Characterization of the MUC1-C Cytoplasmic Domain as a Cancer Target

Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly expressed in diverse human carcinomas and certain hematologic malignancies. The oncogenic MUC1 transmembrane C-terminal subunit (MUC1-C) functions in part by transducing growth and survival signals from cell surface receptors. However, little is known about the structure of the MUC1-C cytoplasmic domain as a potential drug target. Using methods for structural predictions, our results indicate that a highly conserved CQCRRK sequence, which is adjacent to the cell membrane, forms a small pocket that exposes the two cysteine residues for forming disulfide bonds. By contrast, the remainder of the MUC1-C cytoplasmic domain has no apparent structure, consistent with an intrinsically disordered protein. Our studies thus focused on targeting the MUC1 CQCRRK region. The results show that L- and D-amino acid CQCRRK-containing peptides bind directly to the CQC motif. We further show that the D-amino acid peptide, designated GO-203, blocks homodimerization of the MUC1-C cytoplasmic domain in vitro and in transfected cells. Moreover, GO-203 binds directly to endogenous MUC1-C in breast and lung cancer cells. Colocalization studies further demonstrate that GO-203 predominantly binds to MUC1-C at the cell membrane. These findings support the further development of agents that target the MUC1-C cytoplasmic domain CQC motif and thereby MUC1-C function in cancer cells.     

DJ-1 Protects Pancreatic Beta Cells from Cytokine- and Streptozotocin-Mediated Cell Death

A hallmark feature of type 1 and type 2 diabetes mellitus is the progressive dysfunction and loss of insulin-producing pancreatic beta cells, and inflammatory cytokines are known to trigger beta cell death. Here we asked whether the anti-oxidant protein DJ-1 encoded by the Parkinson’s disease gene PARK7 protects islet cells from cytokine- and streptozotocin-mediated cell death. Wild type and DJ-1 knockout mice (KO) were treated with multiple low doses of streptozotocin (MLDS) to induce inflammatory beta cell stress and cell death. Subsequently, glucose tolerance tests were performed, and plasma insulin as well as fasting and random blood glucose concentrations were monitored. Mitochondrial morphology and number of insulin granules were quantified in beta cells. Moreover, islet cell damage was determined in vitro after streptozotocin and cytokine treatment of isolated wild type and DJ-1 KO islets using calcein AM/ethidium homodimer-1 staining and TUNEL staining. Compared to wild type mice, DJ-1 KO mice became diabetic following MLDS treatment. Insulin concentrations were substantially reduced, and fasting blood glucose concentrations were significantly higher in MLDS-treated DJ-1 KO mice compared to equally treated wild type mice. Rates of beta cell apoptosis upon MLDS treatment were twofold higher in DJ-1 KO mice compared to wild type mice, and in vitro inflammatory cytokines led to twice as much beta cell death in pancreatic islets from DJ-1 KO mice versus those of wild type mice. In conclusion, this study identified the anti-oxidant protein DJ-1 as being capable of protecting pancreatic islet cells from cell death induced by an inflammatory and cytotoxic setting.     

Generating Recombinant Antibodies against Putative Biomarkers of Retinal Injury

Candidate biomarkers, indicative of disease or injury, are beginning to overwhelm the process of validation through immunological means. Recombinant antibodies developed through phage-display offer an alternative means of generating monoclonal antibodies faster than traditional immunization of animals. Peptide segments of putative biomarkers of laser induced injury in the rabbit, discovered through mass spectrometry, were used as targets for a selection against a library of phage-displayed human single-chain variable fragment (scFv) antibodies. Highly specific antibodies were isolated to four of these unique peptide sequences. One antibody against the retinal protein, Guanine Nucleotide-Binding Protein Beta 5 (GBB5), had a dissociation constant ~300 nM and recognized the full-length endogenous protein in retinal homogenates of three different animal species by western blot. Alanine scanning of the peptide target identified three charged and one hydrophobic amino acid as the critical binding residues for two different scFvs. To enhance the utility of the reagent, one scFv was dimerized through a Fragment-crystallizable hinge region (i.e., Fc) and expressed in HEK-293 cells. This dimeric reagent yielded a 25-fold lower detection limit in western blots.     

The Multi-Drug Resistant Tuberculosis Diagnosis and Treatment Cascade in Bangladesh

Objectives

To determine, in areas supported by BRAC, Bangladesh i) the pre-diagnosis and pre-treatment attrition among presumptive and confirmed Multi-Drug Resistant Tuberculosis (MDR-TB) patients and ii) factors associated with attrition.

Methods

This was a retrospective cohort study involving record review. Presumptive MDR-TB patients from peripheral microscopy centres serving 60% of the total population of Bangladesh were included in the study. Attrition and turnaround time for MDR-TB diagnosis by Xpert MTB/RIF and treatment initiation were calculated between July 2012 and June 2014.

Results

Of 836 presumptive MDR-TB patients referred from 398 peripheral microscopy centres, 161 MDR-TB patients were diagnosed. The number of diagnosed MDR-TB patients was less than country estimates of MDR-TB patients (2000 cases) during the study period. Among those referred, pre-diagnosis and pre-treatment attrition was 17% and 21% respectively. Median turnaround time for MDR-TB testing, result receipt and treatment initiation was four, zero and five days respectively. Farmers (RR=2.3, p=0.01) and daily wage laborers (RR=2.1, p=0.04) had twice the risk of having pre-diagnosis attrition. Poor record-keeping and unreliable upkeep of presumptive MDR-TB patient databases were identified as challenges at the peripheral microscopy centres.

Conclusion

There was a low proportion of pre-diagnosis and pre-treatment attrition in patients with presumptive and confirmed MDR-TB under programmatic conditions. However, the recording and reporting system did not detect all presumptive MDR-TB patients, highlighting the need to improve the system in order to prevent morbidity, mortality and transmission of MDR-TB in the community.     

Expression,purification and substrate specificities of 3-nitrotoluene dioxygenase from Diaphorobacter sp. strain DS2

3-Nitotoluene dioxygenase (3-NTDO) is the first enzyme in the degradation pathway of 3-nitrotoluene (3-NT) by Diaphorobacter sp. strain DS2. The complete gene sequences of 3-NTDO were PCR amplified from genomic DNA of Diaphorobacter sp., cloned, sequenced and expressed. The 3-NTDO gene revealed a multi component structure having a reductase, a ferredoxin and two oxygenase subunits. Clones expressing the different subunits were constructed in pET21a expression vector system and overexpressed in E. coli BL21(DE3) host. Each subunit was individually purified separately to homogeneity. The active recombinant enzyme was reconstituted in vitro by mixing all three purified subunits. The reconstituted recombinant enzyme could catalyse biotransformations on a variety of organic aromatics.     

TLR4‐mediated skin carcinogenesis is dependent on immune and radioresistant cells

Skin cancers are the most commonly diagnosed cancers. Understanding what are the factors contributing to skin tumour development can be instrumental to identify preventive therapies. The myeloid differentiation primary response gene (MyD)88, the downstream adaptor protein of most Toll‐like receptors (TLR), has been shown to be involved in several mouse tumourigenesis models. We show here that TLR4, but not TLR2 or TLR9, is upstream of MyD88 in skin tumourigenesis. TLR4 triggering is not dependent on lipopolysaccharide associated to skin‐colonizing bacteria, but on the high mobility group box‐1 protein (HMGB1), an endogenous ligand of TLR4. HMGB1 is released by necrotic keratinocytes and is required for the recruitment of inflammatory cells and for the initiation of inflammation. The expression of TLR4 on both bone marrow‐derived and radioresistant cells is necessary for carcinogenesis. Consistently, a human tissue microarray analysis showed that melanoma and colon cancer display an over‐expression of TLR4 and its downstream adaptor protein MyD88 within tumours. Together, our results suggest that the initial release of HMGB1 triggers a TLR4‐dependent inflammatory response that leads to tumour development.     

Synthesis, cytotoxicity, and structure-activity relationship (SAR) studies of andrographolide analogues as anti-cancer agent

A series of analogues of andrographolide, prepared through chemo-selective functionalization at C14 hydroxy, have been evaluated for in vitro cytotoxicities against human leukemic cell lines. Two of the analogues (6a, 9b) exhibited significant potency. Preliminary studies on structure-activity relationship (SAR) revealed that the α-alkylidene-γ-butyrolactone moiety of andrographolide played a major role in the activity profile. The structures of the analogues were established through spectroscopic and analytical data.     

Azaindole N-methyl hydroxamic acids as HIV-1 integrase inhibitors-II. The impact of physicochemical properties on ADME and PK

HIV-1 integrase is one of three enzymes encoded by the HIV genome and is essential for viral replication, and HIV-1 IN inhibitors have emerged as a new promising class of therapeutics. Recently, we reported the discovery of azaindole hydroxamic acids that were potent inhibitors of the HIV-1 IN enzyme. N-Methyl hydroxamic acids were stable against oxidative metabolism, however were cleared rapidly through phase 2 glucuronidation pathways. We were able to introduce polar groups at the β-position of the azaindole core thereby altering physical properties by lowering calculated log D values (c Log D) which resulted in attenuated clearance rates in human hepatocytes. Pharmacokinetic data in dog for representative compounds demonstrated moderate oral bioavailability and reasonable half-lives. These ends were accomplished without a large negative impact on enzymatic and antiviral activity, thus suggesting opportunities to alter clearance parameters in future series.     

Overexpression of γ-tocopherol methyl transferase gene in transgenic Brassica juncea plants alleviates abiotic stress: Physiological and chlorophyll a fluorescence measurements

Tocopherols (vitamin E) are lipid soluble antioxidants synthesized by plants and some cyanobacteria. We have earlier reported that overexpression of the γ-tocopherol methyl transferase (γ-TMT) gene from Arabidopsis thaliana in transgenic Brassica juncea plants resulted in an over six-fold increase in the level of α-tocopherol, the most active form of all the tocopherols. Tocopherol levels have been shown to increase in response to a variety of abiotic stresses. In the present study on Brassica juncea, we found that salt, heavy metal and osmotic stress induced an increase in the total tocopherol levels. Measurements of seed germination, shoot growth and leaf disc senescence showed that transgenic Brassica juncea plants overexpressing the γ-TMT gene had enhanced tolerance to the induced stresses. Analysis of the chlorophyll a fluorescence rise kinetics, from the initial “O” level to the “P” (the peak) level, showed that there were differential effects of the applied stresses on different sites of the photosynthetic machinery; further, these effects were alleviated in the transgenic (line 16.1) Brassica juncea plants. We show that α-tocopherol plays an important role in the alleviation of stress induced by salt, heavy metal and osmoticum in Brassica juncea.