Biology subjects

Zhong, C.

Publications and source records attributed to Zhong, C..

69 records · Page 4Linked to original sources

bioRxiv2019

CRISPR/Cas9-mediated integration of large transgene into pig potential safe harbor

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (Cas9) is a precise genome manipulating tool which can produce targeted gene mutations in various cells and organisms. Although CRISPR/Cas9 can efficiently generate gene knock-out, the gene knock-in efficiency mediated by homology-directed repair (HDR) remains low, especially for large fragment integration. In this study, we established an efficient method for CRISPR/Cas9-mediated integration of large transgene cassette carrying salivary gland-expressing multiple digestion enzymes ({approx} 20 kbp) in CEP112 locus in pig fetal fibroblasts. Our results showed that using homologous donor with a short left arm and a long right arm yielded the best CRISPR/Cas9-mediated knock-in efficiency, and the targeting efficiency in potential safe harbor CEP112 locus are higher than ROSA26 locus. The CEP112 knock-in cell lines were used as nuclear donors for somatic cell nuclear transfer to create genetically modified pigs. We found that knock-in pig (705) successfully expressed three microbial enzymes ({beta}-glucanase, xylanase, and phytase) in salivary gland, suggesting potential safe harbor CEP112 locus supports exogenous genes expression by a tissue-specific promoter. In summary, we successfully targeted CEP112 locus using our optimal homology arm system for precise modification of pigs, and established a modified pig model for foreign digestion enzyme expression in saliva.

bioengineering↗

bioRxiv2019

Dosage effect of multiple genes accounts for multisystem disorder of myotonic dystrophy type 1

Multisystem manifestations in myotonic dystrophy type 1 (DM1) may be due to dosage reduction in multiple genes induced by aberrant expansion of CTG repeats in DMPK, including Dmpk and its neighboring genes (Six5 or Dmwd) and downstream Mbnl1. However, the direct evidences are lack. Here, we develop a new strategy to generate mice carrying multigene mutations in one step by injection of haploid embryonic stem cells with mutant Dmpk, Six5 and Mbnl1 into oocytes. The triple heterozygous mutant mice exhibit adult-onset DM1 phenotypes. With the additional mutation in Dmwd, quadruple heterozygous mutant mice recapitulate many major manifestations in congenital DM1. Moreover, muscle stem cells in both models display reduced stemness. Our results suggest that the complex symptoms of DM1 result from the reduced gene dosage of multiple genes.

genetics↗

bioRxiv2019

Genomic islets in a sea of introgressions between sympatric species

In the conventional view, species are separate gene pools delineated by reproductive isolation (RI). However, species may also be delineated by merely a small set of "speciation genes" without full RI. It is thus important to know whether "good species" (defined by the "secondary sympatry" test) do continue to exchange genes. Here, we carry out sequencing and de novo high-quality assembly of the genomes of two closely related mangrove species (Rhizophora mucronata and R. stylosa). Whole-genome re-sequencing of individuals across their range on the tropical coasts shows their genomes to be well delineated in allopatry. They became sympatric in northeastern Australia but remain distinct species in contact. Nevertheless, their genomes harbor [~] 4,000 to 10,000 introgression blocks, each averaging only about 3-4 Kb. These fine-grained introgressions indicate that gene flow has continued long after speciation. Non-introgressable "genomic islets," averaging only 1.4 Kb, may contribute to speciation as they often harbor diverging genes underlying flower development and gamete production. In conclusion, RI needs not be the main criterion of species delineation even though all species would eventually be fully reproductively isolated.

evolutionary biology↗

bioRxiv2019

3D cell culture stimulates the secretion of in vivo like exosomes

For studying cellular communications ex-vivo, a two-dimensional (2D) cell culture model is currently used as the "gold standard". 2D culture models are also widely used in the study of RNA expression profiles from tumor cells secreted extracellular vesicles (EVs) for tumor biomarker discovery. Although the 2D culture system is simple and easily accessible, the culture environment is unable to represent in vivo extracellular matrix (ECM) microenvironment. Our study observed that 2D culture-derived EVs showed significantly different profiles in terms of secretion dynamics and essential signaling molecular contents (RNAs and DNAs), when compared to the three-dimensional (3D) culture-derived EVs. By performing small RNA next-generation sequencing (NGS) analysis of cervical cancer cells and their EVs compared with cervical cancer patient plasma EV-derived small RNAs, we observed that 3D culture-derived EV small RNAs differ from their parent cell small RNA profile which may indicate a specific sorting process. Most importantly, the 3D culture derived EV small RNA profile exhibited a much higher similarity (~96%) to in vivo circulating EVs derived from cervical cancer patient plasma. However, 2D culture derived EV small RNA profile correlated better with only their parent cells cultured in 2D. On the other hand, DNA sequencing analysis suggests that culture and growth conditions do not affect the genomic information carried by EV secretion. This work also suggests that tackling EV molecular alterations secreted into interstitial fluids can provide an alternative, non-invasive approach for investigating 3D tissue behaviors at the molecular precision. This work could serve as a foundation for building precise models employed in mimicking in vivo tissue system with EVs as the molecular indicators or transporters. Such models could be used for investigating tumor biomarkers, drug screening, and understanding tumor progression and metastasis.

bioengineering↗

bioRxiv2018

Identification of the bacterial biosynthetic gene clusters of the oral microbiome illuminates the unexplored social language of bacteria during health and disease

Small molecules are the primary communication media of the microbial world. Recent bioinformatics studies, exploring the biosynthetic gene clusters (BGCs) which produce many small molecules, have highlighted the incredible biochemical potential of the signaling molecules encoded by the human microbiome. Thus far, most research efforts have focused on understanding the social language of the gut microbiome, leaving crucial signaling molecules produced by oral bacteria, and their connection to health versus disease, in need of investigation. In this study, a total of 4,915 BGCs were identified across 461 genomes representing a broad taxonomic diversity of oral bacteria. Sequence similarity networking provided a putative product class for over 100 unclassified novel BGCs. The newly identified BGCs were cross-referenced against 254 metagenomes and metatranscriptomes derived from individuals with either good oral health, dental caries, or periodontitis. This analysis revealed 2,473 BGCs, which were differentially represented across the oral microbiomes associated with health versus disease. Co-abundance network analysis identified numerous inverse correlations between BGCs and specific oral taxa. These correlations were present in health, but greatly reduced in dental caries, which may suggest a defect in colonization resistance. Finally, corroborating mass spectrometry identified several compounds with homology to products of the predicted BGC classes. Together, these findings greatly expand the number of known biosynthetic pathways present in the oral microbiome and provide an atlas for experimental characterization of these abundant, yet poorly understood, molecules and socio-chemical relationships, which impact the development of caries and periodontitis, two of the worlds most common chronic diseases.\n\nIMPORTANCEThe healthy oral microbiome is symbiotic with the human host, importantly providing colonization resistance against potential pathogens. Dental caries and periodontitis are two of the worlds most common and costly chronic infectious diseases, and are caused by a localized dysbiosis of the oral microbiome. Bacterially produced small molecules, often encoded by BGCs, are the primary communication media of bacterial communities, and play a crucial, yet largely unknown, role in the transition from health to dysbiosis. This study provides a comprehensive mapping of the BGC repertoire of the human oral microbiome and identifies major differences in health compared to disease. Furthermore, BGC representation and expression is linked to the abundance of particular oral bacterial taxa in health versus dental caries and periodontitis. Overall, this study provides a significant insight into the chemical communication network of the healthy oral microbiome, and how it devolves in the case of two prominent diseases.

microbiology↗

bioRxiv2018

Nitric oxide synthase-mediated early nitric oxide-burst alleviates drought-induced oxidative damage in ammonium supplied-rice roots

Ammonium (NH4+) can enhance rice drought tolerance in comparison to nitrate (NO3-). The mechanism underpinning this relationship was investigated based on the time-dependent nitric oxide (NO) production and its protective role in oxidative stress of NH4+-/NO3--supplied rice under drought. An early burst of NO was induced by drought 3h after root NH4+ treatment but not after NO3- treatment. Root oxidative damage induced by drought was significantly higher in NO3- than in NH4+-treatment due to its reactive oxygen species accumulation. Inducing NO production by applying NO donor 3h after NO3- treatment alleviated the oxidative damage, while inhibiting the early NO burst increased root oxidative damage in NH4+ treatment. Application of nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) completely suppressed NO synthesis in roots 3h after NH4+ treatment and aggravated drought-induced oxidative damage, indicating the aggravation of oxidative damage might have resulted from changes in NOS-mediated early NO burst. Drought also increased root antioxidant enzymes activities, which were further induced by NO donor but repressed by NO scavenger and NOS inhibitor in NH4+-treated roots. Thus, the NOS-mediated early NO burst plays an important role in alleviating oxidative damage induced by drought by enhancing antioxidant defenses in NH4+-supplied rice roots.\n\nHighlightNOS-mediated early NO burst plays an important role in alleviating oxidative damage induced by water stress, by enhancing the antioxidant defenses in roots supplemented with NH4+

physiology↗

bioRxiv2018

Speciation with gene flow via cycles of isolation and migration: Insights from multiple mangrove taxa

Allopatric speciation requiring an unbroken period of geographical isolation has been the standard model of neo-Darwinism. While doubts have been repeatedly raised, strict allopatry without any gene flow remains a plausible mechanism in most cases. To rigorously reject strict allopatry, genomic sequences superimposed on the geological records of a well-delineated geographical barrier will be necessary. The Strait of Malacca, narrowly connecting the Pacific and Indian Ocean coasts, serves at different times either as a geographical barrier or a conduit of gene flow for coastal/marine species. We surveyed 1,700 plants from 29 populations of five common mangrove species by large scale DNA sequencing and added several whole-genome assemblies. Speciation between the two oceans is driven by cycles of isolation and gene flow due to the fluctuations in sea level leading to the opening/closing of the Strait to ocean currents. Because the time required for speciation in mangroves is longer than the isolation phases, speciation in these mangroves has proceeded through many cycles of mixing-isolation-mixing, or MIM cycles. The MIM mechanism, by relaxing the condition of no gene flow, can promote speciation in many more geographical features than strict allopatry can. Finally, the MIM mechanism of speciation is also efficient, potentially yielding mn (m>1) species after n cycles.\n\nSignificance statementMechanisms of species formation have always been a conundrum. Speciation between populations that are fully geographically isolated, or allopatric speciation, has been the standard solution in the last 50 years. Complete geographical isolation with no possibility of gene flow, however, is often untenable and is inefficient in generating the enormous biodiversity. By studying mangroves on the Indo-Malayan coasts, a global hotspot of coastal biodiversity, we were able to combine genomic data with geographical records on the Indo-Pacific barrier that separates Pacific and Indian Ocean coasts. We discovered a novel mechanism of speciation, that we call mixing-isolation-mixing (MIM) cycles. By permitting intermittent gene flow during speciation, MIM can potentially generate species at an exponential rate, thus combining speciation and biodiversity in a unified framework.

evolutionary biology↗

bioRxiv2018

Generation of a novel growth-enhanced and reduced environmental impact transgenic pig strain

In pig production, insufficient feed digestion causes excessive nutrients such as phosphorus and nitrogen, which are then released to the environment. To address the issue of environmental emissions, we have established transgenic pigs harboring a single-copy quad-cistronic transgene and simultaneously expressing three microbial enzymes, {beta}-glucanase, xylanase, and phytase in the salivary glands. All the transgenic enzymes were successfully expressed, and the digestion of non-starch polysaccharides (NSPs) and phytate in the feedstuff was enhanced. Fecal nitrogen and phosphate outputs were reduced by 23%-46%, and growth rate improved by 23.4% (gilts) and 24.4% (boars) when the pigs were fed on a corn and soybean-based diet and high-NSP diet. The transgenic pigs showed a 11.5%- 14.5% improvement in feed conversion rate compared to the age-matched wild-type littermates. These findings indicate that transgenic pigs are promising resources for improving feed efficiency and reducing nutrient emissions to the environment.

biochemistry↗

bioRxiv2018

Agricultural Pollution Risks Influence Microbial Ecology in Honghu Lake

BackgroundAgricultural activities, such as stock-farming, planting industry, and fish aquaculture, can influence the physicochemistry and biology of freshwater lakes. However, the extent to which these agricultural activities, especially those that result in eutrophication and antibiotic pollution, effect water and sediment-associated microbial ecology, remains unclear.\n\nMethodsWe performed a geospatial analysis of water and sediment associated microbial community structure, as well as physicochemical parameters and antibiotic pollution, across 18 sites in Honghu lake, which range from impacted to less-impacted by agricultural pollution. Furthermore, the co-occurrence network of water and sediment were built and compared accorded to the agricultural activities.\n\nResultsPhysicochemical properties including TN, TP, NO3--N, and NO2--N were correlated with microbial compositional differences in water samples. Likewise, in sediment samples, Sed-OM and Sed-TN correlated with microbial diversity. Oxytetracycline and tetracycline concentration described the majority of the variance in taxonomic and predicted functional diversity between impacted and less-impacted sites in water and sediment samples, respectively. Finally, the structure of microbial co-associations was influenced by the eutrophication and antibiotic pollution.\n\nConclusionThese analyses of the composition and structure of water and sediment microbial communities in anthropologically-impacted lakes are imperative for effective environmental pollution monitoring. Likewise, the exploration of the associations between environmental variables (e.g. physicochemical properties, and antibiotics) and community structure is important in the assessment of lake water quality and its ability to sustain agriculture. These results show agricultural practices can negatively influence not only the physicochemical properties, but also the biodiversity of microbial communities associated with the Honghu lake ecosystem. And these results provide compelling evidence that the microbial community can be used as a sentinel of eutrophication and antibiotics pollution risk associated with agricultural activity; and that proper monitoring of this environment is vital to maintain a sustainable environment in Honghu lake.

microbiology↗

bioRxiv2017

CLAN: the CrossLinked reads ANalysis tool

The crosslinked RNA sequencing technology ligates interacting RNA strands followed by next-generation sequencing. Mapping of the resulting duplex reads allows for functional inference of the corresponding intramolecular/intermolecular RNA-RNA interactions. However, duplex read mapping remains computationally challenging, and the existing best-performing software fails to map a significant portion of the duplex reads. To address this challenge, we develop a novel algorithm for duplex read mapping, called CrossLinked reads ANalysis tool (CLAN). CLAN demonstrates drastically improved sensitivity and high alignment accuracy when applied to real crosslinked RNA sequencing data. CLAN is implemented in GNU C++, and is freely available from http://sourceforge.net/projects/clan-mapping.

bioinformatics↗

bioRxiv2017

Progesterone regulates hypothalamic-pituitary-thyroid axis

Progesterone is a natural steroid hormone excreted by animals and humans, which has been frequently detected in the aquatic ecosystems. The effects of the residual progesterone on fish are unclear. In this study, we aimed to examine the effects of progesterone on the hypothalamic-pituitary-thyroid (HPT) axis by detecting the gene transcriptional expression levels. Zebrafish embryos were treated with different concentrations of progesterone from 12 hours post-fertilization (hpf) to 120 hpf. Total mRNA was extracted and the transcriptional profiles of genes involved in HPT axis were examined using qPCR. The genes related to thyroid hormone metabolism and thyroid hormone synthesis were up-regulated in zebrafish exposed to progesterone. These results indicated that progesterone affected the mRNA expression of genes involved in the HPT axis, which might interrupt the endocrine system in zebrafish. Our data also suggested that zebrafish is a useful tool for evaluating the effects of chemicals on the thyroid endocrine system.

pharmacology and toxicology↗

bioRxiv2017

De novo discovery of structural motifs in RNA 3D structures through clustering

As functional components in three-dimensional conformation of an RNA, the RNA structural motifs provide an easy way to associate the molecular architectures with their biological mechanisms. In the past years, many computational tools have been developed to search motif instances by using the existing knowledge of well-studied families. Recently, with the rapidly increasing number of resolved RNA 3D structures, there is an urgent need to discover novel motifs with the newly presented information. In this work, we classify all the loops in non-redundant RNA 3D structures to detect plausible RNA structural motif families by using a clustering pipeline. Compared with other clustering approaches, our method has two benefits: first, the underlying alignment algorithm is tolerant to the variations in 3D structures; second, sophisticated downstream analysis has been performed to ensure the clusters are valid and easily applied to further research. The final clustering results contain many interesting new variants of known motif families, such as GNAA tetraloop, kink-turn, sarcin-ricin, and T-loop. We have also discovered potential novel functional motifs conserved in ribosomal RNA, sgRNA, SRP RNA, riboswitch, and ribozyme.

bioinformatics↗

bioRxiv2017

Effects of Progesterone on the reproductive physiology in zebrafish

Recent studies have investigated that the synthetic progestins may lead to health issues to the aquatic organisms. Progesterone is a steroidal progestin and has been used as a contraceptive drug, which is detected in the aquatic ecosystem. However, the potential effects of progesterone on the fish reproduction are largely unclear. Here, we tested the effects of progesterone on the fish reproductive and endocrine systems. Adult zebrafish were exposed to progesterone for 10 days at environmental concentrations. The production of eggs was reduced in the exposed fish, and the circulating concentrations of estradiol (E2) and testosterone (T) in female fish or 11-keto testosterone (11-KT) in male fish were significantly diminished. Our results suggested that progesterone may cause adverse health effects on fish by disrupting the endocrine system, and short-term exposure to progesterone could overt affect the fish reproduction.

pharmacology and toxicology↗

bioRxiv2017

Impairment of zebrafish reproduction upon exposure to melengestrol acetate

Synthetic progestins contamination is common in the aquatic ecosystem, which may lead to serious health problem on aquatic animals. Melengestrol acetate (MGA) has been detected in the aquatic environment; however, its potential effects on fish reproduction are largely unclear. Here, we aimed to investigate the endocrine disruption and impact of MGA on zebrafish reproduction. Six-month old reproductive zebrafish were exposed to four nominal concentrations of MGA (1,10, 100 and 200 ng/L) for 15 days. Treatment with MGA reduced the egg production with a significant decrease at 200 ng/L. The circulating concentrations of estradiol and testosterone in female zebrafish or 11-keto testosterone in male zebrafish were significantly diminished compared to the non-exposed control fish. The early embryonic development or hatching rates were unaffected during the MGA exposure. Our results indicated that MGA was a potent endocrine disruptor in fish and the fish reproduction could be impaired even during a short-term exposure to MGA.

developmental biology↗

bioRxiv2016

The origin and evolution of a pandemic lineage of the kiwifruit pathogen Pseudomonas syringae pv. actinidiae

Recurring epidemics of kiwifruit (Actinidia spp.) bleeding canker disease are caused by Pseudomonas syringae pv. actinidiae (Psa), whose emergence coincided with domestication of its host. The most recent pandemic has had a deleterious effect on kiwifruit production worldwide. In order to strengthen understanding of population structure, phylogeography and evolutionary dynamics of Psa, we sampled 746 Pseudomonas isolates from cultivated and wild kiwifruit across six provinces in China, of which 87 were Psa. Of 234 Pseudomonas isolated from wild Actinidia spp. none were identified as Psa. Genome sequencing of fifty isolates and the inclusion of an additional thirty from previous studies show that China is the origin of the recently emerged pandemic lineage. However China harbours only a fraction of global Psa diversity, with greatest diversity found in Korea and Japan. Distinct transmission events were responsible for introduction of the pandemic lineage of Psa into New Zealand, Chile and Europe. Two independent transmission events occurred between China and Korea, and two Japanese isolates from 2014 cluster with New Zealand Psa. Despite high similarity at the level of the core genome and negligible impact of within-lineage recombination, there has been substantial gene gain and loss even within the single clade from which the global pandemic arose.\n\nSIGNIFICANCE STATEMENTBleeding canker disease of kiwifruit caused by Pseudomonas syringae pv. actinidiae (Psa) has come to prominence in the last three decades. Emergence has coincided with domestication of the host plant and provides a rare opportunity to understand ecological and genetic factors affecting the evolutionary origins of Psa. Here, based on genomic analysis of an extensive set of strains sampled from China and augmented by isolates from a global sample, we show, contrary to earlier predictions, that China is not the native home of the pathogen, but is nonetheless the source of the recent global pandemic. Our data identify specific transmission events, substantial genetic diversity and point to non-agricultural plants in either Japan or Korea as home to the source population.

epidemiology↗