Altered Esophageal Histamine Receptor Expression in Eosinophilic Esophagitis (EoE): Implications on Disease Pathogenesis

Eosinophilic Esophagitis (EoE) is a chronic allergic disorder, whose pathobiology is incompletely understood. Histamine-producing cells including mast cells and basophils have been implicated in EoE. However, very little is currently known about the role of histamine and histamine receptor (HR) expression and signaling in the esophageal epithelium. Herein, we characterized HR (H1R, H2R, H3R, and H4R) expression in human esophageal biopsies and investigate the role of histamine signaling in inducible cytokine expression in human esophageal epithelial cells in vitro. HR expression was quantified in esophageal biopsies from non-EoE control (N = 23), inactive EoE (<15 eos/hpf, N = 26) and active EoE (>15 eos/hpf, N = 22) subjects using qRT-PCR and immunofluorescent localization. HR expression and histamine-mediated cytokine secretion were evaluated in human primary and telomerase-immortalized esophageal epithelial cells. H1R, H2R, and H4R expression were increased in active EoE biopsies compared to inactive EoE and controls. H2R was the most abundantly expressed receptor, and H3R expression was negligible in all 3 cohorts. Infiltrating eosinophils expressed H1R, H2R, and H4R, which contributed to the observed increase in HR in active subjects. H1R and H2R, but not H3R or H4R, were constitutively expressed by primary and immortalized cells, and epithelial histamine stimulation induced GM-CSF, TNFα, and IL-8, but not TSLP or eotaxin-3 secretion. Epithelial priming with the TLR3 ligand poly (I:C) induced H1R and H2R expression, and enhanced histamine-induced GM-CSF, TNFα, and IL-8 secretion. These effects were primarily suppressed by H1R antagonists, but unaffected by H2R antagonism. Histamine directly activates esophageal epithelial cytokine secretion in vitro in an H1R dependent fashion. However, H1R, H2R and H4R are induced in active inflammation in EoE in vivo. While systemic antihistamine (anti-H1R) therapy may not induce clinical remission in EoE, our study suggests that further study of histamine receptor signaling in EoE is warranted and that targeting of additional histamine receptors may lead to novel treatment strategies for this important disease.     

Reproductive Strategies of <Emphasis Type="Italic">Trachypithecus pileatus</Emphasis> in Arunachal Pradesh,India

We studied reproductive behavior of free-ranging capped langurs (Trachypithecus pileatus) in the Pakhui Wildlife Sanctuary, Arunachal Pradesh, India. Four species of primates —Trachypithecus pileatus, Macaca mulatta, M. assamensis, and Nycticebus bengalensis— live there. We studied the mating seasons, mating frequency, copulatory attempts, time spent in copulation, and interval between 2 successive copulations, gestation length, and interbirth interval of 4 groups of capped langurs during 2001–2003. We observed 2 mating seasons in a year. The first was larger, comprising 5 months (September–January), and the second was short, April and May. Mating was intensive in the morning session (0600–1000 h); 57% of total mating events occurred then. The average gestation period was 200 d. November was the most favorable month for breeding. In a year, 107 mating events occurred involving 5 adult females. Average time per mounting attempt is 12 s. Duration of mounting was the maximum in November. Interbirth interval was 23 months and 10 d. The birth season was 129 days, December–April; 53% of births occurred in February and March. Average birth rate is 0.386 birth/female/yr.     

Marine bacteroidetes use a conserved enzymatic cascade to digest diatom β-mannan

The polysaccharide β-mannan, which is common in terrestrial plants but unknown in microalgae, was recently detected during diatom blooms. We identified a β-mannan polysaccharide utilization locus (PUL) in the genome of the marine flavobacterium Muricauda sp. MAR_2010_75. Proteomics showed β-mannan induced translation of 22 proteins encoded within the PUL. Biochemical and structural analyses deduced the enzymatic cascade for β-mannan utilization. A conserved GH26 β-mannanase with endo-activity depolymerized the β-mannan. Consistent with the biochemistry, X-ray crystallography showed the typical TIM-barrel fold of related enzymes found in terrestrial β-mannan degraders. Structural and biochemical analyses of a second GH26 allowed the prediction of an exo-activity on shorter manno-gluco oligosaccharides. Further analysis demonstrated exo-α-1,6-galactosidase- and endo-β-1,4-glucanase activity of the PUL-encoded GH27 and GH5_26, respectively, indicating the target substrate is a galactoglucomannan. Epitope deletion assays with mannanases as analytic tools indicate the presence of β-mannan in the diatoms Coscinodiscus wailesii and Chaetoceros affinis. Mannanases from the PUL were active on diatom β-mannan and polysaccharide extracts sampled during a microalgal bloom at the North Sea. Together these results demonstrate that marine microorganisms use a conserved enzymatic cascade to degrade β-mannans of marine and terrestrial origin and that this metabolic pathway plays a role in marine carbon cycling.Subject terms: Water microbiology, Proteomics, Microbial ecology     

Characterisation of downy mildew resistant and susceptible pearl millet (Pennisetum glaucum (L.) R.Br.) genotypes using isozyme,protein, randomly amplified polymorphic DNA and inter-simple sequence repeat markers

In the present investigation, downy mildew resistant and susceptible pearl millet genotypes were characterised using seed protein and isozymes at pre- [45 days after sowing (DAS)] and post-infection (57 DAS, i.e. 7 days after infection) stage, as well as molecular markers at seedling stage without infection. Native polyacrylamide gel electrophoreis (PAGE) isozyme banding pattern of superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), ascorbate peroxidase (APX) and esterase showed some inducible band(s) due to disease infection and differentiated resistant and susceptible genotypes. Total seed protein profiling revealed the presence of two unique protein of ～97 and ～100 kDa in resistant genotypes. Randomly amplified polymorphic DNA (RAPD) analysis did not show any specific marker for disease resistant and susceptible genotypes. However, inter-simple sequence repeat (ISSR) markers showed six markers in resistant genotypes viz., UBC-825 (900 bp), UBC-827 (900 bp), UBC-857 (1000 bp, 700 bp, 375 bp and 200 bp). Moreover, a single unique band UBC-857 (400 bp) was present in only susceptible genotypes. Overall pooled analysis of isozymes, protein profiling, RAPD and ISSR data showed two distinct clusters of resistant and susceptible genotypes. These results suggested that seed protein profiling and ISSR markers may be used for large scale screening of germplasm for disease reaction trait.     

Directed differentiation of mouse P19 embryonal carcinoma cells to neural cells in a serum- and retinoic acid-free culture medium

P19 embryonal carcinoma cells (EC-cells) provide a simple and robust culture system for studying neural development. Most protocols developed so far for directing neural differentiation of P19 cells depend on the use of culture medium supplemented with retinoic acid (RA) and serum, which has an undefined composition. Hence, such protocols are not suitable for many molecular studies. In this study, we achieved neural differentiation of P19 cells in a serum- and RA-free culture medium by employing the knockout serum replacement (KSR) supplement. In the KSR-containing medium, P19 cells underwent predominant differentiation into neural lineage and by day 12 of culture, neural cells were present in 100% of P19-derived embryoid bodies (EBs). This was consistently accompanied by the increased expression of various neural lineage-associated markers during the course of differentiation. P19-derived neural cells comprised of NES⁺ neural progenitors (~?46%), TUBB3⁺ immature neurons (~?6%), MAP2⁺ mature neurons (~?2%), and GFAP⁺ astrocytes (~?50%). A heterogeneous neuronal population consisting of glutamatergic, GABAergic, serotonergic, and dopaminergic neurons was generated. Taken together, our study shows that the KSR medium is suitable for the differentiation of P19 cells to neural lineage without requiring additional (serum and RA) supplements. This stem cell differentiation system could be utilized for gaining mechanistic insights into neural differentiation and for identifying potential neuroactive compounds.     

Molecular basis of ligand recognition by OASS from E. histolytica: Insights from structural and molecular dynamics simulation studies

Background

O-acetyl serine sulfhydrylase (OASS) is a pyridoxal phosphate (PLP) dependent enzyme catalyzing the last step of the cysteine biosynthetic pathway. Here we analyze and investigate the factors responsible for recognition and different conformational changes accompanying the binding of various ligands to OASS.

Methods

X ray crystallography was used to determine the structures of OASS from Entamoeba histolytica in complex with methionine (substrate analog), isoleucine (inhibitor) and an inhibitory tetra-peptide to 2.00 Å, 2.03 Å and 1.87 Å resolutions, respectively. Molecular dynamics simulations were used to investigate the reasons responsible for the extent of domain movement and cleft closure of the enzyme in presence of different ligands.

Results

Here we report for the first time an OASS-methionine structure with an unmutated catalytic lysine at the active site. This is also the first OASS structure with a closed active site lacking external aldimine formation. The OASS-isoleucine structure shows the active site cleft in open state. Molecular dynamics studies indicate that cofactor PLP, N88 and G192 form a triad of energy contributors to close the active site upon ligand binding and orientation of the Schiff base forming nitrogen of the ligand is critical for this interaction.

Conclusions

Methionine proves to be a better binder to OASS than isoleucine. The β branching of isoleucine does not allow it to reorient itself in suitable conformation near PLP to cause active site closure.

General significance

Our findings have important implications in designing better inhibitors against OASS across all pathogenic microbial species.     

FANCA Gene Mutations with 8 Novel Molecular Changes in Indian Fanconi Anemia Patients

Fanconi anemia (FA), a rare heterogeneous genetic disorder, is known to be associated with 19 genes and a spectrum of clinical features. We studied FANCA molecular changes in 34 unrelated and 2 siblings of Indian patients with FA and have identified 26 different molecular changes of FANCA gene, of which 8 were novel mutations (a small deletion c.2500delC, 4 non-sense mutations c.2182C>T, c.2630C>G, c.3677C>G, c.3189G>A; and 3 missense mutations; c.1273G>C, c.3679 G>C, and c.3992 T>C). Among these only 16 patients could be assigned FA-A complementation group, because we could not confirm single exon deletions detected by MLPA or cDNA amplification by secondary confirmation method and due to presence of heterozygous non-pathogenic variations or heterozygous pathogenic mutations. An effective molecular screening strategy should be developed for confirmation of these mutations and determining the breakpoints for single exon deletions.     

Antifungal Dosing in Critically Ill Patients

This article reviews appropriate dosing for antifungals and emphasizes factors specific to the critically ill patient, along with drug pharmacokinetics and pharmacodynamics. The rationale for doses of the echinocandins (caspofungin, micafungin, anidulafungin), triazoles (fluconazole, voriconazole, itraconazole, posaconazole), amphotericin B (including lipid formulations), and flucytosine are discussed.     

Cooperative miRNA-dependent PTEN regulation drives resistance to BTK inhibition in B-cell lymphoid malignancies

Aberrant microRNA (miR) expression plays an important role in pathogenesis of different types of cancers, including B-cell lymphoid malignancies and in the development of chemo-sensitivity or -resistance in chronic lymphocytic leukemia (CLL) as well as diffuse large B-cell lymphoma (DLBCL). Ibrutinib is a first-in class, oral, covalent Bruton’s tyrosine kinase (BTK) inhibitor (BTKi) that has shown impressive clinical activity, yet many ibrutinib-treated patients relapse or develop resistance over time. We have reported that acquired resistance to ibrutinib is associated with downregulation of tumor suppressor protein PTEN and activation of the PI3K/AKT pathway. Yet how PTEN mediates chemoresistance in B-cell malignancies is not clear. We now show that the BTKi ibrutinib and a second-generation compound, acalabrutinib downregulate miRNAs located in the 14q32 miRNA cluster region, including miR-494, miR-495, and miR-543. BTKi-resistant CLL and DLBCL cells had striking overexpression of miR-494, miR-495, miR-543, and reduced PTEN expression, indicating further regulation of the PI3K/AKT/mTOR pathway in acquired BTKi resistance. Additionally, unlike ibrutinib-sensitive CLL patient samples, those with resistance to ibrutinib treatment, demonstrated upregulation of 14q32 cluster miRNAs, including miR-494, miR-495, and miR-543 and decreased pten mRNA expression. Luciferase reporter gene assay showed that miR-494 directly targeted and suppressed PTEN expression by recognizing two conserved binding sites in the PTEN 3′-UTR, and subsequently activated AKT^Ser473. Importantly, overexpression of a miR-494 mimic abrogated both PTEN mRNA and protein levels, further indicating regulation of apoptosis by PTEN/AKT/mTOR. Conversely, overexpression of a miR-494 inhibitor in BTKi-resistant cells restored PTEN mRNA and protein levels, thereby sensitizing cells to BTKi-induced apoptosis. Inhibition of miR-494 and miR-495 sensitized cells by cooperative targeting of pten, with additional miRNAs in the 14q32 cluster that target pten able to contribute to its regulation. Therefore, targeting 14q32 cluster miRNAs may have therapeutic value in acquired BTK-resistant patients via regulation of the PTEN/AKT/mTOR signaling axis.Subject terms: Cancer, Chronic lymphocytic leukaemia