A gain-of-function mutation in DHT synthesis in castration-resistant prostate cancer.

Growth of prostate cancer cells is dependent upon androgen stimulation of the androgen receptor (AR). Dihydrotestosterone (DHT), the most potent androgen, is usually synthesized in the prostate from testosterone secreted by the testis. Following chemical or surgical castration, prostate cancers usually shrink owing to testosterone deprivation. However, tumors often recur, forming castration-resistant prostate cancer (CRPC). Here, we show that CRPC sometimes expresses a gain-of-stability mutation that leads to a gain-of-function in 3ß-hydroxysteroid dehydrogenase type 1 (3ßHSD1), which catalyzes the initial rate-limiting step in conversion of the adrenal-derived steroid dehydroepiandrosterone to DHT. The mutation (N367T) does not affect catalytic function, but it renders the enzyme resistant to ubiquitination and degradation, leading to profound accumulation. Whereas dehydroepiandrosterone conversion to DHT is usually very limited, expression of 367T accelerates this conversion and provides the DHT necessary to activate the AR. We suggest that 3ßHSD1 is a valid target for the treatment of CRPC.

Dual roles of HK3 in regulating the network between tumor cells and tumor-associated macrophages in neuroblastoma.

Neuroblastoma (NB) is the most common and deadliest extracranial solid tumor in children. Targeting tumor-associated macrophages (TAMs) is a strategy for attenuating tumor-promoting states. The crosstalk between cancer cells and TAMs plays a pivotal role in mediating tumor progression in NB. The overexpression of Hexokinase-3 (HK3), a pivotal enzyme in glucose metabolism, has been associated with poor prognosis in NB patients. Furthermore, it correlates with the infiltration of M2-like macrophages within NB tumors, indicating its significant involvement in tumor progression. Therefore, HK3 not only directly regulates the malignant biological behaviors of tumor cells, such as proliferation, migration, and invasion, but also recruits and polarizes M2-like macrophages through the PI3K/AKT-CXCL14 axis in neuroblastoma. The secretion of lactate and histone lactylation alterations within tumor cells accompanies this interaction. Additionally, elevated expression of HK3 in M2-TAMs was found at the same time. Modulating HK3 within M2-TAMs alters the biological behavior of tumor cells, as demonstrated by our in vitro studies. This study highlights the pivotal role of HK3 in the progression of NB malignancy and its intricate regulatory network with M2-TAMs. It establishes HK3 as a promising dual-functional biomarker and therapeutic target in combating neuroblastoma.

Generation of Dopamine Transporter (DAT)-mCherry Knock-in Rats by CRISPR-Cas9 Genome Editing.

Midbrain dopaminergic neurons respond to rewards and have a crucial role in positive motivation and pleasure. Electrical stimulation of dopaminergic neurons and/or their axonal fibers and arborization has been often used to motivate animals to perform cognitive tasks. Still, the electrical stimulation is incompatible with electrophysiological recordings. In this light, optical stimulation following artificial expression of channelrhodopsin-2 (ChR2) in the cell membrane has been also used, but the expression level of ChR2 varies among researchers. Thus, we attempted to stably express ChR2 fused with a red fluorescence protein, mCherry, in dopaminergic neurons. Since dopamine transporter (DAT) gene is known as a marker for dopaminergic neurons, we inserted ChR2-mCherry into the downstream of the DAT gene locus of the rat genome by clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) genome editing and created DAT-ChR2-mCherry knock-in rats. Immunohistochemistry showed that ChR2-mCherry was expressed in dopaminergic neurons in homozygote knock-in rats, whereas whole-cell recordings revealed that ChR2-mCherry-positive neurons did not fire action potentials upon blue light stimulation, indicating that ChR2 was not functional for optogenetics. Nevertheless, fluorescent labeling of dopaminergic neurons mediated by mCherry could help characterize them physiologically and histologically.

Meta-analysis of efficacy, safety, stability and predictability of Small Incision Lenticule Extraction (SMILE) for myopia.

In order to investigate the postoperative efficacy, safety, stability, and predictability of SMILE surgery as a treatment for myopia, a comprehensive search was conducted in April 2023 across prominent databases, including PubMed, EMBASE, Web of Science, and Cochrane Library. The search aimed to select relevant studies of randomized controlled trials (RCTs) comparing clinical outcomes between SMILE and other corneal refractive surgeries for myopia. Upon conducting the initial search, a total of 324 records were retrieved from the aforementioned databases. These records were subjected to a meticulous selection process, adhering to predetermined inclusion criteria, resulting in 17 studies being ultimately included for analysis. By pooling the results of these studies, the comparison between SMILE surgery and alternative corneal refractive surgeries demonstrated similar outcomes in terms of efficacy, safety, stability, predictability, and higher-order aberrations (HOAs) concerning the correction of myopia. Furthermore, it was observed that the SMILE procedure exhibited a lesser impact on corneal sensation and corneal nerve density compared to other corneal refractive surgeries. Based on these findings, SMILE surgery may be considered as a treatment option with a slight superiority over conventional corneal surgery for myopia.

The impact of obesity on lung function measurements and respiratory disease: A Mendelian randomization study.

INTRODUCTION: Observational studies have shown that body mass index (BMI) and waist-to-hip ratio (WHR) are both inversely associated with lung function, as assessed by forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV1). However, observational data are susceptible to confounding and reverse causation. METHODS: We selected genetic instruments based on their relevant large-scale genome-wide association studies. Summary statistics of lung function and asthma came from the UK Biobank and SpiroMeta Consortium meta-analysis (n = 400,102). After examining pleiotropy and removing outliers, we applied inverse-variance weighting to estimate the causal association of BMI and BMI-adjusted WHR (WHRadjBMI) with FVC, FEV1, FEV1/FVC, and asthma. Sensitivity analyses were performed using weighted median, MR-Egger, and MRlap methods. RESULTS: We found that BMI was inversely associated with FVC (effect estimate, -0.167; 95% confidence interval (CI), -0.203 to -0.130) and FEV1 (effect estimate, -0.111; 95%CI, -0.149 to -0.074). Higher BMI was associated with higher FEV1/FVC (effect estimate, 0.079; 95%CI, 0.049 to 0.110) but was not significantly associated with asthma. WHRadjBMI was inversely associated with FVC (effect estimate, -0.132; 95%CI, -0.180 to -0.084) but has no significant association with FEV1. Higher WHR was associated with higher FEV1/FVC (effect estimate, 0.181; 95%CI, 0.130 to 0.232) and with increased risk of asthma (effect estimate, 0.027; 95%CI, 0.001 to 0.053). CONCLUSION: We found significant evidence that increased BMI is suggested to be causally related to decreased FVC and FEV1, and increased BMI-adjusted WHR could lead to lower FVC value and higher risk of asthma. Higher BMI and BMI-adjusted WHR were suggested to be causally associated with higher FEV1/FVC.

The Effect of Bromfenac Sodium Nanopolymer Used in Anterior Segment of the Eye on Corneal Neovascularization.

This study was to explore the inhibitory effect of bromfenac sodium (BF) / chitosan (CS) nanoparticles (NPs) on corneal neovascularization (CNV). 45 New Zealand white rabbits provided by The First Affiliated Hospital of Jinan University were randomly divided into a control group (group A, n = 15), 0.1% BF aqueous solution treatment group (group B, n = 15), and 0.1% BF/CS-NPs suspension treatment group (group C, n = 15). A rabbit corneal alkali burn model was established. The average particle size of BF/CS-NPs with different BF concentrations was mainly 341.6 ± 12.9 nm - 548.7 ± 15.4 nm; and the Zeta potential distribution was 24.3 ± 2.5 mV - 35.7 ± 4.3 mV. When the initial concentration of BF was 1.5 mg/mL, the maximum drug loading was 57.35 ± 5.26%. The area of CNV in group C was significantly lower than that in groups B and A, and the differences were statistically significant (P < 0.05). At 6, 12, 18, and 24 days after surgery, the mRNA expression levels in cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) gene were compared after standardized by ß-actin; group A had the highest expression level, followed by group B, and group C had the lowest expression level, showing statistically significant differences (P < 0.05). The BF/CS-NPs granules prepared in this study had stable physical and chemical properties and had a good sustained-release effect, and the release duration can be as long as 48 hours. BF/CS-NPs can inhibit the formation of CNV at different time points after alkali burn, and reduce the expression of VEGF and COX-2 in corneal tissue after alkali burn.

Development and validation of a nomogram to predict cancer-specific survival in elderly patients with papillary thyroid carcinoma: a population-based study.

OBJECTIVE: Thyroid carcinoma (TC) is the most common endocrine tumor in the human body. Papillary thyroid carcinoma (PTC) accounts for more than 80% of thyroid cancers. Accurate prediction of elderly PTC can help reduce the mortality of patients. We aimed to construct a nomogram predicting cancer-specific survival (CSS) in elderly patients with PTC. METHODS: Patient information was downloaded from the Surveillance, Epidemiology, and End Results (SEER) program. Univariate and multivariate Cox regression models were used to screen the independent risk factors for patients with PTC. The nomogram of elderly patients with PTC was constructed based on the multivariate Cox regression model. We used the concordance index (C-index), the area under the receiver operating characteristic curve (AUC) and the calibration curve to test the accuracy and discrimination of the prediction model. Decision curve analysis (DCA) was used to test the clinical value of the model. RESULTS: A total of 14,138 elderly patients with PTC were included in this study. Patients from 2004 to 2015 were randomly divided into a training set (N = 7379) and a validation set (N = 3141), and data from 2016 to 2018 were divided into an external validation set (N = 3618). Proportional sub-distribution hazard model showed that age, sex, tumor size, histological grade, TNM stage, surgery and chemotherapy were independent risk factors for prognosis. In the training set, validation set and external validation set, the C-index was 0.87(95%CI: 0.852-0.888), 0.891(95%CI: 0.866-0.916) and 0.931(95%CI:0.894-0.968), respectively, indicating that the nomogram had good discrimination. Calibration curves and AUC suggest that the prediction model has good discrimination and accuracy. CONCLUSIONS: We constructed a new nomogram to predict CSS in elderly patients with PTC. Internal cross-validation and external validation indicate that the model has good discrimination and accuracy. The predictive model can help doctors and patients make clinical decisions.

Bone mineral density, bone metabolism-related factors, and microRNA-218 are correlated with disease activities in Chinese ankylosing spondylitis patients.

OBJECTIVE: To investigate bone mineral density (BMD), bone metabolism-related factors, and microRNA-218 in Chinese ankylosing spondylitis (AS) patients and to identify their correlation with disease activities and the treatment with TNF-α inhibitors. METHODS: A total of 89 AS patients were enrolled in the study. Patients' information and laboratory examination results were collected. BMD of the anteroposterior lumbar spine (L2-L4), left femoral neck, and whole body were measured and T-scores were calculated. MicroRNA-218 was extracted from PBMCs of AS patients and detected by RT-PCR. Bone metabolism-related factors were detected using protein chips and flow cytometer. RESULTS: Out of 86 patients undergoing whole-body BMD measurement, 14 had osteopenia and 72 had normal BMD without osteoporosis or high BMD. Compared with short- (disease duration ≤3 years) and long-term groups (disease duration ≥10 years), medium-term group (disease duration ranges from 3 to 10 years) showed lowest BMD. Patients with onset age ≤20 years old had significantly lower BMD than the other groups (p < 0.05). The BMD of femoral neck had negative correlation with CRP (p < 0.05) and no correlation with BASDAI or ESR. Both whole-body BMD and femoral neck BMD were negatively correlated with BASMI (p < 0.05). Dickkopf-1 (DKK-1), platelet-derived growth factor-BB (PDGF-BB), and receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) were significantly increased, while Osteopontin (OPN) was significantly decreased in AS patients. Expression of microRNA-218 in PBMC of AS patients was low and was positively correlated with BASMI (p < 0.05), but it was not correlated with the duration of disease, age of onset, BASDAI, ESR, or BMD. CONCLUSION: Loss of bone mass mainly occurred at the inflammatory sites in AS patients, depending on the severity of inflammation. The alleviation of inflammation can improve loss of bone mass and bone metabolism disorders. Anti-inflammatory treatment is critical for the treatment of secondary osteoporosis caused by AS.

Intense pulsed light (IPL) therapy for meibomian gland dysfunction (MGD)-related dry eye disease (DED): a systematic review and meta-analysis.

To compare the effect of intense pulsed light (IPL) therapy and conventional treatments in meibomian gland dysfunction (MGD)-related dry eye disease (DED). A literature search was conducted in PubMed, Cochrane Central Register of Controlled Trials (CENTRAL), Web of Science, EMBASE, China National Knowledge Infrastructure (CNKI), Wanfang Database, VIP, and China Biology Medicine (CBM) up to January 2022. Randomized controlled trials (RCTs) were included. Mean differences (MDs) and their 95% confidence intervals (CIs) were calculated. A meta-analysis concerning changes in tear break-up time (BUT), changes in Ocular Surface Disease Index (OSDI) scores, changes in non-invasively measured tear break-up time (NIBUT), changes in corneal and conjunctival fluorescein staining (CFS) scores, and changes in Standard Patient Evaluation of Eye Dryness (SPEED) scores was carried out. The initial search identified a total of 1842 records in the databases, and 11 studies were included in the final analysis. Compared to conventional therapies, IPL therapy was associated with significantly reduced OSDI (MD, - 7.49; 95% CI, - 12.47 to - 2.5) and SPEED (MD, - 3.28; 95% CI, - 5.64 to - 0.93) scores, while BUT (MD, 1.94; 95% CI, 1.19 ~ 2.69) and NIBUT (MD, 2.55; 95% CI, 1.07 ~ 4.04) significantly increased. No significant difference was found in the change in CFS between the two groups. Both IPL treatment and traditional treatments are effective in the treatment of MGD-related DED. IPL application seems to be superior to traditional treatments.

Clinicopathologic and molecular features of denosumab-treated giant cell tumour of bone (GCTB): Analysis of 21 cases.

GCTB is an osteolytic, locally-aggressive, rarely-metastasizing tumour, characterized by abundance of osteoclast-like giant cells, induced by neoplastic mononuclear cells expressing high-levels of the receptor activator of nuclear factor Kappa-B ligand (RANKL), a mediator of osteoclast activation. Although the mainstay of treatment is complete tumour removal with preservation of bone, therapy with denosumab, an inhibitor of RANKL, has been introduced for selected cases. OBJECTIVES: Denosumab-treated GCTB (DT-GCTB) was reported to show a wide spectrum of histological changes such as depletion of osteoclast-like giant cells and intralesional bone deposition, which may lead to diagnostic difficulties. We investigated clinicopathologic and molecular features of DT-GCTB, matched with pre-therapy samples. PARTICIPANTS: 21 cases were included (13 females, 8 males), aged 15 to 64 (median, 30 years). RESULTS: DT-GCTB showed development of sclerotic neocortex and varying degrees of osteosclerosis radiographically. Marked depletion of giant cells, different degree of ossification, fibrosis, and proliferation of mononuclear cells was observed. Staining for H3.3G34W was positive in mononuclear cells in 19 cases (90.5%), while one negative case was positive for H3.3G34V. H3F3A G34W mutation was confirmed in 17 of 19 cases (89.5%), corresponding to nuclear staining with H3.3 G34W antibody. G34L mutation was detected in one G34W negative case, in which H3.3 G34V nuclear positive staining was observed, possibly due to cross-reaction. CONCLUSIONS: Post-therapy tumours still exhibit a similar mutation profile, while significantly differing from classic GCTB morphologically. Correlation with history of denosumab administration, awareness of features of DT-GCTB, IHC and molecular studies for histone H3 mutations are important in its assessment.

Matching-Based Resource Allocation for Satellite-Ground Network.

With the vigorous development of information and communication technology, mobile internet has undergone tremendous changes. How to achieve global coverage of the network has become the primary problem to be solved. GEO satellites and LEO satellites, as important components of the satellite-ground network, can offer service for hotspots or distant regions where ground-based base stations' coverage is limited. Therefore, we build a satellite-ground network model, which transforms the satellite-ground network resource allocation problem into a matching issue between GEO satellites, LEO satellites, and users. A GEO satellite provides data backhaul for users, and a LEO satellite provides data transmission services according to users' requests. It is important to consider the relationships between all entities and establish a distributed scheme, so we propose a three-sided cyclic matching algorithm. It is confirmed by a large number of simulation experiments that the method suggested in this research is better than the conventional algorithm in terms of average delay, satellite revenue, and number of users served.

JDAPCOO: Resource Scheduling and Energy Efficiency Optimization in 5G and Satellite Converged Networks for Power Transmission and Distribution Scenarios.

Along with the continuous revolution of energy production and energy consumption structures, the information data of smart grids have exploded, and effective solutions are urgently needed to solve the problem of power devices resource scheduling and energy efficiency optimization. In this paper, we propose a fifth generation (5G) and satellite converged network architecture for power transmission and distribution scenarios, where power transmission and distribution devices (PDs) can choose to forward power data to a cloud server data center via ground networks or space-based networks for power grid regulation and control. We propose a Joint Device Association and Power Control Online Optimization (JDAPCOO) algorithm to maximize the long-term system energy efficiency while guaranteeing the minimum transmission rate requirement of PDs. Since the formulated issue is a mixed integer nonconvex optimization problem with high complexity, we decompose the original problem into two subproblems, i.e., device association and power control, which are solved using a genetic algorithm and improved simulated annealing algorithm, respectively. Numerical simulation results show that when the number of PDs is 50, the proposed algorithm can improve the system energy efficiency by 105%, 545.05% and 835.26%, respectively, compared with the equal power allocation algorithm, random power allocation algorithm and random device association algorithm.

Conversion of abiraterone to D4A drives anti-tumour activity in prostate cancer.

Prostate cancer resistance to castration occurs because tumours acquire the metabolic capability of converting precursor steroids to 5α-dihydrotestosterone (DHT), promoting signalling by the androgen receptor and the development of castration-resistant prostate cancer. Essential for resistance, DHT synthesis from adrenal precursor steroids or possibly from de novo synthesis from cholesterol commonly requires enzymatic reactions by 3ß-hydroxysteroid dehydrogenase (3ßHSD), steroid-5α-reductase (SRD5A) and 17ß-hydroxysteroid dehydrogenase (17ßHSD) isoenzymes. Abiraterone, a steroidal 17α-hydroxylase/17,20-lyase (CYP17A1) inhibitor, blocks this synthetic process and prolongs survival. We hypothesized that abiraterone is converted by an enzyme to the more active Δ(4)-abiraterone (D4A), which blocks multiple steroidogenic enzymes and antagonizes the androgen receptor, providing an additional explanation for abiraterone's clinical activity. Here we show that abiraterone is converted to D4A in mice and patients with prostate cancer. D4A inhibits CYP17A1, 3ßHSD and SRD5A, which are required for DHT synthesis. Furthermore, competitive androgen receptor antagonism by D4A is comparable to the potent antagonist enzalutamide. D4A also has more potent anti-tumour activity against xenograft tumours than abiraterone. Our findings suggest an additional explanation-conversion to a more active agent-for abiraterone's survival extension. We propose that direct treatment with D4A would be more clinically effective than abiraterone treatment.

Isoorientin Inhibits Inflammation in Macrophages and Endotoxemia Mice by Regulating Glycogen Synthase Kinase 3ß.

Isoorientin has anti-inflammatory effects; however, the mechanism remains unclear. We previously found isoorientin is an inhibitor of glycogen synthase kinase 3ß (GSK3ß) in vitro. Overactivation of GSK3ß is associated with inflammatory responses. GSK3ß is inactivated by phosphorylation at Ser9 (i.e., p-GSK3ß). Lithium chloride (LiCl) inhibits GSK3ß and also increases p-GSK3ß (Ser9). The present study investigated the anti-inflammatory effect and mechanism of isoorientin via GSK3ß regulation in lipopolysaccharide- (LPS-) induced RAW264.7 murine macrophage-like cells and endotoxemia mice. LiCl was used as a control. While AKT phosphorylates GSK3ß, MK-2206, a selective AKT inhibitor, was used to activate GSK3ß via AKT inhibition (i.e., not phosphorylate GSK3ß at Ser9). The proinflammatory cytokines TNF-α, IL-6, and IL-1ß were detected by ELISA or quantitative real-time PCR, while COX-2 by Western blotting. The p-GSK3ß and GSK3ß downstream signal molecules, including NF-κB, ERK, Nrf2, and HO-1, as well as the tight junction proteins ZO-1 and occludin were measured by Western blotting. The results showed that isoorientin decreased the production of TNF-α, IL-6, and IL-1ß and increased the expression of p-GSK3ß in vitro and in vivo, similar to LiCl. Coadministration of isoorientin and LiCl showed antagonistic effects. Isoorientin decreased the expression of COX-2, inhibited the activation of ERK and NF-κB, and increased the activation of Nrf2/HO-1 in LPS-induced RAW264.7 cells. Isoorientin increased the expressions of occludin and ZO-1 in the brain of endotoxemia mice. In summary, isoorientin can inhibit GSK3ß by increasing p-GSK3ß and regulate the downstream signal molecules to inhibit inflammation and protect the integrity of the blood-brain barrier and the homeostasis in the brain.

The exploration of disease-specific gene regulatory networks in esophageal carcinoma and stomach adenocarcinoma.

BACKGROUND: Feed-forward loops (FFLs), consisting of miRNAs, transcription factors (TFs) and their common target genes, have been validated to be important for the initialization and development of complex diseases, including cancer. Esophageal Carcinoma (ESCA) and Stomach Adenocarcinoma (STAD) are two types of malignant tumors in the digestive tract. Understanding common and distinct molecular mechanisms of ESCA and STAD is extremely crucial. RESULTS: In this paper, we presented a computational framework to explore common and distinct FFLs, and molecular biomarkers for ESCA and STAD. We identified FFLs by combining regulation pairs and RNA-seq data. Then we constructed disease-specific co-expression networks based on the FFLs identified. We also used random walk with restart (RWR) on disease-specific co-expression networks to prioritize candidate molecules. We identified 148 and 242 FFLs for these two types of cancer, respectively. And we found that one TF, E2F3 was related to ESCA, two genes, DTNA and KCNMA1 were related to STAD, while one TF ESR1 and one gene KIT were associated with both of the two types of cancer. CONCLUSIONS: This proposed computational framework predicted disease-related biomolecules effectively and discovered the correlation between two types of cancers, which helped develop the diagnostic and therapeutic strategies of Esophageal Carcinoma and Stomach Adenocarcinoma.

Opposing Effects of Cyclooxygenase-2 (COX-2) on Estrogen Receptor ß (ERß) Response to 5α-Reductase Inhibition in Prostate Epithelial Cells.

Current pharmacotherapies for symptomatic benign prostatic hyperplasia (BPH), an androgen receptor-driven, inflammatory disorder affecting elderly men, include 5α-reductase (5AR) inhibitors (i.e. dutasteride and finasteride) to block the conversion of testosterone to the more potent androgen receptor ligand dihydrotestosterone. Because dihydrotestosterone is the precursor for estrogen receptor ß (ERß) ligands, 5AR inhibitors could potentially limit ERß activation, which maintains prostate tissue homeostasis. We have uncovered signaling pathways in BPH-derived prostate epithelial cells (BPH-1) that are impacted by 5AR inhibition. The induction of apoptosis and repression of the cell adhesion protein E-cadherin by the 5AR inhibitor dutasteride requires both ERß and TGFß. Dutasteride also induces cyclooxygenase type 2 (COX-2), which functions in a negative feedback loop in TGFß and ERß signaling pathways as evidenced by the potentiation of apoptosis induced by dutasteride or finasteride upon pharmacological inhibition or shRNA-mediated ablation of COX-2. Concurrently, COX-2 positively impacts ERß action through its effect on the expression of a number of steroidogenic enzymes in the ERß ligand metabolic pathway. Therefore, effective combination pharmacotherapies, which have included non-steroidal anti-inflammatory drugs, must take into account biochemical pathways affected by 5AR inhibition and opposing effects of COX-2 on the tissue-protective action of ERß.

Catalytically relevant electrostatic interactions of cytochrome P450c17 (CYP17A1) and cytochrome b5.

Two acidic residues, Glu-48 and Glu-49, of cytochrome b5 (b5) are essential for stimulating the 17,20-lyase activity of cytochrome P450c17 (CYP17A1). Substitution of Ala, Gly, Cys, or Gln for these two glutamic acid residues abrogated all capacity to stimulate 17,20-lyase activity. Mutations E49D and E48D/E49D retained 23 and 38% of wild-type activity, respectively. Using the zero-length cross-linker ethyl-3-(3-dimethylaminopropyl)carbodiimide, we obtained cross-linked heterodimers of b5 and CYP17A1, wild-type, or mutations R347K and R358K. In sharp contrast, the b5 double mutation E48G/E49G did not form cross-linked complexes with wild-type CYP17A1. Mass spectrometric analysis of the CYP17A1-b5 complexes identified two cross-linked peptide pairs as follows: CYP17A1-WT: (84)EVLIKK(89)-b5: (53)EQAGGDATENFEDVGHSTDAR(73) and CYP17A1-R347K: (341)TPTISDKNR(349)-b5: (40)FLEEHPGGEEVLR(52). Using these two sites of interaction and Glu-48/Glu-49 in b5 as constraints, protein docking calculations based on the crystal structures of the two proteins yielded a structural model of the CYP17A1-b5 complex. The appositional surfaces include Lys-88, Arg-347, and Arg-358/Arg-449 of CYP17A1, which interact with Glu-61, Glu-42, and Glu-48/Glu-49 of b5, respectively. Our data reveal the structural basis of the electrostatic interactions between these two proteins, which is critical for 17,20-lyase activity and androgen biosynthesis.

Double substituted variant of Bacillus amyloliquefaciens esterase with enhanced enantioselectivity and high activity towards 1-(3',4'-methylenedioxyphenyl)ethyl acetate.

Bacillus amyloliquefaciens esterase (BAE) was applied to produce (R)-1-(3',4'-methylenedioxyphenyl)ethanol, a chiral drug intermediate. In this study, we improved the enantioselectivity of BAE by protein engineering instead of process engineering as used in our previous work. Saturation mutagenesis was carried out on eight positions of BAE based on structure modeling and substrate docking. A double substituted variant V10 (K358D/A396C) showed an excellent enantioselectivity without decreasing the activity. The functions of these two mutations (K358D and A396C) were investigated, revealing a synergic effect on the BAE enantioselectivity. Using the variant V10, enantiopure (R)-1-(3',4'-methylenedioxyphenyl)ethanol could be readily prepared in >97 % ee, affording a high space-time yield (123 g L(-1) day(-1)) and a high ratio of substrate/catalyst (40 g g(-1)) in 1-L reaction.

Effect of intestinal autochthonous probiotics isolated from the gut of sea cucumber (Apostichopus japonicus) on immune response and growth of A. japonicus.

The study isolated 224 bacteria from the intestine of Apostichopus japonicus, then selected and identified three of the bacteria (HS1, HS7, and HS10) which demonstrated amylase, lipase, and protease production capacity as candidate probiotics for sea cucumbers. The three potential probiotics showed no pathogenicity both in hemolytic assays on sheep blood agar plates and after immersing sea cucumbers in a suspension of the bacteria. To reveal the effects of these three potential probiotics on the innate immunity of sea cucumbers, total coelomocyte counts, respiratory burst activity, superoxide dismutase activity, lysozyme activity, acid phosphatase activity, and phagocytic activity by coelomocytes were examined after feeding with four different diets for up to 28 days. Also the specific growth rate and survival rate were investigated after a 60-day feeding trial. Sea cucumbers were fed with 4 diets: one control, three diets supplemented with 1 × 10(9) cell g(-1) of HS1, HS7, and HS10 for 28-60 days. Results showed that sea cucumbers fed diets containing HS1, HS7, and HS10 had led to an enhanced cellular and humoral immune response, notably higher total coelomocytes counts, respiratory burst activity, lysozyme activity, acid phosphatase activity, and phagocytic activity, as recorded during the four weeks of probiotics administration. On the other hand, the survival rate among dietary treatments ranged from 90.71 to 97.97% with significant improvement (P < 0.05) compared to that of the control; and the growth rate observed in the sea cucumbers fed HS1 and HS7 showed sharp increases after 60 days feeding. The present study confirmed the potential beneficial effects of Pseudoalteromonas elyakovii HS1, Shewanella japonica HS7, and Vibrio tasmaniensis HS10 as dietary probiotics in A. japonicus.