Fungal pathogens have a robust array of multidrug transporters which aid in active expulsion of drugs and xenobiotics to help them evade toxic effects of drugs. Thus, these transporters impose a major impediment to effective chemotherapy. Although the Saccharomyces cerevisiae strain AD1-8u− has catered well to the need of an overexpression system to study drug transport by multidrug transporters of Candida albicans, artifacts associated with a heterologous system could not be excluded. To avoid the issue, we exploited a azole-resistant clinical isolate of C. albicans to develop a new system devoid of three major multidrug transporters (Cdr1p, Cdr2p, and Mdr1p) for the overexpression of multidrug transporters under native hyperactive CDR1 promoter due to gain of function (GOF) mutation in TAC1. The study deals with overexpression and functional characterization of representatives of two major classes of multidrug transporters, Cdr1p and Mdr1p, to prove the functionality of this newly developed endogenous expression system. Expression of native Cdr1 and Mdr1 protein in C. albicans cells was confirmed by confocal microscopy and immunodetection and resulted in increased resistance to the putative substrates as compared to control. The system was further validated by overexpressing a few key mutant variants of Cdr1p and Mdr1p. Together, our data confirms the utility of new endogenous overexpression system which is devoid of artifactual factors as most suited for functional characterization of multidrug transporter proteins of C. albicans.
Candida albicans; multidrug transporters; endogenous overexpression system; Cdr1p; Mdr1p; TAC1 GOF
Injuries and violence are one of the leading causes of mortality worldwide. A substantial portion of these injuries involve the maxillofacial region. Among the concomitant injuries, injuries to the head and cervical spine are amongst those that demand due consideration on account of their life threatening behaviour. Studies have shown that facial fractures have a strong association with traumatic brain injury. Knowledge of the types and mechanisms of traumatic brain injury is crucial for their treatment. Many a times, facial fractures tend to distract our attention from more severe and often life threatening injuries. Early diagnosis of these intracranial haemorrhage leads to prompt treatment which is essential to improve the outcome of these patients. An oral and maxillofacial surgeon should be able to suspect and diagnose head injury and also provide adequate initial management.
Cerebrospinal fluid; Maxillofacial injuries; Diffuse axonal injury; Intracranial pressure; Subdural haematoma; Traumatic brain injury
Diagnosis of extrapulmonary tuberculosis (EPTB) poses serious challenges. A careful selection of appropriate gene targets is essential for designing a multiplex-polymerase chain reaction (M-PCR) assay.
Materials and Methods
We compared several gene targets of Mycobacterium tuberculosis, including IS6110, devR, and genes encoding MPB-64 (mpb64), 38kDa (pstS1), 65kDa (hsp65), 30kDa (fbpB), ESAT-6 (esat6), and CFP-10 (cfp10) proteins, using PCR assays on 105 EPTB specimens. From these data, we chose the two best gene targets to design an M-PCR.
Among all gene targets tested, mpb64 showed the highest sensitivity (84% in confirmed cases and 77.5% in clinically suspected cases), followed by IS6110, hsp65, 38kDa, 30kDa, esat6, cfp10, and devR. We used mpb64+IS6110 for designing an M-PCR assay. Our M-PCR assay demonstrated a high sensitivity of 96% in confirmed EPTB cases and 88.75% in clinically suspected EPTB cases with a high specificity of 100%, taking clinical diagnosis as the gold standard.
These M-PCR results along with the clinical findings may facilitate an early diagnosis of EPTB patients and clinical management of disease.
Mycobacterium tuberculosis; extrapulmonary tuberculosis; PCR; multiplex-PCR; diagnosis
Verbal autopsy (VA) is recognized as the only feasible alternative to comprehensive medical certification of deaths in settings with no or unreliable vital registration systems. However, a barrier to its use by national registration systems has been the amount of time and cost needed for data collection. Therefore, a short VA instrument (VAI) is needed. In this paper we describe a shortened version of the VAI developed for the Population Health Metrics Research Consortium (PHMRC) Gold Standard Verbal Autopsy Validation Study using a systematic approach.
We used data from the PHMRC validation study. Using the Tariff 2.0 method, we first established a rank order of individual questions in the PHMRC VAI according to their importance in predicting causes of death. Second, we reduced the size of the instrument by dropping questions in reverse order of their importance. We assessed the predictive performance of the instrument as questions were removed at the individual level by calculating chance-corrected concordance and at the population level with cause-specific mortality fraction (CSMF) accuracy. Finally, the optimum size of the shortened instrument was determined using a first derivative analysis of the decline in performance as the size of the VA instrument decreased for adults, children, and neonates.
The full PHMRC VAI had 183, 127, and 149 questions for adult, child, and neonatal deaths, respectively. The shortened instrument developed had 109, 69, and 67 questions, respectively, representing a decrease in the total number of questions of 40-55 %. The shortened instrument, with text, showed non-significant declines in CSMF accuracy from the full instrument with text of 0.4 %, 0.0 %, and 0.6 % for the adult, child, and neonatal modules, respectively.
We developed a shortened VAI using a systematic approach, and assessed its performance when administered using hand-held electronic tablets and analyzed using Tariff 2.0. The length of a VA questionnaire was shortened by almost 50 % without a significant drop in performance. The shortened VAI developed reduces the burden of time and resources required for data collection and analysis of cause of death data in civil registration systems.
Electronic supplementary material
The online version of this article (doi:10.1186/s12916-015-0528-8) contains supplementary material, which is available to authorized users.
Verbal autopsy questionnaire; Mortality surveillance; Causes of death
Reliable data on the distribution of causes of death (COD) in a population are fundamental to good public health practice. In the absence of comprehensive medical certification of deaths, the only feasible way to collect essential mortality data is verbal autopsy (VA). The Tariff Method was developed by the Population Health Metrics Research Consortium (PHMRC) to ascertain COD from VA information. Given its potential for improving information about COD, there is interest in refining the method. We describe the further development of the Tariff Method.
This study uses data from the PHMRC and the National Health and Medical Research Council (NHMRC) of Australia studies. Gold standard clinical diagnostic criteria for hospital deaths were specified for a target cause list. VAs were collected from families using the PHMRC verbal autopsy instrument including health care experience (HCE). The original Tariff Method (Tariff 1.0) was trained using the validated PHMRC database for which VAs had been collected for deaths with hospital records fulfilling the gold standard criteria (validated VAs). In this study, the performance of Tariff 1.0 was tested using VAs from household surveys (community VAs) collected for the PHMRC and NHMRC studies. We then corrected the model to account for the previous observed biases of the model, and Tariff 2.0 was developed. The performance of Tariff 2.0 was measured at individual and population levels using the validated PHMRC database.
For median chance-corrected concordance (CCC) and mean cause-specific mortality fraction (CSMF) accuracy, and for each of three modules with and without HCE, Tariff 2.0 performs significantly better than the Tariff 1.0, especially in children and neonates. Improvement in CSMF accuracy with HCE was 2.5 %, 7.4 %, and 14.9 % for adults, children, and neonates, respectively, and for median CCC with HCE it was 6.0 %, 13.5 %, and 21.2 %, respectively. Similar levels of improvement are seen in analyses without HCE.
Tariff 2.0 addresses the main shortcomings of the application of the Tariff Method to analyze data from VAs in community settings. It provides an estimation of COD from VAs with better performance at the individual and population level than the previous version of this method, and it is publicly available for use.
Electronic supplementary material
The online version of this article (doi:10.1186/s12916-015-0527-9) contains supplementary material, which is available to authorized users.
Verbal autopsy questionnaire; Mortality surveillance; Causes of death
In the light of multidrug resistance (MDR) among pathogenic microbes and cancer cells, membrane transporters have gained profound clinical significance. Chemotherapeutic failure, by far, has been attributed mainly to the robust and diverse array of these proteins, which are omnipresent in every stratum of the living world. Candida albicans, one of the major fungal pathogens affecting immunocompromised patients, also develops MDR during the course of chemotherapy. The pivotal membrane transporters that C. albicans has exploited as one of the strategies to develop MDR belongs to either the ATP binding cassette (ABC) or the major facilitator superfamily (MFS) class of proteins. The ABC transporter Candida drug resistance 1 protein (Cdr1p) is a major player among these transporters that enables the pathogen to outplay the battery of antifungals encountered by it. The promiscuous Cdr1 protein fulfills the quintessential need of a model to study molecular mechanisms of multidrug transporter regulation and structure-function analyses of asymmetric ABC transporters. In this review, we cover the highlights of two decades of research on Cdr1p that has provided a platform to study its structure-function relationships and regulatory circuitry for a better understanding of MDR not only in yeast but also in other organisms.
Three structurally related oxathiolone fused chalcone derivatives appeared effective chemosensitizers, able to restore in part sensitivity to fluconazole of multidrug-resistant C. albicans strains. Compound 21 effectively chemosensitized cells resistant due to the overexpression of the MDR1 gene, compound 6 reduced resistance of cells overexpressing the ABC-type drug transporters CDR1/CDR2 and derivative 18 partially reversed fluconazole resistance mediated by both types of yeast drug efflux pumps. The observed effect of sensitization of resistant strains of Candida albicans to fluconazole activity in the presence of active compounds most likely resulted from inhibition of the pump-mediated efflux, as was revealed by the results of studies involving the fluorescent probes, Nile Red, Rhodamine 6G and diS-C3(3).
multidrug resistance; chalcones; antifungals; chemosensitization; Candida albicans
Poly (ADP-ribose) polymerase-1 (PARP-1) is important for the recognition of both endogenous and exogenous DNA damage, and binds to DNA strand-breaks including intermediates of base excision repair (BER). Once DNA-bound, PARP-1 becomes catalytically activate synthesizing poly-ADP-ribose (PAR) polymers onto itself and other repair factors (PARylation). As a result, BER repair proteins such as XRCC1 and DNA polymerase β (pol β) are more efficiently and rapidly recruited to sites of DNA damage. In the presence of an inhibitor of PARP activity (PARPi), PARP-1 binds to sites of DNA damage, but PARylation is prevented. BER enzyme recruitment is hindered, but binding of PARP-1 to DNA is stabilized, impeding DNA repair and leading to double-strand DNA breaks (DSB). Deficiencies in pol β−/− and Xrcc1−/− cells resulted in hypersensitivity to the PARP inhibitor 4-AN and re-expression of pol β or XRCC1, in these contexts, reversed the 4-AN hypersensitivity phenotype. BER deficiencies also showed evidence of replication defects that lead to DSB-induced apoptosis upon PARPi treatment. Finally, the clinically relevant PARP inhibitors olaparib and veliparib also exhibited hypersensitivity in both pol β−/− and Xrcc1−/− BER-deficient cells. These results reveal heightened sensitivity to PARPi as a function of BER deficiency.
DNA polymerase; XRCC1; PARP-1; methyl methanesulfonate; PARP inhibitor; poly(ADP-ribose)
Alkaline phosphatase (ALP; E.C.3.I.3.1.) is an ubiquitous membrane-bound glycoprotein that catalyzes the hydrolysis of phosphate monoesters at basic pH values. Alkaline phosphatase is divided into four isozymes depending upon the site of tissue expression that are Intestinal ALP, Placental ALP, Germ cell ALP and tissue nonspecific alkaline phosphatase or liver/bone/kidney (L/B/K) ALP. The intestinal and placental ALP loci are located near the end of long arm of chromosome 2 and L/B/K ALP is located near the end of the short arm of chromosome 1. Although ALPs are present in many mammalian tissues and have been studied for the last several years still little is known about them. The bone isoenzyme may be involved in mammalian bone calcification and the intestinal isoenzyme is thought to play a role in the transport of phosphate into epithelial cells of the intestine. In this review, we tried to provide an overview about the various forms, structure and functions of alkaline phosphatase with special focus on liver/bone/kidney alkaline phosphatase.
Enzymes; Isoenzymes; Alkaline phosphatase; L/B/K alkaline phosphatase; Liver alkaline phosphatase; Intestinal alkaline phosphatase; Placental alkaline phosphatase
Obstructive sleep apnea (OSA) and obstructive sleep apnea syndrome (OSAS) are subsets of sleep-disordered breathing. Awareness about OSA and its consequences among the general public as well as the majority of primary care physicians across India is poor. This necessitated the development of the Indian initiative on obstructive sleep apnea (INOSA) guidelines under the auspices of Department of Health Research, Ministry of Health and Family Welfare, Government of India. OSA is the occurrence of an average five or more episodes of obstructive respiratory events per hour of sleep with either sleep-related symptoms or co-morbidities or ≥15 such episodes without any sleep-related symptoms or co-morbidities. OSAS is defined as OSA associated with daytime symptoms, most often excessive sleepiness. Patients undergoing routine health check-up with snoring, daytime sleepiness, obesity, hypertension, motor vehicular accidents, and high-risk cases should undergo a comprehensive sleep evaluation. Medical examiners evaluating drivers, air pilots, railway drivers, and heavy machinery workers should be educated about OSA and should comprehensively evaluate applicants for OSA. Those suspected to have OSA on comprehensive sleep evaluation should be referred for a sleep study. Supervised overnight polysomnography is the “gold standard” for evaluation of OSA. Positive airway pressure (PAP) therapy is the mainstay of treatment of OSA. Oral appliances (OA) are indicated for use in patients with mild to moderate OSA who prefer OA to PAP, or who do not respond to PAP or who fail treatment attempts with PAP or behavioral measures. Surgical treatment is recommended in patients who have failed or are intolerant to PAP therapy.
Bariatric surgery; continuous positive airway pressure; Indian guidelines; obstructive sleep apnea; obstructive sleep apnea syndrome; polysomnography; sleep apnea; sleep study; syndrome Z
The ABC transporter Cdr1 protein (Cdr1p) of Candida albicans, which plays a major role in antifungal resistance, has two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) that are interconnected by extracellular (ECLs) and intracellular (ICLs) loops. To examine the communication interface between the NBDs and ICLs of Cdr1p, we subjected all four ICLs to alanine scanning mutagenesis, replacing each of the 85 residues with an alanine. The resulting ICL mutant library was analyzed by biochemical and phenotypic mapping. Only 18% of the mutants from this library displayed enhanced drug susceptibility. Most of the drug-susceptible mutants displayed uncoupling between ATP hydrolysis and drug transport. The two drug-susceptible ICL1 mutants (I574A and S593A) that lay within or close to the predicted coupling helix yielded two chromosomal suppressor mutations that fall near the Q-loop of NBD2 (R935) and in the Walker A motif (G190) of NBD1. Based on a 3D homology model and kinetic analysis of drug transport, our data suggest that large distances between ICL residues and their respective chromosomal suppressor mutations rule out a direct interaction between them. However, they impact the transport cycle by restoring the coupling interface via indirect downstream signaling.
The apurinic/apyrimidinic- (AP-) site in genomic DNA arises through spontaneous base loss and base removal by DNA glycosylases and is considered an abundant DNA lesion in mammalian cells. The base excision repair (BER) pathway repairs the AP-site lesion by excising and replacing the site with a normal nucleotide via template directed gap-filling DNA synthesis. The BER pathway is mediated by a specialized group of proteins, some of which can be found in multiprotein complexes in cultured mouse fibroblasts. Using a DNA polymerase (pol) β immunoaffinity-capture technique to isolate such a complex, we identified five tightly associated and abundant BER factors in the complex: PARP-1, XRCC1, DNA ligase III, PNKP, and Tdp1. AP endonuclease 1 (APE1), however, was not present. Nevertheless, the complex was capable of BER activity, since repair was initiated by PARP-1’s AP lyase strand incision activity. Addition of purified APE1 increased the BER activity of the pol β complex. Surprisingly, the pol β complex stimulated the strand incision activity of APE1. Our results suggested that PARP-1 was responsible for this effect, whereas other proteins in the complex had no effect on APE1 strand incision activity. Studies of purified PARP-1 and APE1 revealed that PARP-1 was able to stimulate APE1 strand incision activity. These results illustrate roles of PARP-1 in BER including a functional partnership with APE1.
Idiopathic pulmonary fibrosis (IPF) accounts for the majority of lung diseases classified as idiopathic interstitial pneumonia (IIP). It is considered to be lethal because prognosis is very poor and far worse than other types of IIP. An early and accurate diagnosis of IPF is critical. The diagnostic process is complex and requires a multidisciplinary approach involving a pulmonologist, radiologist and pathologist.
Idiopathic pulmonary fibrosis; idiopathic interstitial pneumonia; diagnosis
Telomere binding factors viz. TRF1 and TRF2 are a part of sheltrin complex that are present exclusively at the ends of chromosomes. These factors play an important role in maintaining chromosomal integrity at the ends. However, their status and role are not clear in renal cell carcinoma (RCC). Therefore, the present study was conducted to evaluate TRF1 and TRF2 expressions in RCC tissues. Further, the role of these factors involved in tumorigenesis was elucidated by gene silencing using siRNA in RCC cell line (A498). The present study documented a significant over-expression of TRF1 (P = 0.005) and TRF2 (P = 0.0048) mRNAs by real time PCR in RCC tissues as compared with adjacent normal kidney tissues. Immunohistochemistry studies also revealed higher expression of TRF1 and TRF2 proteins in RCC. Moreover, TRF1 or TRF2 gene silencing using siRNA showed marked reduction in proliferation of RCC cells (P = 0.000). Further, significantly induced cell cycle arrest (P = 0.000) and apoptosis of RCC cells (P = 0.000) was documented upon TRF1 or TRF2 gene silencing. Henceforth, the results deduce that TRF1 or TRF2 inhibitions play an important role in the induction of apoptosis in A498 cells, which may serve as a potential therapeutic target in RCC.
Abortive ligation during base excision repair (BER) leads to blocked repair intermediates containing a 5′-adenylated-deoxyribose phosphate (5′-AMP-dRP) group. Aprataxin (APTX) is able to remove the AMP group allowing repair to proceed. Earlier results had indicated that purified DNA polymerase β (pol β) removes the entire 5′-AMP-dRP group through its lyase activity and flap endonuclease 1 (FEN1) excises the 5′-AMP-dRP group along with one or two nucleotides. Here, using cell extracts from APTX-deficient cell lines, human Ataxia with Oculomotor Apraxia Type 1 (AOA1) and DT40 chicken B cell, we found that pol β and FEN1 enzymatic activities were prominent and strong enough to complement APTX deficiency. In addition, pol β, APTX and FEN1 coordinate with each other in processing of the 5′-adenylated dRP-containing BER intermediate. Finally, other DNA polymerases and a repair factor with dRP lyase activity (pol λ, pol ι, pol θ and Ku70) were found to remove the 5′-adenylated-dRP group from the BER intermediate. However, the activities of these enzymes were weak compared with those of pol β and FEN1.
Background and Objectives:
India is among the largest countries to implement the revised National Tuberculosis Control Program (RNTCP). This program provides intermittent regimens to the patients, where the doses of isoniazid and ethambutol are more as compared to the daily regimen, which is a cause of concern, particularly with regard to the ocular toxicity of ethambutol. The present study was undertaken to explore the ocular toxicity in the patients registered under the program.
Materials and Methods:
This was a prospective single center cohort study of 64 patients of categories I and II, coming to the RNTCP-Directly Observed Treatment Strategy (DOTS) center at a tertiary care referral hospital. The detailed history, best corrected visual acuity, fundus examination, and color vision test were carried out in all patients at the start of treatment and then at the first and second month of treatment.
Loss in visual acuity from the baseline was noted at the second month follow up in 12 (9.4%) eyes (P = 0.001), visual field defects were seen in eight (6.3%) eyes (P = 0.0412), and optic disc abnormalities were observed in six (4.7%) (P = 0.013) eyes. Color vision abnormalities were noted in 16 (12.6%) eyes (P = 0.003), four eyes showed impairment in red–green color perception, and the others showed impairment in blue–yellow color perception as well. Patients with ocular symptoms were advised to stop ethambutol and they showed improvement in visual acuity after follow up of one to two months. The overall outcome of treatment was not affected by discontinuation of ethambutol in these patients.
Ethambutol when taken according to program could cause ocular toxicity. The early recognition of ocular symptoms is important to prevent unnecessary delay in diagnosis and probable irreversible visual loss.
Ethambutol toxicity; ocular symptoms; revised national tuberculosis control program; visual defects
A critical question among the researchers working on fungal lipid biology is whether the use of an enriched growth medium can affect the lipid composition of a cell and, therefore, contribute to the observed phenotypes. One presumption is that enriched medias, such as YPD (yeast extract, peptone and dextrose), are likely to contain lipids, which may homogenize with the yeast lipids and play a role in masking the actual differences in the observed phenotypes or lead to an altered phenotype altogether. To address this issue, we compared the lipids of Candida albicans, our fungus of interest, grown in YPD or in a defined media such as YNB (yeast nitrogen base). Mass spectrometry-based lipid analyses showed differences in the levels of phospholipids, including phosphatidylinositol, phosphatidylglycerol, lyso-phospholipids; sphingolipids, such as mannosyldiinositolphosphorylceramide; and sterols, such as ergostatetraenol. Significant differences were observed in 70 lipid species between the cells grown in the two media, but the two growth conditions did not affect the morphological characteristics of C. albicans. The lipid profiles of the YNB- and YPD-grown C. albicans cells did vary, but these differences did not influence their response to the majority of the tested agents. Rather, the observed differences could be attributed to the slow growth rate of the Candida cells in YNB compared to YPD. Notably, the altered lipid changes between the two media did impact the susceptibility to some drugs. This data provided evidence that changes in media can lead to certain lipid alterations, which may affect specific pathways but, in general, do not affect the majority of the phenotypic properties of C. albicans. It was determined that either YNB or YPD may be suitable for the growth and lipid analysis of C. albicans, depending upon the experimental requirements, but additional precautions are necessary when correlating the phenotypes with the lipids.
Poly(ADP-ribose) polymerase-1 (PARP-1) binds intermediates of base excision repair (BER) and becomes activated for poly(ADP-ribose) (PAR) synthesis. PAR mediates recruitment and functions of the key BER factors XRCC1 and DNA polymerase β (pol β) that in turn regulate PAR. Yet, the molecular mechanism and implications of coordination between XRCC1 and pol β in regulating the level of PAR are poorly understood. A complex of PARP-1, XRCC1 and pol β is found in vivo, and it is known that pol β and XRCC1 interact through a redox-sensitive binding interface in the N-terminal domain of XRCC1. We confirmed here that both oxidized and reduced forms of XRCC1 are present in mouse fibroblasts. To further understand the importance of the C120-C20 oxidized form of XRCC1 and the interaction with pol β, we characterized cell lines representing stable transfectants in Xrcc1−/− mouse fibroblasts of wild-type XRCC1 and two mutants of XRCC1, a novel reduced form with the C12-C20 disulfide bond blocked (C12A) and a reference mutant that is unable to bind pol β (V88R). XRCC1-deficient mouse fibroblasts are extremely hypersensitive to methyl methanesulfonate (MMS), and transfected wild-type and C12A mutant XRCC1 proteins similarly reversed MMS hypersensitivity. However, after MMS exposure the cellular PAR level was found to increase to a much greater extent in cells expressing the C12A mutant than in cells expressing wild-type XRCC1. PARP inhibition resulted in very strong MMS sensitization in cells expressing wild-type XRCC1, but this sensitization was much less in cells expressing the C12A mutant. The results suggest a role for the oxidized form of XRCC1 in the interaction with pol β in 1) controlling the PAR level after MMS exposure and 2) enabling the extreme cytotoxicity of PARP inhibition during the MMS DNA damage response.
Obstructive sleep apnoea (OSA) and obstructive sleep apnoea syndrome (OSAS) are subsets of sleep-disordered breathing. Awareness about OSA and its consequences amongst the general public as well as the majority of primary care physcians across India is poor. This necessiated the development of the INdian initiative on Obstructive sleep apnoea (INOSA) guidelines under the auspices of Department of Health Research, Ministry of Health & Family Welfare, Government of India. OSA is the occurrence of an average five or more episodes of obstructive respiratory events per hour of sleep with either sleep related symptoms or co-morbidities or ≥ 15 such episodes without any sleep related symptoms or co-morbidities. OSAS is defined as OSA associated with daytime symptoms, most often excessive sleepiness. Patients undergoing routine health check-up with snoring, daytime sleepiness, obesity, hypertension, motor vehicular accidents and high risk cases should undergo a comprehensive sleep evaluation. Medical examiners evaluating drivers, air pilots, railway drivers and heavy machinery workers should be educated about OSA and should comprehensively evaluate applicants for OSA. Those suspected to have OSA on comprehensive sleep evaluation should be referred for a sleep study. Supervised overnight polysomnography (PSG) is the “gold standard” for evaluation of OSA. Positive airway pressure (PAP) therapy is the mainstay of treatment of OSA. Oral appliances are indicated for use in patients with mild to moderate OSA who prefer oral appliances to PAP, or who do not respond to PAP or who fail treatment attempts with PAP or behavioural measures. Surgical treatment is recommended in patients who have failed or are intolerant to PAP therapy.
Bariatric surgery; CPAP; Indian guidelines; OSA; OSAS; polysomnography; sleep apnoea; sleep study; Syndrome Z
It is now well-known that the enhanced expression of ATP binding cassette (ABC) and major facilitator superfamily (MFS) proteins contribute to the development of tolerance to antifungals in yeasts. For example, the azole resistant clinical isolates of the opportunistic human fungal pathogen Candida albicans show an overexpression of Cdr1p and/or CaMdr1p belonging to ABC and MFS superfamilies, respectively. Hence, azole resistant isolates display reduced accumulation of therapeutic drug due to its rapid extrusion and that facilitates its survival. Considering the importance of major antifungal transporters, the focus of recent research has been to understand the structure and function of these proteins to design inhibitors/modulators to block the pump protein activity so that the drug already in use could again sensitize resistant yeast cells. The review focuses on the structure and function of ABC and MFS transporters of Candida to highlight the recent advancement in the field.
multidrug resistance; ABC transporters; MFS transporters; azoles; efflux pumps; Candida
Candida albicans causes superficial to systemic infections in immuno-compromised individuals. The concomitant use of fungistatic drugs and the lack of cidal drugs frequently result in strains that could withstand commonly used antifungals, and display multidrug resistance (MDR). In search of novel fungicidals, in this study, we have explored a plant alkaloid berberine (BER) for its antifungal potential. For this, we screened an in-house transcription factor (TF) mutant library of C. albicans strains towards their susceptibility to BER. Our screen of TF mutant strains identified a heat shock factor (HSF1), which has a central role in thermal adaptation, to be most responsive to BER treatment. Interestingly, HSF1 mutant was not only highly susceptible to BER but also displayed collateral susceptibility towards drugs targeting cell wall (CW) and ergosterol biosynthesis. Notably, BER treatment alone could affect the CW integrity as was evident from the growth retardation of MAP kinase and calcineurin pathway null mutant strains and transmission electron microscopy. However, unlike BER, HSF1 effect on CW appeared to be independent of MAP kinase and Calcineurin pathway genes. Additionally, unlike hsf1 null strain, BER treatment of Candida cells resulted in dysfunctional mitochondria, which was evident from its slow growth in non-fermentative carbon source and poor labeling with mitochondrial membrane potential sensitive probe. This phenotype was reinforced with an enhanced ROS levels coinciding with the up-regulated oxidative stress genes in BER-treated cells. Together, our study not only describes the molecular mechanism of BER fungicidal activity but also unravels a new role of evolutionary conserved HSF1, in MDR of Candida.
Curcumin (CUR) shows antifungal activity against a range of pathogenic fungi, including Candida albicans. The reported mechanisms of action of CUR include reactive oxygen species (ROS) generation, defects in the ergosterol biosynthesis pathway, decrease in hyphal development, and modulation of multidrug efflux pumps. Reportedly, each of these pathways is independently linked to the cell wall machinery in C. albicans, but surprisingly, CUR has not been previously implicated in cell wall damage. In the present study, we performed transcriptional profiling to identify the yet-unidentified targets of CUR in C. albicans. We found that, among 348 CUR-affected genes, 51 were upregulated and 297 were downregulated. Interestingly, most of the cell wall integrity pathway genes were downregulated. The possibility of the cell wall playing a critical role in the mechanism of CUR required further validation; therefore, we performed specific experiments to establish if there was any link between the two. The fractional inhibitory concentration index values of 0.24 to 0.37 show that CUR interacts synergistically with cell wall-perturbing (CWP) agents (caspofungin, calcofluor white, Congo red, and SDS). Furthermore, we could observe cell wall damage and membrane permeabilization by CUR alone, as well as synergistically with CWP agents. We also found hypersusceptibility in calcineurin and mitogen-activated protein (MAP) kinase pathway mutants against CUR, which confirmed that CUR also targets cell wall biosynthesis in C. albicans. Together, these data provide strong evidence that CUR disrupts cell wall integrity in C. albicans. This new information on the mechanistic action of CUR could be employed in improving treatment strategies and in combinatorial drug therapy.
Metallothioneins (MTs) are a group of low-molecular weight, cysteine-rich proteins. In general, MT is known to modulate three fundamental processes: (1) the release of gaseous mediators such as hydroxyl radical or nitric oxide, (2) apoptosis and (3) the binding and exchange of heavy metals such as zinc, cadmium or copper. Previous studies have shown a positive correlation between the expression of MT with invasion, metastasis and poor prognosis in various cancers. Most of the previous studies primarily used immunohistochemistry to analyze localization of MT in renal cell carcinoma (RCC). No information is available on the gene expression of MT2A isoform in different types and grades of RCC.
Materials and Methods:
In the present study, total RNA was isolated from 38 histopathologically confirmed cases of RCC of different types and grades. Corresponding adjacent normal renal parenchyma was taken as control. Real-time polymerase chain reaction (RT PCR) analysis was done for the MT2A gene expression using β-actin as an internal control. All statistical calculations were performed using SPSS software.
The MT2A gene expression was found to be significantly increased (P < 0.01) in clear cell RCC in comparison with the adjacent normal renal parenchyma. The expression of MT2A was two to three-fold higher in sarcomatoid RCC, whereas there was no change in papillary and collecting duct RCC. MT2A gene expression was significantly higher in lower grade (grades I and II, P < 0.05), while no change was observed in high-grade tumor (grade III and IV) in comparison to adjacent normal renal tissue.
The first report of the expression of MT2A in different types and grades of RCC and also these data further support the role of MT2A in tumorigenesis.
Clear cell carcinoma; grade; metallothionein; renal cell carcinoma; Real time polymerase chain reaction