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.

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.

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.

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.

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.

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.

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.

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.

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.

Objective predictors of delayed-onset post-traumatic stress disorder occurring after military discharge.

BACKGROUND: Post-traumatic stress disorder (PTSD) that develops after military personnel have been discharged may lead to severe impairment. We investigated whether personnel who develop PTSD after discharge can be identified by independent evidence of internalizing signs such as depression or of externalizing signs such as disciplinary offences while still serving. METHOD: Veterans in receipt of a war pension who only developed PTSD post-discharge were compared with matched veterans who developed PTSD in service or never suffered from PTSD. Contemporaneous medical and personnel records were searched for objective evidence of internalizing and externalizing disorder. RESULTS: Service personnel who developed PTSD post-discharge were indistinguishable from controls with no PTSD on their psychiatric presentation in service. Those with post-discharge PTSD had significantly more disciplinary offences, specifically absence without leave, disobedience, and dishonesty, than the no-PTSD group, and this excess of offences was present before any exposure to trauma. CONCLUSIONS: This is the first study to find objective evidence independent of self-report for the claimed link between externalizing disorder and vulnerability to PTSD. Early signs of externalizing disorders may play an important role in helping to identify service personnel at risk of PTSD after military discharge.

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.

Trauma, identity and mental health in UK military veterans.

BACKGROUND: Military service can lead to profound changes in identity, both in servicemen's perception of themselves and in their relationship to the world, but the significance of these changes for psychopathology is unclear. We investigated whether the extent and valence of identity change was related to the degree of military trauma exposure or to post-traumatic stress disorder (PTSD) and suicide attempts. We further sought to describe the nature of such changes using qualitative analysis. METHOD: A total of 153 veterans in receipt of a war pension for PTSD or physical disability were identified. Interviews established retrospectively DSM-IV diagnoses of PTSD and reports of suicidal ideation or behaviour since enlistment were examined. RESULTS: Trauma exposure alone was unrelated to any measure of identity change. By contrast, PTSD was associated with a relationship to the world that had changed in a negative direction. It was also associated with a changed perception of self, which could be either positive or negative. After controlling for trauma exposure and PTSD, suicidal behaviours were associated with more negative perceptions of the world. These perceptions of the world included disillusionment about human nature and a more specific rejection of civilian life. CONCLUSIONS: PTSD and suicidal behaviours in veterans seem not to be associated with significantly more negative views of the self but rather with more alienation from civilian life. This has serious consequences for engaging veterans in National Health Service (NHS) mental health services and for the provision of effective treatment.

Sociodemographic and behavioural characteristics of youth reporting HIV testing in three Caribbean countries.

OBJECTIVES: Human Immunodeficiency Virus (HIV) testing is the gateway to treatment and care of HIV infection, however little is known about the HIV testing behaviours among Caribbean youth. The objective of this study was to determine the prevalence of HIV testing and to examine associations of HIV testing with sociodemographic characteristics and risk behaviours. METHODS: Data were used from nationally representative surveys in three Caribbean countries: Guyana AIDS Indicator Survey 2005-2006; Haiti Demographic and Health Survey 2005-2006 and the Dominican Republic Demographic and Health Survey 2007. Youth 15-24 years who had ever heard of AIDS and ever had sex were selected, yielding samples of 875 in Guyana, 4199 in Haiti and 12 418 in the Dominican Republic. Bivariate tests were conducted to examine the associations between sociodemographic characteristics, risk behaviours and being tested for HIV. RESULTS: The proportion of youth reporting HIV testing ranged from 17% in Haiti to 48% in the Dominican Republic. About 54% of youth in Haiti and less than one-third in the Dominican Republic initiated HIV testing. A greater proportion of females than males had ever tested in each country, ranging from 68% in Guyana to 82% in Haiti. Higher rates of HIV testing were observed among ever married youth and among youth with 2-4 lifetime sexual partners. CONCLUSIONS: Males, rural and never married youth were less likely to be tested. Outreach at individual and community levels and public health messages targeting these youth should be implemented. There is also a need to mainstream gender into the design of programmes aimed at increasing uptake of HIV testing. Programmes which assist youth in accurately assessing their risk behaviours are also required to improve HIV testing.

Spontaneous murine lupus-like syndromes. Clinical and immunopathological manifestations in several strains.

MRL/1 and BXSB male mice have a systemic lupus erythematosus (SLE)-like disease similar to but more acute than that occurring in NZB X W mice. The common elements of lymphoid hyperplasia, B-cell hyperactivity, autoantibodies, circulating immune complex (IC), complement consumption, IC glomerulonephritis with gp70 deposition, and thymic atrophy were found in all three kinds of SLE mice. On the basis of these common elements, SLE seen in these mice can be considered a single disease in the same sense that human SLE is one disease. The differences in the SLE expressed in the different mice are no greater than those found in an unselected series of humans with SLE. However, the significant quantitative and qualitative variations in abnormal immunologic expression suggest that different constellations of factors, genetic and/or pathophysiologic, may operate in the three murine strains and that each constellation is capable of leading, via its particular abnormal immunologic consequences, to the activation of common immunopathologic effector mechanisms that cause quite similar SLE-like syndromes. From an experimental point of view, the availability of several inbred murine strains of commonplace histocompatibility types that express an SLE-like syndrome makes possible innumerable manipulations which should help to elucidate the nature and cause(s) of this disorder.

Extension of the selection of protein chromatography and the rate model to affinity chromatography.

The rational selection of optimal protein purification sequences, as well as mathematical models that simulate and allow optimization of chromatographic protein purification processes have been developed for purification procedures such as ion-exchange, hydrophobic interaction and gel filtration chromatography. This paper investigates the extension of such analysis to affinity chromatography both in the selection of chromatographic processes and in the use of the rate model for mathematical modelling and simulation. Two affinity systems were used: Blue Sepharose and Protein A. The extension of the theory developed previously for ion-exchange and HIC chromatography to affinity separations is analyzed in this paper. For the selection of operations two algorithms are used. In the first, the value of η, which corresponds to the efficiency (resolution) of the actual chromatography and, Σ, which determines the amount of a particular contaminant eliminated after each separation step, which determines the purity, have to be determined. It was found that the value of both these parameters is not generic for affinity separations but will depend on the type of affinity system used and will have to be determined on a case by case basis. With Blue Sepharose a salt gradient was used and with Protein A, a pH gradient. Parameters were determined with individual proteins and simulations of the protein mixtures were done. This approach allows investigation of chromatographic protein purification in a holistic manner that includes ion-exchange, HIC, gel filtration and affinity separations for the first time.

Viral vectors for the treatment of alcoholism: use of metabolic flux analysis for cell cultivation and vector production.

The HEK293 cell line has been used for the production of adenovirus vectors to be used in the potential treatment of alcoholism using a gene therapy strategy. Culture optimization and scale-up has been achieved by first adapting the cells to serum-free media and secondly by growing them in suspension. Adenovirus production after infection was increased, resulting in higher specific glucose consumption and lactate accumulation rates compared to the growth phase. We applied media design tools and Metabolic Flux Analysis (MFA) to compare the metabolic states of cells during growth and adenovirus production and to optimize culture media according to the metabolic demand of the cells in terms of glucose and glutamine concentrations. This allowed obtaining a higher maximum cell concentration and increased adenovirus production by minimizing the production of metabolites that can have an inhibitory effect on cell growth. We have proposed a stoichiometric equation for adenovirus synthesis. MFA results allowed determination of how these changes in composition affected the way cells distribute their nutrient resources during cell growth and virus production. Virus purification was successfully achieved using chromatography and Aqueous Two-Phase Systems (ATPS).

Categorizing neonatal deaths: a cross-cultural study in the United States, Canada, and The Netherlands.

OBJECTIVE: To clarify the process of end-of-life decision-making in culturally different neonatal intensive care units (NICUs). STUDY DESIGN: Review of medical files of newborns >22 weeks gestation who died in the delivery room (DR) or the NICU during 12 months in 4 NICUs (Chicago, Milwaukee, Montreal, and Groningen). We categorized deaths using a 2-by-2 matrix and determined whether mechanical ventilation was withdrawn/withheld and whether the child was dying despite ventilation or physiologically stable but extubated for neurological prognosis. RESULTS: Most unstable patients in all units died in their parents' arms after mechanical ventilation was withdrawn. In Milwaukee, Montreal, and Groningen, 4% to 12% of patients died while receiving cardiopulmonary resuscitation. This proportion was higher in Chicago (31%). Elective extubation for quality-of-life reasons never occurred in Chicago and occurred in 19% to 35% of deaths in the other units. The proportion of DR deaths in Milwaukee, Montreal, and Groningen was 16% to 22%. No DR deaths occurred in Chicago. CONCLUSIONS: Death in the NICU occurred differently within and between countries. Distinctive end-of-life decisions can be categorized separately by using a model with uniform definitions of withholding/withdrawing mechanical ventilation correlated with the patient's physiological condition. Cross-cultural comparison of end-of-life practice is feasible and important when comparing NICU outcomes.

Stoichiometric model and metabolic flux analysis for Leptospirillum ferrooxidans.

A metabolic model for Leptospirillum ferrooxidans was developed based on the genomic information of an analogous iron oxidizing bacteria and on the pathways of ferrous iron oxidation, nitrogen and CO(2) assimilation based on experimental evidence for L. ferrooxidans found in the literature. From this metabolic reconstruction, a stoichiometric model was built, which includes 86 reactions describing the main catabolic and anabolic aspects of its metabolism. The model obtained has 2 degrees of freedom, so two external fluxes were estimated to achieve a determined and observable system. By using the external oxygen consumption rate and the generation flux biomass as input data, a metabolic flux map with a distribution of internal fluxes was obtained. The results obtained were verified with experimental data from the literature, achieving a very good prediction of the metabolic behavior of this bacterium at steady state.

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 17-kD centromere protein (CENP-A) copurifies with nucleosome core particles and with histones.

We have detected and begun to characterize a 17-kD centromere-specific protein, CENP-A (Earnshaw, W. C., and N. Rothfield, 1985, Chromosoma., 91:313-321). Sera from several humans with CREST scleroderma autoimmune disease (CREST: calcinosis, Raynaud's phenomenon, esophageal dsymotility, sclerodactyly, and telangiectasia) bind this protein in immunoblot assays of HeLa whole cell or nuclear extracts. We have affinity purified the anti-17-kD centromere protein (anti-CENP-A) specific antibodies from immunoblots of HeLa nuclear protein. The antibodies react with epitopes present on CENP-A derived from humans but apparently do not recognize specific epitopes in either rat or chicken nuclei. Only human nuclear protein is CENP-A positive by immunoblot. Furthermore, human cells show localization of anti-CENP-A antibody to centromeres by immunofluorescence microscopy, whereas rat cells do not. On extraction from the nucleus, CENP-A copurifies with core histones and with nucleosome core particles. We conclude that this centromere-specific protein is a histone-like component of chromatin. The data suggest that CENP-A functions as a centromere-specific core histone.