mTORC1/rpS6 and mTORC2/PKC regulate spermatogenesis through Arp3-mediated actin microfilament organization in Eriocheir sinensis.

Mammalian target of rapamycin (mTOR) is a crucial signaling protein regulating a range of cellular events. Numerous studies have reported that the mTOR pathway is related to spermatogenesis in mammals. However, its functions and underlying mechanisms in crustaceans remain largely unknown. mTOR exists as two multimeric functional complexes termed mTOR complex 1 (mTORC1) and mTORC2. Herein, we first cloned ribosomal protein S6 (rpS6, a downstream molecule of mTORC1) and protein kinase C (PKC, a downstream effector of mTORC2) from the testis of Eriocheir sinensis. The dynamic localization of rpS6 and PKC suggested that both proteins may be essential for spermatogenesis. rpS6/PKC knockdown and Torin1 treatment led to defects in spermatogenesis, including germ cell loss, retention of mature sperm and empty lumen formation. In addition, the integrity of the testis barrier (similar to the blood-testis barrier in mammals) was disrupted in the rpS6/PKC knockdown and Torin1 treatment groups, accompanied by changing in expression and distribution of junction proteins. Further study demonstrated that these findings may result from the disorganization of filamentous actin (F-actin) networks, which were mediated by the expression of actin-related protein 3 (Arp3) rather than epidermal growth factor receptor pathway substrate 8 (Eps8). In summary, our study illustrated that mTORC1/rpS6 and mTORC2/PKC regulated spermatogenesis via Arp3-mediated actin microfilament organization in E. sinensis.

Tetrandrine slows the radiographic progression of progressive massive fibrosis in pneumoconiosis: a retrospective cohort study.

OBJECTIVE: This study aims to explore the clinical effect of Tetrandrine (Tet) on progressive massive fibrosis (PMF) of pneumoconiosis. METHODS: This retrospective study collected 344 pneumoconiosis patients with PMF, and 127 were eligible for the final analysis, including 57 patients in the Tet group and 70 patients in the control group. The progress of imaging and lung function were compared between the two groups. RESULTS: After 13 months (median) of treatment, the size of PMF was smaller in the Tet group than that in the control group (1526 vs. 2306, p=0.001), and the size was stable in the Tet group (1568 vs. 1526, p= 0.381), while progressed significantly in the control group (2055 vs. 2306, p=0.000). The small nodule profusion and emphysema were also milder than that in the control group (6.0 vs. 7.5, p=0.046 and 8.0 vs. 12, p=0.016 respectively). Pulmonary ventilation function parameters FVC and FEV1 improved in the Tet group (3222 vs. 3301, p=0.021; 2202 vs. 2259, p=0.025 respectively) and decreased in the control group (3272 vs. 3185, p= 0.00; 2094 vs. 1981, p=0.00 respectively). FEV1/FVC was also significantly higher in the Tet group than that in the control group (68.45vs. 60.74, p=0.001). However, similar result was failed to observed for DLco%, which showed a significant decrease in both groups. CONCLUSION: Tet has shown great potential in the treatment of PMF by slowing the progression of pulmonary fibrosis and the decline of lung function.

mTORC1/C2 regulate spermatogenesis in Eriocheir sinensis via alterations in the actin filament network and cell junctions.

Spermatogenesis is a finely regulated process of germ cell proliferation and differentiation that leads to the production of sperm in seminiferous tubules. Although the mammalian target of rapamycin (mTOR) signaling pathway is crucial for spermatogenesis in mammals, its functions and molecular mechanisms in spermatogenesis remain largely unknown in nonmammalian species, particularly in Crustacea. In this study, we first identified es-Raptor (the core component of mTOR complex 1) and es-Rictor (the core component of mTOR complex 2) from the testis of Eriocheir sinensis. Dynamic localization of es-Raptor and es-Rictor implied that these proteins were indispensable for the spermatogenesis of E. sinensis. Furthermore, es-Raptor and es-Rictor knockdown results showed that the mature sperm failed to be released, causing almost empty lumens in the testis. We investigated the reasons for these effects and found that the actin-based cytoskeleton was disrupted in the knockdown groups. In addition, the integrity of the testis barrier (similar to the blood-testis barrier in mammals) was impaired and affected the expression of cell junction proteins. Further study revealed that es-Raptor and es-Rictor may regulate spermatogenesis via both mTORC1- and mTORC2-dependent mechanisms that involve es-rpS6 and es-Akt/es-PKC, respectively. Moreover, to explore the testis barrier in E. sinensis, we established a cadmium chloride (CdCl2)-induced testis barrier damage model as a positive control. Morphological and immunofluorescence results were similar to those of the es-Raptor and es-Rictor knockdown groups. Altogether, es-Raptor and es-Rictor were important for spermatogenesis through maintenance of the actin filament network and cell junctions in E. sinensis.

Follicle-stimulating hormone signaling in Sertoli cells: a licence to the early stages of spermatogenesis.

Follicle-stimulating hormone signaling is essential for the initiation and early stages of spermatogenesis. Follicle-stimulating hormone receptor is exclusively expressed in Sertoli cells. As the only type of somatic cell in the seminiferous tubule, Sertoli cells regulate spermatogenesis not only by controlling their own number and function but also through paracrine actions to nourish germ cells surrounded by Sertoli cells. After follicle-stimulating hormone binds to its receptor and activates the follicle-stimulating hormone signaling pathway, follicle-stimulating hormone signaling will establish a normal Sertoli cell number and promote their differentiation. Spermatogonia pool maintenance, spermatogonia differentiation and their entry into meiosis are also positively regulated by follicle-stimulating hormone signaling. In addition, follicle-stimulating hormone signaling regulates germ cell survival and limits their apoptosis. Our review summarizes the aforementioned functions of follicle-stimulating hormone signaling in Sertoli cells. We also describe the clinical potential of follicle-stimulating hormone treatment in male patients with infertility. Furthermore, our review may be helpful for developing better therapies for treating patients with dysfunctional follicle-stimulating hormone signaling in Sertoli cells.

The potential use of Zymomonas mobilis for the food industry.

Zymomonas mobilis is a gram-negative facultative anaerobic spore, which is generally recognized as a safe. As a promising ethanologenic organism for large-scale bio-ethanol production, Z. mobilis has also shown a good application prospect in food processing and food additive synthesis for its unique physiological characteristics and excellent industrial characteristics. It not only has obvious advantages in food processing and becomes the biorefinery chassis cell for food additives, but also has a certain healthcare effect on human health. Until to now, most of the research is still in theory and laboratory scale, and further research is also needed to achieve industrial production. This review summarized the physiological characteristics and advantages of Z. mobilis in food industry for the first time and further expounds its research status in food industry from three aspects of food additive synthesis, fermentation applications, and prebiotic efficacy, it will provide a theoretical basis for its development and applications in food industry. This review also discussed the shortcomings of its practical applications in the current food industry, and explored other ways to broaden the applications of Z. mobilis in the food industry, to promote its applications in food processing.

Potential applications of Zymomonas mobilis in food industry summarized for the first time.Research status of Z. mobilis in food additive synthesis, fermentation applications, and probiotics are discussed in details.Future research perspectives of Z. mobilis in food industry further proposed.

Kinesin 12 (KIF15) contributes to the development and tumorigenicity of prostate cancer.

In Asia, prostate cancer is becoming a growing concern, impacting both socially and economically, compared with what is seen in western countries. Hence, it is essential to know the mechanisms associated with the development and tumorigenesis of PCa for primary diagnosis, risk management, and development of therapy strategies against PCa. Kinesin family member 15 (KIF15), a kinesin family member, is a plus-end-directed kinesin that functions to form bipolar spindles. There is emerging evidence indicating that KIF15 plays a significant role in several malignancies, such as pancreatic cancer, hepatocellular carcinoma, lung adenocarcinoma, and breast cancer. Still, the function of KIF15 remains unclear in prostate cancer. Here, we study the functional importance of KIF15 in the tumorigenesis of PCa. The bioinformatic analysis from PCa patients revealed high KIF15 expression compared to normal prostate tissues. High expression hinting at a possible functional role of KIF15 in regulating cell proliferation of PCa, which was demonstrated by both in vitro and in vivo assays. Downregulation of KIF15 silenced the expression of CDK2, p-RB, and Cyclin D1 and likewise blocked the cells at the G1 stage of the cell cycle. In addition, KIF15 downregulation inhibited MEK-ERK signaling by significantly silencing p-ERK and p-MEK levels. In conclusion, this study confirmed the functional significance of KIF15 in the growth and development of prostate cancer and could be a novel therapeutic target for the treatment of PCa.

Antitumor Activity of Small Activating RNAs Induced PAWR Gene Activation in Human Bladder Cancer Cells.

Small double-stranded RNAs (dsRNAs) have been proved to effectively up-regulate the expression of particular genes by targeting their promoters. These small dsRNAs were also termed small activating RNAs (saRNAs). We previously reported that several small double-stranded RNAs (dsRNAs) targeting the PRKC apoptosis WT1 regulator (PAWR) promoter can up-regulate PAWR gene expression effectively in human cancer cells. The present study was conducted to evaluate the antitumor potential of PAWR gene induction by these saRNAs in bladder cancer. Promisingly, we found that up-regulation of PAWR by saRNA inhibited the growth of bladder cancer cells by inducing cell apoptosis and cell cycle arrest which was related to inhibition of anti­apoptotic protein Bcl-2 and inactivation of the NF-κB and Akt pathways. The activation of the caspase cascade and the regulation of cell cycle related proteins also supported the efficacy of the treatment. Moreover, our study also showed that these saRNAs cooperated with cisplatin in the inhibition of bladder cancer cells. Overall, these data suggest that activation of PAWR by saRNA may have a therapeutic benefit for bladder cancer.

KIF3A regulates the Wnt/ß-catenin pathway via transporting ß-catenin during spermatogenesis in Eriocheir sinensis.

The Wnt/ß-catenin pathway participates in many important physiological events such as cell proliferation and differentiation in the male reproductive system. We found that Kinesin-2 motor KIF3A is highly expressed during spermatogenesis in Eriocheir sinensis; it may potentially promote the intracellular transport of cargoes in this process. However, only a few studies have focused on the relationship between KIF3A and the Wnt/ß-catenin pathway in the male reproductive system of decapod crustaceans. In this study, we cloned and characterized the CDS of ß-catenin in E. sinensis for the first time. Fluorescence in situ hybridization and immunofluorescence results showed the colocalization of Es-KIF3A and Es-ß-catenin at the mRNA and the protein level respectively. To further explore the regulatory function of Es-KIF3A to the Wnt/ß-catenin pathway, the es-kif3a was knocked down by double-stranded RNA (dsRNA) in vivo and in primary cultured cells in testes of E. sinensis. Results showed that the expression of es-ß-catenin and es-dvl were decreased in the es-kif3a knockdown group. The protein expression level of Es-ß-catenin was also reduced and the location of Es-ß-catenin was changed from nucleus to cytoplasm in the late stage of spermatogenesis when es-kif3a was knocked down. Besides, the co-IP result demonstrated that Es-KIF3A could bind with Es-ß-catenin. In summary, this study indicates that Es-KIF3A can positively regulate the Wnt/ß-catenin pathway during spermatogenesis and Es-KIF3A can bind with Es-ß-catenin to facilitate the nuclear translocation of Es-ß-catenin.

Optoelectronic properties and anisotropic stress of Mo:ZnO thin films deposited on flexible substrates by radio frequency magnetron sputtering.

This research investigated the optoelectronic properties and anisotropic stress of Mo-doped ZnO (MZO) films, which were deposited on polyethylene terephthalate and polycarbonate flexible substrates with radio frequency magnetron sputtering. The optical properties, x-ray diffraction (XRD) spectra, Hall effect measurements, and self-made phase-shift shadow moiré interferometer readings were utilized to evaluate the performances of the MZO films. Based on the results, the transmittance and (002) peak size of the XRD spectra decreased when the substrate temperature increased. However, this took place especially when the oxygen flow was on the increase. Also, carrier mobility, carrier concentration, and anisotropic stresses increased at higher substrate temperatures, but this was not the case when the oxygen flow increased. The energy gap (Eg) of the MZO films showed a blueshift with an increase in the substrate temperatures, but this rather changed to a redshift when the oxygen flow was observed to be on the rise.

A Highly Sensitive Pressure-Sensing Array for Blood Pressure Estimation Assisted by Machine-Learning Techniques.

This work describes the development of a pressure-sensing array for noninvasive continuous blood pulse-wave monitoring. The sensing elements comprise a conductive polymer film and interdigital electrodes patterned on a flexible Parylene C substrate. The polymer film was patterned with microdome structures to enhance the acuteness of pressure sensing. The proposed device uses three pressure-sensing elements in a linear array, which greatly facilitates the blood pulse-wave measurement. The device exhibits high sensitivity (-0.533 kPa-1) and a fast dynamic response. Furthermore, various machine-learning algorithms, including random forest regression (RFR), gradient-boosting regression (GBR), and adaptive boosting regression (ABR), were employed for estimating systolic blood pressure (SBP) and diastolic blood pressure (DBP) from the measured pulse-wave signals. Among these algorithms, the RFR-based method gave the best performance, with the coefficients of determination for the reference and estimated blood pressures being R² = 0.871 for SBP and R² = 0.794 for DBP, respectively.

A novel role of KIF3b in the seminoma cell cycle.

KIF3b is a protein of the kinesin-2 family which plays an important role in intraflagellar transport. Testis cancer is a common cancer among young men. Its diagnostic rate is increasing and over half of the cases are seminomas. Many aspects of the mechanism and gene expression background of this cancer remain unclear. Using western-blotting and semi-quantitative PCR we found high protein levels of KIF3b enrichment in seminoma tissue despite the mRNA levels remaining equivalent to that of normal testicular tissues. The distribution of KIF3b was mainly in cells with division potential. Wound-healing assays and cell counting kit assays showed that the knockdown of KIF3b significantly suppressed cell migration ability, viability and number in HeLa cells. Immunofluorescence images during the cell cycle revealed that KIF3b tended to gather at the spindles and was enriched at the central spindle. This indicated that KIF3b may also have direct impacts upon spindle formation and cytokinesis. By counting the numbers of nuclei, spindles and cells, we found that the rates of multipolar division and multi-nucleation were raised in KIF3b-knockdown cells. In this way we demonstrate that KIF3b functions importantly in mitosis and may be essential to seminoma cell division and proliferation as well as being necessary for normal cell division.

KIFC1 and myosin Va: two motors for acrosomal biogenesis and nuclear shaping during spermiogenesis of Portunus trituberculatus.

To investigate the molecular mechanisms underlying the spermiogenesis of the swimming crab Portunus trituberculatus, full lengths of motor proteins KIFC1 and myosin Va were cloned by rapid-amplification of cDNA ends from P. trituberculatus testes cDNA, and their respective probes and specific antibodies were used to track their localization during sperm maturation. Antisense probes were designed from the gene sequences and used to detect the mRNA levels of each gene. According to the results of fluorescence in situ hybridization (FISH), the transcription of kifc1 and myosin Va began at the mid-stage of spermatids, with the kifc1 mRNA being most active at the location where the acrosome cap was formed and the myosin Va was more concentrated in the acrosome complex. Immunofluorescence results showed that KIFC1 and myosin Va were highly expressed in each stage of spermigenesis. In the early spermatids, they were randomly dispersed in the cytoplasm together with cytoskeletons. At the mid-stage, the motors were gathered above one side of the nucleus where the acrosome would later form. In the late spermatids and mature sperm, the KIFC1 was closely distributed in the perinuclear region, indicating its role in nucleus deformation. Myosin Va was distributed in the acrosome complex until sperm maturity. This suggests myosin Va's potential role in material transportation during acrosome formation and maturation. The above results provide a preliminary illustration of the essential roles of KIFC1 and myosin Va in the spermiogenesis of the swimming crab P. trituberculatus.

The potential function of prohibitin during spermatogenesis in Chinese fire-bellied newt Cynops orientalis.

Prohibitin proteins are multifunctional proteins located mainly at the inner membrane of mitochondria expressed in universal species. They play a vital role in mitochondria's function, cell proteolysis, senescence, apoptosis and as a substrate for ubiquitination. In this study, we used PCR cloning, protein and nucleotide acids alignment, protein structure prediction, western blot, in situ hybridization and immunofluorescence to study the characteristics of the prohibitin gene and the potential role of prohibitin in spermatogenesis and spermiogenesis processes in the Chinese fire-bellied newt Cynops orientalis. First, we cloned a 1452-bp full-length cDNA from the testis of Cynops orientalis. Second, we found that the 272 amino acids of prohibitin have a SPFH family domain. Thirdly, the western blots showed high expression of prohibitin in testis while the protein size was approximately 32 kDa. Fourthly, the results of in situ hybridization and immunofluorescence experiments showed that most of the prohibitins travelled with the mitochondria's migration in Cynops orientalis. The quantities of mRNA decreased as spermiogenesis proceeded, although the signals of prohibitins existed during the whole period of spermatogenesis and spermiogenesis. In the mature germ cells, the signals of prohibitins were weak and aggregated at the end of the cell. Finally, we discovered that the Sertoli cells had a large quantity of prohibitins and we made several assumptions of prohibitins' potential roles in those cells. This is the first time that the relationship between mitochondria and prohibitin in different stages of the sperm cells in Cynops orientalis has been examined, which also revealed that Sertoli cells have abundant prohibitins.

Engineered Zymomonas mobilis for salt tolerance using EZ-Tn5-based transposon insertion mutagenesis system.

BACKGROUND: The cell growth and ethanol yield of Zymomonas mobilis may be detrimentally affected by salt stress frequently present in some biomass-based fermentation systems, leading to a decrease in the rate of sugar conversion to ethanol or other bioproducts. To address this problem, improving the salt tolerance of Z. mobilis is a desirable way. However, limited progress has been made in development of Z. mobilis with higher salt tolerance for some technical challenges in the past decades. Recently, transposon insertion mutant system has been widely used as a novel genetic tool in many organisms to develop mutant strains. In this study, Tn5-based transposon insertion mutagenesis system firstly used for construction of higher salt tolerance strain in Z. mobilis. RESULTS: Approximately 200 Z. mobilis ZM4 mutants were generated by using Tn5-based transposon mutagenesis system. The mutant strain ZMT2 with improved salt tolerance phenotype was obtained by screening on RM agar plates with additional 1 % NaCl. Strain ZMT2 was confirmed to exhibit better fermentation performance under NaCl stress than wild type of strain ZM4. The transposon insertion was located in ZMO1122 (himA) by genome walking. Discruption of himA gene showed that himA may play an important role in response to salt tolerance in Z. mobils. CONCLUSIONS: The mutant strain ZMT2 with a transposon insertion in himA gene of the genome showed obviously higher sugar conversion rate to ethonal under up to 2 % NaCl stress than did the wild ZM4 strain. Besides, ZMT2 exhibited shared fermentative capabilities with wild ZM4 strain under no or low NaCl stress. This report firstly showed that himA played a role in responding to NaCl stress. Furthermore, the result indicated that Tn5-based transposon mutagenesis system was a feasible tool not only for genetic engineering in Z. mobilis strain improvement, but also in tapping resistent genes.

Improving furfural tolerance of Zymomonas mobilis by rewiring a sigma factor RpoD protein.

Furfural from lignocellulosic hydrolysates is the key inhibitor for bio-ethanol fermentation. In this study, we report a strategy of improving the furfural tolerance in Zymomonas mobilis on the transcriptional level by engineering its global transcription sigma factor (σ(70), RpoD) protein. Three furfural tolerance RpoD mutants (ZM4-MF1, ZM4-MF2, and ZM4-MF3) were identified from error-prone PCR libraries. The best furfural-tolerance strain ZM4-MF2 reached to the maximal cell density (OD600) about 2.0 after approximately 30 h, while control strain ZM4-rpoD reached its highest cell density of about 1.3 under the same conditions. ZM4-MF2 also consumed glucose faster and yield higher ethanol; expression levels and key Entner-Doudoroff (ED) pathway enzymatic activities were also compared to control strain under furfural stress condition. Our results suggest that global transcription machinery engineering could potentially be used to improve stress tolerance and ethanol production in Z. mobilis.

Adaptive laboratory evolution of ethanologenic Zymomonas mobilis strain tolerant to furfural and acetic acid inhibitors.

Furfural and acetic acid from lignocellulosic hydrolysates are the prevalent inhibitors to Zymomonas mobilis during cellulosic ethanol production. Developing a strain tolerant to furfural or acetic acid inhibitors is difficul by using rational engineering strategies due to poor understanding of their underlying molecular mechanisms. In this study, strategy of adaptive laboratory evolution (ALE) was used for development of a furfural and acetic acid-tolerant strain. After three round evolution, four evolved mutants (ZMA7-2, ZMA7-3, ZMF3-2, and ZMF3-3) that showed higher growth capacity were successfully obtained via ALE method. Based on the results of profiling of cell growth, glucose utilization, ethanol yield, and activity of key enzymes, two desired strains, ZMA7-2 and ZMF3-3, were achieved, which showed higher tolerance under 7 g/l acetic acid and 3 g/l furfural stress condition. Especially, it is the first report of Z. mobilis strain that could tolerate higher furfural. The best strain, Z. mobilis ZMF3-3, has showed 94.84% theoretical ethanol yield under 3-g/l furfural stress condition, and the theoretical ethanol yield of ZM4 is only 9.89%. Our study also demonstrated that ALE method might also be used as a powerful metabolic engineering tool for metabolic engineering in Z. mobilis. Furthermore, the two best strains could be used as novel host for further metabolic engineering in cellulosic ethanol or future biorefinery. Importantly, the two strains may also be used as novel-tolerant model organisms for the genetic mechanism on the "omics" level, which will provide some useful information for inverse metabolic engineering.

[Penile necrosis resulting from post-circumcision microwave diathermy: A report of 9 cases].

OBJECTIVE: To investigate the pathogenesis and treatment of penile necrosis resulting from microwave diathermy following circumcision. METHODS: We retrospectively analyzed the clinical data about 9 cases of penile necrosis resulting from postoperative microwave diathermy following circumcision. The 9 males, aged 20 - 39 (mean 26) years, underwent traditional circumcision for redundant prepuce or phimosis in other hospitals, followed by microwave diathermy for 30 - 60 minutes daily, which resulted in penile necrosis. With no response to conservative therapy, the patients were referred to our hospital at 3 -30 days postoperatively. Of the 9 patients, 5 presented with dry gangrene and 4 with moist gangrene. Six of the patients underwent partial penectomy, including 1 that received penis lengthening.3 months later, while the other 3 underwent total penectomy for total penile necrosis followed by penile reconstruction 3 months later, with deep inferior epigastric perforator (DIEP) flaps and by implantation of the 12th costal cartilage in 2 cases and with epigastric groin island flaps and by urethroplasty in the other. RESULTS: The patients were followed up for 2 - 8 years, and all could urinate smoothly in the standing position. Of the 6 men treated by partial penectomy, 1 received penis lengthening and achieved a penile length of 7 cm and 5 had the remaining penile length of 3 -5 cm, 4 with erectile function and the other 2 capable of sexual intercourse. The 3 men treated by total penectomy achieved nearly normal external appearance of the penis, with a finalized length of (11.7 ± 1.3) cm, a circumference of (11.4 ± 2.1) cm, and a normal feel of the skin. Of the 3 cases of penile reconstruction, 2 achieved sufficient erectile hardness of the penis (grade 3) for sexual intercourse, while the other 1 remained impotent. CONCLUSION: Post-circumcision microwave diathermy may result in penile necrosis, for the management of which, early debridement is necessitated and penile lengthening or reconstruction can be performed according to the severity of the lesion and needs of the patient.

Detection of DNA damage caused by cryopreservation using a modified SCGE in large yellow croaker, Pseudosciaena crocea.

We used single-cell gel electrophoresis (SCGE) to detect the integrity of sperm DNA of the teleost large yellow croaker, Pseudosciaena crocea, cryopreserved with Cortland solution and a range of 5% to 30% DMSO concentrations in order to test how sperm cryopreservation affected the DNA stability of nuclei. Electrophoresis was conducted for 60 min at 130 mA and 15 V. The comet images were analyzed with software CometScore 1.5, and parameters such as comet length, tail length and percentage DNA in the tail were obtained. Then the comet rate and damage coefficient were calculated. Results demonstrated that there were no significant differences in motility, comet rate and damage coefficient between fresh sperm and cryopreserved sperm stored in 5%, 10%, 15% and 20% DMSO, while the sperm cryopreserved with 25% and 30% DMSO had a lower motility, higher comet length and damage coefficients than those of fresh sperm. There was a positive correlation between comet rate of cryopreserved sperm and the concentration of DMSO. Our results demonstrate that toxicity of the cryoprotectant is the main cause of DNA damage in cryopreserved sperm nuclei.

KIF2A Upregulates PI3K/AKT Signaling through Polo-like Kinase 1 (PLK1) to Affect the Proliferation and Apoptosis Levels of Eriocheir sinensis Spermatogenic Cells.

E. sinensis is an animal model for studying the reproduction and development of crustaceans. In this study, we knocked down the Es-Kif2a gene by injecting dsRNA into E. sinensis and inhibited Es-Plk1 gene expression by injecting PLK1 inhibitor BI6727 into E. sinensis. Then, the cell proliferation level, apoptosis level, and PI3K/AKT signaling expression level were detected. Our results showed that the proliferation level of spermatogenic cells decreased, while the apoptosis level increased after Es-Kif2a knockdown or Es-Plk1 inhibition. In order to verify whether these changes are caused by regulating the PI3K/AKT pathway, we detected the expression of PI3K and AKT proteins after Es-Kif2a knockdown or Es-Plk1 inhibition. Western Blot showed that in both the Es-Kif2a knockdown group and the Es-Plk1 inhibition group, the expression of PI3K and AKT proteins decreased. In addition, immunofluorescence showed that Es-KIF2A and Es-PLK1 proteins were co-localized during E. sinensis spermatogenesis. To further explore the upstream and downstream relationship between Es-KIF2A and Es-PLK1, we detected the expression level of Es-PLK1 after Es-Kif2a knockdown as well as the expression level of Es-KIF2A after Es-Plk1 inhibition. Western Blot showed that the expression of Es-PLK1 decreased after Es-Kif2a knockdown, while there was no significant change of Es-KIF2A after Es-Plk1 inhibition, indicating that Es-PLK1 may be a downstream factor of Es-KIF2A. Taken together, these results suggest that Es-KIF2A upregulates the PI3K/AKT signaling pathway through Es-PLK1 during the spermatogenesis of E. sinensis, thereby affecting the proliferation and apoptosis levels of spermatogenic cells.

Regulation of paracellular permeability: factors and mechanisms.

Epithelial permeability is composed of transcellular permeability and paracellular permeability. Paracellular permeability is controlled by tight junctions (TJs). Claudins and occludin are two major transmembrane proteins in TJs, which directly determine the paracellular permeability to different ions or large molecules. Intracellular signaling pathways including Rho/Rho-associated protein kinase, protein kinase Cs, and mitogen-activated protein kinase, modulate the TJ proteins to affect paracellular permeability in response for diverse stimuli. Cytokines, growth factors and hormones in organism can regulate the paracellular permeability via signaling pathway. The transcellular transporters such as Na-K-ATPase, Na(+)-coupled transporters and chloride channels, can interact with paracellular transport and regulate the TJs. In this review, we summarized the factors affecting paracellular permeability and new progressions of the related mechanism in recent studies, and pointed out further research areas.