Month: January 2020

In contrast, cysteine exposure reduced the biofilm biomass as effectively as HICA at acidic pH, although no reduction in metabolic activity was observed. Cysteine is vital for cellular function, but high levels cause accumulation of toxic sulfite. This is known to be toxic to bacteria and fungi but its impact on biofilms has not been studied. Although a very low concentration of ethanol was used in this study a reduction in biofilm biomass of 33% at pH 5.2 and 22% at pH 7.4 was observed. This is of clinical interest as ethanol is used in antifungal lock therapy against Candida spp. biofilms. In the human body ACH is metabolized into acetate mainly by the mitochondrial Ald enzyme. In our study, ALD5 was the only Ald enzyme encoding gene highly expressed in C. albicans biofilms in all conditions tested. C. albicans ALD5 is an ortholog of S. cerevisiae ALD5, which encodes the mitochondrial Ald enzyme. Previously our group showed that ALD6, encoding the cytosolic counterpart, is highly expressed in hypoxic conditions in planktonically grown cells and the expression correlated well with ACH levels. In early biofilms exposed to cysteine and PBS, a mild increase in the basal expression of ALD6 relative to the reference gene was observed thus supporting the finding of planktonic cultures. There was a correlation between ACH production and ALD5 expression in our study. Impairment of pyruvate bypass downstream metabolism by down-regulation of ALD5 was observed together with high ACH levels in caspofungin biofilms. ADH1 was also highly expressed but not in all conditions. On exposure to caspofungin, both ADH1 and ADH2 were down-regulated potentially resulting in ACH accumulation. This down-regulation correlates with the results of a previous study on 30 h C. albicans biofilms. The positive correlation between expression of ADH1 and ADH2 seen in this study implies a functional role of Adh2p alongside Adh1p. This finding is in line with previous work on planktonic cultures in hypoxia. The highest levels of ADH1 expression were observed after exposure to PBS, HICA and cysteine and mainly at acidic pH. Gene expression from PBS biofilms reflected an induction of fermentation and pyruvate bypass as many genes in the pathway were highly expressed. Considering the toxicity of HICA and cysteine particularly at acidic pH, the up-regulation of ADH1 and ALD5 could be a response to oxidative stress and impairment of other respiratory functions. Interestingly, upregulation of ADH1 has been noted in apoptosis. The significantly high basal expression of ADH1 and ALD5 in this study highlights their role in biofilms and supports the findings by others in vitro and in vivo. Alcohol abuse is the main etiologic agent in upper digestive tract carcinogenesis and multiple studies by our group and others have shown that the carcinogenic effect of alcohol is a result of microbial metabolism of ethanol to ACH in vitro and in vivo.

Understanding the mechanisms of androgen regulation in the prostate gland is important, because the prostate is affected by several different diseases, in particular prostate cancer. Several ways exist to treat prostate cancer and promote cell cycle arrest and/or epithelial cell death. Treatments involving androgen manipulation include surgical castration, antiandrogens, or substances that inhibit androgen synthesis. 17b-estradiol exerts anti-androgen effects by blocking the hypothalamic production of gonadotropinreleasing hormone and thereby inhibiting the production of testosterone by the testes, but also acts locally via interactions with either of the estrogen receptors found in the gland. The two major drawbacks to the use of antiandrogens or androgen deprivation therapies are the systemic side effects, including physiological and behavioral changes on the one hand, and progression to castration-resistant prostate cancer, which is more aggressive than the original disease, on the other. Although androgens are highly important for prostate cancer development, after androgen deprivation the disease progresses to a castration-resistant state that may be driven by AR mutations, amplifications and/or ligand-independent activation, which can keep the prostate epithelial cells alive in an androgen-poor environment. In addition to the mechanisms centered on AR expression and functioning, a variety of chromosomal and physiological changes are associated with PCa progression, and chromosome aberrations, including frequent bridging. Previous analyses of gene expression revealed significant aspects of prostate physiology. These studies employed different strategies to obtain the data, and arrived at different subsets of genes that are differentially expressed in response to challenging hormonal conditions. Given the extreme drop in secretory function in response to androgen, and the complex interactions between the epithelium and the stroma, it is possible that subtle changes in physiologically important factors are obscured in the mass of information obtained. For instance, Desai et al. pointed out a progressive increase in PTEN expression in the epithelial cells and several genes grouped together to characterize an “immune-inflammatory” response, which was validated and correlated with a high concentration of immune-system cells including macrophages, mast cells and lymphocytes. The concentration of these cells is another complicating component in the analyses of gene expression, because they contribute their own mRNA. Asivartham et al. worked with isolated cells in primary cultures, but in these conditions, the contribution of mutual stromal-epithelial interactions is absent. We therefore hypothesized that a better understanding of the nature of the cells that survive castration would benefit the search for strategies to allow a blockade or at least an extension of the time needed for the transition to the CRPC.

Through the flexibility of Ea-Fa loops and Da-Ea loops by the glycine residues at Gly67, Gly59, and Gly26, which support the importance of flexibility in the most dynamically moving structures, EF loops, Ea helix, and Ja helix. On the contrary, residues Tyr52, Gly51, Lys97, and Gly99 spatially clustered near the GH hairpin and CD loops suggest the importance of the structure stabilization role of the salt bridge between K97 and E56. This data further supports the highly conserved salt-bridge in PAS domains, and the only function retaining mutations at these residues are K97R and E56D, which are capable of forming arginine-glutamate salt bridge and lysine-aspartate salt bridge, respectively. We speculated that this salt bridge must be conserved to stabilize the structure in functional form. This notion is further supported by the fact that the salt bridge plays a structure-stabilizing role rather than a role in the receptor activation of the phot1 LOV2 domain in A. thaliana. Our method differs from the general screening of positive phenotype by random mutagenesis, as the negative phenotype is the main target. However, our deep mutational scanning data also indicates 329 unique mutations that preserve the function along with the enrichment of each mutation. Although the variant selection method was based on the presence or absence of function, the analytical approach can be more useful if applied to other mutation induction methods coupled with functional assays. The diverse range of functional variants can be sub-categorized by increase, decrease, retention, and complete loss of function. Consequently, enriched residues for different functional subcategories can be analyzed using our analytical method that allows comparison of different and conserved enrichment locations between subcategories. Thus, allowing functional weight determination of amino acid residues enables more detailed analysis of protein sequence and function relationship. Another application is the functional analysis of co-existing mutations. The synthetic functional rescue library is a library screened for function gain from loss of function mutants. This method coupled with multiple barcoded adaptors for each cycles, allow the analysis of co-existing mutations similar to the synthetic lethality concept. We suspect that this analysis of multiple mutant behaviors adds another dimension to the investigation of protein function. Our study determined that the most sensitive residues of EcFbFP lies among the bond-formation residues of the FMNbinding pocket, dynamic conformational changing turns and loops, and cluster of residues near the E56-K97 salt-bridge formation sites. These results indicate that similar methodology can be applied to find functionally important residues in a wide array of enzymes. Deep mutational scanning was focused on the functionally sensitive residues to find residues that play important roles in EcFbFP as well as the LOV domain.

The TIMI risk score offers clinical applications as it categorizes patients with a wide, about 5- to 10-fold, range of major adverse clinical events risk into different risk groups. Indeed, patients with intermediate and high-risk scores, in particular those with prior history of PCI and CABG, have been shown to benefit most from an early invasive strategy as compared to low-risk patients. Therefore, a modified TIMI risk score devoid of the biomarker component was used in this study for comprehensive clinical risk assessment at patient admission to the emergency department. In this patient cohort, the clinical TIMI risk score outranged the clinical GRACE risk score in predicting CE at 30 days. These findings might at least in part be due to the different clinical criteria incorporated in the two risk scores and the different weighting of each criterion. While the GRACE risk score focuses more on clinical parameters on admission such as heart rate and systolic blood pressure, the TIMI risk score incorporates patient history including risk factors for coronary artery disease, known coronary artery disease, the use of antiplatelet therapy, and severe episodes of angina. Moreover, the endpoint definition of this study varies from the ones used to validate these risk scoring systems, and limited predictive value of the GRACE risk score has previously been described. However, this study was not designed to allow a comparison between different risk scoring systems, and further studies are needed to compare predictive values of risk scores in different subsets of patients. In ST-elevation patients, distinctive ECG patterns usually determine an early invasive strategy with rare contraindications. However, the heterogenous population of Non-ST-elevation patients requires an appropriate patient selection for early revascularization. Although the combination of clinical parameters or risk scores, respectively with several conventional markers such as c-cTnT and NT-proBNP have occasionally been suggested, our findings show for the first time that integrating clinical and novel cardiac biomarker data including continuous hs-cTnT levels best predicted CE at 30 days in Non-ST-elevation patients. Stand-alone, cardiac biomarkers including hs-cTnT were not better predictors of CE compared to clinical judgment using the modified TIMI risk score. These findings further strengthen the value of traditional clinical practice in assessing the probability that the symptoms represent cardiac ischemia. Both safety issues and limitations of health care resources demand the effective targeting of therapy to those who are likely to benefit most in the heterogeneous population of Non-ST-elevation patients. The use of the highly sensitive troponin assays has substantially increased the number of chest pain patients tested troponin positive. This bears the potential of setting off an avalanche of ischemia-related diagnostics.

Ionizing radiation is also an effective method for treating tumors because it can be localized to the tumor and is a potent inducer of DNA double-strand breaks, a highly toxic form of DNA damage. While much has been learned about x-ray and gamma-ray effects on cells and whole organisms, less is known about the biological effects of neutrons. Neutrons are highly energetic uncharged particles that induce more severe DNA damage than photons and are therefore more effective than photons in controlling radioresistant tumors. The relative biological effectiveness of neutrons has been reported to be as low as 1 and perhaps higher than 10 depending on the tissue type, neutron energy and the biological endpoint being measured. Neutrons were listed as a carcinogen for the first time in the Eleventh Report on Carcinogens. High levels of neutron irradiation occur in patients receiving neutron therapy, while low levels of neutron exposure occur in patients treated with high energy photons and protons. Other sources of low level neutron irradiation may include occupational exposure to workers at nuclear power plants and accelerator facilities, astronauts, airline crews and passengers on high altitude flights, as well as radiation incidents such as the Hiroshima-Nagasaki atomic bomb explosions and the tsunami-induced radiation leak at the Fukushima Daiichi site in Japan. One of the major paradigm shifts in the field of radiation biology was the discovery of non-targeted effects such as the bystander effect in which cells in the vicinity of radiation-damaged cells behave as though they were also irradiated. In addition, late effects such as chromosomal instability may increase susceptibility to cancer. Thus, cells that are directly damaged are not the sole targets of radiation exposure. Cells that do not absorb radiation directly may nevertheless be damaged or altered in ways that do not become apparent for many cell generations. Such non-targeted effects may have serious implications for human health and may cause cancer. Therefore, the risks of ionizing radiation need to be analyzed in terms of both direct and non-targeted effects. The bystander effect has been observed repeatedly in mammalian cell lines, including human skin fibroblasts, epithelial cells and leukemic cells in response to ionizing photons. Depending upon the cell and tissue type, bystander signals can be transmitted either through the culture medium or by cellto-cell contact including gap junctional communication. Some of the candidate intercellular signaling molecules that have been implicated in bystander effects are reactive oxygen species, reactive nitrogen species, nitric oxide, cytokines such as TGFb and interleukin 8, and small molecules such as amino acids. The involvement of intracellular signaling molecules including mitogen-activated protein kinases and their downstream proteins, protein kinase C isoforms.