Fangyukangsuan granules ameliorate hyperuricemia and modulate gut microbiota in rats.

Hyperuricaemia (HUA) is a metabolic disorder characterised by high blood uric acid (UA) levels; moreover, HUA severity is closely related to the gut microbiota. HUA is also a risk factor for renal damage, diabetes, hypertension, and dyslipidaemia; however, current treatments are associated with detrimental side effects. Alternatively, Fangyukangsuan granules are a natural product with UA-reducing properties. To examine their efficacy in HUA, the binding of small molecules in Fangyukangsuan granules to xanthine oxidase (XOD), a key factor in UA metabolism, was investigated via molecular simulation, and the effects of oral Fangyukangsuan granule administration on serum biochemical indices and intestinal microorganisms in HUA-model rats were examined. Overall, 24 small molecules in Fangyukangsuan granules could bind to XOD. Serum UA, creatinine, blood urea nitrogen, and XOD levels were decreased in rats treated with Fangyukangsuan granules compared to those in untreated HUA-model rats. Moreover, Fangyukangsuan granules restored the intestinal microbial structure in HUA-model rats. Functional analysis of the gut microbiota revealed decreased amino acid biosynthesis and increased fermentation of pyruvate into short-chain fatty acids in Fangyukangsuan granule-treated rats. Together, these findings demonstrate that Fangyukangsuan granules have anti-hyperuricaemic and regulatory effects on the gut microbiota and may be a therapeutic candidate for HUA.

Cell-free DNA testing for early hepatocellular carcinoma surveillance.

BACKGROUND: Liver cirrhosis (LC) is the highest risk factor for hepatocellular carcinoma (HCC) development worldwide. The efficacy of the guideline-recommended surveillance methods for patients with LC remains unpromising. METHODS: A total of 4367 LCs not previously known to have HCC and 510 HCCs from 16 hospitals across 11 provinces of China were recruited in this multi-center, large-scale, cross-sectional study. Participants were divided into Stage Ⅰ cohort (510 HCCs and 2074 LCs) and Stage Ⅱ cohort (2293 LCs) according to their enrollment time and underwent Tri-phasic CT/enhanced MRI, US, AFP, and cell-free DNA (cfDNA). A screening model called PreCar Score was established based on five features of cfDNA using Stage Ⅰ cohort. Surveillance performance of PreCar Score alone or in combination with US/AFP was evaluated in Stage Ⅱ cohort. FINDINGS: PreCar Score showed a significantly higher sensitivity for the detection of early/very early HCC (Barcelona stage A/0) in contrast to US (sensitivity of 51.32% [95% CI: 39.66%-62.84%] at 95.53% [95% CI: 94.62%-96.38%] specificity for PreCar Score; sensitivity of 23.68% [95% CI: 14.99%-35.07%] at 99.37% [95% CI: 98.91%-99.64%] specificity for US) (P < 0.01, Fisher's exact test). PreCar Score plus US further achieved a higher sensitivity of 60.53% at 95.08% specificity for early/very early HCC screening. INTERPRETATION: Our study developed and validated a cfDNA-based screening tool (PreCar Score) for HCC in cohorts at high risk. The combination of PreCar Score and US can serve as a promising and practical strategy for routine HCC care. FUNDING: A full list of funding bodies that contributed to this study can be found in Acknowledgments section.

Association between diet and the gut microbiome of young captive red-crowned cranes (Grus japonensis).

BACKGROUND: Exploring the association of diet and indoor and outdoor environments on the gut microbiome of red-crowned cranes. We investigated the microbiome profile of the 24 fecal samples collected from nine cranes from day 1 to 35. Differences in the gut microbiome composition were compared across diet and environments. RESULTS: A total of 2,883 operational taxonomic units (OTUs) were detected, with 438 species-specific OTUs and 106 OTUs common to the gut microbiomes of four groups. The abundance of Dietzia and Clostridium XI increased significantly when the red-crowned cranes were initially fed live mealworms. Skermanella and Deinococcus increased after the red-crowned cranes were fed fruits and vegetables and placed outdoors. Thirty-three level II pathway categories were predicted. Our study revealed the mechanism by which the gut microbiota of red-crowned cranes responds to dietary and environmental changes, laying a foundation for future breeding, nutritional and physiological studies of this species. CONCLUSIONS: The gut microbiome of red-crowned cranes could adapt to changes in diet and environment, but the proportion of live mealworms in captive red-crowned cranes can be appropriately reduced at the initial feeding stage, reducing the negative impact of high-protein and high-fat foods on the gut microbiome and growth and development.

Can circulating cell free DNA be a promising marker in ovarian cancer? - a genome-scale profiling study in a single institution.

BACKGROUND: Cell-free DNA (cfDNA) is emerging as a potential biomarker for the detection of ovarian cancer (OC). Recently, we reported a method based upon cfDNA whole-genome sequencing data including the nucleosome distribution (nucleosome footprinting NF), terminal signature sequence (motif), DNA fragmentation (fragment), and copy number variation (CNV).In the present study, we explored whether multiomics early screening technology in cfDNA can be applied for early screening of ovarian cancer. METHODS: Fifty-nine patients with OC and 100 healthy controls were included in this prospective study. Cell-free DNA was extracted from plasma and analyzed by low-pass whole-genome sequencing. Genomic features were obtained for all samples of the cohort, including copy number variation (CNV), 5'-end motifs, fragmentation profiles, and nucleosome footprinting (NF). An integrated scoring system termed the OC score was developed based on the performance of these four features. RESULTS: All four features showed diagnostic potential for OC. Based on the unique genome features of cfDNA, the OC score has high accuracy in distinguishing OC patients from healthy controls (AUC 97.7%; sensitivity 94.7%; specificity 98.0%) as a new comprehensive diagnostic method for OC. The OC score showed a gradual trend from healthy controls to OC patients with different stages, especially for early OC monitoring of concern, which achieved a satisfactory sensitivity (85.7%) at a high specificity. CONCLUSIONS: This is the first study evaluating the potential of cell-free DNA for the diagnosis of primary OC using multidimensional early screening technology. We present a promising method to increase the accuracy of prediction in patients with OC.

The development of a cSMART-based integrated model for hepatocellular carcinoma diagnosis.

BACKGROUND: Hepatocellular carcinoma (HCC) generally arises from a background of liver cirrhosis (LC). Patients with cirrhosis and suspected HCC are recommended to undergo serum biomarker tests and imaging diagnostic evaluation. However, the performance of routine diagnostic methods in detecting early HCC remains unpromising. METHODS: Here, we conducted a large-scale, multicenter study of 1675 participants including 490 healthy controls, 577 LC patients, and 608 HCC patients from nine clinical centers across nine provinces of China, profiled gene mutation signatures of cell-free DNA (cfDNA) using Circulating Single-Molecule Amplification and Resequencing Technology (cSMART) through detecting 931 mutation sites across 21 genes. RESULTS: An integrated diagnostic model called "Combined method" was developed by combining three mutation sites and three serum biomarkers. Combined method outperformed AFP in the diagnosis of HCC, especially early HCC, with sensitivities of 81.25% for all stages and 66.67% for early HCC, respectively. Importantly, the integrated model exhibited high accuracy in differentiating AFP-negative, AFP-L3-negative, and PIVKA-II-negative HCCs from LCs.

Base-Promoted Synthesis of Vinyl Sulfides from Sulfonium Triflates.

A new protocol has been developed for vinyl sulfide synthesis promoted by an alkoxy base under metal-free conditions. In this reaction, aryl and alkenyl sulfonium triflates with diverse functionalities are converted into vinyl sulfides with excellent reactivity. This transformation features mild and safe reaction conditions that avoid catalyst, transition metal, high-pressure gas, and high reaction temperature without compromising efficiency.

Thermo-sensitive electroactive hydrogel combined with electrical stimulation for repair of spinal cord injury.

The strategy of using a combination of scaffold-based physical and biochemical cues to repair spinal cord injury (SCI) has shown promising results. However, integrating conductivity and neurotrophins into a scaffold that recreates the electrophysiologic and nutritional microenvironment of the spinal cord (SC) remains challenging. In this study we investigated the therapeutic potential of a soft thermo-sensitive polymer electroactive hydrogel (TPEH) loaded with nerve growth factor (NGF) combined with functional electrical stimulation (ES) for the treatment of SCI. The developed hydrogel exhibits outstanding electrical conductance upon ES, with continuous release of NGF for at least 24 days. In cultured nerve cells, TPEH loaded with NGF promoted the neuronal differentiation of neural stem cells and axonal growth, an effect that was potentiated by ES. In a rat model of SCI, TPEH combined with NGF and ES stimulated endogenous neurogenesis and improved motor function. These results indicate that the TPEH scaffold that combines ES and biochemical cues can effectively promote SC tissue repair.

The Landscape of Cell-Free HBV Integrations and Mutations in Cirrhosis and Hepatocellular Carcinoma Patients.

PURPOSE: Intratumoral hepatitis B virus (HBV) integrations and mutations are related to hepatocellular carcinoma (HCC) progression. Circulating cell-free DNA (cfDNA) has shown itself as a powerful noninvasive biomarker for cancer. However, the HBV integration and mutation landscape on cfDNA remains unclear. EXPERIMENTAL DESIGN: A cSMART (Circulating Single-Molecule Amplification and Resequencing Technology)-based method (SIM) was developed to simultaneously investigate HBV integration and mutation landscapes on cfDNA with HBV-specific primers covering the whole HBV genome. Patients with HCC (n = 481) and liver cirrhosis (LC; n = 517) were recruited in the study. RESULTS: A total of 6,861 integration breakpoints including TERT and KMT2B were discovered in HCC cfDNA, more than in LC. The concentration of circulating tumor DNA (ctDNA) was positively correlated with the detection rate of these integration hotspots and total HBV integration events in cfDNA. To track the origin of HBV integrations in cfDNA, whole-genome sequencing (WGS) was performed on their paired tumor tissues. The paired comparison of WGS data from tumor tissues and SIM data from cfDNA confirmed most recurrent integration events in cfDNA originated from tumor tissue. The mutational landscape across the whole HBV genome was first generated for both HBV genotype C and B. A region from nt1100 to nt1500 containing multiple HCC risk mutation sites (OR > 1) was identified as a potential HCC-related mutational hot zone. CONCLUSIONS: Our study provides an in-depth delineation of HBV integration/mutation landscapes at cfDNA level and did a comparative analysis with their paired tissues. These findings shed light on the possibilities of noninvasive detection of virus insertion/mutation.

Development and characterization of microsatellite markers in the earthworm Drawida gisti Michaelsen, 1931 and cross-amplification in two other congeners.

The earthworm (Drawida gisti) is an ecologically important sentinel species for soils that is widely distributed throughout Eastern Asia; however, the molecular tools required for genetic diversity studies of this earthworm are still rare. The aim of the study was to develop and characterize microsatellite markers in D. gisti and to evaluate their transferability to other Drawida species. We employed a RAD-seq approach to develop 12 microsatellite markers for D. gisti. The characterization and analysis of loci was achieved using 24 individuals of D. gisti from a natural population. The number of alleles per locus ranged from four to eleven, with an average of 6.5. Observed and expected heterozygosities varied from 0.708 to 0.958, and from 0.568 to 0.883, respectively. No loci presented significant deviations from the Hardy-Weinberg equilibrium, while linkage disequilibrium was detected between three loci. Cross-species amplification tests suggested that the transferability of ten loci was positive for the two congeners D. japonica and D. ghilarovi. This set of microsatellite markers may be used to evaluate the genetic diversity and population structures of D. gisti and related species in the future.

Characterization of the complete mitochondrial genome of Drawida gisti (Metagynophora, Moniligastridae) and comparison with other Metagynophora species.

Here, the complete mitochondrial genome (mitogenome) of Drawida gisti was sequenced and compared with the mitogenomes of other Metagynophora species. The circular mitogenome was 14,648 bp in length and contained two ribosomal RNA genes (rRNAs), 13 protein-coding genes (PCGs), and 22 transfer RNA genes (tRNAs). The types of constitutive genes and the direction of the coding strand that appeared in Drawida mitogenome were identical to those observed in other Metagynophora species, except for a missing lengthy non-coding region. The conservative relationships between Drawida species were supported by the overall analyses of 13 PCGs, two rRNAs, and 22 tRNAs. A comparison of the Metagynophora mitogenomes revealed that the ATP8 gene possessed the highest polymorphism among the 13 PCGs and two rRNAs. Phylogenetic analysis suggested that the Moniligastridae contained Drawida, which is a primitive Metagynophora group. Our study provides a step forward toward elucidating the evolutionary linkages within Drawida and even Metagynophora.

Complete mitogenomic and phylogenetic characteristics of the speckled wood-pigeon (Columba hodgsonii).

The speckled wood-pigeon, Columba hodgsonii, is mainly distributed in Bhutan, China, India, Laos, Myanmar, Nepal, Pakistan, and Thailand. Although there are several studies on birds in the family Columbidae, no study has focused on C. hodgsonii, a member of this family. Therefore, this study aimed to clarify the phylogenetic status of C. hodgsonii. The complete mitochondrial genome (mitogenome) of C. hodgsonii was sequenced and characterized and compared with those of other Columba species. The C. hodgsonii mitogenome was found to be 17,477 bp in size and contained 13 PCGs, two rRNAs, 22 tRNAs, and one CR. Of the 37 genes encoded by the C. hodgsonii mitogenome, 28 were on the heavy strand and nine were on the light strand. Twelve PCGs were initiated by ATN codons and one PCG harbored an incomplete termination codon (T-). The base composition of C. hodgsonii PCGs was A = 29.44%, T = 24.37%, G = 12.43%, and C = 33.76%. For the whole mitogenome, including PCGs, rRNAs, tRNAs, and the control region, the AT-skew was positive, and the GC-skew was negative. Phylogenetic analysis based on the base sequences of 13 PCGs from 28 Columbidae species and one outgroup using maximum likelihood and Bayesian inference indicated that C. hodgsonii belongs to the genus Columba and that the family Columbidae is monophyletic.

Targeted deep sequencing from multiple sources demonstrates increased NOTCH1 alterations in lung cancer patient plasma.

INTRODUCTION: Targeted therapies are based on specific gene alterations. Various specimen types have been used to determine gene alterations, however, no systemic comparisons have yet been made. Herein, we assessed alterations in selected cancer-associated genes across varying sample sites in lung cancer patients. MATERIALS AND METHODS: Targeted deep sequencing for 48 tumor-related genes was applied to 153 samples from 55 lung cancer patients obtained from six sources: Formalin-fixed paraffin-embedded (FFPE) tumor tissues, pleural effusion supernatant (PES) and pleural effusion cell sediments (PEC), white blood cells (WBCs), oral epithelial cells (OECs), and plasma. RESULTS: Mutations were detected in 96% (53/55) of the patients and in 83% (40/48) of the selected genes. Each sample type exhibited a characteristic mutational pattern. As anticipated, TP53 was the most affected sequence (54.5% patients), however this was followed by NOTCH1 (36%, across all sample types). EGFR was altered in patient samples at a frequency of 32.7% and KRAS 10.9%. This high EGFR/ low KRAS frequency is in accordance with other TCGA cohorts of Asian origin but differs from the Caucasian population where KRAS is the more dominant mutation. Additionally, 66% (31/47) of PEC samples had copy number variants (CNVs) in at least one gene. Unlike the concurrent loss and gain in most genes, herein NOTCH1 loss was identified in 21% patients, with no gain observed. Based on the relative prevalence of mutations and CNVs, we divided lung cancer patients into SNV-dominated, CNV-dominated, and codominated groups. CONCLUSIONS: Our results confirm previous reports that EGFR mutations are more prevalent than KRAS in Chinese lung cancer patients. NOTCH1 gene alterations are more common than previously reported and reveals a role of NOTCH1 modifications in tumor metastasis. Furthermore, genetic material from malignant pleural effusion cell sediments may be a noninvasive manner to identify CNV and participate in treatment decisions.

Using plasma cell-free DNA to monitor the chemoradiotherapy course of cervical cancer.

The liquid biopsy is being integrated into cancer diagnostics and surveillance. However, critical questions still remain, such as how to precisely evaluate cancer mutation burden and interpret the corresponding clinical implications. Herein, we evaluated the role of peripheral blood cell-free DNA (cfDNA) in characterizing the dynamic mutation alterations of 48 cancer driver genes from cervical cancer patients. We performed targeted deep sequencing on 93 plasma cfDNA from 57 cervical cancer patients and from this developed an algorithm, allele fraction deviation (AFD), to monitor in an unbiased manner the dynamic changes of genomic aberrations. Differing treatments, including chemotherapy (n = 22), radiotherapy (n = 14) and surgery (n = 15), led to a significant decrease in AFD values (Wilcoxon, p = 0.029). The decrease of cfDNA AFD values was accompanied by shrinkage in the size of the tumor in most patients. However, in a subgroup of patients where cfDNA AFD values did not reflect a reduction in tumor size, there was a detection of progressive disease (metastasis). Furthermore, a low AFD value at diagnosis followed a later increase of AFD value also successfully predicted relapse. These results show that plasma cfDNA, together with targeted deep sequencing, may help predict treatment response and disease development in cervical cancer.

Polymer scaffolds facilitate spinal cord injury repair.

During the past decades, improving patient neurological recovery following spinal cord injury (SCI) has remained a challenge. An effective treatment for SCI would not only reduce fractured elements and isolate developing local glial scars to promote axonal regeneration but also ameliorate secondary effects, including inflammation, apoptosis, and necrosis. Three-dimensional (3D) scaffolds provide a platform in which these mechanisms can be addressed in a controlled manner. Polymer scaffolds with favorable biocompatibility and appropriate mechanical properties have been engineered to minimize cicatrization, customize drug release, and ensure an unobstructed space to promote cell growth and differentiation. These properties make polymer scaffolds an important potential therapeutic platform. This review highlights the recent developments in polymer scaffolds for SCI engineering. STATEMENT OF SIGNIFICANCE: How to improve the efficacy of neurological recovery after spinal cord injury (SCI) is always a challenge. Tissue engineering provides a promising strategy for SCI repair, and scaffolds are one of the most important elements in addition to cells and inducing factors. The review highlights recent development and future prospects in polymer scaffolds for SCI therapy. The review will guide future studies by outlining the requirements and characteristics of polymer scaffold technologies employed against SCI. Additionally, the peculiar properties of polymer materials used in the therapeutic process of SCI also have guiding significance to other tissue engineering approaches.

The complete mitochondrial genome of Lumbricus rubellus (Oligochaeta, Lumbricidae) and its phylogenetic analysis.

The complete mitochondrial genome of Lumbricus rubellus was analyzed by next-generation sequencing. The mitogenome was 15,464 bp in length, comprising 13 protein-coding genes (PCGs), 22 transfer RNAs, 2 ribosomal RNAs, and a non-coding region. The phylogenetic analysis of 13 PCGs within the class Oligochaeta suggested that L. rubellus was placed as sister to L. terrestris of the same genus. The results obtained here can contribute to the phylogenetic analysis of earthworms.

The complete mitogenome of Hong Kong paradise fish (Macropodus hongkongensis), an endemic freshwater fish in South China.

The first complete mitogenome of Hong Kong paradise fish (Macropodus hongkongensis) was determined in this study. The assembled mitogenome is 16,494 bp and consisted of 13 protein-coding genes, 22 tRNAs, 2 rRNAs, and a control region. Nucleotide composition of the complete mitogenome is 30.6% A, 24.8% C, 14.8% G, and 29.8% T, with an A + T bias of 60.4%. The maximum-likelihood tree based on 13 protein-coding genes showed that M. erythropterus was the closest related species to M. hongkongensis.

The complete mitogenome of red-collared lorikeet (Trichoglossus rubritorquis) and its phylogenetic analysis.

The complete mitogenome of a lorikeet, Trichoglossus rubritorquis (Psittaciformes, Loriidae), was determined first in the genus Trichoglossus. The assembled mitogenome was 17,915 bp and composed of 13 protein-coding genes, 22 tRNAs, two rRNAs and two control regions. Nucleotide composition of T. rubritorquis mitogenome was 30.20% A, 33.30% C, 14.04% G, and 22.46% T, with an A + T bias of 52.66%. ML tree based on 13 protein-coding genes indicated that, Melopsittacus undulatus of the family Psittacidae was the closest related species to T. rubritorquis. This result suggested that lorikeets might still be in the family Psittacidae.

Transcriptomic but not genomic variability confers phenotype of breast cancer stem cells.

BACKGROUND: Breast cancer stem cells (BCSCs) are considered responsible for cancer relapse and drug resistance. Understanding the identity of BCSCs may open new avenues in breast cancer therapy. Although several discoveries have been made on BCSC characterization, the factors critical to the origination of BCSCs are largely unclear. This study aimed to determine whether genomic mutations contribute to the acquisition of cancer stem-like phenotype and to investigate the genetic and transcriptional features of BCSCs. METHODS: We detected potential BCSC phenotype-associated mutation hotspot regions by using whole-genome sequencing on parental cancer cells and derived serial-generation spheres in increasing order of BCSC frequency, and then performed target deep DNA sequencing at bulk-cell and single-cell levels. To identify the transcriptional program associated with BCSCs, bulk-cell and single-cell RNA sequencing was performed. RESULTS: By using whole-genome sequencing of bulk cells, potential BCSC phenotype-associated mutation hotspot regions were detected. Validation by target deep DNA sequencing, at both bulk-cell and single-cell levels, revealed no genetic changes specifically associated with BCSC phenotype. Moreover, single-cell RNA sequencing showed profound transcriptomic variability in cancer cells at the single-cell level that predicted BCSC features. Notably, this transcriptomic variability was enriched during the transcription of 74 genes, revealed as BCSC markers. Breast cancer patients with a high risk of relapse exhibited higher expression levels of these BCSC markers than those with a low risk of relapse, thereby highlighting the clinical significance of predicting breast cancer prognosis with these BCSC markers. CONCLUSIONS: Transcriptomic variability, not genetic mutations, distinguishes BCSCs from non-BCSCs. The identified 74 BCSC markers have the potential of becoming novel targets for breast cancer therapy.

Spatial Vulnerabilities of the Escherichia coli Genome to Spontaneous Mutations Revealed with Improved Duplex Sequencing.

Investigation of spontaneous mutations by next-generation sequencing technology has attracted extensive attention lately due to the fundamental roles of spontaneous mutations in evolution and pathological processes. However, these studies only focused on the mutations accumulated through many generations during long-term (possibly be years of) culturing, but not the freshly generated mutations that occur at very low frequencies. In this study, we established a molecularly barcoded deep sequencing strategy to detect low abundant spontaneous mutations in genomes of bacteria cell cultures. Genome-wide spontaneous mutations in 15 Escherichia coli cell culture samples were defined with a high confidence (P < 0.01). We also developed a hotspot-calling approach based on the run-length encoding algorithm to find the genomic regions that are vulnerable to the spontaneous mutations. The hotspots for the mutations appeared to be highly conserved across the bacteria samples. Further biological annotation of these regions indicated that most of the spontaneous mutations were located at the repeat domains or nonfunctional domains of the genomes, suggesting the existence of mechanisms that could somehow prevent the occurrence of mutations in crucial genic areas. This study provides a more faithful picture of mutation occurrence and spectra in a single expansion process without long-term culturing.

Conflicts of CpG density and DNA methylation are proximally and distally involved in gene regulation in human and mouse tissues.

The relationship between CpG content and DNA methylation has attracted considerable interest in recent years. Direct or indirect methods have been developed to investigate their regulatory functions based on various hypotheses, large cohort studies, and meta-analyses. However, all of these analyses were performed at units of CpG blocks and, thus, the influence of finer genome structure has been neglected. Herein, we present a novel algorithm of base-pair resolution to systematically investigate the relationship between CpG contents and DNA methylation. By introducing the concept of 'complementary index' we examined the methylomes of 34 adult and 7 embryonic tissues and successfully fitted the relationship of DNA methylation and CpG density into a nonlinear mathematical model. A further algorithm was developed to locate the regions where CpG density does not match expectations from the model, termed 'conflict of gap' (COG) regions. Interestingly, COGs are highly concordant in human and mouse and their distributions display a tissue-specific pattern. Based on COG methylation patterns we correctly classified tissues according to their function or origin. We demonstrate that COGs based on our method can reveal more and deeper information than traditional differential methylation region (DMR) approaches. We also found that when COGs are located near to transcription start site (TSS), these regions can determine which promoters will be utilized for initiating gene transcription. Furthermore, COGs located far from the TSS perform as enhancers in terms of histone modification, sequence conservation, transcription factor binding, and DNase I-hypersensitivity.