FlgI Is a Sec-Dependent Effector of Candidatus Liberibacter asiaticus That Can Be Blocked by Small Molecules Identified Using a Yeast Screen.

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide. The phloem-restricted bacterium Candidatus Liberibacter asiaticus (CLas) is considered to be the main pathogen responsible for HLB. There is currently no effective practical strategy for the control of HLB. Our understanding of how pathogens cause HLB is limited because CLas has not been artificially cultured. In this study, 15 potential virulence factors were predicted from the proteome of CLas through DeepVF and PHI-base searches. One among them, FlgI, was found to inhibit yeast growth when expressed in Saccharomyces cerevisiae. The expression of the signal peptide of FlgI fused with PhoA in Escherichia coli resulted in the discovery that FlgI was a novel Sec-dependent secretory protein. We further found that the carboxyl-terminal HA-tagged FlgI was secreted via outer membrane vesicles in Sinorhizobium meliloti. Fluoresence localization of transient expression FlgI-GFP in Nicotiana benthamiana revealed that FlgI is mainly localized in the cytoplasm, cell periphery, and nuclear periphery of tobacco cells. In addition, our experimental results suggest that FlgI has a strong ability to induce callose deposition and cell necrosis in N. benthamiana. Finally, by screening a large library of compounds in a high-throughput format, we found that cyclosporin A restored the growth of FlgI-expressing yeast. These results confirm that FlgI is a novel Sec-dependent effector, enriching our understanding of CLas pathogenicity and helping to develop new and more effective strategies to manage HLB.

Denitrification and the controlling factors in Yunnan Plateau Lakes (China): Exploring the role of enhanced internal nitrogen cycling by algal blooms.

Denitrification plays an important role in nitrogen (N) removal in freshwater ecosystems. This internal process regulates the fluctuations of N concentration, especially for lakes with high nutrients concentrations and long residence time. Lakes in Yunnan plateau (southwestern China) provide typical cases, while studies in this region have been rare. Therefore, we studied denitrification of two lakes (Lake Dianchi in hypereutrophic state and Lake Erhai in mesotrophic) in this region. We used acetylene inhibition technique to quantify potential denitrification rate (PDR) of these lakes in April and August, 2015 and 2016. PDR of the sediments ranged 0-1.21 µmol/(N·m2·hr), and that of overlying water ranged 0-0.24 µmol/(N·L·hr). Then, we used Least Angle Regression to determine the controlling factors for denitrification. Nutrients controlled PDR from two aspects: providing essential nitrogen sources; and affecting the richness and metabolism of denitrifying bacteria. In April, both aspects limited PDR; while only nitrogen sources limited PDR in August, due to depleted nitrate and enhanced denitrifying bacteria activity. Ammonia was most significant to denitrification, indicating that nitrate from nitrification transported to the bottom of well-mixed lake provide major N source by denitrification. The high PDR and low nitrate concentrate in August were evidence of an enhanced internal N cycling by algal blooms.

Internal cycling, not external loading, decides the nutrient limitation in eutrophic lake: A dynamic model with temporal Bayesian hierarchical inference.

Lake eutrophication is associated with excessive anthropogenic nutrients (mainly nitrogen (N) and phosphorus (P)) and unobserved internal nutrient cycling. Despite the advances in understanding the role of external loadings, the contribution of internal nutrient cycling is still an open question. A dynamic mass-balance model was developed to simulate and measure the contributions of internal cycling and external loading. It was based on the temporal Bayesian Hierarchical Framework (BHM), where we explored the seasonal patterns in the dynamics of nutrient cycling processes and the limitation of N and P on phytoplankton growth in hyper-eutrophic Lake Dianchi, China. The dynamic patterns of the five state variables (Chla, TP, ammonia, nitrate and organic N) were simulated based on the model. Five parameters (algae growth rate, sediment exchange rate of N and P, nitrification rate and denitrification rate) were estimated based on BHM. The model provided a good fit to observations. Our model results highlighted the role of internal cycling of N and P in Lake Dianchi. The internal cycling processes contributed more than external loading to the N and P changes in the water column. Further insights into the nutrient limitation analysis indicated that the sediment exchange of P determined the P limitation. Allowing for the contribution of denitrification to N removal, N was the more limiting nutrient in most of the time, however, P was the more important nutrient for eutrophication management. For Lake Dianchi, it would not be possible to recover solely by reducing the external watershed nutrient load; the mechanisms of internal cycling should also be considered as an approach to inhibit the release of sediments and to enhance denitrification.

Plasma proteasomal chymotrypsin-like activity correlates with prostate cancer progression.

Previously, we demonstrated that inhibition of proteasomal chymotrypsin-like (CT-like) activity in human prostate cancer (PCa) PC-3 cultures and PC-3 xenografts results in accumulation of ubiquitinated proteins, followed by induction of cell death. Studies have shown that plasma CT-like proteasomal activity may be a powerful biomarker for risk stratification in hematologic malignancies. We hypothesized that circulating proteasomes could also be used to stratify risk for patients with PCa. A total of 109 patients with suspected PCa underwent prostatic biopsies were enrolled. Subjects were divided into non-cancer, low-risk PCa, and high-risk PCa groups. Three different proteasomal activity markers (CT-like, caspase-like, and trypsin-like) were measured and compared among the three groups. The proteasomal target proteins, Ub-prs, Hsp70, Bax, and P27 in plasma and prostate tissues were also evaluated. Multivariate analysis was used to assess whether CT-like activity was a predictor of PCa progression. Only proteasomal CT-like activity in the high-risk group was statistically higher than in the non-cancer group (P < 0.05). The expression of Ub-prs, Hsp70, Bax, and P27 protein was decreased in both plasma and PCa tissue of high-risk patients. CT-like activity was found to be an independent predictor of high-risk PCa. Subjects with CT-like activity ≥55 had a 2.15-fold higher risk of having high-risk PCa as compared to those with a CT-like activity of <55 (P = 0.021). We found CT-like activity to be an independent predictor of high-risk PCa, and as such, it may be a good candidate as a biomarker for high-risk PCa detection and stratification.

PSA density improves the rate of prostate cancer detection in Chinese men with a PSA between 2.5-10.0 ng ml (-1) and 10.1-20.0 ng ml (-1) : a multicenter study.

Chinese men should have a higher prostate-specific antigen (PSA) "gray zone" than the traditional value of 2.5-10.0 ng ml-1 since the incidence of prostate cancer (PCa) in Chinese men is relative low. We hypothesized that PSA density (PSAD) could improve the rate of PCa detection in Chinese men with a PSA higher than the traditional PSA "gray zone." A total of 461 men with a PSA between 2.5 and 20.0 ng ml-1 , who had undergone prostatic biopsy at two Chinese centers were included in the analysis. The men were then further divided into groups with a PSA between 2.5-10.0 ng ml-1 and 10.1-20.0 ng ml-1 . Receiver operating characteristic (ROC) curve was used to evaluate the efficacy of PSA and PSAD for the diagnosis of PCa. In men with a PSA of 2.5-10.0 ng ml-1 or 10.1-20.0 ng ml-1 , the areas under the ROC curve were higher for PSAD than for PSA. This was consistent across both centers and the cohort overall. When the entire cohort was considered, the optimal PSAD cut-off for predicting PCa in men with a PSA of 2.5-10.0 ng ml-1 was 0.15 ng ml-1 ml-1 , with a sensitivity of 64.4% and specificity of 64.6%. The optimal cut-off for PSAD in men with a PSA of 10.1-20.0 ng ml-1 was 0.33 ng ml-1 ml-1 , with a sensitivity of 60.3% and specificity of 82.7%. PSAD can improve the effectiveness for PCa detection in Chinese men with a PSA of 2.5-10.0 ng ml-1 (traditional Western PSA "gray zone") and 10.1-20.0 ng ml-1 (Chinese PSA "gray zone").

MLN2238 synergizes BH3 mimetic ABT-263 in castration-resistant prostate cancer cells by induction of NOXA.

Patients undergoing androgen blockade therapy develop castration-resistant prostate cancer (CRPC), which is associated with Bcl-2 upregulation and results in disease progression and death. In recent years, promising therapeutic agents, such as the BH3-only mimetic ABT-263 and proteasome inhibitors, have been developed and widely evaluated against a broad spectrum of cancer types, including prostate cancer, alone or in combination with other chemotherapeutic agents. In this study, the antitumor efficacy of ABT-263 and MLN2238 were evaluated as single agents and in combination in four CRPC cell lines: PC3, C4-2B, C4-2, and DU145. The viability of the treated cells and markers of apoptosis were assayed. Protein-protein interactions were analyzed by co-immunoprecipitation in drug-treated cells. Lentivirus-mediated short hairpin RNA was used to knockdown Bax, Mcl-1, and NOXA expressions. We found that ABT-263 and MLN2238 alone exhibited a mild cytotoxicity, and in combination, they elicited a synergistic cytotoxic effect in CRPC cells. The cell apoptosis induced by the combination drug treatment was evidenced by enhanced caspase-3 and Poly (ADP-ribose) polymerase (PARP) cleavage, and annexin-V-positive staining was significantly depleted by Bax knockdown. MLN2238 treatment upregulated NOXA and Mcl-1 expression, leading NOXA/Mcl-1 complexes to disassociate Bak from its complexes with Mcl-1 and enhancing ABT263-triggered Bax activation. NOXA knockdown by short hairpin RNA significantly attenuated the cytotoxicity of ABT-263 and MLN2238 co-administration. In conclusion, MLN2238 and ABT-263 synergistically triggered apoptosis in CRPC cells by upregulating NOXA and activating Bax, indicating a promising therapeutic strategy for the treatment of CRPC.

Percent free prostate-specific antigen does not improve the effectiveness of prostate cancer detection in Chinese men with a prostate-specific antigen of 2.5-20.0 ng/ml: a multicenter study.

The prostate-specific antigen (PSA) "gray zone" in Chinese men is likely higher than the traditional value (2.5-10.0 ng/ml) since the incidence of prostate cancer in Chinese men is relative low. The utility of percent free PSA in predicting prostate cancer is based on Western populations and may introduce sizable bias when applied to a Chinese cohort. We assessed the efficacy of percent free PSA in predicting prostate cancer in Chinese men with a PSA of 2.5-10.0 and 10.1-20.0 ng/ml. A total of 558 men with a PSA of 2.5-20.0 ng/ml who had undergone prostatic biopsy to detect prostate cancer from two Chinese centers were included. The rates of prostate cancer in different percent free PSA ranges were evaluated. Receiver operating characteristic curve (ROC) was used to evaluate and compare the efficiency of PSA and percent free PSA in the diagnosis of prostate cancer. The areas under ROC (AUCs) for percent free PSA for predicting prostate cancer were not higher than those for PSA, although prostate cancer detection rates increased with decreased percent free PSA in men with a PSA of 2.5-10.0, 10.1-20.0, and 2.5-20.0 ng/ml. Similarly, for men aged <70 and ≥ 70 years and with prostate volume <40 and ≥ 40 ml, AUCs showed percent free PSA was not better than PSA in predicting prostate cancer. By analyzing multicenter data, we first found that percent free PSA does not improve detection of prostate cancer in Chinese men with a PSA of 2.5-10.0 or 10.1-20.0 ng/ml.