Sestrin2 mediates FOXM1 expression to block the EMT process in non-small cell lung cancer through the AMPK/YAP pathway.

Non-small cell lung cancer (NSCLC) is characterized by high incidence and mortality, severely threatening human health. The infinite growth and metastasis of NSCLC cells result in a poor prognosis. Therefore, our study was to investigate the mechanism of Sestrin2 on the epithelial-mesenchymal transition (EMT) process of NSCLC cells. Human embryonic lung fibroblasts, NSCLC cell lines, and nude mice were experimental subjects in this study. qRT-PCR and western blot were performed to evaluate the mRNA and protein expression of genes. CCK-8 and EdU assay were conducted to detect cell proliferation. The scratch test and Transwell assay were applied to examine cell migration and invasion. The bioinformatics analysis and Co-IP assay were employed to predict and consolidate the interaction between YAP and TEAD. We found the expression of Sestrin2 was declined but the expression of YAP was elevated in NSCLC cells. Sestrin2 sufficiency or YAP silencing could effectively impair cell growth and metastasis. Mechanistically, YAP interacted with TEAD to enhance FOXM1 expression. Additionally, the elevation of FOXM1 abolished the inhibitory influences of Sestrin2 sufficiency on NSCLC cell growth, invasion, and EMT process. Eventually, Sestrin2 elevation attenuated tumor growth in mice via modulation of the AMPK/YAP/FOXM1 axis, which was reversed by FOXM1 overexpression. Our consequences suggested Sestrin2 could inhibit the activation of YAP via prompting AMPK phosphorylation and then suppress FOXM1 expression through the interplay between YAP and TEAD to impair the capacities of NSCLC cell proliferation, migration, invasion, and EMT. This study provided a novel mechanism of Sestrin2 in NSCLC.

A Combination of Genomics, Transcriptomics, and Genetics Provides Insights into the Mineral Weathering Phenotype of Pseudomonas azotoformans F77.

Silicate mineral weathering (dissolution) plays important roles in soil formation and global biogeochemical cycling. In this study, a combination of genomics, transcriptomics, and genetics was used to identify the molecular basis of mineral weathering activity and acid tolerance in Pseudomonas azotoformans F77. Biotite was chosen as a silicate mineral to investigate mineral weathering. The genome of strain F77 was sequenced, and the genes significantly upregulated when grown in the presence of biotite included mineral weathering-related genes associated with gluconic acid metabolism, flagellar assembly, and pilus biosynthesis and acid tolerance-related genes associated with neutralizing component production, reducing power, and proton efflux. The biotite-weathering behaviors of strain F77 and its mutants that were created by deleting the tkt, tal, and gntP genes, which are involved in gluconic acid metabolism, and the potF, nuoF, and gdtO genes, which are involved in acid tolerance, were determined. The Fe and Al concentrations in the strain F77-inoculated medium increased 2.2- to 13.7-fold compared to the controls. The cell numbers of strain F77 increased over time, while the pH values in the medium ranged from 3.75 to 3.90 between 20 and 36 h of incubation. The release of Al and Fe was significantly reduced in the F77 Δtal, F77 ΔgntP, F77 ΔpotF, and F77 ΔnuoF mutants. Bacterial growth was significantly reduced in the presence of biotite in the F77 ΔpotF and F77 ΔnuoF mutants. Our results demonstrated the acid tolerance of strain F77 and suggested that multiple genes and metabolic pathways in strain F77 are involved in biotite weathering and acid tolerance during the mineral weathering process. IMPORTANCE Acid production and tolerance play important roles in effective and persistent mineral weathering in bacteria, although the molecular mechanisms governing acid production and acid tolerance in bacteria have not been fully elucidated. In this study, the molecular mechanisms underlying biotite (as a silicate mineral) weathering (dissolution) and acid tolerance of P. azotoformans F77 were characterized using genomics, transcriptomics, and genetics analyses. Our results showed that the genes and metabolic pathways for gluconic acid metabolism, flagellar assembly, and pilus biosynthesis may play important roles in mineral weathering by strain F77. Notably, the genes associated with neutralizing component production, reducing power, and proton efflux may be related to acid tolerance in strain F77. The expression of these acid production- and acid tolerance-related genes was observed to be increased by biotite in strain F77. Our findings may help to elucidate the molecular mechanisms governing mineral weathering and, especially, acid tolerance in mineral-weathering bacteria.

Interactions between Biotite and the Mineral-Weathering Bacterium Pseudomonas azotoformans F77.

In this study, the mineral-weathering bacterium Pseudomonas azotoformans F77, which was isolated from the soil of a debris flow area, was evaluated for its weathering activity under direct contact with biotite or without contact. Then, biotite-weathering behaviors of strain F77, mutants that had been created by deleting the gcd and adh genes (which are involved in gluconic acid metabolism and pilus formation, respectively), and the double mutant F77ΔgcdΔadh were compared. The relative gene expression levels of F77 and its mutants F77Δgcd and F77Δadh were also analyzed in the presence of biotite. Direct contact with biotite increased Fe and Al release from the mineral in the presence of F77. All strains had similar abilities to release Fe and Al from the mineral except for F77Δgcd and F77Δadh Mobilized Fe and Al concentrations were decreased by up to 72, 26, and 87% in the presence of F77Δgcd, F77Δadh, and F77ΔgcdΔadh, respectively, compared to levels observed in the presence of F77 during the mineral-weathering process. Gluconic acid production was decreased for F77Δgcd and F77ΔgcdΔadh, while decreased cell attachment on the mineral surface was observed for F77Δadh, compared to findings for F77. The F77 genes involved in pilus formation and gluconic acid metabolism showed increased expression levels in the presence of biotite. The results of this study showed important roles for the genes involved in gluconic acid metabolism and pilus formation in mineral weathering by F77 and demonstrated the distinctive effect of these genes on mineral weathering by F77.IMPORTANCE Bacteria play important roles in mineral weathering and soil formation, although the molecular mechanisms underlying the interactions between bacteria and silicate minerals are poorly understood. In this study, the interactions between biotite and the highly effective mineral-weathering bacterium P. azotoformans F77 were characterized. Our results showed that the genes involved in gluconic acid metabolism and pilus formation play important roles in mineral weathering by F77. The presence of biotite could promote the expression of these genes in F77, and a distinctive effect of these genes on mineral weathering by F77 was observed in this study. Our results provide new knowledge and promote better understanding regarding the interaction between silicate minerals and mineral-weathering bacteria, as well as the molecular mechanisms involved in these processes.

Isolation and characterization of mineral-dissolving bacteria from different levels of altered mica schist surfaces and the adjacent soil.

Microorganisms play important role in mineral weathering. However, little is known about rock-associated mineral-dissolving bacteria. In this study, 129 bacterial isolates were obtained from the less and more weathered mica schist surfaces and the adjacent soil and characterized for mineral dissolving activity, population, and the linkage of rock weathering level and distribution of the bacteria. Among the 129 isolates, 112 isolates could dissolve biotite. The relative abundance of the highly effective Fe solubilizers was significantly higher on the more altered rock surface (89.6%) than in the soil (51.2%) and on the less altered rock surface (22.5%), while the relative abundance of the highly effective Si solubilizers was significantly higher in the soil (65.9%) than on the more (41.7%) and less (12.5%) altered rock surfaces. Furthermore, 17.5-42.5%, 87.5%, and 60.9-90.2% of the highly effective acid- and siderophore-producing isolates were obtained in the less and more weathered rocks and the soil, respectively. The mineral-dissolving bacteria belonged to 18 genera and Burkholderia, Bacillus, and Paenibacillus were the dominant and highly effective mineral-dissolving bacteria. Phylogenetic analysis found 2, 9, and 5 bacterial species in the highly effective mineral-dissolving bacteria on the less and more altered rock surfaces and in the soil, respectively. The results showed the abundant and diverse mineral-dissolving bacterial populations on the more weathered rock surfaces. The results also suggested distinct mineral-dissolving activities and mechanisms of the bacteria and highlighted the possibility for the development of bacterial inocula for plant nutrition improvement in silicate mineral-rich soils.

The E3 Ubiquitin Ligase CRL4 Regulates Proliferation and Progression Through Meiosis in Chinese Mitten Crab Eriocheir sinensis.

The Cullin-RING E3s are multisubunit ubiquitin ligases composed of a scaffold protein known as Cullin, a RING finger protein that regulates diverse cellular pathways; however, their contribution to male gonad development, especially the spermatogenesis of the Chinese mitten crab (Eriocheir sinensis), is not well understood. We identified five evolutionarily conserved Cullins from the transcriptome and genome ofE. sinensis that are potentially involved in regulating male gonad development. The aim of the current study was to determine the mechanisms of Cullin4's effects on spermatogenesis. We observed that Cullin4, p53, and proliferating cell nuclear antigen had a specific expression and localization in primary spermatocytes. We also investigated the accumulation of Cullin substrates by treatment with inhibitor of NEDD8-activating enzyme MLN4924 in vitro. Cell cycle inhibitors p27 and p21 accumulated significantly after 24 and 36 h, respectively. We speculated that p53-mediated spontaneous germ cell apoptosis acts as a quality control mechanism to eliminate defective germ cells and that the Cullin4 complex maintains p53, p21, and p27 homeostasis in primary spermatocytes to regulate spermatogenesis ofE. sinensis Given its widespread evolutionary conservation, Cullin4 may regulate germ line development similarly in other organisms.

Morphological alterations of all stages of spermatogenesis and acrosome reaction in Chinese mitten crab Eriocheir sinensis.

The Chinese mitten crab (Eriocheir sinensis) is a seasonally breeding species and its reproductive system comprises paired symmetrical structures: testes, vasa deferentia, seminal vesicles, accessory glands and ejaculatory ducts. Histological examination of the testis of mature males reveals regression of the gonads and inhibition of the process of spermatogenesis during December to April of the following year, the regeneration of the gonads during June to July and the occurrence of the highest level of spermatogenesis during August to October. Microscopic assessments and hematoxylin and eosin (H&E) staining were used to describe all spermatogenic stages (spermatogonia, primary and secondary spermatocytes, spermatids and spermatozoids). To observe the morphological changes during spermiogenesis, we successfully initiated primary cell culture using testis tissue of E. sinensis, which will lay a solid foundation for further work on the immortalization of crab cells. During the interaction between the sperm and oocyte, the fertilizing spermatozoon must undergo a series of terminal morphological changes, called the acrosome reaction (AR). This study also provides a detailed description of the structural alterations of the acrosome reaction of E. sinensis. The acrosome complex and cup-shaped nucleus are located at the anterior and posterior of the spermatozoon, respectively. Male germ cell development involves a tightly controlled sequence of differentiation switches. The purpose of this study is to increase our knowledge of the morphological alterations during spermatogenesis and the acrosome reaction, whose changes are a fundamental requirement for fertilization of E. sinensis.

[Immune Reconstitution after BTKi Treatment in Chronic Lymphocytic Leukemia].

OBJECTIVE: To analyze the immune reconstitution after BTKi treatment in patients with chronic lymphocytic leukemia (CLL). METHODS: The clinical and laboratorial data of 59 CLL patients admitted from January 2017 to March 2022 in Fujian Medical University Union Hospital were collected and analyzed retrospectively. RESULTS: The median age of 59 CLL patients was 60.5(36-78). After one year of BTKi treatment, the CLL clones (CD5 +/CD19 +) of 51 cases (86.4%) were significantly reduced, in which the number of cloned-B cells decreased significantly from (46±6.1)×109/L to (2.3±0.4)×109/L (P =0.0013). But there was no significant change in the number of non-cloned B cells (CD19 + minus CD5 +/CD19 +). After BTKi treatment, IgA increased significantly from (0.75±0.09)g/L to (1.31±0.1)g/L (P <0.001), while IgG and IgM decreased from (8.1±0.2)g/L and (0.52±0.6)g/L to (7.1±0.1)g/L and (0.47±0.1)g/L, respectively (P <0.001, P =0.002). BTKi treatment resulted in a significant change in T cell subpopulation of CLL patients, which manifested as both a decrease in total number of T cells from (2.1±0.1)×109/L to (1.6±0.4)×109/L and NK/T cells from (0.11±0.1)×109/L to (0.07±0.01)×109/L (P =0.042, P =0.038), both an increase in number of CD4 + cells from (0.15±6.1)×109/L to (0.19±0.4)×109/L and CD8 + cells from (0.27±0.01)×109/L to (0.41±0.08)×109/L (both P <0.001). BTKi treatment also up-regulated the expression of interleukin (IL)-2 while down-regulated IL-4 and interferon (IFN)-γ. However, the expression of IL-6, IL-10, and tumor necrosis factor (TNF)-α did not change significantly. BTKi treatment could also restored the diversity of TCR and BCR in CLL patients, especially obviously in those patients with complete remission (CR) than those with partial remission (PR). Before and after BTKi treatment, Shannon index of TCR in patients with CR was 0.02±0.008 and 0.14±0.001 (P <0.001), while in patients with PR was 0.01±0.03 and 0.05±0.02 (P >0.05), respectively. Shannon index of BCR in patients with CR was 0.19±0.003 and 0.33±0.15 (P <0.001), while in patients with PR was 0.15±0.009 and 0.23±0.18 (P <0.05), respectively. CONCLUSIONS: BTKi treatment can shrink the clone size in CLL patients, promote the expression of IgA, increase the number of functional T cells, and regulate the secretion of cytokines such as IL-2, IL-4, and IFN-γ. BTKi also promote the recovery of diversity of TCR and BCR. BTKi treatment contributes to the reconstitution of immune function in CLL patients.

Effects of Alternating Magnetic Fields on the OER of Heterogeneous Core-Shell Structured NiFe2O4@(Ni, Fe)S/P.

Composition optimization, structural design, and introduction of external magnetic fields into the catalytic process can remarkably improve the oxygen evolution reaction (OER) performance of a catalyst. NiFe2O4@(Ni, Fe)S/P materials with a heterogeneous core-shell structure were prepared by the sulfide/phosphorus method based on spinel-structured NiFe2O4 nanomicrospheres. After the sulfide/phosphorus treatment, not only the intrinsic activity of the material and the active surface area were increased but also the charge transfer resistance was reduced due to the internal electric field. The overpotential of NiFe2O4@(Ni, Fe)P at 10 mA cm-2 (iR correction), Tafel slope, and charge transfer resistance were 261 mV, 42 mV dec-1, and 3.163 Ω, respectively. With an alternating magnetic field, the overpotential of NiFe2O4@(Ni, Fe)P at 10 mA cm-2 (without iR correction) declined by 45.5% from 323 mV (0 mT) to 176 mV (4.320 mT). Such enhancement of performance is primarily accounted for the enrichment of the reactive ion OH- on the electrode surface induced by the inductive electric potential derived from the Faraday induction effect of the AMF. This condition increased the electrode potential and thus the charge transfer rate on the one hand and weakened the diffusion of the active substance from the electrolyte to the electrode surface on the other hand. The OER process was dominantly controlled by the charge transfer process under low current conditions. A fast charge transfer rate boosted the OER performance of the catalyst. At high currents, diffusion exerted a significant effect on the OER process and low OH- diffusion rates would lead to a decrease in the OER performance of the catalyst.

Enhanced Alkaline Oxygen Evolution Using Spin Polarization and Magnetic Heating Effects under an AC Magnetic Field.

Renewable electricity from splitting water to produce hydrogen is a favorable technology to achieve carbon neutrality, but slow anodic oxygen evolution reaction (OER) kinetics limits its large-scale commercialization. Electron spin polarization and increasing the reaction temperature are considered as potential ways to promote alkaline OER. Here, it is reported that in the alkaline OER process under an AC magnetic field, a ferromagnetic ordered electrocatalyst can simultaneously act as a heater and a spin polarizer to achieve significant OER enhancement at a low current density. Moreover, its effect obviously precedes antiferromagnetic, ferrimagnetic, and diamagnetic electrocatalysts. In particular, the noncorrected overpotential of the ferromagnetic electrocatalyst Co at 10 mA cm-2 is reduced by a maximum of 36.6% to 243 mV at 4.320 mT. It is found that the magnetic heating effect is immediate, and more importantly, it is localized and hardly affects the temperature of the entire electrolytic cell. In addition, the spin pinning effect established on the ferromagnetic/paramagnetic interface generated during the reconstruction of the ferromagnetic electrocatalyst expands the ferromagnetic order of the paramagnetic layer. Also, the introduction of an external magnetic field further increases the orderly arrangement of spins, thereby promoting OER. This work provides a reference for the design of high-performance OER electrocatalysts under a magnetic field.

[Clinical observation of transforaminal endoscopy combined with interspinous fusion in the treatment of lumbar spinal stenosis with instability in the elderly].

OBJECTIVE: To observe the clinical effect of transforaminal endoscopy combined with interspinous fusion in the treatment of lumbar spinal stenosis with instability in the elderly. METHODS: From February 2018 to February 2019, 82 elderly patients with lumbar spinal stenosis and instability were divided into control group and observation group. In the control group, there were 23 males and 18 females;the age was (68.9±4.1) years;the course of disease was (14.1±5.7) months;there were 5 cases of single segment lesions and 36 cases of multi segment lesions;simple bacfuse interspinous fusion was used. In the observation group, there were 22 males and 19 females;the age was (69.1±4.0) years;the course of diseasewas (14.4±5.5) months;there were 6 cases of single segment lesions and 35 cases of multi segment lesions;they were treated with transforaminal endoscopic surgery combined with Bacfuse interspinous fusion. The clinical efficacy, visual analogue scale (VAS), Japanese Orthopaedic Association scores (JOA), Oswestry disability index (ODI), Lehmann lumbar function score, posterior disc height and intervertebral foramen height, complication rate and recurrence rate of the two groups were compared. RESULTS: The clinical efficacy of the observation group was better than that of the control group;the VAS score of the observation group was lower than that of the control group, the JOA score was higher than that of the control group, and the ODI index at 3 months after operation and at the last follow-up was lower than that of the control group, the Lehmann lumbar function score was higher than that of the control group;the posterior edge height of intervertebral disc and intervertebral foramen height were higher than those of the controlgroup;the incidence of complications and recurrence rate (4.9% and 0.0%) of the observation group were lower than those of the control group (19.5%, 9.8%), the difference was statistically significant (P<0.05). CONCLUSION: The clinical effect of transforaminal endoscopy combined with interspinous process fusion in the treatment of lumbar spinal stenosis with instability in the elderly is ideal. It can reduce postoperative pain, improve lumbar function, improve the height of posterior edge of intervertebral disc and intervertebral foramen, and reduce the incidence and recurrence rate. It is worthy of clinical promotion.

[Modified transverse tibial bone transplantation and microcirculation reconstruction for the treatment of end-stage diabetic foot].

OBJECTIVE: To explore clinical effect of modified transverse tibial bone transfer microcirculation reconstruction in treating end-stage diabetic foot. METHODS: From August 2016 to June 2018, 87 patients with diabetic foot treated with modified tibial transverse bone removal and microcirculation reconstruction, inclduing 54 males and 33 females;aged from 39 to 95 years old with an average of (68.9±11.3) years old;2 patients were grade 2, 37 patients were grade 3 and 50 patients were grade 4 according to Wagner's classification;the courses of diabetic were for 10 to 16 years with an average of (13.0±2.2) years;the courses of diabetic feet were for 21 to 48 days with an avergae of (34.2±8.6) days. Postoperative comlications were observed. Skin temperature, visual analogue scale(VAS) and ankle brachial index(ABI) and wound healing were recorded before and 3 months after operation. RESULTS: All patients were followed up for 4 to 19 months with an average of (12.6±2.8) months. Two patients occurred subcutaneous tissue liquefaction and seepage under needle passage during bone transfer, and scabed without special treatment. One patient was performed amputation above 5 cm of ankle joint because of severe infection, and 1 patient occurred re-ulceration at 1 year after wound healing, bone transfer was performed again at the same site, and was completely healed at 8 weeks after operation. The healing time of wound ranged from 3 to 24 weeks with an average of (11.9± 3.8) weeks. Foot skin temperature before operation was (28.9±0.91) ℃, and increased to (31.70±0.32)℃ at 3 months after operation(t=5.72 P=0.006);VAS score before opertaion was (7.80±0.72), and improved to (2.20±0.13) at 3 months after operation (t=25.38, P=0.000);ABI beforeoperation was(0.48±0.30), increased to(0.98±0.24) at 3 months after oeprtaion(t= 14.68, P=0.000). CONCLUSION: Modified lateral tibial bone transfer could effectively reconstruct microvascular network under lower leg, promote recovery of peripheral blood vessels, and promote wound healing of foot, reduce or avoid amputation. At the same time, the improved osteotomy is one of the effective methods for the treatment of diabetic foot which has advantags of less trauma, simple opertaion.

Fabrication of Magnetic Superstructure NiFe2O4@MOF-74 and Its Derivative for Electrocatalytic Hydrogen Evolution with AC Magnetic Field.

As an ideal hydrogen production route, electrolyzed water still faces the challenges of high cost of noble-metal electrocatalysts and low performance of non-noble-metal catalysts in scalable applications. Recently, introduction of external fields (such as magnetic fields, light fields, etc.) to improve the electrocatalytic water splitting performance of non-noble-metal catalysts has attracted great attention due to their simplicity. Here, a simple method for preparing magnetic superstructure (NiFe2O4@MOF-74) is described, and the hydrogen evolution reaction (HER) behavior of its carbonized derivative, a ferromagnetic superstructure, is revealed in a wide range of applied voltage under an AC magnetic field. The overpotential (@10 mA cm-2) required for the HER of the obtained ferromagnetic superstructure in 1 M KOH was reduced by 31 mV (7.7%) when a much small AC magnetic field (only 2.3 mT) is applied. Surprisingly, the promotion effect of the AC magnetic field is not monotonically increasing with the increase of the applied voltage or the strength of AC magnetic field, but increasing first, then weakening. This unusual behavior is believed to be mainly caused by the enhanced induced electromotive force and the additional energy by the applied AC magnetic field. This discovery provides a new idea for adjusting the performance of electrocatalytic reactions.

[The effect of different instrumentation on idiopathic scoliosis].

OBJECTIVE: To investigate the effectiveness of different instrumentation on idiopathic scoliosis. METHODS: The information before and after the operations of 94 cases of idiopathic scoliosis, which included Harrington, Harrington-Luque (H-L), Cotrel-Dubousset (C-D), and TSRH 3-dimension correction, were collected and analyzed on a long-term basis, and different operative methods were compared for their advantages and disadvantages. RESULTS: Based on 1 to 3 years inquiries following 94 cases, the respective average correcting rates of the above mentioned instrumentations were 36.42%, 45.18%, 55.68%, 63.28%, which proved that H-L, C-D and TSRH were superior to Harrington in the correction rate and that TSRH 3-dimension correction was even more remarkable. CONCLUSION: The effectiveness of 3-dimension correction was superior to those of other instrumentation.

[Seminal parameter analysis in noninflammatory chronic prostatitis/chronic pelvic pain syndrome].

OBJECTIVE: To investigate seminal parameters in noninflammatory chronic prostatitis/chronic pelvic pain syndrome (CAP III B). METHODS: A total of 74 consecutive cases of patients who had been diagnosed as CAP III B and 46 cases of controls were included in the study. Severity of symptoms in men with CAP III B was defined according to the NIH Chronic Prostatitis Symptom Index (NIH-CPSI). All of them underwent a 'four glass-test' including leukocyte determination in expressed prostatic secretions (EPS), voided urine after prostatic massage (VB3) and ejaculate semen followed by analysis according to WHO. The analysis included seminal volume, pH, duration of liquefaction, sperm density, vitality, motility(a + b) and morphology. Correlations between the duration or the severity of symptoms and spermiogram results in patients with CAP III B were assessed respectively. RESULTS: The CAP III B group and the control group differed significantly in ejaculate volume, duration of liquefaction and motility, while the remaining parameters did not differ significantly. The duration of chronic pelvic pain showed apparently positive correlationship with liquefaction time, while the symptom duration negatively correlated with sperm motility. The NIH-CPSI score had no significant relationship with seminal volume, duration of liquefaction and sperm motility. CONCLUSION: Our results indicate that CAP III B can have a significant negative impact on sperm volume, liquefaction and motility. Our data also supports the results that the longer the duration of symptoms, the more influences on semen liquefaction and motility might be.

Profiling microRNAs in the testis during sexual maturation stages in Eriocheir sinensis.

MicroRNAs (miRNAs) have been implicated in several cellular processes of reproductive tissues through post-transcriptional regulation of protein coding genes. The presence of miRNAs, their expression patterns and processing machinery genes in different stages of testicular and ovarian cellular development have demonstrated the potential role of miRNAs in testicular and ovarian physiology. The Chinese mitten crab (Eriocheir sinensis) is one of the most important aquaculture species in China and has high commercial value as a food source. The molecular mechanisms underlying testis development in these and other crustaceans, which migrate to a new habitat to breed, remain poorly understood. We focus on the gonad of E. sinensis and systematically examined the expression profile of miRNAs in testes during sexual maturation stages using the Illumina Solexa sequencing technology. We found that the microRNA transcriptome exhibited dynamic expression during crab testis development. Subsequent bioinformatic analysis on both conserved and 15 novel miRNAs illustrated that some miRNAs demonstrated a tissue-specific expression pattern and were associated with target genes involved in reproductive function. Our study illustrates how detailed profiling of miRNA expression during stages of sexual maturation and in different tissues can lead to elucidate the role of miRNAs in regulating the development and differentiation of reproductive organs.

P38 participates in spermatogenesis and acrosome reaction prior to fertilization in Chinese mitten crab Eriocheir sinensis.

P38 mitogen-activated protein kinases (MAPKs) comprise a family of serine/threonine protein kinases that play important roles in cellular responses to inflammatory cytokines and environmental stresses. These kinases are involved in controlling cell division, differentiation and death in mammalian testes and therefore are critical to spermatogenesis. To explore their functions in male reproduction of Chinese mitten crabs, Eriocheir sinensis p38 (Es-p38) protein expression was determined in different tissues including testes at different developmental stages by Western blot. Es-p38 was expressed in various tissues, with higher levels in the heart, stomach, gills and testes. Total Es-p38 protein levels increased gradually during spermatogenesis, but phosphorylated Es-p38 was much higher in the spermatid (August-October) than the spermatocyte (July-August) and sperm (October-January) stages. Trypan blue staining and hematoxylin/eosin staining were both used to detect sperm motility and changes in sperm morphology during the acrosome reaction (AR) induced by pre-incubation with A23187 in vitro, activated Es-p38 proteins detected by fluorescent microscopy were translocated gradually to nuclear and apical cap regions, accumulating at the anterior of the acrosomal tubule. The results suggest the involvement of p38 MAPK in spermatogenesis and the AR in E. sinensis.

ERK is involved in the process of acrosome reaction in vitro of the Chinese mitten crab, Eriocheir sinensis.

Mitogen-activated protein kinases (MAPKs), also termed extracellular signal-regulated kinases (ERKs), are cytoplasmic and nuclear serine/threonine kinases involved in signal transduction of several extracellular effectors. In mammals, ERKs participate in the regulation of spermatogenesis, mature spermatozoa motility, hyperactivation, and the acrosome reaction. To investigate ERK functions in Eriocheir sinensis reproduction, we successfully cloned the full-length ERK from the testis of E. sinensis (ES-ERK). The 1098-nucleotide open reading frame encodes a 365-amino-acid protein with a predicted molecular weight of 42 kDa. Expressions of ES-ERK in different tissues and testis development stages were detected by the quantitative RT-PCR and Western blotting. ES-ERK is expressed relatively highly in the testis. The expression of ES-ERK protein gradually increased in the spermatid stage, reaching a peak in sperm stage. Western blotting showed a similar expression pattern for the total ES-ERK protein, but phospho-ERK (p-ERK) showed the higher expression in spermatid than sperm stage. We also used trypan blue and hematoxylin and eosin staining to identify structural changes in E. sinensis spermatozoa during the process of acrosome reaction (AR). After stimulating the process of AR, the ES-ERK has translocated from the nucleus to the acrosomal tubule. This result suggested that the ERK MAPK might be involved in the regulation of the E. sinensis acrosome reaction.

Effects of combined exercise on gait variability in community-dwelling older adults.

Training that focuses on strength, balance, and endurance, the so-called combined exercise, can enhance physical function, including gait, according to a literature review. However, the effects of combined exercise on improving gait variability are limited. The objective of this study is to investigate the effects of 12 weeks of combined exercise comprised of resistance, endurance, and balance training on gait performance in older adults. Twenty-nine community-dwelling older adults were recruited and assigned to either the experimental group (n = 17) or the control group (n = 12). The 12-week intervention was a combined exercise program at 1 h per day and 3 days per week. The participants received an assessment for both a 6-min walk and gait during both habitual walking and fast walking conditions at pre-intervention and after 8 and 12 weeks of exercise. The 6-min walk was used to assess gait endurance. GAITRite was used to evaluate gait. An analysis of covariance with the pretest score as the covariate was used to determine the difference in each dependent variable between groups. The level of significance was set as p less than 0.05. Our results showed significant between-group effects in the 6-min walk and velocity, stride time, and stride length in both conditions after 8 weeks of exercise and significant between-group effects in the 6-min walk test and all selected gait parameters in both conditions after 12 weeks of exercise. Our findings demonstrate that a 12-week combined exercise program may positively affect gait endurance and gait performance including gait variability in habitual walking and fast walking conditions among older adults. The current study provides important evidence of short-term combined exercise effects on improvements in gait performance.

[Diagnosis and treatment of adrenal medullary hyperplasia (with report of 8 cases)].

OBJECTIVE: To study the diagnosis and treatment of adrenal medullary hyperplasia (AMH). METHODS: An retrospective analysis of the clinical data of 8 patients with AMH admitted in our hospital from May 1998 to May 2002 were conducted with a review of the follow-up study. RESULTS: CT scanning of the adrenal gland showed unilateral abnormal appearance in all 8 cases. Diagnoses of AMH in 4 patients and pheochromocytoma in the other 4 patients were established before surgery. All the patients underwent unilateral adrenalectomy, among whom 7 were cured and 1 suffered recurrence 1 month after operation because of medullar hyperplasia in the contralateral adrenal gland. CONCLUSIONS: AMH should be differentiated from pheochromocytoma, especially from adrenal nodules shown by catecholamin assay. Definite diagnosis depends on pathological examination and surgical removal through abdominal approach is the best choice of treatment, in which both sides of the adrenal glands should be explored.