Dynamic Changes of the Gut Microbiota and Its Functional Metagenomic Potential during the Development of Non-Small Cell Lung Cancer.

The gut microbiota plays a significant role in tumor pathogenesis by regulating the host metabolism and immune response, and there are few studies focused on tracking changes in the gut microbiota from the onset of lung cancer. Therefore, the aim of our study is combining preclinical and clinical research to thoroughly analyze the signatures of fecal microbiota in lung cancer, which will be useful for early diagnosis and predicting the therapeutic efficacy of lung cancer. The first part of this study analyzed the fecal metagenomic differences between patients with non-small cell lung cancer and healthy subjects, and the second part of this work constructed a murine lung cancer model to monitor changes in mouse fecal metagenomics and T cell immunology during lung cancer progression. We found that the fecal microbiota was altered in both humans and mice with lung cancer, characterized by a significantly reduced microbial diversity and number of beneficial microbes, with increases in potential pathogens. The fecal level of Akkermansia muciniphila and the gut metabolic module of the secondary bile acid metabolism were diminished in both humans and mice with lung cancer compared with healthy subjects. Splenomegaly was observed in the lung cancer mice. Flow cytometer analysis of the splenocytes revealed substantial alterations in the proportions of T cell subsets in the lung cancer mice, characterized by significant increases in CD4+Foxp3+CD25+ T regulatory cells (p < 0.05) while significant decreases in CD3+ T cells (p < 0.001), CD4+ T cells (p < 0.001), and the CD4+/CD8+ ratio (p < 0.01). Vertical and longitudinal analyses of the fecal microbiota of the two mouse groups identified some lung cancer biomarkers (including Acutalibacter timonensis, Lachnospiraceae bacterium NSJ-38 sp014337195, etc.). The fecal microbiota of the lung cancer mice had a reduced metagenomic potential for neurotransmitters (melatonin, γ-aminobutyric acid, and histamine) compared with healthy mice. In summary, this study found that the diversity, structure, and composition of gut microbiota vary between cancer and healthy conditions, ultimately leading to changes in the potential for functional metagenomics.

Most Tibetan weedy barleys originated via recombination between Btr1 and Btr2 in domesticated barley.

Tibetan weedy barleys reside at the edges of qingke (hulless barley) fields in Tibet (Xizang). The spikes of these weedy barleys contain or lack a brittle rachis, with either two- or six-rowed spikes and either hulled or hulless grains at maturity. Although the brittle rachis trait of Tibetan weedy barleys is similar to that of wild barley (Hordeum vulgare ssp. spontaneum Thell.), these plants share genetic similarity with domesticated barley. The origin of Tibetan weedy barleys continues to be debated. Here, we show that most Tibetan weedy barleys originated from cross-pollinated hybridization of domesticated barleys, followed by hybrid self-pollination and recombination between Non-brittle rachis 1 (btr1) and 2 (btr2). We discovered the specific genetic ancestry of these weedy barleys in South Asian accessions. Tibetan weedy barleys exhibit lower genetic diversity than wild and Chinese landraces/cultivars and share a close relationship with qingke, genetically differing from typical eastern and western barley populations. We classified Tibetan weedy barleys into two groups, brittle rachis (BR) and non-brittle rachis (NBR); these traits align with the haplotypes of the btr1 and btr2 genes. Whereas wild barleys carry haplotype combinations of Btr1 and Btr2, each showing lower proportions in a population, the recombinant haplotype BTR2H8+BTR1H24 is predominant in the BR group. Haplotype block analysis based on whole-genome sequencing revealed two recombination breakpoints, which are present in 80.6% and 16.8% of BR accessions according to marker-assisted analysis. Hybridization events between wild and domesticated barley were rarely detected. These findings support the notion that Tibetan weedy barleys originated via recombination between Btr1 and Btr2 in domesticated barley.

Generating homozygous mutant populations of barley microspores by ethyl methanesulfonate treatment.

Induced mutations are important for genetic research and breeding. Mutations induced by physical or chemical mutagenesis are usually heterozygous during the early generations. However, mutations must be fixed prior to phenotyping or field trials, which requires additional rounds of self-pollination. Microspore culture is an effective method to produce double-haploid (DH) plants that are fixed homozygotes. In this study, we conducted ethyl methanesulfonate (EMS)-induced mutagenesis of microspore cultures of barley (Hordeum vulgare) cultivar 'Hua30' and landrace 'HTX'. The EMS concentrations were negatively correlated with the efficiency of callus induction and the frequency of mutant plant regeneration. The two genotypes showed different regeneration efficiencies. The phenotypic variation of the regenerated M1 plants and the presence of genome-wide nucleotide mutations, revealed by whole-genome sequencing, highlight the utility of EMS-induced mutagenesis of isolated microspore cultures for developing DH mutants. Genome-wide analysis of the mutation frequency in the regenerated plants revealed that a considerable proportion of mutations resulted from microspore culture (somaclonal variation) rather than EMS-induced mutagenesis. In addition to producing a population of 1972 homozygous mutant lines that are available for future field trials, this study lays the foundation for optimizing the regeneration efficiency of DH plants and the richness of mutations (mainly by fine-tuning the mutagen dosage).

Gut microbiome-targeted therapies for Alzheimer's disease.

The advent of high-throughput 'omics' technologies has improved our knowledge of gut microbiome in human health and disease, including Alzheimer's disease (AD), a neurodegenerative disorder. Frequent bidirectional communications and mutual regulation exist between the gastrointestinal tract and the central nervous system through the gut-brain axis. A large body of research has reported a close association between the gut microbiota and AD development, and restoring a healthy gut microbiota may curb or even improve AD symptoms and progression. Thus, modulation of the gut microbiota has become a novel paradigm for clinical management of AD, and emerging effort has focused on developing potential novel strategies for preventing and/or treating the disease. In this review, we provide an overview of the connection and causal relationship between gut dysbiosis and AD, the mechanisms of gut microbiota in driving AD progression, and the successes and challenges of implementing available gut microbiome-targeted therapies (including probiotics, prebiotics, synbiotics, postbiotics, and fecal microbiota transplantation) in preventive and/or therapeutic preclinical and clinical intervention studies of AD. Finally, we discuss the future directions in this field.

Quantification of the apical palatal bone index for maxillary incisor immediate implant assessment: A retrospective cross-sectional study.

BACKGROUND: Apical palatal bone is important in immediate implant evaluation. Current consensus gives qualitative suggestions regarding it, limiting its clinical decision-making value. OBJECTIVES: To quantify the apical palatal bone dimension in maxillary incisors and reveal its quantitative correlation with other implant-related hard tissue indices to give practical advice for pre-immediate implant evaluation and design. MATERIAL AND METHODS: A retrospective analysis of immediate implant-related hard tissue indices in maxillary incisors obtained by cone beam computed tomography (CBCT) was conducted. Palatal bone thickness at the apex level (Apical-P) on the sagittal section was selected as a parameter reflecting the apical palatal bone. Its quantitative correlation with other immediate implant-related hard tissue indices was revealed. Clinical advice of pre-immediate implant assessment was given based on the quantitative classification of Apical-P and its other correlated immediate implant-related hard tissue indices. RESULTS: Apical-P positively correlated with cervical palatal bone, whole cervical buccal-palatal bone, sagittal root angle, and basal bone width indices. while negatively correlated with apical buccal bone, cervical buccal bone, and basal bone length indices. Six quantitative categories of Apical-P are proposed. Cases with Apical-P below 4 mm had an insufficient apical bone thickness to accommodate the implant placement, while Apical-P beyond 12 mm should be cautious about the severe implant inclination. Cases with Apical-P of 4-12 mm can generally achieve satisfying immediate implant outcomes via regulating the implant inclination. CONCLUSIONS: Quantification of the apical palatal bone index for maxillary incisor immediate implant assessment can be achieved, providing a quantitative guide for immediate implant placement in the maxillary incisor zone.

Lacticaseibacillus rhamnosus Probio-M9 enhanced the antitumor response to anti-PD-1 therapy by modulating intestinal metabolites.

BACKGROUND: Probiotics have been increasingly proposed for enhancing immune checkpoint blockade (ICB) treatments against cancer. However, its causal relationship with immunotherapeutic efficacy remains unclear, which promoted us to explore if and how probiotic Lacticaseibacillus rhamnosus Probio-M9 manipulates gut microbiome for expected outcomes. METHODS: We evaluated the effects of Probio-M9 on the anti-PD-1 treatment against colorectal cancer in mice via a multi-omics approach. We defined the mechanisms of Probio-M9-mediated antitumor immunity by comprehensive analyses of metagenome and metabolites of commensal gut microbes as well as the immunologic factors and serum metabolome of the host. FINDINGS: The results indicated that Probio-M9 intervention strengthened the anti-PD-1-based tumor inhibition. Both prophylactic and therapeutic administration of Probio-M9 showed conspicuous performance in controlling tumor growth with ICB treatment. The supplement of Probio-M9 modulated enhanced immunotherapy response through promoting beneficial microbes (e.g., Lactobacillus and Bifidobacterium animalis), producing beneficial metabolites including butyric acids in the gut, and accumulating blood-derived α-ketoglutaric acid, N-acetyl-l-glutamic acid and pyridoxine in particular, which promoted the infiltration and activation of cytotoxic T lymphocytes (CTLs) and suppressing the function of regulatory T cells (Tregs) in the tumor microenvironment (TME). Subsequently, we found that enhanced immunotherapeutic response was transmissible by transplanting either post-probiotic-treatment gut microbes or intestinal metabolites to new tumor-bearing mice. INTERPRETATION: This study offered valuable insight into the causal role of Probio-M9 in correcting the defects in gut microbiota that compromised anti-PD-1 therapeutic efficacy, which can be used as an alternative synergetic agent with ICB for clinical cancer treatment. FUNDING: This research was supported by Research Fund for the National Key R&D Program of China (2022YFD2100702), Inner Mongolia Science and Technology Major Projects (2021ZD0014), and China Agriculture Research System of MOF and MARA.

Improving hard metal implant and soft tissue integration by modulating the "inflammatory-fibrous complex" response.

Soft tissue integration is one major difficulty in the wide applications of metal materials in soft tissue-related areas. The inevitable inflammatory response and subsequent fibrous reaction toward the metal implant is one key response for metal implant-soft tissue integration. It is of great importance to modulate this inflammatory-fibrous response, which is mainly mediated by the multidirectional interaction between fibroblasts and macrophages. In this study, macrophages are induced to generate M1 and M2 macrophage immune microenvironments. Their cytokine profiles have been proven to have potentially multi-regulatory effects on fibroblasts. The multi-reparative effects of soft tissue cells (human gingival fibroblasts) cultured on metal material (titanium alloy disks) in M1 and M2 immune microenvironments are then dissected. Fibroblasts in the M1 immune microenvironment tend to aggravate the inflammatory response in a pro-inflammatory positive feedback loop, while M2 immune microenvironment enhances multiple functions of fibroblasts in soft tissue integration, including soft tissue regeneration, cell adhesion on materials, and contraction to immobilize soft tissue. Enlighted by the close interaction between macrophages and fibroblasts, we propose the concept of an "inflammatory-fibrous complex" to disclose possible methods of precisely and effectively modulating inflammatory and fibrous responses, thus advancing the development of metal soft tissue materials.

"Gingival Soft Tissue Integrative" Lithium Disilicate Glass-Ceramics with High Mechanical Properties and Sustained-Release Lithium Ions.

Due to their good mechanical performances and high biocompatibility, all-ceramic materials are widely applied in clinics, especially in orthopedic and dental areas. However, the "hard" property negatively affects its integration with "soft" tissue, which greatly limits its application in soft tissue-related areas. For example, dental implant all-ceramic abutments should be well integrated with the surrounding gingival soft tissue to prevent the invasion of bacteria. Mimicking the gingival soft tissue and dentine integration progress, we applied the modified ion-exchange technology to "activate" the biological capacity of lithium disilicate glass-ceramics, via introducing OH- to weaken the stability of Si-O bonds and release lithium ions to promote multi-reparative functions of gingival fibroblasts. The underlying mechanism was found to be closely related to the activation of mitochondrial activity and oxidative phosphorylation. In addition, during the ion-exchange process, the larger radius sodium ions (Na+) replaced the smaller radius lithium ions (Li+), so that the residual compressive stress was applied to the glass-ceramics surface to counteract the tensile stress, thus improving the mechanical properties. This successful case in simultaneous improvement of mechanical properties and biological activities proves the feasibility of developing "soft tissue integrative" all-ceramic materials with high mechanical properties. It proposes a new strategy to develop advanced bioactive and high strength all-ceramic materials by modified ion-exchange, which can pave the way for the extended applications of such all-ceramic materials in soft tissue-related areas.

A reference-guided TILLING by amplicon-sequencing platform supports forward and reverse genetics in barley.

Barley is a diploid species with a genome smaller than those of other members of the Triticeae tribe, making it an attractive model for genetic studies in Triticeae crops. The recent development of barley genomics has created a need for a high-throughput platform to identify genetically uniform mutants for gene function investigations. In this study, we report an ethyl methanesulfonate (EMS)-mutagenized population consisting of 8525 M3 lines in the barley landrace "Hatiexi" (HTX), which we complement with a high-quality de novo assembly of a reference genome for this genotype. The mutation rate within the population ranged from 1.51 to 4.09 mutations per megabase, depending on the treatment dosage of EMS and the mutation discrimination platform used for genotype analysis. We implemented a three-dimensional DNA pooling strategy combined with multiplexed amplicon sequencing to create a highly efficient and cost-effective TILLING (targeting induced locus lesion in genomes) platform in barley. Mutations were successfully identified from 72 mixed amplicons within a DNA pool containing 64 individual mutants and from 56 mixed amplicons within a pool containing 144 individuals. We discovered abundant allelic mutants for dozens of genes, including the barley Green Revolution contributor gene Brassinosteroid insensitive 1 (BRI1). As a proof of concept, we rapidly determined the causal gene responsible for a chlorotic mutant by following the MutMap strategy, demonstrating the value of this resource to support forward and reverse genetic studies in barley.

Gut Dysbiosis in Pancreatic Diseases: A Causative Factor and a Novel Therapeutic Target.

Pancreatic-related disorders such as pancreatitis, pancreatic cancer, and type 1 diabetes mellitus (T1DM) impose a substantial challenge to human health and wellbeing. Even though our understanding of the initiation and progression of pancreatic diseases has broadened over time, no effective therapeutics is yet available for these disorders. Mounting evidence suggests that gut dysbiosis is closely related to human health and disease, and pancreatic diseases are no exception. Now much effort is under way to explore the correlation and eventually potential causation between the gut microbiome and the course of pancreatic diseases, as well as to develop novel preventive and/or therapeutic strategies of targeted microbiome modulation by probiotics, prebiotics, synbiotics, postbiotics, and fecal microbiota transplantation (FMT) for these multifactorial disorders. Attempts to dissect the intestinal microbial landscape and its metabolic profile might enable deep insight into a holistic picture of these complex conditions. This article aims to review the subtle yet intimate nexus loop between the gut microbiome and pancreatic diseases, with a particular focus on current evidence supporting the feasibility of preventing and controlling pancreatic diseases via microbiome-based therapeutics and therapies.

Adjunctive Probiotic Lactobacillus rhamnosus Probio-M9 Administration Enhances the Effect of Anti-PD-1 Antitumor Therapy via Restoring Antibiotic-Disrupted Gut Microbiota.

Emerging evidence supports that the efficacy of immune checkpoint blockade (ICB) therapy is associated with the host's gut microbiota, as prior antibiotic intake often leads to poor outcome and low responsiveness toward ICB treatment. Therefore, we hypothesized that the efficacy of ICB therapy like anti-programmed cell death protein-1 (PD-1) treatment required an intact host gut microbiota, and it was established that probiotics could enhance the recovery of gut microbiota disruption by external stimuli. Thus, the present study aimed to evaluate the effect of the probiotics, Lactobacillus rhamnosus Probio-M9, on recovering antibiotic-disrupted gut microbiota and its impact on the outcome of ICB therapy in tumor-bearing mice. We first disrupted the mouse microbiota by antibiotics and then remediated the gut microbiota by probiotics or naturally. Tumor transplantation was then performed, followed by anti-PD-1-based antitumor therapy. Changes in the fecal metagenomes and the tumor suppression effect were monitored during different stages of the experiment. Our results showed that Probio-M9 synergized with ICB therapy, significantly improving tumor inhibition compared with groups not receiving the probiotic treatment (P < 0.05 at most time points). The synergistic effect was accompanied by effective restoration of antibiotic-disrupted fecal microbiome that was characterized by a drastically reduced Shannon diversity value and shifted composition of dominating taxa. Moreover, probiotic administration significantly increased the relative abundance of beneficial bacteria (e.g., Bifidobacterium pseudolongum, Parabacteroides distasonis, and some Bacteroides species; 0.0001 < P < 0.05). The gut microbiome changes were accompanied by mild reshaping of the functional metagenomes characterized by enrichment in sugar degradation and vitamin and amino acid synthesis pathways. Collectively, this study supported that probiotic administration could enhance the efficacy and responsiveness of anti-PD-1-based immunotherapy, and Probio-M9 could be a potential candidate of microbe-based synergistic tumor therapeutics. The preclinical data obtained here would support the design of future human clinical trials for further consolidating the current findings and for safety assessment of probiotic adjunctive treatment in ICB therapy.

C23 gene regulates the nucleolin structure and biosynthesis of ribosomes in bovine intraspecific and interspecific somatic cell nuclear transfer embryos.

Somatic cell nuclear transfer (SCNT) can reprogram differentiated somatic cells to produce individual animals, thus having advantages in animal breeding and chromatin reprogramming. Interspecies SCNT (iSCNT) provides extreme cases of reprogramming failure that can be used to understand the basic biological mechanism of genome reprogramming. It is important to understand the possible mechanisms for the failure of zygotic genome activation (ZGA) in iSCNT embryos in order to improve the efficiency of SCNT embryos. In the present study, we compared the development of bovine-bovine (B-B), ovine-ovine (O-O) SCNT, and ovine-bovine (O-B) iSCNT embryos and found that a developmental block existed in the 8-cell stage in O-B iSCNT embryos. RNA sequencing and q-PCR analysis revealed that the large ribosomal subunit genes (RPL) or the small ribosomal subunit genes (RPS) were expressed at lower levels in the O-B iSCNT embryos. The nucleolin (C23) gene that regulates the ribosomal subunit generation was transcribed at a lower level during embryonic development in O-B iSCNT embryos. In addition, the nucleolin exhibited a clear circular-ring structure in B-B 8-cell stage embryos, whereas this was shell-like or dot-like in the O-B embryos. Furthermore, overexpression of C23 could increase the blastocyst rate of both SCNT and iSCNT embryos and partly rectify the ring-like nucleolin structure and the expression of ribosomal subunit related genes were upregulation, while knockdown of C23 increased the shell-like nucleolin-structure in B-B cloned embryos and downregulated the expression of ribosomal subunit related genes. These results implied that abnormal C23 and ribosome subunit gene expression would lead to the developmental block of iSCNT embryos and ZGA failure. Overexpression of the C23 gene could partly improve the blastocyst development and facilitate the nucleolin structure in bovine preimplantation SCNT embryos.

Genome-Wide Characterization of WRKY Transcription Factors Revealed Gene Duplication and Diversification in Populations of Wild to Domesticated Barley.

The WRKY transcription factors (WRKYs) are known for their crucial roles in biotic and abiotic stress responses, and developmental and physiological processes. In barley, early studies revealed their importance, whereas their diversity at the population scale remains hardly estimated. In this study, 98 HsWRKYs and 103 HvWRKYs have been identified from the reference genome of wild and cultivated barley, respectively. The tandem duplication and segmental duplication events from the cultivated barley were observed. By taking advantage of early released exome-captured sequencing datasets in 90 wild barley accessions and 137 landraces, the diversity analysis uncovered synonymous and non-synonymous variants instead of loss-of-function mutations that had occurred at all WRKYs. For majority of WRKYs, the haplotype and nucleotide diversity both decreased in cultivated barley relative to the wild population. Five WRKYs were detected to have undergone selection, among which haplotypes of WRKY9 were enriched, correlating with the geographic collection sites. Collectively, profiting from the state-of-the-art barley genomic resources, this work represented the characterization and diversity of barley WRKY transcription factors, shedding light on future deciphering of their roles in barley domestication and adaptation.

A two-photon excited near-infrared fluorescent probe for imaging peroxynitrite during drug-induced hepatotoxicity and its remediation.

Drug-induced liver injury (DILI) has been a hot issue of public health, owing to its unpredictability and serious harm to public health. Peroxynitrite (ONOO-) is an important biomarker for the assessment and diagnosis of DILI. In this article, based on a kind of rhodamine analogue with a near-infrared (NIR) emission (610 nm-800 nm) and a two-photon absorption cross section (54 GM), a two-photon excited NIR fluorescence probe (NIR-ONOO) for ONOO- was developed. With a high selectivity and a high sensitivity to ONOO-, NIR-ONOO has a linear range for detection of ONOO- from 5.0 × 10-8 to 1.0 × 10-5 M, a good detection limit (15 nM) and a large fluorescence enhancement (340-fold). In addition, NIR-ONOO has been used to monitor ONOO- in cells with satisfactory results. Because of its two-photon excied NIR emission, NIR-ONOO also showed excellent performances for imaging ONOO- including low autofluorescence, stable and persistent fluorescence, and a deep penetration (204 µm). Finally, NIR-ONOO was successfully employed to image ONOO- in inflammatory mouse, drug-induced hepatotoxicity in cells and its remediation. All the results indicated that NIR-ONOO is a powerful chemical tool to image ONOO- and assay drug-induced hepatotoxicity.

Genome-wide diversity analysis of TCP transcription factors revealed cases of selection from wild to cultivated barley.

Plant-specific TEOSINTE BRANCHED 1/CYCLOIDEA/PROLIFERATING CELL FACTORS 1/2 (TCP) transcription factors have known roles in inflorescence architecture. In barley, there are two family members INTERMEDIUM-C (INT-c/HvTB1-1) and COMPOSITUM 1 (COM1/HvTCP24) which are involved in the manipulation of spike architecture, whereas the participation of TCP family genes in selection from wild (Hordeum vulgare subsp. spontaneum, Hs) to cultivated barley (Hordeum vulgare subsp. vulgare, Hv) remains poorly investigated. Here, by conducting a genome-wide survey for TCP-like sequences in publicly-released datasets, 22 HsTCP and 20 HvTCP genes encoded for mature proteins were identified and assigned into two classes (I and II) based on their functional domains and the phylogenetic analysis. Each counterpart of the orthologous gene in wild and cultivated barley usually represented a similarity on the transcriptional profile across the tissues. The diversity analysis of TCPs in 90 wild barley accessions and 137 landraces with geographically-referenced passport information revealed the detectable selection at three loci including INT-c/HvTB1-1, HvPCF2, and HvPCF8. Especially, the HvPCF8 haplotypes in cultivated barley were found correlating with their geographical collection sites. There was no difference observed in either transactivation activity in yeast or subcellular localization in Nicotiana benthamiana among these haplotypes. Nevertheless, the genome-wide diversity analysis of barley TCP genes in wild and cultivated populations provided insight for future functional characterization in plant development such as spike architecture.

Comparative analysis of bovine maternal corpus luteum microRNAs with aberrant and normal developed cloned fetus at late gestation.

BACKGROUND: The development efficiency of cloned cattle is extremely low (< 5%), most of them were aborted at late gestation. Based on our previous studies, some recipient cows with a cloned fetus would present as engorged uterine vessels and enlarged umbilical vessels randomly. Abortion involves both maternal and fetal factors. OBJECTIVE: Our aim was to explore this phenomenon by microRNAs expression profile analysis of maternal corpus luteum (CL), which was related to pregnancy maintenance. METHODS: The present study provided the comparison of maternal CL miRNAs expression of abnormally and normally developed cloned bovine fetus at late gestation (~ 210 days) using RNA-Seq technology. RESULTS: We selected two abnormally pregnant cows (abnormal group, AG) and three normally pregnant cows (normal group, NG) and acquired valid reads of 9317,261-12,327,185 (~ 84.53-91.28%) from five libraries. In total, we identified 981 conserved miRNAs and 223 novel miRNAs. 1052 miRNAs were co-expressed, 124 miRNAs were uniquely expressed in AG, and 93 miRNAs were uniquely expressed in the NG. Compared with NG, 11 were significantly overexpressed, and 22 were downregulated (p < 0.05) at AG among 1052 co-expressed miRNAs. The differentially expressed miRNAs-targeted genes were further analyzed by Gene Ontology and KEGG pathway analysis. Notably, the steroid biosynthesis pathway was a significantly enriched term (p < 0.01), which may affect the secretion of progesterone. CONCLUSION: Our research suggested that abnormal miRNAs expression of bovine maternal CL may affect the pregnant status at late gestation.

Transcriptome-wide analysis of the SCNT bovine abnormal placenta during mid- to late gestation.

The dysfunction of placenta is common in somatic cell nuclear transfer (SCNT) cloned cattle and would cause aberrant fetal development and even abortion, which occurred with highest rate at the mid- to late gestation. However, the mechanism of abnormal placentas was unclear. To analyze the transcriptome-wide characteristics of abnormal placentas in SCNT cloned cattle, the mRNA, lncRNA and miRNA of placental cotyledon tissue at day 180 after gestation were sequenced. A total of 19,055 mRNAs, 30,141 lncRNAs and 684 miRNAs were identified. Compared with control group, 362 mRNAs, 1,272 lncRNAs and nine miRNAs (six known and three novel miRNAs) were differentially expressed (fold change ≥ 2 and P-value < 0.05). The differentially expressed genes were functionally enriched in urea and ions transmembrane transport, which indicated that the maternal-fetal interactions were disturbed in impaired placentas. Furthermore, the competing endogenous RNAs (ceRNAs) networks were identified to illustrate their roles in abnormal placental morphology. The present research would be helpful to discover the mechanism of late gestational abnormality of SCNT cattle by provides important genomic information and insights.

A photostable Si-rhodamine-based near-infrared fluorescent probe for monitoring lysosomal pH during heat stroke.

Heat stroke is a symptom of hyperthermia with a temperature of more than 40 °C, which usually leads to all kinds of physical discomfort and even death. It is necessary to study the mechanism of action of heat stroke on cells or organelles (such as cytotoxicity of heat) and the processes of cells or organelles during heat stroke. Recent studies have shown that there is a certain correlation between heat stroke and lysosome acidity. In order to clarify their relationship, Lyso-NIR-pH, a photostable Si-rhodamine-based near-infrared fluorescent probe, was developed for sensing pH changes in lysosomes during heat stroke in this paper. For Lyso-NIR-pH, a morpholine group is employed as the lysosome-targeting unit and a H+-triggered openable deoxylactam is employed as the response unit to pH. Lyso-NIR-pH can detect pH with a high selectivity and a sensitivity, and its pKa is 4.63. Lyso-NIR-pH also has outstanding imaging performances, such as excellent lysosome-targeting ability, low autofluorescence and photostable fluorescence signal, which are in favor of long-term imaging of pH with accurate fluorescence signals. Moreover, we successfully applied Lyso-NIR-pH to monitor lysosomal pH increases induced by chloroquine and apoptosis in live cells. Finally, we successfully applied Lyso-NIR-pH for monitoring changes of lysosomal pH during heat stroke. These results confirmed that Lyso-NIR-pH is a powerful tool to monitor pH change in lysosomes and study its possible effects.

A mitochondria-targetable two-photon fluorescent probe with a far-red to near-infrared emission for sensing hypochlorite in biosystems.

Hypochlorite (ClO-), one of reactive oxygen species (ROS), is closely related with many physiological and pathological processes. Especially as one of cellular reactive oxygen species in mitochondria, ClO- can induce mitochondrial permeability, which leads to apoptosis. Thus, developing an effective method which is able to sense ClO- in mitochondria is important. Although fluorescent probe has become a powerful tool for imaging ClO- in mitochondria, most of them suffered from phototoxicity to biosamples, autofluorescence, and photobleaching phenomenon due to their short-wavelength excitations and emissions. Based on advantages of two-photon fluorescent probe and far-red to NIR fluorescent probe, a mitochondria-targetable two-photon fluorescent probe with a turn-on signal in far-red to NIR region, Mito-TP-ClO, was developed for ClO- in this paper. Mito-TP-ClO is consisted of a triphenylphosphonium cations as a mitochondria-targetable unit and a structure of dibenzoylhydrazine as a response unit to ClO-. Mito-TP-ClO exhibited a high sensitivity and a high selectivity to ClO-, with a linear range from 6.0 × 10-8 to 1.0 × 10-5 M and a detection limit of 2.5 × 10-8 M. Due to its large two-photon cross section (267 GM) and far-red to NIR emission, Mito-TP-ClO exhibits excellent performances including low autofluorescence, photostable fluorescence signal, and deep tissue penetration (230 µM). Moreover, Mito-TP-ClO was successfully used to detect endogenous ClO- in bacteria-infected cells and inflammatory mouse model, which confirmed that Mito-TP-ClO is a powerful tool to monitor ClO- in mitochondria and study on effects of hypochlorite on mitochondria.

CircRNA expression profile of bovine placentas in late gestation with aberrant SCNT fetus.

BACKGROUNDS: One of the limitations of somatic cell nuclear transfer (SCNT) strategy to generate genetically modified offspring is the low birth rate. Placental dysfunction is one of the causes of abortion. Circular RNA (circRNA) is noncoding RNA which functions as microRNA (miRNA) sponges in biological processes. METHODS: Two aberrant pregnant placenta (aberrant group, AG) and three normal pregnant placenta (normal group, NG) during late gestation (180-210 days) with bovine SCNT fetus were collected for high-throughput sequencing and analyzed. The host genes of differentially expressed (DE) circRNAs were predicted. And the microRNAs (miRNAs) which could interact with DE circRNAs were analyzed. Then, the expressional level of partial DE circRNAs and corresponding host genes was verified through qRT-PCR. At last, the function of host genes was analyzed through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). RESULTS: Altogether 123 differentially expressed circRNAs between two groups were identified, which were found related to 60 host genes and 32 miRNAs. The top 10 upregulated circRNAs were bta_circ_0012985, bta_circ_0013071, bta_circ_0013074, bta_circ_0016024, bta_circ_0013068, bta_circ_0008816, bta_circ_0012982, bta_circ_0013072, bta_circ_0019285, and bta_circ_0013067. The top 10 downregulated circRNAs were bta_circ_0024234, bta_circ_0017528, bta_circ_0008077, bta_circ_0003222, bta_circ_0007500, bta_circ_0020328, bta_circ_0011001, bta_circ_0016364, bta_circ_0008839, and bta_circ_0016049. The qRT-PCR results showed consistent trend with sequencing analysis result, while host genes had no statistic difference. The GO and KEGG analyses of the host genes suggested that abnormal circRNA expression may play multiple roles in placental structure and dysfunction. CONCLUSION: The abnormal circRNA expression may be one of reasons of placental dysfunction, leads to abortion of bovine SCNT fetus.