Kinetic modeling of microbial growth, enzyme activity, and gene deletions: An integrated model of ß-glucosidase function in Cellvibrio japonicus.

Understanding the complex growth and metabolic dynamics in microorganisms requires advanced kinetic models containing both metabolic reactions and enzymatic regulation to predict phenotypic behaviors under different conditions and perturbations. Most current kinetic models lack gene expression dynamics and are separately calibrated to distinct media, which consequently makes them unable to account for genetic perturbations or multiple substrates. This challenge limits our ability to gain a comprehensive understanding of microbial processes towards advanced metabolic optimizations that are desired for many biotechnology applications. Here, we present an integrated computational and experimental approach for the development and optimization of mechanistic kinetic models for microbial growth and metabolic and enzymatic dynamics. Our approach integrates growth dynamics, gene expression, protein secretion, and gene-deletion phenotypes. We applied this methodology to build a dynamic model of the growth kinetics in batch culture of the bacterium Cellvibrio japonicus grown using either cellobiose or glucose media. The model parameters were inferred from an experimental data set using an evolutionary computation method. The resulting model was able to explain the growth dynamics of C. japonicus using either cellobiose or glucose media and was also able to accurately predict the metabolite concentrations in the wild-type strain as well as in ß-glucosidase gene deletion mutant strains. We validated the model by correctly predicting the non-diauxic growth and metabolite consumptions of the wild-type strain in a mixed medium containing both cellobiose and glucose, made further predictions of mutant strains growth phenotypes when using cellobiose and glucose media, and demonstrated the utility of the model for designing industrially-useful strains. Importantly, the model is able to explain the role of the different ß-glucosidases and their behavior under genetic perturbations. This integrated approach can be extended to other metabolic pathways to produce mechanistic models for the comprehensive understanding of enzymatic functions in multiple substrates.

The complex physiology of Cellvibrio japonicus xylan degradation relies on a single cytoplasmic ß-xylosidase for xylo-oligosaccharide utilization.

Lignocellulose degradation by microbes plays a central role in global carbon cycling, human gut metabolism and renewable energy technologies. While considerable effort has been put into understanding the biochemical aspects of lignocellulose degradation, much less work has been done to understand how these enzymes work in an in vivo context. Here, we report a systems level study of xylan degradation in the saprophytic bacterium Cellvibrio japonicus. Transcriptome analysis indicated seven genes that encode carbohydrate active enzymes were up-regulated during growth with xylan containing media. In-frame deletion analysis of these genes found that only gly43F is critical for utilization of xylo-oligosaccharides, xylan, and arabinoxylan. Heterologous expression of gly43F was sufficient for the utilization of xylo-oligosaccharides in Escherichia coli. Additional analysis found that the xyn11A, xyn11B, abf43L, abf43K, and abf51A gene products were critical for utilization of arabinoxylan. Furthermore, a predicted transporter (CJA_1315) was required for effective utilization of xylan substrates, and we propose this unannotated gene be called xntA (xylan transporter A). Our major findings are (i) C. japonicus employs both secreted and surface associated enzymes for xylan degradation, which differs from the strategy used for cellulose degradation, and (ii) a single cytoplasmic ß-xylosidase is essential for the utilization of xylo-oligosaccharides.

CancerInSilico: An R/Bioconductor package for combining mathematical and statistical modeling to simulate time course bulk and single cell gene expression data in cancer.

Bioinformatics techniques to analyze time course bulk and single cell omics data are advancing. The absence of a known ground truth of the dynamics of molecular changes challenges benchmarking their performance on real data. Realistic simulated time-course datasets are essential to assess the performance of time course bioinformatics algorithms. We develop an R/Bioconductor package, CancerInSilico, to simulate bulk and single cell transcriptional data from a known ground truth obtained from mathematical models of cellular systems. This package contains a general R infrastructure for running cell-based models and simulating gene expression data based on the model states. We show how to use this package to simulate a gene expression data set and consequently benchmark analysis methods on this data set with a known ground truth. The package is freely available via Bioconductor: http://bioconductor.org/packages/CancerInSilico/.

Trauma team activation criteria and outcomes of geriatric trauma: 10 year single centre cohort study.

BACKGROUND: With the aging population, the number of older patients with multiple injuries is increasing. The aim of this study was to understand the patterns and outcomes of older patients admitted to a major trauma centre in Hong Kong from 2006 to 2015, and investigate the performance of the trauma team activation (TTA) criteria for these elderly patients. METHODS: This was a retrospective cohort study from a university hospital major trauma centre in Hong Kong from 2006 to 2015. Patients aged 55 or above who entered the trauma registry were included. Patients were divided into those aged 55-70, and above 70. To test the performance of the TTA criteria, we defined injured patients with severe outcomes as those having any of the following: death within 30 days; the need for surgery; or the need for intensive care unit (ICU) care. RESULTS: 2218 patients were included over the 10 year period. The 30-day mortality was 7.5% for aged 55-70 and 17.7% for those aged above 70. The sensitivity of TTA criteria for identifying severe outcomes for those aged 55 or above was 35.6%, with 91.6% specificity. The under-triage rate was 59% for age 55-70, and 69.1% for those aged above 70. CONCLUSION: There is a need to consider alternative TTA criteria for our geriatric trauma population, and to more clearly define the process and standards of care in Hong Kong.

Assessment of the Authenticity of Herbal Dietary Supplements: Comparison of Chemical and DNA Barcoding Methods.

About 7 % of the U. S. population reports using botanical dietary supplements. Increased use of such supplements has led to discussions related to their authenticity and quality. Reports of adulteration with substandard materials or pharmaceuticals are of concern because such substitutions, whether inadvertent or deliberate, may reduce the efficacy of specific botanicals or lead to adverse events. Methods for verifying the identity of botanicals include macroscopic and microscopic examinations, chemical analysis, and DNA-based methods including DNA barcoding. Macroscopic and microscopic examinations may fail when a supplement consists of botanicals that have been processed beyond the ability to provide morphological characterizations. Chemical analysis of specific marker compounds encounters problems when these compounds are not distinct to a given species or when purified reference standards are not available. Recent investigations describing DNA barcoding analysis of botanical dietary supplements have raised concerns about the authenticity of the supplements themselves as well as the appropriateness of using DNA barcoding techniques with finished botanical products. We collected 112 market samples of frequently consumed botanical dietary supplements of ginkgo, soy, valerian, yohimbe, and St. John's wort and analyzed each for specific chemical markers (i.e., flavonol glycosides, total isoflavones, total valerenic acids, yohimbine, and hypericins, respectively). We used traditional DNA barcoding techniques targeting the nuclear ITS2 gene and the chloroplast gene psbA-trnH on the same samples to determine the presence of DNA of the labelled ingredient. We compared the results obtained by both methods to assess the contribution of each in determining the identity of the samples.

Differences in hepatic phenotype between hemochromatosis patients with HFE C282Y homozygosity and other HFE genotypes.

OBJECTIVE: There are limited data comparing hepatic phenotype among hemochromatosis patients with different HFE genotypes. The goal of this study was to compare hepatic histopathologic features and hepatic iron concentration (HIC) among patients with phenotypic hemochromatosis and different HFE genotypes. METHODS: We studied 182 US patients with phenotypic hemochromatosis. Degree of hepatic fibrosis, pattern of iron deposition, presence of steatosis or necroinflammation, and HIC were compared among different HFE genotypes. RESULTS: C282Y/H63D compound heterozygotes and patients with HFE genotypes other than C282Y/C282Y were more likely to have stainable Kupffer cell iron (31.1% vs. 9.5%; P=0.02), portal or lobular inflammation (28.9% vs. 15.6%; P=0.03), and steatosis (33.3% vs. 10.2%; P<0.01) on liver biopsy than C282Y homozygotes. Mean log10 HIC (P<0.05) and log10 ferritin (P<0.05) were higher among C282Y homozygotes than in patients with other HFE genotypes. In a logistic regression analysis using age, sex, HFE genotype, log10 ferritin, and log10 HIC as independent variables, log10 serum ferritin (P=0.0008), male sex (P=0.0086), and log10 HIC (P=0.047), but not HFE genotype (P=0.0554) were independently associated with presence or absence of advanced hepatic fibrosis. CONCLUSIONS: C282Y/H63D compound heterozygotes and other non-C282Y homozygotes which express the hepatic hemochromatosis phenotype frequently have evidence of steatosis or chronic hepatitis and lower body iron stores than C282Y homozygotes. These data suggest that presence of concomitant liver disease may explain expression of the hemochromatosis phenotype among non-C282Y homozygotes. Increased age, HIC, and ferritin are associated with advanced hepatic fibrosis, regardless of HFE genotype.

Comorbidity has no impact on unplanned discharge or functional gains in persons with dysvascular amputation.

OBJECTIVE: To examine how factors associated with infection, organ failure, poor wound healing, or indices of chronic vascular disease are associated with unplanned transfers and functional gains in a population of dysvascular amputees during inpatient rehabilitation. DESIGN: Cross-sectional. SETTING: Inpatient rehabilitation unit at an academic medical centre. PATIENTS: A total of 118 patients with new, dysvascular, lower-extremity, amputation participating in inpatient rehabilitation. METHODS: Logistic regression and indices of change (minimal detectable change; MDC90), standardized response mean and effect size were used to examine the risks of unplanned transfer and functional change. MAIN OUTCOME MEASUREMENTS: Rate of unplanned transfers from rehabilitation, and Functional Independence Measure (FIM). RESULTS: Out of the total of 118 patients 19 had unplanned transfers due to medical complications. Age, creatinine, haemoglobin, white blood cell count, haemodialysis, wound vacuum device use, intravenous antibiotic use, or previous amputations were not independently associated with unplanned transfers, motor FIM change or efficiency. The MDC90 for motor FIM was 17.84, with 21.2% of patients exceeding this value; standardized response mean and effect size were large (1.03 and 1.39, respectively). CONCLUSION: This study suggests that the presence of comorbidities in a population of dysvascular amputees participating in inpatient rehabilitation did not increase the risk of unplanned transfers or affect FIM gains.

Effects of a Proline Endopeptidase on the Detection and Quantitation of Gluten by Antibody-Based Methods during the Fermentation of a Model Sorghum Beer.

The effectiveness of a proline endopeptidase (PEP) in hydrolyzing gluten and its putative immunopathogenic sequences was examined using antibody-based methods and mass spectrometry (MS). Based on the results of the antibody-based methods, fermentation of wheat gluten containing sorghum beer resulted in a reduction in the detectable gluten concentration. The addition of PEP further reduced the gluten concentration. Only one sandwich ELISA was able to detect the apparent low levels of gluten present in the beers. A competitive ELISA using a pepsin-trypsin hydrolysate calibrant was unreliable because the peptide profiles of the beers were inconsistent with that of the hydrolysate calibrant. Analysis by MS indicated that PEP enhanced the loss of a fragment of an immunopathogenic 33-mer peptide in the beer. However, Western blot results indicated partial resistance of the high molecular weight (HMW) glutenins to the action of PEP, questioning the ability of PEP in digesting all immunopathogenic sequences present in gluten.

Mate choice and the evolutionary stability of a fixed threshold in a sequential search strategy.

The sequential search strategy is a prominent model of searcher behavior, derived as a rule by which females might sample and choose a mate from a distribution of prospective partners. The strategy involves a threshold criterion against which prospective mates are evaluated. The optimal threshold depends on the attributes of prospective mates, which are likely to vary across generations or within the lifetime of searchers due to stochastic environmental events. The extent of this variability and the cost to acquire information on the distribution of the quality of prospective mates determine whether a learned or environmentally canalized threshold is likely to be favored. In this paper, we determine conditions on cross-generational perturbations of the distribution of male phenotypes that allow for the evolutionary stability of an environmentally canalized threshold. In particular, we derive conditions under which there is a genetically determined threshold that is optimal over an evolutionary time scale in comparison to any other unlearned threshold. These considerations also reveal a simple algorithm by which the threshold could be learned.

Surgical treatment of epilepsy in children caused by focal cortical dysplasia.

Focal cortical dysplasia (FCD) is a congenital disorder of neuronal migration that is increasingly recognized as a common cause of seizures in children, occurring in 20-30% of all surgically treated cases of epilepsy in the pediatric population. Advances in neuroimaging have contributed to recognition of FCD. We report 15 children (9 female, 6 male) with FCD and surgically treated intractable epilepsy. In 9 cases, a surgical strategy of anatomic (frameless stereotactic) grid placement and physiologic (electrocorticography) resection was employed. Postoperative MRI scans were obtained, the pathologic specimen was graded according to the Brannstrom system, and seizure outcome was defined using the Engel classification. There were no deaths and no permanent morbidity. After, on average, 4 years since treatment, 10 children are seizure free, 2 are 2A, 2 are 2B and 1 is 3A. Predictors of good outcome are an MRI-defined lesion and increased cortical disorganization (higher Brannstrom grade). Subtotal resection did not preclude a seizure-free outcome.