A Prime-Boost Vaccination Approach Induces Lung Resident Memory CD8+ T Cells Derived from Central Memory T Cells That Prevent Tumor Lung Metastasis.

Memory T cells play a key role in immune protection against cancer. Vaccine-induced tissue-resident memory T (TRM) cells in the lung have been shown to protect against lung metastasis. Identifying the source of lung TRM cells can help to improve strategies, preventing tumor metastasis. Here, we found that a prime-boost vaccination approach using intramuscular DNA vaccine priming, followed by intranasal live-attenuated influenza-vectored vaccine (LAIV) boosting induced higher frequencies of lung CD8+ TRM cells compared with other vaccination regimens. Vaccine-induced lung CD8+ TRM cells, but not circulating memory T cells, conferred significant protection against metastatic melanoma and mesothelioma. Central memory T (TCM) cells induced by the DNA vaccination were major precursors of lung TRM cells established after the intranasal LAIV boost. Single-cell RNA sequencing analysis indicated that transcriptional reprogramming of TCM cells for differentiation into TRM cells in the lungs started as early as day 2 post the LAIV boost. Intranasal LAIV altered the mucosal microenvironment to recruit TCM cells via CXCR3-dependent chemotaxis and induced CD8+ TRM-associated transcriptional programs. These results identified TCM cells as the source of vaccine-induced CD8+ TRM cells that protect against lung metastasis. Significance: Prime-boost vaccination shapes the mucosal microenvironment and reprograms central memory T cells to generate lung resident memory T cells that protect against lung metastasis, providing insights for the optimization of vaccine strategies.

Rapid generation of long, chemically modified pegRNAs for prime editing.

The editing efficiencies of prime editing (PE) using ribonucleoprotein (RNP) and RNA delivery are not optimal due to the challenges in solid-phase synthesis of long PE guide RNA (pegRNA) (>125 nt). Here, we develop an efficient, rapid and cost-effective method for generating chemically modified pegRNA (125-145 nt) and engineered pegRNA (epegRNA) (170-190 nt). We use an optimized splint ligation approach and achieve approximately 90% production efficiency for these RNAs, referred to as L-pegRNA and L-epegRNA. L-epegRNA demonstrates enhanced editing efficiencies across various cell lines and human primary cells with improvements of up to more than tenfold when using RNP delivery and several hundredfold with RNA delivery of PE, compared to epegRNA produced by in vitro transcription. L-epegRNA-mediated RNP delivery also outperforms plasmid-encoded PE in most comparisons. Our study provides a solution to obtaining high-quality pegRNA and epegRNA with desired chemical modifications, paving the way for the use of PE in therapeutics and various other fields.

Secondary diabetes mellitus in acromegaly: Case report and literature review.

RATIONALE: Acromegaly, predominantly resulting from a pituitary adenoma, is marked by excessive secretion of growth hormone (GH) and insulin-like growth factor-1 (IGF-1). However, normalization of blood glucose levels posttreatment is rarely achieved. This case study aims to highlight the diagnostic challenges posed by overlapping symptoms of acromegaly and diabetes, emphasizing the importance of precise diagnosis and effective treatment strategies for optimal patient outcomes. PATIENT CONCERNS: A 22-year-old male was hospitalized for diabetic ketoacidosis and exhibited classic signs of acromegaly, such as enlarged hands and feet, and distinct facial changes. DIAGNOSES: The patient's diagnosis of acromegaly, attributed to a pituitary adenoma, was confirmed through clinical observations, laboratory findings (notably raised serum GH and IGF-1 levels, and absence of GH suppression after glucose load during an OGTT), and pituitary MRI scans. INTERVENTIONS: The patient underwent 2 surgical tumor resections followed by gamma knife radiosurgery (GKRS). After treatment, GH, IGF-1, and blood glucose levels normalized without further need for hypoglycemic intervention. OUTCOMES: Posttreatment, the patient achieved stable GH, IGF-1, and blood glucose levels. The hyperglycemia was attributed to the GH-secreting tumor, and its resolution followed the tumor's removal. LESSONS: This case emphasizes the need for comprehensive assessment in patients with acromegaly to address coexisting diabetic complications. Surgical and radiotherapeutic management of acromegaly can lead to significant metabolic improvements, highlighting the importance of interdisciplinary care in managing these complex cases.

Mechanism of low-voltage electrostatic field on flavor retention in refrigerated sturgeon caviar: Insights from phospholipids.

This study investigated the effect of low-voltage electrostatic field on the flavor quality changes and generation pathways of refrigerated sturgeon caviar. Research has found that after storage for 3-6 weeks, the physicochemical properties of caviar in the LVEF treatment group are better than those in the control group. The results of two-dimensional gas chromatography-time-of-flight mass spectrometry showed that the contents of hexanal, nonanal, (E,Z)-2,6-nonadienal, (E)-2-octenal and 1-octene-3-one related to the characteristic flavor of caviar (sweet, fruity and green) increased significantly. The lipidomics results indicated that the effects of LVEF on caviar mainly involve glycerophospholipid metabolism, linoleic acid metabolism, and α-Linolenic acid metabolism. Methanophosphatidylcholine (15:0/18:1), phosphatidylcholine (18:0/20:5), and phosphatidylcholine (18,1e/22:6) were significantly correlated with odor formation. Therefore, low-voltage electrostatic field treatment preserved the quality and enhanced the flavor of sturgeon caviar. This study provided a new theoretical basis for the preservation of sturgeon caviar.

An N-terminal heptad repeat trimer-based peptide fusion inhibitor exhibits potent anti-H1N1 activity.

Influenza viruses are susceptible to seasonal influenza, which has repeatedly caused global pandemics and jeopardized human health. Vaccines are only used as preventive medicine due to the extreme mutability of influenza viruses, and antiviral medication is the most significant clinical treatment to reduce influenza morbidity and mortality. Nevertheless, the clinical application of anti-influenza virus agents is characterized by the narrow therapeutic time window, the susceptibility to drug resistance, and relatively limited effect on severe influenza. Therefore, it is of great significance to develop novel anti-influenza virus drugs to fulfill the urgent clinical needs. Influenza viruses enter host cells through the hemagglutinin (HA) mediated membrane fusion process, and fusion inhibitors function antivirally by blocking hemagglutinin deformation, promising better therapeutic efficacy and resolving drug resistance, with targets different from marketed medicines. Previous studies have shown that unnatural peptides derived from Human Immunodeficiency Virus Type 1 (HIV-1) membrane fusion proteins exhibit anti-HIV-1 activity. Based on the similarity of the membrane fusion protein deformation process between HIV-1 and H1N1, we selected sequences derived from the gp41 subunit in the HIV-1 fusion protein, and then constructed N-trimer spatial structure through inter-helical isopeptide bond modification, to design the novel anti-H1N1 fusion inhibitors. The results showed that the novel peptides could block 6-HB formation during H1N1 membrane fusion procedure, and thus possessed significant anti-H1N1 activity, comparable to the positive control oseltamivir. Our study demonstrates the design viability of peptide fusion inhibitors based on similar membrane fusion processes among viruses, and furthermore provides an important idea for the novel anti-H1N1 inhibitors development.

The influence mechanism of phospholipids structure and composition changes caused by oxidation on the formation of flavor substances in sturgeon caviar.

The oxidation-induced phospholipids (PLs) underwent structural and compositional analysis, alongside the establishment of a simulation system to verify the link between phospholipid oxidation and flavor substances formation in sturgeon caviar. Structural alterations of PLs were tracked using 31P and 1H nuclear magnetic resonance (NMR), electron spin resonance spectroscopy (ESR), and Raman spectroscopy. The findings revealed a reduction in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) from 82.3% and 10.4% to 58.2% and 5.8% respectively. Free radical signals exhibited an initial increase followed by a decrease. The diminished intensity in Raman spectra at 970 and 1080 cm-1 indicated reduced fat unsaturation attributable to PLs oxidation. Correlation analysis highlighted a significant association between PC and PE containing C22:6, C20:5, C20:4, and C18:2 with flavor substances, suggesting their role as key precursors for flavor development. This study established a theoretical basis for understanding the change of flavor quality in sturgeon caviar during storage.

Multi-omics combined approach to analyze the mechanism of flavor evolution in sturgeon caviar (Acipenser gueldenstaedtii) during refrigeration storage.

Multi-omics techniques were combined with microstructure, molecular sensory science and non-volatile matrices for the first time to investigate variations in organic macromolecules and flavor in caviar during preservation. After 4-6 weeks of storage, the peroxide value was 35.38 mg/g and the accumulation of thiobarbiturates was significant with caviar membranes exhibiting a decrease in elasticity and an increase in viscosity. Sixteen key volatile compounds were detected by GC-MS, while the volatile compounds that contributed to the differences in caviar flavor at different storage times were mainly tetradecane, (E)-2-hexenal, and heptanal. The pathways associated with flavor release during storage were mainly abundant in the linolenic acid metabolism, alanine metabolism, and glycerophospholipid metabolism pathways. The correlation of 11 differential proteins and 24 differential lipids with odorants was further explored, such as arginine, proline, alanine, PE (20:4/22:6), PE (16:1/18:2), and PE (20:5/18:2). Overall, Aspartate, glutamate, oleic acid, linoleic acid, and phospholipids enriched in C22:6 and C18:2 chains are potential metabolic markers. This study provides a basis from a multi-omics perspective for the investigation of the relationship between quality deterioration and precursor metabolism in caviar storage process.

Electroacupuncture ameliorates senile osteoporosis by promoting bone remodeling and regulating autophagy.

OBJECTIVES: Osteoporosis is widely regarded as a typical aged-related disease caused by impaired bone remodeling. This research was designed to explore the protective effects of electroacupuncture (EA) on senile osteoporosis in a rat model and investigate the underlying mechanisms. METHODS: Three-month-old rats were randomly selected as the youth group, and 24-month-old rats were randomly assigned to the elderly and EA groups. Rats in the EA group received 30 min of EA at bilateral SP10, ST36, K13 and GB34 daily, 5 days a week for 8 weeks. Bone mineral density (BMD), microstructure of the bone tissue, bone turnover biomarkers and expression level of autophagy-related proteins were detected. RESULTS: Compared with the elderly group, EA treatment significantly increased BMD of the femur and ameliorated the microstructure. EA treatment increased trabecular bone volume ratio (= bone volume / total volume [BV/TV]) and trabecular number (Tb.N) and decreased trabecular separation (Tb.Sp) in senile osteoporosis rats. Compared with the elderly group, the serum N-terminal telopeptide of type I collagen (NTX1) level in the EA group was lower, and the serum procollagen type I N-terminal propeptide (PINP) concentration was higher. In addition, the expression of Beclin 1, microtubule-associated protein I light chain 3 (LC3B) and P62 was inhibited in the senile osteoporosis rats after EA treatment. CONCLUSIONS: EA can effectively alleviate aging-related bone loss and improve the microstructure of bone tissue in senile osteoporosis rats, and the regulation of autophagy might be one of the important mechanisms.

Pd(II)/Pd(IV) redox shuttle to suppress vacancy defects at grain boundaries for efficient kesterite solar cells.

Charge loss at grain boundaries of kesterite Cu2ZnSn(S, Se)4 polycrystalline absorbers is an important cause limiting the performance of this emerging thin-film solar cell. Herein, we report a Pd element assisted reaction strategy to suppress atomic vacancy defects in GB regions. The Pd, on one hand in the form of PdSex compounds, can heterogeneously cover the GBs of the absorber film, suppressing Sn and Se volatilization loss and the formation of their vacancy defects (i.e. VSn and VSe), and on the other hand, in the form of Pd(II)/Pd(IV) redox shuttle, can assist the capture and exchange of Se atoms, thus contributing to eliminating the already-existing VSe defects within GBs. These collective effects have effectively reduced charge recombination loss and enhanced p-type characteristics of the kesterite absorber. As a result, high-performance kesterite solar cells with a total-area efficiency of 14.5% (certified at 14.3%) have been achieved.

Contribution of phospholipase B to the formation of characteristic flavor in steamed sturgeon meat.

Sensory analysis and untargeted lipidomics were employed to study the impact of phospholipase B (PLB) on lipid oxidation and flavor in steamed sturgeon meat, revealing the inherent relationship between lipid oxidation and flavor regulation. The research verified that PLB effectively suppresses fat oxidation and improves the overall taste of steamed sturgeon meat. Furthermore, the PLB group identified 52 compounds, and the content of odor substances such as isoamyl alcohol and hexanal was reduced compared with other groups. Finally, lipid substances containing eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) were screened out from 32 kinds of differential phospholipids. Through Pearson correlation analysis, it was observed that certain differential phospholipids such as PC (22:6) and PC (22:5) exhibited varying correlations with odor substances like hexanal and isovaleraldehyde. These findings suggest that PLB specifically affects certain phospholipids, leading to the production of distinct volatile substances through oxidative degradation.

Novel Peptides from Sturgeon Ovarian Protein Hydrolysates Prevent Oxidative Stress-Induced Dysfunction in Osteoblast Cells: Purification, Identification, and Characterization.

This study aimed to explore antioxidant peptides derived from sturgeon (Acipenser schrenckii) ovaries that exhibit antiosteoporotic effects in oxidative-induced MC3T3-E1 cells. The F3-15 component obtained from sturgeon ovarian protein hydrolysates (SOPHs) via gel filtration and RP-HPLC significantly increased the cell survival rate (from 49.38 ± 2.88 to 76.26 ± 2.09%). Two putative antioxidant-acting peptides, FDWDRL (FL6) and FEGPPFKF (FF8), were screened from the F3-15 faction via liquid chromatography-tandem mass spectrometry (LC-MS/MS) and through prediction by computer simulations. Molecular docking results indicated that the possible antioxidant mechanisms of FL6 and FF8 involved blocking the active site of human myeloperoxidase (hMPO). The in vitro tests showed that FL6 and FF8 were equally adept at reducing intracellular ROS levels, increasing the activity of antioxidant enzymes, and protecting cells from oxidative injuries by inhibiting the mitogen-activated protein kinase (MAPK) pathway and activating the phosphoinositide-3 kinase (PI3K)/protein kinase B (AKT)/glycogen synthase kinase-3ß (GSK-3ß) signaling pathway. Moreover, both peptides could increase differentiation and mineralization abilities in oxidatively damaged MC3T3-E1 cells. Furthermore, FF8 exhibited high resistance to pepsin and trypsin, showcasing potential for practical applications.

Structural Basis of HIV-1 gp41 N-trimer for Designing Bifunctional Peptide-based Fusion Inhibitors.

BACKGROUND: Pathogenic viruses that cause large-scale global or regional outbreaks almost always contain class I fusion proteins. Although the viruses differ in morphology, they all require fusion protein-mediated virus-host cell membranes during the early stages of host cell invasion. METHOD: The CHR region and NHR region of fusion proteins can form the 6-HB structure to drive the fusion pore formation between viruses and host cells through metastable interactions. Here, we obtained bifunctional N-peptides with inhibitory activities against two viruses, HIV-1 and MERS-CoV, based on the sequences in the HIV-1 NHR region by constructing N-trimer conformation interacting with the CHR region. RESULT: This study demonstrates that N-peptides with the coiled triple helix structure obtained from the NHR region in 6-HB are able to target the CHR region and exhibit inhibitory activity against a variety of viruses. CONCLUSION: Moreover, this strategy can be used to investigate antivirals against unknown viruses for future outbreaks.

Anatomical sectionectomy based on Takasaki's segmentation for solitary intrahepatic cholangiocarcinoma: a propensity-matched analysis.

BACKGROUND: Anatomical sectionectomy based on Takasaki's segmentation has shown advantages in hepatocellular carcinoma. However, whether this approach improves the survival of intrahepatic cholangiocarcinoma (ICC) remains unknown. METHODS: A series of 248 consecutive patients with solitary ICCs who underwent hepatectomy were studied retrospectively. The patients were classified into the groups of anatomical sectionectomy based on Takasaki's segmentation (TS group) and non-Takasaki's hepatectomy (NTH group). The bias between the two groups was minimized using propensity score matching (PSM). Recurrence-free survival (RFS) and overall survival (OS) were evaluated with Kaplan-Meier analysis. The Cox proportional hazards model was performed to determine the adverse risk factors associated with survival. RESULTS: After PSM, 67 pairs of patients were compared. Both the RFS and OS rates in the TS group were significantly better than those in the NTH group (23.2 % vs. 16.5 %, and 40.4 % vs. 27.3 %, P = 0.035 and 0.032, respectively). Multivariate analysis showed that NTH was independently associated with worse RFS and OS than TS. The stratified analysis demonstrated that the RFS and OS rates in the TS group with tumor stage I and tumor size ≥3 cm were significantly better than those in the NTH group, while the survival rates for ICC with stage I and tumor size <3 cm or stage II-III showed no significant difference. CONCLUSION: TS was associated with improved RFS and OS in patients with solitary ICC even after PSM. TS may be preferred particularly in patients with tumor stage I and tumor size ≥3 cm.

Conditional splicing system for tight control of viral overlapping genes.

Viral genomes frequently harbor overlapping genes, complicating the development of virus-vectored vaccines and gene therapies. This study introduces a novel conditional splicing system to precisely control the expression of such overlapping genes through recombinase-mediated conditional splicing. We refined site-specific recombinase (SSR) conditional splicing systems and explored their mechanisms. The systems demonstrated exceptional inducibility (116,700-fold increase) with negligible background expression, facilitating the conditional expression of overlapping genes in adenovirus-associated virus (AAV) and human immunodeficiency virus type 1. Notably, this approach enabled the establishment of stable AAV producer cell lines, encapsulating all necessary packaging genes. Our findings underscore the potential of the SSR-conditional splicing system to significantly advance vector engineering, enhancing the efficacy and scalability of viral-vector-based therapies and vaccines. IMPORTANCE: Regulating overlapping genes is vital for gene therapy and vaccine development using viral vectors. The regulation of overlapping genes presents challenges, including cytotoxicity and impacts on vector capacity and genome stability, which restrict stable packaging cell line development and broad application. To address these challenges, we present a "loxp-splice-loxp"-based conditional splicing system, offering a novel solution for conditional expression of overlapping genes and stable cell line establishment. This system may also regulate other cytotoxic genes, representing a significant advancement in cell engineering and gene therapy as well as biomass production.

Modulation of autophagy affected tumorigenesis induced by the envelope glycoprotein of JSRV.

Ovine pulmonary adenocarcinoma (OPA), caused by the jaagsiekte sheep retrovirus (JSRV), is a chronic, progressive, and contagious lung tumor that seriously affects sheep production. It also represents a valuable animal model for several human lung adenocarcinomas. However, little is known about the role of autophagy in OPA tumorigenesis. Here, Western blotting combined with transmission electron microscopy examination and Cyto-ID dye staining was employed for evaluation of changes of autophagic levels. The results of the present study showed that expression of the autophagy marker proteins Beclin-1 and LC3 was decreased in OPA lung tissues, as well as in cells overexpressing the envelope glycoprotein of JSRV (JSRV Env). Reduced numbers of autophagosomes were also observed in cells overexpressing JSRV Env, although assessment of autophagic flux showed that JSRV Env overexpression did not block the formation of autophagosomes, suggesting increased degradation of autolysosomes. Last, mouse xenograft experiments indicated that inhibition of autophagy by 3-methyladenine suppressed both tumor growth and the epithelial-to-mesenchymal transition. In conclusion, JSRV, through JSRV Env, takes advantage of the autophagy process, leading to the development of OPA.

The application of two drainage angles in neurocritical care patients with complicated pneumonia: a randomized controlled trial.

Background: Although head elevation is an early first-line treatment for elevated intracranial pressure (ICP), the use of the head-down or prone position in managing neurocritical patients is controversial because a change in a position directly affects the intracranial and cerebral perfusion pressure, which may cause secondary brain injury and affect patient outcomes. This study compared the effects of two postural drainage positions (30° head-up tilt and 0° head flat) on the prognosis of neurocritical care patients with complicated pneumonia and a clinical pulmonary infection score (CPIS) ≥5 points to provide a reference for selecting appropriate postural drainage positions for patients with pneumonia in neurocritical care units. Methods: A prospective randomized controlled study was conducted with 62 neurocritical care patients with complicated pneumonia. The patients were categorized into control (=31) and experimental (=31) groups in a 1:1 ratio using a simple randomized non-homologous pairing method. Emphasis was placed on matching the baseline characteristics of the two groups, including patient age, sex, height, weight, Glasgow Coma Scale score, heart rate, mean arterial pressure, cough reflex, and mechanical ventilation usage to ensure comparability. Both groups received bundled care for artificial airway management. The control group maintained a standard postural drainage position of 0° head-flat, whereas the experimental group maintained a 30° head-up tilt. The efficacy of the nursing intervention was evaluated by comparing the CPIS and other therapeutic indicators between the two groups after postural drainage. Results: After the intervention, the within-group comparison showed a significant decrease in the CPIS (P < 0.001); procalcitonin levels showed a significant decreasing trend (P < 0.05); the arterial oxygen pressure significantly increased (P < 0.05); the oxygenation index significantly increased (P < 0.001); and the aspiration risk score showed a significant decreasing trend (P < 0.001). A between-group comparison showed no significant differences in any of the indicators before and after the intervention (P < 0.05). Conclusion: Postural drainage positions of 30° head-up tilt and 0° head-flat can improve the CPIS and oxygenation in patients without adverse effects. Therefore, we recommend that patients under neurological intensive care and having pneumonia be drained in a 30° head-up tilt position with good centralized care of the lung infection. Trial registration: The study, "Study of Angles of Postural Drainage in Neurocritical Patients with Pneumonia," was registered in the Protocol Registration Data Element Definitions for Interventional Study database (# ChiCTR2100042155); date of registration: 2021-01-14.

Liver resection versus microwave ablation for solitary and small (≤ 3 cm) HCC with early recurrence in different stages of liver cirrhosis: A propensity score matching study.

BACKGROUND: This study aimed to compare the effectiveness of liver resection (LR) and microwave ablation (MWA) in hepatocellular carcinoma (HCC) patients with early recurrence and varying stages of cirrhosis. METHOD: This study analyzed patients with HCC who underwent hepatectomy and experienced early tumor recurrence (≤3 cm) between December 2002 and December 2020 at the Tongji Hospital. Treatment effectiveness was assessed using a propensity score matching (PSM) analysis. RESULTS: This study included 295 patients (106, LR; 189, MWA), 86 patients in each of the 2 groups were chosen for further comparison, after PSM. After PSM, both LR and MWA demonstrated similar recurrence-free survival (RFS) and overall survival (OS) rates (p = 0.060 and p = 0.118, respectively). However, the LR group had more treatment-related complications. In patients with moderate or severe cirrhosis, no significant differences in RFS or OS rates were found between the LR and MWA groups (p = 0.779 and p = 0.772, respectively). In patients without cirrhosis or with mild cirrhosis, LR showed better RFS and OS rates than MWA (p = 0.024 and p = 0.047, respectively). Multivariate analysis after PSM identified moderate or severe cirrhosis and recurrence intervals ≤12 months as independent predictors of poor RFS and OS in patients with early recurrence of HCC. CONCLUSION: LR is more effective than MWA for early recurrence of HCC in patients without cirrhosis or with mild cirrhosis, showing improved RFS and OS rates. In patients with moderate or severe cirrhosis, the OS and RFS were statistically equal between the two therapies. However, MWA may be preferred owing to its low complication rate.

Diagnostic procedure for localized constrictive pericarditis leading to bilateral pleural effusion: two case reports.

Constrictive pericarditis is a rare disease. Localized constrictive pericarditis leading to bilateral pleural effusion is more difficult to recognize, and the diagnostic procedure can be ambiguous. Here, we report two patients diagnosed with localized constrictive pericarditis who presented with bilateral pleural effusion. A thorough work-up showed that the pleural effusion was nonspecific, as was the pathology of the pleura. One patient had a history of pericardial effusion 2 years ago, and the other had undergone surgery for an anterior mediastinum teratoma. Pericardial scarring was found on their chest CT scans. The patients underwent pericardiectomy, and localized pericardial thickening was excised. The bilateral pleural effusion was effectively cured, and the patients showed satisfactory recovery on follow-up. Physicians should be aware of localized pericarditis leading to bilateral pleural effusion, and pericardiectomy is an effective diagnostic and therapeutic procedure.

Simcryocluster: a semantic similarity clustering method of cryo-EM images by adopting contrastive learning.

BACKGROUND: Cryo-electron microscopy (Cryo-EM) plays an increasingly important role in the determination of the three-dimensional (3D) structure of macromolecules. In order to achieve 3D reconstruction results close to atomic resolution, 2D single-particle image classification is not only conducive to single-particle selection, but also a key step that affects 3D reconstruction. The main task is to cluster and align 2D single-grain images into non-heterogeneous groups to obtain sharper single-grain images by averaging calculations. The main difficulties are that the cryo-EM single-particle image has a low signal-to-noise ratio (SNR), cannot manually label the data, and the projection direction is random and the distribution is unknown. Therefore, in the low SNR scenario, how to obtain the characteristic information of the effective particles, improve the clustering accuracy, and thus improve the reconstruction accuracy, is a key problem in the 2D image analysis of single particles of cryo-EM. RESULTS: Aiming at the above problems, we propose a learnable deep clustering method and a fast alignment weighted averaging method based on frequency domain space to effectively improve the class averaging results and improve the reconstruction accuracy. In particular, it is very prominent in the feature extraction and dimensionality reduction module. Compared with the classification method based on Bayesian and great likelihood, a large amount of single particle data is required to estimate the relative angle orientation of macromolecular single particles in the 3D structure, and we propose that the clustering method shows good results. CONCLUSIONS: SimcryoCluster can use the contrastive learning method to perform well in the unlabeled high-noise cryo-EM single particle image classification task, making it an important tool for cryo-EM protein structure determination.

Correlation investigation between core microbe inoculation and the evolution of flavor characteristics during the storage of sturgeon caviar (Acipenser gueldenstaedtii).

The volatile and non-volatile compounds were monitored to investigate the microbial evolution associated with the characteristic flavors for sturgeon caviar during refrigeration. The results revealed that the composition of volatile compounds changed significantly with prolonged refrigeration time, especially hexanal, nonanal, phenylacetaldehyde, 3-methyl butyraldehyde, and 1-octen-3-ol. The nonvolatile metabolites were mainly represented by the increase of bitter amino acids (Thr. Ser, Gly, Ala, and Pro) and a decrease in polyunsaturated fatty acids, especially an 18.63 % decrease in 5 months of storage. A total of 332 differential metabolites were mainly involved in the biosynthetic metabolic pathways of α-linolenic acid, linoleic acid, and arachidonic acid. The precursors associated with flavor evolution were mainly phospholipids, including oleic, linoleic, arachidonic, eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids. The most abundant at the genus level was Serratia, followed by Arsenophnus, Rhodococcus, and Pseudomonas, as obtained by high-throughput sequencing. Furthermore, seven core microorganisms were isolated and characterized from refrigerated caviar. Among them, inoculation with Mammalian coccus and Bacillus chrysosporium restored the flavor profile of caviar and enhanced the content of nonvolatile precursors, contributing to the characteristic aroma attributes of sturgeon caviar. The study presents a theoretical basis for the exploitation of technologies for quality stabilization and control of sturgeon caviar during storage.