Mutational Analysis of the Shigella flexneri O-Antigen Polymerase Wzy: Identification of Wzz-Dependent Wzy Mutants

The O-antigen (Oag) component of lipopolysaccharide (LPS) is a major virulence determinant of Shigella flexneri and is synthesized by the O-antigen polymerase, Wzy_Sf. Oag chain length is regulated by chromosomally encoded Wzz_Sf and pHS-2 plasmid-encoded Wzz_pHS2. To identify functionally important amino acid residues in Wzy_Sf, random mutagenesis was performed on the wzy_Sf gene in a pWaldo-TEV-GFP plasmid, followed by screening with colicin E2. Analysis of the LPS conferred by mutated Wzy_Sf proteins in the wzy_Sf-deficient (Δwzy) strain identified 4 different mutant classes, with mutations found in periplasmic loop 1 (PL1), PL2, PL3, and PL6, transmembrane region 2 (TM2), TM4, TM5, TM7, TM8, and TM9, and cytoplasmic loop 1 (CL1) and CL5. The association of Wzy_Sf and Wzz_Sf was investigated by transforming these mutated wzy_Sf plasmids into a wzy_Sf- and wzz_Sf-deficient (Δwzy Δwzz) strain. Comparison of the LPS profiles in the Δwzy and Δwzy Δwzz backgrounds identified Wzy_Sf mutants whose polymerization activities were Wzz_Sf dependent. Colicin E2 and bacteriophage Sf6c sensitivities were consistent with the LPS profiles. Analysis of the expression levels of the Wzy_Sf-GFP mutants in the Δwzy and Δwzy Δwzz backgrounds identified a role for Wzz_Sf in Wzy_Sf stability. Hence, in addition to its role in regulating Oag modal chain length, Wzz_Sf also affects Wzy_Sf activity and stability.     

Off-the-shelf mesenchymal stem cells from human umbilical cord tissue can significantly improve symptoms in COVID-19 patients: An analysis of evidential relations

Coronavirus disease-2019 (COVID-19) has affected more than 200 countries worldwide. This disease has hugely affected healthcare systems as well as the economy to an extent never seen before. To date, COVID-19 infection has led to about 165000 deaths in 150 countries. At present, there is no specific drug or efficient treatment for this disease. In this analysis based on evidential relationships of the biological characteristics of MSCs, especially umbilical cord (UC)-derived MSCs as well as the first clinical trial using MSCs for COVID-19 treatment, we discuss the use of UC-MSCs to improve the symptoms of COVID-19 in patients.     

Theoretical study on the adsorption of phenol on activated carbon using density functional theory

Density functional theory (DFT) calculations performed at the PBE/DZP level using the DFT-D2 method were utilized to investigate the adsorption of phenol on pristine activated carbon (AC) and on activated carbon functionalized with OH, CHO, or COOH groups. Over the pristine AC, the phenol molecule undergoes weak physical adsorption due to van der Waals interactions between the aromatic part of the phenol and the basal planes of the AC. Among the three functional groups used to functionalize the AC, the carboxylic group was found to interact most strongly with the hydroxyl group of phenol. These results suggest that functionalized AC-COOH has great potential for use in environmental applications as an adsorbent of phenol molecules in aqueous phases.     

Crystal Structure of the Hendra Virus Attachment G Glycoprotein Bound to a Potent Cross-Reactive Neutralizing Human Monoclonal Antibody

The henipaviruses, represented by Hendra (HeV) and Nipah (NiV) viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb) have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4) was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines.     

An Evolution-Guided Analysis Reveals a Multi-Signaling Regulation of Fas by Tyrosine Phosphorylation and its Implication in Human Cancers

Demonstrations of both pro-apoptotic and pro-survival abilities of Fas (TNFRSF6/CD95/APO-1) have led to a shift from the exclusive “Fas apoptosis” to “Fas multisignals” paradigm and the acceptance that Fas-related therapies face a major challenge, as it remains unclear what determines the mode of Fas signaling. Through protein evolution analysis, which reveals unconventional substitutions of Fas tyrosine during divergent evolution, evolution-guided tyrosine-phosphorylated Fas proxy, and site-specific phosphorylation detection, we show that the Fas signaling outcome is determined by the tyrosine phosphorylation status of its death domain. The phosphorylation dominantly turns off the Fas-mediated apoptotic signal, while turning on the pro-survival signal. We show that while phosphorylations at Y232 and Y291 share some common functions, their contributions to Fas signaling differ at several levels. The findings that Fas tyrosine phosphorylation is regulated by Src family kinases (SFKs) and the phosphatase SHP-1 and that Y291 phosphorylation primes clathrin-dependent Fas endocytosis, which contributes to Fas pro-survival signaling, reveals for the first time the mechanistic link between SFK/SHP-1-dependent Fas tyrosine phosphorylation, internalization route, and signaling choice. We also demonstrate that levels of phosphorylated Y232 and Y291 differ among human cancer types and differentially respond to anticancer therapy, suggesting context-dependent involvement of Fas phosphorylation in cancer. This report provides a new insight into the control of TNF receptor multisignaling by receptor phosphorylation and its implication in cancer biology, which brings us a step closer to overcoming the challenge in handling Fas signaling in treatments of cancer as well as other pathologies such as autoimmune and degenerative diseases.     

Analysis of a Major Phenolic Glucoside and Biochemistry Compounds from Curculigo Orchioides Gaertn

Background:Orcinol-β-D-glucoside, which is also known as orcinol glucoside, is a major phenolic glucoside compound in the rhizome of the Curculigo orchioides Gaertn. This compound has many medicinal properties such as being antioxidant, immunomodulatory, antiosteoporosis, stress relief, antidepressant, etc.Methods:Determination of reducing sugar content by Bertrand’s method, determination of lipid content by Soxhlet method, determination of vitamin C content by iodine titration, determination of enzyme activity catalase by titration with KMnO4. Quantification of Orcinol-β-D-glucoside was conducted by HPLC analysis.Results:The Orcinol-β-D-glucoside of C. orchioides in Thuy Bang mountain was highest. Besides, the content of reducing sugars, vitamin C, enzyme catalase, and lipids of C. orchioides differed significantly among sites. In which, the reducing sugar and vitamin C of C. orchioides in Ngu Binh mountain was highest. Whereas, enzyme catalase was also highest in Thuy Bang mountain. However, the lipid content of C. orchioides was also highest in Huong Tho mountain.Conclusion:The result will contribute to providing the scientific basis for the selection of breeding, planting, development of C. orchioides in Thua Thien Hue province, as well as other provinces in Vietnam and opening new research directions for applications in the future.Key Words: Antioxidants, Hypoxidaceae, Medicinal Plants, Vietnam     

Use of polyethylenimine-adenovirus complexes to examine triplex formation in intact cells

Triplex-forming oligonucleotides (TFOs) show potential for sequence-specific DNA binding and inhibition of gene expression. We have applied this antigene strategy using a TFO incorporating an Auger-emitting radionucleotide, 125I, to study the production of double-strand breaks (dsb) in the rat aquaporin 5 (rAQP5) cDNA. 125I-TFO bound to the pCMVrAQP5 plasmid in vitro in a dose-dependent manner and formed stable triplexes up to 65 degrees C and in the presence of 140 mM KCl. Further, 125I-TFO resulted in a predictable dsb when analyzed by Southern hybridization. To deliver TFOs to epithelial cells, we employed 125I-TFO-polyethyleneimine-adenovirus (125I-TFO-PEI-Ad) complexes. We hypothesized that these complexes would take advantage of adenoviral characteristics to transfer 125I-TFO to the cell nucleus. Adenovirus-containing complexes brought about greater uptake and nuclear localization of TFOs compared with delivery with 125I-TFO-PEI complexes alone. No significant degradation of 125I-TFO was found after delivery into cells using PEI-Ad complexes and freezing and thawing. We next used PEI-Ad complexes to deliver 125I-TFO and pCMVrAQP5 separately to epithelial cells to determine if triplexes can form de novo within cells, resulting in the specific dsb in the rAQP5 cDNA. After delivery, cell pellets were stored at -80 degrees C for more than 60 days. Thereafter, plasmid DNA was isolated from cells and analyzed for dsb by Southern hybridization. However, none were detected. We conclude that under the experimental conditions employed, effective triplexes, with 125I-TFO and pCMVrAQP5, do not form de novo inside cells.