A standalone approach to utilize telomere length measurement as a surveillance tool in oral leukoplakia.

Oral squamous cell carcinoma (OSCC) is often preceded by a white patch on a surface of the mouth, called oral leukoplakia (OL). As accelerated telomere length (TL) shortening in dividing epithelial cells may lead to oncogenic transformation, telomere length measurement could serve as a predictive biomarker in OL. However, due to high variability and lack of a universal reference, there has been a limited translational application. Here, we describe an approach of evaluating TL using paired peripheral blood mononuclear cells (PBMC) as an internal reference and demonstrate its translational relevance. Oral brush biopsy and paired venous blood were collected from 50 male OL patients and 44 male healthy controls (HC). Relative TL was measured by quantitative PCR. TL of each OL or healthy sample was normalized to the paired PBMC sample (TL ratio). In OL patients, the mean TL ratio was significantly smaller not only in the patch but also in distal normal oral tissue, relative to healthy controls without a high-risk oral habit. Dysplasia was frequently associated with a subgroup that showed a normal TL ratio at the patch but significantly smaller TL ratio at a paired normal distal site. Our data suggest that evaluation of TL attrition using a paired PBMC sample eliminates the requirement of external reference DNA, makes data universally comparable and provides a useful marker to define high-risk OL groups for follow-up programs. Larger studies will further validate the approach and its broader application in other premalignant conditions.

A Novel Calcium Uptake Transporter of Uncharacterized P-Type ATPase Family Supplies Calcium for Cell Surface Integrity in Mycobacterium smegmatis.

Ca2+ plays an important role in the physiology of bacteria. Intracellular Ca2+ concentrations are tightly maintained in the nanomolar range. Molecular mechanisms of Ca2+ uptake in bacteria remain elusive. Here we show that CtpE is responsible for Ca2+ uptake in Mycobacterium smegmatis It represents a previously uncharacterized P-type ATPase family in bacteria. Disruption of ctpE in M. smegmatis resulted in a mutant with impaired growth under Ca2+-deficient conditions. The growth defect of the mutant could be rescued by Ca2+ or by ectopic expression of ctpE from M. smegmatis or the orthologous gene (Rv0908) from Mycobacterium tuberculosis H37Rv. Radioactive transport assays revealed that CtpE is a Ca2+-specific transporter. Ca2+ deficiency increased expression of ctpE, resulting in increased 45Ca2+ accumulation in cells. ctpE is a gene that is part of an operon, which is negatively regulated by Ca2+ The ctpE mutant also showed hypersensitivity to polymyxin B, increased biofilm formation, and higher cell aggregation, indicating cell envelope defects. Our work establishes, for the first time, the presence of Ca2+ uptake pumps of the energy-dependent P-type ATPase superfamily in bacteria and also implicates that intracellular Ca2+ is essential for growth and cell envelope integrity in M. smegmatisIMPORTANCE Ca2+ is essential for gene regulation, enzymatic activity, and maintenance of structural integrity of cell walls in bacteria. Bacteria maintain intracellular calcium concentrations in a narrow range, creating a gradient with low cytoplasmic calcium concentration and high extracellular calcium concentration. Due to this steep gradient, active pumps belonging to family 2 of P-type ATPases and antiporters are used for Ca2+ efflux, whereas Ca2+ uptake is usually carried out by channels. Molecular mechanisms of Ca2+ uptake in bacteria are still elusive and are mainly limited to a nonproteinaceous channel in Escherichia coli and a pH-dependent channel protein from Bacillus subtilis Energy-dependent active transporters are not reported for Ca2+ uptake from any organism. Here we show that CtpE belonging to a family of previously uncharacterized bacterial P-type ATPases is involved in specific uptake of Ca2+ into Mycobacterium smegmatis We also demonstrate that intracellular Ca2+ obtained through CtpE is essential for growth and maintenance of cell surface properties under Ca2+-deficient conditions.