Multiple Approaches to Novel Rifamycin Analogs to Combat MDR-TB

According to the Center for Disease Control and Prevention (CDC), in 2015 10.4 million people worldwide became infected with tuberculosis (TB) with 1.8 million TB related deaths. Tuberculosis is a bacterial infectious disease caused by Mycobacterium tuberculosis, a slow growing yet highly infectious bacterium. Among the first line of treatments for TB is the antibiotic rifampin, a synthetic derivative of the natural product rifamycin B. Rifampin selectively inhibits bacterial RNA polymerase by binding to its β-subunit, effectively inhibiting protein synthesis leading to cell death. However, recently the development of multidrug resistant (MDR) and extensively drug resistant (XDR) TB has reduced the effectiveness of commercially available treatments, including rifampin. Bacterial resistance to rifampin is mainly due to mutations in the target protein RNA polymerase. Through a separate bio-computational modeling study, it was hypothesized that making a slight change to the rifamycin backbone may lead to a novel rifamycin analog that more effectively binds and inhibits mutated RNA polymerase. The suggested change, based on the bio-computational modeling study, is the removal of the methyl on C-7, lessening molecular steric hindrance. Methods focused initially on the genetic re-engineering of the bacterial producer of rifamycin, Amycolatopsis mediterranei S699, through the modification of the biosynthetic machinery. This can be achieved by replacing the acyltransferase (AT) domain of module 1 of the rif-PKS (which recognizes methylmalonyl-CoA as the substrate) with the AT domain of module 2 of the rapamycin PKS (which recognizes malonyl-CoA) using a double crossover homologous recombination. However, there were some issues in the transformation of the final construct into the competent A. mediterranei cells leading to the exploration of another approach. By observation, it was recognized that old plates of Amycolatopsis mediterranei S699 began to show evidence of different metabolites, one of which resembled, in mass spectrometry measurements, that of a desmethylrifamycin SV. In order to test this, cells were starved on different media and then tested at various points in time for metabolite production. After analysis, the compound of interest was identified to be 12-desmethylrifamycin SV. This compound did show significant activity against M. tuberculosis, but not against drug resistant forms. The selective removal of the methyl group on C-12 is most likely due to enzyme catalysis. There are two genes, which encode cytochrome P450 monooxygenases (rif-orf13 and rif-orf16), within the rifamycin biosynthetic gene cluster that may be responsible for the oxidation and removal of the methyl group. Therefore, both of them were cloned and heterologously expressed in Escherichia coli. However, the proteins were found to be insoluble. Therefore, detailed investigations of these proteins cannot be done at this point in time, and will have to wait until soluble proteins are available. Future work would include refinement of the heterologous expression methodology and full characterization of Orf13 and Orf16 to determine their involvement in the formation of 12-desmethylrifamycin SV. Keywords: genetics, drug development, tuberculosis, multi-drug resistanc

Isolation and ultrastructural characterization of squid synaptic vesicles

Author Posting. © Marine Biological Laboratory, 2011. This article is posted here by permission of Marine Biological Laboratory for personal use, not for redistribution. The definitive version was published in Biological Bulletin 220 (2011): 89-96.Synaptic vesicles contain a variety of proteins and lipids that mediate fusion with the pre-synaptic membrane. Although the structures of many synaptic vesicle proteins are known, an overall picture of how they are organized at the vesicle surface is lacking. In this paper, we describe a better method for the isolation of squid synaptic vesicles and characterize the results. For highly pure and intact synaptic vesicles from squid optic lobe, glycerol density gradient centrifugation was the key step. Different electron microscopic methods show that vesicle membrane surfaces are largely covered with structures corresponding to surface proteins. Each vesicle contains several stalked globular structures that extend from the vesicle surface and are consistent with the V-ATPase. BLAST search of a library of squid expressed sequence tags identifies 10 V-ATPase subunits, which are expressed in the squid stellate ganglia. Negative-stain tomography demonstrates directly that vesicles flatten during the drying step of negative staining, and furthermore shows details of individual vesicles and other proteins at the vesicle surface.JAD is supported by the RI-INBRE program award # P20RR016457-10 from the National Center for Research Resources (NCRR), NIH

Ultrasonic Wave Propagation Assessment of Native Cartilage Explants and Hydrogel Scaffolds for Tissue Engineering

Non-destructive techniques characterising the mechanical properties of cells, tissues, and biomaterials provide baseline metrics for tissue engineering design. Ultrasonic wave propagation and attenuation has previously demonstrated the dynamics of extracellular matrix synthesis in chondrocyte-seeded hydrogel constructs. In this paper, we describe an ultrasonic method to analyse two of the construct elements used to engineer articular cartilage in real-time, native cartilage explants and an agarose biomaterial. Results indicated a similarity in wave propagation velocity ranges for both longitudinal (1500-1745 m/s) and transverse (350-950 m/s) waveforms. Future work will apply an acoustoelastic analysis to distinguish between the fluid and solid properties including the cell and matrix biokinetics as a validation of previous mathematical models

Monitoring Temporal Changes in the Specificity of an Oral HIV Test: A Novel Application for Use in Postmarketing Surveillance

BACKGROUND: Postmarketing surveillance is routinely conducted to monitor performance of pharmaceuticals and testing devices in the marketplace. However, these surveillance methods are often done retrospectively and, as a result, are not designed to detect issues with performance in real-time. METHODS AND FINDINGS: Using HIV antibody screening test data from New York City STD clinics, we developed a formal, statistical method of prospectively detecting temporal clusters of poor performance of a screening test. From 2005 to 2008, New York City, as well as other states, observed unexpectedly high false-positive (FP) rates in an oral fluid-based rapid test used for screening HIV. We attempted to formally assess whether the performance of this HIV screening test statistically deviated from both local expectation and the manufacturer's claim for the test. Results indicate that there were two significant temporal clusters in the FP rate of the oral HIV test, both of which exceeded the manufacturer's upper limit of the 95% CI for the product. Furthermore, the FP rate of the test varied significantly by both STD clinic and test lot, though not by test operator. CONCLUSIONS: Continuous monitoring of surveillance data has the benefit of providing information regarding test performance, and if conducted in real-time, it can enable programs to examine reasons for poor test performance in close proximity to the occurrence. Techniques used in this study could be a valuable addition for postmarketing surveillance of test performance and may become particularly important with the increase in rapid testing methods

A Membrane-Bound Vertebrate Globin

The family of vertebrate globins includes hemoglobin, myoglobin, and other O2-binding proteins of yet unclear functions. Among these, globin X is restricted to fish and amphibians. Zebrafish (Danio rerio) globin X is expressed at low levels in neurons of the central nervous system and appears to be associated with the sensory system. The protein harbors a unique N-terminal extension with putative N-myristoylation and S-palmitoylation sites, suggesting membrane-association. Intracellular localization and transport of globin X was studied in 3T3 cells employing green fluorescence protein fusion constructs. Both myristoylation and palmitoylation sites are required for correct targeting and membrane localization of globin X. To the best of our knowledge, this is the first time that a vertebrate globin has been identified as component of the cell membrane. Globin X has a hexacoordinate binding scheme and displays cooperative O2 binding with a variable affinity (P50∼1.3–12.5 torr), depending on buffer conditions. A respiratory function of globin X is unlikely, but analogous to some prokaryotic membrane-globins it may either protect the lipids in cell membrane from oxidation or may act as a redox-sensing or signaling protein

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Short-term low-severity spring grassland fire impacts on soil extractable elements and soil ratios in Lithuania.

Spring grassland fires are common in boreal areas as a consequence of slash and burn agriculture used to remove dry grass to increase soil nutrient properties and crop production. However, fewworks have investigated fire impacts on these grassland ecosystems, especially in the immediate period after the fire. The objective of this work was to study the short-termimpacts of a spring grassland fire in Lithuania. Four days after the firewe established a 400 m2 sampling grid within the burned area and in an adjacent unburned area with the same topographical, hydrological and pedological characteristics. Wecollected topsoil samples immediately after the fire (0 months), 2, 5, 7 and 9 months after the fire. We analysed soil pH, electrical conductivity (EC), major nutrients including calcium(Ca), magnesium(Mg), sodium(Na), and potassium(K), and theminor elements aluminium(Al), manganese (Mn), iron (Fe) and zinc (Zn). We also calculated the soil Na and K adsorption ratio (SPAR), Ca:Mg and Ca:Al. The results showed that this low-severity grassland fire significantly decreased soil pH, Al, and Mn but increased EC, Ca,Mg, and K,. There was no effect on Na, Fe, and Zn. Therewas a decrease of EC, Ca,Mg, and Na from 0months after the fire until 7 months after the fire,with an increase during the last sampling period. Fire did not significantly affect SPAR. Ca:Mg decreased significantly immediately after the fire, but not to critical levels. Ca:Al increased after the fire, reducing the potential effects of Al on plants. Overall, fire impactsweremainly limited to the immediate period after the fire

Expansion and Characterization of Human Melanoma Tumor-Infiltrating Lymphocytes (TILs)

Various immunotherapeutic strategies for cancer are aimed at augmenting the T cell response against tumor cells. Adoptive cell therapy (ACT), where T cells are manipulated ex vivo and subsequently re-infused in an autologous manner, has been performed using T cells from various sources. Some of the highest clinical response rates for metastatic melanoma have been reported in trials using tumor-infiltrating lymphocytes (TILs). These protocols still have room for improvement and furthermore are currently only performed at a limited number of institutions. The goal of this work was to develop TILs as a therapeutic product at our institution.TILs from 40 melanoma tissue specimens were expanded and characterized. Under optimized culture conditions, 72% of specimens yielded rapidly proliferating TILs as defined as at least one culture reaching ≥3×10(7) TILs within 4 weeks. Flow cytometric analyses showed that cultures were predominantly CD3+ T cells, with highly variable CD4+:CD8+ T cell ratios. In total, 148 independent bulk TIL cultures were assayed for tumor reactivity. Thirty-four percent (50/148) exhibited tumor reactivity based on IFN-γ production and/or cytotoxic activity. Thirteen percent (19/148) showed specific cytotoxic activity but not IFN-γ production and only 1% (2/148) showed specific IFN-γ production but not cytotoxic activity. Further expansion of TILs using a 14-day "rapid expansion protocol" (REP) is required to induce a 500- to 2000-fold expansion of TILs in order to generate sufficient numbers of cells for current ACT protocols. Thirty-eight consecutive test REPs were performed with an average 1865-fold expansion (+/- 1034-fold) after 14 days.TILs generally expanded efficiently and tumor reactivity could be detected in vitro. These preclinical data from melanoma TILs lay the groundwork for clinical trials of ACT