Using EEG signals to assess workload during memory retrieval in a real-world scenario.

Objective. The electroencephalogram (EEG) is gaining popularity as a physiological measure for neuroergonomics in human factor studies because it is objective, less prone to bias, and capable of assessing the dynamics of cognitive states. This study investigated the associations between memory workload and EEG during participants' typical office tasks on a single-monitor and dual-monitor arrangement. We expect a higher memory workload for the single-monitor arrangement.Approach. We designed an experiment that mimics the scenario of a subject performing some office work and examined whether the subjects experienced various levels of memory workload in two different office setups: (1) a single-monitor setup and (2) a dual-monitor setup. We used EEG band power, mutual information, and coherence as features to train machine learning models to classify high versus low memory workload states.Main results. The study results showed that these characteristics exhibited significant differences that were consistent across all participants. We also verified the robustness and consistency of these EEG signatures in a different data set collected during a Sternberg task in a prior study.Significance. The study found the EEG correlates of memory workload across individuals, demonstrating the effectiveness of using EEG analysis in conducting real-world neuroergonomic studies.

A Phase 2a randomized, single-center, double-blind, placebo-controlled study to evaluate the safety and preliminary efficacy of oral iOWH032 against cholera diarrhea in a controlled human infection model.

Cholera remains a major cause of infectious diarrhea globally. Despite the increased availability of cholera vaccines, there is still an urgent need for other effective interventions to reduce morbidity and mortality. Furthermore, increased prevalence of antibiotic-resistant Vibrio cholerae threatens the use of many drugs commonly used to treat cholera. We developed iOWH032, a synthetic small molecule inhibitor of the cystic fibrosis transmembrane conductance regulator chloride channel, as an antisecretory, host-directed therapeutic for cholera. In the study reported here, we tested iOWH032 in a Phase 2a cholera controlled human infection model. Forty-seven subjects were experimentally infected with V. cholerae El Tor Inaba strain N16961 in an inpatient setting and randomized to receive 500 mg iOWH032 or placebo by mouth every 8 hours for 3 days to determine the safety and efficacy of the compound as a potential treatment for cholera. We found that iOWH032 was generally safe and achieved a mean (± standard deviation) plasma level of 4,270 ng/mL (±2,170) after 3 days of oral dosing. However, the median (95% confidence interval) diarrheal stool output rate for the iOWH032 group was 25.4 mL/hour (8.9, 58.3), compared to 32.6 mL/hour (15.8, 48.2) for the placebo group, a reduction of 23%, which was not statistically significant. There was also no significant decrease in diarrhea severity and number or frequency of stools associated with iOWH032 treatment. We conclude that iOWH032 does not merit future development for treatment of cholera and offer lessons learned for others developing antisecretory therapeutic candidates that seek to demonstrate proof of principle in a cholera controlled human infection model study. Trial registration: This study is registered with ClinicalTrials.gov as NCT04150250.

Risk perception of heat waves and its spatial variation in Nanjing, China.

The intensity, frequency, and duration of heat waves are expected to increase with climate change. In this study, we found a significant difference of public perceived effects of heat waves and trust in government among urban, suburban, and rural districts. Rural residents had a significant higher effect perception than urbanites and also showed stronger willingness to have medical insurance or regular physical examinations. Meanwhile, suburban residents had the lowest trust perception in government among these three districts, which may be due to suburban districts' unique social structure and complex social issues. Besides, we assessed the relationship between the factor effect and demographic variables. The results showed that urban respondents' effect perception was significantly related to heat wave experiences. Suburban respondents' effect perception was significantly related to age, income, and heat wave experiences. And rural respondents' effect perception was significantly related to income and chronic diseases. Based on our results, much more attention needs to be paid to rural districts. The government should strengthen infrastructure construction such as cooling centers, improve emergency response plans and mechanisms, and increase reserves of emergency supplies in rural districts. Also, targeted risk communication is of the equal importance to aid the policy-makers improving the relationship with the public and regaining the public's trust and support.

Synthesis and evaluation of a new phosphorylated ribavirin prodrug.

Ribavirin is an important broad-spectrum antiviral drug. However, its utilization can be limited by its potential to cause hemolytic anemia as well as its variability in dosing levels and efficacy outcomes. To overcome these issues, we report on a new alkoxyalkylphosphodiester prodrug of ribavirin (2) that is designed to release the active ribavirin-monophosphate species selectively in nucleated cells while limiting its exposure in anucleated red blood cells (RBCs). Prodrug 2 displays improved in vitro antiviral activity against the hepatitis C virus replicon and influenza virus. Unlike ribavirin, prodrug 2 does not significantly decrease ATP levels in RBCs. Prodrug 2 demonstrates decreased uptake in RBCs but increased uptake in HepG2 hepatocytes when compared to ribavirin. In vivo, prodrug 2 is orally bioavailable and well-tolerated in rats in which it is processed to ribavirin and accumulates in the liver. These results indicate that prodrug 2 has the potential for safer, lower, less frequent, and less variable administration than ribavirin.

Identification of nuclear export inhibitors with potent anticancer activity in vivo.

The export protein CRM1 is required for the nuclear export of a wide variety of cancer-related "cargo" proteins including p53, c-Abl, and FOXO-3A. Leptomycin B (LMB) is a highly specific inhibitor of CRM1 with significant in vitro potency but limited in vivo efficacy due to toxicity. We now report a series of semisynthetic LMB derivatives showing substantially improved therapeutic windows. Exposure of cancer cells to these compounds leads to a rapid and prolonged block of nuclear export and apoptosis. In contrast to what is observed in cancer cells, these agents induce cell cycle arrest, but not apoptosis, in normal lung fibroblasts. These new nuclear export inhibitors (NEI) maintain the high potency of LMB, are up to 16-fold better tolerated than LMB in vivo, and show significant efficacy in multiple mouse xenograft models. These NEIs show the potential of CRM1 inhibitors as novel and potent anticancer agents.

Robotic radical prostatectomy: operative technique, outcomes, and learning curve.

OBJECTIVE: To report the operative technique, oncologic and therapeutic outcomes, and learning curve from our initial series of over 140 patients treated by robotic radical prostatectomy. METHODS: Between January 2003 and May 2005, 143 patients with clinically localized prostate cancer underwent a robotic radical prostatectomy. Prospective data collection included patient age, body mass index (BMI), clinical T stage, biopsy Gleason score, and prostate-specific antigen (PSA). Operative outcome measures included operative time, estimated blood loss (EBL), and complications. Post-operative outcomes were length of hospital stay, catheter duration, pathology, margin status, biochemical recurrence, and return of continence. RESULTS: Mean operative time was 241 minutes with an EBL of 274 mL. Five patients (3%) required conversion to open surgery. The average hospitalization was 1.8 days, and Foley catheters were removed after 8.9 days. Twenty-four of 141 men (17%) had a positive surgical margin, with a decrease from 23% in the first half of our experience to 11% in the latter half. Patients with an extracapsular extension had a significantly higher positive surgical margin rate than did those with organ-confined disease (47% vs 15%). Over 40% of the positive margins were located posteriorly. At a mean follow-up of 11 months, 96% of patients had a PSA <0.2 ng/mL. The median time to complete continence was 3.5 months, and over 95% of patients were fully continent at 1 year. CONCLUSION: Robotic radical prostatectomy is an effective treatment modality for clinically localized prostate cancer. Although a learning curve needs to be overcome, patients experienced benefits in convalescence with early oncologic and functional outcomes comparable to those of the open approach. Longer-term results are needed; however, patient outcomes in our series are encouraging.

Increasing body mass index negatively impacts outcomes following robotic radical prostatectomy.

OBJECTIVE: To clarify the impact of increasing body mass index (BMI) on outcomes following robotic radical prostatectomy. METHODS: From January 2003 to May 2005, 132 patients with clinically localized prostate cancer underwent a robotic radical prostatectomy. Patients were divided into 3 cohorts based on BMI: 38 normal (range, 18 to 24.9), 60 overweight (range, 25 to 29.9), and 34 obese (BMI>30). RESULTS: The operative time was significantly longer in obese (304 min) men compared with overweight (235 min) and normal (238 min) BMI patients (P<0.001). Estimated blood loss was significantly greater in both the obese (316 mL) and overweight (318 mL) groups compared with men with normal BMI (234 mL) (P<0.005). Three patients (1 obese and 2 overweight) required conversion to open surgery. Twenty-three of 132 men (17%) had a positive surgical margin, with obese (21%) and overweight (20%) men at a greater risk compared with normal BMI men (11%). No significant differences existed between groups with regard to final pathologic stage, Gleason score, biochemical recurrence at 1-year, and postoperative complication rate. CONCLUSION: Overweight and obese men had a longer operative duration, greater blood loss, longer hospital duration, and higher positive surgical margin rate. Robotic prostatectomy in men with elevated BMI is technically more challenging and is associated with more operative morbidity.

Chemobiosynthesis of novel 6-deoxyerythronolide B analogues by mutation of the loading module of 6-deoxyerythronolide B synthase 1.

Chemobiosynthesis (J. R. Jacobsen, C. R. Hutchinson, D. E. Cane, and C. Khosla, Science 277:367-369, 1997) is an important route for the production of polyketide analogues and has been used extensively for the production of analogues of 6-deoxyerythronolide B (6-dEB). Here we describe a new route for chemobiosynthesis using a version of 6-deoxyerythronolide B synthase (DEBS) that lacks the loading module. When the engineered DEBS was expressed in both Escherichia coli and Streptomyces coelicolor and fed a variety of acyl-thioesters, several novel 15-R-6-dEB analogues were produced. The simpler "monoketide" acyl-thioester substrates required for this route of 15-R-6-dEB chemobiosynthesis allow greater flexibility and provide a cost-effective alternative to diketide-thioester feeding to DEBS KS1(o) for the production of 15-R-6-dEB analogues. Moreover, the facile synthesis of the monoketide acyl-thioesters allowed investigation of alternative thioester carriers. Several alternatives to N-acetyl cysteamine were found to work efficiently, and one of these, methyl thioglycolate, was verified as a productive thioester carrier for mono- and diketide feeding in both E. coli and S. coelicolor.

Chalcomycin biosynthesis gene cluster from Streptomyces bikiniensis: novel features of an unusual ketolide produced through expression of the chm polyketide synthase in Streptomyces fradiae.

Chalcomycin, a 16-membered macrolide antibiotic made by the bacterium Streptomyces bikiniensis, contains a 2,3-trans double bond and the neutral sugar D-chalcose in place of the amino sugar mycaminose found in most other 16-membered macrolides. Degenerate polyketide synthase (PKS)-specific primers were used to amplify DNA fragments from S. bikiniensis with very high identity to a unique ketosynthase domain of the tylosin PKS. The resulting amplimers were used to identify two overlapping cosmids encompassing the chm PKS. Sequencing revealed a contiguous segment of >60 kb carrying 25 putative genes for biosynthesis of the polyketide backbone, the two deoxysugars, and enzymes involved in modification of precursors of chalcomycin or resistance to it. The chm PKS lacks the ketoreductase and dehydratase domains in the seventh module expected to produce the 2,3-double bond in chalcomycin. Expression of PKS in the heterologous host Streptomyces fradiae, from which the tyl genes encoding the PKS had been removed, resulted in production of at least one novel compound, characterized as a 3-keto 16-membered macrolactone in equilibrium with its 3-trans enol tautomer and containing the sugar mycaminose at the C-5 position, in agreement with the structure predicted on the basis of the domain organization of the chm PKS. The production of a 3-keto macrolide from the chm PKS indicates that a discrete set of enzymes is responsible for the introduction of the 2,3-trans double bond in chalcomycin. From comparisons of the open reading frames to sequences in databases, a pathway for the synthesis of nucleoside diphosphate-D-chalcose was proposed.

Production of hybrid 16-membered macrolides by expressing combinations of polyketide synthase genes in engineered Streptomyces fradiae hosts.

Combinations of the five polyketide synthase (PKS) genes for biosynthesis of tylosin in Streptomyces fradiae (tylG), spiramycin in Streptomyces ambofaciens (srmG), or chalcomycin in Streptomyces bikiniensis (chmG) were expressed in engineered hosts derived from a tylosin-producing strain of S. fradiae. Surprisingly efficient synthesis of compounds predicted from the expressed hybrid PKS was obtained. The post-PKS tailoring enzymes of tylosin biosynthesis acted efficiently on the hybrid intermediates with the exception of TylH-catalyzed hydroxylation of the methyl group at C14, which was efficient if C4 bore a methyl group, but inefficient if a methoxyl was present. Moreover, for some compounds, oxidation of the C6 ethyl side chain to an unprecedented carboxylic acid was observed. By also expressing chmH, a homolog of tylH from the chalcomycin gene cluster, efficient hydroxylation of the 14-methyl group was restored.

Synthesis and biological activities of novel 17-aminogeldanamycin derivatives.

Geldanamycin interferes with the action of heat shock protein 90 (Hsp90) by binding to the N-terminal ATP binding site and inhibiting an essential ATPase activity. In a program directed toward finding potent, water soluble inhibitors of Hsp90, we prepared a library of over sixty 17-alkylamino-17-demethoxygeldanamycin analogs, and compared their affinity for Hsp90, ability to inhibit growth of SKBr3 mammalian cells, and in selected cases, water solubility. Over 20 analogs showed cell growth inhibition potencies similar to that of 17-allylamino-17-demethoxygeldanamycin (17-AAG), the front-runner geldanamycin analog that is currently in multiple clinical trials. Many of these analogs showed water solubility properties that were desirable for formulation. One of the most potent and water-soluble analogs in the series was 17-(2-dimethylaminoethyl)amino-17-demethoxygeldanamycin (17-DMAG), which was independently prepared by the NCI and will soon enter clinical trials. Importantly, the binding affinity of these analogs to the molecular target Hsp90 does not correlate well with their cytotoxicity in SKBr3 cells.

The fission yeast TFIIB-related factor limits RNA polymerase III to a TATA-dependent pathway of TBP recruitment.

The RNA polymerase (pol) III-transcribed (e.g. tRNA and 5S rRNA) genes of traditionally studied organisms rely on gene-internal promoters that precisely position the initiation factor, TFIIIB, on the upstream promoter-less DNA. This is accomplished by the ability of the TFIIIB subunit, TFIIB-related factor (Brf1), to make stable protein-protein interactions with TATA-binding protein (TBP) and place it on the promoter-less upstream DNA. Unlike traditional model organisms, Schizosaccharomyces pombe tRNA and 5S rRNA genes contain upstream TATA promoters that are required to program functional pol III initiation complexes. In this study we demonstrate that S.pombe (Sp)Brf does not form stable interactions with TBP in the absence of DNA using approaches that do reveal stable association of TBP and S.cerevisiae (Sc)Brf1. Gel mobility analyses demonstrate that a TBP-TATA DNA complex can recruit SpBrf to a Pol III promoter. Consistent with this, overproduction of SpBrf in S.pombe increases the expression of a TATA-dependent, but not a TATA-less, suppressor tRNA gene. Since previous whole genome analysis also revealed TATA elements upstream of tRNA genes in Arabidopsis, this pathway may be more widespread than appreciated previously.

Incorporation of glucose analogs by GtfE and GtfD from the vancomycin biosynthetic pathway to generate variant glycopeptides.

Analogs of the glycopeptide antibiotics vancomycin and teicoplanin with alterations in one or both sugar moieties of the disaccharide have been prepared by tandem action of the vancomycin pathway glycosyltransferases GtfE and GtfD. All four regioisomers (2-, 3-, 4-, 6-) of TDP-deoxyglucoses and UDP/TDP-aminoglucoses were prepared, predominantly by action of D-glucopyranosyl-1-phosphate thymidylyltransferase, E(p). GtfE transferred the deoxyglucoses or aminoglucoses onto the 4-OH of 4-hydroxyphenylglycine of both the vancomycin and teicoplanin aglycone scaffolds. Kinetic analysis indicated the 2-, 3-, 4-, and 6-amino-glucoses were transferred by GtfE with only a 4- to 30-fold drop in k(cat) and no effect on K(m) compared to the native substrate, UDP/TDP-glucose, suggesting preparative utility. The next enzyme, GtfD, could utilize the variant glucosyl-peptides as substrates for transfer of L-4-epi-vancosamine. The aminosugar moieties in these variant glycopeptides introduce sites for acylation or reductive alkylation.

The structural basis for induction of VanB resistance.

Because teicoplanin and vancomycin are the last line of defense for many bacterial infections, the emergence of resistance to glycopeptide antibiotics in enterococci and streptococci has aroused concern. Despite their similarity in terms of structure and mechanism of action, vancomycin induces the expression of genes that leads to bacterial resistance, and teicoplanin does not. We have used a combination of chemical and enzymatic methods to produce sets of vancomycin and teicoplanin analogues that allow us to consider whether the aglycon, the carbohydrate, or other parts of these molecules stimulate VanB resistance. We show that the teicoplanin and vancomycin aglycons are the structural elements that lead to induction of resistance. We think that lipid-containing analogues of vancomycin, like teicoplanin itself, circumvent resistance because the lipid chain changes the periplasmic distribution of the glycopeptide and, therefore, changes the biosynthetic step that it blocks.