Biological Validation Research Articles

Repositioning the Existing Drugs for Neuroinflammation: A Fusion of Computational Approach and Biological Validation to Counter the Parkinson's Disease Progression

Repositioning the Existing Drugs for Neuroinflammation: A Fusion of Computational Approach and Biological Validation to Counter the Parkinson's Disease Progression

Synthesis and biological evaluation of aminobenzamides containing purine moiety as class I histone deacetylases inhibitors

The aminobenzamide is selective to class I histone deacetylases (HDACs) and displays unique tight-binding/slow-off HDAC-binding mechanism. Herein, we report a series of 9-substituted purine aminobenzamides that selectively inhibit class I HDACs. The activities in vitro showed compound 9d exhibited 12 folds more potent than MS-275 against HDAC1 isoform and showed excellent inhibitory activity on cancer cells, including HCT-116, MDA-MB-231, K562 cell lines. The metabolic stability of 9d was much better than that of the well-known HDAC inhibitor SAHA. Pulse exposure test of western blot assay demonstrated that 9a, 9d induced histone acetylation in a similar manner to MS-275. Further biological validation demonstrated that 9d prevented cell transition from G1 phase to S phase by reducing Cyclin D1, CDK2 and lifting p21, induced early apoptosis by upregulating BAX and downregulating Bcl-2 in HCT-116 cells.

Extracorporeal photopheresis (ECP): in vitro assessment of procedure quality

Objectives: The purpose of this work was to validate the procedure of extracorporeal photopheresis (ECP) offline after the addition of 8- MOP and UV-A (PUVA) irradiation by carrying out functional tests as recommended by the GITMO/SIdEM guidelines. There is currently no general agreement on the protocol to be adopted for the biological validation of the ECP; in fact, it is required that the test needs to be reproducible, reliable and standardisable. So, the protocol needs to be replicated in every processing laboratory.Methods: in absence of guidelines and evidence to support ECP, we have chosen to monitor cellular apoptosis induced by 8-MPO and UV-A (PUVA) irradiation, after 1 h, 48 h, and 96 h, using double marking with FITC-Annexine V and 7-AAD and consequent cytofluorimetric evaluation.Results: the functional tests utilized reproduce in vitro the 8-MOP activation effects. Our data suggest that the use of double staining with FITC-Annexine V and 7-AAD and subsequent cytofluorimetric evaluation may represent a reliable and reproducible method for ECP biological validation.Conclusions: the work allowed us to review our ECP validation procedure. We will adopt as functional test the double marking FITC- Annex V and 7-AAD and reading in cytofluorimetry at +48h post PUVA. The test needs to be performed on each enlisted patient, during the first two procedures, with each modification of the set used and at each 8-MOP batch change. We do not know what is the impact of the ECP procedure in terms of clinical efficacy or if reinfused cells are biologically inactivated or not active in the patient with GvHD. Clinical follow-up must remain the key parameter for evaluating the response to ECP treatment because even qualified therapy becomes clinically irrelevant in a patient not responding to treatment.

Characterization of DELUX: Ultraviolet light sterilization device for PFF2 / N95 masks against COVID-19

The World Health Organization (WHO) has declared a public health pandemic state due to the transmission of the new coronavirus on March 11th, 2020. COVID-19, that is caused by SARS-CoV-2, has a very broad clinical spectrum, with predominantly respiratory symptoms developments. The role of the health professionals in fighting the pandemic requires the use of Personal Protective Equipment (PPE). PFF2 / N95 masks are suitable PPEs for this purpose. Due to the high demand for PFF2 / N95 masks to fight the pandemic, there was a shortage of this PPE worldwide. This work aims to present a characterization of the device called DELUX, utilized for UVC (ultraviolet C light spectra) sterilization of PFF2 / N95 masks, and allowing to extend the time of safe use of this PPE in emergency conditions. The photometric validation of the device resulted in the verification of the emission spectrum of the lamps used in the device, and the measurement of the optical power, demonstrating the adequacy of irradiation with UVC light, with a 15-minute cycle, and safely. Biological validation showed that DELUX is capable of inactivating SARS-CoV-2 present on the surface of PFF2 / N95 masks, thus being efficient for their sterilization. The safety offered by the sterilization cycle allows to extend the safe use of those masks.

Forensic DNA phenotyping: Canis familiaris breed classification and skeletal phenotype prediction using functionally significant skeletal SNPs and indels.

This review highlights a novel application of breed identification and prediction of skeletal traits in forensic investigations using canine DNA evidence. Currently, genotyping methods used for canine breed classification involve the application of highly polymorphic short tandem repeats in addition to larger commercially available SNP arrays. Both applications face technical challenges. An additional approach to breed identification could be through genotyping SNPs and indels that characterise the array of skeletal differences displayed across domestic dog populations. Research has shown that a small number of genetic variants of large effect drive differences in skeletal phenotypes among domestic dog breeds. This feature makes functionally significant canine skeletal variants a cost-effective target for forensic investigators to classify individuals according to their breed. Further analysis of these skeletal variants would enable the prediction of external appearance. To date, functionally significant genes with genetic variants associated with differences in size, bulk, skull shape, ear shape, limb length, digit type, and tail morphology have been uncovered. Recommendations of a cost-effective genotyping method that can be readily designed and applied by forensic investigators have been given. Further advances to improve the field of canine skeletal forensic DNA phenotyping include the refinement of phenotyping methods, further biological validation of the skeletal genetic variants and establishing a publicly available database for storage of allele frequencies of the skeletal genetic variants in the wider domestic dog population.

COL17A1 germline variant p.Ser1029Ala and mucosal malignant melanoma: An autopsy study.

Collagen type XVII α1 (COL17A1) encodes a hemidesmosomal protein at the epidermal-dermal junction and its variants are implicated in blistering skin diseases. Recent experiments in rodents revealed that Col17a1 has critical roles in stem cells of epidermal origin and in melanoma carcinogenesis. In the present study, it was investigated whether germline variants in COL17A1 are associated with skin cancer and other cancer types using indexed consecutive autopsy cases from the Japanese Geriatric Single Nucleotide Polymorphism database (n=2,343; mean age, 80 years). The database included 12 patients with skin cancer. A total of 53 COL17A1 missense variants on an exome chip were analyzed. One variant, p.Ser1029Ala (rs118166857), which had a minor allele frequency of 1.0%, exhibited a nominal positive sign of association with skin cancer [Fisher's exact P=0.002, odds ratio (OR)=16.93, 95% CI: 4.44-64.64]. This variant was detected in 2/2 patients with mucosal malignant melanoma (mMM) and 1/3 patients with extramammary Paget's disease, and in none of the patients with non-melanoma cancer, e.g., squamous cell and basal cell carcinoma. Other cancer types were searched in the database and the p.Ser1029Ala variant was indicated to be nominally associated with breast cancer (P=0.006, OR=4.17, 95% CI: 1.72-10.11). In the two mMM cases, targeted exome sequencing of 55 cancer-predisposing genes (including tumor protein 53, BRCA1/2 and mismatch repair genes) detected no apparent pathogenic variants, but revealed variants of unknown significance in axin 2, DNA directed polymerase ζ catalytic subunit and contactin 6. Since COL17A1 provides a niche for melanocyte stem cells, it was hypothesized that the p.Ser1029Ala variant in the COL17A1 ectodomain may affect the microenvironment, e.g., the cell competition. This is a working hypothesis generated from human autopsy cases and warrants further epidemiological and molecular biological validation.

Anticancer Action of Xiaoxianxiong Tang in Non-Small Cell Lung Cancer by Pharmacological Analysis and Experimental Validation

Xiaoxianxiong Tang (XXXT) is a well-known traditional Chinese medicine formula. Evidence is emerging supporting the benefits of XXXT in ameliorating therapy for non-small cell lung cancer (NSCLC). The purpose of this study aimed to explore the effects and mechanisms of XXXT through network pharmacological analysis and biological validation. TCMSP database was used to identify potentially active compounds in XXXT with absorption, distribution, metabolism, excretion screening, and their potential targets. The disease targets related to NSCLC were predicted by searching for Therapeutic Target database, GeneCards database, DrugBank database, and DisGeNET database. Of the 4385 NSCLC-related targets, 156 targets were also the targets of compounds present in XXXT. Subsequently, GO function and KEGG pathway enrichment and PPI network analyses revealed that, of the 95 targets and 20 pathways influenced by 20 ingredients in XXXT, 20 targets were associated with patient survival, and XXXT could exert an inhibitory action on the PI3K-AKT signaling pathway. Moreover, XXXT restrained the proliferation of A549 and H460 cells in a concentration-dependent manner and suppressed the mRNA and protein levels of key targets CCNA2, FOSL2, and BIRC5 closely linked to the PI3K-AKT pathway. Hence, XXXT has the potential to improve therapy for NSCLC by targeting the PI3K-AKT signaling pathway.

Bladder Exposure to Gardnerella Activates Host Pathways Necessary for Escherichia coli Recurrent UTI.

Recurrent urinary tract infections (rUTI) are a costly clinical problem affecting millions of women worldwide each year. The majority of rUTI cases are caused by uropathogenic Escherichia coli (UPEC). Data from humans and mouse models indicate that some instances of rUTI are caused by UPEC emerging from latent reservoirs in the bladder. Women with vaginal dysbiosis, typically characterized by high levels of Gardnerella and other anaerobes, are at increased risk of UTI. Multiple studies have detected Gardnerella in urine collected by transurethral catheterization (to limit vaginal contamination), suggesting that some women experience routine urinary tract exposures. We recently reported that inoculation of Gardnerella into the bladder triggers rUTI from UPEC bladder reservoirs in a mouse model. Here we performed whole bladder RNA-seq to identify host pathways involved in Gardnerella-induced rUTI. We identified a variety host pathways differentially expressed in whole bladders following Gardnerella exposure, such as pathways involved in inflammation/immunity and epithelial turnover. At the gene level, we identified upregulation of Immediate Early (IE) genes, which are induced in various cell types shortly following stimuli like infection and inflammation. One such upregulated IE gene was the orphan nuclear receptor Nur77 (aka Nr4a1). Pilot experiments in Nur77-/- mice suggest that Nur77 is necessary for Gardnerella exposure to trigger rUTI from UPEC reservoirs. These findings demonstrate that bladder gene expression can be impacted by short-lived exposures to urogenital bacteria and warrant future examination of responses in distinct cell types, such as with single cell transcriptomic technologies. The biological validation studies in Nur77-/- mice lay the groundwork for future studies investigating Nur77 and the Immediate Early response in rUTI.

Targeted and untargeted mass spectrometry reveals impact of high fat diet on peripheral amino acid regulation in a mouse model of Alzheimer's disease

AbstractBackgroundMass spectrometry‐based metabolomics analyses were performed to examine metabolic changes under diet‐induced obesity in Alzheimer's Disease (AD) and assess whether these changes are reversible with diet modification. Specifically, amino acid metabolism was investigated because amino acid levels are related to obesity/diabetes and elevated levels have been shown to induce many of the pathophysiological hallmarks of AD.MethodTargeted hydrophilic interaction liquid chromatography‐triple quadrupole mass spectrometry (HILIC‐MS/MS) and untargeted reversed‐phase liquid chromatography‐high resolution tandem mass spectrometry (RPLC‐HRMS/MS) assays were developed to analyze the metabolic changes that occur in AD and obesity. Frozen liver samples were obtained from a previously defined study, in which APPSwe/PS1ΔE9 (APP/PSEN1) transgenic mice (to represent familial or early‐onset AD) and wild‐type litter mater controls were fed either a high‐fat diet (HFD, 60% kcal from lard), low‐fat diet (LFD, 10% kcal from lard), or reversal diet (REV, high‐fat for 7.5 months followed by low‐fat for 2.5 months). Liver samples were collected after sacrifice and were prepared through homogenization and an established protein precipitation protocol.ResultMultiple amino acids (including alanine, glutamic acid, leucine, isoleucine, and phenylalanine), carnitines, and members of the fatty acid oxidation pathway were significantly increased in APP/PSEN1 mice on HFD compared to LFD. More substantial effects and changes were observed in the APP/PSEN1 mice than WT mice, suggesting that they were more sensitive to a HFD. These dysregulated peripheral pathways include numerous amino acid pathways and fatty acid beta oxidation and suggest that obesity combined with AD further enhances cognitive impairment. These dysregulated peripheral pathways include pathways directly linked to the TCA cycle and mitochondrial dysfunction, which suggest that the HFD may contribute to AD pathogenesis by further contributing to this mitochondrial dysfunction. Furthermore, partial reversibility of many altered pathways was observed, which highlights that diet change can mitigate metabolic effects of AD. The same trends in individual amino acids were observed in both strategies, highlighting the biological validity of the results.ConclusionOur targeted and untargeted metabolomics results suggest that numerous peripheral pathways, specifically amino acid metabolism and fatty acid metabolism, were significantly affected in a combinatorial fashion by AD genotype and diet.

PubMed Text Data Mining Automation for Biological Validation on Lists of Genes and Pathways

Abstract — A prognostic cancer marker is helpful in oncology to identify the abnormal cancer cells from the collected sample. This marker can be used as an indicator to determine a disease outcome, cancer treatment, and drug discovery. Identifying cancer markers is also beneficial to improve cancer patients’ survival rate in receiving the treatment decision-making. Cancer markers can be determined by testing every gene or pathway in the wet lab manually or using the text mining automation method. The use of text mining techniques effectively investigates hidden information and gathers new knowledge from many existing sources. Unfortunately, querying relevant text to excavate important information is a challenging task. PubMed text data mining is one of the applications that help explore potential cancer markers as the trend of scientific articles in PubMed is steadily increased. Besides, it can support biologists to concentrate on the identified small set of genes or pathways. PubMed identifiers (PMIDs) are then obtained as evidence to ascertain the relationship between diseases and genes (or pathways) used as biological validation. Thus, this technique can discover the biological relationship between disease and genes or pathways. Therefore, the PubMed text data mining automation is invented to link to the websites for saving time instead of manually.
 Keywords — PubMed, text data mining, biological validation, cancer markers, diseases, genes, pathways.

Pengembangan Modul Biologi Berbasis Problem Solving Pada Materi Sistem Pencernaan Pada Manusia Untuk Kelas VIII SMP Negeri 3 Kodi

One of the alternative learning resources that can be used as a solution to these problems is compiling modules that suit the needs of students. Besides being interesting, the modules applied can be combined with learning models that can further motivate students. One of these models is problem solving. The objectives of this study are, (1) analysis of the need for the development of problem solving-based biology modules on human digestive materials for grade VIII students of SMP Negeri 3 Kodi, and (2) development of problem solving-based biology modules on human digestive materials for grade VIII students of State Junior High Schools. 3 Kodi that meet the criteria are valid, practical and effective. This research method is research and development (Research and Development). The development model used in this research is the Thiagarajan 4D model, which consists of define, design, and develop stages. This research is limited to the develop stage and ends in the validation activities of material experts and media experts. The research instrument used was a needs analysis questionnaire and a validation questionnaire of experts. The data collection technique was by distributing needs analysis questionnaires to biology teachers and validation questionnaires to material experts and media experts. Data analysis technique with qualitative descriptive. The aims of this study were: (1) to determine the feasibility of developing a problem solving-based module on the human digestive system material for class VIII students of SMP Negeri 3 Kodi (2) to describe the development of a learning module on the biology of the digestive system in humans for class VIII students of SMP Negeri 3 Kodi. to determine the ability of class VIII students of SMP Negeri 3 Kodi. The results showed, (1) The results of the validation by the expert obtained a score of 62.5% included in the category worth developing after going through revisions or improvements. (2) In the practical aspect of problem solving-based modules, the overall average percentage of student response data is 3.4 or 85.9%. Based on the criteria that have been set, the student's response to the problem solving-based module developed is in the positive response criteria and the problem solving-based module developed is also included in the high practicality criteria. (3) On the aspect of effectiveness, problem solving-based modules meet the criteria of being effective because seen from the results of the student's material mastery test at the time of product distribution, the results of the student's mastery of the material show that as many as 20 of 23 students or 86.9%.

In Silico search and biological validation of MicroR171 family related to abiotic stress response in mulberry (Morus alba)

miR171 belongs to a highly conserved and ubiquitously expressed microRNA gene family across species that play a critical role in controlling plant growth and development through the regulation of the miR171-SCL (scarecrow-like proteins) module. There is limited research available on the evolutionary relationship and functional diversification of miR171 members. In this study, we identified eight miR171 genes in the mulberry genome by bioinformatics analysis that were subsequently used to compare the evolutionary levels and explore abiotic stress mechanisms mediated by mno-miR171s (Morus notabilis miR171s). The results of phylogenetic analysis showed that the mature mno-miR171 sequences have strong sequence conservation, but their critical sites also exhibit high variation leading to functional diversification. Through quantitative real-time PCR, the expression profile of each mno-miR171 was analyzed under different stress treatments. All mno-miR171s, apart from mno-miR171h, were found to be significantly up-regulated under salt and drought stress conditions. The target genes of mno-miR171a namely, Morus020885 and Morus011800, were predicted and verified using the plural RNA method. 5′-rapid amplification of complementary DNA ends assays further to reveal that the target genes could be degraded by mno-miR171a post-transcriptionally. Overexpression of mno-miR171a in Arabidopsis improved the percentage of seed germination when the seeds were grown in NaCl- and mannitol-containing media. Transgenic plants were observed to grow better under drought conditions. The expression of various stress genes was significantly higher in transgenic plants than in wild type, except ERF11. Taken together, our study confirmed that mno-miR171a enhanced plant resistance to adverse stress environments via the regulation of the SCL targets.

Development and validation of a liquid chromatography-tandem mass spectrometry method with triple-stage fragmentation to determine levetiracetam in epileptic patient serum and its application in therapeutic drug monitoring.

Levetiracetam is an antiepileptic drug that is primarily approved by the Food and Drug Administration for the treatment of focal and generalized seizures. This study describes the development and validation of a highly selective and sensitive liquid chromatography-tandem mass spectrometry method with triple-stage fragmentation to determine levetiracetam in epileptic patient serum. After simple protein precipitation, the analytes were separated on a short reversed-phase column (Agilent Poroshell 120 SB-C18 column, 4.6 × 50mm, 2.7μm) using isocratic elution with 25% 0.1% formic acid in water (solvent A) and 75% methanol (solvent B) at a flow rate of 0.8ml/min. The linear range is 0.5-50 μg/mL (R2 >0.99). All the validation data, such as lower limit of quantification, linearity, specificity, recoveries, matrix effects, and other parameters, fit the request of biological method validation guidance. Passing-Bablok regression coefficients demonstrated that there is no constant bias and no proportional bias between the liquid chromatography-tandem mass spectrometry methods with triple-stage fragmentation and liquid multiple reaction monitoring. Bland-Altman plot showed that the developed liquid chromatography-tandem mass spectrometry method with triple-stage fragmentation method is reliable and accurate to determine levetiracetam in human serum.

Polygenic risk provides biological validity for the ICHD-3 criteria among Finnish migraine families.

BackgroundMigraine is diagnosed using the extensively field-tested International Classification of Headache Disorders (ICHD-3) consensus criteria derived by the International Headache Society. To evaluate the criteria in respect to a measurable biomarker, we studied the relationship between the main ICHD-3 criteria and the polygenic risk score, a measure of common variant burden in migraine.MethodsWe used linear mixed models to study the correlation of ICHD-3 diagnostic criteria, underlying symptoms, and main diagnoses with the polygenic risk score of migraine in a cohort of 8602 individuals from the Finnish Migraine Genome Project.ResultsMain diagnostic categories and all underlying diagnostic criteria formed a consistent continuum along the increasing polygenic burden. Polygenic risk was associated with the heterogeneous clinical picture starting from the non-migraine headache (mean 0.07; 95% CI 0.02–0.12; p = 0.008 compared to the non-headache group), to probable migraine (mean 0.13; 95% CI 0.08–0.18; p < 0.001), migraine headache (mean 0.17; 95% CI 0.14–0.21; p < 0.001) and migraine with typical visual aura (mean 0.29; 95% CI 0.26–0.33; p < 0.001), all the way to the hemiplegic aura (mean 0.37; 95% CI 0.31–0.43; p < 0.001). All individual ICHD-3 symptoms and the total number of reported symptoms, a surrogate of migraine complexity, demonstrated a clear inclination with an increasing polygenic risk.ConclusionsThe complex migraine phenotype progressively follows the polygenic burden from individuals with no headache to non-migrainous headache and up to patients with attacks manifesting all the features of the ICHD-3 headache and aura. Results provide further biological support for the ICHD-3 diagnostic criteria.

The detection of specific hypermethylated WIF1 and NPY genes in circulating DNA by crystal digital PCR\u2122 is a powerful new tool for colorectal cancer diagnosis and screening

BackgroundIn oncology, liquid biopsy is of major relevance from theranostic point of view. The searching for mutations in circulating tumor DNA (ctDNA) in case of colorectal cancers (CRCs) allows the optimization of patient care. In this context, independent of mutation status biomarkers are required for its detection to confirm the presence of ctDNA in liquid biopsies. Indeed, the hypermethylation of NPY and WIF1 genes appear to be an ideal biomarker for the specific detection of ctDNA in CRCs. The objective of this work is to develop the research of hypermethylation of NPY and WIF1 by Crystal Digital PCR™ for the detection of ctDNA in CRCs.MethodsDetection of hypermethylated NPY and WIF1 was developed on Cristal digital PCR™. Biological validation was performed from a local cohort of 22 liquid biopsies and 23 tissue samples from patients with CRC. These patients were treated at the University Hospital of Besancon (France).ResultsThe local cohort study confirmed that NPY and WIF1 were significantly hypermethylated in tumor tissues compared to adjacent non-tumor tissues (WIF1 p < 0.001; NPY p < 0.001; non-parametric Wilcoxon paired-series test). Histological characteristics, tumor stages or mutation status were not correlated to the methylation profiles. On the other hand, hypermethylation of NPY or WIF1 in liquid biopsy had a 95.5% [95%CI 77–100%] sensitivity and 100% [95%CI 69–100%] specificity.ConclusionUsing Crystal digital PCR™, this study shows that hypermethylation of NPY and WIF1 are constant specific biomarkers of CRCs regardless of a potential role in carcinogenesis.

A 10-gene biosignature of tuberculosis treatment monitoring and treatment outcome prediction

The clinical utility of blood transcriptomic biosignatures for the treatment monitoring and outcome prediction of tuberculosis (TB) remains limited. In this study, we aimed to discover and validate biomarkers for pulmonary TB treatment monitoring and outcome prediction based on kinetic responses of gene expression during treatment. In particular, differentially expressed genes (DEGs) were identified by time-series comparison. Subsequently, DEGs with the monotonic expression alterations during the treatment were selected. Ten consistently down-regulated genes (CD274, KIF1B, IL15, TLR1, TLR5, FCGR1A, GBP1, NOD2, GBP2, EGF) exhibited significant potential in treatment monitoring, demonstrated via biological and technical validation. Additionally, the biosignature showed potential in predicting the cured versus relapsed patients. Furthermore, the biosignature could be utilized for TB diagnosis, latent tuberculosis infection/active TB differential diagnosis, and risk of progression to active TB. Benchmarking analysis of the 10-gene biosignature with other biosignatures showed equivalent performance in tested data sets. In conclusion, we established a 10-gene transcriptomic biosignature that represents the kinetic responses of TB treatment. Subsequent studies are warranted to validate, refine and translate the biosignature into a precise assay to assist clinical decisions in a broad spectrum of TB management.

Biological activity validation of a computationally designed Rituximab/CD3 T cell engager targeting CD20+ cancers with multiple mechanisms of action

BackgroundBispecific T cell engaging antibodies (TEAs) with one arm targeting a cancer antigen and another arm binding to CD3 have demonstrated impressive efficacy in multiple clinical studies. However, establishing a safety/efficacy balance remains challenging. For instance, some TEAs have severe safety issues. Additionally, not all patients or all cancer cells of one patient respond equally to TEAs.MethodsHere, we developed a next-generation bispecific TEA with better safety/efficacy balance and expanded mechanisms of action. Using the computer-aided antibody design strategy, we replaced heavy chain complementarity-determining regions (HCDRs) in one Rituximab arm with HCDRs from a CD3 antibody and generated a novel CD20/CD3 bispecific antibody.ResultsAfter series of computer-aided sequence optimization, the lead molecule, GB261, showed great safety/efficacy balance both in vitro and in animal studies. GB261 exhibited high affinity to CD20 and ultra-low affinity to CD3. It showed comparable T cell activation and reduced cytokine secretion compared with a benchmark antibody (BM). ADCC and CDC caused by GB261 only killed CD20+ cells but not CD3+ cells. It exhibited better RRCL cell killing than the BM in a PBMC-engrafted, therapeutic treatment mouse model and good safety in cynomolgus monkeys.ConclusionsThus, GB261 is a promising novel TEA against CD20+ cancers.

A multimodal cell census and atlas of the mammalian primary motor cortex

Here we report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties and cellular resolution input–output mapping, integrated through cross-modal computational analysis. Our results advance the collective knowledge and understanding of brain cell-type organization1–5. First, our study reveals a unified molecular genetic landscape of cortical cell types that integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a consensus taxonomy of transcriptomic types and their hierarchical organization that is conserved from mouse to marmoset and human. Third, in situ single-cell transcriptomics provides a spatially resolved cell-type atlas of the motor cortex. Fourth, cross-modal analysis provides compelling evidence for the transcriptomic, epigenomic and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types. We further present an extensive genetic toolset for targeting glutamatergic neuron types towards linking their molecular and developmental identity to their circuit function. Together, our results establish a unifying and mechanistic framework of neuronal cell-type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties.

Decrease of MYPT1 Is Critical for Impairment of NO-mediated Vasodilation in Mesenteric Artery of the Older Spontaneously Hypertensive Rats.

Nitric oxide (NO)-mediated vasodilatation is a fundamental response of vasculature, however, regulation of NO signaling pathway on resistance vessels in the older adult with hypertension is still unclear. The 16-week-spontaneously hypertensive rats (SHR), the 18-month-SHR (OldSHR), and the age-matched Wistar-Kyoto rats were used to study the changes of mesenteric resistance artery dilatation caused by sodium nitroprusside (SNP). After pre-vasoconstriction by norepinephrine (NE), the response of endothelium-denuded mesenteric artery ring to SNP was observed, and the changes in vascular response after pharmacological interventions of key nodes in the NO/sGC/cGMP/PKG1α signaling pathway were observed as well. RNA sequencing and functional enrichment analyses were used to provide information for conducting validation experiments. Vasodilation of NO in OldSHR was decreased, which significantly correlated with the reduction of PKG-mediated effect. Functional enrichment analysis of RNA sequencing showed that genes encoding important proteins such as sGC and MYPT1 (protein phosphatase 1 regulatory subunit 12A) were downregulated in OldSHR. Molecular biology validation results showed that mRNA expression of both α and β subunits of sGC were reduced, while mRNA and protein expression of PKG1α were reduced in OldSHR. More importantly, the expression of MYPT1 and pS668-MYPT1 was significantly reduced in OldSHR, even under the treatment of SNP. The experiment also revealed an enhanced cAMP system in vasodilation in hypertension, while this function was completely lost in the OldSHR. Therefore, an NO-mediated decrease in vascular smooth muscle relaxation was found in the OldSHR. The dysfunction in cGMP-PKG signaling, in particular, decreased pS668-MYPT1 was mechanistically involved.

Genomic Tools for the Identification of Loci Associated with Facial Eczema in New Zealand Sheep.

Facial eczema (FE) is a significant metabolic disease that affects New Zealand ruminants. Ingestion of the mycotoxin sporidesmin leads to liver and bile duct damage, which can result in photosensitisation, reduced productivity and death. Strategies used to manage the incidence and severity of the disease include breeding. In sheep, there is considerable genetic variation in the response to FE. A commercial testing program is available for ram breeders who aim to increase tolerance, determined by the concentration of the serum enzyme, gamma-glutamyltransferase 21 days after a measured sporidesmin challenge (GGT21). Genome-wide association studies were carried out to determine regions of the genome associated with GGT21. Two regions on chromosomes 15 and 24 are reported, which explain 5% and 1% of the phenotypic variance in the response to FE, respectively. The region on chromosome 15 contains the β-globin locus. Of the significant SNPs in the region, one is a missense variant within the haemoglobin subunit β (HBB) gene. Mass spectrometry of haemoglobin from animals with differing genotypes at this locus indicated that genotypes are associated with different forms of adult β-globin. Haemoglobin haplotypes have previously been associated with variation in several health-related traits in sheep and warrant further investigation regarding their role in tolerance to FE in sheep. We show a strategic approach to the identification of regions of importance for commercial breeding programs with a combination of discovery, statistical and biological validation. This study highlights the power of using increased density genotyping for the identification of influential genomic regions, combined with subsequent inclusion on lower density genotyping platforms.