BRD9-mediated control of the TGF-ß/Activin/Nodal pathway regulates self-renewal and differentiation of human embryonic stem cells and progression of cancer cells.

Bromodomain-containing protein 9 (BRD9) is a specific subunit of the non-canonical SWI/SNF (ncBAF) chromatin-remodeling complex, whose function in human embryonic stem cells (hESCs) remains unclear. Here, we demonstrate that impaired BRD9 function reduces the self-renewal capacity of hESCs and alters their differentiation potential. Specifically, BRD9 depletion inhibits meso-endoderm differentiation while promoting neural ectoderm differentiation. Notably, supplementation of NODAL, TGF-ß, Activin A or WNT3A rescues the differentiation defects caused by BRD9 loss. Mechanistically, BRD9 forms a complex with BRD4, SMAD2/3, ß-CATENIN and P300, which regulates the expression of pluripotency genes and the activity of TGF-ß/Nodal/Activin and Wnt signaling pathways. This is achieved by regulating the deposition of H3K27ac on associated genes, thus maintaining and directing hESC differentiation. BRD9-mediated regulation of the TGF-ß/Activin/Nodal pathway is also demonstrated in the development of pancreatic and breast cancer cells. In summary, our study highlights the crucial role of BRD9 in the regulation of hESC self-renewal and differentiation, as well as its participation in the progression of pancreatic and breast cancers.

Comprehensive Analysis of Tumor Microenvironment Reveals Prognostic ceRNA Network Related to Immune Infiltration in Sarcoma.

PURPOSE: Sarcoma is the second most common solid tumor type in children and adolescents. The high level of tumor heterogeneity as well as aggressive behavior of sarcomas brings serious difficulties to developing effective therapeutic strategies for clinical application. Therefore, it is of great importance to identify accurate biomarkers for early detection and prognostic prediction of sarcomas. EXPERIMENTAL DESIGN: In this study, we characterized three subtypes of sarcomas based on tumor immune infiltration levels (TIIL), and constructed a prognosis-related competing endogenous RNA (ceRNA) network to investigate molecular regulations in the sarcoma tumor microenvironment (TME). We further built a subnetwork consisting of mRNAs and lncRNAs that are targets of key miRNAs and strongly correlated with each other in the ceRNA network. After validation using public data and experiments in vivo and in vitro, we deeply dug the biological role of the miRNAs and lncRNAs in a subnetwork and their impact on TME. RESULTS: Altogether, 5 miRNAs (hsa-mir-125b-2, hsa-mir-135a-1, hsa-mir92a-2, hsa-mir-181a-2, and hsa-mir-214), 3 lncRNAs (LINC00641, LINC01146, and LINC00892), and 10 mRNAs (AGO2, CXCL10, CD86, CASP1, IKZF1, CD27, CD247, CD69, CCR2, and CSF2RB) in the subnetwork were identified as vital regulators to shape the TME. On the basis of the systematic network, we identified that trichostatin A, a pan-HDAC inhibitor, could potentially regulate the TME of sarcoma, thereby inhibiting the tumor growth. CONCLUSIONS: Our study identifies a ceRNA network as a promising biomarker for sarcoma. This system provides a more comprehensive understanding and a novel perspective of how ceRNAs are involved in shaping sarcoma TME.

Optimization strategies of mesenchymal stem cell-based therapy for acute kidney injury.

Considering the high prevalence and the lack of targeted pharmacological management of acute kidney injury (AKI), the search for new therapeutic approaches for it is in urgent demand. Mesenchymal stem cells (MSCs) have been increasingly recognized as a promising candidate for the treatment of AKI. However, clinical translation of MSCs-based therapies is hindered due to the poor retention and survival rates as well as the impaired paracrine ability of MSCs post-delivery. To address these issues, a series of strategies including local administration, three-dimensional culture, and preconditioning have been applied. Owing to the emergence and development of these novel biotechnologies, the effectiveness of MSCs in experimental AKI models is greatly improved. Here, we summarize the different approaches suggested to optimize the efficacy of MSCs therapy, aiming at promoting the therapeutic effects of MSCs on AKI patients.

Homologous recombination deficiency status predicts response to platinum-based chemotherapy in Chinese patients with high-grade serous ovarian carcinoma.

BACKGROUND: Homologous Recombination Deficiency (HRD) is a predictive biomarker for ovarian cancer treated with PARP inhibitors or for breast cancer treated with first-line platinum-based chemotherapy. However, limited research is documented on platinum-based treatment prediction with HRD as a biomarker in ovarian cancer patients, especially in the Chinese population. METHODS: We investigated the association between HRD status and the response of platinum-based chemotherapy in 240 Chinese HGSOC patients. RESULTS: The Pt-sensitive patients showed higher HRD scores than Pt-resistant ones, but this was not significant(median: 42.6 vs. 31.6, p = 0.086). (Pt)-sensitive rate was higher in HRD + BRCAm tumors and in HRD + BRCAwt tumors (HRD + BRCAm: 97%, p = 0.004 and HRD + BRCAwt: 90%, p = 0.04) compared with 74% in the HRD-BRCAwt tumors. We also found Pt-sensitive patients tend to be enriched in patients with BRCA mutations or non-BRCA HRR pathway gene mutations (BRCA: 93.6% vs 75.4%, p < 0.001; non-BRCA HRR: 88.6% vs 75.4%, p = 0.062). Patients with HRD status positive had significantly improved PFS compared with those with HRD status negative (median PFS: 30.5 months vs. 16.8 months, Log-rank p = 0.001). Even for BRCAwt patients, positive HRD was also associated with better PFS than the HRD-negative group (median: 27.5 months vs 16.8 months, Log-rank p = 0.010). Further, we found patients with pathogenic mutations located in the DNA-binding domain (DBD) of BRCA1 had improved FPS, compared to those with mutations in other domains. (p = 0.03). CONCLUSIONS: The HRD status can be identified as an independent significance in Chinese HGSOC patients treated with first-line platinum-based chemotherapy.

Deep-fried Atractylodes lancea rhizome alleviates spleen deficiency diarrhea-induced short-chain fatty acid metabolic disorder in mice by remodeling the intestinal flora.

ETHNOPHARMACOLOGICAL RELEVANCE: Atractylodes lancea (Thunb.) DC. is a Chinese herb that has been commonly used to treat spleen-deficiency diarrhea (SDD) in China for over a thousand years. However, the underlying mechanism of its antidiarrheal activity is not fully understood. AIM OF THE STUDY: The antidiarrheal effects of the ethanol extract of deep-fried A. lancea rhizome (EEDAR) due to spleen deficiency induced by folium sennae (SE) were determined on the regulation of the short-chain fatty acid (SCFA) metabonomics induced by the intestinal flora. MATERIALS AND METHODS: The effects of EEDAR on a SE-induced mouse model of SDD were evaluated by monitoring the animal weight, fecal water content, diarrhea-grade rating, goblet cell loss, and pathological changes in the colon. The expression of inflammatory factors (tumor necrosis factor [TNF]-α, interleukin [IL]-1ß, IL-6, IL-10), aquaporins (AQP3, AQP4, and AQP8), and tight junction markers (ZO-1, occludin, claudin-1) in colon tissues were determined using quantitative polymerase chain reaction and western blotting. SCFA metabonomics in the feces of mice treated with EEDAR was evaluated using gas chromatography-mass spectrometry. Furthermore, 16S rDNA sequencing was used to determine the effect of EEDAR on the intestinal flora of SDD mice, and fecal microbiota transplantation (FMT) was used to confirm whether the intestinal flora was essential for the anti-SDD effect of EEDAR. RESULTS: Treatment with EEDAR significantly improved the symptoms of mice with SDD by inhibiting the loss of colonic cup cells, alleviating colitis, and promoting the expression of AQPs and tight junction markers. More importantly, the effect of EEDAR on the increase of SCFA content in mice with SDD was closely related to the gut microbiota composition. EEDAR intervention did not significantly improve intestinal inflammation or the barrier of germ-free SDD mice, but FMT was effective. CONCLUSION: EEDAR alleviated SE-induced SDD in mice, as well as the induced SCFA disorder by regulating the imbalance of the intestinal microbiota.

Cell-Specific Actions of the Prostaglandin E-Prostanoid Receptor 4 Attenuating Hypertension: A Dominant Role for Kidney Epithelial Cells Compared With Macrophages.

Background A beneficial role for prostanoids in hypertension is suggested by clinical studies showing nonsteroidal anti-inflammatory drugs, which block the production of all prostanoids, cause sodium retention and exacerbate hypertension. Among prostanoids, prostaglandin E2 and its E-prostanoid receptor 4 receptor (EP4R) have been implicated in blood pressure control. Our previous study found that conditional deletion of EP4R from all tissues in adult mice exacerbates angiotensin II-dependent hypertension, suggesting a powerful effect of EP4R to resist blood pressure elevation. We also found that elimination of EP4R from vascular smooth muscle cells did not affect the severity of hypertension, suggesting nonvascular targets of prostaglandin E mediate this antihypertensive effect. Methods and Results Here we generated mice with cell-specific deletion of EP4R from macrophage-specific EP4 receptor knockouts or kidney epithelial cells (KEKO) to assess the contributions of EP4R in these cells to hypertension pathogenesis. Macrophage-specific EP4 receptor knockouts showed similar blood pressure responses to alterations in dietary sodium or chronic angiotensin II infusion as Controls. By contrast, angiotensin II-dependent hypertension was significantly augmented in KEKOs (mean arterial pressure: 146±3 mm Hg) compared with Controls (137±4 mm Hg; P=0.02), which was accompanied by impaired natriuresis in KEKOs. Because EP4R expression in the kidney is enriched in the collecting duct, we compared responses to amiloride in angiotensin II-infused KEKOs and Controls. Blockade of the epithelial sodium channel with amiloride caused exaggerated natriuresis in KEKOs compared with Controls (0.21±0.01 versus 0.15±0.02 mmol/24 hour per 20 g; P=0.015). Conclusions Our data suggest EP4R in kidney epithelia attenuates hypertension. This antihypertension effect of EP4R may be mediated by reducing the activity of the epithelial sodium channel, thereby promoting natriuresis.

ACE inhibitory activities of two peptides derived from Volutharpa ampullacea perryi hydrolysate and their protective effects on H2O2 induced HUVECs injury.

The purpose of this study is to explore the effects of IVTNWDDMEK and VGPAGPRG, two angiotensin I-converting enzyme (ACE) inhibitory peptides purified from Volutharpa ampullacea perryi, on ACE's two domains and on nitric oxide (NO), endothelin-1(ET-1) production in human vascular endothelial cells (HUVECs). In addition, we sought to investigate the effects of these two peptides on HUVECs injury induced by H2O2. The results indicated that the inhibition of the ACE C-domain was significantly higher than that of the ACE N-domain by these two peptides. Molecular dynamics (MD) analysis revealed that the hydrogen bonds interactions between ACE and two peptides, the chelation between peptides and Zn2+ both play important role, which might contribute significantly to the ACE inhibitory activity. Two peptides significantly increase NO and ET-1 production in a dose-dependent manner and protects against hydrogen peroxide-induced HUVEC cell injury. The reported results also show that two peptides up-regulated the expression of nuclear factor erythroid 2-related factor (Nrf2) and heme oxygenase-1 (HO-1), and reduce the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA). Our study indicated that IVTNWDDMEK and VGPAGPRG could be potent ACE inhibitors and Volutharpa ampullacea perryi is a good source of bioactive peptides, which provided a theoretical basis for the broad application of two selected peptides as functional food with anti-hypertensive activity.

Artificial Kidney Capsule Packed with Mesenchymal Stem Cell-Laden Hydrogel for the Treatment of Rhabdomyolysis-Induced Acute Kidney Injury.

Acute kidney injury (AKI) has emerged as a major public health problem affecting millions of people worldwide without specific and satisfactory therapies due to the lack of an effective delivery approach. In the past few decades, hydrogels present infinite potential in localized drug delivery, while their poor adhesion to moist tissue and isotropic diffusion character always restrict the therapeutic efficiency and may lead to unwanted side effects. Herein, we proposed a novel therapeutic strategy for AKI via a customizable artificial kidney capsule (AKC) together with a mesenchymal stem cell (MSC)-laden hydrogel. Specifically, an elastic capsule owning an inner chamber with the same size and shape as the kidney is designed and fabricated through three-dimensional (3D) modeling and printing, serving as an outer wrap for kidney and cell-laden hydrogels. According to the in vitro experiment, the excellent biocompatibility of gelatin-based hydrogel ensures viability and proliferation of MSCs. In vivo mice experiments proved that this concept of AKC-assisted kidney drug delivery could efficiently reduce epithelial cell apoptosis and minimize the damage of the renal tubular structure for mice suffering AKI. Such a strategy not only provides a promising alternative in the treatment of AKI but also offers a feasible and versatile approach for the repair and recovery of other organs.

GSA: an independent development algorithm for calling copy number and detecting homologous recombination deficiency (HRD) from target capture sequencing.

BACKGROUND: The gain or loss of large chromosomal regions or even whole chromosomes is termed as genomic scarring and can be observed as copy number variations resulting from the failure of DNA damage repair. RESULTS: In this study, a new algorithm called genomic scar analysis (GSA) has developed and validated to calculate homologous recombination deficiency (HRD) score. The two critical submodules were tree recursion (TR) segmentation and filtering, and the estimation and correction of the tumor purity and ploidy. Then, this study evaluated the rationality of segmentation and genotype identification by the GSA algorithm and compared with other two algorithms, PureCN and ASCAT, found that the segmentation result of GSA algorithm was more logical. In addition, the results indicated that the GSA algorithm had an excellent predictive effect on tumor purity and ploidy, if the tumor purity was more than 20%. Furtherly, this study evaluated the HRD scores and BRCA1/2 deficiency status of 195 clinical samples, and the results indicated that the accuracy was 0.98 (comparing with Affymetrix OncoScan™ assay) and the sensitivity was 95.2% (comparing with BRCA1/2 deficiency status), both were well-behaved. Finally, HRD scores and 16 genes mutations (TP53 and 15 HRR pathway genes) were analyzed in 17 cell lines, the results showed that there was higher frequency in HRR pathway genes in high HRD score samples. CONCLUSIONS: This new algorithm, named as GSA, could effectively and accurately calculate the purity and ploidy of tumor samples through NGS data, and then reflect the degree of genomic instability and large-scale copy number variations of tumor samples.

Different Anesthetic Drugs Mediate Changes in Neuroplasticity During Cognitive Impairment in Sleep-Deprived Rats via Different Factors.

BACKGROUND Perioperative neuro-cognitive disorders (PND) are preoperative and postoperative complications of multiple nervous systems, typically manifested as decreased memory and learning ability after surgery. It was used to replace the original definition of postoperative cognitive dysfunctions (POCD) from 2018. Our previous studies have shown that sevoflurane inhalation can lead to cognitive dysfunction in Sprague-Dawley rats, but the specific mechanism is still unclear. MATERIAL AND METHODS Thirty-six male Sprague-Dawley rats were randomly divided into 6 groups (n=6): the SD group was given 24-h acute sleep deprivation; Sevoflurane was inhaled for 2 h in the Sevo group. Two mL propofol was injected into the tail vein of rats in the Prop group. The rats in the SD+Sevo group and SD+Prop group were deprived of sleep before intervention in the same way as before. RESULTS We noted significant behavioral changes in rats treated with SIK3 inhibitors or tau phosphorylation agonists before propofol injection or sevoflurane inhalation, with associated protein levels and dendritic spine density documented. Sevoflurane anesthesia-induced cognitive impairment following acute sleep deprivation was more pronounced than sleep deprivation-induced cognitive impairment alone and resulted in increased brain SIK3 levels, increased phosphorylation of total tau and tau, and decreased acetylation modifications. After using propofol, the cognitive function returned to baseline levels with a series of reversals of cognitive dysfunction. CONCLUSIONS These results suggest that sevoflurane inhalation via the SIK3 pathway aggravates cognitive impairment after acute sleep deprivation and that propofol anesthesia reverses the effects of sleep deprivation by affecting modifications of tau protein.

Synthesis and immunological evaluation of N-acyl modified Globo H derivatives as anticancer vaccine candidates.

Globo H is a tumor-associated carbohydrate antigen (TACA), which serves as a valuable target for antitumor vaccine or cancer immunotherapies. However, most TACAs are T-cell-independent, and they cannot induce powerful immune response due to their poor immunogenicity. To address this problem, herein, several Globo H analogues with modification on the N-acyl group were prepared through a preactivation-based glycosylation strategy from the non-reducing end to the reducing end. These modified Globo H derivatives were then conjugated with carrier protein CRM197 to form glycoconjugates as anticancer vaccine candidates, which were used in combination with adjuvant glycolipid C34 for immunological studies. The immunological effects of these synthetic vaccine candidates were evaluated on Balb/c mice. The results showed that the fluorine-modified N-acyl Globo H conjugates can induce higher titers of IgG antibodies that can recognize the naturally occurring Globo H antigen on the surface of cancer cells and can eliminate cancer cells in the presence of a complement, indicating the potential of these synthetic glycoconjugates as anticancer vaccine candidates.

Spinal NR2B phosphorylation at Tyr1472 regulates IRE(-)DMT1-mediated iron accumulation and spine morphogenesis via kalirin-7 in tibial fracture-associated postoperative pain after orthopedic surgery in female mice.

BACKGROUND: Prolonged postoperative pain is a major concern and occurs more frequently in women, but mechanisms remain elusive. NR2B-containging N-methyl-d-aspartate (NMDA) receptor is a key component of nociception transduction. Divalent metal transporter 1 (DMT1)-mediated iron overload involves NMDA-induced neurotoxicity in males. Kalirin-7 is vital in synaptic plasticity underlying pathological pain in males. Herein, the requirement for kalirin-7 in NR2B phosphorylation-dependent iron accumulation and spine plasticity in postoperative pain after tibial fracture in female mice has been examined. METHODS: Pain-related behavior, spinal NR2B phosphorylation at Tyr1472, kalirin-7 expression, DMT1 with/without iron-responsive element (IRE (+) DMT1 and IRE (-) DMT1) level, iron concentration and spine morphology were assessed in females. NR2B antagonist Ro25-6981, iron chelator deferoxamine and kalirin-7 knockdown by short hairpin RNA were employed to assess the potential cascade. RESULTS: Tibial fracture initiates long-term allodynia lasting at least 21 days postoperatively, and upregulates spinal NR2B phosphorylation, kalirin-7 and IRE (-) DMT1 expression, iron overload and spine density. Ro25-6981 reduces postoperative mechanical and cold allodynia, spinal NR2B phosphorylation, kalirin-7 level and IRE (-) DMT1-mediated iron overload. Kalirin-7 knockdown impairs fracture-associated allodynia, IRE (-) DMT1-mediated iron overload and spine plasticity. Deferoxamine also attenuates behavioral allodynia and spine plasticity. Spinal NMDA application elicits NR2B-dependent mechanical allodynia and iron overload, which is reversed by kalirin-7 knockdown or coadministration of deferoxamine. CONCLUSION: Spinal NR2B phosphorylation at Tyr1472 upregulates kalirin-7 expression to facilitate IRE (-) DMT1-mediated iron accumulation and spine morphogenesis in the development of fracture-associated postoperative pain in female mice.

Spinal caspase-6 regulates AMPA receptor trafficking and dendritic spine plasticity through netrin-1 in postoperative pain after orthopedic surgery for tibial fracture in mice.

Chronic postoperative pain hinders functional recovery after bone fracture and orthopedic surgery. Recently reported evidence indicates that caspase-6 is important in excitatory synaptic plasticity and pathological pain. Meanwhile, netrin-1 controls postsynaptic recruitment of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and synaptogenesis. The present work aimed to examine whether caspase-6 and netrin-1 contribute to fracture-induced postoperative allodynia. A mouse model of tibial fracture by intramedullary pinning was generated for inducing postoperative pain. Then, paw withdrawal threshold, spinal caspase-6 activity, netrin-1 secretion, AMPAR trafficking, and spine morphology were examined. Caspase-6 inhibition and netrin-1 knockdown by shRNA were performed to elucidate the pathogenetic mechanism of allodynia and its prevention. Whole-cell patch-clamp recording was performed to assess caspase-6's function in spinal AMPAR-induced current. Tibial fractures after orthopedic operation initiated persistent postsurgical mechanical and cold allodynia, accompanied by increased spinal active caspase-6, netrin-1 release, GluA1-containing AMPAR trafficking, spine density, and AMPAR-induced current in dorsal horn neurons. Caspase-6 inhibition reduced fracture-associated allodynia, netrin-1 secretion, and GluA1 trafficking. Netrin-1 deficiency impaired fracture-caused allodynia, postsynaptic GluA1 recruitment, and spine plasticity. The specific GluA2-lacking AMPAR antagonist NASPM also dose dependently prevented postoperative pain. The reduction of fracture-mediated postoperative excitatory synaptic AMPAR current in the dorsal horn by caspase-6 inhibition was compromised by recombinant netrin-1. Exogenous caspase-6 induced pain hypersensitivity, reversing by netrin-1 knockdown or coapplication of NASPM. Thus, spinal caspase-6 modulation of GluA1-containing AMPAR activation and spine morphology through netrin-1 secretion is important in the development of fracture-related postsurgical pain in the mouse.

Insulin Promotes Mitochondrial Respiration and Survival through PI3K/AKT/GSK3 Pathway in Human Embryonic Stem Cells.

Insulin is an essential growth factor for the survival and self-renewal of human embryonic stem cells (hESCs). Although it is best known as the principal hormone promoting glycolysis in somatic cells, insulin's roles in hESC energy metabolism remain unclear. In this report, we demonstrate that insulin is essential to sustain hESC mitochondrial respiration that is rapidly decreased upon insulin removal. Insulin-dependent mitochondrial respiration is stem cell specific, and mainly relies on pyruvate and glutamine, while glucose suppresses excessive oxidative phosphorylation. Pharmacologic and genetic manipulations reveal that continuous insulin signal sustains mitochondrial respiration through PI3K/AKT activation and downstream GSK3 inhibition. We further show that insulin acts through GSK3 inhibition to suppress caspase activation and rescue cell survival. This study uncovers a critical role of the AKT/GSK3 pathway in the regulation of mitochondrial respiration and cell survival, highlighting insulin as an essential factor for accurate assessment of mitochondrial respiration in hESCs.

Spinal caspase-3 contributes to tibial fracture-associated postoperative allodynia via up-regulation of LRRTM1 expression in mice.

BACKGROUND: Bone fracture may subsequently cause chronic postoperative pain after orthopedic surgery, but mechanisms remain elusive. The necessity of caspase-3 in neuroinflammation and synaptic plasticity has been summarized in pathological pain. Leucine-rich repeat transmembrane protein 1 (LRRTM1) mediates synaptic delivery of AMPA receptor and synaptogenesis. This study evaluated whether caspase-3 and LRRTM1 are required for fracture-associated postoperative allodynia. METHODS: A model of tibial fracture with intramedullary pinning in mice was established for the induction of postoperative pain, verified by measurement of mechanical paw withdrawal threshold and cold scores response to acetone. The caspase-3 specific inhibitor, recombinant caspase-3 and LRRTM1 knockdown by shRNA were utilized for the investigation of pathogenesis as well as the prevention of allodynia. Also, the activity of caspase-3 and the expression of LRRTM1 in the spinal dorsal horn were examined by Western blot and RT-qPCR. RESULTS: This study reported that tibial fracture and orthopedic surgery produced long-lasting mechanical allodynia and cold allodynia, along with the up-modulation of spinal caspase-3 activity (but not caspase-3 expression) and LRRTM1 expression. Spinal caspase-3 inhibition prevented fracture-associated behavioral allodynia in a dose-dependent manner. Caspase-3 inhibitor also reduced the spinal increased LRRTM1 level after tibial fracture with pinning. Spinal LRRTM1 deficiency impaired fracture-caused postoperative pain. Intrathecal recombinant caspase-3 facilitated acute pain hypersensitivity and spinal LRRTM1 expression in naïve mice, reversing by LRRTM1 knockdown. CONCLUSION: Our current results demonstrate the spinal up-regulation of LRRTM1 by caspase-3 activation in the development of tibial fracture-associated postoperative pain in mice.

Antiarrhythmic effects of ginsenoside Rg2 on calcium chloride-induced arrhythmias without oral toxicity.

BACKGROUND: Malignant arrhythmias require drug therapy. However, most of the currently available antiarrhythmic drugs have significant side effects. Ginsenoside Rg2 exhibits excellent cardioprotective effects and appears to be a promising candidate for cardiovascular drug development. So far, the oral toxicity and antiarrhythmic effects of Rg2 have not been evaluated. METHODS: Acute oral toxicity of Rg2 was assessed by the Limit Test method in mice. Subchronic oral toxicity was determined by repeated dose 28-day toxicity study in rats. Antiarrhythmic activities of Rg2 were evaluated in calcium chloride-induced arrhythmic rats. Antiarrhythmic mechanism of Rg2 was investigated in arrhythmic rats and H9c2 cardiomyocytes. RESULTS: The results of toxicity studies indicated that Rg2 exhibited no single-dose (10 g/kg) acute oral toxicity. And 28-day repeated dose treatment with Rg2 (1.75, 3.5 and 5 g/kg/d) demonstrated minimal, if any, subchronic toxicity. Serum biochemical examination showed that total cholesterol in the high-dose cohort was dramatically decreased, whereas prothrombin time was increased at Day 28, suggesting that Rg2 might regulate lipid metabolism and have a potential anticoagulant effect. Moreover, pretreatment with Rg2 showed antiarrhythmic effects on the rat model of calcium chloride induced arrhythmia, in terms of the reduced duration time, mortality, and incidence of malignant arrhythmias. The antiarrhythmic mechanism of Rg2 might be the inhibition of calcium influx through L-type calcium channels by suppressing the phosphorylation of Ca2+/calmodulin-dependent protein kinase II. CONCLUSION: Our findings support the development of Rg2 as a promising antiarrhythmic drug with fewer side effects for clinical use.

Fluorine-modified sialyl-Tn-CRM197 vaccine elicits a robust immune response.

Even though a vaccine that targets tumor-associated carbohydrate antigens on epithelial carcinoma cells presents an attractive therapeutic approach, relatively poor immunogenicity limits its development. In this study, we investigated the immunological activity of a fluoro-substituted Sialyl-Tn (F-STn) analogue coupled to the non-toxic cross-reactive material of diphtheria toxin197 (CRM197). Our results indicate that F-STn-CRM197 promotes a greater immunogenicity than non-fluorinated STn-CRM197. In the presence or absence of adjuvant, F-STn-CRM197 remarkably enhances both cellular and humoral immunity against STn by increasing antigen-specific lymphocyte proliferation and inducing a mixed Th1/Th2 response leading to production of IFN-γ and IL-4 cytokines, as well as STn-specific antibodies. Furthermore, antisera produced from F-STn-CRM197 immunization significantly recognizes STn-positive tumor cells and increases cancer cell lysis induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) pathways. Our data suggest that this F-STn vaccine may be useful for cancer immunotherapy and possibly for prophylactic prevention of cancer.

Selective effects of ginseng pectins on galectin-3-mediated T cell activation and apoptosis.

Galectin-3 (Gal-3) can induce T-cell activation and apoptosis and plays a role in tumor immune tolerance. Here, we demonstrate that ginseng pectins selectively inhibit Gal-3-induced T-cell apoptosis, while not affecting T-cell activation. This finding stands in contrast to that from the use of modified citrus pectin (MCP) and potato galactan (P-galactan) that inhibit both. Whereas PKC/ERK and ROS/ERK pathways are involved in both T-cell activation and apoptosis, the Ras/PI3K/Akt pathway is unique to T-cell activation. Ginseng pectins selectively inhibit the ROS/ERK pathway. Using the Sarcomar-180 mouse model in which Gal-3 expression is increased, we found that ginseng pectins (but not MCP or P-galactan) significantly promote T-cell proliferation and IL-2 expression, and inhibit tumor growth by 45%. These in vivo data correlate well with selective effects of pectins on Gal-3-mediated T-cell apoptosis and activation. Our study suggests a novel approach for the development of polysaccharide-based agents that target Gal-3 function.

NMR-based insight into galectin-3 binding to endothelial cell adhesion molecule CD146: Evidence for noncanonical interactions with the lectin's CRD ß-sandwich F-face.

Galectin-3 (Gal-3) binds to cell adhesion glycoprotein CD146 to promote cytokine secretion and mediate endothelial cell migration. Here, we used Nuclear Magnetic Resonance (NMR) 15N-Heteronuclear Single Quantum Coherence (HSQC) spectroscopy to investigate binding between 15N-labeled Gal-3 and the extracellular domain (eFL) of purified CD146 (five Ig-like ectodomains D1-D5) and a shorter, D5-deleted version of CD146 (D1-D4). Binding of Gal-3 and its carbohydrate recognition domain (CRD) to CD146 D1-D4 is greatly reduced vis-à-vis CD146 eFL, supporting the proposal of a larger number of glycosylation sites on D5. Even though the canonical sugar-binding ß-sheet S-face (ß-strands 1, 10, 3, 4, 5, 6) of the Gal-3 ß-sandwich is involved in interactions with CD146 (e.g. N-linked glycosylation sites), equivalent HSQC spectral perturbations at residues on the opposing Gal-3 F-face ß-sheet (ß-strands 11, 2, 7, 8, 9) indicate involvement of the Gal-3 F-face in binding CD146. This is supported by the observation that addition of lactose, while significantly attenuating Gal-3 binding (primarily with the S-face) to CD146 eFL, does not abolish it. Bio-Layer Interferometry studies with Gal-3 F-face mutants yield KD values to demonstrate a significant decrease (L203A) or increase (V204A, L218A, T243A) in net binding to CD146 eFL compared to wild type Gal-3. However, HSQC lactose titrations show no highly significant effects on sugar binding to the Gal-3 CRD S-face. Overall, our findings indicate that Gal-3 binding to CD146 is more involved than simple interactions with ß-galactoside epitopes on the cell receptor, and that there is a direct role for the lectin's CRD F-face in the CD146 binding process.

A novel linear 3-O-methylated galactan isolated from Cantharellus cibarius activates macrophages.

A novel polysaccharide (WCCP-N-b) with a molecular weight of 18 kDa was isolated and purified from the fruiting bodies of Cantharellus cibarius. Monosaccharide composition, methylation analysis and NMR spectra indicated that WCCP-N-b was a linear α-1,6-galactan, partially methylated at O-3 of galactose. The molar ratio of Gal, 3-methylated-Gal, Glc and Man was 14.4:4.6:1.0:1.2. WCCP-N-b could significantly increase macrophage phagocytosis, release of NO and secretion of TNF-α, IL-6 and IL-1ß. On a cellular mechanistic level, WCCP-N-b activated MAPKs and NF-κB signaling pathway via Toll-like receptor 2 (TLR2). To further elucidate the structure-function relationship, WCCP-N-b was hydrolyzed by acid. Four degraded fragments were obtained, with molecular weights of 16.1 kDa, 11.2 kDa, 5 kDa and 3.5 kDa, respectively. Their macrophage activation effects were significantly decreased along with the molecular weight decrease. Collectively, WCCP-N-b could activate RAW264.7 cells, and the activation effect was related to its molecular weight.