Small-molecule ion channels increase host defences in cystic fibrosis airway epithelia.

Loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) compromise epithelial HCO3- and Cl- secretion, reduce airway surface liquid pH, and impair respiratory host defences in people with cystic fibrosis1-3. Here we report that apical addition of amphotericin B, a small molecule that forms unselective ion channels, restored HCO3- secretion and increased airway surface liquid pH in cultured airway epithelia from people with cystic fibrosis. These effects required the basolateral Na+, K+-ATPase, indicating that apical amphotericin B channels functionally interfaced with this driver of anion secretion. Amphotericin B also restored airway surface liquid pH, viscosity, and antibacterial activity in primary cultures of airway epithelia from people with cystic fibrosis caused by different mutations, including ones that do not yield CFTR, and increased airway surface liquid pH in CFTR-null pigs in vivo. Thus, unselective small-molecule ion channels can restore host defences in cystic fibrosis airway epithelia via a mechanism that is independent of CFTR and is therefore independent of genotype.

Substrate Specificity of the Flavoenzyme BhaC1 That Converts a C-Terminal Trp to a Hydroxyquinone.

The preparation of protein-protein, protein-peptide, and protein-small molecule conjugates is important for a variety of applications, such as vaccine production, immunotherapies, preparation of antibody-drug conjugates, and targeted delivery of therapeutics. To achieve site-selective conjugation, selective chemical or enzymatic functionalization of proteins is required. We have recently reported biosynthetic pathways in which small, catalytic scaffold peptides are utilized for the generation of amino acid-derived natural products called pearlins. In these systems, peptide amino-acyl tRNA ligases (PEARLs) append amino acids to the C-terminus of a scaffold peptide, and tailoring enzymes encoded in the biosynthetic gene clusters modify the PEARL-appended amino acid to generate a variety of natural products. Herein, we investigate the substrate selectivity of one such tailoring enzyme, BhaC1, that participates in pyrroloiminoquinone biosynthesis. BhaC1 converts the indole of a C-terminal tryptophan into an o-hydroxy-p-quinone, a promising moiety for site-selective bioconjugation. Our studies demonstrate that BhaC1 requires a 20-amino acid peptide for substrate recognition. When this peptide was appended at the C-terminus of proteins, the C-terminal Trp was modified by BhaC1. The enzyme is sufficiently selective that only small changes to the sequence of the peptide are tolerated. An AlphaFold model for substrate recognition explains the selectivity of the enzyme, which may be used to install a reactive handle onto the C-terminus of proteins.

Change over time in adolescent smoking, cannabis use and their association: findings from the School Health Research Network in Wales.

BACKGROUND: While tobacco smoking has declined among UK youth in recent decades, cannabis use has begun to show some growth. Given their interrelationship, growth in cannabis use may act as a barrier to continued reduction in youth smoking. This paper assesses recent tobacco and cannabis use trends in Wales, and their association, to explore whether change in cannabis use might have impacted youth tobacco smoking prevalence. METHODS: Repeat cross-sectional data on tobacco and cannabis use were obtained from biennial Welsh Student Health and Wellbeing surveys between 2013 and 2019. Data were pooled and analysed using logistic regression with adjustment for school-level clustering. RESULTS: No change in regular youth tobacco smoking was observed between 2013 and 2019. In contrast, current cannabis use increased during this time, and cannabis users had significantly greater odds of regular tobacco smoking. After adjusting for change in cannabis use, a significant decline in youth tobacco smoking was observed (OR 0.95; 95% confidence intervals: 0.92, 0.97). CONCLUSION: Recent growth in cannabis use among young people in Wales may have offset prospective declines in regular tobacco smoking. Further reductions in youth smoking may require more integrated policy approaches to address the co-use of tobacco and cannabis among adolescents.

Waterborne outbreak of gastroenteritis on the KwaZulu-Natal Coast, South Africa, December 2016/January 2017.

An unexpected increase in gastroenteritis cases was reported by healthcare workers on the KwaZulu-Natal Coast, South Africa, January 2017 with >600 cases seen over a 3-week period. A case-control study was conducted to identify the source and risk factors associated with the outbreak so as to recommend control and prevention measures. Record review identified cases and controls and structured-telephonic interviews were conducted to obtain exposure history. Stool specimens were collected from 20 cases along with environmental samples and both screened for enteric pathogens. A total of 126 cases and 62 controls were included in the analysis. The odds of developing gastroenteritis were 6.0 times greater among holiday makers than residents (95% confidence interval (CI) 2.0-17.7). Swimming in the lagoon increased the odds of developing gastroenteritis by 3.3 times (95% CI 1.06-10.38). Lagoon water samples tested positive for norovirus (NoV) GI.6, GII.3 and GII.6, astrovirus and rotavirus. Eleven (55%) stool specimens were positive for NoV with eight genotyped as GI.1 (n = 2), GI.5 (n = 3), GI.6 (n = 2), and GI.7 (n = 1). A reported sewage contamination event impacting the lagoon was the likely source with person-to-person spread perpetuating the outbreak. Restriction to swimming in the lagoon was apparently ineffective at preventing the outbreak, possibly due to inadequate enforcement, communication and signage strategies.

Links between deprivation and risk of violence-related injury: a qualitative study to identify potential causal mechanisms.

Background: Deprivation has been shown to have a greater effect on risk of violent injury among adolescent girls than boys, but the mechanisms underlying this association have not been identified. Methods: In this qualitative study designed to identify causal mechanisms, focus groups involving girls aged 14-16 years attending secondary schools in South Wales, UK, were convened. Schools were recruited based on a measure of area-level deprivation. Discussions were audio-recorded and transcripts analysed thematically. Results: Girls from more deprived areas tended not to participate in organized activities, obtained alcohol from multiple sources, consumed alcoholic drinks of varying strengths in both supervised and unsupervised settings, and tended not to feel trusted by their parents; this led to poor adolescent-parent communication. Girls from less deprived areas tended to participate in organized activities, obtain alcohol from parents, consume low strength alcohol in supervised settings, and have a trusting and communicative relationship with their parents. Conclusion: Deprivation may increase risk of adolescent girls sustaining violence-related injury by increasing their time spent in unsupervised environments, with alcohol and without parental knowledge.

Norovirus epidemiology in South African children <5 years hospitalised for diarrhoeal illness between 2009 and 2013.

Public health interest in norovirus (NoV) has increased in recent years following improved diagnostics, global burden estimates and the development of NoV vaccine candidates. This study aimed to describe the detection rate, clinical characteristics and environmental features associated with NoV detection in hospitalized children <5 years with diarrhoea in South Africa (SA). Between 2009 and 2013, prospective diarrhoeal surveillance was conducted at four sites in SA. Stool specimens were collected and screened for NoVs and other enteric pathogens using molecular and serological assays. Epidemiological and clinical data were compared in patients with or without detection of NoV. The study detected NoV in 15% (452/3103) of hospitalized children <5 years with diarrhoea with the majority of disease in children <2 years (92%; 417/452). NoV-positive children were more likely to present with diarrhoea and vomiting (odds ratio (OR) 1·3; 95% confidence interval (CI) 1·1-1·7; P = 0·011) with none-to-mild dehydration (adjusted OR 0·5; 95% CI 0·3-0·7) compared with NoV-negative children. Amongst children testing NoV positive, HIV-infected children were more likely to have prolonged hospitalization and increased mortality compared with HIV-uninfected children. Continued surveillance will be important to consider the epidemic trends and estimate the burden and risk of NoV infection in SA.

Norovirus diversity in children with gastroenteritis in South Africa from 2009 to 2013: GII.4 variants and recombinant strains predominate.

From 2009 to 2013 the diversity of noroviruses (NoVs) in children (⩽5 years) hospitalized with gastroenteritis in South Africa was investigated. NoVs were genotyped based on nucleotide sequence analyses of partial RNA-dependent RNA polymerase (RdRp) and capsid genes. Seventeen RdRp genotypes (GI.P2, GI.P3, GI.P6, GI.P7, GI.P not assigned (NA), GI.Pb, GI.Pf, GII.P2, GII.P4, GII.P7, GII.P13, GII.P16, GII.P21, GII.Pc, GII.Pe, GII.Pg, GII.PNA) and 20 capsid genotypes (GI.1, GI.2, GI.3, GI.5, GI.6, GI.7, GI.NA, GII.1, GII.2, GII.3, GII.4, GII.6, GII.7, GII.10, GII.12, GII.13, GII.14, GII.16, GII.17, GII.21) were identified. The combined RdRp/capsid genotype was determined for 275 GII strains. Fifteen confirmed recombinant NoV strains circulated during the study period. NoV GII.P4/GII.4 (47%) and GII.Pe/GII.4 (18%) predominated, followed by GII.PNA/GII.3 (10%) and GII.P21/GII.3 (7%). Other prevalent strains included GII.Pg/GII.12 (6%) and GII.Pg/GII.1 (3%). Two novel recombinants, GII.Pg/GII.2 and GII.Pg/GII.10 were identified. In 2013 the replacement of GII.4 New Orleans 2009 and GII.P21/GII.3, which predominated during the early part of the study, with GII.4 Sydney 2012 and GII.PNA/GII.3 was observed. This study presents the most comprehensive recent data on NoV diversity in Africa.

Applicability of Bio-wipes for the collection of human faecal specimens for detection and characterisation of enteric viruses.

OBJECTIVE: To determine whether gastroenteritis viruses and other enteric viruses could be detected in faecal specimens collected with Bio-wipes. METHODS: Faecal specimens, self-collected with Bio-wipes, from 190 individuals (94 diarrhoeal, 93 non-diarrhoeal, 3 unknown) were screened for eight human enteric viruses (enterovirus, hepatitis A virus, adenovirus, astrovirus, norovirus GI and GII, sapovirus and rotavirus) by real-time (reverse transcription)-polymerase chain reaction. Rotaviruses and noroviruses from positive specimens were genotyped. RESULTS: At least one enteric virus could be detected in 82.6% (157/190) of faecal specimens. Mixed infections of up to four different viruses could be detected in both diarrhoeal and non-diarrhoeal specimens. Enteroviruses were detected most frequently (63.7%), followed by adenoviruses (48.4%) and noroviruses (32.2%). Genotyping was successful for 78.6% of rotaviruses and 44.8% of noroviruses. CONCLUSIONS: Bio-wipes provide a user friendly, easier method for stool collection that facilitates enteric virus detection and genetic characterisation.

Telescope alignment from sparsely sampled wavefront measurements over pupil subapertures.

We present a simple formalism that has proven useful in on-axis alignment of two-element telescopes when wavefront information is available from only a limited region (here two noncontiguous subapertures) of the pupil. Misalignments cause predictable full-aperture aberrations, which in turn cause predictable tip/tilt modes in the subapertures. For the most useful case in which secondary mirror tilts are independently constrained by optical monitoring, the four subaperture tip/tilt modes provide enough information to solve for the state of misalignment uniquely. A practically important and intuitively appealing simplification of this inversion occurs if the tip/tilts of the two subapertures are first transformed into a new basis consisting of differential and common-mode tilts in each of the x and y directions. Then the matrices interpreting subaperture modes as full-aperture aberrations and those in turn as mechanical misalignments become diagonal, so the mechanical adjustment required to align each degree of freedom is just a constant sensitivity multiplying one of the measured differential or common-mode tilt basis modes. Knowing that this simplification occurs allows rapid empirical calibration of sensitivities in the lab and then deterministic alignment, simply and transparently, with no need for ray tracing to model the optical effects of the adjustments at each step of the alignment.

A biosynthetic pathway to aromatic amines that uses glycyl-tRNA as nitrogen donor.

Aromatic amines in nature are typically installed with Glu or Gln as the nitrogen donor. Here we report a pathway that features glycyl-tRNA instead. During the biosynthesis of pyrroloiminoquinone-type natural products such as ammosamides, peptide-aminoacyl tRNA ligases append amino acids to the C-terminus of a ribosomally synthesized peptide. First, [Formula: see text] adds Trp in a Trp-tRNA-dependent reaction and the flavoprotein AmmC1 then carries out three hydroxylations of the indole ring of Trp. After oxidation to the corresponding ortho-hydroxy para-quinone, [Formula: see text] attaches Gly to the indole ring in a Gly-tRNA dependent fashion. Subsequent decarboxylation and hydrolysis results in an amino-substituted indole. Similar transformations are catalysed by orthologous enzymes from Bacillus halodurans. This pathway features three previously unknown biochemical processes using a ribosomally synthesized peptide as scaffold for non-ribosomal peptide extension and chemical modification to generate an amino acid-derived natural product.

A first report on the characterization of rotavirus strains in Sierra Leone.

In an effort to reduce the high mortalities associated with rotavirus infections, a number of African countries are considering introducing human rotavirus vaccines. The demonstrated safety and efficacy of the live-attenuate human rotavirus vaccines in several clinical trials worldwide has accelerated such initiatives. Although the percentage-mortality rates for Sierra Leone are top of the list for rotavirus-associated deaths in Africa, no study has reported the prevalent strains circulating within this country. In this study, stool specimens were collected from 128 Sierra Leonean children presenting with diarrhea in 2005. Almost 37.5% (48/128) were rotavirus positive by EIA, of which 89.6% (43/48) revealed a short electropherotype, and a further 6.98% (3/48) could not be assigned a PAGE pattern. Genotyping analysis revealed G2P[4] (30.23%), G2P[6] (13.95%), G8P[6] (11.63%), G2P[8] (4.65%), G8P[4] (4.65%), and G8P[8] (2%) strains. About 11% were only assigned VP7 genotypes (G2), while 20.9% had mixed G and P types. The frequent detection of G2 rotaviruses could be of concern considering data generated from some studies that suggests lower efficacy of Rotarix® vaccine against G2 rotaviruses. This underscores the need for extensive and continuous regional strain surveillance to support rotavirus vaccines introduction and guide future vaccine development efforts. Such information will be useful before considering administration of specific rotavirus vaccine candidates in countries like Sierra Leone where little is known about circulating rotavirus strains.

Characterization and molecular epidemiology of rotavirus strains recovered in Northern Pretoria, South Africa during 2003-2006.

Rotavirus infection is the most common cause of severe dehydrating gastroenteritis in infants and young children and remains a significant clinical problem worldwide. The severity and the burden of rotavirus disease could be reduced through the implementation of an effective vaccine. The aim of this study was to characterize rotavirus strains circulating in the local community as part of an ongoing hospital burden of disease study when a G1P[8] rotavirus vaccine candidate was being evaluated in the same community. From 2003 through 2006, 729 rotavirus-positive stool specimens were collected from children <5 years of age who were treated for diarrhea at Dr George Mukhari Hospital, Ga-Rankuwa, South Africa. Molecular characterization of the strains was performed by polyacrylamide gel electrophoresis and genotyping of the VP4 and VP7 alleles using well-established seminested multiplex reverse-transcription polymerase chain reaction methods. In 2003, 62% of strains exhibited the short rotavirus electropherotype, and the most common rotavirus strain was G2P[4]. In subsequent years, predominant rotavirus strains included G1P[8] and G1P[6] in 2004, G3P[8] and G3P[6] in 2005, and G1P[8] in 2006. For the 4 years of the study, rotavirus strains with P[6] genotype were detected in 25% of all rotavirus-positive specimens. In addition, unusual G12P[6] and G8 strains were detected at a low frequency. These results reflect the diversity of rotavirus strains circulating in South African communities.

Diversity of rotavirus VP7 and VP4 genotypes in Northwestern Nigeria.

BACKGROUND: Nigeria has recently been ranked third among the 10 countries with the greatest number of rotavirus disease-associated deaths per year. Estimates attribute up to 33,000 deaths annually to rotavirus disease in Nigerian children <5 years old. Although the introduction of the new oral, live attenuated rotavirus vaccines may not occur for another 4-6 years in developing countries, background data on burden of disease, cost of rotavirus disease, and characterization of circulating strains is required to hasten this introduction to children who would clearly benefit from the intervention. METHODS: Between July 2002 and July 2004, fecal specimens were collected from 869 infants and young children <5 years of age presenting with diarrhea in Kaduna, Kebbi, Sokoto, and Zamfara states in northwestern Nigeria. In addition, 194 control specimens were also collected from children matched for age. Specimens were screened for the presence of rotavirus antigens. Rotavirus-positive specimens were further analyzed to determine electropherotype, subgroup specificity, and G and P genotypes. RESULTS: Rotavirus was detected in 18% of children with diarrhea and 7.2% of the age-matched case control subjects. The highest rotavirus burden was detected in children aged <6 months. The majority of the rotavirus-positive specimens revealed viruses of long electropherotypes, subgroup II specificity, and G1P[8] genotypes. Furthermore, more than a quarter of specimens (37%) displayed mixed G and P genotypes, and almost a third could not be genotyped. CONCLUSIONS: The high numbers of mixed rotavirus infections highlight the multitude of enteric pathogens to which children in African countries are exposed. Data on circulating rotavirus strains serve to inform African government officials to the serious health threat posed by rotavirus in their respective countries and to document the diversity of strains before vaccine introduction.

Burden and epidemiology of rotavirus diarrhea in selected African countries: preliminary results from the African Rotavirus Surveillance Network.

Severe rotavirus diarrhea in children <5 years of age is a major public health problem; however, limited regional and country specific data on rotavirus disease burden are available from sub-Saharan Africa. In June 2006, the World Health Organization Regional Office for Africa initiated rotavirus surveillance in selected African countries. With use of standardized methodology developed by the World Health Organization, children <5 years of age who were hospitalized with severe diarrhea were enrolled, and stool specimens were collected for detection of rotavirus strains with use of a commercial enzyme immunoassay. Rotavirus strains were further characterized for G and P types with use of a reverse-transcriptase polymerase chain reaction. From June 2006 through December 2008, rotavirus surveillance was established at 14 sites in 11 African countries. Of 5461 stool samples collected from children enrolled in 8 countries with 1 or 2 complete years of data, 2200 (40%) were positive for rotavirus. Ninety percent of all rotavirus hospitalizations occurred among children aged 3-12 months. Predominant types included G1P[8] (21%), G2P[4] (7%), and P [8] (29%); however, unusual types were also detected, including G8P[6] (5%), G8P[8] (1%), G12P[6] (1%), and G12P[6] (1%). A high percentage of mixed rotavirus infections was also detected. These preliminary results indicate that rotavirus is a major cause of severe diarrheal disease in African children.

The detection of enteric viruses in selected urban and rural river water and sewage in Kenya, with special reference to rotaviruses.

AIM: To determine the occurrence of eight human enteric viruses in surface water and sewage samples from different geographical areas in Kenya. METHODS AND RESULTS: Enteric viruses were recovered from the water and sewage sources by glass-wool adsorption elution and/or polyethylene glycol/NaCl precipitation and detected by singleplex real-time and conventional PCR and reverse transcriptase-PCR assays. One or more enteric viruses were detected in nearly all sewage and river water samples except the urban Mbagathi River. The VP7 (G types) and the VP4 (P types) of the rotaviruses (RV) were characterized by multiplex nested PCR methods. The G and P types could be determined in 95·5% of the RV strains, respectively. Mixed G types were detected with G12 and G1 predominating, and unusual G types, G5 and G10, were present. P[4] predominated in the urban Karen sewage samples, while P[8] predominated in the urban and rural streams. CONCLUSIONS: The high prevalence of RVs in surface water highlights the importance of assessing the water sources used for domestic purposes for viral contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the benefit of environmental surveillance as an additional tool to determine the epidemiology of RVs and other enteric viruses circulating in a given community.

Small Molecule Channels Harness Membrane Potential to Concentrate Potassium in trk1Δtrk2Δ Yeast.

Many protein ion channels harness membrane potential to move ions in opposition to their chemical gradient. Deficiencies of such proteins cause several human diseases, including cystic fibrosis, Bartter Syndrome, and proximal renal tubular acidosis. Using yeast as a eukaryotic model system, we asked whether, in the context of a protein ion channel deficiency in vivo, small molecule channels could similarly harness membrane potential to concentrate ions. Trk potassium transporters use membrane potential to move potassium from a relatively low concentration outside cells (∼15 mM) to one of >10× higher inside (150-500 mM); trk1Δtrk2Δ are unable to concentrate potassium or grow in standard media. Here we show that potassium-permeable, but not potassium-selective, small-molecule ion channels formed by amphotericin B can harness membrane potential to concentrate potassium and thereby restore trk1Δtrk2Δ growth. This finding expands the list of potential human channelopathies that might be addressed by a molecular prosthetics approach.

The detection and molecular characterization of human G12 genotypes in South Africa.

The last decade has seen an increase in the detection of rotavirus strains other than G1-G4 emerging or even predominating in some settings. The performance of the current rotavirus vaccines against unusual or rare circulating rotavirus serotypes cannot be predicted and continuous monitoring of wild type rotaviruses will remain a priority. Routine molecular rotavirus surveillance conducted in the Gauteng Province, South Africa during 2004, resulted in the detection of strains that could not typed using standard G specific genotyping primers. Sequencing of the first round amplicons revealed 19 serotype G12P[6] strains and one G12P[8] strain. Phylogenetic analyses of the G12 strains indicated that these strains are probably a recent introduction into South Africa and emerged from a strain related to the Indian isolate ISO-5. The association of the South African G12s with the P[6] genotype may suggest a mechanism for unusual strains to become more ecologically suited to local population transmission dynamics. This is the first report of serotype G12 strains on the African continent and continued surveillance will be required to track the emergence of G12 strains in Africa.

Genomic characterization of human rotavirus G8 strains from the African rotavirus network: relationship to animal rotaviruses.

Global rotavirus surveillance has led to the detection of many unusual human rotavirus (HRV) genotypes. During 1996-2004 surveillance within the African Rotavirus Network (ARN), six P[8],G8 and two P[6],G8 human rotavirus strains were identified. Gene fragments (RT-PCR amplicons) of all 11-gene segments of these G8 strains were sequenced in order to elucidate their genetic and evolutionary relationships. Phylogenetic and sequence analyses of each gene segment revealed high similarities (88-100% nt and 91-100% aa) for all segments except for gene 4 encoding VP4 proteins P[8] and P[6]. For most strains, almost all of the genes of the ARN strains other than neutralizing antigens are related to typical human strains of Wa genogroup. The VP7, NSP2, and NSP5 genes were closely related to cognate genes of animal strains (83-99% and 97-99% aa identity). This study suggests that the ARN G8 strains might have arisen through VP7 or VP4 gene reassortment events since most of the other gene segments resemble those of common human rotaviruses. However, VP7, NSP2 (likely), and NSP5 (likely) genes are derived potentially from animals consistent with a zoonotic introduction. Although these findings help elucidate rotavirus evolution, sequence studies of cognate animal rotavirus genes are needed to conclusively determine the specific origin of those genes relative to both human and animal rotavirus strains.

Systematic genetic analysis with ordered arrays of yeast deletion mutants.

In Saccharomyces cerevisiae, more than 80% of the approximately 6200 predicted genes are nonessential, implying that the genome is buffered from the phenotypic consequences of genetic perturbation. To evaluate function, we developed a method for systematic construction of double mutants, termed synthetic genetic array (SGA) analysis, in which a query mutation is crossed to an array of approximately 4700 deletion mutants. Inviable double-mutant meiotic progeny identify functional relationships between genes. SGA analysis of genes with roles in cytoskeletal organization (BNI1, ARP2, ARC40, BIM1), DNA synthesis and repair (SGS1, RAD27), or uncharacterized functions (BBC1, NBP2) generated a network of 291 interactions among 204 genes. Systematic application of this approach should produce a global map of gene function.