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Microtubules (MTs) are intrinsically dynamic polymers. In neurons, staggered individual microtubules form stable, polarized acentrosomal MT arrays spanning the axon and dendrite to support long-distance intracellular transport. How the stability and polarity of these arrays are maintained when individual MTs remain highly dynamic is still an open question. Here we visualize MT arrays in vivo in C. elegans neurons with single microtubule resolution. We find that the CRMP family homolog, UNC-33, is essential for the stability and polarity of MT arrays in neurites. In unc-33 mutants, MTs exhibit dramatically reduced rescue after catastrophe, develop gaps in coverage, and lose their polarity, leading to trafficking defects. UNC-33 is stably anchored on the cortical cytoskeleton and forms patch-like structures along the dendritic shaft. These discrete and stable UNC-33 patches concentrate free tubulins and correlate with MT rescue sites. In vitro , purified UNC-33 preferentially associates with MT tips and increases MT rescue frequency. Together, we propose that UNC-33 functions as a microtubule-associated protein (MAP) to promote individual MT rescue locally. Through this activity, UNC-33 prevents the loss of individual MTs, thereby maintaining the coverage and polarity of MT arrays throughout the lifetime of neurons.

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features., (© 2024. The Author(s).)

Background: Although abnormal uterine bleeding is a known adverse effect of anticoagulant drugs, true rates have not been widely studied. Society-backed recommendations and guidelines do not yet exist for prevention and management of abnormal uterine bleeding among anticoagulated patients., Objective: This study aimed to describe the incidence of new-onset abnormal uterine bleeding among patients receiving therapeutic anticoagulation by anticoagulant class, and to evaluate gynecologic treatment patterns., Study Design: We conducted an institutional review board-waived retrospective chart review of female patients aged 18 to 55 years and prescribed therapeutic anticoagulants, including vitamin-K antagonists, low-molecular-weight heparins, and direct oral anticoagulants, in an urban hospital network from January 2015 through January 2020. We excluded patients with antecedent abnormal uterine bleeding and menopause. Associations between abnormal uterine bleeding, anticoagulant class, and other covariates were evaluated with Pearson chi-square and analysis-of-variance tests. The primary outcome, abnormal uterine bleeding odds by anticoagulant class, was modeled with logistic regression. Age, antiplatelet therapy, body mass index, and race were included in our multivariable model. Secondary outcomes included emergency department visits and treatment patterns., Results: Of the 2479 patients who met the inclusion criteria, 645 were diagnosed with abnormal uterine bleeding after initiating therapeutic anticoagulation. After adjusting for age, race, body mass index, and concurrent use of antiplatelet therapy, those receiving all 3 classes of anticoagulants had higher odds of experiencing abnormal uterine bleeding (adjusted odds ratio, 2.63; confidence interval, 1.70-4.08; P<.001), whereas those taking only direct oral anticoagulants had the lowest odds (adjusted odds ratio, 0.70; confidence interval, 0.51-0.97; P=.032), with vitamin-K antagonists as the reference group. Race other than White was associated with higher odds of abnormal uterine bleeding, as was lower age. The most common hormone therapies used among patients with abnormal uterine bleeding were levonorgestrel intrauterine devices (7.6%; 49/645) and oral progestins (7.6%; 49/645). Sixty-eight patients (10.5%; 68/645) had an emergency department visit for abnormal uterine bleeding; 29.5% (190/645) of patients received a blood transfusion; 12.2% (79/645) began any pharmacologic therapy for bleeding; and 18.8% (121/645) underwent any gynecologic procedure., Conclusion: Abnormal uterine bleeding occurs frequently among patients on therapeutic anticoagulation. Incidence in this sample varied considerably by anticoagulant class and race; use of single-agent direct oral anticoagulation carried the lowest risk. Important sequelae such as bleeding-related emergency department visits, blood transfusions, and gynecologic procedures were common. Balancing bleeding and clotting risk in patients on therapeutic anticoagulation requires a nuanced approach and should involve collaborative management between hematologists and gynecologists., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Coercive and disruptive behaviors commonly interfere with cognitive-behavioral therapy (CBT) trials among youths with obsessive-compulsive disorder (OCD). Although evidence supports parent management training (PMT) for reducing disruptive behavior, no group-based PMT interventions exist for OCD-related disruptive behaviors. We studied feasibility and effectiveness of group-based adjunctive PMT among non-randomized, OCD-affected families receiving family-based group CBT. Linear mixed models estimated treatment effects across OCD-related and parenting outcomes at post-treatment and 1-month follow-up. Treatment response for 37 families receiving CBT + PMT (M age  = 13.90) was compared to 80 families receiving only CBT (M age  = 13.93). CBT + PMT was highly accepted by families. Families who received CBT + PMT had improved disruptive behaviors, parental distress tolerance, and other OCD-related outcomes. OCD-related outcomes did not significantly differ between groups. Results support CBT + PMT as effective treatment for pediatric OCD that may not provide incremental benefits beyond CBT alone. Future research should determine feasible and effective ways to incorporate key PMT components into CBT-based interventions., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: The coronavirus disease 2019 (COVID-19) pandemic disrupted healthcare delivery, including prenatal care. The study objective was to assess if timing of routine prenatal testing changed during the COVID-19 pandemic., Methods: Retrospective observational cohort study using claims data from a regional insurer (Highmark) and electronic health record data from two academic health systems (Penn Medicine and Yale New Haven) to compare prenatal testing timing in the pre-pandemic (03/10/2018-12/31/2018 and 03/10/2019-12/31/2019) and early COVID-19 pandemic (03/10/2020-12/31/2020) periods. Primary outcomes were second trimester fetal anatomy ultrasounds and gestational diabetes (GDM) testing. A secondary analysis examined first trimester ultrasounds., Results: The three datasets included 31,474 pregnant patients. Mean gestational age for second trimester anatomy ultrasounds increased from the pre-pandemic to COVID-19 period (Highmark 19.4 vs. 19.6 weeks; Penn: 20.1 vs. 20.4 weeks; Yale: 18.8 vs. 19.2 weeks, all p  < 0.001). There was a detectable decrease in the proportion of patients who completed the anatomy survey <20 weeks' gestation across datasets, which did not persist at <23 weeks' gestation. There were no consistent changes in timing of GDM screening. There were significant reductions in the proportion of patients with first trimester ultrasounds in the academic institutions (Penn: 57.7% vs. 40.6% and Yale: 78.7% vs. 65.5%, both p  < 0.001) but not Highmark. Findings were similar with multivariable adjustment., Conclusion: While some prenatal testing happened later in pregnancy during the pandemic, pregnant patients continued to receive appropriately timed testing. Despite disruptions in care delivery, prenatal screening remained a priority for patients and providers during the COVID-19 pandemic., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2022 Handley, Ledyard, Lundsberg, Passarella, Yang, Son, Mckenney, Greenspan, Dysart, Culhane and Burris.)

Background: Traumatic Brain Injury (TBI) is a leading cause of mortality in the trauma population. Accurate prognosis remains a challenge. Two common Computed Tomography (CT)-based prognostic models include the Marshall Classification and the Rotterdam CT Score. This study aims to determine the utility of the Marshall and Rotterdam scores in predicting mortality for adult patients in coma with severe TBI., Method: Retrospective review of our Level 1 Trauma Center's registry for patients ≥ 18 years of age with blunt TBI and a Glasgow Coma Scale (GCS) of 3-5, with no other significant injuries. Admission Head CT was evaluated for the presence of extra-axial blood (SDH, EDH, SAH, IVH), intra-axial blood (contusions, diffuse axonal injury), midline shift and mass effect on basilar cisterns. Rotterdam and Marshall scores were calculated for all patients; subsequently patients were divided into two groups according to their score (< 4, ≥ 4)., Results: 106 patients met inclusion criteria; 75.5% were males (n = 80) and 24.5% females (n = 26). The mean age was 52. The odds ratio (OR) of dying from severe TBI for patients in coma with a Rotterdam score of ≥ 4 compared to < 4 was OR = 17 (P < 0.05). The odds of dying from severe TBI for patients in coma with a Marshall score of ≥ 4 versus < 4 was OR = 11 (P < 0.05)., Conclusion: Higher scores in the Marshall classification and the Rotterdam system are associated with increased odds of mortality in adult patients in come from severe TBI after blunt injury. The results of our study support these scoring systems and revealed that a cutoff score of < 4 was associated with improved survival., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: To compare the setting, quality, and utility of nutritional education received by general surgery residents and faculty surgeons and their perceptions and challenges in managing patient nutritional needs., Methods: Cross-sectional analysis utilizing anonymous survey data distributed by the Association of Program Directors in Surgery (APDS) to its affiliated general surgery residency programs., Results: 90.2% (n = 65) of residents and 85.7% (n = 24) of faculty surgeons reported having received nutritional education. The majority (78%) of respondents utilize patient nutrition on a regular basis (monthly or more often), with 54% reporting utilization daily or weekly. Overall, 65% of respondents reported experiencing challenges in managing patient nutritional needs, and 86% agreed that additional nutritional education during training would assist with patient care. Residents and faculty surgeons both significantly reported challenges in determining which specific nutritional formula to use ( X 2 = 22.414, P = .049). Residents were associated with reporting challenges in successfully managing oral, enteral, and parenteral routes of nutrition ( X 2 = 16.241, P = .023)., Conclusions: Despite receiving nutritional education, the majority of surgery residents and faculty surgeons report difficulty in managing their patients nutritional needs. Surgery residents report difficulties with all delivery modes of nutrition, including oral, parenteral, and enteral. Revising medical school nutritional education competencies to focus on more practical aspects of nutrition, reform of formal course format, greater interprofessional collaboration with dieticians starting at the student level, and enforcement of nutritional education requirements by medical school and residency program accrediting bodies can serve to advance physicians' nutritional knowledge and improve patient outcomes.

Protection from immunodeficiency virus challenge in nonhuman primates (NHPs) by a first-generation HIV broadly neutralizing antibody (bnAb) b12 has previously been shown to benefit from interaction between the bnAb and Fcγ receptors (FcγRs) on immune cells. To investigate the mechanism of protection for a more potent second-generation bnAb currently in clinical trials, PGT121, we carried out a series of NHP studies. These studies included treating with PGT121 at a concentration at which only half of the animals were protected to avoid potential masking of FcγR effector function benefits by dominant neutralization and using a new variant that more completely eliminated all rhesus FcγR binding than earlier variants. In contrast to b12, which required FcγR binding for optimal protection, we concluded that PGT121-mediated protection is not augmented by FcγR interaction. Thus, for HIV-passive antibody prophylaxis, these results, together with existing literature, emphasize the importance of neutralization potency for clinical antibodies, with effector function requiring evaluation for individual antibodies., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

SARS-CoV-2 is a novel coronavirus which has caused the COVID-19 pandemic. Other known coronaviruses show a strong pattern of seasonality, with the infection cases in humans being more prominent in winter. Although several plausible origins of such seasonal variability have been proposed, its mechanism is unclear. SARS-CoV-2 is transmitted via airborne droplets ejected from the upper respiratory tract of the infected individuals. It has been reported that SARS-CoV-2 can remain infectious for hours on surfaces. As such, the stability of viral particles both in liquid droplets as well as dried on surfaces is essential for infectivity. Here we have used atomic force microscopy to examine the structural stability of individual SARS-CoV-2 virus like particles at different temperatures. We demonstrate that even a mild temperature increase, commensurate with what is common for summer warming, leads to dramatic disruption of viral structural stability, especially when the heat is applied in the dry state. This is consistent with other existing non-mechanistic studies of viral infectivity, provides a single particle perspective on viral seasonality, and strengthens the case for a resurgence of COVID-19 in winter., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cytoskeletal transport in cells is driven by enzymes whose activity shows sensitive, typically Arrhenius, dependence on temperature. Often, the duration and outcome of cargo transport is determined by the relative success of kinesin vs. dynein motors, which can simultaneously bind to individual cargos and move in opposite direction on microtubules. The question of how kinesin and dynein activity remain coupled over the large temperature ranges experienced by some cells is one of clear biological relevance. We report a break in the Arrhenius behavior of both kinesin-1 and kinesin-3 enzymatic activity at 4.7 °C and 10.5 °C, respectively. Further, we report that this transition temperature significantly changes as a function of chemical background: addition of 200 mM TMAO increases transition temperatures by ∼6 °C in all cases. Our results show that Arrhenius trend breaks are common to all cytoskeletal motors and open a broad question of how such activity transitions are regulated in vivo. STATEMENT OF SIGNIFICANCE: Many cytoskeletal motors studied to date follow Arrhenius kinetics, at least from room temperature up to mammalian body temperature. However the thermal dynamic range is typically finite, and breaks in Arrhenius trends are commonly observed at biologically relevant temperatures. Here we report that the thermal dynamic range of kinesins is also limited and moreover that the location of the Arrhenius break for kinesins can shift significantly based on chemical backgrounds. This implies that the balance of multiple motor cargo transport along the cytoskeleton is far more tunable as a function of temperature than previously appreciated., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Introduction: Traumatic intracranial hemorrhages (ICHs) are high priority injuries. Traumatic brain bleeds can be categorized as traumatic subarachnoid hemorrhage (SAH) versus non-SAH-ICH. Non-SAH-ICH includes subdural, epidural, and intraventricular hematomas and brain contusions. We hypothesize that awake patients with SAH will have lower mortality and needless interventions than awake patients with non-SAH-ICHs., Study Design and Methods: A review of data collected from our Level I trauma center was conducted. Awake was defined as an initial Glasgow coma score (GCS) 13-15. Patients were divided into two cohorts: awake SAH and awake non-SAH-ICH. Chi-square and t -test analyses were used with statistical significance defined as P < 0.05., Results: A total of 12,482 trauma patients were admitted during the study period, of which 225 had a SAH and GCS of 13-15 while 826 had a non-SAH-ICH with a GCS of 13-15. There was no significant difference in demographics between the two groups. Predicted survival between the two groups was similar (97.3 vs. 95.7%, P > 0.05). Mortality rates were, however, significantly lower in SAH patients compared to the non-SAH-ICH (4/225 [1.78%] vs. 22/826 [2.66%], P < 0.05). The need for neurosurgical intervention was significantly different comparing the SAH group versus non-SAH-ICH (2/225 [0.89%] vs. 100/826 [12.1%], P < 0.05)., Conclusion: Despite similar predicted mortality rates, awake patients with a SAH are associated with a significantly lower risk of death and need for neurosurgical intervention when compared to other types of awake patients with a traumatic brain bleed., Competing Interests: There are no conflicts of interest.

Darunavir/cobicistat/emtricitabine/tenofovir alafenamide (D/C/F/TAF) is an oral once-daily single-tablet regimen for the treatment of human immunodeficiency virus-1 infection. Different administration modalities for the D/C/F/TAF fixed-dose combination tablet were explored in this phase 1 randomized, open-label, 3-period, 3-treatment crossover study enrolling 30 healthy adults. The primary objective was to assess the relative bioavailability of each component after a single dose of D/C/F/TAF (800/150/200/10 mg) administered as a split or crushed tablet (tests) versus swallowed whole (reference). Pharmacokinetic parameters (noncompartmental analysis; logarithm-transformed) for each component were compared using linear mixed-effects modeling. For the split versus whole tablet, the bioavailabilities (maximum plasma concentration [C max ] and area under the plasma concentration-time curve [AUC last ]) of each D/C/F/TAF component were comparable. For the crushed versus whole tablet, the bioavailabilities of darunavir, cobicistat, and emtricitabine were comparable, except for a 17% decrease in emtricitabine C max ; the relative bioavailability of tenofovir alafenamide decreased by 29% and 19% for C max and AUC last , respectively. All intakes were safe and generally well tolerated. In summary, there was no clinically relevant impact on the bioavailability of D/C/F/TAF components when administered as a split tablet compared with a tablet swallowed whole. Administration of a crushed tablet resulted in a modest decrease in tenofovir alafenamide bioavailability; the clinical relevance of this change has not been assessed but is expected to be minimal based on the wide therapeutic window for this agent., (© 2018, The American College of Clinical Pharmacology.)

Ribosome biosynthesis, best studied in opisthokonts, is a highly complex process involving numerous protein and RNA factors. Yet, very little is known about the early stages of pre-18S rRNA processing even in these model organisms, let alone the conservation of this mechanism in other eukaryotes. Here we extend our knowledge of this process by identifying and characterizing the essential protein TbUTP10, a homolog of yeast U3 small nucleolar RNA-associated protein 10 - UTP10 (HEATR1 in human), in the excavate parasitic protist Trypanosoma brucei. We show that TbUTP10 localizes to the nucleolus and that its ablation by RNAi knock-down in two different T. brucei life cycle stages results in similar phenotypes: a disruption of pre-18S rRNA processing, exemplified by the accumulation of rRNA precursors, a reduction of mature 18S rRNA, and also a decrease in the level of U3 snoRNA. Moreover, polysome profiles of the RNAi-induced knock-down cells show a complete disappearance of the 40S ribosomal subunit, and a prominent accumulation of the 60S large ribosomal subunit, reflecting impaired ribosome assembly. Thus, TbUTP10 is an important protein in the processing of 18S rRNA., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Sleep disturbances, including delayed sleep phase disorder (DSPD) and disorders of sleep initiation and maintenance (DIMS), have repeatedly been identified in adult obsessive-compulsive disorder (OCD). These disturbances have not been well-characterized objectively in pediatric OCD., Methods: Thirty OCD-affected youth (8-18 yrs, 40% male) and 30 age and gender-matched healthy controls (HCs) completed the Sleep Disturbances Scale for Children (SDSC), and one week of continuous actigraphy with concurrent sleep diary documentation. A subsample completed the Children's Sleep Hygiene Scale (CSHS) and Sleep Attitudes and Beliefs Scale (SABS)., Results: Seventy-two percent of OCD participants reported sleep disturbances versus 15% of HC participants (p < 0.001). Convergent actigraphy results suggested DIMS but not DSPD were common., Discussion: The parents of OCD-affected children seem to be successfully controlling bedtimes, preventing circadian rhythm system disruptions. OCD status does adversely impact, however, the perisleep arousal system. These results have important clinical implications, suggesting sleep problems may be best managed through direct treatment of OCD symptoms. It is recommended that all pediatric OCD patients be screened for sleep disturbances to inform treatment plan development., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This open, uncontrolled study examined the efficacy of a group family-based cognitive behavioral therapy (GF-CBT) protocol in treating pediatric obsessive-compulsive disorder (OCD) and explored predictors of symptom improvement. Eighty-five OCD-affected youth aged 8-18 years (M = 13.9 years, SD = 2.49; 46% male) and their parent(s) participated in a weekly, 12-session GF-CBT program. Data from multiple perspectives were gathered at the beginning and end of treatment, as well as at one-month follow-up. A broad range of assessment measures were utilized to capture clinically-relevant domains and a number of potential predictor variables were explored. Paired t-tests indicated that treatment was associated with significant reductions in clinician- and parent-rated OCD severity (d = 1.47, 1.32), youth and parent-rated functional impairment (d = 0.87, 0.67), coercive/disruptive behaviors (d = 0.75), and family accommodation (d = 1.02), as well as improvements in youth-, mother-, and father-rated family functioning (d = 1.05, 0.50, 0.88). Paired t-tests also indicated that youth remained improved at one-month follow-up. Step-wise regression identified greater homework success as a significant predictor of symptom improvement. This study provides evidence that GF-CBT significantly improves a wide range of domains for youth/families that extends beyond OCD symptom severity and supports homework as a core treatment component., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Postpartum depression (PPD), often comorbid with anxiety, is the leading medical complication among new mothers. Latinas have elevated risk of PPD, which has been associated with early breastfeeding cessation. Lower plasma oxytocin (OT) levels have also been associated with PPD in non-Latinas. This pilot study explores associations between PPD, anxiety, breastfeeding, and OT in Latinas., Materials and Methods: Thirty-four Latinas were enrolled during their third trimester of pregnancy and followed through 8 weeks postpartum. Demographic data were collected at enrollment. Depression was assessed using the Edinburgh Postnatal Depression Scale (EPDS) at each time point (third trimester of pregnancy, 4 and 8 weeks postpartum). The Spielberger State-Trait Anxiety Inventory (STAI) was administered postpartum and EPDS anxiety subscale was used to assess anxiety at each time point. Breastfeeding status was assessed at 4 and 8 weeks postpartum. At 8 weeks, OT was collected before, during, and after a 10-minute breast/bottle feeding session from 28 women who completed the procedures. Descriptive statistics are provided and comparisons by mood and breastfeeding status were conducted. Analyses of variance were used to explore associations between PPD, anxiety, breastfeeding status, and OT., Results: Just under one-third of women were depressed at enrollment. Prenatal depression, PPD, and anxiety were significantly associated with early breastfeeding cessation (i.e., stopped breastfeeding before 2 months) (p < 0.05). There was a significant interaction between early breastfeeding cessation and depression status on OT at 8 weeks postpartum (p < 0.05)., Conclusions: Lower levels of OT were observed in women who had PPD at 8 weeks and who had stopped breastfeeding their infant by 8 weeks postpartum. Future studies should investigate the short- and long-term effects of lower OT levels and early breastfeeding cessation on maternal and child well-being.

Background: Pediatric obsessive-compulsive disorder (OCD) is a common, debilitating illness. When childhood OCD symptom onset is described as acute and severe, diagnostic criteria for pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS) and pediatric acute-onset neuropsychiatric syndrome (PANS) should be considered. However, the frequency and differentiating features of these putative syndromes within pediatric OCD remain poorly understood., Objectives: To determine the prevalence and characteristics of those meeting PANDAS and/or PANS criteria within pediatric OCD, as determined by parent report and clinician interview., Methods: Consecutive youth presenting to a subspecialty pediatric OCD clinic were rigorously assessed through the Anxiety Disorders Interview Schedule for DSM-IV, the Children's Yale-Brown Obsessive-Compulsive Scale, and through self- and parent-report measures, including a medical questionnaire. Strict diagnostic criteria for PANDAS and PANS were applied to determine prevalence rates, and comparative analyses were performed between subgroups., Results: Among 136 youth with a lifetime OCD diagnosis, 5% (n = 7; 95% adjusted Wald interval: 1%-10%) met proposed criteria for PANDAS and/or PANS, of whom two met PANDAS criteria, four met PANS criteria, and one met criteria for both. Those in the PANDAS/PANS subgroup were more likely to have autoimmune illness, less likely to report symmetry factor symptoms, and had greater OCD-related family impairment during their worst OCD episode., Conclusion: A small yet significant percentage of pediatric OCD outpatients met criteria for PANDAS and/or PANS, justifying routine screening and attention to related characteristics during assessment and management. Longitudinal studies of these putative subtypes are warranted.

Background: Major depressive disorder (MDD) is a serious, and common psychiatric disorder worldwide. By the year 2020, MDD will be the second cause of disability in the world. The GranadΣp study is the first, to the best of our knowledge, epidemiological study of mental disorders carried out in Andalusia (South Spain), being one of its main objectives to identify genetic and environmental risk factors for MDD and other major psychiatric disorders. In this study, we focused on the possible association of 91 candidate single nucleotide polymorphisms (SNPs) with MDD., Methods: A total of 711 community-based individuals participated in the GranadΣp study. All individuals were extensively assessed for clinical, psychological, sociodemographic, life style, and other environmental variables. A biological sample was also collected for subsequent genetic analyses in 91 candidate SNPs for MDD. DSM-IV diagnosis of MDD was used as the outcome variable. Logistic regression analysis assuming an additive genetic model was performed to test the association between MDD and the genetic data. The experiment-wide significance threshold adjusted with the SNP spectral decomposition method provided a maximum P-value (8×10(-3)) required to identify an association. Haplotype analyses were also performed., Results: One SNP (rs623580) located in the tryptophan hydroxylase 1 gene (TPH1; chromosome 11), one intergenic variant (rs9526236) upstream of the 5-hydroxytryptamine receptor 2A gene (HTR2A; chromosome 13), and five polymorphisms (rs17689966, rs173365, rs7209436, rs110402, and rs242924) located in the corticotropin-releasing hormone receptor 1 gene (CRHR1; chromosome 17), all showed suggestive trends for association with MDD (P<0.05). Within CRHR1 gene, the TATGA haplotype combination was found to increase significantly the risk for MDD with an odds ratio =1.68 (95% CI: 1.16-2.42, P=0.006)., Conclusion: Although limited, perhaps due to insufficient sample size power, our results seem to support the notion that the hypothalamic-pituitary-adrenal and serotonergic systems are likely to be involved in the genetic susceptibility for MDD. Future studies, including larger samples, should be addressed for further validation and replication of the present findings.

The objective of this study was to determine if admission abdominal/pelvic CT on trauma patients can be used as a novel method to evaluate the inferior vena cava (IVC) anatomy and facilitate potential IVC filter placement. Consecutive trauma admission CT's were reviewed. The potential landing zone for filters was determined by the IVC mid portion between the renal and iliac veins. The IVC landmarks were described anatomically using the thoracic and lumbar vertebral bodies as reference points. The IVC diameter and anatomic anomalies which would affect filter placement were also evaluated from the CT. The records of 610 Trauma patients were reviewed. There were 518 (84.9%) that had an admission CT, forming the basis of the study. The CT for 500 of 518 (96.5%) was of sufficient quality to accurately evaluate the IVC. The third lumbar location of the IVC was a safe landing zone in 476 of 500 (95.2%). Anatomic anomalies were present in 47 of 500 (9.4%). We had the following three conclusions. The admission CT in over 96% of trauma patients can be used to determine the IVC filter landing zone. The third lumbar region of the IVC was a safe landing zone in over 95%. Anatomical anomalies affecting IVC filter placement were revealed in 9.4%.

Objective: To critically analyze published literature to determine the effectiveness of myofascial release therapy as a treatment for orthopaedic conditions., Data Sources: We searched the following electronic databases: MEDLINE, CINAHL, Academic Search Premier, Cochrane Library, and Physiotherapy Evidence Database (PEDro), with key words myofascial release, myofascial release therapy, myofascial release treatment, musculoskeletal, and orthopedic. No date limitations were applied to the searches., Study Selection: Articles were selected based upon the use of the term myofascial release in the abstract or key words. Final selection was made by applying the inclusion and exclusion criteria to the full text. Studies were included if they were English-language, peer-reviewed studies on myofascial release for an orthopaedic condition in adult patients. Ten studies were eligible., Data Extraction: Data collected were number of participants, condition being treated, treatment used, control group, outcome measures and results. Studies were analyzed using the PEDro scale and the Center for Evidence-Based Medicine's Levels of Evidence Scale., Data Synthesis: Study scores on the PEDro scale ranged from 6 of 10 to 8 of 10. Based on the Levels of Evidence Scale, the case studies (n = 6) were of lower quality, with a rank of 4. Three of the 4 remaining studies were rated at 2b, and the final study was rated at 1b., Conclusions: The quality of studies was mixed, ranging from higher-quality experimental to lower-quality case studies. Overall, the studies had positive outcomes with myofascial release, but because of the low quality, few conclusions could be drawn. The studies in this review may serve as a good foundation for future randomized controlled trials.

This article analyzes the evolution of alexithymia, anxiety, and depression associated with fibromyalgia (FM) in three age groups in comparison to how they evolve in the same age groups in healthy women. A total of 240 women participated in the study (120 diagnosed with fibromyalgia and 120 healthy women), divided into three age groups (≤35 years,>35 and <65 years, and ≥65 years old). Our analysis revealed that alexithymia, anxiety, and depression in FM patients showed increased levels compared to healthy controls. Also, young women with FM (under 35) show lower alexithymia, anxiety, and depression levels in comparison to older patients (≥65). Alexithymia in FM patients follows a similar pattern as in healthy women (although with significantly higher scores), increasing with age. Our results show that depression, anxiety, and alexithymia develop over age in a different way in FM patients than in healthy individuals, increasing over age.

Objective: To identify the gene responsible for 14q32-linked dominant spinal muscular atrophy with lower extremity predominance (SMA-LED, OMIM 158600)., Methods: Target exon capture and next generation sequencing was used to analyze the 73 genes in the 14q32 linkage interval in 3 SMA-LED family members. Candidate gene sequencing in additional dominant SMA families used PCR and pooled target capture methods. Patient fibroblasts were biochemically analyzed., Results: Regional exome sequencing of all candidate genes in the 14q32 interval in the original SMA-LED family identified only one missense mutation that segregated with disease state-a mutation in the tail domain of DYNC1H1 (I584L). Sequencing of DYNC1H1 in 32 additional probands with lower extremity predominant SMA found 2 additional heterozygous tail domain mutations (K671E and Y970C), confirming that multiple different mutations in the same domain can cause a similar phenotype. Biochemical analysis of dynein purified from patient-derived fibroblasts demonstrated that the I584L mutation dominantly disrupted dynein complex stability and function., Conclusions: We demonstrate that mutations in the tail domain of the heavy chain of cytoplasmic dynein (DYNC1H1) cause spinal muscular atrophy and provide experimental evidence that a human DYNC1H1 mutation disrupts dynein complex assembly and function. DYNC1H1 mutations were recently found in a family with Charcot-Marie-Tooth disease (type 2O) and in a child with mental retardation. Both of these phenotypes show partial overlap with the spinal muscular atrophy patients described here, indicating that dynein dysfunction is associated with a range of phenotypes in humans involving neuronal development and maintenance.

Schizophrenia is a highly disabling and limiting disorder for patients and the possibility that infections by some microorganisms may be associated to its development may allow prevention and recovery. In the current study we have done a meta-analysis of studies that have assessed the possible association between detection of different infectious agents and schizophrenia. We report results that support the idea that there is a statistically significant association between schizophrenia and infection by Human Herpesvirus 2 (OR=1.34; CI 95%: 1.09-1.70; p=0.05), Borna Disease Virus (OR=2.03; CI 95%: 1.35-3.06; p<0.01), Human Endogenous Retrovirus W (OR=19.31; CI 95%: 6.74-55.29; p<0.001), Chlamydophila pneumoniae (OR=6.34; CI 95%: 2.83-14.19; p<0.001), Chlamydophila psittaci (OR=29.05; CI 95%: 8.91-94.70; p<0.001) and Toxoplasma gondii (OR=2.70; CI 95%: 1.34-4.42; p=0.005). The implications of these findings are discussed and further research options are also explicated., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Introduction: We aimed to investigate the nature of the associations between PD clusters and MDs, functionality and mental health services use., Methods: This is a case register study of all cases with a diagnosis of PD detected clinically in a well-defined area in the province of Barcelona covered by 7 Community Mental Health Teams. DSM-IV diagnoses were established by fully trained psychiatrists. Data was also gathered on socio-demographic variables; functional status (GAF) and data on use of health resources, using a systematic computerized method. We performed a non-parametric univariate statistical analysis., Results: We found a higher percentage of major depressive disorder (MDD) among cluster C patients (17%), followed by cluster A (10%) and cluster B (9, 8%). As for the comorbidity between PD clusters and dysthymic disorder, we found that the prevalence was higher among cluster B patients (23,7%) than cluster C (20,2%) or cluster A (7,1%). When considering both MDs together, we found the highest prevalence among cluster C patients (36,87%), followed by cluster B (33,5%) and cluster A (17,1%). Cluster A patients showed worse functioning and visited hospitals most., Conclusions: A high comorbidity between all MDs analyzed and personality disorders was found, being particularly prominent among cluster C PDs., (Copyright © 2010 Sociedad Española de Psiquiatría and Sociedad Española de Psiquiatría Biológica. Published by Elsevier Espana. All rights reserved.)

Introduction: The risk of schizophrenia conferred by cannabis has recently been proposed to be modulated by the Val158Met polymorphism (rs4680) at the COMT gene. To date, these findings have not been replicated in independent samples., Material and Methods: We tested the potential gene-by-environment interaction between Val158Met genotype at the COMT gene and previous use of cannabis in schizophrenia in 192 healthy controls and 91 inpatients with DSM-IV schizophrenia. The functional COMT Val158Met polymorphism was analyzed using TaqMan technology. Cannabis use was measured by taking into account the frequency of intake during the previous month. Logistic regression models were used to test the interaction between genetic and environment factors., Results: Cannabis use was strongly associated with the case condition (p<0.0001). The Val158Met polymorphism at the COMT gene was not associated with schizophrenia, although Val/Val homozygosity tended to be more frequent in the case group than in the control group (34% vs 27%; OR=1.39; 95% CI, 0.78-2.47). Finally, in women we found a non-significant trend toward the association when we tested for the interaction between cannabis use, the number of Val alleles and susceptibility to schizophrenia (p=0.152)., Conclusions: Our results tend to support recent findings suggesting that the Val158Met polymorphism at the COMT gene modifies the risk of schizophrenia conferred by cannabis use. In our study, this possible effect was only detected in women., (Copyright © 2009 Sociedad Española de Psiquiatría and Sociedad Española de Psiquiatría Biológica. Published by Elsevier Espana. All rights reserved.)

The recent surge in mitochondrial research has been driven by the identification of mitochondria-associated diseases and the role of mitochondria in apoptosis. Both of these aspects have identified mitochondrial analysis as a vital component of medical research. Moreover, mitochondria have been implicated in the process of carcinogenesis because of their vital role in energy production, nuclear-cytoplasmic signal integration and control of metabolic pathways. Interestingly, at some point during neoplastic transformation, there is an increase in reactive oxygen species, which damage the mitochondrial genome. This accelerates the somatic mutation rate of mitochondrial DNA. It has been proposed that these mutations may serve as an early indication of potential cancer development and may represent a means for tracking tumour progression. The purpose of this review is to explore the potential utility that these mutations may afford for the identification and monitoring of neoplasia and malignant transformation where appropriate body fluids or non-invasive tissue access is available for mitochondrial DNA recovery. Specifically, prostate, breast, colorectal, skin and lung cancers are discussed.

Background: North central China has some of the highest rates of esophageal squamous cell carcinoma in the world with cumulative mortality surpassing 20%. Mitochondrial DNA (mtDNA) accumulates more mutations than nuclear DNA and because of its high abundance has been proposed as a early detection device for subjects with cancer at various sites. We wished to examine the prevalence of mtDNA mutation and polymorphism in subjects from this high risk area of China., Methods: We used DNA samples isolated from tumors, adjacent normal esophageal tissue, and blood from 21 esophageal squamous cell carcinoma cases and DNA isolated from blood from 23 healthy persons. We completely sequenced the control region (D-Loop) from each of these samples and used a PCR assay to assess the presence of the 4977 bp common deletion., Results: Direct DNA sequencing revealed that 7/21 (33%, 95% CI = 17-55%) tumor samples had mutations in the control region, with clustering evident in the hyper-variable segment 1 (HSV1) and the homopolymeric stretch surrounding position 309. The number of mutations per subject ranged from 1 to 16 and there were a number of instances of heteroplasmy. We detected the 4977 bp 'common deletion' in 92% of the tumor and adjacent normal esophageal tissue samples examined, whereas no evidence of the common deletion was found in corresponding peripheral blood samples., Conclusions: Control region mutations were insufficiently common to warrant attempts to develop mtDNA mutation screening as a clinical test for ESCC. The common deletion was highly prevalent in the esophageal tissue of cancer cases but absent from peripheral blood. The potential utility of the common deletion in an early detection system will be pursued in further studies.

Although intravenous immunoglobulins (IVIG) and other plasma therapeutics have had a relatively good safety record, improved methods for viral clearance are constantly being evaluated and incorporated into new manufacturing processes. Gamma irradiation has been used routinely to assure sterility of healthcare products and medical devices, but it has not been applied successfully as a viral inactivation method for biologics. We examine whether virucidal doses of gamma irradiation (50 kGy) can be delivered to a manufacturing intermediate form of IVIG, a fractionated plasma paste, with negligible effect on structural and functional integrity of purified IgG product. Immunoglobulins from paste were examined for radiation-induced damage by SDS-PAGE and ELISAs utilizing viral antigens specific for rubella, CMV and mumps. Fc domain integrity was assessed by immunoblotting, quantitatively comparing the binding of irradiated and non-irradiated materials to cell surface Fcgamma receptors, and by employing quantitative RT-PCR to study the kinetics of accumulation of mRNA for the immune modulatory cytokines IL-1alpha, IL-1beta, IL-4, IL-8, IFNgamma, and TNFalpha. The results demonstrate that Fab and Fc domains of IVIG remain essentially intact and functional after gamma irradiation to virucidal doses, suggesting that this method could be used to enhance the safety of IVIG products.

A novel Quantitative Nucleic Acid Test (Q-NAT) technology has been developed to demonstrate, quantify and verify pathogen inactivation by methods that break pathogen nucleic acids, specifically, gamma irradiation. The Q-NAT technology provides significant advantages in cost, efficiency and broad applicability compared with traditional methods for pathogen inactivation detection and quantification such as cell culture.

A calmodulin-like protein (CAMLP) from Mycobacterium smegmatis was purified to homogeneity and partially sequenced; these data were used to produce a full-length clone, whose DNA sequence contained a 55-amino-acid open reading frame. M. smegmatis CAMLP, expressed in Escherichia coli, exhibited properties characteristic of eukaryotic calmodulin: calcium-dependent stimulation of eukaryotic phosphodiesterase, which was inhibited by the calmodulin antagonist trifluoperazine, and reaction with anti-bovine brain calmodulin antibodies. Consistent with the presence of nine acidic amino acids (16%) in M. smegmatis CAMLP, there is one putative calcium-binding domain in this CAMLP, compared to four such domains for eukaryotic calmodulin, reflecting the smaller molecular size (approximately 6 kDa) of M. smegmatis CAMLP. Ultracentrifugation and mass spectral studies excluded the possibility that calcium promotes oligomerization of purified M. smegmatis CAMLP.

The CT diagnosis of bowel injury is difficult and warrants an organized approach. Careful scrutiny of CT images for extraluminal gas or fluid and bowel wall thickening is required. Review of images of the entire abdomen and pelvis using lung window settings is recommended, followed by the analysis using soft tissue window settings. Specific search for extraluminal fluid collections with a triangular or matted appearance will help detect bowel injury.

Ultrasonography (US) is becoming increasingly utilized in the United States for the evaluation of blunt abdominal trauma (BAT). The objective of this study was to assess the cost impact of utilizing US in the evaluation of patients with BAT in a major trauma center. All patients sustaining BAT during a 6-month period before US was used at our institution (Jan-Jun 1993) were compared to BAT patients from a recent period in which US has been utilized (Jan-Jun 1995). The numbers of US, computed tomography (CT), and diagnostic peritoneal lavage (DPL) were tabulated for each group. Financial cost for each of these procedures as determined by our finance department were as follows: US $96, CT $494, DPL $137. These numbers are representative of actual hospital expenditures exclusive of physician fees as calculated in 1994 U.S. dollars. Cost analysis was performed with t test and chi squared test, and significance was defined as P < 0.05. There were 890 BAT admissions in the 1993 study period and 1033 admissions in the 1995 study period. During the 1993 period, 642 procedures were performed on the 890 patients to evaluate the abdomen: 0 US, 466 CT, and 176 DPL (see table) [table: see text]. This compares to 801 procedures on the 1,033 patients in 1995: 552 US, 228 CT, and 21 DPL. Total cost was $254,316 for the 1993 group and $168,501 for the 1995 group. Extrapolated to a 1-year period, a significant (P < 0.05) cost savings of $171,630 would be realized. Cost per patient evaluated was significantly reduced from $285.75 in 1993 to $163.12 in 1995 (P < 0.05). This represents a 43 per cent reduction in per patient expenditure for evaluating the abdomen. By effectively utilizing ultrasonography in the evaluation of patients with blunt abdominal trauma, a significant cost savings can be realized. This effect results chiefly from an eight-fold reduction in the use of DPL, and a two-fold reduction in the use of CT.

Purpose: Sonography provides a fast, portable, and noninvasive method for patient assessment. However, the benefit of providing real-time ultrasound (US) imaging and fluid quantification shortly after patient arrival has not been explored. The objective of this study was to prospectively validate a US hemoperitoneum scoring system developed at our institution and determine whether sonography can predict a therapeutic operation., Methods: For 12 months, prospective data on all patients undergoing a trauma sonogram were recorded. All sonograms positive for free fluid were given a hemoperitoneum score. The US score was compared with initial systolic blood pressure and base deficit to assess the ability of sonography to predict a therapeutic laparotomy., Results: Forty of 46 patients (87%) with a US score > or = 3 required a therapeutic laparotomy. Forty-six of 54 patients with a US score < 3 (85%) did not need operative intervention. The sensitivity of sonography was 83% compared with 28% and 49% for systolic blood pressure and base deficit, respectively, in determining the need for therapeutic operation., Conclusion: We conclude that the majority of patients with a score > or = 3 will need surgery. The US hemoperitoneum scoring system was a better predictor of a therapeutic laparotomy than initial blood pressure and/or base deficit.

Background: Determination of intra-abdominal injury following blunt abdominal trauma (BAT) continues to be a diagnostic challenge. Ultrasound (US) has been described as a potentially useful diagnostic tool in this setting and is being used with increasing frequency in trauma centers. We determined the diagnostic capability of US in the evaluation of BAT., Methods: A retrospective analysis of our trauma US database was performed over a 30-month period. Computed tomographic scan, diagnostic peritoneal lavage, or exploratory laparotomy confirmed the presence of intra-abdominal injury., Results: During the study period, 8,197 patients were evaluated at the Ryder Trauma Center. Of this group, 2,576 (31%) had US in the evaluation of BAT. Three hundred eleven (12%) US exams were considered positive. Forty-three patients (1.7%) had a false-negative US; of this group, 10 (33%) required exploratory laparotomy. US had a sensitivity of 86%, a specificity of 98%, and an accuracy of 97% for detection of intra-abdominal injuries. Positive predictive value was 87% and negative predictive value was 98%., Conclusion: Emergency US is highly reliable and may replace computed tomographic scan and diagnostic peritoneal lavage as the initial diagnostic modality in the evaluation of most patients with BAT.

Widely accepted in Europe and Asia as a screening tool for blunt abdominal trauma, sonography is gradually gaining popularity among trauma and emergency physicians in the United States. Sonography has been shown to be comparable with DPL and CT for the detection of hemoperitoneum and superior to both modalities because of its rapidity, noninvasiveness, portability, and low cost. With its ability to demonstrate the amount of intraperitoneal hemorrhage within minutes of a patient's arrival, sonography may be considered the screening modality of choice for blunt abdominal trauma.

Small angle neutron scattering (SANS) measurements were performed on solutions of cAMP receptor protein (CRP) and on solutions of the T127L,S128A double mutant of CRP (CRP*) in D2O K3PO4 buffer containing 0.5 M KCl, in the absence and presence of 3',5' cyclic adenosine monophosphate (cAMP). Energy-minimized structures of the CRP were calculated by minimization of the x-ray crystallographic structure of CRP in either the exclusively "closed" form where the alpha-helices of the carboxyl-terminal domain are folded close to the amino-terminal domain and in the exclusively "open" form where the alpha-helices of the carboxyl-terminal domain are folded away from the amino-terminal domain. Neutron scattering models show that the CRP SANS data follow closely the data curve predicted for unligated CRP in the open form, whereas the cAMP-ligated data are more in agreement with the data predicted for the minimized cAMP-ligated CRP structure in the closed form. Thus, it appears that CRP undergoes a conformational change from the open form to the closed form in solution upon ligation with cAMP. The SANS data from the CRP* and cAMP-ligated CRP* are coincidental, which implies that there is very little structural difference between the two species of CRP*. This is in agreement with in vivo results, which show that whereas CRP activates transcription in the cell only in the presence of cAMP, CRP* activates transcription in the absence of cAMP, implying that CRP* is already in the correct conformation for the activation of transcription.

Mesenteric panniculitis is an extremely rare inflammatory condition of the adipose tissue of unknown etiology in which the mesentery is replaced with fibrosis. Knowledge of this rare syndrome should prevent any unwarranted aggressive therapy and help to use the clinical, radiological, and surgical sources to obtain the diagnosis. This paper is a review of symptomatology, pathology, treatment, and outcome of this disorder. A case report is described that presented with obstruction of the sigmoid colon.

Objective: To determine whether surgeons and residents with minimal training can evaluate accurately emergency ultrasound (US) examinations compared with radiologists for blunt abdominal trauma., Methods: Over 7 months, we conducted a prospective study comparing the evaluation of emergency US for blunt abdominal trauma by surgeons and attending radiologists. US readings from the surgical team and the radiologists were correlated with outcome., Results: One hundred-twelve patients were included in the study. Ninety-two patients had an US read as negative by the surgical and radiology services with no subsequent injuries identified. Eighteen patients had an US deemed positive by the surgical service and radiologists. Injuries were confirmed in this group by operation or computed tomography. One patient had an US deemed positive by the surgical team and subsequently negative by the radiologist. A diagnostic peritoneal lavage was performed which was negative. Another patient had an US interpreted as negative by the surgical evaluator and positive by the radiologist. Exploratory laparotomy was negative for intraabdominal hemorrhage or organ injury. Overall results reveal an accuracy on US reading of 99% for the surgical team and 99% for the attending radiologists., Conclusion: Surgeons and surgical residents at different levels of training can accurately interpret emergency ultrasound examinations for blunt trauma from the real-time images, at a level comparable to attending radiologists.

Objective: The purpose of this study was to determine the accuracy of sonography when used routinely as the primary screening technique for blunt abdominal trauma., Subjects and Methods: From December 1994 through December 1995, we used sonography as the primary screening technique for the initial assessment of blunt abdominal trauma. Sonography was performed by dedicated technologists and overseen and interpreted by radiology residents or attending physicians. A positive study was defined as evidence of free fluid or parenchymal injury. Sonographic findings were correlated with CT, surgery, or clinical follow-up., Results: Of the 899 sonograms obtained for blunt abdominal trauma, the findings of 783 were negative, 101 were positive, and 15 were indeterminate. Of the 783 sonograms with negative findings, 768 (98%) were confirmed by CT, surgery, or clinical follow-up. Similarly, of the 101 sonograms with positive findings, 95 (94%) were confirmed. Interpretations resulted in 15 false-negative and five false-positive examinations. For all sonograms, we calculated a sensitivity of 86%, a specificity of 99%, and an accuracy of 98%., Conclusion: Sonography can be used effectively as the primary screening technique for blunt abdominal trauma.

Archaeoglobus fulgidus is the first sulphur-metabolizing organism to have its genome sequence determined. Its genome of 2,178,400 base pairs contains 2,436 open reading frames (ORFs). The information processing systems and the biosynthetic pathways for essential components (nucleotides, amino acids and cofactors) have extensive correlation with their counterparts in the archaeon Methanococcus jannaschii. The genomes of these two Archaea indicate dramatic differences in the way these organisms sense their environment, perform regulatory and transport functions, and gain energy. In contrast to M. jannaschii, A. fulgidus has fewer restriction-modification systems, and none of its genes appears to contain inteins. A quarter (651 ORFs) of the A. fulgidus genome encodes functionally uncharacterized yet conserved proteins, two-thirds of which are shared with M. jannaschii (428 ORFs). Another quarter of the genome encodes new proteins indicating substantial archaeal gene diversity.

Helicobacter pylori, strain 26695, has a circular genome of 1,667,867 base pairs and 1,590 predicted coding sequences. Sequence analysis indicates that H. pylori has well-developed systems for motility, for scavenging iron, and for DNA restriction and modification. Many putative adhesins, lipoproteins and other outer membrane proteins were identified, underscoring the potential complexity of host-pathogen interaction. Based on the large number of sequence-related genes encoding outer membrane proteins and the presence of homopolymeric tracts and dinucleotide repeats in coding sequences, H. pylori, like several other mucosal pathogens, probably uses recombination and slipped-strand mispairing within repeats as mechanisms for antigenic variation and adaptive evolution. Consistent with its restricted niche, H. pylori has a few regulatory networks, and a limited metabolic repertoire and biosynthetic capacity. Its survival in acid conditions depends, in part, on its ability to establish a positive inside-membrane potential in low pH.

We have developed a new method for isolating translation initiation sites based on the expression of Haemophilus influenzae Rd gene fusions with the Escherichia coli galactokinase (galK) gene. We cloned random DNA fragments of H. influenzae Rd DNA into a plasmid vector containing the galK coding sequence from which the translation initiation site (the ribosome binding site and translation initiation codon) had been removed. A subset of the cloned DNA fragments contained translation initiation sites that, when fused to the galK gene, produced active galactokinase and complemented the host galK mutation. Molecules expressing galactokinase activity were isolated and characterized by DNA sequence analysis, and the sequences were aligned with the recently completed whole genomic sequence of H. influenzae Rd. Translation initiation sites for known, hypothetical, and new genes were identified. Translation initiation sites internal to the coding sequences of a number of genes were identified, suggesting that internal translation initiation sites are common, especially in large genes. This shotgun method provides functional information on translation initiation sites and helps to define gene coding sequences.