Mechanism of 4-aminopyridine inhibition of the lysosomal channel TMEM175.

Transmembrane protein 175 (TMEM175) is an evolutionarily distinct lysosomal cation channel whose mutation is associated with the development of Parkinson's disease. Here, we present a cryoelectron microscopy structure and molecular simulations of TMEM175 bound to 4-aminopyridine (4-AP), the only known small-molecule inhibitor of TMEM175 and a broad K+ channel inhibitor, as well as a drug approved by the Food and Drug Administration against multiple sclerosis. The structure shows that 4-AP, whose mode of action had not been previously visualized, binds near the center of the ion conduction pathway, in the open state of the channel. Molecular dynamics simulations reveal that this binding site is near the middle of the transmembrane potential gradient, providing a rationale for the voltage-dependent dissociation of 4-AP from TMEM175. Interestingly, bound 4-AP rapidly switches between three predominant binding poses, stabilized by alternate interaction patterns dictated by the twofold symmetry of the channel. Despite this highly dynamic binding mode, bound 4-AP prevents not only ion permeation but also water flow. Together, these studies provide a framework for the rational design of novel small-molecule inhibitors of TMEM175 that might reveal the role of this channel in human lysosomal physiology both in health and disease.

Equitable substance use treatment for migrants and ethnic minorities: the entwinement of micro and meso barriers and facilitators.

In Europe, the prerequisites of equitable substance use treatment (SUT) for migrants and ethnic minorities (MEM) remain understudied. This qualitative study maps barriers and facilitators identified by 14 professionals in Flanders, Belgium. The analysis identified micro and meso level barriers and how they intersect. Whereas barriers to treatment are often attributed to the client (vulnerabilities, language, trust, knowledge) our results demonstrate that they are also rooted in services (lack of expertise, issues with interpreters, diversity policies, waiting list and referral bias). These results emphasize the responsibility of meso and macro policymaking in resolving treatment mismatch problems.

Effects of Different Basal Cell Culture Media upon the Osteogenic Response of hMSCs Evaluated by 99mTc-HDP Labeling.

The osteogenic differentiation of mesenchymal stem cells is now a standard procedure in modern bone tissue engineering. As this is a promising field for future clinical applications, many cell culture media exist to promote osteogenic differentiation. Prior to differentiation, cells must be expanded to obtain sufficient numbers for experiments. Little evidence is available regarding the optimal media combination for expansion and differentiation to maximize the osteogenic response. Therefore, human BM-MSCs (n = 6) were expanded in parallel in DMEM (Dulbecco's Modified Eagle Medium) LG (Low Glucose) and α-MEM (Minimum Essential Media alpha-modification), followed by simultaneous monolayer differentiation toward the osteogenic lineage in: 1. DMEM LG (Low Glucose), 2. DMEM HG (High Glucose), 3. α-MEM, 4. "Bernese medium", and 5. "Verfaillie medium", with a corresponding negative control (total 20 groups). As a marker for osteogenic differentiation, hydroxyapatite was accessed using radioactive 99mTc-HDP labeling and quantitative alizarin red staining. The results indicate that all media except "Bernese medium" are suitable for osteogenic differentiation, while there was evidence that DMEM LG is partly superior when used for expansion and differentiation of BM-hMSCs. Using "Verfaillie medium" after DMEM LG expansion led to the highest grade of osteogenic differentiation. Nevertheless, the difference was not significant. Therefore, we recommend using DMEM LG for robust osteogenic differentiation, as it is highly suitable for that purpose, economical compared to other media, and requires little preparation time.

Roles of MEM1 in safeguarding Arabidopsis genome against DNA damage, inhibiting ATM/SOG1-mediated DNA damage response, and antagonizing global DNA hypermethylation.

Arabidopsis methylation elevated mutant 1 (mem1) mutants have elevated levels of global DNA methylation. In this study, such mutant alleles showed increased sensitivity to methyl methanesulfonate (MMS). In mem1 mutants, an assortment of genes engaged in DNA damage response (DDR), especially DNA-repair-associated genes, were largely upregulated without MMS treatment, suggestive of activation of the DDR pathway in them. Following MMS treatment, expression levels of multiple DNA-repair-associated genes in mem1 mutants were generally lower than in Col-0 plants, which accounted for the MMS-sensitive phenotype of the mem1 mutants. A group of DNA methylation pathway genes were upregulated in mem1 mutants under non-MMS-treated conditions, causing elevated global DNA methylation, especially in RNA-directed DNA methylation (RdDM)-targeted regions. Moreover, MEM1 seemed to help ATAXIA-TELANGIECTASIA MUTATED (ATM) and/or SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1) to fully activate/suppress transcription of a subset of genes regulated simultaneously by MEM1 and ATM and/or SOG1, because expression of such genes decreased/increased consistently in mem1 and atm and/or sog1 mutants, but the decreases/increases in the mem1 mutants were not as dramatic as in the atm and/or sog1 mutants. Thus, our studies reveals roles of MEM1 in safeguarding genome, and interrelationships among DNA damage, activation of DDR, DNA methylation/demethylation, and DNA repair.

From lessons on the long-term effects of the preimplantation environment on later health to a "modified ART-DOHaD" animal model.

Background: At its earliest stages, mammalian embryonic development is apparently simple but vulnerable. The environment during the preimplantation period, which only lasts a couple of days, has been implicated in adult health, extending to such early stages the concept of the developmental origin of health and disease (DOHaD). Methods: In this review, we first provide a brief history of assisted reproductive technology (ART) focusing on in vitro culture and its outcomes during subsequent development mainly in mice and humans. Further, we introduce the "MEM mouse," a novel type 2 diabetes mouse model generated by in vitro culture of preimplantation embryos in alpha minimum essential medium (αMEM). Main findings: The association between ART and its long-term effects has been carefully examined for its application in human infertility treatment. The "MEM mouse" develops steatohepatitis and kidney disease with diabetes into adulthood. Conclusion: The close association between the environment of preimplantation and health in postnatal life is being clarified. The approach by which severe mouse phenotypes are successfully induced by manipulating the environment of preimplantation embryos could provide new chronic disease animal models, which we call "modified ART-DOHaD" animal models. This will also offer insights into the mechanisms underlying their long-term effects.

System-Wide Quantitative N-Glycoproteomic Analysis from K562 Cells and Mouse Liver Tissues.

As a key regulator of many biological processes, glycosylation is an essential post-translational modification (PTM) in the living system. Over 50% of human proteins are known to be glycosylated. Alterations in glycoproteins are directly linked to many diseases, making it crucial to understand system-wide glycosylation changes. The majority of known glycoproteins are from plasma membrane; however, glycosylation is a dynamic process that occurs throughout multiple subcellular organelles and involves sets of enzymes, chaperones, transporters, and sugar donor molecules. Many glycoproteins are expressed not only in plasma membranes but also in subcellular organelles. Here, we developed a mass-spectrometry-based quantitative workflow for the system-wide N-glycoproteomic analysis of membrane and cytosolic proteins extracted using a MEM-PER kit. The kit facilitates the extraction and solubilization of both membrane and cytosolic proteins in a simple, efficient, and reproducible manner. We analyzed the K562 cell line and mouse liver tissue to evaluate this approach. A total of 934 glycosites, 5154 glycopeptides, and 536 glycoproteins from the K562 cell line and a total of 1449 glycosites, 7549 glycopeptides, and 660 glycoproteins from mouse liver tissue were identified. This simple and reproducible approach provides a unique way to understand system-wide glycosylation in biological processes and enables the identification and quantitation of glycan profiles at glycosylation sites in proteins.

Evaluation of a personalized functional near infra-red optical tomography workflow using maximum entropy on the mean.

In the present study, we proposed and evaluated a workflow of personalized near infra-red optical tomography (NIROT) using functional near-infrared spectroscopy (fNIRS) for spatiotemporal imaging of cortical hemodynamic fluctuations. The proposed workflow from fNIRS data acquisition to local 3D reconstruction consists of: (a) the personalized optimal montage maximizing fNIRS channel sensitivity to a predefined targeted brain region; (b) the optimized fNIRS data acquisition involving installation of optodes and digitalization of their positions using a neuronavigation system; and (c) the 3D local reconstruction using maximum entropy on the mean (MEM) to accurately estimate the location and spatial extent of fNIRS hemodynamic fluctuations along the cortical surface. The workflow was evaluated on finger-tapping fNIRS data acquired from 10 healthy subjects for whom we estimated the reconstructed NIROT spatiotemporal images and compared with functional magnetic resonance imaging (fMRI) results from the same individuals. Using the fMRI activation maps as our reference, we quantitatively compared the performance of two NIROT approaches, the MEM framework and the conventional minimum norm estimation (MNE) method. Quantitative comparisons were performed at both single subject and group-level. Overall, our results suggested that MEM provided better spatial accuracy than MNE, while both methods offered similar temporal accuracy when reconstructing oxygenated (HbO) and deoxygenated hemoglobin (HbR) concentration changes evoked by finger-tapping. Our proposed complete workflow was made available in the brainstorm fNIRS processing plugin-NIRSTORM, thus providing the opportunity for other researchers to further apply it to other tasks and on larger populations.

Detection of functional connectivity in the brain during visuo-guided grip force tracking tasks: A functional near-infrared spectroscopy study.

The functional connectivity (FC) between multiple brain regions during tasks is currently gradually being explored with functional near-infrared spectroscopy (fNIRS). However, the FC present during grip force tracking tasks performed under visual feedback remains unclear. In the present study, we used fNIRS to measure brain activity during resting states and grip force tracking tasks at 25%, 50%, and 75% of maximum voluntary contraction (MVC) in 11 healthy subjects, and the activity was measured from four target brain regions: the left prefrontal cortex (lPFC), right prefrontal cortex (rPFC), left sensorimotor cortex (lSMC), and right sensorimotor cortex (rSMC). We determined the FC between these regions utilizing three different methods: Pearson's correlation method, partial correlation method, and a pairwise maximum entropy model (MEM). The results showed that the FC of lSMC-rSMC and lPFC-rPFC (interhemispheric homologous pairs) were significantly stronger than those of other brain region pairs. Moreover, FC of lPFC-rPFC was strengthened during the 75% MVC task compared to the other task states and the resting states. The FC of lSMC-lPFC and rSMC-rPFC (intrahemispheric region pairs) strengthened with a higher task load. The results provided new insights into the FC between brain regions during visuo-guided grip force tracking tasks.

Mice derived from in vitro αMEM-cultured preimplantation embryos exhibit postprandial hyperglycemia and higher inflammatory gene expression in peripheral leukocytes.

We examined whether peripheral leukocytes of mice derived from in vitro αMEM-cultured embryos and exhibiting type 2 diabetes had higher expression of inflammatory-related genes associated with the development of atherosclerosis. Also, we examined the impact of a barley diet on inflammatory gene expression. Adult mice were produced by embryo transfer, after culturing two-cell embryos for 48 h in either α minimal essential media (α-MEM) or potassium simplex optimized medium control media. Mice were fed either a barley or rice diet for 10 weeks. Postprandial blood glucose and mRNA levels of several inflammatory genes, including Tnfa and Nox2, in blood leukocytes were significantly higher in MEM mice fed a rice diet compared with control mice. Barley intake reduced expression of S100a8 and Nox2. In summary, MEM mice exhibited postprandial hyperglycemia and peripheral leukocytes with higher expression of genes related to the development of atherosclerosis, and barley intake reduced some gene expression.

Analysis of Stiffness of Clamped Joints versus Bolted Joints in Steel Structures by Means of Accelerometers and Shaking Table Tests.

This work analyzes the difference in stiffness in a steel laboratory structure using clamped joints or bolted joints and analyzes if the stiffness varies in the same way when the frame is subjected to external dynamic loads that bring the joint materials to their yield strength. To make this comparison, the differences between clamp joint and bolted joint were evaluated using a novel methodology based on the analysis of the structure's natural frequencies from accelerometers. To perform this comparison, several laboratory tests were carried out on a frame made by clamped joints and the same frame made by bolted joints, using a set of tests on a medium-scale shake table for this purpose. The results achieved have verified the methodology used as adequate.

A performant bridge between fixed-size and variable-size seeding.

BACKGROUND: Seeding is usually the initial step of high-throughput sequence aligners. Two popular seeding strategies are fixed-size seeding (k-mers, minimizers) and variable-size seeding (MEMs, SMEMs, maximal spanning seeds). The former strategy supports fast seed computation, while the latter one benefits from a high seed uniqueness. Algorithmic bridges between instances of both seeding strategies are of interest for combining their respective advantages. RESULTS: We introduce an efficient strategy for computing MEMs out of fixed-size seeds (k-mers or minimizers). In contrast to previously proposed extend-purge strategies, our merge-extend strategy prevents the creation and filtering of duplicate MEMs. Further, we describe techniques for extracting SMEMs or maximal spanning seeds out of MEMs. A comprehensive benchmarking shows the applicability, strengths, shortcomings and computational requirements of all discussed seeding techniques. Additionally, we report the effects of seed occurrence filters in the context of these techniques. Aside from our novel algorithmic approaches, we analyze hierarchies within fixed-size and variable-size seeding along with a mapping between instances of both seeding strategies. CONCLUSION: Benchmarking shows that our proposed merge-extend strategy for MEM computation outperforms previous extend-purge strategies in the context of PacBio reads. The observed superiority grows with increasing read size and read quality. Further, the presented filters for extracting SMEMs or maximal spanning seeds out of MEMs outperform FMD-index based extension techniques. All code used for benchmarking is available via GitHub at https://github.com/ITBE-Lab/seed-evaluation .

Involvement of MEM1 in DNA demethylation in Arabidopsis.

KEY MESSAGE: MEM1 participates in ROS1-mediated DNA demethylation pathway, and acts functionally as ROS3 to counteract the effects of RdDM pathway.mem1mutation leads to large numbers of hyper-DMRs inArabidopsisgenome. In higher plants, DNA methylation performs important functions in silencing transcribed genes and transposable elements (TEs). Active DNA demethylation mediated by REPRESSOR OF SILENCING 1 (ROS1) is able to antagonize the action of DNA methylation caused by RNA-directed DNA methylation (RdDM) pathway, which plays critical roles in keeping DNA methylation at a proper level. In this study, a new mutant named mem1 (for methylation elevated mutant 1) was isolated from a genetic screen of T-DNA insertional mutant population for lines with elevated DNA methylation at a particular locus through Chop-PCR method. MEM1 possesses a Zf-C3HC domain, and is localized in nucleus as well as highly expressed in cotyledons. Whole-genome bisulfite sequencing data showed that knockout mutation of MEM1 leads to 4519 CG, 1793 CHG and 12739 CHH hyper-DMRs (for differentially methylated regions). Further analysis indicated that there are 2751, 2216 and 2042 overlapped CG hyper-DMRs between mem1-1and three mutants, i.e. ros1-4, rdd and ros3-2, respectively; 797, 2514, and 6766 overlapped CHH hyper-DMRs were observed between mem1-1 and three such mutants, respectively; mem1 nrpd1-3 and mem1 rdm1 double mutants showed nearly complete or partial loss of hypermethylation at 4 tested loci, suggesting that MEM1 performs similar functions as DNA glycosylase/lyases in counteracting excessive DNA methylation, and MEM1 plays important roles as REPRESSOR OF SILENCING 3 (ROS3) in erasing CHH methylation caused by the RdDM pathway. Together, these data demonstrate the involvement of MEM1 in ROS1-mediated DNA demethylation pathway and functional connections between MEM1 and ROS3.

Serum-reduced media impacts on cell viability and protein expression in human lung epithelial cells.

Serum starvation is a widely used condition in molecular biology experiments. Opti-MEM is a serum-reduced media used during transfection of genetic molecules into mammalian cells. However, the impact of such media on cell viability and protein synthesis is unknown. A549 human lung epithelial cell viability and morphology were adversely affected by growing in Opti-MEM. The cellular protein levels of chloride intracellular channel protein 1, proteasome subunit alpha Type 2, and heat shock 70 kDa protein 5 were dysregulated in A549 cells after growing in serum-reduced media. Small interfering RNA transfection was done in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum, and knockdown efficacy was determined compared with Opti-MEM. Similar amounts of knockdown of the target proteins were achieved in DMEM, and cell viability was higher compared with Opti-MEM after transfection. Careful consideration of the impact of Opti-MEM media during the culture or transfection is important for experimental design and results interpretation.

The utility of whole exome sequencing in diagnosing neurological disorders in adults from a highly consanguineous population.

There is increasing evidence that whole exome sequencing (WES) has a high diagnostic yield and is cost-efficient for individuals with neurological phenotypes. However, there is limited data on the use of WES in non-Western populations, including populations with a high rate of consanguinity. Retrospective chart review was performed on 24 adults with undiagnosed neurological symptoms evaluated in genetics and neurology clinics in a tertiary care facility on the Arabian Peninsula, and had WES between 2014 and 2016. Definitive diagnoses were made in 13/24 (54%) of cases. Of these, 5/13 (38%) revealed novel pathogenic variants. Of the known 19/24 (79%) consanguineous cases, diagnostic rate was slightly higher, 11/19 (58%) as compared to 2/5 (40%) among non-consanguineous cases. Autosomal recessive disorders comprised 10/13 (77%) of molecular diagnoses, all found to be due to homozygous pathogenic variants among consanguineous cases. WES in this cohort of adults with neurological symptoms had a high diagnostic rate likely due to high consanguinity rates in this population, as evidenced by the high diagnostic rate of homozygous pathogenic variants.

Optimizing the choice of a spatial weighting matrix in eigenvector-based methods.

Eigenvector-mapping methods such as Moran's eigenvector maps (MEM) are derived from a spatial weighting matrix (SWM) that describes the relations among a set of sampled sites. The specification of the SWM is a crucial step, but the SWM is generally chosen arbitrarily, regardless of the sampling design characteristics. Here, we compare the statistical performances of different types of SWMs (distance-based or graph-based) in contrasted realistic simulation scenarios. Then, we present an optimization method and evaluate its performances compared to the arbitrary choice of the most-widely used distance-based SWM. Results showed that the distance-based SWMs generally had lower power and accuracy than other specifications, and strongly underestimated spatial signals. The optimization method, using a correction procedure for multiple tests, had a correct type I error rate, and had higher power and accuracy than an arbitrary choice of the SWM. Nevertheless, the power decreased when too many SWMs were compared, resulting in a trade-off between the gain of accuracy and the loss of power. We advocate that future studies should optimize the choice of the SWM using a small set of appropriate candidates. R functions to implement the optimization are available in the adespatial package and are detailed in a tutorial.

The main determinants of iodine in cows' milk in Switzerland are farm type, season and teat dipping.

Milk and dairy products are important iodine sources and contribute about 30-40 % of total iodine in the Swiss diet. Information about variation in milk iodine concentration (MIC) in Switzerland is limited. We examined MIC and its potential determinants in milk from organic and conventional farms. We collected bulk milk samples at 3-month intervals over 1 year from thirty-two farms throughout Switzerland and Aosta valley, North-West Italy. We sampled all feed components including tap water, collected information on farm characteristics, feeding and teat disinfection practices by questionnaire and estimated the cows' winter and summer iodine intake. Iodine in milk and feed components was measured using inductively coupled plasma MS. The overall median MIC was 87 (range 5-371) µg/l. In multivariate analysis, predictors of MIC were as follows: (1) farm type: median MIC from organic and conventional farms was 55 and 93 µg/l (P=0·022); (2) season: 53, 97 and 101 µg/l in September, December and March (P<0·002); and (3) teat dipping: 97 µg/l with v. 56 µg/l without (P=0·028). In conclusion, MIC varied widely between farms because of diverse farming practices that result in large differences in dairy cow exposure to iodine via ingestion or skin application. Standardisation of MIC is potentially achievable by controlling these iodine exposures. In order for milk to be a stable iodine source all year round, dietary iodine could be added at a set level to one feed component whose intake is regular and controllable, such as the mineral supplement, and by limiting the use of iodine-containing teat disinfectants.

Comparative study of sequence aligners for detecting antibiotic resistance in bacterial metagenomes.

We aim to compare the performance of Bowtie2, bwa-mem, blastn and blastx when aligning bacterial metagenomes against the Comprehensive Antibiotic Resistance Database (CARD). Simulated reads were used to evaluate the performance of each aligner under the following four performance criteria: correctly mapped, false positives, multi-reads and partials. The optimal alignment approach was applied to samples from two wastewater treatment plants to detect antibiotic resistance genes using next generation sequencing. blastn mapped with greater accuracy among the four sequence alignment approaches considered followed by Bowtie2. blastx generated the greatest number of false positives and multi-reads when aligned against the CARD. The performance of each alignment tool was also investigated using error-free reads. Although each aligner mapped a greater number of error-free reads as compared to Illumina-error reads, in general, the introduction of sequencing errors had little effect on alignment results when aligning against the CARD. Given each performance criteria, blastn was found to be the most favourable alignment tool and was therefore used to assess resistance genes in sewage samples. Beta-lactam and aminoglycoside were found to be the most abundant classes of antibiotic resistance genes in each sample. SIGNIFICANCE AND IMPACT OF THE STUDY: Antibiotic resistance genes (ARGs) are pollutants known to persist in wastewater treatment plants among other environments, thus methods for detecting these genes have become increasingly relevant. Next generation sequencing has brought about a host of sequence alignment tools that provide a comprehensive look into antimicrobial resistance in environmental samples. However, standardizing practices in ARG metagenomic studies is challenging since results produced from alignment tools can vary significantly. Our study provides sequence alignment results of synthetic, and authentic bacterial metagenomes mapped against an ARG database using multiple alignment tools, and the best practice for detecting ARGs in environmental samples.

[Myoclonus of the middle ear]. / Mioklonus myshts srednego ukha.

The objective of the present study was to overview the foreign literature concerning middle ear myoclonus (MEM) known to be the most common cause of the manifestations of objective tinnitus. The authors reports two typical clinical cases of myoclonus of the middle ear. The present article is aimed at the enhancement of the awareness of the otorhinolaryngologists, audiologists, and neurologists of the condition of interest as a way to promote the further progress in its treatment.

A soluble recombinant form of human leucocyte antigen-G 6 (srHLA-G6).

Human Leucocyte Antigen-G (HLA-G) is a non classical major histocompatibility complex (MHC) molecule that through RNA splicing can encode seven isoforms which are membrane bound (-G1, -G2, -G3 and -G4) and soluble (-G5, -G6 and -G7). HLA-G is described as important immune suppressor endogenous molecule to favor maternal-fetal tolerance, transplant survival and tumor immune scape. HLA-G shows low protein variability and a unique structural complexity that is related with the expression of different isoforms followed by biochemical processes, such as, proteolytic cleavage, molecular interactions, and protein ubiquitination. Studies with HLA-G have shown difficult to assess the role of the individual isoforms. Thus, the aim of this work was to obtain a HLA-G6 recombinant form. The results indicated the production of high homogeneous preparations of soluble recombinant HLA-G6 (srHLA-G6) with molecular mass 23,603.76 Da, determined by MALD-TOF/TOF. In addition, native and denatured srHLA-G6 were detected by ELISA, using commercial monoclonal antibodies. Finally, we developed a suitable methodology to express srHLA-G6 that could contribute in structural and functional studies involving specific isoforms.

A MEM1-like motif directs mesophyll cell-specific expression of the gene encoding the C4 carbonic anhydrase in Flaveria.

The first two reactions of C4 photosynthesis are catalysed by carbonic anhydrase (CA) and phosphoenolpyruvate carboxylase (PEPC) in the leaf mesophyll (M) cell cytosol. Translatome experiments using a tagged ribosomal protein expressed under the control of M and bundle-sheath (BS) cell-specific promoters showed transcripts encoding CA3 from the C4 species Flaveria bidentis were highly enriched in polysomes from M cells relative to those of the BS. Localisation experiments employing a CA3-green fluorescent protein fusion protein showed F. bidentis CA3 is a cytosolic enzyme. A motif showing high sequence homology to that of the Flaveria M expression module 1 (MEM1) element was identified approximately 2 kb upstream of the F. bidentis and F. trinervia ca3 translation start sites. MEM1 is located in the promoter of C4 Flaveria ppcA genes, which encode the C4-associated PEPC, and is necessary for M-specific expression. No MEM1-like sequence was found in the 4 kb upstream of the C3 species F. pringlei ca3 translation start site. Promoter-reporter fusion experiments demonstrated the region containing the ca3 MEM1-like element also directs M-specific expression. These results support the idea that a common regulatory switch drives the expression of the C4 Flaveria ca3 and ppcA1 genes specifically in M cells.