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The eyestalk of crustaceans, such as Macrobrachium nipponense, contains many neurosecretory hormones affecting the process of reproduction, molting, metabolism of glucose, and other functions. In this study, important metabolic pathways and candidate genes involved in male sexual development were selected from M. nipponense. The methodology involved performing long-read and next generation transcriptome sequencing of genes from the androgenic gland after eyestalk ablation. qPCR analysis revealed that the mRNA expression of Mn-IAG was significantly increased after ablation of both the single-side (SS) and double-side (DS) eyestalk, compared with the control group (CG). The long-read transcriptome generated 49,840 non-redundant transcripts. A total of 1319, 2092 and 4351 differentially expressed genes (DEGs) were identified between CG versus SS, SS versus DS and CG versus DS, respectively. These data indicated that ablation of the double-sided eyestalk played stronger regulatory roles than the single-side ablation on male sexual development in M. nipponense. This was consistent with the qPCR analysis. Cell Cycle, Cellular Senescence, Oxidative Phosphorylation, Glycolysis/Gluconeogenesis and Steroid Hormone Biosynthesis were the primary enriched metabolic pathways in all three comparisons, and the important genes from these metabolic pathways were also selected. qPCR permitted secondary confirmation of ten DEGs identified through RNA-seq. RNAi-mediated silencing analyses of Hydroxysteroid dehydrogenase like 1 (HSDL1) revealed that HSDL1 has a positive regulatory effect on testes development. This study provides valuable insight into male sexual development in M. nipponense, including metabolic pathways and genes, paving the way for advanced studies on male sexual development in this species and in other crustaceans.

SummaryIn higher plants, fertilization induces many structural and physiological changes in the fertilized egg that reflect the transition from the haploid female gamete to the diploid zygote – the first cell of the sporophyte. After fusion of the egg nucleus with the sperm nucleus, many molecular changes occur in the zygote during the process of zygote activation during embryogenesis. The zygote originates from the egg, from which some pre-stored translation initiation factors transfer into the zygote and function during zygote activation. This indicates that the control of zygote activation is pre-set in the egg. After the egg and sperm nuclei fuse, gene expression is activated in the zygote, and paternal and maternal gene expression patterns are displayed. This highlights the diversity of zygotic genome activation in higher plants. In addition to new gene expression in the zygote, some genes show quantitative changes in expression. The asymmetrical division of the zygote produces an apical cell and a basal cell that have different destinies during plant reconstruction; these destinies are determined in the zygote. This review describes significant advances in research on the mechanisms controlling zygote activation in higher plants.

Liquid–liquid phase separation (LLPS) plays important roles in forming cellular membraneless organelles. However, how host factors regulate LLPS of viral proteins during negative-sense RNA (NSR) virus infections is largely unknown. Here, we used Barley yellow striate mosaic virus (BYSMV) as a model to demonstrate regulation of host casein kinase 1 in phase separation and infection of NSR viruses. We first found that the BYSMV phosphoprotein (P) formed spherical granules with liquid properties and recruited viral nucleotide (N) and polymerase (L) proteins in vivo. Moreover, the P-formed granules were tethered to the ER/actin network for trafficking and fusion. BYSMV P alone formed droplets and incorporated the N protein and genomic RNA in vitro. Interestingly, phase separation of BYSMV P was inhibited by host casein kinase 1 (CK1)-dependent phosphorylation of an intrinsically disordered P protein region. Genetic assays demonstrated that the unphosphorylated mutant of BYSMV P exhibited condensed phase, which promoted virus replication through concentrating the N, L proteins, and genome RNA into viroplasms. Whereas, the phosphorylation-mimic mutant existed in diffuse phase state leading to enhanced virus transcription. Collectively, our results demonstrate that host CK1 modulates phase separation of viral P protein and virus infection.

Calcium dynamics in the developing pollen of Chinese fir (Cunninghamia lanceolata) were investigated using an antimonate calcium precipitation method. After microspores were released from the tetra...

Plant virus-induced diseases cause significant losses to agricultural crop production worldwide. Reverse genetics systems of plant viruses allow gene manipulation on viral genomes, which greatly facilitates studies of viral pathogenesis and interactions with host organisms. In addition, viral infectious cDNA clones have been modified as versatile recombinant vectors for virus-mediated protein overexpression, virus-induced gene silencing, and gene editing. Since genome RNAs of plant positive-strand RNA viruses are directly translatable, recovery of these viruses has been achieved more than three decades ago by simply expressing viral genome RNA or viral genome-derived in vitro synthesized transcripts in planta. In contrast, genomes of plant negative-strand RNA (NSR) viruses are complementary to their mRNAs and cannot be translated directly. Therefore, rescue of infectious plant NSR viruses from cDNA clones strictly requires the core replication proteins together with their genome RNAs which can assemble into nucleocapsid (NC) complexes as minimal infectious units. However, it is a major challenge to deliver multiple essential components in single cells and to assemble the NC complexes in vivo. Major breakthroughs in reverse genetics systems of plant non-segmented and segmented NSR viruses were just achieved in recent 5 years through various strategies, such as agroinfiltration, minireplicon systems, insect transmission and airbrush inoculation assays. In this review, we summarized critical steps toward developing reverse genetics systems for recovery of several plant NSR viruses in plants and insects. We also highlighted important applications of these reverse genetics of NSR viruses in viral gene function analyses, investigation of virus-insect-plant interactions, and genomic studies of insect vectors and host plants.

Isolated sperm cells and egg cells provide useful materials for the study of fertilization in higher plants, under controlled conditions, in the absence of somatic tissue. Viable egg cells from Bauhinia blakeana were isolated using enzymatic digestion and mechanical dissection. Ovules were digested with enzymes for 40–50 min and then transferred to a solution without enzymes for dissection. The three cells of the egg apparatus (i.e., the egg cell and two synergids) were released from a cut ovule by pushing the micropylar region of the ovule with a dissecting needle. Typically, 7–10 egg cells were isolated within 1 h. B. blakeana pollen is bicellular and contains only a vegetative and generative cell at anthesis. Sperm cells were isolated using an in vivo–in vitro method. The isolation of B. blakeana egg and sperm cells potentially provides a basis for in vitro fertilization of this woody dicotyledonous plant and materials for molecular biological investigations of fertilization mechanisms.

Depression affects both women and men, but women are 2 times more susceptible to the incidence of depression. Although a number of studies report sex differences in stress responses, it remains unclear which animal models of depression can better mimic the sex difference in human depression. The majority of stress models used male rodents whereas fewer studies included females. The aims of this study were to determine which rat stress models mimic the sex difference in depression and to identify sex-specific risk factors for depression model-induced depression-like behaviors. Here, we compared subchronic variable stress (SCVS) and chronic unpredictable mild stress (CUMS) models to evaluate the susceptibility versus resilient phenotypes in male and female rats. SCVS induced depression-like behaviors in female rats only. The CUMS paradigm was more likely to induce depression-like behaviors in male rats. Furthermore, to explore the underlying mechanisms, we used quantitative reverse transcriptase polymerase chain reaction to examine and compare the messenger RNA (mRNA) levels of various transcripts previously shown to be involved in psychiatric disorders in RNA-sequencing/microarray studies including serotonin receptor-7, early growth response-2, histone deacetylase-2, roundabout guidance receptor-2 (Robo2), serum/glucocorticoid regulated kinase-2, orthodenticle homeobox-2, parathyroid hormone-2 receptor, and neuronal PAS domain protein-4 in the hippocampus after exposure of rats to SCVS and CUMS. Our results showed that SCVS significantly altered the mRNA levels of neuronal PAS domain protein-4, orthodenticle homeobox-2, Robo2, parathyroid hormone-2 receptor, and serum/glucocorticoid regulated kinase-2 in the female hippocampus only, and histone deacetylase-2 in only the male hippocampus. CUMS significantly changed the mRNA levels of one transcript (Robo2) in the female hippocampus only when compared with SCVS. Overall, this study shows that SCVS can be used to study sex differences in depression-like behaviors in rats. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

SummaryThe isolation of male and female gametes is an effective method to study the fertilization mechanisms of higher plants. An osmotic shock method was used to rupture pollen grains of Allium tuberosum Roxb and release the pollen contents, including generative cells, which were mass collected. The pollinated styles were cut following 3 h of in vivo growth, and cultured in medium for 6–8 h, during which time pollen tubes grew out of the cut end of the style. After pollen tubes were transferred into a solution containing 6% mannitol, tubes burst and released pairs of sperm cells. Ovules of A. tuberosum were incubated in an enzyme solution for 30 min, and then dissected to remove the integuments. Following transfer to a dissecting solution free of enzymes, each nucellus was cut in the middle, and squeezed gently on the micropylar end, resulting in the liberation of the egg, zygote and proembryo from ovules at selected stages. These cells can be used to explore fertilization and embryonic development using molecular biological methods for each cell type and development stage.

A number of studies have revealed that there is an increasing incidence of early-onset colorectal cancer (CRC) in young adults (before the age of 50 years) and a progressive decline in CRC among older patients, after the age of 50 years (late-onset CRC). However, the etiology of early-onset CRC is not fully understood. The aim of the present study was to identify key genes associated with the development of early-onset CRC through weighted gene co-expression network analysis (WGCNA). The GSE39582 dataset was downloaded from the Gene Expression Omnibus database, and the data profiles of tissues from patients diagnosed before the age of 50 years were selected. The top 10,000 genes with the highest variability were used to construct the WGCNA. Hub genes were identified from the modules associated with clinical traits using gene significance >0.2 and module membership >0.8 as the cut-off criteria. Gene Ontology and pathway analyses were subsequently performed on the hub genes and a protein-protein interaction network (PPI) was constructed. The diagnostic value of module hub genes with a degree score >5 in the PPI network was verified in samples from patients with CRC diagnosed before the age of 50 years obtained from The Cancer Genome Atlas. Eight co-expressed gene modules were identified in the WGCNA and two modules (blue and turquoise) were associated with the tumor-node-metastasis stage. A total of 140 module hub genes were identified and found to be enriched in 'mitochondrial large ribosomal subunit', 'structural constituent of ribosome', 'poly (A) RNA binding', 'collagen binding', 'protein ubiquitination' and 'ribosome pathway'. Twenty-six module hub genes were found to have a degree score >5 in the PPI network, seven of which [secreted protein acidic and cysteine rich (SPARC), decorin (DCN), fibrillin 1 (FBN1), WW domain containing transcription regulator 1 (WWTR1), transgelin (TAGLN), DEAD-box helicase 28 (DDX28) and cold shock domain containing C2 (CSDC2)], had good prognostic values for patients with early-onset CRC, but not late-onset CRC. Therefore, SPARC, DCN, FBN1, WWTR1, TAGLN, DDX28 and CSDC2 may contribute to the development of early-onset CRC and may serve as potential diagnostic biomarkers.

Background Ketamine-related cystitis (KC) has been researched in many clinical studies, but its exact mechanism is ambiguous and needs further research. Methods We established a KC rat model and analyzed physiological, biochemical, and urodynamic parameters of ketamine (KET)-related bladder injury. Bladder histologic feature, reactive oxygen species (ROS), autophagy-, apoptosis-, and endoplasmic reticulum stress (ERS)-related markers were examined by hematoxylin and eosin staining, Masson staining, ROS kit, quantitative real-time polymerase chain reaction, and western blot. In vitro, effects of 0.01, 0.1, and 1 mM KET on cell vitality, apoptosis, ROS level, autophagy-, apoptosis-, and ERS-related markers were examined again. Effects of KET-1 and salubrinal on complex formation, autophagy-, apoptosis-, and ERS-related markers were examined by Co-Immunoprecipitation and western blot. After transfection with shIRE1, complex formation, cell biological behaviors, ROS level, autophagy-, apoptosis-, and ERS-related markers were examined again. Results KET induced bladder hyperactivity and injury. KET facilitated urinary frequency, ROS production, and induced bladder histologic injury by activating autophagy-, apoptosis-, and ERS-related markers in rats. In vitro, KET (0.01, 0.1, and 1 mM) restrained cell vitality and elevated apoptosis and ROS level via activating autophagy-, apoptosis-, and ERS-related markers. Moreover, salubrinal reversed the promotion of KET-1 on complex formation, autophagy-, apoptosis-, and ERS-related marker expressions. After transfection with shIRE1, shIRE1 weakened complex formation induced by KET-1, and the effects of KET-1 on cells were offset by shIRE1. Conclusion KET enhanced autophagy and ERS in vivo and in vitro via restraining IRE1-TRAF2-ASK1-JNK pathway.

Previous studies have revealed that SDHB has potential functions in the male sexual differentiation and development in M. nipponense through providing ATP. In this study, the functions of Mn-SDHB were further analyzed in depth using quantitative polymerase chain reaction (qPCR), in situ hybridization, western-blot, and RNA interference (RNAi), combined with the histological observations. The full-genome sequence of Mn-SDHB was 54,608 bp at Chromosome 34, including 7 introns and 6 exons. The full-length cDNA sequence of Mn-SDHB was 1,268 base pairs (bp) long with an open reading frame of 807 bp, encoding for 268 amino acids. The highest expression level of Mn-SDHB in different tissues was observed in the testis, and male prawns at post-larval developmental stage 25 during different developmental stages, indicating that SDHB was potentially involved in the male sexual development in M. nipponense. In situ hybridization and western-blot analysis indicated that SDHB plays essential roles in the testis development. The in situ hybridization analysis also implies the potential roles of Mn-SDHB in ovarian development. The expressions of Mn-IAG were decreased after Mn-SDHB dsRNA injection, indicating SDHB has the positive regulatory effects on IAG in M. nipponese. Thus, SDHB was involved in the mechanism of the male sexual development. The testis development was inhibited, and sperms were rarely observed after 10 days of Mn-SDHB dsRNA injection, indicating SDHB has positive effects on the male sexual development in M. nipponense. This study highlights the functions of SDHB in M. nipponense, which provide new insights for the future studies of the male sexual development in other crustacean species.

Background The miRNAs play critical roles in the progression of various tumors. Our study aimed to screen and identify miRNAs to investigate their diagnostic and prognostic value for papillary thyroid carcinoma (PTC). Methods miRNAs were evaluated in PTC (n = 30) tissues, A‐PTC (n = 30), benign nodules (n = 35) and A‐benign nodules (n = 35). The expression levels of five miRNAs were quantified using real‐time, quantitative PCR. ROC analysis was used to evaluate the miRNA diagnostic value. Results The expression of miR‐1296‐5p, miR‐1301‐3p, and miR‐532‐5p was significantly downregulated (p = 0.0001, p = 0.0006, p = 0.0024, respectively), while miR‐551b‐3p and miR‐455‐3p were significantly upregulated in PTC tissues compared to A‐PTC tissues (p = 0.0005, p = 0.0046, respectively). Interestingly, the expression of miR‐1296‐5p was downregulated, while miR‐551b‐3p and miR‐455‐3p were upregulated in the A‐PTC group compared to the A‐benign group. Moreover, the miR‐1296‐5p expression level was associated with tumor size, the number of foci and the TNM stage; the miR‐455‐3p expression level was correlated with patient age, tumor size, and TNM stage; and the miR‐532‐5p expression level was correlated with patient age, lymph node metastasis and TNM stage correspondingly. ROC analysis revealed that the AUCs for miR‐1301‐3p, miR‐1296‐5p, miR‐455‐3p, miR‐532‐5p, and miR‐551b‐3p were 0.773, 0.790, 0.783, 0.744, and 0.650, respectively. Conclusions Our results indicated that miR‐1296‐5p, miR‐1301‐3p, miR‐532‐5p, miR‐551b‐3p, and miR‐455‐3p are aberrantly expressed in papillary thyroid carcinomas and correlated with clinicopathological features. ROC curve analysis indicated that these five miRNAs have a potential diagnostic value. Consequently, we speculate that the five altered miRNAs may serve as potential diagnostic and prognostic biomarkers for PTC., miR‐1296‐5p, miR‐1301‐3p and miR‐532‐5p were significantly downregulated (p = 0.0001, p = 0.0006, p = 0.0024, respectively), while miR‐551b‐3p and miR‐455‐3p were significantly upregulated in PTC tissues compared to A‐PTC (p = 0.0005, p = 0.0046, respectively). Aberrant expression of miR‐1296‐5p, miR‐532‐5p, and miR‐455‐3p are correlation with clinical‐pathological features. ROC cure indicated that those five miRNAs have diagnostic value.

The eyestalk of crustacean species secretes many hormones, affecting the process of reproduction, molting, metabolism of glucose, and other functions in crustaceans. In this study, important metabolic pathways and candidate genes involved in the male sexual development were identified through performing the transcriptome profiling analysis of the testis after the ablation of eyestalk from Macrobrachium nipponense. The histological observations revealed that the testis development became vigorous after eyestalk ablation, indicating that the hormones secreted by the eyestalk have negative effects on the testis development in M. nipponense. Transcriptome profiling analysis revealed that 1,039, 1,226, and 3,682 differentially expressed genes (DEGs) were identified between normal prawns (CG) vs single-side eyestalk ablation prawns (SS), SS vs double-side eyestalk ablation prawns (DS), and CG vs DS, respectively, indicating that the ablation of double-side eyestalk has more significant regulatory roles on male sexual development than that of single-side ablation, which was consistent with the histological observations. Lysosome, Apoptosis, Glycolysis/Gluconeogenesis, and Insulin signaling pathway were the main enriched metabolic pathways in all of these three comparisons, and the important genes from these metabolic pathways were also selected. The qPCR verifications of 10 DEGs from these metabolic pathways were the same as those of RNA-seq. The qPCR, in situ hybridization, and RNA interference analysis of Mn-NFkBα revealed that NFkBα has a positive regulatory effect on testis development. This study provided new insights on male sexual development in M. nipponense, promoting the studies on male sexual development in other crustaceans as well.

Tick-borne allergies are a growing public health concern and have been associated with the induction of IgE-mediated food allergy to red meat. However, despite the increasing prevalence of tick bite–induced allergies, the mechanisms by which cutaneous exposure to ticks leads to sensitization and the production of IgE Abs are poorly understood. To address this question, an in vivo approach was used to characterize the IgE response to lone star tick proteins administered through the skin of mice. The results demonstrated that tick sensitization and challenge induced a robust production of IgE Abs and supported a role for IgE-mediated hypersensitivity reactions in sensitized animals following oral administration of meat. The induction of IgE responses was dependent on cognate CD4+ T cell help during both the sensitization phase and challenge phase with cutaneous tick exposure. In addition, IgE production was dependent on B cell–intrinsic MyD88 expression, suggesting an important role for TLR signaling in B cells to induce IgE responses to tick proteins. This model of tick-induced IgE responses could be used to study the factors within tick bites that cause allergies and to investigate how sensitization to food Ags occurs through the skin that leads to IgE production.

Microsporongium development in Chinese fir (Cunninghamia lanceolata) was investigated using cytochemical methods with a special attention to the fluctuations (in amount and distribution) of...

Gastric cancer has become the third most common cancer around the world. In patients with gastric cancer, the 5-year survival rate is still low. However, the mechanism underlying gastric cancer remains largely unknown. As a glycolytic enzyme, enolase 1 (ENO1) is widely expressed in most tissues. The functions of ENO1 have been reported in various types of cancer. Here in this study, we identified that ENO1 promoted the growth of gastric cancer cells through diverse mechanisms. Our immunohistochemical, bioinformatic and Western blot data showed that ENO1 was significantly overexpressed in human gastric cancer cell lines and tissues. The survival analysis revealed that ENO1 overexpression predicted poor survival in the patients suffering gastric cancer. Knockdown of ENO1 expression repressed the rate of proliferation and capacity of colony formation in two human gastric cancer cell lines (MGC-803 and MKN-45). In addition, knockdown of the expression of ENO1 led to the arrest of the cell cycle at the G1 phase and promoted the apoptosis of MKN-45 and MGC-803 cells. The further microarray and bioinformatic analysis revealed that ENO1 regulated the expression of diverse genes, many of which are involved in the progress of cancer. Taken together, our data demonstrated that ENO1 was an oncogene-like factor and might serve as a promising target for the treatment of human gastric cancer.

An early step in signaling from activated receptor tyrosine kinases (RTKs) is the recruitment of cytosolic adaptor proteins to autophosphorylated tyrosines in the receptor cytoplasmic domains. Fibroblast growth factor receptor substrate 2α (FRS2α) associates via its phosphotyrosine-binding domain (PTB) to FGF receptors (FGFRs). Upon FGFR activation, FRS2α undergoes phosphorylation on multiple tyrosines, triggering recruitment of the adaptor Grb2 and the tyrosine phosphatase Shp2, resulting in stimulation of PI3K/AKT and MAPK signaling pathways. FRS2α also undergoes N-myristoylation, which was shown to be important for its localization to membranes and its ability to stimulate downstream signaling events (Kouhara et al., 1997). Here we show that FRS2α is also palmitoylated in cells and that cysteines 4 and 5 account for the entire modification. We further show that mutation of those two cysteines interferes with FRS2α localization to the plasma membrane (PM), and we quantify this observation using fluorescence fluctuation spectroscopy approaches. Importantly, prevention of myristoylation by introduction of a G2A mutation also abrogates palmitoylation, raising the possibility that signaling defects previously ascribed to the G2A mutant may actually be due to a failure of that mutant to undergo palmitoylation. Our results demonstrate that FRS2α undergoes coupled myristoylation and palmitoylation. Unlike stable cotranslational modifications, such as myristoylation and prenylation, palmitoylation is reversible due to the relative lability of the thioester linkage. Therefore, palmitoylation may provide a mechanism, in addition to phosphorylation, for dynamic regulation of FRS2 and its downstream signaling pathways.

SummaryIsolated gametes can be used to investigate fertilization mechanisms, and probe distant hybridization between different species. Pollen grains of wheat and Setaria viridis are tricellular, containing sperm cells at anthesis. Sperm from these plants were isolated by breaking open pollen grains in a osmotic solution. Wheat ovules were digested in an enzyme solution for 20 min, and then transferred to an isolation solution without enzymes to separate egg cells from ovules. The fusion of wheat egg cells with wheat and S. viridis sperm was conducted using an electro-fusion apparatus. Under suitable osmotic pressure (10% mannitol), calcium concentration of 0.001% (CaCl2·2H2O), and a 30–35 V alternating electric field for 15 s, egg cells and sperm adhered to each other and became arranged in a line. Electroporation of the plasma membrane of egg cells and sperm using a 300–500 V direct-current electric field (45 µs amplitude pulse) caused them to fuse.

Accumulating evidence suggests that epidermal growth factor receptor (EGFR) plays a role in the progression of pulmonary arterial hypertension (PAH). Clinically-approved epidermal growth factor inhibitors such as gefitinib, erlotinib, and lapatinib have been explored for PAH. However, None of them were able to attenuate PAH. So, we explored the role of dacomitinib, a new pan-EGFR inhibitor, in PAH. Adult male Sprague-Dawley rats were used to study hypoxia- or monocrotaline-induced right ventricular remodeling as well as systolic function and hemodynamics using echocardiography and a pressure-volume admittance catheter. Morphometric analyses of lung vasculature and pressure-volume vessels were performed. Immunohistochemical staining, flow cytometry, and viability, as well as scratch-wound, and Boyden chamber migration assays were used to identify the roles of dacomitinib in pulmonary artery smooth muscle cells (PASMCs). The results revealed that dacomitinib has a significant inhibitory effect on the thickening of the media, adventitial collagen increased. Dacomitinib also has a significant role in attenuating pulmonary artery pressure and right ventricular hypertrophy. Additionally, dacomitinib inhibits hypoxia-induced proliferation, migration, autophagy and cell cycle progression through PI3K-AKT-mTOR signaling in PASMCs. Our study indicates that dacomitinib inhibited hypoxia-induced cell cycle progression, proliferation, migration, and autophagy of PASMCs, thereby attenuating pulmonary vascular remodeling and development of PAH via the PI3K-AKT-mTOR signaling pathway. Overall, dacomitinib may serve as new potential therapeutic for the treatment of PAH.

Calcium distribution in fertile and sterile anthers of a thermosensitive male-sterile wheat genotype was investigated using an antimonate precipitation method. During fertile anther development, be...

SummaryFertilization in higher plants induces many structural and physiological changes in the fertilized egg, and represents the transition from the haploid female gamete to the diploid zygote, the first cell of a sporophyte. Some changes are induced extremely rapidly following fusion with sperm cells and are the preclusions of egg activation. This review focuses on the early changes that occur in the egg after fusion with sperm cells, but before nuclear fusion. Reported changes include cell shrinkage, cell wall formation, polarity change, oscillation in Ca2+ concentration, and DNA synthesis. In addition, the current understanding of egg activation is summarized and the possible functional relevance of the changes is explored.

The eyestalk of crustacean species contained many neurosecretory structures, affected the process of reproduction, molting, metabolism of glucose and other function in crustaceans. In this study, we aimed to selected important metabolic pathways and candidate genes involved in the male sexual development through performing the long-reads and next generation transcriptome sequencing of androgenic gland after the ablation of eyestalk from Macrobrachium nipponense. qPCR analysis revealed that the mRNA expression of Mn-IAG was significantly increased after ablation both of the single-side (SS) and double-side (DS) eyestalk, compared with that of control group (CG). The long-reads transcriptome generated 49,480 non-redundant transcripts. A total of 1,319, 2,092 and 4,351 differentially expressed genes (DEGs) were identified between CG vs SS, SS vs DS and CG vs DS, respectively, indicating the ablation of double-side eyestalk has more important regulatory roles than that of single-side ablation on male sexual development, which was consistent with that of qPCR analysis. Cell cycle, Cellular senescence, Oxidative phosphorylation, Glycolysis/Gluconeogenesis and Steroid hormone biosynthesis were the main enriched metabolic pathways in all of these three comparisons, and the important genes from these metabolic pathways were also selected. The qPCR verifications of 10 GEDs were as the same as that of RNA-seq. The qPCR and RNAi analysis of Hydroxysteroid dehydrogenase like 1 (HSDL1) revealed that HSDL1 has the positive regulatory effected on testis development. This study provided valuable evidences on male sexual development in M. nipponense, promoting the studies on male sexual development in other crustacean as well.

Background Macrobrachium nipponense is an economically important species of freshwater shrimp in China. Unlike other marine shrimps, the ovaries in adult female M. nipponense can mature rapidly and periodically during the reproductive period, but the resulting high stocking densities and environmental deterioration can negatively impact the harvest yield and economic benefits. To better understand ovary development in female M. nipponense, we performed systematic transcriptome sequencing of five different stages of ovarian maturation. Results We obtained 255,966 Gb of high quality transcriptome data from 15 samples. Of the 105,082 unigenes that were selected, 30,878 were successfully annotated. From these unigenes, we identified 17 differentially expressed genes and identified three distinct gene expression patterns related to different biological processes. We found that cathepins, legumains, and cystatin were enriched in the lysosome pathway, and they are related to vitellogenin hydrolysis. Additionally, we found that myosin heavy chain 67 participated in oocyte excretion. Conclusions We provide the first detailed transcriptome data relating to the ovarian maturation cycle in M. nipponense. Our results provide important reference information about the genomics, molecular biology, physiology, and population genetics of M. nipponense and other crustaceans. It is conducive to further solve the problem of M. nipponense rapid ovarian maturation from the aspects of energy supply and cell division.

Cgnz1 on chromosome 1 mapped into a 1.34 Mb region of chromosome 1 in NZM2328 confers the progression of immune complex (IC)-mediated glomerulonephritis (GN) from acute GN (aGN) to chronic GN (cGN) with severe proteinuria and end stage renal disease in female mice. This genetic locus mediates podocyte susceptibility to IC-mediated damage. Taking advantage of the published observation that Cgnz1 is derived from NZW and that NZW is susceptible to orchitis, epididymitis and vasitis while C57L/J is resistant to these diseases, the possibility that this genetic region also confers germ cells susceptible to damage with aspermatogenesis and sterility in an active experimental autoimmune orchitis (EAO) model was investigated. Male mice from multiple intrachromosome (chromosome 1) recombinant strains were subjected to immunization with a sperm homogenate in CFA with concomitant administration of Bordetella pertussis toxin. There was concordance of the progression from aGN to cGN, severe proteinuria and end stage renal disease with susceptibility of EAO in NZM2328 and its congenic strains with various chromosome 1 genetic intervals introgressed from C57L/J to NZM2328. Both resistant and susceptible strains made comparable anti-testis and anti-sperm Abs. Thus the genetic interval that determines susceptibility to EAO is identical to that of Cgnz1 and mapped to the 1.34 Mb region in chromosone 1. This region likely confers germ cells in the male gonad susceptible to damage by immunologically mediated inflammation. This region has been tentatively renamed Cgnz1/Eaoz1. These observations further emphasize the importance of end organ susceptibility to damage in the pathogenesis of both systemic and organ specific autoimmune diseases.

Background The oriental river prawn, Macrobrachium nipponense, is an economically important shrimp in China. Male prawns have higher commercial value than females because the former grow faster and reach larger sizes. It is therefore important to reveal sex-differentiation and development mechanisms of the oriental river prawn to enable genetic improvement. Results We sequenced 293.3 Gb of raw Illumina short reads and 405.7 Gb of Pacific Biosciences long reads. The final whole-genome assembly of the Oriental river prawn was ∼4.5 Gb in size, with predictions of 44,086 protein-coding genes. A total of 49 chromosomes were determined, with an anchor ratio of 94.7% and a scaffold N50 of 86.8 Mb. A whole-genome duplication event was deduced to have happened 109.8 million years ago. By integration of genome and transcriptome data, 21 genes were predicted as sex-related candidate genes. Conclusion The first high-quality chromosome-level genome assembly of the oriental river prawn was obtained. These genomic data, along with transcriptome sequences, are essential for understanding sex-differentiation and development mechanisms in the oriental river prawn, as well as providing genetic resources for in-depth studies on developmental and evolutionary biology in arthropods.

In this study, we identified three neuroparsin (NP) genes in Macrobrachium nipponense: Mn-NP1, Mn-NP2, and Mn-NP3, encoding 99, 100, and 101 amino acid proteins, respectively. Multiple sequence alignments showed that these genes contained 12 cysteine residues, of which 11 were at conserved positions. The total sequence identity between the genes was 47.5%, and they showed a high degree of sequence identity (> 54% similarity) with other crustacean genes. Phylogenetic tree analysis showed that Mn-NPs were clustered at different branches, indicating that Mn-NPs may have different functions. Tissue distribution data revealed that the three genes were present in males and females during the breeding and nonbreeding season, but their expression patterns differed. Mn-NP1 was highly expressed in the breeding season, in the male testis, and highly expressed in the nonbreeding season, in the female ovary. Mn-NP3 exhibited biased female expression in the breeding and nonbreeding season, with dominant expression in the ovary. All Mn-NPs were detected during embryo development, but with different expression patterns. These data indicated that Mn-NP1 may function during embryonic development, and that Mn-NP2 may be expressed during early embryo cell division, and late larval development. Mn-NP3 expression patterns reflected maternal inheritance, and may be associated with ovarian maturation. These expression data suggested that Mn-NP1 and Mn-NP2 are negatively correlated with ovarian development, with inhibition roles during this development. Mn-NP3 may be involved in vitellogenesis.

Integrin beta- like 1 (ITGBL1), an extracellular matrix protein, plays an oncogenic role in diverse forms of cancers. To this end, we examined the importance of ITGBL1 in gastric cancer (GC). The upregulated expression of ITGBL1 in GC was associated with a poor prognosis. Moreover, upregulation of ITGBL1 enhanced cell mobility while silencing it exerted an opposite effect. Up-regulation of ITGBL1 significantly promoted phosphorylation of Akt, decreased the ratio of phosphorylated Akt in AGS/ITGBL1-shRNA and N87/ITGBL1-shRNA cells, enhanced cell mobility and proliferation. Silencing ITGBL1 had an opposite effect on Akt phosphorylation, cell mobility, and proliferation. These findings show that ITGBL1 regulates mobility and proliferation of GC likely through activation of Akt signaling.

A previous study identified slow-tonic S2 tropomyosin and slow tropomyosin isoform as sex-related genes in Macrobrachium nipponense. Their functions were analyzed using RNA interference. However, more than half of the specimens died approximately 8–12 h after injection of the respective double-stranded RNAs (dsRNAs), and HE staining indicated that the heart and gills were the most likely tissues responsible for the resultant deaths. In the current study, we conducted a comparative transcriptomic study of the gills and hearts of M. nipponense to identify potential target genes associated with acute death after dsRNA injection. A total of 68,772 annotated unigenes were generated. In the heart, differentially expressed genes (DEGs) were mainly enriched in glycolysis/gluconeogenesis and oxidative phosphorylation, while the most relevant pathways in the gills were lysosome, phagosome, and peroxisome. Ten DEGs were screened out and analyzed under lethal hypoxic stress. Among these, fructose 1, 6-biphosphate-aldolase (FBA), glyceraldehyde 3-phosphate dehydrogenase (GDPDH), alcohol dehydrogenase class-3 (ADC3), ATP-synthase subunit 9 (ATPS9), and acid ceramidase-like (ACL) were all differentially expressed under hypoxic conditions. This study shed light on the lethal mechanism caused by interference with tropomyosin genes in M. nipponense, and identifies the related pathways and key genes that could help to improve stress resistance and tolerance in M. nipponense.

In invertebrates, neuropeptide F (NPF) has many regulatory functions, similar to NPY, its homologous peptide. In this study, two neuropeptide F genes were identified in Macrobrachium nipponense: Mn-NPF1 and Mn-NPF2. Mn-NPF2 shared the same amino acid sequence with Mn-NPF1, except for a 37 amino acid insert in the middle of the NPF region. The quantitative-PCR (qPCR) results indicated that Mn-NPF1 expression was positively correlated with ovarian maturation, whereas Mn-NPF2 had opposing expression patterns. Both Mn-NPFs were poorly expressed at early embryonic stages, but enhanced expression levels were observed up to day 10 after hatching, when the gonads began to differentiate. Ovary in situ hybridization (ISH) analyses showed that both Mn-NPFs were present at all stages, but were differentially localized to distinct compartments. Temperature gradient studies showed that both Mn-NPFs were implicated in the seasonal regulation of reproduction. A double-stranded (ds) RNA-Mn-NPF2 injection led to a significant 38.5% increase in the vitellogenin (VG) transcript (P < 0.05). These results demonstrated that Mn-NPF2 plays an important role in inhibiting ovarian maturation.

Casein kinase 1 (CK1) family members are conserved Ser/Thr protein kinases that regulate important developmental processes in all eukaryotic organisms. However, the functions of CK1 in plant immunity remain largely unknown. Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, infects cereal crops and is obligately transmitted by the small brown planthopper (SBPH; Laodelphax striatellus). The BYSMV phosphoprotein (P) exists as two forms with different mobilities corresponding to 42 kD (P42) and 44 kD (P44) in SDS-PAGE gels. Mass spectrometric analyses revealed a highly phosphorylated serine-rich (SR) motif at the C-terminal intrinsically disordered region of the P protein. The Ala-substitution mutant (P(S5A)) in the SR motif stimulated virus replication, whereas the phosphorylation-mimic mutant (P(S5D)) facilitated virus transcription. Furthermore, P(S5A) and P(S5D) associated preferentially with nucleocapsid protein–RNA templates and the large polymerase protein to provide optimal replication and transcription complexes, respectively. Biochemistry assays demonstrated that plant and insect CK1 protein kinases could phosphorylate the SR motif and induce conformational changes from P42 to P44. Moreover, overexpression of CK1 or a dominant-negative mutant impaired the balance between P42 and P44, thereby compromising virus infections. Our results demonstrate that BYSMV recruits the conserved CK1 kinases to achieve its cross-kingdom infection in host plants and insect vectors.

Carbon catabolite repression 4 (CCR4) is a conserved mRNA deadenylase regulating posttranscriptional gene expression. However, regulation of CCR4 in virus infections is less understood. Here, we characterized a pro-viral role of CCR4 in replication of a plant cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV). The barley (Hordeum vulgare) CCR4 protein (HvCCR4) was identified to interact with the BYSMV phosphoprotein (P). The BYSMV P protein recruited HvCCR4 from processing bodies (PBs) into viroplasm-like bodies. Overexpression of HvCCR4 promoted BYSMV replication in plants. Conversely, knockdown of the small brown planthopper CCR4 inhibited viral accumulation in the insect vector. Biochemistry experiments revealed that HvCCR4 was recruited into N–RNA complexes by the BYSMV P protein and triggered turnover of N-bound cellular mRNAs, thereby releasing RNA-free N protein to bind viral genomic RNA for optimal viral replication. Our results demonstrate that the co-opted CCR4-mediated RNA decay facilitates cytorhabdovirus replication in plants and insects.

To remedy the lack of human leukocyte antigen (HLA)-matched donors and address the problems bedeviling traditional allogeneic hematopoietic stem cell transplantation which induces the resultant graft-versus-host disease, we designed a scheme called HLA-mismatched hematopoietic stem cell microtransplantation (MST) for patients with acute myeloid leukemia (AML), where encouraging results were achieved. In providing answers to such questions as how to select the donors of MST and which factors were involved in the outcome of MST. One hundred thirty-one AML patients from four centers with lower or standard risk of prognosis after complete remission were given three courses of MST: high dose of cytarabine plus infusion of granulocyte colony-stimulating factor mobilized peripheral blood stem cells from HLA-mismatched donors. Leukemia-free survival (LFS) and overall survival were compared, with respect to gender difference, number of HLA-matched loci, killer cell immunoglobulin-like receptor (KIR), and ligand mismatch between donors and recipients. Median LFS of recipients with different KIR ligands from those of donors was found to be significantly higher than that of recipients having identical ligands with donors (

Sirtuin 6 (SIRT6) has been reported to play a key role in cognitive function and mood regulation, yet its role in mood disorders is not completely understood. Here, we confirmed that knockdown of hippocampal SIRT6 alleviated depression-like behaviors induced by chronic unpredictable stress (CUS) in mice. Our in vitro data showed that SIRT6 negatively regulated protein kinase B (AKT) signaling by deacetylating histone 3 at Lys9 and Lys56. Knockdown of SIRT6 significantly increased AKT phosphorylation activity, while decreased collapsin response mediator protein 2 (CRMP2) phosphorylation activity. Furthermore, pharmacologic inhibition of SIRT6 by ferulic acid (FA) (40 or 80 mg· kg(−1) per day, i.g.) could activate AKT/CRMP2 pathway in vitro, which has been proved to exert an antidepressant-like effect on CUS-induced depressive models. In conclusion, our study suggested that hippocampal SIRT6 contributes to the performance of depression-like behaviors by suppressing AKT/CRMP2 signaling, and FA ameliorates CUS-induced depression-like behaviors in mice as a potential pharmacologic inhibitor of SIRT6.

In this study, a full-length cDNA sequence of SoxE (subgroup E within the Sox family of transcription factors) was cloned from Macrobrachium nipponense and named MnSoxE1. The full-length cDNA of MnSoxE1 is 1748 bp, consisting of a 110 bp 5′ UTR, a 105 bp 3′ UTR, and a 1533 bp ORF that encodes 510 amino acids. Conserved domains showed that MnSoxE1 has a high similarity to the SoxE gene of Penaeus vannamei. Phylogenetic tree analysis classified that MnSoxE1 with the SoxE gene of other arthropods into one clade. These results suggested that MnSoxE1 belongs to the SoxE subgroup. During embryonic development, MnSoxE1 was mainly expressed in the gastrula stage, implicating its involvement in tissue cell differentiation and formation. In the post-larval stages, the expression of MnSoxE1 continued to increase on days 1–10. The expression level in males was significantly higher than that in females. Males are clearly distinguishable from females on post-larval day 25, showing that MnSoxE1 may play a role in promoting early development and germ cell and gonadal differentiation, especially for males. qPCR analysis showed that MnSoxE1 may also be involved in oogonium proliferation during ovary development. Further in situ hybridization analysis revealed that MnSoxE1 was mainly located in oocytes and spermatocytes, especially in sertoli cells, and implies that it may be involved in the development of oocytes and spermatocytes, as well as the maintenance of testes in mature prawns. These results indicate that MnSoxE1 is involved in gonadal differentiation and development in M. nipponense, especially testis development.

The androgenic gland produces hormones that play crucial roles in driving male sexual development. In this study, we investigated the regulatory proteins and pathways affecting male sexual development of M. nipponense, by analyzing the proteomes of their androgenic glands during their reproductive and nonreproductive seasons using isobaric tags for relative and absolute quantitation (iTRAQ). A total of 13 sex-related protein homologs out of 999 total proteins were identified in the proteome of M. nipponense based on comparisons with the published literature. A total of 32 proteins were differentially expressed in the androgenic gland between the reproductive and nonreproductive seasons of M. nipponense. However, only 10 differentially expressed proteins (DEPs) were annotated in the Nr, COG, GO, and KEGG databases. Other non-annotated DEPs may also play vital roles in the male sexual development of M. nipponense. The qPCR analysis indicated that the mRNA expression patterns of the 10 annotated DEPs were consistent with that determined by iTRAQ analysis. Additional qPCR analysis of the 10 DEPs in testes, ovaries, and androgenic glands showed that Gem, Ferritin, and Rev3 were highly expressed in the androgenic gland, implying importance in male sexual development, given the important role of the androgenic gland in male differentiation and development in many crustacean species. Our study provided valuable information about DEPs in androgenic glands between reproductive and nonreproductive seasons for M. nipponense and identified their potential roles in male sexual development, which will improve our understanding of the development process in both M. nipponense and other crustaceans.

Opsins are photoreceptors with important roles in reproductive regulation in birds and fishes. In the present study, we identified an opsin gene from the eyes of the oriental river prawn Macrobrachium nipponense using expressed sequence tag analysis and rapid amplification of cDNA ends. The full-length transcript contained 1382 base pairs, encoding 375 amino acids. It was classified into the long-wavelength opsin group by phylogenetic analysis, and designated Mn-LW. Mn-LW expression demonstrated significant seasonal variation in somatic tissues from both male and female prawns, with the highest expression in the eyes, and expression also shown in the ovary. The expression profiles of Mn-LW in eyes and ovary were positively related to ovarian development. In situ hybridization showed that Mn-LW was present in retinular cells in the eye and oocytes in the ovary. Injection of Mn-LW dsRNA in vivo effectively down-regulated Mn-LW expression levels compared with control levels. Mn-LW dsRNA injection also significantly reduced vitellogenin (Vg) expression, indicating a close relationship between Mn-LW and Vg in ovarian development. These results suggest that Mn-LW may play an important role in Vg synthesis and accumulation during ovarian maturation in M. nipponense.

As an important economical freshwater prawn, Macrobrachium nipponense has difficulty with adapting to hypoxia. In this study, comparative transcriptome analysis was used for the first time to explore the differences between different living states of Macrobrachium nipponense under hypoxia. A total of 94.22 Gb clean reads were obtained and assembled into 54,688 unigenes. A total of 224, 266, and 750 differently expressed genes were found in the comparison of the control and death groups, the control and moribund groups, and the control and survived groups, respectively. Three signal pathways closely related to hypoxia were found by enriching of the signal pathways in three comparison groups. In addition, much attention was focused on the differential genes in these pathways. Oxidative stress related genes, such as 70 kDa heat shock protein, phosphoenolpyruvate carboxykinase and cyclooxygenase were differentially expressed in different comparisons. After comparing with previous studies, cyclooxygenase was found to be an important hypoxia-related gene that is fully involved in the hypoxic response. Interestingly, two new genes with no Nr annotation were found in this manuscript. This manuscript will enrich our understanding of oxidative stress response to hypoxia and provide a theoretical basis for the subsequent solution of apoptosis caused by hypoxia.

The aim of this study was to explore the function of the Mn-Spook gene, which was found in the ovary transcriptome of the Oriental river prawn (Macrobrachium nipponense). The Spook gene, which is the precursor gene of 20-hydroxyecdysone (20E), plays an important role in the process of molting in many arthropods, but its function in M. nipponense is unclear. We cloned the full-length Mn-Spook gene from the ovary of M. nipponense and found that it had the same conserved domains as the P450 gene of the Halloween family of genes. The Mn-Spook gene was highly expressed in ovary and gill tissue during the breeding period. During ovarian development, Mn-spook gene expression was highest at the nearly-ripe stage, and it also was highly expressed in the zoea developmental stage. Cellular localization analysis showed that Mn-Spook signals accumulated in the cytoplasmic membrane and nucleus of oocytes. Finally, we used RNA interference to evaluate the function of the Mn-Spook gene. Compared with the control group, in vivo injection of Mn-Spook dsRNA effectively downregulated the expression of Mn-Spook and the content of 20E. The molting frequency of M. nipponense in the experimental group also was significantly inhibited. These results demonstrated that the Mn-Spook gene played an important role in the molting process of M. nipponense.

In this manuscript, we first found calcium induces vacuoles formation from mitochondria during microspore development in Bauhinia blakeana, which is not reported yet. Potassium antimonite was used to locate calcium in anther cells of Bauhinia blakeana to determine the role of Ca2+ in pollen development. Some calcium-induced precipitates were observed in early sporogenous cells after which numerous small vacuoles containing calcium precipitates appeared following transformation into microspore mother cells. The small vacuoles were then secreted from the plasma membrane to the callose wall. After meiosis, the number of precipitates in the early microspore cytoplasm increased, accumulating mainly in the mitochondria, the inner membrane (cisterns) of which was then destroyed to create small vacuoles. These small vacuoles then fused to form a large vacuole, which subsequently polarized the cell. After microspore division and decomposition of these large vacuoles, numerous calcium precipitates accumulated in the small vacuoles, which were then secreted out of the pollen. These findings suggest a role of calcium in the regulation of vacuole formation and decomposition during pollen development.

Macrobrachium nipponense has the characteristics of fast ovarian development cycle, which leads to the coexistence of multiple generations, the reduction of commodity specifications and the low economic benefit. Therefore, the study on the mechanism of ovarian development is of great significance to the development of industry. Cyclin A (CycA)is a key gene regulating ovarian development in vertebrates, but little information was available for its function in crustaceans. In this study, the full-length cDNA of Mn-CycA was obtained from the ovary. The full-length cDNA (2033 bp) with an open reading frame of 1368 bp, encoded a 456-amino acid protein. qRT-PCR revealed tissue-specific expression pattern of Mn-CycA, with abundant expression in the ovary. Results in different developmental stages of ovary indicated that Mn-CycA expression is positively correlated with ovarian maturation. qRT-PCR In different developmental stages, the expression of Mn-CycA mRNA gradually increased during the embryonic stage and decreased significantly on the first day of the hatching stage. At the 25th day of the metamorphosis stage, the expression level of Mn-CycAmRNA in female shrimp was 3.5 times higher than that in male shrimp, which may be related to the proliferation of oogonia and the formation of oocytes. In situ hybridization (ISH) of ovary showed Mn-CycA was examined in all stages and was mainly located in oogonia and oocytes. Compared with the control group, the obvious change of gonad somatic index (GSI) proved that injection of Mn-CycA dsRNA could delay the ovarian development cycle, which provided strong evidence for the involvement of Mn-CycA in ovarian maturation and oogenesis, and expanded a new perspective for studying the fast ovarian development cycle in M. nipponense.

Multiple synergetic antimicrobial modality (MSAM) platform is a novel approach which has highly advocated recently to meet the challenge of bacterial infection. In this research, we constructed a “Dew-of-Leaf” like hybrid material (Ag-PCN@Ti3C2-BC) with multiple design features that integrated photodynamic, photothermal and silver ion releasing effects. Porphyrinic MOFs (PCN-224) grown on Ti3C2 nanosheets was first filtered onto BC, then nanosilver was sputtered using magnetron sputtering technique. Interestingly, the light-driven singlet oxygen produced by PCN-224 and the heat produced by Ti3C2 not only synergistically enhanced the antibacterial effect, but also promoted the sputtered nanosilver to be degraded to release silver ions, achieving rapid bacterial inactivation and long lasting bacterial inhibition effects. Moreover, after two rounds of bacterial inactivation and 6 months of room environment preservation, an antibacterial inactivation study still demonstrated 99.9999% elimination of Gram-negative Escherichia coli ATCC-8099 and Gram-positive Staphylococcus aureus ATCC-6538 with Ag-PCN@Ti3C2-BC. Taken together, this nanofiber hybrid material permits an effective way to inactivate bacteria rapidly at first and long lasting bacterial inhibition followed, showing great potential for microbial disinfection.

The pollen grains of Anoectochilus roxburghii aggregate in pollinia. The aim of this study was to analyse the structural characteristics of pollinium development in A. roxburghii. Features of the pollinia begin to form in the sporogenous cells, in which some cell walls begin to differentiate to form the outline of the pollinia. During development of the microspore mother cells (MMCs), the nucleus and organelles penetrate the cell walls (cytomixis) between MMCs within a pollinium, but not between the MMCs among different pollinia. A thick callose wall forms on the surface of the pollinia, but not between MMCs inside the pollinium. After meiosis, a sporopollenin exine forms on the surface of the pollinia, but not on the surface of pollen grains inside the pollinium. The mature pollen of A. roxburghii is bicellular and consists of a vegetative cell and a generative cell.

Cathepsin L is an important member of the cysteine protease family and has a central role in the development of ovaries in insects and fish. In this study, we analyzed the characteristics of the cathepsin L1 gene in the female oriental river prawn, Macrobrachium nipponense. The cathepsin L1 cDNA was 1173 bp in length, including a 960 bp open reading frame that encoded 319 amino acids. The cathepsin L1 gene was classified into the cathepsin L group and named Mn-CTS L1 by phylogenetic analysis. qRT-PCR analysis indicated that Mn-CTS L1 was highly expressed in the hepatopancreas and ovaries of female prawns. During the different stages of ovarian development, Mn-CTS L1 expression in the hepatopancreas and ovaries peaked before ovarian maturation. Mn-CTS L1 expression was higher at the post-larval stage of day 15 than at other stages of embryogenesis. In situ hybridization studies revealed that Mn-CTS L1 was localized in the oocyte of the ovary. Injection of Mn-CTS L1 dsRNA significantly reduced Mn-CTS L1 expression and significantly increased the expression of vitellogenin. Changes in the gonad somatic index also confirmed the inhibitory effects of Mn-CTS L1 dsRNA. These results suggest that Mn-CTS L1 has a key role in ovarian development.

Long-term subcellular intravital imaging in mammals is vital to study diverse intercellular behaviors and organelle functions during native physiological processes. However, optical heterogeneity, tissue opacity, and phototoxicity pose great challenges. Here, we propose a computational imaging framework, termed digital adaptive optics scanning light-field mutual iterative tomography (DAOSLIMIT), featuring high-speed, high-resolution 3D imaging, tiled wavefront correction, and low phototoxicity with a compact system. By tomographic imaging of the entire volume simultaneously, we obtained volumetric imaging across 225 × 225 × 16 μm3, with a resolution of up to 220 nm laterally and 400 nm axially, at the millisecond scale, over hundreds of thousands of time points. To establish the capabilities, we investigated large-scale cell migration and neural activities in different species and observed various subcellular dynamics in mammals during neutrophil migration and tumor cell circulation.

Defective in cullin neddylation 1(DCN1) is a co-E3 ligase that is important for cullin neddylation. Dysregulation of DCN1 highly correlates with the development of various cancers. Herein, from the initial high-throughput screening, a novel hit compound 5a containing a phenyltriazole thiol core (IC50 value of 0.95 μM for DCN1-UBC12 interaction) was discovered. Further structure-based optimization leads to the development of SK-464 (IC50 value of 26 nM). We found that SK-464 not only directly bound to DCN1 in vitro, but also engaged cellular DCN1, suppressed the neddylation of cullin3, and hindered the migration and invasion of two DCN1-overexpressed squamous carcinoma cell lines (KYSE70 and H2170). These findings indicate that SK-464 may be a novel lead compound targeting DCN1-UBC12 interaction.

Potassium antimonite was used to investigate the localisation of calcium in developing wheat anthers to examine the relationship between Ca2+ and pollen development. During anther development, calcium precipitate formation increased in anther wall cells prior to microspore mother cell meiosis and appeared in microspores, suggesting the presence of a calcium influx from anther wall cells into the locule. Initially, the precipitates in microspore cytoplasm primarily accumulated in the mitochondria and destroyed their inner membranes (cisterns) to become small vacuoles, which expanded and fused, ultimately becoming a large vacuole during microspore vacuolisation. After microspore division and large vacuole decomposition, many calcium precipitates again accumulated in the small vacuoles, indicating that calcium from the large vacuole moved back into the cytoplasm of bicellular pollen.

Cholesterol is an important nutrient for crustaceans. In this study, we performed comparative transcriptome analyses to explore the transcriptome differences in the intestine, hepatopancreas, and muscle of Macrobrachium nipponense fed either a low cholesterol (LC) or high cholesterol (HC) diet (2.8 or 17.1g cholesterol per kg diet). High-throughput RNA-Seq was conducted, resulting in 7.65, 5.88, and 7.59G clean bases from the intestine, hepatopancreas, and muscle of the LC group, respectively, and 7.59, 6.73, and 6.70G clean bases from the same tissues of the HC group. Assembly of clean reads resulted in 230,946 unigenes. The following enriched pathways were identified: xenobiotic and drug metabolism by cytochrome P450; chloroalkane and chloroalkene degradation; metabolic and biosynthetic pathways; fatty acid metabolism and biosynthesis; and immune-related pathways. To the best of our knowledge, this is the first study to describe how functional unigenes and biosynthetic and metabolic pathways are affected by dietary cholesterol in aquatic crustaceans. Thus, these results contribute to our understanding of the molecular mechanisms underlying the cholesterol requirement of crustaceans.