No abstract

The mycobacteria comprise both pathogenic and nonpathogenic bacteria. Although several features related to pathogenicity in various mycobacterial species, such as , have been studied in great detail, methylotrophy, i.e., the ability of an organism to utilize single-carbon (C) compounds as the sole source of carbon and energy, has remained largely unexplored in mycobacteria. Reports are available that suggest that mycobacteria, including and, are capable of utilizing alternative C compounds to meet their carbon and energy requirements. However, physiological pathways that are functional in mycobacteria to utilize such carbon compounds are only poorly understood. Here we report the identification and characterization of the gene products required for establishing methylotrophy in We present,-dimethyl--nitrosoaniline (NDMA)-dependent methanol oxidase (Mno) as the key enzyme that is essential for the growth of on methanol. We show that Mno has both methanol and formaldehyde dehydrogenase activities Further, is able to utilize methanol even in the absence of the major formaldehyde dehydrogenase MscR, which suggests that Mno is sufficient to dissimilate methanol and the resulting formaldehyde Finally, we show that devoid of phosphoenolpyruvate carboxykinase, which has been shown to fix CO in , does not grow on methanol, suggesting that the final step of methanol utilization requires CO fixation for biomass generation. Our work here thus forms the first comprehensive report that explores methylotrophy in a mycobacterial species. Methylotrophy, the ability to utilize single-carbon (C) compounds as the sole carbon and energy sources, is only poorly understood in mycobacteria. Both pathogenic and nonpathogenic mycobacteria, including , are capable of utilizing C compounds to meet their carbon and energy requirements, although the precise pathways are not well studied. Here we present a comprehensive study of methylotrophy in With several genetic knockouts, we have dissected the entire methanol metabolism pathway in We show that while methanol dissimilation in differs from that in other mycobacterial species, the concluding step of CO fixation is similar to that in It is therefore both interesting and important to examine mycobacterial physiology in the presence of alternative carbon sources.

Oral premalignant lesions (OPLs) such as leukoplakia, erythroplakia, and oral submucous fibrosis, often precede oral cancer. Screening and management of these premalignant conditions can improve prognosis. Raman spectroscopy has previously demonstrated potential in the diagnosis of oral premalignant conditions (in vivo), detected viral infection, and identified cancer in both oral and cervical exfoliated cells (ex vivo). The potential of Raman exfoliative cytology (REC) in identifying premalignant conditions was investigated. Oral exfoliated samples were collected from healthy volunteers (n=20), healthy volunteers with tobacco habits (n=20), and oral premalignant conditions (n=27, OPL) using Cytobrush. Spectra were acquired using Raman microprobe. Spectral acquisition parameters were: λex: 785 nm, laser power: 40 mW, acquisition time: 15 s, and average: 3. Postspectral acquisition, cell pellet was subjected to Pap staining. Multivariate analysis was carried out using principal component analysis and principal component-linear discriminant analysis using both spectra- and patient-wise approaches in three- and two-group models. OPLs could be identified with ∼77% (spectra-wise) and ∼70% (patient-wise) sensitivity in the three-group model while with 86% (spectra-wise) and 83% (patient-wise) in the two-group model. Use of histopathologically confirmed premalignant cases and better sampling devices may help in development of improved standard models and also enhance the sensitivity of the method. Future longitudinal studies can help validate potential of REC in screening and monitoring high-risk populations and prognosis prediction of premalignant lesions.

How animals rewire cellular programs to survive cold is a fascinating problem with potential biomedical implications, ranging from emergency medicine to space travel. Studying a hibernation-like response in the free-living nematode Caenorhabditis elegans, we uncovered a regulatory axis that enhances the natural resistance of nematodes to severe cold. This axis involves conserved transcription factors, DAF-16/FoxO and PQM-1, which jointly promote cold survival by upregulating FTN-1, a protein related to mammalian ferritin heavy chain (FTH1). Moreover, we show that inducing expression of FTH1 also promotes cold survival of mammalian neurons, a cell type particularly sensitive to deterioration in hypothermia. Our findings in both animals and cells suggest that FTN-1/FTH1 facilitates cold survival by detoxifying ROS-generating iron species. We finally show that mimicking the effects of FTN-1/FTH1 with drugs protects neurons from cold-induced degeneration, opening a potential avenue to improved treatments of hypothermia.

We report here a PCR-based cloning methodology that requires no post-PCR modifications such as restriction digestion and phosphorylation of the amplified DNA. The advantage of the present method is that it yields only recombinant clones thus eliminating the need for screening. Two DNA amplification reactions by PCR are performed wherein the first reaction amplifies the gene of interest from a source template, and the second reaction fuses it with the designed expression vector fragments. These vector fragments carry the essential elements that are required for the fusion product selection. The entire process can be completed in less than 8 hours. Furthermore, ligation of the amplified DNA by a DNA ligase is not required before transformation, although the procedure yields more number of colonies upon transformation if ligation is carried out. As a proof-of-concept, we show the cloning and expression of GFP, adh, and rho genes. Using GFP production as an example, we further demonstrate that the E. coli T7 express strain can directly be used in our methodology for the protein expression immediately after PCR. The expressed protein is without or with 6xHistidine tag at either terminus, depending upon the chosen vector fragments. We believe that our method will find tremendous use in molecular and structural biology.