[Curcumin alleviates the manganese-induced neurotoxicity by promoting autophagy in rat models of manganism].

OBJECTIVE: To investigate the protective effects of curcumin(CUR) and its mechanism on a rat model of neurotoxicity induced by manganese chloride (MnCl2), which mimics mangnism. METHODS: Sixty male SD rats were randomly divided into 5 groups, with 12 rats in each group. Control group received 0.9% saline solution intraperitoneally (ip) plus double distilled water (dd) H2O intragastrically (ig), MnCl2 group received 15 mg/kg MnCl2(Mn2+ 6.48 mg/kg) intraperitoneally plus dd H2O intragastrically, CUR group received 0.9% saline solution intraperitoneally plus 300 mg/kg CUR intragastrically, MnCl2+ CUR1 group received 15 mg/kg MnCl2 intraperitoneally plus 100 mg/kg curcumin intragastrically, MnCl2+ CUR2 group received 15 mg/kg MnCl2 intraperitoneally plus 300 mg/kg CUR intragastrically, 5 days/week, 4 weeks. Open-field and rotarod tests were used to detect animals' exploratory behavior, anxiety, depression, movement and balance ability. Morris water maze (MWM) experiment was used to detect animals' learning and memory ability. ICP-MS was used to investigate the Mn contents in striata. The rats per group were perfused in situ, their brains striata were removed by brains model and fixed for transmission electron microscope (TEM), histopathological and immunohistochemistry (ICH) analyses. The other 6 rats per group were sacrificed. Their brains striata were removed and protein expression levels of transcription factor EB (TFEB), mammalian target of rapamycin (mTOR), p-mTOR, Beclin, P62, microtubule-associated protein light chain-3 (LC3) were detected by Western blotting. Terminal deoxynucleotidyl transterase-mediated dUTP nick end labeling (TUNEL) staining was used to determine neurocyte apoptosis of rat striatum. RESULTS: After exposure to MnCl2 for four weeks, MnCl2-treated rats showed depressive-like behavior in open-field test, the impairments of movement coordination and balance in rotarod test and the diminishment of spatial learning and memory in MWM (P < 0.05). The striatal TH+ neurocyte significantly decreased, eosinophilic cells, aggregative α-Syn level and TUNEL-positive neurocyte significantly increased in the striatum of MnCl2 group compared with control group (P < 0.05). Chromatin condensation, mitochondria tumefaction and autophagosomes were observed in rat striatal neurocytes of MnCl2 group by TEM. TFEB nuclear translocation and autophagy occurred in the striatum of MnCl2 group. Further, the depressive behavior, movement and balance ability, spatial learning and memory ability of MnCl2+ CUR2 group were significantly improved compared with MnCl2 group (P < 0.05). TH+ neurocyte significantly increased, the eosinophilic cells, aggregative α-Syn level significantly decreased in the striatum of MnCl2+ CUR2 group compared with MnCl2 group. Further, compared with MnCl2 group, chromatin condensation, mitochondria tumefaction was alleviated and autophagosomes increased, TFEB-nuclear translocation, autophagy was enhanced and TUNEL-positive neurocyte reduced significantly in the striatum of MnCl2+ CUR2 group (P < 0.05). CONCLUSION: Curcumin alleviated the MnCl2-induced neurotoxicity and α-Syn aggregation probably by promoting TFEB nuclear translocation and enhancing autophagy.

[Analysis for the short-term efficacy and long-term prognosis of laparoscopic and laparotomy radical resection for gallbladder cancer].

Objective: To compare the short-term efficacy and long-term prognosis of laparoscopic and laparotomy radical resection for gallbladder cancer(GBC). Methods: From January 2010 to December 2020,the clinical data and survival information for 133 patients who underwent radical resection of GBC at the Department of Hepatopancreatobiliary Surgery,Zhejiang Provincial People's Hospital,were retrospectively collected. Eighty patients(23 males and 57 females) underwent laparoscopic radical resection and had a median age(M(IQR)) of 66.0(12.8)years(range:28.0 to 82.0 years). Fifty-three patients(45 males and 8 females) who received laparotomy were 63.0(6.0)years old(range:45.0 to 80.0 years old). There were no significant differences in age,gender,body mass index,preoperative albumin,preoperative total bilirubin,N stages,vascular invasion,peri-neural invasion or tumor differentiation between the laparoscopic and laparotomy group(all P>0.05). But there were significant differences in preoperative CA19-9(Z=-2.955, P=0.003), preoperative ALT level(Z=-2.801,P=0.031) and T stage (χ2=19.110,P=0.007) between the two groups. A non-parametric test was used for quantitative data. χ2 test or Fisher exact probability method was used for count data. Results: Patients in the laparoscopic group did not differ from those in the laparotomy group in terms of length of operation,number of lymph node yield,number of positive lymph nodes,the incidence of intraoperative gallbladder rupture,incidence of postoperative bile leakage,abdominal bleeding or abdominal infection,30-day mortality,90-day mortality, the incidence of incision implantation or peritoneal cavity metastasis(all P>0.05). Patients in the laparoscopic group showed less intraoperative bleeding(100.0(200.0)ml vs. 400.0(250.0)ml)(Z=-5.260,P<0.01),fewer days with drainage tube indwelling(6.0(3.8)days vs. 7.0(4.0)days)(Z=-3.351, P=0.001), and fewer postoperative days in hospital(8.0(5.0)days vs. 14.0(7.5)days)(Z=-6.079,P<0.01) than those in the laparotomy group. Patients in the laparoscopic group displayed better overall survival (P<0.01) and progression-free survival (P<0.01). Subgroup analysis for GBC of T1b-T2 and T3 stages revealed comparable overall survival and progression-free survival between the laparoscopic and laparotomy groups (P>0.05). Conclusions: Laparoscopic radical resection can achieve long-term survival for GBC comparable to that with open surgery. Laparoscopic radical resection has advantages over open surgery regarding surgical trauma and postoperative recovery.

[The role of lysosomes in manganese-induced toxicity in SK-N-SH cells].

Objective: To investigate the role of lysosomes in manganese-induced toxicity in human neuroblastoma SK-N-SH cells. Methods: SK-N-SH cells were treated with MnCl(2) at doses of 0.062 5, 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 mmol/L for 24 h, and the cell viability was detected by MTT assay. Cells were treated with MnCl(2) at doses of 0.125, 0.25, 0.5 and 1.0mmol/L for 24 h, and lysosomes labeled with lysotracker red were observed by laser confocal microscopy, the expression levels of LAMP1 and CTSD were detected by western blot, and CTSD activity was detected by Cathepsin D Activity Fluorometric Assay Kit. Results: Compared with the control group, the survival rates of SK-N-SH cells were decreased significantly in the 0.5-4.0 mmol/L MnCl(2) treatment groups (P<0.01) , the relative fluorescence intensities of 0.5 and 1.0 mmol/L MnCl(2) treatment groups were increased (P<0.01) . Compared with the control group, the 0.125-0.5 mmol/L MnCl(2) treatment groups had significant increase in the the expression of LAMP1 (P<0.01) . Compared with the control group, the expression of m-CTSD was significantly increased at the does of 0.125-0.25 mmol/L MnCl(2), while it was decreased at the does of 1.0 mmol/L (P<0.01) . Otherwise, it wasn't observed significant difference of the activity of CTSD between different MnCl(2) treatment groups. Conclusion: MnCl(2) could cause cytotoxicity in SK-N-SH cells. Lysosomes may play a normal function at low doses of manganese, but they may be damaged at high doses of manganese. As an organelle that can degradate substrates in autophagy, lysosomes participate in the neurotoxic mechanism of manganese.

[The comparison of neurobehavioral changes and impaired locations between the mouse model of manganism and Parkinson's disease].

Objective: To investigate the effect of manganese chloride (MnCl(2)) or 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) on the neurobehavioral and histopathology in C57BL/6 mice and provide evidence for the diagnosis, treatment and prevention of manganism. Methods: Adult male C57BL/6 mice were treated with MnCl(2) and MPTP respectively by intraperitoneal injection at the doses of 5, 10, 20mg Mn/kg and 30mg MPTP/kg. Controls were injected equivalent normal saline. All animals were administrated 5 times a week for 4 consecutive weeks and sacrificed after behavior tests on the fifth week. Balance ability, anxiety and depression level and cognitive function were tested respectively by vertical pole test, open field locomotion test and Morris swim task. The neuron pathological changes of striatum and substantia nigra were examined through HE-staining pathological section by using optical microscope. Results: Compared with the control group, the high dose of MnCl(2) reduced body weight obviously (P<0.01) . The results of vertical pole test showed that MnCl(2) and MPTP lengthened the pole-climbing time and turnaround time. Open field locomotion test showed that movement distance, stand-up time and central field time were decreased after the exposure of MnCl(2) or MPTP. In the Morris swim task, the escape latency time increased and the target quadrant activity time decreased significantly after the injection of MPTP as well as high-dose MnCl(2), comparing with controls (P<0.05) . Moreover, the escape latency time of high dose MnCl(2) prolonged prominently comparing with MPTP grou (P<0.05) . The results of histopathology showed that acidophilic changes elevated in MnCl(2) and MPTP group, comparing with controls. Furthermore, in striatum the oxyphil cells number increased in MnCl(2) high-dose group comparing with MPTP group (P<0.01) . On the contrary, there were more oxyphil cells in MPTP group comparing with MnCl(2) groups in substantia nigra (P<0.01) . Conclusion: Both manganese and MPTP can induce the impairment of dopaminergic neural system, but the symptons and injured location of manganism are inconsistent with PD models induced by MPTP.

[Oxidative stress and autophagy in SK-N-SH cells induced by manganese chloride or 1-methyl-4-phenylpyridinium: a comparative analysis].

Objective: To investigate the effect of manganese chloride (MnCl(2)) or 1-methyl-4-phenylpyridinium (MPP (+)) on oxidative stress and autophagy in human neuroblastomaSK-N-SH cells and the mechanism of the neurotoxicity of manganese. Methods: SK-N-SH cells were treated with MnCl(2) or MPP(+) at doses of 0.062 5, 0.125, 0.25, 0.5, 1.0, and 2.0 mmol/L for 24 hours, and MTT assay was used to measure cell viability. The cells weretreated with MnCl(2) or MPP(+) at doses of 0.125, 0.25, and 0.5 mmol/L for 24 hours, and flow cytometry was used to measure the content of reactive oxygen species (ROS) in cells, a laser scanning confocal microscope was used to observe autophagosome in cells, and Western blot was used to measure the expression of autophagy-related proteins P62 and LC3-II/LC3-I. Results: Compared with the control group, the 0.0625-2.0 mmol/L MnCl(2) and 0.125-2.0 mmol/L MPP (+) treatment groups had significant reductions in the viability of SK-N-SH cells, and the 0.25-2.0 mmol/L MnCl(2) treatment groups had significantly lower viability than the groups treated with the same doses of MPP(+) (all P<0.05) . Compared with the control group, the 0.125-0.25 mmol/L MnCl(2) and 0.125-0.5 mmol/L MPP(+) treatment groups had significant increases in the content of ROS, and the 0.25-0.5 mmol/L MPP(+) treatment groups had significantly higher content of ROS than the groups treated with the same doses of MnCl(2) (all P<0.05) . Compared with the control group, the 0.25-0.5 mmol/L MnCl(2) andMPP(+) treatment groups had significant increases in autophagy-related proteins LC3-II/LC3-I and significant reductions in P62 expression; the 0.125-0.5 mmol/L MPP(+) treatment groups had significantly higher LC3-II/LC3-I than the groups treated with the same doses of MnCl(2), and the 0.125 and 0.25 mmol/L MPP (+) treatment groups had significantly lower P62 expression than the groups treated with the same doses of MnCl(2) (all P<0.05) . Conclusion: Both MnCl(2) and MPP(+) can induce oxidative stress and autophagy in SK-N-SH cells, and MPP(+) has a significantly greater inductive effect on autophagy of SK-N-SH cells than MnCl(2).

Xanthotoxin prevents bone loss in ovariectomized mice through the inhibition of RANKL-induced osteoclastogenesis.

UNLABELLED: Xanthotoxin (XAT) is extracted from the seeds of Ammi majus. Here, we reported that XAT has an inhibitory effect on osteoclastogenesis in vitro through the suppression of both receptor activator of nuclear factor-κB ligand (RANKL)-induced ROS generation and Ca(2+) oscillations. In vivo studies showed that XAT treatment decreases the osteoclast number, prevents bone loss, and restores bone strength in ovariectomized mice. INTRODUCTION: Excessive osteoclast formation and the resultant increase in bone resorption activity are key pathogenic factors of osteoporosis. In the present study, we have investigated the effects of XAT, a natural furanocoumarin, on the RANKL-mediated osteoclastogenesis in vitro and on ovariectomy-mediated bone loss in vivo. METHODS: Cytotoxicity of XAT was evaluated using bone marrow macrophages (BMMs). Osteoclast differentiation, formation, and fusion were assessed using the tartrate-resistant acid phosphatase (TRAP) stain, the actin cytoskeleton and focal adhesion (FAK) stain, and the fusion assay, respectively. Osteoclastic bone resorption was evaluated using the pit formation assay. Reactive oxygen species (ROS) generation and removal were evaluated using dichlorodihydrofluorescein diacetate (DCFH-DA). Ca(2+) oscillations and their downstream signaling targets were then detected. The ovariectomized (OVX) mouse model was adopted for our in vivo studies. RESULTS: In vitro assays revealed that XAT inhibited the differentiation, formation, fusion, and bone resorption activity of osteoclasts. The inhibitory effect of XAT on osteoclastogenesis was associated with decreased intracellular ROS generation. XAT treatment also suppressed RANKL-induced Ca(2+) oscillations and the activation of the resultant downstream calcium-CaMKK/PYK2 signaling. Through these two mechanisms, XAT downregulated the key osteoclastogenic factors nuclear factor of activated T cells c1 (NFATc1) and c-FOS. Our in vivo studies showed that XAT treatment decreases the osteoclast number, prevents bone loss, rescues bone microarchitecture, and restores bone strength in OVX mice. CONCLUSION: Our findings indicate that XAT is protective against ovariectomy-mediated bone loss through the inhibition of RANKL-mediated osteoclastogenesis. Therefore, XAT may be considered to be a new therapeutic candidate for treating osteoporosis.

Assessing the association between EFEMP1 rs3791679 polymorphism and risk of glioma in a Chinese Han population.

In this study, we assessed the association between the EFEMP1 rs3791679 polymorphism and glioma risk in a Chinese Han population. A total of 94 glioma patients and 206 healthy controls who conformed to the inclusion and exclusion criteria were recruited from Baogang Hospital between March 2012 and October 2014. The EFEMP1 rs3791679 gene polymorphism was assessed using a polymerase chain reaction-restriction fragment length polymorphism assay and the results were statistically analyzed using SPSS Statistics 17.0. The results of unconditional logistic regression analysis revealed that the GG genotype of EFEMP1 rs3791679 was positively correlated with increased susceptibility to glioma (adjusted OR = 2.09, 95%CI = 1.21-7.81). Moreover, the GG genotype of EFEMP1 rs3791679 was correlated with higher risk of glioma compared to the AA+GA genotype (OR = 2.60, 95%CI = 1.08-6.28) in the regressive model. In conclusion, we report that the EFEMP1 rs3791679 polymorphism influences glioma susceptibility in the Chinese Han population.

Effect of electroacupuncture on P2X3 receptor regulation in the peripheral and central nervous systems of rats with visceral pain caused by irritable bowel syndrome.

The aim of this study is to investigate the role of the purinergic receptor P2X3 in the peripheral and central nervous systems during acupuncture treatment for the visceral pain of irritable bowel syndrome (IBS). A total of 24 8-day-old Sprague-Dawley (SD) neonatal male rats (SPF grade) were stimulated using colorectal distention (CRD) when the rats were awake. The modeling lasted for 2 weeks with one stimulation per day. After 6 weeks, the rats were randomly divided into three groups of eight each: (1) the normal group (NG, n = 8); (2) the model group (MG, n = 8); and (3) the model + electroacupuncture group (EA, n = 8) that received electroacupuncture at a needling depth of 5 mm at the Shangjuxu (ST37, bilateral) and Tianshu (ST25, bilateral) acupoints. The parameters of the Han's acupoint nerve stimulator (HANS) were as follows: sparse-dense wave with a frequency of 2/100 Hz, current of 2 mA, 20 min/stimulation, and one stimulation per day; the treatment was provided for seven consecutive days. At the sixth week after the treatment, the abdominal withdrawal reflex (AWR) score was determined; immunofluorescence and immunohistochemistry were used to measure the expression of the P2X3 receptor in myenteric plexus neurons, prefrontal cortex, and anterior cingulate cortex; and, a real-time PCR assay was performed to measure the expression of P2X3 messenger RNA (mRNA) in the dorsal root ganglion (DRG) and spinal cord. After stimulation with CRD, the expression levels of the P2X3 receptor in the inter-colonic myenteric plexus, DRG, spinal cord, prefrontal cortex, and anterior cingulate cortex were upregulated, and the sensitivity of the rats to IBS visceral pain was increased. Electroacupuncture (EA) could downregulate the expression of the P2X3 receptor and ease the sensitivity to visceral pain. The P2X3 receptor plays an important role in IBS visceral pain. The different levels of P2X3 in the peripheral enteric nervous system and central nervous system mediate the effects of the EA treatment of the visceral hyperalgesia of IBS.

Associations between the Expression of micro-RNA 214 and clinicopathologic parameters of glioma.

UNLABELLED: It have been reported that miR-214 reduction facilitates UBC9 expression and is involved in the regulation of glioma cell proliferation. However, the specific role of miR-214 in glioma remains unknown. Thus, we investigated the relationship between expression level of miR-214 and clinico- pathological features and prognosis in patients with glioma in a follow-up of 5years. We used Chi-square tests for the categorical data and Mann-Whitney tests for continuous data. Survival time was calculated from the date of glioma diagnosis to the date of death or last follow-up. Survival analysis was estimated using the Kaplan-Meier method, log-rank test, and Cox-proportional hazards regression model. In the present study, we confirmed that the expression level of miR-214 was increased in glioma tissues compared with the non-neoplastic brain tissues. Next, the Kaplan-Meier analysis revealed that glioma patients with high miR-214 expression tend to have poorer overall survival. In addition, the multivariate analysis clearly demonstrated that high miR-214 expression was a statistically significant risk factor affecting overall survival in glioma patients, suggesting that miR-214 upregulation in gliomas is not only in a grade-dependent fashion, it is also a predictor of overall survival. Finally, subgroup analyses showed the significant prognostic value of miR-214 upregulation for glioma patients in those with low and high pathological grade. The results of this study showed that miR-214 was up-regulated in glioma tissues. The expression of miR-214 was associated with the pathological stages of glioma. The results of 5-years follow-up showed that the expression level of miR-214 is a significant prognostic factor for patients with glioma. KEYWORDS: miR-214, glioma, prognosis.

DC-STAMP, the key fusion-mediating molecule in osteoclastogenesis.

As a member of the mononuclear phagocyte system, osteoclasts (OC) absorb the bone matrix and participate in bone modeling by keeping a balance with osteoblasts (OB) and stromal cells. Mature OC derive from the fusion of mononuclear osteoclasts (mOC) and the fusion is considered as the indispensable process for the osteoclastogenesis and absorbing activity of OC. DC-STAMP (dendritic cell-specific transmembrane protein) has been validated playing a key role in the fusion of mOC. DC-STAMP is mainly expressed in OC, macrophages and dendritic cells (DC). While DC-STAMP was discovered in DC, more attentions have been paid to DC-STAMP in OC in this decade. This review will mainly focus on the function of DC-STAMP in OC. Studies on DC-STAMP in DC may also provide new sight for the study of DC-STAMP in OC. Since the function of DC-STAMP is still poorly understood and few studies have been implemented for illustration, many issues are still unknown and need to be revealed. We will also discuss these questions in this review.

Combination of HIF-1α gene transfection and HIF-1-activated bone marrow-derived angiogenic cell infusion improves burn wound healing in aged mice.

Impaired burn wound healing in the elderly represents a major clinical problem. Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator that orchestrates the cellular response to hypoxia. Its actions in dermal wounds promote angiogenesis and improve healing. In a murine burn wound model, aged mice had impaired wound healing associated with reduced levels of HIF-1. When gene therapy with HIF-1 alone did not correct these deficits, we explored the potential benefit of HIF-1 gene therapy combined with the intravenous infusion of bone marrow-derived angiogenic cells (BMDACs) cultured with dimethyloxalylglycine (DMOG). DMOG is known to reduce oxidative degradation of HIF-1. The mice treated with a plasmid DNA construct expressing a stabilized mutant form of HIF-1α (CA5-HIF-1α)+BMDACs had more rapid wound closure. By day 17, there were more mice with completely closed wounds in the treated group (χ(2), P=0.05). The dermal blood flow measured by laser Doppler showed significantly increased wound perfusion on day 11. Homing of BMDACs to the burn wound was dramatically enhanced by CA5-HIF-1α gene therapy. HIF-1α mRNA expression in the burn wound was increased after transfection with CA5-HIF-1α plasmid. Our findings offer insight into the pathophysiology of burns in the elderly and point to potential targets for developing new therapeutic strategies.

Expression of interleukin-22 in myasthenia gravis.

IL-17 and IL-22 are implicated in the pathogenesis of autoimmune diseases. The roles of IL-22 in the pathophysiology of myasthenia gravis (MG) remain unsettled. The aim of this study was to investigate the possible relationship between serum IL-22, IL-17 levels, anti-acetylcholine receptor antibody (anti-AChR Ab) titres and clinical parameters in patients with MG. The serum IL-22, IL-17 levels and anti-AChR Ab titres were tested by enzyme-linked immunosorbent assay (ELISA), while the expression of IL-22 and IL-17 mRNAs in peripheral blood mononuclear cells (PBMC) from healthy and MG subjects were detected by quantitative real-time PCR (qRT-PCR). Furthermore, PBMC from 12 patients with generalized MG were purified and treated with recombinant human IL-22 (rhIL-22), the IL-17 levels of supernatant were detected by ELISA. We found that the IL-17 levels were significantly increased, but IL-22 levels were significantly decreased in the serum of patients with MG compared with healthy controls. Consistantly, a significant decrease in IL-22 mRNA levels and an increase in IL-17 mRNA levels were detected in PBMC collected from patients with MG, compared with healthy controls. A negative correlation between IL-22 mRNA in PBMC, serum IL-22 and serum anti-AChR Ab levels was found in patients with MG. Moreover, in cultured MG PBMC treated with recombinant human IL-22 (rhIL-22), the IL-17 levels were decreased in a dose-dependent manner. Our findings indicated a possible role of IL-22 as a protective factor in MG.

Graphene overcoats for ultra-high storage density magnetic media.

Hard disk drives (HDDs) are used as secondary storage in digital electronic devices owing to low cost and large data storage capacity. Due to the exponentially increasing amount of data, there is a need to increase areal storage densities beyond ~1 Tb/in2. This requires the thickness of carbon overcoats (COCs) to be <2 nm. However, friction, wear, corrosion, and thermal stability are critical concerns below 2 nm, limiting current technology, and restricting COC integration with heat assisted magnetic recording technology (HAMR). Here we show that graphene-based overcoats can overcome all these limitations, and achieve two-fold reduction in friction and provide better corrosion and wear resistance than state-of-the-art COCs, while withstanding HAMR conditions. Thus, we expect that graphene overcoats may enable the development of 4-10 Tb/in2 areal density HDDs when employing suitable recording technologies, such as HAMR and HAMR+bit patterned media.

Accumulation and detoxification of manganese in hyperaccumulator Phytolacca americana.

Pokeweed (Phytolacca americana) has recently received much attention because of its ability to hyperaccumulate manganese (Mn). The internal mechanism of detoxification of Mn, however, is not fully understood. In the present study, we investigated Mn accumulation, subcellular distribution, chemical speciation and detoxification through oxalate in pokeweed. The plant accumulated excess Mn in the leaves, mainly in the water-soluble fraction, and over 80% of Mn was in a water-soluble form, while accumulation of excess Mn in the cellular organelle and membrane fraction caused phytotoxicity. In addition, pokeweed has an intrinsically high oxalate content. In all experiments, there was sufficient oxalate to chelate Mn in leaf water extracts at all different levels of Mn application. Phase analysis of X-ray diffraction detected oxalate-Mn chelate complexes, and gel chromatography further confirmed the chelation of Mn by oxalate. In conclusion, pokeweed accumulates excess Mn in the soluble fraction of leaf cells, most likely in vacuoles, in which detoxification of Mn could be achieved by chelation with oxalate.

TWH regulates the development of subsets of spinal cord neurons.

Thymocyte winged helix (TWH) is a putative transcription factor expressed in the developing neural tube. At midgestation, TWH expression identifies subsets of spinal cord motor neurons and interneurons. TWH-expressing motor neurons were restricted to specific spinal cord levels, distinguishing motor neurons at lumbar from those at cervical levels. To understand the developmental role of TWH, we replaced the TWH gene with the lacZ reporter gene and generated mice with a homozygous disruption of the TWH gene. TWH(-/-) mutant mice had increased perinatal mortality, retarded postnatal growth, and motor weakness. The TWH(-/-) mutation resulted in alterations in the sizes and position of different neuronal populations. Our results demonstrate that TWH plays a critical role in neuronal development and suggest that TWH regulates the early differentiation of neural progenitors.

FAST-2 is a mammalian winged-helix protein which mediates transforming growth factor beta signals.

The mechanisms by which transforming growth factor beta (TGF-beta) and related ligands regulate transcription remain poorly understood. The winged-helix (WH) transcription factor fork head activin signal transducer 1 (FAST-1) was identified as a mediator of activin signaling in Xenopus embryos (X. Chen, M. J. Rubock, and M. Whitman, Nature 383:691-696, 1996). We have cloned a novel WH gene from the mouse which shares many properties with FAST-1. We find that this gene, which we call FAST-2, is able to mediate transcriptional activation by TGF-beta. FAST-2 also interacts directly with Smad2, a cytoplasmic protein which is translocated to the nucleus in response to TGF-beta, and forms a multimeric complex with Smad2 and Smad4 on the activin response element, a high-affinity binding site for FAST-1. Analysis of the sequences of FAST-1 and FAST-2 reveals substantial protein sequence divergence compared to known vertebrate orthologs in the WH family. This suggests that FAST-2 represents a new WH gene related to FAST-1, which functions to mediate TGF-beta signals in mammals. We have also examined the structure of the FAST-2 gene and find that it overlaps with a kinesin motor protein gene. The genes are transcribed in opposite orientations, and their transcripts overlap in the 3' untranslated region.

BF-1 interferes with transforming growth factor beta signaling by associating with Smad partners.

The winged-helix (WH) BF-1 gene, which encodes brain factor 1 (BF-1) (also known as foxg1), is essential for the proliferation of the progenitor cells of the cerebral cortex. Here we show that BF-1-deficient telencephalic progenitor cells are more apt to leave the cell cycle in response to transforming growth factor beta (TGF-beta) and activin. We found that ectopic expression of BF-1 in vitro inhibits TGF-beta mediated growth inhibition and transcriptional activation. Surprisingly, we found that the ability of BF-1 to function as a TGF-beta antagonist does not require its DNA binding activity. Therefore, we investigated whether BF-1 can inhibit Smad-dependent transcriptional responses by interacting with Smads or Smad binding partners. We found that BF-1 does not interact with Smads. Because the identities of the Smad partners mediating growth inhibition by TGF-beta are not clearly established, we examined a model reporter system which is known to be activated by activin and TGF-beta through Smads and the WH factor FAST-2. We demonstrate that BF-1 associates with FAST-2. This interaction is dependent on the same region of protein which mediates its ability to interfere with the antiproliferative activity of TGF-beta and with TGF-beta-dependent transcriptional activation. Furthermore, the interaction of FAST-2 with BF-1 is mediated by the same domain which is required for FAST-2 to interact with Smad2. We propose a model in which BF-1 interferes with transcriptional responses to TGF-beta by interacting with FAST-2 or with other DNA binding proteins which function as Smad2 partners and which have a common mode of interaction with Smad2.

Dihydroartemisinin attenuates lipopolysaccharide-induced osteoclastogenesis and bone loss via the mitochondria-dependent apoptosis pathway.

Dihydroartemisinin (DHA) is a widely used antimalarial drug isolated from the plant Artemisia annua. Recent studies suggested that DHA has antitumor effects utilizing its reactive oxygen species (ROS) yielding mechanism. Here, we reported that DHA is inhibitory on lipopolysaccharide (LPS)-induced osteoclast (OC) differentiation, fusion and bone-resorption activity in vitro. Intracellular ROS detection revealed that DHA could remarkably increase ROS accumulation during LPS-induced osteoclastogenesis. Moreover, cell apoptosis was also increased by DHA treatment. We found that DHA-activated caspase-3 increased Bax/Bcl-2 ratio during LPS-induced osteoclastogenesis. Meanwhile, the translocation of apoptotic inducing factor (AIF) and the release of cytochrome c from the mitochondria into the cytosol were observed, indicating that ROS-mediated mitochondrial dysfunction is crucial in DHA-induced apoptosis during LPS-induced osteoclastogenesis. In vivo study showed that DHA treatment decreased OC number, prevents bone loss, rescues bone microarchitecture and restores bone strength in LPS-induced bone-loss mouse model. Together, our findings indicate that DHA is protective against LPS-induced bone loss through apoptosis induction of osteoclasts via ROS accumulation and the mitochondria-dependent apoptosis pathway. Therefore, DHA may be considered as a new therapeutic candidate for treating inflammatory bone loss.

Microarray based analysis of gene expression patterns in pancreatic neuroendocrine tumors.

OBJECTIVE: Pancreatic neuroendocrine tumors (PanNETs) are a small subgroup of tumors with a variety of biological behaviors. MATERIALS AND METHODS: We sought to identify the specially expressed genes and characterize significant pathways in PanNETs compared with non-neoplastic samples. Gene expression profile GSE43795 was obtained from Gene Expression Omnibus database, which included 6 PanNETs and 5 non-neoplastic samples. The differentially expressed genes (DEGs) were identified using Limma package. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to enrich the functions and pathways of DEGs. Transcription factors (TFs) and tumor-associated genes (TAGs) were also identified. Finally, a protein-protein interaction (PPI) network was constructed, and hub proteins and functional module were screened out. RESULTS: Total of 821 DEGs (421 down-regulated, 400 up-regulated) were selected. GO and KEGG enrichment analyses showed that up-regulated DEGs were related to several pathways, including type 2 diabetes mellitus, Ca2+ signaling pathway, long-term potentiation, and long-term depression pathways. Down-regulated DEGs were enriched in several pathways, such as pancreatic secretion, protein digestion and absorption, and metabolic pathway. Interferon-stimulated gene protein 15 (ISG15), somatostatin (SST), and synaptosomal-associated protein 25 kDa (SNAP25) were identified as hub proteins. CONCLUSIONS: The genes involved in type 2 diabetes mellitus pathway may play important roles in the development of PanNETs. SNAP25, SST, and ISG15 may be used as potential targets for treatment of PanNETs.