Similarities between Exogenously- and Endogenously-Induced Envelope Stress: The Effects of a New Antibacterial Molecule,TPI1609-10

Antibiotics with novel and/or multiple targets are highly desirable in the face of the steady rise of clinical antibiotic resistance. We have screened and identified small molecules, typified by the compound TPI1609-10 (aka SM10), with antibiotic activity against both gram-positive and gram-negative bacteria. SM10 was screened in vitro to bind branched Holliday junction intermediates of homologous recombination and tyrosine recombinase-mediated recombination; thus, the cellular targets of the small molecules were expected to include the RuvABC Holliday junction resolvasome and the XerCD complex involved in proper segregation of replicated chromosomes to daughter cells. SM10 indeed induces DNA damage and filamentation in E. coli. However, SM10 also induces envelope stress and causes increased production of intracellular reactive oxygen species. In addition, SM10 has similar effects to endogenously-induced envelope stress via overproducing outer membrane proteins (OmpC and OmpF), which also induces the SOS response, chromosome fragmentation, and production of reactive oxygen species. The synergy between SM10, and cerulenin, a fatty acid synthesis inhibitor, together with the SM10 hypersensitivity of cpx and rpoE mutants, further support that SM10''s mode of action damages membrane damage. The lethality of SM10 treatment and of OmpC overproduction are observed in both aerobically- and anaerobically-grown cells, and is accompanied by substantial DNA damage even anaerobically. Thus, only some DNA damage is due to reactive oxygen. We propose that membrane depolarization and the potential reduction in intracellular pH, leading to abasic site formation, cause a substantial amount of the DNA damage associated with both SM10 treatment and endogenous envelope stress. While it is difficult to completely exclude effects related to envelope damage as the sources of DNA damage, trapping intermediates associated with DNA repair and chromosome segregation pathways remains very likely. Thus SM10 may have distinct but synergistic modes of action.     

The temperate marine phage PhiHAP-1 of Halomonas aquamarina possesses a linear plasmid-like prophage genome

A myovirus-like temperate phage, PhiHAP-1, was induced with mitomycin C from a Halomonas aquamarina strain isolated from surface waters in the Gulf of Mexico. The induced cultures produced significantly more virus-like particles (VLPs) (3.73 x 10(10) VLP ml(-1)) than control cultures (3.83 x 10(7) VLP ml(-1)) when observed with epifluorescence microscopy. The induced phage was sequenced by using linker-amplified shotgun libraries and contained a genome 39,245 nucleotides in length with a G+C content of 59%. The PhiHAP-1 genome contained 46 putative open reading frames (ORFs), with 76% sharing significant similarity (E value of <10(-3)) at the protein level with other sequences in GenBank. Putative functional gene assignments included small and large terminase subunits, capsid and tail genes, an N6-DNA adenine methyltransferase, and lysogeny-related genes. Although no integrase was found, the PhiHAP-1 genome contained ORFs similar to protelomerase and parA genes found in linear plasmid-like phages with telomeric ends. Southern probing and PCR analysis of host genomic, plasmid, and PhiHAP-1 DNA indicated a lack of integration of the prophage with the host chromosome and a difference in genome arrangement between the prophage and virion forms. The linear plasmid prophage form of PhiHAP-1 begins with the protelomerase gene, presumably due to the activity of the protelomerase, while the induced phage particle has a circularly permuted genome that begins with the terminase genes. The PhiHAP-1 genome shares synteny and gene similarity with coliphage N15 and vibriophages VP882 and VHML, suggesting an evolutionary heritage from an N15-like linear plasmid prophage ancestor.     

Discovery of thieno[2,3-c]pyridines as potent COT inhibitors

Evaluation of hit chemotypes from high throughput screening identified a novel series of 2,4-disubstituted thieno[2,3-c]pyridines as COT kinase inhibitors. Structural modifications exploring SAR at the 2- and 4-positions resulting in inhibitors with improved enzyme potency and cellular activity are disclosed.     

Genetic Variability of Yersinia pestis Isolates as Predicted by PCR-Based IS100 Genotyping and Analysis of Structural Genes Encoding Glycerol-3-Phosphate Dehydrogenase (glpD)

A PCR-based genotyping system that detects divergence of IS100 locations within the Yersinia pestis genome was used to characterize a large collection of isolates of different biovars and geographical origins. Using sequences derived from the glycerol-negative biovar orientalis strain CO92, a set of 27 locus-specific primers was designed to amplify fragments between the end of IS100 and its neighboring gene. Geographically diverse members of the orientalis biovar formed a homogeneous group with identical genotype with the exception of strains isolated in Indochina. In contrast, strains belonging to the glycerol-positive biovar antiqua showed a variety of fingerprinting profiles. Moreover, strains of the biovar medievalis (also glycerol positive) clustered together with the antiqua isolates originated from Southeast Asia, suggesting their close phylogenetic relationships. Interestingly, a Manchurian biovar antiqua strain Nicholisk 51 displayed a genotyping pattern typical of biovar orientalis isolates. Analysis of the glycerol pathway in Y. pestis suggested that a 93-bp deletion within the glpD gene encoding aerobic glycerol-3-phosphate dehydrogenase might account for the glycerol-negative phenotype of the orientalis biovar. The glpD gene of strain Nicholisk 51 did not possess this deletion, although it contained two nucleotide substitutions characteristic of the glpD version found exclusively in biovar orientalis strains. To account for this close relationship between biovar orientalis strains and the antiqua Nicholisk 51 isolate, we postulate that the latter represents a variant of this biovar with restored ability to ferment glycerol. The fact that such a genetic lesion might be repaired as part of the natural evolutionary process suggests the existence of genetic exchange between different Yersinia strains in nature. The relevance of this observation on the emergence of epidemic Y. pestis strains is discussed.     

Selection of Specific Endophytic Bacterial Genotypes by Plants in Response to Soil Contamination

Plant-bacterial combinations can increase contaminant degradation in the rhizosphere, but the role played by indigenous root-associated bacteria during plant growth in contaminated soils is unclear. The purpose of this study was to determine if plants had the ability to selectively enhance the prevalence of endophytes containing pollutant catabolic genes in unrelated environments contaminated with different pollutants. At petroleum hydrocarbon contaminated sites, two genes encoding hydrocarbon degradation, alkane monooxygenase (alkB) and naphthalene dioxygenase (ndoB), were two and four times more prevalent in bacteria extracted from the root interior (endophytic) than from the bulk soil and sediment, respectively. In field sites contaminated with nitroaromatics, two genes encoding nitrotoluene degradation, 2-nitrotoluene reductase (ntdAa) and nitrotoluene monooxygenase (ntnM), were 7 to 14 times more prevalent in endophytic bacteria. The addition of petroleum to sediment doubled the prevalence of ndoB-positive endophytes in Scirpus pungens, indicating that the numbers of endophytes containing catabolic genotypes were dependent on the presence and concentration of contaminants. Similarly, the numbers of alkB- or ndoB-positive endophytes in Festuca arundinacea were correlated with the concentration of creosote in the soil but not with the numbers of alkB- or ndoB-positive bacteria in the bulk soil. Our results indicate that the enrichment of catabolic genotypes in the root interior is both plant and contaminant dependent.     

Safety Indices during Fetal Echocardiography at the Time of First-Trimester Scan Are Machine Dependent

The aim of our study was to evaluate the thermal index (TI) and mechanical index (MI), during the assessment of the fetal heart at the time of first-trimester scan, with different ultrasound machines. This was part of an observational study conducted in patients undergoing routine first-trimester screening. Cases were examined with Voluson E8 or 730Pro scanners using 2–8 MHz transabdominal probes. TI and MI were retrieved from the saved displays while in gray mode, color flow mapping and pulsed-wave (PW) Doppler examinations of the fetal heart and also from the ductus venosus (DV) assessment. We evaluated 552 fetal cardiac examinations, 303 (55%) performed with Voluson E8 and 249 (45%) with Voluson 730Pro ultrasound machines. The gray-scale exam of the heart and the PW Doppler DV assessment had TI values significantly lower for the Voluson E8 group (median, 0.04 vs. 0.2 and 0.1 vs. 0.2, respectively). The MI values from gray-scale and color flow mapping of the heart were significantly lower (median, 0.6 vs, 1.2 and 0.7 vs. 1) and for PW Doppler exam of the tricuspid flow were significantly higher (median 0.4 vs. 0.2) in the Voluson E8 group. The TI values from Doppler examinations of the heart, either color flow or PW imaging and MI values from DV assessment were not significantly different between the two groups. A different (newer) generation of ultrasound equipment provides lower or at least the same safety indices for most of the first-trimester heart examinations.     

Quantitative Shear-Wave Elastography of the Liver in Preterm Neonates with Intra-Uterine Growth Restriction

The feasibility and reproducibility of liver stiffness measurements using Supersonic Shear-wave Imaging (SSI) in preterm neonate have not been reported. Our aim was to determine if liver stiffness differs between intra-uterine growth restriction (IUGR) and appropriate for gestational age (AGA) preterm infants with/without cholestasis. We measured liver stiffness (in kPa) in 45 AGA and 18 IUGR preterm infants, and assessed reproducibility in 26 preterms using Intraclass Correlation Coefficients (ICC) and Bland-Altman tests. Liver stiffness values were compared between AGA and IUGR with and without cholestasis and correlated with birth weight. Measurements showed high reproducibility (ICC = 0.94–0.98 for intra-operator, 0.86 for inter-operator) with good agreement (95% limits: -1.24 to 1.24 kPa). During the first postnatal week, liver stiffness was higher in IUGR (7.50 ±1.53 kPa) than in AGA infants (5.11 ±0.80 kPa, p<0.001). After day 8, liver stiffness remained unchanged in AGA but increased progressively in IUGR infants (15.57 ±6.49 kPa after day 21). Liver stiffness was higher in IUGR neonates with cholestasis (19.35 ± 9.80 kPa) than without cholestasis (7.72 ± 1.27 kPa, p<0.001). In conclusion, quantitative liver SSI in preterms is feasible and reproducible. IUGR preterms who will develop cholestasis present high liver stiffness even at birth, before biological cholestasis occurs.     

Fibroblast dynamics as an in vitro screening platform for anti‐fibrotic drugs in primary myelofibrosis

Although the cause for bone marrow fibrosis in patients with myelofibrosis remains controversial, it has been hypothesized that it is caused by extensive fibroblast proliferation under the influence of cytokines generated by the malignant megakaryocytes. Moreover, there is no known drug therapy which could reverse the process. We studied the fibroblasts in a novel system using the hanging drop method, evaluated whether the fibroblasts obtain from patients are part of the malignant clone of not and, using this system, we screen a large library of FDA‐approved drugs to identify potential drugs candidates that might be useful in the treatment of this disease, specifically which would inhibit fibroblast proliferation and the development of bone marrow fibrosis. We have found that the BM fibroblasts are not part of the malignant clone, as previously suspected and two immunosuppressive medications—cyclosporine and mycophenolate mophetil, as most potent suppressors of the fibroblast collagen production thus potentially inhibitors of bone marrow fibrosis production in myelofibrosis.     

In Vitro Selection of Integration Host Factor Binding Sites

Integration host factor (IHF) is a bacterial protein that binds and severely bends a specific DNA target. IHF binding sites are approximately 30 to 35 bp long and are apparently divided into two domains. While the 3' domain is conserved, the 5' domain is degenerate but is typically AT rich. As a result of physical constraints that IHF must impose on DNA in order to bind, it is believed that this 5' domain must possess structural characteristics conducive for both binding and bending with little regard for specific contacts between the protein and the DNA. We have examined the sequence requirements of the 5' binding domain of the IHF binding target. Using a SELEX procedure, we randomized and selected variants of a natural IHF site. We then analyzed these variants to determine how the 5' binding domain affects the structure, affinity, and function of an IHF-DNA complex in a native system. Despite finding individual sequences that varied over 100-fold in affinity for IHF, we found no apparent correlation between affinity and function.