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Seed vigor is an important trait closely related to improved seed quality and long-term germplasm conservation, and it gradually decreases during storage, which has become a major concern for agriculture. However, the underlying regulatory mechanisms of seed vigor loss in terms of genes remain largely unknown in quinoa. Here, two cultivars of quinoa seeds with different storage performance, Longli No.4 (L4) and Longli No.1 (L1), were subjected to transcriptome sequencing to decipher the pathways and genes possibly related to vigor loss under artificial aging. Multispectral imaging features and germination phenotypes showed significantly less seed vigor loss in L1 than in L4, indicating L1 seeds having stronger aging resistance and storability. Totally, 272 and 75 differentially expressed genes (DEGs) were, respectively, identified in L4 and L1 during aging. Transcriptomic analysis further revealed the differences in metabolic pathways, especially, flavonoid biosynthesis, TCA cycle, and terpenoid backbone biosynthesis were significantly enriched in L4 seeds, while carbon metabolism in L1 seeds, which involved key genes such as CHS, CHI, AACT, ENO1, IDH, NADP-ME, and HAO2L. It indicated that the adverse effects on flavonoids and terpenoids induced by aging might be the significant reasons for more vigor loss in storage sensitive seeds, whereas storage tolerant seeds had a stronger ability to maintain carbon metabolism and energy supply. These findings elucidated the underlying molecular mechanism of seed vigor loss in quinoa, which also provided novel insights into improving seed vigor through modern molecular breeding strategies.

Legumes are important grains and forages, providing high-quality proteins, vitamins, and micronutrients to humans and animals. Medicago truncatula is a close relative of alfalfa (Medicago sativa). Caffeic acid O-methyltransferase (COMT), a key gene that is identified to be essential for melatonin synthesis, plays a significant role in plant growth, development, and abiotic stress responses. However, a systematic study on the COMT gene family in M. truncatula has still not been reported. In this study, 63 MtCOMT genes were identified and categorized into three groups. Gene structure and conserved motif analyses revealed the relative conservation of closely clustered MtCOMTs within each group. Duplicated events in MtCOMT members were identified, and segmental duplication was the main mean. Cis-acting element prediction revealed the involvement of MtCOMTs in growth and development and response to light, stress, and plant hormones. RNA-seq data analysis showed that 57 MtCOMTs varied under salt and drought stresses. The RT-qPCR expression patterns showed that MtCOMT9, MtCOMT13, MtCOMT22, MtCOMT24, MtCOMT43, and MtCOMT46 were related to salt and drought responses in M. truncatula. Additionally, Arabidopsis thaliana overexpressing MtCOMT13 displayed superior plant growth phenotypes and enhanced tolerance to salt and drought stresses through higher photosynthetic parameters and activities of antioxidant enzymes, which indicated that MtCOMT13 played an important role in positively regulating plant salt and drought tolerance. These findings contribute to an improved understanding of MtCOMTs’ roles in abiotic stress responses in M. truncatula, providing an important theoretical basis and genetic resource for legume species resistance breeding in the future.

Seed physical dormancy (hard-seededness) is an interesting ecological phenomenon and important agronomic trait. The loss of seed coat impermeability/hard-seededness is a key target trait during the domestication of leguminous crops which allows seeds to germinate rapidly and uniformly. In this study, we examined the mutation of quantitative trait locus (QTL) genes, GmHs1-1 and GmqHS1, in 18 wild soybean (G. soja) and 23 cultivated soybean (G. max) accessions. The sequencing results indicate that a G-to-T substitution in GmqHS1 and a C-to-T substitution in GmHs1-1 occurred in all 23 cultivated soybean accessions but not in any of the 18 wild soybean accessions. The mutations in the two genes led to increased seed coat permeability in cultivated soybean. Therefore, we provide evidence that two genes, GmHs1-1 and GmqHS1, simultaneously contribute to the domestication of hard-seededness in soybeans. This finding is of great significance for genetic analysis and improved utilization of the soybean hard-seededness trait.

Low temperature is the most common abiotic factor that usually occurs during the seed germination of alfalfa (Medicago sativa L.). However, the potential regulatory mechanisms involved in alfalfa seed germination under low temperature stress are still ambiguous. Therefore, to determine the relevant key genes and pathways, the phenotypic and transcriptomic analyses of low-temperature sensitive (Instict) and low-temperature tolerant (Sardi10) alfalfa were conducted at 6 and 15 h of seed germination under normal (20 °C) and low (10 °C) temperature conditions. Germination phenotypic results showed that Sardi10 had the strongest germination ability under low temperatures, which was manifested by the higher germination-related indicators. Further transcriptome analysis indicated that differentially expressed genes were mainly enriched in galactose metabolism and carbon metabolism pathways, which were the most commonly enriched in two alfalfa genotypes. Additionally, fatty acid metabolism and glutathione metabolism pathways were preferably enriched in Sardi10 alfalfa. The Weighted Gene Co-Expression Network Analysis (WGCNA) suggested that genes were closely related to galactose metabolism, fatty acid metabolism, and glutathione metabolism in Sardi10 alfalfa at the module with the highest correlation (6 h of germination under low temperature). Finally, qRT-PCR analysis further validated the related genes involved in the above pathways, which might play crucial roles in regulating seed germination of alfalfa under low temperature conditions. These findings provide new insights into the molecular mechanisms of seed germination underlying the low temperature stress in alfalfa.

Brassinosteroids (BRs) play many pivotal roles in plant growth and development, especially in cell elongation and vascular development. Although its biosynthetic and signal transduction pathway have been well characterized in model plants, their biological roles in Eucalyptus grandis, a major hardwood tree providing fiber and energy worldwide, remain unclear. Here, we treated E. grandis plantlets with 24-epibrassinolide (EBL), the most active BR and/or BR biosynthesis inhibitor brassinazole. We recorded the plant growth and analyzed the cell structure of the root and stem with histochemical methods; then, we performed a secondary growth, BR synthesis, and signaling-related gene expression analysis. The results showed that the BRs dramatically increased the shoot length and diameter, and the exogenous BR increased the xylem area of the stem and root. In this process, EgrBRI1, EgrBZR1, and EgrBZR2 expression were induced by the BR treatment, and the expressions of HD-ZIPIII and cellulose synthase genes were also altered. To further verify the effect of BRs in secondary xylem development in Eucalyptus, we used six-month-old plants as the material and directly applied EBL to the xylem and cambium of the vertical stems. The xylem area, fiber cell length, and cell numbers showed considerable increases. Several key BR-signaling genes, secondary xylem development-related transcription factor genes, and cellulose and lignin biosynthetic genes were also considerably altered. Thus, BR had regulatory roles in secondary xylem development and differentiation via the BR-signaling pathway in this woody plant.

BackgroundThe diffuse large B-cell lymphoma (DLBCL) has the highest incidence of all lymphomas worldwide. To investigate the functions of lymphocyte activation gene 3 (LAG-3) and programmed cell death 1 (PD-1) in tissues and peripheral blood of patients with DLBCL, the expression of LAG-3 and PD-1 genes in DLBCL-TCGA were analyzed.MethodsLAG-3 and PD-1 mRNA levels in DLBCL were analyzed using data from The Cancer Genome Atlas (TCGA) database. Utilize the Genotype-Tissue Expression (GTEx) database for assessing the variance in the expression of LAG-3, PD-1, and other associated factors between the tissues of DLBCL patients and healthy individuals. Immunohistochemistry was applied to detect the expression of LAG-3 and PD-1 levels in 137 cases of DLBCL tissues and 20 cases of reactive lymphoid hyperplasia. The prognostic value of LAG-3 and PD-1 were assessed using the Kaplan-Meier curve. The Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE) and ssGSEA algorithm were used to explore the immune microenvironment of DLBCL. Additionally, the expression and co-expression of LAG-3 and PD-1 were detected on CD4 and CD8 T cells in peripheral blood samples from 100 cases of DLBCL tissues and 30 cases of healthy individuals using flow cytometry.ResultsAccording to TCGA database, LAG-3 and PD-1 gene expression levels were significantly up-regulated in DLBCL tissues. LAG-3 and PD-1 levels were also strongly positively correlated with those of most infiltrating immune cells. Overall survival of patients with high LAG-3 and PD-1 co-expression was significantly shorter than that of patients with low co-expression. In DLBCL patients, LAG-3 and PD-1 were highly expressed in peripheral blood CD8+ T cells. In addition, LAG-3 was highly expressed in CD4+ T cells, while the expression of PD-1 in CD4+ T cells of DLBCL patients showed no significant difference compared to healthy individuals. Additionally, CD8+ T cells and SU-DHL6/OCI-LY3 from patients with DLBCL were co-cultured in vitro; after addition of LAG-3 and/or PD-1 inhibitors alone, an increased perforin and granzyme B secretion levels by CD8+ T cells were detected, as well as an increase in the overall proportion of tumor cells undergoing apoptosis.ConclusionHigh LAG-3 and PD-1 levels significantly inhibit CD8+ T cell function, resulting in weakened ability to kill tumor cells. Combined LAG-3 and PD-1 blockade can restore CD8+ T cell function and provides a potential avenue for development of personalized cellular immunotherapy for DLBCL.

Globally, the species of Amanita are key components of ectomycorrhizal ecosystems. Some of them are widely known as poisonous or edible fungi. Although many new Amanita species from China have been described, the species diversity of Yanshan Mountains remains unknown. We here describe three new species, namely, A. borealis sp. nov. (Sect. Amanita), A. brunneola sp. nov. (Sect. Caesareae), and A. yanshanensis sp. nov. (Sect. Validae), based on morphological observations and molecular phylogenetic analyses. In addition, nine known species, namely, A. caesareoides (Sect. Caesareae), A. chiui (Sect. Vaginatae), A. muscaria (Sect. Amanita), A. oberwinklerana (Sect. Roanokenses), A. ovalispora (Sect. Vaginatae), A. subglobosa (Sect. Amanita), A. subjunquillea (Sect. phalloideae), A. vaginata var. vaginata (Sect. Vaginatae), and A. virosa (Sect. phalloideae), were reported from Yanshan Mountains for the first time. Our results emphasize that China has a high diversity of Amanita species and that additional studies are required to understand the exact species number. These findings play a crucial role in Amanita toxin research and ecological conservation. This study investigated the areas where Amanita species-related research is lacking. The study also attempted to better understand Amanita distribution and thus contribute to related research. This study enriches the species diversity of Amanita in Yanshan Mountains and offers additional data supporting the macrofungal systematics, toxin research, and diversity and ecological studies of Amanita in future studies.

Abstract Whole‐exome sequencing of two patients with idiopathic complex neurodevelopmental disorder (NDD) identified biallelic variants of unknown significance within FIBCD1, encoding an endocytic acetyl group‐binding transmembrane receptor with no known function in the central nervous system. We found that FIBCD1 preferentially binds and endocytoses glycosaminoglycan (GAG) chondroitin sulphate‐4S (CS‐4S) and regulates GAG content of the brain extracellular matrix (ECM). In silico molecular simulation studies and GAG binding analyses of patient variants determined that such variants are loss‐of‐function by disrupting FIBCD1‐CS‐4S association. Gene knockdown in flies resulted in morphological disruption of the neuromuscular junction and motor‐related behavioural deficits. In humans and mice, FIBCD1 is expressed in discrete brain regions, including the hippocampus. Fibcd1 KO mice exhibited normal hippocampal neuronal morphology but impaired hippocampal‐dependent learning. Further, hippocampal synaptic remodelling in acute slices from Fibcd1 KO mice was deficient but restored upon enzymatically modulating the ECM. Together, we identified FIBCD1 as an endocytic receptor for GAGs in the brain ECM and a novel gene associated with an NDD, revealing a critical role in nervous system structure, function and plasticity.

Abstract Background The natural history and genotype–phenotype correlation of Pelizaeus–Merzbacher disease (PMD) of Chinese patients has been rarely reported. Method Patients who met the criteria for PMD were enrolled in our study. Genomic analysis was conducted by multiplex ligation probe amplification (MLPA) and Sanger or whole-exome sequencing (WES). Natural history differences and genotype–phenotype correlations were analyzed. Result A total of 111 patients were enrolled in our follow-up study. The median follow-up interval was 53 m (1185). Among PMD patients, developmental delay was the most common sign, and nystagmus and hypotonia were the most common initial symptoms observed. A total of 78.4% of the patients were able to control their head, and 72.1% could speak words. However, few of the patients could stand (9.0%) or walk (4.5%) by themselves. Nystagmus improved in more than half of the patients, and hypotonia sometimes deteriorated to movement disorders. More PLP1 point mutations patients were categorized into severe group, while more patients with PLP1 duplications were categorized into mild group (p

Abstract Background Congenital myopathies are a group of rare neuromuscular diseases characterized by specific histopathological features. The relationship between the pathologies and the genetic causes is complex, and the prevalence of myopathy-causing genes varies among patients from different ethnic groups. The aim of the present study was to characterize congenital myopathies with infancy onset among patients registered at our institution. Method This retrospective study enrolled 56 patients based on the pathological and/or genetic diagnosis. Clinical, histopathological and genetic features of the patients were analysed with long-term follow-up. Results Twenty-six out of 43 patients who received next-generation sequencing had genetic confirmation, and RYR1 variations (12/26) were the most prevalent. Eighteen novel variations were identified in 6 disease-causing genes, including RYR1, NEB, TTN, TNNT1, DNM2 and ACTA1. Nemaline myopathy (17/55) was the most common histopathology. The onset ages ranged from birth to 1 year. Thirty-one patients were followed for 3.83 ± 3.05 years (ranging from 3 months to 11 years). No patient died before 1 year. Two patients died at 5 years and 8 years respectively. The motor abilities were stable or improved in 23 patients and deteriorated in 6 patients. Ten (10/31) patients developed respiratory involvement, and 9 patients (9/31) had mildly abnormal electrocardiograms and/or echocardiograms. Conclusion The severity of congenital myopathies in the neonatal/infantile period may vary in patients from different ethnic groups. More concern should be given to cardiac monitoring in patients with congenital myopathies even in those with static courses.

Abstract Background Global developmental delay (GDD) has a heterogeneous clinical profile among patients, accounting for approximately 1%–3% of cases in children. An increasing number of gene defects have been demonstrated to be associated with GDD; up to now, only limited studies have reported developmental disorders driven by WDR45B. Methods Trio‐whole exome sequencing (Trio‐WES) was performed for the patient and her family. All variants with a minor allele frequency C (p. Arg226Thr)] was identified from the proband. The variant was absent in published databases such as gnomAD and Exome Aggregation Consortium (ExAC). The variant was predicted to be damaging for proteins and classified as VUS according to the ACMG guidelines. We reviewed the literature, and the development delay level in our case was less severe than the other reported cases. Conclusion We reported another case with a novel homozygous variant of WDR45B and showed the heterogeneity of clinical features.

ObjectiveTo study the burden of neurodevelopmental diseases (NDDs) via cost-of-illness analysis of Chinese patients with genetic diagnosis.MethodsWe recruited NDD patients (0–18 years old) with genetic diagnosis (GD) from September 1, 2020 to January 30, 2021. We gathered basic information on the details of diagnosis, as well as the direct medical cost, direct non-healthcare cost and indirect cost before and after receiving GD. We corrected the cost for time biases by calculating the cost per day for each patient.ResultsFor the 502 patients with NDDs, the mean age was 4.08 ± 3.47. The household income was 0.6 (0.4, 1.0) 10,000 CNY per-month on average. The direct medical cost, direct non-healthcare cost and indirect cost were 12.27 (7.36, 22.23) 10,000 CNY, 1.45 (0.73, 2.69)10,000 CNY and 14.14(4.80, 28.25) 10,000 CNY per patient, respectively. Every patient received 1.20 (0.34, 3.60) 10,000 CNY on average (15.91%) from insurance. The daily total cost after receiving GD were ~62.48% lower than those before GD (191.59 CNY vs. 71.45 CNY). The descend range of lab cost (95.77%, P < 0.05) was the largest, followed by drugs (91.39%, P < 0.05), hospitalization (90.85%, P < 0.05), and consultation (57.41%, P < 0.05). The cost of rehabilitation kept slightly increasing but there were no significant differences (P > 0.05). The daily direct medical cost of each patient fell by 75.26% (P < 0.05) from 311.79 CNY to 77.14 CNY when the diagnostic age was younger than 1, and declined by 49.30% (P < 0.05) and 8.97% (P > 0.05) when the diagnostic age was 1–3 and older than 3, respectively.ConclusionsEarly genetic diagnosis is crucial for to reducing the burden of disease because of the amount of money spent was lower when they are diagnosed at younger age. Patients with NDDs can incur a heavy economic burden, especially in rehabilitation cost and indirect cost, because the insurance coverage for patients is low, so it is urgent for governments to pay more attention to these issues.

Salt stress is a major environmental factor that seriously restricts quinoa seed germination. However, the key regulatory mechanisms underlying the effect of salt stress on the initial imbibition stage of quinoa seeds are unclear. In this study, dry seeds (0 h) and imbibed (8 h) seeds with 450 mM NaCl (artificial salt) and 100% brackish water of Yellow River Estuary (BW, natural salt) were used to assess the key salt responses based on germination, transcriptome, and metabolome analyses. The results indicated that the capacity of germinating seeds to withstand these two salt stresses was similar due to the similarities in the germination percentage, germination index, mean germination time, and germination phenotypes. Combined omics analyses revealed that the common and unique pathways were induced by NaCl and BW. Starch and sucrose metabolism were the only commonly enriched pathways in which the genes were significantly changed. Additionally, amino sugar and nucleotide sugar metabolism, and ascorbate and aldarate metabolism were preferably enriched in the NaCl group. However, glutathione metabolism tended to enrich in the BW group where glutathione peroxidase, peroxiredoxin 6, and glutathione S-transferase were significantly regulated. These findings suggest that the candidates involved in carbohydrate metabolism and antioxidant defense can regulate the salt responses of seed initial imbibition, which provide valuable insights into the molecular mechanisms underlying the effect of artificial and natural salt stresses.

Abstract Background Upon water uptake and release of seed dormancy, embryonic plant cells expand, while being mechanically constrained by the seed coat. Cortical microtubules (CMTs) are key players of cell elongation in plants: their anisotropic orientation channels the axis of cell elongation through the guidance of oriented deposition of load-bearing cellulose microfibrils in the cell wall. Interestingly, CMTs align with tensile stress, and consistently, they reorient upon compressive stress in growing hypocotyls. How CMTs first organise in germinating embryos is unknown, and their relation with mechanical stress has not been investigated at such an early developing stage. Results Here, we analysed CMT dynamics in dormant and non-dormant Arabidopsis seeds by microscopy of fluorescently tagged microtubule markers at different developmental time points and in response to abscisic acid and gibberellins. We found that CMTs first appear as very few thick bundles in dormant seeds. Consistently, analysis of available transcriptome and translatome datasets show that limiting amounts of tubulin and microtubule regulators initially hinder microtubule self-organisation. Seeds imbibed in the presence of gibberellic acid or abscisic acid displayed altered microtubule organisation and transcriptional regulation. Upon the release of dormancy, CMTs then self-organise into multiple parallel transverse arrays. Such behaviour matches the tensile stress patterns in such mechanically constrained embryos. This suggests that, as CMTs first self-organise, they also align with shape-derived tensile stress patterns. Conclusions Our results provide a scenario in which dormancy release in the embryo triggers microtubule self-organisation and alignment with tensile stress prior to germination and anisotropic growth.

Diffuse large B cell lymphoma (DLBCL) is the most common pathological subtype of non-Hodgkin lymphoma (NHL) and is the most common type of adult lymphoma. Due to the poor prognosis of relapsed/refractory DLBCL, new drug targets and therapeutic methods are urgently needed. We investigated the expression of programmed death ligand 1 (PD-L1) and 3-monooxygenase/tryptophan 5-monooxygenase activating protein zeta (14-3-3ζ or YWHAZ) in patients with DLBCL. The purpose was to verify the expression levels of YWHAZ and PD-L1 and their relationships with the prognosis of DLBCL and to lay a foundation for further study on the role of YWHAZ and PD-L1 in DLBCL. Immunohistochemistry was used in 140 patients with DLBCL to test protein expression levels of YWHAZ and PD-L1. All patients were followed up in the hospital or by telephone or via WeChat. The positive expression rate of YWHAZ was 62.14% (87/140). The expression was negatively correlated with the positive expression of BAD (r=−0.177, P=0.036) and positively correlated with the positive expression of BCL-2 (r=0.180, P=0.033). When the cut-off value for PD-L1 was established at 5%, 10%, 15%, and 20%, the corresponding positive expression rates of PD-L1 were 79.66% (94/118), 51.69% (61/118), 40.68% (48/118), and 36.44% (43/118). YWHAZ significantly affected the OS of DLBCL (P≤0.001). The prognosis of the patients was related to the positive expression of PD-L1 when the cut-off value of PD-L1 was 5% (P=0.033). However, positive expression of PD-L1 was not associated with the prognosis when the cut-off values of PD-L1 were 10% (P=0.404), 15% (P=0.208), and 20% (P=0.408). The positive expression of YWHAZ (hazard ratio 6.215; 95% confidence interval 3.214-12.017; P

Urinary benzene metabolites trans, trans-muconic acid (t, t-MA), and S-phenyl mercapturic acid (S-PMA) are often used as biomarkers of internal exposure to benzene. However, there are few reports on using urinary benzene metabolites to estimate airborne benzene concentrations in individuals exposed to benzene. In this study, t, t-MA, and S-PMA were analyzed by UPLC-MS/MS, and a simple pharmacokinetic model was used to calculate the daily intake (DI) of benzene based on the levels of urinary t, t-MA, and S-PMA in occupational individuals. The back-calculated airborne benzene levels (BCABL) were obtained from the DI of benzene. Among the exposed subjects (n = 84), the median BCABL (3.67 mg/m3) based on t, t-MA was very close to the median level of measured airborne benzene (3.27 mg/m3, p = 0.171), and there was no effect of smoking or dietary habits on t, t-MA-based BCABL. In the control subjects (n = 49), the levels of measured airborne benzene were all below the quantitation limit (0.024 mg/m3), and the BCABL (0.002–0.25 mg/m3) calculated by S-PMA was close to this background level. Our study suggests that the t, t-MA-based BCABL can reflect the actual airborne benzene level in a range of 1.10–86.91 mg/m3 and that the S-PMA-based BCABL is more reliable for non-professional benzene exposure.

Abstract Background Intellectual disability/developmental delay is a complex condition with extraordinary heterogeneity. A large proportion of patients lacks a specific diagnosis. Next generation sequencing, enabling identification of genetic variations in multiple genes, has become an efficient strategy for genetic analysis in intellectual disability/developmental delay. Methods Clinical data of 112 Chinese families with unexplained intellectual disability/developmental delay was collected. Targeted next generation sequencing of 454 genes related to intellectual disability/developmental delay was performed for all 112 index patients. Patients with promising variants and their other family members underwent Sanger sequencing to validate the authenticity and segregation of the variants. Results Fourteen promising variants in genes EFNB1, MECP2, ATRX, NAA10, ANKRD11, DHCR7, LAMA1, NFIX, UBE3A, ARID1B and PTPRD were identified in 11 of 112 patients (11/112, 9.82%). Of 14 variants, eight arose de novo, and 13 are novel. Nine patients (9/112, 8.03%) got definite molecular diagnoses. It is the first time to report variants in EFNB1, NAA10, DHCR7, LAMA1 and NFIX in Chinese intellectual disability/developmental delay patients and first report about variants in NAA10 and LAMA1 in affected individuals of Asian ancestry. Conclusions Targeted next generation sequencing of 454 genes is an effective test strategy for patients with unexplained intellectual disability/developmental delay. Genetic heterogenicity is significant in this Chinese cohort and de novo variants play an important role in the diagnosis. Findings of this study further delineate the corresponding phenotypes, expand the mutation spectrum and support the involvement of PTPRD in the disease.

Abstract Background Brown-Vialetto-Van Laere Syndrome (BVVLS), a rare neurological disorder characterized by motor, sensory, and cranial neuronopathies, is mainly associated with defective riboflavin transporters encoded by SLC52A2 and SLC52A3 genes. Clinical outcomes have been shown to be improved significantly by high-dose riboflavin supplementation. The aim of this study was to identify genetic causes and further evaluate the clinical course and response to riboflavin in a Chinese pedigree with BVVLS. Case presentation We report the novel compound heterozygous variants c.1328G>A p.(Cys443Tyr) and c.1022_1023insC p. (Leu341Profs*103) of SLC52A2 gene in a female proband who presented in our out-patient clinic at the age of one-year-old with progressive mental and motor regression, breath holding, and brain stem dysfunction including facial weakness, hearing loss, dysphagia. Following high-dose riboflavin supplementation, the respiratory insufficiency and mental, motor, and bulbar function improved. However, sensorineural hearing loss was not improved. The missense variant site was highly conserved. Both variants were not found in the population database gnomAD. The two variants were inherited from her mother and father, respectively. Both variants were predicted to be deleterious by Polyphen2, Mutation taster, and SIFT and were classified as likely pathogenic according to the ACMG guideline. Conclusions Two novel pathogenic variations of SLC52A2 gene were firstly found from a Chinese pedigree with BVVLS. Clinical outcomes could be improved by early diagnosis and riboflavin supplementation.

Objective: Globally, developed countries such as the United States, Canada, Germany, Korea, have carried out long-term and systematic biomonitoring programs for environmental chemicals in their populations. The China National Human Biomonitoring (CNHBM) was to document the extent of human exposure to a wide array of environmental chemicals, to understand exposure profiles, magnitude and ongoing trends in exposure in the general Chinese population, and to establish a national biorepository. Methods: CNHBM adopted three-stage sampling method to obtain a nationally representative sample of the population. A total of 21,888 participants who were permanent residents in 31 provinces were designed to interviewed in this national biomonitoring (152 monitoring sites × 3 survey units × 2 sexes × 6 age groups × 4 persons = 21,888 persons) in 2017–2018. Unlike the US National Health and Nutrition Examination Survey, the CNHBM will follow the same participants in subsequent cycles allowing for dynamic, longitudinal data sets for epidemiologic follow-up. Each survey cycle of CNHBM will last 2 years and each subsequent cycle will occur 3 years after the prior cycle’s completion. Results: In 2017–2018, the CNHBM created a large cohort of Chinese citizens that included districts/counties questionnaire, community questionnaire collecting information on villages/communities, individual questionnaire, household questionnaire, comprehensive medical examination, and collection of blood and urine samples for measurement of clinical and exposure biomarkers. A total of 21,746 participants were finally included in CNHBM, accounting for 99.4% of the designed sample size; and 152 PSUs questionnaires, 454 community questionnaires, 21,619 family questionnaires, 21,712 cases of medical examinations, 21,700 individual questionnaires, 21,701 blood samples and 21,704 urine samples were collected, respectively. Planned analyses of blood and urine samples were to measure both inorganic and organic chemicals, including 13 heavy metals and metalloids, 18 poly- and per-fluorinated alkyl substances, 12 phthalate metabolites, 9 polycyclic aromatic hydrocarbons metabolites, 4 environmental alkylated phenols, and 2 benzene metabolites. Conclusions: CNHBM established the first nationally representative, prospective cohort in the Chinese population to understand the baseline and trend of internal exposure of environmental chemicals in general population, and to understand environmental toxicity.

Hypomyelinating leukodystrophies (HLDs) are a rare group of heterogeneously genetic disorders characterized by persistent deficit of myelin observed on magnetic resonance imaging (MRI). To identify a new disease-associated gene of HLD, trio-based whole exome sequencing was performed for unexplained patients with HLD. Functional studies were performed to confirm the phenotypic effect of candidate protein variants. Two de novo heterozygous variants, c.227T>G p.(L76R) or c.227T>C p.(L76P) in TMEM163 were identified in two unrelated HLD patients. TMEM163 protein is a zinc efflux transporter localized within the plasma membrane, lysosomes, early endosomes, and other vesicular compartments. It has not been associated with hypomyelination. Functional zinc flux assays in HeLa cells stably-expressing TMEM163 protein variants, L76R and L76P, revealed distinct attenuation or enhancement of zinc efflux, respectively. Experiments using a zebrafish model with knockdown of tmem163a and tmem163b (morphants) showed that loss of tmem163 causes dysplasia of the larvae, locomotor disability and myelin deficit. Expression of human wild type TMEM163 mRNAs in morphants rescues the phenotype, while the TMEM163 L76P and L76R mutants aggravated the condition. Moreover, poor proliferation, elevated apoptosis of oligodendrocytes, and reduced oligodendrocytes and neurons were also observed in zebrafish morphants. Our findings suggest an unappreciated role for TMEM163 protein in myelin development and add TMEM163 to a growing list of genes associated with hypomyelination leukodystrophy.

Background: There is a paucity of mechanistic information on the DNA methylation and particulate matter (PM) exposure. This study aimed to investigate the association of PM and its component with DNA methylation, and the roles of DNA methyltransferase (DNMTs). Methods: There were 240 high-exposed, 318 low-exposed and 210 non-exposed participants in this study. Individual concentrations of PM, polycyclic aromatic hydrocarbons (PAHs) and metals were identified by the monitoring data in their workplaces. Urinary 1-OHP and metals were determined as exposure markers. The global DNA methylation (% 5mC) and the mRNA expression of DNMT1, DNMT3A and DNMT3B were measured. We used mediation analysis to evaluate the role of DNMTs expression on DNA methylation alteration induced by PAHs and metals components. Results: The decreasing trend of % 5mC was associated with increment of PM exposure in all subjects. We found that one IQR increase in total PAHs (3.82 μg/m3) and urinary 1-OHP (1.06 μmol/mol creatinine) were associated with a separate 6.08% and 7.26% decrease in % 5mC (P = 0.009, P

Melatonin priming is an effective strategy to improve the germination of aged oat (Avena sativa L.) seeds, but the mechanism involved in its time-course responses has remained largely unknown. In the present study, the phenotypic differences, ultrastructural changes, physiological characteristics, and proteomic profiles were examined in aged and melatonin-primed seed (with 10 μM melatonin treatment for 12, 24, and 36 h). Thus, 36 h priming (T36) had a better remediation effect on aged seeds, reflecting in the improved germinability and seedlings, relatively intact cell ultrastructures, and enhanced antioxidant capacity. Proteomic analysis revealed 201 differentially abundant proteins between aged and T36 seeds, of which 96 were up-accumulated. In melatonin-primed seeds, the restoration of membrane integrity by improved antioxidant capacity, which was affected by the stimulation of jasmonic acid synthesis via up-accumulation of 12-oxo-phytodienoic acid reductase, might be a candidate mechanism. Moreover, the relatively intact ultrastructures enabled amino acid metabolism and phenylpropanoid biosynthesis, which were closely associated with energy generation through intermediates of pyruvate, phosphoenolpyruvate, fumarate, and α-ketoglutarate, thus providing energy, active amino acids, and secondary metabolites necessary for germination improvement of aged seeds. These findings clarify the time-course related pathways associated with melatonin priming on promoting the germination of aged oat seeds.

Although melatonin has been reported to play an important role in regulating metabolic events under adverse stresses, its underlying mechanisms on germination in aged seeds remain unclear. This study was conducted to investigate the effect of melatonin priming (MP) on embryos of aged oat seeds in relation to germination, ultrastructural changes, antioxidant responses, and protein profiles. Proteomic analysis revealed, in total, 402 differentially expressed proteins (DEPs) in normal, aged, and aged + MP embryos. The downregulated DEPs in aged embryos were enriched in sucrose metabolism, glycolysis, β-oxidation of lipid, and protein synthesis. MP (200 μM) turned four downregulated DEPs into upregulated DEPs, among which, especially 3-ketoacyl-CoA thiolase-like protein (KATLP) involved in the β-oxidation pathway played a key role in maintaining TCA cycle stability and providing more energy for protein translation. Furthermore, it was found that MP enhanced antioxidant capacity in the ascorbate-glutathione (AsA-GSH) system, declined reactive oxygen species (ROS), and improved cell ultrastructure. These results indicated that the impaired germination and seedling growth of aged seeds could be rescued to a certain level by melatonin, predominantly depending on β-oxidation, protein translation, and antioxidant protection of AsA-GSH. This work reveals new insights into melatonin-mediated mechanisms from protein profiles that occur in embryos of oat seeds processed by both aging and priming.

Objective. Metachromatic leukodystrophy (MLD) is an inherited disease caused by a deficiency of the enzyme arylsulfatase A (ARSA) that leads to severe physiologic and developmental problems. Our study is aimed at elucidating the clinical and genetic characteristics of Chinese MLD patients. Methods. Clinical data of 21 MLD patients was collected. All coding exons of ARSA and their flanking intronic sequences were amplified by polymerase chain reaction and subjected to direct sequencing. Results. All 21 patients were diagnosed with MLD clinically and genetically, out of which 17 patients were late infantile and 4 were juvenile types. A total of 34 ARSA mutations, including 28 novel mutations (22 missense, 1 splicing, 1 nonsense, 3 small insertions, and 1 small deletion mutation) and 6 known mutations (5 missense and 1 small insertion mutation), were identified. Prenatal diagnosis was performed for four pedigrees. One fetus was a patient, two fetuses were carriers, and two were wild type. Conclusions. The present study discovered 28 novel ARSA mutations and widely expanded the mutation spectrum of ARSA. Four successful prenatal diagnoses provided critical information for MLD families.

The sucrose non-fermentation-related protein kinase (SnRK) is a kind of Ser/Thr protein kinase, which plays a crucial role in plant stress response by phosphorylating the target protein to regulate the interconnection of various signaling pathways. However, little is known about the SnRK family in Eucalyptus grandis. Thirty-four putative SnRK sequences were identified in E. grandis and divided into three subgroups (SnRK1, SnRK2 and SnRK3) based on phylogenetic analysis and the type of domain. Chromosome localization showed that SnRK family members are unevenly distributed in the remaining 10 chromosomes, with the notable exception of chromosome 11. Gene structure analysis reveal that 10 of the 24 SnRK3 genes contained no introns. Moreover, conserved motif analyses showed that SnRK sequences belonged to the same subgroup that contained the same motif type of motif. The Ka/Ks ratio of 17 paralogues suggested that the EgrSnRK gene family underwent a purifying selection. The upstream region of EgrSnRK genes enriched with different type and numbers of cis-elements indicated that EgrSnRK genes are likely to play a role in the response to diverse stresses. Quantitative real-time PCR showed that the majority of the SnRK genes were induced by salt treatment. Genome-wide analyses and expression pattern analyses provided further understanding on the function of the SnRK family in the stress response to different environmental salt concentrations.

VQ genes play important roles in plant development, growth, and stress responses. However, little information regarding the functions of VQ genes is available for Eucalyptus grandis. In our study, genome-wide characterization and identification of VQ genes were performed in E. grandis. Results showed that 27 VQ genes, which divided into seven sub-families (I–VII), were found, and all but two VQ genes showed no intron by gene structure and conserved motif analysis. To further identify the function of EgrVQ proteins, gene expression analyses were also developed under hormone treatments (brassinosteroids, methyl jasmonate, salicylic acid, and abscisic acid) and abiotic conditions (salt stress, cold 4 °C, and heat 42 °C). The results of a quantitative real-time PCR analysis indicated that the EgrVQs were variously expressed under different hormone treatments and abiotic stressors. Our study provides a comprehensive overview of VQ genes in E. grandis, which will be beneficial in the molecular breeding of E. grandis to promote its resistance to abiotic stressors; the results also provide a basis from which to conduct further investigation into the functions of VQ genes in E. grandis.

OBJECTIVE:Megalencephalic leukoencephalopathy with subcortical cysts (MLC, OMIM 604004) is a rare neurological deterioration disease. We aimed to clarify clinical and genetic features of Chinese MLC patients. METHODS:Clinical information and peripheral venous blood of 20 patients and their families were collected, Sanger-sequencing and Multiple Ligation-dependent Probe Amplification were performed to make genetic analysis. Splicing-site mutation was confirmed with RT-PCR. UPD was detected by haplotype analysis. Follow-up study was performed through telephone for 27 patients. RESULTS:Out of 20 patients, macrocephaly, classic MRI features, motor development delay and cognitive impairment were detected in 20(100%), 20(100%), 17(85%) and 4(20%) patients, respectively. 20(100%) were clinically diagnosed with MLC. 19(95%) were genetically diagnosed with 10 novel mutations in MLC1, MLC1 and GlialCAM mutations were identified in 15 and 4 patients, respectively. Deletion mutation from exon4 to exon9 and a homozygous point mutation due to maternal UPD of chromosome22 in MLC1 were found firstly. c.598-2A>C in MLC1 leads to the skip of exon8. c.772-1G>C in MLC1 accounting for 15.5%(9/58) alleles in Chinese patients might be a founder or a hot-spot mutation. Out of 27 patients in the follow-up study, head circumference was ranged from 56cm to 61cm in patients older than 5yeas old, with a median of 57cm. Motor development delay and cognitive impairment were detected in 22(81.5%) and 5(18.5%) patients, respectively. Motor and cognitive deterioration was found in 5 (18.5%) and 2 patients (7.4%), respectively. Improvements and MRI recovery were first found in Chinese patients. Rate of seizures (45.5%), transient motor retrogress (45.5%) and unconsciousness (13.6%) after head trauma was much higher than that after fever (18.2%, 9.1%, 0%, respectively). SIGNIFICANCE:It's a clinical and genetic analysis and a follow-up study for largest sample of Chinese MLC patients, identifying 10 novel mutations, expanding mutation spectrums and discovering clinical features of Chinese MLC patients.

Mitochondria are the source of reactive oxygen species (ROS) in plant cells and play a central role in the mitochondrial electron transport chain (ETC) and tricarboxylic acid cycle (TCA) cycles; however, ROS production and regulation for seed germination, seedling growth, as well as mitochondrial responses to abiotic stress, are not clear. This study was conducted to obtain basic information on seed germination, embryo mitochondrial antioxidant responses, and protein profile changes in artificial aging in oat seeds (Avena sativa L.) exposed to exogenous nitric oxide (NO) treatment. The results showed that the accumulation of H2O2 in mitochondria increased significantly in aged seeds. Artificial aging can lead to a loss of seed vigor, which was shown by a decline in seed germination and the extension of mean germination time (MGT). Seedling growth was also inhibited. Some enzymes, including catalase (CAT), glutathione reductase (GR), dehydroascorbate reductase (DHAR), and monodehydroascorbate reductase (MDHAR), maintained a lower level in the ascorbate-glutathione (AsA-GSH) scavenging system. Proteomic analysis revealed that the expression of some proteins related to the TCA cycle were down-regulated and several enzymes related to mitochondrial ETC were up-regulated. With the application of 0.05 mM NO in aged oat seeds, a protective effect was observed, demonstrated by an improvement in seed vigor and increased H2O2 scavenging ability in mitochondria. There were also higher activities of CAT, GR, MDHAR, and DHAR in the AsA-GSH scavenging system, enhanced TCA cycle-related enzymes (malate dehydrogenase, succinate-CoA ligase, fumarate hydratase), and activated alternative pathways, as the cytochrome pathway was inhibited. Therefore, our results indicated that seedling growth and seed germinability could retain a certain level in aged oat seeds, predominantly depending on the lower NO regulation of the TCA cycle and AsA-GSH. Thus, it could be concluded that the application of 0.05 mM NO in aged oat seeds improved seed vigor by enhancing the mitochondrial TCA cycle and activating alternative pathways for improvement.

Supporting diverse Quality of Service (QoS) performance for heterogeneous data flows generated by multimedia applications has been a challenging issue that is not fully addressed in the Internet. Software Defined Network, which decouples data forwarding and network control, offers a promising approach to QoS guarantee of multimedia flows in the future Internet. However, the currently available QoS mechanisms proposed for SDN have not fully considered the diversity in performance requirements of multimedia data flows. In order to tackle this challenge, we propose a hybrid scheduling scheme in this paper to combine priority queueing with Packet General Processor Sharing (PGPS) algorithm to provide QoS guarantee for multimedia applications in SDN. We apply network calculus to develop modeling and analysis techniques to evaluate the QoS performance of the proposed scheduling scheme. Both analytical and numerical results obtained in this paper show that the proposed scheme can provide QoS guarantee for meeting diverse requirements of multimedia applications.

Background: The diffuse large B-cell lymphoma (DLBCL) has the highest incidence of all lymphomas worldwide. To investigate the functions of lymphocyte activation gene 3 (LAG-3) and programmed cell death 1 (PD-1) in tissues and peripheral blood of patients with DLBCL, the expression of LAG-3 and PD-1 genes in DLBCL-TCGA were analyzed. Methods: LAG-3 and PD-1 mRNA levels in DLBCL were analyzed using data from The Cancer Genome Atlas (TCGA) database. Utilize the Genotype-Tissue Expression (GTEx) database for assessing the variance in the expression of LAG-3, PD-1, and other associated factors between the tissues of DLBCL patients and healthy individuals. Immunohistochemistry was applied to detect the expression of LAG-3 and PD-1 levels in 137 cases of DLBCL tissues and 20 cases of reactive lymphoid hyperplasia. The prognostic value of LAG-3 and PD-1 were assessed using the Kaplan-Meier curve. The Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE) and ssGSEA algorithm were used to explore the immune microenvironment of DLBCL. Additionally, the expression and co-expression of LAG-3 and PD-1 were detected on CD4 and CD8 T cells in peripheral blood samples from 100 cases of DLBCL tissues and 30 cases of healthy individuals using flow cytometry. Results: According to TCGA database, LAG-3 and PD-1 gene expression levels were significantly up-regulated in DLBCL tissues. LAG-3 and PD-1 levels were also strongly positively correlated with those of most infiltrating immune cells. Overall survival of patients with high LAG-3 and PD-1 co-expression was significantly shorter than that of patients with low co-expression. In DLBCL patients, LAG-3 and PD-1 were highly expressed in peripheral blood CD8+ T cells. In addition, LAG-3 was highly expressed in CD4+ T cells, while the expression of PD-1 in CD4+ T cells of DLBCL patients showed no significant difference compared to healthy individuals. Additionally, CD8+ T cells and SUDHL6/OCI-LY3 from patients with DLBCL were co-cultured in vitro; after addition of LAG-3 and/or PD-1 inhibitors alone, an increased perforin and granzyme B secretion levels by CD8+ T cells were detected, as well as an increase in the overall proportion of tumor cells undergoing apoptosis. Conclusion: High LAG-3 and PD-1 levels significantly inhibit CD8+ T cell function, resulting in weakened ability to kill tumor cells. Combined LAG-3 and PD-1 blockade can restore CD8+ T cell function and provides a potential avenue for development of personalized cellular immunotherapy for DLBCL. [ABSTRACT FROM AUTHOR]