Redefining the H-NS protein family: a diversity of specialized core and accessory forms exhibit hierarchical transcriptional network integration.

H-NS is a nucleoid structuring protein and global repressor of virulence and horizontally-acquired genes in bacteria. H-NS can interact with itself or with homologous proteins, but protein family diversity and regulatory network overlap remain poorly defined. Here, we present a comprehensive phylogenetic analysis that revealed deep-branching clades, dispelling the presumption that H-NS is the progenitor of varied molecular backups. Each clade is composed exclusively of either chromosome-encoded or plasmid-encoded proteins. On chromosomes, stpA and newly discovered hlpP are core genes in specific genera, whereas hfp and newly discovered hlpC are sporadically distributed. Six clades of H-NS plasmid proteins (Hpp) exhibit ancient and dedicated associations with plasmids, including three clades with fidelity for plasmid incompatibility groups H, F or X. A proliferation of H-NS homologs in Erwiniaceae includes the first observation of potentially co-dependent H-NS forms. Conversely, the observed diversification of oligomerization domains may facilitate stable co-existence of divergent homologs in a genome. Transcriptomic and proteomic analysis in Salmonella revealed regulatory crosstalk and hierarchical control of H-NS homologs. We also discovered that H-NS is both a repressor and activator of Salmonella Pathogenicity Island 1 gene expression, and both regulatory modes are restored by Sfh (HppH) in the absence of H-NS.

CHARACTERIZATION OF PATIENTS IN THE POST-ACUTE PHASE OF COVID-19 WHO PRESENTED THEMSELVES TO AN EMERGENCY DEPARTMENT.

Background: Patients that recovered from COVID-19 may remain with symptoms which can persist for an uncertain period of time. Aim: The purpose of this study was to investigate the reasons why patients who passed the acute phase of COVID-19 presented themselves to the Emergency Department. Patients and Methods: We selected 87 patients admitted to the Emergency Department of the Bucharest University Emergency Hospital between 01.01.2021-31.05.2021. Patients had pulmonary fibrosis (11.49%), pleural effusion (16.09%) or a history of hypertension (73.56%), type 2 diabetes (42.53%), stroke (24.14%), malignant diseases (10.34%). Results: Association between neutrophil levels and acute stroke and between fibrinogen levels and alveolar condensation were identified. The percentage of deaths was significantly higher in the subgroup of subjects that had maxim 11 days of hospitalization (p=0.004); we observed a trend of association between the age of more than 51 years old and admission in the Emergency Unit at less than a month after the SARS Cov2 infection, the positive result at the RT-PCR test or a lung damage of over 30% (p<0.05). Conclusion: A significant percentage of patients that were admitted to the Emergency Unit post COVID-19 had chronic pathologies and their characteristics were associated with neutrophilia, high fibrinogen levels or length of hospitalization.

Incorporating the dilution effect in group testing regression.

When screening for infectious diseases, group testing has proven to be a cost efficient alternative to individual level testing. Cost savings are realized by testing pools of individual specimens (eg, blood, urine, saliva, and so on) rather than by testing the specimens separately. However, a common concern that arises in group testing is the so-called "dilution effect." This occurs if the signal from a positive individual's specimen is diluted past an assay's threshold of detection when it is pooled with multiple negative specimens. In this article, we propose a new statistical framework for group testing data that merges estimation and case identification, which are often treated separately in the literature. Our approach considers analyzing continuous biomarker levels (eg, antibody levels, antigen concentrations, and so on) from pooled samples to estimate both a binary regression model for the probability of disease and the biomarker distributions for cases and controls. To increase case identification accuracy, we then show how estimates of the biomarker distributions can be used to select diagnostic thresholds on a pool-by-pool basis. Our proposals are evaluated through numerical studies and are illustrated using hepatitis B virus data collected on a prison population in Ireland.

Negative supercoiling of DNA by gyrase is inhibited in Salmonella enterica serovar Typhimurium during adaptation to acid stress.

DNA in intracellular Salmonella enterica serovar Typhimurium relaxes during growth in the acidified (pH 4-5) macrophage vacuole and DNA relaxation correlates with the upregulation of Salmonella genes involved in adaptation to the macrophage environment. Bacterial ATP levels did not increase during adaptation to acid pH unless the bacterium was deficient in MgtC, a cytoplasmic-membrane-located inhibitor of proton-driven F1 F0 ATP synthase activity. Inhibiting ATP binding by DNA gyrase and topo IV with novobiocin enhanced the effect of low pH on DNA relaxation. Bacteria expressing novobiocin-resistant (NovR ) derivatives of gyrase or topo IV also exhibited DNA relaxation at acid pH, although further relaxation with novobiocin was not seen in the strain with NovR gyrase. Thus, inhibition of the negative supercoiling activity of gyrase was the primary cause of enhanced DNA relaxation in drug-treated bacteria. The Salmonella cytosol reaches pH 5-6 in response to an external pH of 4-5: the ATP-dependent DNA supercoiling activity of purified gyrase was progressively inhibited by lowering the pH in this range, as was the ATP-dependent DNA relaxation activity of topo IV. We propose that DNA relaxation in Salmonella within macrophage is due to acid-mediated impairment of the negative supercoiling activity of gyrase.

Integrin-modulating therapy prevents fibrosis and autoimmunity in mouse models of scleroderma.

In systemic sclerosis (SSc), a common and aetiologically mysterious form of scleroderma (defined as pathological fibrosis of the skin), previously healthy adults acquire fibrosis of the skin and viscera in association with autoantibodies. Familial recurrence is extremely rare and causal genes have not been identified. Although the onset of fibrosis in SSc typically correlates with the production of autoantibodies, whether they contribute to disease pathogenesis or simply serve as a marker of disease remains controversial and the mechanism for their induction is largely unknown. The study of SSc is hindered by a lack of animal models that recapitulate the aetiology of this complex disease. To gain a foothold in the pathogenesis of pathological skin fibrosis, we studied stiff skin syndrome (SSS), a rare but tractable Mendelian disorder leading to childhood onset of diffuse skin fibrosis with autosomal dominant inheritance and complete penetrance. We showed previously that SSS is caused by heterozygous missense mutations in the gene (FBN1) encoding fibrillin-1, the main constituent of extracellular microfibrils. SSS mutations all localize to the only domain in fibrillin-1 that harbours an Arg-Gly-Asp (RGD) motif needed to mediate cell-matrix interactions by binding to cell-surface integrins. Here we show that mouse lines harbouring analogous amino acid substitutions in fibrillin-1 recapitulate aggressive skin fibrosis that is prevented by integrin-modulating therapies and reversed by antagonism of the pro-fibrotic cytokine transforming growth factor ß (TGF-ß). Mutant mice show skin infiltration of pro-inflammatory immune cells including plasmacytoid dendritic cells, T helper cells and plasma cells, and also autoantibody production; these findings are normalized by integrin-modulating therapies or TGF-ß antagonism. These results show that alterations in cell-matrix interactions are sufficient to initiate and sustain inflammatory and pro-fibrotic programmes and highlight new therapeutic strategies.

Longitudinal Assessment of Developmental Outcomes in Infants Undergoing Late Craniosynostosis Repair.

Evaluation of infants with craniosynostosis for surgical intervention, as opposed to conservative management, remains a challenge within the field of craniofacial surgery. Studies have consistently demonstrated that surgical repair of craniosynostosis is ideally performed between 3 and 12 months of age. As such, there is limited data regarding neurocognitive development in infants who initially present with uncorrected craniosynostosis after 12 months of age. Moreover, the impact of cranial vault surgery on neurocognitive development at all ages remains under investigation. A prospective, nonrandomized study was performed. All children with nonsyndromic craniosynostosis who presented for initial evaluation after 12 months of age were enrolled. The Bayley Scales of Infant and Toddler Development, Third Edition (Bayley-III) was utilized to assess pre- and postoperative cognitive development and comparisons were made to normative values. Developmental delay is defined as scoring < 85. Five infants, average age 26 months (13-43 months) at initial presentation, underwent cranial vault remodeling and developmental testing. Fused cranial sutures involved: metopic (n = 4), and right coronal (n = 1). Cognitive testing demonstrated that 4 of 5 infants (80%) were developmentally delayed at presentation (scores: 60, 70, 72, and 80), and 1 infant was within normal limits (score: 100). Postoperative testing was performed between 2 and 12 months postoperatively. Universal improvement was observed in infants who were delayed prior to surgery (80, 80, 75, and 90, respectively). The infant who was not delayed prior to surgery remained within normal limits after surgery. This study demonstrates an association between cranial vault surgery and cognitive improvement in infants presenting late with developmental delay.

The Global Regulatory Cyclic AMP Receptor Protein (CRP) Controls Multifactorial Fluoroquinolone Susceptibility in Salmonella enterica Serovar Typhimurium.

Fluoroquinolone antibiotics are prescribed for the treatment of Salmonella enterica infections, but resistance to this family of antibiotics is growing. Here we report that loss of the global regulatory protein cyclic AMP (cAMP) receptor protein (CRP) or its allosteric effector, cAMP, reduces susceptibility to fluoroquinolones. A Δcrp mutation was synergistic with the primary fluoroquinolone resistance allele gyrA83, thus able to contribute to clinically relevant resistance. Decreased susceptibility to fluoroquinolones could be partly explained by decreased expression of the outer membrane porin genes ompA and ompF with a concomitant increase in the expression of the ciprofloxacin resistance efflux pump gene acrB in Δcrp cells. Expression of gyrAB, which encode the DNA supercoiling enzyme GyrAB, which is blocked by fluoroquinolones, and expression of topA, which encodes the dominant supercoiling-relaxing enzyme topoisomerase I, were unchanged in Δcrp cells. Yet Δcrp cells maintained a more relaxed state of DNA supercoiling, correlating with an observed increase in topoisomerase IV (parCE) expression. Surprisingly, the Δcrp mutation had the unanticipated effect of enhancing fitness in the presence of fluoroquinolone antibiotics, which can be explained by the observation that exposure of Δcrp cells to ciprofloxacin had the counterintuitive effect of restoring wild-type levels of DNA supercoiling. Consistent with this, Δcrp cells did not become elongated or induce the SOS response when challenged with ciprofloxacin. These findings implicate the combined action of multiple drug resistance mechanisms in Δcrp cells: reduced permeability and elevated efflux of fluoroquinolones coupled with a relaxed DNA supercoiling state that buffers cells against GyrAB inhibition by fluoroquinolones.

Biomechanical assessment of fixation methods for segmental mandible reconstruction with fibula in the polyurethane model.

BACKGROUND: A variety of plating techniques are employed by microsurgeons to provide rigid fixation for vascularized bone constructs of the mandible. The aim of this study was to biomechanically compare three commonly utilized plating techniques for rigid fixation of fibula bone flaps in reconstructing lateral segmental mandibular defects. MATERIALS AND METHODS: Polyurethane mandibles with 3-cm segmental defects were reconstructed using polyurethane fibula models. Three fixation techniques were compared (n = 5 models per group): Group 1 used two 2.0-mm miniplates at each osteotomy site, Group 2 used a single 2.3-mm plate, and Group 3 used a single 2.7-mm plate. Biomechanical testing of maximum force and displacement at failure for each plating technique was assessed and statistical comparison performed. RESULTS: The average displacement for Group 1 was 14.08 ± 1.42 mm, Group 2 was 5.79 ± 0.89 mm, and Group 3 was 6.03 ± 1.59 mm. Group 1 had significantly greater (P < 0.05) displacement when compared with Group 2 and 3. Analysis of variance demonstrated the three groups varied significantly in mean displacement (0 < 0.01). The average force before failure for Group 1 was 616.4 ± 33.83N, Group 2 was 737.8 ± 72.57N, and Group 3 was 681.0 ± 67.98N. Group 2 withstood significantly greater force than Group 1 (P < 0.05), and withstood greater force than Group 3, although the difference was not significant. Analysis of variance showed the three groups varied significantly in mean force at failure (P < 0.05). CONCLUSION: Reconstruction using a single 2.3-mm plate provided the best rigid fixation for lateral segmental defects of the mandible. © 2016 Wiley Periodicals, Inc. Microsurgery 36:330-333, 2016.

Assessment of Deformational Plagiocephaly Severity and Neonatal Developmental Delay.

Deformational plagiocephaly (DP) in infants has been associated with developmental delay that can last until adolescence. Despite this association and a 5-fold increase in incidence of DP over the past 2 decades, there are currently no guidelines regarding screening for developmental delay or identification of which infants with DP are at the greatest risk of delay. A prospective, nonrandomized study was performed. Infants diagnosed with DP who had no prior intervention were eligible for enrollment. Cranial deformity was measured by cross-cranial measurements using calipers, and developmental delay was measured using the Bayley Scales of Infant and Toddler Development, Third Edition (Bayley-III). Correlation between cranial deformity and developmental delay was analyzed using a linear regression. Twenty-seven infants, ages 4.0 to 11.0 months (mean = 6.61 months) diagnosed with DP were studied. Developmental delay was observed on the composite language (n = 3 of 27, 11%), and composite motor (n = 5 of 23, 22%) scales, but not the cognitive scale. Severity of cranial deformity did not correlate with scores on any Bayley-III scales (cognitive R = 0.058, P = 0.238; composite language R = 0.03, P = 0.399; composite motor R = 0.0195, P = 0.536). This study demonstrates that severity of cranial deformity cannot be used to predict presence or degree of developmental delay. Craniofacial surgeons should be aware of this risk and consider developmental screening based on clinical suspicion.

Mixtures of benzo(a)pyrene, dichlorodiphenyltrichloroethane and tributyltin are more toxic to neotropical fish Rhamdia quelen than isolated exposures.

The effects of benzo(a)pyrene (BaP), dichlorodiphenyltrichloroethane (DDT) and tributyltin (TBT) association were investigated through a multi-biomarker approach. Ten Rhamdia quelen fish per group were exposed through intraperitoneal injections either to BaP (0.3; 3 or 30 mg kg(-1)), DDT or TBT (0.03; 0.3 or 3 mg kg(-1)) or BaP/DDT, BaP/TBT, DDT/TBT or BaP/DDT/TBT on their lowest doses. The experiments were divided in acute (one dose, 5-day) and sub-chronic (3 doses, 15-day). Control groups received an equal volume of PBS or canola oil (1 ml kg(-1)). The three tested contaminants altered AChE activity in brain and muscle in similar ways; the mixtures antagonized the increase evoked by the contaminants alone. BaP and TBT increased GSH content and mixtures reduced it. GPx activity was increased by DDT and TBT in the 15-day experiment and reduced by the mixtures. BaP increased GST activity in sub-chronic experiment while TBT reduced it in the acute experiment. BaP/TBT increased GST activity compared to all groups; the other mixtures reduced it compared to BaP or DDT in the 5-day experiment. BaP, DDT and TBT increased δ-ALAd activity mainly in acute exposure; the mixtures also increased δ-ALAd compared to DDT or TBT in 5 and 15-day. BaP, TBT and BaP/DDT decreased LPO in the acute experiment. In the sub-chronic experiment DDT/TBT increased LPO when compared to TBT. None of the contaminants alone altered PCO, but all mixtures increased it compared to one or another contaminant. Contaminants isolated had a more acute effect in ALT plasma level; their lowest dose, which had no effect alone, in combination has led to an increase of this enzyme, especially after 15 days. DDT increased AST in the acute and sub-chronic experiments, while TBT did the same in the latter. DDT/TBT decreased AST opposing the effect of the contaminants alone in the 5-day experiment. Hepatic lesions index could be explained by a more acute effect of the contaminants alone or combined and by activation of cell defenses after the sub-chronic exposure. TBT increased melanomacrophages counting in the 5-day experiment and the mixtures increased it in the 5 and 15-day experiments. Overall, the majority of the biomarkers pointed to a more toxic effect when these contaminants were combined, leading to unexpected toxicities compared to individual exposure scenarios. These findings are relevant considering environmental exposure conditions, since organisms are often exposed to different combinations of contaminants.

Incidence and prevalence of Parkinson's disease in Buenos Aires City, Argentina.

BACKGROUND AND PURPOSE: Epidemiologic studies of incidence and prevalence in Parkinson's Disease (PD) show highly variable results. Despite the large number of studies performed worldwide during the last decades, little is known about its prevalence in South America and no incidence studies have been performed. The goal of this study is to assess the incidence and prevalence of PD in a health maintenance organization from Buenos Aires City, the capital city of Argentina. METHODS: The population were all members of the 'Plan de Salud, Hospital Italiano de Buenos Aires', a large prepaid health medical organization in Buenos Aires. From 1 January 2003 to 31 December 2008 patients diagnosed with PD according to Brain Bank of London diagnostic criteria were identified retrospectively. Incidence density was calculated with 95% confidence interval. RESULTS: Hundred and forty thousand people were followed for a total of 754,082 person-years. A total of 239 incident cases of PD were identified. Crude incidence density was 31.2/100,000 person-years. Prevalence was 394/100,000 in the population older than 40 years. Male to female ratio was 1.31. CONCLUSIONS: This is the first study in South America that estimates the incidence of PD. Our results are consistent with other studies from other regions using similar methodologies.

For What It's Worth: Medical School Reputation in The Integrated Plastic Surgery Match.

Integrated Plastic and Reconstructive Surgery residency programs may use medical school reputation to help fill the gap of a pass/fail USMLE Step 1 in the match. The main objective of this manuscript was to consider if this shifting emphasis is warranted. Herein, a cross-sectional analysis of academic plastic surgeons found that medical school reputation did not predict career achievement. In the absence of evidence demonstrating its worth, residency programs should exercise caution in using medical school reputation in the match.

Embryotoxicity and biotransformation responses in zebrafish exposed to water-soluble fraction of crude oil.

The toxic effects of water-soluble fraction (WSF) of crude oil (API27, Petrobras Campos Basin, Brazil) were evaluated during the early life stages of zebrafish, as well as its biotransformation in juvenile fish. Embryonic development was studied during 96 h. Reduced heartbeat rate, weak pigmentation, tail defects, and embryo mortality were observed for all of the tested concentrations of the WSF. Activities of the biotransformation enzymes were induced at the highest concentrations, showing that these enzymes played a role in its elimination. As shown in this study the crude oil WSF altered the normal embryonic development of fish.

In vitro processing and secretion of mutant insulin proteins that cause permanent neonatal diabetes.

Permanent neonatal diabetes mellitus is a rare form of insulin-requiring diabetes presenting within the first few weeks or months of life. Mutations in the insulin gene are the second most common cause of this form of diabetes. These mutations are located in critical regions of preproinsulin and are likely to prevent normal processing or folding of the preproinsulin/proinsulin molecule. To characterize these mutations, we transiently expressed proinsulin-GFP fusion proteins in MIN6 mouse insulinoma cells. Our study revealed three groups of mutant proteins: 1) mutations that result in retention of proinsulin in the endoplasmic reticulum (ER) and attenuation of secretion of cotransfected wild-type insulin: C43G, F48C, and C96Y; 2) mutations with partial ER retention, partial recruitment to granules, and attenuation of secretion of wild-type insulin: G32R, G32S, G47V, G90C, and Y108C; and 3) similar to (2) but with no significant attenuation of wild-type insulin secretion: A24D and R89C. The mutant insulin proteins do not prevent targeting of wild-type insulin to secretory granules, but most appear to lead to decreased secretion of wild-type insulin. Each of the mutants triggers the expression of the proapoptotic gene Chop, indicating the presence of ER stress.

In vivo identification of a 107-base pair promoter element mediating neuron-specific expression of mouse gonadotropin-releasing hormone.

To identify regions of the mouse GnRH (mGnRH) promoter that mediate tissue-specific gene expression, transgenic mice have been generated with fragments of mGnRH promoter fused to the luciferase reporter gene. In this manuscript, we examine transgenic mice, generated with -356/+28 bp and -249/+28 bp of the mGnRH gene. Both fragments of mGnRH promoter target ovarian expression of the luciferase transgene, but neuronal luciferase activity is detected only in the mice bearing the -356-bp fragment, suggesting that the DNA sequences essential for directing neuron-specific expression of the GnRH gene are located between -356 and -249 bp. Two consensus binding sites for Otx2 were identified in this promoter region and were confirmed to be functional. EMSAs demonstrated specific binding of Otx2 to the mGnRH promoter, and overexpression of Otx2 increased transcriptional activity of the mGnRH promoter in transient transfection studies. When both Otx2 binding sites were eliminated, overexpression of Otx2 had no effect. GnRH mRNA expression in immortalized GnRH-secreting cell lines was also found to correlate with Otx2 expression. In addition, transgenic mice, bearing the 356 fragment of the mGnRH gene in which the Otx2 binding sites were eliminated, have significantly lower luciferase activity in the neonatal brain compared with mice generated with intact Otx2 binding sites. Luciferase activity was, however, still present in the ovary. Our findings provide evidence that Otx2 may have a critical role in directing tissue-specific expression of the mGnRH gene to the neuron, but not the ovary.

The small RNA RssR regulates myo-inositol degradation by Salmonella enterica.

Small noncoding RNAs (sRNAs) with putative regulatory functions in gene expression have been identified in the enteropathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). Two sRNAs are encoded by the genomic island GEI4417/4436 responsible for myo-inositol (MI) degradation, suggesting a role in the regulation of this metabolic pathway. We show that a lack of the sRNA STnc2160, termed RssR, results in a severe growth defect in minimal medium (MM) with MI. In contrast, the second sRNA STnc1740 was induced in the presence of glucose, and its overexpression slightly attenuated growth in the presence of MI. Constitutive expression of RssR led to an increased stability of the reiD mRNA, which encodes an activator of iol genes involved in MI utilization, via interaction with its 5'-UTR. SsrB, a response regulator contributing to the virulence properties of salmonellae, activated rssR transcription by binding the sRNA promoter. In addition, the absence of the RNA chaperone Hfq resulted in strongly decreased levels of RssR, attenuated S. Typhimurium growth with MI, and reduced expression of several iol genes required for MI degradation. Considered together, the extrinsic RssR allows fine regulation of cellular ReiD levels and thus of MI degradation by acting on the reiD mRNA stability.

Correlative vs. Causative Relationship between Neonatal Cranial Head Shape Anomalies and Early Developmental Delays.

Deformational plagiocephaly and craniosynostosis are two of the most common neonatal cranial head shape anomalies. Traditionally, both entities were thought to cause aesthetic concerns solely. Recently, many groups have demonstrated that both conditions are strongly associated with developmental delays. The relationship between the abnormal neonatal cranial shape and early developmental delays manifested in both conditions remains poorly understood.

Small RNA, Big Effect: Control of Flagellin Production.

Many bacteria move in their environment using a remarkable, rotating nanomachine - the flagellum. In a recent publication, Choi et al. report a new addition to the group of flagellar regulators, a trans-acting small RNA (sRNA).