Maneesh Pingle

Nonsteroidal anti-inflammatory drug sensitizes Mycobacterium tuberculosis to endogenous and exogenous antimicrobials

Existing drugs are slow to eradicate Mycobacterium tuberculosis (Mtb) in patients and have failed to control tuberculosis globally. One reason may be that host conditions impair Mtb’s replication, reducing its sensitivity to most antiinfectives. We devised a high-throughput screen for compounds that kill Mtb when its replication has been halted by reactive nitrogen intermediates (RNIs), acid, hypoxia, and a fatty acid carbon source. At concentrations routinely achieved in human blood, oxyphenbutazone (OPB), an inexpensive anti-inflammatory drug, was selectively mycobactericidal to nonreplicating (NR) Mtb. Its cidal activity depended on mild acid and was augmented by RNIs and fatty acid. Acid and RNIs fostered OPB’s 4-hydroxylation. The resultant 4-butyl-4-hydroxy-1-(4-hydroxyphenyl)-2-phenylpyrazolidine-3,5-dione (4-OH-OPB) killed both replicating and NR Mtb, including Mtb resistant to standard drugs. 4-OH-OPB depleted flavins and formed covalent adducts with N-acetyl-cysteine and mycothiol. 4-OH-OPB killed Mtb synergistically with oxidants and several antituberculosis drugs. Thus, conditions that block Mtb’s replication modify OPB and enhance its cidal action. Modified OPB kills both replicating and NR Mtb and sensitizes both to host-derived and medicinal antimycobacterial agents.

Self-assembly of micro- and nano-scale particles using bio-inspired events

The head and foot ends of a mattress supporting frame or operating table are articulately connected with the piston rods of two hydraulic motors whose double-acting cylinders are articulately connected to a shiftable bottom frame. The lower chambers of the cylinders can be connected with a supply conduit for pressurized fluid by way of discrete working conduits and discrete solenoid operated directional control valves. The upper chambers of the two cylinders are connected to each other by a first control conduit and the first control conduit is connected with the working conduit for the cylinder at the head end by means of a second control conduit. The supply conduit receives pressurized fluid from a single pump and each valve is further connected with a return conduit for spent fluid. The valve members of the two valves are movable independently of each other between first or neutral positions in which they seal the supply conduit and the return conduit from the respective working conduits, second positions in which they connect the supply conduit with the respective working conduits, and third positions in which they connect the return conduit with the respective working conduits. A solenoid operated shutoff valve can be installed in the working conduit for the cylinder at the head end to seal the lower chamber of such cylinder from the supply and return conduits as well as from the respective working conduit and both control conduits when the foot end is to be raised or lowered without any changes in the level of the head end. The single pump can supply pressurized fluid to the motors for one or more additional frames or tables as well as to auxiliary cylinders which can move the head rest and/or the foot rest of a frame or table between different levels and/or different positions of inclination.

Multiplexed Identification of Blood-Borne Bacterial Pathogens by Use of a Novel 16S rRNA Gene PCR-Ligase Detection Reaction-Capillary Electrophoresis Assay

ABSTRACT We have developed a novel high-throughput PCR-ligase detection reaction-capillary electrophoresis (PCR-LDR-CE) assay for the multiplexed identification of 20 blood-borne pathogens (Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Acinetobacter baumannii, Neisseria meningitidis, Bacteroides fragilis, Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Brucella abortus), the last four of which are biothreat agents. The method relies on the amplification of two regions within the bacterial 16S rRNA gene, using universal PCR primers and querying the identity of specific single-nucleotide polymorphisms within the amplified regions in a subsequent LDR. The ligation products vary in color and size and are separated by CE. Each organism generates a specific pattern of ligation products, which can be used to distinguish the pathogens using an automated software program we developed for that purpose. The assay has been verified on 315 clinical isolates and demonstrated a detection sensitivity of 98%. Additionally, 484 seeded blood cultures were tested, with a detection sensitivity of 97.7%. The ability to identify geographically variant strains of the organisms was determined by testing 132 isolates obtained from across the United States. In summary, the PCR-LDR-CE assay can successfully identify, in a multiplexed fashion, a panel of 20 blood-borne pathogens with high sensitivity and specificity.

Detection and Serotyping of Dengue Virus in Serum Samples by Multiplex Reverse Transcriptase PCR-Ligase Detection Reaction Assay

ABSTRACT The detection and successful typing of dengue virus (DENV) from patients with suspected dengue fever is important both for the diagnosis of the disease and for the implementation of epidemiologic control measures. A technique for the multiplex detection and typing of DENV serotypes 1 to 4 (DENV-1 to DENV-4) from clinical samples by PCR-ligase detection reaction (LDR) has been developed. A serotype-specific PCR amplifies the regions of genes C and E simultaneously. The two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The assay was optimized using 38 DENV strains and was evaluated with 350 archived acute-phase serum samples. The sensitivity of the assay was 98.7%, and its specificity was 98.4%, relative to the results of real-time PCR. The detection threshold was 0.017 PFU for DENV-1, 0.004 PFU for DENV-2, 0.8 PFU for DENV-3, and 0.7 PFU for DENV-4. The assay is specific; it does not cross-react with the other flaviviruses tested (West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, and yellow fever virus). All but 1 of 26 genotypic variants of DENV serotypes in a global DENV panel from different geographic regions were successfully identified. The PCR-LDR assay is a rapid, sensitive, specific, and high-throughput technique for the simultaneous detection of all four serotypes of DENV.

Development of Multiplex PCR-Ligase Detection Reaction Assay for Detection of West Nile Virus

ABSTRACT We have developed a novel multiplex reverse transcription-PCR ligase detection reaction (RT-PCR/LDR) assay for the detection of West Nile virus (WNV) in both clinical and mosquito pool samples. The method relies on the amplification of three different genomic regions, one in the coding sequence of nonstructural protein NS2a and two in nonstructural protein NS5, to minimize the risk of detection failure due to genetic variation. The sensitivity of the PCR is complemented by the high specificity of the LDR step, and the detection of the LDR products can be achieved with capillary electrophoresis (CE) or a universal DNA microarray. We evaluated the limit of detection by both one-step and two-step multiplex RT-PCR/LDR/CE approaches, which reached, respectively, 0.005 and 0.017 PFU. The assay demonstrated 99% sensitivity when mosquito pool samples were tested and 100% sensitivity with clinical samples when the one-step approach was used. The broad strain coverage was confirmed by testing 34 WNV isolates belonging to lineages 1 and 2, and the high specificity of the assay was determined by testing other flaviviruses, as well as negative mosquito pool and clinical samples. In summary, the multiplex RT-PCR/LDR assay could represent a valuable complement to WNV serological diagnosis, especially in early symptomatic patients. In addition, the multiplexing capacity of the technique, which can be coupled to universal DNA microarray detection, makes it an amenable tool to develop a more comprehensive assay for viral pathogens.

Synthetic Calanolides with Bactericidal Activity against Replicating and Nonreplicating Mycobacterium tuberculosis

It is urgent to introduce new drugs for tuberculosis to shorten the prolonged course of treatment and control drug-resistant Mycobacterium tuberculosis (Mtb). One strategy toward this goal is to develop antibiotics that eradicate both replicating (R) and nonreplicating (NR) Mtb. Naturally occurring (+)-calanolide A was active against R-Mtb. The present report details the design, synthesis, antimycobacterial activities, and structure-activity relationships of synthetic calanolides. We identified potent dual-active nitro-containing calanolides with minimal in vitro toxicity that were cidal to axenic Mtb and Mtb in human macrophages, while sparing Gram-positive and -negative bacteria and yeast. Two of the nitrobenzofuran-containing lead compounds were found to be genotoxic to mammalian cells. Although genotoxicity precluded clinical progression, the profound, selective mycobactericidal activity of these calanolides will be useful in identifying pathways for killing both R- and NR-Mtb, as well as in further structure-based design of more effective and drug-like antimycobacterial agents.

Ligase Detection Reaction Generation of Reverse Molecular Beacons for Near Real-Time Analysis of Bacterial Pathogens Using Single-Pair Fluorescence Resonance Energy Transfer and a Cyclic Olefin Copolymer Microfluidic Chip

Detection of pathogenic bacteria and viruses require strategies that can signal the presence of these targets in near real-time due to the potential threats created by rapid dissemination into water and/or food supplies. In this paper, we report an innovative strategy that can rapidly detect bacterial pathogens using reporter sequences found in their genome without requiring polymerase chain reaction (PCR). A pair of strain-specific primers was designed based on the 16S rRNA gene and were end-labeled with a donor (Cy5) or acceptor (Cy5.5) dye. In the presence of the target bacterium, the primers were joined using a ligase detection reaction (LDR) only when the primers were completely complementary to the target sequence to form a reverse molecular beacon (rMB), thus bringing Cy5 (donor) and Cy5.5 (acceptor) into close proximity to allow fluorescence resonance energy transfer (FRET) to occur. These rMBs were subsequently analyzed using single-molecule detection of the FRET pairs (single-pair FRET; spFRET). The LDR was performed using a continuous flow thermal cycling process configured in a cyclic olefin copolymer (COC) microfluidic device using either 2 or 20 thermal cycles. Single-molecule photon bursts from the resulting rMBs were detected on-chip and registered using a simple laser-induced fluorescence (LIF) instrument. The spFRET signatures from the target pathogens were reported in as little as 2.6 min using spFRET.

Benzimidazole-based compounds kill Mycobacterium tuberculosis

Tuberculosis remains one of the deadliest infectious diseases, killing 1.4 million people annually and showing a rapid increase in cases resistant to multiple drugs. New antibiotics against tuberculosis are urgently needed. Here we describe the design, synthesis and structure-activity relationships of a series of benzimidazole-based compounds with activity against Mycobacterium tuberculosis (Mtb) in a replicating state, a physiologically-induced non-replicating state, or both. Compounds 49, 67, 68, 69, 70, and 72, which shared a 5-nitrofuranyl moiety, exhibited high potency and acceptable selectivity indices (SI). As illustrated by compound 70 (MIC90 < 0.049 μg/mL, SI > 512), the 5-nitrofuranyl group was compatible with minimal cytotoxicity and good intra-macrophage killing, although it lacked non-replicating activity when assessed by CFU assays. Compound 70 had low mutagenic potential by SOS Chromotest assay, making this class of compounds good candidates for further evaluation and target identification.

Modular microfluidic system fabricated in thermoplastics for the strain-specific detection of bacterial pathogens

The recent outbreaks of a lethal E. coli strain in Germany have aroused renewed interest in developing rapid, specific and accurate systems for detecting and characterizing bacterial pathogens in suspected contaminated food and/or water supplies. To address this need, we have designed, fabricated and tested an integrated modular-based microfluidic system and the accompanying assay for the strain-specific identification of bacterial pathogens. The system can carry out the entire molecular processing pipeline in a single disposable fluidic cartridge and detect single nucleotide variations in selected genes to allow for the identification of the bacterial species, even its strain with high specificity. The unique aspect of this fluidic cartridge is its modular format with task-specific modules interconnected to a fluidic motherboard to permit the selection of the target material. In addition, to minimize the amount of finishing steps for assembling the fluidic cartridge, many of the functional components were produced during the polymer molding step used to create the fluidic network. The operation of the cartridge was provided by electronic, mechanical, optical and hydraulic controls located off-chip and packaged into a small footprint instrument (1 ft(3)). The fluidic cartridge was capable of performing cell enrichment, cell lysis, solid-phase extraction (SPE) of genomic DNA, continuous flow (CF) PCR, CF ligase detection reaction (LDR) and universal DNA array readout. The cartridge was comprised of modules situated on a fluidic motherboard; the motherboard was made from polycarbonate, PC, and used for cell lysis, SPE, CF PCR and CF LDR. The modules were task-specific units and performed universal zip-code array readout or affinity enrichment of the target cells with both made from poly(methylmethacrylate), PMMA. Two genes, uidA and sipB/C, were used to discriminate between E. coli and Salmonella, and evaluated as a model system. Results showed that the fluidic system could successfully identify bacteria in <40 min with minimal operator intervention and perform strain identification, even from a mixed population with the target of a minority. We further demonstrated the ability to analyze the E. coli O157:H7 strain from a waste-water sample using enrichment followed by genotyping.

Novel Cephalosporins Selectively Active on Nonreplicating Mycobacterium tuberculosis

We report two series of novel cephalosporins that are bactericidal to Mycobacterium tuberculosis alone of the pathogens tested, which only kill M. tuberculosis when its replication is halted by conditions resembling those believed to pertain in the host, and whose bactericidal activity is not dependent upon or enhanced by clavulanate, a β-lactamase inhibitor. The two classes of cephalosporins bear an ester or alternatively an oxadiazole isostere at C-2 of the cephalosporin ring system, a position that is almost exclusively a carboxylic acid in clinically used agents in the class. Representatives of the series kill M. tuberculosis within macrophages without toxicity to the macrophages or other mammalian cells.