Staphylococcus aureus SigS Induces Expression of a Regulatory Protein Pair That Modulates Its mRNA Stability.

SigS is the sole extracytoplasmic function sigma factor in Staphylococcus aureus and is necessary for virulence, immune evasion, and adaptation to toxic chemicals and environmental stressors. Despite the contribution of SigS to a myriad of critical phenotypes, the downstream effectors of SigS-dependent pathogenesis, immune evasion, and stress adaptation remain elusive. To address this knowledge gap, we analyzed the S. aureus transcriptome following transient overexpression of SigS. We identified a bicistronic transcript, upregulated 1,000-fold, containing two midsized genes, each containing single domains of unknown function (DUFs). We renamed these genes SigS-regulated orfA (sroA) and SigS-regulated orfB (sroB). We demonstrated that SigS regulation of the sroAB operon is direct by using in vitro transcription analysis. Using Northern blot analysis, we also demonstrated that SroA and SroB have opposing autoregulatory functions on the transcriptional architecture of the sigS locus, with SroA stimulating SigS mRNA levels and SroB stimulating s750 (SigS antisense) levels. We hypothesized that these opposing regulatory effects were due to a direct interaction. We subsequently demonstrated a direct interaction between SroA and SroB using an in vivo surrogate genetics approach via bacterial adenylate cyclase-based two-hybrid (BACTH) analysis. We demonstrated that the SroA effect on SigS is at the posttranscriptional level of mRNA stability, highlighting a mechanism likely used by S. aureus to tightly control SigS levels. Finally, we demonstrate that the sroAB locus promotes virulence in a murine pneumonia model of infection. IMPORTANCE SigS is necessary for S. aureus virulence, immune evasion, and adaptation to chemical and environmental stressors. These processes are critically important for the ability of S. aureus to cause disease. However, the SigS-dependent transcriptome has not been identified, hindering our ability to identify downstream effectors of SigS that contribute to these pathogenic and adaptive phenotypes. Here, we identify a regulatory protein pair that is a major direct target of SigS, known as SroA and SroB. SroA also acts to stimulate SigS expression at the posttranscriptional level of RNA turnover, providing insight into intrinsically low levels of SigS. The discovery of SroA and SroB increases our understanding of SigS and the S. aureus pathogenesis process.

Tongalides, Halogenated Butenolides from an Antarctic Delisea sp. Rhodophyte.

Six new halogenated butenolides, tongalides A-C (1-3) and their acetylated congeners (4-6), were isolated from an extract of the Antarctic rhodophyte Delisea sp. that displayed significant antibiotic activity. The structures of the compounds were determined by analysis of data acquired by spectroscopic and spectrometric techniques including NMR, HRESIMS, optical rotation, and X-ray diffraction studies. The newly isolated compounds were assayed for antibacterial activity, but exhibited no growth inhibition of ESKAPE pathogens. The extract bioactivity was attributed to the previously reported Z-acetoxyfimbrolide A also isolated from the extract, providing further evidence that the exocyclic double bond is essential to the antibacterial activity of the structurally related fimbrolide class of metabolite.

Phenogenomic Characterization of a Newly Domesticated and Novel Species from the Genus Verrucosispora.

The concept of bacterial dark matter stems from our inability to culture most microbes and represents a fundamental gap in our knowledge of microbial diversity. Here, we present the domestication of such an organism: a previously uncultured, novel species from the rare Actinomycetes genus Verrucosispora. Although initial recovery took >4 months, isolation of phenotypically distinct, domesticated generations occurred within weeks. Two isolates were subjected to phenogenomic analyses, revealing domestication correlated with enhanced growth rates in nutrient-rich media but diminished capacity to metabolize diverse amino acids. This is seemingly mediated by genomic atrophy through a mixed approach of pseudogenization and reversion of pseudogenization of amino acid metabolism genes. Conversely, later generational strains had enhanced spore germination rates, potentially through the reversion of a sporulation-associated kinase from pseudogene to true gene status. We observed that our most wild-type isolate had the greatest potential for antibacterial activity, which correlated with extensive mutational attrition of biosynthetic gene clusters in domesticated strains. Comparative analyses revealed wholesale genomic reordering in strains, with widespread single nucleotide polymorphism, indel, and pseudogene-impactful mutations observed. We hypothesize that domestication of this previously unculturable organism resulted from the shedding of genomic flexibility required for life in a dynamic marine environment, parsing out genetic redundancy to allow for a newfound cultivable amenability. IMPORTANCE The majority of environmental bacteria cannot be cultured within the laboratory. Understanding why only certain environmental isolates can be recovered is key to unlocking the abundant microbial dark matter that is widespread on our planet. In this study, we present not only the culturing but domestication of just such an organism. Although initial recovery took >4 months, we were able to isolate distinct, subpassaged offspring from the originating colony within mere weeks. A phenotypic and genotypic analysis of our generational strains revealed that adaptation to life in the lab occurred as a result of wholesale mutational changes. These permitted an enhanced ability for growth in nutrient rich media but came at the expense of reduced genomic flexibility. We suggest that without dynamic natural environmental stressors our domesticated strains effectively underwent genomic atrophy as they adapted to static conditions experienced in the laboratory.

Culture-dependent identification of rare marine sediment bacteria from the Gulf of Mexico and Antarctica.

Laboratory-viable cultivars of previously uncultured bacteria further taxonomic understanding. Despite many years of modern microbiological investigations, the vast majority of bacterial taxonomy remains uncharacterized. While many attempts have been made to decrease this knowledge gap, culture-based approaches parse away at the unknown and are critical for improvement of both culturing techniques and computational prediction efficacy. To this end of providing culture-based approaches, we present a multi-faceted approach to recovering marine environmental bacteria. We employ combinations of nutritional availability, inoculation techniques, and incubation parameters in our recovery of marine sediment-associated bacteria from the Gulf of Mexico and Antarctica. The recovered biodiversity spans several taxa, with 16S-ITS-23S rRNA gene-based identification of multiple isolates belonging to rarer genera increasingly undergoing phylogenetic rearrangements. Our modifications to traditional culturing techniques have not only recovered rarer taxa, but also resulted in the recovery of biotechnologically promising bacteria. Together, we propose our stepwise combinations of recovery parameters as a viable approach to decreasing the bacterial knowledge gap.

Draft genome sequence of a highly proteolytic Staphylococcus aureus USA300 isolate from human urine.

The secreted proteases of Staphylococcus aureus have been shown to be critical during infection. Here, we present the draft genome sequence of S. aureus TGH337, a hyper-proteolytic USA300 strain isolated from human urine.

Colorimetric assays for the rapid and high-throughput screening of antimicrobial peptide activity against diverse bacterial pathogens.

With the post-antibiotic era rapidly approaching, naturally-sourced antimicrobial peptides (AMPs) are a prime resource for restocking our antibiotic medicine cupboard. The efficiency of identification requires high-throughput screens that can identify bioactive peptides present within abundant natural-products chemical-space. While there are multiple amenable and high sensitivity colorimetric-based screening approaches available, resazurin-based assays are cost-effective, peptide compatible, and expedient, allowing one to screen a multitude of AMPs in a high-throughput fashion. Herein, we provide a detailed protocol for the optimization and use of resazurin assays for AMP testing, providing key experimental insight, and highlight pitfalls to be avoided.

Vandetanib plus docetaxel versus docetaxel as second-line treatment for patients with advanced non-small-cell lung cancer (ZODIAC): a double-blind, randomised, phase 3 trial.

BACKGROUND: Vandetanib is a once-daily oral inhibitor of vascular endothelial growth factor receptor (VEGFR), epidermal growth factor receptor (EGFR), and rearranged during transfection (RET) tyrosine kinases. In a randomised phase 2 study in patients with previously treated non-small-cell lung cancer (NSCLC), adding vandetanib 100 mg to docetaxel significantly improved progression-free survival (PFS) compared with docetaxel alone, including a longer PFS in women. These results supported investigation of the combination in this larger, definitive phase 3 trial (ZODIAC). METHODS: Between May, 2006, and April, 2008, patients with locally advanced or metastatic (stage IIIB-IV) NSCLC after progression following first-line chemotherapy were randomly assigned 1:1 through a third-party interactive voice system to receive vandetanib (100 mg/day) plus docetaxel (75 mg/m(2) intravenously every 21 days; maximum six cycles) or placebo plus docetaxel. The primary objective was comparison of PFS between the two groups in the intention-to-treat population. Women were a coprimary analysis population. This study has been completed and is registered with ClinicalTrials.gov, number NCT00312377. FINDINGS: 1391 patients received vandetanib plus docetaxel (n=694 [197 women]) or placebo plus docetaxel (n=697 [224 women]). Vandetanib plus docetaxel led to a significant improvement in PFS versus placebo plus docetaxel (hazard ratio [HR] 0.79, 97.58% CI 0.70-0.90; p<0.0001); median PFS was 4.0 months in the vandetanib group versus 3.2 months in placebo group. A similar improvement in PFS with vandetanib plus docetaxel versus placebo plus docetaxel was seen in women (HR 0.79, 0.62-1.00, p=0.024); median PFS was 4.6 months in the vandetanib group versus 4.2 months in the placebo group. Among grade 3 or higher adverse events, rash (63/689 [9%] vs 7/690 [1%]), neutropenia (199/689 [29%] vs 164/690 [24%]), leukopenia (99/689 [14%] vs 77/690 [11%]), and febrile neutropenia (61/689 [9%] vs 48/690 [7%]) were more common with vandetanib plus docetaxel than with placebo plus docetaxel. The most common serious adverse event was febrile neutropenia (46/689 [7%] in the vandetanib group vs 38/690 [6%] in the placebo group). INTERPRETATION: The addition of vandetanib to docetaxel provides a significant improvement in PFS in patients with advanced NSCLC after progression following first-line therapy.

Baseline vascular endothelial growth factor concentration as a potential predictive marker of benefit from vandetanib in non-small cell lung cancer.

PURPOSE: Vandetanib [vascular endothelial growth factor (VEGF) receptor/epidermal growth factor receptor/RET inhibitor] has shown improvements in progression-free survival (PFS) in advanced non-small cell lung cancer in three randomized phase II studies: vandetanib versus gefitinib (study 3), docetaxel +/- vandetanib (study 6), and carboplatin-paclitaxel and/or vandetanib (study 7). In study 7, vandetanib monotherapy was inferior to carboplatin-paclitaxel. We performed an exploratory retrospective analysis of the relationship between baseline circulating VEGF concentrations and PFS. EXPERIMENTAL DESIGN: Mean baseline VEGF levels were determined by ELISA from two baseline samples of plasma (163 of 168 patients, study 3; 65 of 127, study 6) or serum (144 of 181, study 7). High baseline VEGF values were above the immunoassay reference range for healthy subjects; low baseline VEGF values were within the range. RESULTS: Patients with low baseline VEGF had a lower risk of disease progression with vandetanib versus gefitinib [hazard ratio (HR), 0.55; 95% confidence interval (95% CI), 0.35-0.86; P = 0.01] or vandetanib 100 mg/d + docetaxel versus docetaxel (HR, 0.25; 95% CI, 0.09-0.68; P = 0.01). High VEGF patients had a similar risk of disease progression with vandetanib monotherapy versus gefitinib (HR, 1.03; 95% CI, 0.60-1.75; P = 0.92) or vandetanib 100 mg/d + docetaxel versus docetaxel (HR, 0.95; 95% CI, 0.25-3.61; P = 0.94). In study 7, low VEGF patients had a similar risk of disease progression with vandetanib monotherapy 300 mg/d versus carboplatin-paclitaxel (HR, 0.80; 95% CI, 0.41-1.56; P = 0.51); high VEGF patients progressed more quickly (HR, 1.60; 95% CI, 0.81-3.15; P = 0.17). CONCLUSIONS: These analyses suggest that low baseline circulating VEGF may be predictive of PFS advantage in patients with advanced non-small cell lung cancer receiving vandetanib versus gefitinib or vandetanib + docetaxel versus docetaxel. Moreover, patients with low VEGF levels may have a similar outcome with either vandetanib monotherapy or carboplatin-paclitaxel.

The Effects of a Functional Food Breakfast on Gluco-Regulation, Cognitive Performance, Mood, and Satiety in Adults.

A whole diet which combines multiple functional foods benefits metabolic risk factors and cognition, but evidence supporting meal to meal benefits, which individuals may find easier to implement, is limited. This study developed a functional food breakfast (FB), using polyphenol-rich ingredients selected for their gluco-regulating and cognitive-enhancing properties, and compared it to a control breakfast (CB). For study 1, total polyphenols were determined using the Folin-Ciocalteu method, and sugar release by in vitro digestion, in frozen and fresh samples. In study 2, healthy adults (n = 16) consumed an FB, CB and ready-to-eat breakfast cereal (RTEC) in a randomised crossover design. Glucose (GR) and insulin response (IR), satiety, mood and memory were measured over 180 min. The FB was a rich source of polyphenols (230 mg) compared to the CB (147 mg) (p < 0.05), and using frozen muffins did not compromise the polyphenol content or sugar release. Peak GR was highest after the RTEC (p < 0.05), and the insulin area under the curve (AUC) was lowest in the FB at 60, 120, 180 min and peak (p < 0.05). There were no effects on GR AUC, mood, satiety or memory. Reductions in GR peak and IR following consumption of the FB support the inclusion of functional ingredients at breakfast.

Genetic divergence and evidence of human-mediated translocation of two-fingered sloths (Choloepus hoffmanni) in Costa Rica.

Sloths are notoriously slow and consequently have limited dispersal ability, which makes them particularly vulnerable to the effects of habitat fragmentation and degradation. Sloths in Costa Rica are considered of conservation concern due to habitat loss, livestock production and increasing urbanization. Reintroductions from rescue centres are commonplace across the country, yet their genetic diversity and population structure are unknown, and there is currently little consideration of the genetic background prior to intervention or releases. We used microsatellite analysis to undertake the first exploratory investigation into sloth population genetics in Costa Rica. Using data from 98 two-fingered sloths (Choloepus hoffmanni) from four different geographic regions, we determined the presence of four potential genetic groups, three of them with minimal population structuring despite the limited dispersal ability and presence of physical barriers. Sloths from the North appear to represent a highly distinct population that we propose may require management as a discrete unit for conservation. We stress the need for additional analyses to better understand the genetic structure and diversity of North andWest regions and suggest that rescue facilities in Costa Rica should consider the genetic background of rehabilitated sloths when planning future reintroductions. Our results also highlight the threat posed by physical isolation due to widespread urbanization and agriculture expansion for a species with a weak dispersal ability.

Draft Genome Sequence of Verrucosispora sp. Strain CWR15, Isolated from a Gulf of Mexico Sponge.

Here, we present the draft genome sequence of Verrucosispora sp. strain CWR15, a bacterial symbiont of a Gulf of Mexico sponge. The genome consists of 35 contigs encoding 5,840 genes. The genome is the basis for future study and presents an underexplored taxonomy and biosynthetic potential.

Cheap labor: myosin fiber type expression and enzyme activity in the forelimb musculature of sloths (Pilosa: Xenarthra).

Sloths are canopy-dwelling inhabitants of American neotropical rainforests that exhibit suspensory behaviors. These abilities require both strength and muscular endurance to hang for extended periods of time; however, the skeletal muscle mass of sloths is reduced, thus requiring modifications to muscle architecture and leverage for large joint torque. We hypothesize that intrinsic muscle properties are also modified for fatigue resistance and predict a heterogeneous expression of slow/fast myosin heavy chain (MHC) fibers that utilize oxidative metabolic pathways for economic force production. MHC fiber type distribution and energy metabolism in the forelimb muscles of three-toed ( Bradypus variegatus, n = 5) and two-toed ( Choloepus hoffmanni, n = 4) sloths were evaluated using SDS-PAGE, immunohistochemistry, and enzyme activity assays. The results partially support our hypothesis by a primary expression of the slow MHC-1 isoform as well as moderate expression of fast MHC-2A fibers, whereas few hybrid MHC-1/2A fibers were found in both species. MHC-1 fibers were larger in cross-sectional area (CSA) than MHC-2A fibers and comprised the greatest percentage of CSA in each muscle sampled. Enzyme assays showed elevated activity for the anaerobic enzymes creatine kinase and lactate dehydrogenase compared with low activity for aerobic markers citrate synthase and 3-hydroxyacetyl CoA dehydrogenase. These findings suggest that sloth forelimb muscles may rely heavily on rapid ATP resynthesis pathways, and lactate accumulation may be beneficial. The intrinsic properties observed match well with suspensory requirements, and these modifications may have further evolved in unison with low metabolism and slow movement patterns as means to systemically conserve energy. NEW & NOTEWORTHY Myosin heavy chain (MHC) fiber type and fiber metabolic properties were evaluated to understand the ability of sloths to remain suspended for extended periods without muscle fatigue. Broad distributions of large, slow MHC-1 fibers as well as small, fast MHC-2A fibers are expressed in sloth forelimbs, but muscle metabolism is generally not correlated with myosin fiber type or body size. Sloth muscles rely on rapid, anaerobic pathways to resist fatigue and sustain force production.

A phase I study of Vandetanib in combination with vinorelbine/cisplatin or gemcitabine/cisplatin as first-line treatment for advanced non-small cell lung cancer.

INTRODUCTION: Vandetanib is a once-daily oral agent that selectively inhibits vascular endothelial growth factor receptor, epidermal growth factor receptor, and RET (REarranged during Transfection) signaling. METHODS: This Phase I study investigated the safety, tolerability, and pharmacokinetics of vandetanib when administered with either gemcitabine plus cisplatin (GC) or vinorelbine plus cisplatin (VC) in patients with previously untreated locally advanced or metastatic non-small cell lung cancer. RESULTS: Seventeen patients received vandetanib 100 mg/d plus VC (n = 9) or GC (n = 8). Three dose-limiting toxicities were reported in each treatment group: vandetanib + VC (pulmonary artery thrombosis and asymptomatic QTc prolongation [n = 2]); vandetanib + GC (peripheral ischemia [due to arterial occlusion], pulmonary embolism, and limb venous thrombosis). The protocol definition of a tolerable dose was not met, and no patients were recruited to receive vandetanib 300 mg plus VC or GC. There was no apparent pharmacokinetic interaction between vandetanib and vinorelbine or gemcitabine, but there was an approximate 30% increase in the exposure to cisplatin, which may be due to accumulation of total platinum and/or an interaction with vandetanib. CONCLUSIONS: In this study, in patients with previously untreated advanced non-small cell lung cancer, vandetanib 100 mg/d in combination with either VC or GC was not tolerated.

Vandetanib versus gefitinib in patients with advanced non-small-cell lung cancer: results from a two-part, double-blind, randomized phase ii study.

PURPOSE: Vandetanib is a once-daily oral inhibitor of vascular endothelial growth factor receptor (VEGFR) and epidermal growth factor receptor (EGFR) signaling. In this two-part phase II study, the efficacy and safety of vandetanib was compared with that of gefitinib, an inhibitor of EGFR signaling. PATIENTS AND METHODS: Patients (N = 168) with locally advanced or metastatic (stage IIIB/IV) non-small-cell lung cancer (NSCLC), after failure of first-line with or without second-line platinum-based chemotherapy, received once-daily vandetanib 300 mg (n = 83) or gefitinib 250 mg (n = 85) until disease progression or evidence of toxicity (part A). After a 4-week washout period, eligible patients had the option to switch to the alternative treatment (part B). Progression-free survival (PFS) was the primary efficacy assessment in part A, which was designed to have a higher than 75% power to detect a 33% prolongation of PFS at a one-sided significance level of .2. RESULTS: In part A, vandetanib prolonged PFS compared with gefitinib (hazard ratio = 0.69; 95% CI, 0.50 to 0.96; one-sided P = .013). Patients receiving vandetanib experienced adverse events that were manageable and generally consistent with inhibition of EGFR and VEGFR signaling, including diarrhea, rash, and hypertension. There were no unexpected safety findings with gefitinib. Overall survival, a secondary assessment, was not significantly different between patients initially randomly assigned to either vandetanib or gefitinib. CONCLUSION: The primary efficacy objective was achieved, with vandetanib demonstrating a significant prolongation of PFS versus gefitinib. Vandetanib 300 mg/d is currently being evaluated as a monotherapy in two randomized phase III studies in advanced NSCLC.

Randomized, placebo-controlled phase II study of vandetanib plus docetaxel in previously treated non small-cell lung cancer.

PURPOSE: Vandetanib is a once-daily oral inhibitor of vascular endothelial growth factor receptor-2 and epidermal growth factor receptor kinase activity. The activity of vandetanib plus docetaxel was assessed in patients with previously treated non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: This two-part study comprised an open-label run-in phase and a double-blind randomized phase. Eligible patients had locally advanced or metastatic (stage IIIB/IV) NSCLC after failure of first-line platinum-based chemotherapy. The primary objective of the randomized phase was to prolong progression-free survival (PFS) in patients receiving vandetanib (100 or 300 mg/d) plus docetaxel (75 mg/m2 intravenous infusion every 21 days) versus placebo plus docetaxel. The study was designed to have more than 75% power to detect 50% prolongation at a one-sided significance level of P < .20. Secondary objectives included objective response rate, overall survival, safety and tolerability. RESULTS: In the randomized phase (n = 127), median PFS was 18.7 weeks for vandetanib 100 mg plus docetaxel (n = 42; hazard ratio v docetaxel = 0.64; one-sided P = .037); 17.0 weeks for vandetanib 300 mg plus docetaxel (n = 44; hazard ratio v docetaxel = 0.83; one-sided P = .231); and 12 weeks for docetaxel (n = 41). There was no statistically significant difference in overall survival among the three treatment arms. Common adverse events included diarrhea, rash, and asymptomatic prolongation of corrected QT (QTC) interval. CONCLUSION: The primary objective was achieved, with vandetanib 100 mg plus docetaxel demonstrating a significant prolongation of PFS compared with docetaxel in relation to the prespecified significance level. On the basis of these encouraging data, phase III evaluation of vandetanib 100 mg plus docetaxel in second-line NSCLC has been initiated.