Prognostic Significance of Grade Discrepancy Between Primary Tumor and Venous Thrombus in Nonmetastatic Clear-cell Renal Cell Carcinoma: Analysis of the REMEMBER Registry and Implications for Adjuvant Therapy.

BACKGROUND: Further stratification of the risk of recurrence of clear-cell renal cell carcinoma (ccRCC) with venous tumor thrombus (VTT) will facilitate selection of candidates for adjuvant therapy. OBJECTIVE: To assess the impact of tumor grade discrepancy (GD) between the primary tumor (PT) and VTT in nonmetastatic ccRCC on disease-free survival (DFS), overall survival (OS), and cancer-specific survival (CSS). DESIGN, SETTING, AND PARTICIPANTS: This was a retrospective analysis of a multi-institutional nationwide data set for patients with pT3N0M0 ccRCC who underwent radical nephrectomy and thrombectomy. OUTCOMES MEASUREMENTS AND STATISTICAL ANALYSIS: Pathology slides were centrally reviewed. GD, a bidirectional variable (upgrading or downgrading), was numerically defined as the VTT grade minus the PT grade. Multivariable models were built to predict DFS, OS, and CSS. RESULTS AND LIMITATIONS: We analyzed data for 604 patients with median follow-up of 42 mo (excluding events). Tumor GD between VTT and PT was observed for 47% (285/604) of the patients and was an independent risk factor with incremental value in predicting the outcomes of interest (all p < 0.05). Incorporation of tumor GD significantly improved the performance of the ECOG-ACRIN 2805 (ASSURE) model. A GD-based model (PT grade, GD, pT stage, PT sarcomatoid features, fat invasion, and VTT consistency) had a c index of 0.72 for DFS. The hazard ratios were 8.0 for GD = +2 (p < 0.001), 1.9 for GD = +1 (p < 0.001), 0.57 for GD = -1 (p = 0.001), and 0.22 for GD = -2 (p = 0.003) versus GD = 0 as the reference. According to model-converted risk scores, DFS, OS, and CSS significantly differed between subgroups with low, intermediate, and high risk (all p < 0.001). CONCLUSIONS: Routine reporting of VTT upgrading or downgrading in relation to the PT and use of our GD-based nomograms can facilitate more informed treatment decisions by tailoring strategies to an individual patient's risk of progression. PATIENT SUMMARY: We developed a tool to improve patient counseling and guide decision-making on other therapies in addition to surgery for patients with the clear-cell type of kidney cancer and tumor invasion of a vein.

Development and validation of a prognostic model incorporating tumor thrombus grading for nonmetastatic clear cell renal cell carcinoma with tumor thrombus: A multicohort study.

There is significant variability with respect to the prognosis of nonmetastatic clear cell renal cell carcinoma (ccRCC) patients with venous tumor thrombus (VTT). By applying multiregion whole-exome sequencing on normal-tumor-thrombus-metastasis quadruples from 33 ccRCC patients, we showed that metastases were mainly seeded by VTT (81.8%) rather than primary tumors (PTs). A total of 706 nonmetastatic ccRCC patients with VTT from three independent cohorts were included in this study. C-index analysis revealed that pathological grading of VTT outperformed other indicators in risk assessment (OS: 0.663 versus 0.501-0.610, 0.667 versus 0.544-0.651, and 0.719 versus 0.511-0.700 for Training, China-Validation, and Poland-Validation cohorts, respectively). We constructed a risk predicting model, TT-GPS score, based on four independent variables: VTT height, VTT grading, perinephric fat invasion, and sarcomatoid differentiation in PT. The TT-GPS score displayed better discriminatory ability (OS, c-index: 0.706-0.840, AUC: 0.788-0.874; DFS, c-index: 0.691-0.717, AUC: 0.771-0.789) than previously reported models in risk assessment. In conclusion, we identified for the first-time pathological grading of VTT as an unheeded prognostic factor. By incorporating VTT grading, the TT-GPS score is a promising prognostic tool in predicting the survival of nonmetastatic ccRCC patients with VTT.

Long non-coding RNA ENST00000503625 is a potential prognostic biomarker and metastasis suppressor gene in prostate cancer.

BACKGROUND: Dysregulation of Long Non-coding RNAs (lncRNAs) emerges to be a hallmark of cancers. Metastatic prostate cancer and localized disease that recurs after treatment are clinical challenges, it remains unclear how lncRNA plays a role in those processes. METHODS: From previous RNA-Seq data on 65 prostate cancer and adjacent normal tissues. We identified a novel lncRNA ENST00000503625 down-regulated in prostate cancer and correlated with tumor progression characteristics. Public datasets were examined for associations between ENST00000503625 expression and clinical parameters and prognoses. Subsequently, we constructed and externally validated a nomogram for predicting biochemical recurrence (BCR). Finally, in vitro experiments were carried out to determine how ENST00000503625 functions biologically in prostate cancer. RESULTS: Low ENST00000503625 in tumor was associated with poor clinical features and prognoses. TCGA pan-cancer analysis found that ENST00000503625 was deregulated in a variety of tumors and correlated with overall survival, disease-specific survival, and progression-free survival. The nomogram for predicting BCR was constructed using TCGA data, which exhibited excellent accuracy in external validation with Chinese Prostate Cancer Genome and Epigenome Atlas data. Gene Ontology and KEGG pathway analysis found that genes related to ENST00000503625 were enriched in multiple tumor progression related pathways. When ENST00000503625 was knocked down in vitro, the epithelial-mesenchymal transition was induced, by which cancer cells migrated and invaded more readily. CONCLUSION: Our data suggested that ENST00000503625 may serve as a potential prognostic marker or a therapeutic target for prostate cancer metastases.

Catheter-based adrenal ablation: an alternative therapy for patients with aldosterone-producing adenoma.

Unilateral adrenalectomy is the standard treatment for patients with aldosterone-producing adenoma (APA), but it lacks an option for patients with APA who refuse or are not suitable for surgery. In this study, we studied whether catheter-based adrenal ablation for APA is comparable to adrenalectomy. A total of 2185 hypertensive patients were screened, and 112 patients with APA were recruited and counselled on the treatment options. Fifty-two patients opted for catheter-based adrenal ablation, and 60 opted for adrenalectomy. Clinical and biochemical outcomes were assessed at 6 months after treatment. Factors associated with hypertension remission and the advantages and limitations of this approach were evaluated. According to the primary aldosteronism surgical outcome (PASO) criteria, complete and partial clinical success was achieved in 21 (40.4%) and 23 (44.2%) patients in the ablation group vs. 33 (55.0%) and 23 (38.3%) patients in the adrenalectomy group, respectively. Complete and partial biochemical success was achieved in 30 (57.7%) and 17 (32.7%) patients in the ablation group vs. 51 (85.0%) and 5 (8.3%) patients in the adrenalectomy group, respectively. The complete clinical success rate was not (P > 0.05), but the complete biochemical success rate was significantly different between the two groups (P < 0.01). Factors associated with adrenal ablation-mediated hypertension remission were hypertension duration and serum potassium level at baseline. Compared with surgery, adrenal ablation requires a shorter operating time and time to resume physical activity. Catheter-based adrenal ablation may be an alternative and feasible option for APA patients unwilling to receive surgical treatment.

Exploration of the driving factors and distribution of fecal coliform in rivers under a traditional agro-pastoral economy in Kyrgyzstan, Central Asia.

Fecal coliform (FC) in river water is one of the threats to human health. To explore the pollution status of FC in rivers of Kyrgyzstan, a mountainous country with traditional agro-pastoral economy, 184 water samples from the rivers of Kyrgyzstan in low and high river flow period were analyzed. Spatial autocorrelation and classical statistical methods were used to analyze the spatiotemporal distribution and driving factors of FC. The results showed that the surface water quality of Kyrgyz rivers was good, and the concentration range of FC was 0-23 MPN/100 mL. Temporally, the maximum FC concentration was 4 MPN/100 mL in low river flow period, while in the period of high river flow, the highest value reached to 23 MPN/100 mL. Spatially, the concentration of FC in high altitude areas was low, while that in the lowland areas was relatively high, which indicated that animal husbandry in high altitude areas contributed little to FC in rivers, and urban domestic sewage and agricultural activities in lowlands were the main pollution sources of FC in rivers. There was no correlation between FC and hardness, electrical conductivity (EC), pH and total organic carbon (TOC) in river water of Kyrgyzstan, and the distribution of FC in high river flow period was mainly driven by population and human modification of terrestrial systems. The results can provide a basis for the prevention and control of surface water FC pollution and related diseases in Kyrgyzstan.

Genome-wide CRISPR-Cas9 screen identified KLF11 as a druggable suppressor for sarcoma cancer stem cells.

Cancer stem cells (CSCs) are involved in tumorigenesis, recurrence, and therapy resistance. To identify critical regulators of sarcoma CSCs, we performed a reporter-based genome-wide CRISPR-Cas9 screen and uncovered Kruppel-like factor 11 (KLF11) as top candidate. In vitro and in vivo functional annotation defined a negative role of KLF11 in CSCs. Mechanistically, KLF11 and YAP/TEAD bound to adjacent DNA sites along with direct interaction. KLF11 recruited SIN3A/HDAC to suppress the transcriptional output of YAP/TEAD, which, in turn, promoted KLF11 transcription, forming a negative feedback loop. However, in CSCs, this negative feedback was lost because of epigenetic silence of KLF11, causing sustained YAP activation. Low KLF11 was associated with poor prognosis and chemotherapy response in patients with sarcoma. Pharmacological activation of KLF11 by thiazolidinedione effectively restored chemotherapy response. Collectively, our study identifies KLF11 as a negative regulator in sarcoma CSCs and potential therapeutic target.

Hydrochemical composition and potentially toxic elements in the Kyrgyzstan portion of the transboundary Chu-Talas river basin, Central Asia.

Water chemistry and the assessment of health risks of potentially toxic elements have important research significance for water resource utilization and human health. However, not enough attention has been paid to the study of surface water environments in many parts of Central Asia. Sixty water samples were collected from the transboundary river basin of Chu-Talas during periods of high and low river flow, and the hydrochemical composition, including major ions and potentially toxic elements (Zn, Pb, Cu, Cr, and As), was used to determine the status of irrigation suitability and risks to human health. The results suggest that major ions in river water throughout the entire basin are mainly affected by water-rock interactions, resulting in the dissolution and weathering of carbonate and silicate rocks. The concentrations of major ions change to some extent with different hydrological periods; however, the hydrochemical type of calcium carbonate remains unchanged. Based on the water-quality assessment, river water in the basin is classified as excellent/good for irrigation. The relationship between potentially toxic elements (Zn, Pb, Cu, Cr, and As) and major ions is basically the same between periods of high and low river flow. There are significant differences between the sources of potentially toxic elements (Zn, Pb, Cu, and As) and major ions; however, Cr may share the same rock source as major ions. The risk assessment revealed low non-carcinogenic and carcinogenic risks for human health; however, the maximum carcinogenic risk for As exceeded the allowable value, which requires further consideration. These results provide a scientific basis for the management of agricultural irrigation uses and also infill existing gaps regarding the hydrochemical composition in the Chu-Talas river basin, Central Asia.

Novel Long Non-coding RNA lncAMPC Promotes Metastasis and Immunosuppression in Prostate Cancer by Stimulating LIF/LIFR Expression.

Long non-coding RNAs (lncRNAs) participate in the development and progression of prostate cancer (PCa). We aimd to identify a novel lncRNA, named lncRNA activated in metastatic PCa (lncAMPC), and investigate its mechanisms and clinical significance in PCa. First, the biological capacity of lncAMPC in PCa was demonstrated both in vitro and in vivo. The lncAMPC was overexpressed in tumor tissue and urine of metastatic PCa patients and promoted PCa tumorigenesis and metastasis. Then, a mechanism study was conducted to determine how the lncAMPC-activated pathway contributed to PCa metastasis and immunosuppression. In the cytoplasm, lncAMPC upregulated LIF expression by sponging miR-637 and inhibiting its activity. In the nucleus, lncAMPC enhanced LIFR transcription by decoying histone H1.2 away from the upstream sequence of the LIFR gene. The lncAMPC-activated LIF/LIFR expressions stimulated the Jak1-STAT3 pathway to simultaneously maintain programmed death-ligand 1 (PD-L1) protein stability and promote metastasis-associated gene expression. Finally, the prognostic value of the expression of lncAMPC and its downstream genes in PCa patients was evaluated. High LIF/LIFR levels indicated shorter biochemical recurrence-free survival among patients who underwent radical prostatectomy. Therefore, the lncAMPC/LIF/LIFR axis plays a critical role in PCa metastasis and immunosuppression and may serve as a prognostic biomarker and potential therapeutic target.

Heavy Metals and Related Human Health Risk Assessment for River Waters in the Issyk-Kul Basin, Kyrgyzstan, Central Asia.

The water resources of Central Asia play an important role in maintaining the fragile balance of ecosystems and the sustainable development of human society. However, the lack of research on the heavy metals in river waters has a far-reaching influence on public health and the sustainable development in Central Asia. In order to reveal the possible sources of the heavy metals and to assess the associated human health risks, thirty-eight water samples were collected from the rivers of the Issyk-Kul Basin during the period with low river flow (May) and the period with high river flow (July and August), and the hydrochemical compositions and major ions of heavy metals were analyzed. No changes in hydrochemical facies were observed between the two periods and the river water type was calcium bicarbonate. Carbonate dissolution and silicate weathering controlled the variation of cations and anions in river waters from the Issyk-Kul Basin. There were some differences in the sources of heavy metals in water bodies between the two periods. During the period with low river flow, heavy metals (Cr) were closely clustered with major ions, indicating that they were mainly affected by water-rock interactions. During the period with high river flow, all heavy metals studied in this paper had different sources of major ions, and the heavy metals maybe influenced by human activities. From the human health risk assessment, the hazard quotients for all samples were less than 1, reflecting that there was no noncarcinogenic risk in the river waters of the Issyk-Kul Basin during the two sampling periods. However, the water samples with carcinogenic risk of arsenic exceeding the threshold (10-4) accounted for 21.1% of the total, indicating that there were some certain carcinogenic hazards for human health via water drinking with direct oral ingestion. The results are of certain significance for the utilization and protection of water resources in the basin as well as the protection of public health.

The previously uncharacterized lncRNA APP promotes prostate cancer progression by acting as a competing endogenous RNA.

Long noncoding RNAs (lncRNAs) promote cell proliferation, migration, invasion and castration resistance in prostate cancer (PCa). Understanding the inherited molecular mechanisms by which lncRNAs contribute to the progression of PCa to a lethal disease could have an important impact on cancer detection, diagnosis and prognosis. In our study, PCa-associated lncRNA transcripts from RNA-seq data were identified and screened via bioinformatics analysis, NCBI annotations and literature review. We identified a novel lncRNA, lncAPP (lncRNA activated in PCa progression), which activates in PCa progression and is expressed in primary tumor tissues and urine samples of patients with localized or advanced PCa. Urinary-based lncAPP is a promising biomarker for predicting PCa progression. In vitro and in vivo studies demonstrated that lncAPP enhanced cell proliferation and promoted migration and invasion. The underlying mechanism of lncRNA was investigated by RNA immunoprecipitation, dual-luciferase reporter system assay, etc. Upregulation of lncAPP promoted cell migration and invasion via competitively binding miR218 to facilitate ZEB2/CDH2 expression. In addition, in vivo subcutaneous tumor xenograft models and tail intravenously injection metastatic models were constructed to evaluate lncRNA function. Targeting lncAPP/miR218 axis in cell lines and tumor xenografts restrained tumor progression properties both in vitro and in vivo. These results establish that lncAPP/miR218 axis plays a critical role in PCa progression, and they also suggest new strategies to prevent tumor progression for therapeutic purposes.

Intranasal immunization with recombinant Vaccinia virus Tiantan harboring Zaire Ebola virus gp elicited systemic and mucosal neutralizing antibody in mice.

Accumulating literature revealed that human mucosa was likely one of the important routes for EBOV attachment and further infection. Therefore inducing effective mucosal immune responses play key role in preventing the virus infection. Vaccinia virus Tiantan strain (VV) was a remarkably attenuated poxvirus, which has been broadly exploited as a multifunctional vector during the development of genetically recombinant vaccine and cancer therapeutic agent. In this study, we generated a recombinant VV harboring EBOV gp (VV-Egp) that was used to immunize mice, followed by assessing immune responses, particularly the mucosal immune responses to EBOV GP. A stable and further attenuated VV-Egp, in which the VV ha gene was replaced with the EBOV gp, was generated. In BALB/c mouse model, intranasal immunization with VV-Egp elicited robust humoral and cellular immune responses, including high level of neutralizing serum IgG and IgA against EBOV, and a large amount of GP-specific IFN-γ secreting lymphocytes. More importantly, EBOV GP-specific neutralizing secreted IgA (sIgA) in nasal wash and both sIgA and IgG in vaginal wash were induced. In summary, immunization with a safe and stable recombinant VV carrying a single EBOV gp conferred robust systemic immune response and mucosal neutralizing antibodies, indicating that the recombinant virus could be utilized as a viral vector for plug-and-play universal platform in mucosal vaccine development.

BnaMPK3 Is a Key Regulator of Defense Responses to the Devastating Plant Pathogen Sclerotinia sclerotiorum in Oilseed Rape.

The disease caused by Sclerotinia sclerotiorum has traditionally been difficult to control, resulting in tremendous economic losses in oilseed rape (Brassica napus). Identification of important genes in the defense responses is critical for molecular breeding, an important strategy for controlling the disease. Here, we report that a B. napus mitogen-activated protein kinase gene, BnaMPK3, plays an important role in the defense against S. sclerotiorum in oilseed rape. BnaMPK3 is highly expressed in the stems, flowers and leaves, and its product is localized in the nucleus. Furthermore, BnaMPK3 is highly responsive to infection by S. sclerotiorum and treatment with jasmonic acid (JA) or the biosynthesis precursor of ethylene (ET), but not to treatment with salicylic acid (SA) or abscisic acid. Moreover, overexpression (OE) of BnaMPK3 in B. napus and Nicotiana benthamiana results in significantly enhanced resistance to S. sclerotiorum, whereas resistance is diminished in RNAi transgenic plants. After S. sclerotiorum infection, defense responses associated with ET, JA, and SA signaling are intensified in the BnaMPK3-OE plants but weakened in the BnaMPK3-RNAi plants when compared to those in the wild type plants; by contrast the level of both H2O2 accumulation and cell death exhibits a reverse pattern. The candidate gene association analyses show that the BnaMPK3-encoding BnaA06g18440D locus is a cause of variation in the resistance to S. sclerotiorum in natural B. napus population. These results suggest that BnaMPK3 is a key regulator of multiple defense responses to S. sclerotiorum, which may guide the resistance improvement of oilseed rape and related economic crops.

Dual monoclonal antibody-based sandwich ELISA for detection of in vitro packaged Ebola virus.

BACKGROUND: Rapid transmission and high mortality of Ebola virus disease (EVD) highlight a urgent need of large scale, convenient and effective measure for Ebola virus screening. Application of monoclonal antibodies (mAbs) are crucial for establishment of an enzyme-linked immunosorbent assay (ELISA) with high sensitivity and specificity. METHODS: The traditional cell fusion technique was used to generate a panel of hybridomas. Two mAbs were characterized by SDS-PAGE, Western blot, Indirect immunofluorescence assay (IFA). A sandwich ELISA was established using the two mAbs. The detection capability of the ELISA was evaluated. RESULTS: In the current study, we produced two murine-derived mAbs (designated as 6E3 and 3F21) towards Zaire Ebola virus glycoprotein (GP), the major viral transmembrane spike protein associated with viral attachment. It was shown that 6E3 and 3F21 recognized GP1 and GP2 subunits of the GP respectively. Furthermore, 6E3 and 3F21 bound to corresponding epitopes on GP without reciprocal topographical interpretation. Subsequently, a sandwich ELISA based on the two mAbs were established and evaluated. The detection limit was 3.6 ng/ml, with a linear range of 3.6-100 ng/ml. More importantly, Ebola virus like particles (eVLPs) were able to be detected by this established virus detection measure. CONCLUSIONS: We produced and characterized two murine-derived mAbs (designated as 6E3 and 3F21) towards Zaire Ebola virus glycoprotein (GP), and established a sandwich ELISA based on the mAbs. It was suggested that the sandwich ELISA provided an alternative method for specific and sensitive detection of Ebola virus in the field setting.

Evidence for Multiple Applications of Monoclonal Antibody 5G10.

We had earlier obtained a murine monoclonal antibody (mAb), termed 5G10, that bound to Salmonella flagellin (SF) and subsequently impaired the latter property of Toll-like receptor 5 (TLR5) signaling activation. Besides interrupting SF-mediated TLR5 activation, mAb 5G10 probably had other potential applications. In this study, we explored multiple functions of 5G10. A short peptide QRVRELAV (designated T5) derived from SF in either terminal of proteins was specifically recognized by 5G10. T5 tag expressed in eukaryotic cell was also detected by 5G10 when analyzed by Western blot, immunofluorescence assay (IFA), and fluorescent-activated cell sorting (FACS). The result of the co-immunoprecipitation assay showed that 5G10 as a bait antibody dragged out the complex of enterovirus 71 (EV71) 2A and mitochondrial antiviral signaling (MAVS) protein. More importantly, 5G10 helped to purify fusion proteins T5-tagged (EV71) 2A and T5-Japanese encephalitis virus NS5 methyltransferase (MTase). Thus, it has been suggested that mAb 5G10 could be useful in several biological applications, including protein identification, location, and affinity purification.

Prognostic Value of a Long Non-coding RNA Signature in Localized Clear Cell Renal Cell Carcinoma.

BACKGROUND: Long non-coding RNAs (lncRNAs) can be used as prognostic biomarkers in many types of cancer. OBJECTIVE: We sought to establish an lncRNA signature to improve postoperative risk stratification for patients with localized clear cell renal cell carcinoma (ccRCC). DESIGN, SETTING, AND PARTICIPANTS: Based on the RNA-seq data of 444 stage I-III ccRCC tumours from The Cancer Genome Atlas project, we built a four-lncRNA-based classifier using the least absolute shrinkage and selection operation (LASSO) Cox regression model in 222 randomly selected samples (training set) and validated the classifier in the remaining 222 samples (internal validation set). We confirmed this classifier in an external validation set of 88 patients with stage I-III ccRCC from a Japan cohort and using quantitative reverse transcription polymerase chain reaction (RT-PCR) in another three independent sets that included 1869 patients from China with stage I-III ccRCC. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Univariable and multivariable Cox regression, Harrell's concordance index (c-index), and time-dependent receiver operating characteristic curves were used to evaluate the association of the classifier with overall survival, disease-specific survival, and disease-free survival. RESULTS AND LIMITATIONS: Using the LASSO Cox regression model, we built a classifier named RCClnc4 based on four lncRNAs: ENSG00000255774, ENSG00000248323, ENSG00000260911, and ENSG00000231666. In the RNA-seq and RT-PCR data sets, the RCClnc4 signature significantly stratified patients into high-risk versus low-risk groups in terms of clinical outcome across and within subpopulations and remained as an independent prognostic factor in multivariate analyses (hazard ratio range, 1.34 [95% confidence interval {CI}: 1.03-1.75; p=0.028] to 1.89 [95% CI, 1.55-2.31; p<0.001]) after adjusting for clinical and pathologic factors. The RCClnc4 signature achieved a higher accuracy (mean c-index, 0.72) than clinical staging systems such as TNM (mean c-index, 0.62) and the stage, size, grade, and necrosis (SSIGN) score (mean c-index, 0.64), currently reported prognostic signatures and biomarkers for the estimation of survival. When integrated with clinical characteristics, the composite clinical and lncRNA signature showed improved prognostic accuracy in all data sets (TNM + RCClnc4 mean c-index, 0.75; SSIGN + RCClnc4 score mean c-index, 0.75). The RCClnc4 classifier was able to identify a clinically significant number of both high-risk stage I and low-risk stage II-III patients. CONCLUSIONS: The RCClnc4 classifier is a promising and potential prognostic tool in predicting the survival of patients with stage I-III ccRCC. Combining the lncRNA classifier with clinical and pathological parameters allows for accurate risk assessment in guiding clinical management. PATIENT SUMMARY: The RCClnc4 classifier could facilitate patient management and treatment decisions.

Improved immune response against HIV-1 Env antigen by enhancing EEV production via a K151E mutation in the A34R gene of replication-competent vaccinia virus Tiantan.

The development of an effective HIV-1 vaccine is still a global priority. In recent years, vaccinia virus (VV) has been widely used as an HIV-1 vaccine vector, but its immune efficacy against HIV-1 antigens needs to be optimized. The extracellular enveloped virus (EEV) of VV is capable of faster entry, earlier release, and long-range dissemination. We hypothesized that an improvement in EEV formation by the manipulation of VV genes involved in the EEV release would consequently cause an improved expression of the VV carrying HIV-1 Env antigen and a subsequent enhanced immune response. To this end, an A34R K151E mutant (rVTT-A34Rmut) from VV Tiantan strain (VTT) with robustly increased EEV release was selected to serve as an optimized vaccine vector. The results were consistent with our hypothesis: the A34R mutant-based HIV-1 vaccine candidate rVTT-A34Rmut-Env produced more HIV-1 Env antigen in vitro and in vivo, and thus led to an improved HIV-1 Env-specific T cell immune response, binding antibody, and even the neutralizing antibody response in mice without increased virulence. Meanwhile, the application of the A34R mutation on another VV-based HIV-1 vaccine candidate, VTKgpe, also exhibited a similar immune enhancement effect with no enhanced virulence. The results in this study suggested that rVTT-A34Rmut is a potentially improved vaccine vector candidate for human application. In addition, the improvement of the EEV formation via the A34R gene mutation may also be potent in other poxvirus vector-based vaccines against HIV-1 or other pathogens and even cancer in the future.

A novel germline ARMC5 mutation in a patient with bilateral macronodular adrenal hyperplasia: a case report.

BACKGROUND: Bilateral macronodular adrenal hyperplasia (BMAH) is a rare cause of Cushing's syndrome (CS). BMAH is predominantly believed to be caused by two mutations, a germline and somatic one, respectively, as described in the two-hit hypothesis. In many familial cases of BMAH, mutations in armadillo repeat containing 5 (ARMC5), a putative tumor suppressor gene, are thought to induce the disorder. The objective of this study was to report a case in which the patient presented with BMAH induced by a novel heterozygous germline ARMC5 mutation (c. 517C > T, p. Arg173*) alone rather than a two-hit mutation. CASE PRESENTATION: A 51-year-old woman was identified with masses in the bilateral adrenals. Serum cortisol levels were increased significantly both in the morning (08:00 AM) and late at night (24:00 AM), while plasma adrenocorticotropic hormone was normal. The patient underwent a left adrenalectomy and histopathology substantiated the BMAH diagnosis. WES of the germline DNA discovered a novel heterozygous germline ARMC5 mutation (c. 517C > T, p. Arg173*) and in silico analysis predicted that the mutation significantly impaired protein function, resulting in inactivated ARMC5. Subsequently, WES of the tumor specimen identified 79 somatic single nucleotide polymorphisms (SNPs)/insertion-deletion (indel) mutations, including 32 missense/nonsense/splice/stop-loss mutations. None of these mutations were CS-related. CONCLUSIONS: A novel germline ARMC5 mutation (c. 517C > T, p. Arg173*) was identified that induced BMAH alone without a second mutation. ARMC5 sequencing may improve the identification of clinical forms of BMAH and allow earlier diagnosis of this disease.

HIF2A germline-mutation-induced polycythemia in a patient with VHL-associated renal-cell carcinoma.

In this study, we report here a rare case of polycythemia and cRCC in the same patient, which may be helpful in understanding clinical features and molecular mechanisms underlying VHL-mutation-associated cRCC and polycythemia induced by germline mutation of HIF2A. Firstly, we identified a rare but well studied germline mutation resulting in polycythemia in HIF2A (c.1609G>A, p.Gly537Arg) in the blood of the patient and his daughter. Meanwhile, we identified an inactivating VHL mutation (c.391A>T, p.N131Y), as well as TP53 mutation(c.977A>T, p.E326V) and mTOR mutation(c.7498A>T, p.I2500F) in renal cancer tissue. Moreover, protein levels of VHL, HIF1A, HIF2A, EPO, and VEGF estimated by immunohistochemical staining substantiated hyperactivation of the oxygen-sensing pathway. In addition, we identified 158 somatic SNP/indel mutations, including 90 missense/nonsense/splice/stop-loss mutations by whole-exome sequencing (WES) of the tumor specimen and matched normal DNA.

Metformin reverses prostate cancer resistance to enzalutamide by targeting TGF-ß1/STAT3 axis-regulated EMT.

Although the newly developed second-generation anti-androgen drug enzalutamide can repress prostate cancer progression significantly, it only extends the survival of prostate cancer patients by 4-6 months mainly due to the occurrence of enzalutamide resistance. Most of the previous studies on AR antagonist resistance have been focused on AR signaling. Therefore, the non-AR pathways on enzalutamide resistance remain largely unknown. By using C4-2, CWR22Rv1 and LNCaP cell lines, as well as mice bearing CWR22Rv1 xenografts treated with either enzalutamide or metformin alone or in combination, we demonstrated that metformin is capable of reversing enzalutamide resistance and restores sensitivity of CWR22Rv1 xenografts to enzalutamide. We showed that metformin alleviated resistance to enzalutamide by inhibiting EMT. Furthermore, based on the effect of metformin on the activation of STAT3 and expression of TGF-ß1, we propose that metformin exerts its effects by targeting the TGF-ß1/STAT3 axis. These findings suggest that combination of metformin with enzalutamide could be a more efficacious therapeutic strategy for the treatment of castration-resistant prostate cancer.