Wnt5a participates in distal lung morphogenesis

Operational parallels in overall mechanisms of three-dimensional patterning of vertebrate organs are becoming increasingly apparent. Many key mediators, such as FGFs, BMPs, and sonic hedgehog, participate in organization of a number of organs, including the lungs, which exhibit a defined proximodistal (P-D) polarity. Recently, Wnt5a a member of the wingless family of signaling molecules involved in cell proliferation, differentiation, and organogenesis, was shown to underlie the outgrowth and P-D morphogenesis of the vertebrate limb. In the current study, we show that Wnt5a is expressed in the mouse lung and plays an important role in lung distal morphogenesis. Analysis of the mutant phenotype in mice carrying a targeted disruption of the Wnt5a locus shows distinct abnormalities in distal lung morphogenesis as manifested by distinct truncation of the trachea and overexpansion of the distal respiratory airways. In the face of deleted WNT5a activity, both epithelial and mesenchymal cell compartments of the Wnt5a(-/-) lungs exhibit increased cell proliferation. The overall architecture of the mutant lungs is characterized by overexpansion of the distal airways and inhibition of lung maturation as reflected by persistence of thickened intersaccular interstitium. Absence of WNT5a activity in the mutant lungs leads to increased expression of Fgf-10, Bmp4, Shh, and its receptor Ptc, raising the possibility that WNT5a, FGF-10, BMP4, and SHH signaling pathways are functionally interactive.     

The conserved active site proline determines the reducing power of Staphylococcus aureus thioredoxin

Nature uses thioredoxin-like folds in several disulfide bond oxidoreductases. Each of them has a typical active site Cys-X-X-Cys sequence motif, the hallmark of thioredoxin being Trp-Cys-Gly-Pro-Cys. The intriguing role of the highly conserved proline in the ubiquitous reducing agent thioredoxin was studied by site-specific mutagenesis of Staphylococcus aureus thioredoxin (Sa_Trx). We present X-ray structures, redox potential, pK(a), steady-state kinetic parameters, and thermodynamic stabilities. By replacing the central proline to a threonine/serine, no extra hydrogen bonds with the sulphur of the nucleophilic cysteine are introduced. The only structural difference is that the immediate chemical surrounding of the nucleophilic cysteine becomes more hydrophilic. The pK(a) value of the nucleophilic cysteine decreases with approximately one pH unit and its redox potential increases with 30 mV. Thioredoxin becomes more oxidizing and the efficiency to catalyse substrate reduction (k(cat)/K(M)) decreases sevenfold relative to wild-type Sa_Trx. The oxidized form of wild-type Sa_Trx is far more stable than the reduced form over the whole temperature range. The driving force to reduce substrate proteins is the relative stability of the oxidized versus the reduced form Delta(T(1/2))(ox/red). This driving force is decreased in the Sa_Trx P31T mutant. Delta(T(1/2))(ox/red) drops from 15.5 degrees C (wild-type) to 5.8 degrees C (P31T mutant). In conclusion, the active site proline in thioredoxin determines the driving potential for substrate reduction.     

The SARS-CoV-2 Delta variant induces an antibody response largely focused on class 1 and 2 antibody epitopes

Exposure histories to SARS-CoV-2 variants and vaccinations will shape the specificity of antibody responses. To understand the specificity of Delta-elicited antibody immunity, we characterize the polyclonal antibody response elicited by primary or mRNA vaccine-breakthrough Delta infections. Both types of infection elicit a neutralizing antibody response focused heavily on the receptor-binding domain (RBD). We use deep mutational scanning to show that mutations to the RBD’s class 1 and class 2 epitopes, including sites 417, 478, and 484–486 often reduce binding of these Delta-elicited antibodies. The anti-Delta antibody response is more similar to that elicited by early 2020 viruses than the Beta variant, with mutations to the class 1 and 2, but not class 3 epitopes, having the largest effects on polyclonal antibody binding. In addition, mutations to the class 1 epitope (e.g., K417N) tend to have larger effects on antibody binding and neutralization in the Delta spike than in the D614G spike, both for vaccine- and Delta-infection-elicited antibodies. These results help elucidate how the antigenic impacts of SARS-CoV-2 mutations depend on exposure history.     

RNA Binding Protein Rbms1 Enables Neuronal Differentiation and Radial Migration during Neocortical Development by Binding and Stabilizing the RNA Message for Efr3a

Various RNA-binding proteins (RBPs) are key components in RNA metabolism and contribute to several neurodevelopmental disorders. To date, only a few of such RBPs have been characterized for their roles in neocortex development. Here, we show that the RBP, Rbms1, is required for radial migration, polarization and differentiation of neuronal progenitors to neurons in the neocortex development. Rbms1 expression is highest in the early development in the developing cortex, with its expression gradually diminishing from embryonic day 13.5 (E13.5) to postnatal day 0 (P0). From in utero electroporation (IUE) experiments when Rbms1 levels are knocked down in neuronal progenitors, their transition from multipolar to bipolar state is delayed and this is accompanied by a delay in radial migration of these cells. Reduced Rbms1 levels in vivo also reduces differentiation as evidenced by a decrease in levels of several differentiation markers, meanwhile having no significant effects on proliferation and cell cycle rates of these cells. As an RNA binding protein, we profiled the RNA binders of Rbms1 by a cross-linked-RIP sequencing assay, followed by quantitative real-time polymerase chain reaction verification and showed that Rbms1 binds and stabilizes the mRNA for Efr3a, a signaling adapter protein. We also demonstrate that ectopic Efr3a can recover the cells from the migration defects due to loss of Rbms1, both in vivo and in vitro migration assays with cultured cells. These imply that one of the functions of Rbms1 involves the stabilization of Efr3a RNA message, required for migration and maturation of neuronal progenitors in radial migration in the developing neocortex.     

Proteolytic activities in Phaseolus vulgaris cotyledons under copper stress

The changes in the protease activities of bean cotyledons were investigated in response to copper stress. Assays using synthetic substrates and specific protease inhibitors followed by activity measurements and electrophoresis analysis allowed to study the classes of enzymes involved in the storage protein mobilization during the germination of bean (Phaseolus vulgaris L) seeds, and then identify which ones were affected in the presence of 200 μM CuCl₂ in the imbibition medium. Copper treatment affected embryo growth and total protease activity. The results of SDS-gelatin-PAGE show that Cu excess led to a decrease in protease activity of 45 to 66 kDa. Moreover, cysteine-, aspartic- and metallo-protease activities were markedly lowered under copper stress, while serine-protease one was enhanced as well as its activity dependent abundance in comparison with control. However, the relative distribution of major cysteine protease in H₂O-germinated seeds was significantly diminished after Cu exposure. Thus, copper excess can disturb the nitrogen freeing from reserve tissues at enzymatic level; differential responses of protease classes are discussed, notably, cysteine protease in the way of storage protein mobilization and serine protease in protective mechanism one.     

High occurrence of carbapenem-resistant Escherichia coli isolates from healthy rabbits (Oryctolagus cuniculus): first report of blaIMI and blaVIM type genes from livestock in Tunisia

We aimed to study the antibiotic susceptibility and possible occurrence of extended-spectrum beta-lactamases (ESBL)/carbapenemase-producing Escherichia coli isolates collected from rabbits in Tunisia. In all, 35 faecal samples from healthy rabbits were collected from one farm and E. coli were isolated from three media: antibiotic-free TBX agar, TBX+2 mg l⁻¹ cefotaxime and TBX+1 mg l⁻¹ imipenem. In total, 39 E. coli isolates were recovered; the majority showed resistance to at least one antibiotic and none was ESBL producer. Carbapenem resistance was detected in 16 isolates from either selective or un-selective media. Phenotypic methods used to detect carbapenemase production showed two positive isolates by Modified Hodge Test, six metallo-carbapenemase producers (Imipenem disc+EDTA) and all were temocillin resistant (possible OXA-48 carbapenemase). bla_VIM and bla_IMP type genes were detected in two and one isolates, respectively; one of them harboured both genes. Isolates contained common genes encoding resistance to sulphonamides (sul1, sul2), tetracycline (tetA, tetB, tetC) and fluoroquinolones (qnrS, aac(6′)-Ib-cr). Class 1 and 2 integrons were detected in five and four isolates, respectively. These findings highlight the importance of rabbit production as reservoir of carbapenem-resistant E. coli and argument the first report of bla_VIM and bla_IMP genes in livestock in Tunisia.     

Semicarbazide-sensitive amine oxidase in annulo-aortic ectasia disease: relation to elastic lamellae-associated proteins.

Lysyl oxidases (Lox), which are members of the amine oxidase family, are involved in the maturation of elastic lamellae and collagen fibers. Modifications of amine oxidases in idiopathic annulo-aortic ectasia disease (IAAED) have never been investigated. Our aim was to examine the expression of several proteins that might interfere with elastic fiber organization in control (n=10) and IAAED (n=18) aortic tissues obtained at surgery. Expression of amine oxidases and semicarbazide-sensitive amine oxidase (SSAO), and cellular phenotypic markers were examined by immunohistopathology and confocal microscopy. The expression of these proteins was assessed in relation to clinical and histomorphological features of the arterial wall. In control aorta, SSAO staining was expressed along elastic lamellae, whereas in aneurysmal areas of IAAED, SSAO was markedly decreased, in association with severe disorganization of elastic lamellae. Smooth muscle myosin heavy chain was also decreased in IAAED compared with controls, indicating smooth muscle cell dedifferentiation. Multiple regression analysis showed that elastic lamellar thickness (ELT) was correlated positively with the SSAO:elastin ratio and negatively with the Lox:elastin ratio, and that the clinical features of IAAED (aneurysm, thoracic aorta diameter, and aortic insufficiency) were positively correlated with ELT but not with SSAO. The relationship between SSAO expression and ELT suggests that this amine oxidase may be involved in elastic fiber organization. However, in advanced IAAED, the deficit in SSAO expression could be secondary to the decrease and fragmentation of elastic fibers and/or to vascular smooth muscle cell dedifferentiation.     

Genetic Variation in the Interleukin-28B Gene Is Associated with Spontaneous Clearance and Progression of Hepatitis C Virus in Moroccan Patients

Background

Genetic variation in the IL28B gene has been strongly associated with treatment outcomes, spontaneous clearance and progression of the hepatitis C virus infection (HCV). The aim of the present study was to investigate the role of polymorphisms at this locus with progression and outcome of HCV infection in a Moroccan population.

Methods

We analyzed a cohort of 438 individuals among them 232 patients with persistent HCV infection, of whom 115 patients had mild chronic hepatitis and 117 had advanced liver disease (cirrhosis and hepatocellular carcinoma), 68 individuals who had naturally cleared HCV and 138 healthy subjects. The IL28B SNPs rs12979860 and rs8099917 were genotyped using a TaqMan 5′ allelic discrimination assay.

Results

The protective rs12979860-C and rs8099917-T alleles were more common in subjects with spontaneous clearance (77.9% vs 55.2%; p = 0.00001 and 95.6% vs 83.2%; p = 0.0025, respectively). Individuals with clearance were 4.69 (95% CI, 1.99–11.07) times more likely to have the C/C genotype for rs12979860 polymorphism (p = 0.0017) and 3.55 (95% CI, 0.19–66.89) times more likely to have the T/T genotype at rs8099917. Patients with advanced liver disease carried the rs12979860-T/T genotype more frequently than patients with mild chronic hepatitis C (OR = 1.89; 95% CI, 0.99–3.61; p = 0.0532) and this risk was even more pronounced when we compared them with healthy controls (OR = 4.27; 95% CI, 2.08–8.76; p = 0.0005). The rs8099917-G allele was also associated with advanced liver disease (OR = 2.34; 95% CI, 1.40–3.93; p = 0.0100).

Conclusions

In the Moroccan population, polymorphisms near the IL28B gene play a role both in spontaneous clearance and progression of HCV infection.