In Vitro Reconstitution of an Escherichia coli RNA-guided Immune System Reveals Unidirectional,ATP-dependent Degradation of DNA Target

Many prokaryotes utilize small RNA transcribed from clustered, regularly interspaced, short palindromic repeats (CRISPRs) to protect themselves from foreign genetic elements, such as phage and plasmids. In Escherichia coli, this small RNA is packaged into a surveillance complex (Cascade) that uses the RNA sequence to direct binding to invasive DNA. Once bound, Cascade recruits the Cas3 nuclease-helicase, which then proceeds to progressively degrade the invading DNA. Here, using individually purified Cascade and Cas3 from E. coli, we reconstitute CRISPR-mediated plasmid degradation in vitro. Analysis of this reconstituted assay suggests that Cascade recruits Cas3 to a single-stranded region of the DNA target exposed by Cascade binding. Cas3 then nicks the exposed DNA. Recruitment and nicking is stimulated by the presence, but not hydrolysis, of ATP. Following nicking and powered by ATP hydrolysis, the concerted actions of the helicase and nuclease domains of Cas3 proceed to unwind and degrade the entire DNA target in a unidirectional manner.     

A Swedish subfossil find of a bowhead whale from the late Pleistocene: shore displacement,paleoecology in south-west Sweden and the identity of the Swedenborg whale (Balaena swedenborgii Liljeborg, 1867)

The Swedenborg whale Balaena swedenborgii Liljeborg, 1867, is a baleen whale species believed to have existed in the North Sea from the period when the inland ice melted around 13,000 before present (BP) until about 8000 years ago. The first bones attributed to this species were found in Sweden in 1705. When whale remains were discovered on the Swedish west coast during the extension work of a motorway extension, it was speculated that this could be a specimen of the extinct Swedenborg whale. The bones were found 72 m above the present-day sea level embedded in glacial mud. Shelly remains of marine organisms were present in the deposit surrounding the whale-fall, and sediments with the associated specimens were therefore collected for further analyses. We applied radiocarbon dating, thin sectioning, morphological analyses, ancient DNA typing and analyses of the associated shelly assemblage in an interdisciplinary effort to understand the circumstances of this fossil whale-fall. Our results show that the whale is not the putative species B. swedenborgii, but a bowhead whale Balaena mysticetus. The results also indicate that the whale must have been rapidly covered by glacial sediments, highlighting the speed of the deglacial process in the area.     

8-Methoxyquinolines as PDE4 inhibitors

The synthesis and pharmacological profile of a novel series of 2-substituted 8-methoxyquinolines is described. The 2-trifluoromethyl compound was found to be a potent inhibitor of phosphodiesterase type 4 (PDE4).     

Magnetic biosensor for the detection of Yersinia pestis

A novel type of magnetic-beads based magnetic biosensor is described for the detection of Yersinia pestis. Experiments were performed with the antigen fraction F1 of these bacteria. The magnetic sensor platform offers easy and reliable detection of Y. pestis by the use of magnetic beads for labelling and quantification in a single step due to their paramagnetic features. The system uses antiYPF1 antibodies as capture element on ABICAP columns as core element of the magnetic sensor. Several immobilization methods for antibodies on polyethylene were exploited. The established biosensor has a linear detection range of 25-300 ng/ml Y. pestis antigen F1 and a detection limit of 2.5 ng/ml in buffer and human blood serum. The presented sensor system is small, simple, portable and therefore usable as off-lab detection unit for medical and warfare analytes.     

An exact sampling formula for the Wright-Fisher model and a solution to a conjecture about the finite-island model

An exact sampling formula for a Wright-Fisher population of fixed size N under the infinitely many neutral alleles model is deduced. This extends the Ewens formula for the configuration of a random sample to the case where the sample is drawn from a population of small size, that is, without the usual large-N and small-mutation-rate assumption. The formula is used to prove a conjecture ascertaining the validity of a diffusion approximation for the frequency of a mutant-type allele under weak selection in segregation with a wild-type allele in the limit finite-island model, namely, a population that is subdivided into a finite number of demes of size N and that receives an expected fraction m of migrants from a common migrant pool each generation, as the number of demes goes to infinity. This is done by applying the formula to the migrant ancestors of a single deme and sampling their types at random. The proof of the conjecture confirms an analogy between the island model and a random-mating population, but with a different timescale that has implications for estimation procedures.     

Fatty acid-induced defects in insulin signalling, in myotubes derived from children, are related to ceramide production from palmitate rather than the accumulation of intramyocellular lipid

The elevation of free fatty acids (FFAs), observed in childhood obesity results in intramyocellular lipid (IMCL) accumulation with consequent insulin resistance. Using in vitro differentiated myotubes from normal weight pre-pubertal children (n = 8), we examined the effects of saturated (palmitate) and unsaturated (oleate) FFAs on insulin-stimulated AKT phosphorylation (pAKT) and IMCL accumulation. Palmitate decreased pAKT (Mean [SEM] % change pAKT with palmitate 750 microM vs. control; pThr308 site -50.5% [28.7] and pSer473 site -38.7% [11.7]; P < 0.001) with no effect on IMCL formation. Equimolar bromopalmitate did not effect pAKT and blocking ceramide production abolished the palmitate-induced reduction in signalling, suggesting that ceramide synthesis is critical for palmitate's actions. Oleate did not effect pAKT (1,000 microM oleate; pSer473 site -3.4% [11.4]; P = NS) but increased IMCL accumulation (+32.3% [7.1%]; P < 0.001). Co-administration of oleate diminished the reduction in pAKT seen with palmitate (+36.4% [23.6] vs. -13.3% [13.6]; P = 0.28), with similar IMCL levels to oleate alone. Co-administration also caused a significant reduction in 14C-ceramide synthesis from 14C-palmitate (101.6 [21.6] vs. 371.5 [122.4] DPM/mg protein; P < 0.001). In summary, palmitate appears to cause insulin resistance in children's myotubes via its metabolism to ceramide, and this process appears unrelated to IMCL formation and is ameliorated by oleate.     

A Genomic Approach to Resolving Relapse versus Reinfection among Four Cases of Buruli Ulcer

Background

Increased availability of Next Generation Sequencing (NGS) techniques allows, for the first time, to distinguish relapses from reinfections in patients with multiple Buruli ulcer (BU) episodes.

Methodology

We compared the number and location of single nucleotide polymorphisms (SNPs) identified by genomic screening between four pairs of Mycobacterium ulcerans isolates collected at the time of first diagnosis and at recurrence, derived from a collection of almost 5000 well characterized clinical samples from one BU treatment center in Benin.

Principal Findings

The findings suggest that after surgical treatment—without antibiotics—the second episodes were due to relapse rather than reinfection. Since specific antibiotics were introduced for the treatment of BU, the one patient with a culture available from both disease episodes had M. ulcerans isolates with a genomic distance of 20 SNPs, suggesting the patient was most likely reinfected rather than having a relapse.

Conclusions

To our knowledge, this study is the first to study recurrences in M. ulcerans using NGS, and to identify exogenous reinfection as causing a recurrence of BU. The occurrence of reinfection highlights the contribution of ongoing exposure to M. ulcerans to disease recurrence, and has implications for vaccine development.     

Global assessment of existing HIV and key population stigma indicators: A data mapping exercise to inform country-level stigma measurement

BackgroundStigma is an established barrier to the provision and uptake of HIV prevention, diagnostic, and treatment services. Despite consensus on the importance of addressing stigma, there are currently no country-level summary measures to characterize stigma and track progress in reducing stigma around the globe. This data mapping exercise aimed to assess the potential for existing data to be used to summarize and track stigma, including discrimination, related to HIV status, or key population membership at the country level.Methods and findingsThis study assessed existing indicators of stigma related to living with HIV or belonging to 1 of 4 key populations including gay men and other men who have sex with men, sex workers, people who use drugs, and transgender persons. UNAIDS Strategic Information Department led an initial drafting of possible domains, subdomains, and indicators, and a 3-week e-consultation was held to provide feedback. From the e-consultation, 44 indicators were proposed for HIV stigma; 14 for sexual minority stigma (including sexual behavior or orientation) related to men who have sex with men; 12 for sex work stigma; 10 for drug use stigma; and 17 for gender identity stigma related to transgender persons. We conducted a global data mapping exercise to identify and describe the availability and quality of stigma data across countries with the following sources: UNAIDS National Commitments and Policies Instrument (NCPI) database; Multiple Indicator Cluster Surveys (MICS); Demographic and Health Surveys (DHS); People Living with HIV Stigma Index surveys; HIV Key Populations Data Repository; Integrated Biological and Behavioral Surveys (IBBS); and network databases. Data extraction was conducted between August and November 2020. Indicators were evaluated based on the following: if an existing data source could be identified; the number of countries for which data were available for the indicator at present and in the future; variation in the indicator across countries; and considerations of data quality or accuracy. This mapping exercise resulted in the identification of 24 HIV stigma indicators and 10 key population indicators as having potential to be used at present in the creation of valid summary measures of stigma at the country level. These indicators may allow assessment of legal, societal, and behavioral manifestations of stigma across population groups and settings. Study limitations include potential selection bias due to available data sources to the research team and other biases due to the exploratory nature of this data mapping process.ConclusionsBased on the current state of data available, several indicators have the potential to characterize the level and nature of stigma affecting people living with HIV and key populations across countries and across time. This exercise revealed challenges for an empirical process reliant on existing data to determine how to weight and best combine indicators into indices. However, results for this study can be combined with participatory processes to inform summary measure development and set data collection priorities going forward.

Carrie Lyons and co-workers report on population-level indicators of stigma for people with or at risk of HIV infection.     

A comparison of blood pressure measurement over a sleeved arm versus a bare arm

Background

The measurement of blood pressure is a common clinical exam with important health consequences. We sought to determine whether the measurement of blood pressure over a sleeved arm varies from that taken on a bare arm.

Methods

We recruited 376 patients between 18 and 85 years of age from a family medicine clinic between September 2004 and November 2006. They all had their blood pressure recorded using the same automatic oscillometric device, with the cuff placed over their bare arms for the first reading. Each patient was then randomly assigned to either the bare-arm group, for which the second blood pressure reading was also taken on a bare arm, or the sleeved-arm group, for which the second reading was taken with the cuff placed over the patient''s sleeve.

Results

The mean age of the 376 participants was 61.6 years (standard deviation 15.0), 61% of the participants were male, 41% had hypertension and 11.7% had diabetes. We found no clinically important differences between the bare-arm group (n = 180) and the sleeved-arm group (n = 196) in age, sex or body mass index. The mean differences between the first and second readings for patients in the bare-arm group were 4.1 mm Hg (95% confidence interval [CI] 2.8 to 5.5) for systolic blood pressure and 0.1 mm Hg (95% CI –0.7 to 0.9) for diastolic blood pressure. The mean differences between the first and second readings for patients in the sleeved-arm group were 3.4 mm Hg (95% CI 2.1 to 4.7) for systolic blood pressure and 0.4 mm Hg (95% CI –0.4 to 1.3) for diastolic blood pressure. The between-group differences in these values was 0.76 mm Hg (95% CI –1.13 to 2.65) for systolic and –0.31 mm Hg (95% CI –1.48 to 0.86) for diastolic blood pressure; neither of these differences was clinically important or statistically significant.

Interpretation

We found that there was no significant difference in blood pressure recorded over a sleeve or on a bare arm. For practical purposes, the decision to measure blood pressure on a bare arm or over a sleeved arm should be left to the judgment of the health care professional taking the blood pressure.The measurement of blood pressure is one of the most common examinations undertaken in family practice and has important health and management consequences for the patient. Accurate assessment of blood pressure, therefore, is very important. Current guidelines outline standards for obtaining accurate and reproducible blood pressure measurements.¹ These standards include guidelines for the sizes of blood pressure cuffs and the position of the patient''s arm, as well as a recommendation that the patient be seated for 5 minutes before his or her blood pressure is taken. In addition, it has been recommended that blood pressure be measured over the patient''s bare arm.¹ However, the current recommended method to determine blood pressure² has several limitations (e.g., high variability of blood pressure at different times of day or if measured only once, loss of proper technique post-training).Three previous studies have assessed whether blood pressure varies significantly when taken over a sleeved arm compared with a bare arm. Details of the literature review are described in Appendix 1, available online at www.cmaj.ca/cgi/content/full/178/5/585/DC2. In one study, involving 36 patients, Holleman et al³ found no significant differences in systolic or diastolic blood pressure readings taken over the sleeved or bare arm of each patient. However, this study was limited because of its small sample and because blood pressure measurements were taken on both arms simultaneously. A study by Kahan et al,⁴ involving 201 patients, compared blood pressure measurements taken over a sleeved arm, a bare arm and below a rolled-up sleeve. They found that the degree of clothing under the sphygmomanometer cuff did not have a clinically important effect on the reading. Although they found no significant difference in the effect of clothing on blood pressure readings, the study was limited by their design of measuring blood pressure below a rolled-up sleeve. The third study, conducted by Liebl et al,⁵ was published while our study was in progress. Their study, which involved 201 patients, compared blood pressure measurements taken over a sleeved arm and a bare arm with both sphygmomanometric and oscillometric devices. They concluded no significant difference in readings between the sleeved and bare-arm groups. In contrast to previous studies, we sought to determine whether the measurement of blood pressure over a sleeved arm varies from that taken over a bare arm through the use of a control group in which patients'' blood pressure was taken only over a bare arm.