Construction of carbon quantum dots/riboflavin fluorescent probe and its application in the detection of ciprofloxacin.

The research outlined a novel approach for creating a sensitive and efficient ratio fluorescent probe for ciprofloxacin (CIP) detection. The method used the biomass materials passionfruit shell and diethylenetriamine as carbon and nitrogen sources, respectively, to prepare blue fluorescent carbon quantum dots (b-CQDs) with an average size of 3.29 nm and a quantum yield of 19.6% by a hydrothermal method. The newly designed b-CQDs/riboflavin ratio fluorescent probe demonstrates a distinct advantage for CIP monitoring, exhibiting a marked increase in fluorescence intensity at 445 nm upon interaction with CIP, while maintaining a stable intensity at 510 nm. In the water system, the I445 nm/I510 nm ratio of the fluorescent probe showed a significant linear relationship with CIP at the concentrations of 0-250 µmol·L-1, and the probe boasts a low detection limit of 0.86 µmol·L-1. The outstanding selectivity, broad detection range, low detection limits, and high quantum yield of the b-CQDs highlight their significant potential in the development of advanced sensing probes for efficient detection of ciprofloxacin, offering promising insights for future sensor technology advancements.

The protective function of non-coding DNA in DNA damage accumulation with age and its roles in age-related diseases.

Aging is a progressive decline of physiological function in tissue and organ accompanying both accumulation of DNA damage and reduction of non-coding DNA. Peripheral non-coding DNA/heterochromatin has been proposed to protect the genome and centrally-located protein-coding sequences in soma and male germ cells against radiation and the invasion of exogenous nucleic acids. Therefore, this review summarizes the reduction of non-coding DNA/heterochromatin (including telomeric DNA and rDNA) and DNA damage accumulation during normal physiological aging and in various aging-related diseases. Based on analysis of data, it is found that DNA damage accumulation is roughly negatively correlated with the reduction of non-coding DNA and therefore speculated that DNA damage accumulation is likely due to the reduction of non-coding DNA protection in genome defense during aging. Therefore, it is proposed here that means to increase the total amount of non-coding DNA and/or heterochromatin prior to the onset of these diseases could potentially better protect the genome and protein-coding DNA, reduce the incidence of aging-related diseases, and thus lead to better health during aging.

Nutrition and water temperature regulate the expression of heat-shock proteins in golden pompano larvae (Trachinotus ovata, Limmaeus 1758).

Understanding fish larval development is of a great interest for aquaculture production efficiency. Identifying possible indicators of fish larvae stress could improve the production and limit the mortality rate that larval stage is subjected to. Heat-shock proteins (HSPs) and heat-shock factors (HSFs) are well known as indicators of response to many kinds of stressor (e.g., environmental, morphological, or pathological changes). In this study, golden pompano larvae were raised at different temperatures (23 °C, 26 °C, and 29 °C), as well as three different diets (Artemia nauplii unenriched, Artemia nauplii enriched with Nannochloropsis sp., and Artemia nauplii enriched with Algamac 3080), and the expression of HSP60, HSP70, HSF1, HSP2, and GRP94 were monitored. While stress genes were widely expressed in the larval tissues, HSP60 and HSP70 were principally from the gills and heart; HSF1 principally from the muscle, brain, and heart; and GRP94 principally from the head kidney and spleen. Golden pompano larvae were found to be more sensitive to thermal changes at later larval stage, and 29 °C was showed to likely be the best condition for golden pompano larval development. Nannochloropsis sp.-enriched Artemia nauplii treatment was found to be the most appropriate feed type with moderate relative expressions of HSP60, HSP70, HSF1, HSF2, and GRP94.

The immune response of the C-Jun in the black tiger shrimp (Penaeus monodon) after bacterial infection.

The transcription factor C-Jun widely exists in vertebrates and invertebrates and plays an important role in various kinds of stimulus response. In this study, PmC-jun gene was first cloned from Penaeus monodon. The full-length cDNA of PmC-jun was 1857 bp in length and included an 879 bp open reading frame (ORF), which encoded 293 amino acids. qRT-PCR analysis results showed that PmC-jun mRNAs were ubiquitously expressed in all the examined tissues. The highest expression level was observed in gill, followed by hepatopancreas. The expression patterns of PmC-jun after Vibrio harveyi and Streptococcus agalactiae injections were studied by qRT-PCR experiment. PmC-jun increased obviously in the gill and hepatopancreas. The expression pattern of PmC-jun in the hepatopancreas was further studied using in situ hybridization (ISH) method. The mRNA expression level of PmC-jun significantly increased in the hepatopancreas after bacterial infection. The expression sites of PmC-jun were almost unchanged. PmC-jun played a regulatory role in pathogen invasion.

Molecular cloning, characterization and expression analysis of Toll-like receptor 5M gene in Japanese sea perch (Lateolabrax japonicas) after bacterial infection.

Toll-like receptor 5M belongs to Toll-like receptors (TLRs) family, which plays a crucial role in innate immunity due to its important role in the recognition of bacteria invasion and in the activation of immune related pathways downstream. In the present study, we firstly cloned the full-length cDNAs of TLR 5M (LjTLR 5M) from Japanese sea perch (Lateolabrax japonicas). The full-length cDNAs of LjTLR 5M include an open reading frame (ORF) of 2676 bp encoding a polypeptide of 891 amino acid residues. The deduced amino acid sequence analysis showed that LiTLR 5M contains LRRs (extracellular leucine rich repeats), transmembrane and TIR (Toll/interleukin-1 receptor) domain. Transcriptional expression analysis indicated that LiTLR 5M mRNAs were ubiquitously expressed in wide array of tissues and the peak level was observed in the head-kidney. The expression patterns of LjTLR 5M after Vibro harveyi and Streptococus agalactiae infection were detected by qRT-PCR, and the results showed that LjTLR 5M was significant up-regulated in spleen, liver and head-kidney. Additionally, the expression patterns of LjTLR 5M in infected spleen and head-kidney were further validated by in situ hybridization (ISH). In summary, these findings indicate that LjTLR 5M is significant induced after different bacterial infection and is involved in immune response. Furthermore, this study will provide foundational information for other TLRs research of L. japonicas against different bacterial pathogens invasion.

RNA-Seq analysis of immune-relevant genes in Lateolabrax japonicus during Vibrio anguillarum infection.

Lateolabrax japonicus is one of the main marine aquatic fish species, and is popularly cultured in East Asia due to its highly commercial value. In recent years, because of large-scale and intensive farming and seawater pollution, fish diseases keep breaking out. However, systematic study on L. japonicus immunogenetics is limited due to the deficiency of deep sequencing technologies and genome backgrounds. In this study, the widely analysis at the transcriptome level for L. japonicus that infected with Vibrio anguillarum was performed. In total, 334,388,688 high quality reads were obtained in six libraries (HK-VA, HK-PBS, LI-VA, LI-PBS, SP-VA and SP-PBS) and de novo assembled into 101,860 Unigenes with an average unigene length of 879 bp. Based on sequence similarity 30,142 unigenes (29.59%) were annotated in the public databases. Comparative analysis revealed, 1,202, 3034 and 3519 differentially expressed genes (DEGs) were identified in three comparisons (HK-PBS VS HK-VA, LI-PBS VS LI-VA and SP-PBS VS SP-VA). Enrichment and pathway analysis of the DEGs was also carried out to excavate the candidate genes related to immunity. In conclusion, this study identifies and evaluates dozen of potential immune related pathways and candidate genes, which are indispensable for padding genomic resources of L. japonicus, and would lay the foundation for further studying and illuminating the mechanism of host-pathogen interactions.

Cloning, characterization, and expression of the macrophage migration inhibitory factor gene from the black tiger shrimp (Penaeus monodon).

Macrophage migration inhibitory factor (MIF) is an ancient cytokine that engages in innate immune system of vertebrates and invertebrates. In this study, the MIF gene homologue (PmMIF) was cloned from the black tiger shrimp, Penaeus monodon. The full-length cDNA sequence of PmMIF was 838 bp and contained 78 bp 5' untranslated region (UTR) and 397 bp 3' UTR, and an open reading frame (ORF) of 363 bp which coded 120 amino acids (aa). Multiple alignment analysis showed that the deduced amino acid sequence shared 98% identities with MIF from closely related species of Litopenaeus vannamei. Quantitative real-time PCR (qRT-PCR) analysis indicated that PmMIF was highly expression observed in hepatotpancreas and gills. After Vibrio harveyi challenge, PmMIF mRNA level in hepatopancreas and gills were sharply up-regulated at 6 h post-injection, and reached the maximum at 12 h. PmMIF expression level in the hepatopancreas and gills were up-regulated markedly under low (2.3%) and high (4.3%) salinity exposure, respectively. PmMIF expression level in gills increased significantly at 12 h and reached peak values (2.5- fold, 6.4-fold and 1.8-fold compared with the control) at 12 h, 48 h and 12 h after zinc, cadmium and copper exposure, respectively. In the hepatopancreas, the expression of PmMIF reached maximum levels (8.5- fold, 6.2-fold and 2.1-fold compared with the control) at 24 h, 6 h and 48 h after zinc, cadmium and copper exposure, respectively. All the results indicate that PmMIF plays an important role in responding in the innate immune system of shrimps.

Characterization, expression and silencing by RNAi of p53 from Penaeus monodon.

The tumor suppressor p53 is a sequence-specific transcription factor, whose target genes can regulate genomic stability, the cellular response to DNA damage and cell-cycle progression. In the present study, the full-length complementary DNA (cDNA) sequence of p53 gene from Penaeus monodon (Pmp53) was cloned by the technology of rapid amplification of cDNA ends (RACE). The cDNA of Pmp53 was 2239 bp, encoding a protein of 450 amino acids with calculated molecular weight of 50.62 kDa. The temporal expression of Pmp53 in different tissues (ovary, heart, intestine, brain, muscles, stomach and gills) and different developmental stages of ovary was investigated by real-time quantitative PCR (RT-qPCR). The lowest expression level of Pmp53 was observed in the stomach, while the highest expression level was detected in the brain. During the ovary development stages, the expression level of Pmp53 reached the peak at stage III. RNA interference (RNAi) and serotonin (5-hydroxytryptamine, 5-HT) injection experiments were conducted to study the expression profile of Pmp53 and PmCDK2 (cyclin-dependent kinase 2, CDK2). Knocked down of Pmp53 by dsRNA-p53 was sequence-specific and successful. Expression levels of Pmp53 and PmCDK2 in ovary of P. monodon were significantly increased at 12-96 h post 5-HT injection. These results indicate that Pmp53 may be involved in the regulation of ovarian development of P. monodon.

Identification and expression analysis of Dicer2 in black tiger shrimp (Penaeus monodon) responses to immune challenges.

Dicer is a key initiative protein of the RNA interference (RNAi) pathway that produces small interfering RNAs (siRNAs) or micro RNAs (miRNA), which then leads to RNA-directed gene regulation or viral immunity. In the present study, we identified and characterized a Dicer2 cDNA from black tiger shrimp Penaeus monodon (designated as PmDcr2). The full length cDNA of PmDcr2 contains a 5' untranslated region (UTR) of 109 bp, an open reading frame (ORF) of 4509 bp and a 3' UTR of 842 bp. The molecular weight (MW) of predicted PmDcr2 protein is 171.7 KDa with the theoretical isoelectric point of 6.23. PmDcr2 amino acid shared the highest similarity of 91.8% and 90.7% with Dicer2 of Litopenaeus vannamei and Marsupenaeus japonicas, respectively. Phylogenic analysis showed PmDcr2 was clustering with shrimp Dicer2, and closed to the insect group including Tribolium castaneum Dicer2. Real-time quantitative PCR showed that PmDcr2 was widely expressed in almost all examined tissues except muscle, with high expression in gill, hemocyte and lymph. The expression of PmDcr2 in hepatopancreas was up-regulated by Vibrio vulnificus and White Spot Syndrome Virus (WSSV), but not by Staphylococcus aureus. Furthermore, the viral nucleotide homologue dsRNA poly (I:C) and ssRNA R484 also remarkably induced PmDcr2 mRNA expression more efficient and stronger. These data reflect that PmDcr2 is not only response to the gram negative bacteria infection, but also specially to the viral infection in black tiger shrimp.

Selenium-dependent glutathione peroxidase gene expression during gonad development and its response to LPS and H2O2 challenge in Scylla paramamosain.

A selenium-dependent glutathione peroxidase cDNA was obtained from green mud crab Scylla paramamosain (SpGPx) by homology PCR technique and rapid amplification of cDNA ends (RACE) methods. The 1135 bp full-length cDNA contains a 9 bp 5'-untranslated region (UTR), an open reading frame (ORF) of 564 bp encoded a deduced protein of 187 amino acids (aa), and a 562 bp 3'-UTR with a 100 bp conserved eukaryotic selenocysteine insertion sequence (SECIS). It involves a putative selenocysteine (Sec4°, or U4°) residue which is encoded by an opal codon, ¹²7TGA¹²9, and forms an active site with residues Q74 and W¹4². Sequence characterization revealed that SpGPx contain a characteristic GPx signature motif 2 (64LAFPCNQF7¹), an active site motif (¹5²WNFEKF¹57), a potential N-glycosylation site (76NTT78), and two residues (R9° and R¹68) which contribute to the electrostatic architecture by directing the glutathione donor substrate. Multiple sequence alignment and phylogenetic analysis showed that SpGPx share a high level of identities and closer relationship with other selected invertebrate GPxs and vertebrate GPx1 and GPx2. Molecular modelling analysis results also supported these observations. Real time quantitative PCR analysis revealed that SpGPx was constitutively expressed in 10 selected tissues, and its expression level in gill and testis was higher than that in the other tissues (p < 0.05). The SpGPx expression increased and then declined during ovarian and testicular development implying thatnscrpits yowed that SpGPx might play an important role in gonad development by protecting them from oxidative stress. The expression of SpGPx mRNA was induced by lipopolysaccharide (LPS) and hydrogen peroxide (H2O2) in hepatopancreas and haemocytes. The results suggested that SpGPx was implicated in the immune response induced by LPS and H2O2.

The decreased exclusion of nuclear eccDNA: From molecular and subcellular levels to human aging and age-related diseases.

Extrachromosomal circular DNA (eccDNA) accumulates within the nucleus of eukaryotic cells during physiological aging and in age-related diseases (ARDs) and the accumulation could be caused by the declined exclusion of nuclear eccDNA in these states. This review focuses on the formation of eccDNA and the roles of some main factors, such as nuclear pore complexes (NPCs), nucleoplasmic reticulum (NR), and nuclear actin, in eccDNA exclusion. eccDNAs are mostly formed from non-coding DNA during DNA damage repair. They move to NPCs along nuclear actin and are excluded out of the nucleus through functional NPCs in young and healthy cells. However, it has been demonstrated that defective NPCs, abnormal NPC components and nuclear actin rods are increased in aged cells, various cancers and certain other ARDs such as cardiovascular diseases, premature aging, neurodegenerative diseases and myopathies. Therefore, mainly resulting from the increase of dysfunctional NPCs, the exclusion of nuclear eccDNAs may be reduced and eccDNAs thus accumulate within the nucleus in aging and the aforementioned ARDs. In addition, the protective function of non-coding DNA in tumorigenesis is further discussed.

Digenean parasites of Chinese marine fishes: a list of species, hosts and geographical distribution.

In the literature, 630 species of Digenea (Trematoda) have been reported from Chinese marine fishes. These belong to 209 genera and 35 families. The names of these species, along with their hosts, geographical distribution and records, are listed in this paper.

Molecular cloning, expression and functional analysis of three subunits of protein phosphatase 2A (PP2A) from black tiger shrimps (Penaeus monodon).

Protein phosphatase 2A (PP2A) is a cellular serine-threonine (Ser/Thr) phosphatase that plays a crucial role in regulating most cellular functions. In the present study, the full-length cDNAs of three subunits of PmPP2A (PmPP2A-A, PP2A-B and PP2A-C) were cloned from Penaeus monodon, which are the first available for shrimps. Sequence analysis showed that PmPP2A-A, PmPP2A-B and PmPP2A-C encoded polypeptides of 591, 443, and 324 amino acids, respectively. The mRNAs of three subunits of PmPP2A were expressed constitutively in all tissues examined, and predominantly in the ovaries. In ovarian maturation stages, the three subunits of PmPP2A were continuously but differentially expressed. Dopamine and 5-hydroxytryptamine injection experiments were conducted to study the expression profile of three subunits of PmPP2A, and the results indicated that PmPP2A played a negative regulatory role in the process of ovarian maturation. In addition, the recombinant proteins of three subunits of PmPP2A were successfully obtained, and the phosphatase activity of PmPP2A was tested in vitro. The results of this study will advance our understanding about the molecular mechanisms of PmPP2A in Penaeus monodon.

Water temperature induces jaw deformity and bone morphogenetic proteins (BMPs) gene expression in golden pompano Trachinotus ovatus larvae.

Golden pompano Trachinotus ovatus larvae were kept at 26, 29 and 33 °C for 15 days from 3-day post hatching (DPH) to 18 DPH to test temperature-dependent growth and jaw malformation. The growth, survival, jaw deformity and the gene expressions of bone morphogenetic proteins (BMPs) were used as criteria to examine the fish response to temperature manipulation. The growth rate of fish at 29 or 33 °C was significantly faster than fish at 26 °C, while fish survival at 29 °C was significantly higher than fish at 33 °C. Jaw deformity was significantly affected by water temperature. The highest jaw deformity occurred on fish at 33 °C, and the lowest jaw deformity was at 26 °C. The expressions of all BMP genes except BMP10 were significantly affected by water temperature. The highest gene expression of BMP2 was on fish at 29 °C, and the lowest expression was at 33 °C. For the BMP4 gene, the highest and lowest expressions were found on fish at 33 and 26 °C, respectively. The present study indicates that jaw deformity of golden pompano larvae increases with increasing temperature, and the gene expression of BMP4 proteins coincides with high jaw deformity and water temperature elevation.

Characterization and function analysis of Hsp60 and Hsp10 under different acute stresses in black tiger shrimp, Penaeus monodon.

Heat shock proteins (Hsps) are a class of highly conserved proteins produced in virtually all living organisms from bacteria to humans. Hsp60 and Hsp10, the most important mitochondrial chaperones, participate in environmental stress responses. In this study, the full-length complementary DNAs (cDNAs) of Hsp60 (PmHsp60) and Hsp10 (PmHsp10) were cloned from Penaeus monodon. Sequence analysis showed that PmHsp60 and PmHsp10 encoded polypeptides of 578 and 102 amino acids, respectively. The expression profiles of PmHsp60 and PmHsp10 were detected in the gills and hepatopancreas of the shrimps under pH challenge, osmotic stress, and heavy metal exposure, and results suggested that PmHsp60 and PmHsp10 were involved in the responses to these stimuli. ATPase and chaperone activity assay indicated that PmHsp60 could slow down protein denaturation and that Hsp60/Hsp10 may be combined to produce a chaperone complex with effective chaperone and ATPase activities. Overall, this study provides useful information to help further understand the functional mechanisms of the environmental stress responses of Hsp60 and Hsp10 in shrimp.

Response of a Mu-class glutathione S-transferase from black tiger shrimp Penaeus monodon to aflatoxin B1 exposure.

Glutathione S-transferases (GSTs) are a family of multifunctional phase II enzymes that are involved in the detoxification of exogenous and endogenous compounds. In this study, a full-length cDNA of Mu-class GST (PmMuGST) was isolated from the hepatopancreas of Penaeus monodon using rapid amplification of cDNA ends method. The full length cDNA of PmMuGST is 867 bp, contains an open read frame of 660 bp, and encodes a polypeptide of 219 amino acids with a molecular mass of 25.61 kDa and pI of 6.15. Sequence analysis indicated that the predicted protein sequence of PmMuGST was very similar to (86 %) that of Litopenaeus vannamei. A conserved domain of GST_N_Mu_like (PSSM: cd03075) and GST_C_family_superfamily_like (PSSM: cl02776) was indentified in PmMuGST. Real time quantitative RT-PCR analysis indicated that PmMuGST was present in all of the tested tissues. PmMuGST transcripts both in the hepatopancreas and in the muscle were significantly induced after 14 days of treatment with a low dosage of AFB1 (50 µg/kg) exposure and were significantly inhibited after 42 and 56 days of a high dosage of AFB1 (1000, 2500 µg/kg AFB1) exposure. Taken together, the Mu-class GST from P. monodon was inducible and was involved in the response to AFB1 exposure.

Molecular characterization and expression profiles of cdc2 and cyclin B during oogenesis and spermatogenesis in green mud crab (Scylla paramamosain).

The maturation promoting factor (MPF) is a key regulator of controlling G2/M phase transition in the meiotic maturation of oocyte and spermatocyte in animals, which is a complex of CDC2 (CDK1) and cyclin B. To better understand the molecular mechanism of oocyte and spermatocyte maturation in mud crab (Scylla paramamosain), the full length cDNA of cdc2 (Sp-cdc2) and cyclin B (Sp-cyclin B) were cloned and characterized. The full length cDNA of Sp-cdc2 gene is of 1593 bp encoding a protein of 299 amino acids. Real-time quantitative PCR analysis revealed that the expression level of Sp-cdc2 in the ovary was higher than in other tissues (P<0.01); and its expression level was not significantly different in different stages of ovary development (P>0.05), meanwhile there was higher expression in T3 stage than in T1 and T2 stages (P<0.05). The full length cDNA of Sp-cyclin B is 1492 bp encoding a protein of 391 amino acids. The real-time PCR results showed that its expression level in the ovary was the highest in all examined tissues (P<0.01), and the gonad expression level in O5 stage was significantly higher than in previous 4 stages and the testis (P<0.05), and was also significantly higher in T2 stage than in T1 stage (P<0.05). In situ hybridization analysis showed that the expressions of Sp-cdc2 and Sp-cyclin B transcripts were presented in similar distribution patterns in different developing stages of ovary and testis. The positive signals of Sp-cdc2 and Sp-cyclin B mRNA were detected in the oocytoplasm of oogonia and pre-vitellogenic and primary vitellogenic oocytes, while these two genes had higher expression level in the spermatid and secondary spermatocyte following primary spermatocyte. These results suggested that Sp-cdc2 and Sp-cyclin B may play essential roles in the oogenesis and spermatogenesis of the crab.

Erk2 in ovarian development of green mud crab Scylla paramamosain.

We identified extracellular signal-regulated kinase 2 (erk2) from green mud crab, Scylla paramamosain, in this article. It was originally identified from an expressed sequence tag fragment from a normalized gonadal cDNA library. 5' Rapid amplification of cDNA end (RACE) technique was used to obtain the 5' untranslated region (UTR). The full-length cDNA of Sp-erk2 is 1516 bp, including a 5'-terminal UTR of 19 bp, an open-reading frame of 1098 bp, and a 3'-terminal UTR of 399 bp. The translated protein is 365 amino acids in length with a predicted molecular weight of 42 kDa, which is the same as other species. It is the first time that the expression of Sp-erk2 in different stages of ovary development of crustacean was analyzed, and the result showed that the expression of Sp-erk2 increased gradually with ovarian development, with a peak in the mature phase. In situ hybridization histochemistry was used to clarify the detail of expression. Positive signals illustrated that Sp-erk2 mRNA is present in follicular cells when the ovary is in early stages, and in both follicular cells and oocytes when it is in mature phases. All above suggest that Sp-erk2 is important for ovarian development.

EST analysis on the gonad development related organs and microarray screen for differentially expressed genes in mature ovary and testis of Scylla paramamosain.

A total of 5160 high quality ESTs (expressed sequence tags) averaging 357 bp were collected from normalized cDNA libraries created from testis, ovary and mixed organs of mud crab Scylla paramamosain. Clustering and assembly of these ESTs resulted in a total of 3837 unique sequences with 576 overlapping contigs and 3261 singletons. Comparisons with the GenBank non-redundant (Nr) protein database (BLASTx, e-values <10(-5)) revealed putative functions or matched homologs from other organisms for 847 (22%) of the ESTs. Several gonad development related genes such as cathepsin C, thioredoxin peroxidase, vitellogenin receptor precursor, 50S ribosomal protein L24 and ubiquitin-conjugating enzyme E2 isoform 2 were identified from this EST project and demonstrated as gonad differential expression genes by rqRT-PCR. Sixty five different types of SSRs (simple sequence repeats) were identified from the total 411 EST-SSR motifs. A home-made cDNA microarray containing 5664 spots was developed and the hybridization results indicated that 39 unique transcripts were differentially expressed in testis and ovaries (P<0.05). The expression levels of eleven unique transcripts examined by rqRT-PCR were matched with microarray fairly. These results will provide a useful resource for functional genomic studies on the biology of reproduction of mud crab.