Thermophilic PHP Protein Tyrosine Phosphatases (Cap8C and Wzb) from Mesophilic Bacteria.

Protein tyrosine phosphatases (PTPs) of the polymerase and histidinol phosphatase (PHP) superfamily with characteristic phosphatase activity dependent on divalent metal ions are found in many Gram-positive bacteria. Although members of this family are co-purified with metal ions, they still require the exogenous supply of metal ions for full activation. However, the specific roles these metal ions play during catalysis are yet to be well understood. Here, we report the metal ion requirement for phosphatase activities of S. aureus Cap8C and L. rhamnosus Wzb. AlphaFold-predicted structures of the two PTPs suggest that they are members of the PHP family. Like other PHP phosphatases, the two enzymes have a catalytic preference for Mn2+, Co2+ and Ni2+ ions. Cap8C and Wzb show an unusual thermophilic property with optimum activities over 75 °C. Consistent with this model, the activity-temperature profiles of the two enzymes are dependent on the divalent metal ion activating the enzyme.

Role of the C-Terminal ß Sandwich of Thermoanaerobacter tengcongensis Thermophilic Esterase in Hydrolysis of Long-Chain Acyl Substrates.

To search for a novel thermostable esterase for optimized industrial applications, esterase from a thermophilic eubacterium species, Thermoanaerobacter tengcongensis MB4, was purified and characterized in this work. Sequence analysis of T. tengcongensis esterase with other homologous esterases of the same family revealed an apparent tail at the C-terminal that is not conserved across the esterase family. Hence, it was hypothesized that the tail is unlikely to have an essential structural or catalytic role. However, there is no documented report of any role for this tail region. We probed the role of the C-terminal domain on the catalytic activity and substrate preference of T. tengcongensis esterase EstA3 with a view to see how it could be engineered for enhanced properties. To achieve this, we cloned, expressed, and purified the wild-type and the truncated versions of the enzyme. In addition, a naturally occurring member of the family (from Brevibacillus brevis) that lacks the C-terminal tail was also made. In vitro characterization of the purified enzymes showed that the C-terminal domain contributes significantly to the catalytic activity and distinct substrate preference of T. tengcongensis esterase EstA3. All three recombinant enzymes showed the highest preference for paranitrophenyl butyrate (pNPC4), which suggests they are true esterases, not lipases. Kinetic data revealed that truncation had a slight effect on the substrate-binding affinity. Thus, the drop in preference towards long-chain substrates might not be a result of substrate binding affinity alone. The findings from this work could form the basis for future protein engineering allowing the modification of esterase catalytic properties through domain swapping or by attaching a modular protein domain.

A Genetic Circuit Design for Targeted Viral RNA Degradation.

Advances in synthetic biology have led to the design of biological parts that can be assembled in different ways to perform specific functions. For example, genetic circuits can be designed to execute specific therapeutic functions, including gene therapy or targeted detection and the destruction of invading viruses. Viral infections are difficult to manage through drug treatment. Due to their high mutation rates and their ability to hijack the host's ribosomes to make viral proteins, very few therapeutic options are available. One approach to addressing this problem is to disrupt the process of converting viral RNA into proteins, thereby disrupting the mechanism for assembling new viral particles that could infect other cells. This can be done by ensuring precise control over the abundance of viral RNA (vRNA) inside host cells by designing biological circuits to target vRNA for degradation. RNA-binding proteins (RBPs) have become important biological devices in regulating RNA processing. Incorporating naturally upregulated RBPs into a gene circuit could be advantageous because such a circuit could mimic the natural pathway for RNA degradation. This review highlights the process of viral RNA degradation and different approaches to designing genetic circuits. We also provide a customizable template for designing genetic circuits that utilize RBPs as transcription activators for viral RNA degradation, with the overall goal of taking advantage of the natural functions of RBPs in host cells to activate targeted viral RNA degradation.

Influence of ethanolic extract of Allium sativum on TP53 gene and its anticancer potential in N-Nitrosodiethylamine (NDEA)-induced hepatocellular carcinoma in male albino rats.

Objectives: Cancer is a group of genetic disorders in which the behavior of the cell is disturbed by mutation and other abnormalities thereby posing as the leading cause of morbidity and mortality globally. Hepatocellular Carcinoma (HCC) is the most common form of liver cancer, highly aggressive with high mortality and incidence rate; and has limited therapeutic options. Most of the conventional cancer chemotherapeutics are associated with undesirable side effects, toxicity, chemoresistance, and high treatment cost, driving the need for a safer and more effective treatment alternative. Medicinal plants and herbs have shown very promising anti-cancer properties which are important for cancer treatment due to their multiple chemical compounds. Materials and Methods: Qualitative screening of the ethanolic extractof Allium sativum was conducted showing the different phytochemicalspresent. The levels of liver function and hematological parameters wasdetermined via spectrophotometric analysis. Polymerase Chain Reaction techniquewas used to assess the gene patterns of Tumorsuppressor p53 (TP53). Results: Phytochemical analysis revealed that Allium sativum has properties that antagonize the proliferating process of carcinogenesis in the liver. The NDEA-group showed significant distortion in the liver architecture characterized by vascular congestion of blood sinusoids, cirrhosis, and congestive hepatopathy while the treated groups showed a reduction in the abnormalities and malignant formation. The treated group showed a significant (P<0.05) increase and restored activities of Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Bilirubin and hematological parameters (RBCs, WBCs, and Platelets). TP53 gene amplification was significantly (P<0.05) visible after treatment. Conclusion: Ethanolic plant extract of A. sativum demonstrates its anticancer properties by improving the liver architecture, increasing the antioxidant defense systems, and activation of the tumor suppressor (TP53) gene. Garlic extract has anti-proliferating properties and can be used as an alternative mode of treatment and prevention for hepatocellular carcinoma.

Evaluation of HIV infection in febrile patients visiting health centers in Lagos, Nigeria.

OBJECTIVE: Acute febrile infections compatible with malaria are the most prevalent presentation at sub-Saharan African health clinics, accounting for 30-50% of outpatient visits. Acute human immunodeficiency virus (HIV) infection can mimic acute malaria symptoms. As a result, screening people with malaria symptoms for HIV infection is critical. The goal of our study was to find out how common HIV infection was among feverish patients. RESULTS: Out of the 310 individuals screened, 9 (3.0%) had HIV-1 infection, with 5 (55.5%) being females and 4 (44.4%) being males. This study found no evidence of HIV-2 infection or HIV-1/HIV-2 co-infection. HIV infection was found in 1-3% of patients with probable malaria at different sites in Lagos, Nigeria.

HhAntag, a Hedgehog Signaling Antagonist, Suppresses Chondrogenesis and Modulates Canonical and Non-Canonical BMP Signaling.

Chondrogenesis subtends the development of most skeletal elements and involves mesenchymal cell condensations differentiating into growth plate chondrocytes that proliferate, undergo hypertrophy, and are replaced by bone. In the pediatric disorder Hereditary Multiple Exostoses, however, chondrogenesis occurs also at ectopic sites and causes formation of benign cartilaginous tumors--exostoses--near the growth plates. No treatment is currently available to prevent or reverse exostosis formation. Here, we asked whether chondrogenesis could be stopped by targeting the hedgehog pathway, one of its major regulators. Micromass cultures of limb mesenchymal cells were treated with increasing amounts of the hedgehog inhibitor HhAntag or vehicle. The drug effectively blocked chondrogenesis and did so in a dose-dependent manner as monitored by: alcian blue-positive cartilage nodule formation; gene expression of cartilage marker genes; and reporter activity in Gli1-LacZ cell cultures. HhAntag blocked chondrogenesis even when the cultures were co-treated with bone morphogenetic protein 2 (rhBMP-2), a strong pro-chondrogenic factor. Immunoblots showed that HhAntag action included modulation of canonical (pSmad1/5/8) and non-canonical (pp38) BMP signaling. In cultures co-treated with HhAntag plus rhBMP-2, there was a surprising strong up-regulation of pp38 levels. Implantation of rhBMP-2-coated beads near metacarpal elements in cultured forelimb explants induced formation of ectopic cartilage that however, was counteracted by HhAntag co-treatment. Collectively, our data indicate that HhAntag inhibits not only hedgehog signaling, but also modulates canonical and non-canonical BMP signaling and blocks basal and rhBMP2-stimulated chondrogenesis, thus representing a potentially powerful drug-based strategy to counter ectopic cartilage growth or induce its involution.