A novel nanobody broadly neutralizes SARS-CoV-2 via induction of spike trimer dimers conformation.

The ongoing mutations of the SARS-CoV-2 pose serious challenges to the efficacy of the available antiviral drugs, and new drugs with fantastic efficacy are always deserved investigation. Here, a nanobody called IBT-CoV144 is reported, which exhibits broad neutralizing activity against SARS-CoV-2 by inducing the conformation of spike trimer dimers. IBT-CoV144 was isolated from an immunized alpaca using the RBD of wild-type SARS-CoV-2, and it showed strong cross-reactive binding and neutralizing potency against diverse SARS-CoV-2 variants, including Omicron subvariants. Moreover, the prophylactically and therapeutically intranasal administration of IBT-CoV144 confers fantastic protective efficacy against the challenge of Omicron BA.1 variant in BALB/c mice model. The structure analysis of the complex between spike (S) protein, conducted using Cryo-EM, revealed a special conformation known as the trimer dimers. This conformation is formed by two trimers, with six RBDs in the "up" state and bound by six VHHs. IBT-CoV144 binds to the lateral region of the RBD on the S protein, facilitating the aggregation of S proteins. This aggregation results in steric hindrance, which disrupts the recognition of the virus by ACE2 on host cells. The discovery of IBT-CoV144 will provide valuable insights for the development of advanced therapeutics and the design of next-generation vaccines.

A novel three-microRNA signature for predicting survival in patients with nasopharyngeal carcinoma.

BACKGROUND/PURPOSE: Nasopharyngeal carcinoma (NPC) is a malignant neoplasm of the head and neck. This study aims to use integrated bioinformatics technologies to develop a predictive miRNA-signature correlated with the prognosis of NPC. MATERIALS AND METHODS: Initially, the differentially expressed miRNAs (DEMs) in NPC were identified, and then DEMs related to the prognosis of NPC were further screened. Subsequently, the relatively important DEMs identified by random forest algorithm were used to construct a predictive signature by multivariate COX regression analysis. Moreover, PCA, Kaplan-Meier analysis, time-dependent ROC analysis, and univariate and multivariate COX regression analysis were performed to evaluate the ability of the signature in risk identification and prognosis prediction in NPC. RESULTS: Hsa-miR-29c, hsa-miR-30e and hsa-miR-93 were selected from DEMs to construct a signature, and their abnormal expression was significantly associated with poor prognosis of NPC. The average AUC values of 1- to 5-year OS, DFS and DMFS predicted by the signature were all above 0.7, and showed better clinical independence than other indexes. In addition, 295 differentially expressed mRNAs could be used as potential target genes of the 3 DEMs. Among them, 56 differentially expressed mRNAs were related to PFS. GO and KEGG enrichment analysis indicated that the poor prognosis of NPC was related to the abnormality of chromosomes, cytokines, and chemokines. CONCLUSION: We constructed a three-miRNA signature with good independent performance in predicting the prognosis for NPC. This study may lay the foundation for exploring new therapeutic targets and improving survival outcomes in NPC patients.

Identifying Prognostic Significance of RCL1 and Four-Gene Signature as Novel Potential Biomarkers in HCC Patients.

BACKGROUND: Hepatocellular carcinoma (HCC) is a highly malignant disease, and it is characterized by rapid progression and low five-year survival rate. At present, there are no effective methods for monitoring the treatment and prognosis of HCC. METHODS: The transcriptome and gene expression profiles of HCC were obtained from the Cancer Genome Atlas (TCGA) program, International Cancer Genome Consortium (ICGC), and Gene Expression Omnibus (GEO) databases. The random forest method was applied to construct a four-gene prognostic model based on RNA terminal phosphate cyclase like 1 (RCL1) expression. The Kaplan-Meier method was performed to evaluate the prognostic value of RCL1, long noncoding RNAs (AC079061, AL354872, and LINC01093), and four-gene signature (SPP1, MYBL2, TRNP1, and FTCD). We examined the relationship between RCL1 expression and immune cells infiltration, tumor mutation burden (TMB), and microsatellite instability (MSI). RESULTS: The results of multiple databases indicated that the aberrant expression of RCL1 was associated with clinical outcome, immune cells infiltration, TMB, and MSI in HCC patients. Meanwhile, we found that long noncoding RNAs (AC079061, AL354872, and LINC01093) and RCL1 were significantly coexpressed in HCC patients. We also confirmed that the four-gene signature was an independent prognostic factor for HCC patients. Ferroptosis potential index, immune checkpoint molecules, and clinical feature were found to have obvious correlations with risk score. The area under the receiver operating characteristic curve values for the model were 0.7-0.8 in the training set and the validation set, suggesting high robustness of the four-gene signature. We then built a nomogram for facilitating the use in clinical practice. CONCLUSION: Our study demonstrated that RCL1 and a novel four-gene signature can be used as prognostic biomarkers for predicting clinical outcome in HCC patients; and this model may assist in individualized treatment monitoring of HCC patients in clinical practice.

Cell-bound IgE and plasma IgE as a combined clinical diagnostic indicator for allergic patients.

Allergic responses are mainly caused by IgE, which is often located on the cell surface. The current diagnostic method detects both allergen-specific IgE and total IgE levels, but a number of allergic patients have a normal serum IgE level, which is a poor clinical correlate for allergy. Here, we developed a simple method to detect the level of cell-bound IgE by dissociating it from blood cells with lactic acid. Dissociated cell-bound IgE and plasma IgE levels were detected using the same ELISA kit at the same time. We established two clinical cohorts: an allergic patient group and a healthy participant group. In general, cell-bound IgE correlated well with plasma IgE; however, some patients exhibited high cell-bound IgE levels but low plasma IgE levels. We recommended 350 ng/mL peripheral blood total IgE (cell-bound IgE + plasma IgE) as the cut-off value for allergy diagnosis. Using this indicator, 90.32% of our allergic patients were correctly diagnosed. The peripheral blood total IgE level is a promising clinical diagnostic indicator in allergic patients and will provide more guidance for allergy diagnosis and therapeutic evaluation.

A Comprehensive Prognostic and Immune Analysis of SLC41A3 in Pan-Cancer.

SLC41A3, as a member of the 41st family of solute carriers, participates in the transport of magnesium. The role of SLC41A3 in cancer prognosis and immune regulation has rarely been reported. This study was designed to analyze the expression status and prognostic significance of SLC41A3 in pan-cancers. The mRNA expression profiles of SLC41A3 were obtained from The Cancer Genome Atlas (TCGA), the Genotype-Tissue Expression (GTEx), the Broad Institute Cancer Cell Line Encyclopedia (CCLE), and the International Cancer Genome Consortium (ICGC). The Cox regression and Kaplan-Meier analyses were used to evaluate the prognostic value of SLC41A3 in pan-cancer. Furthermore, the correlation between SLC41A3 expression and immune cells infiltration, immune checkpoint, mismatch repair (MMR), DNA methyltransferase (DNMT), tumor mutation burden (TMB), and microsatellite instability (MSI) were calculated using data form TCGA database. The results showed that the expression of SLC41A3 was down-regulated in kidney renal clear cell carcinoma (KIRC), and was associated with poor overall survival and tumor-specific mortality. Whereas, the expression of SLC41A3 was up-regulated in liver hepatocellular carcinoma (LIHC), and the results of Cox regression analysis revealed that SLC41A3 was an independent factor for LIHC prognosis. Meanwhile, a nomogram including SLC41A3 and stage was built and exhibited good predictive power for the overall survival of LIHC patients. Additionally, correlation analysis suggested a significant correlation between SLC41A3 and TMB, MSI, MMR, DNMT, and immune cells infiltration in various cancers. The overall survival and disease-specific survival analysis revealed that the combined SLC41A3 expression and immune cell score, TMB, and MSI were significantly associated with clinical outcomes in ACC, LIHC, and UVM patients. Therefore, we proposed that SLC41A3 may serve as a potential prognostic biomarker for cancer.

Downregulation of Gabarapl1 significantly attenuates antibody binding to porcine aortic endothelial cells.

After hyperacute rejection in pig-to-primate xenotransplantation had been overcome by the introduction of α1,3-galactosyltransferase gene-knockout (GTKO) pigs, acute and chronic antibody-mediated rejection became one of the major barriers to long-term graft survival. This was associated with exposure of non-Gal antigens to the recipient's immune system and indicated that further genetic engineering of the pigs would be necessary. We here report that Gabarapl1, a regulator of tumorigenesis, plays a role in the regulation of immunogenicity of porcine aortic endothelial cells (PAECs). Knockdown of Gabarapl1 in PAECs results in a remarkable reduction in binding of serum antibody from PAEC-immunized monkeys, associated with decreased serum cytotoxicity of pig cells. Expression of swine leukocyte antigens (SLA) II DR was downregulated by Gabarapl1 knockdown. However, suppression of expression of SLA II is associated with less reduction of antibody binding than achieved by Gabarapl1 knockdown, suggesting that other Gabarapl1-regulated xenoantigens may be more important. These findings indicate a hitherto unknown relationship between Gabarapl1 and xenoimmunogenicity, suggesting a potential new strategy to reduce rejection initiated by the presence of non-Gal antigens.

Circulating pig-specific DNA as a novel biomarker for monitoring xenograft rejection.

Monitoring for immune rejection is crucial for long-term survival of pig xenografts. Circulating DNA is a promising non-invasive biomarker for either organ injury or response to therapy. In this study, circulating pig-specific DNA (cpsDNA) was monitored during xenograft rejection. Potential targets of cpsDNA were selected by in silico analysis, and species specificity of selected primers was confirmed by PCR. Subsequently, cpsDNA as a biomarker was evaluated using a complement-dependent cytotoxicity (CDC) assay in vitro. Then, early diagnosis and response to rapamycin were assessed by an in vivo imaging model of pig-to-mouse cell transplantation. Finally, cpsDNA was monitored in a pig-to-monkey artery patch transplantation model. The results showed that (a) a method of cpsDNA quantitation was established for application in mouse and nonhuman primate models; (b) cpsDNA reflected CDC in vitro; (c) cpsDNA in vivo mirrored xenograft rejection, and correlated with xenograft loss in pig-to-mouse cell transplantation; (d) cpsDNA was significantly reduced when rapamycin was administered; and (e) dynamic cpsDNA was detectable in pig-to-monkey artery patch transplantation. In conclusion, measurement of cpsDNA could prove to be a less invasive, but more specific and sensitive low-cost biomarker enabling monitoring of xenograft rejection and the response to immunosuppressive therapy.

Potential Antigens Involved in Delayed Xenograft Rejection in a Ggta1/Cmah Dko Pig-to-Monkey Model.

When hyperacute rejection is avoided by deletion of Gal expression in the pig, delayed xenograft rejection (DXR) becomes a major immunologic barrier to successful xenotransplantation. This study was to investigate the potential antigens involved in DXR. We isolated primary renal microvascular endothelial cells (RMEC) and aortic endothelial cells (AEC) from a GGTA1/CMAH double-knockout (DKO) pig (and a GGTA1-KO pig) and immunized cynomolgus monkeys with both of these cells. After sensitization, monkey serum antibody binding and cytotoxicity to RMEC was significantly higher than to AEC(p < 0.05), suggesting that RMEC are more immunogenic than AEC. Transcriptome sequencing of GGTA1/CMAH DKO pigs indicated that the expression of 1,500 genes was higher in RMEC than in AEC, while expression of 896 genes was lower. Next, we selected 101 candidate genes expressed only in pig RMEC, but not in pig AEC or in monkey or human RMEC. When these genes were knocked out individually in GGTA1/CMAH DKO RMEC, 32 genes were associated with reduced antibody binding, indicating that these genes might be primary immunologic targets involved in DXR. These genes may be important candidates for deletion in producing pigs against which there is a reduced primate immune response in pig kidney xenograft.

EphA6 promotes angiogenesis and prostate cancer metastasis and is associated with human prostate cancer progression.

Metastasis is the primary cause of prostate cancer (CaP)-related death. We investigate the molecular, pathologic and clinical outcome associations of EphA6 expression and CaP metastasis. The expression profiling of Eph receptors (Ephs) and their ephrin ligands was performed in parental and metastatic CaP cell lines. Among Ephs and ephrins, only EphA6 is consistently overexpressed in metastatic CaP cells. Metastatic potential of EphA6 is assessed by RNAi in a CaP spontaneous metastasis mouse model. EphA6 knock-down in human PC-3M cells causes decreased invasion in vitro and reduced lung and lymph node metastasis in vivo. In addition, knock-down of EphA6 decreases tube formation in vitro and angiogenesis in vivo. EphA6 mRNA expression is higher in 112 CaP tumor samples compared with benign tissues from 58 benign prostate hyperplasia patients. Positive correlation was identified between EphA6 expression and vascular invasion, neural invasion, PSA level, and TNM staging in CaP cases. Further, genome-wide gene expression analysis in EphA6 knock-down cells identified a panel of differentially regulated genes including PIK3IPA, AKT1, and EIF5A2, which could contribute to EphA6-regulated cancer progression. These findings identify EphA6 as a potentially novel metastasis gene which positively correlates with CaP progression. EphA6 may be a therapeutic target in metastatic CaP.

Gabarapl1 mediates androgen-regulated autophagy in prostate cancer.

Autophagy plays an important role in prostate cancer development. It promotes tumor cell survival and was found to be associated with androgen pathway. In the present study, we found that GABA(A) receptor-associated protein like 1 (Gabarapl1), a ubiquitin-like modifier, participates in the regulation of autophagy. Gabarapl1 is transcriptionally regulated by androgen receptor (AR) and has a repressive role in autophagy. Androgen deprivation downregulates Gabarapl1 in an AR dependent manner, resulting in the increase of autophagy flux. Elevated Gabarapl1 also represses the proliferation of prostate cancer cells. In summary, our study provides evidence to show that Gabarapl1 is a mediator involved in androgen-regulated autophagy process.

RUVBL2 is a novel repressor of ARF transcription.

ARF is the second most commonly inactivated tumor suppressor behind p53. It has been implicated in the control of cell proliferation, cell senescence, and tumor suppression. However, the detailed mechanism underlying the transcriptional control of ARF remains largely unknown. Here we report RUVBL2 as a novel transcriptional repressor of ARF. Ectopic expression of RUVBL2 decreases the levels of ARF, whereas knockdown of RUVBL2 results in a marked increase in ARF levels. In addition, RUVBL2 down-regulates the levels of p53 in an ARF-dependent manner. Mechanistically, RUVBL2 binds to the distal region of ARF promoter, thus leading to the repression of ARF transcription. These results suggest an important role of RUVBL2 in the regulation of ARF-p53 pathway.

Noxa/Mcl-1 balance regulates susceptibility of cells to camptothecin-induced apoptosis.

Although camptothecin (CPT) has been reported to induce apoptosis in various cancer cells, the molecular details of this regulation remain largely unknown. In this study, we demonstrate that BH3-only protein Noxa is upregulated during CPT-induced apoptosis, which is independent of p53. In addition, we show that phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is responsible for Noxa's induction. Luciferase assay and cAMP response element binding protein (CREB) knockdown experiments further demonstrate that CREB is involved in the transcriptional upregulation of Noxa. Moreover, blocking Noxa expression using specific small interfering ribonucleic acid (siRNA) significantly reduces the apoptosis in response to CPT, indicating that Noxa is an essential mediator for CPT-induced apoptosis. Interestingly, antiapoptotic Mcl-1 was also upregulated through PI3K/Akt signaling pathway upon CPT treatment. Using immunoprecipitation assay, Noxa was found to interact with Mcl-1 in the presence or absence of CPT. Knockdown of Mcl-1 expression by short hairpin ribonucleic acid (shRNA) was shown to potentiate CPT-induced apoptosis. Consistently, ectopic overexpression of Mcl-1 rescued cells from apoptosis induced by CPT. Cells coexpressing Noxa and Mcl-1 at different ratio correlates well with the extent of apoptosis, suggesting that the balance between Noxa and Mcl-1 may determine the susceptibility of HeLa cells to CPT-induced apoptosis.

Effects of cross correlation on the relaxation time of a bistable system driven by cross-correlated noise.

We study the effects of correlations between additive and multiplicative noise on relaxation time in a bistable system driven by cross-correlated noise. Using the projection-operator method, we derived an analytic expression for the relaxation time T(c) of the system, which is the function of additive (alpha) and multiplicative (D) noise intensities, correlation intensity lambda of noise, and correlation time tau of noise. After introducing a noise intensity ratio and a dimensionless parameter R=D/alpha, and then performing numerical computations, we find the following: (i) For the case of R<1, the relaxation time T(c) increases as R increases. (ii) For the cases of R>/=1, there is a one-peak structure on the T(c)-R plot and the effects of cross-correlated noise on the relaxation time are very notable. (iii) For the case of R<1, T(c) almost does not change with both lambda and tau, and for the cases of R>/=1, T(c) decreases as lambda increases, however T(c) increases as tau increases. lambda and tau play opposite roles in T(c), i.e., lambda enhances the fluctuation decay of dynamical variable and tau slows down the fluctuation decay of dynamical variable.