Field utility of phenolic glycolipid coated latex agglutination test for rapid detection of bacilliferous leprosy cases.

Serum samples were collected from eighty-three leprosy patients and twenty-five healthy controls supposedly not exposed to Mycobacterium leprae infection. Phenolic glycolipid-1 coated latex agglutination test (PGL-LAT) was carried out with the serum samples to detect antibodies specific to M. leprae. Samples showing positive agglutination were 50% in the lepromatous leprosy (LL) group showing no erythema nodosum leprosum (ENL) complications, 66.6% in LL group with ENL complication, 60% in borderline lepromatous (BL) group, 50% in borderline (BB) and 33.3% in borderline tuberculoid (BT). The patients belonging to the tuberculoid (TT) group and most of the long-term treated patients were interestingly negative, and so were sera from all the healthy controls. PGL-LAT developed by us therefore is specific and a fairly sensitive technique to detect antibodies specific to M. leprae and will be very useful in field conditions.     

Disulfide conformation and design at helix N‐termini

To understand structural and thermodynamic features of disulfides within an α‐helix, a non‐redundant dataset comprising of 5025 polypeptide chains containing 2311 disulfides was examined. Thirty‐five examples were found of intrahelical disulfides involving a CXXC motif between the N‐Cap and third helical positions. GLY and PRO were the most common amino acids at positions 1 and 2, respectively. The N‐Cap residue for disulfide bonded CXXC motifs had average (?,ψ) values of (?112 ± 25.2°, 106 ± 25.4°). To further explore conformational requirements for intrahelical disulfides, CYS pairs were introduced at positions N‐Cap‐3; 1,4; 7,10 in two helices of an Escherichia coli thioredoxin mutant lacking its active site disulfide (nSS Trx). In both helices, disulfides formed spontaneously during purification only at positions N‐Cap‐3. Mutant stabilities were characterized by chemical denaturation studies (in both oxidized and reduced states) and differential scanning calorimetry (oxidized state only). All oxidized as well as reduced mutants were destabilized relative to nSS Trx. All mutants were redox active, but showed decreased activity relative to wild‐type thioredoxin. Such engineered disulfides can be used to probe helix start sites in proteins of unknown structure and to introduce redox activity into proteins. Conversely, a protein with CYS residues at positions N‐Cap and 3 of an α‐helix is likely to have redox activity. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

DNA site-specific N3-adenine methylation targeted to estrogen receptor-positive cells

A compound that can target cells expressing the estrogen receptor (ER), and produce predominantly 3-MeA adducts in those cells has been designed and synthesized. This compound produces mainly the 3-MeA adduct upon reaction with calf thymus DNA, and binds to the ER with a relative binding affinity of 51% (estradiol = 100%). The compound is toxic to ER-expressing MCF-7 breast cancer cells, and pre-treatment with the ER antagonist fulvestrant abrogates the toxicity. Pre-treatment of MCF-7 cells with netropsin, which inhibits N3-adenine methylation by the compound, resulted in a threefold decrease in the toxicity. These results demonstrate the feasibility of this strategy for producing 3-MeA adducts in targeted cells.     

A genome-wide RNAi screen identifies novel targets of neratinib sensitivity leading to neratinib and paclitaxel combination drug treatments

ErbB2 is frequently activated in tumors, and influences a wide array of cellular functions, including proliferation, apoptosis, cell motility and adhesion. HKI-272 (neratinib) is a small molecule pan-kinase inhibitor of the ErbB family of receptor tyrosine kinases, and shows strong antiproliferative activity in ErbB2-overexpressing breast cancer cells. We undertook a genome-wide pooled lentiviral RNAi screen to identify synthetic lethal or enhancer (synthetic modulator screen) genes that interact with neratinib in a human breast cancer cell line (SKBR-3). These genes upon knockdown would modulate cell viability in the presence of subeffective concentrations of neratinib. We discovered a diverse set of genes whose depletion selectively impaired or enhanced the viability of SKBR-3 cells in the presence of neratinib. We observed diverse pathways including EGFR, hypoxia, cAMP, and protein ubiquitination that, when co-treated with RNAi and neratinib, resulted in arrest of cell proliferation. Examining the changes of these genes and their protein products also led to a rationale for clinically relevant drug combination treatments. Treatment of cells with either paclitaxel or cytarabine in combination with neratinib resulted in a strong antiproliferative effect. The identification of novel mediators of cellular response to neratinib and the development of potential drug combination treatments have expanded our understanding of neratinib's mode-of-action for the development of more effective therapeutic regimens. Notably, our findings support a paclitaxel and neratinib phase III clinical trial in breast cancer patients.     

Global gene disruption in human cells to assign genes to phenotypes by deep sequencing

Insertional mutagenesis in a haploid background can disrupt gene function. We extend our earlier work by using a retroviral gene-trap vector to generate insertions in >98% of the genes expressed in a human cancer cell line that is haploid for all but one of its chromosomes. We apply phenotypic interrogation via tag sequencing (PhITSeq) to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of cells, rather than individual clones enables rapid assessment of the spectrum of genes involved in the phenotypes under study. This facilitates comparative screens as illustrated here for the family of cytolethal distending toxins (CDTs). CDTs are virulence factors secreted by a variety of pathogenic Gram-negative bacteria responsible for tissue damage at distinct anatomical sites. We identify 743 mutations distributed over 12 human genes important for intoxication by four different CDTs. Although related CDTs may share host factors, they also exploit unique host factors to yield a profile characteristic for each CDT.     

Conformational changes of the chaperone SecB upon binding to a model substrate--bovine pancreatic trypsin inhibitor (BPTI)

SecB is a homotetrameric cytosolic chaperone that forms part of the protein translocation machinery in E. coli. Due to SecB, nascent polypeptides are maintained in an unfolded translocation-competent state devoid of tertiary structure and thus are guided to the translocon. In vitro SecB rapidly binds to a variety of ligands in a non-native state. We have previously investigated the bound state conformation of the model substrate bovine pancreatic trypsin inhibitor (BPTI) as well as the conformation of SecB itself by using proximity relationships based on site-directed spin labeling and pyrene fluorescence methods. It was shown that SecB undergoes a conformational change during the process of substrate binding. Here, we generated SecB mutants containing but a single cysteine per subunit or an exposed highly reactive new cysteine after removal of the nearby intrinsic cysteines. Quantitative spin labeling was achieved with the methanethiosulfonate spin label (MTS) at positions C97 or E90C, respectively. Highfield (W-band) electron paramagnetic resonance (EPR) measurements revealed that with BPTI present the spin labels are exposed to a more polar/hydrophilic environment. Nanoscale distance measurements with double electron-electron resonance (DEER) were in excellent agreement with distances obtained by molecular modeling. Binding of BPTI also led to a slight change in distances between labels at C97 but not at E90C. While the shorter distance in the tetramer increased, the larger diagonal distance decreased. These findings can be explained by a widening of the tetrameric structure upon substrate binding much like the opening of two pairs of scissors.     

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials^1-4. There are several advantages to using CAR⁺ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR⁺ T cells in the event of host toxicity⁵.Delineating the optimal functions of CAR⁺ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo^6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets⁸.Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes⁹. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.     

Expression of cell surface zinc transporter LIV1 in triple negative breast cancer is an indicator of poor prognosis and therapy failure

Triple negative breast cancers (TNBC) are an aggressive molecular subtype of breast carcinoma (BC) identified by the lack of receptor expression for estrogen, progesterone, & human epidermal growth factor receptor-2. Lack of tangible drug targets warrants further research in TNBC. LIV1, is a zinc (Zn) transporter known to be overexpressed in few cancer types including BCs. Recently, in the United States of America, FDA approved the use of a new drug targeting LIV1, antibody drug conjugate SGN-LIV1A for treatment of TNBC patients. Though LIV1 also has a role in modulating immune cells by its differential transport of Zn, a correlation between the tumor cell expression of LIV1 and immune cell infiltrations were scantily reported. Further adequate baseline data on LIV1 expression in other populations have not been documented. Our objective was to screen a large Indian cohort of TNBC patient samples for LIV1, categorize the immune cell infiltration using CD4/CD8 expression and correlate the findings with therapy outcomes. Further, we also investigated for LIV1 expression in matched samples of primary & secondary tumors; pre & postchemotherapy in TNBC patients. Results showed an elevated expression of LIV1 in TNBC samples as compared to adjacent normal, the mean Q scores being 183.06 ± 6.39 and 120.78 ± 7.37 (p < 0.0001), respectively. Similarly, LIV1 levels were elevated in secondary tumors than primary & in patient samples postchemotherapy as compared to naïve. In the TNBC cohort, using automated method, cell morphology parameters were computed and analysis showed LIV1 levels were elevated in grade 3 TNBC samples presenting with altered cell morphology parameters namely cell size, cell perimeter, & nucleus size. Thus indicating LIV1 expressing TNBC samples portrayed an aggressive phenotype. Finally, TNBC patients with 3+ staining intensity showed poor survival (4.44 year) as compared to patients with 2+ LIV1 expression (5.47 year), emphasizing that LIV1 expression is a poor prognostic factor in TNBC. In conclusion, the study reports elevated expression of LIV1 in a large Indian TNBC cohort; high expression is a poor prognostic factor and correlated with aggressive disease and indicating the need for LIV1 targeted therapies.