A novel cell biological tool to explain mechanics and dynamics in fission yeast.

The Rho guanosine triphosphatase hydrolase enzyme (GTPase) is required for the control of the actin cytoskeleton, but its activation in vivo condition is unknown. The study's goal was to find a new synthetic nanobody VHH (P-36 tagged with mNeonGreen) that interacts strongly with the Rho GTPase. We present the first novel synthetic nanobody, VHH (P-36 tagged with mNeonGreen), tested in fission yeast cells and found to have a particular interaction with Rho1GTPase. Plasmids were constructed by using of certain enzymes to digest the pDUAL-pef1a vector plasmid to produce a protein that was encoded by cloned genes. A varied VHH library was created synthetically, then transformed into yeast cells, and positive clones were chosen using chemical agents. To investigate protein interactions and cellular reactions, several studies were carried out, such as live cell imaging, growth curve analysis, coimmunoprecipitation, structural analysis, and cell therapies. Prism and RStudio were used for the statistical analysis. The presence of VHH (P-36) has no effect on the growth pattern making it an appropriate model for studying cytokinesis in vivo. According to a computational biological study, its affinity to interact with Rho1GTPase with all the complementarity-determining region (CDR) regions found on VHH (P-36) is extremely strong. We were able to track its subcellular target by localization using a fluorescent confocal microscope, ensuring the maintenance of cell polarity and morphology. Spheroplast analysis revealed a circular-shaped cell with an even distribution of Rho1 tagged VHH (P-36), indicating that the interaction occurs near the plasma membrane. The introduction of latrunculin-A (Lat-A) disrupted Rho GTPase localization, demonstrating the control over actin production, and the cell did not show evidence of mitotic phase commencement while Lat-A was present. Finally, this important biological tool can aid in our understanding of the mechanics and dynamics of cytokinesis in relation to Rho1GTPase.

Detection of Mycobacterium tuberculosis in AFB smear-negative sputum specimens through MTB culture and GeneXpert® MTB/RIF assay.

Tuberculosis (TB) is an important public health issue around the globe which is a chronic infectious disease and is still one of the major challenges for developing countries. The emergence of drug-resistant TB makes the condition worse and there is an urgent need of fast, highly sensitive diagnostic methods. This study was undertaken to evaluate the performance of GeneXpert® MTB/RIF assay and MTB culture for the detection of Mycobacterium tuberculosis (MTB) in sputum smear-negative pulmonary TB/drug-resistant tuberculosis (DR-TB) suspects. A total of 168 sputum smear-negative TB suspects were recruited for the study. Among the suspected TB cases, 52.98% were male and 47.02% were females with the mean age of 42 ± 17.6 years. All the sputum specimens collected from the study population were subjected to Ziehl-Neelsen (ZN) smear microscopy, GeneXpert MTB/RIF assay, and MTB culture. The results revealed that, out of 168 acid-fast bacilli (AFB)/ZN smear microscopy-negative sputum specimens, 48 (28.57%) and 58 (34.52%) were detected MTB positive by GeneXpert MTB/RIF assay and MTB culture, respectively, while 120 (71.43%) and 110 (65.48%) suspected TB cases were confirmed negative by GeneXpert MTB/RIF assay and MTB culture, respectively. The study concluded that GeneXpert assay was found to be a rapid and accurate tool for MTB detection in smear-negative sputum specimens. GeneXpert has advantage over ZN smear microscopy and MTB culture as it detects MTB and rifampicin resistance simultaneously within 2 h with minimal biohazards.