Oncogenic lncRNA downregulates cancer cell antigen presentation and intrinsic tumor suppression.

How tumor cells genetically lose antigenicity and evade immune checkpoints remains largely elusive. We report that tissue-specific expression of the human long noncoding RNA LINK-A in mouse mammary glands initiates metastatic mammary gland tumors, which phenotypically resemble human triple-negative breast cancer (TNBC). LINK-A expression facilitated crosstalk between phosphatidylinositol-(3,4,5)-trisphosphate and inhibitory G-protein-coupled receptor (GPCR) pathways, attenuating protein kinase A-mediated phosphorylation of the E3 ubiquitin ligase TRIM71. Consequently, LINK-A expression enhanced K48-polyubiquitination-mediated degradation of the antigen peptide-loading complex (PLC) and intrinsic tumor suppressors Rb and p53. Treatment with LINK-A locked nucleic acids or GPCR antagonists stabilized the PLC components, Rb and p53, and sensitized mammary gland tumors to immune checkpoint blockers. Patients with programmed ccll death protein-1(PD-1) blockade-resistant TNBC exhibited elevated LINK-A levels and downregulated PLC components. Hence we demonstrate lncRNA-dependent downregulation of antigenicity and intrinsic tumor suppression, which provides the basis for developing combinational immunotherapy treatment regimens and early TNBC prevention.

CD56 Expression Marks Human Group 2 Innate Lymphoid Cell Divergence from a Shared NK Cell and Group 3 Innate Lymphoid Cell Developmental Pathway.

According to the established model of murine innate lymphoid cell (ILC) development, helper ILCs develop separately from natural killer (NK) cells. However, it is unclear how helper ILCs and NK cells develop in humans. Here we elucidated key steps of NK cell, ILC2, and ILC3 development within human tonsils using ex vivo molecular and functional profiling and lineage differentiation assays. We demonstrated that while tonsillar NK cells, ILC2s, and ILC3s originated from a common CD34-CD117+ ILC precursor pool, final steps of ILC2 development deviated independently and became mutually exclusive from those of NK cells and ILC3s, whose developmental pathways overlapped. Moreover, we identified a CD34-CD117+ ILC precursor population that expressed CD56 and gave rise to NK cells and ILC3s but not to ILC2s. These data support a model of human ILC development distinct from the mouse, whereby human NK cells and ILC3s share a common developmental pathway separate from ILC2s.

Evidence from 2100 index cases supports genome sequencing as a first-tier genetic test.

PURPOSE: Genome sequencing (GS) is one of the most comprehensive assays that interrogate single-nucleotide variants, copy number variants, mitochondrial variants, repeat expansions, and structural variants in a single assay. Despite the clear technical superiority, the full clinical utility of GS has yet to be determined. METHODS: We systematically evaluated 2100 clinical GS index cases performed in our laboratory to explore the diagnostic yield of GS as first-tier and as follow-up testing. RESULTS: The overall diagnostic yield was 28% (585/2100). The diagnostic yield for GS as the first-tier test was 26% (294/1146). Among cases with prior non-diagnostic genetic tests, GS provided a diagnosis for 27% (247/910) of cases, including 56 cases with prior exome sequencing (ES). Although re-analysis of previous ES might have resolved the diagnosis in 29 cases, diagnoses for 27 cases would have been missed because of the technical inferiority of ES. Moreover, GS further disclosed additional genetic etiology in 3 out of 44 cases with existing partial diagnosis. CONCLUSION: We present the largest-to-date GS data set of a clinically heterogeneous cohort from a single clinical laboratory. Our data demonstrate that GS should be considered as the first-tier genetic test that has the potential to shorten the diagnostic odyssey.

Pompe disease ascertained through The Lantern Project, 2018-2021: Next-generation sequencing and enzymatic testing to overcome obstacles to diagnosis.

The Lantern Project is an ongoing complimentary diagnostic program for patients in the United States sponsored by Sanofi and implemented by PerkinElmer Genomics. It combines specific enzymatic, biomarker, and genetic testing to facilitate rapid, accurate laboratory diagnosis of Pompe disease and several other lysosomal storage diseases, and a multigene next-generation sequencing panel including Pompe disease, LGMD, and other neuromuscular disorders. This article reports data for Pompe disease collected from October 2018 through December 2021, including acid α-glucosidase (GAA) enzyme assay and GAA sequencing (standard or expedited for positive newborn screening [NBS] to rule out infantile-onset Pompe disease [IOPD]) and the Focused Neuromuscular Panel, which includes GAA. One hundred forty patients (12 received only GAA enzyme testing, 128 had GAA sequencing alone or in addition to enzyme assay) have been confirmed with Pompe disease in this project. Eight of the 140 had a variant of unknown significance, but GAA activity ≤2.10 µmol/L/h, thus were confirmed with Pompe disease. Three diagnosed patients 0-2 years old had cross-reactive immunologic material (CRIM)-negative GAA variants and thus IOPD. One additional infant with presumptive IOPD had a homozygous frameshift c.1846del, likely CRIM-negative; symptoms were not provided. Among the 128 patients with molecular results, the c.-32-13T>G splice variant was homozygous in 11, compound-heterozygous in 98, and absent in 19. Proximal muscle weakness (58 patients) was the most common sign reported at testing; elevated creatine kinase (29 patients) was the most common laboratory result. The most common symptom categories were muscular (73 patients), musculoskeletal (13 patients), and respiratory (23 patients). Clinical information was not available for 42 samples, and 17 infants had only "abnormal NBS" or "low GAA" reported. Cardiac symptoms in 7 included potentially age-related conditions in five c.-32-13T>G-compound-heterozygous adults (myocardial infarction, heart murmur/palpitations, congestive heart failure: 1 each; 2 with atrial fibrillation) and hypertrophic cardiomyopathy in 2 children (1 and 2 years old) with presumptive IOPD. One novel GAA variant was observed in a patient with enzyme activity 0.31 µmol/L/h: c.1853_1854ins49, a frameshift pathogenic variant. The Lantern Project demonstrates the combinatorial utility of enzyme assay, targeted single-gene testing, and a focused neuromuscular next-generation sequencing panel in diagnosing Pompe disease.

Variant Classification for Pompe disease; ACMG/AMP specifications from the ClinGen Lysosomal Diseases Variant Curation Expert Panel.

Accurate determination of the clinical significance of genetic variants is critical to the integration of genomics in medicine. To facilitate this process, the NIH-funded Clinical Genome Resource (ClinGen) has assembled Variant Curation Expert Panels (VCEPs), groups of experts and biocurators which provide gene- and disease- specifications to the American College of Medical Genetics & Genomics and Association for Molecular Pathology's (ACMG/AMP) variation classification guidelines. With the goal of classifying the clinical significance of GAA variants in Pompe disease (Glycogen storage disease, type II), the ClinGen Lysosomal Diseases (LD) VCEP has specified the ACMG/AMP criteria for GAA. Variant classification can play an important role in confirming the diagnosis of Pompe disease as well as in the identification of carriers. Furthermore, since the inclusion of Pompe disease on the Recommended Uniform Screening Panel (RUSP) for newborns in the USA in 2015, the addition of molecular genetic testing has become an important component in the interpretation of newborn screening results, particularly for asymptomatic individuals. To date, the LD VCEP has submitted classifications and supporting data on 243 GAA variants to public databases, specifically ClinVar and the ClinGen Evidence Repository. Here, we describe the ACMG/AMP criteria specification process for GAA, an update of the GAA-specific variant classification guidelines, and comparison of the ClinGen LD VCEP's GAA variant classifications with variant classifications submitted to ClinVar. The LD VCEP has added to the publicly available knowledge on the pathogenicity of variants in GAA by increasing the number of expert-curated GAA variants present in ClinVar, and aids in resolving conflicting classifications and variants of uncertain clinical significance.

12-h clock regulation of genetic information flow by XBP1s.

Our group recently characterized a cell-autonomous mammalian 12-h clock independent from the circadian clock, but its function and mechanism of regulation remain poorly understood. Here, we show that in mouse liver, transcriptional regulation significantly contributes to the establishment of 12-h rhythms of mRNA expression in a manner dependent on Spliced Form of X-box Binding Protein 1 (XBP1s). Mechanistically, the motif stringency of XBP1s promoter binding sites dictates XBP1s's ability to drive 12-h rhythms of nascent mRNA transcription at dawn and dusk, which are enriched for basal transcription regulation, mRNA processing and export, ribosome biogenesis, translation initiation, and protein processing/sorting in the Endoplasmic Reticulum (ER)-Golgi in a temporal order consistent with the progressive molecular processing sequence described by the central dogma information flow (CEDIF). We further identified GA-binding proteins (GABPs) as putative novel transcriptional regulators driving 12-h rhythms of gene expression with more diverse phases. These 12-h rhythms of gene expression are cell autonomous and evolutionarily conserved in marine animals possessing a circatidal clock. Our results demonstrate an evolutionarily conserved, intricate network of transcriptional control of the mammalian 12-h clock that mediates diverse biological pathways. We speculate that the 12-h clock is coopted to accommodate elevated gene expression and processing in mammals at the two rush hours, with the particular genes processed at each rush hour regulated by the circadian and/or tissue-specific pathways.

Differential gene expression in layer 3 pyramidal neurons across 3 regions of the human cortical visual spatial working memory network.

Visual spatial working memory (vsWM) is mediated by a distributed cortical network composed of multiple nodes, including primary visual (V1), posterior parietal (PPC), and dorsolateral prefrontal (DLPFC) cortices. Feedforward and feedback information is transferred among these nodes via projections furnished by pyramidal neurons (PNs) located primarily in cortical layer 3. Morphological and electrophysiological differences among layer 3 PNs across these nodes have been reported; however, the transcriptional signatures underlying these differences have not been examined in the human brain. Here we interrogated the transcriptomes of layer 3 PNs from 39 neurotypical human subjects across 3 critical nodes of the vsWM network. Over 8,000 differentially expressed genes were detected, with more than 6,000 transcriptional differences present between layer 3 PNs in V1 and those in PPC and DLPFC. Additionally, over 600 other genes differed in expression along the rostral-to-caudal hierarchy formed by these 3 nodes. Moreover, pathway analysis revealed enrichment of genes in V1 related to circadian rhythms and in DLPFC of genes involved in synaptic plasticity. Overall, these results show robust regional differences in the transcriptome of layer 3 PNs, which likely contribute to regional specialization in their morphological and physiological features and thus in their functional contributions to vsWM.

Proteomic Analysis Revealed Different Molecular Mechanisms of Response to PEG Stress in Drought-Sensitive and Drought-Resistant Sorghums.

Drought is the major limiting factor that directly or indirectly inhibits the growth and reduces the productivity of sorghum (Sorghum bicolor (L.) Moench). As the main vegetative organ of sorghum, the response mechanism of the leaf to drought stress at the proteomic level has not been clarified. In the present study, nano-scale liquid chromatography mass spectrometry (nano-LC-MS/MS) technology was used to compare the changes in the protein expression profile of the leaves of drought-sensitive (S4 and S4-1) and drought-resistant (T33 and T14) sorghum varieties at the seedling stage under 25% PEG-6000 treatment for 24 h. A total of 3927 proteins were accurately quantitated and 46, 36, 35, and 102 differentially abundant proteins (DAPs) were obtained in the S4, S4-1, T14, and T33 varieties, respectively. Four proteins were randomly selected for parallel reaction monitoring (PRM) assays, and the results verified the reliability of the mass spectrometry (MS) results. The response mechanism of the drought-sensitive sorghum leaves to drought was attributed to the upregulation of proteins involved in the tyrosine metabolism pathway with defense functions. Drought-resistant sorghum leaves respond to drought by promoting the TCA cycle, enhancing sphingolipid biosynthesis, interfering with triterpenoid metabolite synthesis, and influencing aminoacyl-tRNA biosynthesis. The 17 screened important candidate proteins related to drought stress were verified by quantitative real-time PCR (qRT-PCR), the results of which were consistent with the results of the proteomic analysis. This study lays the foundation for revealing the drought-resistance mechanism of sorghum at the protein level. These findings will help us cultivate and improve new drought-resistant sorghum varieties.

Comparative proteomic analysis reveals that the Heterosis of two maize hybrids is related to enhancement of stress response and photosynthesis respectively.

BACKGROUND: Heterosis refers to superior traits exhibiting in a hybrid when compared with both parents. Generally, the hybridization between parents can change the expression pattern of some proteins such as non-additive proteins (NAPs) which might lead to heterosis. 'Zhongdan808' (ZD808) and 'Zhongdan909' (ZD909) are excellent maize hybrids in China, however, the heterosis mechanism of them are not clear. Proteomics has been wildly used in many filed, and comparative proteomic analysis of hybrid and its parents is helpful for understanding the mechanism of heterosis in the two maize hybrids. RESULTS: Over 2000 protein groups were quantitatively identified from second seedling leaves of two hybrids and their parents by label-free quantification. Statistical analysis of total identified proteins, differentially accumulated proteins (DAPs) and NAPs of the two hybrids revealed that both of them were more similar to their female parents. In addition, most of DAPs were up-regulated and most of NAPs were high parent abundance or above-high parent abundance in ZD808, while in ZD909, most of DAPs were down-regulated and most of NAPs were low parent abundance or below-low parent abundance. Pathway enrichment analysis showed that more of stress response-related NAPs in ZD808 were high parent abundance or above-high parent abundance, and most of PS related NAPs in ZD909 were high parent abundance or above-high parent abundance. Finally, four stress response-related proteins and eight proteins related to PS were verified by PRM, ten of them had significant differences between hybrid and midparent value. CONCLUSIONS: Even though every one of the two hybrids were more similar to its female parent at proteome level, the biological basis of heterosis is different in the two maize hybrids. In comparison with their parents, the excellent agronomic traits of hybrid ZD808 is mainly correlated with the high expression levels of some proteins related to stress responses and metabolic functions, while traits of ZD909 is mainly correlated with high expressed proteins related to photosynthesis. Our proteomics results support previous physiological and morphological research and have provided useful information in understanding the reason of valuable agronomic traits.

[Research progress on Chinese medicinal material-derived active polypeptides against ischemic cardiovascular and cerebrovascular diseases].

Ischemic cardiovascular and cerebrovascular diseases threatening human health and survival have high morbidity and mortality. The common cause of them is reduced blood supply caused by vascular stenosis, atherosclerosis, and infarction. However,the pathological processes of ischemic cardiovascular and cerebrovascular diseases are complex, involving oxidative stress, calcium overload, inflammation, apoptosis, autophagy and other mechanisms. Protein drugs such as recombinant tissue plasminogen activator(rt-PA) and urokinase have been proved with excellent therapeutic effects and huge economic and social benefits in the clinical treatment and interventional therapy. Among them, peptide drugs have shown unique advantages and potential prospects owing to their strong biological activity, high target specificity, biochemical diversity, and low toxicity. Chinese medicinal materials, characterized by multi-component and multi-target therapy, have also shown excellent clinical efficacy against ischemic cardiovascular and cerebrovascular diseases. However, the research and development of related peptides in Chinese medicinal materials is at the initial stage. Therefore, this paper reviewed the targets and action mechanisms of a variety of Chinese medicinal material-derived polypeptides with activities against ischemic cardiovascular and cerebrovascular diseases, aiming to provide support for the in-depth research as well as the clinical development and application of these polypeptides.

[Chronic heart failure due to Qi deficiency and blood stasis and intervention mechanism of Compound Renshen Buqi Granules:a proteomics-based study].

Compound Renshen Buqi Granules have been widely used to treat chronic heart failure(CHF) due to Qi deficiency and blood stasis, but the mechanism of action remains unclear. This paper explored the pathogenesis of CHF due to Qi deficiency and blood stasis and the intervention mechanism of Compound Renshen Buqi Granules based on quantitative proteomics for uncovering the biological basis. SD rats were divided into the normal control(N) group, normal+Compound Renshen Buqi Granules(ND) group, model(M) group, model+Compound Renshen Buqi Granules(D) group, and positive control(Y) group. The rat model of CHF due to Qi deficiency and blood stasis was established by ligation of the left anterior descending(LAD) coronary artery and chronic sleep deprivation. The rats in the ND group and D group were provided with Compound Renshen Buqi Granules, while those in the Y group received valsartan. Six weeks later, the serum was sampled and the data-dependent acquisition(DDA) was employed for the non-targeted quantitative proteomics analysis of the differences in protein expression among groups, followed by the targeted analysis of differentially expressed proteins(DEPs) generated by data-independent acquisition(DIA). Compared with the N group, the rats in the M group pre-sented with decreased body weight, grip strength, and pulse amplitude and increased RGB value on the tongue surface. The pathomorphological examination revealed inflammatory cell infiltration, cell degeneration and necrosis, tissue fibrosis, etc. After the intervention with Compound Renshen Buqi Granules, multiple indicators were reversed. As demonstrated by proteomics results, there were 144 and 111 DEPs found in the M group and ND group in comparison with the N group. Compared with the M group, 107 and 194 DEPs were found in the D group and the Y group, respectively. Compared with the ND group, 119 DEPs were detected in the D group. As illustrated by DIA-based verification, the quantitative results of six proteins in each group were consistent with those by DDA. The syndrome indicators and pathomorphological examination results demonstrated that the protein expression profile of rats with CHF due to Qi deficiency and blood stasis changed obviously. However, Compound Renshen Buqi Granules were able to reverse the differential expression of immune proteins to regulate CHF of Qi deficiency and blood stasis syndrome, which has provided clues for figuring out the pathogenesis of CHF due to Qi deficiency and blood stasis and the intervention mechanism of Compound Renshen Buqi Granules.

Activating p53 family member TAp63: A novel therapeutic strategy for targeting p53-altered tumors.

BACKGROUND: Over 96% of high-grade ovarian carcinomas and 50% of all cancers are characterized by alterations in the p53 gene. Therapeutic strategies to restore and/or reactivate the p53 pathway have been challenging. By contrast, p63, which shares many of the downstream targets and functions of p53, is rarely mutated in cancer. METHODS: A novel strategy is presented for circumventing alterations in p53 by inducing the tumor-suppressor isoform TAp63 (transactivation domain of tumor protein p63) through its direct downstream target, microRNA-130b (miR-130b), which is epigenetically silenced and/or downregulated in chemoresistant ovarian cancer. RESULTS: Treatment with miR-130b resulted in: 1) decreased migration/invasion in HEYA8 cells (p53 wild-type) and disruption of multicellular spheroids in OVCAR8 cells (p53-mutant) in vitro, 2) sensitization of HEYA8 and OVCAR8 cells to cisplatin (CDDP) in vitro and in vivo, and 3) transcriptional activation of TAp63 and the B-cell lymphoma (Bcl)-inhibitor B-cell lymphoma 2-like protein 11 (BIM). Overexpression of TAp63 was sufficient to decrease cell viability, suggesting that it is a critical downstream effector of miR-130b. In vivo, combined miR-130b plus CDDP exhibited greater therapeutic efficacy than miR-130b or CDDP alone. Mice that carried OVCAR8 xenograft tumors and were injected with miR-130b in 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) liposomes had a significant decrease in tumor burden at rates similar to those observed in CDDP-treated mice, and 20% of DOPC-miR-130b plus CDDP-treated mice were living tumor free. Systemic injections of scL-miR-130b plus CDDP in a clinically tested, tumor-targeted nanocomplex (scL) improved survival in 60% and complete remissions in 40% of mice that carried HEYA8 xenografts. CONCLUSIONS: The miR-130b/TAp63 axis is proposed as a new druggable pathway that has the potential to uncover broad-spectrum therapeutic options for the majority of p53-altered cancers.

Single-Cell RNA-Sequencing Identifies Activation of TP53 and STAT1 Pathways in Human T Lymphocyte Subpopulations in Response to Ex Vivo Radiation Exposure.

Detrimental health consequences from exposure to space radiation are a major concern for long-duration human exploration missions to the Moon or Mars. Cellular responses to radiation are expected to be heterogeneous for space radiation exposure, where only high-energy protons and other particles traverse a fraction of the cells. Therefore, assessing DNA damage and DNA damage response in individual cells is crucial in understanding the mechanisms by which cells respond to different particle types and energies in space. In this project, we identified a cell-specific signature for radiation response by using single-cell transcriptomics of human lymphocyte subpopulations. We investigated gene expression in individual human T lymphocytes 3 h after ex vivo exposure to 2-Gy gamma rays while using the single-cell sequencing technique (10X Genomics). In the process, RNA was isolated from ~700 irradiated and ~700 non-irradiated control cells, and then sequenced with ~50 k reads/cell. RNA in each of the cells was distinctively barcoded prior to extraction to allow for quantification for individual cells. Principal component and clustering analysis of the unique molecular identifier (UMI) counts classified the cells into three groups or sub-types, which correspond to CD4+, naïve, and CD8+/NK cells. Gene expression changes after radiation exposure were evaluated using negative binomial regression. On average, BBC3, PCNA, and other TP53 related genes that are known to respond to radiation in human T cells showed increased activation. While most of the TP53 responsive genes were upregulated in all groups of cells, the expressions of IRF1, STAT1, and BATF were only upregulated in the CD4+ and naïve groups, but were unchanged in the CD8+/NK group, which suggests that the interferon-gamma pathway does not respond to radiation in CD8+/NK cells. Thus, single-cell RNA sequencing technique was useful for simultaneously identifying the expression of a set of genes in individual cells and T lymphocyte subpopulation after gamma radiation exposure. The degree of dependence of UMI counts between pairs of upregulated genes was also evaluated to construct a similarity matrix for cluster analysis. The cluster analysis identified a group of TP53-responsive genes and a group of genes that are involved in the interferon gamma pathway, which demonstrate the potential of this method for identifying previously unknown groups of genes with similar expression patterns.

Proteomic Analysis of the Function of a Novel Cold-Regulated Multispanning Transmembrane Protein COR413-PM1 in Arabidopsis.

The plasma membrane is the first subcellular organ that senses low temperature, and it includes some spanning transmembrane proteins that play important roles in cold regulation. COR413-PM1 is a novel multispanning transmembrane cold-regulated protein; however, the related functions are not clear in Arabidopsis. We found the tolerance to freezing stress of cor413-pm1 was lower than wild-type (WT). A proteomics method was used to analyze the differentially abundant proteins (DAPs) between cor413-pm1 and WT. A total of 4143 protein groups were identified and 3139 were accurately quantitated. The DAPs associated with COR413-PM1 and freezing treatment were mainly involved in the metabolism of fatty acids, sugars, and purine. Quantitative real-time PCR (qRT-PCR) confirmed the proteomic analysis results of four proteins: fatty acid biosynthesis 1 (FAB1) is involved in fatty acid metabolism and might affect the plasma membrane structure; fructokinase 3 (FRK3) and sucrose phosphate synthase A1 (SPSA1) play roles in sugar metabolism and may influence the ability of osmotic adjustment under freezing stress; and GLN phosphoribosyl pyrophosphate amidotransferase 2 (ASE2) affects freezing tolerance through purine metabolism pathways. In short, our results demonstrate that the multispanning transmembrane protein COR413-PM1 regulates plant tolerance to freezing stress by affecting the metabolism of fatty acids, sugars, and purine in Arabidopsis.

[Quantitative proteomic research of biological basis of Buyang Huanwu decoction therapy for cerebral infarction combined with Qi-deficiency and blood-stasis syndrome].

Chinese medicine Buyang Huanwu decoction (BYHW) is widely used in treating cerebral infarction combined with Qi-deficiency and blood-stasis syndrome, but the pharmacological basis is still not clear. This study aims to uncover the biological basis of BYHW therapy for cerebral infarction combined with Qi-deficiency and blood-stasis syndrome using label-free proteomic technology. Using Qi deficiency and blood stasis rat cerebral infarction model as the research object, the protein expression of rat brain tissue was compared among the sham operation group, the model group and the drug group. Quantitative analysis of the 3 groups of tissue samples detected 3 959, 3 996 and 4 055 proteins in the sham operation group, the model group and the drug group, respectively. Take model group as the control group, 391 proteins were identified to be upregulated or downregulated for more than 2 folds. Biological analysis and functional enrichment of the differentially expressed proteins revealed that BYHW may treat cerebral infarction combined with Qi-deficiency and blood-stasis syndrome through energy metabolism, nervous system and several signal pathways. This study preliminarily revealed the pharmacological mechanism of BYHW at the protein level, and provided a molecular basis for clinical treatment and traditional Chinese medicine research on cerebral infarction combined with Qi-deficiency and blood-stasis syndrome.

Epigenetic regulation of global proteostasis dynamics by RBBP5 ensures mammalian organismal health.

Proteostasis is vital for cellular health, with disruptions leading to pathologies including aging, neurodegeneration and metabolic disorders. Traditionally, proteotoxic stress responses were studied as acute reactions to various noxious factors; however, recent evidence reveals that many proteostasis stress-response genes exhibit ~12-hour ultradian rhythms under physiological conditions in mammals. These rhythms, driven by an XBP1s-dependent 12h oscillator, are crucial for managing proteostasis. By exploring the chromatin landscape of the murine 12h hepatic oscillator, we identified RBBP5, a key subunit of the COMPASS complex writing H3K4me3, as an essential epigenetic regulator of proteostasis. RBBP5 is indispensable for regulating both the hepatic 12h oscillator and transcriptional response to acute proteotoxic stress, acting as a co-activator for proteostasis transcription factor XBP1s. RBBP5 ablation leads to increased sensitivity to proteotoxic stress, chronic inflammation, and hepatic steatosis in mice, along with impaired autophagy and reduced cell survival in vitro. In humans, lower RBBP5 expression is associated with reduced adaptive stress-response gene expression and hepatic steatosis. Our findings establish RBBP5 as a central regulator of proteostasis, essential for maintaining mammalian organismal health.

Combined sequence and copy number analysis improves diagnosis of limb girdle and other myopathies.

OBJECTIVE: Clinical and genetic heterogeneities make diagnosis of limb-girdle muscular dystrophy (LGMD) and other overlapping disorders of muscle weakness complicated and expensive. We aimed to develop a comprehensive next generation sequence-based multi-gene panel ("The Lantern Focused Neuromuscular Panel") to detect both sequence variants and copy number variants in one assay. METHODS: Patients with clinical diagnosis of LGMD or other overlapping muscular dystrophies in the United States were tested by PerkinElmer Genomics in 2018-2021 via "The Lantern Project," a sponsored diagnostic testing program. Sixty-six genes related to LGMD subtypes- and other myopathies were investigated. Main outcomes were diagnostic yield, gene-variant spectrum, and LGMD subtypes' prevalence. RESULTS: Molecular diagnosis was established in 19.6% (1266) of 6473 cases. Major genes contributing to LGMD were identified including CAPN3 (5.4%, 68), DYSF (4.0%, 51), GAA (3.7%, 47), ANO5 (3.6%, 45), and FKRP (2.7%, 34). Genes of other overlapping MD subtypes identified included PABPN1 (10.5%, 133), VCP (2.2%, 28), MYOT (1.2% 15), LDB3 (1.0%, 13), COL6A1 (1.5%, 19), FLNC (1.1%, 14), and DNAJB6 (0.8%, 10). Different sizes of copy number variants including single exon, multi-exon, and whole genes were identified in 7.5% (95) cases in genes including DMD, EMD, CAPN3, ANO5, SGCG, COL6A2, DOK7, and LAMA2. INTERPRETATION: "The Lantern Focused Neuromuscular Panel" enables identification of LGMD subtypes and other myopathies with overlapping clinical features. Prevalence of some MD subtypes was higher than previously reported. Widespread deployment of this comprehensive NGS panel has the potential to ensure early, accurate diagnosis as well as re-define MD epidemiology.

To explore the regulatory effect of Buyang Huanwu Decoction on cerebral infarction based on quantitative proteomics.

Buyang Huanwu Decoction (BYHW) contains chemical components such as ligustrazine, oxypaeoniflora, chlorogenic acid, and others. To explore the neuroprotective effect and potential target protein of BYHW in cerebral infarction (CI). A double-blind, randomized controlled trial was established and patients with CI were divided into the BYHW group (n = 35) and the control group (n = 30). To evaluate the efficacy by TCM syndrome score and clinical indicators, and to explore the changes of serum proteins by proteomics technology, so as to explore the mechanism of BYHW and potential target proteins. The study found that compared with the control group, the TCM syndrome score, including Deficiency of Vital Energy (DVE), Blood Stasis (BS), and NIHSS in the BYHW group decreased significantly (p < 0.05), and the Barthel Index (BI) score was significantly higher. A total of 99 differential regulatory proteins were identified by proteomics, which act on lipids and atherosclerosis, complement and coagulation cascade, and TNF-α signaling pathway. In addition, Elisa verified the results of proteomics and found that BYHW can reduce the neurological impairments focus on IL-1ß, IL-6, TNF-α, MCP-1, MMP-9, and PAI-1. Significance: In this study, quantitative proteomics was used in combination with liquid chromatography-mass spectrometry (LC-MS/MS) to study the therapeutic effect of BYHW on cerebral infarction (CI) and potential changes in serum proteomics. In addition, the public proteomics database was used for bioinformatics analysis, and Elisa experiment verified the results of proteomics, further clarifying the potential protection mechanism of BYHW on CI.

Neuroprotective effect of Astragali Radix on cerebral infarction based on proteomics.

Objective: Astragali Radix (AR, Huangqi in Chinese) has a neuroprotective effect on cerebral infarction (CI). In order to explore the biological basis and therapeutic mechanism of AR in CI, a double-blind randomized controlled trial was established in this study, and proteomics analysis was carried out on serum samples of patients. Methods: The patients were divided into the AR group (n = 35) and the control group (n = 30). The curative effect was evaluated by the traditional Chinese medicine (TCM) syndrome score and clinical indicators, and the serum of the two groups was analyzed by proteomics. Based on bioinformatics analysis methods, the changes in differential proteins between two groups of samples were explored, and the key proteins were validated through enzyme-linked immunosorbent assay (ELISA). Results: The results of this study showed that the scores of deficiency of vital energy (DVE), blood stasis (BS), and NIH Stroke Scale (NIHSS) decreased significantly (p < 0.05), while the scores of the Barthel Index (BI) increased, indicating that AR could significantly improve the symptoms of CI patients. In addition, we found that compared with the control group, AR upregulated 43 proteins and downregulated 20 proteins, especially focusing on anti-atherosclerosis and neuroprotective effects. Moreover, ELISA indicated the levels of IL-6, TNF-α, VCAM-1, MCP-1, and ICAM-1 were significantly decreased in the serum of the AR group (p < 0.05, p < 0.01). Conclusion: This study found that AR can significantly recover the clinical symptoms of CI. Serum proteomics research results show that AR may act on IL-6, TNF-α, VCAM-1, MCP-1, and ICAM-1, and play anti-atherosclerosis and neuroprotective roles. Clinical Trial Registration: [clinicaltrials.gov], identifier [NCT02846207].

Melatonin enhances drought tolerance by affecting jasmonic acid and lignin biosynthesis in wheat (Triticum aestivum L.).

Drought severely affects the yield of wheat (Triticum aestivum L.), which is mainly grown in arid and semi-arid regions. Melatonin plays an important role in various types of stress resistance in plants, including drought resistance. However, the molecular mechanism through which melatonin affects drought tolerance remains largely unknown. In this study, we revealed that melatonin (100 µM) significantly improved drought resistance during the maturation stage of Chinese Spring, Shi4185, and Hanxuan10 varieties, but not Chang6878. Further physiological, transcriptomic, and proteomic data analysis at the wheat seedling stage revealed that melatonin increased jasmonic acid (JA) content, upregulating the expression of JA genes (LOX1.5 and LOX2.1) and two transcription factors (HY5 and MYB86) under drought conditions. It also upregulated genes related to lignin biosynthesis (4CL2, P5CS1, and CCR2) as well as starch and sucrose metabolism (PME53 and SUS4). Additionally, melatonin alleviated photosynthetic and cell membrane damage caused by drought stress through maintaining low levels of hydrogen peroxide. The current results elucidate melatonin-regulated pathways in wheat and provide evidence for using melatonin as a potential biostimulant to improve wheat drought resistance under field conditions in the future.