A Survey of Two-Component Systems in Coxiella burnetii Reveals Redundant Regulatory Schemes and a Requirement for an Atypical PhoBR System in Mammalian Cell Infection.

Coxiella burnetii is an obligate intracellular bacterium and the etiological agent of Q fever in humans. C. burnetii transitions between a replicative, metabolically active large-cell variant (LCV) and a spore-like, quiescent small-cell variant (SCV) as a likely mechanism to ensure survival between host cells and mammalian hosts. C. burnetii encodes three canonical two-component systems, four orphan hybrid histidine kinases, five orphan response regulators, and a histidine phosphotransfer protein, which have been speculated to play roles in the signaling required for C. burnetii morphogenesis and virulence. However, very few of these systems have been characterized. By employing a CRISPR interference system for genetic manipulation of C. burnetii, we created single- and multigene transcriptional knockdown strains targeting most of these signaling genes. Through this, we revealed a role for the C. burnetii PhoBR canonical two-component system in virulence, regulation of [Pi] maintenance, and Pi transport. We also outline a novel mechanism by which PhoBR function may be regulated by an atypical PhoU-like protein. We also determined that the GacA.2/GacA.3/GacA.4/GacS orphan response regulators coordinately and disparately regulate expression of SCV-associated genes in C. burnetii LCVs. These foundational results will inform future studies on the role of C. burnetii two-component systems in virulence and morphogenesis. IMPORTANCE C. burnetii is an obligate intracellular bacterium with a spore-like stability allowing it to survive long periods of time in the environment. This stability is likely due to its biphasic developmental cycle, whereby it can transition from an environmentally stable small-cell variant (SCV) to a metabolically active large-cell variant (LCV). Here, we define the role of two-component phosphorelay systems (TCS) in C. burnetii's ability to survive within the harsh environment contained in the phagolysosome of host cells. We show that the canonical PhoBR TCS has an important role in C. burnetii virulence and phosphate sensing. Further examination of the regulons controlled by orphan regulators indicated a role in modulating gene expression of SCV-associated genes, including genes essential for cell wall remodeling.

Surgical repair of massive hemorrhage secondary to button battery ingestion causing aortoesophageal fistula.

BACKGROUND: In pediatrics, foreign body ingestion poses unique challenges. Each case is unique given variability in timing, type, and size of object, compounded by underlying comorbidities and age. In the mid-1990s, mortality and morbidity associated with button battery (BB) ingestion (BBI) emerged corresponding to modification in battery fabrication towards higher voltage, large-diameter lithium cells. AIMS: To describe the case and management of a BBI in a pediatric patient necessitating the use of cardiopulmonary bypass and deep hypothermic circulatory arrest (DHCA). MATERIALS AND METHODS: A 17-month-old female presented with the sudden loss of consciousness at home. Chest X-ray revealed an esophageal foreign body suspicious for BBI. A massive upper gastrointestinal bleeding was temporized with packing. The patient was urgently taken to the operating room for sternotomy, establishment of cardiopulmonary bypass (CPB) with deep hypothermic circulatory arrest (DHCA), removal of BB, repair of the left common carotid artery, esophageal, and tracheal injuries. DISCUSSION: Successful management of BBI requires coordinated care and a multidisciplinary approach. A high degree of clinical suspicion for BBI is imperative to facilitate early aggressive interventions. Lateral and anteroposterior chest films should be obtained in any suspected BBI. CONCLUSION: This case demonstrates the utility of CPB and DHCA where control of bleeding secondary to BBI is not otherwise possible.

Assessing how global health partnerships function: an equity-informed critical interpretive synthesis.

BACKGROUND: Global health partnerships (GHPs) are situated in complex political and economic relationships and involve partners with different needs and interests (e.g., government agencies, non-governmental organizations, corporations, universities, professional associations, philanthropic organizations and communities). As part of a mixed methods study designed to develop an equity-sensitive tool to support more equity-centred North-South GHPs, this critical interpretive synthesis examined reported assessments of GHPs. RESULTS: We examined 30 peer-reviewed articles for power dynamics, equity and inequities, and contradictions or challenges encountered in North-South partnerships. Among articles reviewed, authors most often situated GHPs around a topical focus on research, capacity-building, clinical, or health services issues, with the 'work' of the partnership aiming to foster skills or respond to community needs. The specific features of GHPs that were assessed varied widely, with consistently-reported elements including the early phases of partnering; governance issues; the day-to-day work of partnerships; the performance, impacts and benefits of GHPs; and issues of inclusion. Articles shared a general interest in partnering processes and often touched briefly on issues of equity; but they rarely accounted for the complexity of sociopolitical and historical contexts shaping issues of equity in GHPs. Further, assessments of GHPs were often reported without inclusion of voices from all partners or named beneficiaries. GHPs were frequently portrayed as inherently beneficial for Southern partners, without attention to power dynamics and inequities (North-South, South-South). Though historical and political dynamics of the Global North and South were inconsistently examined as influential forces in GHPs, such dynamics were frequently portrayed as complex and characterized by asymmetries in power and resources. Generally, assessments of GHPs paid little attention to the macroeconomic forces in the power and resource dynamics of GHPs highlights the importance of considering the broader political. Our findings suggest that GHPs can serve to entrench both inequitable relationships and unfair distributions of power, resources, and wealth within and between countries (and partners) if inequitable power relationships are left unmitigated. CONCLUSIONS: We argue that specific practices could enhance GHPs' contributions to equity, both in their processes and outcomes. Enhancing partnering practices to focus on inclusion, responsiveness to North-South and South-South inequities, and recognition of GHPs as situated in a broader (and inequitable) political economy. A relational and equity-centred approach to assessing GHPs would place social justice, humility and mutual benefits as central practices-that is, regular, routine things that partners involved in partnering do intentionally to make GHPs function well. Practicing equity in GHPs requires continuous efforts to explicitly acknowledge and examine the equity implications of all aspects of partnering.

Actin polymerization in the endosomal pathway, but not on the Coxiella-containing vacuole, is essential for pathogen growth.

Coxiella burnetii is an intracellular bacterium that replicates within an expansive phagolysosome-like vacuole. Fusion between the Coxiella-containing vacuole (CCV) and late endosomes/multivesicular bodies requires Rab7, the HOPS tethering complex, and SNARE proteins, with actin also speculated to play a role. Here, we investigated the importance of actin in CCV fusion. Filamentous actin patches formed around the CCV membrane that were preferred sites of vesicular fusion. Accordingly, the mediators of endolysosomal fusion Rab7, VAMP7, and syntaxin 8 were concentrated in CCV actin patches. Generation of actin patches required C. burnetii type 4B secretion and host retromer function. Patches decorated with VPS29 and VPS35, components of the retromer, FAM21 and WASH, members of the WASH complex that engage the retromer, and Arp3, a component of the Arp2/3 complex that generates branched actin filaments. Depletion by siRNA of VPS35 or VPS29 reduced CCV actin patches and caused Rab7 to uniformly distribute in the CCV membrane. C. burnetii grew normally in VPS35 or VPS29 depleted cells, as well as WASH-knockout mouse embryo fibroblasts, where CCVs are devoid of actin patches. Endosome recycling to the plasma membrane and trans-Golgi of glucose transporter 1 (GLUT1) and cationic-independent mannose-6-phosphate receptor (CI-M6PR), respectively, was normal in infected cells. However, siRNA knockdown of retromer resulted in aberrant trafficking of GLUT1, but not CI-M6PR, suggesting canonical retrograde trafficking is unaffected by retromer disruption. Treatment with the specific Arp2/3 inhibitor CK-666 strongly inhibited CCV formation, an effect associated with altered endosomal trafficking of transferrin receptor. Collectively, our results show that CCV actin patches generated by retromer, WASH, and Arp2/3 are dispensable for CCV biogenesis and stability. However, Arp2/3-mediated production of actin filaments required for cargo transport within the endosomal system is required for CCV generation. These findings delineate which of the many actin related events that shape the endosomal compartment are important for CCV formation.

Moral Distress of Clinicians in Canadian Pediatric and Neonatal ICUs.

OBJECTIVE: To quantify moral distress in neonatal ICU and PICU clinicians and to identify associated factors. DESIGN: A national cross-sectional survey of clinicians working in an neonatal ICU or PICU. Moral distress was assessed with the Moral Distress Scale-Revised and by self-rating. Depersonalization was assessed on the subscale of the Maslach Burnout Inventory. Respondents reported their attendance at each of six hospital supports that may serve to mitigate moral distress in frontline staff. Analyses compared outcomes across respondent characteristics and hierarchical linear regression evaluated individual, ICU, hospital, and regional effects. SETTING: Eligible ICUs were PICUs and level-3 neonatal ICUs in Canada. SUBJECTS: Eligible participants had worked in the participating ICU for more than 3 months. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We identified 54 eligible ICUs from 31 hospitals. Forty-nine Canadian neonatal ICUs and PICUs (91%) contributed 2,852 complete responses for a 45.2% response rate. Most respondents were nurses (64.9%) or from a neonatal ICU (66.5%). The median and interquartile range Moral Distress Scale-Revised were 79 (52-113); 997 respondents (34.2%) had Moral Distress Scale-Revised scores greater than or equal to 100, and 234 respondents (8.3%) strongly agreed that work caused them significant moral distress. Nurses had a median (interquartile range) Moral Distress Scale-Revised score of 85 (57-121), 19 points higher than physicians and 8 points higher than respiratory therapists (p < 0.0001). Moral Distress Scale-Revised scores increased from 53 (35-79) for those working in ICU less than 1 year to 83 (54-120) in those working in ICU more than 30 years (p < 0.0001); 22.5% reported high degrees of depersonalization, which was associated with moral distress (p < 0.0001). Variability in Moral Distress Scale-Revised scores was explained by individual-level (92%), hospital-level (5%), and ICU-level effects (1%). Frequency of participation in potentially mitigating hospital supports had small effects (< 10 points) on mean Moral Distress Scale-Revised scores. CONCLUSIONS: Moral distress is common in clinicians working in ICUs for children. Addressing moral distress will require interventions tailored to individuals in higher-risk groups.

Dependency of Coxiella burnetii Type 4B Secretion on the Chaperone IcmS.

Macrophage parasitism by Coxiella burnetii, the cause of human Q fever, requires the translocation of proteins with effector functions directly into the host cell cytosol via a Dot/Icm type 4B secretion system (T4BSS). Secretion by the analogous Legionella pneumophila T4BSS involves signal sequences within the C-terminal and internal domains of effector proteins. The cytoplasmic chaperone pair IcmSW promotes secretion and binds internal sites distinct from signal sequences. In the present study, we investigated requirements of C. burnetii IcmS for host cell parasitism and effector translocation. A C. burnetiiicmS deletion mutant (ΔicmS) exhibited impaired replication in Vero epithelial cells, deficient formation of the Coxiella-containing vacuole, and aberrant T4BSS secretion. Three secretion phenotypes were identified from a screen of 50 Dot/Icm substrates: IcmS dependent (secreted by only wild-type bacteria), IcmS independent (secreted by both wild-type and ΔicmS bacteria), or IcmS inhibited (secreted by only ΔicmS bacteria). Secretion was assessed for N-terminal or C-terminal truncated forms of CBU0794 and CBU1525. IcmS-inhibited secretion of CBU1525 required a C-terminal secretion signal whereas IcmS-dependent secretion of CBU0794 was directed by C-terminal and internal signals. Interchange of the C-terminal 50 amino acids of CBU0794 and CBU1525 revealed that sites within the C terminus regulate IcmS dependency. Glutathione S-transferase-tagged IcmSW bound internal sequences of IcmS-dependent and -inhibited substrates. Thus, the growth defect of the C. burnetii ΔicmS strain is associated with a loss of T4BSS chaperone activity that both positively and negatively regulates effector translocation.IMPORTANCE The intracellular pathogen Coxiella burnetii employs a type 4B secretion system (T4BSS) that promotes growth by translocating effectors of eukaryotic pathways into host cells. T4BSS regulation modeled in Legionella pneumophila indicates IcmS facilitates effector translocation. Here, we characterized type 4B secretion by a Coxiella ΔicmS mutant that exhibits intracellular growth defects. T4BSS substrates demonstrated increased, equivalent, or decreased secretion by the ΔicmS mutant relative to wild-type Coxiella Similar to the Legionella T4BSS, IcmS dependency in Coxiella was determined by C-terminal and/or internal secretion signals. However, IcmS inhibited secretion of some effectors by Coxiella that were previously shown to be translocated by Legionella Thus, Coxiella has a unique IcmS regulatory mechanism that both positively and negatively regulates T4BSS export.

Targeted antibiotic discovery through biosynthesis-associated resistance determinants: target directed genome mining.

Intense competition between microbes in the environment has directed the evolution of antibiotic production in bacteria. Humans have harnessed these natural molecules for medicinal purposes, magnifying them from environmental concentrations to industrial scale. This increased exposure to antibiotics has amplified antibiotic resistance across bacteria, spurring a global antimicrobial crisis and a search for antibiotics with new modes of action. Genetic insights into these antibiotic-producing microbes reveal that they have evolved several resistance strategies to avoid self-toxicity, including product modification, substrate transport and binding, and target duplication or modification. Of these mechanisms, target duplication or modification will be highlighted in this review, as it uniquely links an antibiotic to its mode of action. We will further discuss and propose a strategy to mine microbial genomes for these genes and their associated biosynthetic gene clusters to discover novel antibiotics using target directed genome mining.

Bayesian Methods for Exposure Misclassification Adjustment in a Mediation Analysis: Hemoglobin and Malnutrition in the Association Between Ascaris and IQ.

BACKGROUND: Soil-transmitted helminth infections have been found to be associated with child development. The objective was to investigate hemoglobin levels and malnutrition as mediators of the association between Ascaris infection and intelligence quotient (IQ) scores in children. METHODS: We conducted a longitudinal cohort study in Iquitos, Peru, between September 2011 and July 2016. A total of 1760 children were recruited at 1 year of age and followed up annually to 5 years. We measured Ascaris infection and malnutrition at each study visit, and hemoglobin levels were measured as of age 3. The exposure was defined as the number of detected Ascaris infections between age 1 and 5. We measured IQ scores at age 5 and used Bayesian models to correct exposure misclassification. RESULTS: We included a sample of 781 children in the analysis. In results adjusted for Ascaris misclassification, mean hemoglobin levels mediated the association between Ascaris infection and IQ scores. The natural direct effects (not mediated by hemoglobin) (95% CrI) and natural indirect effects (mediated by hemoglobin) (95% CrI) were compared with no or one infection: -0.9 (-4.6, 2.8) and -4.3 (-6.9, -1.6) for the effect of two infections; -1.4 (-3.8, 1.0) and -1.2 (-2.0, -0.4) for three infections; and -0.4 (-3.2, 2.4) and -2.7 (-4.3, -1.0) for four or five infections. CONCLUSION: Our results are consistent with the hypothesis that hemoglobin levels mediate the association between Ascaris infection and IQ scores. Additional research investigating the effect of including iron supplements in STH control programs is warranted.

Iron status and inherited haemoglobin disorders modify the effects of micronutrient powders on linear growth and morbidity among young Lao children in a double-blind randomised trial.

Some studies found that providing micronutrient powder (MNP) causes adverse health outcomes, but modifying factors are unknown. We aimed to investigate whether Fe status and inherited Hb disorders (IHbD) modify the impact of MNP on growth and diarrhoea among young Lao children. In a double-blind controlled trial, 1704 children of age 6-23 months were randomised to daily MNP (with 6 mg Fe plus fourteen micronutrients) or placebo for about 36 weeks. IHbD, and baseline and final Hb, Fe status and anthropometrics were assessed. Caregivers provided weekly morbidity reports. At enrolment, 55·6 % were anaemic; only 39·3 % had no sign of clinically significant IHbD. MNP had no overall impact on growth and longitudinal diarrhoea prevalence. Baseline Hb modified the effect of MNP on length-for-age (LAZ) (P for interaction = 0·082). Among children who were initially non-anaemic, the final mean LAZ in the MNP group was slightly lower (-1·93 (95 % CI -1·88, -1·97)) v. placebo (-1·88 (95 % CI -1·83, -1·92)), and the opposite occurred among initially anaemic children (final mean LAZ -1·90 (95 % CI -1·86, -1·94) in MNP v. -1·92 (95 % CI -1·88, -1·96) in placebo). IHbD modified the effect on diarrhoea prevalence (P = 0·095). Among children with IHbD, the MNP group had higher diarrhoea prevalence (1·37 (95 % CI 1·17, 1·59) v. 1·21 (95 % CI 1·04, 1·41)), while it was lower among children without IHbD who received MNP (1·15 (95 % CI 0·95, 1·39) v. 1·37 (95 % CI 1·13, 1·64)). In conclusion, there was a small adverse effect of MNP on growth among non-anaemic children and on diarrhoea prevalence among children with IHbD.

Modulation of auditory-vocal feedback control due to planned changes in voice fo.

Previous studies have demonstrated that voice fundamental frequency (fo), or pitch, relies on auditory feedback to monitor and correct for errors in production. When voice-pitch auditory feedback is unexpectedly perturbed, individuals typically produce a compensatory change in fo that opposes the direction of the pitch-perturbation. Studies comparing steady vowel vocalizations and speech tasks have demonstrated task-dependent modulation of the compensatory response, but the effects of planning to volitionally change fo during active vocalization have yet to be explored. Ten musicians and ten non-musicians were asked to perform two vocal tasks. Both tasks started off at a conversational fo. In one task, pitch-shifted feedback was presented when the participants were planning to hold fo constant (steady fo), and in the other, feedback was shifted while participants were in the planning stage prior to raising fo (raised fo) from a steady state. Acoustical analyses of fo were performed to measure the peak magnitude and latency of both the compensatory response as well as the voluntary fo change. Results showed that planning to change pitch modulates the mechanisms controlling feedback-based error correction of fo, and musicality affects how individuals incorporate modulations in auditory feedback with the feedforward plans to increase voice fo.

Comparison of volitional opposing and following responses across speakers with different vocal histories.

Research has shown that people who are instructed to volitionally respond to pitch-shifted feedback either produce responses that follow the shift direction with a short latency of 100-200 ms or oppose the shift direction with longer latencies of 300-400 ms. This difference in response latencies prompted a comparison of three groups of vocalists with differing abilities, non-trained English-speaking subjects, non-trained Mandarin-speaking subjects, and trained English-speaking singers. All subjects produced short latency following responses and long latency opposing responses, and in most cases the opposing responses were preceded by a shorter latency following response. Across groups, the magnitudes of the opposing and following responses were largest for the Mandarin speakers. Singers produced the smallest opposing response magnitudes, suggesting differences in the pitch goals of the two groups. Opposing response latencies were longest for the English and Mandarin speaking subjects and shortest for the trained singers, demonstrating that musical training increases the speed of producing the opposing responses. The presence of similar latencies of small following responses preceding larger opposing responses in all groups suggests that the tendency to mimic changes in sounds to which a person is attending are not influenced by vocal training or experience.

Neural Correlates of Vocal Production and Motor Control in Human Heschl's Gyrus.

The present study investigated how pitch frequency, a perceptually relevant aspect of periodicity in natural human vocalizations, is encoded in Heschl's gyrus (HG), and how this information may be used to influence vocal pitch motor control. We recorded local field potentials from multicontact depth electrodes implanted in HG of 14 neurosurgical epilepsy patients as they vocalized vowel sounds and received brief (200 ms) pitch perturbations at 100 Cents in their auditory feedback. Event-related band power responses to vocalizations showed sustained frequency following responses that tracked voice fundamental frequency (F0) and were significantly enhanced in posteromedial HG during speaking compared with when subjects listened to the playback of their own voice. In addition to frequency following responses, a transient response component within the high gamma frequency band (75-150 Hz) was identified. When this response followed the onset of vocalization, the magnitude of the response was the same for the speaking and playback conditions. In contrast, when this response followed a pitch shift, its magnitude was significantly enhanced during speaking compared with playback. We also observed that, in anterolateral HG, the power of high gamma responses to pitch shifts correlated with the magnitude of compensatory vocal responses. These findings demonstrate a functional parcellation of HG with neural activity that encodes pitch in natural human voice, distinguishes between self-generated and passively heard vocalizations, detects discrepancies between the intended and heard vocalization, and contains information about the resulting behavioral vocal compensations in response to auditory feedback pitch perturbations. SIGNIFICANCE STATEMENT: The present study is a significant contribution to our understanding of sensor-motor mechanisms of vocal production and motor control. The findings demonstrate distinct functional parcellation of core and noncore areas within human auditory cortex on Heschl's gyrus that process natural human vocalizations and pitch perturbations in the auditory feedback. In addition, our data provide evidence for distinct roles of high gamma neural oscillations and frequency following responses for processing periodicity in human vocalizations during vocal production and motor control.

PCR-Independent Method of Transformation-Associated Recombination Reveals the Cosmomycin Biosynthetic Gene Cluster in an Ocean Streptomycete.

The transformation-associated recombination cloning methodology facilitates the genomic capture and heterologous expression of natural product biosynthetic gene clusters (BGCs). We have streamlined this procedure by introduction of synthetic DNA gene blocks for the efficient capture of BGCs. We show the successful capture and expression of the aromatic polyketide antitumor agent cosmomycin from streptomycete bacteria and the discovery of new cosmomycin analogues by mass spectral molecular networking.

Prioritizing Natural Product Diversity in a Collection of 146 Bacterial Strains Based on Growth and Extraction Protocols.

In order to expedite the rapid and efficient discovery and isolation of novel specialized metabolites, while minimizing the waste of resources on rediscovery of known compounds, it is crucial to develop efficient approaches for strain prioritization, rapid dereplication, and the assessment of favored cultivation and extraction conditions. Herein we interrogated bacterial strains by systematically evaluating cultivation and extraction parameters with LC-MS/MS analysis and subsequent dereplication through the Global Natural Product Social Molecular Networking (GNPS) platform. The developed method is fast, requiring minimal time and sample material, and is compatible with high-throughput extract analysis, thereby streamlining strain prioritization and evaluation of culturing parameters. With this approach, we analyzed 146 marine Salinispora and Streptomyces strains that were grown and extracted using multiple different protocols. In total, 603 samples were analyzed, generating approximately 1.8 million mass spectra. We constructed a comprehensive molecular network and identified 15 molecular families of diverse natural products and their analogues. The size and breadth of this network shows statistically supported trends in molecular diversity when comparing growth and extraction conditions. The network provides an extensive survey of the biosynthetic capacity of the strain collection and a method to compare strains based on the variety and novelty of their metabolites. This approach allows us to quickly identify patterns in metabolite production that can be linked to taxonomy, culture conditions, and extraction methods, as well as informing the most valuable growth and extraction conditions.

Moral Distress in PICU and Neonatal ICU Practitioners: A Cross-Sectional Evaluation.

OBJECTIVES: To measure the level of moral distress in PICU and neonatal ICU health practitioners, and to describe the relationship of moral distress with demographic factors, burnout, and uncertainty. DESIGN: Cross-sectional survey. SETTING: A large pediatric tertiary care center. SUBJECTS: Neonatal ICU and PICU health practitioners with at least 3 months of ICU experience. INTERVENTIONS: A 41-item questionnaire examining moral distress, burnout, and uncertainty. MEASUREMENTS AND MAIN RESULTS: The main outcome was moral distress measured with the Revised Moral Distress Scale. Secondary outcomes were frequency and intensity Revised Moral Distress Scale subscores, burnout measured with the Maslach Burnout Inventory depersonalization subscale, and uncertainty measured with questions adapted from Mishel's Parent Perception of Uncertainty Scale. Linear regression models were used to examine associations between participant characteristics and the measures of moral distress, burnout, and uncertainty. Two-hundred six analyzable surveys were returned. The median Revised Moral Distress Scale score was 96.5 (interquartile range, 69-133), and 58% of respondents reported significant work-related moral distress. Revised Moral Distress Scale items involving end-of-life care and communication scored highest. Moral distress was positively associated with burnout (r = 0.27; p < 0.001) and uncertainty (r = 0.04; p = 0.008) and inversely associated with perceived hospital supportiveness (r = 0.18; p < 0.001). Nurses reported higher moral distress intensity than physicians (Revised Moral Distress Scale intensity subscores: 57.3 vs 44.7; p = 0.002). In nurses only, moral distress was positively associated with increasing years of ICU experience (p = 0.02) and uncertainty about whether their care was of benefit (r = 0.11; p < 0.001) and inversely associated with uncertainty about a child's prognosis (r = 0.03; p = 0.03). CONCLUSIONS: In this single-center, cross-sectional study, we found that moral distress is present in PICU and neonatal ICU health practitioners and is correlated with burnout, uncertainty, and feeling unsupported.

Application of Sepsis Definitions to Pediatric Patients Admitted With Suspected Infections in Uganda.

OBJECTIVES: Acute infectious diseases are the most common cause of under-5 mortality. However, the hospital burden of nonneonatal pediatric sepsis has not previously been described in the resource poor setting. The objective of this study was to determine the prevalence of sepsis among children 6 months to 5 years old admitted with proven or suspected infection and to evaluate the presence of sepsis as a predictive tool for mortality during admission. DESIGN: In this prospective cohort study, we used the pediatric International Consensus Conference definition of sepsis to determine the prevalence of sepsis among children admitted to the pediatric ward with a proven or suspected infection. The diagnosis of sepsis, as well as each individual component of the sepsis definition, was evaluated for capturing in-hospital mortality. SETTING: The pediatric ward of two hospitals in Mbarara, Uganda. PATIENTS: Admitted children between 6 months and 5 years with a confirmed or suspected infection. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: One thousand three hundred seven (1,307) subjects with a confirmed or suspected infection were enrolled, and 65 children died (5.0%) during their admission. One thousand one hundred twenty-one (85.9%) met the systemic inflammatory response syndrome criteria, and therefore, they were defined as having sepsis. The sepsis criteria captured 61 deaths, demonstrating a sensitivity and a specificity of 95% (95% CI, 90-100%) and 15% (95% CI, 13-17%), respectively. The most discriminatory individual component of the systemic inflammatory response syndrome criteria was the leukocyte count, which alone had a sensitivity of 72% and a specificity of 56% for the identification of mortality in hospital. CONCLUSIONS: This study is among the first to quantify the burden of nonneonatal pediatric sepsis in children with suspected infection, using the international consensus sepsis definition, in a typical resource-constrained setting in Africa. This definition was found to be highly sensitive in identifying those who died but had very low specificity as most children who were admitted with infections had sepsis. The systemic inflammatory response syndrome-based sepsis definition offers little value in identification of children at high risk of in-hospital mortality in this setting.

Coxiella burnetii effector protein subverts clathrin-mediated vesicular trafficking for pathogen vacuole biogenesis.

Successful macrophage colonization by Coxiella burnetii, the cause of human Q fever, requires pathogen-directed biogenesis of a large, growth-permissive parasitophorous vacuole (PV) with phagolysosomal characteristics. The vesicular trafficking pathways co-opted by C. burnetii for PV development are poorly defined; however, it is predicted that effector proteins delivered to the cytosol by a defective in organelle trafficking/intracellular multiplication (Dot/Icm) type 4B secretion system are required for membrane recruitment. Here, we describe involvement of clathrin-mediated vesicular trafficking in PV generation and the engagement of this pathway by the C. burnetii type 4B secretion system substrate Coxiella vacuolar protein A (CvpA). CvpA contains multiple dileucine [DERQ]XXXL[LI] and tyrosine (YXXΦ)-based endocytic sorting motifs like those recognized by the clathrin adaptor protein (AP) complexes AP1, AP2, and AP3. A C. burnetii ΔcvpA mutant exhibited significant defects in replication and PV development, confirming the importance of CvpA in infection. Ectopically expressed mCherry-CvpA localized to tubular and vesicular domains of pericentrosomal recycling endosomes positive for Rab11 and transferrin receptor, and CvpA membrane interactions were lost upon mutation of endocytic sorting motifs. Consistent with CvpA engagement of the endocytic recycling system, ectopic expression reduced uptake of transferrin. In pull-down assays, peptides containing CvpA-sorting motifs and full-length CvpA interacted with AP2 subunits and clathrin heavy chain. Furthermore, depletion of AP2 or clathrin by siRNA treatment significantly inhibited C. burnetii replication. Thus, our results reveal the importance of clathrin-coated vesicle trafficking in C. burnetii infection and define a role for CvpA in subverting these transport mechanisms.

Effects of Change in Tongue Pressure and Salivary Flow Rate on Swallow Efficiency Following Chemoradiation Treatment for Head and Neck Cancer.

Patients treated with chemoradiation for head and neck cancer frequently develop dysphagia. Tissue damage to the oral tongue causing weakness along with decreases in saliva production may contribute to dysphagia. Yet, effects of these variables on swallowing-related measures are unclear. The purpose of this study was (1) to determine effects of chemoradiation on tongue pressures, as a surrogate for strength, and salivary flow rates and (2) to elucidate relationships among tongue pressures, saliva production, and swallowing efficiency by bolus type. Twenty-one patients with head and neck cancer treated with chemoradiation were assessed before and after treatment and matched with 21 healthy control participants who did not receive chemoradiation. Each participant was given a questionnaire to rate dysphagia symptoms. Videofluoroscopic Evaluation of Swallowing (VFES) was used to determine swallowing efficiency; the Saxon test measured salivary flow rate; and the Iowa Oral Performance Instrument (IOPI) was used for oral tongue maximum and endurance measures. Results revealed significantly lower tongue endurance measures for patients post-treatment as compared to controls (p = .012). Salivary flow rates also were lower compared to pre-treatment (p = .000) and controls (p = .000). Simple linear regression analyses showed that change in salivary flow rate was predictive of change in swallow efficiency measures from pre- to post-treatment for 1 mL thin liquid (p = .017), 3 mL nectar-thick liquid (p = .026), and 3 mL standard barium pudding (p = .011) boluses. Based on these findings, it appears that chemoradiation treatment affects tongue endurance and salivary flow rate, and these changes may impact swallow efficiency. These factors should be considered when planning treatment for dysphagia.

Coxiella burnetii effector proteins that localize to the parasitophorous vacuole membrane promote intracellular replication.

The intracellular bacterial pathogen Coxiella burnetii directs biogenesis of a parasitophorous vacuole (PV) that acquires host endolysosomal components. Formation of a PV that supports C. burnetii replication requires a Dot/Icm type 4B secretion system (T4BSS) that delivers bacterial effector proteins into the host cell cytosol. Thus, a subset of T4BSS effectors are presumed to direct PV biogenesis. Recently, the PV-localized effector protein CvpA was found to promote C. burnetii intracellular growth and PV expansion. We predict additional C. burnetii effectors localize to the PV membrane and regulate eukaryotic vesicle trafficking events that promote pathogen growth. To identify these vacuolar effector proteins, a list of predicted C. burnetii T4BSS substrates was compiled using bioinformatic criteria, such as the presence of eukaryote-like coiled-coil domains. Adenylate cyclase translocation assays revealed 13 proteins were secreted in a Dot/Icm-dependent fashion by C. burnetii during infection of human THP-1 macrophages. Four of the Dot/Icm substrates, termed Coxiella vacuolar protein B (CvpB), CvpC, CvpD, and CvpE, labeled the PV membrane and LAMP1-positive vesicles when ectopically expressed as fluorescently tagged fusion proteins. C. burnetii ΔcvpB, ΔcvpC, ΔcvpD, and ΔcvpE mutants exhibited significant defects in intracellular replication and PV formation. Genetic complementation of the ΔcvpD and ΔcvpE mutants rescued intracellular growth and PV generation, whereas the growth of C. burnetii ΔcvpB and ΔcvpC was rescued upon cohabitation with wild-type bacteria in a common PV. Collectively, these data indicate C. burnetii encodes multiple effector proteins that target the PV membrane and benefit pathogen replication in human macrophages.

The intrinsic resting state voice network in Parkinson's disease.

Over 90 percent of patients with Parkinson's disease experience speech-motor impairment, namely, hypokinetic dysarthria characterized by reduced pitch and loudness. Resting-state functional connectivity analysis of blood oxygen level-dependent functional magnetic resonance imaging is a useful measure of intrinsic neural functioning. We utilized resting-state functional connectivity modeling to analyze the intrinsic connectivity in patients with Parkinson's disease within a vocalization network defined by a previous meta-analysis of speech (Brown et al., 2009). Functional connectivity of this network was assessed in 56 patients with Parkinson's disease and 56 gender-, age-, and movement-matched healthy controls. We also had item 5 and 18 of the UPDRS, and the PDQ-39 Communication subscale available for correlation with the voice network connectivity strength in patients. The within-group analyses of connectivity patterns demonstrated a lack of subcortical-cortical connectivity in patients with Parkinson's disease. At the cortical level, we found robust (homotopic) interhemispheric connectivity but only inconsistent evidence for many intrahemispheric connections. When directly contrasted to the control group, we found a significant reduction of connections between the left thalamus and putamen, and cortical motor areas, as well as reduced right superior temporal gyrus connectivity. Furthermore, most symptom measures correlated with right putamen, left cerebellum, left superior temporal gyrus, right premotor, and left Rolandic operculum connectivity in the voice network. The results reflect the importance of (right) subcortical nodes and the superior temporal gyrus in Parkinson's disease, enhancing our understanding of the neurobiological underpinnings of vocalization impairment in Parkinson's disease.