NhaA facilitates the maintenance of bacterial envelope integrity and the evasion of complement attack contributing to extraintestinal pathogenic Escherichia coli virulence.

Extraintestinal pathogenic Escherichia coli (ExPEC) is responsible for severe bloodstream infections in humans and animals. However, the mechanisms underlying ExPEC's serum resistance remain incompletely understood. Through the transposon-directed insertion-site sequencing approach, our previous study identified nhaA, the gene encoding a Na+/H+ antiporter, as a crucial factor for infection in vivo. In this study, we investigated the role of NhaA in ExPEC virulence utilizing both in vitro models and systemic infection models involving avian and mammalian animals. Genetic mutagenesis analysis revealed that nhaA deletion resulted in filamentous bacterial morphology and rendered the bacteria more susceptible to sodium dodecyl sulfate, suggesting the role of nhaA in maintaining cell envelope integrity. The nhaA mutant also displayed heightened sensitivity to complement-mediated killing compared to the wild-type strain, attributed to augmented deposition of complement components (C3b and C9). Remarkably, NhaA played a more crucial role in virulence compared to several well-known factors, including Iss, Prc, NlpI, and OmpA. Our findings revealed that NhaA significantly enhanced virulence across diverse human ExPEC prototype strains within B2 phylogroups, suggesting widespread involvement in virulence. Given its pivotal role, NhaA could serve as a potential drug target for tackling ExPEC infections.

Citrate utilization under anaerobic environment in Escherichia coli is under direct control of Fnr and indirect control of ArcA and Fnr via CitA-CitB system.

Most Escherichia coli (E. coli) strains do not cause disease, naturally living in the lower intestine and is expelled into the environment within faecal matter. Escherichia coli can utilize citrate under anaerobic conditions but not aerobic conditions. However, the underlying regulatory mechanisms are poorly understood. In this study, we explored regulatory mechanisms of citrate fermentation genes by global regulators ArcA and Fnr under anaerobic conditions. A gel mobility shift assay showed that the regulator proteins ArcA and Fnr binded to the promoter region localized between the citAB and citCDEFXGT operons. Subsequent assays confirmed that ArcA indirectly controled the expression of citrate fermentation genes via regulating CitA-CitB system, while Fnr directly regulated but also indirectly modulated citrate fermentation genes via controling CitA-CitB system. Deletions of arcA and fnr significantly reduced the growth of Escherichia coli in M9 medium with a citrate carbon source. We conclude that both ArcA and Fnr can indirectly control the citrate utilization via CitA-CitB system, while Fnr can also directly regulate the expression of citrate fermentation genes in E. coli under anaerobic conditions.

Role of enterotoxigenic Escherichia coli prophage in spreading antibiotic resistance in a porcine-derived environment.

Enterotoxigenic Escherichia coli (ETEC) cause acute secretory diarrhoea in pigs, posing a great economic loss to the swine industry. This study analysed the prevalence and genetic characteristics of prophages from 132 ETEC isolates from symptomatic pigs to determine their potential for spreading antibiotic resistance. A total of 1105 potential prophages were identified, and the distribution of the genome size showed three 'overlapping' trends. Similarity matrix comparison showed that prophages correlated with the ETEC lineage distribution, and further identification of these prophages corroborated the lineage specificity. In total, 1206 antibiotic resistance genes (ARGs) of 52 different categories were identified in 132 ETEC strains; among these, 2.65% (32/1206) of ARGs were found to be carried by prophages. Analysis of flanking sequences showed that almost all the ARGs could be grouped into two types: 'blaTEM-1B ' and 'classic class 1 integron (IntI1)'. They co-occurred with a strictly conserved recombinase and transposon Tn3 family but with a difference: the 'blaTEM-1B type' prophages exhibited a classic Tn2 transposon structure with 100% sequence identity, whereas the 'IntI1 type' co-occurred with the TnAs2 transposon with only 84% sequence identity. These results imply that ARGs might be pervasive in natural bacterial populations through transmission by transposable bacteriophages.

Extraintestinal pathogenic Escherichia coli increase extracytoplasmic polysaccharide biosynthesis for serum resistance in response to bloodstream signals.

Extraintestinal pathogenic Escherichia coli (ExPEC) is one of the leading causes of bloodstream infections. Characteristically, these organisms exhibit strong resistance to the bactericidal action of host serum. Although numerous serum resistance factors in ExPEC have been identified, their regulatory mechanisms during in vivo infection remain largely unknown. Here, RNA sequencing analyses together with quantitative reverse-transcription PCR revealed that ExPEC genes involved in the biosynthesis of extracytoplasmic polysaccharides (ECPs) including K-capsule, lipopolysaccharide (LPS), colanic acid, peptidoglycan and Yjb exopolysaccharides were significantly upregulated in response to serum under low oxygen conditions and during bloodstream infection. The oxygen sensor FNR directly activated the expression of K-capsule and colanic acid and also indirectly modulated the expression of colanic acid, Yjb exopolysaccharides and peptidoglycan via the known Rcs regulatory system. The global regulator Fur directly or indirectly repressed the expression ofECP biosynthesis genes in iron replete media, whereas the low iron conditions in the bloodstream could relieve Fur repression. Using in vitro and animal models, FNR, Fur and the Rcs system were confirmed as contributing to ExPEC ECP production, serum resistance and virulence. Altogether, these findings indicated that the global regulators FNR andFur and the signaling transduction system Rcs coordinately regulated the expression of ECP biosynthesis genes leading to increased ExPEC serum resistance in response to low oxygen and low iron levels in the bloodstream.

Genotypes and Antimicrobial Susceptibility Profiles of Hemolytic Escherichia coli from Diarrheic Piglets.

Hemolytic Escherichia coli are important pathogens in neonatal and weaned pigs. In this study, we analyzed 95 hemolytic E. coli isolated from intestinal contents or fecal samples of diarrheic piglets in 15 states of the United States between November 2013 and December 2014. Phenotypic antimicrobial susceptibility was determined through Sensititre BOFO6F plates for all the strains. They were all resistant to clindamycin, penicillin, tiamulin, tilmicosin, and highly resistant to oxytetracycline (91.6%), chlortetracycline (78.9%), ampicillin (75.8%), and sulfadimethoxine (68.4%). 86.2% of them were multidrug resistant. Whole genome sequencing (WGS) showed that 55 strains were enterotoxigenic E. coli (ETEC) and 40 strains were non-ETEC, and the strains belonged to 22 known and 2 novel sequence types (STs). ST100 and ST10 were the main and predominant STs in ETEC strains, whereas the non-ETEC strains were diverse with ST23 and ST761 as the main STs. Antibiotic resistance gene/mutation profiling of the genomes confirmed the results of antimicrobial susceptibility test. Notably, significant differences were found in the susceptibility to enrofloxacin between ETEC and non-ETEC (58.2% vs. 5.0%) and gentamicin (32.7% vs. 7.5%). ampH, ampC2, and ampC1 were the most common beta-lactamase genes in all E. coli strains, and extended-spectrum beta-lactamase (ESBL) genes were rare in these isolates. This study provides new insights into antibiotic resistance and genotypes of intestinal pathogenic E. coli associated with swine disease in the United States, and support the utility of WGS in accurate prediction of resistance to most antibiotics.

Effect of Thyrotropin on Osteopontin, Integrin αvß3, and VCAM-1 in the Endothelium via Activation of Akt.

Numerous epidemiological studies have shown that subclinical hypothyroidism (SCH) can impair endothelial function and cause dyslipidemia. Studies have evaluated the effects of thyroid stimulating hormone (TSH) on endothelial cells, but the mechanism underlying the proatherosclerotic effect of increased TSH levels remains unclear. In the present study, SCH rat models were established in thyroidectomized Wistar rats that were given ʟ-T4 daily. The results showed that in vivo, the expression of osteopontin (OPN) vascular cell adhesion molecule (VCAM-1), and levels of integrin αvß3 in the aortic tissue in SCH and Hypothyroidism (CH) groups was higher than in the control group. However, the effect in the SCH group was higher than in the CH group. In vitro, results showed that different concentration and time gradients of TSH stimulation could increase the expression of OPN, VCAM-1, and integrin αvß3, and this was accompanied by extracellular signal regulated kinase 1/2 (Erk1/2) and Akt activation in human umbilical vein endothelial cells (HUVECs). TSH induced elevation of these proatherosclerotic factors was partially suppressed by a specific Akt inhibitor but not by a specific Erk inhibitor. Findings suggested that the endothelial dysfunction caused by SCH was related to increased proatherosclerotic factors induced by TSH via Akt activation.

ArcA Controls Metabolism, Chemotaxis, and Motility Contributing to the Pathogenicity of Avian Pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) strains cause one of the three most significant infectious diseases in the poultry industry and are also potential food-borne pathogens threating human health. In this study, we showed that ArcA (aerobic respiratory control), a global regulator important for E. coli's adaptation from anaerobic to aerobic conditions and control of that bacterium's enzymatic defenses against reactive oxygen species (ROS), is involved in the virulence of APEC. Deletion of arcA significantly attenuates the virulence of APEC in the duck model. Transcriptome sequencing (RNA-Seq) analyses comparing the APEC wild type and the arcA mutant indicate that ArcA regulates the expression of 129 genes, including genes involved in citrate transport and metabolism, flagellum synthesis, and chemotaxis. Further investigations revealed that citCEFXG contributed to APEC's microaerobic growth at the lag and log phases when cultured in duck serum and that ArcA played a dual role in the control of citrate metabolism and transportation. In addition, deletion of flagellar genes motA and motB and chemotaxis gene cheA significantly attenuated the virulence of APEC, and ArcA was shown to directly regulate the expression of motA, motB, and cheA. The combined results indicate that ArcA controls metabolism, chemotaxis, and motility contributing to the pathogenicity of APEC.

Molecular characterization of insulin-like androgenic gland hormone-binding protein gene from the oriental river prawn Macrobrachium nipponense and investigation of its transcriptional relationship with the insulin-like androgenic gland hormone gene.

Insulin-like androgenic gland hormone-binding protein (IAGBP) has been investigated in crustaceans in vitro. However, the relationship between IAGBP and its putative binding protein partner insulin-like androgenic gland hormone (IAG) has not been studied at the transcriptional level in vivo. In the current study, we cloned the full-length cDNA of IAGBP from the oriental river prawn Macrobrachium nipponense (Mn-IAGBP) and investigated the transcriptional patterns of Mn-IAGBP and the M. nipponense IAG gene (Mn-IAG) at different developmental stages and in different tissues. Mn-IAGBP mRNA was detected in all examined tissues from adult male prawns, with the highest transcriptional levels in the testis. Mn-IAG mRNA was detected in the androgenic gland and hepatopancreas. The genomic sequences of Mn-IAGBP and Mn-IAG were isolated by genome walking and two gene copies were found in both Mn-IAGBP and Mn-IAG. The relationship between Mn-IAGBP and Mn-IAG at the transcriptional level was studied by RNA interference. Injection of Mn-IAGBP double-stranded RNA (dsRNA) significantly reduced the transcription of Mn-IAG, while injection of Mn-IAG dsRNA significantly reduced the transcription of Mn-IAGBP in testis, muscle, androgenic gland, and hepatopancreas. These results demonstrate the involvement of the IAGBP gene in IAG signaling in M. nipponense.

ArcA positively regulates the expression of virulence genes and contributes to virulence of porcine Shiga toxin-producing enterotoxigenic Escherichia coli.

IMPORTANCE: Enterotoxigenic Escherichia coli (ETEC) cause severe diarrhea in humans and animals, leading to death and huge economic loss worldwide. Thus, elucidation of ETEC's pathogenic mechanisms will provide powerful data for the discovery of drugs serving as prevention or therapeutics against ETEC-caused diarrheal diseases. Here, we report that ArcA plays an essential role in the pathogenicity and virulence regulation in ETEC by positively regulating the expression of several key virulence factors including F18 fimbriae, heat-labile and heat-stable toxins, Shiga toxin 2e, and hemolysin, under microaerobic conditions and in vivo. Moreover, we found that positive regulation of several virulence genes by ArcA requires a global repressor H-NS (histone-like nucleoid structuring), implying that ArcA may exert positive effects by antagonizing H-NS. Collectively, our data established a key role for ArcA in the pathogenicity of porcine ETEC and ETEC strains isolated from human infections. Moreover, our work reveals another layer of regulation in relation to oxygen control of virulence factors in ETEC.

Genomic Island-Encoded Histidine Kinase and Response Regulator Coordinate Mannose Utilization with Virulence in Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is a highly adaptive pathogen and has acquired diverse genetic elements, such as genomic islands and prophages, via horizontal gene transfer to promote fitness in vivo. Two-component signaling systems (TCSs) allow bacteria to sense, respond to, and adapt to various environments. This study identified a putative two-component signaling system composed of the histidine kinase EDL5436 (renamed LmvK) and the response regulator EDL5428 (renamed LmvR) in EHEC. lmvK and lmvR along with EDL5429 to EDL5434 (EDL5429-5434) between them constitute the OI167 genomic island and are highly associated with the EHEC pathotype. EDL5429-5434 encode transporters and metabolic enzymes that contribute to growth on mannose and are directly upregulated by LmvK/LmvR in the presence of mannose, as revealed by quantitative PCR (qPCR) and DNase I footprint assays. Moreover, LmvR directly activates the expression of the type III secretion system in response to mannose and promotes the formation of attaching and effacing lesions on HeLa cells. Using human colonoid and mouse infection models, we show that lmvK and lmvR contributed greatly to adherence and microcolony (MC) formation ex vivo and colonization in vivo. Finally, RNA sequencing and chromatin immunoprecipitation coupled with sequencing analyses identified additional direct targets of LmvR, most of which are involved in metabolism. Given that mannose is a mucus-derived sugar that induces virulence and is preferentially used by EHEC during infection, our data revealed a previously unknown mechanism by which EHEC recognizes the host metabolic landscape and regulates virulence expression accordingly. Our findings provide insights into how pathogenic bacteria evolve by acquiring genetic elements horizontally to adapt to host environments. IMPORTANCE The gastrointestinal tract represents a complex and challenging environment for enterohemorrhagic Escherichia coli (EHEC). However, EHEC is a highly adaptable pathogen, requiring only 10 to 100 CFUs to cause infection. This ability was achieved partially by acquiring mobile genetic elements, such as genomic islands, that promote overall fitness. Mannose is an intestinal mucus-derived sugar that stimulates virulence and is preferentially used by EHEC during infection. Here, we characterize the OI167 genomic island of EHEC, which encodes a novel two-component signaling system (TCS) and transporters and metabolic enzymes (EDL5429-5434) involved in mannose utilization. The TCS directly upregulates EDL5429-5434 and genes encoding the type III secretion system in the presence of mannose. Moreover, the TCS contributes greatly to EHEC virulence ex vivo and in vivo. Our data demonstrate an elegant example in which EHEC strains evolve by acquiring genetic elements horizontally to recognize the host metabolic landscape and regulate virulence expression accordingly, leading to successful infections.

An epidemiological study of the serum thyrotropin reference range and factors that influence serum thyrotropin levels in iodine sufficient areas of China.

The aims of this study performed in 2007 were to verify the selection criteria proposed by the National Academy of Clinical Biochemistry (NACB) guidelines, to investigate factors that influence thyrotropin (TSH) levels, and to determine serum TSH reference range in iodine sufficient areas of China. After excluding 291 subjects, a total of 5,348 inhabitants from three iodine sufficient areas of Liaoning province were asked to fulfill the questionnaire, and take TSH, thyroid peroxidase antibody (TPOAb), and thyroglobulin antibody (TgAb) measurements and a thyroid ultrasound examination. The distribution of TSH was right skewed in normal people. It has been customary to log transform the values to observe the Gaussian distribution. In the subjects 12-19 years of age, the TSH level was significantly higher than in the other age groups (p<0.001), while there were no significant difference in the TSH values of the other age groups. The TSH levels in females(1.68±1.90mIU/L) were significantly higher than in males (1.45±1.92mIU/L) (p<0.001). Therefore, the normal TSH range in males over age 20 was 0.43-4.74mIU/L, and in females the range was 0.48-5.39 mIU/L. A family history of thyroid disease, abnormal thyroid ultrasound, a thyroid antibody-positive status were the factors that influenced the TSH reference range. Non-thyroid disease did not impact the TSH reference range significantly. We recommend use of a TSH reference range 0.46-5.19mIU/L in iodine sufficient areas of China for males and females over 20 years old. We suggest using a normal thyroid ultrasound as a new criterion in addition to the NACB guidelines to determine the TSH reference range.

The relationship between serum thyrotropin and components of metabolic syndrome.

To explore the relationship between serum thyrotropin and components of metabolic syndrome in a Chinese cohort. A total of 1534 adult inhabitants in DaDong district of Shenyang were asked to fulfill the questionnaire, complete physical examination and OGTT. Blood samples were collected to test thyrotropin (TSH), fasting plasma glucose (FPG), OGTT 2h PG, fasting insulin (FINS), triglyceride (TG) and high density lipoprotein cholesterol (HDL-C). Serum TSH in metabolic syndrome group was higher than that in the non-metabolic syndrome group (2.54 mIU/L vs. 2.22 mIU/L, p<0.05). TG level increased significantly in subclinical hypothyroid group compared with euthyroid subjects (1.73±0.12 mmol/L vs. 1.47±0.03 mmol/L, p<0.05), and HDL-C decreased significantly in patients with subclinical hypothyroidism compared with euthyroid subjects (1.26±0.27 mmol/L vs. 1.33±0.27 mmol/L, p<0.05). The prevalence of hypertension was higher in the subclinical hypothyroid group than that in euthyroid group (42.86% vs. 33.2%, p<0.05). The serum TSH within the reference range was positively related with the prevalence of overweight/obesity. Slight increase in serum TSH maybe a risk factor for metabolic syndrome.

The correlation between thyrotropin and dyslipidemia in a population-based study.

This study investigated the relationship between serum thyrotrophin levels and dyslipidemia in subclinical hypothyroid and euthyroid subjects. A total of 110 subjects with subclinical hypothyroidism and 1,240 euthyroid subjects enrolled in this study. Patients with subclinical hypothyroidism had significantly lower high density lipoprotein cholesterol (HDL-C) levels than those who were euthyroid. The lipid profiles were each categorized and mean thyrotrophin levels were higher in subjects in the dyslipidemia subclasses than subjects in the normal subclasses. Thyrotrophin was positively associated with serum triglyceride and negatively associated with serum HDL-C in women. Thyrotrophin was also positively associated with total cholesterol (TC) in the overweight population along with TC and LDL-C in overweight women. In the euthyroid population, thyrotrophin was positively associated with TC in the overweight population. In conclusion, serum thyrotrophin was correlated with dyslipidemia in subclinical hypothyroid and euthyroid subjects; the correlation was independent of insulin sensitivity.

Transcriptomic and Metabolomic Profiling Reveals That KguR Broadly Impacts the Physiology of Uropathogenic Escherichia coli Under in vivo Relevant Conditions.

Urinary tract infections are primarily caused by uropathogenic Escherichia coli (UPEC). In contrast to the intestinal E. coli strains that reside in nutrient-rich gut environment, UPEC encounter distinct niches, for instance human urine, which is an oxygen- and nutrient-limited environment. Alpha-ketoglutarate (KG) is an abundant metabolite in renal proximal tubule cells; and previously we showed that two-component signaling system (TCS) KguS/KguR contributes to UPEC colonization of murine urinary tract by promoting the utilization of KG as a carbon source under anaerobic conditions. However, knowledge about the KguR regulon and its impact on UPEC fitness is lacking. In this work, we analyzed transcriptomic and metabolomic changes caused by kguR deletion under anaerobiosis when KG is present. Our results indicated that 620 genes were differentially expressed in the ΔkguR mutant, as compared to the wild type; of these genes, 513 genes were downregulated and 107 genes were upregulated. Genes with substantial changes in expression involve KG utilization, acid resistance, iron uptake, amino acid metabolism, capsule biosynthesis, sulfur metabolism, among others. In line with the transcriptomics data, several amino acids (glutamate, lysine, etc.) and uridine 5'-diphosphogalactose (involved in capsule biosynthesis) were significantly less abundant in the ΔkguR mutant. We then confirmed that the ΔkguR mutant, indeed, was more sensitive to acid stress than the wild type, presumably due to downregulation of genes belonging to the glutamate-dependent acid resistance system. Furthermore, using gene expression and electrophoretic mobility shift assays (EMSAs), we demonstrate that KguR autoregulates its own expression by binding to the kguSR promoter region. Lastly, we performed a genome-wide search of KguR binding sites, and this search yielded an output of at least 22 potential binding sites. Taken together, our data establish that in the presence of KG, KguR broadly impacts the physiology of UPEC under anaerobiosis. These findings greatly further our understanding of KguS/KguR system as well as UPEC pathobiology.

Whole genome sequencing analysis of avian pathogenic Escherichia coli from China.

Avian pathogenic Escherichia coli (APEC) can cause localized or systemic infection in poultry herds, i.e., colibacillosis, which is an economically devastating bacterial disease of the poultry industry worldwide. Additionally, some APEC may have zoonotic potential. In this study, we sequenced 125 APEC isolates from chickens and ducks with obvious clinical symptoms in poultry farms in China and performed genomic epidemiological analysis along with 16 APEC reference genomes downloaded from NCBI. The phylogenetic analysis indicated a great diversity of APEC isolates, and a total of 35 different O types, 22 H types, and 29 ST types were identified. Several virulence-associated genes (VAGs), such as ompT (96.45 %), iss (97.87 %), and hlyF (90.78 %), as well as four complete siderophore gene clusters, including the Sit transport system (86.52 %), aerobactin (89.36 %), salmochelin (79.43 %), and yersiniabactin (54.61 %), were detected in APEC isolates with high prevalence, which could serve as virulence markers of APEC. Several virulence-associated gene clusters, including the two T6SS systems and the K1 capsule biosynthesis gene clusters, were significantly associated with APEC of phylogroups B2, D, and F but very rarely encoded by the APEC from phylogroups C and E. In addition, several virulence-associated genes, which have been reported in other E. coli pathotypes but have not been reported in APEC, were identified in this study. Our findings in this study have implications for a better understanding of APEC evolution and pathogenesis and may lead to the development of new diagnostic tools for APEC.

Cyclooxygenase-2 Inhibition Reduces Autophagy of Macrophages Enhancing Extraintestinal Pathogenic Escherichia coli Infection.

Extraintestinal pathogenic Escherichia coli (ExPEC) is one of the top pathogens responsible for bloodstream infection and severe, often fatal, sepsis. Although the virulence factors and host immune responses to ExPEC infection have been investigated, the responses to a particular ExPEC strain could be very different. In this study, we investigated the mechanisms of Cyclooxygenase-2 (COX-2) up-regulation in influencing the host defenses against infection of ExPEC XM O2:K1:H7. Our results demonstrated that ExPEC XM O2:K1:H7 infection in mouse and RAW264.7 macrophages leads to COX-2 up-regulation, and COX-2 inhibition significantly enhances ExPEC infection. The up-regulation of COX-2 in macrophages was mediated by Toll-like receptor 4 (TLR4) through the activation of p38 and extracellular signal-regulated kinase/Mitogen-activated protein kinase (ERK/MAPK) pathways. Further studies showed that COX-2 inhibition significantly decreased autophagy in macrophages during ExPEC XM O2:K1:H7 infection. Autophagy inhibition significantly enhanced, while induction reduced ExPEC XM O2:K1:H7 survival in macrophages. In addition, COX-2 inhibition significantly increased macrophage cell death during ExPEC XM O2:K1:H7 infection and increased the expression of anti-inflammatory cytokine interleukin-10 (IL-10). Our results indicate that COX-2 up-regulation benefits host defense against ExPEC XM O2:K1:H7 infection by increasing autophagy in macrophages and by reducing IL-10 expression and macrophage cell death during ExPEC infection.

Thyrotropin Regulates eNOS Expression in the Endothelium by PGRN Through Akt Pathway.

To investigate the expression of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) in the aorta of subclinical hypothyroidism (SCH) rat model. The mechanisms underlying thyrotropin (TSH) affecting eNOS and PGRN expression in human umbilical vein endothelial cells (HUVECs) cultured in vitro were investigated. In the current study, SCH rat models were established by the administration of L-T4 injection after thyroidectomy in Wistar rats, as opposed to that in the normal and clinical hypothyroidism (CH) groups. The concentrations of NO (pmol/µL) in the SCH and CH groups were significantly lower than that in the normal group (40.8 ± 7.6 and 32.9 ± 10.8 vs. 51.2 ± 12.1, P < 0.05). However, the expression level of eNOS is increased significantly (P < 0.05) in both SCH and CH groups; a similar result was observed for the PGRN protein. In cultured HUVECs, TSH can also up-regulate the expression of eNOS; however, it is accompanied by a reduced concentration of NO and increased level of superoxide anion, thereby indicating uncoupled eNOS. As eNOS is increased, we found that Akt in HUVECs were upregulated by TSH, as well as PGRN expression. While inhibiting the expression of PGRN in HUVECs using siRNA, the expression of eNOS, as well as Akt were also inhibited. In conclusion, SCH can induce vascular endothelial dysfunction in rats, and PGRN participated in the process of TSH-induced expression of Akt/eNOS in the endothelium.

Characterization, expression patterns of molt-inhibiting hormone gene of Macrobrachium nipponense and its roles in molting and growth.

The oriental river prawn, Macrobrachium nipponense, is an important commercial aquaculture resource in China. In order to overwinter, M. nipponense displays decreased physiological activity and less consumption of energy. Sudden warming would trigger molting and cause an extensive death, resulting in huge economic losses. Therefore, it is of great practical significance to study the molting mechanism of oriental river prawns. Molt-inhibiting hormone gene (MIH) plays a major role in regulating molting in crustaceans. In this study, a full length MIH cDNA of M. nipponense (Mn-MIH) was cloned from the eyestalk. The total length of the Mn-MIH was 925 bp, encoding a protein of 119 amino acids. Tissue distribution analysis showed that Mn-MIH was highly expressed in the eyestalk, and that it had relatively low expression in gill, ovary, and abdominal ganglion. Mn-MIH was detected in all developmental stages, and changed regularly in line with the molting cycle of the embryo and larva. Mn-MIH varied in response to the molting cycle, suggesting that Mn-MIH negatively regulates ecdysteroidogenesis. Mn-MIH inhibition by RNAi resulted in a significant acceleration of molting cycles in both males and females, confirming the inhibitory role of MIH in molting. After long-term RNAi males, but not females had significant weight gain, confirming that Mn-MIH plays an important role in growth of M. nipponense. Our work contributes to a better understanding of the role of Mn-MIH in crustacean molting and growth.

Cloning of genomic sequences of three crustacean hyperglycemic hormone superfamily genes and elucidation of their roles of regulating insulin-like androgenic gland hormone gene.

The insulin-like androgenic gland hormone (IAG) gene in crustaceans plays an important role in male sexual differentiation, metabolism, and growth. However, the upstream regulation of IAG signaling schemes remains poorly studied. In the present study, we cloned the 5' flanking sequence of IAG and full-length genomic sequences of gonad-inhibiting hormone (Mn-GIH), molt-inhibiting hormone (Mn-MIH) and crustacean hyperglycemic hormone (Mn-CHH) in Macrobrachium nipponense. We identified the transcription factor-binding sites in the 5' flanking sequence of IAG and investigated the exon-intron patterns of the three CHH superfamily genes. Each CHH superfamily gene consisted of two introns separating three exons. Mn-GIH and Mn-MIH shared the same intron insertion sites, which differed from Mn-CHH. We provided DNA-level evidence for the type definition. We also identified two cAMP response elements in the 5' untranslated region. We further investigated the regulatory relationships between Mn-GIH, Mn-MIH, and Mn-CHH and IAG at the transcriptional level by injection of double-stranded RNA (dsRNA). IAG transcription levels were significantly increased to 660.2%, 472.9%, and 112.4% of control levels in the Mn-GIH dsRNA, Mn-MIH dsRNA, and Mn-CHH dsRNA groups, respectively. The results clearly demonstrated that Mn-GIH and Mn-MIH, but not Mn-CHH, negatively regulate the expression of the IAG gene.

Six chitinases from oriental river prawn Macrobrachium nipponense: cDNA characterization, classification and mRNA expression during post-embryonic development and moulting cycle.

Chitinase plays crucial physiological roles in crustaceans, including the digestion of chitin-containing food, moulting and the defense of shrimp against viruses. However, in contrast to insect species, no genome-wide analysis has been carried out in crustacean species and cDNAs encoding chitinase and chitinase-like proteins have been characterized in relatively few species. In this study, we identified six chitinase genes in the oriental river prawn, Macrobrachium nipponense, according to the established expressed sequence tag (EST) information using Rapid Amplification of the cDNA Ends (RACE) technique and homology cloning. We assigned these genes to three different chitinase groupings, which were designated MnCht1A, 1B, 3A, 3B, 3C and 4. The domain organization analysis of the six MnCht proteins revealed that only MnCht3C and MnCht4 possessed full structure, while MnCht1A, 1B, 3A and 3B lacked the serine/threonine (S/T)-rich linker and chitin-binding domains (CBDs). Their expression in different tissues and different developmental stages suggested that all of them have a function in the digestion of chitinous foods, modification of gut peritrophic membrane and degradation of the chitin exoskeleton. Analysis of the stage-specific moulting cycle and different temperature stimulation provided further evidence that MnCht1A, 1B and 3B have pivotal roles in the moulting cycle, while MnCht 4 only assists in the moulting process. This study provides important information for further investigations on the functions of chitinase in M. nipponense and other crustaceans.