17 years' experience of surgical management of thoracic outlet syndrome at a district general hospital.

INTRODUCTION: Thoracic outlet syndrome (TOS) is caused by compression of the neurovascular structures passing through the thoracic inlet. It is categorised into three subtypes: neurogenic TOS (NTOS), venous TOS (VTOS) and arterial TOS (ATOS). This study evaluates the outcomes of patients who underwent first rib resection (FRR) for TOS during a period of 17 years at a single district general hospital. METHODS: Retrospective review of patient notes of individuals treated with FRR from August 2004 to August 2021. RESULTS: A total of 62 FRRs were performed on 51 individual patients. Indications for FRR included 42 NTOS (68%), 6 VTOS (10%) and 14 ATOS (23%). Thirty-four patients (64%) were female and the mean age at time of surgery was 39 years (range 27 to 64 years). Eleven patients (21%) underwent bilateral FRR and seven cases of cervical ribs were observed. The mean time from initial symptoms to diagnosis was 18 months (range 2 to 60 months). Overall, outcomes after surgery were positive across all subtypes of TOS. Based on Derkash's classification, 52 patients (84%) reported excellent/good, 8 (13%) reported fair and 2 (3%) reported poor resolution of symptoms at 6 month follow-up. Complications included four (9%) pneumothorax, two (4%) wound infections, two (4%) haematoma, one (2%) haemothorax, three (5%) phrenic nerve complications and one (2%) brachial neuropraxia. CONCLUSIONS: FRR for TOS can be performed safely and effectively in a district general hospital environment with excellent patient clinical outcomes.

A Nationwide Enumeration of the Surgical Workforce, its Production and Disparities in Operative Productivity in Liberia.

BACKGROUND: Any health care system that strives to deliver good health and well-being to its population relies on a trained workforce. The aim of this study was to enumerate surgical provider density, describe operative productivity and assess the association between key surgical system characteristics and surgical provider productivity in Liberia. METHODS: A nationwide survey of operation theatre logbooks, available human resources and facility infrastructure was conducted in 2018. Surgical providers were counted, and their productivity was calculated based on operative numbers and full-time equivalent positions. RESULTS: A total of 286 surgical providers were counted, of whom 67 were accredited specialists. This translated into a national density of 1.6 specialist providers per 100,000 population. Non-specialist physicians performed 58.3 percent (3607 of 6188) of all operations. Overall, surgical providers performed a median of 1.0 (IQR 0.5-2.7) operation per week, and there were large disparities in operative productivity within the workforce. Most operations (5483 of 6188) were categorized as essential, and each surgical provider performed a median of 2.0 (IQR 1.0-5.0) different types of essential procedures. Surgical providers who performed 7-14 different types of essential procedures were more than eight times as productive as providers who performed 0-1 essential procedure (operative productivity ratio = 8.66, 95% CI 6.27-11.97, P < 0.001). CONCLUSION: The Liberian health care system struggles with an alarming combination of few surgical providers and low provider productivity. Disaggregated data can provide a high-resolution picture of local challenges that can lead to local solutions.

Breath testing for intra-abdominal infection: appendicitis, a preliminary study.

In the current pilot study we aimed to determine whether breath analysis could be used to help recognise intra-abdominal infection, using acute appendicitis as an exemplar condition. Our study included 53 patients (aged 18-88 years) divided into three groups: appendix group, 26 (13 male) patients suffering from acute appendicitis; control group 20 (seven male) patients undergoing elective abdominal surgery; normal group, seven patients who were clinically diagnosed with appendicitis, but whose appendix was normal on histological examination. Samples of breath were analysed using ion molecule reaction mass spectroscopy measuring the concentration of volatile compounds (VCs) with molecular masses 27-123. Intraperitoneal gas samples were collected from a subset of 23 patients (nine diagnosed with acute appendicitis). Statistically significant differences in the concentration of VCs in breath were found between the three groups. Acetone, isopropanol, propanol, butyric acid, and further unassigned VCs with molecular mass/charge ratio (m/z) 56, 61 and 87 were all identified with significant endogenous contributions. Principle component analysis was able to separate the control and appendicitis groups for seven variables: m/z = 56, 58, 59, 60, 61, 87 and 88. Comparing breath and intraperitoneal samples showed significant relationships for acetone and the VC with m/z = 61. Our data suggest that it may be possible to help diagnose acute appendicitis by breath analysis; however, factors such as length of starvation remain to be properly accounted for and the management or mitigation of background levels needs to be properly addressed, and larger studies relating breath VCs to the causative organisms may help to highlight the relative importance of individual VCs.

A Genocentric Approach to Discovery of Mendelian Disorders.

The advent of inexpensive, clinical exome sequencing (ES) has led to the accumulation of genetic data from thousands of samples from individuals affected with a wide range of diseases, but for whom the underlying genetic and molecular etiology of their clinical phenotype remains unknown. In many cases, detailed phenotypes are unavailable or poorly recorded and there is little family history to guide study. To accelerate discovery, we integrated ES data from 18,696 individuals referred for suspected Mendelian disease, together with relatives, in an Apache Hadoop data lake (Hadoop Architecture Lake of Exomes [HARLEE]) and implemented a genocentric analysis that rapidly identified 154 genes harboring variants suspected to cause Mendelian disorders. The approach did not rely on case-specific phenotypic classifications but was driven by optimization of gene- and variant-level filter parameters utilizing historical Mendelian disease-gene association discovery data. Variants in 19 of the 154 candidate genes were subsequently reported as causative of a Mendelian trait and additional data support the association of all other candidate genes with disease endpoints.

Direct evaluation of the force constant matrix in quantum Monte Carlo.

We develop a formalism to directly evaluate the matrix of force constants within a Quantum Monte Carlo calculation. We utilize the matrix of force constants to accurately relax the positions of atoms in molecules and determine their vibrational modes, using a combination of variational and diffusion Monte Carlo. The computed bond lengths differ by less than 0.007 Šfrom the experimental results for all four tested molecules. For hydrogen and hydrogen chloride, we obtain fundamental vibrational frequencies within 0.1% of experimental results and ∼10 times more accurate than leading computational methods. For carbon dioxide and methane, the vibrational frequency obtained is on average within 1.1% of the experimental result, which is at least 3 times closer than results using restricted Hartree-Fock and density functional theory with a Perdew-Burke-Ernzerhof functional and comparable or better than density functional theory with a semi-empirical functional.

Measurement of breath acetone in patients referred for an oral glucose tolerance test.

Breath acetone concentrations were measured in 141 subjects (aged 19-91 years, mean = 59.11 years, standard deviation = 12.99 years), male and female, undergoing an oral glucose tolerance test (OGTT), having been referred to clinic on suspicion of type 2 diabetes. Breath samples were measured using an ion-molecule-reaction mass spectrometer, at the commencement of the OGTT, and after 1 and 2 h. Subjects were asked to observe the normal routine before and during the OGTT, which includes an overnight fast and ingestion of 75 g glucose at the beginning of the routine. Several groups of diagnosis were identified: type 2 diabetes mellitus positive (T2DM), n = 22; impaired glucose intolerance (IGT), n = 33; impaired fasting glucose, n = 14; and reactive hypoglycaemia, n = 5. The subjects with no diagnosis (i.e. normoglycaemia) were used as a control group, n = 67. Distributions of breath acetone are presented for the different groups. There was no evidence of a direct relationship between blood glucose (BG) and acetone measurements at any time during the study (0 h: p = 0.4482; 1 h: p = 0.6854; and 2 h: p = 0.1858). Nor were there significant differences between the measurements of breath acetone for the control group and the T2DM group (0 h: p = 0.1759; 1 h: p = 0.4521; and 2 h: p = 0.7343). However, the ratio of breath acetone at 1 h to the initial breath acetone was found to be significantly different for the T2DM group compared to both the control and IGT groups (p = 0.0189 and 0.011, respectively). The T2DM group was also found to be different in terms of ratio of breath acetone after 1 h to that at 2 h during the OGTT. And was distinctive in that it showed a significant dependence upon the level of BG at 2 h (p = 0.0146). We conclude that single measurements of the concentrations of breath acetone cannot be used as a potential screening diagnostic for T2DM diabetes in this cohort, but monitoring the evolution of breath acetone could open a non-invasive window to aid in the diagnosis of metabolic conditions.

The Low-Energy Neutral Imager (LENI).

To achieve breakthroughs in the areas of heliospheric and magnetospheric energetic neutral atom (ENA) imaging, a new class of instruments is required. We present a high angular resolution ENA imager concept aimed at the suprathermal plasma populations with energies between 0.5 and 20 keV. This instrument is intended for understanding the spatial and temporal structure of the heliospheric boundary recently revealed by Interstellar Boundary Explorer instrumentation and the Cassini Ion and Neutral Camera. The instrument is also well suited to characterize magnetospheric ENA emissions from low-altitude ENA emissions produced by precipitation of magnetospheric ions into the terrestrial upper atmosphere, or from the magnetosheath where solar wind protons are neutralized by charge exchange, or from portions of the ring current region. We present a new technique utilizing ultrathin carbon foils, 2-D collimation, and a novel electron optical design to produce high angular resolution (≤2°) and high-sensitivity (≥10-3 cm2 sr/pixel) ENA imaging in the 0.5-20 keV energy range.

Characterization of Ferroplasma acidiphilum growing in pure and mixed culture with Leptospirillum ferriphilum.

Biomining is defined as biotechnology for metal recovery from minerals, and is promoted by the concerted effort of a consortium of acidophile prokaryotes, comprised of members of the Bacteria and Archaea domains. Ferroplasma acidiphilum and Leptospirillum ferriphilum are the dominant species in extremely acid environments and have great use in bioleaching applications; however, the role of each species in this consortia is still a subject of research. The hypothesis of this work is that F. acidiphilum uses the organic matter secreted by L. ferriphilum for growth, maintaining low levels of organic compounds in the culture medium, preventing their toxic effects on L. ferriphilum. To test this hypothesis, a characterization of Ferroplasma acidiphilum strain BRL-115 was made with the objective of determining its optimal growth conditions. Subsequently, under the optimal conditions, L. ferriphilum and F. acidiphilum were tested growing in each other's supernatant, in order to define if there was exchange of metabolites between the species. With these results, a mixed culture in batch cyclic operation was performed to obtain main specific growth rates, which were used to evaluate a mixed metabolic model previously developed by our group. It was observed that F. acidiphilum, strain BRL-115 is a chemomixotrophic organism, and its growth is maximized with yeast extract at a concentration of 0.04% wt/vol. From the experiments of L. ferriphilum growing on F. acidiphilum supernatant and vice versa, it was observed that in both cases cell growth is favorably affected by the presence of the filtered medium of the other microorganism, proving a synergistic interaction between these species. Specific growth rates were obtained in cyclic batch operation of the mixed culture and were used as input data for a Flux Balance Analysis of the mixed metabolic model, obtaining a reasonable behavior of the metabolic fluxes and the system as a whole, therefore consolidating the model previously developed. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1390-1396, 2016.

The effect of FES-rowing training on cardiac structure and function: pilot studies in people with spinal cord injury.

STUDY DESIGN: Two studies were conducted: Study-1 was cross-sectional; and Study-2 a longitudinal repeated measures design. OBJECTIVES: To examine the influence of functional electrical stimulation (FES) rowing training on cardiac structure and function in people with spinal cord injury (SCI). SETTING: A university sports science department and home-based FES-training. METHODS: Fourteen participants with C4-T10 SCI (American Spinal Injury Association Impairment Scale A or B) were recruited for the studies. Cardiac structure and function, and peak: oxygen uptake ([Vdot ]O2peak), power output (POpeak) and heart rate (HRpeak), were compared between two FES-untrained groups (male n=3, female n=3) and an FES-trained group (male n=3) in Study-1 and longitudinally assessed in an FES-naive group (male n=1, female n=4) in Study-2. Main outcome measures left ventricular-dimensions, volumes, mass, diastolic and systolic function, and [Vdot ]O2peak, POpeak and HRpeak. In Study-2, in addition to peak values, the [Vdot ]O2 sustainable over 30 min and the related PO and HR were also assessed. RESULTS: Sedentary participants with chronic SCI had cardiac structure and function at the lower limits of non-SCI normal ranges. Individuals with chronic SCI who habitually FES-row have cardiac structure and function that more closely resemble non-SCI populations. A programme of FES-rowing training improved cardiac structure and function in previously FES-naive people. CONCLUSION: FES-rowing training appears to be an effective stimulus for positive cardiac remodelling in people with SCI. Further work, with greater participant numbers, should investigate the impact of FES-rowing training on cardiac health in SCI. SPONSORSHIP: We thank the INSPIRE Foundation, UK, for funding these studies.

A Systematic Review of the Effects of Exercise Interventions on Body Composition in HIV+ Adults.

Over the years, physical activity and exercise have been used to positively impact the health and quality of life of persons infected with HIV and, more recently, has been associated with a spectrum of body composition changes. The aim of this review was to examine the effects of various exercise interventions on body composition in HIV positive adults, using a search strategy of randomized, controlled trials (RCTs). A systematic review was performed by five independent reviewers using a predetermined protocol adapted from previous research for assessing the articles for inclusion, the extracted data, and methodological quality. Eight RCTs involving 430 (26% female) HIV positive adults performing exercise a minimum of thrice weekly for at least six weeks were finally selected: Four were progressive resistance training (PRT) studies, three were aerobic training (AT) studies, and one involved yoga. In the PRT studies, there were significant increases in three anthropometric measures, namely, body mass, sum of skinfolds and sum of limb girths. In the AT studies, significant decreases were found in seven anthropometric measures, namely, body mass index, waist-hip ratio, body mass, triceps skinfold, waist circumference and sum of skinfolds. With yoga, the changes were non-significant. Exercise contributes to improved body composition and, when applied safely, appears to be beneficial for adults living with HIV/AIDS. However, these findings should be interpreted cautiously due to the relatively few RCTs published to date. Future studies would benefit from increased attention to sample size, female participants, participant follow-up, complete statistical analysis and intention-to-treat analysis.

Genome-scale reconstruction of Salinispora tropica CNB-440 metabolism to study strain-specific adaptation.

The first manually curated genome-scale metabolic model for Salinispora tropica strain CNB-440 was constructed. The reconstruction enables characterization of the metabolic capabilities for understanding and modeling the cellular physiology of this actinobacterium. The iCC908 model was based on physiological and biochemical information of primary and specialised metabolism pathways. The reconstructed stoichiometric matrix consists of 1169 biochemical conversions, 204 transport reactions and 1317 metabolites. A total of 908 structural open reading frames (ORFs) were included in the reconstructed network. The number of gene functions included in the reconstructed network corresponds to 20% of all characterized ORFs in the S. tropica genome. The genome-scale metabolic model was used to study strain-specific capabilities in defined minimal media. iCC908 was used to analyze growth capabilities in 41 different minimal growth-supporting environments. These nutrient sources were evaluated experimentally to assess the accuracy of in silico growth simulations. The model predicted no auxotrophies for essential amino acids, which was corroborated experimentally. The strain is able to use 21 different carbon sources, 8 nitrogen sources and 4 sulfur sources from the nutrient sources tested. Experimental observation suggests that the cells may be able to store sulfur. False predictions provided opportunities to gain new insights into the physiology of this species, and to gap fill the missing knowledge. The incorporation of modifications led to increased accuracy in predicting the outcome of growth/no growth experiments from 76 to 93%. iCC908 can thus be used to define the metabolic capabilities of S. tropica and guide and enhance the production of specialised metabolites.

Analyzing redox balance in a synthetic yeast platform to improve utilization of brown macroalgae as feedstock.

Macroalgae have high potential to be an efficient, and sustainable feedstock for the production of biofuels and other more valuable chemicals. Attempts have been made to enable the co-fermentation of alginate and mannitol by Saccharomyces cerevisiae to unlock the full potential of this marine biomass. However, the efficient use of the sugars derived from macroalgae depends on the equilibrium of cofactors derived from the alginate and mannitol catabolic pathways. There are a number of strong metabolic limitations that have to be tackled before this bioconversion can be carried out efficiently by engineered yeast cells. An analysis of the redox balance during ethanol fermentation from alginate and mannitol by Saccharomyces cerevisiae using metabolic engineering tools was carried out. To represent the strain designed for conversion of macroalgae carbohydrates to ethanol, a context-specific model was derived from the available yeast genome-scale metabolic reconstructions. Flux balance analysis and dynamic simulations were used to determine the flux distributions. The model indicates that ethanol production is determined by the activity of 4-deoxy-l-erythro-5-hexoseulose uronate (DEHU) reductase (DehR) and its preferences for NADH or NADPH which influences strongly the flow of cellular resources. Different scenarios were explored to determine the equilibrium between NAD(H) and NADP(H) that will lead to increased ethanol yields on mannitol and DEHU under anaerobic conditions. When rates of mannitol dehydrogenase and DehRNADH tend to be close to a ratio in the range 1-1.6, high growth rates and ethanol yields were predicted. The analysis shows a number of metabolic limitations that are not easily identified through experimental procedures such as quantifying the impact of the cofactor preference by DEHU reductase in the system, the low flux into the alginate catabolic pathway, and a detailed analysis of the redox balance. These results show that production of ethanol and other chemicals can be optimized if a redox balance is achieved. A possible methodology to achieve this balance is presented. This paper shows how metabolic engineering tools are essential to comprehend and overcome this limitation.

Stoichiometric model and flux balance analysis for a mixed culture of Leptospirillum ferriphilum and Ferroplasma acidiphilum.

The oxidation process of sulfide minerals in natural environments is achieved by microbial communities from the Archaea and Bacteria domains. A metabolic reconstruction of two dominant species, Leptospirillum ferriphilum and Ferroplasma acidiphilum, which are always found together as a mixed culture in this natural environments, was made. The metabolic model, composed of 152 internal reactions and 29 transport reactions, describes the main interactions between these species, assuming that both use ferrous iron as energy source, and F. acidiphilum takes advantage of the organic compounds secreted by L. ferriphilum for chemomixotrophic growth. A first metabolic model for a mixed culture used in bacterial leaching is proposed in this article, which pretends to represent the characteristics of the mixed culture in a simplified manner. It was evaluated with experimental data through flux balance analysis (FBA) using as objective function the maximization of biomass. The growth yields on ferrous iron obtained for each microorganism are consistent with experimental data, and the flux distribution obtained allows understanding of the metabolic capabilities of both microorganisms growing together in a bioleaching process. The model was used to simulate the growth of F. acidiphilum on different substrates, to determine in silico which compounds maximize cell growth, and which are essential. Knockout simulations were carried out for L. ferriphilum and F. acidiphilum metabolic models, predicting key enzymes of central metabolism. The results of this analysis are consistent with experimental data from literature, showing a robust behavior of the metabolic model.

Can FES-rowing mediate bone mineral density in SCI: a pilot study.

STUDY DESIGN: A single case study. OBJECTIVES: To compare proximal tibia trabecular bone mineral density (BMD) of a participant with complete spinal cord injury (SCI), long-termed functional electrical stimulation-rowing (FES-R) trained, with previously reported SCI and non-SCI group norms. To estimate lower limb joint contact forces (JCFs) in the FES-R trained participant. SETTING: UK University and orthopaedic hospital research centre. METHODS: Bilateral proximal tibial trabecular BMD of the FES-R trained participant was measured using peripheral quantitative computerised tomography, and the data were compared with SCI and non-SCI groups. An instrumented four-channel FES-R system was used to measure the lower limb JCFs in the FES-R trained participant. RESULTS: Structurally, proximal tibial trabecular BMD was higher in the FES-R trained participant compared with the SCI group, but was less than the non-SCI group. Furthermore, left (184.7 mg cm(-3)) and right (160.7 mg cm(-3)) BMD were well above the threshold associated with non-traumatic fracture. The knee JCFs were above the threshold known to mediate BMD in SCI, but below threshold at the hip and ankle. CONCLUSION: As pathological fractures predominate in the distal femur and proximal tibia in chronic SCI patients, the fact that the FES-R trained participant's knee JCFs were above those known to partially prevent bone loss, suggests that FES-R training may provide therapeutic benefit. Although the elevated bilateral proximal tibial BMD of the FES-R participant provides circumstantial evidence of osteogenesis, this single case precludes any statement on the clinical significance. Further investigations are required involving larger numbers and additional channels of FES to increase loading at the hip and ankle.

FES-rowing in tetraplegia: a preliminary report.

STUDY DESIGN: A training intervention study using functional electrical stimulation-rowing (FES-R) in a group of eight individuals with tetraplegia. OBJECTIVES: To assess the feasibility of a structured progressive FES-R training programme in people with tetraplegia, and to explore the number and type of FES-training sessions required to enable continuous FES-R for 30 min. SETTING: A fully integrated sports centre, elite rowing training centre and university sport science department. METHODS: Eight participants with chronic complete and incomplete tetraplegia (C4 to C7, American Spinal Injury Association Impairment Scale A, B and C) who had not previously used any form of FES-assisted exercise, participated in the study. Participants completed a progressive FES-assisted training programme building to three continuous 30-min FES-R sessions per week at 60-80% of their predetermined peak power output. Thereafter, rowing performance was monitored for 12 months. MAIN OUTCOME MEASURES: number and type of FES-training sessions required before achieving 30-min continuous FES-R, and FES-R average power output (POav) pre and post 12 months training. Participant feedback of perceived benefits was also documented. RESULTS: All participants were able to continuously FES-row for 30 min after completing 13±7 FES-R training sessions. Each individual POav during 30 min FES-R increased over 12 months FES-training. FES-R was found safe and well tolerated in this group of individuals with tetraplegia. CONCLUSION: Individuals with tetraplegia are able to engage in a progressive programme of FES-R training. Future research examining FES-R training as an adjunctive therapy in people with tetraplegia is warranted.

Medical students' clerkship experiences and self-perceived competence in clinical skills.

INTRODUCTION: In a traditional curriculum, medical students are expected to acquire clinical competence through the apprenticeship model using the Halstedian "see one, do one, and teach one, approach". The University of Zambia School of Medicine used a traditional curriculum model from 1966 until 2011 when a competence-based curriculum was implemented. OBJECTIVE: To explore medical students' clerkships experiences and self-perceived competence in clinical skills. METHODS: A cross-sectional survey was conducted on 5th, 6th, and 7th year medical students of the University of Zambia, School of Medicine two months prior to final examinations. Students were asked to rate their clerkship experiences with respect to specific skills on a scale of 1 to 4 and their level of self-perceived competence on a scale of 1 to 3. Skills evaluated were in four main domains: history taking and communication, physical examination, procedural, and professionalism, team work and medical decision making. Using Statistical Package for Social Scientist (SPSS), correlations were performed between experiences and self-perceived competence on specific skills, within domains and overall. RESULTS: Out of 197 clinical students 138 (70%) participated in the survey. The results showed significant increase in the proportion of students performing different skills and reporting feeling very competent with each additional clinical year. Overall correlations between experience and self-perceived competence were moderate (0.55). On individual skills, the highest correlation between experience and self-perceived competence were observed on mainly medical and surgical related procedural skills with the highest at 0.82 for nasal gastric tube insertion and 0.76 for endotracheal intubation. CONCLUSION: Despite the general improvement in skills experiences and self-perceived competence, some deficiencies were noted as significant numbers of final year students had never attempted common important procedures especially those performed in emergency situations. Deficiencies in certain skills may call for incorporation of teaching/learning methods that broaden students' exposure to such skills.

Trends and challenges in United States neonatal intensive care units follow-up clinics.

OBJECTIVE: A mandate exists that all level III neonatal intensive care units (NICUs) provide a means to assess and follow their high-risk neonates after discharge. However, no standardized guidelines exist for the follow-up services provided. To determine trends of structure and care provided in NICU follow-up clinics in both the academic and private clinical setting. STUDY DESIGN: We sent an Internet survey to NICU follow-up clinic directors at both academically affiliated and private centers. This study received institutional review board exemption. RESULT: We received 89 surveys from academic institutions and 94 from private level III follow-up programs. These responses represent 55% of academic programs and 40% of private programs in the United States. Similar to academic institutions, 18% of private NICU follow-up clinics provide primary care services to patients. In both settings, the hospital supports 60% of the funding required for clinic activities. Forty-five percent of NICU graduates seen in both private and academic follow-up clinics have public aid as their primary insurance. Eighty-five percent of NICUs in both settings have guidelines outlining requirements for referrals to the follow-up clinic. Academic programs find feeding difficulties the most difficult, whereas private programs find bronchopulmonary dysplasia and feeding difficulties equally as difficult. CONCLUSION: The care and struggles of NICU follow-up clinics are similar in both the academic affiliated and private settings. Similar referrals, clinical evaluation and medical care occur with varying struggles.

Cloning, expression and decoding of the cold adaptation of a new widely represented thermolabile subtilisin-like protease.

AIMS: Cloning, expression and characterization of a new cold-adapted protease with potential biotechnological application, isolated from Antarctic bacteria. METHOD AND RESULTS: A subtilisin-like gene was isolated from several Antarctic bacterial genus using CODPEHOP-designed primers and a genome walking method. This gene encodes a precursor protein, which undergoes an autocatalytic cleavage resulting in a 34.6 kDa active cold-adapted protease with a maximum activity at 25-35°C and optimum pH of 8.0-9.0. It showed a higher catalytic efficiency at lower temperatures compared to its mesophilic counterpart. Heat-induced inactivation resulted in a very low melting point. Local packing analysis using the homology model indicated Ala284 as an important cold-adaptation determinant, which was corroborated by the site-directed mutagenesis. CONCLUSIONS: A new thermolabile subtilisin-like protease has been successfully cloned and analysed, and an important hot spot in the evolution of the cold adaptation and substrate specificity of this enzyme was identified and tested. SIGNIFICANCE AND IMPACT OF THE STUDY: This work reports a new cold-adapted protease with a vast representation amongst Antarctic genus, suggesting therefore its evolutionary success in this cold environment. Likewise, important sites for genetic potentiation have been identified, which are extrapolated to other enzymes of the same kind.

Continuous modeling of metabolic networks with gene regulation in yeast and in vivo determination of rate parameters.

A continuous model of a metabolic network including gene regulation to simulate metabolic fluxes during batch cultivation of yeast Saccharomyces cerevisiae was developed. The metabolic network includes reactions of glycolysis, gluconeogenesis, glycerol and ethanol synthesis and consumption, the tricarboxylic acid cycle, and protein synthesis. Carbon sources considered were glucose and then ethanol synthesized during growth on glucose. The metabolic network has 39 fluxes, which represent the action of 50 enzymes and 64 genes and it is coupled with a gene regulation network which defines enzyme synthesis (activities) and incorporates regulation by glucose (enzyme induction and repression), modeled using ordinary differential equations. The model includes enzyme kinetics, equations that follow both mass-action law and transport as well as inducible, repressible, and constitutive enzymes of metabolism. The model was able to simulate a fermentation of S. cerevisiae during the exponential growth phase on glucose and the exponential growth phase on ethanol using only one set of kinetic parameters. All fluxes in the continuous model followed the behavior shown by the metabolic flux analysis (MFA) obtained from experimental results. The differences obtained between the fluxes given by the model and the fluxes determined by the MFA do not exceed 25% in 75% of the cases during exponential growth on glucose, and 20% in 90% of the cases during exponential growth on ethanol. Furthermore, the adjustment of the fermentation profiles of biomass, glucose, and ethanol were 95%, 95%, and 79%, respectively. With these results the simulation was considered successful. A comparison between the simulation of the continuous model and the experimental data of the diauxic yeast fermentation for glucose, biomass, and ethanol, shows an extremely good match using the parameters found. The small discrepancies between the fluxes obtained through MFA and those predicted by the differential equations, as well as the good match between the profiles of glucose, biomass, and ethanol, and our simulation, show that this simple model, that does not rely on complex kinetic expressions, is able to capture the global behavior of the experimental data. Also, the determination of parameters using a straightforward minimization technique using data at only two points in time was sufficient to produce a relatively accurate model. Thus, even with a small amount of experimental data (rates and not concentrations) it was possible to estimate the parameters minimizing a simple objective function. The method proposed allows the obtention of reasonable parameters and concentrations in a system with a much larger number of unknowns than equations. Hence a contribution of this study is to present a convenient way to find in vivo rate parameters to model metabolic and genetic networks under different conditions.