Multi-organ proteomic landscape of COVID-19 autopsies.

The molecular pathology of multi-organ injuries in COVID-19 patients remains unclear, preventing effective therapeutics development. Here, we report a proteomic analysis of 144 autopsy samples from seven organs in 19 COVID-19 patients. We quantified 11,394 proteins in these samples, in which 5,336 were perturbed in the COVID-19 patients compared to controls. Our data showed that cathepsin L1, rather than ACE2, was significantly upregulated in the lung from the COVID-19 patients. Systemic hyperinflammation and dysregulation of glucose and fatty acid metabolism were detected in multiple organs. We also observed dysregulation of key factors involved in hypoxia, angiogenesis, blood coagulation, and fibrosis in multiple organs from the COVID-19 patients. Evidence for testicular injuries includes reduced Leydig cells, suppressed cholesterol biosynthesis, and sperm mobility. In summary, this study depicts a multi-organ proteomic landscape of COVID-19 autopsies that furthers our understanding of the biological basis of COVID-19 pathology.

Proteomic and Metabolomic Characterization of COVID-19 Patient Sera.

Early detection and effective treatment of severe COVID-19 patients remain major challenges. Here, we performed proteomic and metabolomic profiling of sera from 46 COVID-19 and 53 control individuals. We then trained a machine learning model using proteomic and metabolomic measurements from a training cohort of 18 non-severe and 13 severe patients. The model was validated using 10 independent patients, 7 of which were correctly classified. Targeted proteomics and metabolomics assays were employed to further validate this molecular classifier in a second test cohort of 19 COVID-19 patients, leading to 16 correct assignments. We identified molecular changes in the sera of COVID-19 patients compared to other groups implicating dysregulation of macrophage, platelet degranulation, complement system pathways, and massive metabolic suppression. This study revealed characteristic protein and metabolite changes in the sera of severe COVID-19 patients, which might be used in selection of potential blood biomarkers for severity evaluation.

metaExpertPro: A Computational Workflow for Metaproteomics Spectral Library Construction and Data-Independent Acquisition Mass Spectrometry Data Analysis.

Analysis of large-scale data-independent acquisition mass spectrometry metaproteomics data remains a computational challenge. Here, we present a computational pipeline called metaExpertPro for metaproteomics data analysis. This pipeline encompasses spectral library generation using data-dependent acquisition MS, protein identification and quantification using data-independent acquisition mass spectrometry, functional and taxonomic annotation, as well as quantitative matrix generation for both microbiota and hosts. By integrating FragPipe and DIA-NN, metaExpertPro offers compatibility with both Orbitrap and timsTOF MS instruments. To evaluate the depth and accuracy of identification and quantification, we conducted extensive assessments using human fecal samples and benchmark tests. Performance tests conducted on human fecal samples indicated that metaExpertPro quantified an average of 45,000 peptides in a 60-min diaPASEF injection. Notably, metaExpertPro outperformed three existing software tools by characterizing a higher number of peptides and proteins. Importantly, metaExpertPro maintained a low factual false discovery rate of approximately 5% for protein groups across four benchmark tests. Applying a filter of five peptides per genus, metaExpertPro achieved relatively high accuracy (F-score = 0.67-0.90) in genus diversity and showed a high correlation (rSpearman = 0.73-0.82) between the measured and true genus relative abundance in benchmark tests. Additionally, the quantitative results at the protein, taxonomy, and function levels exhibited high reproducibility and consistency across the commonly adopted public human gut microbial protein databases IGC and UHGP. In a metaproteomic analysis of dyslipidemia patients, metaExpertPro revealed characteristic alterations in microbial functions and potential interactions between the microbiota and the host.

Multitissue Circadian Proteome Atlas of WT and Per1-/-/Per2-/- Mice.

The molecular basis of circadian rhythm, driven by core clock genes such as Per1/2, has been investigated on the transcriptome level, but not comprehensively on the proteome level. Here we quantified over 11,000 proteins expressed in eight types of tissues over 46 h with an interval of 2 h, using WT and Per1/Per2 double knockout mouse models. The multitissue circadian proteome landscape of WT mice shows tissue-specific patterns and reflects circadian anticipatory phenomena, which are less obvious on the transcript level. In most peripheral tissues of double knockout mice, reduced protein cyclers are identified when compared with those in WT mice. In addition, PER1/2 contributes to controlling the anticipation of the circadian rhythm, modulating tissue-specific cyclers as well as key pathways including nucleotide excision repair. Severe intertissue temporal dissonance of circadian proteome has been observed in the absence of Per1 and Per2. The γ-aminobutyric acid might modulate some of these temporally correlated cyclers in WT mice. Our study deepens our understanding of rhythmic proteins across multiple tissues and provides valuable insights into chronochemotherapy. The data are accessible at https://prot-rhythm.prottalks.com/.

Identification of SLC7A11-AS1/SLC7A11 pair as a ferroptosis-related therapeutic target for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), a prevalent malignancy worldwide, poses significant challenges in terms of prognosis, necessitating innovative therapeutic approaches. Ferroptosis offers notable advantages over apoptosis, holding promise as a novel therapeutic approach for HCC complexities. Moreover, while the interaction between long non-coding RNAs (lncRNAs) and mRNAs is pivotal in various physiological and pathological processes, their involvement in ferroptosis remains relatively unexplored. In this study, we constructed a ferroptosis-related lncRNA-mRNA correlation network in HCC using Pearson correlation analysis. Notably, the SLC7A11-AS1/SLC7A11 pair, exhibiting high correlation, was identified. Bioinformatics analysis revealed a significant correlation between the expression levels of this pair and key clinical characteristics of HCC patients, including gender, pathology, Ishak scores and tumour size. And poor prognosis was associated with high expression of this pair. Functional experiments demonstrated that SLC7A11-AS1, by binding to the 3'UTR region of SLC7A11 mRNA, enhanced its stability, thereby promoting HCC cell growth and resistance to erastin- induced ferroptosis. Additionally, in vivo studies confirmed that SLC7A11-AS1 knockdown potentiated the inhibitory effects of erastin on tumour growth. Overall, our findings suggest that targeting the SLC7A11-AS1/SLC7A11 pair holds promise as a potential therapeutic strategy for HCC patients.

Safety, tolerability, pharmacokinetics, and pharmacodynamics of TG103 injection in participants who are overweight or obese: a randomized, double-blind, placebo-controlled, multiple-dose phase 1b study.

BACKGROUND: TG103, a glucagon-like peptide-1 analog, is being investigated as an option for weight management. We aimed to determine the safety, tolerability, pharmacokinetics, and pharmacodynamics of TG103 injection in participants who are overweight or obese without diabetes. METHODS: In this randomized, double-blind, placebo-controlled, multiple-dose phase 1b study, participants aged 18-75 years with a body-mass index (BMI) ≥ 26.0 kg/m2 and body weight ≥ 60 kg were enrolled from three centers in China. The study included three cohorts, and in each cohort, eligible participants were randomly assigned (3:1) to one of three once-weekly subcutaneous TG103 groups (15.0, 22.5 and 30.0 mg) or matched placebo, without lifestyle interventions. In each cohort, the doses of TG103 were escalated in 1-week intervals to the desired dose over 1 to 4 weeks. Then participants were treated at the target dose until week 12 and then followed up for 2 weeks. The primary endpoint was safety and tolerability assessed by the incidence and severity of adverse events (AEs) from baseline to the end of the follow-up period. Secondary endpoints included pharmacokinetic and pharmacodynamic profiles of TG103 and the occurrence of anti-drug antibodies to TG103. RESULTS: A total of 147 participants were screened, and 48 participants were randomly assigned to TG103 (15.0, 22.5 and 30.0 mg groups, n = 12 per group) or placebo (n = 12). The mean (standard deviation, SD) age of the participants was 33.9 (10.0) years; the mean bodyweight was 81.65 (10.50) kg, and the mean BMI was 29.8 (2.5) kg/m2. A total of 466 AEs occurred in 45 of the 48 participants, with 35 (97.2%) in the TG103 group and 10 (83.3%) in the pooled placebo group. Most AEs were grade 1 or 2 in severity, and there were no serious adverse events (SAEs), AEs leading to death, or AEs leading to discontinuation of treatment. The steady-state exposure of TG103 increased with increasing dose and was proportional to Cmax,ss, AUCss, AUC0-t and AUC0-inf. The mean values of Cmax,ss ranged from 951 to 1690 ng/mL, AUC0-t ranged from 150 to 321 µg*h/mL, and AUC0-inf ranged from 159 to 340 µg*h/mL. TG103 had a half-life of 110-116 h, with a median Tmax of 36-48 h. After treatment for 12 weeks, the mean (SD) values of weight loss from baseline in the TG103 15.0 mg, 22.5 mg and 30.0 mg groups were 5.65 (3.30) kg, 5.35 (3.39) kg and 5.13 (2.56) kg, respectively, and that in the placebo group was 1.37 (2.13) kg. The least square mean percent weight loss from baseline to D85 in all the TG103 groups was more than 5% with p < 0.05 for all comparisons with placebo. CONCLUSIONS: In this trial, all three doses of once-weekly TG103 were well tolerated with an acceptable safety profile. TG103 demonstrated preliminary 12-week body weight loss without lifestyle interventions, thus showing great potential for the treatment of overweight and obesity. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04855292. Registered on April 22, 2021.

Type 2 diabetic mellitus related osteoporosis: focusing on ferroptosis.

With the aging global population, type 2 diabetes mellitus (T2DM) and osteoporosis(OP) are becoming increasingly prevalent. Diabetic osteoporosis (DOP) is a metabolic bone disorder characterized by abnormal bone tissue structure and reduced bone strength in patients with diabetes. Studies have revealed a close association among diabetes, increased fracture risk, and disturbances in iron metabolism. This review explores the concept of ferroptosis, a non-apoptotic cell death process dependent on intracellular iron, focusing on its role in DOP. Iron-dependent lipid peroxidation, particularly impacting pancreatic ß-cells, osteoblasts (OBs) and osteoclasts (OCs), contributes to DOP. The intricate interplay between iron dysregulation, which comprises deficiency and overload, and DOP has been discussed, emphasizing how excessive iron accumulation triggers ferroptosis in DOP. This concise overview highlights the need to understand the complex relationship between T2DM and OP, particularly ferroptosis. This review aimed to elucidate the pathogenesis of ferroptosis in DOP and provide a prospective for future research targeting interventions in the field of ferroptosis.

Heteryunine A, an amidated tryptophan-catechin-spiroketal hybrid with antifibrotic activity from Heterosmilax yunnanensis.

An unprecedented spiro-C-glycoside adduct, heteryunine A (1), along with two uncommon alkaloids featuring a 2,3-diketopiperazine skeleton, heterpyrazines A (2) and B (3), were discovered in the roots of Heterosmilax yunnanensis. The detailed spectroscopic analysis helped to clarify the planar structures of these compounds. Compound 1, containing 7 chiral centers, features a catechin fused with a spiroketal and connects with a tryptophan derivative by a CC bond. Its complex absolute configuration was elucidated by rotating frame overhauser enhancement spectroscopy (ROESY), specific rotation, and the 13C nuclear magnetic resonance (NMR) and electronic circular dichroism (ECD) calculation. The possible biosynthetic routes for 1 were deduced. Compounds 1 and 2 showed significant antifibrotic effects and further research revealed that they inhibited the activation, migration and proliferation of hepatic stellate cells (HSCs) through suppressing the activity of Ras homolog family member A (RhoA).

Six new iridoid glycosides from the whole plants of Hedyotis diffusa.

Six new iridoid glycosides were isolated from the ethyl acetate fraction of the whole plants of Hedyotis diffusa Willd. They were identified as E-6-O-p-methoxycinnamoyl-10-O-acetyl scandoside acid methyl ester (1), Z-6-O-p-methoxycinnamoyl-10-O-acetyl scandoside acid methyl ester (2), E-6-O-caffeoyl scandoside methyl ester (3), E-6-O-p-coumaroyl-6'-O-acetyl scandoside methyl ester (4), Z-6-O-p-coumaroyl-6'-O-acetyl scandoside methyl ester (5), and E-6-O-p-coumaroyl-4'-O-acetyl scandoside methyl ester (6). The structures of them were elucidated based on unambiguous spectroscopic data (UV, IR, HRESIMS, and NMR). They were screened for anti-inflammatory effect, antioxidant effect, antitumor effect, and neuroprotective effect and did not show potent activities.

Profiling of Volatile Compounds in 'Muscat Hamburg' Contaminated with Aspergillus carbonarius before OTA Biosynthesis Based on HS-SPME-GC-MS and DLLME-GC-MS.

Aspergillus carbonarius is known to produce the carcinogenic ochratoxin A (OTA) in grapes. The metabolism process before OTA biosynthesis influences the content and composition of the volatile compounds in grapes. In this study, a self-established method based on QuEChERS coupled with high-performance liquid chromatography-fluorescence detection (HPLC-FLD) was used to determine the OTA levels during a seven-day contamination period. The results showed that OTA was detected on the second day after contamination with A. carbonarius. Thus, the first day was considered as the critical sampling timepoint for analyzing the volatiles in grapes before OTA biosynthesis. Additionally, the volatile compounds in grapes were analyzed using headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) and dispersive liquid-liquid microextraction gas chromatography-mass spectrometry (DLLME-GC-MS). The corresponding data were evaluated via multivariate data analysis using projection methods, including PCA and OPLS-DA. The results indicated significant differences in the nine volatile compounds in grapes contaminated with A. carbonarius before OTA biosynthesis. The results of the Pearson correlation analysis showed positive correlations between ethyl acetate, styrene, 1-hexanol and OTA; (E)-2-hexenal and nerolic acid were negatively correlated with OTA. Overall, these findings provide a theoretical basis for the early prediction of OTA formation in grape and grape products using GC-MS technology.

End-extended Conjugation Strategy to Reduce the Efficiency-Stability-Mechanical Robustness Gap in Binary All-Polymer Solar Cells.

Concurrently achieving high efficiency, mechanical robustness and thermal stability is critical for the commercialization of all-polymer solar cells (APSCs). However, APSCs usually demonstrate complicated morphology, primarily attributed to the polymer chain entanglement which has a detrimental effect on their fill factors (FF) and morphology stability. To address these concerns, an end-group extended polymer acceptor, PY-NFT, was synthesized and studied. The morphology analysis showed a tightly ordered molecular packing mode and a favorable phase separation was formed. The PM6:PY-NFT-based device achieved an exceptional PCE of 19.12% (certified as 18.45%), outperforming the control PM6:PY-FT devices (17.14%). This significant improvement highlights the record-high PCE for binary APSCs. The thermal aging study revealed that the PM6:PY-NFT blend exhibited excellent morphological stability, thereby achieving superior device stability, retaining 90% of initial efficiency after enduring thermal stress (65 °C) for 1500 hours. More importantly, the PM6:PY-NFT blend film exhibited outstanding mechanical ductility with a crack onset strain of 24.1%. Overall, rational chemical structure innovation, especially the conjugation extension strategy to trigger appropriate phase separation and stable morphology, is the key to achieving high efficiency, improved thermal stability and robust mechanical stability of APSCs.

Local Structure-Induced Selective Interactions Enables High-Performance and Burn-in-Free Organic Photovoltaics.

Oligomeric acceptors (OAs) have attracted considerable attention in the organic photovoltaics (OPV) field owing to their capacity in balancing the merits from both monomeric and polymeric acceptors. A delicate control over the distortion between blocks of OAs usually determines the performance and stability of relevant OPV devices. However, it imposes great complexity to realize a controllable degree of distortion by tuning the skeleton of blocks and the position of linker between blocks. Herein, we developed a facile strategy to rationally control the geometry distortion of OAs via a straightforward substitution of alkoxy side-chains on their blocks. This helps elucidate the integrated influences of molecular distortion and non-bonded contacts on the selective interactions between OA molecules and between OA and host acceptor in ternary blend. We demonstrate the alkoxy-OA molecules having stronger self-interactions would mitigate their interactions with host acceptor, therefore alleviating the kinetic diffusion and excessive aggregation of total acceptors. Combining with a composite-interlayer strategy by introducing a phenyl-substituted self-assembled monolayer to enhance the doping with polyoxometalate, an impressive efficiency of 20.1% is achieved accompanied by a negligible burn-in loss against physical aging. This study demonstrates the validation of tuning of selective interactions towards high-performance and burn-in-free OPV.

Recent Progress in All-Small-Molecule Organic Solar Cells.

Active layer material plays a critical role in promoting the performance of an organic solar cell (OSC). Small-molecule (SM) materials have the merits of well-defined chemical structures, few batch-to-batch variations, facile synthesis and purification procedures, and easily tuned properties. SM-donor and non-fullerene acceptor (NFA) innovations have recently produced all-small-molecule (ASM) devices with power conversion efficiencies that exceed 17% and approach those of their polymer-based counterparts, thereby demonstrating their great future commercialization potential. In this review, recent progress in both SM donors and NFAs to illustrate structure-property relationships and various morphology-regulation strategies are summarized. Finally, ASM-OSC challenges and outlook are discussed.

Enhanced extracellular production of raw starch-degrading α-amylase in Bacillus subtilis through expression regulatory element modification and fermentation optimization.

BACKGROUND: Raw starch-degrading α-amylase (RSDA) can hydrolyze raw starch at moderate temperatures, thus contributing to savings in starch processing costs. However, the low production level of RSDA limits its industrial application. Therefore, improving the extracellular expression of RSDA in Bacillus subtilis, a commonly used industrial expression host, has great value. RESULTS: In this study, the extracellular production level of Pontibacillus sp. ZY raw starch-degrading α-amylase (AmyZ1) in B. subtilis was enhanced by expression regulatory element modification and fermentation optimization. As an important regulatory element of gene expression, the promoter, signal peptide, and ribosome binding site (RBS) sequences upstream of the amyZ1 gene were sequentially optimized. Initially, based on five single promoters, the dual-promoter Pveg-PylB was constructed by tandem promoter engineering. Afterward, the optimal signal peptide SPNucB was obtained by screening 173 B. subtilis signal peptides. Then, the RBS sequence was optimized using the RBS Calculator to obtain the optimal RBS1. The resulting recombinant strain WBZ-VY-B-R1 showed an extracellular AmyZ1 activity of 4824.2 and 41251.3 U/mL during shake-flask cultivation and 3-L fermenter fermentation, which were 2.6- and 2.5-fold greater than those of the original strain WBZ-Y, respectively. Finally, the extracellular AmyZ1 activity of WBZ-VY-B-R1 was increased to 5733.5 U/mL in shake flask by optimizing the type and concentration of carbon source, nitrogen source, and metal ions in the fermentation medium. On this basis, its extracellular AmyZ1 activity was increased to 49082.1 U/mL in 3-L fermenter by optimizing the basic medium components as well as the ratio of carbon and nitrogen sources in the feed solution. This is the highest production level reported to date for recombinant RSDA production. CONCLUSIONS: This study represents a report on the extracellular production of AmyZ1 using B. subtilis as a host strain, and achieved the current highest expression level. The results of this study will lay a foundation for the industrial application of RSDA. In addition, the strategies employed here also provide a promising way for improving other protein production in B. subtilis.

AtHSPR functions in gibberellin-mediated primary root growth by interacting with KNAT5 and OFP1 in Arabidopsis.

KEY MESSAGE: AtHSPR forms a complex with KNAT5 and OFP1 to regulate primary root growth through GA-mediated root meristem activity. KNAT5-OFP1 functions as a negative regulator of AtHSPR in response to GA. Plant root growth is modulated by gibberellic acid (GA) signaling and depends on root meristem maintenance. ARABIDOPSIS THALIANA HEAT SHOCK PROTEIN-RELATED (AtHSPR) is a vital regulator of flowering time and salt stress tolerance. However, little is known about the role of AtHSPR in the regulation of primary root growth. Here, we report that athspr mutant exhibits a shorter primary root compared to wild type and that AtHSPR interacts with KNOTTED1-LIKE HOMEOBOX GENE 5 (KNAT5) and OVATE FAMILY PROTEIN 1 (OFP1). Genetic analysis showed that overexpression of KNAT5 or OFP1 caused a defect in primary root growth similar to that of the athspr mutant, but knockout of KNAT5 or OFP1 rescued the short root phenotype in the athspr mutant by altering root meristem activity. Further investigation revealed that KNAT5 interacts with OFP1 and that AtHSPR weakens the inhibition of GIBBERELLIN 20-OXIDASE 1 (GA20ox1) expression by the KNAT5-OFP1 complex. Moreover, root meristem cell proliferation and root elongation in 35S::KNAT5athspr and 35S::OFP1athspr seedlings were hypersensitive to GA3 treatment compared to the athspr mutant. Together, our results demonstrate that the AtHSPR-KNAT5-OFP1 module regulates root growth and development by impacting the expression of GA biosynthetic gene GA20ox1, which could be a way for plants to achieve plasticity in response to the environment.

EZH2 Expression in Retinoblastoma: A Potential Therapeutic Target.

INTRODUCTION: The enhancer of zeste homolog 2 (EZH2) is a member of the polycomb repressive complex 2 (PRC2) and is important in cell-cycle regulation. Increased expression of EZH2 has been reported in retinoblastoma (RB). The aim of the study was to determine EZH2 expression, compare this with clinicopathological parameters in RB, and assess its relationship with tumor cell proliferation. METHODS: Ninety-nine retrospective cases of enucleated RB were included in the present study. Expression of EZH2 and the marker of cell proliferation, Ki67, were investigated by immunohistochemistry. RESULTS: Among the 99 cases of RB in this study, EZH2 was found highly expressed (positive expression rate ≥70%) in 92 cases. EZH2 was expressed in tumor cells but absent in normal retinal tissues. The expression of EZH2 was positively linked to Ki67 expression (r = 0.65, p < 0.001). CONCLUSION: Elevated EZH2 expression was found in most RB cases, indicating that EZH2 could be a potential therapeutic target for RB.

Nursing adherence, barriers and facilitators to conduct post-stroke dysphagia screening and assessment: A study based on theoretical domain framework.

BACKGROUND: There are an increasing number of evidence-based recommendations for managing dysphagia in post-stroke patients. However, it is unclear whether nurses adopt these recommendations in their daily nursing practices. AIMS: This study aimed to explore nurses' adherence, barriers, facilitators and views on dysphagia screening and assessment of post-stroke dysphagia. METHODS: In this study, multiple methods were adopted. In Phase 1, a general information questionnaire and a knowledge-attitude-practice and barriers/facilitators questionnaire for dysphagia screening and assessment were distributed in 55 hospitals online. In Phase 2, semi-structured interviews were conducted to explore nurses' views on barriers. Descriptive and one-way variance analyses were used to analyse the quantitative data, while content analysis was used to analyse the qualitative data. This study adheres to STROBE and COREQ guidelines. RESULTS: Nine hundred and forty-two completed questionnaires were collected. Only 36.52% of the nurses screened for swallow function in patients as a guideline. The biggest barrier was 'memory, attention and decision process', with an average score of 3.22 (.74). The different stages of implementation had various types and degrees of barriers (p < .001). Five themes were extracted after interviews, namely 'Inadequate environment and resource support', 'Increased workload', 'Professional value perception', 'Organisational culture', and 'Poor knowledge and skill'. CONCLUSIONS: Nurses' practice of dysphagia screening and assessment of patients with dysphagia after stroke were inadequate, and the barriers originated from patients, leadership and the nurses themselves. RELEVANCE TO CLINICAL PRACTICE: This research extracted five barriers of guidance adherence for post-stroke dysphagia screening and assessment and identified the different kinds and degrees of barriers in five implementation stages, providing a basis for nursing managers to break through the bottleneck of guideline implementation. PATIENT OR PUBLIC CONTRIBUTION: The nurses recruited in this study completed validated questionnaires in the survey and suggestive answers in interviews.

Exploring the Role of the Plant Actin Cytoskeleton: From Signaling to Cellular Functions.

The plant actin cytoskeleton is characterized by the basic properties of dynamic array, which plays a central role in numerous conserved processes that are required for diverse cellular functions. Here, we focus on how actins and actin-related proteins (ARPs), which represent two classical branches of a greatly diverse superfamily of ATPases, are involved in fundamental functions underlying signal regulation of plant growth and development. Moreover, we review the structure, assembly dynamics, and biological functions of filamentous actin (F-actin) from a molecular perspective. The various accessory proteins known as actin-binding proteins (ABPs) partner with F-actin to finely tune actin dynamics, often in response to various cell signaling pathways. Our understanding of the significance of the actin cytoskeleton in vital cellular activities has been furthered by comparison of conserved functions of actin filaments across different species combined with advanced microscopic techniques and experimental methods. We discuss the current model of the plant actin cytoskeleton, followed by examples of the signaling mechanisms under the supervision of F-actin related to cell morphogenesis, polar growth, and cytoplasmic streaming. Determination of the theoretical basis of how the cytoskeleton works is important in itself and is beneficial to future applications aimed at improving crop biomass and production efficiency.

[Analysis of frequency and molecular genetics of Jk (a-b-) phenotype among blood donors from Jining area].

OBJECTIVE: To screen for Jk(a-b-) phenotype among blood donors from Jining area and explore its molecular basis to enrich the rare blood group bank for the region. METHODS: The population who donated blood gratuitously at Jining Blood Center from July 2019 to January 2021 were selected as the study subjects. The Jk(a-b-) phenotype was screened with the 2 mol/L urea lysis method, and the result was confirmed by using classical serological methods. Exons 3 to 10 of the SLC14A1 gene and its flanking regions were subjected to Sanger sequencing. RESULTS: Among 95 500 donors, urea hemolysis test has identified three without hemolysis, which was verified by serological method as the Jk(a-b-) phenotype and demonstrated no anti-Jk3 antibody. The frequency of the Jk(a-b-) phenotype in Jining area is therefore 0.0031%. Gene sequencing and haplotype analysis showed that the genotypes of the three samples were JK*02N.01/JK*02N.01, JK*02N.01/JK-02-230A and JK*02N.20/JK-02-230A, respectively. CONCLUSION: The splicing variant of c.342-1G>A in intron 4, missense variants of c.230G>A in exon 4, and c.647_ 648delAC in exon 6 probably underlay the Jk(a-b-) phenotype in the local population, which is different from other regions in China. The c.230G>A variant was unreported previously.

Optimization of Microflow LC Coupled with Scanning SWATH and Its Application in Hepatocellular Carcinoma Tissues.

Scanning SWATH coupled with normal-flow LC has been recently introduced for high-content, high-throughput proteomics analysis, which requires a relatively large amount of sample injection. Here we established the microflow LC coupled with Scanning SWATH for samples with relatively small quantities. First, we optimized several key parameters of the LC and MS settings, including C18 particle size for the analytical column, LC gradient and flow rate, as well as effective ion accumulation time and isolation window width for MS acquisition. We then compared the optimized Scanning SWATH method with the conventional variable window SWATH (referred to as SWATH) method. Results showed that the total ion chromatogram signals in Scanning SWATH were 10 times higher than that of SWATH, and Scanning SWATH identified 12.2-22.2% more peptides than SWATH. Finally, we employed 120 min Scanning SWATH to acquire the proteomes of 62 formalin-fixed, paraffin-embedded (FFPE) tissue samples from 31 patients with hepatocellular carcinoma (HCC). Altogether, 92 334 peptides and 8516 proteins were quantified. Besides the reported biomarkers, including ANXA2, MCM7, SUOX, and AKR1B10, we identified new potential HCC biomarkers such as CST5, TP53, CEBPB, and E2F4. Taken together, we present an optimal workflow integrating microflow LC and Scanning SWATH that effectively improves the protein identification and quantitation.