Subsequent maternal sleep deprivation aggravates cognitive impairment by modulating hippocampal neuroinflammatory responses and synaptic function in maternal isoflurane-exposed offspring mice.

INTRODUCTION: Pregnant women may need to undergo non-obstetric surgery under general anesthesia owing to medical needs, and pregnant women frequently experience sleep disturbances during late gestation. Preclinical studies demonstrated that maternal isoflurane exposure (MISO) or maternal sleep deprivation (MSD) contributed to cognitive impairments in offspring. Research studies in mice have revealed that SD can aggravate isoflurane-induced cognitive deficits. However, it remains unclear whether MSD aggravates MISO-induced cognitive deficits in offspring. The purpose of this research was to explore the combined effects of MSD and MISO on offspring cognitive function and the role of neuroinflammation and synaptic function in the process of MSD + MISO. METHODS: Pregnant mice were exposed to 1.4% isoflurane by inhalation for 4 h on gestational day (GD) 14. Dams were then subjected to SD for 6 h (12:00-18:00 h) during GD15-21. At 3 months of age, the offspring mice were subjected to the Morris water maze test to assess cognitive function. Then the levels of inflammatory and anti-inflammatory markers and synaptic function-related proteins were assessed using molecular biology methods. RESULTS: The results of this study demonstrated that MISO led to cognitive dysfunction, an effect that was aggravated by MSD. In addition, MSD exacerbated the maternal isoflurane inhalation, leading to an enhancement in the expression levels of interleukin (IL)-1ß, IL-6, and tumor necrosis factor-alpha and a reduction in the hippocampal levels of IL-10, synaptophysin, post-synaptic density-95, growth-associated protein-43, and brain-derived neurotrophic factor. CONCLUSION: Our findings revealed that MSD aggravated the cognitive deficits induced by MISO in male offspring mice, and these results were associated with neuroinflammation and alternations in synaptic function.

In vivo imaging reveals a synchronized correlation among neurotransmitter dynamics during propofol and sevoflurane anesthesia.

General anesthesia is widely applied in clinical practice. However, the precise mechanism of loss of consciousness induced by general anesthetics remains unknown. Here, we measured the dynamics of five neurotransmitters, including γ-aminobutyric acid, glutamate, norepinephrine, acetylcholine, and dopamine, in the medial prefrontal cortex and primary visual cortex of C57BL/6 mice through in vivo fiber photometry and genetically encoded neurotransmitter sensors under anesthesia to reveal the mechanism of general anesthesia from a neurotransmitter perspective. Results revealed that the concentrations of γ-aminobutyric acid, glutamate, norepinephrine, and acetylcholine increased in the cortex during propofol-induced loss of consciousness. Dopamine levels did not change following the hypnotic dose of propofol but increased significantly following surgical doses of propofol anesthesia. Notably, the concentrations of the five neurotransmitters generally decreased during sevoflurane-induced loss of consciousness. Furthermore, the neurotransmitter dynamic networks were not synchronized in the non-anesthesia groups but were highly synchronized in the anesthetic groups. These findings suggest that neurotransmitter dynamic network synchronization may cause anesthetic-induced loss of consciousness.

Genome-wide identification, phylogeny, expression and eyestalk neuroendocrine regulation of vitellogenin gene family in the freshwater giant prawn Macrobrachium rosenbergii.

Vitellogenin (Vg) is the precursor of vitellin, which is an important female-specific protein stored in oocytes as the major nutrient and energy sources for embryogenesis in oviparous animals. In this study, we performed comprehensive genome-wide analysis of Vg gene family in the prawn Macrobrachium rosenbergii, and eight Vg genes designated as MrVg1a, MrVg1b and MrVg2-7 were identified. MrVg1a clusters with the previously described MrVg1b near the end of chromosome 46 and MrVg2 is on the chromosome 42 while MrVg3-7 cluster on the chromosome 23. All the putative MrVg proteins are characterized by the presence of three conserved functional domains: LPD-N, DUF1943 and vWD. Phylogenetic analysis revealed that MrVg1a shares 93% identity with MrVg1b and groups together into a branch while MrVg2-7 group into another branch, suggesting that MrVg1a, 1b and MrVg2-7 might diversify from a common ancestral gene. All the corresponding MrVg transcripts especially for MrVg1 exhibit high expression in the female hepatopancreas at late vitellogensis stage but extremely low in the ovaries except MrVg5, indicating that hepatopancreas is the major site of MrVgs synthesis. In the male, interestingly, MrVg5 and MrVg6 are also highly expressed in the testis, suggesting their potential involvement in testicular development. Bilateral ablation of eyestalk significantly upregulate all the MrVgs mRNA in the female hepatopancreas and the MrVg1 in ovary, but have no effect on the expression of MrVg2-7 in the ovary, demonstrating that eyestalk hormones could promote the ovarian development mostly by inducing the synthesis of MrVgs in the hepatopancreas but rarely in the ovary. Our results provide new insights into the prawn MrVgs family and improve our understanding of the potential role for each member of the family in the gonadal development of M. rosenbergii.

Identification of GnRH-like peptide and its potential signaling pathway involved in the oocyte meiotic maturation in the Chinese mitten crab, Eriocheir sinensis.

Gonadotropin-releasing hormone (GnRH) plays a pivotal role in reproductive regulation in vertebrates. However, GnRH was rarely isolated and its function remains poorly characterized in invertebrates. The existence of GnRH in ecdysozoa has been controversial for a long. Here, we isolated and identified two GnRH-like peptides from brain tissues in Eriocheir sinensis. Immunolocalization showed that the presence of EsGnRH-like peptide in brain, ovary and hepatopancreas. Synthetic EsGnRH-like peptides can induce germinal vesicle breakdown (GVBD) of oocyte. Similar to vertebrates, ovarian transcriptomic analysis revealed a GnRH signaling pathway in the crab, in which most genes exhibited dramatically high expression at GVBD. RNAi knockdown of EsGnRHR suppressed the expression of most genes in the pathway. Co-transfection of the expression plasmid for EsGnRHR with reporter plasmid bearing CRE-luc or SRE-luc response element into 293T cells showed that EsGnRHR transduces its signal via cAMP and Ca2+ signaling transduction pathways. In vitro incubation of the crab oocyte with EsGnRH-like peptide confirmed the cAMP-PKA cascade and Ca2+ mobilization signaling cascade but lack of a PKC cascade. Our data present the first direct evidence of the existence of GnRH-like peptides in the crab and demonstrated its conserved role in the oocyte meiotic maturation as a primitive neurohormone.

Propofol exerts anti-anhedonia effects via inhibiting the dopamine transporter.

Lasker's award-winning drug propofol is widely used in general anesthesia. The recreational use of propofol is reported to produce a well-rested feeling and euphoric state; yet, the neural mechanisms underlying such pleasant effects remain unelucidated. Here, we report that propofol actively and directly binds to the dopamine transporter (DAT), but not the serotonin transporter (SERT), which contributes to the rapid relief of anhedonia. Then, we predict the binding mode of propofol by molecular docking and mutation of critical binding residues on the DAT. Fiber photometry recording on awake freely moving mice and [18F] FP-CIT-PET scanning further establishes that propofol administration evokes rapid and lasting dopamine accumulation in nucleus accumbens (NAc). The enhanced dopaminergic tone drives biased activation of dopamine-receptor-1-expressing medium spiny neurons (D1-MSNs) in NAc and reverses anhedonia in chronically stressed animals. Collectively, these findings suggest the therapeutic potential of propofol against anhedonia, which warrants future clinical investigations.

Potential role for the germ cell-specific Rad21 in early meiosis of oocyte and spermatocyte in the Chinese mitten crab Eriocheir sinensis.

Rad21/Rec8 family proteins are vital for sister chromatid segregation in mitosis and homologous recombination in meiosis, but no molecular data are available in crustacean species. In this study, a germ cell-specific Rad21 named EsRad21 was identified in the crab Eriocheir sinensis. EsRad21 mRNA has an open reading frame of 2310 base pairs (bp) encoding a 769 amino acids (aa) protein. RT-PCR showed that EsRad21 mRNA was particularly expressed in testis and ovary. The RT-qPCR results further revealed that the EsRad21 mRNA exhibited similar expression pattern in gonads at various developmental stages. EsRad21 mRNA expression level was the highest in testis at early spermatogenesis stage and ovaries at previtellogenesis stage, thereafter decreased significantly at middle spermatogenesis and vitellogenesis, and finally reach the lowest level at late spermatogenesis and vitellogenesis. In situ hybridization (ISH) analysis showed that EsRad21 mRNA was exclusively expressed in germline cells, but not in gonadal somatic cells. Notably, hybridized signal was detected on chromosomes of metaphase spermatocytes. EsRad21 is thus an underlying helpful indicator of the early phases of germ cell development. RNAi knockdown of EsRad21 downregulated the expression of other meiosis-related genes like Smc5-Smc6 and SPO11 and resulted in high mortality of individuals after 24 h post injection of EsRad21 dsRNA. Taken together, our results showed a potential role for EsRad21 in early meiosis of oocytes and spermatocytes in E. sinensis. This is the first report on the molecular characterization of the Rad21 transcript in a crustacean species.

Comprehensive Transcriptome Analysis of Gonadal and Somatic Tissues for Identification of Sex-Related Genes in the Largemouth Bass Micropterus salmoides.

Largemouth bass (Micropterus salmoides) is an economically important fish. It can spawn many times during a breeding season, and there are no obvious morphological characteristics to distinguish male and female juvenile fish. So far, little is known about the genes regulating their sexual development in this species. Here, we performed RNA sequencing (RNA-Seq) analysis of the testis, ovary, and somatic tissue to identify sex-related genes in the largemouth bass. A total of 51,672 unigenes were obtained via the transcriptome analysis, and 5900 differential expression genes (DEGs), including 3028 up-regulated and 2872 down-regulated DEGs, were obtained in the somatic tissue, testis, and ovary. DEGs were retrieved by making comparisons: somatic tissue vs testis (1733-up and 1382-down), testis vs ovary (841-up and 807-down), and ovary vs somatic tissue (454-up and 683-down). Finally, functional annotation identified 22 key sex-related DEGs, including 13 testis-biased DEGs (dmrt1, cyp11b1, sox9, spata4, spata22, spata17, fshr, fem-1a, wt1, daz1, amh, vasa, and piwi1) and 9 ovary-biased DEGs (foxl2, gdf9, zp3, sox3, cyp19a, bmp15, fem-1b, fig. la, and piwi2). This result was further confirmed by the tissue expression detection via RT-PCR and RT-qPCR. Protein-protein interacting (PPI) network analysis revealed that the testis-specific dmrt1 interacts directly with the testis-biased DEGs (cyp11b1 and spata4) and the ovary-biased DEGs (foxl2, gdf9, zp3, sox3, cyp19a, and bmp15), suggesting that the dmrt1 as a sex-determining gene can play a dual role through inducing the testis-biased DEGs and inhibiting the ovary-biased DEGs during the testicular development. Our present results provide useful molecular data for a better understanding of sexual development in the largemouth bass.

Identification and characterization of a new germline-specific marker vasa gene and its promoter in the giant freshwater prawn Macrobrachium rosenbergii.

Vasa gene encodes a protein member of DEAD-box superfamily of ATP-dependent RNA helicases, which plays a key role in germline development in metazoans. In present study, we identified a new germline-specific marker Mrvasa in the prawn Macrobrachium rosenbergii, whose genomic DNA sequence consists of 14 exons and 13 introns. A 2516 bp of full-length Mrvasa cDNA encodes a protein of 603 amino acids. It contains nine conserved motifs, a zinc-finger motif, and RGG repeats. RT-PCR indicated that Mrvasa mRNA was specifically expressed in gonads. QPCR analysis further revealed that the expression of Mrvasa mRNA is much higher in testis than in ovary. In testis, the relative expression level of Mrvasa mRNA in late developing stage is significantly higher than that in early-middle developing stage. During ovarian development, no significant difference in expression was found. In situ hybridization demonstrated that Mrvasa mRNA was localized in germline cells including spermatogonia, spermatocytes, and spermatozoa in testes, and previtellogenic and vitellogenic oocytes in ovary. We then isolated the Mrvasa promoter and determined the transcription core region of this promoter. This is the first report on identification of vasa core promoter in crustaceans. Our results will provide a useful germline-specific marker Mrvasa for tracing germline cell formation and development in M. rosenbergii.

Germ plasm and the origin of the primordial germ cells in the oriental river prawn Macrobrachium nipponense.

Germ plasm is a special cytoplasmic component containing special RNAs and proteins, and is located in specific regions of oocytes and embryos. Only the blastomeres inheriting the germ plasm can develop into primordial germ cells (PGCs). By using Vasa mRNA as a germline marker, we previously demonstrated that germline specification followed the preformation mode in the prawn Macrobrachium nipponense. In this study, we raised the Vasa antibody to identify germ plasm in the oocyte and trace the origin and migration of PGCs. In previtellogenic oocytes, Vasa protein was detected in the perinuclear region, in which electron-dense granules associated with numerous mitochondria were mostly visualized under a transmission electron microscope. In mature oocytes, immunosignal was localized to a large granule under the plasma membrane. During early embryogenesis, the granule was inherited by a single blastomere from 1-cell to 16-cell stages, and thereafter was segregated into two daughter blastomeres at the 32-cell stage. In gastrula, the Vasa-positive cells were large with typical PGC characteristics, containing a big round nucleus and a prominent nucleolus. The immunosignal was localized to the perinuclear region again. In the zoea stage, the Vasa-positive cells migrated toward the genital ridge and clustered in the dorsomedial region close to the yolk portion. Accordingly, we concluded that the prawn PGCs could be specified from the 16-cell stage by inheriting the germplasm. To our knowledge, this is the first report on the identification of the prawn germ plasm and PGCs. The continuous expression of Vasa protein throughout oogenesis and embryogenesis suggests that Vasa protein could be an important factor in germ plasm that functions in early germ cell specification.

A trade-off between antifouling and the electrochemical stabilities of PEDOTs.

Strong nonspecific protein/cell adhesion on conducting polymer (CP)-based bioelectronic devices can cause an increase in the impedance or the malfunction of the devices. Incorporating oligo(ethylene glycol) or zwitterionic functionalities with CPs has demonstrated superior performance in the reduction of nonspecific adhesion. However, there is no report on the evaluation of the antifouling stability of oligo(ethylene glycol) and zwitterion-functionalized CPs under electrical stimulation as a simulation of the real situation of device operation. Moreover, there is a lack of understanding of the correlation between the molecular structure of antifouling CPs and the antifouling and electrochemical stabilities of the CP-based electrodes. To address the aforementioned issue, we fabricated a platform with antifouling poly(3,4-ethylenedioxythiophene) (PEDOT) featuring tri(ethylene glycol), tetra(ethylene glycol), sulfobetaine, or phosphorylcholine (PEDOT-PC) to evaluate the stability of the antifouling/electrochemical properties of antifouling PEDOTs before and after electrical stimulation. The results reveal that the PEDOT-PC electrode not only exhibits good electrochemical stability, low impedance, and small voltage excursion, but also shows excellent resistance toward proteins and HAPI microglial cells, as a cell model of inflammation, after the electrical stimulation. The stable antifouling/electrochemical properties of zwitterionic PEDOT-PC may aid its diverse applications in bioelectronic devices in the future.

Identification and characterization of sex-biased and differentially expressed miRNAs in gonadal developments of the Chinese mitten crab, Eriocheir sinensis.

MicroRNA (miRNA) is a posttranscriptional downregulator that plays a vital role in a wide variety of biological processes. In this study, we constructed five ovarian and testicular small RNA libraries using two somatic libraries as reference controls for the identification of sex-biased miRNAs and gonadal differentially expressed miRNAs (DEMs) of the Chinese mitten crab, Eriocheir sinensis. A total of 535 known and 243 novel miRNAs were identified, including 312 sex-biased miRNAs and 402 gonadal DEMs. KEGG pathway analysis showed that DEM target genes were statistically enriched in MAPK, Wnt, and GnRH signaling pathway, and so on. A number of the sex-biased miRNAs target genes associated with sex determination/differentiation, such as IAG, Dsx, Dmrt1, and Fem1, while others target the genes related to gonadal development, such as P450s, Wnt, Ef1, and Tra-2c. Dual-luciferase reporter assay in vitro further confirmed that miR-34 and let-7b can downregulate IAG expression, miR-9-5p, let-7d, let-7b, and miR-8915 can downregulate Dsx. Taken together, these data strongly suggest a potential role for the sex-biased miRNAs in sex determination/differentiation and gonadal development in the crab.

Evidences for Red Pigment Concentrating Hormone (RPCH) and Beta-Pigment Dispersing Hormone (ß-PDH) Inducing Oocyte Meiotic Maturation in the Chinese Mitten Crab, Eriocheir sinensis.

Red pigment concentrating hormone (RPCH) and pigment dispersing hormone (PDH) are crustacean neuropeptides involved in broad physiological processes including body color changes, circadian rhythm, and ovarian growth. In this study, the full-length cDNA of RPCH and PDH were identified from the brain of the Chinese mitten crab Eriocheir sinensis. The deduced RPCH and PDH mature peptides shared identical sequence to the adipokinetic hormone/RPCH peptides family and the ß-PDH isoforms and were designated as Es-RPCH and Es-ß-PDH, respectively. Es-RPCH and Es-ß-PDH transcripts were distributed in the brain and eyestalks. The positive signals of Es-RPCH and Es-ß-PDH were localized in the neuronal clusters 6, 8, 9, 10, and 17 of the brain as revealed by in situ hybridization. The expression level of Es-RPCH and Es-ß-PDH mRNA in nervous tissues were all significantly increased at vitellogenic stage, and then decreased at the final meiotic maturation stage. The administrated with synthesized Es-RPCH peptide results in germinal vesicles shift toward the plasma membrane in vitellogenic oocyte, and significant decrease of the gonad-somatic index (GSI) and mean oocyte diameter as well as the expression of vitellogenin mRNA at 30 days post injection in vivo. Similar results were also found when injection of the Es-ß-PDH peptide. In vitro culture demonstrated that Es-RPCH and Es-ß-PDH induced germinal vesicle breakdown of the late vitellogenic oocytes. Comparative ovarian transcriptome analysis indicated that some reproduction/meiosis-related genes such as cdc2 kinase, cyclin B, 5-HT-R and retinoid-X receptor were significantly upregulated in response to Es-RPCH and Es-ß-PDH treatments. Taken together, these results provided the evidence for the inductive effect of Es-RPCH and Es-ß-PDH on the oocyte meiotic maturation in E. sinensis.

Identification of a novel germ cell marker MnTdrd from the oriental river prawn Macrobrachium nipponense.

Germ cell-specific genes play an important role in establishing the reproductive system in sexual organisms and have been used as valuable markers for studying gametogenesis and sex differentiation. Previously, we isolated a vasa transcript as a germ cell marker to trace the origin and migration of germ cells in the oriental river prawn Macrobrachium nipponense. Here, we identified a new germ cell-specific marker MnTdrd RNA and assessed its temporal and spatial expression during oogenesis and embryogenesis. MnTdrd transcripts were expressed in high abundance in unfertilized eggs and embryos at cleavage stage and then dropped significantly during late embryogenesis, suggesting that MnTdrd mRNA is maternally inherited. In situ hybridization of ovarian tissue showed that MnTdrd mRNA was initially present in the cytoplasm of previtellogenic oocyte and localized to the perinuclear region as the accumulation of yolk in vitellogenic oocyte. Whole-mount in situ hybridization of embryos showed that MnTdrd-positive signals were only localized in one blastomere until 16-cell stage. In the blastula, there were approximately 16 MnTdrd-positive blastomeres. During embryonized-zoea stage, the MnTdrd-positive cells aggregated as a cluster and migrated to the genital rudiment which would develop into primordial germ cells (PGCs). The localized expression pattern of MnTdrd transcripts resembled that of the previously identified germ cell marker vasa, supporting the preformation mode of germ cell specification. Therefore, we concluded that MnTdrd, together with vasa, is a component of the germ plasm and might have critical roles in germ cell formation and differentiation in the prawn. Thus, MnTdrd can be used as a novel germ cell marker to trace the origin and migration of germ cells.

Prenatal ultrasonic diagnosis of umbilical cord ulcer: analysis of three cases and literature review / 中华围产医学杂志

Objective:To investigate the features, diagnostic value and clinical significance of prenatal ultrasound for umbilical cord ulcer (UCU).Methods:Ultrasonographic characteristics of three fetuses with UCU diagnosed at Guangzhou Women and Children's Medical Center from January 2014 to June 2020 were reviewed. Relevant articles published from January 1, 1990, to June 1, 2020, were retrieved from Wanfang, CNKI and PubMed databases and analyzed. Likelihood ratio, adjusted Chi-square test and Fisher's exact test were used to analyze the association between the pathological results and fetal outcomes. Results:(1) Among the three fetuses with UCU, two were complicated by upper gastrointestinal obstruction and one by intrauterine infection. Two of them survived and one died after birth. Prenatal ultrasound examination of the umbilical cord and amniotic cavity showed one case was normal, while the other two showing features such as flocculent hypoechoic sign around the umbilical cord, cloudy hyperechoic sign ejecting from the umbilical cord lesion in dynamic observation, exposed umbilical vessels and ribbon-like echo around the umbilical cord. (2) No reported case of UCU had been found in any publications in China. A total of 39 reported cases of UCU complicated by duodenal or jejunal obstruction were retrieved from English literature, among which intrauterine fetal death, neonatal death and live births accounted for 30.8% (12/39), 28.2% (11/39) and 41.0% (16/39), respectively. Six cases with intrauterine abnormalities detected by prenatal ultrasound survived after emergent cesarean section, showing a positive detection rate of 15.4% (6/39), of which three obtained a snapshot of umbilical cord bleeding and three were characterized by lamellar and linear echoes around the amniotic cavity or umbilical cord. Hemorrhagic amniotic fluid was observed in 66.7% (26/39) of the fetuses and decreased heart rate occurred in 35.9%(14/39). The site, number and pathological grade of UCU were not related to the pregnancy outcomes (all P>0.05). Conclusions:Prenatal ultrasound for detecting abnormalities in umbilical cord and amniotic cavity is of a certain value in the diagnosis of UCU. Lamellar and linear echoes around the umbilical cord may be the ultrasonographic characteristics of UCU. The real-time detection of UCU bleeding by prenatal ultrasound can provide direct evidence for timely clinical treatment.

Transcriptome analysis of the growth performance of hybrid mandarin fish after food conversion.

During recent years, China has become a hotspot for the domestication of mandarin fish, and this is of great commercial value. Although the food preference of domesticated mandarin fish has been studied, little is known about genes regulating their growth. We raised hybrid mandarin fish on artificial feed for 3 months, the results showed that the survival rate of hybrid mandarin fish was 60.00%. Their total length and body weight were 18.34 ±0.43 cm and 100.44 ±4.87 g. The absolute length and weight gain rates were 0.14 cm/d and 1.08 g/d, respectively. Finally, RNA sequencing (RNA-Seq) was performed to identify potential genes and pathways activated in response to growth performance. The transcriptome analysis generated 68, 197 transcripts and 45,871 unigenes. Among them, 1025 genes were up-regulated and 593 genes were down-regulated between the fast- and slow-growth fish. Finally, we obtained 32 differentially expressed genes, which were mainly related to fatty acid biosynthesis (e.g. FASN and ACACB), collecting duct acid secretion (e.g. ATP6E and KCC4), cell cycle (e.g. CDC20 and CCNB), and the insulin-like growth factor (IGF) system (IGFBP1). These pathways might be related to the growth of hybrid mandarin fish. In addition, more potential single nucleotide polymorphisms (SNPs) were detected in the fast-growth fish than in the slow-growth fish. The results suggest that the interaction of metabolism and abundant alleles might determine the growth of hybrid mandarin fish after food conversion.

Cyclin B protein undergoes increased expression and nuclear relocation during oocyte meiotic maturation of the freshwater prawn Macrobrachium rosenbergii and the Chinese mitten crab Eriocheir sinensis.

Cyclin B functions as a regulatory protein through association with its catalytic partner Cdc2 kinase forming M-phase promoting factor (MPF), which plays a central role in the meiotic maturation of oocyte. To gain insight into the molecular events, we here cloned a cyclin B cDNA from the ovary of the prawn Macrobrachium rosenbergii and compared its spatial-temporal expression patterns during oocyte maturation with those of crab Eriocheir sinensis. The prawn cyclin B cDNA encodes a 398 amino acid protein with predicted molecular weight of 45.16 kDa. Immunodetection of cyclin B protein by Western blot showed that a target band of approximately 53 kDa protein in the prawn ovaries at both late vitellogenesis (lVt) and germinal vesicle breakdown (GVBD) stages, whereas a 41 kDa band was present in the crab ovaries. Cyclin B protein expression changes indicating that the newly synthesis of cyclin B proteins could be required for GVBD in both prawn and crab. Immunohistochemical analysis revealed that both the prawn and crab cyclin B proteins, were localized in the ooplasm of previtellogenic oocytes, then relocated into germinal vesicle at vitellogenesis stage and localized on meiotic spindle at M phase. These similar behaviors suggested that the prawn and the crab cyclin B proteins associated with Cdc2 kinase have conserved roles in inducing GVBD and regulating the formation of meiotic spindle. The similar expression patterns of the cyclin B proteins during oocyte maturation implicated that the molecular mechanisms for MPF activation could be identical between the prawn and the crab.

Molecular characterization of ovary-specific gene Mrfem-1 and siRNA-mediated regulation on targeting Mrfem-1 in the giant freshwater prawn, Macrobrachium rosenbergii.

Characterized by ankyrin repeat motifs, the feminization-1 (fem-1) gene plays an essential role in sex determination/differentiation in Caenorhabditis elegans. However, there are only a few reports on fem-1 in crustaceans. In this study, a fem-1 gene (Mrfem-1) was first isolated from the giant freshwater prawn Macrobrachium rosenbergii. The full-length cDNA of Mrfem-1 was 2607 bp long, containing an open reading frame encoding 615 amino acids, and presenting eight ankyrin repeats. The full-length cDNA has been submitted to GenBank with the accession no. MT160093. According to the RT-PCR results, Mrfem-1 was exclusively expressed in the ovary. The expression level of Mrfem-1 had increased with ovarian maturation and reached the highest peak at vitellogenic stage. In situ hybridization results showed that positive signals were concentrated in the cytoplasm of previtellogenic stage, and scattered in the cytoplasm and follicular cells at vitellogenic stage, suggesting that Mrfem-1 might be associated with ovarian maturation. Moreover, two effective siRNAs targeting Mrfem-1 were found and their effectiveness verified in vitro. These results on Mrfem-1 will help us to better understand the fem family and provide a new resource for subsequent investigations of siRNA-mediated regulation on ovarian development in M. rosenbergii.

Dexmedetomidine inhibits the invasion, migration, and inflammation of rheumatoid arthritis fibroblast-like synoviocytes by reducing the expression of NLRC5.

Rheumatoid arthritis (RA) is a chronic, autoimmune disease characterized by inflammatory synovitis, but its pathogenesis remains unclear. NLRC5 is a newly discovered member of the NLR family that is effective in regulating autoimmunity, inflammatory responses, and cell death processes. Dexmedetomidine (DEX) has been reported to have a variety of pharmacological effects, including anti-inflammatory and analgesic effects. However, the role of DEX in RA has not been explored. In adjuvant-induced arthritis (AA) rat models, DEX (10 µg/kg and 20 µg/kg) reduced the pathological score, the arthritis score, paw swelling volume, and the serum levels of IL-1ß, IL-6, IL-17A, and TNF-α. Moreover, by using Western blot and real-time quantitative PCR (RT-qPCR), it was demonstrated that DEX can inhibit the expression of IL-1ß, IL-6, MMP-3, MMP-9 and P-P65 in the synovial tissue of AA rats. In human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs), DEX (250 nM and 500 nM) was found to inhibit the expression of IL-1ß, IL-6, MMP-3, MMP-9, and P-P65 following stimulation with TNF-α. Moreover, DEX can inhibit the invasion and migration of RA-FLSs stimulated by TNF-α. Finally, the expression of NLRC5 in RA-FLSs and AA rat models was also reduced by DEX. After silencing NLRC5 in RA-FLSs, the expression of IL-1ß, IL-6, MMP-3, MMP-9, and P-P65, as well as the invasion and migration of cells, were significantly reduced. These results indicate that DEX inhibits the invasion, migration, and inflammation of RA-FLSs by reducing the expression of NLRC5 and inhibiting the NF-κB activation.

Tunable Protein/Cell Binding and Interaction with Neurite Outgrowth of Low-Impedance Zwitterionic PEDOTs.

Zwitterionic poly(3,4-ethylenedioxythiophene) (PEDOT) is an effective electronic material for bioelectronics because it exhibits efficient electrical trade-off and diminishes immune response. To promote the use of zwitterionic PEDOTs in bioelectronic devices, especially for cell alignment control and close electrocoupling, features such as tunable interaction of PEDOTs with proteins/cells and spatially modulating cell behavior are required. However, there is a lack of reliable methods to assemble zwitterionic EDOTs with other functionalized EDOT materials, having different polarities and oxidation potentials, to prepare PEDOTs with the aforementioned surface properties. In this study, we have developed a surfactant-assisted electropolymerization to assemble phosphorylcholine (PC)-functionalized EDOT with other functionalized EDOTs. By adjusting compositions, the interaction of PEDOT copolymers with proteins/cells can be finely tuned; the composition adjustment has an ignorable influence on the impedance of the copolymers. We also demonstrate that the cell-repulsive force generated from PC can spatially guide the neurite outgrowth to form a neuron network at single-cell resolution and greatly enhance the neurite outgrowth by 179%, which is significantly more distinctive than the reported topography effect. We expect that the derived tunable protein/cell interaction and the PC-induced repulsive guidance for the neurite outgrowth can make low-impedance zwitterionic PEDOTs more useful in bioelectronics.

Alternative splice variants and differential relative abundance patterns of vasa mRNAs during gonadal development in the Chinese mitten crab Eriocheir sinensis.

Gonadal development usually involves alternative splicing of sex-related genes. Vasa, a highly conserved ATP-dependent RNA helicase present mainly in germ cells, has an important function in gonadal development. As an important sex-related gene, recent evidence indicates that different splice variants of vasa exist in many species. In this study, there was identification of two types of vasa splice variants in the Chinese mitten crab Eriocheir sinensis, termed Esvasa-l and Esvasa-s, respectively. Furthermore, splice variants of Esvasa-s were sub-divided into Esvasa-s1, Esvasa-s2, Esvasa-s3, Esvasa-s4, and Esvasa-s5, based on differing numbers of TGG repeats. Results from genomic structure analyses indicated that these forms are alternatively spliced transcripts from a single vasa gene. Results from tissue distribution assessments indicate the vasa splice variants were exclusively expressed in the gonads of male and female adult crabs. In situ hybridization results indicate Esvasa mRNA was mainly present in the cytoplasm of previtellogenic oocytes. As oocyte size increased, relative abundance of Esvasa mRNA decreased and became distributed near the cellular membrane. The Esvasa mRNA was not detectable in mature oocytes. In testis, Esvasa mRNA was detected in spermatids and spermatozoa, but not in spermatogonia and spermatocytes. Notably, results from qPCR analysis of Esvasa-l and Esvasa-s indicate there are different relative proportions during gametogenesis, implying that splice variants of the Esvasa gene may have different biological functions during crab gonadal development.