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Abstract Proximity labeling (PL) via biotinylation coupled with mass spectrometry (MS) captures spatial proteomes in cells. Large-scale processing requires a workflow minimizing hands-on time and enhancing quantitative reproducibility. We introduced a scalable PL pipeline integrating automated enrichment of biotinylated proteins in a 96-well plate format. Combining this with optimized quantitative MS based on data-independent acquisition (DIA), we increased sample throughput and improved protein identification and quantification reproducibility. We applied this pipeline to delineate subcellular proteomes across various compartments. Using the 5HT2A serotonin receptor as a model, we studied temporal changes of proximal interaction networks induced by receptor activation. In addition, we modified the pipeline for reduced sample input to accommodate CRISPR-based gene knockout, assessing dynamics of the 5HT2A network in response to perturbation of selected interactors. This PL approach is universally applicable to PL proteomics using biotinylation-based PL enzymes, enhancing throughput and reproducibility of standard protocols.

Summary: Artificial intelligence (AI) is rarely directly used in patent foramen ovale (PFO) diagnosis. In this study, an AI model was developed to detect the presence of PFO automatically in both contrast transthoracic echocardiography (cTTE) images and videos. The whole intelligent diagnosis neural network framework consists of two functional modules of image segmentation (Unet, n = 1866) and image classification (ResNet 101, n = 9152). Finally, another test databases, including 20 cTTE videos (4609 cTTE images), was used to compare the RLS classification model accuracy between AI model and different levels of physicians. The Dice similarity coefficient of left chamber segmentation model of cTTE images was 91.41%, the accuracy of PFO-RLS classification model of cTTE images was 83.55%, the accuracy of PFO-RLS classification model of cTTE videos was 90%. Besides, the AI diagnosis time was significantly shorter than doctors (at only 1.3 s).

The rise of nanotechnology has opened new horizons for cancer immunotherapy. However, most nanovaccines fabricated with nanomaterials suffer from carrier-related concerns, including low drug loading capacity, unpredictable metabolism, and potential systemic toxicity, which bring obstacles for their clinical translation. Herein, we developed an antigen self-assembled nanovaccine, which was resulted from a simple acryloyl modification of the antigen to induce self-assembly. Furthermore, a dendritic cell targeting head mannose monomer and a mevalonate pathway inhibitor zoledronic acid (Zol) were integrated or absorbed onto the nanoparticles (denoted as MEAO-Z) to intensify the immune response. The synthesized nanovaccine with a diameter of around 70 nm showed successful lymph node transportation, high dendritic cell internalization, promoted costimulatory molecule expression, and preferable antigen cross-presentation. In virtue of the above superiorities, MEAO-Z induced remarkably higher titers of serum antibody, stronger cytotoxic T lymphocyte immune responses and IFN-γ secretion than free antigen and adjuvants. In vivo, MEAO-Z significantly suppressed EG7-OVA tumor growth and prolonged the survival time of tumor-bearing mice. These results indicated the translation promise of our self-assembled nanovaccine for immune potentiation and cancer immunotherapy.

COCH (coagulation factor C homology) is one of the most frequently mutated genes of autosomal dominant non-syndromic hearing loss. Variants in COCH could cause DFNA9, which is characterized by late-onset hearing loss with variable degrees of vestibular dysfunction. In this study, we report a Chinese family with a novel COCH variant (c.1687delA) causing p.D544Vfs*3 in the cochlin. Comprehensive audiometric tests and vestibular function assessments were taken to acquire the phenotypic profile of the subjects. Next-generation sequencing was conducted and segregation analysis was carried out using Sanger sequencing. The proband presented mild vestibular symptoms and normal functional assessment results in almost every test, while the variant co-segregated with hearing impairment in the pedigree. The variant was located beyond the vWFA2 domain, which was predicted to affect the post-translational cleavage of the cochlin via molecular modeling analysis. Notably, in the overexpressing study, by transient transfecting the HEK 293T cells, we found that the p.D544Vfs*3 variant increased the formation of multimeric cochlin. Our result enriched the spectrum of DFNA9-linked pathological COCH variants and suggested that variants, causative of cochlin multimerization, could be related to DFNA9 with sensorineural hearing loss rather than serious vestibular symptoms.

The small GTPase ADP-ribosylation factor 6 (ARF6) mediates chemokine (C-C motif) ligand 18 (CCL18)-induced activation of breast cancer (BC) metastasis through its downstream effector AMAP1. However, the molecular mechanisms underlying CCL18 up-regulating ARF6 remain largely unclear. Here, microRNAs (miRNAs) that target ARF6 were predicted and selected in high metastatic BC cells treated with CCL18. Next, we assessed the role of exosomal miR-760 in vitro and in vivo. We further analyzed the expression of ARF6, AMAP1, and phosphorylated (p)-AMAP1 in tumor and adjacent normal tissues. We first observed that CCL18 increased the expression of ARF6 and p-AMAP1 and activated the Src/phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. ARF6 knockdown significantly impaired CCL18-induced malignant cellular behaviors and the Src/PI3K/Akt signaling pathway. Next, ARF6 was confirmed as a target gene of miR-760 in exosomes derived from CCL18-stimulated high metastatic BC cells. Moreover, recipient MCF-7 cells could effectively uptake these miR-760-rich exosomes that significantly promoted proliferation, tumor growth in vivo, migration, invasion, and chemoresistance by activating ARF6-mediated Src/PI3K/Akt signaling and the epithelial-mesenchymal transition (EMT) pathway. Together, our results support that exosomal miR-760 secreted by CCL18-stimulated high metastatic BC cells promoted the malignant behaviors in low metastatic BC cells by up-regulating the ARF6-mediated Src/PI3K/Akt signaling pathway.

Soybean seeds show great potential as a safe and cost-effective host for the large-scale production of biopharmaceuticals and industrially important macromolecules. However, the yields of desired recombinant proteins in soybean seeds are usually lower than the economic threshold for their potential commercialization. Our previous study demonstrated that polypeptide fusion such as maize γ-zein or elastin-like polypeptide (ELP) could significantly increase the accumulation of foreign proteins. In the present study, a recombination strategy of polypeptide fusions (γ-zein or ELP) and suppression of intrinsic storage proteins (glycinin or conglycinin) via RNA interference was further exploited to improve the yield of the target protein in soybean seeds. Transgenic soybean plants harboring both polypeptide-fused green fluorescent protein (GFP) and glycinin/conglycinin RNAi expression cassettes were generated and confirmed by molecular analysis. The results showed that on both the glycinin and conglycinin suppression backgrounds, the average accumulation levels of recombinant zein-GFP and GFP-ELP proteins were significantly increased as compared to that of their counterparts without such suppressions in our previous study. Moreover, zein-GFP and GFP-ELP accumulation was also remarkably higher than unfused GFP on the glycinin suppression background. However, no significant differences were detected in the glycinin or conglycinin suppression backgrounds for the same polypeptide fusion constructs, though suppression of one of the storage proteins in soybean seeds led to a significant increase in the other. Additionally, the increases in the recombinant protein yield did not affect the total protein content and the protein/oil ratio in soybean seeds. Taken together, the results indicate that both the fusion of the foreign protein with polypeptide tags together with the depletion of endogenous storage proteins contributed to a higher accumulation of the recombinant proteins without affecting the total protein content or the protein/oil ratio in soybean seeds.

Abstract As a monocotyledonous plant in family Cyperaceae, yellow nutsedge (Cyperus esculentus L.) is unique in accumulating a substantial amount of oil in underground tubers and provides a model system for studying oil accumulation in nonseed tissues. However, no data on the mitochondrial and nuclear genome sequences of this species are available, which greatly limits our understanding of its evolutionary characteristics and some essential biological mechanisms. In the present study, we report the first complete mitochondrial genome sequence of the cultivated yellow nutsedge. The analysis of the genome showed that the yellow nutsedge mitochondrial genome is 1,002,696 bp in size and encodes 62 genes consisting of 36 protein‐coding genes (PCGs), 20 transfer RNA (tRNA) genes, and six ribosomal RNA (rRNA) genes. Compared with other angiosperms, yellow nutsedge mitochondrial genome contains much higher percentage of noncoding sequences (95.36%). Sixteen plastid‐derived fragments were identified to be strongly associated with mitochondrial genes including one intact plastid‐related gene (ndhH). Comparative analysis with seven other sequenced plant mitochondrial genomes revealed that two syntenic gene clusters, rps3‐rpl16 and rps12‐nad3, are highly conserved in all plant mitochondrial genomes, and the mitochondrial genome of yellow nutsedge is more similar to those of monocotyledons in the gene order. Phylogenetic analysis based on 13 shared protein‐encoding genes in eight plant species showed that yellow nutsedge is evolutionarily more closely related to monocotyledonary species. Overall, the species‐specific features of the cultivated yellow nutsedge mitochondrial genome provide additional information for the evolutionary and comparative genomic studies in the yellow nutsedge and other Cyperus species of the Cyperaceae family.

Abstract Background Antimicrobial peptides play important roles in both plant and animal defense systems. Moreover, over-expression of CaAMP1 (Capsicum annuum antimicrobial protein 1), an antimicrobial protein gene isolated from C. annuum leaves infected with Xanthomonas campestris pv. vesicatoria, confers broad-spectrum resistance to hemibiotrophic bacterial and necrotrophic fungal pathogens in Arabidopsis. Phytophthora root and stem rot (PRR), caused by the fungus Phytophthora sojae, is one of the most devastating diseases affecting soybean (Glycine max) production worldwide. Results In this study, CaAMP1 was transformed into soybean by Agrobacterium-mediated genetic transformation. Integration of the foreign gene in the genome of transgenic soybean plants and its expression at the translation level were verified by Southern and western blot analyses, respectively. CaAMP1 over-expression (CaAMP1-OX) lines inoculated with P. sojae race 1 exhibited enhanced and stable PRR tolerance through T2–T4 generations compared with the wild-type Williams 82 plants. Gene expression analyses in the transgenic plants revealed that the expression of salicylic acid-dependent, jasmonic acid-dependent, and plant disease resistance genes (R-genes) were significantly up-regulated after P. sojae inoculation. Conclusions These results indicate that CaAMP1 over-expression can significantly enhance PRR tolerance in soybean by eliciting resistance responses mediated by multiple defense signaling pathways. This provides an alternative approach for developing soybean varieties with improved tolerance against soil-borne pathogenic PRR.

BackgroundBreast cancer patients who achieve pathological complete response (pCR) after neoadjuvant chemotherapy (NAC) have favorable outcomes. Reliable predictors for pCR help to identify patients who will benefit most from NAC. The pretreatment serum albumin-to-alkaline phosphatase ratio (AAPR) has been shown to be a prognostic predictor in several malignancies, but its predictive value for pCR in breast cancer is still unknown. This study aims to investigate the predictive role of AAPR in breast cancer patients and develop an AAPR-based nomogram for pCR rate prediction.MethodsA total of 780 patients who received anthracycline and taxane-based NAC from January 2012 to March 2018 were retrospectively analyzed. Univariate and multivariate analyses were performed to assess the predictive value of AAPR and other clinicopathological factors. A nomogram was developed and calibrated based on multivariate logistic regression. A validation cohort of 234 patients was utilized to further validate the predictive performance of the model. The C-index, calibration plots and decision curve analysis (DCA) were used to evaluate the discrimination, calibration and clinical value of the model.ResultsPatients with a lower AAPR (

Abstract Lactic acid bacteria (LAB), a diverse family of gram‐positive bacteria, has been proven effective in delivering varieties of therapeutic and prophylactic molecules such as antigens and cytokines. Featuring the properties of acid‐resistant, high uptake into Peyer's patches, and superior capacity for inducing secretory IgA antibodies, LAB have good potential to be used as vaccine vectors for mucosal vaccination. Mucosal immunization enables both mucosal and systemic immune responses, which are critical for resisting pathogens that invade the host through the mucosal surfaces. With the development of genetic engineering, LAB strains, primarily Lactococcus and Lactobacillus have been exploited to express a range of heterologous antigens. Numerous studies have demonstrated that LAB mucosal vaccines can stimulate all arms of the immune system to provide adequate protection against pathogen infections. Additionally, several LAB‐based human papillomavirus vaccines have entered the clinical trial studies, which suggest the great promise of LAB vaccines for new interventions in mucosal transport diseases. Herein, we will discuss the factors that influence the immunogenicity of LAB vaccines, including LAB strains, the location of antigens, and administration routes, and focus on the current strategies that have been reported for optimizing LAB vaccines.

The Valsalva maneuver (VM) is the most sensitive auxiliary method for the detection of patent foramen ovale (PFO), but it is difficult to assess whether the maneuver is adequately performed during transesophageal echocardiography (TEE). In this study, we tried to use aortic root downward movement as a novel method for judging whether VM was adequate or not, and to evaluate whether this novel method can increase the sensitivity of detecting PFO. A total of 224 patients with clinically suspected PFO were enrolled in this study. These patients were injected with activated normal saline to detect the right-to-left shunt (RLS), in the following three conditions: contrast-enhanced transthoracic echocardiography under adequate VM (AVM cTTE), contrast-enhanced TEE under non-adequate VM (non-AVM cTEE), and cTEE under adequate VM (AVM cTEE). A novel method in which the aorta root moves downward (movement range ≥16 mm) in the cTEE judged whether AVM was performed. The PFO detection rate and sensitivity of AVM cTEE were better than that of non-AVM cTEE (detection rate: 108 PFOs [48.2%] vs. 86 PFOs [38.4%], p = 0.036; sensitivity: 100% vs. 79.6%). Among AVM cTTE, non-AVM cTEE, and AVM cTEE, the RLS grade evaluation results were inconsistent, with significant differences (p < 0.05). Non-AVM cTEE had RLS underestimation or false negatives. Compared with non-AVM cTEE, AVM cTEE and AVM cTTE had better consistency in evaluating PFO RLS (kappa value = 0.675). Aortic root downward movement could be used as a novel method for judging the effectiveness of VM, which is critical for the detection of PFO in cTEE. Concerning effectiveness and convenience, this method should be promoted during the clinical detection of PFO.

Tiger nut (Cyperus esculentus L.) has recently attracted increasing interest from scientific and technological communities because of its potential for serving as additional source of food, oil, and feed. The present study reports morphology and biochemical characterization of 42 tiger nut accessions collected from China and other counties performed in the 2020 and 2021 growing seasons at Nongan, Jilin Province. Assessment of variability of 14 agronomic traits including plant height, maturation, leaf width, tilling number, color, size, and shape: 100-tuber weight showed a wide range of phenotypic variation. The color, size, and shape and maturation of the tubers, as well as the leaf width, were the most distinct characteristics describing variation among the accessions. Compositional analyses of major nutritional components of the tubers reveals that this crop could be a source of high-value proteins, fatty acids, and carbohydrates. Specifically, tiger nut tubers contained high levels of starch, oil, and sugars, and significant amounts of fiber, Ca, P, and Na. Furthermore, the tubers appeared to be a good source of proteins as they contain 16 amino acids, including the essential ones. Amino acid profiles were dominated by aspartic acid followed by glutamic acid, leucine, alanine, and arginine. Overall, these results demonstrated that tiger nut is well adapted to the temperature and light conditions in the north temperate zone of China, even with a shorter growth season. The tiger nut accessions collected here exhibited wide variations for agronomical and biochemical traits, suggesting potential for potential for breeding improvement by maximizing the fresh tuber and grass yield based on the optimal selection of genetic characteristics in climate and soil conditions of northern China.

(1) Objective: To evaluate myocardial injury by observing the different parameters of global myocardial work (MW) by left ventricular pressure-strain loop (PSL) analysis in patients with chronic kidney disease (CKD). (2) Methods: According to the left ventricular mass index, the study patients with CKD were further divided into two groups: the left ventricular normal group (CKDN-LVH, 59) and left ventricular hypertrophy group (CKDLVH, 46). Thirty-three healthy controls (CON) matched in age and sex with the CKD group were recruited. The routine ultrasonic parameters were obtained by routine TTE, and the strain index and different parameters of the left ventricular MW were obtained by dynamic image offline analysis. (3) Results: This study found that (1) compared with the CON group, the CKDN-LVH group had a significantly increased global waste work (GWW) and significantly decreased global work efficiency (GWE), the GWW further increased, and GWE further decreased in the CKDLVH group. There was no significant change in the global work index (GWI) and global constructive work index (GCW) in the CKDN-LVH group, but the GWI and GCW in the CKDLVH group were significantly increased. (2) According to the grouping analysis of systolic blood pressure (SBP), we found that the GWW increased and GWE decreased in CKD patients with an elevated SBP. (3) Correlation analysis showed that the increase of the peak strain dispersion, SBP, and left ventricular mass index and the decrease of the estimated glomerular filtration rate were significantly correlated with the decrease of the GWE and the increase of the GWW. (4) Receiver operating characteristic curve analysis showed that the area under the curve (AUC) of myocardial damage induced by the GWE and GWW in the CKD group and CON group was higher than that of left ventricular global longitudinal strain (AUCs: 0.87 and 0.878 versus 0.72, respectively). (4) Conclusions: Noninvasive left ventricular PSL analysis can be used to evaluate the global MW in patients with CKD. The study justified the role of GWW in the noninvasive assessment of myocardial function in patients with CKD.

Cyperus esculentus produces large amounts of oil as one of the main oil storage reserves in underground tubers, making this crop species not only a promising resource for edible oil and biofuel in food and chemical industry, but also a model system for studying oil accumulation in non-seed tissues. In this study, we determined the chloroplast genome sequence of the cultivated C. esculentus (var. sativus Boeckeler). The results showed that the complete chloroplast genome of C. esculentus was 186,255 bp in size, and possessed a typical quadripartite structure containing one large single copy (100,940 bp) region, one small single copy (10,439 bp) region, and a pair of inverted repeat regions of 37,438 bp in size. Sequence analyses indicated that the chloroplast genome encodes 141 genes, including 93 protein-coding genes, 40 transfer RNA genes, and 8 ribosomal RNA genes. We also identified 396 simple-sequence repeats and 49 long repeats, including 15 forward repeats and 34 palindromes within the chloroplast genome of C. esculentus. Most of these repeats were distributed in the noncoding regions. Whole chloroplast genome comparison with those of the other four Cyperus species indicated that both the large single copy and inverted repeat regions were more divergent than the small single copy region, with the highest variation found in the inverted repeat regions. In the phylogenetic trees based on the complete chloroplast genomes of 13 species, all five Cyperus species within the Cyperaceae formed a clade, and C. esculentus was evolutionarily more related to C. rotundus than to the other three Cyperus species. In summary, the chloroplast genome sequence of the cultivated C. esculentus provides a valuable genomic resource for species identification, evolution, and comparative genomic research on this crop species and other Cyperus species in the Cyperaceae family.

Alzheimer’s disease (AD), a progressive neurodegenerative disease, is characterized by the accumulation of senile plaques, neurofibrillary tangles, and loss of synapses and neurons in the brain. The pathophysiological process of AD begins with a long asymptomatic phase, which provides a potential opportunity for early therapeutic intervention. Therefore, it is crucial to define putative biomarkers via reliable and validated methods for early diagnosis of AD. Here, we characterized candidate biomarkers by discovery proteomics analysis of cerebrospinal fluid (CSF), revealing that 732 and 704 proteins with more than one unique peptide were identified in healthy controls and preclinical AD patients, respectively. Among them, 79 and 98 proteins were significantly altered in preclinical AD for women and men, respectively, many of which have been demonstrated with consistent regulation pattern in patients with mild cognitive impairment or AD dementia. In-house developed 5-plex isotopic N,N-dimethyl leucine (iDiLeu) tags were further utilized to verify candidate biomarkers, neurosecretory protein VGF (VGF) and apolipoprotein E (apoE). By labeling peptide standards with different iDiLeu tags, a four-point internal calibration curve was constructed to allow for determination of the absolute amount of target analytes in CSF through a single liquid chromatography-mass spectrometry run.

Heterosis has been widely exploited as an approach to enhance crop traits during breeding. However, its underlying molecular genetic mechanisms remain unclear. Recent advances in RNA sequencing technology (RNA-seq) have provided an opportunity to conduct transcriptome profiling for heterosis studies. We used RNA-seq to analyze the flower transcriptomes of two F1 hybrid soybeans (HYBSOY-1 and HYBSOY-5) and their parents. More than 385 million high-quality reads were generated and aligned against the soybean reference genome. A total of 681 and 899 genes were identified as being differentially expressed between HYBSOY-1 and HYBSOY-5 and their parents, respectively. These differentially expressed genes (DEGs) were categorized into four major expression categories with 12 expression patterns. Furthermore, gene ontology (GO) term analysis showed that the DEGs were enriched in the categories metabolic process and catalytic activity, while Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis found that metabolic pathway and biosynthesis of secondary metabolites were enriched in the two F1 hybrids. Comparing the DEGs of the two F1 hybrids by GO term and KEGG pathway analyses identified 26 common DEGs that showed transgressive up-regulation, and which could be considered potential candidate genes for heterosis in soybean F1 hybrids. This identification of an extensive transcriptome dataset gives a comprehensive overview of the flower transcriptomes in two F1 hybrids, and provides useful information for soybean hybrid breeding. These findings lay the foundation for future studies on molecular mechanisms underlying soybean heterosis.