Intracellular Uropathogenic E. coli Exploits Host Rab35 for Iron Acquisition and Survival within Urinary Bladder Cells

Recurrent urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC) are common and morbid infections with limited therapeutic options. Previous studies have demonstrated that persistent intracellular infection of bladder epithelial cells (BEC) by UPEC contributes to recurrent UTI in mouse models of infection. However, the mechanisms employed by UPEC to survive within BEC are incompletely understood. In this study we aimed to understand the role of host vesicular trafficking proteins in the intracellular survival of UPEC. Using a cell culture model of intracellular UPEC infection, we found that the small GTPase Rab35 facilitates UPEC survival in UPEC-containing vacuoles (UCV) within BEC. Rab35 plays a role in endosomal recycling of transferrin receptor (TfR), the key protein responsible for transferrin–mediated cellular iron uptake. UPEC enhance the expression of both Rab35 and TfR and recruit these proteins to the UCV, thereby supplying UPEC with the essential nutrient iron. Accordingly, Rab35 or TfR depleted cells showed significantly lower intracellular iron levels and reduced ability to support UPEC survival. In the absence of Rab35, UPEC are preferentially trafficked to degradative lysosomes and killed. Furthermore, in an in vivo murine model of persistent intracellular infection, Rab35 also colocalizes with intracellular UPEC. We propose a model in which UPEC subverts two different vesicular trafficking pathways (endosomal recycling and degradative lysosomal fusion) by modulating Rab35, thereby simultaneously enhancing iron acquisition and avoiding lysosomal degradation of the UCV within bladder epithelial cells. Our findings reveal a novel survival mechanism of intracellular UPEC and suggest a potential avenue for therapeutic intervention against recurrent UTI.     

Nitric oxide/cGMP protects endothelial cells from hypoxia-mediated leakiness

Leakiness of the endothelial bed is attributed to the over-perfusion of the pulmonary bed, which leads to high altitude pulmonary edema (HAPE). Inhalation of nitric oxide has been successfully employed to treat HAPE patients. We hypothesize that nitric oxide intervenes in the permeability of the pulmonary macrovascular endothelial bed to rectify the leaky bed under hypoxia. Our present work explores the underlying mechanism of 'hypoxia-mediated' endothelial malfunction by using human umbilical cord-derived immortalized endothelial cells, ECV-304, and bovine pulmonary artery primary endothelial cells. The leakiness of the endothelial monolayer was increased by two-fold under hypoxia in comparison to cells under normoxia, while optical tweezers-based tethering assays reported a higher membrane tension of endothelial cells under hypoxia. Phalloidin staining demonstrated depolymerization of F-actin stress fibers and highly polarized F-actin patterns in endothelial cells under hypoxia. Nitric oxide, 8-Br-cGMP and sildenafil citrate (phosphodiesterase type 5 inhibitor) led to recovery from hypoxia-induced leakiness of the endothelial monolayers. Results of the present study also suggest that 'hypoxia-induced' cytoskeletal rearrangements and membrane leakiness are associated with the low nitric oxide availability under hypoxia. We conclude that nitric oxide-based recovery of hypoxia-induced leakiness of endothelial cells is a cyclic guanosine monophosphate (cGMP)-dependent phenomenon.     

Phosphatases and phosphate affect the formation of glucose from pentoses in Escherichia coli

Metabolically engineered Escherichia coli MEC143 with deletions of the ptsG, manZ, glk, pfkA, and zwf genes converts pentoses such as arabinose and xylose into glucose, with the dephosphorylation of glucose‐6‐phosphate serving as the final step. To determine which phosphatase mediates this conversion, we examined glucose formation from pentoses in strains containing knockouts of six different phosphatases singly and in combination. Deletions of single phosphatases and combinations of multiple phosphatases did not eliminate the accumulation of glucose from xylose or arabinose. Overexpression of one phosphatase, haloacid dehalogenase‐like phosphatase 12 coded by the ybiV gene, increased glucose yield significantly from 0.26 to 0.30 g/g (xylose) and from 0.32 to 0.35 g/g (arabinose). Growing cells under phosphate‐limited steady‐state conditions increased the glucose yield to 0.39 g glucose/g xylose, but did not affect glucose yield from arabinose (0.31 g/g). No single phosphatase is exclusively responsible for the conversion of glucose‐6‐phosphate to glucose in E. coli MEC143. Phosphate‐limited conditions are indeed able to enhance glucose formation in some cases, with this effect likely influenced by the different phosphate demands when E. coli metabolizes different carbon sources.     

Protective effect of Withania somnifera (Solanaceae) on collagen glycation and cross-linking

Modification of collagen such as non-enzymatic glycation and cross-linking plays an important role in diabetic complications and age-related diseases. We evaluate the effect of Withania somnifera on glucose-mediated collagen glycation and cross-linking in vitro. Extent of glycation, viscosity, collagen-linked fluorescence and pepsin solubility were assessed in different experimental procedures to investigate the effect of W. somnifera. Tail tendons obtained from rats (Rattus norvegicus) weighing 250-275 g were incubated with 50 mM glucose and 100 mg of metformin or Withania root powder or ethanolic extract of Withania under physiological conditions of temperature and pH for 30 days. Formation of advanced glycation end products (AGE) was measured by fluorescent method whereas the cross-linking of collagen was assessed by pepsin digestion and viscosity measurements. Tendon collagen incubated with glucose showed an increase in glycation, AGE and cross-linking of collagen. The collagen incubated with W. somnifera and metformin ameliorates these modifications. The ethanolic extract of Withania showed more prominent effect than Withania root powder. The activity of ethanolic extract of Withania is comparable to metformin, a known antiglycating agent. In conclusion, Withania could have therapeutic role in the prevention of glycation induced pathogenesis in diabetes mellitus and aging.     

Validation of Dolphin dosimetry in three dimensional patient-specific quality assurance programme

AimThe aim of this study is to commission and validate Dolphin-Compass dosimetry as a patient-specific Quality Assurance (QA) device.BackgroundThe advancement of radiation therapy in terms of highly conformal delivery techniques demands a novel method of patient-specific QA. Dolphin-Compass system is a dosimetry solution capable of doing different QA in radiation therapy.Materials and methodsDolphin, air-vented ionization detector array mounted on Versa-HD Linear Accelerator (LINAC) was used for measurements. The Compass is a dose computation algorithm which requires modelling of LINAC head similar to other Treatment Planning Systems (TPS). The dosimetry system was commissioned after measuring the required beam data. The validation was performed by comparison of treatment plans generated in Monaco TPS against the measurement data. Different types of simple, complex, static and dynamic radiation fields and highly conformal treatment plans of patients were used in this study.ResultsFor all field sizes, point doses obtained from Dolphin-Compass dosimetry were in good agreement with the corresponding TPS calculated values in most of the regions, except the penumbra, outside field and at build-up depth. The results of gamma passing rates of measurements by using different Multi-leaf Collimator patterns and Intensity Modulated Radiation Therapy fluence were also found to be in good correlation with the corresponding TPS values.ConclusionsThe commissioning and validation of dosimetry was performed with the help of various fields, MLC patterns and complex treatment plans. The present study also evaluated the efficiency of the 3D dosimetry system for the QA of complex treatment plans.