Studies showed that manipulation of gut microbiota (GM) composition through the treatment of prebiotics could be a novel preventive measure against colorectal cancer (CRC) development. In this study, for the first time, we assessed the non-toxic doses of the triterpene saponins (ginsenoside-Rb3 and ginsenoside-Rd) - as prebiotics - that effectively reinstated the dysbiotic-gut microbial composition and intestinal microenvironment in an ApcMin/+ mice model. Rb3 and Rd effectively reduced the size and the number of the polyps that accompanied with the downregulation of oncogenic signaling molecules (iNOS, STAT3/pSTAT3, Src/pSrc). Both the compounds improved the gut epithelium by promoting goblet and Paneth cells population and reinstating the E-cadherin and N-Cadherin expression. Mucosal immunity remodeled with increased in anti-inflammatory cytokines and reduced in pro-inflammatory cytokines in treated mice. All these changes were correlating with the promoted growth of beneficial bacteria such as Bifidobacterium spp., Lactobacillus spp., Bacteroides acidifaciens, and Bacteroides xylanisolvens. Whereas, the abundance of cancer cachexia associated bacteria, such as Dysgonomonas spp. and Helicobacter spp., was profoundly lower in Rb3/Rd-treated mice. In conclusion, ginsenosides Rb3 and Rd exerted anti-cancer effects by holistically reinstating mucosal architecture, improving mucosal immunity, promoting beneficial bacteria, and down-regulating cancer-cachexia associated bacteria.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a highly heterogeneous disease with different host defence responses. However, whether periostin and vascular endothelial growth factor (VEGF) are similarly impaired in patients with eosinophilic CRSwNP (ENP) and those with non-eosinophilic CRSwNP (nENP) remains unclear. We sought to evaluate the expression and possible modulation of periostin and VEGF, regulated on activation normal T expressed and secreted (RANTES) and eotaxin-2 in the polyp tissues from 30 patients with ENP and from 36 patients with nENP and in middle turbinate tissues from 12 control subjects. We found that ENP tissues exhibited a significantly increased expression of periostin and VEGF compared with tissues from patients with nENP and control subjects (P < 0.05, respectively). Accordingly, the expression of VEGF, RANTES, and eotaxin-2 in ENP fibroblasts was significantly up-regulated after stimulation with up-regulated periostin in vitro, but the expression of VEGF and RANTES was significantly inhibited by stimulation with down-regulated periostin. Our findings suggest that periostin might play an important role in the occurrence and progression of ENP and might be a potential therapeutic target.

Gaucher disease is caused by mutations in GBA1 encoding acid β-glucosidase (GCase). Saposin C enhances GCase activity and protects GCase from intracellular proteolysis. Structure simulations indicated that the mutant GCases, N370S (0 S), V394L (4L) and D409V(9V)/H(9H), had altered function. To investigate the in vivo function of Gba1 mutants, mouse models were generated by backcrossing the above homozygous mutant GCase mice into Saposin C deficient (C*) mice. Without saposin C, the mutant GCase activities in the resultant mouse tissues were reduced by ~50% compared with those in the presence of Saposin C. In contrast to 9H and 4L mice that have normal histology and life span, the 9H;C* and 4L;C* mice had shorter life spans. 9H;C* mice developed significant visceral glucosylceramide (GC) and glucosylsphingosine (GS) accumulation (GC»GS) and storage macrophages, but lesser GC in the brain, compared to 4L;C* mice that presents with a severe neuronopathic phenotype and accumulated GC and GS primarily in the brain. Unlike 9V mice that developed normally for over a year, 9V;C* pups had a lethal skin defect as did 0S;C* mice resembled that of 0S mice. These variant Gaucher disease mouse models presented a mutation specific phenotype and underscored the in vivo role of Saposin C in the modulation of Gaucher disease.

The objective of this study was to estimate overall survival in children with extremity rhabdomyosarcoma (RMS). In addition, we attempted to construct a nomogram to predict the prognosis in such patients using a population-based cohort. The national Surveillance, Epidemiology, and End Results (SEER) registry was used to identify a cohort of childhood RMS patients. A total of 197 patients with RMS were ultimately included. Multivariable analysis identified age group, N classification, M classification, and treatment combinations as independent predictive factors for patient overall survival. Candidate variables such as age group, N classification, M classification, and treatment combinations were used to fit the model. For overall survival, the bootstrap-adjusted c-index was 0.76 (95% CI, 0.73-0.80) for the nomogram. Furthermore, we performed recursive partitioning analysis for risk stratification according to overall survival, and 3 prognostic subgroups were generated (low, intermediate and high risk). Finally, we evaluated multimodal treatment based on the risk stratification according to the nomogram and IRSG prognostic stratification model. With regard to the entire cohort, overall survival in patients who received surgery and radiation was superior to that in patients who received surgery or radiation (p = 0.001). Regarding RPA and IRSG prognostic stratification, we found that the differences remained significant (p < 0.05) in patients with low-intermediate risk. However, the difference disappeared in patients with high risk (p > 0.05). We performed a population-based analysis of data from the SEER registry in an effort to identify prognostic factors and develop a nomogram in children with extremity RMS. The nomogram appears to be suitable for the survival stratification of children with RMS and will help clinicians identify patients who may be at a reduced probability of survival and assist them in making treatment and surveillance decisions. More studies concerning overall survival in children with RMS are needed to confirm and update our findings.

Although KIF4A has been found to play an important role in a variety of tumors and is closely associated with the activation of immunocytes, its role in bladder cancer (BC) remains unclear. Here, we report increased expression of KIF4A in both lymph node-positive and high grade BC tissues. High expression of KIF4A has been significantly correlated with fewer CD8+ tumor-infiltrating lymphocytes (TILs) and a much worse prognosis in patients with BC. With respect to promoting tumor growth, the expression of KIF4A in promoting tumor growth was more pronounced in immune-competent mice (C57BL/6) than in immunodeficient mice (BALB/C). In addition, the more increased accumulation of myeloid-derived suppressor cells (MDSCs) was observed in tumor-bearing mice with KIF4A overexpression than in the control group. Transwell chemotaxis assays revealed that KIF4A overexpression in T24 cells increased MDSC recruitment. Furthermore, according to ELISA results, CXCL5 was the most noticeably increased cytokine in the KIF4A-transduced BC cells. Additional studies in vitro and in vivo showed that the capability of KIF4A to promote BC cells to recruit MDSCs could be significantly inhibited by anti-CXCL5 antibody. Therefore, our results demonstrated that KIF4A-mediated BC production of CXCL5 led to an increase in MDSC recruitment, which contributed to tumor progression.

Meningioma was the most primary intracranial tumor, but the molecular characteristics and the treatment of malignant meningioma were still unclear. Nine malignant progression-related genes based prognostic signatures were identified by transcriptome analysis between benign meningioma and malignant meningioma. The external dataset GEO136661 and quantitative Real-time Polymerase Chain Reaction were used to verify the prognostic factors. has-miR-3605-5p, hsa-miR-664b-5p, PNRC2, BTBD8, EXTL2, SLFN13, DGKD, NSD2, and BVES were closed with malignant progression. Moreover, Doxorubicin was identified by Connectivity Map website with the differential malignant progression-related genes. CCK-8 assay, Edu assay, wound healing assay, and trans-well experiment were used to reveal that Doxorubicin could inhibit proliferation, migration and invasion of IOMM-Lee Cells.

Botulinum neurotoxin (BoNT) shows high lethality and toxicity, marking it as an important biological threat. The only effective post-exposure therapy is botulinum antitoxin; however, such products have great potential for improvement. To prevent or treat BoNT, monoclonal antibodies (mAbs) are promising agents. Herein, we aimed to construct a bispecific antibody (termed LUZ-A1-A3) based on the anti-BoNT/A human monoclonal antibodies (HMAb) A1 and A3. LUZ-A1-A3 binds to the Hc and L-HN domains of BoNT/A, displaying potent neutralization activity against BoNT/A (124 × higher than that of HMAb A1 or HMAb A3 alone and 15 × higher than that of the A1 + A3 combination). LUZ-A1-A3 provided effective protection against BoNT/A in an in vivo mouse model. Mice were protected from infection with 500 × LD50 of BoNT/A by LUZ-A1-A3 from up to 7 days before intraperitoneal administration of BoNT/A. We also demonstrated the effective therapeutic capacity of LUZ-A1-A3 against BoNT/A in a mouse model. LUZ-A1-A3 (5 μg/mouse) neutralized 20 × LD50 of BoNT/A at 3 h after intraperitoneal BoNT/A administration and complete neutralized 20 × LD50 of BoNT/A at 0.5 h after intraperitoneal BoNT/A administration. Thus, LUZ-A1-A3 is a promising agent for the pre-exposure prophylaxis and post-exposure treatment of BoNT/A.

Recently, the efficacy of two low-invasive treatments, ablation, and radiotherapy, has been fully compared for the patients with the early-stage hepatocellular carcinoma (HCC). However, the comparison between radiotherapy plus ablation and ablation alone has been less frequently reported. Data from the Surveillance, Epidemiology, and End Results (SEER) database were searched for early-stage HCC patients treated with ablation plus radiotherapy or ablation alone. The outcome measures were overall survival (OS) and cancer-specific survival (CSS). The propensity score matching (PSM) was used to reduce selection bias. We included 240 and 6619 patients in the radiotherapy plus ablation group and ablation group before the PSM. After PSM, 240 pairs of patients were included. The median OS (mOS) and median CSS (mCSS) of patients receiving ablation alone were longer than that of receiving radiotherapy plus ablation (mOS: 47 vs. 34 months, P = 0.019; mCSS: 77 vs. 40 months, P = 0.018, after PSM) before and after PSM. The multivariate analysis indicated that radiotherapy plus ablation independent risk factor for OS and CSS before PSM, but the significance disappeared after PSM. The detailed subgroup analyses indicated ablation alone brought more benefit in very early-stage HCC and older patients. In addition, we found different types of radiotherapy might lead to different outcomes when combined with ablation. In conclusion, ablation alone is noninferior to radiotherapy plus ablation in patients with early-stage HCC. The additional radiation prior to ablation may bring survival benefits in the patients with higher tumor stage. However, due to the risk of selection bias in that study, the results should be interpreted cautiously.

Necroptosis has been shown as an alternative form of cell death in many diseases, but the detailed mechanisms of the neuron loss after traumatic brain injury (TBI) in rodents remain unclear. To investigate whether necroptosis is induced after TBI and gets involved in the neuroprotecton of therapeutic hypothermia on the TBI, we observed the pathological and biochemical change of the necroptosis in the fluid percussion brain injury (FPI) model of the rats. We found that receptor-interacting protein (RIP) 1 and 3, and mixed lineage kinase domain-like protein (MLKL), the critical downstream mediators of necroptosis recently identified in vivo, as well as HMGB1 and the pro-inflammation cytokines TNF-α, IL-6 and IL-18, were increased at an early phase (6 h) in cortex after TBI. Posttraumatic hypothermia (33 °C) led to the decreases in the necroptosis regulators, inflammatory factors and brain tissue damage in rats compared with normothermia-treated TBI animals. Immunohistochemistry studies showed that posttraumatic hypothermia also decreased the necroptosis-associated proteins staining in injured cortex and hippocampal CA1. Therefore, we conclude that the RIP1/RIP3-MLKL-mediated necroptosis occurs after experimental TBI and therapeutic hypothermia may protect the injured central nervous system from tissue damage and the inflammatory responses by targeting the necroptosis signaling after TBI.

Dentin sialophosphoprotein (DSPP) is a dentin extracellular matrix protein that is processed into dentin sialoprotein (DSP), dentin glycoprotein (DGP) and dentin phosphoprotein (DPP). DSP is mainly expressed in odontoblasts. We hypothesized that DSP interacts with cell surface receptors and subsequently activates intracellular signaling. Using DSP as bait for screening a protein library, we demonstrate that DSP acts as a ligand and binds to integrin β6. The 36 amino acid residues of DSP are sufficient to bind to integrin β6. This peptide promoted cell attachment, migration, differentiation and mineralization of dental mesenchymal cells. In addition, DSP (aa183-219) stimulated phosphorylation of ERK1/2 and P38 kinases. This activation was inhibited by an anti-integrin β6 antibody and siRNA. Furthermore, we demonstrate that this DSP fragment induces SMAD1/5/8 phosphorylation and nuclear translocation via ERK1/2 and P38 signaling. SMAD1/5/8 binds to SMAD binding elements (SBEs) in the DSPP gene promoter. SBE mutations result in a decrease in DSPP transcriptional activity. Endogenous DSPP expression was up-regulated by DSP (aa183-219) in dental mesenchymal cells. The data in the current study demonstrate for the first time that this DSP domain acts as a ligand in a RGD-independent manner and is involved in intracellular signaling via interacting with integrin β6. The DSP domain regulates DSPP expression and odontoblast homeostasis via a positive feedback loop.

Transgenic sheep can be used to achieve genetic improvements in breeds and as an important large-animal model for biomedical research. In this study, we generated a TALEN plasmid specific for ovine MSTN and transfected it into fetal fibroblast cells of STH sheep. MSTN biallelic-KO somatic cells were selected as nuclear donor cells for SCNT. In total, cloned embryos were transferred into 37 recipient gilts, 28 (75.7%) becoming pregnant and 15 delivering, resulting in 23 lambs, 12 of which were alive. Mutations in the lambs were verified via sequencing and T7EI assay, and the gene mutation site was consistent with that in the donor cells. Off-target analysis was performed, and no off-target mutations were detected. MSTN KO affected the mRNA expression of MSTN relative genes. The growth curve for the resulting sheep suggested that MSTN KO caused a remarkable increase in body weight compared with those of wild-type sheep. Histological analyses revealed that MSTN KO resulted in muscle fiber hypertrophy. These findings demonstrate the successful generation of MSTN biallelic-KO STH sheep via gene editing in somatic cells using TALEN technology and SCNT. These MSTN mutant sheep developed and grew normally, and exhibited increased body weight and muscle growth.

Transient receptor potential vanilloid 4 (TRPV4) is a Ca2+-permeable nonselective cation channel and can be activated during ischemia/reperfusion (I/R). This study tested whether blockade of TRPV4 can alleviate myocardial I/R injury in mice. TRPV4 expression began to increase at 1 h, reached statistically at 4 h, and peaked at 24-72 h. Treatment with the selective TRPV4 antagonist HC-067047 or TRPV4 knockout markedly ameliorated myocardial I/R injury as demonstrated by reduced infarct size, decreased troponin T levels and improved cardiac function at 24 h after reperfusion. Importantly, the therapeutic window for HC-067047 lasts for at least 12 h following reperfusion. Furthermore, treatment with HC-067047 reduced apoptosis, as evidenced by the decrease in TUNEL-positive myocytes, Bax/Bcl-2 ratio, and caspase-3 activation. Meanwhile, treatment with HC-067047 attenuated the decrease in the activation of reperfusion injury salvage kinase (RISK) pathway (phosphorylation of Akt, ERK1/2, and GSK-3β), while the activation of survival activating factor enhancement (SAFE) pathway (phosphorylation of STAT3) remained unchanged. In addition, the anti-apoptotic effects of HC-067047 were abolished by the RISK pathway inhibitors. We conclude that blockade of TRPV4 reduces apoptosis via the activation of RISK pathway, and therefore might be a promising strategy to prevent myocardial I/R injury.

Microbial syntrophic metabolism has been well accepted as the heart of how methanogenic and other anaerobic microbial communities function. In this work, we applied a single-cell RT-qPCR approach to reveal gene-expression heterogeneity in a model syntrophic system of Desulfovibrio vulgaris and Methanosarcina barkeri, as compared with the D. vulgaris monoculture. Using the optimized primers and single-cell analytical protocol, we quantitatively determine gene-expression levels of 6 selected target genes in each of the 120 single cells of D. vulgaris isolated from its monoculture and dual-culture with M. barkeri. The results demonstrated very significant cell-to-cell gene-expression heterogeneity for the selected D. vulgaris genes in both the monoculture and the syntrophic dual-culture. Interestingly, no obvious increase in gene-expression heterogeneity for the selected genes was observed for the syntrophic dual-culture when compared with its monoculture, although the community structure and cell-cell interactions have become more complicated in the syntrophic dual-culture. In addition, the single-cell RT-qPCR analysis also provided further evidence that the gene cluster (DVU0148-DVU0150) may be involved syntrophic metabolism between D. vulgaris and M. barkeri. Finally, the study validated that single-cell RT-qPCR analysis could be a valuable tool in deciphering gene functions and metabolism in mixed-cultured microbial communities.

Selective inhibition of function-specific β-GlcNAcase has great potential in terms of drug design and biological research. The symmetrical bis-naphthalimide M-31850 was previously obtained by screening for specificity against human glycoconjugate-lytic β-GlcNAcase. Using protein-ligand co-crystallization and molecular docking, we designed an unsymmetrical dyad of naphthalimide and thiadiazole, Q2, that changes naphthalimide specificity from against a human glycoconjugate-lytic β-GlcNAcase to against insect and bacterial chitinolytic β-GlcNAcases. The crystallographic and in silico studies reveal that the naphthalimide ring can be utilized to bind different parts of these enzyme homologs, providing a new starting point to design specific inhibitors. Moreover, Q2-induced closure of the substrate binding pocket is the structural basis for its 13-fold increment in inhibitory potency. Q2 is the first non-carbohydrate inhibitor against chitinolytic β-GlcNAcases. This study provides a useful example of structure-based rationally designed inhibitors as potential pharmaceuticals or pesticides.

A clear perception of gene essentiality in bacterial pathogens is pivotal for identifying drug targets to combat emergence of new pathogens and antibiotic-resistant bacteria, for synthetic biology, and for understanding the origins of life. We have constructed a comprehensive set of deletion mutants and systematically identified a clearly defined set of essential genes for Streptococcus sanguinis. Our results were confirmed by growing S. sanguinis in minimal medium and by double-knockout of paralogous or isozyme genes. Careful examination revealed that these essential genes were associated with only three basic categories of biological functions: maintenance of the cell envelope, energy production, and processing of genetic information. Our finding was subsequently validated in two other pathogenic streptococcal species, Streptococcus pneumoniae and Streptococcus mutans and in two other gram-positive pathogens, Bacillus subtilis and Staphylococcus aureus. Our analysis has thus led to a simplified model that permits reliable prediction of gene essentiality.

The prognostic value of diabetes remains unknown in nasopharyngeal carcinoma (NPC) treated with intensity-modulated radiation therapy (IMRT). We retrospectively reviewed medical records of 1489 patients with non-metastatic, histologically-proven NPC treated using IMRT. 81/1489 (5.4%) patients were diabetic, 168/1489 (11.3%) were prediabetic, and 1240/1489 (83.3%) were normoglycemic. The 4-year disease-free survival (DFS), overall survival (OS), loco-regional relapse-free survival (LRRFS) and distant metastasis-free survival (DMFS) rates were 77.1% vs. 82.4% (P = 0.358), 85.8% vs. 91.0% (P = 0.123), 90.9% vs. 91.7% (P = 0.884), and 85.5% vs. 89.2% (P = 0.445) for diabetic vs. normoglycemic patients, and 82.4% vs. 82.4% (P = 0.993), 88.7% vs. 91.0% (P = 0.285), 90.6% vs. 91.7% (P = 0.832) and 91.5% vs. 89.2% (P = 0.594) for preidabetic vs. normoglycemic patients. Multivariate analysis did not established diabetes as poor prognostic factors in NPC patients treated with IMRT (P = 0.332 for DFS, P = 0.944 for OS, P = 0.977 for LRRFS, P = 0.157 for DMFS), however, triglycerides and low density lipoprotein cholesterol were independent prognostic factors. In conclusion, diabetes does not appear to be a prognostic factor in NPC patients treated with IMRT, and attention should be paid to hyperglycemia-associated hyperlipaemia.

The Wnt/β-catenin signaling is an evolutionarily conserved pathway that regulates a wide range of physiological functions, including embryogenesis, organ maintenance, cell proliferation and cell fate decision. Dysregulation of Wnt/β-catenin signaling has been implicated in various cancers, but its role in cell death has not yet been fully elucidated. Here we show that activation of Wg signaling induces cell death in Drosophila eyes and wings, which depends on dFoxO, a transcription factor known to be involved in cell death. In addition, dFoxO is required for ectopic and endogenous Wg signaling to regulate wing patterning. Moreover, dFoxO is necessary for activated Wg signaling-induced target genes expression. Furthermore, Arm is reciprocally required for dFoxO-induced cell death. Finally, dFoxO physically interacts with Arm both in vitro and in vivo. Thus, we have characterized a previously unknown role of dFoxO in promoting Wg signaling, and that a dFoxO-Arm complex is likely involved in their mutual functions, e.g. cell death.

Coat color is an important characteristic and economic trait in domestic sheep. Aiming at alteration of Chinese merino sheep coat color by genome manipulation, we disrupted sheep agouti signaling protein gene by CRISPR/Cas9. A total of seven indels were identified in 5 of 6 born lambs. Each targeted lamb happened at least two kinds of modifications, and targeted lambs with multiple modifications displayed variety of coat color patterns. Three lambs with 4 bp deletion showed badgerface with black body coat color in two lambs, and brown coat color with light ventral pigmentation in another one. The black-white spotted color was observed in two lambs with 2 bp deletion. Further analysis unraveled that modifications happened in one or more than two copies of ASIP gene, and moreover, the additional spontaneous mutations of D9 and/or D5 preceding the targeting modification could also involve the formation of coat color patterns. Taken together, the entanglement of ASIP modifications by CRISPR/Cas9, spontaneous D9/D5 mutations, and ASIP gene duplications contributed to the variety of coat color patterns in targeted lambs.

Biomarkers are measurable changes associated with the disease. Urine can reflect the changes of the body while blood is under control of the homeostatic mechanisms; thus, urine is considered an important source for early and sensitive disease biomarker discovery. A comprehensive profile of the urinary proteome will provide a basic understanding of urinary proteins. In this paper, we present an in-depth analysis of the urinary proteome based on different separation strategies, including direct one dimensional liquid chromatography-tandem mass spectrometry (LC/MS/MS), two dimensional LC/MS/MS, and gel-eluted liquid fraction entrapment electrophoresis/liquid-phase isoelectric focusing followed by two dimensional LC/MS/MS. A total of 6085 proteins were identified in healthy urine, of which 2001 were not reported in previous studies and the concentrations of 2571 proteins were estimated (spanning a magnitude of 106) with an intensity-based absolute quantification algorithm. The urinary proteins were annotated by their tissue distribution. Detailed information can be accessed at the "Human Urine Proteome Database" (www.urimarker.com/urine).

Altered gut microbiota is associated with autism spectrum disorders (ASD), a group of complex, fast growing but difficult-to-diagnose neurodevelopmental disorders worldwide. However, the role of the oral microbiota in ASD remains unexplored. Via high-throughput sequencing of 111 oral samples in 32 children with ASD and 27 healthy controls, we demonstrated that the salivary and dental microbiota of ASD patients were highly distinct from those of healthy individuals. Lower bacterial diversity was observed in ASD children compared to controls, especially in dental samples. Also, principal coordinate analysis revealed divergences between ASD patients and controls. Moreover, pathogens such as Haemophilus in saliva and Streptococcus in plaques showed significantly higher abundance in ASD patients, whereas commensals such as Prevotella, Selenomonas, Actinomyces, Porphyromonas, and Fusobacterium were reduced. Specifically, an overt depletion of Prevotellaceae co-occurrence network in ASD patients was obtained in dental plaques. The distinguishable bacteria were also correlated with clinical indices, reflecting disease severity and the oral health status (i.e. dental caries). Finally, diagnostic models based on key microbes were constructed, with 96.3% accuracy in saliva. Taken together, this study characterized the habitat-specific profile of the oral microbiota in ASD patients, which might help develop novel strategies for the diagnosis of ASD.