Neuronal MeCP2 in the dentate gyrus regulates mossy fiber sprouting of mice with temporal lobe epilepsy.

Sprouting of mossy fibers, one of the most consistent findings in tissue from patients with mesial temporal lobe epilepsy, exhibits several uncommon axonal growth features and has been considered a paradigmatic example of circuit plasticity that occurs in the adult brain. Clarifying the mechanisms responsible may provide new insight into epileptogenesis as well as axon misguidance in the central nervous system. Methyl-CpG-binding protein 2 (MeCP2) binds to methylated genomic DNA to regulate a range of physiological functions implicated in neuronal development and adult synaptic plasticity. However, exploring the potential role of MeCP2 in the documented misguidance of axons in the dentate gyrus has not yet been attempted. In this study, a status epilepticus-induced decrease of neuronal MeCP2 was observed in the dentate gyrus (DG). An essential regulatory role of MeCP2 in the development of functional mossy fiber sprouting (MFS) was confirmed through stereotaxic injection of a recombinant adeno-associated virus (AAV) to up- or down-regulate MeCP2 in the dentate neurons. Chromatin immunoprecipitation sequencing (ChIP-seq) was performed to identify the binding profile of native MeCP2 using micro-dissected dentate tissues. In both dentate tissues and HT22 cell lines, we demonstrated that MeCP2 could act as a transcription repressor on miR-682 with the involvement of the DNA methylation mechanism. Further, we found that miR-682 could bind to mRNA of phosphatase and tensin homolog (PTEN) in a sequence specific manner, thus leading to the suppression of PTEN and excessive activation of mTOR. This study therefore presents a novel epigenetic mechanism by identifying MeCP2/miR-682/PTEN/mTOR as an essential signal pathway in regulating the formation of MFS in the temporal lobe epileptic (TLE) mice. SIGNIFICANCE STATEMENT: Understanding the mechanisms that regulate axon guidance is important for a better comprehension of neural disorders. Sprouting of mossy fibers, one of the most consistent findings in patients with mesial temporal lobe epilepsy, has been considered a paradigmatic example of circuit plasticity in the adult brain. Although abnormal regulation of DNA methylation has been observed in both experimental rodents and humans with epilepsy, the potential role of DNA methylation in this well-documented example of sprouting of dentate axon remains elusive. This study demonstrates an essential role of methyl-CpG-binding protein 2 in the formation of mossy fiber sprouting. The underlying signal pathway has been also identified. The data hence provide new insight into epileptogenesis as well as axon misguidance in the central nervous system.

Incidence trends and survival of metastatic prostate cancer with bone and visceral involvement: 2010-2019 surveillance, epidemiology, and end results.

Background: The incidence of prostate cancer (PCa) has continued to increase since the US Preventive Services Task Force (USPSTF) recommendations against prostate-specific antigen (PSA)-based screening for all men in 2012, approximately half of additional diagnosed cases are advanced-stage, including regional PCa and metastatic PCa (mPCa). It is very important to investigate the shift in mPCa incidence and mPCa-related mortality risk, as the survival of mPCa remains poor. Objective: To investigate the incidence temporal trend of mPCa stratified by metastatic site, including bone and visceral metastatic involvement, and potential survival improvements. Materials: Based on the recently released Surveillance, Epidemiology, and End Results (SEER) data (2010-2019), the age-adjusted incidence rates of mPCa with bone and visceral involvement with annual percentage changes (APCs) were assessed by a joinpoint regression model in men aged 45 years and older by age and race groups, and potential recent improvements in overall survival (OS) and cancer-specific survival (CSS) were estimated by the Kaplan-Meier method and Cox regression model. Results: From 2010 to 2019, a total of 19081 (84.8%) and 3413 (15.2%) mPCa patients with bone and visceral involvement, respectively, were recorded in the SEER database. Considering all races and age groups, the incidence rate of mPCa with bone metastasis remained stable during 2017-2019 (APC, 0.9%; p=0.421) after increasing during 2010-2017 (APC, 5.8%; p<0.001). For visceral metastasis, the incidence rate increased by 12.3% (p<0.001) per year from 2010-2019. Non-Hispanic Black men have higher incidence rates than other populations, and the Non-Hispanic Black to Non-Hispanic White incidence rates ratios of mPCa declined with the greater increasing pace of incidence of Non-Hispanic White men. There was a slight improvement in both OS and CSS among men with bone and visceral metastasis involvement when comparing the 2013-2016 period to the pre-2013 period. Conclusion: Our findings show that the incidence of mPCa with bone and visceral involvement has increased in recent years and that there has been a potential improvement in survival. Future efforts are still needed to watch closely if the rising incidence trends continue.

Case report and literature review: Treatment of multiple meningiomas combined with multiple unruptured aneurysms in a single operation.

Background: Although the incidence of a single meningioma or a single aneurysm is common, cases of multiple meningiomas combined with multiple aneurysms are rarely reported, and surgical treatment of the coexisting situation is also relatively uncommon. Case presentation: A 38-year-old male patient presented to the neurosurgery department of our center with a headache. Examination revealed only symptoms of headache. Laboratory tests showed only decreased total protein and albumin. Magnetic resonance imaging showed preoccupation with the frontal lobe and the right temple bone. Magnetic resonance angiography and digital subtraction angiography showed two aneurysms in the anterior communicating artery and right anterior cerebral artery. Based on a combination of the patient's history and imaging, we hypothesized that the patient was simultaneously suffering from meningioma and an aneurysm, and both of them are multiple. The patient underwent tumor resection and clipping procedure based on this hypothesis in one surgery. Intraoperative biopsy proved to be a meningioma. The patient was discharged on the 10th postoperative day, and a postoperative follow-up suggested no complications. Conclusion: Multiple meningiomas combined with multiple aneurysms are rare to be reported in the same patient. For those unruptured intracranial aneurysms (UIAs) located in the visual field of craniotomy prepared for brain tumorlike meningioma, it is possible to do the clipping as well. When the meningiomas are multiple, fitted with the surgical indication, and located in a position that cannot be treated in one surgery, this may lead to a two-stage operation, no matter where the UIAs are located.

CT psoas calculations on the prognosis prediction of emergency laparotomy: a single-center, retrospective cohort study in eastern Asian population.

BACKGROUND: Emergency laparotomy (EL) has a high mortality rate. Clinically, frail patients have a poor tolerance for EL. In recent years, sarcopenia has been used as an important indicator of frailty and has received much attention. There have been five different calculation methods of psoas for computed tomography (CT) to measure sarcopenia, but lack of assessment of these calculation methods in Eastern Asian EL patients. METHODS: We conducted a 2-year retrospective cohort study of patients over 18 years of age who underwent EL in our institution. Five CT measurement values (PMI: psoas muscle index, PML3: psoas muscle to L3 vertebral body ratio, PMD: psoas muscle density, TPG: total psoas gauge, PBSA: psoas muscle to body face area ratio) were calculated to define sarcopenia. Patients with sarcopenia defined by the sex-specific lowest quartile of each measurement were compared with the rest of the cohort. The primary outcome was "ideal outcome", defined as: (1) No postoperative complications of Clavien-Dindo Grade ≥ 4; (2) No mortality within 30 days; (3) When discharged, no need for fluid resuscitation and assisted ventilation, semi-liquid diet tolerated, and able to mobilize independently. The second outcome was mortality at 30-days. Multivariate logistic regression and receiver operating characteristic (ROC) analysis were used. RESULTS: Two hundred and twenty-eight patients underwent EL met the inclusion criteria, 192 (84.2%) patients had an ideal outcome after surgery; 32 (14%) patients died within 30 days. Multivariate analysis showed that, except PMD, each calculation method of psoas was independently related to clinical outcome (ideal outcome: PML3, P < 0.001; PMI, P = 0.001; PMD, P = 0.157; TPG, P = 0.006; PBSA, P < 0.001; mortality at 30-days: PML3, P < 0.001; PMI, P = 0.002; PMD, P = 0.088; TPG, P = 0.002; PBSA, P = 0.001). In ROC analysis, the prediction model containing PML3 had the largest area under the curve (AUC) value (AUC value = 0.922 and 0.920, respectively). CONCLUSION: The sarcopenia determined by CT psoas measurements is significantly related to the clinical outcome of EL. The calculation of CT psoas measurement is suitable for application in outcome prediction of EL. In the future, it is necessary to develop a scoring tool that includes sarcopenia to evaluate the risk of EL better.

The molecular biology of pancreatic adenocarcinoma: translational challenges and clinical perspectives.

Pancreatic cancer is an increasingly common cause of cancer mortality with a tight correspondence between disease mortality and incidence. Furthermore, it is usually diagnosed at an advanced stage with a very dismal prognosis. Due to the high heterogeneity, metabolic reprogramming, and dense stromal environment associated with pancreatic cancer, patients benefit little from current conventional therapy. Recent insight into the biology and genetics of pancreatic cancer has supported its molecular classification, thus expanding clinical therapeutic options. In this review, we summarize how the biological features of pancreatic cancer and its metabolic reprogramming as well as the tumor microenvironment regulate its development and progression. We further discuss potential biomarkers for pancreatic cancer diagnosis, prediction, and surveillance based on novel liquid biopsies. We also outline recent advances in defining pancreatic cancer subtypes and subtype-specific therapeutic responses and current preclinical therapeutic models. Finally, we discuss prospects and challenges in the clinical development of pancreatic cancer therapeutics.

Status epilepticus induced Gadd45b is required for augmented dentate neurogenesis.

In animal models with temporal lobe epilepsy (TLE), the status epilepticus (SE) leads to a dramatic increase in number of newly born neuron in the subgranular zone (SGZ) of dentate gyrus. How the SE confers a modulation in the dentate neurogenesis is mostly unknown. Gadd45b is involved in epigenetic gene activation by DNA demethylation. This study was performed to present a novel mechanism underling SE-induced dentate neurogenesis. A transient induction (12 hrs to 3 days) of Gadd45b was observed in dentate gyrus of mice after pilocarpine-induced SE. Labeling the dividing cells with BrdU, we next found that the induction of Gadd45b was required to increase the rate of cell proliferation in the dentate gyrus at 7 and 14 days after SE. Afterward, the DNA methylation levels for candidate growth factor genes critical for the adult neurogenesis were assayed with Sequenom MassARRAY Analyzer. The results indicated that Gadd45b was necessary for SE-induced DNA demethylation of specific promoters and expression of corresponding genes in the dentate gyrus, including brain-derived neurotrophic factor (BDNF) and fibroblast growth factor-2 (FGF-2). Using Timm staining, we further suggested that SE-induced Gadd45b might contribute to the subsequent mossy fiber sprouting (MFS) in the chronically epileptic hippocampus via epigenetic regulation of dentate neurogenesis at early stage after SE. Together, Gadd45b links pilocarpine-induced SE to epigenetic DNA modification of secreted factors in the dentate gyrus, leading to extrinsic modulation on the neurogenesis.

Simultaneous detection of transcribed functional assA gene and the corresponding metabolites of linear alkanes (C4, C5, and C7) in production water of a low-temperature oil reservoir.

Methanogenic hydrocarbon degradation is an important biogeochemical process in oil reservoirs; however, genomic DNA-based analysis of microorganisms and metabolite detection are not conclusive for identification of the ongoing nature of this bioprocess. In this study, a suite of analyses, involving the study of microbial community and selective gene quantification of both genomic DNA and RNA together with signature metabolites, were performed to comprehensively advance the understanding of the methanogenic biodegradation of hydrocarbons in a low-temperature oilfield. The fumarate addition products for alkanes-C4, C5, and C7-alkylsuccinates-and transcribed assA and mcrA genes were simultaneously detected in the production water sample, providing robust and convincing evidence for both the initial activation of n-alkanes and methane metabolism in this oilfield. The clone library of assA gene transcripts showed that Smithella was active and most likely responsible for the addition of fumarate to n-alkanes, whereas Methanoculleus and Methanothrix were the dominant and active methane-producers via CO2 reduction and acetoclastic pathways, respectively. Additionally, qPCR results of assA and mcrA genes and their transcribed gene copy numbers revealed a roughly similar transcriptional activity in both n-alkanes-degraders and methane producers, implying that they were the major participants in the methanogenic degradation of n-alkanes in this oilfield. To the best of our knowledge, this is the first report presenting sufficient speculation, through detection of signature intermediates, corresponding gene quantification at transcriptional levels, and microbial community analysis, of methanogenic degradation of n-alkanes in production water of an oil reservoir.

Long-chain n-alkane biodegradation coupling to methane production in an enriched culture from production water of a high-temperature oil reservoir.

Paraffinic n-alkanes (C22-C30), crucial portions of residual oil, are generally considered to be difficult to be biodegraded owing to their general solidity at ambient temperatures and low water solubility, rendering relatively little known about metabolic processes in different methanogenic hydrocarbon-contaminated environments. Here, we established a methanogenic C22-C30 n-alkane-degrading enrichment culture derived from a high-temperature oil reservoir production water. During two-year incubation (736 days), unexpectedly significant methane production was observed. The measured maximum methane yield rate (164.40 µmol L-1 d-1) occurred during the incubation period from day 351 to 513. The nearly complete consumption (> 97%) of paraffinic n-alkanes and the detection of dicarboxylic acids in n-alkane-amended cultures indicated the biotransformation of paraffin to methane under anoxic condition. 16S rRNA gene analysis suggested that the dominant methanogen in n-alkane-degrading cultures shifted from Methanothermobacter on day 322 to Thermoplasmatales on day 736. Bacterial community analysis based on high-throughput sequencing revealed that members of Proteobacteria and Firmicutes exhibiting predominant in control cultures, while microorganisms affiliated with Actinobacteria turned into the most dominant phylum in n-alkane-dependent cultures. Additionally, the relative abundance of mcrA gene based on genomic DNA significantly increased over the incubation time, suggesting an important role of methanogens in these consortia. This work extends our understanding of methanogenic paraffinic n-alkanes conversion and has biotechnological implications for microbial enhanced recovery of residual hydrocarbons and effective bioremediation of hydrocarbon-containing biospheres.

Effects of AAV2-mediated co-transfection of CTGF and TIMP1 genes on degenerative lumbar intervertebral discs in rhesus monkeys in vivo.

OBJECTIVE: This study aims to investigate the effects of co-transfection of the genes for connective tissue growth factor (CTGF) and tissue inhibitor of metalloproteinase-1 (TIMP1) mediated by adeno-associated virus 2 (AAV2) on degenerative lumbar intervertebral discs in a primate model. METHODS: Twelve 4-7 year-old rhesus monkeys weighing 4.5-7.0 kg were utilized. CTGF and TIMP1 genes carried by AAV2 were injected into the degenerative lumbar intervertebral discs. Cytokine expression and biological effects were determined using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and 35S-sulfate incorporation assays. A rhesus monkey model of intervertebral disc degeneration was successfully established. RESULTS: At post-transfection, CTGF mRNA expression was higher in the transfection group than in the control group (P < 0.05). Furthermore, TIMP1 mRNA expression in the transfection group was several times the levels observed in the control group (P < 0.05). Moreover, type-II collagen mRNA expression was higher in the transfection group than in the control group (P < 0.05). In addition, higher aggrecan mRNA expression and synthesis were observed in the transfection group, compared to that in the control group (P < 0.05). CONCLUSION: The stable expression of CTGF and TIMP1 genes in vivo promoted the synthesis of aggrecan and type II collagen in the nucleus pulposus in the rhesus monkey model of intervertebral disc degeneration, which has a potential for intervertebral disc regeneration.

[Correlation of Gemin 3 SNP in the microRNA Biosynthesis Pathway with Risk and Prognosis of Patients with Non-Hodgkin's Lymphoma].

OBJECTIVE: To evaluate the correlation of single nucleotide polymorphisms (SNP) of Gemin 3(rs197412) in the miRNA biosynthesis with NHL cancer risk and overall prognosis. METHODS: miR-SNP were genotyped using PCR-ligase detection reaction(LAR, LCR) in NHL group of 230 non-Hodgkin lymphoma (NHL) patients and in control group of 120 healthy persons. The survival curves were drawn using the Kaplan-Meier method, and comparisons between the curves were made using the log-rank test. Multivariate survival analysis was performed by using a Cox proportional hazards model. RESULTS: The rs197412 genotype distribution difference was not statistically significant, in NHL and control group; the survival time of patients carrying the rs197412 TT genotype was significantly longer than that of the patients carrying the CC+CT genotype (P=0.007). In addition, rs197412 was independent from the survival of NHL patients by multivariate analysis (RR: 2.138,95% CI: 1.303-3.508, P<0.01). CONCLUSION: The single nucleotide polymorphisms of Gemin 3 (rs197412) in the miRNA processing are not related with NHL risk, but that may affect NHL survival.

A case report and literature review of barium sulphate aspiration during upper gastrointestinal examination.

RATIONALE: Even though barium sulphate aspiration during upper gastrointestinal examination is a well-known phenomenon, complication such as long-term lung injury and death may still occur. This may depend upon the concentration, amount, anatomy, or certain predisposing factors. PATIENT CONCERNS: A 47-year-old woman who had a barium swallow to screen for foreign body in esophagus. DIAGNOSES: Chest radiographs demonstrated massive barium sulphate depositions in her trachea and inferior lobe of right lung. INTERVENTIONS: A chest x-ray was done that revealed massive barium sulphate depositions in her trachea and lower lobe of right lung. As the patient did not have further complaints, she requested a transfer to West China Hospital of Sichuan University, the hospital being near her residence, for further treatment. She eventually recovered and was discharged after 1 week. OUTCOMES: There were 23 articles (22 English and 1 Chinese with 17 men and 11 women) included in the study. The risk factors of barium sulphate aspiration are dysphagia (10/28, 35.71%) followed by esophageal obstruction caused by tumor (5/28, 17.86%) and foreign body in esophagus (3/28, 10.71%). Infants (5/28, 17.86%) are also one of the high-risk population. Both the lungs were affected in most of the patients (21/28, 75%). Majority of the presentation in patients (21/28, 75%) were dyspnea, hypoxemia, acute respiratory distress syndrome (ARDS), or respiratory failure. Few patients (7/28, 25%) showed no symptoms or mild symptoms such as cough and fever. Barium sulphate aspiration can be life-threatening with a high risk of death (nearly 40%). LESSONS: When performing an upper gastrointestinal examination with barium sulphate, careful consideration of concentration and amount of barium sulphate and that of risk factors should be undertaken so as to avoid life-threatening aspiration.

Treatment strategies for huge central neurocytomas.

Central neurocytomas (CNs), initially asymptomatic, sometimes become huge before detection. We described and analyzed the clinical, radiological, operational and outcome data of 13 cases of huge intraventricular CNs, and discussed the treatment strategies in this study. All huge CNs (n=13) in our study were located in bilateral lateral ventricle with diameter ≥5.0 cm and had a broad-based attachment to at least one side of the ventricle wall. All patients received craniotomy to remove the tumor through transcallosal or transcortical approach and CNs were of typical histologic and immunohistochemical features. Adjuvant therapies including conventional radiation therapy (RT) or gamma knife radiosurgery (GKRS) were also performed postoperatively. Transcallosal and transcortical approaches were used in 8 and 5 patients, respectively. Two patients died within one month after operation and 3 patients with gross total resection (GTR) were additionally given a decompressive craniectomy (DC) and/or ventriculoperitoneal shunt (VPS) as the salvage therapy. Six patients received GTR(+RT) and 7 patients received subtotal resection (STR)(+GKRS). Eight patients suffered serious complications such as hydrocephalus, paralysis and seizure after operation, and patients who underwent GTR showed worse functional outcome [less Karnofsky performance scale (KPS) scores] than those having STR(+GKRS) during the follow-up period. The clinical outcome of huge CNs seemed not to be favorable as that described in previous reports. Surgical resection for huge CNs should be meticulously considered to guarantee the maximum safety. Better results were achieved in STR(+GKRS) compared with GTR(+RT) for huge CNs, suggesting that STR(+GKRS) may be a better treatment choice. The recurrent or residual tumor can be treated with GKRS effectively.

Deficiency of adiponectin protects against ovariectomy-induced osteoporosis in mice.

Adipokine adiponectin (APN) has been recently reported to play a role in regulating bone mineral density (BMD). To explore the mechanism by which APN affects BMD, we investigated BMD and biomechanical strength properties of the femur and vertebra in sham-operated (Sham) and ovariectomized (OVX) APN knockout (KO) mice as compared to their operated wild-type (WT) littermates. The results show that APN deficiency has no effect on BMD but induces increased ALP activity and osteoclast cell number. While OVX indeed leads to significant bone loss in both femora and vertebras of WT mice with comparable osteogenic activity and a significant increase in osteoclast cell number when compared to that of sham control. However, no differences in BMD, ALP activity and osteoclast cell number were found between Sham and OVX mice deficient for APN. Further studies using bone marrow derived mesenchymal stem cells (MSCs) demonstrate an enhanced osteogenic differentiation and extracellular matrix calcification in APN KO mice. The possible mechanism for APN deletion induced acceleration of osteogenesis could involve increased proliferation of MSCs and higher expression of Runx2 and Osterix genes. These findings indicate that APN deficiency can protect against OVX-induced osteoporosis in mice, suggesting a potential role of APN in regulating the balance of bone formation and bone resorption, especially in the development of post-menopausal osteoporosis.

Reactomes of porcine alveolar macrophages infected with porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) has devastated pig industries worldwide for many years. It is caused by a small RNA virus (PRRSV), which targets almost exclusively pig monocytes or macrophages. In the present study, five SAGE (serial analysis of gene expression) libraries derived from 0 hour mock-infected and 6, 12, 16 and 24 hours PRRSV-infected porcine alveolar macrophages (PAMs) produced a total 643,255 sequenced tags with 91,807 unique tags. Differentially expressed (DE) tags were then detected using the Bayesian framework followed by gene/mRNA assignment, arbitrary selection and manual annotation, which determined 699 DE genes for reactome analysis. The DAVID, KEGG and REACTOME databases assigned 573 of the DE genes into six biological systems, 60 functional categories and 504 pathways. The six systems are: cellular processes, genetic information processing, environmental information processing, metabolism, organismal systems and human diseases as defined by KEGG with modification. Self-organizing map (SOM) analysis further grouped these 699 DE genes into ten clusters, reflecting their expression trends along these five time points. Based on the number one functional category in each system, cell growth and death, transcription processes, signal transductions, energy metabolism, immune system and infectious diseases formed the major reactomes of PAMs responding to PRRSV infection. Our investigation also focused on dominant pathways that had at least 20 DE genes identified, multi-pathway genes that were involved in 10 or more pathways and exclusively-expressed genes that were included in one system. Overall, our present study reported a large set of DE genes, compiled a comprehensive coverage of pathways, and revealed system-based reactomes of PAMs infected with PRRSV. We believe that our reactome data provides new insight into molecular mechanisms involved in host genetic complexity of antiviral activities against PRRSV and lays a strong foundation for vaccine development to control PRRS incidence in pigs.

Autoinhibitory mechanism for the mutation-induced impaired FGF9 signaling.

Fibroblast growth factor 9 (FGF9), an important member of the fibroblast growth factor (FGF) family, can bind with high affinity to FGFR3 in a heparin-dependent approach. In humans, the deletions and mutations resulting in dysfunction of the FGF9 signaling can cause human skeletal dysplasia and cancers. A mutation (S99N) in this protein has been identified to be associated with significantly impaired FGF signaling considered as a potential cause of synostoses syndrome. However, the detailed mechanism for this observation still remains unknown. In this study, we used molecular dynamics simulations and free energy calculations to study the interactions of FGF9(WT/S99N), FGFR3c, and heparin, with an aim of providing atomic sights into the detailed mechanism for the impaired FGF signaling caused by the S99N mutation. We found that the S99N mutation has a well-ordered C-terminal structure, which can reduce its homodimerization ability so as to break the monomer-dimer equilibrium in the FGF signaling, which is considered as a key factor to regulate extracellular matrix affinity and tissue diffusion in the FGF signaling pathway. The FGF9(WT) monomer can preferentially form a homodimer owing to its comparatively favorable binding free energy. In contrast, the FGF9(S99N) monomer is preferred to bind with the FGFR3c receptor to form an inactive complex, leading to impair FGF signaling. To support our computational findings, we also performed biochemical experiments, which confirm the computational results mentioned above. The impaired FGF signaling is believed to be a potential cause of human synostoses syndrome, implicating an important role for FGF9 in normal joint development.

Multiple synostoses syndrome is due to a missense mutation in exon 2 of FGF9 gene.

Fibroblast growth factors (FGFs) play diverse roles in several developmental processes. Mutations leading to deregulated FGF signaling can cause human skeletal dysplasias and cancer.(1,2) Here we report a missense mutation (Ser99Asp) in exon 2 of FGF9 in 12 patients with multiple synostoses syndrome (SYNS) in a large Chinese family. In vitro studies demonstrate that FGF9(S99N) is expressed and secreted as efficiently as wild-type FGF9 in transfected cells. However, FGF9(S99N) induces compromised chondrocyte proliferation and differentiation, which is accompanied by enhanced osteogenic differentiation and matrix mineralization of bone marrow-derived mesenchymal stem cells (BMSCs). Biochemical analysis reveals that S99N mutation in FGF9 leads to significantly impaired FGF signaling, as evidenced by diminished activity of Erk1/2 pathway and decreased beta-catenin and c-Myc expression when compared with wild-type FGF9. Importantly, the binding of FGF9(S99N) to its receptor is severely impaired although the dimerization ability of mutant FGF9 itself or with wild-type FGF9 is not detectably affected, providing a basis for the defective FGFR signaling. Collectively, our data demonstrate a previously uncharacterized mutation in FGF9 as one of the causes of SYNS, implicating an important role of FGF9 in normal joint development.

Bisphenol A exposure induces apoptosis and upregulation of Fas/FasL and caspase-3 expression in the testes of mice.

There are controversies about adverse effects of bisphenol A (BPA), a ubiquitous xenoestrogen, on reproduction and development of male animals. To understand BPA action and assess its risk more completely, we examined the impact of BPA at high doses on the testes of pubertal male Kunming (China) mice. BPA at 0 (control), 160, 480, and 960 mg/kg/day was given by gavage to mice from postnatal days (PND) 31-44, followed by observation of morphology and detection of apoptosis and expressions of Fas/FasL and active caspase-3 on PND 45, 60, and 90 by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling, immunohistochemistry, and Western blotting. There was no effect of BPA at 160 mg/kg/day, however, at 480 and 960 mg/kg/day there was underdevelopment of testes and disruption of spermatogenesis. There were many apoptotic Leydig and germ cells in the testes with apoptotic indices being significantly increased compared with controls. The expression of Fas and active caspase-3 was localized in the same cell types as apoptosis occurred, and expression levels of Fas, FasL, and active caspase-3 were significantly increased compared with controls. The disturbed spermatogenesis, apoptosis and upregulation of Fas, FasL, and active caspase-3 expression persisted to PND 90. The results suggest that high-dose BPA induces apoptosis of Leydig and germ cells in the mouse testis through the Fas-signaling pathway. Therefore, there is concern about reproductive health for humans occupationally exposed to high levels of BPA.

Overexpression of PTEN suppresses growth and induces apoptosis by inhibiting the expression of survivin in bladder cancer cells.

The tumor suppressor gene PTEN, which encodes a multifunctional phosphatase protein, is mutated in a variety of human cancers. Several reports have indicated that it has growth-suppressive and proapoptosis properties and displayed an altered expression pattern during human oncogenesis. Overexpression of PTEN leads to decreasing cell growth and tumorigenicity in vitro and in vivo. In the present study, we further demonstrated that overexpression of PTEN mediated by adenovirus suppressed bladder cancer cell growth and significantly induced apoptosis, through downregulating of survivin and activating of caspase cascades. Our results indicate that Ad-PTEN exerts its tumor suppressive effect on bladder cancer cells through inhibiting survivin and upregulating caspase-related proteins. Thus Ad-PTEN may be potentially therapeutic for the treatment of bladder cancers.

[Caspase-3 antisense oligodeoxynucleotides inhibit caspase-3 expression and apoptosis of gamma-irradiated HL-60 cells].

It has been shown that gamma -irradiation induces apoptosis of the human promyeloid leukemia cell line HL-60, but the mechanism remains unclear. To explore the effect of caspase-3 in this apoptotic model, antisense oligodeoxynucleotides (ASODNs) targeting 5'-noncoding region (ASODN-1) and initial translation region (ASODN-2) of caspase-3 mRNA were designed, synthesized and introduced into HL-60 cells by means of liposome-mediated transfection followed by gamma-irradiation in the present study. The TUNEL assay was used for morphological analysis of HL-60 cell apoptosis. Immunocytochemical staining, Western blotting and RT-PCR were, respectively, performed for detecting expression of caspase-3 and its mRNA. HL-60 cells transfected with mismatched oligodeoxynucleotide (MODN) or untransfected were taken as the control groups. The TUNEL assay showed that the percentages of HL-60 cell apoptosis induced by gamma-Irradiation in both ASODN-1 and ASODN-2 groups were significantly reduced compared with those in the control group (P<0.01) when the final transfection concentration was > or = 3 micromol/L. Immunocytochemistry demonstrated that in the ASODN-1 and ASODN-2 groups, caspase-3 positive cell percentages were reduced and average gray values of the positive cells increased significantly compared with those in the control group (P<0.01). Western blotting found that procaspase-3 expression in HL-60 cells of the ASODNs groups was decreased,and it was lower in the ASODN-1 group than in the ASODN-2 group. RT-PCR revealed marked expression of caspase-3 mRNA in HL-60 cells of the control group. Expression of caspase-3 mRNA was decreased after ASODNs transfection. Furthermore, ASODN-1 was more effective in inhibiting HL-60 cell apoptosis (P<0.05) and caspase-3 expression (P<0.01) than ASODN-2. These results indicate that caspase-3 mRNA ASODNs prevent HL-60 cells from apoptosis induced by gamma-radiation,and reduce expression of caspase-3 and its mRNA. These effects are dose dependent in a certain range.