High Expression Levels Research Articles

Functional Characterization of the Gibberellin (GA) Receptor ScGID1 in Sugarcane.

Sugarcane smut caused by Sporisorium scitamineum represents the most destructive disease in the sugarcane industry, causing host hormone disruption and producing a black whip-like sorus in the apex of the stalk. In this study, the gibberellin metabolic pathway was found to respond to S. scitamineum infection, and the contents of bioactive gibberellins were significantly reduced in the leaves of diseased plants. The gibberellin receptor gene ScGID1 was identified and significantly downregulated. ScGID1 localized in both the nucleus and cytoplasm and had the highest expression level in the leaves. Eight proteins that interact with ScGID1 were screened out using a yeast two-hybrid assay. Novel DELLA proteins named ScGAI1a and ScGA20ox2, key enzymes in GA biosynthesis, were both found to interact with ScGID1 in a gibberellin-independent manner. Transcription factor trapping with a yeast one-hybrid system identified 50 proteins that interacted with the promoter of ScGID1, among which ScS1FA and ScPLATZ inhibited ScGID1 transcription, while ScGDSL promoted transcription. Overexpression of ScGID1 in transgenic Nicotiana benthamiana plants could increase plant height and promote flowering. These results not only contribute to improving our understanding of the metabolic regulatory network of sugarcane gibberellin but also expand our knowledge of the interaction between sugarcane and pathogens.

Exploring the Relationship Between Gene Expression and Low-Frequency Somatic Mutations in Arabidopsis with Duplex Sequencing.

Intragenomic mutation rates can vary dramatically due to transcription-associated mutagenesis or transcription-coupled repair, which vary based on local epigenomic modifications that are nonuniformly distributed across genomes. One feature associated with decreased mutation is higher expression level, which depends on environmental cues. To understand the magnitude of expression-dependent mutation rate variation, we perturbed expression through a heat treatment in Arabidopsis thaliana. We quantified gene expression to identify differentially expressed genes, which we then targeted for mutation detection using duplex sequencing. This approach provided a highly accurate measurement of the frequency of rare somatic mutations in vegetative plant tissues, which has been a recent source of uncertainty. Somatic mutations in plants may be useful for understanding drivers of DNA damage and repair in the germline since plants experience late germline segregation and both somatic and germline cells share common repair machinery. We included mutant lines lacking mismatch repair (MMR) and base excision repair (BER) capabilities to understand how repair mechanisms may drive biased mutation accumulation. We found wild-type (WT) and BER mutant mutation frequencies to be very low (mean variant frequency 1.8 × 10-8 and 2.6 × 10-8, respectively), while MMR mutant frequencies were significantly elevated (1.13 × 10-6). Interestingly, in the MMR mutant lines, there was no difference in the somatic mutation frequencies between temperature treatments or between highly versus lowly expressed genes. The extremely low somatic variant frequencies in WT plants indicate that larger datasets will be needed to address fundamental evolutionary questions about whether environmental change leads to gene-specific changes in mutation rate.

Exploring the prognostic and diagnostic value of lactylation-related genes in sepsis

The discovery of Lactylation may pave the way for novel approaches to investigating sepsis. This study focused on the prognostic and diagnostic significance of lactylated genes in sepsis. RNA sequencing was performed on blood samples from 20 sepsis patients and 10 healthy individuals at Southwest Medical University in Luzhou, Sichuan, China. Genes associated with sepsis were identified through analysis of RNA sequencing data. Afterward, the genes that were expressed differently were compared with the lactylation genes, resulting in the identification of 55 lactylation genes linked to sepsis. The overlapping genes underwent analysis using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Protein-Protein Network Interactions were used to screen for the core genes. The datasets GSE65682, GSE69528, GSE54514, GSE63042, and GSE95233 were obtained from the GEO database to validate core genes. Survival analysis evaluated the predictive significance of central genes in sepsis, while Receiver Operating Characteristic (ROC) Curve analysis was employed to establish the diagnostic value of genes. Additionally, a meta-analysis was conducted to confirm the precision of RNA-seq data. We obtained five peripheral blood samples, including two from healthy individuals, one from a patient with systemic inflammatory response syndrome (SIRS), and two from patients with sepsis. These samples were used to identify the specific location of core genes using 10×single-cell sequencing. High-throughput sequencing and bioinformatics techniques identified two lactylation-related genes (S100A11 and CCNA2) associated with sepsis. Survival analysis indicated that septic patients with reduced levels of S100A11 had a decreased 28-day survival rate compared to those with elevated levels. Conversely, individuals exhibiting decreased CCNA2 levels demonstrated a greater likelihood of surviving for 28 days than those in the high expression category, indicating a favorable association with survival rates among sepsis patients (P < 0.05). Both genes showed high sensitivity and specificity based on the ROC curve, with AUC values of 0.961 for S100A11 and 0.890 for CCNA2. The meta-analysis revealed that S100A11 exhibited high levels of expression in the sepsis survivors, whereas it displayed low levels of expression in the non-survivors; on the other hand, CCNA2 demonstrated low expression in the sepsis survivors and high expression in the non-survivors (P < 0.05). Single-cell RNA sequencing ultimately showed that monocyte macrophages, T cells, and B cells exhibited high expression levels of the crucial genes associated with sepsis-induced lactylation. In conclusion, the lactylation genes S100A11 and CCNA2 are strongly linked to sepsis and could be valuable markers for diagnosing, predicting outcomes, and providing guidance for sepsis.

Production of active human iduronate-2-sulfatase (IDS) enzyme in Nicotiana benthamiana

Many strategies have been developed to produce high levels of biologically active recombinant proteins in plants for biopharmaceutical purposes. However, the production of an active form of human iduronate-2-sulfatase (hIDS) for the treatment of Hunter syndrome by enzyme replacement therapy (ERT) is challenging due to the requirement for cotranslational modification by a formylglycine-producing enzyme encoded by sulfatase modifying factor 1 (hSUMF1) at the Cys84 residue, which converts it to C(alpha)-formylglycine. In this study, we have shown that hIDS can be highly expressed in N. benthamiana by using different constructs. Among them, BiP-GB1-L-dCBD1-2L-8xHis-L-6xHis-3L-EK-hIDS-HDEL (GB1-CBD1-hIDS) showed a high expression level when transiently co-expressed with the turnip crinkle virus gene silencing suppressor P38 and GB1-fused human calreticulin (GB1-CRT1) as a folding enhancer. The hSUMF1 was co-expressed with hIDS for cotranslational modification. The full-length recombinant proteins were purified using Ni2+-NTA affinity resin followed by enterokinase treatment to obtain tag-free hIDS. The N-terminal fragment was removed using microcrystalline cellulose (MCC) beads. The purified active form of hIDS can successfully cleave the sulfate group from an artificial substrate, 4-nitrocatechol sulfate, at a similar level to commercial hIDS expressed in animal cells. These results suggest that plants could be a promising platform for the production of recombinant hIDS.

Mitotic spindle positioning protein serves as prognostic biomarker in patients with colorectal cancer

Background Colorectal cancer (CRC) ranks among the most aggressive types of cancer globally. Currently, clinical tumor prognostic biomarkers still lack accuracy. Mitotic spindle positioning (MISP) protein connects microtubules to the actin cytoskeleton and adhesive plaques, playing a critical role in spindle positioning, orientation, and the process of cell division. MISP can regulate the malignant biological functions of pancreatic cancer and intrahepatic cholangiocarcinoma and it acts as biomarker for prognosis, but its role in CRC remains unclear. Methods This study has collected 37 CRC tissue samples and 37 corresponding adjacent nontumor tissue samples, and 57 additional CRC tissues samples. Clinical data were obtained from the patients with CRC. MISP mRNA and protein expression levels were analyzed in normal control and CRC tissues using the GEPIA and Human Protein Atlas website. MISP protein levels in the collected tissues were analyzed using immunohistochemistry. Results MISP mRNA and protein expression levels were significantly increased in CRC tissues compared to adjacent nontumor tissues. Higher MISP protein levels were associated with distant metastasis, recurrence, and lower survival rates. Kaplan–Meier analysis showed that high expression levels of MISP protein were associated with recurrence and death in CRC patients. In addition, a high expression level of MISP protein, lymph node metastasis, and distance metastasis were risk factors for recurrence and a poor prognosis in patients with CRC. Conclusion Elevated MISP protein correlated with tumor metastasis, recurrence, and lower survival rates in patients with CRC, and thus, MISP has the potential to become a prognostic marker for CRC.

Expression of thermostable MMLV reverse transcriptase in Escherichia coli by directed mutation

The functionality of Moloney murine leukemia virus reverse transcriptase (MMLV RT) will increase with the improvement of its solubility and thermal stability. Introduce directed mutation at specific positions of the MMLV RT sequence and codon optimization is needed to achieve these properties. The two RT coding sequences with (rRT-K) and without directed mutations (rRT-L) were versatility optimized and expressed to analyze the ribonuclease H (RNase H) inactivity and thermostable polymerase activity. For this purpose, the five-point mutations (438–442aa) and three-point mutations (530, 568, and 659 aa) were done at the RT connection domain and RNase H active site, respectively. High expression levels of rRT-L and rRT-K were obtained in E. coli BL21(DE3) and BL21(shuffle) strains, 0.5 mM IPTG concentration at 37 °C, and 8 hours’ post-induction condition. Then, recombinant enzymes were purified and verified by Ni-NTA resin and western blotting. Insilico analysis (IUpred 3.0) showed that the directed mutation in the RNase H domain caused the formation of disorder regions or instability in the RNase H domain of rRT-K compared to rRT-L. The modified RT-PCR and the RT-LAMP reactions proved the RNase H inactivity of rRT-K. In addition, increasing of thermostability of rRT-K compared to rRT-L and commercial RT was evaluated by the RT-PCR and RT-LAMP reactions. The results showed that rRT-K could successfully tolerate 60 ºC in the two methods. This study revealed that the directed mutations and the versatile sequence optimization can promise to produce thermostable commercial enzymes to decrease non-specific one-step RT-PCR and RT-LAMP products.

The Association of Neuroendocrine Differentiation with MicroRNA 21 and MicroRNA let7f Expression and the Clinicopathological Parameters in Primary Invasive Breast Carcinomas with Neuroendocrine Features.

MiRNAs have been reported as biomarkers with diagnostic, prognostic, and predictive value for many different diseases. Therapeutic agents targeting some miRNAs are currently being developed. We aimed to compare BC-NEFs (carcinoma of the breast with neuroendocrine features) with IDC (invasive ductal carcinoma) cases without neuroendocrine features in terms of the level of miRNA expression known to show the oncogenic (miR-21) and tumor-suppressor effects (miR-let7f) and the clinicopathological features. A total of 29 patients with a diagnosis of BC-NEFs (15 cases with neuroendocrine differentiation >50% of the whole section of tumor and 14 cases with neuroendocrine differentiation 10-50% of the tumor) and 30 patients with a diagnosis of IDC (no neuroendocrine differentiation) were retrospectively re-evaluated. Expression levels of miR-21 and miR-let7f were determined by the qRT-PCR method in paraffin tissue blocks. MiR-21 expression was significantly higher in the IDC group than in the group with BC-NEFs. miR-let7f expression was significantly lower in the group with BC-NEFs compared to the IDC group. A high expression level of miR-21 was found to be associated with progesterone receptor (PR) negativity. Our findings show that the presence of NEFs in breast carcinomas makes a significant difference in the expression levels of the investigated oncogenic (miR-21) and tumor-suppressor (miR-let7f) miRNAs. These findings suggest that miRNAs may be a potential biomarker in BC-NEFs and would benefit from targeted therapy.

Investigation of Cell Mechanics and Migration on DDR2-Expressing Neuroblastoma Cell Line.

Neuroblastoma is a devastating disease accounting for ~15% of all childhood cancer deaths. Collagen content and fiber association within the tumor stroma influence tumor progression and metastasis. High expression levels of collagen receptor kinase, Discoidin domain receptor II (DDR2), are associated with the poor survival of neuroblastoma patients. Additionally, cancer cells generate and sustain mechanical forces within their environment as a part of their normal physiology. Despite this, evidence regarding whether collagen-activated DDR2 signaling dysregulates these migration forces is still elusive. To address these questions, a novel shRNA DDR2 knockdown neuroblastoma cell line (SH-SY5Y) was engineered to evaluate the consequence of DDR2 on cellular mechanics. Atomic force microscopy (AFM) and traction force microscopy (TFM) were utilized to unveil the biophysical altercations. DDR2 downregulation was found to significantly reduce proliferation, cell stiffness, and cellular elongation. Additionally, DDR2-downregulated cells had decreased traction forces when plated on collagen-coated elastic substrates. Together, these results highlight the important role that DDR2 has in reducing migration mechanics in neuroblastoma and suggest DDR2 may be a promising novel target for future therapies.

Hexokinase 2 as an independent risk factor for worse patient survival in esophageal adenocarcinoma and as a potential therapeutic target protein: A retrospective, single‑center cohort study.

Cancer cells exhibit a distinct metabolic profile that features an upregulation of less efficient glycolysis accompanied by lactate production for energy generation, in contract to the characteristic metabolism of normal cells. Consequently, cancer research has focused on the enzymes that participate in these cancer metabolic pathways. Among them, hexokinase 2 (HK2) has an important position as the initial enzyme in the glycolytic pathway. Increased expression levels of HK2 have been correlated with an increased risk of poor patient outcomes and advanced tumor stages in a number of malignant tumors, such as gastric carcinoma. The present study aimed to investigate the specific role of HK2 in patients diagnosed with esophageal adenocarcinoma. A total of 643 patients with esophageal adenocarcinoma were included. Immunohistochemical staining and HK2 mRNA in situ probes were used to investigate the association of HK2 expression levels with clinical and molecular tumor characteristics. Patients who exhibited high HK2 expression levels demonstrated significantly reduced overall survival (OS) times compared with patients who exhibited low HK2 expression levels (29.6 vs. 39.9 months, respectively; P=0.027). Furthermore, high HK2 expression levels were demonstrated to be an independent risk factor for reduced patient survival (hazard ratio, 1.65; 95% CI, 1.09-2.50; P=0.018). Significantly reduced patient survival was also demonstrated in the subgroups of male patients, patients with primarily resected tumors, patients with HER2-negative tumors and patients with tumors exhibiting Y chromosome loss. Elevated expression of HK2 was identified as a risk factor for unfavorable patient survival in esophageal adenocarcinoma. This revelation suggests the potential for future diagnostic and therapeutic avenues tailored to this specific patient subset. Identifying patients with high HK2 expression may pinpoint a higher-risk cohort, paving the way for comprehensive prospective studies that could advocate for intensified monitoring and more aggressive therapeutic regimens. Furthermore, the targeted inhibition of HK2 could hold promise as a strategy to potentially enhance patient outcomes.

Enrichment of Allelic Editing Outcomes by Prime Editing in Induced Pluripotent Stem Cells.

Gene editing in human induced pluripotent stem (iPS) cells with programmable nucleases facilitates reliable disease models, but methods using double-strand break repair often produce random on-target by-products. Prime editing (PE) combines Cas9 nickase with reverse transcriptase and PE guide RNA (pegRNA) encoding a repair template to reduce by-products. We implemented a GMP-compatible protocol for transfecting Cas9- or PE-2A-mCherry plasmids to track and fractionate human iPS cells based on PE expression level. We compared the editing outcomes of Cas9- and PE-based methods in a GFP-to-BFP conversion assay at the HEK3 benchmark locus and at the APOE Alzheimer's risk locus, revealing superior precision of PE at high expression levels. Moreover, sorting cells for PE expression level influenced allelic editing outcomes at the target loci. We expect that our findings will aid in the creation of gene-edited human iPS cells with intentional heterozygous and homozygous genotypes.

Genome-wide identification of the grapevine β-1,3-glucanase gene (VviBG) family and expression analysis under different stresses.

The β-1,3-glucanase gene is widely involved in plant development and stress defense. However, an identification and expression analysis of the grape β-1,3-glucanase gene (VviBG) family had not been conducted prior to this study. Here, 42 VviBGs were identified in grapevine, all of which contain a GH-17 domain and a variable C-terminal domain. VviBGs were divided into three clades α, β and γ, and six subgroups A-F, with relatively conserved motifs/domains and intron/exon structures within each subgroup. The VviBG gene family contained four tandem repeat gene clusters. There were intra-species synteny relationships between two pairs of VviBGs and inter-species synteny relationships between 20 pairs of VviBGs and AtBGs. The VviBG promoter contained many cis-acting elements related to stress and hormone responses. Tissue-specific analysis showed that VviBGs exhibited distinct spatial and temporal expression patterns. Transcriptome analysis indicated that many VviBGs were induced by wounds, UV, downy mildew, cold, salt and drought, especially eight VviBGs in subgroup A of the γ clade. RT-qPCR analysis showed that these eight VviBGs were induced under abiotic stress (except for VviBG41 under cold stress), and most of them were induced at higher expression levels by PEG6000 and NaCl than under cold treatment. The chromosome localization, synteny and phylogenetic analysis of the VviBG members were first conducted. The cis-acting elements, transcriptome data and RT-qPCR analysis showed that VviBG genes play a crucial role in grape growth and stress (hormone, biotic and abiotic) responses. Our study laid a foundation for understanding their functions in grape resistance to different stresses.

Identification, expression, and function analysis of Rbpms2 splicing variants in Japanese flounder gonad

Rbpms2, an RNA-binding protein with multiple splicing (Rbpms), can interact with RNAs to involve oocyte development, thereby influencing female sex differentiation in vertebrates. Here, two splicing variants of the Rbpms2 gene from Japanese flounder (Paralichthys olivaceus) were identified, namely Rbpms2.1 and Rbpms2.2. The two variants exhibited 98.22 % amino acid homology, both featuring an RNA recognition motif (RRM) domain spanning positions 98–170 amino acids. They were relatively conserved throughout phylogenetic evolution. Differently, the C-terminal region of the Rbpms2.1 contains five additional sequential amino acids (–VRDQP–) compared to Rbpms2.2. The real-time qPCR results demonstrated that Rbpms2.1 and Rbpms2.2 had relatively abundant expression in the gonads of adult Japanese flounder, with higher expression levels in the ovary compared to the testis (P < 0.05). In situ hybridization results showed strong positive expression of Rbpms2 mRNA in oocytes at stages I-III during the V stage of ovarian development. In the testis atstage IV, the expression of Rbpms2 mRNA was mainly concentrated on primary spermatocytes. Importantly, Rbpms2 binding sites were found in the 3′UTR, 5′UTR, and ORF regions of the sex-related genes including dmrt1, sox9, amh, foxl2, and wnt4. siRNA interference and overexpression analysis of Rbpms2.1 and Rbpms2.2 in primary cells of the ovary and testis showed that Rbpms2 can repress the expression of male-related genes (dmrt1, sox9, and amh) and significantly promote the expression of female-related genes (foxl2 and wnt4). Our results revealed that Rbpms2 may play a critical role by targeting the sex-related genes in the gonad development of Japanese flounder.

Mechanical and structural features of three AcSp proteins underlie the diverse material properties of aciniform silks of Neoscona spiders

Spider silks are desirable multicomponent biomaterials characterized by great tensile strength, extensibility, and biocompatibility. Of all spider silk types, aciniform silk has highest toughness due to its combination of high tensile strength and elsticity. Here, we identify three major spidroin components (AcSp1A, AcSp1B, and AcSp2) from aciniform silk of orbweb weaving spider, Neoscona scylloides, and present their full-length coding gene sequences. Comparative sequence and expression level analysis show that AcSp1B has highest expression level and higher serine content than other two AcSp proteins, while the AcSp2 shows very low mRNA level. Furthermore, three recombinant minimalist AcSp proteins are produced and could be induced to form fibers by shear forces in a physiological buffer. The manual-drawn AcSp1B fiber shows strongest tensile strength among three AcSp fibers because of its higher β-sheet formed by abundant serine content. We also compare mechanical properties of aciniform silks between two Neoscona species (N. theisi and N. scylloides) and found that aciniform silks from N. theisi exhibit higher tensile strength than those of N. scylloides, which may result from altering expression levels of two AcSp1 proteins. Collectively, our results provide insights into the mechanical features of each component in aciniform silk from N. scylloides and reveal the molecular mechanism of diverse material properties of aciniform silk among species.

Passive immunoprophylaxis with Ccombodies against Vibrio parahaemolyticus in Pacific white shrimp (Penaeus vannamei)

The Vibrio parahaemolyticus strain causing acute hepatopancreatic necrosis disease (AHPND) in shrimp secretes toxins A and B (PirAVp/PirBVp). These toxins have been implicated in pathogenesis and are targets for developing anti-AHPND therapeutics or prophylactics that include passive immunization. We have previously reported that Ccombodies (recombinant hagfish variable lymphocyte receptor B antibodies; VLRB) targeting PirBVp conferred protection against V. parahaemolyticus in shrimp when administered as a feed supplement. In this study, we screened a phage-displayed library of engineered VLRBs for PirAVp-targeting Ccombodies that were mass-produced in a bacterial expression system. We then introduced these Ccombodies into the diet of Pacific white shrimp (Penaeus vannamei) over a seven-day period. Subsequently, the shrimp were exposed to a challenge with V. parahaemolyticus. Mortality rates were then observed and recorded over the following seven days. Administering shrimp feed supplemented with Ccombodies at a high dose (100 mg per 100 g feed) reduced mortality in recipient animals (2.96-5.19%) statistically similar to mock-challenged control (1.48%), but significantly different from the Ccombody-deficient control (74.81%). This suggests that the Ccombodies provided strong protection against the bacterium. Feeding shrimp with a median dose (10 mg/100 g feed) gave statistically comparable low mortality (5.93-6.67%) as the high dose. Reducing the Ccombody dose to 1 mg/100 g feed showed variable effects. Ccombody A2 showed mortality (11.85%) significantly lower than that of the Ccombody-deficient group (74.81%), suggesting that it can effectively protect against the bacterial challenge at a low dose. Our results demonstrate the ability of the phage-displayed VLRB library to generate antigen-specific Ccombodies rapidly and simply, with the expression of high protein levels in bacteria. The protective effect provided by these Ccombodies aligns with our earlier results, strongly supporting the use of VLRB antibodies as a substitute for IgY in passive immunoprophylaxis against AHPND in shrimp.

ω-9 monounsaturated fatty acids in Sapindus mukorossi seed oil enhance calcium deposition expression of Wharton’s jelly mesenchymal stem cells

Promoting osteogenesis is crucial to improve successful bone regeneration in bone tissue engineering. Several studies on osteogenesis have reported positive effects of ω-9 monounsaturated fatty acids (MUFA) on bone regeneration. This study examined the potential of ω-9 monounsaturated fatty acid abundant seed oil mechanically extracted from Sapindus mukorossi (S. mukorossi) fruit to improve osteogeneses. After showing the presence of ω-9 MUFA in S. mukorossi seed oil (SM oil) through GC-MS spectrum analysis, the proliferation and differentiation of human umbilical cord Wharton’s jelly mesenchymal stem cells (WJMSCs) under SM treatment was evaluated. Our results indicate that WJMSC differentiation was induced by adding an osteogenesis-induced medium combined with SM oil. A high level of calcium deposition expression in WJMSCs induced by SM oil appears to be due to the effect of oleic acid and eicosenoic acid, both of which are ω-9 monounsaturated fatty acids. In addition, we found major contributors of SM oil-promoted WJMSC osteogenesis to be extracellular signal-regulated kinase (ERKs), c-Jun N-terminal kinase (JNKs), and the p38 MAPK pathway. The enhancement of WJMSC osteogenesis via ERK/MAPK pathways as demonstrated by qPCR analysis indicates the promise of SM oil for stem cell-based bone tissue engineering applications.

UBE2Q1 as a novel cancer biomarker for lung adenocarcinoma: Short Title: Oncogenic function of UBE2Q1 in lung adenocarcinoma

PurposeUbiquitin-conjugating enzymes (E2s) participate in various tumor-promoting processes. UBE2Q1 is a member of the E2 family. This research aimed to detect the expression level of UBE2Q1 in human lung adenocarcinoma and to study its malignant biological function. MethodsWestern blot, qRT-PCR and immunohistochemistry was used to measure the expression of UBE2Q1 in human lung adenocarcinoma tissues. The association between UBE2Q1 expression and clinic-pathological variables in 99 lung adenocarcinoma samples was analyzed by immunohistochemistry. In vitro experiment, establishing UBE2Q1 knockdown pattern, the markers of apoptosis, cell cycle and epithelial-mesenchymal transition (EMT) were analyzed by Western blot. CCK8, colony formation, Transwell and invasion assay analyzed the effect of UBE2Q1 knockdown on the proliferation, metastasis and invasion of lung cancer cells. ResultsUBE2Q1 was overexpressed in lung adenocarcinoma, and the expression level of UBE2Q1 was related with TNM stage, tumor size, and lymph node metastasis. The high level of UBE2Q1 expression was also associated with poor survival and was an independent risk factor. In vitro, It was also confirmed that steady downregulation of UBE2Q1 could promote apoptosis, induce G2/M cell cycle arrest and regulate EMT. UBE2Q1 silencing dramatically reduce lung tumor cells proliferation, migration and invasion capacities. ConclusionsUBE2Q1 may serve as a prognostic biomarker and a new therapeutic target of lung adenocarcinoma.

Development and validation of a droplet digital PCR method for quantifying lentiviral vector infectious titer

Lentiviruses, with their high transduction efficiency and gene expression levels, are widely used as gene delivery vectors in the development of chimeric antigen receptor T cells (CAR-T) and other genetically modified cell therapies. Accurate determination of the lentiviral vector infectious titer is essential to ensure effective transduction and product consistency. In this study, we developed an efficient method for lentiviral vector titration based on digital droplet polymerase chain reaction (ddPCR) technology, enabling absolute quantification of the target gene. Benzonase treatment of non-transduced plasmids substantially shortened the experimental period, reducing cell culture duration from 10-14 days–3 days. The method was rigorously validated by assessing specificity, working range, limit of quantification, precision, accuracy, and robustness. This study demonstrates the feasibility of combining enzymatic digestion with ddPCR to quantify lentiviral vector infectious titer and provides a detailed and readily adaptable methodology for the scientific community.

High expression of COL8A1 predicts poor prognosis and promotes EMT in papillary thyroid cancer.

Collagen type VIII α 1 chain (COL8A1), a collagen type VIII protein, has been suggested to exert various functions in progression of multiple cancers. However, the effect of COL8A1 in papillary thyroid cancer (PTC) has not been elucidated. The Cancer Genome Atlas (TCGA) databases were applied to investigate the COL8A1 expression and its clinical significance in PTC. The COL8A1 expression level was further validated using Gene Expression Omnibus (GEO) data and clinical paired PTC tissues. Additionally, Kaplan-Meier curve was used to analyze the prognosis. The cell migrative and invasive abilities were evaluated by wound healing assay and Transwell assay. CCK8 assays were used to evaluate proliferation of PTC cells. Western blotting was conducted to explore the potential mechanisms involved in the pro-tumor role of COL8A1. The correlation between immune cell infiltration and COL8A1 was analyzed using Tumor Immune Estimation Resource (TIMER) database and the single-sample GSEA (ssGSEA) method. We found that COL8A1 was upregulated in PTC (P<0.05). High COL8A1 expression level was significantly associated with advanced T stage (P<0.01), N stage (P<0.001) and poor prognosis (P=0.0142) in PTC. Furthermore, cell migration and invasion were significantly reduced following COL8A1 knockdown (P<0.001). Mechanistic studies demonstrated that the epithelial-to-mesenchymal transition (EMT) related proteins (FN1, MMP9, MMP7, ZEB2 and Twist1) and phosphorylation of AKT and ERK were obviously down-regulated after COL8A1 knockdown (P<0.01). Moreover, COL8A1 expression was correlated with immune cell infiltration. Our study demonstrates that COL8A1 may function as an oncogene and a potential prognostic biomarker for PTC patients.

Gram-negative microflora dysbiosis facilitates tumor progression and immune evasion by activating CCL3/CCL5-CCR1-MAPK-PD-L1 pathway in esophageal squamous cell carcinoma.

Gram-negative micro-flora dysbiosis occurs in multiple digestive tumors and is found to be the dominant micro-flora in esophageal squamous cell carcinoma (ESCC) micro-environment. The continuous stimulation of G- bacterium metabolites may cause tumorigenesis and reshape the micro-immune environment in ESCC. However, the mechanism of G- bacilli causing immune evasion in ESCC remains underexplored. We identified CC Chemokine receptor 1 (CCR1) as a tumor-indicating gene in ESCC. Interestingly, expression levels of CCR1 and PD-L1 were mutually up regulated after G- bacilli metabolites lipopolysaccharide (LPS) stimulation. Firstly, we found CCR1 high expression level to be associated with poor overall survival in ESCC. Importantly, we found that high level expression of CCR1 up-regulated PD-L1 expression by activating MAPK phosphorylation in ESCC and induced tumor malignant behavior. Finally, we found that T cells exhaustion and cytotoxicity suppression were associated with CCR1 expression in ESCC, which were decreased after CCR1 inhibiting. Our work identifies CCR1 as a potential immune check point regulator of PD-L1 and may cause T cell exhaustion and cytotoxicity suppression in ESCC micro-environment and highlights the potential value of CCR1 as therapeutic target of immunotherapy. Implications: The esophageal microbial environment and its metabolites significantly affect the outcome of immunotherapy for ESCC.

Comprehensive Proteomic Analysis of Dysferlinopathy Unveiling Molecular Mechanisms and Biomarkers Linked to Pathological Progression.

Previous proteomics studies in dysferlinopathy muscle have been limited in scope, often utilizing 2D-electrophoresis and yielding only a small number of differential expression calls. To address this gap, this study aimed to employ high-resolution proteomics to explore the proteomic landscapes of dysferlinopathy and analyze the correlation between muscle pathological changes and alterations in protein expression in muscle biopsies. We conducted a comprehensive approach to investigate the proteomic profile and disease-associated changes in the muscle tissue proteome from 15 patients with dysferlinopathy, exhibiting varying degrees of dystrophic pathology, alongside age-matched controls. Our methodology encompasses tandem mass tag (TMT)-labeled liquid chromatography-mass spectrometry (LC-MS/MS)-based proteomics, protein-protein interaction (PPI) network analysis, weighted gene co-expression network analysis, and differential expression analysis. Subsequently, we examined the correlation between the expression of key proteins and the clinical characteristics of the patients to identify pathogenic targets associated with DYSF mutations in dysferlinopathy. A total of 1600 differentially expressed proteins were identified, with 1321 showing high expression levels and 279 expressed at lower levels. Our investigation yields a molecular profile delineating the altered protein networks in dysferlinopathy-afflicted skeletal muscle, uncovering dysregulation across numerous cellular pathways and molecular processes, including mRNA metabolic processes, regulated exocytosis, immune response, muscle system processes, energy metabolic processes, and calcium transmembrane transport. Moreover, we observe significant associations between the protein expression of ANXA1, ANXA2, ANXA4, ANXA5, LMNA, PYGM, and the extent of histopathologic changes in muscle biopsies from patients with dysferlinopathy, validated through immunoblotting and immunofluorescence assays. Through the aggregation of expression data from dysferlinopathy-impacted muscles exhibiting a range of pathological alterations, we identified multiple key proteins associated with the dystrophic pathology of patients with dysferlinopathy. These findings provide novel insights into the pathogenesis of dysferlinopathy and propose promising targets for future therapeutic endeavors.