Soluble Angiotensin-Converting Enzyme 2 Protein Improves Survival and Lowers Viral Titers in Lethal Mouse Model of Severe Acute Respiratory Syndrome Coronavirus Type 2 Infection with the Delta Variant.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) utilizes angiotensin-converting enzyme 2 (ACE2) as its main receptor for cell entry. We bioengineered a soluble ACE2 protein termed ACE2 618-DDC-ABD that has increased binding to SARS-CoV-2 and prolonged duration of action. Here, we investigated the protective effect of this protein when administered intranasally to k18-hACE2 mice infected with the aggressive SARS-CoV-2 Delta variant. k18-hACE2 mice were infected with the SARS-CoV-2 Delta variant by inoculation of a lethal dose (2 × 104 PFU). ACE2 618-DDC-ABD (10 mg/kg) or PBS was administered intranasally six hours prior and 24 and 48 h post-viral inoculation. All animals in the PBS control group succumbed to the disease on day seven post-infection (0% survival), whereas, in contrast, there was only one casualty in the group that received ACE2 618-DDC-ABD (90% survival). Mice in the ACE2 618-DDC-ABD group had minimal disease as assessed using a clinical score and stable weight, and both brain and lung viral titers were markedly reduced. These findings demonstrate the efficacy of a bioengineered soluble ACE2 decoy with an extended duration of action in protecting against the aggressive Delta SARS-CoV-2 variant. Together with previous work, these findings underline the universal protective potential against current and future emerging SARS-CoV-2 variants.

Processing of LtaS restricts LTA assembly and YSIRK preprotein trafficking into Staphylococcus aureus cross-walls.

Septal membranes of Staphylococcus aureus serve as the site of secretion for precursors endowed with the YSIRK motif. Depletion of ltaS, a gene required for lipoteichoic acid (LTA) synthesis, results in the loss of restricted trafficking of YSIRK precursors to septal membranes. Here, we seek to understand the mechanism that ties LTA assembly and trafficking of YSIRK precursors. We confirm that catalytically inactive lipoteichoic acid synthase (LtaS)T300A does not support YSIRK precursor trafficking to septa. We hypothesize that the enzyme's reactants [gentiobiosyldiacylglycerol (Glc2-DAG) and phosphatidylglycerol (PG)] or products [LTA and diacylglycerol (DAG)], not LtaS, must drive this process. Indeed, we observe that septal secretion of the staphylococcal protein A YSIRK precursor is lost in ypfP and ltaA mutants that produce glycerophosphate polymers [poly(Gro-P)] without the Glc2-DAG lipid anchor. These mutants display longer poly(Gro-P) chains, implying enhanced PG consumption and DAG production. Our experiments also reveal that in the absence of Glc2-DAG, the processing of LtaS to the extracellular catalytic domain, eLtaS, is impaired. Conversely, LTA polymerization is delayed in a strain producing LtaSS218P, a variant processed more slowly than LtaS. We conclude that Glc2-DAG binding to the enzyme couples catalysis by LtaS and the physical release of eLtaS. We propose a model for the temporal and localized assembly of LTA into cross-walls. When LtaS is not processed in a timely manner, eLtaS no longer diffuses upon daughter cell splitting, LTA assembly continues, and the unique septal-lipid pool, PG over DAG ratio, is not established. This results in profound physiological changes in S. aureus cells, including the inability to restrict the secretion of YSIRK precursors at septal membranes.IMPORTANCEIn Staphylococcus aureus, peptidoglycan is assembled at the septum. Dedicated cell division proteins coordinate septal formation and the fission of daughter cells. Lipoteichoic acid (LTA) assembly and trafficking of preproteins with a YSIRK motif also occur at the septum. This begs the question as to whether cell division components also recruit these two pathways. This study shows that the processing of lipoteichoic acid synthase (LtaS) to extracellular LtaS by signal peptidase is regulated by gentiobiosyldiacylglycerol (Glc2-DAG), the priming substrate for LTA assembly. A model is proposed whereby a key substrate controls the temporal and spatial activity of an enzyme. In turn, this mechanism enables the establishment of a unique and transient lipid pool that defines septal membranes as a targeting site for the secretion of YSIRK preproteins.

Protein coopted from a phage restriction system dictates orthogonal cell division plane selection in Staphylococcus aureus.

The spherical bacterium Staphylococcus aureus, a leading cause of nosocomial infections, undergoes binary fission by dividing in two alternating orthogonal planes, but the mechanism by which S. aureus correctly selects the next cell division plane is not known. To identify cell division placement factors, we performed a chemical genetic screen that revealed a gene which we termed pcdA. We show that PcdA is a member of the McrB family of AAA+ NTPases that has undergone structural changes and a concomitant functional shift from a restriction enzyme subunit to an early cell division protein. PcdA directly interacts with the tubulin-like central divisome component FtsZ and localizes to future cell division sites before membrane invagination initiates. This parallels the action of another McrB family protein, CTTNBP2, which stabilizes microtubules in animals. We show that PcdA also interacts with the structural protein DivIVA and propose that the DivIVA/PcdA complex recruits unpolymerized FtsZ to assemble along the proper cell division plane. Deletion of pcdA conferred abnormal, non-orthogonal division plane selection, increased sensitivity to cell wall-targeting antibiotics, and reduced virulence in a murine infection model. Targeting PcdA could therefore highlight a treatment strategy for combatting antibiotic-resistant strains of S. aureus.

Neuroprotective Effects of Some Nutraceuticals against Manganese-Induced Parkinson's Disease in Rats: Possible Modulatory Effects on TLR4/NLRP3/NF-κB, GSK-3ß, Nrf2/HO-1, and Apoptotic Pathways.

Parkinson's disease (PD) is a progressive neurodegenerative disorder affecting the substantia nigra where functions controlling body movement take place. Manganese (Mn) overexposure is linked to a neurologic syndrome resembling PD. Sesamol, thymol, wheat grass (WG), and coenzyme Q10 (CoQ10) are potent antioxidants, anti-inflammatory, and anti-apoptotic nutraceuticals. We investigated the potential protective effects of these nutraceuticals alone or in combinations against MnCl2-induced PD in rats. Seven groups of adult male Sprague Dawley rats were categorized as follows: group (I) was the control, while groups 2-7 received MnCl2 either alone (Group II) or in conjunction with oral doses of sesamol (Group III), thymol (Group IV), CoQ10 (Group V), WG (Group VI), or their combination (Group VII). All rats were subjected to four behavioral tests (open-field, swimming, Y-maze, and catalepsy tests). Biochemical changes in brain levels of monoamines, ACHE, BDNF, GSK-3ß, GABA/glutamate, as well as oxidative stress, and apoptotic and neuroinflammatory biomarkers were evaluated, together with histopathological examinations of different brain regions. Mn increased catalepsy scores, while decreasing neuromuscular co-ordination, and locomotor and exploratory activity. It also impaired vigilance, spatial memory, and decision making. Most behavioral impairments induced by Mn were improved by sesamol, thymol, WG, or CoQ10, with prominent effect by sesamol and thymol. Notably, the combination group showed more pronounced improvements, which were confirmed by biochemical, molecular, as well as histopathological findings. Sesamol or thymol showed better protection against neuronal degeneration and some behavioral impairments induced by Mn than WG or CoQ10, partly via interplay between Nrf2/HO-1, TLR4/NLRP3/NF-κB, GSK-3ß and Bax/Bcl2 pathways.

Error-prone PCR mutagenesis and reverse bacterial two-hybrid screening identify a mutation in asparagine 53 of the Staphylococcus aureus ESAT6-like component EsxB that perturbs interaction with EsxD.

The ESAT6-like Secretion System (ESS) of the human pathogen Staphylococcus aureus secretes heterodimeric virulence effectors such as EsxB and EsxD. To gain insights into the nature of EsxB-EsxD interaction, randomly mutated esxB generated by error-prone PCR was co-transformed together with esxD as adenylate cyclase fusion constructs into cyclase-deficient Escherichia coli, followed by reverse bacterial two-hybrid screening. Three color species were observed: dark blue, light blue, and white (no EsxB-EsxD interaction). The esxB from white colonies was subjected to standard PCR to check for gene signal, followed by SDS-PAGE for variant stability assessment. The gene coding for a stable EsxB variant that perturbed interaction with EsxD was further subjected to DNA sequencing. A single point mutation in esxB at position 157 was identified, leading to an amino acid change from asparagine to aspartic acid at position 53 in the resulting protein. Structural modeling of EsxB reveals that N53 is surface exposed. Whereas N53S substitution by site-directed mutagenesis retained heterodimerization with EsxD, N53A substitution abrogated such interaction. In addition, N53D change in EsxB did not alter interaction with EssG, another soluble component of the ESS pathway, suggesting minimal impact of the N53D substitution on EsxB stability and solubility. Taken together, these data provide new insights into the nature of EsxB-EsxD interaction and offer a systematic approach for in vivo analysis of protein-protein interactions of pathogenic bacteria in non-pathogenic hosts.

MicroRNA-199b expression level and coliform count in irritable bowel syndrome.

Irritable bowel syndrome (IBS) is a common intestinal disorder. The pathophysiology of IBS may involve an altered intestinal microbiota. Recent studies have shown that alterations in microRNA (miRNA) levels have affected IBS and its subtypes. We aimed to compare both the count of Coliform and serum level of miRNA-199b between patients with IBS and healthy controls and to find the relationship between the Coliform and miRNAs in patients with IBS. Patients with IBS were classified into three subgroups based on their predominant bowel pattern as defined by Rome III criteria. Quantitative culture of Coliform and determination of serum miRNA-199b expression level by quantitative real-time PCR in IBS group versus healthy controls were performed. There was a significant increase in the count of Coliform in patients with IBS and its different subtypes when compared with healthy controls. There was a significant decrease of serum miR-199b expression level in patients with IBS and its different subtypes when compared with healthy controls with the highest level (1.9 ± 0.53 log scale) in healthy controls and lowest one (0.71 ± 0.27 log scale) in IBS with diarrhea (IBS-D) subtype. Moreover, there was a negative correlation between the count of Coliform and the serum miRNA-199b expression level in IBS. This study reported that there was a significant increase in the count of Coliform and a decrease in the serum miRNA-199b expression level. In addition, there was a negative correlation between them in patients with IBS and its different subtypes when compared with healthy controls. © 2016 IUBMB Life, 68(5):335-342, 2016.

Glutathione S-Transferase M1 and T1 Gene Polymorphisms and the Outcome of Chronic Hepatitis C Virus Infection in Egyptian Patients.

We analysed the distribution of GSTM1 and GSTT1 gene polymorphisms in Egyptian patients with chronic hepatitis C, and investigated their relationship to the clinical outcome of chronic hepatitis C virus (HCV) infection. This study included 169 patients with chronic HCV infection and 145 healthy and matched controls.GSTM1 and GSTT1 polymorphisms were genotyped by multiplex polymerase chain reaction. Individual GSTM1 null and GSTT1 null genotypes were more frequent in patients versus control subjects [OR, 4 (95% CI, 2.5-6.4); P ˂ 0.001] and [OR, 1.7 (95% CI, 1.1-2.6); P = 0.025], respectively. The patient group showed a higher frequency of the combined GSTM1/GSTT1 double-null genotype than the control group [OR, 1.8 (95% CI, 1.1-2.9); P = 0.016]. The distribution frequencies of the combined GSTM1/GSTT1 double-null genotype were significantly different [OR, 0.5 (95% CI, 0.25-0.99); P = 0.049] between F0-F3 and F4. There were no significant differences between the two groups with regard to other genotypes. The combined GSTM1/GSTT1 double-null genotype was significantly increased in Child-Pugh C patients in comparison to Child-Pugh A+B (P = 0.02). There was no significant difference between different classes with regard to other genotypes. In conclusion, we identified an association between the combined GSTM1/GSTT1 double-null genotype and advanced liver fibrosis and outcome of chronic HCV infection in Egyptian patients.

Spectroscopic and kinetic studies on the degradation of methylene blue using the supramolecular coordination polymer [(Ph3Sn)4FeCN(6)] as catalyst.

The structure of the supramolecular coordination polymer (SCP), [(Ph(3)Sn)(4)Fe(CN)(6)], 1, consists of octahedral [Fe(CN)(6)](4-) building blocks which are connected by the TBPY-5 configured Ph(3)Sn(CN…)(2) fragments creating 3D-network structure that contains terminal cyanide groups. The catalytic behavior of the SCP 1 was utilized for Fenton and photo-Fenton degradation of methylene blue dye (MB). The plot of kinetic degradation indicates pseudo first-order rate with respect to the MB dye concentration, K(obs.)=0.071 min(-1). On the other hand, the observed rate constant of the photo catalytic degradation of MB equals to 1.45 min(-1) indicating that irradiation enhances, significantly, the rate of degradation of MB dye. Discoloration of the dye was obtained in less than 3h. Meanwhile, the conjugated structure and the phenyl rings of the MB molecule were destroyed or even broken down into small organic acids and inorganic ions, as indicated by FT-IR spectra. Disodium salt of terephthalic acid photoluminescence probing technology and radical scavenging measurements were carried out to identify the reactive oxygen species. The different parameters that affect MB degradation rate were evaluated. Moreover, the efficiency of recycled the SCP 1 and the mechanism of degradation of MB dye was investigated.

[CuCN.Me(3)SnCN.0.5bpy] (bpy = 4,4'-Bipyridine): A Novel Supramolecular Assembly Involving the No Longer Unusual {Cu(2)(&mgr;-CN)(2)(CN)(2)} Motif.

Reaction of K(3)[Cu(CN)(4)] with Me(3)SnCl and 4,4'-bipyridine (bpy) affords the novel ternary adduct [CuCN.Me(3)SnCN.0.5bpy], 2, as yellow, cubelike crystals. The supramolecular architecture of 2 consists, according to a single-crystal X-ray study, of remarkably porous, approximately planar sheets held together by almost perpendicular bpy pillars. Basic building blocks of the sheets are {Cu(2)(&mgr;-CN)(2)(CN)(2)} fragments whose N atoms are connected, via Me(3)Sn bridges, with the N atoms of adjacent fragments. 2 crystallizes in space group P2(1)/c with (at 20 degrees C) a = 10.035(4) Å, b = 11.980(3) Å, c = 12.425(5) Å, beta = 110.94(3) degrees, and Z = 4. The dinuclear building blocks resemble strongly those of the long known, but chemically more labile, layered coordination polymer [CuCN.NH(3)], 1. Although the lattice of 2 consists of two equivalent, ideally interpenetrating 3D frameworks, spacious, straight channels of a cross section of ca. 10 Å x 5 Å persist in this unprecedented structure. The likewise polymeric new adduct [CuCN.Me(3)SnCN.H(2)O] (3) obtained from K(3)[Cu(CN)(4)] and Me(3)SnCl in the absence of bpy may be considered as a formal precursor of 2.