Filament Assembly by Spire: Key Residues and Concerted Actin Binding

The most recently identified class of actin nucleators, WASp homology domain 2 (WH2) nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or SC) plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of SC in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within SC that are critical for its activity. Using this information, we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that SC binds actin filaments, in addition to monomers

Filament Assembly by Spire: Key Residues and Concerted Actin Binding

The most recently identified class of actin nucleators, WASp homology domain 2 (WH2) nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or SC) plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of SC in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within SC that are critical for its activity. Using this information, we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that SC binds actin filaments, in addition to monomers

The burden and characteristics of enteric fever at a healthcare facility in a densely populated area of Kathmandu

Enteric fever, caused by Salmonella enterica serovars Typhi and Paratyphi A (S. Typhi and S. Paratyphi A) remains a major public health problem in many settings. The disease is limited to locations with poor sanitation which facilitates the transmission of the infecting organisms. Efficacious and inexpensive vaccines are available for S. Typhi, yet are not commonly deployed to control the disease. Lack of vaccination is due partly to uncertainty of the disease burden arising from a paucity of epidemiological information in key locations. We have collected and analyzed data from 3,898 cases of blood culture-confirmed enteric fever from Patan Hospital in Lalitpur Sub-Metropolitan City (LSMC), between June 2005 and May 2009. Demographic data was available for a subset of these patients (n = 527) that were resident in LSMC and who were enrolled in trials. We show a considerable burden of enteric fever caused by S. Typhi (2,672; 68.5%) and S. Paratyphi A (1,226; 31.5%) at this Hospital over a four year period, which correlate with seasonal fluctuations in rainfall. We found that local population density was not related to incidence and we identified a focus of infections in the east of LSMC. With data from patients resident in LSMC we found that the median age of those with S. Typhi (16 years) was significantly less than S. Paratyphi A (20 years) and that males aged 15 to 25 were disproportionately infected. Our findings provide a snapshot into the epidemiological patterns of enteric fever in Kathmandu. The uneven distribution of enteric fever patients within the population suggests local variation in risk factors, such as contaminated drinking water. These findings are important for initiating a vaccination scheme and improvements in sanitation. We suggest any such intervention should be implemented throughout the LSMC area.This work was supported by The Wellcome Trust, Euston Road, London, United Kingdom. MFB is supported by the Medical Research Council (grant G0600718). SB is supported by an OAK foundation fellowship through Oxford University

The burden and characteristics of enteric fever at a healthcare facility in a densely populated area of Kathmandu.

Enteric fever, caused by Salmonella enterica serovars Typhi and Paratyphi A (S. Typhi and S. Paratyphi A) remains a major public health problem in many settings. The disease is limited to locations with poor sanitation which facilitates the transmission of the infecting organisms. Efficacious and inexpensive vaccines are available for S. Typhi, yet are not commonly deployed to control the disease. Lack of vaccination is due partly to uncertainty of the disease burden arising from a paucity of epidemiological information in key locations. We have collected and analyzed data from 3,898 cases of blood culture-confirmed enteric fever from Patan Hospital in Lalitpur Sub-Metropolitan City (LSMC), between June 2005 and May 2009. Demographic data was available for a subset of these patients (n = 527) that were resident in LSMC and who were enrolled in trials. We show a considerable burden of enteric fever caused by S. Typhi (2,672; 68.5%) and S. Paratyphi A (1,226; 31.5%) at this Hospital over a four year period, which correlate with seasonal fluctuations in rainfall. We found that local population density was not related to incidence and we identified a focus of infections in the east of LSMC. With data from patients resident in LSMC we found that the median age of those with S. Typhi (16 years) was significantly less than S. Paratyphi A (20 years) and that males aged 15 to 25 were disproportionately infected. Our findings provide a snapshot into the epidemiological patterns of enteric fever in Kathmandu. The uneven distribution of enteric fever patients within the population suggests local variation in risk factors, such as contaminated drinking water. These findings are important for initiating a vaccination scheme and improvements in sanitation. We suggest any such intervention should be implemented throughout the LSMC area

The Sudden Dominance of blaCTX–M Harbouring Plasmids in Shigella spp. Circulating in Southern Vietnam

Shigellosis is a disease caused by bacteria belonging to Shigella spp. and is a leading cause of bacterial gastrointestinal infections in infants in unindustrialized countries. The Shigellae are dynamic and capable of rapid change when placed under selective pressure in a human population. Extended spectrum beta lactamases (ESBLs) are enzymes capable of degrading cephalosporins (a group of antimicrobial agents) and the genes that encode them are common in pathogenic E. coli and other related organisms in industrialized countries. In southern Vietnam, we have isolated multiple cephalosporin-resistant Shigella that express ESBLs. Furthermore, over two years these strains have replaced strains isolated from patients with shigellosis that cannot express ESBLs. Our work describes the genes responsible for this characteristic and we investigate one of the elements carrying one of these genes. These finding have implications for treatment of shigellosis and support the growing necessity for vaccine development. Our findings also may be pertinent for other countries undergoing a similar economic transition to Vietnam's and the corresponding effect on bacterial populations

A Multi-Center Randomized Trial to Assess the Efficacy of Gatifloxacin versus Ciprofloxacin for the Treatment of Shigellosis in Vietnamese Children

The bacterial genus Shigella is the most common cause of dysentery (diarrhea containing blood and/or mucus) and the disease is common in developing countries with limitations in sanitation. Children are most at risk of infection and frequently require hospitalization and antimicrobial therapy. The WHO currently recommends the fluoroquinolone, ciprofloxacin, for the treatment of childhood Shigella infections. In recent years there has been a sharp increase in the number of organisms that exhibit resistance to nalidixic acid (an antimicrobial related to ciprofloxacin), corresponding with reduced susceptibility to ciprofloxacin. We hypothesized that infections with Shigella strains that demonstrate resistance to nalidixic acid may prevent effective treatment with ciprofloxacin. We performed a randomized controlled trial to compare 3 day ciprofloxacin therapy with 3 days of gatifloxacin, a newer generation fluoroquinolone with greater activity than ciprofloxacin. We measured treatment failure and time to the cessation of individual disease symptoms in 249 children with dysentery treated with gatifloxacin and 245 treated with ciprofloxacin. We could identify no significant differences in treatment failure between the two groups or in time to the cessation of individual symptoms. We conclude that, in Vietnam, ciprofloxacin and gatifloxacin are similarly effective for the treatment of acute dysentery

An evaluation of purified Salmonella Typhi protein antigens for the serological diagnosis of acute typhoid fever.

OBJECTIVES: The diagnosis of typhoid fever is a challenge. Aiming to develop a typhoid diagnostic we measured antibody responses against Salmonella Typhi (S. Typhi) protein antigens and the Vi polysaccharide in a cohort of Bangladeshi febrile patients. METHODS: IgM against 12 purified antigens and the Vi polysaccharide was measured by ELISA in plasma from patients with confirmed typhoid fever (n = 32), other confirmed infections (n = 17), and healthy controls (n = 40). ELISAs with the most specific antigens were performed on plasma from 243 patients with undiagnosed febrile disease. RESULTS: IgM against the S. Typhi protein antigens correlated with each other (rho > 0.8), but not against Vi (rho 0.85, respectively. Applying a dynamic cut-off to patients with undiagnosed febrile disease suggested that 34-58% had an IgM response indicative of typhoid. CONCLUSIONS: We evaluated the diagnostic potential of several S. Typhi antigens; our assays give good sensitivity and specificity, but require further assessment in differing patient populations

Phylogeographical analysis of the dominant multidrug-resistant H58 clade of Salmonella Typhi identifies inter- and intracontinental transmission events.

The emergence of multidrug-resistant (MDR) typhoid is a major global health threat affecting many countries where the disease is endemic. Here whole-genome sequence analysis of 1,832 Salmonella enterica serovar Typhi (S. Typhi) identifies a single dominant MDR lineage, H58, that has emerged and spread throughout Asia and Africa over the last 30 years. Our analysis identifies numerous transmissions of H58, including multiple transfers from Asia to Africa and an ongoing, unrecognized MDR epidemic within Africa itself. Notably, our analysis indicates that H58 lineages are displacing antibiotic-sensitive isolates, transforming the global population structure of this pathogen. H58 isolates can harbor a complex MDR element residing either on transmissible IncHI1 plasmids or within multiple chromosomal integration sites. We also identify new mutations that define the H58 lineage. This phylogeographical analysis provides a framework to facilitate global management of MDR typhoid and is applicable to similar MDR lineages emerging in other bacterial species

Global, regional, and national burden of disorders affecting the nervous system, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BACKGROUND: Disorders affecting the nervous system are diverse and include neurodevelopmental disorders, late-life neurodegeneration, and newly emergent conditions, such as cognitive impairment following COVID-19. Previous publications from the Global Burden of Disease, Injuries, and Risk Factor Study estimated the burden of 15 neurological conditions in 2015 and 2016, but these analyses did not include neurodevelopmental disorders, as defined by the International Classification of Diseases (ICD)-11, or a subset of cases of congenital, neonatal, and infectious conditions that cause neurological damage. Here, we estimate nervous system health loss caused by 37 unique conditions and their associated risk factors globally, regionally, and nationally from 1990 to 2021. METHODS: We estimated mortality, prevalence, years lived with disability (YLDs), years of life lost (YLLs), and disability-adjusted life-years (DALYs), with corresponding 95% uncertainty intervals (UIs), by age and sex in 204 countries and territories, from 1990 to 2021. We included morbidity and deaths due to neurological conditions, for which health loss is directly due to damage to the CNS or peripheral nervous system. We also isolated neurological health loss from conditions for which nervous system morbidity is a consequence, but not the primary feature, including a subset of congenital conditions (ie, chromosomal anomalies and congenital birth defects), neonatal conditions (ie, jaundice, preterm birth, and sepsis), infectious diseases (ie, COVID-19, cystic echinococcosis, malaria, syphilis, and Zika virus disease), and diabetic neuropathy. By conducting a sequela-level analysis of the health outcomes for these conditions, only cases where nervous system damage occurred were included, and YLDs were recalculated to isolate the non-fatal burden directly attributable to nervous system health loss. A comorbidity correction was used to calculate total prevalence of all conditions that affect the nervous system combined. FINDINGS: Globally, the 37 conditions affecting the nervous system were collectively ranked as the leading group cause of DALYs in 2021 (443 million, 95% UI 378–521), affecting 3·40 billion (3·20–3·62) individuals (43·1%, 40·5–45·9 of the global population); global DALY counts attributed to these conditions increased by 18·2% (8·7–26·7) between 1990 and 2021. Age-standardised rates of deaths per 100 000 people attributed to these conditions decreased from 1990 to 2021 by 33·6% (27·6–38·8), and age-standardised rates of DALYs attributed to these conditions decreased by 27·0% (21·5–32·4). Age-standardised prevalence was almost stable, with a change of 1·5% (0·7–2·4). The ten conditions with the highest age-standardised DALYs in 2021 were stroke, neonatal encephalopathy, migraine, Alzheimer's disease and other dementias, diabetic neuropathy, meningitis, epilepsy, neurological complications due to preterm birth, autism spectrum disorder, and nervous system cancer. INTERPRETATION: As the leading cause of overall disease burden in the world, with increasing global DALY counts, effective prevention, treatment, and rehabilitation strategies for disorders affecting the nervous system are needed

Safety and efficacy of fluoxetine on functional outcome after acute stroke (AFFINITY): a randomised, double-blind, placebo-controlled trial

Background Trials of fluoxetine for recovery after stroke report conflicting results. The Assessment oF FluoxetINe In sTroke recoverY (AFFINITY) trial aimed to show if daily oral fluoxetine for 6 months after stroke improves functional outcome in an ethnically diverse population. Methods AFFINITY was a randomised, parallel-group, double-blind, placebo-controlled trial done in 43 hospital stroke units in Australia (n=29), New Zealand (four), and Vietnam (ten). Eligible patients were adults (aged ≥18 years) with a clinical diagnosis of acute stroke in the previous 2–15 days, brain imaging consistent with ischaemic or haemorrhagic stroke, and a persisting neurological deficit that produced a modified Rankin Scale (mRS) score of 1 or more. Patients were randomly assigned 1:1 via a web-based system using a minimisation algorithm to once daily, oral fluoxetine 20 mg capsules or matching placebo for 6 months. Patients, carers, investigators, and outcome assessors were masked to the treatment allocation. The primary outcome was functional status, measured by the mRS, at 6 months. The primary analysis was an ordinal logistic regression of the mRS at 6 months, adjusted for minimisation variables. Primary and safety analyses were done according to the patient's treatment allocation. The trial is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12611000774921. Findings Between Jan 11, 2013, and June 30, 2019, 1280 patients were recruited in Australia (n=532), New Zealand (n=42), and Vietnam (n=706), of whom 642 were randomly assigned to fluoxetine and 638 were randomly assigned to placebo. Mean duration of trial treatment was 167 days (SD 48·1). At 6 months, mRS data were available in 624 (97%) patients in the fluoxetine group and 632 (99%) in the placebo group. The distribution of mRS categories was similar in the fluoxetine and placebo groups (adjusted common odds ratio 0·94, 95% CI 0·76–1·15; p=0·53). Compared with patients in the placebo group, patients in the fluoxetine group had more falls (20 [3%] vs seven [1%]; p=0·018), bone fractures (19 [3%] vs six [1%]; p=0·014), and epileptic seizures (ten [2%] vs two [<1%]; p=0·038) at 6 months. Interpretation Oral fluoxetine 20 mg daily for 6 months after acute stroke did not improve functional outcome and increased the risk of falls, bone fractures, and epileptic seizures. These results do not support the use of fluoxetine to improve functional outcome after stroke