Inhibition of 5-α-Reductase (TYPE-II) Expression by Antisense 3′-Deoxy-(2′-5′) Oligonucleotide Chimeras

Abstract

ABSTRACT: 3′-Deoxy-(2′-5′) oligonucleotides bind selectively to complementary RNA but not to DNA. 3′-Deoxy-(2′-5′) phosphorothioate ODN chimeras embedded with a short stretch of 3′-5′ phosphorothioate cassette are potent inhibitors of steroid 5-α-reductasc expression with significantly less non-specific interactions in cell culture.     

The Synthesis of Two Novel Ring-Expanded Xanthine Nucleosides Containing the Imidazo[4,5-e][1,4]Diazepine Ring System

Abstract

The synthesis of two regioisomeric nucleosides, 4,5,7,8-tetrahydro-6H-3-(β-D-ribofuranosyl)imidazo[4,5-e][1,4]diazepin-5,8-dione (2) and its 1-glycosyl analogue (3) is reported. They were prepared by ribosydation of the heterocyclic aglycon 4 which in turn was synthesized in three steps from 4(5)-nitroimidazole-5(4)-carboxylic acid (5). Distinction between the two isomers was achieved by an unequivocal synthesis of 2.     

A Next-Generation Cleaved,Soluble HIV-1 Env Trimer,BG505 SOSIP.664 gp140, Expresses Multiple Epitopes for Broadly Neutralizing but Not Non-Neutralizing Antibodies

A desirable but as yet unachieved property of a human immunodeficiency virus type 1 (HIV-1) vaccine candidate is the ability to induce broadly neutralizing antibodies (bNAbs). One approach to the problem is to create trimeric mimics of the native envelope glycoprotein (Env) spike that expose as many bNAb epitopes as possible, while occluding those for non-neutralizing antibodies (non-NAbs). Here, we describe the design and properties of soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A transmitted/founder strain, BG505. These trimers are highly stable, more so even than the corresponding gp120 monomer, as judged by differential scanning calorimetry. They are also homogenous and closely resemble native virus spikes when visualized by negative stain electron microscopy (EM). We used several techniques, including ELISA and surface plasmon resonance (SPR), to determine the relationship between the ability of monoclonal antibodies (MAbs) to bind the soluble trimers and neutralize the corresponding virus. In general, the concordance was excellent, in that virtually all bNAbs against multiple neutralizing epitopes on HIV-1 Env were highly reactive with the BG505 SOSIP.664 gp140 trimers, including quaternary epitopes (CH01, PG9, PG16 and PGT145). Conversely, non-NAbs to the CD4-binding site, CD4-induced epitopes or gp41_ECTO did not react with the trimers, even when their epitopes were present on simpler forms of Env (e.g. gp120 monomers or dissociated gp41 subunits). Three non-neutralizing MAbs to V3 epitopes did, however, react strongly with the trimers but only by ELISA, and not at all by SPR and to only a limited extent by EM. These new soluble trimers are useful for structural studies and are being assessed for their performance as immunogens.     

Ethnic Variation in Inflammatory Profile in Tuberculosis

Distinct phylogenetic lineages of Mycobacterium tuberculosis (MTB) cause disease in patients of particular genetic ancestry, and elicit different patterns of cytokine and chemokine secretion when cultured with human macrophages in vitro. Circulating and antigen-stimulated concentrations of these inflammatory mediators might therefore be expected to vary significantly between tuberculosis patients of different ethnic origin. Studies to characterise such variation, and to determine whether it relates to host or bacillary factors, have not been conducted. We therefore compared circulating and antigen-stimulated concentrations of 43 inflammatory mediators and 14 haematological parameters (inflammatory profile) in 45 pulmonary tuberculosis patients of African ancestry vs. 83 patients of Eurasian ancestry in London, UK, and investigated the influence of bacillary and host genotype on these profiles. Despite having similar demographic and clinical characteristics, patients of differing ancestry exhibited distinct inflammatory profiles at presentation: those of African ancestry had lower neutrophil counts, lower serum concentrations of CCL2, CCL11 and vitamin D binding protein (DBP) but higher serum CCL5 concentrations and higher antigen-stimulated IL-1 receptor antagonist and IL-12 secretion. These differences associated with ethnic variation in host DBP genotype, but not with ethnic variation in MTB strain. Ethnic differences in inflammatory profile became more marked following initiation of antimicrobial therapy, and immunological correlates of speed of elimination of MTB from the sputum differed between patients of African vs. Eurasian ancestry. Our study demonstrates a hitherto unappreciated degree of ethnic heterogeneity in inflammatory profile in tuberculosis patients that associates primarily with ethnic variation in host, rather than bacillary, genotype. Candidate immunodiagnostics and immunological biomarkers of response to antimicrobial therapy should be derived and validated in tuberculosis patients of different ethnic origin.     

The Effect of Riboflavin/UVA Collagen Cross-linking Therapy on the Structure and Hydrodynamic Behaviour of the Ungulate and Rabbit Corneal Stroma

Purpose

To examine the effect of riboflavin/UVA corneal crosslinking on stromal ultrastructure and hydrodynamic behaviour.

Methods

One hundred and seventeen enucleated ungulate eyes (112 pig and 5 sheep) and 3 pairs of rabbit eyes, with corneal epithelium removed, were divided into four treatment groups: Group 1 (28 pig, 2 sheep and 3 rabbits) were untreated; Group 2 (24 pig) were exposed to UVA light (3.04 mW/cm²) for 30 minutes and Group 3 (29 pig) and Group 4 (31 pig, 3 sheep and 3 rabbits) had riboflavin eye drops applied to the corneal surface every 5 minutes for 35 minutes. Five minutes after the initial riboflavin instillation, the corneas in Group 4 experienced a 30 minute exposure to UVA light (3.04 mW/cm²). X-ray scattering was used to obtain measurements of collagen interfibrillar spacing, spatial order, fibril diameter, D-periodicity and intermolecular spacing throughout the whole tissue thickness and as a function of tissue depth in the treated and untreated corneas. The effect of each treatment on the hydrodynamic behaviour of the cornea (its ability to swell in saline solution) and its resistance to enzymatic digestion were assessed using in vitro laboratory techniques.

Results

Corneal thickness decreased significantly following riboflavin application (p<0.01) and also to a lesser extent after UVA exposure (p<0.05). With the exception of the spatial order factor, which was higher in Group 4 than Group 1 (p<0.01), all other measured collagen parameters were unaltered by cross-linking, even within the most anterior 300 microns of the cornea. The cross-linking treatment had no effect on the hydrodynamic behaviour of the cornea but did cause a significant increase in its resistance to enzymatic digestion.

Conclusions

It seems likely that cross-links formed during riboflavin/UVA therapy occur predominantly at the collagen fibril surface and in the protein network surrounding the collagen.     

First International Conference on “Biotechnology in Agriculture”

The 1st International Conference on “Biotechnology in Agriculture” was held at the Agricultural University of Tirana, Albania, from the 22nd to the 23rd of April 2014. The conference was organized as collaboration between the Agricultural University of Tirana (AUT) and the Friedrich Schiller University in Jena, Germany. About 200 scientists from different countries presented their work orally or as posters covering the three main topics of the conference: plant, animal and food biotechnology. The conference included a field trip as well to the Experimental Didactic Economy Station and a Fish Farm, both belonging to the Agricultural University of Tirana. In this report, we describe the main topics of the Conference and a summary of talks and posters presented in different sessions. The main conclusions provided at the end of the activity, the objectives for the future and a brief overview of the field trips and pre- and post-conference tours are displayed in this current report. Since the conference on “Biotechnology in Agriculture” was the first one organized in Albania and also an international one, it was of a great interest for the participants and also for the scientific community working in the biotechnology area. Therefore, one of the decisions made at the end of the conference was to hold similar conferences periodically, every two years, at AUT.     

Alteration in Endometrial Proteins during Early- and Mid-Secretory Phases of the Cycle in Women with Unexplained Infertility

Background

Compromised receptivity of the endometrium is a major cause of unexplained infertility, implantation failure and subclinical pregnancy loss. In order to investigate the changes in endometrial protein profile as a cause of unexplained infertility, the current study was undertaken to analyze the differentially expressed proteins of endometrium from early-secretory (LH+2) to mid-secretory phase (LH+7), in women with unexplained infertility.

Methods

2-D gel electrophoresis was performed to analyze the proteomic changes between early- (n = 8) and mid-secretory (n = 8) phase endometrium of women with unexplained infertility. The differentially expressed protein spots were identified by LC-MS analysis and validated by immunoblotting and immuno-histochemical analysis in early- (n = 4) and mid-secretory (n = 4) phase endometrium of infertile women. Validated proteins were also analyzed in early- (n = 4) and mid-secretory (n = 4) phase endometrium of fertile women.

Results

Nine proteins were found to be differentially expressed between early- and mid- secretory phases of endometrium of infertile women. The expression of Ras-related protein Rap-1b, Protein disulfide isomerase A3, Apolipoprotein-A1 (Apo-A1), Cofilin-1 and RAN GTP-binding nuclear protein (Ran) were found to be significantly increased, whereas, Tubulin polymerization promoting protein family member 3, Superoxide dismutase [Cu-Zn], Sorcin, and Proteasome subunit alpha type-5 were significantly decreased in mid- secretory phase endometrium of infertile women as compared to early-secretory phase endometrium of infertile women. Validation of 4 proteins viz. Sorcin, Cofilin-1, Apo-A1 and Ran were performed in separate endometrial biopsy samples from infertile women. The up-regulated expression of Sorcin and down-regulated expression of Cofilin-1 and Apolipoprotein-A1, were observed in mid-secretory phase as compared to early-secretory phase in case of fertile women.

Conclusions

De-regulation of the expression of Sorcin, Cofilin-1, Apo-A1 and Ran, during early- to mid-secretory phase may have physiological significance and it may be one of the causes for altered differentiation and/or maturation of endometrium, in women with unexplained infertility.     

Rabies healthcare-seeking behaviors of urban and peri-urban residents: Results from a rabies knowledge,attitudes, and practices survey,Bangladesh, 2018

Rabies is one of the most lethal infectious diseases, with those living in Asia and Africa having the highest risk of dying from rabies. We conducted a knowledge, attitudes and practices survey in urban and peri-urban areas of Bangladesh to describe canine bite rates, rabies knowledge, and healthcare seeking behaviors and barriers to human and dog vaccination. A bite risk assessment score (BRAS) and healthcare-seeking behavior score (HSBS) was calculated for each bite victim. Respondents were given two hypothetical situations to assess potential behaviors after a bite and willingness to pay for rabies vaccine and immunoglobulin. In total, 2,447 households participated in the survey and 85 bite victims were identified. The BRAS identified that 31% of bites posed no risk of rabies transmission. Multivariate analyses showed that living in Chittagong (β = 1.4; 95% CI: 0.1, 2.7) was associated with a higher HSBS. Findings presented here provide useful information regarding bite occurrences, healthcare-seeking behaviors, and a need for strategies to increase rabies awareness.     

Glucose-induced activation of H+-ATPase in Dunaliella salina and its role in hygromycin resistance

Dunaliella salina, a eukaryotic microalga, is known for its highly halophilic nature. The high level of salts in growth medium for this alga has made its genetic transformation a comparatively difficult procedure, particularly during the selection stage. The high salt content decreases the efficiency of most antibiotics which are being used as selection markers. Studies pertaining to the interrelationship between salt concentration and antibiotic sensitivity are scarce in Dunaliella. During our previous experiment at genetic transformation of Dunaliella, an inverse relationship between the amount of antibiotic hygromycin and sodium chloride in the medium was revealed. A possible link between plasma membrane activity and the hygromycin sensitivity was investigated in the present study by modulating plasma membrane H⁺-ATPase activity using glucose. Glucose-induced activation of H⁺-ATPase, reduced the tolerance of D. salina to the antibiotic hygromycin. Hygromycin concentration required for selection during genetic transformation of Dunaliella was lowered from 100 to 25 mg L^?1 in the presence of 10 mM glucose. Conversely, the inhibitors of the plasma membrane H⁺-ATPase, orthovanadate and diethylstilbestrol were found to inhibit the glucose activation at concentrations of 10 and 15 μM, respectively. The activation of H⁺-ATPase by glucose was further confirmed through H⁺-ATPase assay and medium acidification experiments. The results indicated that the sensitivity of Dunaliella to antibiotic is related to H⁺-ATPase and the possible involvement of pH gradient, created through H⁺-ATPase activation during drug transport.