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Background The antipsychotic aripiprazole is often used in the treatment of first-episode psychosis. Measuring aripiprazole blood levels provides an objective measure of treatment adherence, but this currently involves taking a venous blood sample and sending to a laboratory for analysis. Aims To detail the development, validation and utility of a new point of care (POC) test for finger-stick capillary blood concentrations of aripiprazole. Method Analytical performance (sensitivity, precision, recovery and linearity) of the assay were established using spiked whole blood and control samples of varying aripiprazole concentration. Assay validation was performed over a 14-month period starting in July 2021. Eligible patients were asked to provide a finger-stick capillary sample in addition to their usual venous blood sample. Capillary blood samples were tested by the MyCare™ Insite POC analyser, which provided measurement of aripiprazole concentration in 6 min, and the venous blood sample was tested by the standard laboratory method. Results A total of 101 patients agreed to measurements by the two methods. Venous blood aripiprazole concentrations as assessed by the laboratory method ranged from 17 to 909 ng/mL, and from 1 to 791 ng/mL using POC testing. The correlation coefficient between the two methods (r) was 0.96 and there was minimal bias (slope 0.91, intercept 4 ng/ml). Conclusions The MyCare Insite POC analyser is sufficiently accurate and reliable for clinical use. The availability of this technology will improve the assessment of adherence to aripiprazole and the optimising of aripiprazole dosing.

Onur Hur&scedil;ito&gbreve;lu,1 Ergul Belge Kurutas,2 Rebecca Strawbridge,3 Omer Faruk Uygur,4 Emrah Yildiz,5 Thomas J Reilly6 1Department of Psychiatry, Sular Academy Hospital, Kahramanmaras, Turkey; 2Department of Biochemistry, Faculty of Medicine, Kahramanmaras Sutcu Imam University, Kahramanmaras, Turkey; 3Centre for Affective Disorders, Department of Psychological Medicine, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, London, UK; 4Department of Psychiatry, Faculty of Medicine, Ataturk University, Erzurum, Turkey; 5Private Clinic, Department of Psychiatry, Gaziantep, Turkey; 6Department of Psychosis Studies, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, London, UKCorrespondence: Onur Hur&scedil;ito&gbreve;lu, Department of Psychiatry, Sular Academy Hospital, Kahramanmaras, 46100, Turkey, Tel +90 507 420 68 03, Email onurhursitoglu@gmail.comIntroduction: There is increasing evidence that oxidative stress (OS) and neuroinflammation play a role in the neuroprogression of schizophrenia (SCZ). Promising novel candidates which have been proposed in the search for biomarkers of psychotic illness include NADPH oxidase 1,2 (NOX1,2) and raftlin. NOX1 from the NOX family is the main source of physiological reactive oxygen species (ROS) and raftlin, the main lipid raft protein, is associated with inflammatory processes. The aim of the present study was to evaluate serum NOX1 and raftlin levels in chronic stable patients with SCZ.Methods: We measured serum NOX1 and raftlin levels from 45 clinically stable patients with SCZ and 45 healthy controls (HCs) matched for age, sex, and body-mass index. The Positive and Negative Syndrome Scale was applied to the patient group to evaluate the severity of psychotic symptoms.Results: NOX1 and raftlin levels in the patients were statistically significantly higher than the HCs (NOX1 p< 0.001, raftlin p< 0.001). Both parameters showed very good diagnostic performance (NOX1 AUC = 0.931, raftlin AUC = 0.915). We obtained positive and significant correlations between serum levels of both biomarkers and symptom severity.Discussion: This preliminary study indicating elevations in serum NOX1 and raftlin levels in patients with SCZ supports the importance of OS and inflammatory processes in the etiopathogenesis of the illness.Keywords: oxidative stress, oxidant, neuroinflammation, reactive oxygen species, lipid raft, diagnostic performance

Psychotic disorders can be exacerbated by the hormonal changes associated with childbirth, but the extent to which exacerbations occur with the menstrual cycle is unclear. We addressed this issue by conducting a systematic review. Embase, Medline, and PsychINFO databases were searched for studies that measured exacerbations of psychotic disorders in relation to the menstrual cycle. We extracted exacerbation measure, definition of menstrual cycle phase, and measurement of menstrual cycle phase. Standard incidence ratios were calculated for the perimenstrual phase based on the observed admissions during this phase divided by the expected number of admissions if the menstrual cycle had no effect. Random effects models were used to examine pooled rates of psychiatric admission in the perimenstrual phase. Nineteen studies, comprising 1193 participants were eligible for inclusion. Eleven studies examined psychiatric admission rates, 5 examined symptoms scores, 2 examined self-reported exacerbation, and 1 examined both admission rates and symptom scores. A random effects model demonstrated the rate of admissions during the perimenstrual phase was 1.48 times higher than expected (95% CI: 1.31–1.67), with no significant heterogeneity detected. Four of six symptom score studies reported perimenstrual worsening, but lack of consistency in timepoints precluded meta-analysis. Two studies examining self-reported menstrual exacerbations reported prevalences ranging from 20% to 32.4%. Psychiatric admission rates are significantly higher than expected during the perimenstrual phase. There is some evidence that a worsening of psychotic symptoms also occurs during this phase, but further research with more precise measurement of the menstrual cycle and symptomatology is required.

Abnormal gamma oscillations, measured by electroencephalography (EEG), have been associated with chronic psychotic disorders, but their prevalence in the early phase of psychosis is less clear. We sought to address this by systematically reviewing the relevant literature. We searched for EEG studies of gamma band oscillations in subjects at high risk for psychosis and in patients with first episode psychosis. The following measures of gamma oscillations were extracted: resting power, evoked power, induced power, connectivity and peak frequency. Forty-five studies with a total of 3099 participants were included. There were potential sources of bias in the study designs and potential artefacts. Although there were few consistent findings, several studies reported decreased evoked or induced power in both high risk subjects and first episode patients. Studies using larger samples with serial EEG measurements, and designs that minimise artefacts that occur at the gamma frequency may advance work in this area.

The purpose of this study was to investigate non-aureus Staphylococcus spp. intramammary infections (IMI) in periparturient heifers and determine the relationship of precalving body site isolation with precalving IMI and postcalving IMI using molecular speciation and strain-typing methods. Primiparous heifers were enrolled at approximately 14 d before expected calving date. Precalving mammary quarter secretions and body site swabbing samples (teat skin, inguinal skin, muzzle, and perineum) were collected. Postcalving, mammary quarter milk samples were collected for culture and somatic cell counting. Precalving body site samples were cultured, and up to 10 staphylococcal colonies were saved for characterization. Staphylococcal isolates were speciated using matrix-assisted laser/desorption ionization time-of-flight mass spectrometry or sequencing of rpoB or tuf. Pulsed-field gel electrophoresis was used to strain type a subset of isolates. Overall, Staphylococcus chromogenes, Staphylococcus agnetis, and Staphylococcus simulans were the most common species identified in precalving mammary secretions, whereas S. chromogenes, Staphylococcus xylosus, and S. agnetis were the most common species found in postcalving milk samples. The most common species identified from body site samples were S. chromogenes, S. xylosus, and Staphylococcus haemolyticus. Mammary quarters that had a precalving mammary secretion that was culture positive for S. agnetis, S. chromogenes, or Staphylococcus devriesei had increased odds of having an IMI with the same species postcalving. A S. chromogenes IMI postcalving was associated with higher somatic cell count when compared with postcalving culture-negative quarters. Among heifers identified with a non-aureus Staphylococcus spp. IMI either precalving or postcalving, heifers that had S. agnetis or S. chromogenes isolated from their teat skin had increased odds of having the same species found in their precalving mammary secretions, and heifers with S. chromogenes, S. simulans, and S. xylosus isolated from their teat skin precalving were at increased odds of having an IMI with the same species postcalving. Overall, 44% of all heifers with a S. chromogenes IMI around the time of parturition had the same strain isolated from a body site. Based on pulsed-field gel electrophoresis, a high level of strain diversity was found.

The purpose of this study was to describe the prevalence and distribution of staphylococcal species on the teat and inguinal skin of dairy heifers across the various stages of the heifer life cycle. The cross-sectional study included 106 Holstein heifers with an age range of 0 d to 27 mo that were selected from 11 different groups, based on housing type and age, on a single dairy operation. A composite swabbing sample including all 4 teats and a second composite sample including both inguinal regions of each heifer were collected using gas-sterilized electrostatic dusters (Swiffers; Procter and Gamble, Cincinnati, OH). Swabbing samples were mixed with 10 mL of sterile saline, agitated, and cultured on mannitol salt agar plates. At 24 h, plates were read and up to 10 staphylococcal colonies were saved for further analysis. Staphylococcal isolates were speciated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or PCR amplification and partial sequencing of rpoB or tuf. The prevalence of staphylococci was compared between the inguinal and teat regions using the chi-squared or Fisher's exact test, as applicable. Logistic regression models were used to investigate the relationship between a heifer's age (treated as a quantitative continuous variable) and the probability of isolating a given staphylococcal species from a given body site (inguinal region or teats). Overall, the most common species identified were Staphylococcus haemolyticus followed by Staphylococcus chromogenes, Staphylococcus xylosus, Staphylococcus devriesei, and Staphylococcus sciuri. Staphylococcus aureus was more prevalent on the teat than in the inguinal region, whereas Staphylococcus arlettae was more prevalent in the inguinal region than on the teat. All other staphylococcal species were as likely to be found on the teat skin as the inguinal region skin. Isolation from the inguinal and teat skin was associated with age for Staphylococcus agnetis, S. chromogenes, S. devriesei, Staphylococcus equorum, S. haemolyticus, Staphylococcus lentus, S. sciuri, Staphylococcus vitulinus, and S. xylosus. The probability of finding S. chromogenes and S. agnetis on the teat and inguinal region increased with age, whereas the probability of S. devriesei and S. haemolyticus decreased with age. This study provides further insight into the ecology of staphylococcal species involved in heifer mastitis.

Twenty percent of individuals at clinical high risk for psychosis (CHR-P) develop the disorder within 2 years. Extensive research has explored the factors that differentiate those who develop psychosis and those who do not, but the results are conflicting.The current systematic review and meta-analysis comprehensively addresses the consistency and magnitude of evidence for non-purely genetic risk and protective factors associated with the risk of developing psychosis in CHR-P individuals. Random effects meta-analyses, standardized mean difference (SMD) and odds ratio (OR) were used, in combination with an established stratification of evidence that assesses the association of each factor and the onset of psychotic disorders (from class I, convincing evidence to class IV weak evidence), while controlling for several types of biases.A total of 128 original controlled studies relating to 26 factors were retrieved. No factors showed class I-convincing evidence. Two further factors were associated with class II-highly suggestive evidence: attenuated positive psychotic symptoms (SMD = 0.348, 95% CI: 0.280, 0.415) and global functioning (SMD = −0.291, 95% CI: −0.370, −0.211). There was class III-suggestive evidence for negative psychotic symptoms (SMD = 0.393, 95% CI: 0.317, 0.469). There was either class IV-weak or no evidence for all other factors.Our findings suggest that despite the large number of putative risk factors investigated in the literature, only attenuated positive psychotic symptoms, global functioning, and negative psychotic symptoms show suggestive evidence or greater for association with transition to psychosis. The current findings may inform the refinement of clinical prediction models and precision medicine in this field.

Histidine acid phosphatases (HAPs) utilize a nucleophilic histidine residue to catalyze the transfer of a phosphoryl group from phosphomonoesters to water. HAPs function as protein phosphatases and pain suppressors in mammals, are essential for Giardia lamblia excystation, and contribute to virulence of the category A pathogen Francisella tularensis. Herein we report the first crystal structure and steady-state kinetics measurements of the HAP from Legionella pneumophila (LpHAP), also known as Legionella major acid phosphatase. The structure of LpHAP complexed with the inhibitor l(+)-tartrate was determined at 2.0 Å resolution. Kinetics assays show that l(+)-tartrate is a 50-fold more potent inhibitor of LpHAP than of other HAPs. Electrostatic potential calculations provide insight into the basis for the enhanced tartrate potency: the tartrate pocket of LpHAP is more positive than other HAPs because of the absence of an ion pair partner for the second Arg of the conserved RHGXRXP HAP signature sequence. The structure also reveals that LpHAP has an atypically expansive active site entrance and lacks the nucleotide substrate base clamp found in other HAPs. These features imply that nucleoside monophosphates may not be preferred substrates. Kinetics measurements confirm that AMP is a relatively inefficient in vitro substrate of LpHAP.

Effective management of depression is predicated upon reliable assessment. The Quick Inventory of Depressive Symptomatology (QIDS) is a depression severity scale with both self-rated (QIDS-SR16) and clinician-rated (QIDS-C16) versions. Although widely used in research, the psychometric properties of the QIDS16 have not been systematically reviewed. We performed a systematic review of studies of the psychometric properties (factor structure, internal consistency, convergent validity, discriminant validity, test-retest reliability and responsiveness to change) of the QIDS-SR16 or QIDS-C16. Six databases were searched: MEDLINE, EMBASE, PsycINFO, CinAHL, Web of Science and the Cochrane Central Register of Controlled Trials. Findings were summarised, bias assessed and correlations with reference standards were pooled. 37 studies (17,118 participants) were included in the review. Both versions of the QIDS16 were unidimensional. Cronbach's alpha ranged from 0.69 to 0.89 for the QIDS-SR16 and 0.65 to 0.87 for the QIDS-C16. The QIDS-SR16 correlated moderately to highly with several depression severity scales. Seven studies were pooled where QIDS-SR16 was correlated with the HRSD-17 (r = 0.76, CI 0.69, 0.81) in patients diagnosed with depression. Four studies examined convergent validity with the QIDS-C16. Four studies examined discriminant validity, for the QIDS-SR16 alone. Eighteen studies had at least one author who was a co-author of the original QIDS16 study. Most studies were conducted in the USA (n = 26). The QIDS-SR16 and the QIDS-C16 are unidimensional rating scales with acceptable internal consistency. To justify the use of the QIDS16 scale in clinical practice, more research is needed on convergent and discriminant validity, and in populations outside the USA.

Importance: The overall effect of At Risk Mental State (ARMS) services for the detection of individuals who will develop psychosis in secondary mental health care is undetermined.Objective: To measure the proportion of individuals with a first episode of psychosis detected by ARMS services in secondary mental health services, and to develop and externally validate a practical web-based individualized risk calculator tool for the transdiagnostic prediction of psychosis in secondary mental health care.Design, Setting, and Participants: Clinical register-based cohort study. Patients were drawn from electronic, real-world, real-time clinical records relating to 2008 to 2015 routine secondary mental health care in the South London and the Maudsley National Health Service Foundation Trust. The study included all patients receiving a first index diagnosis of nonorganic and nonpsychotic mental disorder within the South London and the Maudsley National Health Service Foundation Trust in the period between January 1, 2008, and December 31, 2015. Data analysis began on September 1, 2016.Main Outcomes and Measures: Risk of development of nonorganic International Statistical Classification of Diseases and Related Health Problems, Tenth Revision psychotic disorders.Results: A total of 91 199 patients receiving a first index diagnosis of nonorganic and nonpsychotic mental disorder within South London and the Maudsley National Health Service Foundation Trust were included in the derivation (n = 33 820) or external validation (n = 54 716) data sets. The mean age was 32.97 years, 50.88% were men, and 61.05% were white race/ethnicity. The mean follow-up was 1588 days. The overall 6-year risk of psychosis in secondary mental health care was 3.02 (95% CI, 2.88-3.15), which is higher than the 6-year risk in the local general population (0.62). Compared with the ARMS designation, all of the International Statistical Classification of Diseases and Related Health Problems, Tenth Revision diagnoses showed a lower risk of psychosis, with the exception of bipolar mood disorders (similar risk) and brief psychotic episodes (higher risk). The ARMS designation accounted only for a small proportion of transitions to psychosis (n = 52 of 1001; 5.19% in the derivation data set), indicating the need for transdiagnostic prediction of psychosis in secondary mental health care. A prognostic risk stratification model based on preselected variables, including index diagnosis, age, sex, age by sex, and race/ethnicity, was developed and externally validated, showing good performance and potential clinical usefulness.Conclusions and Relevance: This online individualized risk calculator can be of clinical usefulness for the transdiagnostic prediction of psychosis in secondary mental health care. The risk calculator can help to identify those patients at risk of developing psychosis who require an ARMS assessment and specialized care. The use of this calculator may eventually facilitate the implementation of an individualized provision of preventive focused interventions and improve outcomes of first episode psychosis.

The purpose of our study was to determine the relative frequencies of causes of widespread ground-glass opacity (GGO) in an unselected, consecutive patient population and to identify any associated imaging findings that can narrow or reorganize the differential.The study was approved by the center's IRB and is HIPPA compliant. Cases with widespread GGO in the radiology report were identified by searching the Radiology Information System. Medical records and CT scan examinations were reviewed for the causes of widespread GGO. Associations between a less dominant imaging finding and a particular diagnosis were analyzed with the chi square test. Our study group consisted of 234 examinations with 124 women and 110 men and a mean age of 53.7 years.A cause was established in 204 (87.2%) cases. Hydrostatic pulmonary edema was most common with 131 cases (56%). Interstitial lung diseases (ILD) were the next most common, most often hypersensitivity pneumonitis (HP) (n=12, 5%) and connective tissue disease related ILD (n=7, 3%). Infection accounted for 5% (12 cases). A few miscellaneous diseases accounted for 5 cases (2.1%). The combination of septal thickening and pleural effusions had a specificity of 0.91 for hydrostatic pulmonary edema (P.001) while centrilobular nodules and air trapping had a specificity of 1.0 for HP. In 24 (10.2%) patients, increased opacification from expiration was incorrectly interpreted as representing widespread ground glass opacity. The relative frequency of disease dramatically changed according to the setting. In the inpatient setting, diffuse alveolar disease and diffuse infection accounted for all of the known diagnoses. Pulmonary edema accounted for 75% of the diagnoses and diffuse infection accounted for approximately 7%. In the outpatient setting, interstitial lung disease was the most common cause, accounting for 26 of 53 cases (49%). Regarding immunocompromised patients, hydrostatic pulmonary edema was still the most common cause (46%) with diffuse infection (24%) the next most likely diagnosis. For patients with bone marrow suppression, 80% of the cases were due to opportunistic viral infection.Widespread GGO is most commonly a manifestation of hydrostatic pulmonary edema. Outpatients will most often have HP or connective tissue disease related ILD. Associated findings are helpful for the diagnosis of hydrostatic pulmonary edema and HP.

E-mental health is an emerging area of research that has the potential to overcome some of the current barriers to progress in working with people at clinical high risk for psychosis (CHR-P). This article provides an overview of how e-mental health could be used in the detection, prediction, and treatment in the CHR-P population. Specifically, we evaluate e-detection, e-prediction, and e-therapeutics for this clinical population. E-mental health holds great promise to improve current management of CHR-P individuals.

Schizophrenia has historically been considered to be a deteriorating disease, a view reinforced by recent MRI findings of progressive brain tissue loss over the early years of illness. On the other hand, the notion that recovery from schizophrenia is possible is increasingly embraced by consumer and family groups. This review critically examines the evidence from longitudinal studies of (1) clinical outcomes, (2) MRI brain volumes, and (3) cognitive functioning. First, the evidence shows that although approximately 25% of people with schizophrenia have a poor long-term outcome, few of these show the incremental loss of function that is characteristic of neurodegenerative illnesses. Second, MRI studies demonstrate subtle developmental abnormalities at first onset of psychosis and then further decreases in brain tissue volumes; however, these latter decreases are explicable by the effects of antipsychotic medication, substance abuse, and other secondary factors. Third, while patients do show cognitive deficits compared with controls, cognitive functioning does not appear to deteriorate over time. The majority of people with schizophrenia have the potential to achieve long-term remission and functional recovery. The fact that some experience deterioration in functioning over time may reflect poor access, or adherence, to treatment, the effects of concurrent conditions, and social and financial impoverishment. Mental health professionals need to join with patients and their families in understanding that schizophrenia is not a malignant disease that inevitably deteriorates over time but rather one from which most people can achieve a substantial degree of recovery.

Aberrant motor behaviour (AMB) in Alzheimer's disease shares behavioural correlates with obsessive compulsive disorder (OCD). We investigated whether AMB was also comparable in terms of metabolic activity in the orbitofrontal cortex (OFC), an area shown to be hyperactive in OCD. In this study 135 patients meeting research criteria for Alzheimer's disease were identified from a database of patients recruited as part of a phase II drug trial. These patients were assessed using the Neuropsychiatric Inventory, the Alzheimer's disease assessment scale, cognitive subscale and perfusion SPECT performed with 99Tc(m) hexamethylpropyleneamine oxime. Regions of interest were created for orbitofrontal cortices and basal ganglia. In 35 patients with AMB, adjusted tracer uptake was greater in the OFC. This reached statistical significance in right superior, left superior, right medial and left medial orbital gyri (p0.05). The association between AMB and hyperactivity in the OFC remained significant after adjusting for the presence of anxiety. These results parallel the OFC hypermetabolism consistently seen in OCD. One model of OCD, proposes that dysfunctional interactions between frontal regions, including the OFC, produce the characteristic symptoms of OCD. The behaviour is though to be brought about by a perceived incompleteness of performing a task and is caused by an error in normal reward signals initiated upon task completion. These finding indicate that AMB in Alzheimer's disease are brought about by the same mechanistic failure.

Francisella tularensis contains four putative acid phosphatases that are conserved in Francisella novicida. An F. novicida quadruple mutant (AcpA, AcpB, AcpC, and Hap [ΔABCH]) is unable to escape the phagosome or survive in macrophages and is attenuated in the mouse model. We explored whether reduced survival of the ΔABCH mutant within phagocytes is related to the oxidative response by human neutrophils and macrophages. F. novicida and F. tularensis subspecies failed to stimulate reactive oxygen species production in the phagocytes, whereas the F. novicida ΔABCH strain stimulated a significant level of reactive oxygen species. The ΔABCH mutant, but not the wild-type strain, strongly colocalized with p47phox and replicated in phagocytes only in the presence of an NADPH oxidase inhibitor or within macrophages isolated from p47phox knockout mice. Finally, purified AcpA strongly dephosphorylated p47phox and p40phox, but not p67phox, in vitro. Thus, Francisella acid phosphatases play a major role in intramacrophage survival and virulence by regulating the generation of the oxidative burst in human phagocytes.

Summary Histidine acid phosphatases catalyze the transfer of a phosphoryl group from phosphomonoesters to water at acidic pH using an active-site histidine. The histidine acid phosphatase from the category A pathogen Francisella tularensis (FtHAP) has been implicated in intramacrophage survival and virulence, motivating interest in understanding the structure and mechanism of this enzyme. Here, we report a structure-based study of ligand recognition by FtHAP. The 1.70-A-resolution structure of FtHAP complexed with the competitive inhibitor l (+)-tartrate was solved using single-wavelength anomalous diffraction phasing. Structures of the ligand-free enzyme and the complex with inorganic phosphate were determined at resolutions of 1.85 and 1.70 A, respectively. The structure of the Asp261Ala mutant enzyme complexed with the substrate 3′-AMP was determined at 1.50 A resolution to gain insight into substrate recognition. FtHAP exhibits a two-domain fold similar to that of human prostatic acid phosphatase, consisting of an α/β core domain and a smaller domain that caps the core domain. The structures show that the core domain supplies the phosphoryl binding site, catalytic histidine (His17), and an aspartic acid residue (Asp261) that protonates the leaving group, while the cap domain contributes residues that enforce substrate preference. FtHAP and human prostatic acid phosphatase differ in the orientation of the crucial first helix of the cap domain, implying differences in the substrate preferences of the two enzymes. 3′-AMP binds in one end of a 15-A-long tunnel, with the adenine clamped between Phe23 and Tyr135, and the ribose 2′-hydroxyl interacting with Gln132. The importance of the clamp is confirmed with site-directed mutagenesis; mutation of Phe23 and Tyr135 individually to Ala increases Km by factors of 7 and 10, respectively. The structural data are consistent with a role for FtHAP in scavenging phosphate from small molecules present in host macrophage cells.

Clostridium perfringens is a gram-positive anaerobe and a pathogen of medical importance. The detection of acid phosphatase activity is a powerful diagnostic indicator of the presence of C. perfringens among anaerobic isolates; however, characterization of the enzyme has not previously been reported. Provided here are details of the characterization of a soluble recombinant form of this cell-associated enzyme. The denatured enzyme was ∼31 kDa and a homodimer in solution. It catalyzed the hydrolysis of several substrates, including para -nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 3′ and 5′ nucleoside monophosphates at pH 6. Calculated K m s ranged from 0.2 to 0.6 mM with maximum velocity ranging from 0.8 to 1.6 μmol of P i /s/mg. Activity was enhanced in the presence of some divalent cations but diminished in the presence of others. Wild-type enzyme was detected in all clinical C. perfringens isolates tested and found to be cell associated. The described enzyme belongs to nonspecific acid phosphatase class C but is devoid of lipid modification commonly attributed to this class.

Francisella tularensis is a facultative intracellular pathogen and the etiologic agent of tularemia. It is capable of escape from macrophage phagosomes and replicates in the host cell cytosol. Bacterial acid phosphatases are thought to play a major role in the virulence and intracellular survival of a number of intracellular pathogens. The goal of this study was to delete the four primary acid phosphatases (Acps) from Francisella novicida and examine the interactions of mutant strains with macrophages, as well as the virulence of these strains in mice. We constructed F. novicida mutants with various combinations of acp deletions and showed that loss of the four Acps (AcpA, AcpB, AcpC, and histidine acid phosphatase [Hap]) in an F. novicida strain (ΔABCH) resulted in a 90% reduction in acid phosphatase activity. The ΔABCH mutant was defective for survival/growth within human and murine macrophage cell lines and was unable to escape from phagosome vacuoles. With accumulation of Acp deletions, a progressive loss of virulence in the mouse model was observed. The ΔABCH strain was dramatically attenuated and was an effective single-dose vaccine against homologous challenge. Furthermore, both acpA and hap were induced when the bacteria were within host macrophages. Thus, the Francisella acid phosphatases cumulatively play an important role in intracellular trafficking and virulence.

Discriminating subjects at clinical high risk (CHR) for psychosis who will develop psychosis from those who will not is a prerequisite for preventive treatments. However, it is not yet possible to make any personalized prediction of psychosis onset relying only on the initial clinical baseline assessment. Here, we first present a systematic review of prognostic accuracy parameters of predictive modeling studies using clinical, biological, neurocognitive, environmental, and combinations of predictors. In a second step, we performed statistical simulations to test different probabilistic sequential 3-stage testing strategies aimed at improving prognostic accuracy on top of the clinical baseline assessment. The systematic review revealed that the best environmental predictive model yielded a modest positive predictive value (PPV) (63%). Conversely, the best predictive models in other domains (clinical, biological, neurocognitive, and combined models) yielded PPVs of above 82%. Using only data from validated models, 3-stage simulations showed that the highest PPV was achieved by sequentially using a combined (clinical + electroencephalography), then structural magnetic resonance imaging and then a blood markers model. Specifically, PPV was estimated to be 98% (number needed to treat, NNT = 2) for an individual with 3 positive sequential tests, 71%–82% (NNT = 3) with 2 positive tests, 12%–21% (NNT = 11–18) with 1 positive test, and 1% (NNT = 219) for an individual with no positive tests. This work suggests that sequentially testing CHR subjects with predictive models across multiple domains may substantially improve psychosis prediction following the initial CHR assessment. Multistage sequential testing may allow individual risk stratification of CHR individuals and optimize the prediction of psychosis.

AcpA is a respiratory burst-inhibiting acid phosphatase from the Centers for Disease Control and Prevention Category A bioterrorism agent Francisella tularensis and prototype of a superfamily of acid phosphatases and phospholipases C. We report the 1.75-A resolution crystal structure of AcpA complexed with the inhibitor orthovanadate, which is the first structure of any F. tularensis protein and the first for any member of this superfamily. The core domain is a twisted 8-stranded beta-sheet flanked by three alpha-helices on either side, with the active site located above the carboxyl-terminal edge of the beta-sheet. This architecture is unique among acid phosphatases and resembles that of alkaline phosphatase. Unexpectedly, the active site features a serine nucleophile and metal ion with octahedral coordination. Structure-based sequence analysis of the AcpA superfamily predicts that the hydroxyl nucleophile and metal center are also present in AcpA-like phospholipases C. These results imply a phospholipase C catalytic mechanism that is radically different from that of zinc metallophospholipases.

Francisella tularensis is a highly infectious bacterial pathogen that is considered by the Centers for Disease Control and Prevention to be a potential bioterrorism weapon. Here, the crystallization of a 37.2 kDa phosphatase encoded by the genome of F. tularensis subsp. holarctica live vaccine strain is reported. This enzyme shares 41% amino-acid sequence identity with Legionella pneumophila major acid phosphatase and contains the RHGXRXP motif that is characteristic of the histidine acid phosphatase family. Large diffraction-quality crystals were grown in the presence of Tacsimate, HEPES and PEG 3350. The crystals belong to space group P4(1)2(1)2, with unit-cell parameters a = 61.96, c = 210.78 A. The asymmetric unit is predicted to contain one protein molecule, with a solvent content of 53%. A 1.75 A resolution native data set was recorded at beamline 4.2.2 of the Lawrence Berkeley National Laboratory Advanced Light Source. Molecular-replacement trials using the human prostatic acid phosphatase structure as the search model (28% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of F. tularensis histidine acid phosphatase will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.

The Haemophilus influenzae P4 lipoprotein ( hel ) is a potential component of a nontypeable H. influenzae otitis media vaccine. Since P4 is known to be an enzyme, nonenzymatically active forms of recombinant P4 are required. After site-directed mutagenesis of the hel gene, three of the mutated proteins were shown to be vaccine candidates.

In 80-year-old woman presented with dyspnea on exertion, present for the last 2 years. She said she became short of breath after walking 1 block. Her medical history was significant only for 35 pack-years of smoking. On examination, her lung fields were clear, with no audible murmurs, and she had

The results from purification and characterization studies of the hppA gene product of Helicobacter pylori confirm its identification as a class C acid phosphatase. The hppA gene of H. pylori ATCC strain 49503 was amplified and modified by PCR, cloned into pET21b, and overexpressed in Escherichia coli. The recombinant protein was liberated from membranes and purified (16x) to apparent homogeneity with cation exchange and Ni-chelate chromatography resulting in a recovery of 39% of total starting activity. The recombinant acid phosphatase exhibited a denatured molecular mass of 24 kDa by SDS-PAGE. Phosphatase activity in both crude and purified samples could be renatured and detected after SDS-PAGE. The native molecular mass of recombinant enzyme was approximately 72 kDa by gel filtration chromatography on Superdex 75. While phosphate and tartrate had little effect on phosphatase activity, molybdate, vanadate, and EDTA had significant inhibitory effects on enzymatic activity. Phosphomonoesterase activity for hydrolysis of p-nitrophenylphosphate (pNPP) as well as other substrates was enhanced in the presence of divalent cations including Cu(2+), Ni(2+), Co(2+), and Mg(2+). Recombinant HppA had narrow substrate specificity with highest activity for arylphosphates and significant activity for 5' nucleoside monophosphates. The pH optimum for enzyme activity was 4.6 and 5.2 for purine and pyrimidine 5' monophosphates, respectively. The affinity constants for the 5' nucleoside monophosphates were found to be 0.5-1 mM. Results from this study confirm HppA inclusion in the class C acid phosphatases and led to its identification as a 5' nucleotidase.

The intermediary metabolism of Haemophilus influenzae strain Rd KW20 was studied by a combination of protein expression analysis using a recently developed direct proteomics approach, mutational analysis, and mathematical modeling. Special emphasis was placed on carbon utilization, sugar fermentation, TCA cycle, and electron transport of H. influenzae cells grown microaerobically and anaerobically in a rich medium. The data indicate that several H. influenzae metabolic proteins similar to Escherichia coli proteins, known to be regulated by low concentrations of oxygen, were well expressed in both growth conditions in H. influenzae. An in silico model of the H. influenzae metabolic network was used to study the effects of selective deletion of certain enzymatic steps. This allowed us to define proteins predicted to be essential or non-essential for cell growth and to address numerous unresolved questions about intermediary metabolism of H. influenzae. Comparison of data from in vivo protein expression with the protein list associated with a genome-scale metabolic model showed significant coverage of the known metabolic proteome. This study demonstrates the significance of an integrated approach to the characterization of H. influenzae metabolism.

The proteome of Haemophilus influenzae strain Rd KW20 was analyzed by liquid chromatography (LC) coupled with ion trap tandem mass spectrometry (MS/MS). This approach does not require a gel electrophoresis step and provides a rapidly developed snapshot of the proteome. In order to gain insight into the central metabolism of H. influenzae , cells were grown microaerobically and anaerobically in a rich medium and soluble and membrane proteins of strain Rd KW20 were proteolyzed with trypsin and directly examined by LC-MS/MS. Several different experimental and computational approaches were utilized to optimize the proteome coverage and to ensure statistically valid protein identification. Approximately 25% of all predicted proteins (open reading frames) of H. influenzae strain Rd KW20 were identified with high confidence, as their component peptides were unambiguously assigned to tandem mass spectra. Approximately 80% of the predicted ribosomal proteins were identified with high confidence, compared to the 33% of the predicted ribosomal proteins detected by previous two-dimensional gel electrophoresis studies. The results obtained in this study are generally consistent with those obtained from computational genome analysis, two-dimensional gel electrophoresis, and whole-genome transposon mutagenesis studies. At least 15 genes originally annotated as conserved hypothetical were found to encode expressed proteins. Two more proteins, previously annotated as predicted coding regions, were detected with high confidence; these proteins also have close homologs in related bacteria. The direct proteomics approach to studying protein expression in vivo reported here is a powerful method that is applicable to proteome analysis of any (micro)organism.

Hydrogen peroxide is a cell signaling agent that inactivates protein tyrosine phosphatases (PTPs) via oxidation of their catalytic cysteine residue. PTPs are inactivated rapidly during H(2)O(2)-mediated cellular signal transduction processes, but, paradoxically, hydrogen peroxide is a rather sluggish PTP inactivator in vitro. Here we present evidence that the biological buffer bicarbonate/CO(2) potentiates the ability of H(2)O(2) to inactivate PTPs. The results of biochemical experiments and high-resolution crystallographic analysis are consistent with a mechanism involving oxidation of the catalytic cysteine residue by peroxymonocarbonate generated via the reaction of H(2)O(2) with HCO(3)(-)/CO(2).

The assembled and annotated genome of Actinobacillus suis ATCC 33415 T is reported here. The 2,501,598-bp genome encodes 2,246 open reading frames (ORFs) with strain variable incursion of an integrative conjugative element into a tRNA locus. Comparative analysis of the deduced gene set should inform our understanding of pathogenesis, genomic plasticity, and serotype variation.

Moraxella bovoculi is a recently identified species, recovered from the bovine eye, which is under investigation as an etiological agent of infectious bovine keratoconjunctivitis. A draft genome sequence of the Moraxella bovoculi type strain 237 T has been determined to identify features that may be important during host colonization.

Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks almost all the biosynthetic enzymes necessary for the de novo synthesis of that cofactor. Factor V can be provided as either nicotinamide adenosine dinucleotide (NAD), nicotinamide mononucleotide (NMN), or nicotinamide riboside (NR) in vitro, but little is known about the source or the mechanism of uptake of these substrates in vivo. As shown by us earlier, at least two gene products are involved in the uptake of NAD, the outer membrane lipoprotein e (P4), which has phosphatase activity and is encoded by hel , and a periplasmic NAD nucleotidase, encoded by nadN . It has also been observed that the latter gene product is essential for H. influenzae growth on media supplemented with NAD. In this report, we describe the functions and substrates of these two proteins as they act together in an NAD utilization pathway. Data are provided which indicate that NadN harbors not only NAD pyrophosphatase but also NMN 5′-nucleotidase activity. The e (P4) protein is also shown to have NMN 5′-nucleotidase activity, recognizing NMN as a substrate and releasing NR as its product. Insertion mutants of nadN or deletion and site-directed mutants of hel had attenuated growth and a reduced uptake phenotype when NMN served as substrate. A hel and nadN double mutant was only able to grow in the presence of NR, whereas no uptake of NMN was observed.

Haemophilus influenzae lipoprotein e (P4) is a member of the DDDD phosphohydrolase superfamily and mediates heme transport. Each of the aspartate residues of the signature motif is required for phosphomonoesterase activity, as none of the e (P4) single D mutants (D64A, D66A, D181N, and D185A) possessed detectable phosphomonoesterase activity. These results suggest that the signature motif is essential to the phosphomonoesterase activity of lipoprotein e (P4). When assessed for phosphomonoesterase-dependent heme transport activity in Escherichia coli hemA strains, plasmids containing D181N and D185A retained heme transport as indicated by aerobic growth while D64A and D66A did not. We conclude that phosphomonoesterase activity is not required for heme transport.

Haemophilus influenzae is a common inhabitant of the upper respiratory tract and can cause serious infections of mucosal surfaces. Results from recent studies indicate that this pathogen possesses copious amounts of surface-localized phosphomonoesterase activity mediated by the bacterial lipoprotein e (P4). While the enzyme has previously been purified to apparent homogeneity, purification of large amounts of protein has been prevented by presence of N-terminal lipid modification. Recombinant DNA technology was employed to simultaneously replace the N-terminal lipid modification signal sequence with one for protein secretion without such modification and to place expression of the protein under the control of the T7-inducible promoter. Results from this work show that high levels of phosphomonoesterase activity were achieved after IPTG induction and purified to apparent homogeneity after two chromatography steps. Consistent with loss of the N-terminal lipid modification, the recombinant enzyme was easily extracted from the bacterial membrane and partitioned within the matrix of gel filtration chromatography resin while retaining a denatured molecular weight similar to that of wild-type e (P4). Results from physicochemical characterization suggest that the recombinant protein was similar to wild-type protein in SDS-PAGE-derived molecular weight, primary structure, substrate specificity, pH optimum, and sensitivity or resistance to various inhibitors. Acquisition of sufficient amounts of recombinant P4 was a prelude for studies to elucidate the structure and function of this unusual phosphomonoesterase.

Haemophilus influenzae exists as a commensal of the upper respiratory tract of humans but also causes infections of contiguous structures. We describe the identification, localization, purification, and characterization of a novel, surface-localized phosphomonoesterase from a nontypeable H. influenzae strain, R2866. Sequences obtained from two CNBr-derived fragments of this protein matched lipoprotein e (P4) within the H. influenzae sequence database. Escherichia coli DH5α transformed with plasmids containing the H. influenzae hel gene, which encodes lipoprotein e (P4), produced high levels of a membrane-associated phosphomonoesterase. The isolated ∼28-kDa enzyme was tartrate resistant and displayed narrow substrate specificity with the highest activity for arylphosphates, excluding 5-bromo-4-chloro-3-indolylphosphate. Optimum enzymatic activity was observed at pH 5.0 and only in the presence of divalent copper. The enzyme was inhibited by vanadate, molybdate, and EDTA but was resistant to inorganic phosphate. The association of phosphomonoesterase activity with a protein that has also been recognized as a heme transporter suggests a unique role for this unusual phosphohydrolase.

SummaryDiseases of the brain are generally classified as either neurological or psychiatric. However, these two groups of illnesses cannot be readily separated on the basis of pathophysiology or symptomatology. It is difficult to rationally explain to someone with no prior frame of reference why we have the split between neurological and psychiatric illness. This demonstrates that the division is untenable, which has implications for training in both psychiatry and neurology.

SummaryAlthough translational medicine has become a priority for medical science, advances in neuroscience have failed to be translated for the benefit of patients. In populations at high risk of psychosis, neuroimaging could stratify those mostly likely to develop psychosis. This is an example of potentially translatable psychiatry.

Although many pathogenic copy number variations (CNVs) are associated with neuropsychiatric diseases, few of them have been functionally characterised. Here we report multiple schizophrenia cases with CNV abnormalities specific to unc-51-like kinase 4 ( ULK4 ), a serine/threonine kinase gene. Deletions spanning exons 21–34 of ULK4 were present in 4 out of 3391 schizophrenia patients from the International Schizophrenia Consortium, but absent in 3181 controls. Deletions removing exons 33 and 34 of the large splice variant of ULK4 also were enriched in Icelandic schizophrenia and bipolar patients compared with 98,022 controls ( P = 0.0007 for schizophrenia plus bipolar disorder). Combining the two cohorts gives a P -value less than 0.0001 for schizophrenia, or for schizophrenia plus bipolar disorder. The expression of ULK4 is neuron-specific and developmentally regulated. ULK4 modulates multiple signalling pathways that include ERK, p38, PKC and JNK, which are involved in stress responses and implicated in schizophrenia. Knockdown of ULK4 disrupts the composition of microtubules and compromises neuritogenesis and cell motility. Targeted Ulk4 deletion causes corpus callosum agenesis in mice. Our findings indicate that ULK4 is a rare susceptibility gene for schizophrenia.

A 3-year-old Texas Longhorn steer had a long history of progressive swelling of the soft tissues of the jaw and neck. At necropsy, multifocal to coalescing dermal and subcutaneous pyogranulomas were surrounded by fibrous tissue. Microscopically, the pyogranulomas contained aggregates of gram-negative coccobacilli surrounded by Splendore-Hoeppli material and were separated by bands of fibrovascular tissue (botryomycosis). Phylogenetic analysis of multilocus sequence-typing data revealed that the bacteria recovered in pure culture from swabs of submandibular tissue were most closely related to Bibersteinia [ Pasteurella] trehalosi. The bacterial colonies were immunohistochemically reactive with a rabbit polyclonal anti-Pasteurella class C acid phosphatase antibody. Botryomycosis is a pyogranulomatous inflammation caused by a variety of nonbranching, nonfilamentous bacteria that elicit the formation of Splendore-Hoeppli material. This case of botryomycosis is unique for its association with Bibersteinia trehalosi.

Class C acid phosphatases (CCAPs) are 25–30 kDa bacterial surface proteins that are thought to function as broad-specificity 5′,3′-nucleotidases. Analysis of the newly published complete genome sequence of Mycoplasma bovis PG45 revealed a putative CCAP with a molecular weight of 49.9 kDa. The expression, purification and crystallization of this new family member are described here. Standard purification procedures involving immobilized metal-ion affinity chromatography and ion-exchange chromatography yielded highly pure and crystallizable protein. Crystals were grown in sitting drops at room temperature in the presence of PEG 3350 and HEPES buffer pH 7.5 and diffracted to 2.3 A resolution. Analysis of diffraction data suggested a primitive monoclinic space group, with unit-cell parameters a = 78, b = 101, c = 180 A, β = 92°. The asymmetric unit is predicted to contain six molecules, which are likely to be arranged as three dimers.