Improved self-cleaving precipitation tags for efficient column free bioseparations.

Current biological research requires simple protein bioseparation methods capable of purifying target proteins in a single step with high yields and purities. Conventional affinity tag-based approaches require specific affinity resins and expensive proteolytic enzymes for tag removal. Purification strategies based on self-cleaving aggregating tags have been previously developed to address these problems. However, these methods often utilize C-terminal cleaving contiguous inteins which suffer from premature cleavage, resulting in significant product loss during protein expression. In this work, we evaluate two novel mutants of the Mtu RecA ΔI-CM mini-intein obtained through yeast surface display for improved protein purification. When used with the elastin-like-polypeptide (ELP) precipitation tag, the novel mutants - ΔI-12 and ΔI-29 resulted in significantly higher precursor content, product purity and process yield compared to the original Mtu RecA ΔI-CM mini-intein. Product purities ranging from 68 % to 94 % were obtained in a single step for three model proteins - green fluorescent protein (GFP), maltose binding protein (MBP) and beta-galactosidase (beta-gal). Further, high cleaving efficiency was achieved after 5 h under most conditions. Overall, we have developed improved self-cleaving precipitation tags which can be used for purifying a wide range of proteins cheaply at laboratory scale.

Hunting imaging biomarkers in pulmonary fibrosis: Benchmarks of the AIIB23 challenge.

Airway-related quantitative imaging biomarkers are crucial for examination, diagnosis, and prognosis in pulmonary diseases. However, the manual delineation of airway structures remains prohibitively time-consuming. While significant efforts have been made towards enhancing automatic airway modelling, current public-available datasets predominantly concentrate on lung diseases with moderate morphological variations. The intricate honeycombing patterns present in the lung tissues of fibrotic lung disease patients exacerbate the challenges, often leading to various prediction errors. To address this issue, the 'Airway-Informed Quantitative CT Imaging Biomarker for Fibrotic Lung Disease 2023' (AIIB23) competition was organized in conjunction with the official 2023 International Conference on Medical Image Computing and Computer Assisted Intervention (MICCAI). The airway structures were meticulously annotated by three experienced radiologists. Competitors were encouraged to develop automatic airway segmentation models with high robustness and generalization abilities, followed by exploring the most correlated QIB of mortality prediction. A training set of 120 high-resolution computerised tomography (HRCT) scans were publicly released with expert annotations and mortality status. The online validation set incorporated 52 HRCT scans from patients with fibrotic lung disease and the offline test set included 140 cases from fibrosis and COVID-19 patients. The results have shown that the capacity of extracting airway trees from patients with fibrotic lung disease could be enhanced by introducing voxel-wise weighted general union loss and continuity loss. In addition to the competitive image biomarkers for mortality prediction, a strong airway-derived biomarker (Hazard ratio>1.5, p < 0.0001) was revealed for survival prognostication compared with existing clinical measurements, clinician assessment and AI-based biomarkers.

CRISPR/Cas9-mediated enhancement of semi-dwarf glutinous traits in elite Xiangdaowan rice (Oryza sativa L.): targeting SD1 and Wx genes for yield and quality improvement.

In rice cultivation, the traits of semi-dwarfism and glutinous texture are pivotal for optimizing yield potential and grain quality, respectively. Xiangdaowan (XDW) rice, renowned for its exceptional aromatic properties, has faced challenges due to its tall stature and high amylose content, resulting in poor lodging resistance and suboptimal culinary attributes. To address these issues, we employed CRISPR/Cas9 technology to precisely edit the SD1 and Wx genes in XDW rice, leading to the development of stable genetically homozygous lines with desired semi-dwarf and glutinous characteristics. The sd1-wx mutant lines exhibited reduced gibberellin content, plant height, and amylose content, while maintaining hardly changed germination rate and other key agronomic traits. Importantly, our study demonstrated that exogenous GA3 application effectively promoted growth by compensating for the deficiency of endogenous gibberellin. Based on this, a semi-dwarf glutinous elite rice (Oryza sativa L.) Lines was developed without too much effect on most agronomic traits. Furthermore, a comparative transcriptome analysis unveiled that differentially expressed genes (DEGs) were primarily associated with the anchored component of the membrane, hydrogen peroxide catabolic process, peroxidase activity, terpene synthase activity, and apoplast. Additionally, terpene synthase genes involved in catalyzing the biosynthesis of diterpenoids to gibberellins were enriched and significantly down-regulated. This comprehensive study provides an efficient method for simultaneously enhancing rice plant height and quality, paving the way for the development of lodging-resistant and high-quality rice varieties.

Exposure levels and determinants of placental polybrominated diphenyl ethers in Chinese pregnant women.

BACKGROUND: Polybrominated diphenyl ethers (PBDEs) are a group of widely used chemicals and humans are exposed to them in their daily life. PBDEs exposure during pregnancy may have adverse effects on pregnant women and their fetuses. Nevertheless, limited information is available on the levels and determinants of PBDEs exposure in Chinese pregnant women. METHODS: The internal exposure levels of eight PBDEs (BDE-28, 47, 99, 100, 153, 154, 183, and 209) in placental samples of 1280 pregnant women from Zunyi birth cohort were analyzed using gas chromatography tandem mass spectrometry. All PBDEs concentrations were lipid adjusted (ng/g lw). Determinants of exposure were assessed by multivariable logistic regression model. RESULTS: Eight PBDE homologues were quantifiable in more than 70% of the samples. The highest median concentrations were found for BDE-209 (2.78 ng/g lw), followed by BDE-153 (1.00 ng/g lw) and BDE-183 (0.93 ng/g lw). The level of ΣPBDEs ranged from 0.90 to 308.78 ng/g lw, with a median concentration of 10.02 ng/g lw. Multivariate logistic regression analysis showed that maternal age older than 30 years old (OR: 1.59; 95% CI: 1.14, 2.23), pre-pregnancy obesity (1.51; 1.08, 2.10), home renovation within 2 years (1.43; 1.08, 1.91), spending more time outdoors during pregnancy (0.70; 0.55, 0.89), high consumption of fish/seafood (1.46; 1.13, 1.90) and eggs (1.44; 1.04, 2.00), male infant sex (1.69; 1.18, 2.42) were associated with PBDEs exposure. CONCLUSION: The study population is generally exposed to PBDEs, of which BDE-209 is the dominant congener, indicating extensive application of products containing deca-BDE mixtures. Maternal age, pre-pregnancy BMI, home decoration, average outdoor time during pregnancy, fish, seafood, eggs consumption, and fetal sex were exposure-determinning factors. This study contributes to the knowledge on region-specific PBDEs contamination in pregnant women and related risk factors.

Effects of multiple anesthetic exposures on rhesus macaque brain development: a longitudinal structural MRI analysis.

Concerns about the potential neurotoxic effects of anesthetics on developing brain exist. When making clinical decisions, the timing and dosage of anesthetic exposure are critical factors to consider due to their associated risks. In our study, we investigated the impact of repeated anesthetic exposures on the brain development trajectory of a cohort of rhesus monkeys (n = 26) over their first 2 yr of life, utilizing longitudinal magnetic resonance imaging data. We hypothesized that early or high-dose anesthesia exposure could negatively influence structural brain development. By employing the generalized additive mixed model, we traced the longitudinal trajectories of brain volume, cortical thickness, and white matter integrity. The interaction analysis revealed that age and cumulative anesthetic dose were variably linked to white matter integrity but not to morphometric measures. Early high-dose exposure was associated with increased mean, axial, and radial diffusivities across all white matter regions, compared to late-low-dose exposure. Our findings indicate that early or high-dose anesthesia exposure during infancy disrupts structural brain development in rhesus monkeys. Consequently, the timing of elective surgeries and procedures that require anesthesia for children and pregnant women should be strategically planned to account for the cumulative dose of volatile anesthetics, aiming to minimize the potential risks to brain development.

Hypoxia-induced TMTC3 expression in esophageal squamous cell carcinoma potentiates tumor angiogenesis through Rho GTPase/STAT3/VEGFA pathway.

BACKGROUND: Hypoxia is one of most typical features in the tumor microenvironment of solid tumor and an inducer of endoplasmic reticulum (ER) stress, and HIF-1α functions as a key transcription factor regulator to promote tumor angiogenesis in the adaptive response to hypoxia. Increasing evidence has suggested that hypoxia plays an important regulatory role of ER homeostasis. We previously identified TMTC3 as an ER stress mediator under nutrient-deficiency condition in esophageal squamous cell carcinoma (ESCC), but the molecular mechanism in hypoxia is still unclear. METHODS: RNA sequencing data of TMTC3 knockdown cells and TCGA database were analyzed to determine the association of TMTC3 and hypoxia. Moreover, ChIP assay and dual-luciferase reporter assay were performed to detect the interaction of HIF-1α and TMTC3 promoter. In vitro and in vivo assays were used to investigate the function of TMTC3 in tumor angiogenesis. The molecular mechanism was determined using co-immunoprecipitation assays, immunofluorescence assays and western blot. The TMTC3 inhibitor was identified by high-throughput screening of FDA-approved drugs. The combination of TMTC3 inhibitor and cisplatin was conducted to confirm the efficiency in vitro and in vivo. RESULTS: The expression of TMTC3 was remarkably increased under hypoxia and regulated by HIF-1α. Knockdown of TMTC3 inhibited the capability of tumor angiogenesis and ROS production in ESCC. Mechanistically, TMTC3 promoted the production of GTP through interacting with IMPDH2 Bateman domain. The activity of Rho GTPase/STAT3, regulated by cellular GTP levels, decreased in TMTC3 knockdown cells, whereas reversed by IMPDH2 overexpression. Additionally, TMTC3 regulated the expression of VEGFA through Rho GTPase/STAT3 pathway. Allopurinol inhibited the expression of TMTC3 and further reduced the phosphorylation and activation of STAT3 signaling pathway in a dose-dependent manner in ESCC. Additionally, the combination of allopurinol and cisplatin significantly inhibited the cell viability in vitro and tumor growth in vivo, comparing with single drug treatment, respectively. CONCLUSIONS: Collectively, our study clarified the molecular mechanism of TMTC3 in regulating tumor angiogenesis and highlighted the potential therapeutic combination of TMTC3 inhibitor and cisplatin, which proposed a promising strategy for the treatment of ESCC.

Physiological and transcriptomic analysis of OsLHCB3 knockdown lines in rice.

The photosystem II (PSII) outer antenna LHCB3 protein plays critical roles in distributing the excitation energy and modulating the rate of state transition for photosynthesis. Here, OsLHCB3 knockdown mutants were produced using the RNAi system. Phenotypic analyses showed that OsLHCB3 knockdown led to pale green leaves and lower chlorophyll contents at both tillering and heading stages. In addition, mutant lines exhibited decreased non-photochemical quenching (NPQ) capacity and net photosynthetic rate (Pn) by downregulating the expression of PSII-related genes. Moreover, RNA-seq experiments were performed at both tillering and heading stages. The differentially expressed genes (DEGs) mainly involved in chlorophyll binding response to abscisic acid, photosystem II, response to chitin, and DNA-binding transcription factor. Besides, our transcriptomic and physiological data indicated that OsLHCB3 was essential for binding chlorophyll, but not for the metabolism of chlorophyll in rice. OsLHCB3 RNAi knockdown plants affected the expression of PS II-related genes, but not PS I-related genes. Overall, these results suggest that OsLHCB3 also plays vital roles in regulating photosynthesis and antenna proteins in rice as well as responses to environment stresses. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01387-z.

Diagnostic and prognostic performance of plasma neurofilament light chain in multiple system atrophy: a cross-sectional and longitudinal study.

BACKGROUND: The longitudinal dynamics of neurofilament light chain (NfL) in multiple system atrophy (MSA) were incompletely illuminated. This study aimed to explore whether the plasma NfL (pNfL) could serve as a potential biomarker of clinical diagnosis and disease progression for MSA. METHODS: We quantified pNfL concentrations in both a large cross-sectional cohort with 214 MSA individuals, 65 PD individuals, and 211 healthy controls (HC), and a longitudinal cohort of 84 MSA patients. Propensity score matching (PSM) was used to balance the age between the three groups. The pNfL levels between groups were compared using Kruskal-Wallis test. Linear mixed models were performed to explore the disease progression-associated factors in longitudinal MSA cohort. Random forest model as a complement to linear models was employed to quantify the importance of predictors. RESULTS: Before and after matching the age by PSM, the pNfL levels could reliably differentiate MSA from HC and PD groups, but only had mild potential to distinguish PD from HC. By combining linear and nonlinear models, we demonstrated that pNfL levels at baseline, rather than the change rate of pNfL, displayed potential prognostic value for progression of MSA. The combination of baseline pNfL levels and other modifiers, such as subtypes, Hoehn-Yahr stage at baseline, was first shown to improve the diagnosis accuracy. CONCLUSIONS: Our study contributed to a better understanding of longitudinal dynamics of pNfL in MSA, and validated the values of pNfL as a non-invasive sensitive biomarker for the diagnosis and progression. The combination of pNfL and other factors is recommended for better monitoring and prediction of MSA progression.

The mitochondrial genome of the diploid oat Avena longiglumis.

BACKGROUND: Avena longiglumis Durieu (2n = 2x = 14) is a wild relative of cultivated oat (Avena sativa, 2n = 6x = 42) with good agronomic and nutritional traits. The plant mitochondrial genome has a complex organization and carries genetic traits of value in exploiting genetic resources, not least male sterility alleles used to generate F1 hybrid seeds. Therefore, we aim to complement the chromosomal-level nuclear and chloroplast genome assemblies of A. longiglumis with the complete assembly of the mitochondrial genome (mitogenome) based on Illumina and ONT long reads, comparing its structure with Poaceae species. RESULTS: The complete mitochondrial genome of A. longiglumis can be represented by one master circular genome being 548,445 bp long with a GC content of 44.05%. It can be represented by linear or circular DNA molecules (isoforms or contigs), with multiple alternative configurations mediated by long (4,100-31,235 bp) and medium (144-792 bp) size repeats. Thirty-five unique protein-coding genes, three unique rRNA genes, and 11 unique tRNA genes are identified. The mitogenome is rich in duplications (up to 233 kb long) and multiple tandem or simple sequence repeats, together accounting for more than 42.5% of the total length. We identify homologous sequences between the mitochondrial, plastid and nuclear genomes, including the exchange of eight plastid-derived tRNA genes, and nuclear-derived retroelement fragments. At least 85% of the mitogenome is duplicated in the A. longiglumis nuclear genome. We identify 269 RNA editing sites in mitochondrial protein-coding genes including stop codons truncating ccmFC transcripts. CONCLUSIONS: Comparative analysis with Poaceae species reveals the dynamic and ongoing evolutionary changes in mitochondrial genome structure and gene content. The complete mitochondrial genome of A. longiglumis completes the last link of the oat reference genome and lays the foundation for oat breeding and exploiting the biodiversity in the genus.

Risk assessment and environmental determinants of urinary phthalate metabolites in pregnant women in Southwest China.

Pregnant women are widely exposed to phthalic acid esters (PAEs) that are commonly used in most aspects of modern life. However, few studies have examined the cumulative exposure of pregnant women to a variety of PAEs derived from the living environmental conditions in China. Therefore, this study aimed to determine the urinary concentrations of nine PAE metabolites in pregnant women, examine the relationship between urinary concentrations and residential characteristics, and conduct a risk assessment analysis. We included 1,888 women who were in their third trimester of pregnancy, and we determined their urinary concentrations of nine PAE metabolites using high-performance gas chromatography-mass spectrometry. The risk assessment of exposure to PAEs was calculated based on the estimated daily intake. A linear regression model was used to analyze the relationship between creatinine-adjusted PAE metabolite concentrations and residential characteristics. The detection rate of five PAE metabolites in the study population was > 90%. Among the PAE metabolites adjusted by creatinine, the urinary metabolite concentration of monobutyl phthalate was found to be the highest. Residential factors, such as housing type, proximity to streets, recent decorations, lack of ventilation in the kitchen, less than equal to three rooms, and the use of coal/kerosene/wood/wheat straw fuels, were all significantly associated with high PAE metabolite concentrations. Due to PAE exposure, ~ 42% (n = 793) of the participants faced potential health risks, particularly attributed to dibutyl phthalate, diisobutyl phthalate, and di(2-ethyl)hexyl phthalate exposure. Living in buildings and using coal/kerosene/wood/wheat straw as domestic fuel can further increase the risks.

Characteristics of exposure to 10 polycyclic aromatic hydrocarbon metabolites among pregnant women: cohort of pregnant women in Zunyi, southwest China.

OBJECTIVE: Our aim was to elucidate the polycyclic aromatic hydrocarbon (PAH) metabolites exposure levels of pregnant women in the underdeveloped region of Zunyi, southwest China. METHODS: Sociodemographic information was collected via questionnaires, and urine samples were collected at the same time. A total of 3047 pregnant women participated in the study. Gas chromatography/mass spectrometry was used to detect the urine concentrations of 10 PAH metabolites. A generalised linear model (GLM) was used to identify predictive factors of PAH metabolites. RESULTS: All PAH metabolites had a detection rate greater than 60% (67.21%-90.57%) except for 4-OH-PHE at 55.54%. The median concentrations were 0.02-0.11 µg/g Cre except for 1-OH-NAP, 2-OH-NAP, 2-OH-FLU and 9-OH-FLU (0.36-0.50 µg/g Cre). The cluster analysis identified the phenanthrene and fluorene metabolite clusters (containing no other metabolites), while naphthalene metabolites (1-OH-NAP, 2-OH-NAP) could not be clustered without other metabolites. GLM analysis identified that pregnant women with the following characteristics have high urinary concentration of PAH metabolites: overweight, in the last trimester of pregnancy, distance between their house and main traffic lines as <5 m, use fuel for cooking, passive smoking, renovated their residence for less than 3 years, middle family income and office workers. CONCLUSION: The results clarified pregnant women from the economically underdeveloped area could be the victims of PAHs. In addition, PAHs present a demographic and seasonal differential distribution, which will aid in the development of targeted interventions and reduce exposure to PAHs during pregnancy.

Effects of exposure to phthalate during early pregnancy on gestational diabetes mellitus: a nested case-control study with propensity score matching.

Owing to the complexity of phthalates (PAEs) components and the diversity of their sources, the health hazards of their metabolites to pregnant women remain unclear. This study aimed to explore the relationship between exposure to PAEs during early pregnancy and gestational diabetes mellitus (GDM) in rural pregnant women. We assessed pregnant women with (n = 338) or without (n = 3082) GDM from the ongoing Zunyi Birth Cohort. Participants' urine samples were collected to measure the levels of 10 metabolites of PAEs. GDM was diagnosed using the 75-g oral glucose tolerance test at 24-28 weeks of gestation. We adopted propensity score matching based on GDM-related factors and pregnant women's backgrounds to establish two groups of 338 patients: those with or without GDM. In the cohort, we included 5734 pregnant women; 519 of them developed GDM, yielding a GDM incidence rate of 9.05%. Urinary concentrations of monooctyl phthalate (MOP), mono-benzyl phthalate (MBzP), mono(2-ethyl-5-oxyhexyl) phthalate (MEOHP), and mono(2-ethyl-5-carboxypentyl) phthalate (MECPP) during early pregnancy were significantly associated with GDM (P < 0.05). Logistic regression models revealed that MEOHP in the urine was positively associated with GDM (odds ratio [OR] = 1.55; 95% confidence interval [CI]: 1.00-2.39). Furthermore, restricted cubic spline models revealed that urine MEOHP concentrations greater than 15.6 µg/L were positively associated with GDM, and approximately 23.5% pregnant women had urine MEOHP concentrations greater than 15.6 µg/L. Thus, approximately 23.5% of pregnant women were at the risk of developing GDM due to MEOHP, which suggested that pregnant women should reduce the use of packaged food and cosmetics to reduce the risk of GDM. However, further molecular biology experiments are required to confirm these findings and to elucidate the underlying mechanisms.

Maternal urine phthalate metabolite exposure and miscarriage risk: a nested case-control study of the Zunyi Birth Cohort.

Phthalates (PAEs) are widespread persistent organic pollutants and endocrine disruptors. However, the associations between PAE exposure and the risk of miscarriage in humans are unclear, and an insufficient number of studies have evaluated the possible threshold or dose-dependent effects of first trimester PAE exposure on miscarriage risk. Our research measured the levels of mono-methyl phthalate (MMP), mono-ethyl phthalate, mono-isobutyl phthalate, MiBP mono-butyl phthalate (MBP), mono-octyl phthalate, mono-benzyl phthalate, mono(2-ethylhexyl) phthalate, mono(2-ethyl-5-oxohexyl) phthalate, and mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) in maternal urine collected in early gestation between 150 pregnancies ending in miscarriage and 150 pregnancies with live birth. We also estimated the odds ratios (ORs) and 95% confidence intervals (CIs) for miscarriage and each PAE as a continuous variable or quartile. A restrictive cubic splines was used to assess dose-dependent effects after controlling for maternal characteristics (e.g., age, educational level). we identified monotonically increasing dose-dependent effects of MEHHP and MMP on the risk of miscarriage. The largest effect estimates were approximately threefold higher for the highest MBP (OR = 2.57; 95% CI = 1.32-5.01) or MMP quartile (OR = 3.57; 95% CI = 1.82-7.00) and two-fold higher for the highest MEHHP quartile (OR = 2.12; 95% CI = 1.10-4.11). Our research preliminarily obtained possible thresholds of MBP, MEHHP, and MMP which were 18.07, 2.38, and 0.80 µg/g Cr for the risk of miscarriage, respectively. First-trimester exposure to MBP, MEHHP, and MMP exceeding certain thresholds increases the risk of miscarriage.

Effect of prior drought and heat stress on Camellia sinensis transcriptome changes to Ectropis oblique (Lepidoptera: Geometridae) resistance.

Tea plants are continuously confronted with a wide range of biotic and abiotic stressors in the field, which can occur concurrently or sequentially. To elucidate the molecular mechanisms in responses to such individual and combined stresses, we used RNAseq to compare the temporal changes in the transcriptome of Camellia sinensis to Ectropis oblique Prout alone or in combination with exposure to drought and heat. Compared with the individual stress, tea plants exhibit significant differences in transcriptome profiles under the combined stresses. Additionally, many unique genes exhibited significant differences in expression in individual and combined stress conditions. Our research showed novel insights into the molecular mechanisms of E. oblique Prout resistance in tea plants and provided a valuable resource for developing tea varieties with broad spectrum stress tolerance.

Ephedra sinica polysaccharide alleviates airway inflammations of mouse asthma-like induced by PM2.5 and ovalbumin via the regulation of gut microbiota and short chain fatty acid.

OBJECTIVES: Epidemiological investigations show that long-term exposure to PM2.5 is directly related to asthma-like and other respiratory diseases. This study aims to further explore the pharmacological effect of Ephedra sinica polysaccharide (ESP) on lung injury caused by atmospheric PM2.5. METHODS: To achieve the aim, we explored the therapeutic effect of ESP on an aggravated asthma-like mouse induced by PM2.5 combined with ovalbumin (OVA), and explored mechanisms underlying the connection between gut microbiota and lung function. KEY FINDINGS: Preliminary results showed that ESP alleviated the symptoms of aggravated allergic asthma-like in mice; reduced the number of eosinophils in BALF; reduced the levels of serum Ig-E, IL-6, TNF-α, and IL-1ß. Further qRT-PCR detected that ESP inhibited the NF-κB pathway. The final analysis detected by 16S rRNA and short chain fatty acid (SCFA) confirmed that ESP increased relative proportions of Bacteroides, Lactobacillus, Prevotella, Butyricicoccus and Paraprevotella, but decreased that of Enterococcus and Ruminococcus; increased acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and isohexanic acid in the meanwhile. CONCLUSIONS: The study showed that ESP has a potential for future therapeutical applications in the prevention and treatment of asthma-like disease induced by PM2.5 and OVA via regulation of gut microbiota and SCFA.

A Novel ER Stress Mediator TMTC3 Promotes Squamous Cell Carcinoma Progression by Activating GRP78/PERK Signaling Pathway.

During tumor progression, tumor cells are exposed to various stress conditions, which result in endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) to restore ER homeostasis. Accumulating evidence reported the orchestrating role of ER stress in epithelial-mesenchymal transition (EMT) progress, but the detailed mechanism was unclear. Here, we identified ectopic expression of TMTC3 in cells undergoing ER stress and verified the association with EMT markers through the cellular model of ER stress and database analysis. TMTC3 was abnormally highly expressed in squamous cell carcinomas (SCCs), and regulated by TP63, an SCCs-specific transcription factor. Biological function experiments indicated that TMTC3 promoted a malignant phenotype in vitro, and accelerated tumor growth and metastasis in vivo. RNA-seq analyses and further experiments revealed that TMTC3 promoted the expression of EMT markers via interleukin-like EMT inducer (ILEI, FAM3C). Further studies on the mechanism showed that TMTC3 disrupted the interaction between PERK and GRP78 to activate the PERK pathway and promote the nuclear translocation of ATF4, which increased the transcriptional activity of ILEI. These findings indicated that TMTC3 activates GRP78/PERK signaling pathway during ER stress-induced EMT, which might serve as a potential therapeutic target in SCCs.

The Natural History of Spinocerebellar Ataxia Type 3 in Mainland China: A 2-Year Cohort Study.

Objective: The natural history of spinocerebellar ataxia type 3 (SCA3) has been reported in several populations and shows heterogeneity in progression rate and affecting factors. However, it remains unexplored in the population of Mainland China. This study aimed to identify the disease progression rate and its potential affecting factors in patients with SCA3 in Mainland China. Participants and Methods: We enrolled patients with genetically confirmed SCA3 in Mainland China. Patients were seen at three visits, i.e., baseline, 1 year, and 2 years. The primary outcome was the Scale for the Assessment and Rating of Ataxia (SARA), and the secondary outcomes were the Inventory of Non-Ataxia Signs (INAS) as well as the SCA Functional Index (SCAFI). Results: Between 1 October 2015, and 30 September 2016, we enrolled 263 patients with SCA3. We analyzed 247 patients with at least one follow-up visit. The annual progression rate of SARA was 1.49 points per year (SE 0.08, 95% confidence interval [CI] 1.33-1.65, p < 0.0001). The annual progression rates of INAS and SCAFI were 0.56 points per year (SE 0.05, 95% CI 0.47-0.66, p < 0.001) and -0.30 points per year (SE 0.01, 95% CI -0.33∼-0.28, p < 0.001), respectively. Faster progression in SARA was associated with longer length of the expanded allele of ATXN3 (p < 0.0001); faster progression in INAS was associated with lower INAS at baseline (p < 0.0001); faster decline in SCAFI was associated with shorter length of the normal allele of ATXN3 (p = 0.036) and higher SCAFI at baseline (p < 0.0001). Conclusion: Our results provide quantitative data on the disease progression of patients with SCA3 in Mainland China and its corresponding affecting factors, which could facilitate the sample size calculation and patient stratification in future clinical trials. Trial Registration: This study was registered with Chictr.org on 15 September 2015, number ChiCTR-OOC-15007124.

Dynamic Variation of Amino Acid Contents and Identification of Sterols in Xinyang Mao Jian Green Tea.

As important biomolecules in Camellia sinensis L., amino acids (AAs) are considered to contribute to the overall green tea sensory quality and undergo dynamic changes during growth. However, limited by analytical capacity, detailed AAs composition in different growth stages remains unclear. To address this question, we analyzed the dynamic changes of 23 AAs during leaf growth in Xinyang Mao Jian (XYMJ) green tea. Using amino acid analyzer, we demonstrated that most AAs are abundant on Pure Brightness Day and Grain Rain Day. After Grain Rain, 23 AAs decreased significantly. Further analysis shows that theanine has a high level on the day before Spring Equinox and Grain Rain, accounting for 44-61% of the total free AAs content in tea leaves. Glu, Pro, and Asp are the second most abundant AAs. Additionally, spinasterol and 22,23-dihydrospinasterol are first purified and identified in ethanol extract of XYMJ by silica gel column chromatography method. This study reveals the relationship between plucking days and the dynamic changes of AAs during the growth stage and proves the rationality of the traditional plucking days of XYMJ green tea.

Identification and Expression Analysis of CAMTA Genes in Tea Plant Reveal Their Complex Regulatory Role in Stress Responses.

Calmodulin-binding transcription activators (CAMTAs) are evolutionarily conserved transcription factors and have multi-functions in plant development and stress response. However, identification and functional analysis of tea plant (Camellia sinensis) CAMTA genes (CsCAMTAs) are still lacking. Here, five CsCAMTAs were identified from tea plant genomic database. Their gene structures were similar except CsCAMTA2, and protein domains were conserved. Phylogenetic relationship classified the CsCAMTAs into three groups, CsCAMTA2 was in group I, and CsCAMTA1, 3 and CsCAMTA4, 5 were, respectively, in groups II and III. Analysis showed that stress and phytohormone response-related cis-elements were distributed in the promoters of CsCAMTA genes. Expression analysis showed that CsCAMTAs were differentially expressed in different organs and under various stress treatments of tea plants. Three-hundred and four hundred-one positive co-expressed genes of CsCAMTAs were identified under cold and drought, respectively. CsCAMTAs and their co-expressed genes constituted five independent co-expression networks. KEGG enrichment analysis of CsCAMTAs and the co-expressed genes revealed that hormone regulation, transcriptional regulation, and protein processing-related pathways were enriched under cold treatment, while pathways like hormone metabolism, lipid metabolism, and carbon metabolism were enriched under drought treatment. Protein interaction network analysis suggested that CsCAMTAs could bind (G/A/C)CGCG(C/G/T) or (A/C)CGTGT cis element in the target gene promoters, and transcriptional regulation might be the main way of CsCAMTA-mediated functional regulation. The study establishes a foundation for further function studies of CsCAMTA genes in stress response.

First Report of Alternaria alternata Inciting Leaf Spot of Dangshen (Codonopsis pilosula) in China.

As a commonly traditional Chinese medicine, the perennial herb Dangshen (Codonopsis pilosula) has superior curative effects including regulating immunity, strengthening the spleen, and tonifying lungs (Bai et al. 2020). To imitate natural ecological conditions, plants were grown on hillside fields with stems prostrate on the ground, tangle-up with each other. In August 2020, leaf spots were observed on C. pilosula in Wutai county, Shanxi province, China, and indicated a high disease incidence (70%-80%) in investigated fields (6.67 ha). Small brown necrotic spots, occasionally enclosed by chlorotic halos, were observed on leaves, stems, and sepals. For identification of the pathogen, 15 small pieces (5×5 mm) of symptomatic tissues from 5 randomly-collected diseased plants were surface sterilized, placed on potato dextrose agar plates, and incubated for 4 d in darkness at 25 °C to obtain the colonies. Cultures were purified by single spore isolation from these colonies. A total of 15 isolates named as Dcp-3, and Dcp-5~Dcp-18 were recovered. They produced ovoid or obclavate spores with 15.9-57.5×9.1-20.1 µm in size, 1-6 transverse septa, and 0-4 longitudinal septa. The conidial chains with 4 to 6 spores had numerous secondary and occasionally tertiary chains on potato carrot agar plates. Because all isolates had identical morphological traits, five genes from the representative isolate Dcp-3, actin (ACT), Alternaria major allergen (Alt a1), plasma membrane ATPase (ATP), histone 3 (H3), and rDNA ITS, were amplified with primer pairs ACTDF1/ACTDR1, Alt-for/Alt-rev, ATPDF1/ATPDR1, H3-1a/H3-1b, and ITS1/ITS4, respectively (Hong et al. 2005; Lawrence et al. 2013; Ma et al. 2020). BLASTn searches indicated species of Dcp-3 could not be accurately confirmed by rDNA ITS, ATP, ACT, and Alt a1 (GenBank accession nos. OM334894, OM362504, OM326344, OM362500). Phylogenetic analysis showed it was most closely related to Alternaria alternata, A. arborescens, and A. tenuissima based on concatenated sequences of above four genes. The H3 sequence (OM362508) shared 100% homology with that of A. alternata (MN481948). The phylogenetic tree using H3 also confirmed Dcp-3 as A. alternata. Heathly, two-year-old C. pilosula were transplanted to a greenhouse. A surface-sterilized leaf was sprayed with 50 µL spore suspension (106 conidia/mL) of Dcp-3. A leaf sprayed with isometric sterile water was used as controls. Each treatment used six plants (five leaves per plant). Plants were covered with sterilized plastic bags and incubated at 22 ℃. The test was repeated twice. A week later, control leaves were healthy, but brown necrotic spots similar to field symptoms emerged on treated leaves. The A. alternata isolates were re-isolated from the border of lesions, and confirmed by morphological and molecular characteristics mentioned above, fulfilling Koch's postulates. Leaf spot of C. pilosula caused by Septoria codonopsidis has been reported in China (Wang et al. 2011). However, to our knowledge, this is the first report of A. alternata inciting leaf spot of C. pilosula in China. Our report would promote growers to enhance the field management and consider associated strategies on controlling Alternaria leaf spot of C. pilosula.