Direct detection of methylation in genomic DNA

The identification of methylated sites on bacterial genomic DNA would be a useful tool to study the major roles of DNA methylation in prokaryotes: distinction of self and nonself DNA, direction of post-replicative mismatch repair, control of DNA replication and cell cycle, and regulation of gene expression. Three types of methylated nucleobases are known: N(6)-methyladenine, 5-methylcytosine and N(4)-methylcytosine. The aim of this study was to develop a method to detect all three types of DNA methylation in complete genomic DNA. It was previously shown that N(6)-methyladenine and 5-methylcytosine in plasmid and viral DNA can be detected by intersequence trace comparison of methylated and unmethylated DNA. We extended this method to include N(4)-methylcytosine detection in both in vitro and in vivo methylated DNA. Furthermore, application of intersequence trace comparison was extended to bacterial genomic DNA. Finally, we present evidence that intrasequence comparison suffices to detect methylated sites in genomic DNA. In conclusion, we present a method to detect all three natural types of DNA methylation in bacterial genomic DNA. This provides the possibility to define the complete methylome of any prokaryote

KAI407, a potent non-8-aminoquinoline compound that kills Plasmodium cynomolgi early dormant liver stage parasites in vitro.

Preventing relapses of Plasmodium vivax malaria through a radical cure depends on use of the 8-aminoquinoline primaquine, which is associated with safety and compliance issues. For future malaria eradication strategies, new, safer radical curative compounds that efficiently kill dormant liver stages (hypnozoites) will be essential. A new compound with potential radical cure activity was identified using a low-throughput assay of in vitro-cultured hypnozoite forms of Plasmodium cynomolgi (an excellent and accessible model for Plasmodium vivax). In this assay, primary rhesus hepatocytes are infected with P. cynomolgi sporozoites, and exoerythrocytic development is monitored in the presence of compounds. Liver stage cultures are fixed after 6 days and stained with anti-Hsp70 antibodies, and the relative proportions of small (hypnozoite) and large (schizont) forms relative to the untreated controls are determined. This assay was used to screen a series of 18 known antimalarials and 14 new non-8-aminoquinolines (preselected for blood and/or liver stage activity) in three-point 10-fold dilutions (0.1, 1, and 10 μM final concentrations). A novel compound, designated KAI407 showed an activity profile similar to that of primaquine (PQ), efficiently killing the earliest stages of the parasites that become either primary hepatic schizonts or hypnozoites (50% inhibitory concentration [IC50] for hypnozoites, KAI407, 0.69 μM, and PQ, 0.84 μM; for developing liver stages, KAI407, 0.64 μM, and PQ, 0.37 μM). When given as causal prophylaxis, a single oral dose of 100 mg/kg of body weight prevented blood stage parasitemia in mice. From these results, we conclude that KAI407 may represent a new compound class for P. vivax malaria prophylaxis and potentially a radical cure

Promoting HIV indicator condition-guided testing in hospital settings (PROTEST 2.0): study protocol for a multicentre interventional study

BACKGROUND: Late presentation remains a key barrier towards controlling the HIV epidemic. Indicator conditions (ICs) are those that are AIDS-defining, associated with a prevalence of undiagnosed HIV > 0.1%, or whose clinical management would be impeded if an HIV infection were undiagnosed. IC-guided HIV testing is an effective strategy in identifying undiagnosed HIV, but opportunities for earlier HIV diagnosis through IC-guided testing are being missed. We present a protocol for an interventional study to improve awareness of IC-guided testing and increase HIV testing in patients presenting with ICs in a hospital setting. METHODS: We designed a multicentre interventional study to be implemented at five hospitals in the region of Amsterdam, the Netherlands. Seven ICs were selected for which HIV test ratios (proportion of patients with an IC tested for HIV) will be measured: tuberculosis, cervical/vulvar cancer or high-grade cervical/vulvar dysplasia, malignant lymphoma, hepatitis B and C, and peripheral neuropathy. Prior to the intervention, a baseline assessment of HIV test ratios across ICs will be performed in eligible patients (IC diagnosed January 2015 through May 2020, ≥18 years, not known HIV positive) and an assessment of barriers and facilitators for HIV testing amongst relevant specialties will be conducted using qualitative (interviews) and quantitative methods (questionnaires). The intervention phase will consist of an educational intervention, including presentation of baseline results as competitive graphical audit and feedback combined with discussion on implementation and opportunities for improvement. The effect of the intervention will be assessed by comparing HIV test ratios of the pre-intervention and post-intervention periods. The primary endpoint is the HIV test ratio within ±3 months of IC diagnosis. Secondary endpoints are the HIV test ratio within ±6 months of diagnosis, ratio ever tested for HIV, HIV positivity percentage, proportion of late presenters and proportion with known HIV status prior to initiating treatment for their IC. DISCUSSION: This protocol presents a strategy aimed at increasing awareness of the benefits of IC-guided testing and increasing HIV testing in patients presenting with ICs in hospital settings to identify undiagnosed HIV in Amsterdam, the Netherlands. TRIAL REGISTRATION: Dutch trial registry: NL7521 . Registered 14 February 2019

Comparison of haemodynamic- and electroencephalographic-monitored effects evoked by four combinations of effect-site concentrations of propofol and remifentanil, yielding a predicted tolerance to laryngoscopy of 90%

This prospective study evaluates haemodynamic and electroencephalographic effects observed when administering four combinations of effect-site concentrations of propofol (Ce-PROP) and remifentanil (Ce-REMI), all yielding a single predicted probability of tolerance of laryngoscopy of 90% (P-TOL = 90%) according to the Bouillon interaction model. We aimed to identify combinations of Ce-PROP and Ce-REMI along a single isobole of P-TOL that result in favourable hypnotic and haemodynamic conditions. This knowledge could be of advantage in the development of drug advisory monitoring technology. 80 patients (18-90 years of age, ASA I-III) were randomized into four groups and titrated towards Ce-PROP (Schnider model, ug.ml(-1)) and Ce-REMI (Minto model, ng.ml(-1)) of respectively 8.6 and 1, 5.9 and 2, 3.6 and 4 and 2.0 and 8. After eleven minutes of equilibration, baseline measurements of haemodynamic endpoints and bispectral index were compared with three minutes of responsiveness measurements after laryngoscopy. Before laryngoscopy, bispectral index differed significantly (p < 0.0001) between groups in concordance with Ce-PROP. Heart rate decreased with increasing Ce-REMI (p = 0.001). The haemodynamic and arousal responses evoked by laryngoscopy were not significantly different between groups, but Ce-PROP = 3.6 mu g.ml(-1) and Ce-REMI = 4 ng.ml(-1) evoked the lowest median value for increment HR and increment SAP after laryngoscopy. This study provides clinical insight on the haemodynamic and hypnotic consequences, when a model based predicted P-TOL is used as a target for combined effect-site controlled target- controlled infusion of propofol and remifentanil. Heart rate and bispectral index were significantly different between groups despite a theoretical equipotency for P-TOL, suggesting that each component of the anaesthetic state (immobility, analgesia, and hypnotic drug effect) should be considered as independent neurophysiological and pharmacological phenomena. However, claims of (in)accuracy of the predicted P-TOL must be considered preliminary because larger numbers of observations are required for that goal

Randomized Trial of Time-Limited Interruptions of Protease Inhibitor-Based Antiretroviral Therapy (ART) vs. Continuous Therapy for HIV-1 Infection

Background The clinical outcomes of short interruptions of PI-based ART regimens remains undefined. Methods A 2-arm non-inferiority trial was conducted on 53 HIV-1 infected South African participants with viral load/ml and CD4 T cell count \u3e450 cells/µl on stavudine (or zidovudine), lamivudine and lopinavir/ritonavir. Subjects were randomized to a) sequential 2, 4 and 8-week ART interruptions or b) continuous ART (cART). Primary analysis was based on the proportion of CD4 count \u3e350 cells(c)/ml over 72 weeks. Adherence, HIV-1 drug resistance, and CD4 count rise over time were analyzed as secondary endpoints. Results The proportions of CD4 counts \u3e350 cells/µl were 82.12% for the intermittent arm and 93.73 for the cART arm; the difference of 11.95% was above the defined 10% threshold for non-inferiority (upper limit of 97.5% CI, 24.1%; 2-sided CI: −0.16, 23.1). No clinically significant differences in opportunistic infections, adverse events, adherence or viral resistance were noted; after randomization, long-term CD4 rise was observed only in the cART arm. Conclusion We are unable to conclude that short PI-based ART interruptions are non-inferior to cART in retention of immune reconstitution; however, short interruptions did not lead to a greater rate of resistance mutations or adverse events than cART suggesting that this regimen may be more forgiving than NNRTIs if interruptions in therapy occur

The Dynamic Landscape of Novel Psychoactive Substance (NPS) Use in Ireland: Results from an Expert Consultation

In Ireland, legislators encountered a new phenomenon in 2005 onwards with the advent of ‘legal highs’ sold in headshops. Use of ‘legal highs’ containing herbal and synthetic new psychoactive substances (NPS) was not confined to problematic drug users, and included social recreational users. Legislative controls were enacted in 2010, 2011 and 2015. The study aimed to investigate expert perspectives on the NPS situation with regard to changing and emergent trends in use, health and social consequences and service implications. This brief report presents descriptive findings from a national consultation using a structured guide with experts in 2016. Four themes emerged and centred on; ‘Definitions of NPS used within Professional Roles’; ‘Professional Experiences of NPS‘; ‘Types of NPS Users, Sourcing and Consequences of Use’; and ‘Service Response.’ Findings underscored the mental health and addiction related consequences of NPS use, with prevention, clinical and treatment services ill- equipped to deal with the particular characteristics of this form of drug abuse. Enhanced strategies, services and clinical responses are warranted to address the challenges encountered. © 2016 Springer Science+Business Media New Yor

The Identification of Zebrafish Mutants Showing Alterations in Senescence-Associated Biomarkers

There is an interesting overlap of function in a wide range of organisms between genes that modulate the stress responses and those that regulate aging phenotypes and, in some cases, lifespan. We have therefore screened mutagenized zebrafish embryos for the altered expression of a stress biomarker, senescence-associated β-galactosidase (SA-β-gal) in our current study. We validated the use of embryonic SA-β-gal production as a screening tool by analyzing a collection of retrovirus-insertional mutants. From a pool of 306 such mutants, we identified 11 candidates that showed higher embryonic SA-β-gal activity, two of which were selected for further study. One of these mutants is null for a homologue of Drosophila spinster, a gene known to regulate lifespan in flies, whereas the other harbors a mutation in a homologue of the human telomeric repeat binding factor 2 (terf2) gene, which plays roles in telomere protection and telomere-length regulation. Although the homozygous spinster and terf2 mutants are embryonic lethal, heterozygous adult fish are viable and show an accelerated appearance of aging symptoms including lipofuscin accumulation, which is another biomarker, and shorter lifespan. We next used the same SA-β-gal assay to screen chemically mutagenized zebrafish, each of which was heterozygous for lesions in multiple genes, under the sensitizing conditions of oxidative stress. We obtained eight additional mutants from this screen that, when bred to homozygosity, showed enhanced SA-β-gal activity even in the absence of stress, and further displayed embryonic neural and muscular degenerative phenotypes. Adult fish that are heterozygous for these mutations also showed the premature expression of aging biomarkers and the accelerated onset of aging phenotypes. Our current strategy of mutant screening for a senescence-associated biomarker in zebrafish embryos may thus prove to be a useful new tool for the genetic dissection of vertebrate stress response and senescence mechanisms

Metal-responsive gene regulation and metal transport in Helicobacter species

Helicobacter species are among the most successful colonizers of the mammalian gastrointestinal and hepatobiliary tract. Colonization is usually lifelong, indicating that Helicobacter species have evolved intricate mechanisms of dealing with stresses encountered during colonization of host tissues, like restriction of essential metal ions. The recent availability of genome sequences of the human gastric pathogen Helicobacter pylori, the murine enterohepatic pathogen Helicobacter hepaticus and the unannotated genome sequence of the ferret gastric pathogen Helicobacter mustelae has allowed for comparitive genome analyses. In this review we present such analyses for metal transporters, metal-storage and metal-responsive regulators in these three Helicobacter species, and discuss possible contributions of the differences in metal metabolism in adaptation to the gastric or enterohepatic niches occupied by Helicobacter species

A genome-wide DNA methylation signature for SETD1B-related syndrome

SETD1B is a component of a histone methyltransferase complex that specifically methylates Lys-4 of histone H3 (H3K4) and is responsible for the epigenetic control of chromatin structure and gene expression. De novo microdeletions encompassing this gene as well as de novo missense mutations were previously linked to syndromic intellectual disability (ID). Here, we identify a specific hypermethylation signature associated with loss of function mutations in the SETD1B gene which may be used as an epigenetic marker supporting the diagnosis of syndromic SETD1B-related diseases. We demonstrate the clinical utility of this unique epi-signature by reclassifying previously identified SETD1B VUS (variant of uncertain significance) in two patients