Smartphone multiplex microcapillary diagnostics using Cygnus: Development and evaluation of rapid serotype-specific NS1 detection with dengue patient samples

Laboratory diagnosis of dengue virus (DENV) infection including DENV serotyping requires skilled labor and well-equipped settings. DENV NS1 lateral flow rapid test (LFT) provides simplicity but lacks ability to identify serotype. A simple, economical, point-of-care device for serotyping is still needed. We present a gravity driven, smartphone compatible, microfluidic device using microcapillary film (MCF) to perform multiplex serotype-specific immunoassay detection of dengue virus NS1. A novel device–termed Cygnus–with a stackable design allows analysis of 1 to 12 samples in parallel in 40 minutes. A sandwich enzyme immunoassay was developed to specifically detect NS1 of all four DENV serotypes in one 60-μl plasma sample. This test aims to bridge the gap between rapid LFT and laboratory microplate ELISAs in terms of sensitivity, usability, accessibility and speed. The Cygnus NS1 assay was evaluated with retrospective undiluted plasma samples from 205 DENV infected patients alongside 50 febrile illness negative controls. Against the gold standard RT-PCR, clinical sensitivity for Cygnus was 82% in overall (with 78, 78, 80 and 76% for DENV1-4, respectively), comparable to an in-house serotyping NS1 microplate ELISA (82% vs 83%) but superior to commercial NS1-LFT (82% vs 74%). Specificity of the Cygnus device was 86%, lower than that of NS1-microplate ELISA and NS1-LFT (100% and 98%, respectively). For Cygnus positive samples, identification of DENV serotypes DENV2-4 matched those by RT-PCR by 100%, but for DENV1 capillaries false positives were seen, suggesting an improved DENV1 capture antibody is needed to increase specificity. Overall performance of Cygnus showed substantial agreement to NS1-microplate ELISA (κ = 0.68, 95%CI 0.58–0.77) and NS1-LFT (κ = 0.71, 95%CI 0.63–0.80). Although further refinement for DENV-1 NS1 detection is needed, the advantages of multiplexing and rapid processing time, this Cygnus device could deliver point-of-care NS1 antigen testing including serotyping for timely DENV diagnosis for epidemic surveillance and outbreak prediction.     

Patient-level interventions to reduce alcohol-related harms in low- and middle-income countries: A systematic review and meta-summary

BackgroundDisease and disability from alcohol use disproportionately impact people in low- and middle-income countries (LMICs). While varied interventions have been shown to reduce alcohol use in high-income countries, their efficacy in LMICs has not been assessed. This systematic review describes current published literature on patient-level alcohol interventions in LMICs and specifically describes clinical trials evaluating interventions to reduce alcohol use in LMICs.Methods and findingsIn accordance with PRISMA, we performed a systematic review using an electronic search strategy from January 1, 1995 to December 1, 2020. Title, abstract, as well as full-text screening and extraction were performed in duplicate. A meta-summary was performed on randomized controlled trials (RCTs) that evaluated alcohol-related outcomes. We searched the following electronic databases: PubMed, EMBASE, Scopus, Web of Science, Cochrane, WHO Global Health Library, and PsycINFO. Articles that evaluated patient-level interventions targeting alcohol use and alcohol-related harm in LMICs were eligible for inclusion. No studies were excluded based on language.After screening 5,036 articles, 117 articles fit our inclusion criteria, 75 of which were RCTs. Of these RCTs, 93% were performed in 13 middle-income countries, while 7% were from 2 low-income countries. These RCTs evaluated brief interventions (24, defined as any intervention ranging from advice to counseling, lasting less than 1 hour per session up to 4 sessions), psychotherapy or counseling (15, defined as an interaction with a counselor longer than a brief intervention or that included a psychotherapeutic component), health promotion and education (20, defined as an intervention encouraged individuals’ agency of taking care of their health), or biologic treatments (19, defined as interventions where the biological function of alcohol use disorder (AUD) as the main nexus of intervention) with 3 mixing categories of intervention types. Due to high heterogeneity of intervention types, outcome measures, and follow-up times, we did not conduct meta-analysis to compare and contrast studies, but created a meta-summary of all 75 RCT studies. The most commonly evaluated intervention with the most consistent positive effect was a brief intervention; similarly, motivational interviewing (MI) techniques were most commonly utilized among the diverse array of interventions evaluated.ConclusionsOur review demonstrated numerous patient-level interventions that have the potential to be effective in LMICs, but further research to standardize interventions, populations, and outcome measures is necessary to accurately assess their effectiveness. Brief interventions and MI techniques were the most commonly evaluated and had the most consistent positive effect on alcohol-related outcomes.Trial registrationProtocol Registry: PROSPERO CRD42017055549

Catherine Staton and co-workers report on evidence about interventions against harmful alcohol use in low- and middle-income countries.     

Sequence and structure of the mouse gene coding for the largest neurofilament subunit

We have determined the complete nucleotide sequence of the mouse gene encoding the neurofilament NF-H protein. The C-terminal domain of NF-H is very rich in charged amino acids (aa) and contains a 3-aa sequence, Lys-Ser-Pro, that is repeated 51 times within a stretch of 368 aa. The location of this serine-rich repeat in the phosphorylated domain of NF-H indicates that it represents the major protein kinase recognition site. The nfh gene shares two common intron positions with the nfl and nfm genes, but has an additional intron that occurs at a location equivalent to one of the introns in non-neuronal intermediate filament-coding genes. This additional nfh intron may have been acquired via duplication of a primordial intermediate filament gene.     

RB signaling prevents replication-dependent DNA double-strand breaks following genotoxic insult

Cell cycle checkpoints induced by DNA damage play an integral role in preservation of genomic stability by allowing cells to limit the propagation of deleterious mutations. The retinoblastoma tumor suppressor (RB) is crucial for the maintenance of the DNA damage checkpoint function because it elicits cell cycle arrest in response to a variety of genotoxic stresses. Although sporadic loss of RB is characteristic of most cancers and results in the bypass of the DNA damage checkpoint, the consequence of RB loss upon chemotherapeutic responsiveness has been largely uninvestigated. Here, we employed a conditional knockout approach to ablate RB in adult fibroblasts. This system enabled us to examine the DNA damage response of adult cells following acute RB deletion. Using this system, we demonstrated that loss of RB disrupted the DNA damage checkpoint elicited by either cisplatin or camptothecin exposure. Strikingly, this bypass was not associated with enhanced repair, but rather the accumulation of phosphorylated H2AX (γH2AX) foci, which indicate DNA double-strand breaks. The formation of γH2AX foci was due to ongoing replication following chemotherapeutic treatment in the RB-deficient cells. Additionally, peak γH2AX accumulation occurred in S-phase cells undergoing DNA replication in the presence of damage, and these γH2AX foci co-localized with replication foci. These results demonstrate that acute RB loss abrogates DNA damage-induced cell cycle arrest to induce γH2AX foci formation. Thus, secondary genetic lesions induced by RB loss have implications for the chemotherapeutic response and the development of genetic instability.     

Polyamine derivatives as selective RNaseA mimics

Site-selective scission of ribonucleic acids (RNAs) has attracted considerable interest, since RNA is an intermediate in gene expression and the genetic material of many pathogenic viruses. Polyamine-imidazole conjugates for site-selective RNA scission, without free imidazole, were synthesized and tested on yeast phenylalanine transfer RNA. These molecules catalyze RNA hydrolysis non-randomly. Within the polyamine chain, the location of the imidazole residue, the numbers of nitrogen atoms and their relative distances have notable influence on cleavage selectivity. A norspermine derivative reduces the cleavage sites to a unique location, in the anticodon loop of the tRNA, in the absence of complementary sequence. Experimental results are consistent with a cooperative participation of an ammonium group of the polyamine moiety, in addition to it’s binding to the negatively charged ribose-phosphate backbone, as proton source, and the imidazole moiety as a base. There is correlation between the location of the magnesium binding sites and the RNA cleavage sites, suggesting that the protonated nitrogens of the polycationic chain compete with some of the magnesium ions for RNA binding. Therefore, the cleavage pattern is specific of the RNA structure. These compounds cleave at physiological pH, representing novel reactive groups for antisense oligonucleotide derivatives or to enhance ribozyme activity.     

Phenylpropanoids, phenylalanine ammonia lyase and peroxidases in elicitor-challenged cassava (Manihot esculenta) suspension cells and leaves

BACKGROUND AND AIMS: Control of diseases in the key tropical staple, cassava, is dependent on resistant genotypes, but the innate mechanisms are unknown. The aim was to study phenylpropanoids and associated enzymes as possible defence components. METHODS: Phenylalanine ammonia-lyase (PAL), phenylpropanoids and peroxidases (POD) were investigated in elicited cassava suspension cells and leaves. Yeast elicitor was the most effective of several microbial and endogenous elicitors. Fungitoxicity was determined against the cassava pathogens Fusarium solani, F. oxysporum and the saprotroph Trichoderma harzianum. KEY RESULTS: A single and rapid (> or =2-3 min) oxidative burst, measured as hydrogen peroxide, occurred in elicited cells. PAL activity was induced maximally at 15 h and was preceded by PAL mRNA accumulation, which peaked at 9 h. Symplasmic POD activity increased four-fold in cells, 48 h post-elicitation. POD isoforms (2-7 isoforms, pI 3.1-8.8) were detected in elicited and unelicited cells, extracellular medium and leaves but two extracellular isoforms were enhanced post-elicitation. Also expression of a cassava peroxidase gene MecPOD1 increased in elicited cells. Only anionic forms oxidized scopoletin, with highest activity by isoform pI 3.6, present in all samples. Unidentified phenolics and possibly scopolin increased post-elicitation, but there was no enhancement of scopoletin, rutin or kaempferol-3-O-rutinoside concentration. Fungal germ tube elongation was inhibited more than germination by esculetin, ferulic acid, quercetin and scopoletin. T. harzianum was generally more sensitive than the pathogens and was inhibited by > or =50 microg mL(-1) of ferulic acid and quercetin and > or =10 microg mL(-1) of scopoletin. CONCLUSIONS: Phenolic levels in cells were not enhanced and were, theoretically, too low to be inhibitory. However, in combination and when oxidized they may contribute to defence, because oxidation of esculetin and scopoletin by peroxidase and of esculetin by tyrosinase enhanced their fungitoxicity up to 20-fold.     

Phase I/II trial of immunogenicity of a human papillomavirus (HPV) type 16 E7 protein–based vaccine in women with oncogenic HPV-positive cervical intraepithelial neoplasia

Purpose: Infection with oncogenic human papillomavirus (HPV) and HPV-16 in particular is a leading cause of anogenital neoplasia. High-grade intraepithelial lesions require treatment because of their potential to progress to invasive cancer. Numerous preclinical studies have demonstrated the therapeutic potential of E7-directed vaccination strategies in mice tumour models. In the present study, we tested the immunogenicity of a fusion protein (PD-E7) comprising a mutated HPV-16 E7 linked to the first 108 amino acids of Haemophilus influenzae protein D, formulated in the GlaxoSmithKline Biologicals adjuvant AS02B, in patients bearing oncogenic HPV-positive cervical intraepithelial neoplasia (CIN). Methods: Seven patients, five with a CIN3 and two with a CIN1, received three intramuscular injections of adjuvanted PD-E7 at 2-week intervals. Three additional CIN1 patients received a placebo. CIN3 patients underwent conization 8 weeks postvaccination. Cytokine flow cytometry and ELISA were used to monitor antigen-specific cellular and antibody responses from blood taken before and after vaccine or placebo injection. Results: Some patients had preexisting systemic IFN- CD4⁺ (1/10) and CD8⁺ (5/10) responses to PD-E7. Vaccination, not placebo injection, elicited systemic specific immune responses in the majority of the patients. Five vaccinated patients (71%) showed significantly increased IFN- CD8⁺ cell responses upon PD-E7 stimulation. Two responding patients generated long-term T-cell immunity toward the vaccine antigen and E7 as well as a weak H. influenzae protein D (PD)–directed CD4⁺ response. All the vaccinated patients, but not the placebo, made significant E7- and PD-specific IgG. Conclusions: The encouraging results obtained from this study performed on a limited number of subjects justify further analysis of the efficacy of the PD-E7/AS02B vaccine in CIN patients.     

Development of a gene-based radiation hybrid map of chicken Chromosome 7 and comparison to human and mouse

To validate the ChickRH6 whole-genome radiation hybrid (WGRH) panel, we constructed a map of chicken Chromosome 7 based on 19 microsatellite markers from the genetic map and 76 ESTs (expressed sequence tags), whose efficient targeted development was made possible by using the ICCARE software. This high-density radiation hybrid (RH) map of a chicken macrochromosome gives us indications on characteristics of ChickRH6. The potential resolution of the panel is 325 kb and the practical resolution of our framework map is 1.3 Mb. Based on these results, a complete framework map of the chicken genome would comprise 1000 markers. The marker order is in good agreement with the genetic map and comparison with the human and mouse sequence maps revealed a number of internal rearrangements.