A Platform for Generation of Chamber-Specific Cardiac Tissues and Disease Modeling.

Tissue engineering using cardiomyocytes derived from human pluripotent stem cells holds a promise to revolutionize drug discovery, but only if limitations related to cardiac chamber specification and platform versatility can be overcome. We describe here a scalable tissue-cultivation platform that is cell source agnostic and enables drug testing under electrical pacing. The plastic platform enabled on-line noninvasive recording of passive tension, active force, contractile dynamics, and Ca2+ transients, as well as endpoint assessments of action potentials and conduction velocity. By combining directed cell differentiation with electrical field conditioning, we engineered electrophysiologically distinct atrial and ventricular tissues with chamber-specific drug responses and gene expression. We report, for the first time, engineering of heteropolar cardiac tissues containing distinct atrial and ventricular ends, and we demonstrate their spatially confined responses to serotonin and ranolazine. Uniquely, electrical conditioning for up to 8 months enabled modeling of polygenic left ventricular hypertrophy starting from patient cells.

metGWAS 1.0: an R workflow for network-driven over-representation analysis between independent metabolomic and meta-genome-wide association studies.

MOTIVATION: The method of genome-wide association studies (GWAS) and metabolomics combined provide an quantitative approach to pinpoint metabolic pathways and genes linked to specific diseases; however, such analyses require both genomics and metabolomics datasets from the same individuals/samples. In most cases, this approach is not feasible due to high costs, lack of technical infrastructure, unavailability of samples, and other factors. Therefore, an unmet need exists for a bioinformatics tool that can identify gene loci-associated polymorphic variants for metabolite alterations seen in disease states using standalone metabolomics. RESULTS: Here, we developed a bioinformatics tool, metGWAS 1.0, that integrates independent GWAS data from the GWAS database and standalone metabolomics data using a network-based systems biology approach to identify novel disease/trait-specific metabolite-gene associations. The tool was evaluated using standalone metabolomics datasets extracted from two metabolomics-GWAS case studies. It discovered both the observed and novel gene loci with known single nucleotide polymorphisms when compared to the original studies. AVAILABILITY AND IMPLEMENTATION: The developed metGWAS 1.0 framework is implemented in an R pipeline and available at: https://github.com/saifurbd28/metGWAS-1.0.

Assessing Guidelines on the Need for Colonoscopy After Initial Flexible Sigmoidoscopy in Young Patients With Outlet-Type Rectal Bleeding.

BACKGROUND: Although young-age-of-onset colorectal cancer is increasing in incidence, lack of screening leads to symptomatic presentation, often with rectal bleeding. Because most cancers in patients younger than 50 years are left-sided, flexible sigmoidoscopy is a reasonable way of investigating bleeding in these patients. OBJECTIVE: To predict which patients undergoing flexible sigmoidoscopy for outlet-type rectal bleeding need a full colonoscopy. DESIGN: Findings at colonoscopy were compared with published indications for colonoscopy after flexible sigmoidoscopy, which were as follows: 1) any number of advanced adenomas defined as a tubular adenoma of >9 mm diameter, a tubulovillous or villous adenoma of any size, or any adenoma with high-grade dysplasia; 2) 3 or more tubular adenomas of any size or histology; 3) any sessile serrated lesion; and 4) 20 or more hyperplastic polyps. SETTING: Charity Hospital with volunteer specialists. PATIENTS: Patients were included if they were younger than 57 years, had outlet-type rectal bleeding, and underwent flexible sigmoidoscopy at least to the descending colon followed by colonoscopy with biopsy of all resected lesions. INTERVENTIONS: Flexible sigmoidoscopy and colonoscopy with excision of all removable lesions. MAIN OUTCOME MEASURES: Findings at colonoscopy. RESULTS: There were 66 patients who had a colonoscopy between 5 and 811 days after sigmoidoscopy and also had complete data. There were 43 men and 23 women with a mean age of 39.5 years. Analysis of flexible sigmoidoscopy criteria for finding proximal high-risk lesions on colonoscopy showed a sensitivity of 76.9%, a specificity of 67.9%, a positive predictive value of 37%, a negative predictive value of 92.3%, and an accuracy of 69.7%. LIMITATIONS: A large number of exclusions for inadequate colonoscopy or inadequate data resulted in a reduced patient number in the study. CONCLUSIONS: Our criteria for follow-up colonoscopy based on the findings at initial flexible sigmoidoscopy in young patients with outlet-type rectal bleeding are reliable enough to be used in routine clinical practice, provided this is audited. See Video Abstract. GUA DE EVALUACIN PARA LA NECESIDAD DE COLONOSCOPIA DESPUS DE UNA SIGMOIDOSCOPIA FLEXIBLE INICIAL EN PACIENTES JVENES CON RECTORRAGIA: ANTECEDENTES:Si bien la edad de aparición temprana del cáncer colorrectal está aumentando en incidencia, la falta de pruebas de detección conduce a una presentación sintomática, a menudo con sangrado rectal. Debido a que la mayoría de los cánceres en pacientes menores de 50 años son del lado izquierdo, la sigmoidoscopia flexible es una forma razonable de investigar el sangrado en estos pacientes.OBJETIVO:Predecir qué pacientes sometidos a sigmoidoscopia flexible por rectorragia necesitan una colonoscopia completa.DISEÑO:Los resultados de la colonoscopia se compararon con las indicaciones publicadas para la colonoscopia después de una sigmoidoscopia flexible. Estos fueron: 1. Cualquier número de adenomas avanzados, definidos como un adenoma tubular > 9 mm, un adenoma tubulovelloso o velloso de cualquier tamaño, o cualquier adenoma con displasia de alto grado. 2. Tres o más adenomas tubulares de cualquier tamaño o histología. 3. Cualquier lesión serrada sésil. 4. Veinte o más pólipos hiperplásicos.ENTORNO CLINICO:Hospital de Caridad con especialistas voluntarios.PACIENTES:Menores de 57 años, con rectorragia, sometidos a sigmoidoscopia flexible al menos hasta el colon descendente, seguida de colonoscopia con biopsia de todas las lesiones resecadas.INTERVENCIONES:sigmoidoscopia flexible y colonoscopia con escisión de todas las lesiones removibles.PRINCIPALES MEDIDAS DE VALORACIÓN:Hallazgos en la colonoscopia.RESULTADOS:66 casos a los que se les realizó una colonoscopia entre 5 y 811 días después de la sigmoidoscopia, que también tenían datos completos. 43 hombres y 23 mujeres con una edad media de 39,5 años. El análisis de los criterios de sigmoidoscopia flexible para encontrar lesiones proximales de alto riesgo en la colonoscopia mostró una sensibilidad del 76,9 %, una especificidad del 67,9 %, un valor predictivo positivo del 37 %, un valor predictivo negativo del 92,3 % y una precisión del 69,7 %.LIMITACIONES:Gran número de exclusiones por colonoscopia inadecuada o datos inadecuados que causan un número reducido de pacientes en el estudio.CONCLUSIÓN:Nuestros criterios para la colonoscopia de seguimiento basados en los hallazgos de la sigmoidoscopia flexible inicial en pacientes jóvenes con rectorragia son lo suficientemente confiables para ser utilizados en la práctica clínica habitual, siempre que se audite. (Traducción- Dr. Ingrid Melo ).

Cancer screening programs in South-east Asia and Western Pacific.

BACKGROUND: The burden of cancer can be altered by screening. The field of cancer screening is constantly evolving; from the initiation of program for new cancer types as well as exploring innovative screening strategies (e.g. new screening tests). The aim of this study was to perform a landscape analysis of existing cancer screening programs in South-East Asia and the Western Pacific. METHODS: We conducted an overview of cancer screening in the region with the goal of summarizing current designs of cancer screening programs. First, a selective narrative literature review was used as an exploration to identify countries with organized screening programs. Second, representatives of each country with an organized program were approached and asked to provide relevant information on the organizations of their national or regional cancer screening program. RESULTS: There was wide variation in the screening strategies offered in the considered region with only eight programs identified as having an organized design. The majority of these programs did not meet all the essential criteria for being organized screening. The greatest variation was observed in the starting and stopping ages. CONCLUSIONS: Essential criteria of organized screening are missed. Improving organization is crucial to ensure that the beneficial effects of screening are achieved in the long-term. It is strongly recommended to consider a regional cancer screening network.

Substance Use Screening in Geriatric Primary Care: Cultural Issues and Alcohol Consumption in the Deep South.

OBJECTIVES: To investigate indicators of potentially hazardous alcohol use among older adults living in a region with high substance use stigma. METHODS: Patients at a university-affiliated geriatrics clinic in the Deep South of theUS completed behavioral health screenings including self-reported alcohol use, symptoms of depression or anxiety, and cognitive functioning between 2018 and 2022. RESULTS: Participants (N = 278) averaged 76.04 years of age (SD = 9.25), were predominantly female (70.9%), and non-Hispanic white (84.5%), with an averageof 6.08 comorbid diagnoses (SD = 2.86). Race/ethnicity, age, and symptoms of anxiety were associated with alcohol use and hazardous alcohol use, with non-Hispanic whites, younger individuals, and those with more anxiety symptoms reporting more alcohol use. Notably, alcohol use and hazardous alcohol use were associated with cognitive functioning in the dementia range. CONCLUSION: Self-reported alcohol use is low in geriatric primary care in the Deep South, US, differs by race/ethnicity, and is predictive of cognitive impairment when alcohol use is hazardous. Issues of trust and stigma may play a role in self-report ofstigmatized behaviors. CLINICAL IMPLICATIONS: Self-reported alcohol intake must be considered within the cultural context of regional stigma. Recommendations to address this are provided.

The neurotransmitter receptor Gabbr1 regulates proliferation and function of hematopoietic stem and progenitor cells.

Hematopoietic and nervous systems are linked via innervation of bone marrow (BM) niche cells. Hematopoietic stem/progenitor cells (HSPCs) express neurotransmitter receptors, such as the γ-aminobutyric acid (GABA) type B receptor subunit 1 (GABBR1), suggesting that HSPCs could be directly regulated by neurotransmitters like GABA that directly bind to GABBR1. We performed imaging mass spectrometry and found that the endogenous GABA molecule is regionally localized and concentrated near the endosteum of the BM niche. To better understand the role of GABBR1 in regulating HSPCs, we generated a constitutive Gabbr1-knockout mouse model. Analysis revealed that HSPC numbers were significantly reduced in the BM compared with wild-type littermates. Moreover, Gabbr1-null hematopoietic stem cells had diminished capacity to reconstitute irradiated recipients in a competitive transplantation model. Gabbr1-null HSPCs were less proliferative under steady-state conditions and upon stress. Colony-forming unit assays demonstrated that almost all Gabbr1-null HSPCs were in a slow or noncycling state. In vitro differentiation of Gabbr1-null HSPCs in cocultures produced fewer overall cell numbers with significant defects in differentiation and expansion of the B-cell lineage. To determine whether a GABBR1 agonist could stimulate human umbilical cord blood (UCB) HSPCs, we performed brief ex vivo treatment prior to transplant into immunodeficient mice, with significant increases in long-term engraftment of HSPCs compared with GABBR1 antagonist or vehicle treatments. Our results indicate a direct role for GABBR1 in HSPC proliferation, and identify a potential target to improve HSPC engraftment in clinical transplantation.

Practice patterns for reporting digestive system neuroendocrine neoplasms: results from a large, comprehensive international survey.

AIMS: Criteria for the interpretation of digestive system neuroendocrine neoplasms (NENs) continue to evolve. Although there are some literature recommendations regarding workup and diagnosis of these lesions, different practice patterns exist among pathologists when signing out these specimens. The aim of this study was to assess practice trends among pathologists worldwide when reporting these neoplasms. METHODS AND RESULTS: We created an online survey with multiple questions pertaining to digestive NENs. The results were analysed based on type of practice setting, years of sign-out experience, and practice location. Respondents included 384 practicing pathologists: 70% academic, 30% private practice; 63% gastrointestinal (GI) pathology-subspecialised, 37% not; 39% North American, 42% European, 19% others; 45% with ≤10 years in practice; 55% with >10 years. Some question responses were chosen by the majority (e.g. 85% use both mitotic count and Ki67 index for grading NENs, 82% complete a synoptic, and Ki67 stain even for small incidental appendiceal neuroendocrine tumours [NETs], and 96% utilize the diagnosis of grade 3 NET). However, some questions showed varying responses, including counting mitotic figures, Ki67 stain interpretation, and pancreatic grade 3 NEN workup. Pathologists also had some variability in interpreting regional metastatic foci of small bowel NETs and in choosing blocks for Ki67 staining in multifocal lesions. CONCLUSION: There existed scenarios wherein practice patterns varied despite recommendations in the literature, and there were also scenarios lacking clear guidelines wherein pathologists used varying judgement. This survey highlights current key grey areas in digestive system NEN evaluation, leading to variation in practice patterns.

Proinflammatory changes in the maternal circulation, maternal-fetal interface, and placental transcriptome in preterm birth.

BACKGROUND: Preterm birth remains a leading obstetrical complication because of the incomplete understanding of its multifaceted etiology. It is known that immune alterations toward a proinflammatory profile are observed in women with preterm birth, but therapeutic interventions are still lacking because of scarcity of evidence in the integration of maternal and placental interrelated compartments. OBJECTIVE: This study aimed to obtain an integrated view of the maternal and placental contribution to preterm birth compared with normal term pregnancies for an in-depth understanding of the immune/inflammatory involvement, intending to identify novel strategies to mitigate the negative impact of inflammation. STUDY DESIGN: We prospectively recruited 79 women with preterm or term deliveries and collected placentas for RNA sequencing, histologic analyses, and to assess levels of inflammatory mediators. Blood samples were also collected to determine the circulating immune profiles by flow cytometry and to evaluate the circulating levels of inflammatory mediators. RESULTS: Placental transcriptomic analyses revealed 102 differentially expressed genes upregulated in preterm birth, including known and novel targets, which were highly enriched for inflammatory biological processes according to gene ontology analyses. Analysis of maternal immune cells revealed distinct profiles in preterm births vs term births, including an increased percentage of CD3- cells and monocyte subsets and decreased CD3+ cells along with Th17 subsets of CD4+ lymphocytes. Supporting our bioinformatic findings, we found increases in proinflammatory mediators in the plasma, placenta, and fetal membranes (primarily the amnion) of women with preterm birth, such as interleukin-6 and tumor necrosis factor-α. These findings were not distinct between spontaneous and iatrogenic preterm births except at a molecular level where spontaneous preterm birth presented with an elevated inflammatory profile compared with iatrogenic preterm birth. Analysis of placental histology revealed increased structural and inflammatory lesions in preterm vs term births. We found that genes upregulated in placentas with inflammatory lesions have enrichment of proinflammatory pathways. CONCLUSION: This work sheds light on changes within the immune system in preterm birth on multiple levels and compartments to help identify pregnancies at high risk of preterm birth and to discover novel therapeutic targets for preterm birth.

Here and there a trophoblast, a transcriptional evaluation of trophoblast cell models.

A recent explosion of methods to produce human trophoblast and stem cells (hTSCs) is fuelling a renewed interest in this tissue. The trophoblast is critical to reproduction by facilitating implantation, maternal physiological adaptations to pregnancy and the growth of the fetus through transport of nutrients between the mother and fetus. More broadly, the trophoblast has phenotypic properties that make it of interest to other fields. Its angiogenic and invasive properties are similar to tumours and could identify novel drug targets, and its ability to regulate immunological tolerance of the allogenic fetus could lead to improvements in transplantations. Within this review, we integrate and assess transcriptomic data of cell-based models of hTSC alongside in vivo samples to identify the utility and applicability of these models. We also integrate single-cell RNA sequencing data sets of human blastoids, stem cells and embryos to identify how these models may recapitulate early trophoblast development.

Biased signaling by endogenous opioid peptides.

Opioids, such as morphine and fentanyl, are widely used for the treatment of severe pain; however, prolonged treatment with these drugs leads to the development of tolerance and can lead to opioid use disorder. The "Opioid Epidemic" has generated a drive for a deeper understanding of the fundamental signaling mechanisms of opioid receptors. It is generally thought that the three types of opioid receptors (µ, δ, κ) are activated by endogenous peptides derived from three different precursors: Proopiomelanocortin, proenkephalin, and prodynorphin. Posttranslational processing of these precursors generates >20 peptides with opioid receptor activity, leading to a long-standing question of the significance of this repertoire of peptides. Here, we address some aspects of this question using a technical tour de force approach to systematically evaluate ligand binding and signaling properties ([35S]GTPγS binding and ß-arrestin recruitment) of 22 peptides at each of the three opioid receptors. We show that nearly all tested peptides are able to activate the three opioid receptors, and many of them exhibit agonist-directed receptor signaling (functional selectivity). Our data also challenge the dogma that shorter forms of ß-endorphin do not exhibit receptor activity; we show that they exhibit robust signaling in cultured cells and in an acute brain slice preparation. Collectively, this information lays the groundwork for improved understanding of the endogenous opioid system that will help in developing more effective treatments for pain and addiction.

Glycogenic hepatopathy is associated with type 1 diabetes mellitus in only a minority of cases in a contemporary adult population.

OBJECTIVES: This study examines the clinical-pathological profiles of patients with glycogenic hepatopathy in a contemporary cohort of patients at an adult acute care hospital. METHODS: Liver biopsies with glycogenic hepatopathy were retrieved from the departmental surgical pathology database, the histological findings were studied, and the clinical findings were reviewed. RESULTS: Five cases of glycogenic hepatopathy were found, including cases associated with type 1 diabetes mellitus (n = 1), type 2 diabetes mellitus (n = 1), corticosteroids (n = 2), and anorexia (n = 2, including the patient with type 1 diabetes). AST and ALT were normal to mildly elevated (13-115 U/L and 7-126 U/L, respectively). Trace ascites was present in two patients. Hepatomegaly was only present in the patient with type 1 diabetes at the time of diagnosis. CONCLUSIONS: Four of five cases were associated with etiologies other than type 1 diabetes, which is widely reported as the most common etiology of glycogenic hepatopathy. This study suggests that etiologies currently only rarely recognized may actually be more common causes of glycogenic hepatopathy than type 1 diabetes in a contemporary adult population. It is important not only to recognize that these rarely reported causes of glycogenic hepatopathy may be underrecognized, but that the clinical presentation may also be mild.

Elf5-centered transcription factor hub controls trophoblast stem cell self-renewal and differentiation through stoichiometry-sensitive shifts in target gene networks.

Elf5 is a transcription factor with pivotal roles in the trophoblast compartment, where it reinforces a trophoblast stem cell (TSC)-specific transcriptional circuit. However, Elf5 is also present in differentiating trophoblast cells that have ceased to express other TSC genes such as Cdx2 and Eomes. In the present study, we aimed to elucidate the context-dependent role of Elf5 at the interface between TSC self-renewal and the onset of differentiation. We demonstrate that precise levels of Elf5 are critical for normal expansion of the TSC compartment and embryonic survival, as Elf5 overexpression triggers precocious trophoblast differentiation. Through integration of protein interactome, transcriptome, and genome-wide chromatin immunoprecipitation data, we reveal that this abundance-dependent function is mediated through a shift in preferred Elf5-binding partners; in TSCs, Elf5 interaction with Eomes recruits Tfap2c to triply occupied sites at TSC-specific genes, driving their expression. In contrast, the Elf5 and Tfap2c interaction becomes predominant as their protein levels increase. This triggers binding to double- and single-occupancy sites that harbor the cognate Tfap2c motif, causing activation of the associated differentiation-promoting genes. These data place Elf5 at the center of a stoichiometry-sensitive transcriptional network, where it acts as a molecular switch governing the balance between TSC proliferation and differentiation.

Robotic colorectal procedures: does operative start time impact short-term outcome?

BACKGROUND: Previous publications revealed more complications in afternoon versus morning surgeries. With much attention given to robotic surgery outcomes, we sought to evaluate whether morning versus afternoon start time matters. METHODS: In a retrospective review of a prospective database, 210 robotic colorectal surgeries were grouped into 97 morning versus 113 afternoon cases. Preoperative risk factors, intraoperative events, and 30-day postoperative outcomes were compared. An independent samples t-test, Fisher's exact test, and linear regression were used for categorical and continuous variables. RESULTS: Morning patients were significantly younger than afternoon patients (59.5 vs. 65.5, p = 0.004), but there were no significant differences in gender, mean BMI, Charlson Comorbidity Index score, total operative time, console time, estimated blood loss, indications for surgery, and resection type. Morning patients had a significantly shorter mean length of stay (6.0 vs. 8.0 days, p = 0.021), but no significant differences in overall postoperative complications (0.30 vs. 0.30, p = 0.715), wound infection (5.2% vs. 7.1%, p = 0.564), anastomotic leak (0% vs. 2.7%, p = 0.251), ileus/small bowel obstruction (29.9% vs. 22.1%, p = 0.199), and 30-day readmission (8.2% vs. 7.1%, p = 1.000). When analyzing time of day as a continuous variable, we found no significant associations with intra- or postoperative complications. CONCLUSION: We found no correlation between surgery start time and intra- or postoperative outcomes. This can be partly attributed to these cases being non-emergent and performed primarily by two experienced surgeons with highly trained operating room robotic staff in a large volume tertiary center. This, along with decreased fatigue attributed to superior ergonomics of robotic surgery, may have mitigated previously reported differences between morning and afternoon procedures.

Esophageal OCT Imaging Using a Paddle Probe Externally Attached to Endoscope.

BACKGROUND AND AIMS: Endoscopic surveillance of Barrett's esophagus (BE) by white light examination is insufficient to diagnose dysplastic change. In this work, we describe an optical imaging method to obtain high-resolution cross-sectional imaging using a paddle-shaped probe affixed to the endoscope tip. METHODS: We integrated Optical Coherence Tomography (OCT), an optical imaging method that produces cross-sectional images, into a paddle probe attached to video endoscope. We acquired images of esophageal epithelium from patients undergoing routine upper GI endoscopy. Images were classified by a reviewer blinded to patient identity and condition, and these results were compared with clinical diagnosis. RESULTS: We successfully captured epithelial OCT images from 30 patients and identified features consistent with both squamous epithelium and Barrett's esophagus. Our blinded image reviewer classified BE versus non-BE with 91.5% accuracy (65/71 image regions), including sensitivity of 84.6% for BE (11/13) and a specificity of 93.1% (54/58). However, in 16 patients, intubation of the probe into the esophagus could not be achieved. CONCLUSIONS: A paddle probe is a feasible imaging format for acquiring cross-sectional OCT images from the esophagus and can provide a structural assessment of BE and non-BE tissue. Probe form factor is the current limiting obstacle, but could be addressed by further miniaturization.

Sleep Dysfunction in Adolescents With Prolonged Postconcussion Symptoms: A Reciprocal Coupling of Traumatic Brain Injury and Sleep-Related Problems.

CLINICAL SCENARIO: Concussions are often neglected injuries that affect children and adolescents. Two physiological responses to a concussion are an ionic flux and an increased indiscriminate release of glutamate, which leads to an increase of intracellular calcium and extracellular potassium. This can ultimately result in sleep dysfunction, which often occurs after concussion and has long been thought of as simply another concussion symptom. FOCUSED CLINICAL QUESTION: Does the likelihood of prolonged postconcussion symptoms increase with reported sleep-related problems (SRPs) in young athletes (8-18 y) compared to concussed young athletes without SRPs and healthy controls? SUMMARY OF KEY FINDINGS: Four cohort studies with level 2/3 evidence measured subjective and objective sleep dysregulations in concussed and healthy populations. Overall, there was a difference in subjective SRPs between concussed and healthy patients. This correlated with other studies where worse sleep scores during the acute phase of concussion and increased SRPs led to worse ImPACT scores in patients 3 to 12 months postconcussion and longer overall recovery. Objective sleep dysfunction measures were significantly worse in concussed patients than in healthy controls, but no significant difference existed in melatonin measures. CLINICAL BOTTOM LINE: There is strong evidence that sleep dysfunction is both a symptom of concussion as well as a causal factor of prolonged postconcussion symptoms. These studies show that sleep dysregulation is not always evident in objective measurements, leading to the strong possibility of a functional dysregulation of the sleep-wake cycle that is evident solely from subjective reports. STRENGTH OF RECOMMENDATION: While there are strong cohort studies researching the role of sleep in those with postconcussion symptoms, the nature of sleep studies prevents the production of strong, high-level evidence studies such as randomized control trials. Thus, there is level B evidence that the likelihood of prolonged postconcussion symptoms is increased by a higher amount of SRPs.

The plant pathogen enzyme AldC is a long-chain aliphatic aldehyde dehydrogenase.

Aldehyde dehydrogenases are versatile enzymes that serve a range of biochemical functions. Although traditionally considered metabolic housekeeping enzymes because of their ability to detoxify reactive aldehydes, like those generated from lipid peroxidation damage, the contributions of these enzymes to other biological processes are widespread. For example, the plant pathogen Pseudomonas syringae strain PtoDC3000 uses an indole-3-acetaldehyde dehydrogenase to synthesize the phytohormone indole-3-acetic acid to elude host responses. Here we investigate the biochemical function of AldC from PtoDC3000. Analysis of the substrate profile of AldC suggests that this enzyme functions as a long-chain aliphatic aldehyde dehydrogenase. The 2.5 Å resolution X-ray crystal of the AldC C291A mutant in a dead-end complex with octanal and NAD+ reveals an apolar binding site primed for aliphatic aldehyde substrate recognition. Functional characterization of site-directed mutants targeting the substrate- and NAD(H)-binding sites identifies key residues in the active site for ligand interactions, including those in the "aromatic box" that define the aldehyde-binding site. Overall, this study provides molecular insight for understanding the evolution of the prokaryotic aldehyde dehydrogenase superfamily and their diversity of function.

Subclinical in utero Zika virus infection is associated with interferon alpha sequelae and sex-specific molecular brain pathology in asymptomatic porcine offspring.

Zika virus (ZIKV) infection during human pregnancy may lead to severe fetal pathology and debilitating impairments in offspring. However, the majority of infections are subclinical and not associated with evident birth defects. Potentially detrimental life-long health outcomes in asymptomatic offspring evoke high concerns. Thus, animal models addressing sequelae in offspring may provide valuable information. To induce subclinical infection, we inoculated selected porcine fetuses at the mid-stage of development. Inoculation resulted in trans-fetal virus spread and persistent infection in the placenta and fetal membranes for two months. Offspring did not show congenital Zika syndrome (e.g., microcephaly, brain calcifications, congenital clubfoot, arthrogryposis, seizures) or other visible birth defects. However, a month after birth, a portion of offspring exhibited excessive interferon alpha (IFN-α) levels in blood plasma in a regular environment. Most affected offspring also showed dramatic IFN-α shutdown during social stress providing the first evidence for the cumulative impact of prenatal ZIKV exposure and postnatal environmental insult. Other eleven cytokines tested before and after stress were not altered suggesting the specific IFN-α pathology. While brains from offspring did not have histopathology, lesions, and ZIKV, the whole genome expression analysis of the prefrontal cortex revealed profound sex-specific transcriptional changes that most probably was the result of subclinical in utero infection. RNA-seq analysis in the placenta persistently infected with ZIKV provided independent support for the sex-specific pattern of in utero-acquired transcriptional responses. Collectively, our results provide strong evidence that two hallmarks of fetal ZIKV infection, altered type I IFN response and molecular brain pathology can persist after birth in offspring in the absence of congenital Zika syndrome.

Lymphogranuloma venereum (LGV) of the anorectum: evaluation of clinicopathological associations and the utility of a novel RNA in-situ hybridisation stain.

AIMS: Recent studies from multiple global regions have reported a resurgence of lymphogranuloma venereum (LGV) proctitis, which is caused by Chlamydia trachomatis (CT). LGV proctitis is histologically indistinguishable from other forms of sexually transmitted proctitis and is difficult to differentiate from inflammatory bowel disease. While immunohistochemical stains are available for syphilis, there is no commonly available stain for the tissue identification of CT. MATERIALS AND METHODS: From 200 positive CT nucleic acid tests (NAT) from anorectal swabs, we identified 12 patients with biopsies collected from the distal colorectum or anus within 90 days of the positive NAT. We collected basic demographic information and tabulated clinical and histological findings. We examined the performance of a novel RNA in-situ hybridisation (ISH) stain targeting CT 23s rRNA on these 12 cases and 10 controls from the anorectum. RESULTS: All 12 patients were male; nine were HIV+, two had concurrent gonococcal infection, one had concurrent syphilis and one had cytomegalovirus co-infection. The majority of biopsies (11 of 12) showed mild or moderate acute inflammation, had a prominent lymphoplasmacytic infiltrate (eight of 11) and lacked marked crypt distortion (10 of 10). The RNA ISH stain was positive in 10 of 12 cases (sensitivity 83%). One case showed equivocal staining. No controls showed definitive positive staining (specificity 100%). One had equivocal staining. CONCLUSION: Our series showed that anorectal LGV had similar histological findings to those of prior STI proctitis series predominantly comprised of syphilis. The novel RNA ISH stain was sensitive and specific and may show utility in differentiating types of STI proctitis.

Structural and Electronic Control of the Bidentate 1-(2-pyridyl)benzotriazole Ligand in Copper Chemistry with Application to Catalysis in the A3 Coupling Reaction.

The hybrid bidentate 1-(2-pyridyl)benzotriazole (pyb) ligand was introduced into 3d transition metal catalysis. Specifically, [CuII (OTf)2 (pyb)2 ]⋅2 CH3 CN (1) enables the synthesis of a wide range of propargylamines by the A3 coupling reaction at room temperature in the absence of additives. Experimental and high-level theoretical calculations suggest that the bridging N atom of the ligand imposes exclusive trans coordination at Cu and allows ligand rotation, while the N atom of the pyridine group modulates charge distribution and flux, and thus orchestrates structural and electronic precatalyst control permitting alkyne binding with simultaneous activation of the C-H bond via a transient CuI species.

Aversive Stress Reduces Mu Opioid Receptor Expression in the Intercalated Nuclei of the Rat Amygdala.

The amygdala plays an important role in the integration of responses to noxious and fearful stimuli. Sensory information from many systems is integrated in the lateral and basolateral amygdala and transmitted to the central amygdala, the major output nucleus of the amygdala regulating both motor and emotional responses. The network of intercalated cells (ITC) which surrounds the lateral and basolateral amygdala and serves to modulate information flow from the lateral amygdala to the central nucleus, express a very high local concentration of mu-type opioid receptors. Loss of the ITC neurons impairs fear extinction. We demonstrate here that exposure of rats to a severe stress experience resulted in a marked downregulation of the level of expression of mu opioid receptors in the ITC nuclei over a period of at least 24 h after the end of the stress exposure. The endogenous opioid dynorphin is also expressed in the central and ITC nuclei of the amygdala. Following stress exposure, we also observed an increase in the expression in the more lateral regions of the central amygdala of pro-dynorphin mRNA and a peptide product of pro-dynorphin with known affinity for mu opioid receptors. It is possible that the downregulation of mu receptors in ITC neurons after stress may result from sustained activation and internalization of mu receptors following a stress-induced increase in the release of endogenous opioid peptides.