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Herbicide resistance in weeds and environmental pollution resulting from excessive application of chemical herbicides keeps increasing. Development of environment-friendly and effective weed management strategies are required for sustainable agricultural production. In this study we investigated the effects of duckweeds (Landoltia punctata (G. Meyer) LesD. J. Crawford and Spirodela polyrhiza (Linnaeus) Schle iden) introduction on the weed community and rice growth in paddy fields.The study was conducted in the two rice-growing seasons (2018 and 2019) with three treatments: rice grown without duckweed introduction (CK), with L. punctata introduction (LP), and with S. polyrhiza introduction (SP). On average, LP and SP significantly reduced total weed density by more than 90% and 97%, respectively. Early in the rice-growing season, both duckweed species completely prevented weed growth. Further, both species significantly promoted rice plant growth in the advanced stages. SP significantly improved grain yield of rice by 23%. Light transmittance, temperature of the floodwater and soil, floodwater pH, and dissolved oxygen content significantly decreased following introduction of the duckweeds, indicating that the duckweeds introduction might inhibit weeds growth by altering environmental factors.This study provides a possible environment-friendly way to inhibit weed biomass in the paddy field by introducing duckweeds and interpreted the possible reasons of the impacts of duckweed on environmental variables. Weed control is beneficial for rice growth. Duckweed coverage might be limited in open fields and the associated practice requires additional investigation. © 2022 Society of Chemical Industry.

Traditional Chinese medicine (TCM) has been used to treat infectious diseases and could offer potential drug leads. This study evaluates the in vitro antimicrobial activities from commercially sourced Dryopteris crassirhizoma Nakai (Polypodiaceae) whose authenticity was confirmed by DNA barcoding based on the ribulose bisphosphate carboxylase (rbcL) gene. Powdered rhizomes were sequentially extracted using n-hexane, dichloromethane, ethyl acetate, and methanol at ambient temperature. The dried extracts at different concentrations were tested for antimicrobial activities against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and Mycobacterium smegmatis. D. crassirhizoma extracts exhibited significant antimicrobial activities only against MRSA (minimum inhibitory concentration: 3.125 μg/ml n-hexane extract). Activity-led fractionations of D. crassirhizoma and characterization by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) targeted a fraction (A3), with two anti-MRSA phloroglucinol derivatives, flavaspidic acid AB and norflavaspidic acid AB—being greatly enriched in the latter. The impact of A3 on MRSA cells was examined using untargeted metabolomic analysis and compared to that of other established antibiotics (all treatments normalized to MIC50 at 6 h). This suggested that norflavaspidic acid AB had distinctive effects, one of which involved targeting bioenergetic transformation, metabolism, and particularly acetyl-CoA, on MRSA cells. No cytotoxicity was observed for the norflavaspidic acid AB-enriched fraction against murine HepG2 cells. This study requires further experimental validation but can have indicated a naturally available compound that could help counter the threat of clinically relevant strains with antibiotic resistance.

Traditional Chinese Medicine (TCM) has been used to treat infectious diseases and could offer potential drug leads. This study evaluates the in vitro antimicrobial activities from commercially sourced Dryopteris crassirhizoma Nakai (Polypodiaceae) whose authenticity was confirmed by DNA barcoding based on the ribulose bisphosphate carboxylase (rbcL) gene. Powdered rhizomes were sequentially extracted using n-hexane, dichloromethane, ethyl acetate and methanol at ambient temperature. The dried extracts at different concentrations were tested for antimicrobial activities against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus (MRSA), and Mycobacterium smegmatis. D. crassirhizoma extracts exhibited significant antimicrobial activities only against MRSA (minimum inhibitory concentration: 3.125 µg/mL n-hexane extract). Activity-led fractionations of D. crassirhizoma and characterisation by Ultra performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) targeted a fraction (A3), with two anti MRSA phloroglucinol derivatives, Flavaspidic acid AB and Norflavaspidic acid AB – being greatly enriched with the latter. The impact of A3 on MRSA cells was examined using untargeted metabolomic analysis and compared to that of other established antibiotics (all treatments normalized to MIC50 at 6 h). This suggested that norflavaspidic acid AB had a distinctive effects one of which involved targeting bioenergetic transformation, metabolism, and particularly Acetyl CoA, in MRSA cells. No cytotoxicity was observed for the norflavaspidic acid AB enriched fraction against murine HepG2 cells. This study requires further experimental validation but can have indicated a naturally available compound that could help counter the threat of clinically relevant strains with antibiotic resistance. The uploaded data is the result of non-targeted metabolite profiling in both negative and positive ionisation modes&nbsp

Traditional Chinese medicine (TCM) has been used to treat infectious diseases and could offer potential drug leads. This study evaluates the

Background. Traditional Chinese medicine (TCM) typically attributes the etiopathogenesis of perimenopausal syndrome (PMS) to kidney deficiency in the TCM stratification system for diagnosis. However, the molecular basis of this classical attribution remains to be investigated. Aim of the Study. By unraveling the responses to TCM treatment for kidney deficiency, the metabolomic link between PMS and kidney deficiency can be evaluated for in-depth understanding of the mechanism of TCM treatment and development of better treatment protocols. Materials and Methods. With naturally aged rats as a model for PMS, the metabolomic response to TCM treatment for kidney deficiency was investigated by 1H NMR. Results. 1H NMR metabolomic evidence of plasma samples demonstrates that treatments with two classical TCM prescriptions for kidney deficiency, decoctions of Yougui and Zuogui, result in modulating the metabolic state of the disease model towards that of rats of younger age. Conclusion. The data support the notion that kidney deficiency is responsible, in part at least, for PMS, and the relevant prescriptions are helpful in dampening the changes in the body’s metabolic states to alleviate symptoms of the disorder.

Traditional Chinese Medicine (TCM) has been used to treat infectious diseases and could offer potential drug leads. This study evaluates the in vitro antimicrobial activities commercially sourced Dryopteris crassirhizoma Nakai whose authenticity was confirmed by DNA barcoding based on the ribulose bisphosphate carboxylase (rbcL) gene. Powdered rhizomes were sequentially extracted using n-hexane, dichloromethane, ethyl acetate and methanol at ambient temperature. The dried extracts at different concentrations were tested for antimicrobial activities against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus (MRSA), and Mycobacterium smegmatis. D. crassirhizoma extracts exhibited significant antimicrobial activities only against MRSA. Activity-led fractionations of D. crassirhizoma and characterisation by Ultra performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) identified two potent anti-MRSA phloroglucinol derivatives: Norflavaspidic acid AB and flavaspidic acid AB. The impact of norflavaspidic acid AB on MRSA cells was examined using untargeted metabolomic analysis and compared to that of other established antibiotics (all treatments normalized to MIC50 at 6 h). This suggested that norflavaspidic acid AB had a distinctive effect which involved targeting bioenergetic transformation, metabolism, and particularly acetyl CoA, in MRSA cells. No cytotoxicity was observed for norflavaspidic acid AB against murine HepG2 cells. This study requires further experimental validation but can have indicated a naturally available compound that could help counter the threat of clinically relevant strains with antibiotic resistance.

Ethnopharmacological relevance: Two types of traditional Chinese formulas of botanical drugs are prescribed for treating perimenopausal syndrome (PMS), a disorder in middle-aged women during their transition to menopause. One is for treating PMS as kidney deficiency (KD) due to senescence and declining reproductive functions, and the other is for treating it as liver qi stagnation (LQS) in association with stress and anxiety. Despite the time-tested prescriptions, an objective attestation to the effectiveness of the traditional Chinese treatment of PMS is still to be established and the associated molecular mechanism is still to be investigated.Materials and methods: A model for PMS was generated from perimenopausal rats with chronic restraint stress (CRS). The effectiveness of traditional Chinese formulas of botanical drugs and a combination of two of the formulas was evaluated based on 1H NMR plasma metabolomic, as well as behavioral and physiological, indicators. To investigate whether the formulas contained ligands that could compensate for the declining level of estrogen, the primary cause of PMS, the ligand-based NMR technique of saturation transfer difference (STD) was employed to detect possible interacting molecules to estrogen receptors in the decoction.Results: Each prescription of the classical Chinese formula moderately attenuated the metabolomic state of the disease model. The best treatment strategy however was to combine two traditional Chinese formulas, each for a different etiology, to adjust the metabolomic state of the disease model to that of rats at a much younger age. In addition, this attenuation of the metabolomics of the disease model was by neither upregulating the estrogen level nor supplementing an estrogenic compound.Conclusion: Treatment of PMS with a traditional Chinese formula of botanical drugs targeting one of the two causes separately could ameliorate the disorder moderately. However, the best outcome was to treat the two causes simultaneously with a decoction that combined ingredients from two traditional prescriptions. The data also implicated a new paradigm for phytotherapy of PMS as the prescribed decoctions contained no interacting compound to modulate the activity of estrogen receptors, in contrast to the treatment strategy of hormone replacement therapy.

Ethnopharmacological relevance Tougu Xiaotong capsule (TXC) is a Chinese herbal compound that belongs to a range of Chinese herbs functioning as ‘kidney invigorators and liver softeners’ commonly used to treat osteoarthritis (OA) in China. Aims of the study: The aims of the present study are to confirm the therapeutic effects of TXC in an OA cell model and to determine the mechanisms involved in such effects. Materials and methods A tunicamycin (Tm)-exposed OA cell model was employed, and the effects of TXC were confirmed by observing cell viability and apoptosis. The reduced cell viability and increased apoptosis caused by Tm were improved by TXC, confirming the cellular protection of TXC. We then investigated the expression of biomarkers related to the endoplasmic reticulum (ER) stress pathway, including microRNA-211 (miR-211), a regulator in the ER stress pathway. Results Downregulation of X-box binding protein 1 (Xbp-1) and miR-211 expression following Tm administration was reversed by TXC. Moreover, the upregulation by Tm of the expression levels of binding immunoglobulin protein, Xbp-1, activating transcription factor 4, C/EBP-homologous protein, Caspase-9 and Caspase-3 was downregulated by TXC. These results indicated that the ER stress pathway-related mechanism may play a potential role in the therapeutic effects of TXC. Conclusions The present study provides evidence of the therapeutic effects of TXC at the cell level and describes a cellular model for establishing the mechanisms of the effects of TXC used in the treatment of OA.

Arsenic trioxide (ATO, Trisenox) is an ancient Chinese medicine which have good clinical effects for acute promyelocytic leukemia (APL). However, ATO has serious cardiotoxicity which has restricted its widely applied in clinic. Saposhnikovia divaricata (Trucz.) Schischk was an antidote to ATO in Chinese medical book. Sec-O-Glucosylhamaudol (SOG) is one of its four main active ingredients. So we explore if SOG can detoxify the ATO’s cardiotoxicity through the Nuclear factor erythroid 2-related factor 2(Nrf2) signal pathway and apoptosis pathway. We added 10 μM ATO and 10, 50, 100 μM SOG in Rat cardiac myocyte-like H9c2 cells. The results indicate SOG can active Nrf2 signal pathway, improve Nrf2 phosphorylation levels, promote the expression of Nrf2, HO-1, NQO1 gene and protein, increase the contents of antioxidant enzyme SOD, CAT and GSH-PX, reduce the ROS levels, increase cell activity. By testing the apoptosis-related protein Bax, Bcl-2 and caspase-3, also proved that 10 μM SOG can improve ATO-induced cell apoptosis. So SOG can plays a role in detoxifying ATO cardiac toxicity.

Alchornea cordifolia Müll. Arg. (commonly known as Christmas Bush) has been used traditionally in Africa to treat sickle cell anaemia (a recessive disease, arising from the S haemoglobin (Hb) allele), but the active compounds are yet to be identified. Herein, we describe the use of sequential fractionation coupled with in vitro anti-sickling assays to purify the active component. Sickling was induced in HbSS genotype blood samples using sodium metabisulphite (Na2S2O5) or through incubation in 100% N2. Methanol extracts of A. cordifolia leaves and its sub-fractions showed >70% suppression of HbSS erythrocyte sickling. The purified compound demonstrated a 87.2 ± 2.39% significant anti-sickling activity and 93.1 ± 2.69% erythrocyte sickling-inhibition at 0.4 mg/mL. Nuclear magnetic resonance (NMR) spectra and high-resolution mass spectroscopy identified it as quercitrin (quercetin 3-rhamnoside). Purified quercitrin also inhibited the polymerisation of isolated HbS and stabilized sickle erythrocytes membranes. Metabolomic comparisons of blood samples using flow-infusion electrospray-high resolution mass spectrometry indicated that quercitrin could convert HbSS erythrocyte metabolomes to be like HbAA. Sickling was associated with changes in antioxidants, anaerobic bioenergy, and arachidonic acid metabolism, all of which were reversed by quercitrin. The findings described could inform efforts directed to the development of an anti-sickling drug or quality control assessments of A. cordifolia preparations.

The discovery of antimalarial artemisinin from Artemisia annua L. is an example of how Traditional Chinese Medicine (TCM) may be exploited to meet a recognized need. In this study, we systemically investigated A. annua L. for its antimicrobial activity and assessed it as a source of bioactive natural products for anti-mycobacterial activity.We used a silica gel column to perform antimicrobial activity-guided purification of the A. annua leaf, whose identity was confirmed by rbcL DNA barcoding, and used UHPLC-HRMS and NMR to elucidate the structure of purified active compounds. The antimicrobial activity of crude extracts, isolated compounds and the control artemisinin (Apollo Scientific Ltd) was assessed against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Mycobacterium smegmatis strains by serial micro dilution method (31.25-1000 μg/mL). The isolated compounds were tested for synergistic effects against mycobacterium.Bioactive compounds were purified and identified as deoxyartemisinin and artemisinic acid. Artemisinic acid (MIC 250 μg/mL) was more effective in comparison to deoxyartemisinin (MIC 500 μg/mL) and artemisinin (MIC 1000 μg/mL) against M. smegmatis. We used a molecular docking approach to investigate the interactions between selected anti-mycobacterial compounds and proteins involved in vital physiological functions in M. tuberculosis, namely MtPks13, MtPknB, MtPanK, MtKasA, MtInhA and MtDprE1 and found artemisinic acid showed docking scores superior to the control inhibiters for MtKasA, suggesting it to be a potential nick for further in vitro biological evaluation and anti-TB drug design.

Women are vulnerable to adverse stress events, especially during perimenopause. Substantial evidence has associated the impaired neuronal plasticity with abnormal behaviors under stressful conditions in animals. The Notch signaling pathway is critical for neuronal plasticity in the structure and function of brain areas. In this study, the mid-aged female rats were subjected to chronic restraint stress(CRS) in combination with isolated rearing for 6 weeks. The behavior tests and HPA activity were conducted to evaluate the model. The mRNA and protein levels of Notch1 signaling related genes in the hippocampus(HIP) and prefrontal cortex(PFC) were analyzed by RT-qPCR and western blotting. The promoter methylation levels were measured by bisulfite sequencing PCR analysis. CRS induced depression-like and anxiety-like behaviors in mid-aged stressed females, as shown by decreased locomotor activity, sucrose consumption and increased HPA activity. Moreover, after CRS, the rats exhibited decreased mRNA and protein levels in Jagged1, Notch1 and Hes5 in the HIP and Notch1, Hes1 and Hes5 in the PFC. However, there were no significant promotor methylation changes between the stressed and control female rats. These findings suggest that Notch1 signaling pathway may contribute to the behavioral changes following CRS in mid-aged female rats and the upstream cause of the gene expression changes needs to be further investigated.

Using an enzyme-linked immunosorbent assay (ELISA) and limited dilution methods to screen and clone antigen-specific hybridoma cells is extremely time-consuming and labor-intensive. This work features a simple and rapid cell surface fluorescence immunosorbent assay (CSFIA), designed for the detection and isolation of antigen-specific hybridoma clones. In this assay, antigens are first anchored to the hybridoma cell surface through a dual-functioning molecular Oleyl-PEG4000-NHS. Specific antibodies secreted from hybridoma cells are then captured by the antigens on the cell surface. Positive hybridoma cells are stained using a fluorescently labeled anti-mouse IgG-Fc antibody. After the addition of a methylcellulose semisolid medium, positive clones are easily picked using a pipet. These positive cell clones can be used to produce monoclonal antibodies after direct expansion. Using this method, positive hybridoma clones against both malachite green and porcine epidemic diarrhea virus are selected with high efficiency. Compared to the ELISA-based method, the CSFIA-based method achieved the capability of isolating2-fold more hybridoma clones in25% of the corresponding processing time. In brief, the CSFIA-based method is highly efficient and inexpensive with a simple and direct operation, which is an excellent candidate method for antigen-specific positive clone isolation in a monoclonal antibody preparation.

Expanded hexanucleotide GGGGCC repeat in a noncoding region of C9ORF72 is the most common cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). However, its molecular pathogenesis remains unclear. In our previous study, the expanded GGGGCC repeats have been shown to be sufficient to cause neurodegeneration. In order to investigate the further role of expanded GGGGCC repeats in the neuron, the normal r(GGGGCC)3 and mutant-type expanded r(GGGGCC)30 expression vectors were transfected into Neuro-2a cells. Cell proliferation, dendrite development, and the proteins’ levels of microtubule-associated protein-2 (MAP2) and cyclin-dependent kinase-5 (CDK5) were used to evaluate the cell toxicity of GGGGCC repeats on Neuro-2a cells. The results were shown that expression of expanded GGGGCC repeats caused neuronal cell toxicity in Neuro-2a cells, enhanced the expression of pMAP2 and pCDK5. Moreover, overexpression of Purα repaired expanded GGGGCC repeat-inducing neuronal toxicity in Neuro-2a cells and reduced the expression of pMAP2 and pCDK5. In all, our findings suggested that the expanded GGGGCC repeats might cause neurodegeneration through destroyed neuron cells. And the GGGGCC repeat-induced neuronal cell toxicity was inhibited by upregulation of Purα. We inferred that Purα inhibits expanded GGGGCC repeat-inducing neurodegeneration, which might reveal a novel mechanism of neurodegenerative diseases ALS and FTD.

Background Aconitum plants have been widely used in China for thousands of years. Recent evidences indicate that aconitine, the main active ingredient of Aconitum, has immunomodulatory properties that might be useful for treating autoimmune diseases, such as rheumatoid arthritis. In this study, we conducted a pilot study to explore the effect and mechanisms of aconitine on the treatment of systemic lupus erythematosus. Methods A pristane-induced murine model was used. The pristane-induced mice were treated with aconitine (25, 75 μg kg −1 d −1 , po ) for 9 weeks. Every three weeks, proteinuria was detected to monitor the kidney damage and blood was collected to measure serum levels of autoantibodies, besides the kidney pathological examination. The major B cell activating factor and major pro-inflammatory mediators, PGE2, IL-17a and IL-6, were also detected. Results We found that aconitine significantly improved the mouse health, decreased the elevated blood leukocyte counts, reduced the serum level of anti-double-stranded DNA (anti-dsDNA) antibody, greatly ameliorated renal histopathologic damage and reduced IgG deposit in glomerular. Furtherly, the levels of PGE2, IL-17a and IL-6, were found to have decreased in aconitine treated mice. Conclusion We have demonstrated that aconitine can inhibit the progression of disease and ameliorate the pathologic lesion of systemic lupus erythematosus.

The upregulation of ELTD1 ([epidermal growth factor (EGF), latrophilin and seven transmembrane domain-containing 1] on chromosome 1) in tumor cells has been reported in several types of cancer and correlates with poor cancer prognosis. However, the role of ELTD1 in glioma progression remains unknown. In this study, we examined ELTD1 expression levels in human glioma cell lines and in sixteen human gliomas of different grades. The molecular effects of ELTD1 in glioma cells were measured using quantitative polymerase chain reaction (qRT-PCR), Western blotting, Cell proliferation assays, Matrigel migration and invasion assays and brain orthotopic xenografts. We found that high expression levels of ELTD1 were positively associated with cancer progression and poor prognosis in human glioma. Mechanistically, ELTD1 activated the JAK/STAT3/HIF-1α signaling axis and p-STAT3 bound with HIF-1α. Taken together, our data provide a plausible mechanism for ELTD1-modulated glioma progression and suggest that ELTD1 may represent a potential therapeutic target in the prevention and therapy of glioma.

Receptor tyrosine kinases are believed to be activated through ligand-induced dimerization. We now demonstrate that in cultured neurons, a substantial amount of endogenous TrkB, the receptor for brain-derived neurotrophic factor (BDNF), exists as an inactive preformed dimer, and the application of BDNF activates the pre-existing dimer. Deletion of the extracellular juxtamembrane motif (EJM) of TrkB increased the amount of preformed dimer, suggesting an inhibitory role of EJM on dimer formation. Further, binding of an agonistic antibody (MM12) specific to human TrkB-EJM activated the full-length TrkB and unexpectedly also truncated TrkB lacking ECD (TrkBdelECD365), suggesting that TrkB is activated by attenuating the inhibitory effect of EJM through MM12 binding-induced conformational changes. Finally, in cells co-expressing rat and human TrkB, MM12 could only activate TrkB human-human dimer but not TrkB human-rat TrkB dimer, indicating that MM12 binding to two TrkB monomers is required for activation. Our results support a model that TrkB preforms as an inactive dimer and BDNF induces TrkB conformation changes leading to its activation.

The temporomandibular joint (TMJ), a unique synovial joint whose development differs from that of other synovial joints, develops from two distinct mesenchymal condensations that grow toward each other and ossify through different mechanisms. The short stature homeobox 2 (Shox2) gene serves an important role in TMJ development and previous studies have demonstrated that Shox2SHOX KI/KI mice display a TMJ defective phenotype, congenital dysplasia and premature eroding of the articular disc, which is clinically defined as a TMJ disorder. In the present study, Shox2SHOX KI/KI mouse models were used to investigate the mechanisms of congenital osteoarthritis (OA)‑like disease during postnatal TMJ growth. Shox2SHOX KI/KI mice were observed to develop a severe muscle wasting syndrome from day 7 postnatal. Histological examination indicated that the condyle and glenoid fossa of Shox2SHOX KI/KI mice was reduced in size in the second week after birth. The condyles of Shox2SHOX KI/KI mice exhibited reduced expression levels of collagen type II and Indian hedgehog, and increased expression of collagen type I. A marked increase in matrix metalloproteinase 9 (MMP9) and MMP13 in the condyles was also observed. These cellular and molecular defects may contribute to the observed (OA)‑like phenotype of Shox2SHOX KI/KI mouse TMJs.

Recovery of common agricultural weeds after burial by soil was studied in four greenhouse and three field experiments. Species studied included velvetleaf, Powell amaranth, common lambsquarters, barnyardgrass, and giant foxtail. Seedlings were bent over before burial to simulate the effect of the impact of soil thrown by a cultivator. Altogether, more than 35,000 seedlings were marked and observed for recovery. No seedlings recovered from 4 cm of burial. Recovery from complete burial under 2 cm of soil ranged from 0 to 24% depending on the experiment, species, and watering treatment, but recovery greater than 5% was rare. Large-seeded species tended to recover from complete burial under 2 cm of soil better than small-seeded species. The study did not reveal a difference in recovery of grasses relative to broadleaf weeds. Overall, seedlings tended to recover best when water was applied daily after burial, worst when water was applied once on the day of burial, and to an intermediate extent when no water was applied. However, difference in recovery between the no-water and watering-once treatments were usually small. Also, many experiment by species combinations showed no significant differences among watering treatments. When even a small portion of the seedling was left exposed, recovery generally exceeded 50%. Organic weed management systems commonly use burial of weed seedlings with tine weeders and soil thrown by sweeps and hilling disks to control weeds in crop rows. Recovery from burial could pose a substantial weed management problem in some circumstances, particularly for large-seeded weed species. Maximizing burial depth is important for limiting recovery. Recovery from burial can be minimized by withholding irrigation for several days after hilling-up operations.

Objective: We constructed 3D-model of ONFH in computer according to three-dimensional computerized tomography (3D-CT) data. We determined the location and volume of necrosis to investigate its clinical efficacy. Method: Totally 92 hips (59 cases) with ONFH (44 males, 15 females) were included, with mean age of 37.5 years (range from 26 to 58). Totally 20 cases (35 hips) were induced by corticosteroid (CTSs), 31 (49 hips) induced by alcohol, 4 (4 hips) induced by trauma and 4 (4 hips) idiopathic. All the hips were categorized into stage ARCO II. Finally diagnosed by MRI, all hips were scanned by CT to acquire data in DICOM format. The images were imported into software to extract 3D-shape of femoral heads, necrotic foci, their volumes and distribution in each quadrant. Deviation of volumes between digital image and biopsy specimen was analyzed by SAS9.1 package. Correlativity between collapse and volume of necrosis under specific pathogeneses was also analyzed. Among the cases necessitating total hip arthroplasty (THA) due to advancing to ARCO III, we randomly selected 8 of them to perform 3D-CT scanning thrice prior to surgical operation. Total femoral heads harvested were torn asunder. Cubic capacity of femoral heads and necrotic foci were hereby measured and compared with those acquired from digital models. Result: Through the digital model, necrotic foci were found mainly locating within the super lateral portion of femoral head, coinciding with those observed in biopsy specimen. Average volumetric ratio of digitally acquired necrosis focus/femoral head in 58 collapsed hips was 36.8%. The ratio of the 34 hips without collapse was 17.3%. In collapsed femoral heads, the distribution of necrosis focus was 23.4% in quadrant 1 (q1), 23.6% in q2, 12.1% in q3, 14.4% in q4, 9.0% in q5, 11.8% in q6, 1.6% in q7 and 3.9% in q8. In femoral heads without collapse, the distribution was 34.2% in q1, 29.6% in q2, 11.8% in q3, 11.3% in q4, 6.0% in q5, 6.0% in q6, 0.5% in q7 and 0.4% in q8. As for the average cubic capacities of femoral heads and necrotic foci, those acquired from the digital model and biopsy specimen had no significant difference in matched-pairs test (t = -1.49, P = 0.179 for femoral heads and t = -1.52, P = 0.172 for necrotic foci). There was significant difference (F = 2.720, P = 0.035 < 0.05) among volumetric ratios of necrotic focus/total femoral head under different pathogeneses, which were 33.55% in alcohol-induced cases, 26.74% in the CTSs-induced, 40.41% in the trauma-induced, and 18.39% in idiopathic cases. Shapes of necrotic foci could be subdivided into 5 types: vault, sublobe, dumbbell, ball and petal. The distribution of these 5 types of shape was different among necroses caused by CTSs, alcohol, trauma and idiopathic pathogeneses. The distribution in 58 collapsed femoral heads was also distinct from that in 34 femoral heads without collapse. Through multinomial logistic regression, we analyzed the correlativity among volumetric ratios of necrotic tissue, its distribution in q2(superioanteriomedial portion of femoral head) and collapse. The result showed statistical significance (P was respectively 0.0001 and 0.0005). Decision tree model showed that 94.6% (53/56) hips would progress into collapse if the volumetric ratio of necrotic tissue was over 23.48%. Otherwise, if distribution in q2 was over 45.13%, 83.3% (5/6) hips would progress into collapse. No collapse (0/30) would occur if the distribution of necrotic tissue in q2 was under 45.13%. Conclusion: Digital 3D-model reconstructed from CT scanning can precisely incarnate spatial orientation of necrotic foci in femoral head. Multinomial logistic regression and decision-making tree shows that volumetric ratio of necrotic tissues plays an important role in anticipating collapse of femoral head.

Women are vulnerable to adverse stress events, especially during perimenopause. Substantial evidence has associated the impaired neuronal plasticity with abnormal behaviors under stressful conditions in animals. The Notch signaling pathway is critical for neuronal plasticity in the structure and function of brain areas. In this study, the mid-aged female rats were subjected to chronic restraint stress(CRS) in combination with isolated rearing for 6 weeks. The behavior tests and HPA activity were conducted to evaluate the model. The mRNA and protein levels of Notch1 signaling related genes in the hippocampus(HIP) and prefrontal cortex(PFC) were analyzed by RT-qPCR and western blotting. The promoter methylation levels were measured by bisulfite sequencing PCR analysis. CRS induced depression-like and anxiety-like behaviors in mid-aged stressed females, as shown by decreased locomotor activity, sucrose consumption and increased HPA activity. Moreover, after CRS, the rats exhibited decreased mRNA and protein levels in Jagged1, Notch1 and Hes5 in the HIP and Notch1, Hes1 and Hes5 in the PFC. However, there were no significant promotor methylation changes between the stressed and control female rats. These findings suggest that Notch1 signaling pathway may contribute to the behavioral changes following CRS in mid-aged female rats and the upstream cause of the gene expression changes needs to be further investigated.

While Brain-derived Neurotrophic Factor (BDNF) has long been implicated in treating neurological diseases, recombinant BDNF protein has failed in multiple clinical trials. In addition to its unstable and adhesive nature, BDNF can activate p75NTR, a receptor mediating cellular functions opposite to those of TrkB. We have now identified TrkB agonistic antibodies (TrkB-agoAbs) with several properties superior to BDNF: They exhibit blood half-life of days instead of hours, diffuse centimeters in neural tissues instead millimeters, and bind and activate TrkB, but not p75NTR. In addition, TrkB-agoAbs elicit much longer TrkB activation, reduced TrkB internalization and less intracellular degradation, compared with BDNF. More importantly, some of these TrkB-agoAbs bind TrkB epitopes distinct from that by BDNF, and work cooperatively with endogenous BDNF. Unlike BDNF, the TrkB-agoAbs exhibit a half-life of days/weeks and diffused readily in nerve tissues. We tested one of TrkB-agoAbs further and showed that it enhanced motoneuron survival in the spinal-root avulsion model for motoneuron degeneration in vivo. Thus, TrkB-agoAbs are promising drug candidates for the treatment of neural injury.

Plasma membrane (PM) and mitochondrial membrane (MM) proteins of rat hippocampal neurons were identified and comparatively characterized on the basis of a sequential digestion-centered combinative strategy for sample treatment. A total of 478 membrane proteins were identified, of which 240 had PM localization, 170 had MM localization, and 33 had both of the two subcellular localizations. Compared with the PM proteome, the MM proteome not only was smaller, more basic, and more hydrophobic, but also had a narrower protein molecular mass distribution range and a higher proportion of transmembrane proteins. By functional enrichment analysis, 287 molecular function terms for the PM proteome and 173 for the MM proteome were obtained. The MM proteome had a lower percentage of binding function terms and a higher percentage of catalysis function terms than the PM proteome, suggesting that mitochondrial proteins were more inclined to affect the physiological and biochemical processes by binding various molecules and as enzymes. Biological process enrichment showed that the genes of the PM and MM proteomes were mapped to 1104 and 460 biological processes, respectively. The biological processes with the most mapped genes of the PM proteome included those involved in vesicle recycling, transmitter release, neuronal development, protein and ion transport, etc., whereas those involved in electron transport, ATP synthesis, mitochondrial transport, mitochondrial apoptosis, etc., were the most gene-mapped biological processes for the MM proteome. The present work has deepened our understanding of the structure and function of hippocampal neurons and provided reference methods for research in the related field. Graphical abstract Functional comparison of the plasma membrane and mitochondrial membrane proteomes.

Background Contrast-induced nephropathy (CIN) after coronary angiography is frequently observed in patients with chronic renal insufficiency and no effective measures have been developed for prevention of CIN. There is evidence showing that trimetazidine (TMZ) has renoprotective effect on CIN. This study was to evaluate the role of TMZ in the prevention of CIN in renal dysfunction patients undergoing coronary angiography. Methods A total of 132 patients with renal dysfunction who underwent coronary angiography were enrolled in our study and divided into control group (n = 70) and TMZ group (n = 62). Standard hydration was administered in all the patients. In TMZ group, patients were administered TMZ orally for 48 hours before and 24 hours after coronary angiography. Serum creatinine (SCr) and cystatin (CysC) were detected before and after contrast media injection, and the incidence of CIN was evaluated according to the elevation of SCr. Adverse events were observed in 12 months. Results In both groups, CysC and SCr increased significantly after coronary angiography and peaked at 24 and 48 hours, respectively. CysC and SCr were significantly lower in TMZ group than in control group after coronary angiography. The incidence of CIN and adverse events was reduced in TMZ group when compared with control group ( P = 0.034 and P = 0.043, respectively). Conclusions TMZ in combination with standard hydration is more effective than isotonic saline alone in protecting renal function in patients with renal dysfunction who undergo coronary angiography and can reduce the adverse events within 12 months.

A novel acetolactate synthase (ALS) gene was cloned from Anabaena azotica , which was isolated from China and is regarded as a vital and beneficial photosynthetic microorganism that can contribute to soil fertility and maintain ecosystem stability. The full-length gene consists of an open reading frame (ORF) of 1644 bp and encodes a 59.2 kDa 547 amino acid protein. Sequence alignment using the BLAST similarity search in the GenBank database revealed that the amino acid sequence was similar to ALS derived from Anabaena sp. PCC 7120. The ALS gene was subcloned into the plasmid pET28a (+) and expressed in Escherichia coli BL21 (DE3) by IPTG induction. The recombinant protein was purified and subsequently characterized. The properties of ALS from the E. coli transformant are similar to that of an enzyme from the original A. azotica . Conserved amino acid sequence and the predicted structure of the active site were consistent with those observed for the bacterial enzyme Klebsiella pneumoniae ALS in similar experiments. Our results represent a significant functional demonstration of the existence of an algae ALS subunit and provide a solid basis for further studies on the structure, function and properties of ALS, as well as its response mechanism to inhibitors.

Magnetic manipulation of cell activity offers advantages over optical manipulation but an ideal tool remains elusive. The MagR protein was found through its interaction with cryptochrome (Cry) and the protein in solution appeared to respond to magnetic stimulation (MS). After we initiated an investigation on the specific role of MagR in cellular response to MS, a subsequent study claimed that MagR expression alone could achieve cellular activation by MS. Here we report that despite systematically testing different ways of measuring intracellular calcium and different magnetic stimulation protocols, it was not possible to detect any cellular or neuronal responses to magnetic stimulation in MagR-expressing HEK cells or primary neurons from the dorsal root ganglion and the hippocampus. By contrast, in neurons co-expressing MagR and channelrhodopin, optical but not magnetic stimulation increased calcium influx in hippocampal neurons. Our results indicate that MagR alone is not sufficient to confer cellular magnetic responses.

Amyloid β-peptide (Aβ) has been implicated as a key molecule in the neurodegenerative cascades of Alzheimer's disease (AD). Humanin (HN) is a secretory peptide that inhibits the neurotoxicity of Aβ. However, the mechanism(s) by which HN exerts its neuroprotection against Aβ-induced AD-like pathological changes and memory deficits are yet to be completely defined. In the present study, we provided evidence that treatment of rats with HN increases the number of dendritic branches and the density of dendritic spines, and upregulates pre- and post-synaptic protein levels; these effects lead to enhanced long-term potentiation and amelioration of the memory deficits induced by Aβ(1-42). HN also attenuated Aβ(1-42)-induced tau hyperphosphorylation, apparently by inhibiting the phosphorylation of Tyr307 on the inhibitory protein phosphatase-2A (PP2A) catalytic subunit and thereby activating PP2A. HN also inhibited apoptosis and reduced the oxidative stress induced by Aβ(1-42). These findings provide novel mechanisms of action for the ability of HN to protect against Aβ(1-42)-induced AD-like pathological changes and memory deficits.

Tongue inspection is a unique and important method of diagnosis in traditional Chinese medicine (TCM). It is a diagnostic approach which involves observing the changes in the tongue proper and tongue coating in order to understand the physiological functions and pathological changes of the body. However, the biological basis of TCM tongue diagnosis remains to be poorly understood and lacks systematic investigation at the molecular level. In this study, we evaluated the effects of tongue coating microbiome on changes in the tongue texture and coating in patients with post-menopausal osteoporosis (PMO) of Gan‑shen deficiency syndrome type. Our aim was to delineate the mechanisms of tongue coating microbiome-induced changes in the tongue texture and coating by investigating the histomorphological changes and performing a bacterial analysis of the tongue coating. We found that the number of intermediate cells in the red tongue with a thin coating was higher, while the number of superficial cells in the red tongue with a thin coating was lower. The maturation value (MV) of tongue exfoliated cells in the red tongue with a thin coating decreased, compared with that in the pale red tongue with a thin white coating. Furthermore, the total bacterial count, oral streptococcus, Gram‑positive (G+) and Gram‑negative (G-) anaerobic bacteria in the red tongue with a thin coating was significantly decreased compared with the pale red tongue with a thin white coating. The results of ultrastructural examination demonstrated that the number of epithelial cells and bacteria in the red tongue with a thin coating decreased compared with that in the pale red tongue with a thin white coating. These observations indicate that the tongue coating microbiome may be an important factor contributing to changes in the tongue in patients with PMO of Gan‑shen deficiency syndrome type.

Ruthenium‐based complexes have emerged as promising anticancer, especially antimetastatic agents. Among them, NAMI‐A (trans‐[Ru(III)Cl4 (DMSO)(Im)][ImH], Im = imidazole, DMSO = dimethyl sulfoxide) was well studied. In this study, we studied the antimetastatic activities of two novel NAMI‐A derivatives containing pyridine, G26b and G94a, using cultured cells and tumor‐bearing mice. Same to NAMI‐A, these two complexes displayed little direct cytotoxicity to the cancer cells in vitro and in vivo, but they, especially G26b, significantly reduced the occurrence and development of lung metastases in mice bearing the 4T1 mammary carcinoma. In vitro, these two complexes displayed significant suppressive effect on invasion and migration of cells and tube formation of human umbilical vein endothelial cell, to the same extent of NAMI‐A. The transcription of important molecules involved in metastasis, matrix metalloproteinase 2 and 9 (MMP‐2 and ‐9), and vascular endothelial growth factor, was suppressed by the two complexes, as well as NAMI‐A. Plasma atomic emission spectrometer showed G26b had a longer Ru‐elimination time in lung, which may be a reason for better antimetastatic effect of G26b than NAMI‐A. Our results have demonstrated that G26b is a more effective antimetastatic agent than NAMI‐A.

Nogo-66 plays a central role in the myelin-mediated inhibition of neurite outgrowth. Tau is a microtubule-associated protein involved in microtubule assembly and stabilization. It remains unverified whether tau interacts directly with growth factor receptors, or engages in cross-talk with regeneration inhibitors like Nogo-66. Here, we report that plasmid overexpression of tau significantly elevated the protein levels of total tau, phosphorylated tau, and microtubule-affinity regulating kinase (MARK). Nogo-66 transiently elevated the total tau protein level and persistently reduced the level of p-S262 tau (tau phosphorylated at serine 262), whereas it had little influence on the level of p-T205 tau (tau phosphorylated at threonine 205). Nogo-66 significantly decreased the protein level of MARK. Hymenialdisine, an inhibitor of MARK, significantly reduced the level of p-S262 tau. Overexpression of tau rescued the Nogo-66-induced inhibition of neurite outgrowth in neuroblastoma 2a (N2a) cells and primary cortical neurons. However, concomitant inhibition of MARK abolished the rescue of neurite outgrowth by tau in N2a cells. We conclude that dephosphorylation of tau at S262 is able to regulate Nogo-66 signaling, and that overexpression of tau can rescue the Nogo-66-induced inhibition of neurite outgrowth in vitro.

The ability of neurons in the adult mammalian central nervous system (CNS) to regenerate after injury is limited by inhibitors in CNS myelin. Nogo-66 is the most important myelin inhibitor but the mechanisms of Nogo-66 inhibition of neurite outgrowth remain poorly understood. Particularly, the relationship between Nogo-66 and microtubule-affinity regulating kinase 2 (MARK2) has not been examined. This study investigated the role of MARK2 in Nogo-66 inhibition and the function of MARK2 in neurite elongation in neurons in vitro. MARK2 and phosphorylated MARK2 at Ser212 (p-Ser212) alterations in Neuro 2a cells were assessed at different Nogo-66 exposure times; the relationships between MARK2 and microtubule-associated proteins (MAPs) were determined via the overexpression or interference of MARK2. Our study reports that Nogo-66 inhibited the expression of total MARK2 but also reduced Ser212 phosphorylation of MARK2, whereas levels of MAP1-b and tau varied depending on MARK2 overexpression or reduced expression. Furthermore, MARK2 increased the proportion of tyrosinated α-tubulin, thereby disrupting the stability of tubulin, most likely affecting axonal growth. In line with these results, overexpression of MARK2 promoted neurite elongation and therefore is able to rescue the inhibitory effect of Nogo-66 on neurite growth. In conclusion, the intracellular PKB/MARK2/MAPs/α-tubulin pathway appears to be essential for neurite elongation in neurons in vitro. These results suggest a critical role for MARK2 in overcoming Nogo-66-induced inhibition of axon outgrowth in neurons. Pharmacological activators of MARK2 may be applicable to promote successful axonal outgrowth following many types of CNS injuries.

Nogo-A, a member of the reticulon family, is one of the most important myelin-associated inhibitors for axonal growth, regeneration, and plasticity in the central nervous system. RhoA has been targeted pharmacologically to promote neurite outgrowth and functional recovery in the brain and spinal cord. However, the underlying mechanism of the inhibition of neurite outgrowth by Nogo-A has not yet been fully defined. Protein kinase B (PKB, also known as Akt) is a protein serine/threonine kinase that plays a key role in intracellular signaling and cellular homeostasis. This study reports the role of PKB signaling on Nogo-A-treated PC12 neuronal cells. An inhibitory fragment of Nogo-A (Nogo-66) activated RhoA and reduced the phosphorylation of PKB at Ser473 in a time-dependent manner. In contrast, pretreatment with Y27632, a specific inhibitor of Rho-A, resulted in an increase of the phosphorylation of PKB. Nogo-66 also inhibited the neurite outgrowth of PC12 cells, whereas pretreatment with LY294002, a specific inhibitor of PKB, ameliorated the neurite outgrowth. These data suggest that PKB is involved in the inhibition of neurite outgrowth by Nogo-A in PC12 cells.

The tropomyosin-related kinase A (TrkA) receptor and its ligand, nerve growth factor (NGF), play crucial roles in the development and function of the nervous system. NGF is believed to activate TrkA by bridging two TrkA monomers, leading to TrkA transphosphorylation. However, here we show that the majority of TrkA receptors exist as preformed, yet inactive, homodimers prior to NGF binding by using three different approaches such as chemical crosslinking and enzyme fragment complementation assay. Furthermore, TrkA homodimers are formed in endoplasmic reticulum before newly synthesized receptors reach the cell surface. These findings shed light on molecular mechanisms underlying transmembrane signaling by TrkA.Structured summaryTrkA physically interacts with TrkA by protein complementation assay (View interaction)TrkA physically interacts with TrkA by bimolecular fluorescence complementation (View interaction)TrkA physically interacts with TrkA by cross-linking study (View interaction)

When developing malaria vaccines, the most crucial step is to elucidate the mechanisms involved in protective immunity against the parasites. We found that CD8(+) T cells contribute to protective immunity against infection with blood-stage parasites of Plasmodium yoelii. Infection of C57BL/6 mice with P. yoelii 17XL was lethal, while all mice infected with a low-virulence strain of the parasite 17XNL acquired complete resistance against re-infection with P. yoelii 17XL. However, the host mice transferred with CD8(+) T cells from mice primed only with P. yoelii 17XNL failed to acquire protective immunity. On the other hand, the irradiated host mice were completely resistant to P. yoelii 17XL infection, showing no grade of parasitemia when adoptively transferred with CD8(+) T cells from immune mice that survived infection with both P. yoelii XNL and, subsequently, P. yoelii 17XL. These protective CD8(+) T cells from immune WT mice had the potential to generate IFN-gamma, perforin (PFN) and granzyme B. When mice deficient in IFN-gamma were used as donor mice for CD8(+) T cells, protective immunity in the host mice was fully abrogated, and the immunity was profoundly attenuated in PFN-deficient mice. Thus, CD8(+) T cells producing IFN-gamma and PFN appear to be involved in protective immunity against infection with blood-stage malaria.

Nitrogen-fixing cyanobacteria are vital photosynthetic microorganisms that contribute to soil fertility by fixing atmospheric nitrogen and are also important for maintaining ecosystem stability. These microorganisms can be very sensitive to herbicides because they possess many characteristics of higher plants. Six days after the application of monosulfuron at 0.03 to 0.3 nmol L 21 under laboratory conditions, growth of the nitrogen-fixing cyanobacteria Anabaena flos-aquae, Anabaena azollae, and Anabaena azotica was stimulated, but at higher concentrations (30 to 300 nmol L 21 ) protein synthesis was inhibited. The production of 16 amino acids in A. flos-aquae was reduced from 7 to 69% with increasing monosulfuron concentration. Application of monosulfuron at 3 to 300 nmol L 21 substantially inhibited in vitro acetolactate synthase (ALS) activity as indicated by 50% inhibition index values of 3.3, 65.2, and 101.3 nmol L 21 for A. flos-aquae, A. azollae, and A. azotica, respectively. In contrast, extractable ALS activity was not affected in these algal species with monosulfuron treatments ranging from 0.03 to 300 nmol L 1 except in A. flos-aquae at higher concentrations (30 to 300 nmol L 21 ). The most sensitive species to monosulfuron was A. flos-aquae, followed by A. azollae and A. azotica. Molecular analyses showed that the genomic DNA of A. azollae and A. azotica differed in only one amino acid. Results from photogenetic analyses revealed a high degree of homology between these algae. In contrast, the genomic DNA of A. flos-aquae differed from that of A. azollae and A. azotica in 44 and 45 amino acids, respectively. Our findings support the view that monosulfuron toxicity in these three nitrogen-fixing cyanobacteria is due to interference with protein metabolism via inhibition of branch-chain amino acid biosynthesis, and particularly ALS activity. Nomenclature: Monosulfuron; Anabaena flos-aquae (Lyngb) Breb; Anabaena azollae Strasb; Anabaena azotica Ley. Nitrogen-fixing cyanobacteria are vital photosynthetic microorganisms that contribute to soil fertility by fixing atmospheric nitrogen (Habte and Alexander 1980; Sinha and Kumar 1992) and maintaining ecosystem stability (Irisarri et al. 2001; Shen and Lu 2005). The agronomic importance of free-living symbiotic cyanobacteria as biofertilizers has been recognized (Habte and Alexander 1980). Some cyanobacterial strains that thrive in fields release small quantities of ammonia, as well as other small nitrogenous polypeptides during active growth. Recent research by Shen and Lu (2005) demonstrated that the application of a mixture of 12 species of nitrogen-fixing cyanobacteria (cultured for 90 d; 1 g dry algae weight per liter) after the sowing of a rice (Oryza sativa L.) crop was more beneficial to rice yield and soil fertility than the application (0.78 g L 21 ) of a urea fertilizer [CO(NH2)2] or a nonfertilizer control treatment. After the addition of the algal mixture to the rice paddies, total nitrogen content of rice stems and leaves increased by 27 and 39%, and rice yields increased by 9 and 12% relative to the urea fertilizer and control treatments, respectively. Three of the most common cyanobacteria in agricultural fields in China are the filamentous nitrogen-fixing species Anabaena azotica Ley (a free-living soil cyanobacterium with a high nitrogen-fixing capability), Anabaena flos-aquae (Lyngb) Breb. (a free-living soil cyanobacterium that may be toxic to some organisms), and Anabaena azollae Strasb (a cyanobacterium that forms a symbiotic relationship with the water fern, Azolla spp.) (Li 1962; Shen and Li 1993; Wu and Zhou 2004).

The effects of monosulfuron on growth and photosynthetic pigments of the nitrogen-fixing cyanobacterium Anabaena flos-aquae grown exposed to 2000-, 3000-, and 4000-lux light intensity were studied. Exposed to three light intensities, the seven concentrations of monosulfuron tested can significantly inhibit algal growth in a dose-dependent manner. The cell numbers and growth rate were decreased with the increase in mono-sulfuron concentration, and A. flos-aquae had different degrees of sensitivity to monosulfuron with the most sensitive light intensity being 4000-lux followed by 3000-lux and 2000-lux. The herbicide monosulfuron appeared to have different effects on the synthesis of photosynthetic pigments. The chlorophyll appeared to tackle monosulfuron concentrations. The caroteniod content of algae treated with 0.008 and 0.08 mg/L monosulfuron exposed to 2000-lux had a different stimulatory effect from that of treatments exposed to 3000-lux and 4000-lux, but an inhibitory effect at concentration above 0.8 mg/L. The effect of monosulfuron on biliprotein in cells of A. flos-aquae exposed three light intensities displayed contrary dose dependence.

Acquired immunity against infection with Trypanosoma cruzi is dependent on CD8 + T cells. Here, to develop a vaccine strategy taking advantage of activated CD8 + T cells, we constructed a DNA vaccine, designated pGFP-TSA1, encoding a fusion protein linking GFP to a single CTL epitope of TSA1, a leading candidate for vaccine against T. cruzi . C57BL/6 mice vaccinated with this plasmid showed suppressed parasitemia and prolonged survival. Vaccination with pGFP-TSA1 enhanced epitope-specific cytotoxicity and IFN-γ secretion by CD8 + T cells. Furthermore, the depletion of CD8 + T cells prior to challenge infection with T. cruzi completely abolished this protection, indicating that CD8 + T cells are the principal effector T cells involved. When mice deficient in the proteasome activator PA28α/β or the immunoproteasome subunits LMP2 and LMP7 were used, the protective immunity against infection was profoundly attenuated. Our findings clearly demonstrate that vaccination with pGFP-TSA1 successfully induces protection dependent on CD8 + T cell activation, in which immunoproteasomes play a crucial role. It is noteworthy to document that physical binding of the epitope and GFP is required for induction of this protection, since mice vaccinated with pTSA1-IRES-GFP failed to acquire resistance, probably because the epitope and GFP are separately expressed in the antigen-presenting cells.

Analysis of membrane proteins, particularly integral membrane proteins, still presents a great challenge due to their poor water solubility and low abundance though much effort has been devoted to the solubilization and enrichment of the protein class. In this paper, a two-phase, on-membrane digestion method was developed and applied in the analysis of rat liver membrane proteome. The two-phase system was constituted by mixing n-butanol and 25 mM NH4HCO3. Comparative experiments indicated that the proteins on membranes could be digested in the two-phase system more efficiently than in both 60% methanol and 25 mM NH4HCO3 solutions under the same conditions, thereby improving the identification of the membrane proteins. When the established two-phase system and CapLC-MS/MS was used to analyze rat liver membrane proteome, a total of 411 membrane proteins were identified, more than 80% of which were transmembrane proteins with 1-12 mapped transmembrane domains (TMDs). Because of its extraction and dissolution actions, the two-phase on-membrane digestion system we developed could efficiently improve the digestion and removal of adsorbed nonmembrane proteins, and remarkably increase the number and coverage of identified membrane proteins, particularly the transmembrane proteins. Using our procedure to identify a complementary protein set from all fractions of the two-phase system could achieve a higher coverage of the membrane proteome.

Appropriate gene expression patterns form the basis for bone microvascular endothelial cells' function in femoral head. Although previous studies have elucidated the impact of glucocorticoids on these cells' specific gene expression the exact differential transcriptomes and comprehensive gene expression profiles remain unknown. Using microarray-based platforms we investigated the transcriptome patterns before and after hydrocortisone administration of bone microvascular endothelial cells from human femoral head. Our results highlight the involvement of development differentiation and apoptosis in the bone microvascular endothelial cells. Elucidation of differential gene expression before and after hydrocortisone administration emphasizes the importance of regulatory networks to gene co-expression within biological processes induced by glucocorticoids. With Benjamini–Hochberg characterization we identified 73 up-regulated and 166 down-regulated long noncoding RNAs the expression of 107 of which significantly correlated with 172 mRNAs after administration of hydrocortisone. Transcriptome analysis of bone microvascular endothelial cells from human femoral head samples is highly informative because it is deduced from data comprised of large number of genes expressed above background. The data have been submitted to the repository of Gene Expression Omnibus (Series GSE60332).

Objective: This study aimed to explore the relationship between different risk factors (especially serum alanine aminotransferase [ALT] and aspartate aminotransferase [AST]) and coronary heart disease (CHD). Methods: A total of 610 inpatients were recruited. Initial coronary angiography (CAG) was performed to evaluate the severity of coronary lesions. On the basis of findings from CAG, patients were divided into control group (n=260) and CHD group (n=350). Logistic regression analysis was employed for the evaluation of clinical characteristics and biochemical parameters, aiming to explore the relationship between risk factors (including AST and ALT) and CHD. Results: Results showed type 2 diabetes, hypertension, dyslipidemia, smoking and family history of CHD were clinical risk factors of CHD. Laboratory examinations showed the serum levels of triglycerides, low-density lipoprotein, AST and ALT in CHD group were significantly higher than those in control group (P

MUT1 is an H-2Kb-restricted 8-mer CTL epitope expressed in Lewis lung carcinoma (3LL) tumor cells derived from C57BL/6 (B6) mice. We constructed a chimeric gene encoding ubiquitin-fused MUT1 (pUB-MUT1). By using a gene gun, B6 mice were immunized with the gene prior to challenge with 3LL tumor cells. Tumor growth and lung metastasis were prominently suppressed in mice immunized with pUB-MUT1 but only slightly in those immunized with the MUT1 gene (pMUT) alone. CD8+ T cells were confirmed to be the final effector by in vitro experiments and in vivo removal of the cells with a corresponding antibody. Anti-tumor immunity was profoundly suppressed in mice deficient in an immuno-subunit of proteasome, LMP7. Furthermore, mice deficient in a proteasome regulator, PA28alpha/beta, failed to acquire protective immunity. Thus, application of the ubiquitin-fusion degradation pathway was useful even in immunization with genes encoding a single CTL epitope for induction of specific and active CD8+ T cells.