scPRAM accurately predicts single-cell gene expression perturbation response based on attention mechanism.

MOTIVATION: With the rapid advancement of single-cell sequencing technology, it becomes gradually possible to delve into the cellular responses to various external perturbations at the gene expression level. However, obtaining perturbed samples in certain scenarios may be considerably challenging, and the substantial costs associated with sequencing also curtail the feasibility of large-scale experimentation. A repertoire of methodologies has been employed for forecasting perturbative responses in single-cell gene expression. However, existing methods primarily focus on the average response of a specific cell type to perturbation, overlooking the single-cell specificity of perturbation responses and a more comprehensive prediction of the entire perturbation response distribution. RESULTS: Here, we present scPRAM, a method for predicting perturbation responses in single-cell gene expression based on attention mechanisms. Leveraging variational autoencoders and optimal transport, scPRAM aligns cell states before and after perturbation, followed by accurate prediction of gene expression responses to perturbations for unseen cell types through attention mechanisms. Experiments on multiple real perturbation datasets involving drug treatments and bacterial infections demonstrate that scPRAM attains heightened accuracy in perturbation prediction across cell types, species, and individuals, surpassing existing methodologies. Furthermore, scPRAM demonstrates outstanding capability in identifying differentially expressed genes under perturbation, capturing heterogeneity in perturbation responses across species, and maintaining stability in the presence of data noise and sample size variations. AVAILABILITY AND IMPLEMENTATION: https://github.com/jiang-q19/scPRAM and https://doi.org/10.5281/zenodo.10935038.

NAV-003, a bispecific antibody targeting a unique mesothelin epitope and CD3ε with improved cytotoxicity against humoral immunosuppressed tumors.

Mesothelin (MSLN) is a cell surface protein overexpressed in a number of cancer types. Several antibody- and cellular-based MSLN targeting agents have been tested in clinical trials where their therapeutic efficacy has been moderate at best. Previous studies using antibody and Chimeric Antigen Receptor-T cells (CAR-T) strategies have shown the importance of particular MSLN epitopes for optimal therapeutic response, while other studies have found that certain MSLN-positive tumors can produce proteins that can bind to subsets of IgG1-type antibodies and suppress their immune effector activities. In an attempt to develop an improved anti-MSLN targeting agent, we engineered a humanized divalent anti-MSLN/anti-CD3ε bispecific antibody that avoids suppressive factors, can target a MSLN epitope proximal to the tumor cell surface, and is capable of effectively binding, activating, and redirecting T cells to the surface of MSLN-positive tumor cells. NAV-003 has shown significantly improved tumor cell killing against lines producing immunosuppressive proteins in vitro and in vivo. Moreover, NAV-003 demonstrated good tolerability in mice and efficacy against patient-derived mesothelioma xenografts co-engrafted with human peripheral blood mononuclear cells. Together these data support the potential for NAV-003 clinical development and human proof-of-concept studies in patients with MSLN-expressing cancers.

Isolation and the pathogenicity characterization of Decapod iridescent virus 1 (DIV1) from diseased Macrobrachium nipponense and its activation on host immune response.

The high morbidity and mortality of Macrobrachium nipponense occurred in several farms in China, with cardinal symptoms of slow swimming, loss of appetite, empty of intestine, reddening of the hepatopancreas and gills. The pathogen has been confirmed as Decapod Iridescent Virus 1 (DIV1), namely DIV1-mn, by molecular epidemiology, histopathological examination, TEM observation, challenge experiment, and viral load detection. Histopathological analysis showed severe damage in hepatopancreas and gills of diseased prawns, exhibited few eosinophilic inclusions and pyknosis, and TEM of diseased prawns revealed that icosahedral virus particles existed in hepatopancreas and gill, which confirmed the disease of the farmed prawns caused by the DIV1 infection. Besides, challenge tests showed LD50 of DIV1 to M. nipponense was determined to be 2.14 × 104 copies/mL, and real-time PCR revealed that M. nipponense had a very high DIV1 load in the hemocytes, gills and hepatopancreas after infection. Furthermore, qRT-PCR was undertaken to investigated the expression of six immune-related genes in DIV1-infected M. nipponense after different time points, and the results revealed UCHL3, Relish, Gly-Cru2, CTL, MyD88 and Hemocyanin were significantly up-regulated in hemocytes, gills and hepatopancreas, which revealed various expression patterns in response to DIV1 infection. This study revealed that DIV1 infection is responsible for the mass mortality of M. nipponense, one of the important crustacean species, indicating its high susceptibility to DIV1. Moreover, this study will contribute to exploring the interaction between the host and DIV1 infection, specifically in terms of understanding how M. nipponense recognizes and eliminates the invading of DIV1.

rpoS involved in immune response of Macrobrachium nipponens to Vibrio mimicus infection.

Vibrio mimicus is a pathogenic bacterium that cause red body disease in Macrobrachium nipponense, leading to high mortality and financial loss. Based on previous studies, rpoS gene contribute to bacterial pathogenicity during infection, but the role of RpoS involved in the immune response of M. nipponense under V. mimicus infection remains unclear. In this study, the pathogen load and the RNA-seq of M. nipponense under wild-type and ΔrpoS strain V. mimicus infection were investigated. Over the entire infection period, the ΔrpoS strain pathogen load was always lower than that of the wild-type strain in the M. nipponense hemolymph, hepatopancreas, gill and muscle. Furthermore, the expression level of rpoS gene in the hepatopancreas was the highest at 24 hours post infection (hpi), then the samples of hepatopancreas tissue infected with the wild type and ΔrpoS strain at 24 hpi were selected for RNA-seq sequencing. The results revealed a significant change in the transcriptomes of the hepatopancreases infected with ΔrpoS strain. In contrast to the wild-type infected group, the ΔrpoS strain infected group exhibited differentially expressed genes (DEGs) enriched in 181 KEGG pathways at 24 hpi. Among these pathways, 8 immune system-related pathways were enriched, including ECM-receptor interaction, PI3K-Akt signaling pathway, Rap1 signaling pathway, Gap junction, and Focal adhesion, etc. Among these pathways, up-regulated genes related to Kazal-type serine protease inhibitors, S-antigen protein, copper zinc superoxide dismutase, tight junction protein, etc. were enriched. This study elucidates that rpoS can affect tissue bacterial load and immune-related pathways, thereby impacting the survival rate of M. nipponense under V. mimicus infection. These findings validate the potential of rpoS as a promising target for the development of a live attenuated vaccine against V. mimicus.

Long-term carbaryl exposure leads to behavioral abnormalities and reproductive toxicity in male marine medaka through apoptosis-mediated HPA and HPG axes dysregulation.

Carbaryl is a widely used carbamate pesticide that has been detected in the marine environment, but its effects on marine fish are still unknown. This study was aimed to investigate the effects of long-term exposure of carbaryl on male marine medaka. For this purpose, we set up five exposure concentration groups of 0, 0.1, 1, 10, and 100 µg/L for 180 days. On the one hand, we observed increased aggression and decreased ability to avoid predators in males after exposure, which was affected by the levels of HPA-axis hormones, especially decreased cortisol level. On the other hand, after exposure, HPG axis hormone levels and gene transcription levels were disturbed. Males exhibited a decreased gonadosomatic index and a notable reduction in mature sperm proportion and the F1 generation displayed a significant increase in malformation rate. Additionally, the number of apoptotic cells and the transcription level of apoptosis-related genes in the brains of male marine medaka substantially increased after exposure. Apoptosis of brain cells may be responsible for the disturbance of HPA and HPG axes, consequently leading to behavioral and reproductive abnormalities. These findings provide novel insights into evaluating the toxic effects of carbaryl on male marine medaka and emphasizing the criticality of exploring the potential environmental risks posed by carbaryl in the marine environment, thus providing toxicity value basis for further strengthening marine environmental monitoring and the protection of biological resources.

A 14-3-3 protein positively regulates rice salt tolerance by stabilizing phospholipase C1.

The phosphatidylinositol-specific phospholipase Cs (PI-PLCs) catalyze the hydrolysis of phosphatidylinositols, which play crucial roles in signaling transduction during plant development and stress response. However, the regulation of PI-PLC is still poorly understood. A previous study showed that a rice PI-PLC, OsPLC1, was essential to rice salt tolerance. Here, we identified a 14-3-3 protein, OsGF14b, as an interaction partner of OsPLC1. Similar to OsPLC1, OsGF14b also positively regulates rice salt tolerance, and their interaction can be promoted by NaCl stress. OsGF14b also positively regulated the hydrolysis activity of OsPLC1, and is essential to NaCl-induced activation of rice PI-PLCs. We further discovered that OsPLC1 was degraded via ubiquitin-proteasome pathway, and OsGF14b could inhibit the ubiquitination of OsPLC1 to protect OsPLC1 from degradation. Under salt stress, the OsPLC1 protein level in osgf14b was lower than the corresponding value of WT, whereas overexpression of OsGF14b results in a significant increase of OsPLC1 stability. Taken together, we propose that OsGF14b can interact with OsPLC1 and promote its activity and stability, thereby improving rice salt tolerance. This study provides novel insights into the important roles of 14-3-3 proteins in regulating protein stability and function in response to salt stress.

OsCYBDOMG1, a cytochrome b561 domain-containing protein, regulates salt tolerance and grain yield in rice.

KEY MESSAGE: OsCYBDOMG1 positively regulates salt tolerance, plant growth, and grain yield by affecting ascorbate biosynthesis and redox state. Soil salinity is a major abiotic stress affecting rice growth and productivity. Many genes involved in the salt stress response have been identified, but the precise mechanisms underlying salt tolerance remain unclear. In this study, we isolated a salt-sensitive mutant of rice, rss5, which exhibited more severe wilting and chlorosis with a significant increase in lipid peroxidation, electrolyte leakage, and shoot Na+ concentration compared to wild-type plants. Map-based cloning, MutMap analysis, and genetic complementation revealed that a single-nucleotide mutation in a gene encoding a cytochrome b561 domain-containing protein (OsCYBDOMG1) was responsible for the mutant phenotype of rss5. The OsCYBDOMG1 gene was mainly expressed in young shoots and nodes, and the encoded protein was principally located in the plasma membrane and endoplasmic reticulum. Mutations of OsCYBDOMG1 resulted in decreased ascorbic acid (AsA) content and AsA/DHA (dehydroascorbate) ratio, which led to increased H2O2 accumulation and reduced salt tolerance. Moreover, plant growth and grain yield of rss5 and the OsCYBDOMG1 knockout mutant (cr-1) were significantly decreased compared to wild-type plants under normal conditions. The elite haplotype of OsCYBDOMG1 associated with higher salt tolerance and grain width and weight was mainly existed in japonica varieties. These results suggest that OsCYBDOMG1 plays an important role in the regulation of salt tolerance, plant growth, and grain yield in rice.

Pathogenicity of Plesiomonas shigelloides causing mass mortalities of largemouth bass (Micropterus salmoides) and its induced host immune response.

The outbreak of mass mortality of M. salmoides occurred in an aquaculture farm in Jiangsu province of China, showing signs of skin ulceration and haemorrhages. The bacteria were isolated from diseased largemouth bass, and identified as Plesiomonas shigelloides based on morphological, physiological and biochemical features, as well as 16S rRNA gene sequence analysis. The pathogenicity of P. shigelloides was determined by challenge experiments, and the median lethal dosage (LD50) of the isolate NJS1 for M. salmoides was calculated as 1.6 × 105 CFU/mL at 7 d post-infection. Histopathological analysis revealed that extensive necrosis, vacuolization and inflammation were presented in the kidney, liver and gill of the diseased fish. Detection of virulence-related genes showed that P. shigelloides NJS1 was positive for astA, astB, astD, astE, actP and 6 ahpA. Additionally, the host defensive response of M. salmoides infected by P. shigelloides was analyzed by quantitive real-time PCR (qRT-PCR), and the results showed that the expression levels of Cas3, Hep1, HIF, IgM, IL15 and TGF were significantly up-regulated in head kidney, liver and spleen in different hours post-infection, which revealed varying expression profiles and clear transcriptional activation of immune related genes. The results suggested that P. shigelloides was an etiological element in the mass mortalities of M. salmoides and this study provided deeper insights for the pathogenesis and host defensive system in P. shigelloides invasion.

The Empty-Nest Power User Management Based on Data Mining Technology.

With the aging of the social population structure, the number of empty-nesters is also increasing. Therefore, it is necessary to manage empty-nesters with data mining technology. This paper proposed an empty-nest power user identification and power consumption management method based on data mining. Firstly, an empty-nest user identification algorithm based on weighted random forest was proposed. Compared with similar algorithms, the results indicate that the performance of the algorithm is the best, and the identification accuracy of empty-nest users is 74.2%. Then a method for analyzing the electricity consumption behavior of empty-nest users based on fusion clustering index adaptive cosine K-means was proposed, which can adaptively select the optimal number of clusters. Compared with similar algorithms, the algorithm has the shortest running time, the smallest Sum of the Squared Error (SSE), and the largest mean distance between clusters (MDC), which are 3.4281 s, 31.6591 and 13.9513, respectively. Finally, an anomaly detection model with an Auto-regressive Integrated Moving Average (ARIMA) algorithm and an isolated forest algorithm was established. The case analysis shows that the recognition accuracy of abnormal electricity consumption for empty-nest users was 86%. The results indicate that the model can effectively detect the abnormal behavior of empty-nest power users and help the power department to better serve empty-nest users.

The pathogenic characterization of Citrobacter freundii and its activation on immune related genes in Macrobrachium nipponense.

Out breaks of mass mortalities occurred in Macrobrachium nipponense farms in Jintan county, Jiangsu Province. The bacterial isolates from M. nipponense exhibited the same phenotypic traits and biochemical characteristics, and were identified as Citrobacter freundii according to biochemical characteristics and molecular identification. The infection test revealed that the strain YG2 was pathogenic to M. nipponense, and the half lethal dose (LD50) was 3.35 × 105 CFU/mL at 7 d post-infection. Detection of virulence genes indicated that YG2 was positive for cfa, ureG, ureF, ureE, ureD, viaB, ompX, and LDH. Furthermore, the results of extracellular enzyme analysis revealed that the strain can produce protease, amylase, lecithin, urease, and hemolysin. Antibiotic resistance results showed that the isolate was resistant to ampicillin, cefazolin, cephalothin, cefoxitin, aboren, doxycycline, neomycin, penicillin, erythromycin, and vancomycin. The expression level of MyD88, α2M, CDSP, and Relish were detected in hepatopancreas, hemolymph, gills and intestine tissues by quantitive real-time PCR (qRT-PCR), and clear transcriptional activation of these genes were observed in M. nipponense after C. freundii infection. These results revealed pathogenicity of C. freundii and its activation of host immune response, which will provide a scientific reference for the breeding and disease prevention in M. nipponense culture.

Pathogenicity of Aeromonas veronii causing mass mortalities of Odontobutis potamophila and its induced host immune response.

Aeromonas veronii is a freshwater bacterium associated with many diseases in aquatic animals. However, few cases of A. veronii infection were reported in Odontobutis potamophila, which has been becoming a promising fish species in China in recent years. In this study, the dominant bacteria were isolated from diseased O. potamophila showing signs of hemorrhage on fins, ulceration on the dorsal and abdomen. The representative isolate Stl3-1was identified as A. veronii based on analysis of its morphological, physiological, and biochemical features, as well as 16S rRNA and gyrB gene sequences. The median lethal dosage (LD50) of the isolate Stl3-1 for O. potamophila was determined as 4.5 × 105 CFU/mL. Histopathological analysis revealed that the isolate Stl3-1caused considerable histological lesions in the fish, including tissue cell degeneration, necrosis, and inflammatory cell infiltrating. Detection of virulence-related genes showed that A. veronii Stl3-1 was positive for exu, ompA, lip, flaH, hlyA, aer, flgM, tapA, act, flgA, gcaT and flgN. Additionally, quantitive real-time PCR (qRT-PCR) was also undertaken to analyses the host defensive response in O. potamophila infected by A. veronii. The immune-related gene expressions in O. potamophila during experimental infection were monitored at different point of time, and the results showed that the expression levels of MHC II, Myd88, TLR, and SOD were significantly up-regulated in liver, gill, spleen, and head kidney. The results revealed that A. veronii was a pathogen causing mass mortalities of O. potamophila and will contribute to better understanding the host defensive response against A. veronii infection.

[Analysis and suggestions on harvest period standards of plant medicinal materials in 2020 edition of Chinese Pharmacopoeia].

The improvement of the harvest period standards is critical in the quality control of Chinese medicinal materials. The present study statistically analyzed the harvest period standards of plant medicinal materials in the 2020 edition of Chinese Pharmacopoeia(Vol.Ⅰ) and put forward the existing problems and suggestions based on herbal records and modern research to provide references for the improvement of the standards. According to the statistical analysis, in 499 types of plant medicinal materials, harvest period standards are recorded under 486 types, accounting for 97.4%, and are lacking in the remaining. Only one medicinal material(Stellariae Radix) is recorded with the standard of the harvest year. The standards of the harvest season and phenological period are recorded under 233 types, accounting for 46.7%. For 237 types, only harvest season is specified, accounting for 47.5%, and for 15 types, only harvest phenological period is specified, accounting for 3.0%. Among 222 types mainly derived from cultivation and 51 types from wild resources and cultivation, only 11 types are recorded with harvest period of cultivated products. Only Stellariae Radix is recorded with the harvest period standards for the wild and cultivated products separately. The harvest period standards of plant medicinal materials with different medicinal parts have certain rules to follow. The main problems about the harvest period standards are discovered. Specifically, no harvest period standards are recorded under 13 types of plant medicinal materials. Almost all perennial cultivated medicinal materials are not recorded with harvest year standard. No phenological period standard is found under 250 types of plant medicinal materials. There is no clear distinction between the harvest period standards of cultivated and wild products. The evidence for harvest period standards of 26 types of plant medicinal materials that can be harvested all year round is insufficient. As a result, it is proposed to strengthen basic research in response to the above-mentioned problems and improve the harvest period standards as soon as possible to ensure the quality of Chinese medicinal materials.

Kin17 facilitates thyroid cancer cell proliferation, migration, and invasion by activating p38 MAPK signaling pathway.

Kin17 DNA and RNA binding protein (Kin17) is an extremely conserved nuclear protein that is almost expressed in every type of mammal cells. Recently, Kin17 has been implicated into the regulation of tumorigenesis of diverse human cancers. However, its functions in thyroid cancer (TC) are still largely unexplored. Kin17 mRNA and protein level were tested by qRT-PCR and western blot, respectively. Effects of Kin17 on TC cell proliferation were estimated by colony formation assay and flow cytometry analysis in vitro as well as by in vivo tumor growth experiment. TC cell migratory and invasive capacities were assessed via wound-healing and transwell experiments. Epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin and N-cadherin) and p38 MAPAK signaling pathway-related proteins (p-p38, p38, Cyclin D1, and p27) were examined via western blot. Kin17 was remarkably increased in TC tissue samples and cell lines at both mRNA and protein levels compared to normal tissue and control cell line. Knockdown of Kin17 obviously repressed TC cell proliferation, arrested cell cycle, and inhibited TC cell migration and invasion in vitro, while overexpression of Kin17 produced opposite effects. Kin17 knockdown suppressed p38 MAPK signaling pathway, while Kin17 overexpression activated this pathway. Treatment of p38 agonist (p79350) abolished the repressive effects of sh-Kin17 on TC cell proliferation, migration, and invasion, as well as on p38 pathway. Kin17 knockdown was also found to enhance the sensitivity of Doxorubicin of TC cells. In addition, Kin17 knockdown in vivo also markedly repressed TC tumor growth and p38 pathway. Kin17 functioned as an oncogene of TC by activating p38 MAPK signaling pathway.

Aeromonas hydrophila associated with red spot disease in Macrobrachium nipponense and host immune-related gene expression profiles.

In September 2018, a serious disease causing high mortality with red spot syndrome occurred in a Macrobrachium nipponense aquaculture farm in Jintan County, Jiangsu Province, China. In this study, a pathogenic isolate 5-S3 was isolated from diseased M. nipponense and was identified as Aeromonas hydrophila by phenotypically and molecularly. The pathogenicity of the isolate 5-S3 to M. nipponense was determined by challenge experiments. Results of artificial challenge showed A. hydrophila was pathogenic to M. nipponense, the LD50 was 9.58 × 104 CFU/mL, and histopathological analysis revealed that the hepatopancreas of infected M. nipponense exhibited obvious inflammatory responses to A. hydrophila infection. The isolate showed significant phenotypical activities such as the lecithinase, esterase, caseinase and hemolysin which are indicative of their virulence potential. Besides, virulence genes such as aerA, act, fla, ahpß, alt, lip, eprCAI, hlyA, acg and gcaT were detected in the isolate 5-S3. Subsequently, the immune-related genes expression in M. nipponense were evaluated by quantitative real-time PCR (qRT-PCR), and the results showed that the expression levels of dorsal, relish, crustin1, crustin2, anti-lipopolysaccharide factors 1 (ALF1), anti-lipopolysaccharide factors 2 (ALF2), hemocyanin, i-lysozyme and prophenoloxidase were significantly up-regulated in hepatopancreas of M. nipponense after A. hydrophila infection, the stat, p38, crustin3, anti-lipopolysaccharide factors 3 (ALF3) genes had no significant change during the infection. The present results reveal that A. hydrophila was an etiological agent causing red spot syndrome and mass mortality of M. nipponense and the influence of A. hydrophila infection on host immune genes.

Multi-biomarker assessment in the giant freshwater prawn Macrobrachium rosenbergii after deltamethrin exposure.

Deltamethrin (DM) is a synthetic pyrethroid used for agricultural purposes to control insects. However, its extensive use contaminates the aquatic environment and results in serious health problems in aquatic organisms. Knowledge about the toxic effect of DM in freshwater prawns is limited; therefore, this study aims to assess the toxicity of DM in Macrobrachium rosenbergii based on multiple biomarkers. Four-day acute toxicity tests showed that DM was highly toxic to M. rosenbergii with the 24 h, 48 h, 72 h and 96 h LC50 values to be 1.919, 0.603, 0.539, and 0.449 µg/L, respectively. According to 96 h LC50, prawns were exposed to DM at three concentrations (0.02, 0.08, and 0.32 µg/L) for 4 days, and then moved into fresh water for decontamination to investigate the toxic effect of DM in M. rosenbergii. At low concentration (0.02 µg/L and 0.08 µg/L), DM did not cause obvious histopathological damage to hepatopancreas and gill tissue, while at high concentration (0.32 µg/L), the histopathological harm was serious and the damage did not recover to the initial level after 7-day decontamination. 0.02 µg/L DM exposure did not induce significant changes in most of the biomarkers except the increased lactate dehydrogenase (LDH) activity, lactic acid (LD) level, and the first increased then decreased mRNA expression of immune-related genes, indicating the stimulation of DM on energy production and immunity. 0.08 µg/L and 0.32 µg/L DM exposure resulted in varying degrees of damage on prawns, but overall, their toxic effects showed similar trends based on the biomarkers. Increase in malonaldehyde (MDA) and hydrogen peroxide (H2O2) content and decrease in superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity after DM exposure demonstrated the oxidative stress caused by DM. The significantly increased acid phosphatase (ACP), alkaline phosphatase (AKP), LDH activity and LD level indicated hepatopancreatic dysfunction and respiration disruption. The first increased and then decreased expression pattern of immune-related genes indicated the immunosuppression caused by DM. After 7-day decontamination in freshwater, the activity/level of the biomarkers partly recovered. This study revealed the severe toxic effect of DM on Macrobrachium rosenbergii based on multiple biomarkers, providing fundamental knowledge for the establishment of DM toxicity assessment system with proper parameters in freshwater crustaceans.

Pathogenicity of Aeromonas hydrophila causing mass mortalities of Procambarus clarkia and its induced host immune response.

Outbreaks of mass mortalities among cultured Procambarus clarkia occurred in a commercial hatchery during the spring of 2019 in Jiangsu province of China. Here, we exploit the pathogenicity and immune response of Aeromonas hydrophila (GPC1-2), which was isolated from diseased P. clarkia. Crayfish challenged showed similar pathological signs to the naturally diseased P. clarkia, lethal dose 50% (LD50) of the strain GPC1-2 to P. clarkia was 3.8 × 106 CFU/mL. Detection of virulence-associated genes by PCR indicated that the strain GPC1-2 carried hlyA, aerA, alt, ast, act, aha, ahp, ahpA, and ahpB. Histopathological analysis of hepatopancreas revealed that the hepatic tubule lumen and the gap between the hepatic tubules became larger, and the brush border disappeared in the P. clarkia infected by GPC1-2. Quantitive real-time PCR (qRT-PCR) was undertaken to measure mRNA expression levels for six immune-related genes in P. clarkia after A. hydrophila infection. The expression level of proPO, NOS, ALF1, TLR2, PX, and AST were detected in hemolymph, hepatopancreas, gill and intestine tissues, and clear transcriptional activation of these genes were observed in the infected individuals. These results revealed pathogenicity of A. hydrophila and its activation of host immune response, which will provide a scientific reference for the breeding and disease prevention in P. clarkia culture.

New Synthesis for the Monobactam Antibiotic-LYS228.

A new synthesis of LYS228, fitting for further process development for commercial manufacture, is described. The key features of this synthesis include development of new protocols for acylation reactions, application of an asymmetric hydrogenation via dynamic kinetic resolution, and a late-stage ring closure to form ß-lactam 1.

Transcriptome analysis and immune-related genes expression reveals the immune responses of Macrobrachium rosenbergii infected by Enterobacter cloacae.

Macrobrachium rosenbergii is an important cultural species in China and other Southeast Asian countries. However, Enterobacter cloacae infection has caused a great economic loss in M. rosenbergii culture industry. The immune responses of M. rosenbergii to the E. cloacae infection is not fully characterized. To investigate the immune response of M. rosenbergii against E. cloacae, we performed transcriptome analysis of the M. rosenbergii hepatopancreas with and without E. cloacae infection using RNA-seq. After assembly and annotation, 29,731 high quality unigenes were obtained from RNA-seq data. Differential expression analysis revealed the existence of 2498 significantly differently expressed genes (DEGs) at 12 h post infection, with 1365 up-regulated and 1133 down-regulated genes. Among these DEGs, some well-known immune-related genes were up-regulated significantly, including C-type lectin 1, lectin 3, anti-lipopolysaccharide factor 2, Cu/Zn superoxide dismutase and heat shock protein 70. GO analysis demonstrated 24 biological process subcategories, 14 cellular component subcategories, and 12 molecular function subcategories that were enriched among these DEGs, and some DEGs were clustered into immune related subcategories such as immune system process, response to stimulus, biological adhesion, and antioxidant activity. These DEGs were enriched into 216 KEGG pathways including a core set of immune correlated pathways notably in phagosome and lysosome. In addition, 5 up-regulated and 5 down-regulated immune-related DEGs were selected for further validation by quantitative real-time PCR and the results showed consistence with the RNA-seq data. Additionally, the expression level of six selected immune-related genes (ALF2, CLEC1, LEC3, hemocyanin1, HSP70 and SOD) based on the transcriptomic data were monitored at different point of time in hepatopancreas, gill, hemolymph and intestine. Results revealed these immune-related genes were significantly up-regulated in different tissues from 6 to 24 h after E. cloacae infection. Overall, these results provided valuable information for further studying the immune response of M. rosenbergii against E. cloacae infection.

Insights into carbohydrate metabolism from an insulin-like peptide in Macrobrachium rosenbergii.

This study identified an insulin-like peptide (ILP) in Macrobrachium rosenbergii termed Mr-ILP and further investigated its function through glucose injection and RNAi. With the analysis of five other glucose metabolism related genes, this study shed light on the molecular mechanism of carbohydrate metabolism in crustaceans. Mr-ILP shared the typical skeleton with six conserved cysteine and mainly expressed in neuroendocrine system. In M. rosenbergii, the elevated hemolymph glucose concentration after glucose injection returned to basal levels in short time, implying an efficient regulatory system in carbohydrate metabolism. Hyperglycemic related genes answered the elevated hemolymph glucose concentration quickly with significant decreased expression level, while Mr-ILP showed delayed response. Instead, glycolysis increased after glucose injection, which indicated glycolysis might play an important role in lowering the abnormally high glucose level. In vivo silencing of Mr-ILP, by injecting the prawns with double-stranded RNA (dsRNA) for 21 days reduced its expression by approximately 75%. Accordingly, glycogen synthase decreased and the trehalose and glycogen level in the hepatopancreas were significantly reduced, indicating the function of Mr-ILP in oligosaccharide and polysaccharide accumulation. When Mr-ILP was silenced, the expression of hyperglycemic related genes were enhanced, but the hemolymph glucose level was not elevated significantly, which might attribute to the increased glycolysis to keep a balanced glucose level in hemolymph.

Sevoflurane Postconditioning Reduces Hypoxia-Reoxygenation Injury in H9C2 Embryonic Rat Cardiomyocytes and Targets the STRADA Gene by Upregulating microRNA-107.

BACKGROUND Sevoflurane as a widely used inhalational general anesthetic that also has a cardioprotective role in hypoxia-reoxygenation (H/R) injury. This study aimed to investigate the effects of microRNA-107 (miR-107) on sevoflurane postconditioning (SpostC) in H9C2 embryonic rat cardiomyocytes and to use bioinformatics analysis to identify the molecular basis of cardioprotection from sevoflurane in human cardiac tissue. MATERIAL AND METHODS The STRADA gene was identified from the Gene Expression Omnibus (GEO) database. H9C2 embryonic rat cardiomyocytes were cultured with sevoflurane. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to measure the mRNA expression and protein expression of STRADA and miR-107 in H9C2 cells. TargetScanHuman version 7.2 was used to identify the target gene of miR-107 and to predict the STRADA 3'-UTR binding site of miR-107. The dual-luciferase reporter assay measured the relative luciferase activity. The cell proliferation rate and cell apoptosis were measured using the MTT assay and flow cytometry, respectively. RESULTS H/R injury in H9C2 cells following SpostC resulted in increased expression of miR-107 and reduced expression of STRADA. Specific binding of miR-107 was identified to STRADA 3'-UTR. Upregulation of the miR-107 in SpostC H/R injured H9C2 cells promoted cell proliferation, reduced cell apoptosis, and downregulating the protein expression of caspase-3. STRADA overexpression reduced the effects of a miR-107 mimic on SpostC. CONCLUSIONS SpostC reduced H/R injury in H9C2 embryonic rat cardiomyocytes by targeting the STRADA gene and by upregulating the expression of microRNA-107.