Analysis of mutational variations in TP53 tumour suppressor gene among Pakistani head and neck cancer patients.

The aim of this study was to determine the frequency of TP53 mutation among Pakistani head and neck cancer (HNC) patients who visited Nishtar Hospital Multan and Nishtar Institute of Dentistry (NID), Multan, Pakistan. While significant research has been conducted on the role of p53 in HNC throughout the world, this study is the first of its kind in Southern Punjab, Pakistan. A total of 242 samples (121 cases and 121 controls) were collected from Nishtar Hospital Multan and NID, Multan, Pakistan. After histopathological analysis to determine the stage type and grade of malignancy, DNA extraction and sequencing were carried out to assess any mutations in the TP53 region (exons 5-8). Genetic screening was performed by the polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) technique and Chromas 2.6.6 was used to visualise the sequencing results. The mean age of cases was 50.73 ±16.41 years and controls were 37.55 ± 15.51 years. The frequency of HNC was higher in male patients (65.28%) than in female patients (34.71%). Overall, this cancer was found to be significantly more prevalent in the age group of >35-55 years (p < 0.001). Smoking (51% versus 14%), naswar usage (15.7% versus 6.6%), poor oral hygiene (52.9% versus 29.8%) and anemic status (57.0% versus 4.1%) were significantly associated with cases (p ≤ 0.05) compared to controls. Only 04 samples exon 5 (1 sample), exon 7 (2 samples) and exon 8 (1 sample) with changed mobility patterns were found on the SSCP gel. All mutations were missense and heterozygous. Out of four mutant samples, three mutations were in the hotspot regions (codon 175, 245 and 248) of p53. Based on this study, there may be a weak association between the TP53 exon 5-8 mutation and HNC patients in Southern Punjab, Pakistan.

High-throughput screening identifies broad-spectrum Coronavirus entry inhibitors.

The COVID-19 pandemic highlighted the need for antivirals against emerging coronaviruses (CoV). Inhibiting spike (S) glycoprotein-mediated viral entry is a promising strategy. To identify small molecule inhibitors that block entry downstream of receptor binding, we established a high-throughput screening (HTS) platform based on pseudoviruses. We employed a three-step process to screen nearly 200,000 small molecules. First, we identified hits that inhibit pseudoviruses bearing the SARS-CoV-2 S glycoprotein. Counter-screening against pseudoviruses with the vesicular stomatitis virus glycoprotein (VSV-G), yielded sixty-five SARS-CoV-2 S-specific inhibitors. These were further tested against pseudoviruses bearing the MERS-CoV S glycoprotein, which uses a different receptor. Out of these, five compounds, which included the known broad-spectrum inhibitor Nafamostat, were subjected to further validation and tested against pseudoviruses bearing the S glycoprotein of the Alpha, Delta, and Omicron variants as well as bona fide SARS-CoV-2. This rigorous approach revealed an unreported inhibitor and its derivative as potential broad-spectrum antivirals.

SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses.

On 24th November 2021, the sequence of a new SARS-CoV-2 viral isolate Omicron-B.1.1.529 was announced, containing far more mutations in Spike (S) than previously reported variants. Neutralization titers of Omicron by sera from vaccinees and convalescent subjects infected with early pandemic Alpha, Beta, Gamma, or Delta are substantially reduced, or the sera failed to neutralize. Titers against Omicron are boosted by third vaccine doses and are high in both vaccinated individuals and those infected by Delta. Mutations in Omicron knock out or substantially reduce neutralization by most of the large panel of potent monoclonal antibodies and antibodies under commercial development. Omicron S has structural changes from earlier viruses and uses mutations that confer tight binding to ACE2 to unleash evolution driven by immune escape. This leads to a large number of mutations in the ACE2 binding site and rebalances receptor affinity to that of earlier pandemic viruses.

Extracellular vesicle fusion visualized by cryo-electron microscopy.

Extracellular vesicles (EVs) transfer bioactive molecules between cells in a process reminiscent of enveloped viruses. EV cargo delivery is thought to occur by protein-mediated and pH-dependent membrane fusion of the EV and the cellular membrane. However, there is a lack of methods to identify the fusion proteins and resolve their mechanism. We developed and benchmarked an in vitro biophysical assay to investigate EV membrane fusion. The assay was standardized by directly comparing EV and viral fusion with liposomes. We show that EVs and retroviruses fuse with liposomes mimicking the membrane composition of the late endosome in a pH- and protein-dependent manner. Moreover, we directly visualize the stages of membrane fusion using cryo-electron tomography. We find that, unlike most retroviruses, EVs remain fusogenic after acidification and reneutralization. These results provide novel insights into the EV cargo delivery mechanism and an experimental approach to identify the EV fusion machinery.

Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses.

On the 24 th November 2021 the sequence of a new SARS CoV-2 viral isolate spreading rapidly in Southern Africa was announced, containing far more mutations in Spike (S) than previously reported variants. Neutralization titres of Omicron by sera from vaccinees and convalescent subjects infected with early pandemic as well as Alpha, Beta, Gamma, Delta are substantially reduced or fail to neutralize. Titres against Omicron are boosted by third vaccine doses and are high in cases both vaccinated and infected by Delta. Mutations in Omicron knock out or substantially reduce neutralization by most of a large panel of potent monoclonal antibodies and antibodies under commercial development. Omicron S has structural changes from earlier viruses, combining mutations conferring tight binding to ACE2 to unleash evolution driven by immune escape, leading to a large number of mutations in the ACE2 binding site which rebalance receptor affinity to that of early pandemic viruses.

Golgi organization is regulated by proteasomal degradation.

The Golgi is a dynamic organelle whose correct assembly is crucial for cellular homeostasis. Perturbations in Golgi structure are associated with numerous disorders from neurodegeneration to cancer. However, whether and how dispersal of the Golgi apparatus is actively regulated under stress, and the consequences of Golgi dispersal, remain unknown. Here we demonstrate that 26S proteasomes are associated with the cytosolic surface of Golgi membranes to facilitate Golgi Apparatus-Related Degradation (GARD) and degradation of GM130 in response to Golgi stress. The degradation of GM130 is dependent on p97/VCP and 26S proteasomes, and required for Golgi dispersal. Finally, we show that perturbation of Golgi homeostasis induces cell death of multiple myeloma in vitro and in vivo, offering a therapeutic strategy for this malignancy. Taken together, this work reveals a mechanism of Golgi-localized proteasomal degradation, providing a functional link between proteostasis control and Golgi architecture, which may be critical in various secretion-related pathologies.

One-Pot Synthesis and Evaluation of Antileishmanial Activities of Functionalized S-Alkyl/Aryl Benzothiazole-2-carbothioate Scaffold.

The synthesis of hitherto unreported S-alkyl/aryl benzothiazole-2-carbothioate is reported from thiols, oxalyl chloride, and 2-aminothiophenols using 10 mol % n-tetrabutylammonium iodide (TBAI) as catalyst in acetonitrile through multicomponent reaction (MCR) strategy. The present protocol favored formation of benzothiazoles and thioesters via simultaneous formation of C-N and C-S bonds in good yields with a wide range of substrates. A few of the synthesized derivatives were evaluated for their antimicrobial activity against the protozoan parasite Leishmania donovani, a causative agent of visceral leishmaniasis (VL). Further, these compounds displayed no toxicity toward macrophage RAW 264.7 cells and are therefore nontoxic and effective antileishmanial leads. In silico docking studies were performed to understand the possible binding site interaction with trypanothione reductase (TryR).