TREX2 enables efficient genome disruption mediated by paired CRISPR-Cas9 nickases that generate 3'-overhanging ends.

Paired SpCas9 nickases (SpCas9n) are an effective strategy to reduce off-target effect in genome editing. However, this approach is not efficient with 3'-overhanging ends, limiting its applications. In order to expand the utility of paired SpCas9n in genome editing, we tested the effect of the TREX2 3'-5' exonuclease on repair of 3'-overhanging ends. We found ectopic overexpression of Trex2 stimulates the efficiency of paired SpCas9n in genome disruption with 3'-overhanging ends up to 400-fold with little stimulation of off-target editing. TREX2 overexpressed preferentially deletes entire 3' overhangs but has no significant effect on 5' overhangs. Trex2 overexpression also stimulates genome disruption by paired SpCas9n that potentially generate short 3'-overhanging ends at overlapping SpCas9n target sites, suggesting sequential nicking of overlapping target sites by SpCas9n. This approach is further simplified with improved efficiency and safety by fusion of TREX2 and particularly its DNA-binding-deficient mutant to SpCas9n. Junction analysis at overlapping targets revealed the different extent of end resection of 3' single-stranded DNA (ssDNA) by free TREX2 and TREX2 fused to SpCas9n. SpCas9n-TREX2 fusion is more convenient and safer than overexpression of free TREX2 to process 3'-overhanging ends for efficient genome disruption by paired SpCas9n, allowing practical use of this TREX2-based strategy in genome editing.

Construction and evaluation of intrauterine adhesion model in rats by different methods of mechanical injury.

PURPOSE: The study aimed to establish a stable and effective animal model for the experimental study of intrauterine adhesion (IUA) by evaluating various mechanical injury methods. METHODS: A total of 140 female rats were divided into four groups according to the extent and area of endometrial injury: group A (excision area: 2.0 × 0.5 cm2), group B (excision area: 2.0 × 0.25 cm2), group C (endometrial curettage) and group D (sham operation). On the 3rd, 7th, 15th and 30th day after the operation, the tissue samples of each group were collected, and the uterine cavity stenosis and histological changes were recorded by HE and Masson staining. Immunohistochemistry of CD31 was applied to visualize microvessel density (MVD). The pregnancy rate and the number of gestational sacs were used to evaluate the reproductive outcome. RESULTS: The results showed that endometrium injured by small-area endometrial excision or simple curettage could be repaired. The ratio of fibrosis in groups A and B was higher than that in groups C and group D 30 days after modeling (P < 0.001). The number of endometrial glands and MVD in group A was significantly lower than those in groups B, C and D (P < 0.05). The pregnancy rate in group A was 20%, which was lower than that in groups B (33.3%), C (89%) and D (100%) (P < 0.05). CONCLUSION: Full-thickness endometrial excision has a high rate of success in constructing stable and effective IUA models in rats.

Proximal binding of dCas9 at a DNA double strand break stimulates homology-directed repair as a local inhibitor of classical non-homologous end joining.

In CRISPR/Cas9 genome editing, the tight and persistent target binding of Cas9 provides an opportunity for efficient genetic and epigenetic modification on genome. In particular, technologies based on catalytically dead Cas9 (dCas9) have been developed to enable genomic regulation and live imaging in a site-specific manner. While post-cleavage target residence of CRISPR/Cas9 could alter the pathway choice in repair of Cas9-induced DNA double strand breaks (DSBs), it is possible that dCas9 residing adjacent to a break may also determine the repair pathway for this DSB, providing an opportunity to control genome editing. Here, we found that loading dCas9 onto a DSB-adjacent site stimulated homology-directed repair (HDR) of this DSB by locally blocking recruitment of classical non-homologous end-joining (c-NHEJ) factors and suppressing c-NHEJ in mammalian cells. We further repurposed dCas9 proximal binding to increase HDR-mediated CRISPR genome editing by up to 4-fold while avoiding exacerbation of off-target effects. This dCas9-based local inhibitor provided a novel strategy of c-NHEJ inhibition in CRISPR genome editing in place of small molecule c-NHEJ inhibitors, which are often used to increase HDR-mediated genome editing but undesirably exacerbate off-target effects.

Prediction of 3-year recurrence rate of hepatocellular carcinoma after resection based on contrast-enhanced CT: a single-centre study.

OBJECTIVE: We present a new artificial intelligence-powered method to predict 3-year hepatocellular carcinoma (HCC) recurrence by analysing the radiomic profile of contrast-enhanced CT (CECT) images that was validated in patient cohorts. METHODS: This retrospective cohort study of 224 HCC patients with follow-up for at least 3 years was performed at a single centre from 2012 to 2019. Two groups of radiomic signatures were extracted from the arterial and portal venous phases of pre-operative CECT. Then, the radiological model (RM), deep learning-based radiomics model (DLRM), and clinical & deep learning-based radiomics model (CDLRM) were established and validated in the area under curve (AUC), calibration curve, and clinical decision curve. RESULTS: Comparison of the clinical baseline variables between the non-recurrence (n = 109) and recurrence group (n = 115), three clinical independent factors (Barcelona Clinic Liver Cancer staging, microvascular invasion, and α-fetoprotein) were incorporated into DLRM for the CDLRM construction. Among the 30 radiomic features most crucial to the 3 year recurrence rate, the selection from deep learning-based radiomics (DLR) features depends on CECT. through the Gini index. In most cases, CDLRM has shown superior accuracy and distinguished performance than DLRM and RM, with the 0.98 AUC in the training cohorts and 0.83 in the testing. CONCLUSION: This study proposed that DLR-based CDLRM construction would be allowed for the predictive utility of 3-year recurrence outcomes of HCCs, providing high-risk patients with an effective and non-invasive method to possess extra clinical intervention. ADVANCES IN KNOWLEDGE: This study has highlighted the predictive value of DLR in the 3-year recurrence rate of HCC.

[Experimental study of improved sclerotherapy injection in rat model of vertebral arteriocervical spondylopathy].

OBJECTIVE: To improve the rat model of cervical spondylosis of vertebral artery type (CSA) induced by injecting sclerosing agent. To evaluate the efficacy of injecting sclerosing agent to induce CSA. METHODS: Forty Health SPF SD rats(20 males and 20 females), were randomly divided into two groups:the model group (20) and the blank group (20). All the animals were followed up for 4 weeks for the observation of general situation, transcranial Doppler(TCD) detection of blood flow velocity, pulsatility index and resistive index of the vertebral artery, measurement of mental distress by open-field test. RESULTS: One to two days after establish the animal model, rats in the model group appeared apathetic with decreased autonomic activities, trembling, squinting, increased eye excrement, etc., and no rats died during the experiment. The mean blood flow velocity of the model group was lower than that of the blank group (P<0.05), and the pulsatilit index and resistive index of the model group were higher than that of the blank group (P<0.05). The mental distress of the model group was significantly higher than that of the blank group. CONCLUSION: The modified injection of sclerosing agent is a practical method to establish the rat model of CSA, with high success rate, high stability, low mortality and simple operation.

Genomic and phenotypic biology of a novel Dickeya zeae WH1 isolated from rice in China: Insights into pathogenicity and virulence factors.

Soft rot caused by Dickeya zeae is an important bacterial disease affecting rice and other plants worldwide. In this study, Nanopore and Illumina sequencing platforms were used to sequence the high-quality complete genome of a novel D. zeae strain WH1 (size: 4.68 Mb; depth: 322.37x for Nanopore, 243.51x for Illumina; GC content: 53.59%), which was isolated from healthy rice root surface together with Paenibacillus polymyxa, a potential biocontrol bacterium against D. zeae strain WH1. However, the pure WH1 culture presented severe pathogenicity. Multilocus sequence analysis (MLSA) indicated that strains WH1, EC1, and EC2 isolated from rice were grouped into a clade differentiated from other D. zeae strains. The average nucleotide identity (ANI) and DNA-DNA hybridization (DDH) analyses demonstrated that WH1 was phylogenetically closest to EC2. Furthermore, the pathogenicity determinants and virulence factors of WH1 were mainly analyzed through genomic comparison with complete genomes of other D. zeae strains with high virulence (EC1, EC2, MS1, and MS2). The results revealed that plant cell wall-degrading extracellular enzymes (PCWDEs), flagellar and chemotaxis, and quorum sensing were highly conserved in all analyzed genomes, which were confirmed through phenotypic assays. Besides, WH1 harbored type I, II, III, and VI secretion systems (T1SS, T2SS, T3SS, and T6SS), but lost T4SS and T5SS. Like strains MS1 and MS2 isolated from bananas, WH1 harbored genes encoding both capsule polysaccharide (CPS) and exopolysaccharide (EPS) biosynthesis. The results of pathogenicity assays demonstrated that WH1 produced severe soft rot symptoms on potato tubers, carrots, radishes, and Chinese cabbage. Meanwhile, WH1 also produced phytotoxin(s) to inhibit rice seed germination with an 87% inhibitory rate in laboratory conditions. More importantly, we confirmed that phytotoxin(s) produced by WH1 are different from zeamines produced by EC1. Comparative genomics analyses and phenotypic and pathogenicity assays suggested that WH1 likely evolved through a pathway different from the other D. zeae strains from rice, producing a new type of rice foot rot pathogen. These findings highlight the emergence of a new type of D. zeae strain with high virulence, causing soft rot in rice and other plants.

Target residence of Cas9-sgRNA influences DNA double-strand break repair pathway choices in CRISPR/Cas9 genome editing.

BACKGROUND: Due to post-cleavage residence of the Cas9-sgRNA complex at its target, Cas9-induced DNA double-strand breaks (DSBs) have to be exposed to engage DSB repair pathways. Target interaction of Cas9-sgRNA determines its target binding affinity and modulates its post-cleavage target residence duration and exposure of Cas9-induced DSBs. This exposure, via different mechanisms, may initiate variable DNA damage responses, influencing DSB repair pathway choices and contributing to mutational heterogeneity in genome editing. However, this regulation of DSB repair pathway choices is poorly understood. RESULTS: In repair of Cas9-induced DSBs, repair pathway choices vary widely at different target sites and classical nonhomologous end joining (c-NHEJ) is not even engaged at some sites. In mouse embryonic stem cells, weakening the target interaction of Cas9-sgRNA promotes bias towards c-NHEJ and increases target dissociation and reduces target residence of Cas9-sgRNAs in vitro. As an important strategy for enhancing homology-directed repair, inactivation of c-NHEJ aggravates off-target activities of Cas9-sgRNA due to its weak interaction with off-target sites. By dislodging Cas9-sgRNA from its cleaved targets, DNA replication alters DSB end configurations and suppresses c-NHEJ in favor of other repair pathways, whereas transcription has little effect on c-NHEJ engagement. Dissociation of Cas9-sgRNA from its cleaved target by DNA replication may generate three-ended DSBs, resulting in palindromic fusion of sister chromatids, a potential source for CRISPR/Cas9-induced on-target chromosomal rearrangements. CONCLUSIONS: Target residence of Cas9-sgRNA modulates DSB repair pathway choices likely through varying dissociation of Cas9-sgRNA from cleaved DNA, thus widening on-target and off-target mutational spectra in CRISPR/Cas9 genome editing.

Fluorescence in situ hybridization-based confirmation of acute graft-vs-host disease diagnosis following liver transplantation: A case report.

BACKGROUND: Although acute graft-vs-host disease (aGvHD) is a rare complication of liver transplantation, it is poorly understood and has an extremely high mortality rate. No standardized diagnostic criteria or treatment regimens currently exist. CASE SUMMARY: The present study investigated the etiology, diagnosis, and treatment of aGvHD following liver transplantation. Presentation, diagnosis, disease course, histology, and treatment of an aGvHD case are reported, and associated literature is reviewed. A 64-year-old female required LTx due to primary biliary cirrhosis. The donor was a 12-year-old male. Three weeks following liver transplantation, the recipient developed pyrexia, diarrhea, rashes, and antibiotic-unresponsive pancytopenia. Clinical symptoms together with laboratory investigations suggested a diagnosis of aGvHD, which was confirmed via peripheral blood fluorescent in situ hybridization. Donor XY chromosome fluorescent in situ hybridization indicating early chimerism achieved 93% sensitivity in the detection of GvHD. Existing immunosuppressants were discontinued, and high-dose intravenous methylprednisolone was initiated along with antibiotics. While diarrhea resolved, the patient's general condition continued to deteriorate until demise due to multi-system organ failure at 37 d post-liver transplantation. This case illustrates the life-threatening nature of aGvHD. CONCLUSION: Herein, we have summarized a post-LTx aGvHD case and reviewed associated literature in order to increase awareness and provide potentially risk-mitigating recommendations.

CaFtsH06, A Novel Filamentous Thermosensitive Protease Gene, Is Involved in Heat, Salt, and Drought Stress Tolerance of Pepper (Capsicum annuum L.).

Harsh environmental factors have continuous negative effects on plant growth and development, leading to metabolic disruption and reduced plant productivity and quality. However, filamentation temperature-sensitive H protease (FtsH) plays a prominent role in helping plants to cope with these negative impacts. In the current study, we examined the transcriptional regulation of the CaFtsH06 gene in the R9 thermo-tolerant pepper (Capsicum annuum L.) line. The results of qRT-PCR revealed that CaFtsH06 expression was rapidly induced by abiotic stress treatments, including heat, salt, and drought. The CaFtsH06 protein was localized to the mitochondria and cell membrane. Additionally, silencing CaFtsH06 increased the accumulation of malonaldehyde content, conductivity, hydrogen peroxide (H2O2) content, and the activity levels of superoxide dismutase and superoxide (·O2-), while total chlorophyll content decreased under these abiotic stresses. Furthermore, CaFtsH06 ectopic expression enhanced tolerance to heat, salt, and drought stresses, thus decreasing malondialdehyde, proline, H2O2, and ·O2- contents while superoxide dismutase activity and total chlorophyll content were increased in transgenic Arabidopsis. Similarly, the expression levels of other defense-related genes were much higher in the transgenic ectopic expression lines than WT plants. These results suggest that CaFtsH06 confers abiotic stress tolerance in peppers by interfering with the physiological indices through reducing the accumulation of reactive oxygen species, inducing the activities of stress-related enzymes and regulating the transcription of defense-related genes, among other mechanisms. The results of this study suggest that CaFtsH06 plays a very crucial role in the defense mechanisms of pepper plants to unfavorable environmental conditions and its regulatory network with other CaFtsH genes should be examined across variable environments.

Inhibition of cell proliferation and radioresistance by miR-383-5p through targeting RNA binding protein motif (RBM3) in nasopharyngeal carcinoma.

BACKGROUND: RNA binding protein motif (RBM3) is associated with radioresistance in nasopharyngeal carcinoma (NPC), and miR-383-5p was predicted to target the 3'-untranslated region (3'UTR) of RBM3 messenger RNA (mRNA). Our study aimed to investigate the role and the mechanisms of miR-383-5p targeting RBM3 in NPC cell proliferation and radioresistance (RR). METHODS: The expression of miR-383-5p was detected by Real-time quantitative PCR (qRT-PCR) between RS (Radiosensitivity) and RR (Radioresistance) NPC patient- tissue specimens and cell lines. Cell Counting Kit-8 (CCK-8) and Clonogenic survival assay were applied to analyze the effect of miR-383-5p on NPC cell proliferation and radioresistance. Possible downstream target of miR-383-5p in NPC cells, RBM3was evaluated by luciferase assay and qRT-PCR. miR-383-5p inhibited NPC cell proliferation and radioresistance through RBM3 by rescue experiments. The effect of miR-383-5p on radiation-induced apoptosis was explored through Flow cytometric analysis and Western blotting. Western blotting was analyzed the molecular of RBM3-mediated Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) signaling pathways. RESULTS: The expression of miR-383-5p was decreased in radioresistant NPC tissues and cells. miR-383-5p inhibited cell proliferation and radioresistance in CNE1/IR cells. We also observed that therapeutic administration of a miR-383-5p agomir dramatically sensitized NPC xenografts to radiation in a mouse model. Conversely, in the same xenograft model, administration of a miR-383-5p antagomir dramatically increased NPC resistance to radiation. miR-383-5p targeted the 3'UTR of RBM3. miR-383-5p inhibited NPC cell proliferation and radioresistance through RBM3. Finally, we found that miR-383-5p increased radiation-induced apoptosis, activated JNK signaling, and inhibited ERK signaling. CONCLUSIONS: Our study revealed that miR-383-5p targeted the 3'UTR of RBM3 and contributed to the efficacy of NPC radiation therapy by altering the RBM3-mediated JNK and ERK signaling pathways.

CaHsfA1d Improves Plant Thermotolerance via Regulating the Expression of Stress- and Antioxidant-Related Genes.

Heat shock transcription factor (Hsf) plays an important role in regulating plant thermotolerance. The function and regulatory mechanism of CaHsfA1d in heat stress tolerance of pepper have not been reported yet. In this study, phylogenetic tree and sequence analyses confirmed that CaHsfA1d is a class A Hsf. CaHsfA1d harbored transcriptional function and predicted the aromatic, hydrophobic, and acidic (AHA) motif mediated function of CaHsfA1d as a transcription activator. Subcellular localization assay showed that CaHsfA1d protein is localized in the nucleus. The CaHsfA1d was transcriptionally up-regulated at high temperatures and its expression in the thermotolerant pepper line R9 was more sensitive than that in thermosensitive pepper line B6. The function of CaHsfA1d under heat stress was characterized in CaHsfA1d-silenced pepper plants and CaHsfA1d-overexpression Arabidopsis plants. Silencing of the CaHsfA1d reduced the thermotolerance of the pepper, while CaHsfA1d-overexpression Arabidopsis plants exhibited an increased insensitivity to high temperatures. Moreover, the CaHsfA1d maintained the H2O2 dynamic balance under heat stress and increased the expression of Hsfs, Hsps (heat shock protein), and antioxidant gene AtGSTU5 (glutathione S-transferase class tau 5) in transgenic lines. Our findings clearly indicate that CaHsfA1d improved the plant thermotolerance via regulating the expression of stress- and antioxidant-related genes.

Palladium-Catalyzed Regioselective Coupling Cyclohexenone into Indoles: Atom-Economic Synthesis of ß-Indolyl Cyclohexenones and Derivatization Applications.

Herein, we report a palladium-catalyzed dehydrogenative cross-coupling of indoles with cyclic enones to give ß-indolyl cyclic enones under mild and neutral reaction conditions. The key to the success is to explore a mild condition, which ensures the indole C-H activation and subsequent syn ß-hydride elimination through rapid enolization isomerization of Pd(II)-enolate while suppressing other undesired side reactions. Synthetic utility has also been demonstrated in the flexible transformation of the coupling products to meta-phenols and benzo[a]carbazoles.

Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing.

BACKGROUND: Many applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ. RESULTS: In the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent + 1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient for XRCC4 and in mouse liver. Due to high frequencies of precise deletions of defined "3n"-, "3n + 1"-, or "3n + 2"-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to "3n + 1"-bp, "3n + 2"-bp can overcome + 1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions. CONCLUSIONS: NHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length.

RNA binding motif protein 3 (RBM3) drives radioresistance in nasopharyngeal carcinoma by reducing apoptosis via the PI3K/AKT/Bcl-2 signaling pathway.

Radioresistance is an important obstacle to nasopharyngeal carcinoma (NPC) therapy. In this study, we explored the role of RNA-binding motif protein 3 (RBM3) in the radioresistance of NPC and its underlying mechanism. We measured the expression of RBM3 in 20 clinical NPC tissues and in NPC cell lines. We found that RBM3 was upregulated in radioresistant NPC tissues and cells. Radioresistant NPC cells (CNE1/IR) and parental NPC cells (CNE1) were subjected to RBM3-shRNA knockdown and RBM3 overexpression, respectively. RBM3 depletion in CNE1/IR cells sensitized cells to radiotherapy, increased DNA damage, and accelerated the rate of apoptosis. In contrast, RBM3 overexpression in CNE1 cells significantly enhanced radioresistance and reduced the rate of apoptosis. Additionally, radioresistance conferred by RBM3 was attributed to the activation of the AKT/Bcl-2 signaling pathway and reduction of caspase 3. Inhibition of AKT signaling attenuated RBM3-mediated radioresistance. Furthermore, RBM3 directly interacted with PI3K subunit p85 in NPC cell lines. Altogether, our data demonstrate that RBM3 enhances radioresistance by inhibiting the apoptotic response to radiotherapy through the PI3K/AKT/Bcl-2 signaling pathway. RBM3 may serve as a novel factor for predicting radioresistance and as a molecular target in the treatment of NPC.

Excessive glucocorticoid-induced muscle MuRF1 overexpression is independent of Akt/FoXO1 pathway.

The ubiquitin-proteasome system (UPS)-dependent proteolysis plays a major role in the muscle catabolic action of glucocorticoids (GCs). Atrogin-1 and muscle-specific RING finger protein 1 (MuRF1), two E3 ubiquitin ligases, are uniquely expressed in muscle. It has been previously demonstrated that GC treatment induced MuRF1 and atrogin-1 overexpression. However, it is yet unclear whether the higher pharmacological dose of GCs induced muscle protein catabolism through MuRF1 and atrogin-1. In the present study, the role of atrogin-1 and MuRF1 in C2C12 cells protein metabolism during excessive dexamethasone (DEX) was studied. The involvement of Akt/forkhead box O1 (FoXO1) signaling pathway and the cross-talk between anabolic regulator mammalian target of rapamycin (mTOR) and catabolic regulator FoXO1 were investigated. High concentration of DEX increased MuRF1 protein level in a time-dependent fashion (P<0.05), while had no detectable effect on atrogin-1 protein (P>0.05). FoXO1/3a (Thr24/32) phosphorylation was enhanced (P<0.05), mTOR phosphorylation was suppressed (P<0.05), while Akt protein expression was not affected (P>0.05) by DEX. RU486 treatment inhibited the DEX-induced increase of FoXO1/3a phosphorylation (P<0.05) and MuRF1 protein; LY294002 (LY) did not restore the stimulative effect of DEX on the FoXO1/3a phosphorylation (P>0.05), but inhibited the activation of MuRF1 protein induced by DEX (P<0.05); rapamycin (RAPA) inhibited the stimulative effect of DEX on the FoXO1/3a phosphorylation and MuRF1 protein (P<0.05).

Establishment and Clinical Application of an Electronic Database for Breast Cancer in China.

PURPOSE: To establish a database for breast cancer patients to save and manage clinical data and to preliminarily investigate its clinical application. MATERIALS AND METHODS: Information on breast cancer patients hospitalized in our department from 2008.01 to 2013.01 were input into our breast cancer management system. SPSS 16.0 software was used as a convenient reference to evaluate the accuracy of the newly built database. RESULTS: A database of 2403 breast cancer patients was successfully established. Information in the database clearly displayed capabilities of storage, addition, retrieval, statistical analysis and other functions. As the continuously updated database showed, the distribution of age, sex, nationality, allergy history, pausimenia and marriage of patients was identical to that achieved by SPSS analysis, indicating reliable and accurate data analysis. CONCLUSIONS: The described database is easy and convenient to operate and manage, and should prove suitable for application in clinical research and treatment.

Exome sequencing identifies novel compound heterozygous IFNA4 and IFNA10 mutations as a cause of impaired function in Crohn's disease patients.

Previous studies have highlighted the role of genetic predispositions in disease, and several genes had been identified as important in Crohn's disease (CD). However, many of these genes are likely rare and not associated with susceptibility in Chinese CD patients. We found 294 shared identical variants in the CD patients of which 26 were validated by Sanger sequencing. Two heterozygous IFN variants (IFNA10 c.60 T > A; IFNA4 c.60 A > T) were identified as significantly associated with CD susceptibility. The single-nucleotide changes alter a cysteine situated before the signal peptide cleavage site to a stop code (TGA) in IFNA10 result in the serum levels of IFNA10 were significantly decreased in the CD patients compared to the controls. Furthermore, the IFNA10 and IFNA4 mutants resulted in an impairment of the suppression of HCV RNA replication in HuH7 cells, and the administration of the recombinant IFN subtypes restored DSS-induced colonic inflammation through the upregulation of CD4(+) Treg cells. We identified heterozygous IFNA10 and IFNA4 variants as a cause of impaired function and CD susceptibility genes in Chinese patients from multiple center based study. These findings might provide clues in the understanding of the genetic heterogeneity of CD and lead to better screening and improved treatment.

Structural identification and sulfated modification of an antiglycation Dendrobium huoshanense polysaccharide.

Dendrobium huoshanense, an important food material, has been used to make teas and soups in the folk of China for centuries. In the present study, an antiglycation polysaccharide DHPD2 with molecular weight of 8.09 × 10(6)Da was extracted from the protocorm-like bodies of D. huoshanense. The backbone of DHPD2 contained (1→5)-linked α-l-Araf, (1→6)-linked α-d-Glcp, (1→6)-linked ß-d-Glcp, (1→4)-linked ß-d-Glcp, (1→3,6)-linked ß-d-Galp and (1→6)-linked ß-d-Galp, with the branches of terminal α-d-Xlyp and ß-d-Manp. DHPD2 was further modified using chlorosulfonic acid-pyridine method, giving two sulfated derivatives with the substitution degree of 0.475 and 0.940. The appearance of two new characteristic absorption bands at near 1250 and 822cm(-1) in FT-IR spectra revealed the success of sulfation occurred to DHPD2. Moreover, the sulfated derivatives exhibited stronger inhibitory abilities on protein glycation than those of DHPD2. NMR analysis disclosed that the sulfation on C2 and C6 of sugar residues was beneficial to enhance this activity.

Sulfated modification can enhance antiglycation abilities of polysaccharides from Dendrobium huoshanense.

Dendrobium huoshanense is an important edible-medicinal plant with high nutritional values and health functions. A homogenous polysaccharide (DHPD1) with molecular weight of 3.2 × 10(3)Da was extracted from D. huoshanense, which was mainly composed of glucose, arabinose, galactose, mannose and xylose. Chlorosulfonic acid-pyridine (CSA-Pyr) method was performed to modify the structure of DHPD1. In order to get a high degree of substitution (DS), sulfated modification conditions were optimized by response surface methodology. The maximum DS of 1.473 was obtained when the reaction condition was fixed at reaction temperature 60°C, reaction time 160 min and volume ratio of Pyr to CSA 2:1. NMR spectra revealed that this sulfation occurred to C-2 and C-6 of glycosyl residues in DHPD1. After 28 days of incubation, the sulfated DHPD1 at 1.0mg/mL showed the inhibitory ability of 58.5%, which increased by 16.2% and 52.5% than that of aminoguanidine and DHPD1 at the same dosage.

Using next-generation sequencing as a genetic diagnostic tool in rare autosomal recessive neurologic Mendelian disorders.

Next-generation sequencing was used to investigate 9 rare Chinese pedigrees with rare autosomal recessive neurologic Mendelian disorders. Five probands with ataxia-telangectasia and 1 proband with chorea-acanthocytosis were analyzed by targeted gene sequencing. Whole-exome sequencing was used to investigate 3 affected individuals with Joubert syndrome, nemaline myopathy, or spastic ataxia Charlevoix-Saguenay type. A list of known and novel candidate variants was identified for each causative gene. All variants were genetically verified by Sanger sequencing or quantitative polymerase chain reaction with the strategy of disease segregation in related pedigrees and healthy controls. The advantages of using next-generation sequencing to diagnose rare autosomal recessive neurologic Mendelian disorders characterized by genetic and phenotypic heterogeneity are demonstrated. A genetic diagnostic strategy combining the use of targeted gene sequencing and whole-exome sequencing with the aid of next-generation sequencing platforms has shown great promise for improving the diagnosis of neurologic Mendelian disorders.