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Microplastics (MPs) are a new type of environmental pollutant. To investigate the response of shrimp and their microflora to MPs, Litopenaeus vannamei (L. vannamei) was exposed to different concentrations of MPs (0, 50, 500, and 5000 μg/L, i.e., C, L, M and H groups) for 48 h. The survival rate, intake of MPs, immune-related gene expression and microbial response under MP exposure were detected. The results showed that the survival rate in the H group was significantly lower than those in the C, L and M groups, while the relative expression levels of proPO, TLR and ALF in the M and H groups were significantly higher than those in the C and L groups. For the microbial response, microbial community richness in the L group was significantly decreased, while community richness and diversity in the H group were significantly increased compared with those in the C group. The relative abundances of 3, 4 and 11 taxa were significantly changed after MP treatment at the phylum, class and genus levels, respectively. The results suggested that short-term exposure to low concentrations of MPs did not cause immune defense responses or death but affected the balance of bacterial composition in shrimp. Exposure to high concentrations of MPs can induce immune responses and microbial changes and can even cause death in shrimp. These findings increase our understanding of MP impacts on aquatic organisms.

Temperature is a significant environmental factor contributing to the success of aquaculture. To investigate the immune defense response during temperature fluctuation, Litopenaeus vannamei (L. vannamei) was treated under conditions of gradual cooling from an acclimation temperature of 28 °C (C group) to 13 °C (T group) in 2 days with a cooling rate of 7.5 °C/d and then rewarmed to 28 °C (R group) using the same rate. Relative expression of immune defense system-related genes and intestinal microbial composition in L. vannamei were investigated. The results showed that with a decrease in temperature, the expression of TLR, IMD, proPO and Casp3 in intestine was significantly decreased, while the expression of Muc-3A, Muc-5AC, Muc-17, IAP, p53, HSP70, MT and Fer was significantly increased after cooling. After temperature recovery, gene expression generally showed a trend of recovering to the normal level (C). Intestinal microbial analysis showed that, compared with the C group, the Chao and Ace indexes, the relative abundance of microflora from the Phylum Bacteroidetes, Class Alphaproteobacteria, and Class Bacteroidia, significantly decreased in the R group. The results revealed that cold-stress may decrease microbial community richness, alter the bacterial community in general and reduce shrimp immunity to pathogens and antibacterial activity. As a result, during temperature fluctuation shrimp may mobilize the immune defense system through upregulating the expression of Muc genes, anti-apoptosis related genes, and antioxidant related genes in order to maintain organism homeostasis as well as to repair damaged intestinal tissue.

MicroRNAs affect various pathological pathways by binding to multiple target mRNAs. Recently, it was reported that eukaryotic RNA G-quadruplexes were enriched in mRNA 3'-UTR, where microRNAs can also bind. To determine the roles of the G-quadruplex within mRNA 3'-UTR in microRNA binding sites, a bioinformatics approach was utilized to identify candidate microRNA target mRNAs with potential G-quadruplex formation. Circular dichroism spectrometry demonstrated the formation of RNA G-quadruplexes in vitro in candidate G-rich sequences. Mutated guanosines that are critical for G-quadruplex formation significantly decreased luciferase activities. Moreover, a G-quadruplex ligand TMPyP4 was used to destabilize the KCNJ11 RNA G-quadruplex in cardiomyocytes, resulting in binding of the microRNA to mRNA and subsequent suppression of KCNJ11 expression. In conclusion, our study showed that the G-quadruplex structure affects microRNA binding to its target mRNA. Thus, our study reveals a novel mechanism for G-quadruplex-dependent regulation of microRNA-mRNA interaction, which is essential to maintain normal gene expression.

Microorganisms sense environmental fluctuations in nutrients and light, coordinating their growth and development accordingly. Despite their critical roles in fungi, only a few G-protein coupled receptors (GPCRs) have been characterized. The Aspergillus nidulans genome encodes 86 putative GPCRs. Here, we characterise a carbon starvation-induced GPCR-mediated glucose sensing mechanism in A. nidulans. This includes two class V (gprH and gprI) and one class VII (gprM) GPCRs, which in response to glucose promote cAMP signalling, germination and hyphal growth, while negatively regulating sexual development in a light-dependent manner. We demonstrate that GprH regulates sexual development via influencing VeA activity, a key light-dependent regulator of fungal morphogenesis and secondary metabolism. We show that GprH and GprM are light-independent negative regulators of sterigmatocystin biosynthesis. Additionally, we reveal the epistatic interactions between the three GPCRs in regulating sexual development and sterigmatocystin production. In conclusion, GprH, GprM and GprI constitute a novel carbon starvation-induced glucose sensing mechanism that functions upstream of cAMP-PKA signalling to regulate fungal development and mycotoxin production., Author summary Sensing and responding to the environment is essential for the survival of all fungi. Despite our detailed knowledge of the signalling events that coordinate these adaptations, the extracellular receptors and stimuli that trigger these adaptations remain elusive. G-protein coupled receptors (GPCRs) are the largest class of receptors in fungi and regulate multiple aspects of their biology. Here, we characterized a novel GPCR-mediated glucose sensing mechanism in Aspergillus nidulans, which influences fungal development and toxin production. This work suggests fungal GPCRs sense glucose and propagate their signals through the cAMP-PKA and light-responsive VeA pathways to promote vegetative growth, while repressing sexual development and toxin production. Then we show how light also impacts on these nutrient sensing mechanisms. Our work reflects the complex and integrated structure of fungal mechanism to perceive different environmental stimuli, and how these mechanisms provide developmental and metabolic versatility to enable fungi to adapt to a fluctuating environment. Fungal GPCRs are poorly conserved in animals and plants, making them relevant antifungal drug targets to intervene in fungal disease and toxin contamination.

The present study aimed to evaluate the effect of kai xin san (KXS, at doses of 500, 250, and 125 mg/kg body weight per day), a well-known traditional Chinese medicine, on learning and memory in paradoxical sleep deprivation (PSD)-induced cognition deficit rats. Two behavior tests (the Open Field test and the Morris water maze task) were used for testing the effects of KXS on a PSD-induced learning and memory deficit model. Furthermore, its effect on the glutamic acid (GLU) and γ-amino-butyric acid (GABA) levels in the brain tissue, brain-derived neurotrophic factor (BDNF), cyclic AMP response element binding protein (CREB), and phosphorylated-CREB (p-CREB) expression in the hippocampus was also tested. KXS exerted the greatest cognition against the 48 h PSD-induced cognitive deficit and these effects may be mediated by decreasing the GLU and GABA levels and increasing the levels of BDNF, CREB, and p-CREB. This study indicates that the effect of KXS on learning and memory in a rat model of PSD could be associated with the modulation of neurotransmitter levels and the expression of some genes in the brain that contribute to memory functions.

Helicobacter pylori (H. pylori) is a spiral-shaped Gram-negative bacterium that causes the most common chronic infection in the human stomach. Approximately 1%-3% of infected individuals develop gastric cancer. However, the mechanisms by which H. pylori induces gastric cancer are not completely understood. The available evidence indicates a strong link between the virulence factor of H. pylori, cytotoxin-associated gene A (CagA), and gastric cancer. To further characterize H. pylori virulence, we established three cell lines by infecting the gastric cancer cell lines SGC-7901 and AGS with cagA+ H. pylori and transfecting SGC-7901 with a vector carrying the full-length cagA gene. We detected 135 differently expressed proteins from the three cell lines using proteome technology, and 10 differential proteins common to the three cell lines were selected and identified by LC-MS/MS as well as verified by western blot: β-actin, L-lactate dehydrogenase (LDH), dihydrolipoamide dehydrogenase (DLD), pre-mRNA-processing factor 19 homolog (PRPF19), ATP synthase, calmodulin (CaM), p64 CLCP, Ran-specific GTPase-activating protein (RanGAP), P43 and calreticulin. Detection of the expression of these proteins and genes encoding these proteins in human gastric cancer tissues by real-time PCR (RT-qPCR) and western blot revealed that the expression of β-ACTIN, LDH, DLD, PRPF19 and CaM genes were up-regulated and RanGAP was down-regulated in gastric cancer tissues and/or metastatic lymph nodes compared to peri-cancerous tissues. High gene expression was observed for H. pylori infection in gastric cancer tissues. Furthermore, the LDH, DLD and CaM genes were demethylated at the promoter -2325, -1885 and -276 sites, respectively, and the RanGAP gene was highly methylated at the promoter -570 and -170 sites in H. pylori-infected and cagA-overexpressing cells. These results provide new insights into the molecular pathogenesis and treatment targets for gastric cancer with H. pylori infection.

Chemosensory proteins (CSPs) are a class of soluble proteins present in high concentrations in the sensilla of insect antennae. It has been proposed that they play an important role in insect olfaction by mediating interactions between odorants and odorant receptors. Here we report, for the first time, the presence of five CSP genes in the tsetse fly Glossina morsitans morsitans, a major vector transmitting nagana in livestock. Real-time quantitative reverse transcription PCR showed that three of the CSPs are expressed in antennae. One of them, GmmCSP2, is transcribed at a very high level and could be involved in olfaction. We also determined expression in the antennae of both males and females at different life stages and with different blood feeding regimes. The transcription of GmmCSP2 was lower in male antennae than in females, with a sharp increase in 10-week-old flies, 48 h after a bloodmeal. Thus there is a clear relationship between CSP gene transcription and host searching behaviour. Genome annotation and phylogenetic analyses comparing G. morsitans morsitans CSPs with those of other Diptera showed rapid evolution after speciation of mosquitoes.

T-helper type 1 (Th1) immune response is involved in the development of protective immunity against Mycobacterium tuberculosis. Thus, an increase in Th1 and cellular immune responses should lead to enhanced anti-mycobacterial activity. In this study, we aimed to improve Th1 immune responses to a DNA vaccine by adding potentially immunostimulatory nucleotide sequences into the transcribed region downstream of the antigen. The Mycobacterium leprae gene for hsp65, codon-optimized for expression in mammalian cells, was inserted into pVAX1 with and without 3'-sequences containing CpG and dsRNA motifs. When the plasmid contained both motifs, transfected murine macrophage-like RAW264.7 cells showed markedly increased levels of mRNA for immune molecules of Th1 (IFN-α, IL-12) and Th17 (IL-17, IL-23 and IL-6) responses and for T cell co-stimulatory molecules (CD80 and CD86) but not for a Th2 response (IL-4 and IL-10). Immunized mice showed substantially increased serum anti-Hsp65 IgG2a antibody levels and IFN-γ production by spleen cells, confirming enhancement of the Th1 response in vivo. Furthermore, when non-vaccinated mice were infected with H37Rv by low-dose aerosol challenge, and then 4 weeks later were treated with plasmids by intramuscular injection, the mice that had been treated with plasmids containing immunostimulatory motifs showed an enhanced reduction in mycobacterial loads in lung and spleen. We conclude that DNA vaccines may be made more highly immunogenic and more effective for treatment by including transcribed stimulatory sequences.

Odorant-binding proteins (OBPs) play an important role in insect olfaction by mediating interactions between odorants and odorant receptors. We report for the first time 20 OBP genes in the tsetse fly Glossina morsitans morsitans. qRT-PCR revealed that 8 of these genes were highly transcribed in the antennae. The transcription of these genes in the antennae was significantly lower in males than in females and there was a clear correlation between OBP gene transcription and feeding status. Starvation over 72 h post-blood meal (PBM) did not significantly affect the transcription. However, the transcription in the antennae of 10-week-old flies was much higher than in 3-day-old flies at 48 h PBM and decreased sharply after 72 h starvation, suggesting that the OBP gene expression is affected by the insect’s nutritional status. Sequence comparisons with OBPs of other Dipterans identified several homologs to sex pheromone-binding proteins and OBPs of Drosophila melanogaster. Electronic supplementary material The online version of this article (doi:10.1007/s00018-009-0221-1) contains supplementary material, which is available to authorised users.

The Mediterranean fruit fly, Ceratitis capitata (medfly), is an extremely invasive agricultural pest due to its extremely wide host range and its ability to adapt to a broad range of climatic conditions and habitats. Chemosensory behaviour plays an important role in many crucial stages in the life of this insect, such as the detection of pheromone cues during mate pursuit and odorants during host plant localisation. Thus, the analysis of the chemosensory gene repertoire is an important step for the interpretation of the biology of this species and consequently its invasive potential. Moreover, these genes may represent ideal targets for the development of novel, effective control methods and pest population monitoring systems. Expressed sequence tag libraries from C. capitata adult heads, embryos, male accessory glands and testes were screened for sequences encoding putative odorant binding proteins (OBPs). A total of seventeen putative OBP transcripts were identified, corresponding to 13 Classic, three Minus-C and one Plus-C subfamily OBPs. The tissue distributions of the OBP transcripts were assessed by RT-PCR and a subset of five genes with predicted proteins sharing high sequence similarities and close phylogenetic affinities to Drosophila melanogaster pheromone binding protein related proteins (PBPRPs) were characterised in greater detail. Real Time quantitative PCR was used to assess the effects of maturation, mating and time of day on the transcript abundances of the putative PBPRP genes in the principal olfactory organs, the antennae, in males and females. The results of the present study have facilitated the annotation of OBP genes in the recently released medfly genome sequence and represent a significant contribution to the characterisation of the medfly chemosensory repertoire. The identification of these medfly OBPs/PBPRPs permitted evolutionary and functional comparisons with homologous sequences from other tephritids of the genera Bactrocera and Rhagoletis.

Moths depend on olfactory cues such as sex pheromones to find and recognize mating partners. Pheromone receptors (PRs) and Pheromone binding proteins (PBPs) are thought to be associated with olfactory signal transduction of pheromonal compounds in peripheral olfactory reception. Here six candidate pheromone receptor genes in the diamondback moth, Plutella xyllostella were identified and cloned. All of the six candidate PR genes display male-biased expression, which is a typical characteristic of pheromone receptors. In the Xenopus-based functional study and in situ hybridization, PxylOR4 is defined as another pheromone receptor in addition to the previously characterized PxylOR1. In the study of interaction between PRs and PBPs, PxylPBPs could increase the sensitivity of the complex expressing oocyte cells to the ligand pheromone component while decreasing the sensitivity to pheromone analogs. We deduce that activating pheromone receptors in olfactory receptor neurons requires some role of PBPs to pheromone/PBP complex. If the chemical signal is not the pheromone component, but instead, a pheromone analog with a similar structure, the complex would have a decreased ability to activate downstream pheromone receptors.

Two heterologous expression systems using thioredoxin (trxA) as a gene fusion part in Escherichia coli were developed to produce recombinant pediocin PA-1. Pediocin PA-1 structural gene pedA was isolated from Pediococcus acidilactici PA003 by the method of polymerase chain reaction (PCR), then cloned into vector pET32a(+), and expressed as thioredoxin-PedA fusion protein in the host strain E. coli BL21 (DE3). The fusion protein was in the form of inclusion body and was refolded before purification by nickel-iminodiacetic acid (Ni-IDA) agarose resin column. Biological activity of recombinant pediocin PA-1 was analyzed after cleavage of the fusion protein by enterokinase. Agar diffusion test revealed that 512-arbitrary unit (AU) recombinant pediocin PA-1 was obtained from 1 ml culture medium of E. coli (pPA003PED1) using Listeria monocytogenes as the indicator strain. Thioredoxin-PedA fusion gene was further cloned into pET20b(+). Thioredoxin-PedA fusion protein was detected in both the periplasmic and cytoplasmic spaces. The recombinant pediocin PA-1 from the soluble fraction attained 384 AU from 1 ml culture medium of E. coli (pPA003PED2). Therefore, biologically active pediocin PA-1 could be obtained by these two hybrid gene expression methods.

Host preference and blood feeding are restricted to female mosquitoes. Olfaction plays a major role in host-seeking behaviour, which is likely to be associated with a subset of mosquito olfactory genes. Proteins involved in olfaction include the odorant receptors (ORs) and the odorant-binding proteins (OBPs). OBPs are thought to function as a carrier within insect antennae for transporting occurs to the olfactory receptors. Here we report the annotation of 32 genes encoding putative OBPs in the malaria mosquito Anopheles gambiae and their tissue-specific expression in two mosquito species of the Anopheles complex, a highly anthropophilic species An. gambioe sensu stricto and an opportunistic, but more zoophilic species, An. arobiensis. RTPCR shows that some of the genes are expressed mainly in head tissue and a subset of these show highest expression in female heads. One of the genes (agCP1588) which has not been identified as an OBP, has a high similarity (40%) to the Drosophila pheromone- binding protein 4 (PBPRP4) and is only expressed in heads of both An. gombiae and An. arabiensis, and at higher levels in female heads. Two genes (agCP3071 and agCP15554) are expressed only in female heads and agC15554 also shows higher expression levels in An. gambiae. The expression profiles of the genes in the two members of the Anopheles complex provides the first step towards further molecular analysis of the mosquito olfactory apparatus. Arch. Insect Bio(hem. Physiol. 58:175-189, 2005. (C) 2005 Wiley-Liss, Inc.

To compare the expression patterns of cholecystokinin-B (CCK-B)/gastrin receptor genes in matched human gastric carcinoma and adjacent non-neoplastic mucosa of patients with gastric cancer, inflammatory gastric mucosa from patients with gastritis, normal stomachs from 2 autopsied patients and a gastric carcinoma cell line (SGC-7901), and to explore their relationship with progression to malignancy of human gastric carcinomas.RT-PCR and sequencing were employed to detect the mRNA expression levels of CCK-B receptor and gastrin gene in specimens from 30 patients with gastric carcinoma and healthy bordering non-cancerous mucosa, 10 gastritis patients and normal stomachs from 2 autopsied patients as well as SGC-7901. The results were semi-quantified by normalizing it to the mRNA level of beta-actin gene using Lab Image software. The sequences were analyzed by BLAST program.CCK-B receptor transcripts were detected in all of human gastric tissues in this study, including normal, inflammatory and malignant tissues and SGC-7901. However, the expression levels of CCK-B receptor in normal gastric tissues were higher than those in other groups (P0.05), and its expressions did not correlate with the differentiation and metastasis of gastric cancer (P0.05). On the other hand, gastrin mRNA was detected in SGC-7901 and in specimens obtained from gastric cancer patients (22/30) but not in other gastric tissues, and its expression was highly correlated with the metastases of gastric cancer (P0.05).Human gastric carcinomas and gastric cancer cell line SGC-7901 cells coexpress CCK-B receptor and gastrin mRNA. Gastrin/CCK-B receptor autocrine or paracrine pathway may possibly play an important role in the progression of gastric cancer.

To clone and construct the expression vector for human vascular endothelial growth factor (VEGF) genes.Total RNAs were extracted from human lung tissue of a 4-month-old fetus and subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) with the amplified products cloned into pMD18-T vector. Sequence analysis was performed before the amplified products were cloned into the expression plasmid pcDNA3.1-, the recombinant of which was verified by endonuclease digestion.After RT-PCR using a pair of primers (sense -21/7 bp and antisense 554/576 bp), two bands were identified. The band (487 bp) shorter in length was confirmed as VEGF121 (with the full length of VEGF121 being 444 bp) while the longer band (619 bp) was normal VEGF165 (with the full length of VEGF165 being 576 bp). Interestingly, another slightly longer VEGF165 nucleotide sequence was identified by sequencing analysis, which featured an unique 20 bp insertion precisely between exon 3 and exon 4 from the first ATG of human VEGF165 cDNA. The 20 bp insert was identified as the retaining intron 3 terminal nucleotides containing the splicing signal, which caused frame shift mutation in the reading frame and could probably give rise to a short polypeptides consisting of only 97 amino acid residuals due to the early appearance of stop code UAG in the middle of exon 4.We have successfully constructed the expression vector for VEGF121 and VEGF165 genes, and a new possible alternative splicing isoform of VEGF is identified in normal fetus whose molecular mechanism and physiological function needs further investigation.

To construct eukaryotic expression vector of thrombospondin-1 type I repeat sequences.Thrombospondin-1 type I repeat sequence gene was amplified from human fetal lung tissue by reverse transcriptase-PCR (RT-PCR) to construct both recombinant clone vector and expression vector through coupling reaction, followed by transforming these vectors into E.coli DH5alpha. The positive clones were selected for verification by double enzyme digestion and sequence analysis.The expected amplification product, thrombospondin-1 type I repeat gene sequence, was acquired by RT-PCR, which had a consistency up to 99% with the sequence from the GenBank.The eukaryotic expression vector PcDNA3.1(+)/TSP-1 type I repeat sequence was successfully constructed.

The Xenopus oocyte provides a powerful system for the expression and characterisation of plant membrane proteins. Many different types of plant membrane proteins have been expressed and characterised using this system. As there are already several general reviews on the methodology for oocyte expression of channel proteins, we have summarised the particular advantages and disadvantages of using the system for the characterisation of plant cotransporter proteins. As an example of how the system can be used to identify transporters, we describe evidence for a low affinity nitrate transporter in oocytes injected with poly(A) RNA extracted from nitrate-induced barley roots. Furthermore, we describe evidence that the expression of some transporters in oocytes can modify the properties of endogenous membrane proteins. We conclude that although care must be taken in the interpretation of results and in choosing appropriate controls for experiments, oocyte expression is an excellent tool which will have an important role in characterising plant membrane proteins.

BACKGROUND: A large number of insect chemosensory genes from different gene subfamilies have been identified and annotated, but their functional diversity and complexity are largely unknown. A systemic examination of expression patterns in chemosensory organs could provide important information. METHODOLOGY/PRINCIPAL FINDINGS: We identified 92 putative chemosensory genes by analysing the transcriptome of the antennae and female sex pheromone gland of the purple stem borer Sesamia inferens, among them 87 are novel in this species, including 24 transcripts encoding for odorant binding proteins (OBPs), 24 for chemosensory proteins (CSPs), 2 for sensory neuron membrane proteins (SNMPs), 39 for odorant receptors (ORs) and 3 for ionotropic receptors (IRs). The transcriptome analyses were validated and quantified with a detailed global expression profiling by Reverse Transcription-PCR for all 92 transcripts and by Quantitative Real Time RT-PCR for selected 16 ones. Among the chemosensory gene subfamilies, CSP transcripts are most widely and evenly expressed in different tissues and stages, OBP transcripts showed a clear antenna bias and most of OR transcripts are only detected in adult antennae. Our results also revealed that some OR transcripts, such as the transcripts of SNMP2 and 2 IRs were expressed in non-chemosensory tissues, and some CSP transcripts were antenna-biased expression. Furthermore, no chemosensory transcript is specific to female sex pheromone gland and very few are found in the heads. CONCLUSION: Our study revealed that there are a large number of chemosensory genes expressed in S. inferens, and some of them displayed unusual expression profile in non-chemosensory tissues. The identification of a large set of putative chemosensory genes of each subfamily from a single insect species, together with their different expression profiles provide further information in understanding the functions of these chemosensory genes in S. inferens as well as other insects.

Maize is one of the most important cereal crops worldwide and one of the primary targets of genetic manipulation, which provides an excellent way to promote its production. However, the obvious difference of the dedifferentiation frequency of immature maize embryo among various genotypes indicates that its genetic transformation is dependence on genotype and immature embryo-derived undifferentiated cells. To identify important genes and metabolic pathways involved in forming of embryo-derived embryonic calli, in this study, DGE (differential gene expression) analysis was performed on stages I, II, and III of maize inbred line 18-599R and corresponding control during the process of immature embryo dedifferentiation. A total of ∼21 million cDNA tags were sequenced, and 4,849,453, 5,076,030, 4,931,339, and 5,130,573 clean tags were obtained in the libraries of the samples and the control, respectively. In comparison with the control, 251, 324 and 313 differentially expressed genes (DEGs) were identified in the three stages with more than five folds, respectively. Interestingly, it is revealed that all the DEGs are related to metabolism, cellular process, and signaling and information storage and processing functions. Particularly, the genes involved in amino acid and carbohydrate transport and metabolism, cell wall/membrane/envelope biogenesis and signal transduction mechanism have been significantly changed during the dedifferentiation. To our best knowledge, this study is the first genome-wide effort to investigate the transcriptional changes in dedifferentiation immature maize embryos and the identified DEGs can serve as a basis for further functional characterization.