Pubertal exposure to Microcystin-LR arrests spermatogonia proliferation by inducing DSB and inhibiting SIRT6 dependent DNA repair in vivo and in vitro.

The reproduction toxicity of pubertal exposure to Microcystin-LR (MC-LR) and the underlying mechanism needs to be further investigated. In the current study, pubertal male ICR mice were intraperitoneally injected with 2 µg/kg MC-LR for four weeks. Pubertal exposure to MC-LR decreased epididymal sperm concentration and blocked spermatogonia proliferation. In-vitro studies found MC-LR inhibited cell proliferation of GC-1 cells and arrested cell cycle in G2/M phase. Mechanistically, MC-LR exposure evoked excessive reactive oxygen species (ROS) and induced DNA double-strand break in GC-1 cells. Besides, MC-LR inhibited DNA repair by reducing PolyADP-ribosylation (PARylation) activity of PARP1. Further study found MC-LR caused proteasomal degradation of SIRT6, a monoADP-ribosylation enzyme which is essential for PARP1 PARylation activity, due to destruction of SIRT6-USP10 interaction. Additionally, MG132 pretreatment alleviated MC-LR-induced SIRT6 degradation and promoted DNA repair, leading to the restoration of cell proliferation inhibition. Correspondingly, N-Acetylcysteine (NAC) pre-treatment mitigated the disturbed SIRT6-USP10 interaction and SIRT6 degradation, causing recovered DNA repair and subsequently restoration of cell proliferation inhibition in MC-LR treated GC-1 cells. Together, pubertal exposure to MC-LR induced spermatogonia cell cycle arrest and sperm count reduction by oxidative DNA damage and simultaneous SIRT6-mediated DNA repair failing. This study reports the effect of pubertal exposure to MC-LR on spermatogenesis and complex mechanism how MC-LR induces spermatogonia cell proliferation inhibition.

Limonin delays the progression of intervertebral disc degeneration in vivo and in vitro: the key role of the MAPK/NF-κB and necroptosis pathways.

OBJECTIVES: Limonin has received significant attention due to its multiple biological effects, intervertebral disc degeneration (IDD) is also of interest due to the high prevalence of this disease. In this study, we determined the effects of limonin on IDD and the underlying mechanism of action to find novel ways to treat IDD. METHODS: An IL-1ß-induced cell inflammation model and a lumbar instability model inducing IDD were established to assess the progression of IDD with or without limonin treatment. We further evaluated MAPK/NF-κB and necroptosis pathways and alterations in the extracellular matrix specific within the disc. KEY FINDINGS: Limonin suppresses inflammation in the nucleus pulposus in vitro by reducing the production of pro-inflammatory markers such as iNOS and COX-2. Limonin reduced the activation of the MAPK/NF-κB signalling pathway and the RIP1/RIP3/MLKL necroptosis pathway in the NP cells. Moreover, limonin delays the IDD progression in the lumbar instability model. CONCLUSIONS: Limonin could potentially delay IDD by inhibiting NP cell necroptosis and modulating peripheral matrix proteins within the intervertebral disc and is a potential pharmacological research direction for the therapy in patients with IDD.

Transcriptomic Responses to Chilling Reveal Potential Chilling Tolerance Mechanisms in Cucumber.

Chilling is a devastating stress that has led to a crisis of production for cucumber (Cucumis sativus L.). To determine the molecular mechanisms underlying chilling responses in cucumber, we investigated physiological changes and transcriptomic responses to chilling stress in the chilling-tolerant inbred line CC and chilling-susceptible inbred line R1461. Physiological analysis showed that CC had a higher survival rate, lower H2O2 accumulation, and ion leakage than R1461 after chilling treatment. RNA-seq analysis identified 938 differentially expressed genes (DEGs) in response to chilling and revealed that chilling stress regulated the transcript levels of genes related to hormones, including auxin, salicylic acid (SA), jasmonic acid (JA), and ethylene. RT-qPCR and pharmacological analysis suggested that cucumber chilling tolerance was associated with variation in the gene expression involved in ethylene biosynthesis and signaling. Exogenously applying 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, improved the chilling tolerance of cucumber, while the exogenous application of the ethylene inhibitor AgNO3 impaired the chilling tolerance of cucumber. After ACC treatment, the difference in chilling tolerance between CC and R1461 disappeared, suggesting that the different chilling tolerance level between CC and R1461 is dependent on the ethylene biosynthesis and signaling pathway. In addition, a comparison of cucumber lines with different chilling tolerances revealed that chilling tolerance is highly associated with the up-regulation of C-repeat binding factor (CBF) genes, while natural variation in the promoter of CsCBF1 is associated with chilling response. This study thus provides information on transcriptomic responses in different varieties of chilling-tolerant cucumber and reveals potential chilling tolerance mechanisms that could be used to improve chilling tolerance in cucumber.

AZ-628 delays osteoarthritis progression via inhibiting the TNF-α-induced chondrocyte necroptosis and regulating osteoclast formation.

As a degenerative disease, the pathogenesis and treatment of osteoarthritis (OA) are still being studied. The prevailing view is that articular cartilage dysfunction plays an essential role in the development of osteoarthritis. Similarly, dynamic bone remodeling dramatically influences the development of osteoarthritis. The inflammatory response is caused by the overexpression of inflammatory factors, among which tumor necrosis factor-α is one of the main causes of OA, and its sources include the secretion of chondrocytes themselves and osteoclast secretion of subchondral bone. Moreover, TNF-α-induced activation of RIP1, RIP3, and MLKL has been shown to play an important role in cell necroptosis and inflammatory responses. In vitro, AZ-628 alleviates chondrocyte inflammation and necroptosis by inhibiting the NF-κB signaling pathway and RIP3 activation instead of RIP1 activation. AZ-628 also reduces osteoclast activity, proliferation and differentiation, and release of inflammatory substances by inhibiting autophagy, MAPK, and NF-κB pathways. Similarly, the in vivo study demonstrated that AZ-628 could inhibit chondrocyte breakdown and lower osteoclast formation and bone resorption, thereby slowing down subchondral bone changes induced by dynamic bone remodeling and reversing the progression of osteoarthritis in mice. The results of this study indicate that AZ-628 could be used to treat OA byinhibiting chondrocyte necroptosis and regulating osteoclast formation.

Dl-3-n-butylphthalide Attenuates Spinal Cord Injury via Regulation of MMPs and Junction Proteins in Mice.

As a serious trauma of the neurological system, spinal cord injury (SCI) results in permanent disability, gives rise to immediate vascular damage and a wide range of matters that induce the breakage of blood spinal cord barrier (BSCB). SCI activates the expression of MMP-2/9, which are considered to accelerate the disruption of BSCB. Recent research shows that Dl-3-n-butylphthalide (NBP) exerted protective effects on blood spinal cord barrier in animals after SCI, but the underlying molecular mechanism of NBP on the BSCB undergoing SCI is unknown. Here, our research show that NBP inhibited the expression of MMP-2/9, then improved the permeability of BSCB following SCI. After the T9 level of spinal cord performed with a moderate injury, NBP was managed by intragastric administration and further performed once a day. NBP remarkably improved the permeability of BSCB and junction proteins degration, then promoted locomotion recovery. The protective effect of NBP on BSCB destruction is related to the regulation of MMP-2/9 induced by SCI. Moreover, NBP obviously inhibited the MMP-2/9 expression and junction proteins degradation in microvascular endothelial cells. In conclusion, our results indicate that MMP-2/9 are relevant to the breakdown of BSCB, NBP impairs BSCB destruction through inhibiting MMP-2/9 and promotes functional recovery subjected to SCI. NBP is likely to become a new nominee as a therapeutic to treat SCI via a transigent BSCB.

[Regulatory effect of traditional Chinese medicine on intestinal microbiota].

As a large micro-ecosystem in the human body,the intestinal microbiota is closely associated with the occurrence of many diseases.The clinical investigations and animal experiments have showed that traditional Chinese medicine(TCM) could maintain the balance of the intestinal micro-ecological system.This review summarized the research methods and literatures on the regulation effects of TCM,including different effective ingredients,extracts and Chinese herbal formulae,on intestinal microflora in recent five years,in order to provide a reference for the further research and development of TCM.

[Expression of TLR4/MyD88/NF-κB pathway genes and its related inflammatory factors in secondary spinal cord injury].

OBJECTIVE: To investigate the expression of Toll-like receptor 4 (TLR4)/myeloid differentiation factor (MyD88)/nuclear factor-κB (NF-κB) pathway genes and related inflammatory factors tumor necrosis factor-α (TNF-α), interleukin (IL)-12, IL-6 in patients with secondary spinal cord injury (SSCI) and the correlations with prognosis. METHODS: The clinical data of 105 SSCI patients and 40 healthy subjects were reviewed. According to Frankel's classification of spinal cord injury, the patients were divided into complete injury group and incomplete injury group, and according to the improvement of Japanese Orthopedic Association (JOA) scores, the patients were divided into good prognosis group and poor prognosis group. The expression of TLR4, MyD88, NF-κB in peripheral blood mononuclear cells (PBMC) and serum TNF-α, IL-12, IL-6 levels were compared between SSCI patients and healthy controls, between patients with complete and incomplete injury, between patients with poor and good prognosis. Logistic regression analysis was used to analyze the risk factors leading to poor prognosis of SSCI, and Pearson's correlation analysis was used to analyze the correlation between JOA score and the above indicators. RESULTS: The expressions of TLR4, MyD88, NF-κB in PBMC and serum TNF-α, IL-12, IL-6 levels in SSCI patients were significantly higher than those in healthy subjects (all P<0.01), those in complete injury group were higher than those in incomplete injury group, and those in poor prognosis group were higher than those in good prognosis group (all P<0.01). The proportions of patients with Frankel grade A, spinal cord edema or hemorrhage, spinal cord injury length longer than 4 cm in poor prognosis group was significantly higher than those in good prognosis group (all P<0.01). Logistic regression analysis showed that Frankel grade, spinal cord edema or hemorrhage, length of spinal cord injury, relative expressions of TLR4, MyD88, NF-κB in PBMC, serum levels of TNF-α, IL-12 and IL-6 were risk factors for poor prognosis in SSCI patients (P<0.05 or P<0.01). Pearson's correlation analysis showed that JOA improvement rate was negatively correlated with the relative expressions of TLR4, MyD88, NF-κB mRNA in PBMC and serum TNF-α, IL-12, IL-6 levels (P<0.05 or P<0.01). CONCLUSIONS: The activation of TLR4/MyD88/NF-κB pathway and the up-regulation of the expression of related inflammatory factors TNF-α, IL-12 and IL-6 are involved in the progression of SSCI, which are closely related to the neuroinflammatory injury, and can be used as reference indexes for evaluating prognosis in SSCI patients.

Accuracy of pedicle screw placement in posterior lumbosacral instrumentation by computer tomography evaluation: A multi-centric retrospective clinical study.

BACKGROUND: Pedicle screw misplacement rates are relatively high with fluoroscopically guided techniques. However, breach rates in the 5th lumbar and the 1st sacral spines in conventional operations have not been specifically concerned because of their broad cross sections. It's a retrospective study to evaluate the accuracy and safety of pedicle screw placement in posterior lumbosacral instrumentation under CT scan with reconstruction. MATERIALS AND METHODS: 401 patients were evaluated under CT scan with reconstruction in 3 hospitals by 2 professional observers after posterior lumbosacral instrumentation including 152 3rd lumbar spines (L3), 219 4th lumbar spines (L4), 270 5th lumbar spines (L5) and 95 1st sacral spines (S1) with screws placed. Patients were followed for potential clinical symptoms. RESULTS: In a total of 1467 instrumented pedicles, there were 371 pedicle breaches. Of these, the segment of the breached pedicles were L3: 91/301 (30.2%), L4: 126/436 (29.0%), L5: 132/539 (24.5%), S1: 22/191 (11.5%). For severe violation from L3 to S1, 8/91 (8.8%), 8/126 (6.3%), 19/132 (14.4%), 8/22 (36.4%) were confirmed respectively. Furthermore, the inferomedial breach sites quantified from L3 to S1 were 43/91 (47.3%), 74/126 (58.7%), 99/132 (75%), 19/22 (86.4%) respectively. And there were 9 cases of cerebrospinal fluid leakage and 3 cases of neurological deficit. CONCLUSION: L3, L4 and L5 have no significant differences in pedicle breach rates. The incidences of high risk pedicle breach (grade III, grade IV) are higher in L5 and S1, and the breach sites are more common in the inferomedial wall of L5 and S1 than that of L3 and L4. Many surgeons took it for granted that L5 and S1 were safe when inserting pedicle screws, but they are not safe as expected actually.

Increases of ferrous iron oxidation activity and arsenic stressed cell growth by overexpression of Cyc2 in Acidithiobacillus ferrooxidans ATCC19859.

Acidithiobacillus ferrooxidans plays an important role in bioleaching in reproducing the mineral oxidant of ferric iron (Fe(3+) ) by oxidization of ferrous iron (Fe(2+) ). The high-molecular-weight c-type cytochrome Cyc2 that is located in the external membrane is postulated as the first electron carrier in the Fe(2+) oxidation respiratory pathway of A. ferrooxidans. To increase ferrous iron oxidation activity, a recombinant plasmid pTCYC2 containing cyc2 gene under the control of Ptac promoter was constructed and transferred into A. ferrooxidans ATCC19859. The transcriptional level of cyc2 gene was increased by 2.63-fold and Cyc2 protein expression was observed in the recombinant strain compared with the control. The ferrous iron oxidation activity and the arsenic stressed cell growth of the recombinant strain were also elevated.

Complete genome of Leptospirillum ferriphilum ML-04 provides insight into its physiology and environmental adaptation.

Leptospirillum ferriphilum has been identified as the dominant, moderately thermophilic, bioleaching microorganism in bioleaching processes. It is an acidic and chemolithoautrophic bacterium that gains electrons from ferrous iron oxidation for energy production and cell growth. Genetic information about this microorganism has been limited until now, which has hindered its further exploration. In this study, the complete genome of L. ferripilum ML-04 is sequenced and annotated. The bacterium has a single circular chromosome of 2,406,157 bp containing 2,471 coding sequences (CDS), 2 rRNA operons, 48 tRNA genes, a large number of mobile genetic elements and 2 genomic islands. In silico analysis shows L. ferriphilum ML-04 fixes carbon through a reductive citric acid (rTCA) cycle, and obtains nitrogen through ammonium assimilation. The genes related to "cell envelope biogenesis, outer membrane" (6.9%) and "DNA replication, recombination and repair" (5.6%) are abundant, and a large number of genes related to heavy metal detoxification, oxidative and acidic stress defense, and signal transduction pathways were detected. The genomic plasticity, plentiful cell envelope components, inorganic element metabolic abilities and stress response mechanisms found the base for this organism's survival in the bioleaching niche.

A weighted multi-scale morphological gradient filter for rolling element bearing fault detection.

This paper presents a novel signal processing scheme, named the weighted multi-scale morphological gradient filter (WMMG), for rolling element bearing fault detection. The WMMG can depress the noise at large scale and preserve the impulsive shape details at small scale. Both a simulated signal and vibration signals from a bearing test rig are employed to evaluate the performance of the proposed technique. The traditional envelope analysis and a multi-scale enveloping spectrogram algorithm combining continuous wavelet transform and envelope analysis (WT-EA) are also studied and compared with the presented WMMG. Experimental results have demonstrated the effectiveness of the WMMG to extract the impulsive components from the raw vibration signal with strong background noise. We also investigated the classification performance on identifying bearing faults based on the WMMG and statistical parameters with varied noise levels. Application results reveal that the WMMG achieves the same or better performance as EA and WT-EA. Meanwhile, the WMMG requires low computation cost and is very suitable for on-line condition monitoring of bearing operating states.

Overexpression of rusticyanin in Acidithiobacillus ferrooxidans ATCC19859 increased Fe(II) oxidation activity.

A wide-host-range plasmid pTRUS containing a homologous rus gene under the control of Ptac promoter was constructed and transferred into Acidithiobacillus ferrooxidans ATCC19859 using conjugation gene transfer method to generate the engineered strain of A. ferrooxidans(pTRUS). The plasmid-based recombinant rus gene was successfully expressed. According to the results of real-time quantitative PCR assay, not only the transcription level of recombinant rus gene but also the transcription levels of the other genes of rus operon (cyc1, orf, coxB, coxA) were increased in A. ferrooxidans(pTRUS). The ferrous ion (Fe(2+)) oxidation activity was increased in A. ferrooxidans(pTRUS).

Arsenic resistance operon structure in Leptospirillum ferriphilum and proteomic response to arsenic stress.

The response of Leptospirillum ferriphilum ML-04 to arsenic stress was analyzed using two-dimensional electrophoresis (2-DE), matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Thirty-eight of 65 significantly differentially expressed arsenic response proteins were identified, and 25 of them have known functions. Three proteins are arsenic resistance system (ARS) member proteins. Two ars operons appear to be present in this strain. In addition to the ARS system, phosphate regulation and glutathione (GSH) synthesis appear involved in As[V] and As[III] tolerance, respectively. These findings provide information potentially useful for the genetic engineering of arsenic resistant organisms.

Y55 and D78 are crucial amino acid residues of a new IgE epitope on trichosanthin.

Trichosanthin (TCS) possesses many biological and pharmaceutical activities, but its strong immunogenicity limits its clinical application. To reduce the immunogenicity of TCS, we modified the reported method for the prediction of antigenic site and identified two crucial amino acid residues (Y55 and D78) for a new epitope. We mutated these two residues into glycine and serine, respectively, and obtained three mutants, Y55G, D78S, and Y55G/D78S. These mutants induced less amount of Ig and IgG antibodies in C57BL/6J mice than wild-type TCS (wTCS) (p<0.01) and almost lost the ability to induce IgE antibody production. The mutants stimulated fewer TCS-specific B cells in C57BL/6J mice than wTCS (p<0.01). Compared with wTCS, Y55G, D78S, and Y55G/D78S lost 26.9%, 17.9%, and 98.7% specific binding ability to anti-TCS monoclonal antibody TCS4E9, respectively. These mutants still retained RNA N-glycosidase activity. In conclusion, Y55 and D78 are two crucial amino acid residues of a new IgE epitope on TCS, and their mutation reduces the immunogenicity of TCS, but still retained the enzymatic activity.

Enhanced green fluorescence protein tracks trichosanthin in human choriocarcinoma cells as a feasible and stable reporter.

Trichosanthin (TCS) is a ribosome-inactivating protein (RIP) which can inhibit the growth of human choriocarcinoma (JAR) cells. There are no clear mechanisms to discover the interaction pathway and cytotoxicity of TCS in JAR cells. In this paper, we showed the distribution and transport of endogenously expressed TCS in JAR cells. Enhanced Green Fluorescence Protein (EGFP), fused with TCS, was applied as a reporter to track the behavior of TCS in JAR cells. Firstly, we investigated the expression stability of EGFP and physiological effects on JAR cells. A stable cell line expressing EGFP was created, which could reproduce and express EGFP even if transplanted into nude mice. Based on the proved stability and feasibility of EGFP in cultured cells and in vivo, the fusion gene of EGFP and TCS was constructed and transfected into JAR cells by liposome. The fluorescence microscopy showed that TCS-EGFP fusion gene was expressed in JAR cells in 24 to 48 hours and the fluorescence spread in cytoplasm mainly and in nucleus partially, which could trace the distribution and transport of TCS-EGFP in JAR cells. Most of fluorescent cells died after 48 hours for the cytotoxicity of expressed TCS-EGFP. These results first reported a stable expression and tracing method by EGFP in JAR cells, and provided theoretical basis to apply TCS in cancer therapy.

Trichomislin, a novel ribosome-inactivating protein, induces apoptosis that involves mitochondria and caspase-3.

Trichomislin, a novel ribosome-inactivating protein, was cloned from the genome of Trichosanthes kirilowii Maxim. The gene was recombined to prokaryotic expression vector and the protein was purified by cation-exchange chromatography. The secondary structure of trichomislin was measured by circular-dichroism analysis and the ratios of alpha-helices and beta-sheets were calculated. Trichomislin could inhibit the synthesis of protein in rabbit reticulocyte lysate systems and its reaction mechanism was to inactivate ribosome as an rRNA N-glycosidase. Antitumor analyses indicated trichomislin induced the apoptosis and inhibited the growth of choriocarcinoma cells. Further investigation showed that trichomislin could bind to and enter choriocarcinoma cells, and then increase the caspase-3 activity in a time-dependent manner. At the same time, the concentration of cytochrome c in cytosol increased while that in mitochondria decreased. These results suggested that trichomislin induced apoptosis by releasing cytochrome c from mitochondria which then triggered the caspase family member activation.