Single-cell RNA-sequencing of zebrafish hair cells reveals novel genes potentially involved in hearing loss.

Hair cells play key roles in hearing and balance, and hair cell loss would result in hearing loss or vestibular dysfunction. Cellular and molecular research in hair cell biology provides us a better understanding of hearing and deafness. Zebrafish, owing to their hair cell-enriched organs, have been widely applied in hair cell-related research worldwide. Similar to mammals, zebrafish have inner ear hair cells. In addition, they also have lateral line neuromast hair cells. These different types of hair cells vary in morphology and function. However, systematic analysis of their molecular characteristics remains lacking. In this study, we analyzed the GFP+ cells isolated from Tg(Brn3c:mGFP) larvae with GFP expression in all hair cells using single-cell RNA-sequencing (scRNA-seq). Three subtypes of hair cells, namely macula hair cell (MHC), crista hair cell (CHC), and neuromast hair cell (NHC), were characterized and validated by whole-mount in situ hybridization analysis of marker genes. The hair cell scRNA-seq data revealed hair cell-specific genes, including hearing loss genes that have been identified in humans and novel genes potentially involved in hair cell formation and function. Two novel genes were discovered to specifically function in NHCs and MHCs, corresponding to their specific expression in NHCs and MHCs. This study allows us to understand the specific genes in hair cell subpopulations of zebrafish, which will shed light on the genetics of both human vestibular and cochlear hair cell function.

MSCs-Derived Exosomes Attenuate Acute Brain Injury and Inhibit Microglial Inflammation by Reversing CysLT2R-ERK1/2 Mediated Microglia M1 Polarization.

Inflammatory responses play a major role in the pathophysiology of cerebral ischemia. Mesenchymal stem cell-derived exosomes (MSC-exos) have important anti-inflammatory effects on the treatment of organ injury. This study aimed to determine the anti-inflammatory effect and furtherly investigate the potential mechanism of MSC-exos on acute cerebral ischemia. MSC-exos were isolated by ultracentrifugation, characterized by transmission electron microscopy and FACS. Rats subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) surgery were administered MSC-exos through the tail vein. In vitro, microglia exposed to oxygen- and glucose-deprivation (OGD) and leukotrienes were used to study the protective mechanism of exosomes against ischemia/reperfusion-induced inflammation. The intake of exosomes into microglia was visualized through immunofluorescence staining. The results showed that MSC-exos treatment significantly improved motor, learning and memory abilities of MCAO/R rats 7 days later. The production of pro-inflammatory factors decreased, while the anti-inflammatory cytokines and neurotrophic factors increased both in the cortex and hippocampus of ischemic hemisphere as well as in the culture supernatant of microglia treated with OGD and NMLTC4. MSC-exos treatment also significantly inhibited M1 microglia polarization and increased M2 microglia cells. Furthermore, western blot analysis demonstrated that CysLT2R expression and ERK1/2 phosphorylation were downregulated both in vivo and in vitro. Thus, MSC-exos attenuated brain injury and inhibited microglial inflammation by reversing CysLT2R-ERK1/2 mediated microglia M1 polarization.

Hydrogen-assisted post-growth substitution of tellurium into molybdenum disulfide monolayers with tunable compositions.

Herein we report the successful doping of tellurium (Te) into molybdenum disulfide (MoS2) monolayers to form MoS2x Te2(1-x) alloy with variable compositions via a hydrogen-assisted post-growth chemical vapor deposition process. It is confirmed that H2 plays an indispensable role in the Te substitution into as-grown MoS2 monolayers. Atomic-resolution transmission electron microscopy allows us to determine the lattice sites and the concentration of introduced Te atoms. At a relatively low concentration, tellurium is only substituted in the sulfur sublattice to form monolayer MoS2(1-x)Te2x alloy, while with increasing Te concentration (up to ∼27.6% achieved in this study), local regions with enriched tellurium, large structural distortions, and obvious sulfur deficiency are observed. Statistical analysis of the Te distribution indicates the random substitution. Density functional theory calculations are used to investigate the stability of the alloy structures and their electronic properties. Comparison with experimental results indicate that the samples are unstrained and the Te atoms are predominantly substituted in the top S sublattice. Importantly, such ultimately thin Janus structure of MoS2(1-x)Te2x exhibits properties that are distinct from their constituents. We believe our results will inspire further exploration of the versatile properties of asymmetric 2D TMD alloys.

Distinct molecular basis for endothelial differentiation: gene expression profiles of human mesenchymal stem cells versus umbilical vein endothelial cells.

The capacity for endothelial differentiation has been described in mesenchymal stem cells (MSC) from human bone marrow. To identify genes associated with the endothelial differentiation potential of this cell-type, and search for the optimal regulatory factors, the expression profile of MSC was compared with cDNA from primary human umbilical vein endothelial cells as controls, using cDNA chips with 4096 genes. The data were corroborated by quantitative PCR, Western blotting, and immunocytochemistry. Among the 3948 effective genes, ∼84% (3321) were co-expressed in both cell-types, and 627 were differentially expressed more than twofold in MSC versus EC. MSC highly expressed numerous stem-cell-like genes. Early development genes of endothelial cells, though not up-regulated, had a high expression in MSC, such as EDF1, MDG1, and EDG2. In contrast, mature endothelial growth and signal pathway genes, like VEGF, CXCR4, and CTNNB1, were down-regulated in MSC. In conclusion, human MSC have a distinct molecular basis for endothelial differentiation.

bFGF promotes adipocyte differentiation in human mesenchymal stem cells derived from embryonic stem cells.

In this work we describe the establishment of mesenchymal stem cells (MSCs) derived from embryonic stem cells (ESCs) and the role of bFGF in adipocyte differentiation. The totipotency of ESCs and MSCs was assessed by immunofluorescence staining and RT-PCR of totipotency factors. MSCs were successfully used to induce osteoblasts, chondrocytes and adipocytes. MSCs that differentiated into adipocytes were stimulated with and without bFGF. The OD/DNA (optical density/content of total DNA) and expression levels of the specific adipocyte genes PPARγ2 (peroxisome proliferator activated receptor γ2) and C/EBPs were higher in bFGF cells. Embryonic bodies had a higher adipocyte level compared with cells cultured in plates. These findings indicate that bFGF promotes adipocyte differentiation. MSCs may be useful cells for seeding in tissue engineering and have enormous therapeutic potential for adipose tissue engineering.

ftr82 is necessary for hair cell morphogenesis and auditory function during zebrafish development.

Damages of sensory hair cells (HCs) are mainly responsible for sensorineural hearing loss, while the pathological mechanism remains not fully understood due to the many potential deafness genes unidentified. ftr82, a member of the largely TRIMs family in fish, has been found specifically expressed in the otic vesicle while its function is still unclear. Here, we investigate the roles of ftr82 in HC development and hearing function utilizing the zebrafish model. The results of in situ hybridization illustrate that ftr82 is always restricted to localize in otic vesicles at different stages. The defects of HCs are observed both in ftr82 morphants and mutants, including significantly decreased crista HCs, shortened cilia as well as remarkably reduced functional HCs in neuromasts, which could be successfully rescued by co-injection of exogenous ftr82 mRNA. The behavior assay of startle response indicates that larvae lacking of ftr82 exhibits lower sensitivity to external sound stimuli. Further research reveals that the loss of HCs is mainly caused by cell apoptosis mediated by caspase-3 activation. Our study demonstrates that ftr82 is a crucial hearing-related gene that regulates the HC morphogenesis and auditory function performing, which provides new insight into the rapid identification of the deafness gene.

Potential Effect of DIMBOA (2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one) on Alleviating the Autotoxic Coumarin Stress in Alfalfa (Medicago sativa) Seedlings.

The allelopathic theory has garnered considerable attention in the field of agricultural production for its efficient plant protection, rapid crop yield increase, and scientific establishment of the crop rotation system. To study the effects of the main maize allelochemical DIMBOA (2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one) on the growth and development of alfalfa under autotoxic coumarin stress, we treated alfalfa seedlings with DIMBOA under coumarin stress and non-stress conditions in this study. Results show that 0.0342 mM coumarin significantly inhibited alfalfa seed germination percentage(Gp), germination potential(GP), radicle length, germ length, seeding height, and simple viability index (SVI), with decreases of 37.29%, 59.91%, 7.60%, 30.90%, 13.27%, and 45.70%, respectively. An amount of 0.6 mM DIMBOA could promote alfalfa seed Gp, GP, radicle length, germ length, seeding height, dry fresh ratio, and SVI, with increases of 12.38%, 23.91%, 48.69%, 48.65%, 48.68%, 295.12%, and 67.17%, respectively. However, the addition of DIMBOA under conditions of coumarin stress could effectively alleviate coumarin effects on alfalfa seedlings. Coumarin + DIMBOA treatment for 24 h mainly decreased reactive oxygen species (ROSs) and malondialdehyde (MDA) as well as soluble protein and soluble sugar, increasing some antioxidant enzyme activities and antioxidant content to alleviate the oxidative damage of alfalfa caused by coumarin stress. Administration of treatment for 72 h significantly promoted the morphological development of alfalfa seeding roots. Administration of treatment for 96 h significantly enhanced the photosynthetic capacity of alfalfa seedlings. The results of principal component analysis demonstrated that chlorophyll b(Chl b)and net photosynthetic rate(Pn) were the key indicators for coumarin + DIMBOA treatment to promote photosynthesis in alfalfa seedlings. Additionally, root length, mean root diameter, and root volume were the key indicators of root growth and development. Coumarin + DIMBOA treatment primarily increased catalase(CAT), peroxidase (POD), and ascorbate peroxidase (APX) activity and antioxidants(ASA) while reducing MDA and superoxide anion radical(O2•-). This study strongly suggested that DIMBOA can effectively improve the tolerance of alfalfa seedlings to coumarin stress through a combination of effects on root morphology, photosynthesis, and physiological indicators.

Drought tolerance in alfalfa (Medicago sativa L.) varieties is associated with enhanced antioxidative protection and declined lipid peroxidation.

Drought stress is considered the most adverse factor restricting plant survival, growth, and productivity. The identification of the key adaptive mechanisms to drought stress is essential to enhance the drought resistance of plants. In this study, differential responses of three alfalfa varieties to drought, including Medicago sativa L. cv. Longzhong (drought-tolerant), Longdong (moderate drought-tolerant), and Gannong No. 3 (drought-sensitive), were comparatively studied at morphological, physio-biochemical, and transcriptional levels after a 12-day period of drought stress simulated by -1.2 MPa polyethylene glycol (PEG-6000). The results showed that prolonged drought stress dramatically decreased growth and photosynthetic capacity of three alfalfa varieties while it increased the accumulation of malondialdehyde (MDA), reactive oxygen species (ROS), osmolytes and antioxidants including reduced ascorbate and glutathione, ascorbate peroxidase (APX) activities, and gene expression of antioxidative enzymes (MsCu/Zn-SOD, MsFeSOD, MtPOD, MsGPX, MsAPX, MsMDAR, MtDHAR, and MsGR). Nine days of treatment and some key traits, including the maximum quantum yield of photosystem II (Fv/Fm), the levels of MDA, O2-, and H2O2, the redox states of ascorbate and glutathione, APX activity, and the transcript levels of MsFeSOD, MsGR, and MsMDAR, might contribute to differentiating the drought stress tolerance in alfalfa. Overall, drought-tolerant Longzhong showed the highest water retention, photosynthetic performance, and osmoregulation capacity, the lowest lipid peroxidation, and the highest antioxidant enzyme activities and gene expression, which were mainly involved in the ascorbate-glutathione cycle to maintain the balance between the generation and scavenging of intracellular ROS. These findings highlight that enhanced antioxidative protection and declined lipid peroxidation play an important role in alfalfa tolerance against drought.

Ligament regeneration using a knitted silk scaffold combined with collagen matrix.

This study was aimed to develop a new practical ligament scaffold by synergistic incorporation of silk fibers, a knitted structure, and a collagen matrix. The efficacy for ligament tissue engineering was investigated in vitro and in animal models. Cells cultured on a collagen substrate expressed ligament matrix genes at higher levels than those on a silk substrate. The silk scaffold elicited little inflammatory reaction and degraded slowly after subcutaneous implantation in a mouse model. In the rabbit MCL defect model, MCLs treated with a silk+collagen scaffold deposited more collagen, had better mechanical properties, and showed more native microstructure with larger diameter collagen fibrils and stronger scaffold-ligament interface healing than untreated MCLs and those treated with silk scaffolds. These results demonstrated that the knitted silk+collagen sponge scaffold improves structural and functional ligament repair by regulating ligament matrix gene expression and collagen fibril assembly. The findings are the first to highlight the important roles of biomaterials in ligament regeneration biology. Also, the concept of an "internal-space-preservation" scaffold is proposed for the tissue repair under physical loading.

A male reproduction-related Kazal-type peptidase inhibitor gene in the prawn, Macrobrachium rosenbergii: molecular characterization and expression patterns.

Peptidase inhibitors in the male reproductive tract are well known in mammals, in which they play roles in protecting the tract epithelium against proteolytic damage or in regulating the fertilization process. By screening the subtracted cDNA clones enriched for male reproductive tract-specific transcripts, one clone encoding a putative protein that showed significant similarity to Kazal-type peptidase inhibitor (KPI) was obtained. This is the first report of an invertebrate in which a male reproductive tract-specific KPI gene has been identified and characterized. The gene contains a 405-bp open reading frame (ORF), a 72 bp 5' untranslated region (UTR), and a 259 bp 3' UTR. The conceptually translated protein consisted of a 21-amino-acid signal peptide and a 113-amino-acid mature polypeptide with two Kazal-type domains (named after the discoverer). Significant levels of the mRNA were observed only in the male reproductive tract, while mRNA expression was not detected in any other tissues tested. The transcription of the gene remained constant during maturation, although not in the postlarval stage. In situ hybridization demonstrated the presence of the mRNA in the secretory epithelial cells of vas deferens and terminal ampullae.

Naturally occurring antisense RNA of allatostatin gene in the prawn, Macrobrachium rosenbergii.

Allatostatins are important regulatory neuropeptides which are widely distributed in invertebrates and execute their functions through either neural or humoral routes. However, the regulatory mechanism of the gene expression is unclear. In this paper, we report a naturally occurring antisense transcript, named as asMacro-AST A, of the crustacean FGLamide allatostatin gene (Macro-AST A) from the prawn, Macrobrachium rosenbergii. The asMacro-AST A contains an 843-bp sequence fully complementary to the 3' end of the Macro-AST A. To our knowledge, this is the first report of a natural antisense transcript in crustacean and the first endogenous antisense transcript of all identified allatostatin genes. Northern blotting analysis demonstrated that the gene was expressed in the thoracic ganglia where the sense gene was also expressed. Furthermore, we have detected a RNA-RNA duplex between the sense-antisense complementary region by using RNase protection analysis and RT-PCR. These results suggest that the antisense gene may play a role in the regulation of Macro-AST A gene expression.

Atomic process of oxidative etching in monolayer molybdenum disulfide.

The microscopic process of oxidative etching of two-dimensional molybdenum disulfide (2D MoS2) at an atomic scale is investigated using a correlative transmission electron microscope (TEM)-etching study. MoS2 flakes on graphene TEM grids are precisely tracked and characterized by TEM before and after the oxidative etching. This allows us to determine the structural change with an atomic resolution on the edges of the domains, of well-oriented triangular pits and along the grain boundaries. We observe that the etching mostly starts from the open edges, grain boundaries and pre-existing atomic defects. A zigzag Mo edge is assigned as the dominant termination of the triangular pits, and profound terraces and grooves are observed on the etched edges. Based on the statistical TEM analysis, we reveal possible routes for the kinetics of the oxidative etching in 2D MoS2, which should also be applicable for other 2D transition metal dichalcogenide materials like MoSe2 and WS2.

Combination therapy with dendritic cell-based vaccine and anti-CD69 antibody enhances antitumor efficacy in renal cell carcinoma-bearing mice.

BACKGROUND/AIM: Dendritic cell-based vaccine therapy for renal cell carcinoma is effective but requires improvement. Here we explored whether combination therapy with dendritic cell-based vaccine and anti-CD69 antibody can enhance antitumor efficacy in renal cell carcinoma-bearing mice. MATERIALS AND METHODS: Balb/c mice were challenged subcutaneously with murine renal cell carcinoma (Renca) cells. On day 3 after tumor cell inoculation, tumor-bearing mice either were left untreated or were treated with Renca tumor lysate-pulsed dendritic cells (i.e. dendritic cell-based vaccine), anti-CD69 antibody, or a combination of Renca tumor lysate-pulsed dendritic cells with anti-CD69 antibody. The mice were sacrificed on day 28. Tumor volume was measured for analysis of antitumor efficacy. Spleens were excised to evaluate antitumor immunological responses by measuring the proliferation and activation of T cells, which have the capacity to recognize and destroy tumor cells. RESULTS: Combination treatment with Renca tumor lysate-pulsed dendritic cells and anti-CD69 antibody resulted in significant decreases in tumor volume and significant increases in T-cell proliferation and activity, compared with no treatment or either treatment alone. CONCLUSION: These findings indicate that anti-CD69 antibody can potentiate antitumor efficacy of dendritic cell-based vaccine. The augmented therapeutic efficacy conferred by the combination therapy may be associated with increased T-cell proliferation and activity.

Identification of a novel male reproduction-related gene and its regulated expression patterns in the prawn, Macrobrachium rosenbergii.

To identify male-specific genes that could be involved in male development, we screened a subtracted male reproductive tract library and isolated a novel gene named Mar-Mrr (M. rosenbergii male reproduction-related gene). The Mar-Mrr cDNA sequence consists of 683 nucleotides with a 333 nucleotide open reading frame, encoding putative 110 amino acids (11.7473 kDa) precursor protein and a signal peptide consisting of 24 amino acids. Significant developmentally dependent accumulation of the mRNA was observed in the male reproductive tract, specifically in epithelial cells of vas deferens and terminal ampullae.

Molecular cloning and characterization of FGLamide allatostatin gene from the prawn, Macrobrachium rosenbergii.

Allatostatins are important regulatory neuropeptides that inhibit juvenile hormone (JH) biosynthesis by the corpora allata (CA) in insects. However, to date, the structure and expression of the gene encoding allatostatins have not been reported in any species other than insects. In this study, we used a combination of a semi-nested polymerase chain reaction (PCR) and screening of a central nervous system cDNA library of Macrobrachium rosenbergii to isolate and sequence a cDNA clone (2885 bp) encoding a 701 amino acid FGLamide allatostatin precursor polypeptide. This is the first reported allatostatin gene in crustacean. The deduced precursor was conceptually split into at least 35 FGLamide allatostatins at dibasic cleavage sites (Lys and Lys/Arg), far more than reported for any other known FGLamide allatostatin precursors from insects (13-14 allatostatins). Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that the gene was expressed in the brain, gut, thoracic and abdominal ganglia, but not in the heart, muscle, ovary, gill, or hepatopancreas. Furthermore, developmentally-dependent expression of the gene was observed in the brain and thoracic ganglia of the prawn by using semi-quantitative RT-PCR analysis.

Aspirin can elicit the recurrence of gastric ulcer induced with acetic acid in rats.

UNLABELLED: This study was supported by grants of New Ideas Capability for Backbone Teachers in Universities of Heilongjiang and of Scientific Research foundation in Qiqihar Medical College. BACKGROUND/AIMS: Ulcer recurrence and poor healing may be critically important to the development of serious gastrointestinal complications in patients with long-term non-steroid anti-inflammatory drugs (NSAIDs). The present study is to investigate the effects of aspirin on ulcer recurrence and healing quality and to explore the mechanism. METHODS: Gastric ulcers were induced with acetic acid in rats; aspirin was administrated by gavage from day 25 to day 54 after ulcer induction. The gastric juice volume, pH, gastric mucus, gastric mucosal blood flow (GMBF) and prostaglandin E(2) (PGE(2)) were measured. The mRNA transcription of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) were analyzed with RT-PCR and protein expression with Western blot. RESULTS: The gastric juice volume was significantly increased in aspirin group compared with those of fasting or saline control groups (P<0.01); while the pH, mucus, GMBF and PGE(2) were significantly decreased in aspirin treated rats compared with those of other two groups (P<0.01). COX-2, evaluated with mRNA and protein expression, was significantly augmented in aspirin group compared with others. The quality of ulcer healing (QOUH) in Aspirin group was poorer than that of fasting or saline control groups. CONCLUSIONS: Aspirin enhance the recurrence of gastric ulcer. The inhibition of cycloxygenase, mucus secretion and mucosal blood flow may be involved. Aspirin also impair the quality of ulcer healing.