Genome-wide high-throughput chromosome conformation capture analysis reveals hierarchical chromatin interactions during early somatic embryogenesis.

Somatic embryogenesis (SE), like zygotic embryo development, is a progressive process. Early SE is the beginning of a switch from a somatic to an embryogenic state and is an important stage for initiating chromatin reprogramming of SE. Previous studies suggest that changes in chromatin accessibility occur during early SE, although information on the 3D structure of chromatin is not yet available. Here, we present a chromosome-level genome assembly of longan (Dimocarpus longan) using PacBio combined with high-through chromosome conformation capture scaffolding, which resulted in a 446 Mb genome assembly anchored onto 15 scaffolds. During early SE, chromatin was concentrated and then decondensed, and a large number of long terminal repeat retrotransposons (LTR-RTs) were enriched in the local chromatin interaction region, suggesting LTR-RTs were involved in chromatin reorganization. Early SE was accompanied by the transformation from A to B compartments, and the interactions between B compartments were enhanced. Results from chromatin accessibility, monomethylation of histone H3 at lysine 4 (H3K4me1) modification, and transcription analyses further revealed a gene regulatory network for cell wall thickening during SE. Particularly, we found that the H3K4me1 differential peak binding motif showed abnormal activation of ethylene response factor transcription factors and participation in SE. The chromosome-level genomic and multiomics analyses revealed the 3D conformation of chromatin during early SE, providing insight into the molecular mechanisms underlying cell wall thickening and the potential regulatory networks of TFs during early SE in D. longan. These results provide additional clues for revealing the molecular mechanisms of plant SE.

Quantitative Magnetic Resonance Imaging of Femoral Head Articular Cartilage Change in Patients with Hip Osteonecrosis Treated with Extracorporeal Shock Wave Therapy.

Background: Multiple reports have demonstrated the therapeutic potential of extracorporeal shock wave (ESWT) in osteonecrosis of the femoral head (ONFH). However, few studies reported the changes in hip articular cartilage after the intervention. This study aimed to investigate the effect of ESWT on femoral head cartilage using a novel technique, quantitative T2-mapping magnetic resonance imaging. Methods: A total of 143 eligible patients with unilateral early-stage ONFH were randomized into the ESWT group and control group. Seventy-three patients in the ESWT group received two sessions of ESWT with oral drug treatment, while seventy patients in the control group received oral drug treatment only. The visual analog pain scale (VAS) and Harris hip score (HHS) at 3-month, 6-month, and 12-month follow-up were used as the clinical evaluation index. The radiological evaluation index used the T2 mapping values, necrotic size, and China-Japan Friendship Hospital (CJFH) classification. Results: A total of 143 patients (62 females and 81 males) were finally included, and the characteristics before treatment were comparable between the two groups. At the last follow-up (12 months), the T2 values and ΔT2 changes in the ESWT group were all smaller than those in the control group (p=0.042; p=0.039), while the CJFH classification of ONFH and necrotic lesion size were not statistically significant. At 3 months and 6 months, the VAS in the ESWT group was lower than that in the control group (p=0.021; p=0.046) and the HHS in the ESWT group was higher (p=0.028; p=0.039). However, there were no significant differences in the VAS and HHS at 12 months between the ESWT and control groups. Conclusions: The results of the current study indicated that, based on drug treatment, ESWT is an effective treatment method for nontraumatic ONFH, which could result in significant pain relief and function restoration. Furthermore, it could delay the injury of femoral head cartilage during the progression of ONFH.

Identification and Genetic Analysis of a Factor IX Gene Intron 3 Mutation in a Hemophilia B Pedigree in China.

OBJECTIVE: Hemophilia B is caused by coagulation defects in the factor IX gene located in Xq27.1 on the X chromosome. A wide range of mutations, showing extensive molecular heterogeneity, have been described in hemophilia B patients. Our study was aimed at genetic analysis and prenatal diagnosis of hemophilia B in order to further elucidate the pathogenesis of the hemophilia B pedigree in China. MATERIALS AND METHODS: Polymerase chain reaction amplification and direct sequencing of all the coding regions was conducted in hemophilia B patients and carriers. Prenatal diagnosis of the proband was conducted at 20 weeks. RESULTS: We identified the novel point mutation 10.389 A>G, located upstream of the intron 3 acceptor site in hemophilia B patients. The fetus of the proband's cousin was identified as a carrier. CONCLUSION: Our identification of a novel mutation in the F9 gene associated with hemophilia B provides novel insight into the pathogenesis of this genetically inherited disorder and also represents the basis of prenatal diagnosis.

Genome-Wide Identification and Expression Analysis Reveal bZIP Transcription Factors Mediated Hormones That Functions during Early Somatic Embryogenesis in Dimocarpus longan.

The basic leucine zip (bZIP) transcription factors (TFs) are a group of highly conserved gene families that play important roles in plant growth and resistance to adversity stress. However, studies on hormonal regulatory pathways and functional analysis during somatic embryogenesis (SE) in Dimocarpus longan is still unavailable. In this study, a total of 51 bZIP family members were systematically identified in the whole genome of longan, a comprehensive bioinformatics analysis of DlbZIP (bZIP family members of D. longan) was performed, and subcellular localization and profiles patterns after transiently transformed DlbZIP60 were analyzed. The combined analysis of RNA-seq, ATAC-seq and ChIP-seq showed that four members have different H3K4me1 binding peaks in early SE and differentially expressed with increased chromatin accessibility. Comparative transcriptome analysis of bZIPs expression in early SE, different tissues and under 2,4-D treatment revealed that DlbZIP family might involved in growth and development during longan early SE. The qRT-PCR results implied that DlbZIP family were subjected to multiple hormonal responses and showed different degrees of up-regulated expression under indole-3-acetic acid (IAA), abscisic acid (ABA) and methyl jasmonate (MeJA) treatments, which indicated that they played an important role in the hormone synthesis pathways associated with the early SE of longan. Subcellular localization showed that DlbZIP60 was located in the nucleus, and the contents of endogenous IAA, MeJA and ABA were up-regulated in transiently DlbZIP60 overexpressed cell lines. These results suggest that DlbZIP60 may mediate hormones pathways that functions the development during early SE in longan.

Genome-wide analysis of the SAUR gene family and function exploration of DlSAUR32 during early longan somatic embryogenesis.

The early auxin responsive small auxin up-regulated RNA (SAUR) family is an important gene family in the auxin signal transduction pathway. This study focused on the regulatory mechanism of DlSAUR genes during early somatic embryogenesis (SE) and its response to hormone treatment and abiotic stress. Mining of the available Dimocarpus longan Lour. (D. longan) genome sequence yielded 68 putative SAUR genes. Transcript profiles based on RNA-seq data showed that most of the 24 detected DlSAUR genes were highly expressed in the globular embryos (GE) (10) and most of them responded to heat stress and 2,4-D treatment. The results of qRT-PCR showed that most of DlSAUR genes were up-regulated under auxin inhibitor N-1-naphthylphthalamic acid (NPA) and auxin indole-3-acetic acid (IAA) treatments. Moreover, NPA could promote longan SE. The assay for ATAC-seq data analysis showed that chromatin accessibility of 19 of the 24 DlSAUR genes were open during early SE, and most DlSAUR genes differentially expressed during early SE were not associated with H3K4me1 signal enrichment. The DlSAUR32 was selected for subcellular localization and RNA-seq analysis, which encode a cell nuclear-localized protein. Dual-luciferase assays and transient transformation showed that the transcription factors (TFs) DlWRKY75-1 and DlWRKY75-2 might bind to the DlSAUR32 promoters to inhibition gene transcription. Transient overexpression of DlWRKY75-1 and DlWRKY75-2 decreased IAA content in N. benthamiana leaves. Thus, the regulatory network composed of DlSAUR32 and its related TFs may regulate the early longan SE and be involved in the auxin response regulatory pathway of longan.

Yeast enrichment facilitated lipid removal and bioconversion by black soldier fly larvae in the food waste treatment.

Food waste can be converted into insectile fatty acids (FAs) by the larvae of black soldier fly (BSFL), Hermetia illucens, for use in the feed sector or as a source of biodiesel. However, waste oil was less decomposed than carbohydrate or protein in frass due to the limitation of larval lipid metabolism. In this study, 10 yeast strains were screened, corresponding to six species, to examine their capacity of improving lipid transformation performance by BSFL. The species of Candida lipolytica was superior to the other five species, which exhibited significantly higher lipid reduction rate (95.0-97.1 %) than the control (88.7 %), and the larval FA yields achieved 82.3-115.5 % of the food waste FA matters, suggesting that BSFL not only transformed waste oil but also biosynthesized FAs from waste carbohydrate and other substances. Further, the CL2 strain of Candida lipolytica was examined for treating food waste containing high lipid content (16-32 %). The lipid removal rate was found improved from 21.4 to 42.3 % (control) to 80.5-93.3% in the waste containing 20-32 % lipid. The upper limit of lipid content that could be endured by BSFL was ≈16 %, and the CL2-enrichment elevated the upper limit to ≈24 %. Fungal community analysis indicated that Candida spp. accounted for the lipid removal improvement. The Candida spp. CL2 strain may facilitate the lipid reduction and transformation by BSFL through microbial catabolizing and assimilation of waste FAs. Altogether, this study suggests that yeast enrichment is feasible in improving lipid transformation by BSFL especially for food waste exhibiting high lipid content.

Genome-wide analysis of the XTH gene family and functional analysis of DlXTH23.5/25 during early longan somatic embryogenesis.

Introduction: Xyloglucan endotransglucosylase (XET)/hydrolase (XTH) is a cell wall-modifying protein that affects cell expansion and loosening of the cell wall. Results: This study focused on the regulatory mechanism of DlXTH genes during early somatic embryogenesis (SE) and the heat stress response in longan. Mining of the available D. longan genome sequence yielded 25 putative XTH genes. Transcript profiles based on RNA sequencing (RNA-seq) data showed that most of the 17 detected DlXTH genes were highly expressed in the embryogenic callus (EC) (8) and globular embryo (GE) (8), and 13 of them responded significantly to heat stress. The assay for transposase-accessible chromatin sequencing (ATAC-seq) data analysis showed that in terms of chromatin accessibility, 22 of the 25 DlXTH genes were open during early SE, and most of the peak DlXTH genes with transcription differences during early SE were associated with high levels of H3K4me1. The most differentially expressed genes, DlXTH23.5 and DlXTH25, were selected for analysis. According to subcellular localization and quantitative real-time PCR (qRT-PCR) analysis, DlXTH23.5/25, which encode cell membrane-localized proteins, were expressed at the highest level in the GE and significantly responded to heat stress. Dual-luciferase assays and transient transformation showed that the transcription factors (TFs) DlWRKY31, DlERF1, and DlERF5 might bind to the DlXTH23.5/25 promoters to activate gene transcription. Transient overexpression of TFs and DlXTH23.5/25 induced XET activity in Nicotiana benthamiana leaves. Under heat stress in the longan EC, the XET activities and expression levels of TFs and DlXTH23.5/25 were significantly increased, and a high concentration of XET might inhibit longan SE. Discussions: Thus, the regulatory network composed of DlXTH23.5/25 and its related TFs may regulate early longan SE and participate in the regulatory pathway of longan under heat stress via cell wall repair through the action of XET.

The correlation between high-risk HPV infection and precancerous lesions and cervical cancer.

OBJECTIVE: To analyze the correlation between high-risk human papillomavirus (HPV) infection and precancerous lesions and cervical cancer. METHODS: Patients with cervicitis (N=100), cervical intraepithelial neoplasia grade I (CIN I) (N=100), cervical intraepithelial neoplasia grades II-III (CIN II-III) (N=100) and cervical cancer (N=100) were enrolled. The exfoliated cervical cells were collected with the same method, and the detection of the HPV types was carried out by PCR-reverse dot blot (RDB) assay. RESULTS: The top 5 HPV types in stage I-II cervical cancer were 16, 18, 52, 58, and 53, with a HPV positivity rate of 83.61%, while top 5 HPV types in stage III-IV cervical cancer were 16, 18, 58, 52, and 33, with a HPV positivity rate of 82.05%. The rate of high-risk HPV positivity for cervicitis was 5%, with HPV types of 16, 18, 52, and 33, 12% for CIN I, with HPV types of 16, 58, 52, 33, 56, 66, and 68, and 42% for CIN II-III, with HPV types of 16, 18, 58, 52, 33, 66, and 68. The prevalence of single, dual, and multiple HPV infection was 8.00%, 1.00%, and 0.00% for CIN I, 24.00%, 7.00%, and 1.00% for CIN II-III, and 57.00%, 25.00%, and 3.00% for cervical cancer, respectively. The age of patients with CIN I was mainly ≤24 and 25-34 years while CIN II-III in 25-34 and 35-44 years, and cervical cancer in 35-44 and 45-54 years. CONCLUSION: The distribution of HPV subtypes in cervical cancer is closely related to the pathological types, lesion grades, and stages of cervical cancer. The incidence of cervical lesions varies with age, suggesting that high-risk groups should be well monitored and receive regular screening and timely HPV vaccination to effectively prevent cervical cancer.

A Bioassay-Based Approach for the Batch-To-Batch Consistency Evaluation of Xuesaitong Injection on a Zebrafish Thrombosis Model.

Quality control of Chinese medicine (CM) is mainly based on chemical testing, which sometimes shows weak correlation to pharmacological effects. Thus, there is a great demand to establish bioactivity-based assays to ensure the quality of CM. The aim of the present study was to establish a bioassay-based approach to evaluate the biological activity of Xuesaitong injection (XST) based on an in vivo zebrafish model. Zebrafish larvae with arachidonic acid (AA)-induced thrombus were applied to evaluate anti-thrombosis effects of XST and explore the potential mechanism of XST. Analysis of major components in normal and abnormal XST samples was performed by high performance liquid chromatography (HPLC). The results indicate that XST could significantly restore heart red blood cells (RBCs) intensity of thrombotic zebrafish in a dose-dependent manner, whilst decreasing RBCs accumulation in the caudal vein. The results were confirmed using a green fluorescence protein (GFP)-labeled zebrafish thrombosis model. Moreover, we could show that XST downregulates the expression of the fibrinogen alpha chain (fga) gene to inhibit the coagulation cascade during the process of thrombosis in zebrafish. Notoginsenoside R1, ginsenoside Rg1, ginsenoside Rb1 and ginsenoside Rd, which were considered to be the major components of XST, also showed moderate anti-thrombosis efficacy. Further results showed that the zebrafish thrombosis model could efficiently distinguish five abnormal batches of XST from 24 normal batches. Furthermore, the inhibition rates of different batches were correlated with the content level of major components. Our results suggested that the proposed zebrafish thrombosis model could be successfully used to evaluate the batch-to-batch consistency of XST, which provided an alternative way for the quality control of CM.

Serum miR-26a as a diagnostic and prognostic biomarker in cholangiocarcinoma.

In order to determine the diagnostic and prognostic value of miR-26a in Cholangiocarcinoma (CCA), we compared miR-26a levels in serum from 66 CCA patients and 66 healthy controls, which was followed by serum analysis between the pre-operative serum and post-operative serum of these CCA patients. We found the concentration levels of miR-26a in serum of CCA patients were significantly higher than that from healthy controls (P < 0.01). Furthermore, the concentration levels of miR-26a in the post-operative serum were significantly reduced when compared to the pre-operative serum (P < 0.001). High miR-26a in serum was correlated significantly with clinical stage, distant metastasis, differentiation status, and poor survival of CCA patients. More importantly, serum miR-26a was an independent prognostic marker for CCA. In conclusion, our results suggested that miR-26a in serum might be a potential and useful noninvasive biomarker for the early detection of CCA.

[Clinical application of primary tumor contralateral facial artery musculocutaneous flap to reconstruct oral and maxillofacial defects].

OBJECTIVE: This study aims to explore the method that uses primary tumor contralateral facial artery musculocutaneous (FAMM) flap to reconstruct defects of the tongue and floor of mouth. METHODS: Six cases were selected for the use of primary tumor contralateral FAMM flap to reconstruct tongue and floor of mouth defects after tumor resection. RESULTS: The FAMM flap of the six cases had a long pedicle that could reach the contralateral tongue and floor of mouth. All flaps were intact until post-operation. All patients experienced post-operation complications, such as temporary facial tension and limited mouth opening, which improved after 3 months. Half a year later, the flaps still did not show signs of shrinking. CONCLUSION: Features of the primary tumor contralateral FAMM flap include the tissue-like material provided for reconstructing tongue or floor of mouth defects, easy acquisition, and high survival rate with minimal donor site morbidity. As such, it is an ideal material for repairing tongue and floor of mouth defects.