The first Toll receptor from the triangle-shell pearl mussel Hyriopsis cumingii.

Toll receptor was first discovered in Drosophila and has an important function in the innate immunity of invertebrates. In this study, the Toll receptor HcToll1 from Hyriopsis cumingii with a full length of 3810 bp consisting of a 3687 bp ORF that encodes a total of 1228 amino acids protein was selected for further study. The HcToll1 protein consisted of a signal peptide, 17 LRR domains, 2 LRRCT domains, 1 LRRNT domain, 1 TM domain, and 1 TIR domain. Phylogenetic analysis results showed that HcToll1 was clustered in one group together with other mollusca tolls. RT-PCR analysis results showed that HcToll1 was expressed in all tested tissues such as hemocytes, hepatopancreas, gills, and mantle. qRT-PCR analysis results showed that HcToll1 expression was increased by the presence of Escherichia coli, Vibrio anguillarum, Staphyloccocus aureus, and White Spot Syndrome Virus (WSSV). Over-expression of HcTIR could up-regulate expression of drosomycin gene in Drosophila S2 cells. The results of our study indicated that HcToll1 is a functional Toll and it has an important function in the generation of innate immune responses of H. cumingii against microbial challenge.

Cloning and identification of four Mu-type glutathione S-transferases from the giant freshwater prawn Macrobrachium rosenbergii.

Glutathione S-transferases (GSTs) are essential components of the cellular detoxification system because of their capability to protect organisms against the toxicity of reactive oxygen species (ROSs). Four different GSTs (MrMuGST1-MrMuGST4) showing similarities with Mu-type GSTs were cloned from the hepatopancreas of Macrobrachium rosenbergii. These four GSTs have 219, 216, 218 and 219 amino acids in length, respectively. MrMuGST1-MrMuGST4 proteins all have a G-site in the N-terminus and an H-site in the C-terminus. Phylogenetic analysis reveals that four Mu-type GSTs are classified into two different clades (MrMuGST2 one clade; MrMuGST1, MrMuGST3 and MrMuGST4 other clades). Nonetheless, no site under positive selection was detected but rapid evolution was found in the few of MuGST genes. Reverse transcription-polymerase chain reaction (RT-PCR) results showed that MrMuGST1 and MrMuGST2 transcripts were expressed in all detected tissues, however, MrMuGST3 and MrMuGST4 were just mainly expressed in hepatopancreas and intestines. Quantitative RT-PCR analysis showed that MrMuGST1 and MrMuGST2 were down-regulated upon Vibrio anguillarum challenge, whereas MrMuGST3 and MrMuGST4 were quickly up-regulated 2 h after the Vibrio challenge. Our results imply that different Mu-type GSTs may respond to Vibrio challenge with different manners.

Curcumin inhibits the metastasis of K1 papillary thyroid cancer cells via modulating E-cadherin and matrix metalloproteinase-9 expression.

The anti-metastatic effect of curcumin on papillary thyroid cancer K1 cells and its underlying mechanisms were investigated. Curcumin at 12.5, 25 and 50 µM promoted mesenchymal-epithelial transition and decreased the migration rate of K1 cells by 24-87%. Its mechanism may involve the up-regulation of E-cadherin expression levels and down-regulation of the activity and expression of matrix metalloproteinase-9.

Inverse CO2/C2H2 separation assisted by coordinated water in a dysprosium(III) metal-organic framework.

An ultramicroporous metal-organic framework (MOF) constructed from dysprosium(III) and oxalate, termed Dy-F-oxa, is carefully studied for inverse separation of CO2 from C2H2. Adsorption experiments and modeling studies reveal that the high CO2 adsorption is attributed to the preferential sites for CO2 by coordinated water. After the equimolar gas mixture breakthrough experiment, C2H2 can be directly produced as a pure effluent.

Immune response of four dual-CRD C-type lectins to microbial challenges in giant freshwater prawn Macrobrachium rosenbergii.

C-type lectins (CTLs) are believed to play important roles in the innate immunity of invertebrates and serve as pattern recognition receptors, opsonins, or effector molecules. In this study, the full-lengths cDNA of 4 CTL genes from giant freshwater prawn Macrobrachium rosenbergii were cloned and designated as MrLec1, MrLec2, MrLec3, and MrLec4. All of these 4 lectin cDNAs encode proteins with 2 carbohydrate recognition domains (CRDs). While MrLec1, MrLec3, and MrLec4 had signal peptides, no signal peptide was detected in MrLec2. Two carbohydrate recognition motifs within two CRDs of each lectin were predicted (QPE, EPG in MrLec1; EPT, EPA in MrLec2; QPT, NPR in MrLec3; KPN, EPD in MrLec4). Phylogenetic analysis showed that MrLec4 belongs to group A whereas MrLec1, MrLec2, and MrLec3 belong to group B. Positive selection in dual-CRD lectins suggested their probable roles in innate immunity, and positively selected induced amino acid diversity of lectins may confer their ability to recognize a broad range of microbes. The qRT-PCR analysis in adult prawns showed that MrLec1 is mainly expressed in the hepatopancreas, gills, and stomach, MrLec2 and MrLec4 are mainly distributed in the hepatopancreas, and MrLec3 is mainly expressed in the hepatopancreas and stomach. Time-course analysis using qRT-PCR showed that MrLec1 to MrLec4 are all upregulated by the Vibrio anguillarum challenge. MrLec1 is upregulated after 2, 12, and 24 h of white spot syndrome virus (WSSV) challenge. The expression of MrLec2 increases after 12 and 24 h of WSSV challenge, and the transcript of MrLec3 and MrLec4 are downregulated after 2 h of WSSV challenge. The results suggest the potential roles of dual-CRD lectins in the innate immunity of M. rosenbergii.

Four invertebrate-type lysozyme genes from triangle-shell pearl mussel (Hyriopsis cumingii).

Lysozymes in animals have three types, namely chicken-type, goose-type, and invertebrate-type (i-type) lysozymes and all these 3 types have been found in bivalve mollusks. The i-type lysozymes in mollusks are involved in digestion and innate immunity. In this study, four different lysozyme genes that belong to i-type were identified from Hyriopsis cumingii. The HcLyso1 to HcLyso4 genes encode proteins with 144, 144, 161, and 228 amino acids, respectively, and contain a destabilase domain. HcLyso4 also contains SH3b domain in addition to its destabilase domain. Multiple alignments showed that two catalytic residues of Glu and Asp which were necessary for enzyme activity were present in i-type lysozymes. Phylogenetic analysis using CDS sequences of i-type lysozymes showed that these lysozymes can be divided into mollusk and crustacean clades, and that HcLyso1 to HcLyso4 all belong to the mollusk clades. Although there was no positive selection predicted in i-type lysozymes, some branches suffered rapid evolution. HcLyso1 is mainly expressed in hepatopancreas and can be detected in hemocytes. HcLyso2 is primarily expressed in hepatopancreas and can be detected in hemocytes Whereas, HcLyso3 can be detected mainly in hemocytes, hepatopancreas, gills, and mantle. HcLyso4 is expressed in hemocytes and hepatopancreas. qRT-PCR analysis showed that HcLyso1 to HcLyso4 were all nearly down-regulated by Vibrio or Staphylococcus aureus challenge. Moreover, our research indicated that HcLyso1 to HcLyso4 might play a key role in the innate immunity of mussel.

Three different anti-lipopolysaccharide factors identified from giant freshwater prawn, Macrobrachium rosenbergii.

Anti-lipopolysaccharide factor (ALF) is a type of basic protein and an important antimicrobial peptide that can bind and neutralize lipopolysaccharides (LPS). This protein shows a broad spectrum of antimicrobial activity. In this study, three forms of ALF designated as MrALF5, MrALF6, and MrALF7 were identified from giant freshwater prawn, Macrobrachium rosenbergii. MrALF5, MrALF6, and MrALF7 genes encode 133, 121, and 120 amino acids of the corresponding proteins, respectively. All these ALF proteins contain LPS-binding domain with two conserved cysteine residues. The genomic sequences of MrALF5 and MrALF7 were amplified. The genomic structures of MrALF5 and MrALF7 comprise three exons interrupted by two introns. Phylogenetic analysis showed that MrALF5, MrALF6, and MrALF7 were clustered into clade II. Evolutionary analysis showed that ALF genes from M. rosenbergii may suffer a rapid evolution. MrALF5 was expressed mainly in the hepatopancreas, gills, and heart. MrALF6 was mainly distributed in the intestine and hepatopancreas. The highest expression level of MrALF7 was detected in the hepatopancreas. MrALF6, as well as MrALF7, was downregulated by Escherichia coli challenge, and all three ALF genes were upregulated by Vibrio or white spot syndrome virus challenge. MrALF6 was also upregulated by Staphylococcus aureus challenge. In summary, the three isoforms of ALF genes may participate in the innate immune response against bacteria and virus infecting the giant fresh water prawn.

Cloning and expression analysis of an anti-lipopolysaccharide factor from giant freshwater prawn, Macrobrachium rosenbergii.

Anti-lipopolysaccharide factors (ALFs) are a group of effector molecules that are classified as antimicrobial peptides (AMPs). They are found in limulids and crustaceans and show a broad range of antimicrobial activity. In the current study, an ALF gene from Macrobrachium rosenbergii (MrALF) was identified. Its full length was 690 bp and it encoded a 124 amino acid protein. A signal peptide and a conserved LPS-binding domain with two conserved cysteine residues that comprise a cluster of positive charged residues within a disulfide loop were predicted in MrALF. The M. rosenbergii ALF clusters with the Macrobrachium olfersii ALF and further clusters with most crustacean ALFs, suggesting that they should originate from one common ancestor. Positive selections should have sharpen the evolution of M. rosenbergii and M. olfersii ALF genes. Reverse transcriptase polymerase chain reaction analysis showed that MrALF was expressed in all detected tissues. In the epidermis, MrALF was obviously upregulated 24 h after the LPS challenge. In the stomach and gills, MrALF was upregulated upon LPS challenge. The results show that MrALF might have important roles in the immune defense against invading bacteria. The positive selections that occur in the ALFs of crustaceans might have resulted from a Red Queen's race with its pathogens. We found evidence of positive selection acting to drive functional divergence during the evolution crustacean ALF genes, especially in the M. rosenbergii ALF gene. The evolutionary changes might correspond to the challenges induced by pathogens that infect crustaceans.

Metagenomic analysis reveals presence of different animal viruses in commercial fetal bovine serum and trypsin.

Animal-derived biological products, such as fetal bovine serum (FBS) and trypsin, are important supplements for scientific, pharmaceutical, and medical use. Although preventive guidelines and tests are implemented to reduce potential viral contamination in these biologicals, they do not target unusual or emerging viruses, leading to safety concerns. Using unbiased metagenomics, we investigated the presence of viruses in recently collected commercial FBS and trypsin samples from different geographic regions. In total, we detected viral sequences belonging to Parvoviridae, Anelloviridae, Flaviviridae, Herpesviridae, Caliciviridae, Nodaviridae, Rhabdoviridae, and Paramyxoviridae, including several viruses related to bovine diseases, viruses of potential human and insect origin, and viruses of unknown origin. Bovine parvovirus 3 and bosavirus were detected with high frequency and abundance in FBS, necessitating more stringent testing for these parvoviruses during production. Both bovine norovirus and bovine viral diarrhea virus 1 displayed relatively high genetic distance to closest hits, indicating the presence of new genotypes in farm animals. While the origin of novel lyssavirus and Nipah virus is unclear, their presence raises the possibility of the introduction of pathogenic animal-derived viruses into biologicals. Our results showed relatively widespread contamination of different viruses in biologicals, underscoring the need for robust safety protocol alternatives, such as metagenomic sequencing, to monitor emerging viruses.

Six defensins from the triangle-shell pearl mussel Hyriopsis cumingii.

Antimicrobial peptides (AMPs) are the first line of defense of invertebrates against invading pathogens. Defensins, unique AMPs, have a cysteine-stabilized α-helix and ß-sheet (CSαß) motif. In invertebrates, defensins have been reported in arthropods and mussels. Recently, six defensins were identified from Hyriopsis cumingii for the first time, and were designated as HcDef1, HcDef2, HcDef3, HcDef4, HcDef5, and HcDef6. HcDef1 and HcDef2 encode a protein containing 61 and 60 amino acids, respectively. HcDef3, HcDef4, and HcDef6 have 65 amino acids each. HcDef5 is longer than the other five defensins, comprising 83 amino acids. HcDef3 and HcDef4 have three pairs of disulfide bonds. HcDef1, HcDef5, and HcDef6 are exceptions; each has four pairs of disulfide bonds. Evolutionary analysis revealed that only purifying selection and no positive selection could be detected in defensin genes; purifying selection might be the major evolutionary driving force in the evolution of defensin genes. The present study reveals for the first time that the defensins from H. cumingii are diverse and phylogenetic analysis showed that these 6 defensins from H. cumingii were clustered into one group. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that HcDef1-HcDef4 could be detected in the hepatopancreas and gills whereas HcDef5-HcDef6 could only be detected in gills. In addition, the expression levels of HcDef2, HcDef3, and HcDef5 in H. cumingii with pearls were higher than that in H. cumingii without pearls. Quantitative RT-PCR analysis showed that HcDef1, HcDef2, HcDef3, and HcDef5 were downregulated by Vibrio anguillarum challenge whereas HcDef4 and HcDef6 were upregulated under Vibrio challenge. Our results suggest the roles of defensins in the innate immunity of H. cumingii.

Adaptive evolution of the spike gene of SARS coronavirus: changes in positively selected sites in different epidemic groups.

BACKGROUND: It is believed that animal-to-human transmission of severe acute respiratory syndrome (SARS) coronavirus (CoV) is the cause of the SARS outbreak worldwide. The spike (S) protein is one of the best characterized proteins of SARS-CoV, which plays a key role in SARS-CoV overcoming species barrier and accomplishing interspecies transmission from animals to humans, suggesting that it may be the major target of selective pressure. However, the process of adaptive evolution of S protein and the exact positively selected sites associated with this process remain unknown. RESULTS: By investigating the adaptive evolution of S protein, we identified twelve amino acid sites (75, 239, 244, 311, 479, 609, 613, 743, 765, 778, 1148, and 1163) in the S protein under positive selective pressure. Based on phylogenetic tree and epidemiological investigation, SARS outbreak was divided into three epidemic groups: 02-04 interspecies, 03-early-mid, and 03-late epidemic groups in the present study. Positive selection was detected in the first two groups, which represent the course of SARS-CoV interspecies transmission and of viral adaptation to human host, respectively. In contrast, purifying selection was detected in 03-late group. These indicate that S protein experiences variable positive selective pressures before reaching stabilization. A total of 25 sites in 02-04 interspecies epidemic group and 16 sites in 03-early-mid epidemic group were identified under positive selection. The identified sites were different between these two groups except for site 239, which suggests that positively selected sites are changeable between groups. Moreover, it was showed that a larger proportion (24%) of positively selected sites was located in receptor-binding domain (RBD) than in heptad repeat (HR)1-HR2 region in 02-04 interspecies epidemic group (p = 0.0208), and a greater percentage (25%) of these sites occurred in HR1-HR2 region than in RBD in 03-early-mid epidemic group (p = 0.0721). These suggest that functionally different domains of S protein may not experience same positive selection in each epidemic group. In addition, three specific replacements (F360S, T487S and L665S) were only found between 03-human SARS-CoVs and strains from 02-04 interspecies epidemic group, which reveals that selective sweep may also force the evolution of S genes before the jump of SARS-CoVs into human hosts. Since certain residues at these positively selected sites are associated with receptor recognition and/or membrane fusion, they are likely to be the crucial residues for animal-to-human transmission of SARS-CoVs, and subsequent adaptation to human hosts. CONCLUSION: The variation of positive selective pressures and positively selected sites are likely to contribute to the adaptive evolution of S protein from animals to humans.

The key role for local base order in the generation of multiple forms of China HIV-1 B'/C intersubtype recombinants.

BACKGROUND: HIV-1 is a retrovirus with high rate of recombination. Increasing experimental studies in vitro indicated that local hairpin structure of RNA was associated with recombination by favoring RT pausing and promoting strand transfer. A method to estimate the potential to form stem-loop structure by calculating the folding of randomized sequence difference (FORS-D) has been used to investigate the relationship between secondary structure and evolutionary pressure in some genome. It showed that gene regions under strong positive "Darwinian" selection were associated with positive FORS-D values. In the present study, the sequences of HIV-1 subtypes B' and C, both of which represent the parent strains of CRF07_BC, CRF08_BC and China URFs, were selected to investigate the relationship between natural recombination and secondary structure by calculating the FORS-D values. RESULTS: The apparent higher negative FORS-D value region appeared in the gag-pol gene region (nucleotide 0-3000) of HIV-1 subtypes B' and C. Thirteen (86.7%) of 15 mosaic fragments and 17 (81%) of 21 recombination breakpoints occurred in this higher negative FORS-D region. This strongly suggested that natural recombination did not occur randomly throughout the HIV genome, and that there might be preferred (or hot) regions or sites for recombination. The FORS-D analysis of breakpoints showed that most breakpoints of recombinants were located in regions with higher negative FORS-D values (P = 0.0053), and appeared to have a higher negative average FORS-D value than the whole genome (P = 0.0007). The regression analysis also indicated that FORS-D values correlated negatively with breakpoint overlap. CONCLUSION: High negative FORS-D values represent high, base order determined stem-loop potentials and influence mainly the formation of stem-loop structures. Therefore, the present results suggested for the first time that occurrence of natural recombination was associated with high base order-determined stem-loop potential, and that local base order might play a key role in the initiation of natural recombination by favoring the formation of stable stem-loop structures.

[Research progress in HIV auxiliary proteins counteracting host restriction factors].

Identification and functional analyses of antiviral restriction factors in hosts have become hot research topics. Four HIV restriction factors, APOBEC3G, Trim5alpha, Tetherin, and SAMHD1, have been identified in recent years. By encoding auxiliary proteins, lentiviruses can counteract host restriction factors. For example, the auxiliary proteins Vif, Vpu, and Vpx of HIV antagonize APOBEC3G, Tetherin, and SAMHD1, respectively. Furthermore, these auxiliary proteins enable the entry of HIV into host cells and influence the replication and pathogenicity of HIV. In this paper, we review the research progress in the functions of the three HIV auxiliary proteins that can antagonize the host restriction factors.

Curcumin inhibits invasion and metastasis in K1 papillary thyroid cancer cells.

Curcumin, the active constituent of dietary spice turmeric, possesses a strong potential for cancer prevention and treatment. However, there is no study to address the effects of curcumin on invasion and metastasis of thyroid cancers. Thyroid cancer is the most common malignancy of endocrine organs, and its incidence rates have steadily increased over recent decades. Although most indolent tumours can be effectively managed, metastatic tumours at distant secondary sites behave aggressively and currently there is no effective form of treatment. Here, for the first time it has been reported that curcumin inhibit multiple metastasis steps of K1 papillary thyroid cancer cells. Curcumin dose-dependently suppressed viability of K1 cells as well as its cell attachment, spreading, migration and invasion abilities. Moreover, curcumin could also down-regulate the expression and activity of matrix metalloproteinase-9 (MMP-9). The findings showed that curcumin might be an effective tumouristatic agent for the treatment of aggressive papillary thyroid carcinomas.

[SAMHD1 − a HIV-1 restriction factor derived from Myeloid lineage monocytes].

HIV-1 restriction factors have became one of the hottest fields of AIDS researches. In 2011, SAMHD1 was demonstrated to be a novel HIV-1 restriction factor, adding to a list of HIV-1 restriction factors that include APOBEC3G, TRIM5α and Tetherin. SAMHD1 is highly expressed in myeloid-lineage monocytes, such as macrophages and dendritic cells. In this paper, we review the current research progress on the structure of SAMHD1, its antiviral mechanism, interaction with the lentivirus Vpx, and evolution. The identification of SAMHD1 opens the door towards understanding the role of SAMHD1 in lentiviral pathogenesis.

Evaluation of FORS-D analysis: a comparison with the statistically significant stem-loop potential.

The stem-loop potential of a nucleic acid segment (expressed as a FONS value), decomposes into base composition-dependent and base order-dependent components. The latter, expressed as a FORS-D value, is derived by subtracting the value of the base composition-dependent component (FORS-M) from the FONS value. FORS-D analysis is the use of FORS-D values to estimate the potential of local base order to contribute to a stem-loop structure, and it has been used to investigate the relationship between stem-loop structure and other selective pressures on genomes. In the present study, we evaluated the reliability of FORS-D analysis by comparing it with statistically significant stem-loop potential, another robust method developed by Le and Maizel for examining stem-loop structure. We found that FORS-M values calculated using 10 randomized sequences are as reliable as those calculated using 100 randomized sequences. The resulting FORS-D values have a similar trend and distribution as statistically significant stem-loop potential, implying that FORS-D analysis is as reliable as the latter in measuring the distribution of base order-dependent stem-loop potential. Since the calculation of the FORS-M values is time consuming, the integrated program Bodslp developed by us will become a convenient tool for large-scale FORS-D analysis. The results also suggest that for some purposes the online program SigStb developed by Le and Maizel may be used as an alternative tool for FORS-D analysis.

Local base order influences the origin of ccr5 deletions mediated by DNA slip replication.

CCR5 is a seven-transmembrane G-protein-coupled receptor that binds the CC-chemokines including RANTES, eotaxin, MIP-1alpha and beta. CCR5 serves as an essential coreceptor for cell entry of R5 (macrophage-tropic, nonsyncytium-inducing) strains of HIV-1. To date, four deletions have been found in human and primate ccr5. There is little evidence, however, on how these deletion mutations occur. In the present study, we analyzed ccr5 sequences of both mutants and wild type and found that direct repeats flanked the breakpoints of the deletions, suggesting that these deletions resulted from slipped mispairing during DNA replication. Of particular interest was the location of these deletions in or near the regions with higher negative FORS-D values. High negative FORS-D values stand for high stem-loop potential determined by base order and influence mainly the formation of stem-loop structures. Therefore, the particular location of these deletions suggests that the local sequence of bases might be important in the initiation of deletions mediated by DNA slip replication in concert with direct repeats.