Structure-Based Design and Synthesis of Covalent Inhibitors for Deubiquitinase and Acetyltransferase ChlaDUB1 of Chlamydia trachomatis.

Upon infection by an intracellular pathogen, host cells activate apoptotic pathways to limit pathogen replication. Consequently, efficient proliferation of the obligate intracellular pathogen Chlamydia trachomatis, a major cause of trachoma and sexually transmitted diseases, depends on the suppression of host cell apoptosis. C. trachomatis secretes deubiquitinase ChlaDUB1 into the host cell, leading among other interactions to the stabilization of antiapoptotic proteins and, thus, suppression of host cell apoptosis. Targeting the bacterial effector protein may, therefore, lead to new therapeutic possibilities. To explore the active site of ChlaDUB1, an iterative cycle of computational docking, synthesis, and enzymatic screening was applied with the aim of lead structure development. Hereby, covalent inhibitors were developed, which show enhanced inhibition with a 22-fold increase in IC50 values compared to previous work. Comprehensive insights into the binding prerequisites to ChlaDUB1 are provided, establishing the foundation for an additional specific antichlamydial therapy by small molecules.

Dual role for microbial short-chain fatty acids in modifying SIV disease trajectory following anti-α4ß7 antibody administration.

BACKGROUND: Human Immunodeficiency Virus (HIV)/Simian Immunodeficiency Virus (SIV) infection is associated with significant gut damage, similar to that observed in patients with inflammatory bowel disease (IBD). This pathology includes loss of epithelial integrity, microbial translocation, dysbiosis, and resultant chronic immune activation. Additionally, the levels of all-trans-retinoic acid (atRA) are dramatically attenuated. Data on the therapeutic use of anti-α4ß7 antibodies has shown promise in patients with ulcerative colitis and Crohn's disease. Recent evidence has suggested that the microbiome and short-chain fatty acid (SCFA) metabolites it generates may be critical for anti-α4ß7 efficacy and maintaining intestinal homeostasis. MATERIALS AND METHODS: To determine whether the microbiome contributes to gut homeostasis after anti-α4ß7 antibody administered to SIV-infected rhesus macaques, faecal SCFA concentrations were determined, 16S rRNA sequencing was performed, plasma viral loads were determined, plasma retinoids were measured longitudinally, and gut retinoid synthesis/response gene expression was quantified. RESULTS: Our results suggest that anti-α4ß7 antibody facilitates the return of retinoid metabolism to baseline levels after SIV infection. Furthermore, faecal SCFAs were shown to be associated with retinoid synthesis gene expression and rebound viral loads after therapy interruption. CONCLUSIONS: Taken together, these data demonstrate the therapeutic advantages of anti-α4ß7 antibody administration during HIV/SIV infection and that the efficacy of anti-α4ß7 antibody may depend on microbiome composition and SCFA generation.

Eukaryotic Clathrin Adapter Protein and Mediator of Cholesterol Homeostasis, PICALM, Affects Trafficking to the Chlamydial Inclusion.

The obligate intracellular pathogen Chlamydia trachomatis has unique metabolic requirements as it proceeds through its biphasic developmental cycle from within the inclusion within the host cell. In our previous study, we identified a host protein, PICALM, which localizes to the chlamydial inclusion. PICALM functions in many host pathways including the recycling of receptors, specific SNARE proteins, and molecules like transferrin, and maintaining cholesterol homeostasis. Hence, we hypothesized that PICALM functions to maintain the cholesterol content and to moderate trafficking from the endosomal recycling pathway to the inclusion, which controls chlamydial access to this pathway. In uninfected cells, siRNA knockdown of PICALM resulted in increased cholesterol within the Golgi and transferrin receptor (TfR) positive vesicles (recycling endosomes). PICALM knockdown in cells infected with C. trachomatis resulted in increased levels of Golgi-derived lipid and protein, TfR, transferrin, and Rab11-FIP1 localized to inclusions and a decrease of Golgi fragmentation at and Rab11 trafficking to the inclusion. Interestingly, chlamydial infection alone also increases cholesterol in TfR and Rab11-associated vesicles, and PICALM knockdown reverses this effect. Our data suggest that PICALM functions to balance or limit chlamydial access to multiple subcellular trafficking pathways to maintain the health of the host cell during chlamydial infection.

Early treatment with anti-α4ß7 antibody facilitates increased gut macrophage maturity in SIV-infected rhesus macaques.

Despite advances in combination antiretroviral therapy (cART), people living with HIV (PLWH) continue to experience gastrointestinal dysfunction. Infusions of anti-α4ß7 monoclonal antibodies (mAbs) have been proposed to increase virologic control during simian immunodeficiency virus (SIV) infection in macaques with mixed results. Recent evidences suggested that therapeutic efficacy of vedolizumab (a humanized anti-α4ß7 mAb), during inflammatory bowel diseases depends on microbiome composition, myeloid cell differentiation, and macrophage phenotype. We tested this hypothesis in SIV-infected, anti-α4ß7 mAb-treated macaques and provide flow cytometric and microscopic evidence that anti-α4ß7 administered to SIV-infected macaques increases the maturity of macrophage phenotypes typically lost in the small intestines during SIV disease progression. Further, this increase in mature macrophage phenotype was associated with tissue viral loads. These phenotypes were also associated with dysbiosis markers in the gut previously identified as predictors of HIV replication and immune activation in PLWH. These findings provide a novel model of anti-α4ß7 efficacy offering new avenues for targeting pathogenic mucosal immune response during HIV/SIV infection.

Histopathology of laryngomalacia.

BACKGROUND: Laryngomalacia is the commonest laryngeal anomaly and cause of stridor in children. Although most cases are self-limiting, failure to thrive, hypoxaemia or significant apnoeic episodes may warrant surgical intervention in the form of aryepiglottoplasty. Opinion is divided as to the pathophysiological mechanisms involved in the disease process. AIMS AND OBJECTIVES: This study explores the aetiology of laryngomalacia by reviewing the histology of aryepiglottoplasty resection specimens. MATERIAL AND METHODS: The histology reports of 61 aryepiglottoplasty specimens resected between 1 October 2014 and 31 October 2018 were reviewed. RESULTS: Age of patients ranged from 3 weeks to 36 months. 36 patients were male and 25 female. 43 of 61 (70.5%) cases had inflammation, most of which were mild. 3 (4.9%) cases had histological specimens with detectable eosinophils. None of the specimens had signs of granulomatous change, ulceration or calcification. Cartilage was present in the resected specimen in 47 (77%) cases. Over half of these (59.6%) were immature cartilage. CONCLUSION AND SIGNIFICANCE: The results suggest a mild concurrent laryngitis/supraglottitis in most cases. Eosinophilia is rare and does not support eosinophilic oesophageal reflux as part of the aetiology. The high proportion of immature cartilage in the specimens supports the theory of chondropathic aetiology.

NF-κB Duplications in the Promoter-Variant HIV-1C LTR Impact Inflammation Without Altering Viral Replication in the Context of Simian Human Immunodeficiency Viruses and Opioid-Exposure.

Recent spread of the promoter variant (4-κB) Human immunodeficiency virus-1 clade C (HIV-1C) strain is attributed to duplication of the Nuclear Factor Kappa B (NF-κB) binding sites and potential increased heroin consumption in India. To study the underlying biology of 4-κB HIV-1C in rhesus macaques, we engineered a promoter-chimera variant (4NF-κB) Simian Human Immunodeficiency Virus (SHIV) by substituting the HIV-1C Long Terminal Repeat (LTR) region consisting of 4 NF-κB and 3 Sp-1 sites with the corresponding segment in the LTR of SHIV AD8EO. The wild-type (3NF-κB) promoter-chimera SHIV was generated by inactivating the 5' proximal NF-κB binding site in SHIV 4NF-κB. CD8-depleted rhesus macaque PBMCs (RM-PBMCs) were infected with the promoter-chimera and AD8EO SHIVs to determine the effects of opioid-exposure on inflammation, NF-κB activation, neurotoxicity in neuronal cells and viral replication. Morphine-exposure of RM-PBMCs infected with SHIVs 4NF-κB, 3NF-κB, and AD8EO altered cellular transcript levels of monocyte chemoattractant protein 1, interleukin 6, interleukin 1ß, and Tumor Necrosis Factor α. Of note, divergent alteration of the cytokine transcript levels was observed with these promoter-chimera wild-type and variant SHIVs. NF-κB activation was observed during infection of all three SHIVs with morphine-exposure. Finally, we observed that SHIV AD8EO infection and exposure to both morphine and naloxone had the greatest impact on the neurotoxicity. The promoter-chimera SHIV 4NF-κB and SHIV 3NF-κB did not have a similar effect on neurotoxicity as compared to SHIV AD8EO. All SHIVs replicated efficiently at comparable levels in RM-PBMCs and morphine-exposure did not alter viral replication kinetics. Future in vivo studies in rhesus macaques will provide greater understanding of 4-κB HIV-1C viral immunopathogenesis and onset of disease in the central nervous system during morphine-exposure.

Comparing the effects of atrazine and an environmentally relevant mixture on estrogen-responsive gene expression in the northern leopard frog and the fathead minnow.

In Nebraska, fish are exposed to herbicides in agricultural runoff. The study objectives were to determine 1) if fathead minnows and northern leopard frogs exposed to atrazine experience alterations in gene expression, and 2) whether these changes are elicited by a simulated herbicide mixture. Following a 7-d exposure to atrazine, female minnows were defeminized, whereas male frogs were feminized. The mixture did not elicit statistically significant effects in either species. Environ Toxicol Chem 2018;37:1182-1188. © 2018 SETAC.

De novo Assembly and Analysis of the Northern Leopard Frog Rana pipiens Transcriptome.

The northern leopard frog Rana (Lithobates) pipiens is an important animal model, being used extensively in cancer, neurology, physiology, and biomechanical studies. R. pipiens is a native North American frog whose range extends from northern Canada to southwest United States, but over the past few decades its populations have declined significantly and is now considered uncommon in large portions of the United States and Canada. To aid in the study and conservation of R. pipiens, this paper describes the first R. pipiens transcriptome. The R. pipiens transcriptome was annotated using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Eukaryotic Orthologous Groups (KOG). Differential expression analysis revealed universal and tissue specific genes, and endocrine-related genes were identified. Transcriptome assemblies and other sequence data are available for download.

The spring runoff in Nebraska's (USA) Elkhorn River watershed and its impact on two sentinel organisms.

The objectives of the present study were to determine 1) if temporal variability influenced the toxicity of Elkhorn River, Nebraska, USA, water, and 2) if the toxic effect was consistent between 2 sentinel organisms, the fathead minnow (Pimephales promelas) and the northern leopard frog (Rana pipiens). During spring 2012, atrazine indicator strips were used to document the occurrence of agrichemical pulses in the Elkhorn River. Polar organic chemical integrative samplers (POCIS) were deployed for 14 d during both a pulse and a postpulse period as indicated by the atrazine strips. Pesticide concentrations detected in the POCIS extracts ranged from 1.6-fold to 281-fold higher during the pulse period compared to the postpulse period. Fish and frog bioassays were conducted for 7 d, and hepatic mRNA expression of vitellogenin (VTG) and estrogen receptor alpha (ERα) was determined by quantitative real-time polymerase chain reaction. Compared with lab water controls, fish exposed to water collected during an agrichemical pulse experienced significant reductions in VTG and ERα, whereas exposed female frogs did not. Male leopard frogs, in contrast, experienced significant increases in the expression of ERα, whereas pulse-exposed male minnows did not. The significant effects observed following agrichemical pulse exposure demonstrate that episodic agrichemical runoff adversely impacts sentinel organisms, and that the adverse impacts observed depend on the sex and species of the sentinel organism.

Severe obstructive sleep apnoea due to adenotonsillar hypertrophy after liver transplantation.

Epstein-Barr virus-related adenotonsillar hypertrophy is a precursor to post-transplantation lymphoproliferative disorder. We report a case of a 4-year-old child with severe obstructive sleep apnoea, who had liver transplantation at the age of 7 months. She had gross lymphoid hypertrophy in the oropharynx and supraglottis. We performed an adenotonsillectomy and aryepiglottoplasty which improved her symptoms. We emphasise the importance to consider the diagnosis of post-transplantation lymphoproliferative disorder in post-transplantation immunosuppressed patients who present with adenotonsillar hypertrophy.

Congenital cysts of the infant larynx.

OBJECTIVES: Congenital laryngeal cysts are a rare cause of neonatal airway obstruction. Two varieties can be distinguished, ductal cysts and saccular cysts. Treatment remains controversial, particularly with regard to saccular cysts, with some believing that endoscopic procedures leave cysts more prone to recurrence. We reviewed our management of congenital laryngeal cysts to determine the most effective intervention. METHODS: A review of all children treated by our unit for congenital laryngeal cysts between 2001 and 2011. Demographic data, age at and mode of presentation were noted. Operation notes were reviewed to determine the nature of each laryngeal cyst and treatment methodology. RESULTS: Ten children were identified, six with a ductal cyst of the vallecula and four with a saccular cyst, giving an overall incidence of 3.49 cases per 100,000 live births. Children of Pakistani ancestry were disproportionately represented, accounting for 40% of cases. Saccular cysts presented earlier with symptoms of more severe airway compromise, whilst vallecular cysts tended to present later with feeding difficulties and failure to thrive. Two-thirds of children with vallecular cysts had co-existing laryngomalacia. Treatment by single-stage endoscopic marsupialisation was effective in all cases at a mean follow-up of thirteen months. CONCLUSIONS: Congenital laryngeal cysts are rare, although they appear to be more common in the British-Pakistani population. Presentation depends on cyst size and proximity to the glottis and can be associated with laryngomalacia. Endoscopic marsupialisation is an effective means of treatment for ductal cysts and saccular cysts confined to the larynx.

The anti-estrogenic activity of sediments from agriculturally intense watersheds: assessment using in vivo and in vitro assays.

The goal of the current study was to determine whether sediments from agriculturally intense watersheds can act as a potential source of anti-estrogenic endocrine-disrupting compounds. The specific objectives of the current study were to determine (1) whether female fathead minnows (Pimephales promelas) experience alterations in endocrine function when exposed to sediments collected from agriculturally intense watersheds and (2) if these sediments display anti-estrogenic activity in an in vitro assay. In addition, sediment samples were analyzed for the presence of steroid hormones and pesticides associated with local agricultural practices. To accomplish this, sediments and water were collected from three sites within two agriculturally intense Nebraska watersheds (Bow Creek and the Elkhorn River). In 2009, minnows were exposed to sediment and/or water collected from the two Bow Creek sites (East Bow Creek and the Confluence) in the laboratory, while in 2010, minnows were exposed to sediment and/or water from East Bow Creek, the Confluence and the Elkhorn River. Following the 7-day exposure period, the hepatic mRNA expression of two-estrogen responsive genes, estrogen receptor α (ERα) and vitellogenin (Vtg) was determined. In 2009, females exposed to Confluence sediments, in the presence of laboratory water or Confluence water, experienced significant reductions in ERα expression relative to unexposed and Confluence water-exposed females. The defeminization of these females suggests the presence of a biologically available anti-estrogenic compound in sediments collected from this site. In 2010, sediments were assessed for anti-estrogenic activity on days 0 and 7 of the exposure period using a 4-h yeast estrogen screen. Lipophilic extracts (LEs) of day 0 sediments collected from the Confluence and the Elkhorn River induced significant reductions in the estrogenic reporter activity of treated yeast cultures suggesting the presence of a lipophilic anti-estrogenic compound in these extracts. Chemical analysis revealed the presence of a variety of steroid hormones, including those associated with the production of beef cattle (i.e. ß-trenbolone, α-zearalanol and α-zearalenol), in sediments indicating that compounds utilized by local beef cattle operations are capable of entering nearby watersheds. Overall, the results of this study indicate that an environmentally relevant anti-estrogenic compound is present in sediments from agriculturally intense watersheds and that this compound is bioavailable to fish. Furthermore, the presence of steroid hormones in sediments from these watersheds provides evidence indicating that steroids are capable of sorbing to sediments.

US and MRI of a pharyngeal hairy polyp with pathological correlation.

Hairy polyps are uncommon developmental malformations of the oropharynx. They are frequently pedunculated and can cause respiratory distress in the neonate. Hairy polyps are usually diagnosed with MRI but can be well seen at US. To our knowledge, the sonographic features have not been previously reported.

Multiple cervical bronchogenic cysts.

Bronchogenic cysts are rare, benign, congenital lesions that occur as a result of aberrant development of the tracheobronchial tree during embryogenesis. They usually present during the first decade of life and are encountered predominantly within the mediastinum or the lung parenchyma. In a few instances, they appear within the neck mimicking a neoplasm and, depending on their size and site, may also cause acute upper respiratory obstruction. We describe a case of two cervical bronchogenic cysts adjacent to the larynx in a child who presented with a hoarse voice.