Discovery and Engineering of a Novel Baeyer-Villiger Monooxygenase with High Normal Regioselectivity.

Baeyer-Villiger monooxygenases (BVMOs) are remarkable biocatalysts for the Baeyer-Villiger oxidation of ketones to generate esters or lactones. The regioselectivity of BVMOs is essential for determining the ratio of the two regioisomeric products ("normal" and "abnormal") when catalyzing asymmetric ketone substrates. Starting from a known normal-preferring BVMO sequence from Pseudomonas putida KT2440 (PpBVMO), a novel BVMO from Gordonia sihwensis (GsBVMO) with higher normal regioselectivity (up to 97/3) was identified. Furthermore, protein engineering increased the specificity constant (kcat /KM ) 8.9-fold to 484 s-1 mM-1 for 10-ketostearic acid derived from oleic acid. Consequently, by using the variant GsBVMOC308L as an efficient biocatalyst, 10-ketostearic acid was efficiently transformed into 9-(nonanoyloxy)nonanoic acid, with a space-time yield of 60.5 g L-1 d-1 . This study showed that the mutant with higher regioselectivity and catalytic efficiency could be applied to prepare medium-chain ω-hydroxy fatty acids through biotransformation of long-chain aliphatic keto acids derived from renewable plant oils.

Phenomenon of dual- and single-retention behaviors of solutes and its validation by computational simulation in linear programmed temperature gas chromatography.

The current theory of programmed temperature gas chromatography considers that solutes are focused by the stationary phase at the column head completely and does not explicitly recognize the different effects of initial temperature (To ) and heating rate (rT ) on the retention time or temperature of a homologue series. In the present study, n-alkanes, 1-alkenes, 1-alkyl alcohols, alkyl benzenes, and fatty acid methyl esters standards were used as model chemicals and were separated on two nonpolar columns, one moderately polar column and one polar column. Effects of To and rT on the retention of nonstationary phase focusing solutes can be explicitly described with isothermal and cubic equation models, respectively. When the solutes were in the stationary phase focusing status, the single-retention behavior of solutes was observed. It is simple, dependent upon rT only and can be well described by the cubic equation model that was visualized through four sequential slope analyses. These observed dual- and single-retention behaviors of solutes were validated by various experimental data, physical properties, and computational simulation.

Genome-Wide Transcriptome Profiling of Mycobacterium smegmatis MC² 155 Cultivated in Minimal Media Supplemented with Cholesterol, Androstenedione or Glycerol.

Mycobacterium smegmatis strain MC² 155 is an attractive model organism for the study of M. tuberculosis and other mycobacterial pathogens, as it can grow well using cholesterol as a carbon resource. However, its global transcriptomic response remains largely unrevealed. In this study, M. smegmatis MC² 155 cultivated in androstenedione, cholesterol and glycerol supplemented media were collected separately for a RNA-Sequencing study. The results showed that 6004, 6681 and 6348 genes were expressed in androstenedione, cholesterol and glycerol supplemented media, and 5891 genes were expressed in all three conditions, with 237 specially expressed in cholesterol added medium. A total of 1852 and 454 genes were significantly up-regulated by cholesterol compared with the other two supplements. Only occasional changes were observed in basic carbon and nitrogen metabolism, while almost all of the genes involved in cholesterol catabolism and mammalian cell entry (MCE) were up-regulated by cholesterol, but not by androstenedione. Eleven and 16 gene clusters were induced by cholesterol when compared with glycerol or androstenedione, respectively. This study provides a comprehensive analysis of the cholesterol responsive transcriptome of M. smegmatis. Our results indicated that cholesterol induced many more genes and increased the expression of the majority of genes involved in cholesterol degradation and MCE in M. smegmatis, while androstenedione did not have the same effect.

[The Determination of Tungsten Extracted from Prefi llable Syringes by ICP-MS].

First, simply expounded the purpose and meaning of detecting tungsten extracted from prefi lable syringes: And then, designed simulation experiment for extracting tungsten from prefi lable glass syringes. and established determination methods of tungsten content by ICP-MSL Finaly, evaluated the limit of content of tungsten ?extracting from simulation experiment.

The Progression of Bone Mineral Density Abnormalities After Chemotherapy for Childhood Acute Lymphoblastic Leukemia.

Although reduced bone mineral density in survivors of childhood acute lymphoblastic leukemia (ALL) is well documented, the degree of demineralization and relation to age are not well described. This is a retrospective chart analysis of 58 patients consecutively treated for ALL without relapse, cranial irradiation, or transplantation. Bone mineral densities were measured by dual-energy x-ray absorptiometry and patients were divided by sex and age (≤5, 6 to 10, and >10 y) at diagnosis. Serial scans for 6 years after therapy were analyzed as Z-scores. Over 6 years after therapy, 93.1% of patients exhibited a decreased Z-score in at least 1 anatomic site. The difference in Z-score among the age cohorts was significant at both the lumbar spine and femoral neck. Patients older than 10 years at diagnosis had the lowest Z-scores: -2.78 and -2.87 for boys and -2.39 and -2.91 for girls at the lumbar spine and femoral neck, respectively. Children after ALL therapy exhibit a significant bone mineral deficit shortly after completion of therapy that persists for at least 6 years. The degree of bone demineralization can be followed up by a dual-energy x-ray absorptiometry scan and is most severe in patients older than 10 years at the initiation of therapy.

Treatment intensification with maraviroc (CCR5 antagonist) leads to declines in CD16-expressing monocytes in cART-suppressed chronic HIV-infected subjects and is associated with improvements in neurocognitive test performance: implications for HIV-associated neurocognitive disease (HAND).

HIV-associated neurocognitive disorders (HAND) continues to be prevalent (30-50%) despite plasma HIV-RNA suppression with combination antiretroviral therapy (cART). There is no proven therapy for individuals on suppressive cART with HAND. We have shown that the degree of HIV reservoir burden (HIV DNA) in monocytes appear to be linked to cognitive outcomes. HIV infection of monocytes may therefore be critical in the pathogenesis of HAND. A single arm, open-labeled trial was conducted to examine the effect of maraviroc (MVC) intensification on monocyte inflammation and neuropsychological (NP) performance in 15 HIV subjects on stable 6-month cART with undetectable plasma HIV RNA (<48 copies/ml) and detectable monocyte HIV DNA (>10 copies/10(6) cells). MVC was added to their existing cART regimen for 24 weeks. Post-intensification change in monocytes was assessed using multiparametric flow cytometry, monocyte HIV DNA content by PCR, soluble CD163 (sCD163) by an ELISA, and NP performance over 24 weeks. In 12 evaluable subjects, MVC intensification resulted in a decreased proportion of circulating intermediate (median; 3.06% (1.93, 6.45) to 1.05% (0.77, 2.26)) and nonclassical (5.2% (3.8, 7.9) to 3.2% (1.8, 4.8)) CD16-expressing monocytes, a reduction in monocyte HIV DNA content to zero log10 copies/10(6) cells and in levels of sCD163 of 43% by 24 weeks. This was associated with significant improvement in NP performance among six subjects who entered the study with evidence of mild to moderate cognitive impairment. The results of this study suggest that antiretroviral therapy with potency against monocytes may have efficacy against HAND.

Identification of gene expression profiles in the actinomycete Gordonia neofelifaecis grown with different steroids.

Gordonia neofelifaecis NRRL B-59395 was initially isolated from the fresh feces of a clouded leopard based on its ability to degrade cholesterol. The transcriptome profiles of G. neofelifaecis NRRL B-59395 grown with cholesterol, androstenedione (AD), and pyruvic acid were compared by RNA-Seq. The sterol catabolic genes are highly conserved in G. neofelifaecis, Rhodococcus jostii RHA1, and Mycobacterium tuberculosis. The RNA-Seq results indicated that the genes involved in the sterol side chain cleavage were exclusively induced by cholesterol, while the genes involved in the degradation of rings A/B and C/D were up-regulated by both cholesterol and AD. It appears that the induction mechanisms for the genes responsible for side chain cleavage and those for degradation of rings are different. There are approximately 21 genes encoding transporter proteins that are differentially expressed in cholesterol or AD compared with pyruvic acid. The genes camABCD and camM encode two systems that take up cholate, and they have been shown to be cholesterol- and AD-inducible. The potential biological functions of other differentially expressed genes are also discussed. These results will promote the functional characterization of the sterol catabolic genes and also provide important clues in understanding the mechanisms of their gene expression, and they may help us understand the mechanism underlying microbial cholesterol catabolism.

A family psychosocial risk questionnaire for use in pediatric practice.

The objective of this study is to develop new methods to better identify psychosocial risk such that children with the greatest risk of poor future outcomes receive more intensive preventive health services. Based on structured literature review and secondary data analysis, a 52-item psychosocial risk questionnaire was administered to 2,083 families of children (<36 months). To quantify the questionnaire's construct validity, developmental concern was assessed with the Ages and Stages Questionnaire version II (ASQ) [n = 1,163]. An iterative model selection process was used to produce the most parsimonious predictive model. Model fit was examined using c-statistics, the Hosmer-Lemeshow test, and a heuristic measure of model overfit based on the fitted log-likelihood values and associated number of degrees of freedom. We found 13 items easily obtained from parental report produced a regression model with a c-statistic of 0.70. Using an integer scoring system derived from the regression model, we calculated stratum specific likelihood ratios to revise a given prior probability of ASQ failure. The posterior probability of ASQ failure was 44.9 % for a child in the highest risk group (score >25) on the questionnaire, more than double our observed average failure rate of 19.5 %, while it was less than 7 % for a child with the lowest possible score on the questionnaire. Thirteen parent-reported items can be compiled into a summary psychosocial risk questionnaire that predicts failure on developmental screening among preschool children. With further validation, this questionnaire could conceivably be used by clinicians to tailor pediatric preventive care to children at varying levels of risk.

Adaptive Fitting of Linear Mixed-Effects Models with Correlated Random-effects.

Linear mixed-effects model has been widely used in longitudinal data analyses. In practice, the fitting algorithm can fail to converge due to boundary issues of the estimated random-effects covariance matrix G, i.e., being near-singular, non-positive definite, or both. Current available algorithms are not computationally optimal because the condition number of matrix G is unnecessarily increased when the random-effects correlation estimate is not zero. We propose an adaptive fitting (AF) algorithm using an optimal linear transformation of the random-effects design matrix. It is a data-driven adaptive procedure, aiming at reducing subsequent random-effects correlation estimates down to zero in the optimal transformed estimation space. Simulations show that AF significantly improves the convergent properties, especially under small sample size, relative large noise and high correlation settings. One real data for Insulin-like Growth Factor (IGF) protein is used to illustrate the application of this algorithm implemented with software package R (nlme).

The role of the CNA Pain Assessment Tool (CPAT) in the pain management of nursing home residents with dementia.

Implementation and testing of the CPAT in two hundred fifteen dementia residents of three regional skilled nursing facilities. To examine the effect of incorporating the CPAT into an AMDA long-term care pain management clinical practice guideline on nursing home residents with dementia. To evaluate changes in CPAT scores after treatment for pain. A non-randomized pre and post intervention design was used. Main outcome measures of the number of falls, episodes of distressed behavior and rates of antipsychotic usage were compared pre and post CPAT/AMDA guideline implementation. CPAT score changes were calculated after pain management. Falls and verbally aggressive behavior were reduced post intervention but did not achieve statistical significance. Antipsychotic usage declined significantly post intervention. CPAT scores declined significantly after treatment for pain. The implementation of a CPAT/AMDA guideline in skilled nursing facilities may reduce falls, verbally aggressive behaviors and antipsychotic usage in residents with dementia. The CPAT is useful in evaluating the effects of pain treatment in nursing home residents with dementia.

Draft genome sequence of Gordonia neofelifaecis NRRL B-59395, a cholesterol-degrading actinomycete.

We report a draft sequence of the genome of Gordonia neofelifaecis NRRL B-59395, a cholesterol-degrading actinomycete isolated from fresh feces of a clouded leopard (Neofelis nebulosa). As predicted, the reported genome contains several gene clusters for cholesterol degradation. This is the second available genome sequence of the family Gordoniaceae.

Anti-breast cancer potential of SS5020, a novel benzopyran antiestrogen.

Treatment with tamoxifen (TAM) increases the risk of developing endometrial cancer in women. The carcinogenic effect is thought to involve initiation and/or promotion resulting from DNA damage induced by TAM as well as its estrogenic action. To minimize this serious side-effect while increasing the anti-breast cancer potential, a new benzopyran antiestrogen, 2E-3-{4-[(7-hydroxy-2-oxo-3-phenyl-2H-chromen-4-yl)-methyl]-phenyl}-acrylic acid (SS5020), was synthesized. Unlike TAM, SS5020 exhibits no genotoxic activity to damage DNA. Furthermore, SS5020 does not present significant uterotrophic potential in rats; in contrast, the structurally related compounds, TAM, toremifene, raloxifene (RAL) and SP500263 all have uterotrophic activity. At the human equivalent molar dose of TAM (0.33 or 1.0 mg/kg), SS5020 had much stronger antitumor potential than those same antiestrogens against 7,12-dimethylbenz(a)anthracene-induced mammary carcinoma in rats. The growth of human MCF-7 breast cancer xenograft implanted into athymic nude mice was also effectively suppressed by SS5020. SS5020, lacking genotoxic and estrogenic actions, could be a safer and stronger antiestrogen alternative to TAM and RAL for breast cancer therapy and prevention.

Gordonia neofelifaecis sp. nov., a cholesterol side-chain-cleaving actinomycete isolated from the faeces of Neofelis nebulosa.

A cholesterol side-chain-cleaving bacterial strain, AD-6(T), was isolated from fresh faeces of a clouded leopard (Neofelis nebulosa) and was studied using a polyphasic taxonomic approach. 16S rRNA gene sequence analysis showed that the novel strain formed a distinct subline within the genus Gordonia, its closest neighbours being the type strains of Gordonia cholesterolivorans, Gordonia sihwensis and Gordonia hydrophobica, with sequence similarity values of 98.2, 97.8 and 97.6 %, respectively. The gyrB gene sequence of strain AD-6(T) exhibited similarities of 77-91 % with those of the type strains of recognized species of the genus Gordonia, being most similar to the type strains of G. sihwensis, G. hydrophobica and Gordonia hirsuta (91, 87 and 84 % similarity, respectively). The results of whole-cell fatty acid analyses and DNA-DNA relatedness data readily distinguished the new isolate from its nearest neighbours. Strain AD-6(T) is therefore considered to represent a novel species of the genus Gordonia, for which the name Gordonia neofelifaecis sp. nov. is proposed. The type strain is AD-6(T) (=NRRL B-59395(T)=CCTCC AB-209144(T)).

Anti-breast cancer potential of SS1020, a novel antiestrogen lacking estrogenic and genotoxic actions.

Long-term treatment with tamoxifen (TAM) increases the risk of developing endometrial cancer in women. Several antiestrogens developed in last decades have been discontinued from clinical testing because of their undesirable effects on the uterus. To avoid such serious side-effect while increasing the drug's anti-breast cancer potential, new triphenylethylene antiestrogens, 2E-3-{4-[(E)-4-chloro-1-(4-hydroxyphenyl)-2-phenylbut-1-enyl]-phenyl} acrylic acid (SS1020) and 2E-3-{4-[(Z)-4-chloro-1,2-diphenylbut-1-enyl]phenyl}acrylic acid (SS1010), were designed as safer alternatives. Unlike TAM, SS1020 does not present significant uterotrophic potential in rats; in contrast, SS1010, a compound removing a 4-OH moiety from SS1020, represented weak uterotrophic activity. The structurally related compounds 4-hydroxytamoxifen, toremifene, ospemifene, raloxifene (RAL) and GW5638 all have uterotrophic activity. In addition, SS1020 and SS1010 exhibit no genotoxic activity to damage hepatic DNA in rats. Therefore, SS1020 was selected as a safer antiestrogen candidate and used for evaluating the antitumor potential in animals. At the human equivalent doses of TAM, SS1020 had antitumor potential much higher than that of TAM, RAL and GW5638 against 7,12-dimethylbenz(a)anthracene-induced mammary carcinoma in rats. The growth of human MCF-7 breast cancer xenograft implanted into athymic nude mice was also effectively suppressed by SS1020. SS1020, lacking estrogenic and genotoxic actions and having strong antitumor potency superior to that of TAM and RAL, could be a safer alternative for breast cancer therapy and prevention.

Exploration of Patients' Spiritual/Religious Beliefs and Resuscitation Decisions.

Artificial resuscitation has potential to reverse a premature death or to prolong the dying process. The resuscitation decision is one of life and death making it imperative that healthcare providers understand patients' beliefs. Making the decision to resuscitate has been associated with patients' spiritual/religious beliefs. Clinicians' assumptions based upon a patients' religion or spiritual beliefs may bias the resuscitation decision. The purpose of this study was to determine associations between hospitalized patients' spiritual/religious beliefs and their resuscitation decisions. A single-site, correlational study was conducted with a convenience sample of hospitalized patients in Honolulu, HI. Patients were enrolled November 2015 to January 2016. Spiritual/religious beliefs were assessed using two validated metrics. Two questions were used to determine the resuscitation decision (chest compressions and intubation). The sample of 84 patients represented no ethnic majority among Caucasian, Asian, and Native Hawaiian/Pacific Islander. Seventy-nine percent of the participants identified theistic spiritual beliefs. No associations were found between resuscitation decisions with either spiritual/religious beliefs or demographic characteristics of this study sample. Interestingly, 20% of the participants answered yes to only one of the resuscitation decision questions. Thus, providers' assumptions should not be made about an association between spiritual/religious beliefs and resuscitation decisions. It is imperative that patients are aware of the necessity for both medical interventions of chest compressions and intubation. Further research should address the complexity of the resuscitation decision, including patients understanding of medical interventions and anticipated prognosis, and other influencing factors.

Temporal trends of cirrhosis associated conditions.

BACKGROUND: Chronic liver disease and cirrhosis is the 12th leading cause of death in the United States. Patients with decompensated-cirrhosis, especially with hepatic encephalopathy/coma (HC), have a higher rate of early readmission and contribute to higher healthcare cost. AIM: To evaluate the national inpatient trends of discharges, mortalities and financial impacts associated with four common conditions of cirrhosis. METHODS: The publicly available Healthcare Cost and Utilization Project National Inpatient Sample database was utilized to examine the temporal trends of total number of discharges, mortalities and inpatient costs related to hospitalization with a primary diagnosis of HC, transjugular intrahepatic portosystemic shunt (TIPS), esophageal varices with bleeding (EV) and spontaneous bacterial peritonitis (SBP) from 2005 to 2014. The ten-year temporal trends were assessed using simple linear regressions and multiple regression analysis. Two-sided P < 0.05 was considered statistically significant. RESULTS: From 2005 to 2014, the total number of discharges with cirrhosis-associated complications trended up for HC, SBP and EV (HC by 70% increase, P < 0.0001; SBP by 819% increase, P = 0.0002; EV by 9% increase, P = 0.016), but not for TIPS (P = 0.90). HC related to viral hepatitis showed faster increase by 357% (P < 0.0001) in comparison to HC not related to viral hepatitis by 33 % (P = 0.0006). Overall, in-hospital mortality rates for each condition decreased from 2005 to 2014 (HC by 29% reduction, P = 0.0024; SBP by 26% reduction, P = 0.0038; TIPS by 32% reduction, P = 0.021) except for EV (P = 0.34). After adjustment for inflation, aggregate cost of hospitalization for EV, HC, and SBP significantly increased by 20%, 86%, and 980%, respectively, from 2005 to 2014 (all P < 0.02), while TIPS had trend toward decreasing cost by 3% (P = 0.95). CONCLUSION: The number of hospitalizations and costs for some of the cirrhosis-associated conditions increased. However, the inpatient mortality rates for most of these conditions decreased.

Temporal Trends of Hospitalization, Mortality, and Financial Impact Related to Preeclampsia with Severe Features in Hawai'i and the United States.

The temporal trend of hospitalizations, cost, and outcomes associated with preeclampsia with severe features have been inadequately studied. The publicly available Healthcare Cost and Utilization Project (HCUP) National Inpatient Sample (NIS) database was accessed to examine the temporal trend of total number of discharges, age, death, and mean charges per admission associated with preeclampsia with severe features. Eleven-year temporal trends (2004 to 2014) of these measures were compared using linear regression and run charts using the statistical process control rule. From 2004 to 2014, the total number of discharges related to preeclampsia with severe features increased both for Hawai'i and the U.S. (United States) (Hawai'i: 104 to 231; U.S.: 35,082 to 55,235; both P<.0001). The corresponding rates of discharges per 100,000 population also both increased (Hawai'i: 8.2 to 16.3; U.S.: 12.0 to 17.3; both P<.0001). Comparing the temporal trends between Hawai'i and the U.S., Hawai'i had a significantly higher average annual increase in the rate of incidence than the national level (an annual increase rate of 9.2% in Hawai'i vs 4.2% nationally; P=.0004). The cost of hospitalization for preeclampsia with severe features also showed an increased trend for both Hawai'i and the U.S. (Hawai'i: 33.1% increase, P=.0005; U.S.: 41.1% increase, P<.0001). In the U.S., in-hospital mortality rates associated with this condition decreased from 0.09% in 2004 to 0.02% in 2014 (P=.03). In conclusion, the number of discharges related to preeclampsia with severe features increased over an 11-year period in Hawai'i and the U.S., and the rate of increase was higher in Hawai'i than the U.S. Maternal mortality rates from this condition also declined over the study period.

Patients with acute cholecystitis should be admitted to a surgical service.

BACKGROUND: In bowel obstruction and biliary pancreatitis, patients receive more expedient surgical care when admitted to surgical compared with medical services. This has not been studied in acute cholecystitis. METHODS: Retrospective analysis of clinical and cost data from July 2013 to September 2015 for patients with cholecystitis who underwent laparoscopic cholecystectomy in a tertiary care inpatient hospital. One hundred ninety lower-risk (Charlson-Deyo) patients were included. We assessed admitting service, length of stay (LOS), time from admission to surgery, time from surgery to discharge, number of imaging studies, and total cost. RESULTS: Patients admitted to surgical (n = 106) versus medical (n = 84) service had shorter mean LOS (1.4 days vs. 2.6 days), shorter time from admission to surgery (0.4 days vs. 0.8 days), and shorter time from surgery to discharge (0.8 days vs. 1.1 days). Surgical service patients had fewer CT (38% vs. 56%) and magnetic resonance imaging (MRI) (5% vs. 16%) studies. Cholangiography (30% vs. 25%) and endoscopic retrograde cholangiopancreatography (ERCP) (3 vs. 8%) rates were similar. Surgical service patients had 39% lower median total costs (US $7787 vs. US $12572). CONCLUSION: Nonsurgical admissions of patients with cholecystitis are common, even among lower-risk patients. Routine admission to the surgical service should decrease LOS, resource utilization and costs. LEVEL OF EVIDENCE: Therapeutic/care management, level IV.

Recess environment and curriculum intervention on children's physical activity: IPLAY.

Understanding the impacts of the built environment on physical activity (PA) is essential to promoting children's PA. The purpose of this study was to investigate the effects of schoolyard renovations and a PA recess curriculum alone and in combination on children's PA. This was a 2 (learning landscape [LL] vs. non-LL) × 2 (curriculum intervention vs. no curriculum intervention) factorial design with random assignment to the curriculum intervention, and six elementary schools per condition. PA outcomes were assessed preprogram, mid-program, immediate postprogram, and one year postprogram. No meaningful intervention effects were found. Lack of an effect may be due to the brief dose of recess, the curriculum not being integrated within the schoolyard, the LL implementation occurring prior to the study, or the already high levels of PA. Potential avenues to promote PA include making recess longer, integrating recess into the school curricula, and developing recess PA curricula integrating schoolyards.

An experimental study on the noise properties of x-ray CT sinogram data in Radon space.

Computed tomography (CT) has been well established as a diagnostic tool through hardware optimization and sophisticated data calibration. For screening purposes, the associated x-ray exposure risk must be minimized. An effective way to minimize the risk is to deliver fewer x-rays to the subject or lower the mAs parameter in data acquisition. This will increase the data noise. This work aims to study the noise property of the calibrated or preprocessed sinogram data in Radon space as the mAs level decreases. An anthropomorphic torso phantom was scanned repeatedly by a commercial CT imager at five different mAs levels from 100 down to 17 (the lowest value provided by the scanner). The preprocessed sinogram datasets were extracted from the CT scanner to a laboratory computer for noise analysis. The repeated measurements at each mAs level were used to test the normality of the repeatedly measured samples for each data channel using the Shapiro-Wilk statistical test merit. We further studied the probability distribution of the repeated measures. Most importantly, we validated a theoretical relationship between the sample mean and variance at each channel. It is our intention that the statistical test and particularly the relationship between the first and second statistical moments will improve low-dose CT image reconstruction for screening applications.