Evolutionary dynamics of SARS-CoV-2 circulating in Yogyakarta and Central Java,Indonesia: sequence analysis covering furin cleavage site (FCS) region of the spike protein

International Microbiology - Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new virus responsible for the COVID-19 pandemic. The emergence of the new SARS-CoV-2 has been...     

Animal models of Duchenne muscular dystrophy: from basic mechanisms to gene therapy

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder. It is caused by loss-of-function mutations in the dystrophin gene. Currently, there is no cure. A highly promising therapeutic strategy is to replace or repair the defective dystrophin gene by gene therapy. Numerous animal models of DMD have been developed over the last 30 years, ranging from invertebrate to large mammalian models. mdx mice are the most commonly employed models in DMD research and have been used to lay the groundwork for DMD gene therapy. After ~30 years of development, the field has reached the stage at which the results in mdx mice can be validated and scaled-up in symptomatic large animals. The canine DMD (cDMD) model will be excellent for these studies. In this article, we review the animal models for DMD, the pros and cons of each model system, and the history and progress of preclinical DMD gene therapy research in the animal models. We also discuss the current and emerging challenges in this field and ways to address these challenges using animal models, in particular cDMD dogs.KEY WORDS: Duchenne muscular dystrophy, Dystrophin, Animal model, Canine DMD, Gene therapy     

What factors account for black–white differences in anti-Muslim sentiment in the contemporary USA?

I use data from the 2004 General Social Survey (N=719) and multivariate analyses to: explore the effects of race on attitudes toward Muslims; evaluate the extent to which the racial differences were mediated by psychological and religious factors; and assess whether the race effects differed significantly by gender. The findings show that blacks report significantly more favourable feelings toward Muslims than whites. Those respondents who are female, more educated and Catholic also hold significantly higher scores on the 100-point scale assessing feelings toward Muslims. After controlling for religious and psychological factors, I find that the racial difference in feelings toward Muslims is increased, indicating that the race effect is suppressed by these factors. Moderation analysis reveals that white men hold the highest level of negative feelings toward Muslims, compared to women and black men. The findings suggest challenging the misconstrued perceptions of Muslims through education and endorsement of positive images.     

Nonrandom structure in the urea-unfolded Escherichia coli outer membrane protein X (OmpX)

On the basis of sequence-specific resonance assignments for the complete polypeptide backbone and most of the amino acid side chains by heteronuclear nuclear magnetic resonance (NMR) spectroscopy, the urea-unfolded form of the outer membrane protein X (OmpX) from Escherichia coli has been structurally characterized. (1)H-(1)H nuclear Overhauser effects (NOEs), dispersion of the chemical shifts, amide proton chemical shift temperature coefficients, amide proton exchange rates, and (15)N[(1)H]-NOEs show that OmpX in 8 M urea at pH 6.5 is globally unfolded, but adopts local nonrandom conformations in the polypeptide segments of residues 73-82 and 137-145. For these two regions, numerous medium-range and longer-range NOEs were observed, which were used as the input for structure calculations of these polypeptide segments with the program DYANA. The segment 73-82 forms a quite regular helical structure, with only loosely constrained amino acid side chains. In the segment 137-145, the tryptophan residue 140 forms the core of a small hydrophobic cluster. Both nonrandom structures are present with an abundance of about 25% of the protein molecules. The sequence-specific NMR assignment and the physicochemical characterization of urea-denatured OmpX presented in this paper are currently used as a platform for investigations of the folding mechanism of this integral membrane protein.     

“Inclonals”: IgGs and IgG-enzyme fusion proteins produced in an E. coli expression-refolding system

Full-length antibodies and antibodies that ferry a cargo to target cells are desired biopharmaceuticals. We describe the production of full-length IgGs and IgG-toxin fusion proteins in E. coli. In the presented examples of anti CD30 and anti EGF-receptor antibodies, the antibody heavy and light chains or toxin fusions thereof were expressed in separate bacterial cultures, where they accumulated as insoluble inclusion bodies. Following refolding and purification, high yields (up to 50 mg/L of shake flask culture) of highly purified (>90%) full-length antibodies and antibody-toxin fusions were obtained. The bacterially produced antibodies, named “Inclonals,” equaled the performance of the same IgGs that were produced using conventional mammalian cell culture in binding properties as well as in cell killing potency. The rapid and cost effective IgG production process and the high quality of the resultant product may make the bacterial production of full-length IgG and IgG-drug fusion proteins an attractive option for antibody production and a significant contribution to recombinant antibody technology.Key words: IgG, IgG-toxin fusion protein, CD30, EGFR, PE38, inclusion bodies, refolding     

Effects of lung volume and alveolar surface tension on pulmonary vascular resistance

Utilizing the arterial and venous occlusion technique, the effects of lung inflation and deflation on the resistance of alveolar and extraalveolar vessels were measured in the dog in an isolated left lower lobe preparation. The lobe was inflated and deflated slowly (45 s) at constant speed. Two volumes at equal alveolar pressure (Palv = 9.9 +/- 0.6 mmHg) and two pressures (13.8 +/- 0.8 mmHg, inflation; 4.8 +/- 0.5 mmHg, deflation) at equal volumes during inflation and deflation were studied. The total vascular pressure drop was divided into three segments: arterial (delta Pa), middle (delta Pm), and venous (delta Pv). During inflation and deflation the changes in pulmonary arterial pressure were primarily due to changes in the resistance of the alveolar vessels. At equal Palv (9.9 mmHg), delta Pm was 10.3 +/- 1.2 mmHg during deflation compared with 6.8 +/- 1.1 mmHg during inflation. At equal lung volume, delta Pm was 10.2 +/- 1.5 mmHg during inflation (Palv = 13.8 mmHg) and 5.0 +/- 0.7 mmHg during deflation (Palv = 4.8 mmHg). These measurements suggest that the alveolar pressure was transmitted more effectively to the alveolar vessels during deflation due to a lower alveolar surface tension. It was estimated that at midlung volume, the perimicrovascular pressure was 3.5-3.8 mmHg greater during deflation than during inflation.     

Occlusion pressures vs. micropipette pressures in the pulmonary circulation

Because of the discrepancies between the arterial and venous occlusion technique and the micropuncture technique in estimating pulmonary capillary pressure gradient, we compared measurements made with the two techniques in the same preparations (isolated left lower lobe of dog lung). In addition, we also obtained direct and reliable measurements of pressures in 0.9-mm arteries and veins using a retrograde catheterization technique, as well as a microvascular pressure made with the double-occlusion technique. The following conclusions were made from dog lobes perfused with autologous blood at normal flow rate of 500-600 ml/min and pressure gradient of 12 mmHg. 1) The double-occlusion technique measures pressure in the capillaries, 2) a small pressure gradient (0.5 mmHg) exists between 30- to 50-micron arteries and veins, 3) a large pressure gradient occurs in arteries and veins greater than 0.9 mm, 4) the arterial and venous occlusion techniques measure pressures in vessels that are less than 900 microns diam but greater than 50 microns, very likely close to 100 microns, 5) serotonin constricts arteries (larger and smaller than 0.9 mm) whereas histamine constricts veins (larger and smaller than 0.9 mm). Thus three different techniques (small retrograde catheter, arterial and venous occlusion, and micropuncture) show consistent results, confirming the presence of significant resistance in large arteries and veins with minimal resistance in the microcirculation.     

Characterization of the human nicotinic acetylcholine receptor subunit alpha (alpha) 9 (CHRNA9) and alpha (alpha) 10 (CHRNA10) in lymphocytes

Though the nicotinic acetylcholine receptor (nAChR) subunits alpha9 and alpha 10 have been thoroughly characterized within hair cells of the organ of Corti in the inner ear, prior studies have shown that they are also expressed in lymphocytes. In this report, we sought to more definitively characterize the nAChR subunits alpha9 and alpha10 within various populations of human lymphocytes. Using a combination of techniques, including RT-PCR, single-cell RT-PCR, Northern and western blot analysis, and immunofluorescence, expression of both alpha9 and alpha 10 was demonstrated in purified populations of T-cells (CD3+, CD4+, CD8+ and the Jurkat, MT2 and CEM T-cell lines) and B-cells (CD19+, CD80+ and EBV-immortalized B-cells). Single-lymphocyte recording techniques failed to identify an ionic current in response to applied acetylcholine in either T-cells or B-cells. These results clearly demonstrate the presence of these nicotinic receptor subunits within several populations of human lymphocytes, implicating their role in the immune response. However, a lack of demonstrated response to applied acetylcholine using standard single-cell recording techniques suggests a physiology different than that seen in hair cells of the inner ear.     

The lack of CB1 receptors prevents neuroadapatations of both NMDA and GABA(A) receptors after chronic ethanol exposure

As the contribution of cannabinoid (CB1) receptors in the neuroadaptations following chronic alcohol exposure is unknown, we investigated the neuroadaptations induced by chronic alcohol exposure on both NMDA and GABA(A) receptors in CB1-/- mice. Our results show that basal levels of hippocampal [(3)H]MK-801 ((1)-5-methyl-10,11-dihydro-5Hdibenzo[a,d]cyclohepten-5,10-imine) binding sites were decreased in CB1-/- mice and that these mice were also less sensitive to the locomotor effects of MK-801. Basal level of both hippocampal and cerebellar [(3)H]muscimol binding was lower and sensitivity to the hypothermic effects of diazepam and pentobarbital was increased in CB1-/- mice. GABA(A)alpha1, beta2, and gamma2 and NMDA receptor (NR) 1 and 2B subunit mRNA levels were altered in striatum of CB1-/- mice. Our results also showed that [(3)H]MK-801 binding sites were increased in cerebral cortex and hippocampus after chronic ethanol ingestion only in wild-type mice. Chronic ethanol ingestion did not modify the sensitivity to the locomotor effects of MK-801 in both genotypes. Similarly, chronic ethanol ingestion reduced the number of [(3)H]muscimol binding sites in cerebral cortex, but not in cerebellum, only in CB1+/+ mice. We conclude that lifelong deletion of CB1 receptors impairs neuroadaptations of both NMDA and GABA(A) receptors after chronic ethanol exposure and that the endocannabinoid/CB1 receptor system is involved in alcohol dependence.     

Retentissement de l’infection genitale a chlamydia trachomatis sur le sperme chez les hommes consultant pour infertilite du couple

Chlamydia trachomatis (CT) genital infection is one of the most frequent causes of infertility. Its repercution on semen parameters and male infertility is controversial. The objective of this study was to evaluate the impact of CT genital infection on semen parameters in male partners of infertile couples. Ninety-seven infertile couples were studied. Semen, urethral and cervical samples were tested for CT by means of direct fluorescence antibodies assay (DFA), cell culture, polymerase chain reaction (PCR) and FLISA. Sera from both parteners were tested for immunoglobulin M, A and G antibodies to Chlamydia by means of the microimmunofluorescence MIF). For all mens, standard semen parameters were analysed according to the guidlines of the word health organisation. CT infection was identified in 34% of the male partners. In 76% of cases, the infection was asymptomatic. 60,6% of infected patients’s wives were also infected by CT. There was no significant difference between the mean values of concentration, motility and morphology of spermatozoa in both groups of male patients, infected by CT (CT+ group) and lacked infection (CT-group). The mean values of motility, vitality, concentration and normal forms of spermatozoa, in both CT+ and CT- groups were respectively: 39,6%±17,5% vs 40,4% ± 14,9%, 61,9% ±18,1% vs 62,4% ± 18,5%, 80,7×10⁶±67,5×10⁶ vs 67,1×10⁶ ±65,2×10⁶ and 34,7% ± 16,7% vs 33% ± 0,1%. Oligospermia was significantly more frequent in CT+ group (54,9%) than in CT-group (26,9%). High levels of coiled flagella (≥20) were more frequently observed in CT+ group (18,5%) than in CT-group (7,4%), but the difference was not significant. We found in this study a high prevalence of genital chlamydial infection into infertile couples. This infection has no repercution on sperm quality, suggesting that there is no effect of CT upon the spermatozoa. But, we can not exclude any impact on fertilisation ability and/or ultrastructure of these gametes. The finding that oligospermia was more frequent in CT+group, leds us to suggest thas chlamydial infection has a repercution on the gametogenesis or on genital ducts permeability. Another hypothesis would be that oligospermia, reflect of spermatogenesis disorder would be associated with reduction of local immunity. Other studies with wide exploration of spermatic functions and of different parts of genital tract are needed to specify the real impact of genital chlamydial infection upon men reproduction function.