Identity by descent fine mapping of familial adult myoclonus epilepsy (FAME) to 2p11.2–2q11.2

Familial adult myoclonus epilepsy (FAME) is a rare autosomal dominant disorder characterized by adult onset, involuntary muscle jerks, cortical myoclonus and occasional seizures. FAME is genetically heterogeneous with more than 70 families reported worldwide and five potential disease loci. The efforts to identify potential causal variants have been unsuccessful in all but three families. To date, linkage analysis has been the main approach to find and narrow FAME critical regions. We propose an alternative method, pedigree free identity-by-descent (IBD) mapping, that infers regions of the genome between individuals that have been inherited from a common ancestor. IBD mapping provides an alternative to linkage analysis in the presence of allelic and locus heterogeneity by detecting clusters of individuals who share a common allele. Succeeding IBD mapping, gene prioritization based on gene co-expression analysis can be used to identify the most promising candidate genes. We performed an IBD analysis using high-density single nucleotide polymorphism (SNP) array data followed by gene prioritization on a FAME cohort of ten European families and one Australian/New Zealander family; eight of which had known disease loci. By identifying IBD regions common to multiple families, we were able to narrow the FAME2 locus to a 9.78 megabase interval within 2p11.2–q11.2. We provide additional evidence of a founder effect in four Italian families and allelic heterogeneity with at least four distinct founders responsible for FAME at the FAME2 locus. In addition, we suggest candidate disease genes using gene prioritization based on gene co-expression analysis.     

PSITE vectors for stable integration or transient expression of autofluorescent protein fusions in plants: probing Nicotiana benthamiana-virus interactions

Plant functional proteomics research is increasingly dependent upon vectors that facilitate high-throughput gene cloning and expression of fusions to autofluorescent proteins. Here, we describe the pSITE family of plasmids, a new set of Agrobacterium binary vectors, suitable for the stable integration or transient expression of various autofluorescent protein fusions in plant cells. The pSITE vectors permit single-step Gateway-mediated recombination cloning for construction of binary vectors that can be used directly in transient expression studies or for the selection of transgenic plants on media containing kanamycin. These vectors can be used to express native proteins or fusions to monmeric red fluorescent protein or the enhanced green fluorescent protein and its cyan and yellow-shifted spectral variants. We have validated the vectors for use in transient expression assays and for the generation of transgenic plants. Additionally, we have generated markers for fluorescent highlighting of actin filaments, chromatin, endoplasmic reticulum, and nucleoli. Finally, we show that pSITE vectors can be used for targeted gene expression in virus-infected cells, which should facilitate high-throughput characterization of protein dynamics in host-virus interactions.     

Optimizing recombinant protein expression via automated induction profiling in microtiter plates at different temperatures

Background

Escherichia coli (E. coli) is the most abundant expression host for recombinant proteins. The production efficiency is dependent on a multitude of parameters. Therefore, high-throughput applications have become an increasingly frequent technique to investigate the main factors. Within this study, the effects of temperature, induction time and inducer concentration on the metabolic state and the product formation were extensively examined. Induction profiling of E. coli Tuner(DE3) pRhotHi-2-EcFbFP was performed in 48-well Flowerplates and standard 96-well plates using a robotic platform. In parallel shake flask cultivations, the respiration activity of the microorganisms was analyzed. Therefore, two online-monitoring systems were applied: the BioLector for microtiter plates and the RAMOS-device for shake flasks. The impact of different induction conditions on biomass and product formation as well as on the oxygen transfer rate was surveyed.

Results

Different optimal induction conditions were obtained for temperatures of 28, 30, 34, and 37 °C. The best inducer concentrations were determined to be between 0.05 and 0.1 mM IPTG for all investigated temperatures. This is 10–20 times lower than conventional guidelines suggest. The induction time was less relevant when the correct inducer concentration was chosen. Furthermore, there was a stronger impact on growth and respiration activity at higher temperatures. This indicated a higher metabolic burden. Therefore, lower IPTG concentrations were advantageous at elevated temperatures. Very similar results were obtained in standard 96-well plates.

Conclusion

Two online-monitoring systems were successfully used to investigate the optimal induction conditions for the E. coli Tuner(DE3) pRhotHi-2-EcFbFP strain (lacY deletion mutant) at four different temperatures. The experimental effort was reduced to a minimum by integrating a liquid handling robot. To reach the maximum product formation, a detailed induction analysis was necessary. Whenever the cultivation temperature was changed, the induction conditions have to be adapted. Due to the experimental options provided by the BioLector technology, it was found that the higher the cultivation temperature, the lower the inducer concentration that has to be applied.

     

RADX interacts with single‐stranded DNA to promote replication fork stability

Single‐stranded DNA (ssDNA) regions form as an intermediate in many DNA‐associated transactions. Multiple cellular proteins interact with ssDNA via the oligonucleotide/oligosaccharide‐binding (OB) fold domain. The heterotrimeric, multi‐OB fold domain‐containing Replication Protein A (RPA) complex has an essential genome maintenance role, protecting ssDNA regions from nucleolytic degradation and providing a recruitment platform for proteins involved in responses to replication stress and DNA damage. Here, we identify the uncharacterized protein RADX (CXorf57) as an ssDNA‐binding factor in human cells. RADX binds ssDNA via an N‐terminal OB fold cluster, which mediates its recruitment to sites of replication stress. Deregulation of RADX expression and ssDNA binding leads to enhanced replication fork stalling and degradation, and we provide evidence that a balanced interplay between RADX and RPA ssDNA‐binding activities is critical for avoiding these defects. Our findings establish RADX as an important component of cellular pathways that promote DNA replication integrity under basal and stressful conditions by means of multiple ssDNA‐binding proteins.     

Scaling up target regimens for tuberculosis preventive treatment in Brazil and South Africa: An analysis of costs and cost-effectiveness

BackgroundShorter, safer, and cheaper tuberculosis (TB) preventive treatment (TPT) regimens will enhance uptake and effectiveness. WHO developed target product profiles describing minimum requirements and optimal targets for key attributes of novel TPT regimens. We performed a cost-effectiveness analysis addressing the scale-up of regimens meeting these criteria in Brazil, a setting with relatively low transmission and low HIV and rifampicin-resistant TB (RR-TB) prevalence, and South Africa, a setting with higher transmission and higher HIV and RR-TB prevalence.Methods and findingsWe used outputs from a model simulating scale-up of TPT regimens meeting minimal and optimal criteria. We assumed that drug costs for minimal and optimal regimens were identical to 6 months of daily isoniazid (6H). The minimal regimen lasted 3 months, with 70% completion and 80% efficacy; the optimal regimen lasted 1 month, with 90% completion and 100% efficacy. Target groups were people living with HIV (PLHIV) on antiretroviral treatment and household contacts (HHCs) of identified TB patients. The status quo was 6H at 2019 coverage levels for PLHIV and HHCs. We projected TB cases and deaths, TB-associated disability-adjusted life years (DALYs), and costs (in 2020 US dollars) associated with TB from a TB services perspective from 2020 to 2035, with 3% annual discounting. We estimated the expected costs and outcomes of scaling up 6H, the minimal TPT regimen, or the optimal TPT regimen to reach all eligible PLHIV and HHCs by 2023, compared to the status quo. Maintaining current 6H coverage in Brazil (0% of HHCs and 30% of PLHIV treated) would be associated with 1.1 (95% uncertainty range [UR] 1.1–1.2) million TB cases, 123,000 (115,000–132,000) deaths, and 2.5 (2.1–3.1) million DALYs and would cost $1.1 ($1.0–$1.3) billion during 2020–2035. Expanding the 6H, minimal, or optimal regimen to 100% coverage among eligible groups would reduce DALYs by 0.5% (95% UR 1.2% reduction, 0.4% increase), 2.5% (1.8%–3.0%), and 9.0% (6.5%–11.0%), respectively, with additional costs of $107 ($95–$117) million and $51 ($41–$60) million and savings of $36 ($14–$58) million, respectively. Compared to the status quo, costs per DALY averted were $7,608 and $808 for scaling up the 6H and minimal regimens, respectively, while the optimal regimen was dominant (cost savings, reduced DALYs). In South Africa, maintaining current 6H coverage (0% of HHCs and 69% of PLHIV treated) would be associated with 3.6 (95% UR 3.0–4.3) million TB cases, 843,000 (598,000–1,201,000) deaths, and 36.7 (19.5–58.0) million DALYs and would cost $2.5 ($1.8–$3.6) billion. Expanding coverage with the 6H, minimal, or optimal regimen would reduce DALYs by 6.9% (95% UR 4.3%–95%), 15.5% (11.8%–18.9%), and 38.0% (32.7%–43.0%), respectively, with additional costs of $79 (−$7, $151) million and $40 (−$52, $140) million and savings of $608 ($443–$832) million, respectively. Compared to the status quo, estimated costs per DALY averted were $31 and $7 for scaling up the 6H and minimal regimens, while the optimal regimen was dominant. Study limitations included the focus on 2 countries, and no explicit consideration of costs incurred before the decision to prescribe TPT.ConclusionsOur findings suggest that scale-up of TPT regimens meeting minimum or optimal requirements would likely have important impacts on TB-associated outcomes and would likely be cost-effective or cost saving.

Placide Nsengiyumva and colleagues analyze costs and cost-effectiveness of scaling up target regimens for Tuberculosis Preventive Treatment among persons living with HIV and household contacts of TB patients in Brazil and South Africa.