Consensus Practice Guidelines on Postdural Puncture Headache From a Multisociety, International Working Group: A Summary Report.

Importance: Postdural puncture headache (PDPH) can follow unintentional dural puncture during epidural techniques or intentional dural puncture during neuraxial procedures, such as a lumbar puncture or spinal anesthesia. Evidence-based guidance on the prevention, diagnosis, and management of this condition is, however, currently lacking. Objective: To fill the practice guidelines void and provide comprehensive information and patient-centric recommendations for preventing, diagnosing, and managing PDPH. Evidence Review: With input from committee members and stakeholders of 6 participating professional societies, 10 review questions that were deemed important for the prevention, diagnosis, and management of PDPH were developed. A literature search for each question was performed in MEDLINE on March 2, 2022. Additional relevant clinical trials, systematic reviews, and research studies published through March 2022 were also considered for practice guideline development and shared with collaborator groups. Each group submitted a structured narrative review along with recommendations that were rated according to the US Preventive Services Task Force grading of evidence. Collaborators were asked to vote anonymously on each recommendation using 2 rounds of a modified Delphi approach. Findings: After 2 rounds of electronic voting by a 21-member multidisciplinary collaborator team, 47 recommendations were generated to provide guidance on the risk factors for and the prevention, diagnosis, and management of PDPH, along with ratings for the strength and certainty of evidence. A 90% to 100% consensus was obtained for almost all recommendations. Several recommendations were rated as having moderate to low certainty. Opportunities for future research were identified. Conclusions and Relevance: Results of this consensus statement suggest that current approaches to the treatment and management of PDPH are not uniform due to the paucity of evidence. The practice guidelines, however, provide a framework for individual clinicians to assess PDPH risk, confirm the diagnosis, and adopt a systematic approach to its management.

Evidence-based clinical practice guidelines on postdural puncture headache: a consensus report from a multisociety international working group.

INTRODUCTION: Postdural puncture headache (PDPH) can follow unintentional dural puncture during epidural techniques or intentional dural puncture during neuraxial procedures such as a lumbar puncture or spinal anesthesia. Evidence-based guidance on the prevention, diagnosis or management of this condition is, however, currently lacking. This multisociety guidance aims to fill this void and provide practitioners with comprehensive information and patient-centric recommendations to prevent, diagnose and manage patients with PDPH. METHODS: Based on input from committee members and stakeholders, the committee cochairs developed 10 review questions deemed important for the prevention, diagnosis and management of PDPH. A literature search for each question was performed in MEDLINE (Ovid) on 2 March 2022. The results from each search were imported into separate Covidence projects for deduplication and screening, followed by data extraction. Additional relevant clinical trials, systematic reviews and research studies published through March 2022 were also considered for the development of guidelines and shared with contributors. Each group submitted a structured narrative review along with recommendations graded according to the US Preventative Services Task Force grading of evidence. The interim draft was shared electronically, with each collaborator requested to vote anonymously on each recommendation using two rounds of a modified Delphi approach. RESULTS: Based on contemporary evidence and consensus, the multidisciplinary panel generated 50 recommendations to provide guidance regarding risk factors, prevention, diagnosis and management of PDPH, along with their strength and certainty of evidence. After two rounds of voting, we achieved a high level of consensus for all statements and recommendations. Several recommendations had moderate-to-low certainty of evidence. CONCLUSIONS: These clinical practice guidelines for PDPH provide a framework to improve identification, evaluation and delivery of evidence-based care by physicians performing neuraxial procedures to improve the quality of care and align with patients' interests. Uncertainty remains regarding best practice for the majority of management approaches for PDPH due to the paucity of evidence. Additionally, opportunities for future research are identified.

Angelman and Prader-Willi Syndromes: Sister Imprinting Disorders With High Complication Rates Following Spinal Deformity Surgery.

We sought to examine the modern surgical treatment of spinal deformity associated with sister imprinting disorders, Prader-Willi syndrome (PWS) and Angelman syndrome (AS), with emphasis on the specific complications encountered in these patient populations. Fifteen patients with PWS and 5 patients with AS who underwent surgical intervention for spinal deformity between 2000 and 2018 were identified. Postoperative complications were classified using the modified Clavien-Dindo-Sink (CDS) system and further categorized into specific subtypes including excessive drainage, dehiscence, implant failure, infection, and delayed wound healing. Perioperative and final follow-up radiographic data were analyzed. Mean age at surgery was 12.9 years (range, 4-21 years) with mean follow-up of 46.1 months (range, 1-145 months). There were postoperative complications in 17 patients (85%). Ten major complications (CDS ≥ 3) occurred in 9 patients (45%). These included 5 infections requiring reoperation, 1 seroma requiring drainage, 2 severe cervical-thoracic deformities requiring reoperation, 1 implant failure requiring reoperation, and 1 death secondary to fungal sepsis and thromboembolic disease. Eight additional patients (40%) had minor complications (CDS 1 or 2). Eight intraoperative complications occurred in 5 patients (25%), including loss of neuromonitoring signals and cerebrospinal fluid leaks. Surgical intervention for scoliosis in PWS and AS continues to have high complication rates secondary to medical and behavioral comorbidities found in these patient populations. The exact etiology of the high complication rates encountered cannot be definitively stated, but both syndromes frequently present with a number of unique features that may predispose patients to develop surgical complications. [Orthopedics. 2023;46(4):e223-e229.].

Retraining Convolutional Neural Networks for Specialized Cardiovascular Imaging Tasks: Lessons from Tetralogy of Fallot.

Ventricular contouring of cardiac magnetic resonance imaging is the gold standard for volumetric analysis for repaired tetralogy of Fallot (rTOF), but can be time-consuming and subject to variability. A convolutional neural network (CNN) ventricular contouring algorithm was developed to generate contours for mostly structural normal hearts. We aimed to improve this algorithm for use in rTOF and propose a more comprehensive method of evaluating algorithm performance. We evaluated the performance of a ventricular contouring CNN, that was trained on mostly structurally normal hearts, on rTOF patients. We then created an updated CNN by adding rTOF training cases and evaluated the new algorithm's performance generating contours for both the left and right ventricles (LV and RV) on new testing data. Algorithm performance was evaluated with spatial metrics (Dice Similarity Coefficient (DSC), Hausdorff distance, and average Hausdorff distance) and volumetric comparisons (e.g., differences in RV volumes). The original Mostly Structurally Normal (MSN) algorithm was better at contouring the LV than the RV in patients with rTOF. After retraining the algorithm, the new MSN + rTOF algorithm showed improvements for LV epicardial and RV endocardial contours on testing data to which it was naïve (N = 30; e.g., DSC 0.883 vs. 0.905 for LV epicardium at end diastole, p < 0.0001) and improvements in RV end-diastolic volumetrics (median %error 8.1 vs 11.4, p = 0.0022). Even with a small number of cases, CNN-based contouring for rTOF can be improved. This work should be extended to other forms of congenital heart disease with more extreme structural abnormalities. Aspects of this work have already been implemented in clinical practice, representing rapid clinical translation. The combined use of both spatial and volumetric comparisons yielded insights into algorithm errors.

Anterior insular thickness predicts speech sound learning ability in bilinguals.

A previous fMRI study of novel speech sound learning, tied to the methods and results presented here, identified groups of advanced and novice learners and related their classification to neural activity. To complement those results and better elucidate the role of the entire neural system in speech learning, the current study analyzed the neuroanatomical data with the goals of 1) uncovering the regions of interest (ROIs) that predicted speech learning performance in a sample of monolingual and bilingual adults, and 2) examining if the relationship between cortical thickness from selected ROIs and individual learning ability depends on language group. The ROIs selected were brain regions well-established in the literature as areas associated with language and speech processing (i.e., Transverse Superior Temporal Gyrus, anterior insula and posterior insula, all bilaterally). High-resolution brain scans (T1-weighted) were acquired from 23 Spanish-English bilinguals and 20 English monolingual adults. The thickness of the left anterior insula significantly predicted speech sound learning ability in bilinguals but not monolinguals. These results suggest that aptitude for learning a new language is associated with variations in the cortical thickness of the left anterior insula in bilinguals. These findings may provide insight into the higher order mechanisms involved in speech perception and advance our understanding of the unique strategies employed by the bilingual brain during language learning.

Real-world outcomes of paliperidone palmitate compared to daily oral antipsychotic therapy in schizophrenia: a randomized, open-label, review board-blinded 15-month study.

OBJECTIVE: The Paliperidone Palmitate Research in Demonstrating Effectiveness (PRIDE) study compared the effects of once-monthly paliperidone palmitate with daily oral antipsychotics on treatment failure in adults with schizophrenia. METHOD: The PRIDE study is a 15-month, randomized, multicenter study (May 5, 2010, to December 9, 2013) of adult subjects with a DSM-IV diagnosis of schizophrenia and a history of incarceration. Subjects were randomly assigned to once-monthly paliperidone palmitate injections or daily oral antipsychotics (randomly assigned from 7 acceptable, prespecified oral antipsychotics) for 15 months. The primary end point was time to first treatment failure, defined as arrest/incarceration; psychiatric hospitalization; suicide; treatment discontinuation or supplementation due to inadequate efficacy, safety, or tolerability; or increased psychiatric services to prevent hospitalization. Time to first treatment failure was determined by a blinded event-monitoring board and analyzed with the Kaplan-Meier method. RESULTS: In this study, 450 patients were randomly assigned, and 444 were included in the intent-to-treat population. Paliperidone palmitate was associated with significant delay in time to first treatment failure versus oral antipsychotics (hazard ratio, 1.43; 95% CI, 1.09-1.88; log rank P = .011). Observed treatment failure rates over 15 months were 39.8% and 53.7%, respectively. Arrest/incarceration and psychiatric hospitalization were the most common reasons for treatment failure in the paliperidone palmitate and oral antipsychotic groups (21.2% vs 29.4% and 8.0% vs 11.9%, respectively). The 5 most common treatment-emergent adverse events for the paliperidone palmitate treatment group were injection site pain (18.6% of subjects), insomnia (16.8%), weight increased (11.9%), akathisia (11.1%), and anxiety (10.6%). CONCLUSIONS: In a trial designed to reflect real-world management of schizophrenia, once-monthly paliperidone palmitate demonstrated superiority compared to oral antipsychotics in delaying time to treatment failure. TRIAL REGISTRATION: Clinicaltrials.gov Identifier: NCT01157351.

Design and rationale of the Paliperidone Palmitate Research in Demonstrating Effectiveness (PRIDE) study: a novel comparative trial of once-monthly paliperidone palmitate versus daily oral antipsychotic treatment for delaying time to treatment failure in persons with schizophrenia.

BACKGROUND: Public health considerations require that clinical trials address the complex "real-world" needs of patients with chronic illnesses. This is particularly true for persons with schizophrenia, whose management is frequently complicated by factors such as comorbid substance abuse, homelessness, and contact with the criminal justice system. In addition, barriers to obtaining health care in the United States often prevent successful community reentry and optimal patient management. Further, nonadherence to treatment is common, and this reinforces cycles of relapse and recidivism. Long-acting injectable antipsychotic therapy may facilitate continuity of treatment and support better outcomes, particularly in patients who face these challenges. Clinical trials with classical explanatory designs may not be the best approaches for evaluating these considerations. We describe the design and rationale of a novel trial that combines both explanatory and pragmatic design features and studies persons with schizophrenia who face these challenges. DESIGN AND RATIONALE: The Paliperidone Palmitate Research in Demonstrating Effectiveness (PRIDE) study is a prospective, open-label, randomized, 15-month study conducted between May 5, 2010, and December 9, 2013, comparing long-acting injectable paliperidone palmitate and oral antipsychotic medications in subjects with schizophrenia (according to DSM-IV criteria). Investigators and subjects had broad flexibility for treatment decision-making, thus making it a model that better reflects real-world practice. The primary end point was time to treatment failure, defined as arrest/incarceration psychiatric hospitalization; suicide; treatment discontinuation or supplementation due to inadequate efficacy, safety, or tolerability; or increased psychiatric services to prevent hospitalization. This end point was adjudicated by a blinded event monitoring board. Patients were followed to the 15-month end point, regardless of whether they were maintained on their initial randomized treatment. This article provides some of the reasoning behind the authors' choices when combining features from both explanatory and pragmatic approaches to this trial's design. CONCLUSIONS: The PRIDE study incorporates real-world design features in a novel, prospective, comparative study of long-acting injectable and oral antipsychotics in persons with schizophrenia who have had recent contact with the criminal justice system. Insights provided should help the reader to better understand the need for more real-world approaches for clinical studies and how a broader approach can better aid clinical treatment and public health decision-making. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01157351.

Lipidation of the FPI protein IglE contributes to Francisella tularensis ssp. novicida intramacrophage replication and virulence.

Francisella tularensis is a Gram-negative bacterium responsible for the human disease tularemia. The Francisella pathogenicity island (FPI) encodes a secretion system related to type VI secretion systems (T6SS) which allows F. tularensis to escape the phagosome and replicate within the cytosol of infected macrophages and ultimately cause disease. A lipoprotein is typically found encoded within T6SS gene clusters and is believed to anchor portions of the secretion apparatus to the outer membrane. We show that the FPI protein IglE is a lipoprotein that incorporates (3)H-palmitate and localizes to the outer membrane. A C22G IglE mutant failed to be lipidated and failed to localize to the outer membrane, consistent with C22 being the site of lipidation. Francisella tularensis ssp. novicida expressing IglE C22G is defective for replication in macrophages and unable to cause disease in mice. Bacterial two-hybrid analysis demonstrated that IglE interacts with the C-terminal portion of the FPI inner membrane protein PdpB, and PhoA fusion analysis indicated the PdpB C-terminus is located within the periplasm. We predict this interaction facilitates channel formation to allow secretion through this system.

Safety of paliperidone extended-release in patients with schizophrenia or schizoaffective disorder and hepatic disease.

BACKGROUND/PURPOSE: Patients with schizophrenia often suffer from comorbid hepatic disease. This multicenter, open-label, single-arm, crossover study evaluated the safety and efficacy of paliperidone extended-release (ER) in patients with schizophrenia or schizoaffective disorder and hepatic disease. METHODS: The study comprised a screening period, followed by 9 weeks' open-label treatment, divided into 2 phases. Phase 1 (4 weeks) was a continuation of usual antipsychotic treatment (UAT); phase 2 (5 weeks) consisted of a 1-week cross-titration from UAT to flexibly dosed paliperidone ER (3-12 mg/d), followed by 4 weeks of paliperidone ER alone. Treatment-emergent adverse events (TEAEs), including those considered more relevant to antipsychotic treatment (prespecified adverse events [AEs]), were analyzed. RESULTS: Although more subjects reported TEAEs during the paliperidone ER alone period than during the UAT period, no significant differences occurred in prespecified AE rates. No new safety signals were detected, and minimal shifts in liver function test values were observed. Improvements in psychiatric symptoms and functioning were observed after 4 weeks' paliperidone ER treatment. CONCLUSIONS: This study suggests that paliperidone ER is well tolerated in patients with schizophrenia or schizoaffective disorder and hepatic disease. To the best of our knowledge, this is the largest prospective study to date in this population.

Antibiotic-free production of a herpes simplex virus 2 DNA vaccine in a high yield cGMP process.

Two DNA vaccine plasmids encoding Herpes simplex virus type 2 (HSV-2) glycoprotein D, NTC8485-O2-gD2 and NTC8485-O2-UgD2tr, were produced at large scale under current good manufacturing practice (cGMP) for use in a Phase I human clinical trial. These DNA vaccines incorporate the regulatory agency compliant, minimal, antibiotic-free (AF) NTC8485 mammalian expression vector. Plasmid yields of>1 g/L were achieved using the HyperGRO™ fed-batch fermentation process, with successful scale up from 10 L process development scale to 320 L culture volume for cGMP production. The DNA vaccines were purified using a low residence time, high shear lysis process and AIRMIX(TM) technology, followed by chromatographic purification. This combination of optimized plasmid vector, high yield upstream production, and efficient downstream purification resulted in purified HSV-2 DNA vaccines with>99% total supercoiled plasmid, ≤ 0.2% RNA, ≤ 0.1% host cell genomic DNA, and ≤ 0.1 endotoxin units per mg.

Assessment of effectiveness measures in patients with schizophrenia initiated on risperidone long-acting therapy: the SOURCE study results.

BACKGROUND: To evaluate effectiveness outcomes in a real-world setting in patients with schizophrenia initiating risperidone long-acting therapy (RLAT). METHODS: This was a 24-month, multicenter, prospective, longitudinal, observational study in patients with schizophrenia who were initiated on RLAT. Physicians could change treatment during the study as clinically warranted. Data were collected at baseline and subsequently every 3 months up to 24 months. Effectiveness outcomes included changes in illness severity as measured by Clinical Global Impression-Severity (CGI-S) scale; functional scores as measured by Personal and Social Performance (PSP) scale, Global Assessment of Functioning (GAF), and Strauss-Carpenter Levels of Functioning (LOF); and health status (Medical Outcomes Survey Short Form-36 [SF-36]). Life-table methodology was used to estimate the cumulative probability of relapse over time. Adverse events were evaluated for safety. RESULTS: 532 patients were enrolled in the study; 209 (39.3%) completed the 24-month study and 305 (57.3%) had at least 12 months of follow-up data. The mean (SD) age of patients was 42.3 (12.8) years. Most patients were male (66.4%) and either Caucasian (60.3%) or African American (23.7%). All changes in CGI-S from baseline at each subsequent 3-month follow-up visit were statistically significant (p < .0001), indicating improvement in disease severity. Improvements were also noted for the PSP, GAF, and total LOF, indicating improvement in daily functioning and health outcome. CONCLUSIONS: Patients with schizophrenia who were initiated on RLAT demonstrated improvements in measures of effectiveness within 3 months, which persisted over 24 months. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00246194.

Evasion of IFN-γ signaling by Francisella novicida is dependent upon Francisella outer membrane protein C.

BACKGROUND: Francisella tularensis is a Gram-negative facultative intracellular bacterium and the causative agent of the lethal disease tularemia. An outer membrane protein (FTT0918) of F. tularensis subsp. tularensis has been identified as a virulence factor. We generated a F. novicida (F. tularensis subsp. novicida) FTN_0444 (homolog of FTT0918) fopC mutant to study the virulence-associated mechanism(s) of FTT0918. METHODS AND FINDINGS: The ΔfopC strain phenotype was characterized using immunological and biochemical assays. Attenuated virulence via the pulmonary route in wildtype C57BL/6 and BALB/c mice, as well as in knockout (KO) mice, including MHC I, MHC II, and µmT (B cell deficient), but not in IFN-γ or IFN-γR KO mice was observed. Primary bone marrow derived macrophages (BMDM) prepared from C57BL/6 mice treated with rIFN-γ exhibited greater inhibition of intracellular ΔfopC than wildtype U112 strain replication; whereas, IFN-γR KO macrophages showed no IFN-γ-dependent inhibition of ΔfopC replication. Moreover, phosphorylation of STAT1 was downregulated by the wildtype strain, but not the fopC mutant, in rIFN-γ treated macrophages. Addition of NG-monomethyl-L-arginine, an NOS inhibitor, led to an increase of ΔfopC replication to that seen in the BMDM unstimulated with rIFN-γ. Enzymatic screening of ΔfopC revealed aberrant acid phosphatase activity and localization. Furthermore, a greater abundance of different proteins in the culture supernatants of ΔfopC than that in the wildtype U112 strain was observed. CONCLUSIONS: F. novicida FopC protein facilitates evasion of IFN-γ-mediated immune defense(s) by down-regulation of STAT1 phosphorylation and nitric oxide production, thereby promoting virulence. Additionally, the FopC protein also may play a role in maintaining outer membrane stability (integrity) facilitating the activity and localization of acid phosphatases and other F. novicida cell components.

Effectiveness of injectable risperidone long-acting therapy for schizophrenia: data from the US, Spain, Australia, and Belgium.

BACKGROUND: Because wide variations in mental health care utilization exist throughout the world, determining long-term effectiveness of psychotropic medications in a real-world setting would be beneficial to physicians and patients. The purpose of this analysis was to describe the effectiveness of injectable risperidone long-acting therapy (RLAT) for schizophrenia across countries. METHODS: This was a pragmatic analysis of data from two prospective observational studies conducted in the US (Schizophrenia Outcomes Utilization Relapse and Clinical Evaluation [SOURCE]; ClinicalTrials.gov registration number for the SOURCE study: NCT00246194) and Spain, Australia, and Belgium (electronic Schizophrenia Treatment Adherence Registry [eSTAR]). Two separate analyses were performed to assess clinical improvement during the study and estimate psychiatric hospitalization rates before and after RLAT initiation. Clinical improvement was evaluated using the Clinical Global Impressions-Severity (CGI-S) and Global Assessment of Functioning (GAF) scales, and change from baseline was evaluated using paired t tests. Psychiatric hospitalization rates were analyzed using incidence densities, and the bootstrap resampling method was used to examine differences between the pre-baseline and post-baseline periods. RESULTS: The initial sample comprised 3,069 patients (US, n = 532; Spain, n = 1,345; Australia, n = 784; and Belgium, n = 408). In all, 24 months of study participation, completed by 39.3% (n = 209), 62.7% (n = 843), 45.8% (n = 359), and 64.2% (n = 262) of patients from the US, Spain, Australia, and Belgium, respectively, were included in the clinical analysis. Improvements compared with baseline were observed on both clinical assessments across countries (P < 0.001 at all post-baseline visits). The mean improvement was approximately 1 point on the CGI-S and 15 points on the GAF. A total of 435 (81.8%), 1,339 (99.6%), 734 (93.6%), and 393 (96.3%) patients from the US, Spain, Australia, and Belgium, respectively, had ≥1 post-baseline visit and were included in the analysis of psychiatric hospitalization rates. Hospitalization rates decreased significantly in all countries regardless of hospitalization status at RLAT initiation (P < 0.0001) and decreased significantly in the US and Spain (P < 0.0001) when the analysis was limited to outpatients only. CONCLUSIONS: RLAT in patients with schizophrenia was associated with improvements in clinical and functional outcomes and decreased hospitalization rates in the US, Spain, Australia, and Belgium, despite differences in health care delivery systems.

Production of pharmaceutical-grade plasmids at high concentration and high supercoiled percentage.

The increased use of plasmid-based vaccines to replace their more challenging viral counterparts has increased the demand for high purity and high concentration plasmids. Here we report the production of plasmids encoding different transgenes for DNA vaccine candidates at gram scale with an integrated process consisting of batch fermentation and limited steps of purification. Plasmid products encoding for eight smallpox antigens that were combined into a bioterrorism DNA vaccine exhibited high purity with undetectable RNA, protein and endotoxin, concentration of up to 13.6mg/mL and supercoiled percentage of 94.5+/-1.1% after storage at -80 degrees C for over 1 year. The process has been scaled up for the cGMP manufacture of pharmaceutical-grade human papillomavirus and influenza DNA vaccines up to a 50g scale, also demonstrating high purity and high concentration.

DNA vaccine manufacture: scale and quality.

The demand for plasmid DNA in large quantities at high purity and concentration is expected to escalate as more DNA vaccines are entering clinical trial status and becoming closer to market approval. This review outlines different methods for DNA vaccine manufacture and discusses the challenges that hinder large-scale production. Current technologies are summarized, focusing on novel approaches that have the potential to address downstream bottlenecks and adaptability for large-scale application. Product quality in terms of supercoiled percentage and impurity levels are compared at the different production levels.

Genomic and systems evolution in Vibrionaceae species.

BACKGROUND: The steadily increasing number of prokaryotic genomes has accelerated the study of genome evolution; in particular, the availability of sets of genomes from closely related bacteria has facilitated the exploration of the mechanisms underlying genome plasticity. The family Vibrionaceae is found in the Gammaproteobacteria and is abundant in aquatic environments. Taxa from the family Vibrionaceae are diversified in their life styles; some species are free living, others are symbiotic, and others are human pathogens. This diversity makes this family a useful set of model organisms for studying bacterial evolution. This evolution is driven by several forces, among them gene duplication and lateral gene transfer, which are believed to provide raw material for functional redundancy and novelty. The resultant gene copy increase in one genome is then detected as lineage-specific expansion (LSE). RESULTS: Here we present the results of a detailed comparison of the genomes of eleven Vibrionaceae strains that have distinct life styles and distinct phenotypes. The core genome shared by all eleven strains is composed of 1,882 genes, which make up about 31%-50% of the genome repertoire. We further investigated the distribution and features of genes that have been specifically expanded in one unique lineage of the eleven strains. Abundant duplicate genes have been identified in the eleven Vibrionaceae strains, with 1-11% of the whole genomes composed lineage specific radiations. These LSEs occurred in two distinct patterns: the first type yields one or more copies of a single gene; we call this a single gene expansion. The second pattern has a high evolutionary impact, as the expansion involves two or more gene copies in a block, with the duplicated block located next to the original block (a contiguous block expansion) or at some distance from the original block (a discontiguous block expansion). We showed that LSEs involve genes that are tied to defense and pathogenesis mechanisms as well as in the fundamental life cycle of Vibrionaceae species. CONCLUSION: Our results provide evidence of genome plasticity and rapid evolution within the family Vibrionaceae. The comparisons point to sources of genomic variation and candidates for lineage-specific adaptations of each Vibrionaceae pathogen or nonpathogen strain. Such lineage specific expansions could reveal components in bacterial systems that, by their enhanced genetic variability, can be tied to responses to environmental challenges, interesting phenotypes, or adaptive pathogenic responses to host challenges.

Targeted gene disruption in Francisella tularensis by group II introns.

Francisella tularensis is a highly infectious Gram-negative bacterium that is the causative agent of tularemia. Very little is known about the molecular mechanisms responsible for F. tularensis virulence, in part due to the paucity of genetic tools available for the study of F. tularensis. We have developed a gene knockout system for F. tularensis that utilizes retargeted mobile group II introns, or "targetrons". These targetrons disrupt both single and duplicated target genes at high efficiency in three different F. tularensis subspecies. Here we describe in detail the targetron-based method for insertional mutagenesis of F. tularensis genes, which should facilitate a better understanding of F. tularensis pathogenesis. Group II introns can be adapted to inactivate genes in bacteria for which few genetic tools exist, thus providing a powerful tool to study the genetic basis of bacterial pathogenesis.

The Francisella tularensis pathogenicity island encodes a secretion system that is required for phagosome escape and virulence.

Francisella tularensis causes the human disease tularemia. F. tularensis is able to survive and replicate within macrophages, a trait that has been correlated with its high virulence, but it is unclear the exact mechanism(s) this organism uses to escape killing within this hostile environment. F. tularensis virulence is dependent upon the Francisella pathogenicity island (FPI), a cluster of genes that we show here shares homology with type VI secretion gene clusters in Vibrio cholerae and Pseudomonas aeruginosa. We demonstrate that two FPI proteins, VgrG and IglI, are secreted into the cytosol of infected macrophages. VgrG and IglI are required for F. tularensis phagosomal escape, intramacrophage growth, inflammasome activation and virulence in mice. Interestingly, VgrG secretion does not require the other FPI genes. However, VgrG and other FPI genes, including PdpB (an IcmF homologue), are required for the secretion of IglI into the macrophage cytosol, suggesting that VgrG and other FPI factors are components of a secretion system. This is the first report of F. tularensis FPI virulence proteins required for intramacrophage growth that are translocated into the macrophage.

Targeted inactivation of francisella tularensis genes by group II introns.

Studies of the molecular mechanisms of pathogenesis of Francisella tularensis, the causative agent of tularemia, have been hampered by a lack of genetic techniques for rapid targeted gene disruption in the most virulent subspecies. Here we describe efficient targeted gene disruption in F. tularensis utilizing mobile group II introns (targetrons) specifically optimized for F. tularensis. Utilizing a targetron targeted to blaB, which encodes ampicillin resistance, we showed that the system works at high efficiency in three different subspecies: F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, and "F. tularensis subsp. novicida." A targetron was also utilized to inactivate F. tularensis subsp. holarctica iglC, a gene required for virulence. The iglC gene is located within the Francisella pathogenicity island (FPI), which has been duplicated in the most virulent subspecies. Importantly, the iglC targetron targeted both copies simultaneously, resulting in a strain mutated in both iglC genes in a single step. This system will help illuminate the contributions of specific genes, and especially those within the FPI, to the pathogenesis of this poorly studied organism.

Abnormal Involuntary Movement Scale (AIMS) and Extrapyramidal Symptom Rating Scale (ESRS): cross-scale comparison in assessing tardive dyskinesia.

Assessing tardive dyskinesia (TD) has been complicated by the use of different research criteria and rating scales. We studied concordance between two commonly used scales, the Abnormal Involuntary Movement Scale (AIMS) and Extrapyramidal Symptom Rating Scale (ESRS), to study interscale concordance and criteria to define TD. Patients with schizophrenia or schizoaffective disorder (N = 374) were rated at baseline with both scales. Linear and logistic regression models explored relationships between scale ratings and mapped scores for corresponding items. TD was defined as at least mild in > or = 2 anatomical areas, or moderate or greater symptoms in > or = 1 area at baseline. Logistic regression was used to find simplified criteria for predicting AIMS-defined TD by ESRS scores. There was a strong association on corresponding item ratings. "Mild" was defined as AIMS score of 2 and ESRS 2 or 3, and "moderate or greater" as AIMS score > or = 3 and ESRS > or = 4. Using these criteria, there was 96.0% (359/374) agreement between AIMS- and ESRS-defined TD cases. The ESRS Clinical Global Impressions of severity of dyskinesia (CGI-SD) best predicted AIMS-defined TD. An ESRS CGI-SD > or = 4 (95% CI: 3.61, 4.76) was associated with > or = 95% probability of AIMS-defined TD. High concordance between the scales for dyskinesia scores was found. Findings suggest that the ESRS CGI-SD score can serve as a simplified criterion for identifying AIMS-defined TD, and may be a useful tool for future research-based TD analyses, when occurring in the context of a full movement disorder assessment.