Biology subjects

Yu, G.

Publications and source records attributed to Yu, G..

At least 145 records · Page 8Linked to original sources

bioRxiv2021

Analysis of Spleen Histopathology, Splenocyte composition and Hematological Parameters in Mice Infected with Plasmodium berghei K173

Malaria is a fatal disease that presents clinically as a continuum of symptoms and severity, which are determined by complex host-parasite interactions. Clearance of infection is believed to be accomplished by the spleen and mononuclear phagocytic system (MPS), both in the presence and absence of artemisinin treatment. The spleen filters infected RBCs from circulation through immune-mediated recognition of the infected RBCs followed by phagocytosis. Using different strains of mice infected with P. berghei K173 (PbK173), the mechanisms leading to splenomegaly, histopathology, splenocyte activation and proliferation, and their relationship to control of parasitemia and host mortality were examined. Survival time of mice infected with PbK173 varied, although the infection was uniformly lethal. Mice of the C57BL/6 strain were the most resistant, while mice of the strain ICR were the most susceptible. BALB/c and KM mice were intermediate. In the course of PbK173 infection, both strains of mice experienced significant splenomegaly. Parasites were observed in the red pulp at 3 days post infection in all animals. All spleens retained late trophozoite stages as well as a fraction of earlier ring-stage parasites. The percentages of macrophages in infected C57BL/6 and KM mice were higher than uninfected mice on 8 dpi. Spleens of infected ICR and KM mice exhibited structural disorganization and remodeling. Furthermore, parasitemia was significantly higher in KM versus C57BL/6 mice at 8 dpi. The percentages of macrophages in ICR infected mice were lower than uninfected mice, and the parasitemia was higher than other strains. The results presented here demonstrate the rate of splenic mechanical filtration and the splenic macrophages likely contribute to an individuals total parasite burden. This in turn can influence the pathogenesis of malaria. Finally, different genetic backgrounds of mice have different splenic mechanisms for controlling malaria infection.

microbiology↗

bioRxiv2021

Prime editing enables precise genome editing in mouse liver and retina

Prime editing can induce any small-sized genetic change without donor DNA or double strand breaks. However, it has not been investigated whether prime editing is possible in postnatal animals. Here we delivered prime editors 2 and 3 into a mouse model of hereditary tyrosinemia, a genetic liver disease, using hydrodynamic injection, which corrected the disease-causing mutation and rescued the phenotype. We also achieved prime editing in the retina and retina pigment epithelium in wild-type mice by delivering prime editor 3 using trans-splicing adeno-associated virus. Deep sequencing showed that unintended edits at or near the target site or off-target effects were not detectable except for low levels (0% to 1.2%) of indels when PE3, but not PE2, was used. Our study suggests that precise, prime editor-mediated genome editing is possible in somatic cells of adult animals.

bioengineering↗

bioRxiv2021

Sample-wise unsupervised deconvolution of complex tissues

MotivationComplex biological tissues are often a heterogeneous mixture of several molecularly distinct cell or tissue subtypes. Both subtype compositions and expressions in individual samples can vary across different biological states or conditions. Computational deconvolution aims to dissect patterns of bulk gene expression data into subtype compositions and subtype-specific expressions. Typically, existing deconvolution methods can only estimate averaged subtype-specific expressions in a population, while detecting differential expressions or co-expression networks in particular subtypes requires unique subtype expression estimates in individual samples. Different from population-level deconvolution, however, individual-level deconvolution is mathematically an underdetermined problem because there are more variables than observations. ResultsWe report a sample-wise Convex Analysis of Mixtures (swCAM) method that can estimate subtype proportions and subtype-specific expressions in individual samples from bulk tissue transcriptomes. We extend our previous CAM framework to include a new term accounting for between-sample variations and formulate swCAM as a nuclear-norm and{ell} 2,1-norm regularized matrix factorization problem. We determine hyperparameter values using a cross-validation scheme with random entry exclusion and obtain a swCAM solution using an efficient alternating direction method of multipliers. The swCAM is implemented in open-source R scripts. Experimental results on realistic simulation data show that swCAM can accurately estimate subtype-specific expressions in individual samples and successfully extract co-expression networks in particular subtypes that are otherwise unobtainable using bulk expression data. Application of swCAM to bulk-tissue data of 320 samples from bipolar disorder patients and controls identified changes in cell proportions, expression and coexpression modules in patient neurons. Mitochondria related genes showed significant changes suggesting an important role of energy dysregulation in bipolar disorder. Availability and implementationThe R Scripts of swCAM is freely available at https://github.com/Lululuella/swCAM. A users guide and a vignette are provided. Contactyuewang@vt.edu Supplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2020

IOBR: Multi-omics Immuno-Oncology Biological Research to decode tumor microenvironment and signatures

MotivationRecent advance in next generation sequencing has triggered the rapid accumulation of publicly available multi-omics datasets. The application of integrated omics to exploring robust signatures for clinical translation is increasingly highlighted, attributed to the clinical success of immune checkpoint blockade in diverse malignancies. However, effective tools to comprehensively interpret multi-omics data is still warranted to provide increased granularity into intrinsic mechanism of oncogenesis and immunotherapeutic sensitivity. ResultsWe developed a computational tool for effective Immuno-Oncology Biological Research (IOBR), providing comprehensive investigation of estimation of reported or user-built signatures, TME deconvolution and signature construction base on multi-omics data. Notably, IOBR offers batch analyses of these signatures and their correlations with clinical phenotypes, lncRNA profiling, genomic characteristics and signatures generated from single-cell RNA sequencing data in different cancer settings. Additionally, IOBR also integrates multiple existing microenvironmental deconvolution methodologies and signature construction tools for convenient comparison and selection. Collectively, IOBR is a user-friendly tool, to leverage multi-omics data to facilitate immuno-oncology exploration and unveiling of tumor-immune interactions and accelerating precision immunotherapy.

bioinformatics↗

bioRxiv2020

The Arabidopsis E3 ubiquitin ligase PUB4 regulates BIK1 homeostasis and is targeted by a bacterial type-III effector

Plant immunity is tightly controlled by a complex and dynamic regulatory network, which ensures optimal activation upon detection of potential pathogens. Accordingly, each component of this network is a potential target for manipulation by pathogens. Here, we report that RipAC, a type III-secreted effector from the bacterial pathogen Ralstonia solanacearum, targets the plant E3 ubiquitin ligase PUB4 to inhibit pattern-triggered immunity (PTI). PUB4 plays a positive role in PTI by regulating the homeostasis of the central immune kinase BIK1. Before PAMP perception, PUB4 promotes the degradation of non-activated BIK1, while, after PAMP perception, PUB4 contributes to the accumulation of activated BIK1. RipAC leads to BIK1 degradation, which correlates with its PTI-inhibitory activity. RipAC causes a reduction in pathogen-associated molecular pattern (PAMP)-induced PUB4 accumulation and phosphorylation. Our results shed light on the role played by PUB4 in immune regulation, and illustrate an indirect targeting of the immune signalling hub BIK1 by a bacterial effector.

plant biology↗

bioRxiv2020

Allantoin induces pruritus by activating MrgprD in chronic kidney disease

Chronic kidney disease is a disease with decreased, irreversible renal function. Pruritus is the most common skin symptom in patients with chronic kidney disease, especially in end-stage renal disease (AKA chronic kidney disease-associated pruritus [CKD-aP]); however, the underlying molecular and neural mechanism of the CKD-aP in patients remains obscure. Our data show that the level of allantoin increases in the serum of CKD-aP and CKD model mice. Allantoin could induce scratching behavior in mice and active DRG neurons; the calcium influx and the action potential were significantly reduced in DRG neurons of MrgprD KO or TRPV1 KO mice. U73122, an antagonist of PLC, could also block calcium influx in DRG neurons induced by allantoin. Thus, our results concluded that allantoin plays an important role in CKD-aP, mediated by MrgprD and TrpV1, in CKD patients.

pathology↗

bioRxiv2020

Cell group analysis reveals changes in upper-layer neurons associated with schizophrenia

Genome-wide association studies (GWAS) of schizophrenia (SCZ) have revealed over 100 risk loci. We investigated whether these SCZ-associated variants regulate gene expression by cell type. Using a fully unsupervised deconvolution method, we calculated gene expression by clusters of estimated cell types (cell-groups, CGs). Five CGs emerged in the dorsolateral prefrontal cortices (DLPFC) of 341 donors with and without SCZ. By mapping expression quantitative trait loci (eQTL) per CG, we partitioned the heritability of SCZ risk in GWAS by CGs. CG-specific expressions and eQTLs were replicated in both a deconvoluted bulk tissue data set with a different method and also in sorted-cell expression data. Further, we characterized CG-specific gene differential expression and cell proportion changes in SCZ brains. We found upper-layer neurons in the DLPFC to be associated with SCZ based on enrichment of SCZ heritability in eQTLs, disease-related transcriptional signatures, and decreased cell proportion. Our study suggests that neurons and related anomalous circuits in the upper layers of the DLPFC may have a major contribution to SCZ risk.

genomics↗

bioRxiv2020

Ggtree: a serialized data object for visualization of phylogenetic tree and annotation data

Ggtree supports mapping and visualizing associated external data on phylogeny with two general methods. The output of ggtree is a ggtree graphic object that can be rendered as a static image. Most importantly, the input tree and associated data that used in visualization can be extracted from the graphic object, making it an ideal data structure for publishing tree (image, tree and data in one single object) and thus enhance data reuse and analytic reproducibility.

bioinformatics↗

bioRxiv2020

A bacterial effector uncovers a metabolic pathway involved in resistance to bacterial wilt disease

Bacterial wilt caused by the soil-borne pathogen Ralstonia solanacearum is a devastating disease worldwide. Upon plant colonization, R. solanacearum replicates massively, causing plant wilting and death; collapsed infected tissues then serve as a source of inoculum. In this work, we show that the metabolic pathway mediated by pyruvate decarboxylases (PDCs), activated in response to low oxygen and involved in drought stress tolerance, contributes to resistance against bacterial wilt disease. Arabidopsis and tomato plants with deficient PDC activity are more susceptible to bacterial wilt, and treatment with either pyruvic acid or acetic acid (substrate and product of the PDC pathway, respectively) enhances resistance. An effector protein secreted by R. solanacearum, RipAK, interacts with PDCs and inhibits their oligomerisation and enzymatic activity. This work reveals a metabolic pathway involved in resistance to biotic and abiotic stresses, and a bacterial virulence strategy to promote disease and the completion of the pathogenic life cycle.

plant biology↗

bioRxiv2020

AD-linked R47H-TREM2 mutation induces disease-enhancing proinflammatory microglial states in mice and humans

The hemizygous R47H variant of TREM2, a microglia-specific gene in the brain, increases risk for late-onset Alzheimers disease (AD). In this study, we identified a subpopulation of microglia with disease-enhancing proinflammatory signatures associated with the R47H mutation in human AD brains and tauopathy mouse brains. Using transcriptomic analysis at the single-nuclei level from AD patients with the R47H or the common variant (CV)-TREM2 with matched sex, pathology and APOE status, we found that the R47H mutation was associated with cell type- and sex-specific transcriptional changes in human AD brains, with microglia exhibiting the most robust alterations. Further characterization revealed that R47H-associated microglial subpopulations had enhanced inflammatory signatures including hyperactivation of Akt, one of the signaling pathways downstream of TREM2. In a newly-generated tauopathy knock-in mouse model expressing one allele of human TREM2 (hTREM2) with either the R47H mutation or CV, R47H induced and exacerbated tau-mediated spatial memory deficits in female mice. Single-cell transcriptomic analysis of microglia from these mice also revealed transcriptomic changes induced by R47H that had significant overlaps with R47H microglia in human AD brains, including robust increases in proinflammatory cytokines, activation of Syk-Akt-signaling, and elevation of a subset of disease-associated microglial signatures. Strikingly, pharmacological Akt inhibition largely reversed the enhanced inflammatory signatures induced by R47H in primary microglia treated with tau fibrils. By unraveling the disease-enhancing properties of the R47H mutation in mouse and human, our findings shed light on an immune-linked AD subtype and provide new directions for modulating microglial immune responses to treat AD.

neuroscience↗

bioRxiv2020

Quantitative proteomics reveals the protective effects of ESD against osteoarthritis via attenuating inflammation and modulating immune response

Epimedium, Salvia miltiorrhiza, and Dioscorea nipponica Makino (ESD) have been combined to treat osteoarthritis (OA) for a long time. In this study we used quantitative proteomics to find the protective effects of ESD against OA and possible mechanism. After papain-induced rats OA model established ESD was intragastrically administrated to rats for four weeks. Label-free quantitative proteomics was used to screen the comprehensive protein profiling changes in both OA and ESD groups. After stringent filtering, 62 proteins were found to be significantly up-regulated and 208 proteins were down-regulated in OA model compared with sham-operated control. Functional analysis revealed that these OA up-regulated proteins were enriched in the activation of humoral immunity response, complement activation, leukocyte mediated immunity, acute inflammatory, endocytosis regulation, and proteolysis regulation. ESD partially recovered the protein profiling changes in OA model. The effects of ESD were also assessed by measurement of behavioral activity and pathologic changes in the joints. ESD showed protective effects in suppressing inflammation, releasing joint pain, and attenuating cartilage degradation. Our study presented that ESD as a potential candidate to alleviate OA damage by reducing inflammation and modulating of immune system.

pharmacology and toxicology↗

bioRxiv2020

Accurate recognition of colorectal cancer with semi-supervised deep learning on pathological images

BackgroundMachine-assisted recognition of colorectal cancer (CRC) has been mainly focused on supervised deep learning that suffers from a significant bottleneck of requiring massive labeled data. We hypothesize that semi-supervised deep learning leveraging a small amount of labeled data with abundant available unlabeled data can provide a powerful alternative strategy. MethodWe proposed a semi-supervised model based on the mean teacher architecture that provides pathological predictions at both patch- and patient-levels. We demonstrated the general utility of the model utilizing 13,111 CRC whole slide images from 8,803 subjects gathered from 13 independent centers. We compared our proposed method with the prevailing supervised learning and six pathologists. Two extended evaluations on 15,000 lung and 294,912 lymph node images were also performed to confirm the generality of utility of semi-supervised learning for different cancers. ResultsWith a small amount of labeled training patches ([~]3,150 labeled, [~]40,950 unlabeled or [~]6,300 labeled, [~]37,800 unlabeled), the semi-supervised learning (SSL) performed significantly better than the supervised learning (SL, which only used the labeled data) (area under the curve, AUC: 0.90 {+/-} 0.06 vs. 0.84 {+/-} 0.07, P value = 0.02 or AUC: 0.98 {+/-} 0.01 vs. 0.92 {+/-} 0.04, P value = 0.0004). Moreover, we found no significant difference between SL using massive [~]44,100 labeled patches and SSL ([~]6,300 labeled, [~]37,800 unlabeled) at patch-level diagnoses (AUC:0.98 {+/-} 0.01 vs. 0.987 {+/-} 0.01, P value = 0.134) and patient-level diagnoses (average AUC: 0.974 vs. 0.980, P value = 0.117). SSL was close to human pathologists in diagnosis performance (average AUC: 0.972 vs. 0.969). This extended evaluation on lung and lymph node also confirmed when a small amount of labeled data were used, SSL was better than SL, and achieved similar performance as that of SL with massive labeling. ConclusionsWe reported that SSL can achieve excellent performance through a multi-center study. Because SSL dramatically reduces the need and cost of pathological image annotation, it has great potential to effectively build pathological artificial intelligence (AI) platforms in practice.

pathology↗

bioRxiv2020

SGT1 is not required for bacterial PAMP-triggered immunity

Plant immune signaling activated by the perception of pathogen-associated molecular patterns (PAMPs) or effector proteins is mediated by PRRs and NLRs, respectively, and often share cellular components and downstream responses. The suppressor of the G2 allele of skp1 (SGT1) is a core immune regulator required for the activation of NLR-mediated immunity. In this work, we examined the requirement of SGT1 for PRR-mediated immune responses in both Nicotiana benthamiana and Arabidopsis. Using complementary genetic approaches, we found that SGT1 is not limiting for early PRR-dependent responses or anti-bacterial immunity. Therefore, we conclude that SGT1 does not play a significant role in bacterial PAMP-triggered immunity.

plant biology↗

bioRxiv2020

Accurate Diagnosis of Colorectal Cancer Based on Histopathology Images Using Artificial Intelligence

BackgroundAccurate and robust pathological image analysis for colorectal cancer (CRC) diagnosis is time-consuming and knowledge-intensive, but is essential for CRC patients treatment. The current heavy workload of pathologists in clinics/hospitals may easily lead to unconscious misdiagnosis of CRC based on their daily image analyses. MethodsBased on a state-of-the-art transfer-learned deep convolutional neural network in artificial intelligence (AI), we proposed a novel patch aggregation strategy for clinic CRC prediction/diagnosis using weakly labeled pathological whole slide image (WSI) patches. This approach was trained and validated using an unprecedented and enormously large number of 170,099 patches, >14,680 WSIs, from >9,631 subjects that covered diverse and representative clinical cases from multi-independent-sources across China, U.S., and Germany. ResultsOur innovative AI tool was consistently nearly perfectly agreed with (average Kappa-statistic 0.896) and even often better than most of the experienced expert pathologists when tested in diagnosing CRC WSIs from multi-centers. The average area under the receiver operating characteristics curve (AUC) of AI was greater than that of the pathologists (0.981 vs 0.970) and achieved the best performance among the application of other AI methods to CRC diagnosis. Our AI-generated heatmap highlights the image regions of cancer tissue/cells. ConclusionsThis first-ever generalizable AI system can handle large amounts of WSIs consistently and robustly without potential bias due to fatigue commonly experienced by clinical pathologists. Hence, it will drastically alleviate the heavy clinical burden of daily pathology diagnosis, and improve the treatment for CRC patients. This tool is generalizable to other cancer diagnosis based on image recognition.

pathology↗

bioRxiv2020

Global Screening of Sentrin-Specific Protease Family Substrates in SUMOylation

Post-translational modification of proteins by the addition of small ubiquitin-related modifier (SUMO) is a dynamic process, in which deSUMOylation is carried out by members of the Sentrin/SUMO-specific protease (SENP) family. While identification of SUMOylation sites at global scale has made great progress, much less effort has been made on the SENP family-dependent deSUMOylation. Here we report a dataset of 3,763 high confident SUMO1 modification sites and their dependence on the 6 members of SENP proteins. Interrogation of the dataset led to the discovery that SENP3 regulates the innate immune response via deSUMOylation of DHX15 and PCBP2 and recruitment of inflammatory molecules. Collectively, this dataset presents a site-resolved network of the SUMO-SENP system, providing information for potential substrates of the SENP proteins.

biochemistry↗

bioRxiv2020

Perturbation of GABA Biosynthesis Links Cell Cycle to Control Arabidopsis thaliana Leaf Development

To investigate the molecular mechanism underlying increasing leaf area in {gamma}-Aminobutyric acid (GABA) biosynthetic mutants, the first pair of true leaves of GABA biosynthetic mutants was measured. The results showed that the leaf blade area in GABA biosynthetic mutants was larger than that of the wild type to different extents, and the area of the leaf epidermal cells in mutants was larger than that of the wild type. DNA polyploid analysis showed that polyploid cells in GABA biosynthetic mutants were appearing earlier and more abundant than in the wild type. To check the correlation between cell size and endoreplication, the expression of factors involving endocycles, including D-type cyclin gene (CYCD3;1, CYCD3;2, CYCD3;3, and CYCD4;1) and kinase CKDA;1, were analysed by qRT-PCR. The results showed that CKDA;1 in GABA biosynthetic mutants was downregulated, and four types of CYCDs showed different expression patterns in different GABA biosynthetic mutants. Inconsistent with this result, for CCS52A (CELL CYCLE SWITCH 52A) (controlling the endocycle entry) in gad2 and gad1/gad2 mutants, the expression of CCS52A2 was significantly higher than that in the wild type. The expression of SIM (SIAMESE) and SMR (SIAMESE-RELATED), which inhibit kinase activity, were also upregulated compared with the control. To further study the possible potential relationship between GABA metabolism and endoreplication, we analysed the reactive oxygen species (ROS) levels in guard cells using ROS fluorescent probes. ROS levels were significantly higher in GABA biosynthetic mutants than the control. All results indicated that cyclin, the cyclin-dependent kinase, and its inhibitory protein were coordinated to participate in endoreplication control at the transcription level in the leaves of GABA biosynthetic mutant Arabidopsis. Contribution to the field statement{gamma}-Aminobutyric acid (GABA) metabolic pathway plays a dual role in plant development. This research investigated the perturbation of GABA biosynthesis on Arabidopsis leave endoreplication for the first time. In the GABA biosynthetic mutants, many genes, participating in cell division regulation, are coordinately transcriptionally expressed to trigger the onset and maintenance of endoreplication, and this led to the cell expansion and the increase leaf blade area. However, this initiation of endoreplication links with the decrease of endogenous GABA level and the increase Reactive oxygen species (ROS). This may be a compensation mechanism to adapt to abnormal GABA level in plant leaf development. Present evidence provided hypothesized that the normal GABA level in plant leaf development plays a brake to inhibit the immature cell expansion and differentiation, and this negative regulation functions a guarantee mechanism to watchdog the normal leaf development. In all, this contribution provides an updated perspective on the role of GABA in plant development.

plant biology↗

bioRxiv2019

The Dopamine Receptor Antagonist TFP Prevents Phenotype Conversion and Improves Survival in Mouse Models of Glioblastoma

Glioblastoma (GBM) is the deadliest adult brain cancer and all patients ultimately succumb to the disease. Radiation therapy (RT) provides survival benefit of 6 months over surgery alone but these results have not improved in decades. We report that radiation induces a glioma-initiating cell phenotype and we have identified trifluoperazine (TFP) as a compound that interferes with this phenotype conversion. TFP caused loss of radiation-induced Nanog mRNA expression, activation of GSK3 with consecutive post-translational reduction in p-Akt, Sox2 and {beta}-catenin protein levels. TFP did not alter the intrinsic radiation sensitivity of glioma-initiating cells (GICs). Continuous treatment with TFP and a single dose of radiation reduced the number of GICs in vivo and prolonged survival in syngeneic and patient-derived orthotopic xenograft (PDOX) mouse models of GBM. Our findings suggest that combination of a dopamine receptor antagonist with radiation enhances the efficacy of RT in GBM by preventing radiation-induced phenotype conversion of radiosensitive non-GICs into treatment resistant, induced GICs. SignificanceGBM is the most common and most deadly adult brain cancer. The current standard-of-care is surgery followed by RT and temozolomide, which results in a median survival time of only 15 months. The efficacy of chemotherapies and targeted therapies in GBM is very limited because most of these drugs do not pass the blood-brain-barrier. Ultimately, all patients succumb to the disease. Our study describes radiation-induced cellular plasticity as a novel resistance mechanism in GBM. We identified a dopamine receptor antagonist as a readily available, FDA-approved drug known to penetrate the blood-brain-barrier which prevents phenotype conversion of glioma cells into glioma-initiating cells and prologs survival in mouse models of GBM, thus suggesting that it will improve the efficacy of RT without increasing toxicity.

neuroscience↗

bioRxiv2019

Inland-coastal bifurcation of southern East Asians revealed by Hmong-Mien genomic history

The early history of the Hmong-Mien language family and its speakers is elusive. A good variety of Hmong-Mien-speaking groups distribute in Central China. Here, we report 903 high-resolution Y-chromosomal, 624 full-sequencing mitochondrial, and 415 autosomal samples from 20 populations in Central China, mainly Hunan Province. We identify an autosomal component which is commonly seen in all the Hmong-Mien-speaking populations, with nearly unmixed composition in Pahng. In contrast, Hmong and Mien respectively demonstrate additional genomic affinity to Tibeto-Burman and Kra-Dai speakers. We also discover two prevalent uniparental lineages of Hmong-Mien speakers. Y-chromosomal haplogroup O2a2a1b1a1b-N5 diverged [~]2,330 years before present (BP), approximately coinciding with the estimated time of Proto-Hmong-Mien ([~]2,500 BP), whereas mitochondrial haplogroup B5a1c1a significantly correlates with Pahng and Mien. All the evidence indicates a founding population substantially contributing to present-day Hmong-Mien speakers. Consistent with the two distinct routes of agricultural expansion from southern China, this Hmong-Mien founding ancestry is phylogenetically closer to the founding ancestry of Neolithic Mainland Southeast Asians and present-day isolated Austroasiatic-speaking populations than Austronesians. The spatial and temporal distribution of the southern East Asian lineage is also compatible with the scenario of out-of-southern-China farming dispersal. Thus, our finding reveals an inland-coastal genetic discrepancy related to the farming pioneers in southern China and supports an inland southern China origin of an ancestral meta-population contributing to both Hmong-Mien and Austroasiatic speakers.

genetics↗