Toxicity of benzyl paraben on aquatic as well as terrestrial life.

Parabens are derivatives of alkyl esters of p-hydroxybenzoic acid and come in different classes. These compounds are primarily used as antimicrobial preservative agents in many commercial products, including cosmetics and pharmaceuticals. Accordingly, Benzyl paraben (BeP) is known to be a potential endocrine disruptor. The aim of this study was to determine the toxicity of benzyl paraben (BeP) on aquatic and terrestrial organisms, specifically Scenedesmus sp., Moina macrocopa, and Eisenia fetida. All the organisms were treated with different concentrations of BeP (0.025 mg/L and 1000 mg/L), and LC25, LC50, and LC90 values were used to measure the toxicity levels. Results showed the LC values of BeP for M. macrocopa (3.3 mg/L, 4.7 mg/L, 7.3 mg/L) and E. fetida (173.2 mg/L, 479.8 mg/L, 1062 mg/L), respectively. Toxicity tests on green algae (Scenedesmus sp.) were conducted, the green algae were subjected to various BeP concentration. At 50 mg/L of BeP, cell viability was reduced to 56.2% and the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay indicated 35.4% viable cells. The chlorophyll value and the biochemical parameters of the algal cells were corroborative with the cell viability test. Lethal indices (LC50) for M. macrocopa and E. fetida were evaluated for their toxicity on biochemical properties and were found to be catalase (0.111 mg/L, 0.5 mg/L), lipid peroxidation (0.072 mg/L, 0.056 mg/L), and total protein (0.309 mg/L, 0.314 mg/L), respectively. Overall, this study demonstrated the toxic impact of BeP on non-target aquatic as well as terrestrial species.

Assessment of Safety and Biodistribution of AAVrh.10hCLN2 Following Intracisternal Administration in Nonhuman Primates for the Treatment of CLN2 Batten Disease.

CLN2 disease is a fatal, childhood autosomal recessive disorder caused by mutations in ceroid lipofuscinosis type 2 (CLN2) gene, encoding tripeptidyl peptidase 1 (TPP-1). Loss of TPP-1 activity leads to accumulation of storage material in lysosomes and resultant neuronal cell death with neurodegeneration. Genotype/phenotype comparisons suggest that the phenotype should be ameliorated with increase of TPP-1 levels to 5-10% of normal with wide central nervous system (CNS) distribution. Our previous clinical study showed that intraparenchymal (IPC) administration of AAVrh.10hCLN2, an adeno-associated vector serotype rh.10 encoding human CLN2, slowed, but did not stop disease progression, suggesting that this may be insufficient to distribute the therapy throughout the CNS (Sondhi 2020). In this study, we assessed whether the less invasive intracisternal delivery route would be safe and provide a wider distribution of TPP-1. A study was conducted in nonhuman primates (NHPs) with intracisternal delivery to cerebrospinal fluid (CSF) of AAVrh.10hCLN2 (5 × 1013 genome copies) or phosphate buffered saline (PBS). No abnormal behavior was noted. CNS magnetic resonance imaging and clinical chemistry data were all unremarkable. Histopathology of major organs had no abnormal finding attributable to the intervention or the vector, except that in one out of two animals treated with AAVrh.10hCLN2, dorsal root ganglia showed mild-to-moderate mononuclear cell infiltrates and neuronal degeneration. In contrast to our previous NHP study (Sondhi 2012) with IPC administration where TPP-1 activity was >2 × above controls in 30% of treated brains, in the two intracisternal treated NHPs, the TPP-1 activity was >2 × above controls in 50% and 41% of treated brains, and 52% and 84% of brain had >1,000 vector genomes/µg DNA, compared to 0% in the two PBS NHP. CSF TPP1 levels in treated animals were 43-62% of normal human levels. Collectively, these data indicate that AAVrh.10hCLN2 delivered by intracisternal route is safe and widely distributes TPP-1 in brain and CSF at levels that are potentially therapeutic. Clinical Trial Registration: NCT02893826, NCT04669535, NCT04273269, NCT03580083, NCT04408625, NCT04127578, and NCT04792944.

Bioconjugation of Meldrum's acid activated furan: A detergent compatible assay for protein quantitation.

A simple yet efficient assay for the quantitation of proteins ranging from plasma proteins to purified proteins from whole cell lysate, based on the bioconjugation reaction between protein and Meldrum's acid Activated Furan (MAF) is described. This easy to use, sensitive method is based on the conjugation of amine functionalities present on the protein with MAF to form the corresponding Donor Acceptor Stenhouse Adducts (DASAs) with characteristic absorption in the visible region. The reaction is rapid as well as reproducible and shows a proportionate increase in color change over a broad range of protein concentration. The assay was found to be sensitive up to 0.125 mg/mL concentration of the protein and was compatible with most of the commonly employed detergents and isolation protocols which makes it ideal for the estimation of protein samples containing detergents. Another striking feature of this protocol is its tolerance towards other major interference contributors such as chelating agents, reducing agents, carbohydrates and protease inhibitors.

Blocking drug efflux mechanisms facilitate genome engineering process in hypercellulolytic fungus, Penicillium funiculosum NCIM1228.

BACKGROUND: Penicillium funiculosum NCIM1228 is a non-model filamentous fungus that produces high-quality secretome for lignocellulosic biomass saccharification. Despite having desirable traits to be an industrial workhorse, P. funiculosum has been underestimated due to a lack of reliable genetic engineering tools. Tolerance towards common fungal antibiotics had been one of the major hindrances towards development of reliable transformation tools against the non-model fungi. In this study, we sought to understand the mechanism of drug tolerance of P. funiculosum and the provision to counter it. We then attempted to identify a robust method of transformation for genome engineering of this fungus. RESULTS: Penicillium funiculosum showed a high degree of drug tolerance towards hygromycin, zeocin and nourseothricin, thereby hindering their use as selectable markers to obtain recombinant transformants. Transcriptome analysis suggested a high level expression of efflux pumps belonging to ABC and MFS family, especially when complex carbon was used in growth media. Antibiotic selection medium was optimized using a combination of efflux pump inhibitors and suitable carbon source to prevent drug tolerability. Protoplast-mediated and Agrobacterium-mediated transformation were attempted for identifying efficiencies of linear and circular DNA in performing genetic manipulation. After finding Ti-plasmid-based Agrobacterium-mediated transformation more suitable for P. funiculosum, we improvised the system to achieve random and homologous recombination-based gene integration and deletion, respectively. We found single-copy random integration of the T-DNA cassette and could achieve 60% efficiency in homologous recombination-based gene deletions. A faster, plasmid-free, and protoplast-based CRISPR/Cas9 gene-editing system was also developed for P. funiculosum. To show its utility in P. funiculosum, we deleted the gene coding for the most abundant cellulase Cellobiohydrolase I (CBH1) using a pair of sgRNA directed towards both ends of cbh1 open reading frame. Functional analysis of ∆cbh1 strain revealed its essentiality for the cellulolytic trait of P. funiculosum secretome. CONCLUSIONS: In this study, we addressed drug tolerability of P. funiculosum and developed an optimized toolkit for its genome modification. Hence, we set the foundation for gene function analysis and further genetic improvements of P. funiculosum using both traditional and advanced methods.

In Situ Decoration of Gold Nanoparticles on Graphene Oxide via Nanosecond Laser Ablation for Remarkable Chemical Sensing and Catalysis.

Gold decorated graphene-based nano-hybrids find extensive research interest due to their enhanced chemical catalytic performance and biochemical sensing. The unique physicochemical properties and the very large surface area makes them propitious platform for the rapid buildouts of science and technology. Graphene serves as an outstanding matrix for anchoring numerous nanomaterials because of its atomically thin 2D morphological features. Herein, we have designed a metal-graphene nano-hybrid through pulsed laser ablation. Commercially available graphite powder was employed for the preparation of graphene oxide (GO) using modified Hummers' method. A solid, thin gold (Au) foil was ablated in an aqueous suspension of GO using second harmonic wavelength (532 nm) of the Nd:YAG laser for immediate generation of the Au-GO nano-hybrid. The synthesis strategy employed here does not entail any detrimental chemical reagents and hence avoids the inclusion of reagent byproducts to the reaction mixture, toxicity, and environmental or chemical contamination. Optical and morphological characterizations were performed to substantiate the successful anchoring of Au nanoparticles (Au NPs) on the GO sheets. Remarkably, these photon-generated nano-hybrids can act as an excellent surface enhanced Raman spectroscopy (SERS) platform for the sensing/detection of the 4-mercaptobenzoic acid (4-MBA) with a very low detection limit of 1 × 10-12 M and preserves better reproducibility also. In addition, these hybrid materials were found to act as an effective catalyst for the reduction of 4-nitrophenol (4-NP). Thus, this is a rapid, mild, efficient and green synthesis approach for the fabrication of active organometallic sensors and catalysts.

Barriers to the utilization of genetic testing and genetic counseling in patients with suspected hereditary breast and ovarian cancers.

A heritable condition is the identified cause of cancer in 5% to 10% of women with breast cancer and in 25% of women with ovarian cancer. It is critical to identify patients at risk for inherited genetic mutations to implement risk-reducing screening and interventions; however, reports in the medical literature indicate that an alarming number of patients with inherited genetic mutations do not receive recommended genetic counseling, testing, or interventions. In order to improve outcomes for these high-risk patients, barriers to genetic testing and counseling must be identified. We analyzed approximately 200 patients seen at our institution with breast or ovarian cancer who met criteria of the National Comprehensive Cancer Network for genetic counseling and testing. Of these patients, almost 70% had appropriate genetic testing and counseling. Review of the remaining 30% revealed that the largest obstacle to receiving genetic testing and/or counseling was lack of referral from the treating oncologist. Of the patients diagnosed with a pathogenic heritable mutation, most underwent appropriate risk-reducing procedures and surveillance. Thus, the initial referral to genetic counseling is the most significant barrier for at-risk patients at our institution and likely in this population at large. Additional study is needed to identify ways to improve appropriate use of genetic testing and counseling.

Improved efficacy of a next-generation ERT in murine Pompe disease.

Pompe disease is a rare inherited disorder of lysosomal glycogen metabolism due to acid α-glucosidase (GAA) deficiency. Enzyme replacement therapy (ERT) using alglucosidase alfa, a recombinant human GAA (rhGAA), is the only approved treatment for Pompe disease. Although alglucosidase alfa has provided clinical benefits, its poor targeting to key disease-relevant skeletal muscles results in suboptimal efficacy. We are developing an rhGAA, ATB200 (Amicus proprietary rhGAA), with high levels of mannose-6-phosphate that are required for efficient cellular uptake and lysosomal trafficking. When administered in combination with the pharmacological chaperone AT2221 (miglustat), which stabilizes the enzyme and improves its pharmacokinetic properties, ATB200/AT2221 was substantially more potent than alglucosidase alfa in a mouse model of Pompe disease. The new investigational therapy is more effective at reversing the primary abnormality - intralysosomal glycogen accumulation - in multiple muscles. Furthermore, unlike the current standard of care, ATB200/AT2221 dramatically reduces autophagic buildup, a major secondary defect in the diseased muscles. The reversal of lysosomal and autophagic pathologies leads to improved muscle function. These data demonstrate the superiority of ATB200/AT2221 over the currently approved ERT in the murine model.

In Situ Synthesis of Silver Nanospheres, Nanocubes, and Nanowires over Boron-Doped Graphene Sheets for Surface-Enhanced Raman Scattering Application and Enzyme-Free Detection of Hydrogen Peroxide.

An effective in situ synthesis strategy is demonstrated for the preparation of silver nanostructures (nanospheres (NSs), nanocubes (NCs), and nanowires (NWs)) on the surface of boron-doped graphene (BG). Further, these functional nanomaterials are employed for the surface-enhanced Raman scattering (SERS) and non-enzymatic electrochemical detection of H2O2. The results confirm the superior performance of BG-Ag nanostructures as SERS platform. Among various geometries of silver nanoparticles studied in this work, we find that the AgNCs over BG (BG-AgNC) present outstanding SERS performance for detecting 4-mercaptobenzoic acid, with a limit of detection of 1.0 × 10-13 M. Furthermore, BG-AgNC exhibits excellent capability to detect melamine as low as 1.0 × 10-9 M. Electrochemical results confirm that the BG-AgNW-based platform exhibits a superior biosensing performance toward H2O2 detection. The enhanced performance is due to the presence of graphene, which improves the conductivity and provides more active sites. The synthesis of doped graphene with metallic nanoparticles described in this work is expected to be a key strategy for the development of an efficient SERS and electrochemical sensor that offers simplicity, cost-effectiveness, long-term stability, and better reproducibility.

The incidence of cardiomyopathy in BRCA1 and BRCA2 mutation carriers after anthracycline-based adjuvant chemotherapy.

PURPOSE: Breast cancer remains the fourth-leading cause of death in the United States. Nearly 10% of breast cancers are hereditary, with deleterious mutations in BRCA1 and BRCA2 genes being the leading cause. Anthracycline chemotherapy, used commonly for breast cancer, carries cardiotoxicity risk. Recent studies demonstrated anthracycline-induced cardiac failure in homozygous BRCA2-deficient mice and increased rates of heart failure in homozygous BRCA1-deficient mice following ischemic insult. Therefore, we conducted a retrospective matched cohort study to determine the rates of anthracycline-induced cardiomyopathy in breast cancer patients with germline mutation in BRCA1 or BRCA2 genes compared to age-matched patients without a BRCA1 or BRCA2 gene mutation. METHODS: The primary endpoint was to determine the rate of cardiomyopathy defined as either congestive heart failure or asymptomatic decline in ejection fraction to <50%. A total of 102 breast cancer patients who were BRCA gene mutation carriers (55 BRCA1, 45 BRCA2, and two with both), who received anthracycline-based chemotherapy were compared to a matched cohort of breast cancer patients with wild-type BRCA gene status. RESULTS: We found a 4.9% rate of cardiomyopathy in the BRCA mutation carriers and 5.2% in the matched controls (p = 0.99). Cox proportional hazards model showed that only trastuzumab and hypertension were significantly associated with the development of cardiomyopathy in both groups (p < 0.05). CONCLUSIONS: Given the limitations of a retrospective study, we saw no increased risk of cardiotoxicity among breast cancer patients with BRCA1 and/or BRCA2 gene mutations treated with standard doses of anthracycline compared to the general population.

Boron doped graphene wrapped silver nanowires as an efficient electrocatalyst for molecular oxygen reduction.

Metal nanowires exhibit unusually high catalytic activity towards oxygen reduction reaction (ORR) due to their inherent electronic structures. However, controllable synthesis of stable nanowires still remains as a daunting challenge. Herein, we report the in situ synthesis of silver nanowires (AgNWs) over boron doped graphene sheets (BG) and demonstrated its efficient electrocatalytic activity towards ORR for the first time. The electrocatalytic ORR efficacy of BG-AgNW is studied using various voltammetric techniques. The BG wrapped AgNWs shows excellent ORR activity, with very high onset potential and current density and it followed four electron transfer mechanism with high methanol tolerance and stability towards ORR. The results are comparable to the commercially available 20% Pt/C in terms of performance.

Characterization and biological activities of cyclic (1 → 3, 1 → 6)-ß-glucans from Bradyrhizobium japonicum.

OBJECTIVE: To isolate cyclic (1 â†’ 3, 1 â†’ 6)-ß-glucan from Bradyrhizobium japonicum MTCC120, to characterize its structure and to study its biological activities. RESULTS: The degree of polymerization of cyclic (1 â†’ 3, 1 â†’ 6)-ß-glucan varied between 10 and 13 and with substituents acetyl, succinyl and phosphocholine. The cyclic glucans showed bimodal particle size distribution, with hydrodynamic diameters of 1.92 and 231 nm corresponding to monomeric and aggregated cyclic glucans, respectively. SEM and TEM images showed that the glucans formed aggregates of nanorods. The glucans were biocompatible, exhibited good antioxidant activity and had the abilities to bind to Aniline Blue dye to form a fluorescence complex which was concentration dependent. CONCLUSION: The glucans isolated are cyclic and have good antioxidant activities, hence have potential application in food and pharmaceutical industries. Their dye binding ability could be exploited in medical imaging to reduce the cytotoxicity of the dyes.

Process optimization and kinetic modelling of cyclic (1→3, 1→6)-ß-glucans production from Bradyrhizobium japonicum MTCC120.

Cyclic (1→3, 1→6)-ß-glucans are water soluble, biocompatible polymers with potential applications in food and pharmaceutical industries but have not yet been exploited due to their poor yield. In the present study statistical experimental design methodology was employed to improve their production. Initial screening indicated arabinose and peptone as best carbon and nitrogen source respectively, for glucan production. Arabinose and osmolyte concentrations as well as pH significantly contributed to the glucan production. Central composite design indicated a significant interaction between osmolyte concentration and pH on glucan production. The maximum amount of cyclic glucan produced was 6.7g/L in a 2.5L reactor in batch conditions. The logistic equation for cell growth and Luedeking-Piret equation for glucan production could satisfactorily simulate the batch kinetics data. Cyclic ß-glucans could efficiently encapsulate a hydrophobic molecule, curcumin and increase its solubility in water, thus indicating that these glucans have potential as drug delivery systems.

Severe Refractory Immune Thrombocytopenia Successfully Treated with High-Dose Pulse Cyclophosphamide and Eltrombopag.

Severe refractory ITP is clinically challenging and a variety of single or combination chemotherapies have been tried with limited outcome. We report a case of ITP that was unresponsive to multiple agents including high-dose steroid, IVIG, Rho(D) immune globulin, rituximab, cyclosporine, azathioprine, vincristine, mycophenolate mofetil, romiplostim, and eltrombopag; however, it achieved complete remission with combination treatment of cyclophosphamide and eltrombopag.

Nanoparticle assembly following Langmuir-Hinshelwood kinetics on a Langmuir film and chain networks captured in LB films.

The Langmuir-Blodgett (LB) technique is an elegant protocol for the steered assembly of metal nanoparticles, the deposition pressure serving as a convenient parameter to tune the assembly. Adsorption of nanoparticles from the subphase to the air-water interface can provide further control of the process. Citrate-stabilized gold nanoparticles in the aqueous subphase are shown to assemble into extended 2-dimensional chain networks following adsorption on a cationic amphiphile Langmuir film at the air-water interface. Kinetic investigations show that the process can be visualized as a surface-catalyzed reaction and explained in terms of the Langmuir-Hinshelwood mechanism. The LB deposition proves to be a unique route to capture the reaction product together with the amphiphile film. The deposition pressure is used to tune the density of nanoparticle chain networks in the LB film, and their optical extinction spectrum. The unusual blue shift of the extinction observed with increasing deposition pressure is attributed to the impact of the amphiphile monolayer environment. The extent of formation of the chain network is analyzed in terms of the pathways in the corresponding graph representation, and shown to scale with the deposition pressure. The current investigation highlights the use of a charged monolayer as a heterogeneous catalyst surface, provides fundamental insight into the kinetics of nanoparticle assembly at interfaces, and demonstrates the utility of the LB technique in tuning the formation of 2-dimensional nanoparticle chain networks.

Patient and provider attitudes toward the use of patient portals for the management of chronic disease: a systematic review.

BACKGROUND: Patient portals provide patients with the tools to better manage and understand their health status. However, widespread adoption of patient portals faces resistance from patients and providers for a number of reasons, and there is limited evidence evaluating the characteristics of patient portals that received positive remarks from patients and providers. OBJECTIVE: The objectives of this systematic review are to identify the shared characteristics of portals that receive favorable responses from patients and providers and to identify the elements that patients and providers believe need improvement. METHODS: The authors conducted a systematic search of the CINAHL and PubMed databases to gather data about the use of patient portals in the management of chronic disease. Two reviewers analyzed the articles collected in the search process in order remove irrelevant articles. The authors selected 27 articles to use in the literature review. RESULTS: Results of this systematic review conclude that patient portals show significant improvements in patient self-management of chronic disease and improve the quality of care provided by providers. The most prevalent positive attribute was patient-provider communication, which appeared in 10 of 27 articles (37%). This was noted by both patients and providers. The most prevalent negative perceptions are security (concerns) and user-friendliness, both of which occurred in 11 of 27 articles (41%). The user-friendliness quality was a concern for patients and providers who are not familiar with advanced technology and therefore find it difficult to navigate the patient portal. The high cost of installation and maintenance of a portal system, not surprisingly, deters some providers from implementing such technology into their practice, but this was only mentioned in 3 of the 27 articles (11%). It is possible that the incentives for meaningful use assuage the barrier of cost. CONCLUSIONS: This systematic review revealed mixed attitudes from patients and their providers regarding the use of patient portals to manage their chronic disease. The authors suggest that a standard patient portal design providing patients with the resources to understand and manage their chronic conditions will promote the adoption of patient portals in health care organizations.

A rare presentation of in situ mantle cell lymphoma and follicular lymphoma: a case report and review of the literature.

A 65-year-old gentleman presented with left groin swelling over the course of two months. Physical exam revealed nontender left inguinal adenopathy, and computed tomography scans detected multiple lymph nodes in the mesenteric, aortocaval, and right common iliac regions. An excisional lymph node biopsy was performed. Pathologic evaluation demonstrated follicular center site which stained positive for PAX5, CD20, CD10, Bcl-2, Bcl-6, and mantle zone cells. These findings demonstrated CCND1 and CD5 positivity, suggesting composite lymphoma comprising follicular lymphoma (FL) with in situ mantle cell lymphoma (MCLIS). FL is known as indolent non-Hodgkin lymphoma; however, the clinical significance of a coexisting MCLIS continues to be elusive, and optimal management of these patients remains largely unknown. This case illustrates the diagnostic and therapeutic challenges of composite lymphomas. This paper also discusses advances in molecular pathogenesis and lymphoma genomics which offer novel insights into these rare diseases.

Evaluation of a novel hexavalent humanized anti-IGF-1R antibody and its bivalent parental IgG in diverse cancer cell lines.

A major mechanism of monoclonal antibodies that selectively target the insulin-like growth factor type 1 receptor (IGF-1R) to inhibit tumor growth is by downregulating the receptor, regardless whether they are capable (antagonistic) or incapable (agonistic) of blocking the binding of cognate ligands. We have developed and characterized a novel agonistic anti-IGF-1R humanized antibody, hR1, and used the Dock-and-Lock (DNL) method to construct Hex-hR1, the first multivalent antibody comprising 6 functional Fabs of hR1, with the aim of enhancing potency of hR1. Based on cross-blocking experiments, hR1 recognizes a region of cysteine-rich domain on the α-subunit, different from the epitopes mapped for existing anti-IGF-1R antibodies, yet hR1 is similar to other anti-IGF-1R antibodies in downregulating IGF-1R and inhibiting proliferation, colony formation, or invasion of selected cancer cell lines in vitro, as well as suppressing growth of the RH-30 rhabdomyosarcoma xenograft in nude mice when combined with the mTOR inhibitor, rapamycin. Hex-hR1 and hR1 are generally comparable in their bioactivities under the in-intro and in-vivo conditions investigated. Nevertheless, in selective experiments involving a direct comparison of potency, Hex-hR1 demonstrated a stronger effect on inhibiting cell proliferation stimulated by IGF-1 and could effectively downregulate IGF-1R at a concentration as low as 20 pM.

Efflux pumps of Mycobacterium tuberculosis play a significant role in antituberculosis activity of potential drug candidates.

Active efflux of drugs mediated by efflux pumps that confer drug resistance is one of the mechanisms developed by bacteria to counter the adverse effects of antibiotics and chemicals. To understand these efflux mechanisms in Mycobacterium tuberculosis, we generated knockout (KO) mutants of four efflux pumps of the pathogen belonging to different classes. We measured the MICs and kill values of two different compound classes on the wild type (WT) and the efflux pump (EP) KO mutants in the presence and absence of the efflux inhibitors verapamil and l-phenylalanyl-l-arginyl-ß-naphthylamide (PAßN). Among the pumps studied, the efflux pumps belonging to the ABC (ATP-binding cassette) class, encoded by Rv1218c, and the SMR (small multidrug resistance) class, encoded by Rv3065, appear to play important roles in mediating the efflux of different chemical classes and antibiotics. Efflux pumps encoded by Rv0849 and Rv1258c also mediate the efflux of these compounds, but to a lesser extent. Increased killing is observed in WT M. tuberculosis cells by these compounds in the presence of either verapamil or PAßN. The efflux pump KO mutants were more susceptible to these compounds in the presence of efflux inhibitors. We have shown that these four efflux pumps of M. tuberculosis play a vital role in mediating efflux of different chemical scaffolds. Inhibitors of one or several of these efflux pumps could have a significant impact in the treatment of tuberculosis. The identification and characterization of Rv0849, a new efflux pump belonging to the MFS (major facilitator superfamily) class, are reported.

Rv1218c, an ABC transporter of Mycobacterium tuberculosis with implications in drug discovery.

Efflux systems are important in determining the efficacy of antibiotics used in the treatment of bacterial infections. In the last decade much attention has been paid to studying the efflux pumps of mycobacteria. New classes of compounds are under investigation for development into potential candidate drugs for the treatment of tuberculosis. Quite often, these have poor bactericidal activities but exhibit excellent target (biochemical) inhibition. Microarray studies conducted in our laboratories for deciphering the mode of action of experimental drugs revealed the presence of putative ABC transporters. Among these transporters, Rv1218c was chosen for studying its physiological relevance in mediating efflux in Mycobacterium tuberculosis. A ΔRv1218c mutant of M. tuberculosis displayed a 4- to 8-fold increase in the inhibitory and bactericidal potency for different classes of compounds. The MICs and MBCs were reversed to wild-type values when the full-length Rv1218c gene was reintroduced into the ΔRv1218c mutant on a multicopy plasmid. Most of the compound classes had significantly better bactericidal activity in the ΔRv1218c mutant than in the wild-type H37Rv, suggesting the involvement of Rv1218c gene product in effluxing these compounds from M. tuberculosis. The implication of these findings on tuberculosis drug discovery is discussed.