Distinct effects of cardiac mitochondrial calcium uniporter inactivation via EMRE deletion in the short and long term.

Transport of Ca2+ into mitochondria is thought to stimulate the production of ATP, a critical process in the heart's fight or flight response, but excess Ca2+ can trigger cell death. The mitochondrial Ca2+ uniporter complex is the primary route of Ca2+ transport into mitochondria, in which the channel-forming protein MCU and the regulatory protein EMRE are essential for activity. In previous studies, chronic Mcu or Emre deletion differed from acute cardiac Mcu deletion in response to adrenergic stimulation and ischemia/reperfusion (I/R) injury, despite equivalent inactivation of rapid mitochondrial Ca2+ uptake. To explore this discrepancy between chronic and acute loss of uniporter activity, we compared short-term and long-term Emre deletion using a novel conditional cardiac-specific, tamoxifen-inducible mouse model. After short-term Emre deletion (3 weeks post-tamoxifen) in adult mice, cardiac mitochondria were unable to take up Ca2+, had lower basal mitochondrial Ca2+ levels, and displayed attenuated Ca2+-induced ATP production and mPTP opening. Moreover, short-term EMRE loss blunted cardiac response to adrenergic stimulation and improved maintenance of cardiac function in an ex vivo I/R model. We then tested whether the long-term absence of EMRE (3 months post-tamoxifen) in adulthood would lead to distinct outcomes. After long-term Emre deletion, mitochondrial Ca2+ handling and function, as well as cardiac response to adrenergic stimulation, were similarly impaired as in short-term deletion. Interestingly, however, protection from I/R injury was lost in the long-term. These data suggest that several months without uniporter function are insufficient to restore bioenergetic response but are sufficient to restore susceptibility to I/R.

Orthovoltage X-Rays Exhibit Increased Efficacy Compared with γ-Rays in Preclinical Irradiation.

Radionuclide irradiators (137Cs and 60Co) are commonly used in preclinical studies ranging from cancer therapy to stem cell biology. Amidst concerns of radiological terrorism, there are institutional initiatives to replace radionuclide sources with lower energy X-ray sources. As researchers transition, questions remain regarding whether the biological effects of γ-rays may be recapitulated with orthovoltage X-rays because different energies may induce divergent biological effects. We therefore sought to compare the effects of orthovoltage X-rays with 1-mm Cu or Thoraeus filtration and 137Cs γ-rays using mouse models of acute radiation syndrome. Following whole-body irradiation, 30-day overall survival was assessed, and the lethal dose to provoke 50% mortality within 30-days (LD50) was calculated by logistic regression. LD50 doses were 6.7 Gy, 7.4 Gy, and 8.1 Gy with 1-mm Cu-filtered X-rays, Thoraeus-filtered X-rays, and 137Cs γ-rays, respectively. Comparison of bone marrow, spleen, and intestinal tissue from mice irradiated with equivalent doses indicated that injury was most severe with 1-mm Cu-filtered X-rays, which resulted in the greatest reduction in bone marrow cellularity, hematopoietic stem and progenitor populations, intestinal crypts, and OLFM4+ intestinal stem cells. Thoraeus-filtered X-rays provoked an intermediate phenotype, with 137Cs showing the least damage. This study reveals a dichotomy between physical dose and biological effect as researchers transition to orthovoltage X-rays. With decreasing energy, there is increasing hematopoietic and intestinal injury, necessitating dose reduction to achieve comparable biological effects. SIGNIFICANCE: Understanding the significance of physical dose delivered using energetically different methods of radiation treatment will aid the transition from radionuclide γ-irradiators to orthovoltage X-irradiators.

Cellular Prion Protein PrPC and Ecto-5'-Nucleotidase Are Markers of the Cellular Stress Response to Aneuploidy.

Aneuploidy is a hallmark of most human tumors, but the molecular physiology of aneuploid cells is not well characterized. In this study, we screened cell surface biomarkers of approximately 300 proteins by multiparameter flow cytometry using multiple aneuploid model systems such as cell lines, patient samples, and mouse models. Several new biomarkers were identified with altered expression in aneuploid cells, including overexpression of the cellular prion protein CD230/PrPC and the immunosuppressive cell surface enzyme ecto-5'-nucleotidase CD73. Functional analyses associated these alterations with increased cellular stress. An increased number of CD73+ cells was observed in confluent cultures in aneuploid cells relative to their diploid counterparts. An elevated expression in CD230/PrPC was observed in serum-deprived cells in association with increased generation of reactive oxygen species. Overall, our work identified biomarkers of aneuploid karyotypes, which suggest insights into the underlying molecular physiology of aneuploid cells. Cancer Res; 77(11); 2914-26. ©2017 AACR.

Interaction of chitin/chitosan with salivary and other epithelial cells-An overview.

Chitin and its deacetylated form, chitosan, have been widely used for tissue engineering of both epithelial and mesenchymal tissues. Epithelial cells characterised by their sheet-like tight cellular arrangement and polarised nature, constitute a major component in various organs and play a variety of roles including protection, secretion and maintenance of tissue homeostasis. Regeneration of damaged epithelial tissues has been studied using biomaterials such as chitin, chitosan, hyaluronan, gelatin and alginate. Chitin and chitosan are known to promote proliferation of various embryonic and adult epithelial cells. However it is not clearly understood how this activity is achieved or what are the mechanisms involved in the chitin/chitosan driven proliferation of epithelial cells. Mechanistic understanding of influence of chitin/chitosan on epithelial cells will guide us to develop more targeted regenerative scaffold/hydrogel systems. Therefore, current review attempts to elicit a mechanistic insight into how chitin and chitosan interact with salivary, mammary, skin, nasal, lung, intestinal and bladder epithelial cells.

Antibiotic overusage and resistance: A cross-sectional survey among pediatric dentists.

INTRODUCTION: Most human orofacial infections originate from odontogenic infections and prescribing antibiotics has become a ubiquitous phenomenon. The World Health Organization (WHO) has recognized the inappropriate, indiscriminate, and irrational use of antibiotics leading to antibiotic resistance as a global problem. OBJECTIVE: The objective of this survey is to compare the antibiotic prescription pattern and the awareness of antibiotic resistance among Bachelor of Dental Surgery (BDS) practitioners and pediatric dentists. MATERIALS AND METHODS: A hundred BDS practitioners and 100 pediatric dentists included in the study were given a questionnaire containing both open-ended and closed-ended questions. The questionnaire comprised information pertaining to antibiotic prescription for most common oral conditions, commonly prescribed antibiotics, their dosage, etc. RESULTS: The majority of the practitioners prescribed antibiotics for managing oral diseases. On comparing the prescription patterns between the BDS practitioners and pediatric dentists, there was an overprescription in the BDS group for many conditions, which was statistically significant. Amoxicillin was the most commonly prescribed drug in both the groups. In the presence of an anaerobic infection, the most preferred drug was a combination of amoxicillin and clavulanic acid with metronidazole. With regard to the duration of antibiotic prescription, 74% BDS practitioners prescribed antibiotics as a 3-day course and 60% pediatric dentists resorted to a 5-day course, which was statistically significant. The awareness regarding antibiotic prophylaxis and antibiotic resistance was found to be adequate in both the groups. However, there was a general lack of awareness with regard to the guidelines for antibiotic prescribing in both the groups. CONCLUSION: Practitioners should prescribe antibiotics in accordance with the guidelines to curb antibiotic resistance, an emerging public health problem.

Multiple nontuberculous scrofulodermas showing dramatic response to clarithromycin.

Atypical mycobacteria are distinct from the Mycobacterium tuberculosis. Mycobacterium chelonae, a non-pigment producing rapid grower, can be found in many cutaneous sites; infection occurs most commonly after skin trauma from surgery, injections, or minor injuries. In immune competent patients, the infection is more frequently localized as a cellulitis or a nodule, whereas, in the immunocompromised patient, dissemination (more than five lesions) can occur. Because the organism is resistant to antituberculous therapy, abscess can develop and follow a chronic, indolent course. We report a case of multiple scrofuloderma due to nontuberculous infection caused by M. chelonae showing dramatic response to clarithromycin.

Purification and ex vivo expansion of fully functional salivary gland stem cells.

Hyposalivation often leads to irreversible and untreatable xerostomia. Salivary gland (SG) stem cell therapy is an attractive putative option to salvage these patients but is impeded by the limited availability of adult human tissue. Here, using murine SG cells, we demonstrate single-cell self-renewal, differentiation, enrichment of SG stem cells, and robust in vitro expansion. Dependent on stem cell marker expression, SG sphere-derived single cells could be differentiated in vitro into distinct lobular or ductal/lobular organoids, suggestive of progenitor or stem cell potency. Expanded cells were able to form miniglands/organoids containing multiple SG cell lineages. Expansion of these multipotent cells through serial passaging resulted in selection of a cell population, homogenous for stem cell marker expression (CD24(hi)/CD29(hi)). Cells highly expressing CD24 and CD29 could be prospectively isolated and were able to efficiently restore radiation-damaged SG function. Our approach will facilitate the use of adult SG stem cells for a variety of scientific and therapeutic purposes.

Salisphere derived c-Kit+ cell transplantation restores tissue homeostasis in irradiated salivary gland.

INTRODUCTION: During radiotherapy salivary glands of head and neck cancer patients are unavoidably co-irradiated, potentially resulting in life-long impairment. Recently we showed that transplantation of salisphere-derived c-Kit expressing cells can functionally regenerate irradiated salivary glands. This study aims to select a more potent subpopulation of c-Kit(+) cells, co-expressing stem cell markers and to investigate whether long-term tissue homeostasis is restored after stem cell transplantation. METHODS AND RESULTS: Salisphere derived c-Kit(+) cells that co-expressed CD24 and/or CD49f markers, were intra-glandularly injected into 15 Gy irradiated submandibular glands of mice. Particularly, c-Kit(+)/CD24(+)/CD49f(+) cell transplanted mice improved saliva production (54.59 ± 11.1%) versus the irradiated control group (21.5 ± 8.7%). Increase in expression of cells with differentiated duct cell markers like, cytokeratins (CK8, 18, 7 and 14) indicated functional recovery of this compartment. Moreover, ductal stem cell marker expression like c-Kit, CD133, CD24 and CD49f reappeared after transplantation indicating long-term functional maintenance potential of the gland. Furthermore, a normalization of vascularization as indicated by CD31 expression and reduction of fibrosis was observed, indicative of normalization of the microenvironment. CONCLUSIONS: Our results show that stem cell transplantation not only rescues hypo-salivation, but also restores tissue homeostasis of the irradiated gland, necessary for long-term maintenance of adult tissue.

Regeneration of irradiated salivary glands with stem cell marker expressing cells.

BACKGROUND: Stem cell therapy could be a potential way for reducing radiation-induced hyposalivation and improving the patient's quality of life. However, the identification and purification of salivary gland stem cells have not been accomplished. This study aims to better characterize the stem/progenitor cell population with regenerative potential residing in the mouse salivary gland. METHODS: Mouse submandibular gland tissue, isolated cells and cultured 3 day old salispheres were tested for their expression of stem cell markers c-Kit, CD133, CD49f, and CD24 using immunohistochemistry for tissue and flow cytometry for cells. Mice were locally irradiated with a single dose of 15 Gy and transplanted with cells expressing defined markers. RESULTS: Cells expressing known stem cell markers are localized in the larger ducts of the mouse salivary gland. Isolated cells and cells from day 3 salispheres also express these markers: c-Kit (0.058% vs. 0.65%), CD133 (6% vs. 5%), CD49f (78% vs. 51%), and CD24 (60% vs. 60%, respectively). Intraglandular transplantation of these cells into irradiated salivary glands of mice resulted in stem cell marker-specific recovery of salivary gland function. CONCLUSIONS: Different stem cell-associated markers are expressed in mouse salivary gland cells, which upon transplantation are able to regenerate the irradiation damaged salivary gland.

Isolation of mouse salivary gland stem cells.

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process. In the manifestation of diseases such as Sjögren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer. The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

Effectiveness of recombinant soybean cysteine proteinase inhibitors against selected crop pests.

Three recombinant soybean cysteine proteinase inhibitors (rSCPIs), L1, R1 and N2, were assessed for their potential to inhibit the growth and development of three major agricultural crop pests known to utilize digestive cysteine proteinases: Western corn rootworm (Diabrotica virgifera virgifera, WCR), Colorado potato beetle (Leptinotarsa decemlineata, CPB) and cowpea weevil (Callosobruchus maculatus, CW). In vitro experiments showed that cysteine proteinase activities in the crude gut extracts of the WCR, CPB, and CW were inhibited to various degrees by the three rSCPIs. Of the three rSCPIs tested, N2 was most effective in inhibiting the crude gut extract of WCR, CPB, and CW (50% inhibition at 5 x 10(-8), 5 x 10(-8), and 3 x 10(-7) M, respectively). The L1 was the least potent of the three CPIs tested, with 50% inhibition at 5 x 10(-6) M of the crude gut extracts of WCR. Results of in vivo experiments conducted to assess the effect of the three rSCPIs on the vital growth parameters of WCR, CPB and CW were consistent with results of the in vitro experiments.

Comparison of chemical characteristics of three soybean cysteine proteinase inhibitors.

Three recombinant soybean cysteine proteinase inhibitors (rSCPIs) L1, R1, and N2 were chemically characterized. These inhibitors have the potential to inhibit the growth and development of three major agricultural crop pests known to utilize cysteine proteinases (CPs) for protein digestion: Western corn rootworm, Colorado potato beetle, and cowpea weevil. Characterization data obtained show differences between the inhibitors and will be needed to consider the use of rSCPIs to create insect resistance in plants.

Plasma and RBCs antioxidant status in occupational male pesticide sprayers.

A total of forty-one (n=41) male, healthy agricultural sprayers, exposed to pesticides for 5 years, were compared with twenty one (n=21) controls matched for age and economic status with respect to free radical generation, lipid peroxidation, antioxidant status and concentration of cellular enzymes were determined. Significantly increased TBARS (thiobarbituric acid reactive substances) were observed (P<0.001) in sprayer populations when compared to controls. The concentration of antioxidants such as glutathione (GSH), alpha-tocopherol, ascorbic acid and ceruloplasmin were significantly altered when compared to controls, and the activities of antioxidant enzymes were remarkably elevated (P<0.001) in sprayer populations, when compared to controls.

Lipid peroxidation, osmotic fragility and antioxidant status in children with acute post-streptococcal glomerulonephritis.

Plasma and erythrocyte samples from acute post-streptococcal glomerulonephritis (APSGN) children and control children were enrolled in this study. Lipid peroxidation (LPO), measured in terms of thiobarbituric acid-reactive substances (TBARS) was found to be significantly increased in plasma and RBCs of APSGN children (P<0.05) than in control children. Osmotic fragility of erythrocytes was examined. RBCs of APSGN patients were found to be osmotically more sensitive towards hypotonic saline (50% hemolysis at 7 g/l saline) when compared to control RBCs (50% hemolysis at 4 g/l saline). The activities of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST) were significantly lowered (P<0.05) in APSGN RBCs when compared to control RBCs. Plasma ascorbic acid, reduced glutathione (GSH), RBC ascorbic acid, GSH and RBC total sulphydryl content (TSH) were significantly depleted in APSGN children relative to controls. The susceptibility of RBCs of APSGN children to lipid peroxidation was confirmed in this study.