulmonary involvement in tuberculosis (TB) patients is necessary to prevent TB transmission. We describe frequency and characteristics of patients with extrapulmonary TB (EPTB), normal chest radiographs, and positive sputum culture. METHODS: We analyzed data of patients ≥15 years of age with EPTB reported to the California TB registry during 2011-2017 with cultured sputum and normal chest radiographs using generalized linear modeling to estimate prevalence ratios associated with positive sputum culture. Demographic, behavioral, clinical characteristics, and testing were compared for patients with positive and negative sputum culture. RESULTS: Of 1635 patients with EPTB and normal chest radiographs, 937 (57%) had sputum culture performed, and 127 (13%) patients had positive results for Mycobacterium tuberculosis complex. Patients with positive results were more likely to: be male, experience homelessness, use substances, have HIV, and have >1 disease site. Among 85 patients with HIV co-infection, 54% had positive culture results compared with 9.5% among 852 patients without HIV co-infection. Patients with EPTB in more than 1 site were also more likely to have a positive sputum culture. CONCLUSIONS: Culturing sputum from patients with EPTB identified pulmonary cases not detected by chest radiograph, particularly among patients with HIV or >1 disease site.

Gut Resistome of Preschool Children After Prolonged Mass Azithromycin Distribution: A Cluster-randomized Trial.

We evaluated the gut resistome of children from communities treated with 10 twice-yearly azithromycin distributions. Although the macrolide resistance remained higher in the azithromycin arm, the selection of non-macrolide resistance observed at earlier time points did not persist. Longitudinal resistance monitoring should be a critical component of mass distribution programs. CLINICAL TRIALS REGISTRATION: NCT02047981.

Changes in microarchitecture of atherosclerotic calcification assessed by 18F-NaF PET and CT after a progressive exercise regimen in hyperlipidemic mice.

BACKGROUND: Despite the association of physical activity with improved cardiovascular outcomes and the association of high coronary artery calcification (CAC) scores with poor prognosis, elite endurance athletes have increased CAC. Yet, they nevertheless have better cardiovascular survival. We hypothesized that exercise may transform vascular calcium deposits to a more stable morphology. METHODS: To test this, hyperlipidemic mice (Apoe-/-) with baseline aortic calcification were separated into 2 groups (n = 9/group) with control mice allowed to move ad-lib while the exercise group underwent a progressive treadmill regimen for 9 weeks. All mice underwent blood collections and in vivo 18F-NaF µPET/µCT imaging both at the start and end of the exercise regimen. At euthanasia, aortic root specimens were obtained for histomorphometry. RESULTS: Results showed that, while aortic calcification progressed similarly in both groups based on µCT, the fold change in 18F-NaF density was significantly less in the exercise group. Histomorphometric analysis of the aortic root calcium deposits showed that the exercised mice had a lower mineral surface area index than the control group. The exercise regimen also raised serum PTH levels twofold. CONCLUSION: These findings suggest that weeks-long progressive exercise alters the microarchitecture of atherosclerotic calcium deposits by reducing mineral surface growth, potentially favoring plaque stability.

Age-based targeting of biannual azithromycin distribution for child survival in Niger: an adaptive cluster-randomized trial protocol (AVENIR).

BACKGROUND: Biannual distribution of azithromycin to children 1-59 months old reduced mortality by 14% in a cluster-randomized trial. The World Health Organization has proposed targeting this intervention to the subgroup of children 1-11 months old to reduce selection for antimicrobial resistance. Here, we describe a trial designed to determine the impact of age-based targeting of biannual azithromycin on mortality and antimicrobial resistance. METHODS: AVENIR is a cluster-randomized, placebo-controlled, double-masked, response-adaptive large simple trial in Niger. During the 2.5-year study period, 3350 communities are targeted for enrollment. In the first year, communities in the Dosso region will be randomized 1:1:1 to 1) azithromycin 1-11: biannual azithromycin to children 1-11 months old with placebo to children 12-59 months old, 2) azithromycin 1-59: biannual azithromycin to children 1-59 months old, or 3) placebo: biannual placebo to children 1-59 months old. Regions enrolled after the first year will be randomized with an updated allocation based on the probability of mortality in children 1-59 months in each arm during the preceding study period. A biannual door-to-door census will be conducted to enumerate the population, distribute azithromycin and placebo, and monitor vital status. Primary mortality outcomes are defined as all-cause mortality rate (deaths per 1000 person-years) after 2.5 years from the first enrollment in 1) children 1-59 months old comparing the azithromycin 1-59 and placebo arms, 2) children 1-11 months old comparing the azithromycin 1-11 and placebo arm, and 3) children 12-59 months in the azithromycin 1-11 and azithromycin 1-59 arms. In the Dosso region, 50 communities from each arm will be followed to monitor antimicrobial resistance. Primary resistance outcomes will be assessed after 2 years of distributions and include 1) prevalence of genetic determinants of macrolide resistance in nasopharyngeal samples from children 1-59 months old, and 2) load of genetic determinants of macrolide resistance in rectal samples from children 1-59 months old. DISCUSSION: As high-mortality settings consider this intervention, the results of this trial will provide evidence to support programmatic and policy decision-making on age-based strategies for azithromycin distribution to promote child survival. TRIAL REGISTRATION: This trial was registered on January 13, 2020 (clinicaltrials.gov: NCT04224987 ).

Updating Molecular Diagnostics for Detecting Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus Isolates in Blood Culture Bottles.

Molecular diagnostic tests can be used to provide rapid identification of staphylococcal species in blood culture bottles to help improve antimicrobial stewardship. However, alterations in the target nucleic acid sequences of the microorganisms or their antimicrobial resistance genes can lead to false-negative results. We determined the whole-genome sequences of 4 blood culture isolates of Staphylococcus aureus and 2 control organisms to understand the genetic basis of genotype-phenotype discrepancies when using the Xpert MRSA/SA BC test (in vitro diagnostic medical device [IVD]). Three methicillin-resistant S. aureus (MRSA) isolates each had a different insertion of a genetic element in the staphylococcal cassette chromosome (SCCmec)-orfX junction region that led to a misclassification as methicillin-susceptible S. aureus (MSSA). One strain contained a deletion in spa, which produced a false S. aureus-negative result. A control strain of S. aureus that harbored an SCCmec element but no mecA (an empty cassette) was correctly called MSSA by the Xpert test. The second control contained an SCCM1 insertion. The updated Xpert MRSA/SA BC test successfully detected both spa and SCCmec variants of MRSA and correctly identified empty-cassette strains of S. aureus as MSSA. Among a sample of 252 MSSA isolates from the United States and Europe, 3.9% contained empty SCCmec cassettes, 1.6% carried SCCM1, <1% had spa deletions, and <1% contained SCCmec variants other than those with SCCM1 These data suggest that genetic variations that may interfere with Xpert MRSA/SA BC test results remain rare. Results for all the isolates were correct when tested with the updated assay.

Streptococcus agalactiae Strains with Chromosomal Deletions Evade Detection with Molecular Methods.

Surveillance of circulating microbial populations is critical for monitoring the performance of a molecular diagnostic test. In this study, we characterized 31 isolates of Streptococcus agalactiae (group B Streptococcus [GBS]) from several geographic locations in the United States and Ireland that contain deletions in or adjacent to the region of the chromosome that encodes the hemolysin gene cfb, the region targeted by the Xpert GBS and GBS LB assays. PCR-negative, culture-positive isolates were recognized during verification studies of the Xpert GBS assay in 12 laboratories between 2012 and 2018. Whole-genome sequencing of 15 GBS isolates from 11 laboratories revealed four unique deletions of chromosomal DNA ranging from 181 bp to 49 kb. Prospective surveillance studies demonstrated that the prevalence of GBS isolates containing deletions in the convenience sample was <1% in three geographic locations but 7% in a fourth location. Among the 15 isolates with chromosomal deletions, multiple pulsed-field gel electrophoresis types were identified, one of which appears to be broadly dispersed across the United States.

dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression.

Distinct epigenomic profiles of histone marks have been associated with gene expression, but questions regarding the causal relationship remain. Here we investigated the activity of a broad collection of genomically targeted epigenetic regulators that could write epigenetic marks associated with a repressed chromatin state (G9A, SUV39H1, Krüppel-associated box (KRAB), DNMT3A as well as the first targetable versions of Ezh2 and Friend of GATA-1 (FOG1)). dCas9 fusions produced target gene repression over a range of 0- to 10-fold that varied by locus and cell type. dCpf1 fusions were unable to repress gene expression. The most persistent gene repression required the action of several effector domains; however, KRAB-dCas9 did not contribute to persistence in contrast to previous reports. A 'direct tethering' strategy attaching the Ezh2 methyltransferase enzyme to dCas9, as well as a 'recruitment' strategy attaching the N-terminal 45 residues of FOG1 to dCas9 to recruit the endogenous nucleosome remodeling and deacetylase complex, were both successful in targeted deposition of H3K27me3. Surprisingly, however, repression was not correlated with deposition of either H3K9me3 or H3K27me3. Our results suggest that so-called repressive histone modifications are not sufficient for gene repression. The easily programmable dCas9 toolkit allowed precise control of epigenetic information and dissection of the relationship between the epigenome and gene regulation.

Effect of biannual azithromycin distribution on antibody responses to malaria, bacterial, and protozoan pathogens in Niger.

The MORDOR trial in Niger, Malawi, and Tanzania found that biannual mass distribution of azithromycin to children younger than 5 years led to a 13.5% reduction in all-cause mortality (NCT02048007). To help elucidate the mechanism for mortality reduction, we report IgG responses to 11 malaria, bacterial, and protozoan pathogens using a multiplex bead assay in pre-specified substudy of 30 communities in the rural Niger placebo-controlled trial over a three-year period (n = 5642 blood specimens, n = 3814 children ages 1-59 months). Mass azithromycin reduces Campylobacter spp. force of infection by 29% (hazard ratio = 0.71, 95% CI: 0.56, 0.89; P = 0.004) but serological measures show no significant differences between groups for other pathogens against a backdrop of high transmission. Results align with a recent microbiome study in the communities. Given significant sequelae of Campylobacter infection among preschool aged children, our results support an important mechanism through which biannual mass distribution of azithromycin likely reduces mortality in Niger.

Rhamnan sulfate reduces atherosclerotic plaque formation and vascular inflammation.

OBJECTIVE: While lipid-lowering drugs have become a mainstay of clinical therapy these treatments only slow the progression of the disease and can have side effects. Thus, new treatment options are needed to supplement the effects of lipid lowering therapy for treating atherosclerosis. We examined the use of an inexpensive and widely available marine polysaccharide rhamnan sulfate as an oral therapeutic for limiting vascular inflammation and atherosclerosis. METHODS AND RESULTS: We found rhamnan sulfate enhanced the barrier function of endothelial cells, preventing the deposition of LDL and maintaining barrier function even in the presence of glycocalyx-degrading enzymes. Rhamnan sulfate was also found to bind directly to FGF-2, PDGF-BB and NF-κB subunits with high affinity. In addition, rhamnan sulfate was a potent inhibitor of NF-κB pathway activation in endothelial cells by TNF-α. We treated ApoE-/- mice with a high fat diet for 4 weeks and then an addition 9 weeks of high fat diet with or without rhamnan sulfate. Rhamnan sulfate reduced vascular inflammation and atherosclerosis in both sexes of ApoE-/- mice but had a stronger therapeutic effect in female mice. Oral consumption of rhamnan sulfate induced a significant decrease in cholesterol plasma levels in female mice but not in male mice. In addition, there was a marked reduction in inflammation for female mice in the liver and aortic root in comparison to male mice. CONCLUSIONS: Rhamnan sulfate has beneficial effects in reducing inflammation, binding growth factors and NF-κB, enhancing endothelial barrier function and reducing atherosclerotic plaque formation in ApoE-/- mice.

Decreased aortic diameter and compliance precedes blood pressure increases in postnatal development of elastin-insufficient mice.

Increased arterial stiffness and blood pressure are characteristic of humans and adult mice with reduced elastin levels caused by aging or genetic disease. Direct associations have been shown between increased arterial stiffness and hypertension in humans, but it is not known whether changes in mechanical properties or increased blood pressure occur first. Using genetically modified mice with elastin haploinsufficiency (Eln(+/-)), we investigated the temporal relationship between arterial mechanical properties and blood pressure throughout postnatal development. Our results show that some mechanical properties are maintained constant regardless of elastin amounts. The peak diameter compliance for both genotypes occurs near the physiologic pressure at each age, which acts to provide maximum pulse dampening. The stress-strain relationships are similar between genotypes and become nonlinear near the systolic pressure for each age, which serves to limit distension under high pressure. Our results also show that some mechanical properties are affected by reduced elastin levels and that these changes occur before measurable changes in blood pressure. Eln(+/-) mice have decreased aortic diameter and compliance in ex vivo tests that are significant by postnatal day 7 and increased blood pressure that is not significant until postnatal day 14. This temporal relationship suggests that targeting large arteries to increase diameter or compliance may be an effective treatment for human hypertension.

Effect of storage duration on the mechanical behavior of mouse carotid artery.

Determining arterial mechanical properties is important for understanding the work done by the heart and how it changes with cardiovascular disease. Ex vivo tests are necessary to apply various loads to the artery and obtain data to model and predict the behavior under any load. Most ex vivo tests are performed within 24 h of dissection, so the tissue is still "alive." For large elastic arteries; however, the passive mechanical behavior is attributed mostly to the very stable proteins, elastin, and collagen. If the testing equipment fails, is in use, or is located at another facility, it would be useful to store the vessels and postpone the tests until the equipment is available. The goal of this study is to determine the effects of storage time on the mechanical behavior of the common carotid artery from adult mice. Each artery was tested after storage for 1-28 days in physiologic saline at 4°C. There were no significant effects of storage time on the arterial diameter or force at each pressure, but there were significant effects on the stretch ratio and stress at each pressure. The significant effects on the stretch ratio and stress were due to decreases in the unloaded dimensions with storage time, when measured from cut arterial rings. When the unloaded dimensions were measured instead from histology sections, there were no significant changes with storage time. We conclude that histology sections yield a more consistent measurement of the unloaded dimensions and that there are no significant changes in the mechanical behavior of mouse carotid artery with storage up to 28 days.

Characterization of carbapenem-resistant gram-negative bacterial isolates from Nigeria by whole genome sequencing.

This study characterized the mechanisms of carbapenem resistance in gram-negative bacteria isolated from patients in Yola, Nigeria. Whole genome sequencing (WGS) was performed on 66 isolates previously identified phenotypically as carbapenem-non-susceptible. The patterns of beta-lactamase resistance genes identified were primarily species-specific. However, blaNDM-7 and blaCMY-4 were detected in all Escherichia coli and most Providencia rettgeri isolates; blaNDM-7 was also detected in 1 Enterobacter cloacae. The E. coli and E. cloacae isolates also shared blaOXA-1, while blaOXA-10 was found in all P. rettgeri, one Pseudomonas aeruginosa and 1 E. coli. Except for Stenotrophomonas maltophilia isolates, which only contained blaL1, most species carried multiple beta-lactamase genes, including those encoding extended-spectrum beta-lactamases, AmpC and OXA in addition to a carbapenemase gene. Carbapenemase genes were either class B or class D beta-lactamases. No carbapenemase gene was detected by WGS in 13.6% of isolates.

Molecular tension in syndecan-1 is regulated by extracellular mechanical cues and fluidic shear stress.

The endothelium plays a central role in regulating vascular homeostasis and is key in determining the response to materials implanted in the vascular system. Endothelial cells are uniquely sensitive to biophysical cues from applied forces and their local cellular microenvironment. The glycocalyx is a layer of proteoglycans, glycoproteins and glycosaminoglycans that lines the luminal surface of the vascular endothelium, interacting directly with the components of the blood and the forces of blood flow. In this work, we examined the changes in mechanical tension of syndecan-1, a cell surface proteoglycan that is an integral part of the glycocalyx, in response to substrate stiffness and fluidic shear stress. Our studies demonstrate that syndecan-1 has higher mechanical tension in regions of cell adhesion, on and in response to nanotopographical cues. In addition, we found that substrate stiffness also regulated the mechanical tension of syndecan-1 and altered its binding to actin, myosin iiB and signaling intermediates including Src, PKA and FAK. Application of fluidic shear stress created a gradient in tension in syndecan-1 and led to enhanced association with actin, Src, myosin IIb and other cytoskeleton related molecules. Overall, our studies support that syndecan-1 is responsive to the mechanical environment of the cells and alters its association with actin and signaling intermediates in response to mechanical stimuli.

Mobile genetic elements responsible for discordant Staphylococcus aureus phenotypes and genotypes in the same blood culture bottle.

Approximately 15-20% of the S. aureus genome contains mobile genetic elements that can cause discrepancies between phenotypic and genotypic identification methods. Three blood culture bottles (each from a different patient) that showed discordant results, were shown to contain 2 S. aureus isolates after additional subcultures. One bottle had MRSA and MSSA that by DNA sequence analysis differed only by 31 kb; however, the deletions encompassed parts of SCCmec including mecA and SCCM1. The second bottle contained MRSA and MSSA that differed by 124 kb; the MSSA was missing the entire SCCmec and spa regions. The last bottle contained 2 MRSA, one with ACME II disrupting SCCmec and a 24 bp spa deletion. The deletions in SCCmec and the other elements gave rise to the discrepancies between molecular and the original culture results. Such discrepancies should prompt a search for additional strains in the blood culture bottle.