CRISPLD1 promotes gastric cancer progression by regulating the Ca2+/PI3K-AKT signaling pathway.

Gastric cancer (GC) is a malignant tumor with poor prognosis. Studies have shown that cysteine-rich secretory protein LCCL domain containing 1 (CRISPLD1) is associated with tumor progression. However, its role in GC is unclear. The present study aimed to determine the pathogenic mechanism of CRISPLD1 in GC. Analysis of public databases revealed high mRNA expression of CRISPLD1 in GC, which was associated with poor prognosis. Additionally, CRISPLD1 expression levels showed significant correlations with T stage, overall survival events, and stage. Knockdown of CRISPLD1 reduced cell proliferation, invasion, and migration. Furthermore, CRISPLD1 knockdown decreased intracellular calcium levels in GC cells and inhibited the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway. Treatment with an AKT activator reversed the inhibitory effect of CRISPLD1 knockdown on GC cell migration and invasion. Our findings suggest that CRISPLD1 promotes tumor cell progression in GC by mediating intracellular calcium levels and activating the PI3K-AKT pathway, highlighting CRISPLD1 as a potential therapeutic target for GC.

A DNA-based and bifunctional nanomedicine for alleviating multi-organ injury in sepsis under diabetic conditions.

Sepsis, defined as a life-threatening organ dysfunction, is associated with increased mortality in individuals with diabetes mellitus. In sepsis under diabetic conditions (SUDC), the superimposed inflammatory response and excessive production of reactive oxygen species (ROS) can cause severe damage to the kidney and liver, making it challenging to effectively repair multi-organ injury. In this study, we report the development of a DNA-based bifunctional nanomedicine, termed IL10-rDON, generated by assembling interleukin 10 (IL10) with rectangular DNA origami nanostructures (rDON) to address multi-organ dysfunction in SUDC. IL10-rDON was shown to predominantly accumulate in the kidney and liver of diabetic mice in vivo and effectively alleviate inflammatory responses through its anti-inflammatory IL10 component. In addition, the consumption of rDON itself significantly reduced excessive ROS in the liver and kidney. Serum and histological examinations further confirmed that IL10-rDON treatment could effectively improve liver and kidney function, as well as the survival of mice with SUDC. This study demonstrates an attractive antioxidant and anti-inflammatory nanomedicine for addressing acute liver and renal failure. The integration of rDON with therapeutic agents using DNA nanotechnology is a promising strategy for generating multifunctional nanomedicine to treat multi-organ dysfunction and other complicated diseases. STATEMENT OF SIGNIFICANCE: Sepsis under diabetic conditions (SUDC) leads to high mortality due to multiple organ failure such as acute liver and kidney injury. The anti-inflammatory cytokine interleukin 10 (IL10) holds great potential to treat SUDC, while disadvantages of IL-10 such as short half-life, non-specific distribution and lack of antioxidant activities limit its wide clinical applications. In this study, we developed a DNA-based, bifunctional nanomedicine (IL10-rDON) by assembling IL10 with rectangular DNA origami nanostructures (rDON). We found that IL10-rDON preferentially accumulated and sufficiently attenuated the increased levels of ROS and inflammation in the kidney and liver injury sites, and eventually improved the survival rate of mice with SUDC. Our finding provides new insights into the application of DNA-based nanomedicine in treating multi-organ failure.

Multifunctional DNA-Based Hydrogel Promotes Diabetic Alveolar Bone Defect Reconstruction.

Diabetic alveolar bone defect (DABD) causes persistent bacterial infection, prolonged inflammation, and delayed bone healing, making it a considerable clinical challenge. In this study, by integrating silver nanoclusters (AgNCs) and M2 macrophage-derived extracellular vesicles (M2EVs), a multifunctional DNA-based hydrogel, called Agevgel, is developed with antibacterial, anti-inflammatory, immunomodulatory, and osteogenic properties to promote DABD rebuilding. AgNCs are tightly embedded into the DNA scaffolds and exhibit effective anti-bacterial activity, while immunomodulatory M2EVs are encapsulated within the shape-variable DNA scaffolds and exhibit potent anti-inflammatory and osteogenic properties. The results reveal that Agevgel effectively prolongs the local retention time and bioactivity of M2EVs in vivo. In particular, the sustained release of M2EVs can last for at least 7 days when applying Agevgel to DABD. Compared to free M2EVs or Aggel (AgNCs encapsulated within the DNA hydrogel) treatments, the Agevgel treatment accelerates the defect healing rate of alveolar bone and dramatically improves the trabecular architecture. Mechanistically, Agevgel plays a key role in regulating macrophage polarization and promoting the expression of proliferative and osteogenic factors. In summary, Agevgel provides a comprehensive treatment strategy for DABD with a great clinical translational value, highlighting the application of DNA hydrogels as an ideal bioscaffolds for periodontal diseases.

Antibacterial and DNA-Based Hydrogels In Situ Block TNF-α to Promote Diabetic Alveolar Bone Rebuilding.

Alveolar bone injury under diabetic conditions can severely impede many oral disease treatments. Rebuilding diabetic alveolar bone in clinics is currently challenging due to persistent infection and inflammatory response. Here, an antibacterial DNA-based hydrogel named Agantigel is developed by integrating silver nanoclusters (AgNCs) and tumor necrosis factor-alpha (TNF-α) antibody into DNA hydrogel to promote diabetic alveolar bone regeneration. Agantigel can effectively inhibit bacterial growth through AgNCs while exhibiting negligible cytotoxicity in vitro. The sustained release of TNF-α antibody from Agantigel effectively blocks TNF-α and promotes M2 polarization of macrophages, ultimately accelerating diabetic alveolar bone regeneration in vivo. After 21 days of treatment, Agantigel significantly accelerates the defect healing rate of diabetic alveolar bone up to 82.58 ± 8.58% and improves trabecular architectures compared to free TNF-α (42.52 ± 15.85%). The results imply that DNA hydrogels are potential bio-scaffolds helping the sustained release of multidrug for treating DABI or other oral diseases.

Characterization of site-specific N-glycosylation signatures of isolated uromodulin from human urine.

Uromodulin (Umod, Tamm-Horsfall protein) is the most abundant urinary N-glycoprotein produced exclusively by the kidney. It can form filaments to antagonize the adhesion of uropathogens. However, the site-specific N-glycosylation signatures of Umod in healthy individuals and patients with IgA nephropathy (IgAN) remain poorly understood due to the lack of suitable isolation and analytical methods. In this study, we first presented a simple and fast method based on diatomaceous earth adsorption to isolate Umod. These isolated glycoproteins were digested by trypsin and/or Glu-C. Intact N-glycopeptides with or without HILIC enrichment were analyzed using our developed EThcD-sceHCD-MS/MS. Based on the optimized workflow, we identified a total of 780 unique intact N-glycopeptides (7 N-glycosites and 152 N-glycan compositions) from healthy individuals. As anticipated, these glycosites exhibited glycoform heterogeneity. Almost all N-glycosites were modified completely by the complex type, except for one N-glycosite (N275), which was nearly entirely occupied by the high-mannose type for mediating Umod's antiadhesive activity. Then, we compared the N-glycosylation of Umod between healthy controls (n = 9) and IgAN patients (n = 9). The N-glycosylation of Umod in IgAN patients will drastically decrease and be lost. Finally, we profiled the most comprehensive site-specific N-glycosylation map of Umod and revealed its alterations in IgAN patients. Our method provides a high-throughput workflow for characterizing the N-glycosylation of Umod, which can aid in understanding its roles in physiology and pathology, as well as serving as a potential diagnostic tool for evolution of renal tubular function.

The RNA binding protein MEX3A promotes tumor progression of breast cancer by post-transcriptional regulation of IGFBP4.

PURPOSE: Breast cancer (BC) is the most frequent malignant tumor in women worldwide with exceptionally high morbidity. The RNA-binding protein MEX3A plays a crucial role in genesis and progression of multiple cancers. We attempted to explore its clinicopathological and functional significance in BC in which MEX3A is expressed. METHODS: The expression of MEX3A detected by RT-qPCR and correlated the results with clinicopathological variables in 53 BC patients. MEX3A and IGFBP4 profile data of BC patients were downloaded from TCGA and GEO database. Kaplan-Meier (KM) analysis was used to estimate the survival rate of BC patients. Western Blot, CCK-8, EdU, colony formation and flow cytometry were performed to investigate the role of MEX3A and IGFBP4 in BC cell proliferation, invasion and cell cycle in vitro. A subcutaneous tumor mouse model was constructed to analyze in vivo growth of BC cells after MEX3A knockdown. The interactions among MEX3A and IGFBP4 were measured by RNA pull-down and RNA immunoprecipitation. RESULTS: The expression of MEX3A was upregulated in BC tissues compared to adjacent tissues and high expression of MEX3A was associated with poor prognosis. Subsequent in vitro studies demonstrated that MEX3A knockdown inhibited BC cells proliferation and migration, as well as xenograft tumor growth in vivo. The expression of IGFBP4 was significantly negatively correlated with MEX3A in BC tissues. Mechanistic investigation showed that MEX3A binds to IGFBP4 mRNA in BC cells, decreasing IGFBP4 mRNA levels, which further activated the PI3K/AKT and other downstream signaling pathways implicated cell cycle progression and cell migration. CONCLUSION: Our results indicate that MEX3A plays a prominent oncogenic role in BC tumorigenesis and progression by targeting IGFBP4 mRNA and activating PI3K/AKT signaling, which can be used as a novel therapeutic target for BC.

Recent progress of nanomedicine in the treatment of Alzheimer's disease.

Alzheimer's disease (AD) is the most common cause of memory disruption in elderly subjects, with the prevalence continuing to rise mainly because of the aging world population. Unfortunately, no efficient therapy is currently available for the AD treatment, due to low drug potency and several challenges to delivery, including low bioavailability and the impediments of the blood-brain barrier. Recently, nanomedicine has gained considerable attention among researchers all over the world and shown promising developments in AD treatment. A wide range of nano-carriers, such as polymer nanoparticles, liposomes, solid lipid nanoparticles, dendritic nanoparticles, biomimetic nanoparticles, magnetic nanoparticles, etc., have been adapted to develop successful new treatment strategies. This review comprehensively summarizes the recent advances of different nanomedicine for their efficacy in pre-clinical studies. Finally, some insights and future research directions are proposed. This review can provide useful information to guide the future design and evaluation of nanomedicine in AD treatment.

Annexin A9 promotes cell proliferation by regulating the Wnt signaling pathway in colorectal cancer.

Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality worldwide. Expression of Annexin A9 (ANXA9), a member of the annexin A family, is upregulated in CRC. However, the molecular role of ANXA9 in CRC remains unknown. In the present study, we aimed to investigate the function of ANXA9 and to elucidate the mechanisms underlying its regulation in CRC. In this study, mRNA expression data and clinical information were downloaded from The Cancer Genome Atlas (TCGA) and GEPIA database, respectively. Kaplan-Meier analysis was used to analyze the survival rates. LinkedOmics and Metascape databases were used to explore the potential mechanisms of regulation of ANXA9 and to identify genes co-expressed with ANXA9. Finally, in vitro experiments were used to evaluate the function of ANXA9 and explore potential mechanisms. We found that ANXA9 expression was significantly elevated in CRC tissue and cells. High ANXA9 expression was associated with shorter overall survival, poorer disease specific survival, as well as with patient age, clinical stage, M stage, and OS events in CRC. Knockdown of ANXA9 inhibited cell proliferation, invasion, migratory potential, and cell cycle arrest. Mechanistically, functional analysis revealed that genes co-expressed with ANXA9 were mainly enriched in the Wnt signaling pathway. ANXA9 deletion suppressed cell proliferation via the Wnt signaling pathway, while Wnt activation reversed the effects of ANXA9. In conclusion, ANXA9 may promote CRC progression by regulating the Wnt signaling pathway and may be a potential diagnostic biomarker in the clinical management of CRC.

Temporal Proteomic and Lipidomic Profiles of Cerulein-Induced Acute Pancreatitis Reveal Novel Insights for Metabolic Alterations in the Disease Pathogenesis.

The pathophysiological mechanisms of acute pancreatitis (AP) are complex and have remained a mystery to date, but metabolism is gradually recognized as an important driver of AP onset and development. We used a cerulein-induced AP mouse model to conduct liquid chromatography-mass spectrometry (LC-MS/MS)-based time-course proteomics and lipidomics in order to better understand the underlying metabolic alterations linked with AP. Results showed that a series of significant changes in proteins over time with a boost in expression were enriched in lipase activity, lipoprotein, and lipids absorption and transport regulation. Furthermore, 16 proteins associated with lipid metabolism and signaling pathways together with the whole lipid species changing profile led to the vital identification of changing law in glycerides, phosphoglycerides, and free fatty acids. In addition to lipid metabolism and regulation-associated proteins, several digestive enzymes and adaptive anti-trypsin, stress response, and energy metabolism-related proteins showed an increment in abundance. Notably, central carbon and branched chain amino acid metabolism were enhanced during 0-24 h from the first cerulein stimulation. Taken together, this integrated proteomics and lipidomics revealed a novel metabolic insight into metabolites transforming rules that might be relevant to their function and drug targets investigation. (Created with Biorender.com.).

Function of stem cells in radiation-induced damage.

PURPOSE: The aim of this review is to discuss previous studies on the function of stem cells in radiation-induced damage, and the factors affecting these processes, in the hope of improving our understanding of the different stem cells and the communication networks surrounding them. This is essential for the development of effective stem cell-based therapies to regenerate or replace normal tissues damaged by radiation. CONCLUSION: In salivary glands, senescence-associated cytokines and inflammation-associated cells have a greater effect on stem cells. In the intestinal glands, Paneth cells strongly affect stem cell-mediated tissue regeneration after radiation treatment. In the pancreas, ß-cells as well as protein C receptor positive (Procr) cells are expected to be key cells in the treatment of diabetes. In the bone marrow, a variety of cytokines such as CXC-chemokine ligand 12 (CXCL12) and stem cell factor (SCF), contribute to the functional recovery of hematopoietic stem cells after irradiation.

Sini Decoction Inhibits Tumor Progression and Enhances the Anti-Tumor Immune Response in a Murine Model of Colon Cancer.

BACKGROUND: Sini decoction (SND) is a widely used Traditional Chinese Medicine (TCM). The reports of SND application in colorectal cancer (CRC) is limited. OBJECTIVE: The objective of this study is to investigate the anti-tumor activity of SND in the treatmeant of CRC. METHODS: SND was analyzed using high-performance liquid chromatography. A CRC metastasis model was established using murine CT-26 cells. Whole-body fluorescence imaging was used to observe CRC liver metastasis. Liver morphology was determined using hematoxylin-eosin staining. Cytokine mRNA expression (interleukin-2 (IL-2), interleukin-10 (IL-10), interferon-gamma (IFN-γ), and tumor necrosis factor beta (TNF-ß)) were determined using real-time reverse transcription polymerase chain reaction. Spectral flow cytometry was used to detect mouse tumor immune subgroups. Databases were used to find potential target genes of SND. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes were used to identify potential signaling pathways of target genes. RESULTS: SND suppressed CRC liver metastasis and alleviated liver injury in vivo. After SND treatment, IL-2 and IFN-γ were upregulated, whereas IL-10 and TGF-ß were downregulated. Moreover, CD3+, CD8+T cells, natural killer T cells, and macrophages increased significantly after SND treatment, while CD4+CD25+T cells decreased significantly. Importantly, increasing the aconite concentration had a better anti-tumor effect. Fifty-50 compounds in SND were screened, and 611 potential target genes were identified. Functional analyses showed that the genes were associated with the PI3K-Akt signaling pathway, EGFR tyrosine kinase inhibitor resistance, and HIF-1 signaling pathway. CONCLUSION: SND exerts anti-tumor activity by inhibiting tumor progression and enhancing antitumor immunity in mice, suggesting its application to prevent and treat CRC.

Action mechanism of hypoglycemic principle 9-(R)-HODE isolated from cortex lycii based on a metabolomics approach.

The 9-(R)-HODE is an active compound isolated from cortex lycii that showed significant hypoglycemic effects in our previous in vitro study. In this study, 9-(R)-HODE's in vivo hypoglycemic activity and effect on alleviating diabetic complications, together with its molecular mechanism, was investigated using a metabolomics approach. The monitored regulation on dynamic fasting blood glucose, postprandial glucose, body weight, biochemical parameters and histopathological analysis confirmed the hypoglycemic activity and attenuation effect, i.e., renal lesions, of 9-(R)-HODE. Subsequent metabolomic studies indicated that 9-(R)-HODE induced metabolomic alterations primarily by affecting the levels of amino acids, organic acids, alcohols and amines related to amino acid metabolism, glucose metabolism and energy metabolism. By mediating the related metabolism or single molecules related to insulin resistance, e.g., kynurenine, myo-inositol and the branched chain amino acids leucine, isoleucine and valine, 9-(R)-HODE achieved its therapeutic effect. Moreover, the mediation of kynurenine displayed a systematic effect on the liver, kidney, muscle, plasma and faeces. Lipidomic studies revealed that 9-(R)-HODE could reverse the lipid metabolism disorder in diabetic mice mainly by regulating phosphatidylinositols, lysophosphatidylcholines, lysophosphatidylcholines, phosphatidylserine, phosphatidylglycerols, lysophosphatidylglycerols and triglycerides in both tissues and plasma. Treatment with 9-(R)-HODE significantly modified the structure and composition of the gut microbiota. The SCFA-producing bacteria, including Rikenellaceae and Lactobacillaceae at the family level and Ruminiclostridium 6, Ruminococcaceae UCG 014, Mucispirillum, Lactobacillus, Alistipes and Roseburia at the genus level, were increased by 9-(R)-HODE treatment. These results were consistent with the increased SCFA levels in both the colon content and plasma of diabetic mice treated with 9-(R)-HODE. The tissue DESI‒MSI analysis strongly confirmed the validity of the metabolomics approach in illustrating the hypoglycemic and diabetic complications-alleviation effect of 9-(R)-HODE. The significant upregulation of liver glycogen in diabetic mice by 9-(R)-HODE treatment validated the interpretation of the metabolic pathways related to glycogen synthesis in the integrated pathway network. Altogether, 9-(R)-HODE has the potential to be further developed as a promising candidate for the treatment of diabetes.

Metabolomics-based evidence of the hypoglycemic effect and alleviation of diabetic complications by Ficus racemosa fruit in diabetic mice.

The hypoglycemic and metabolic effects of Ficus racemosa fruit were studied in diabetic mice, and its potential mechanisms of hypoglycemic activity and its alleviation of diabetic complications were explored using a metabolomics approach. The histopathological effect of Ficus racemosa fruit was characterized by hematoxylin and eosin histological staining. Dynamic fasting blood glucose (FBG), postprandial glucose (PPG), body weight, and biochemical parameters, including hepatic-renal function and lipid levels, were monitored to confirm the hypoglycemic activity and attenuation effect. The metabolomics analysis was performed using the established platform, combining liquid chromatography-tandem mass spectrometry with statistical analysis to identify the metabolites internally regulated by Ficus racemosa fruit. Desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) was employed to explore the presence and spatial distribution patterns of differential molecules further. An inhibition of blood glucose levels and improvements in tissue lesions were observed after Ficus racemosa fruit treatment, especially with high-dose treatment. Ficus racemosa fruit primarily induced metabolomic alterations in amino acids, organic acids and nucleotides, and displayed a systematic effect, which involved the mediation of amino acid metabolism, glucose metabolism, energy metabolism and lipid accumulation. The effect of Ficus racemosa fruit on the liver was primarily discussed in this study, and it regulated purine metabolism, glycolysis/gluconeogenesis, arginine biosynthesis, histidine metabolism, alanine, aspartate and glutamate metabolism, and the citrate cycle. Through the mediation of related pathways or single molecules that could affect insulin resistance, insulin secretion or FBG, e.g., the amino acid histidine or the organic acid uric acid in the liver, Ficus racemosa fruit achieved its hypoglycemic effect and alleviated diabetic complications in the liver. The results of the tissue metabolomic analysis, histopathological analysis, plasma biochemical parameters, plasma metabolite analysis and tissue DESI-MSI analysis were consistent with one another. The present study provides the evidence of the hypoglycemic effect and its alleviation of diabetic complications for Ficus racemosa fruit as well as the scientific support for its traditional use.

Temporal metabolic trajectory analyzed by LC-MS/MS based targeted metabolomics in acute pancreatitis pathogenesis and Chaiqin Chengqi decoction therapy.

BACKGROUND: Acute pancreatitis (AP) is an inflammatory disorder of pancreas that lacks effective specific drugs as well as gold standard laboratory tests for diagnosis and severity assessment. Chaiqin chengqi decoction (CQCQD) has been proven to alleviate the severity and mortality of AP, but its underlying mechanisms remain incompletely understood. PURPOSE: To investigate the correlation between metabolic trajectories of the serum and pancreas, the metabolic pathways with respect to the onset and progression of AP, and investigate the effect of CQCQD in modulating the dysregulated pancreatic metabolism of AP. METHODS: Serum and pancreas samples from cerulein-induced AP mice were collected for pathology, biochemical index assessment, LC-MS/MS based metabolomics and functional validation over the course of 1 - 24 h. The temporal trends of pancreatic and serum metabolites in AP were analyzed using Mfuzz clustering algorithm, and their associations were revealed by Pearson correlation analysis. The metabolic trajectories and pathways across multi-timepoints were analyzed by univariate and multivariate statistical analyses, and the AP-related metabolic pathways were further screened by metabolite correlation and network interaction analyses. Finally, the changes in metabolite levels and metabolic trajectory after CQCQD therapy were identified, and the altered expression of related metabolic enzymes was verified by RT-qPCR, western blotting, and immunohistochemistry. RESULTS: Amino acid metabolism was significantly altered in the pancreas and serum of AP, but with different trends. The unsynchronized "open" and "closed" metabolic trajectories in pancreas and serumrevealed that metabolic processes occur earlier in peripheral rather than local tissue, with the most obvious changes occuring at 12 h in the pancreas which were also consistent with the inflammation score results. Several amino acid intermediates showed strong positive correlation between serum and pancreas, and therein serum cystathionine was positively correlated to 33 pancreatic metabolites. In particular, the correlations between the levels of pancreatic cystathionine and methionine, serine, and glutathione (GSH) emphasized the importance of trans-sulfuration to GSH metabolism for AP progression. CQCQD treatment reversed the metabolic trajectory of the pancreas, and also restored the levels of cystathionine and glutathione synthase. CONCLUSION: Our results have defined a unique time-course metabolic trajectory for AP progression in both the serum and pancreas; it has also revealed a key role of CQCQD in reversing AP-associated metabolic alterations, thus providing new metabolic targets for the treatment and prognosis of AP.

Stereochemically altered cephalosporins as potent inhibitors of New Delhi metallo-ß-lactamases.

Antibiotic resistance caused by ß-lactamases, particularly metallo-ß-lactamases, has been a major threat to public health globally. New Delhi metallo-ß-lactamase-1 (NDM-1) represents one of the most important metallo-ß-lactamases; the production of NDM-1 in bacterial pathogen significantly reduces the efficacy of ß-lactam antibiotics, including life-saving carbapenems. Herein, we have demonstrated stereochemically altered cephalosporins as potent inhibitors against NDM-1, as well as mutants of NDM. The structure and activity relationship (SAR) study on over twenty cephalosporin analogues discloses the stereochemistry and the substituents on 7-position and 3'-position of cephalosporin are critical to suppress the activity of NDM-1 and the optimal compound 1u exhibited an IC50 of 0.13 µM. Furthermore, a crystal complex of NDM-1 and 1u has been obtained, suggesting this cephalosporin derivative inhibits enzyme activity by the formation of a relatively stable hydrolytic product-NDM-1 intermediate. The discovery in this study may pave the way to turn cephalosporin, a natural substrate of ß-lactamase, into an effective NDM-1 inhibitor to combat antibiotic resistance.

LncRNA LIMT (LINC01089) contributes to sorafenib chemoresistance via regulation of miR-665 and epithelial to mesenchymal transition in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is one of the most malignant tumors worldwide and HCC patients often develop drug resisitene. Long non-coding RNAs (LncRNAs) are closely related to cell cycle, growth, development, differentiation, and apoptosis. Abnormally expressed lncRNAs have been proved to mediate drug resistance in tumor cells. However, the effect of LIMT on drug resistance has not been explored in HCC. In this study, we explored the effect of long non-coding RNA LIMT on drug resistance and its underlying mechanism in hepatocellular carcinoma (HCC). Our results showed that LncRNA LINC01089 (LIMT) expression is downregulated in 78.57% (44/56) of 56 HCC tumor tissue samples. LIMT expression is also downregulated in HCC cells compared with that in normal liver LO2 cells. Inhibition of LIMT increases the resistance to sorafenib and promotes cell invasion via regulation of epithelial to mesenchymal transition (EMT) in HCC. StarBase V3.0 was used to predict the potential binding site of miR-665 in . Furthermore, miR-665 participates in sorafenib resistance and also regulates the level of EMT-related proteins in HCC cells. A rescue experiment demonstrated that silencing of eliminats the inhibitory effect of the miR-665 inhibitor on sorafenib resistance in HCC cells. Taken together, our findings revealed that downregulation of LIMT increases the resistance of HCC to sorafenib via miR-665 and EMT. Therefore, LIMT, which serves as a therapeutically effective target, will provide new hope for the treatment of HCC.

Specific detection of IMP-1 ß-lactamase activity using a trans cephalosporin-based fluorogenic probe.

A fluorogenic probe for the specific detection of IMP-1 ß-lactamase activity has been developed. This imaging reagent features a unique trans-acetylamino cephalosporin as an enzymatic recognition moiety, exhibiting excellent selectivity to IMP-1 ß-lactamase over other ß-lactamases, including serine- and metallo-ß-lactamases. The selective activation of the probe by IMP-1 ß-lactamase leads to over 30-fold enhancement in the fluorescence intensity, which allows enzyme activity to be reported with high sensitivity.

Sequential Analysis of the N/O-Glycosylation of Heavily Glycosylated HIV-1 gp120 Using EThcD-sceHCD-MS/MS.

Deciphering the glycosylation of the viral envelope (Env) glycoprotein is critical for evaluating viral escape from the host's immune response and developing vaccines and antiviral drugs. However, it is still challenging to precisely decode the site-specific glycosylation characteristics of the highly glycosylated Env proteins, although glycoproteomics have made significant advances in mass spectrometry techniques and data analysis tools. Here, we present a hybrid dissociation technique, EThcD-sceHCD, by combining electron transfer/higher-energy collisional dissociation (EThcD) and stepped collision energy/higher-energy collisional dissociation (sceHCD) into a sequential glycoproteomic workflow. Following this scheme, we characterized site-specific N/O-glycosylation of the human immunodeficiency virus type 1 (HIV-1) Env protein gp120. The EThcD-sceHCD method increased the number of identified glycopeptides when compared with EThcD, while producing more comprehensive fragment ions than sceHCD for site-specific glycosylation analysis, especially for accurate O-glycosite assignment. Finally, eighteen N-glycosites and five O-glycosites with attached glycans were assigned unambiguously from heavily glycosylated gp120. These results indicate that our workflow can achieve improved performance for analysis of the N/O-glycosylation of a highly glycosylated protein containing numerous potential glycosites in one process. Knowledge of the glycosylation landscape of the Env glycoprotein will be useful for understanding of HIV-1 infection and development of vaccines and drugs.

Microbiome Profiling Using Shotgun Metagenomic Sequencing Identified Unique Microorganisms in COVID-19 Patients With Altered Gut Microbiota.

COVID-19 is mainly associated with respiratory distress syndrome, but a subset of patients often present gastrointestinal (GI) symptoms. Imbalances of gut microbiota have been previously linked to respiratory virus infection. Understanding how the gut-lung axis affects the progression of COVID-19 can provide a novel framework for therapies and management. In this study, we examined the gut microbiota of patients with COVID-19 (n = 47) and compared it to healthy controls (n = 19). Using shotgun metagenomic sequencing, we have identified four microorganisms unique in COVID-19 patients, namely Streptococcus thermophilus, Bacteroides oleiciplenus, Fusobacterium ulcerans, and Prevotella bivia. The abundances of Bacteroides stercoris, B. vulgatus, B. massiliensis, Bifidobacterium longum, Streptococcus thermophilus, Lachnospiraceae bacterium 5163FAA, Prevotella bivia, Erysipelotrichaceae bacterium 6145, and Erysipelotrichaceae bacterium 2244A were enriched in COVID-19 patients, whereas the abundances of Clostridium nexile, Streptococcus salivarius, Coprococcus catus, Eubacterium hallii, Enterobacter aerogenes, and Adlercreutzia equolifaciens were decreased (p < 0.05). The relative abundance of butyrate-producing Roseburia inulinivorans is evidently depleted in COVID-19 patients, while the relative abundances of Paraprevotella sp. and the probiotic Streptococcus thermophilus were increased. We further identified 30 KEGG orthology (KO) modules overrepresented, with 7 increasing and 23 decreasing modules. Notably, 15 optimal microbial markers were identified using the random forest model to have strong diagnostic potential in distinguishing COVID-19. Based on Spearman's correlation, eight species were associated with eight clinical indices. Moreover, the increased abundance of Bacteroidetes and decreased abundance of Firmicutes were also found across clinical types of COVID-19. Our findings suggest that the alterations of gut microbiota in patients with COVID-19 may influence disease severity. Our COVID-19 classifier, which was cross-regionally verified, provides a proof of concept that a set of microbial species markers can distinguish the presence of COVID-19.

O-Glycosylation Landscapes of SARS-CoV-2 Spike Proteins.

The densely glycosylated spike (S) proteins that are highly exposed on the surface of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) facilitate viral attachment, entry, and membrane fusion. We have previously reported all the 22 N-glycosites and site-specific N-glycans in the S protein protomer. Herein, we report the O-glycosylation landscapes of SARS-CoV-2 S proteins, which were characterized through high-resolution mass spectrometry. Following digestion with trypsin and trypsin/Glu-C, and de-N-glycosylation using PNGase F, we determined the GalNAc-type O-glycosylation pattern of S proteins, including O-glycosites and the six most common O-glycans occupying them, via Byonic identification and manual validation. Finally, 255 intact O-glycopeptides composed of 50 peptides sequences and 43 O-glycosites were discovered by higher energy collision-induced dissociation (HCD), and three O-glycosites were confidently identified by electron transfer/higher energy collision-induced dissociation (EThcD) in the insect cell-expressed S protein. Most glycosites were modified by non-sialylated O-glycans such as HexNAc(1) and HexNAc(1)Hex (1). In contrast, in the human cell-expressed S protein S1 subunit, 407 intact O-glycopeptides composed of 34 peptides sequences and 30 O-glycosites were discovered by HCD, and 11 O-glycosites were unambiguously assigned by EThcD. However, the measurement of O-glycosylation occupancy hasn't been made. Most glycosites were modified by sialylated O-glycans such as HexNAc(1)Hex (1)NeuAc (1) and HexNAc(1)Hex (1)NeuAc (2). Our results reveal that the SARS-CoV-2 S protein is an O-glycoprotein; the O-glycosites and O-glycan compositions vary with the host cell type. These comprehensive O-glycosylation landscapes of the S protein are expected to provide novel insights into the viral binding mechanism and present a strategy for the development of vaccines and targeted drugs.