Fabrication and application of carrier-free and carrier-based nanopesticides in pest management.

Pesticides are widely used for pest control to promote sustained and stable growth of agricultural production. However, indiscriminate pesticide usage poses a great threat to environmental and human health. In recent years, nanotechnology has shown the ability to increase the performance of conventional pesticides and has great potential for improving adhesion to crop foliage, solubility, stability, targeted delivery, and so forth. This review discusses two types of nanopesticides, namely, carrier-free nanopesticides and carrier-based nanopesticides, that can precisely release necessary and sufficient amounts of active ingredients. At first, the basic characterization and preparation methods of these two distinct types of nanopesticides are briefly summarized. Subsequently, current applications and future perspectives on scientific examples and strategies for promoting the usage efficacy and reducing the environmental risks of these nanopesticides were also described. Overall, nanopesticides can promote higher crop yields and lay the foundation for sustainable agriculture and global food security.

Identification and expression patterns of somatic piRNAs and PIWI genes in Riptortus pedestris (Hemiptera: Alydidae).

RNA interference (RNAi)-based gene silencing is a feasible and sustainable technology for the management of hemipteran pests by double-stranded RNA involvement, including small-interfering RNA, microRNA, and Piwi-interacting RNA (piRNA) pathways, that may help to decrease the usage of chemical insecticides. However, only a few data are available on the somatic piRNAs and their biogenesis genes in Riptortus pedestris, which serves as a significant pest of soybean (Glycine max). In this study, two family members of the PIWI gene were identified and characterized in R. pedestris, containing Argonaute3 (RpAgo3) and Aubergine (RpAub) genes with conserved protein domains, and their clusters were validated by phylogenetic analysis. In addition, they were widely expressed in all developmental stages of the whole body of R. pedestris and had lower expression levels in R. pedestris guts under different rearing conditions based on previous transcriptome sequencing. Furthermore, abundant clean reads were filtered to a total number of 45,998 piRNAs with uridine bias at the first nucleotide (nt) position and 26-32 nt in length by mapping onto the reference genome of R. pedestris according to our previous whole-transcriptome sequencing. Finally, our data revealed that gut bacterial changes were significantly positively or negatively associated with differentially expressed piRNAs among the five comparison groups with Pearson correlation analysis. In conclusion, these findings paved new avenues for the application of RNAi-based biopesticides for broad-spectrum hemipteran pest control.

Neural habituation during acute stress signals a blunted endocrine response and poor resilience.

BACKGROUND: A blunted hypothalamic-pituitary-adrenal (HPA) axis response to acute stress is associated with psychiatric symptoms. Although the prefrontal cortex and limbic areas are important regulators of the HPA axis, whether the neural habituation of these regions during stress signals both blunted HPA axis responses and psychiatric symptoms remains unclear. In this study, neural habituation during acute stress and its associations with the stress cortisol response, resilience, and depression were evaluated. METHODS: Seventy-seven participants (17-22 years old, 37 women) were recruited for a ScanSTRESS brain imaging study, and the activation changes between the first and last stress blocks were used as the neural habituation index. Meanwhile, participants' salivary cortisol during test was collected. Individual-level resilience and depression were measured using questionnaires. Correlation and moderation analyses were conducted to investigate the association between neural habituation and endocrine data and mental symptoms. Validated analyses were conducted using a Montreal Image Stress Test dataset in another independent sample (48 participants; 17-22 years old, 24 women). RESULTS: Neural habituation of the prefrontal cortex and limbic area was negatively correlated with cortisol responses in both datasets. In the ScanSTRESS paradigm, neural habituation was both positively correlated with depression and negatively correlated with resilience. Moreover, resilience moderated the relationship between neural habituation in the ventromedial prefrontal cortex and cortisol response. CONCLUSIONS: This study suggested that neural habituation of the prefrontal cortex and limbic area could reflect motivation dysregulation during repeated failures and negative feedback, which might further lead to maladaptive mental states.

Application of computer-aided design and 3D-printed template for accurate bone augmentation in the aesthetic region of anterior teeth.

PURPOSE: To explore the outcomes of bone augmentation in the aesthetic zone of the anterior teeth using computer-aided design and a 3D-printed template. METHODS: Ten patients with severe bone defects in the aesthetic zone of anterior teeth were included in the study; CT data were collected before surgery. The design of the osteotomy line in the bone defect area was determined under computer simulation. The position parameters and osteotomy line of the free bone were determined via virtual surgery. A 3D-printed template was prepared to guide the accurate placement of the bone graft. Reexamination was conducted to evaluate the position of the bone graft immediately after the operation and the resorbed capacity of the bone graft before implant restoration. RESULTS: The position of the bone graft was consistent with the preoperative design. The amount of bone graft resorbed was within the acceptable range three months after the operation, and the effect of implant restoration was satisfactory. CLINICAL SIGNIFICANCE: Use of computer-aided design and a 3D-printed template can be an effective approach for accurate bone augmentation in the aesthetic zone of the anterior teeth.

The Hippocampal-Ventral Medial Prefrontal Cortex Neurocircuitry Involvement in the Association of Daily Life Stress With Acute Perceived Stress and Cortisol Responses.

OBJECTIVE: Daily life stressors include everyday irritants, hassles, and inconveniences, such as problems in traffic and unexpected work deadlines. A growing body of research has suggested higher daily stress is associated with blunted cortisol response to acute psychosocial stressors. However, so far, the neural mechanism underlying this association has not been elucidated. The current study aimed to examine the role of stress neurocircuitry between the hippocampus and the ventral medial prefrontal cortex in this relationship. METHODS: To this end, as an index of daily stress in 44 young healthy individuals (23 females; mean [standard deviation] age = 19.07 [1.11] years), the total stressful rating score of daily life stress events that occurred in a 24-hour period was quantified. Individuals were then administered a modified version of the Montreal Imaging Stress Task while undergoing functional magnetic resonance imaging scans, and their saliva samples were collected for assessment of the stress hormone cortisol. RESULTS: Results revealed that a higher level of daily stress was associated with lower salivary cortisol secretion (r = -0.39, p = .008) and lower activation of the left hippocampus (tpeak = -5.51) in response to the Montreal Imaging Stress Task. Furthermore, a higher level of daily stress was associated with stronger functional connectivity between the left hippocampus and the ventral medial prefrontal cortex/subgenual anterior cingulate cortex (tpeak = 4.91, R2= 0.365). CONCLUSIONS: Taken together, the current study suggested a possible neurocircuitry of the hippocampus and ventral medial prefrontal cortex in the relationship between daily life stress and acute psychosocial stress.

Predictive value of systemic inflammatory response-related indices for survival in tongue cancer.

OBJECTIVES: We assessed the potential prognostic significance of the preoperative systemic inflammation index, platelet-to-lymphocyte ratio, and neutrophil-to-lymphocyte ratio in patients who underwent surgical resection. SUBJECTS AND METHODS: This retrospective study included 224 patients with clinicopathologically confirmed squamous carcinoma of the tongue who underwent surgery between August 2009 and December 2017. The optimal cut-off values for the indices were determined by receiver operating characteristic curves. Correlations between the indices and clinicopathological variables were determined by Pearson chi-square or Fisher exact tests. The Kaplan-Meier test was used to compare overall survival between groups (high and low values); the log-rank or Breslow test was used to assess differences in survival. Univariate and multivariate Cox regression models were used to analyze predictive values of the indices as independent indicators of overall survival. Bilateral p values of <0.05 were considered statistically significant. RESULTS: Significant association was found between the indices and sex, tissue grade, tumor location, and lymph nodes metastases (p < 0.05). On Kaplan-Meier analysis, patients with lower values of the indices had longer overall survival (p < 0.05). Univariate and multivariate Cox regression models identified age, lymph node metastases, and neutrophil-to-lymphocyte ratio as independent predictors of overall survival. CONCLUSION: The studied indices have potential prognostic significance in patients with squamous tongue cancer.

Transcriptome-wide identification and characterization of toll-like receptors response to Vibrio anguillarum infection in Manila clam (Ruditapes philippinarum).

The Manila clam (Ruditapes philippinarum), one of the major marine aquaculture species in China, is susceptible to infection with the pathogen Vibrio, which results in massive mortality and economic losses. Toll-like receptors (TLRs) are significant pattern recognition receptors (PRRs) of innate immunity that are involved in immune regulation against pathogenic invasion. Molecular characterization of Manila clam TLRs and investigations of their immune functions are essential to prevent and control Vibrio infection. In the present research, eight cDNA sequences of R. philippinarum TLRs (RpTLRs) were identified from previous transcriptome libraries and then classified into four groups, namely, P-TLR (one sequence), V-TLR (one sequence), Ls-TLR (two sequences) and sP-TLR (four sequences), based on the corresponding LRR domain arrangement of their protein structures within the typical TLR motifs. A selective pressure test firstly suggested that the molluscan P-TLR, V-TLR, Ls-TLR and sP-TLR families underwent positive selection, and different numbers of positive selection sites (PSSs) were identified in different domains of the four types of RpTLRs, as determined by PAML and analysis of website data. These findings indicated that the evolution of RpTLRs may be associated with their immune recognition and function. Furthermore, tissue-specific expression analysis showed that all RpTLRs were ubiquitously expressed in all test tissues and were dominant in hemocytes. Quantitative real-time PCR revealed that the cDNA expression of all eight RpTLRs was upregulated after injection with Vibrio anguillarum (P < 0.01) in R. philippinarum hemocytes, revealing that these RpTLRs play important roles in responding to pathogenic stimulation. In summary, these findings provide a foundation for future investigations of the molecular classification and evolutionary patterns of Toll-like receptors in invertebrates, and the innate immune responses of TLR signaling pathways in Mollusca.

Retraction Note: DNA methylome profiling at single-base resolution through bisulfite sequencing of 5mC-immunoprecipitated DNA.

An amendment to this paper has been published and can be accessed via the original article.

Presentation, Management, and Outcome of Lingual Thyroglossal Duct Cyst in Pediatric and Adult Populations.

Lingual thyroglossal duct cysts (LTDCs) are rare congenital anomalies of the neck. The authors described the presentation, management, and outcome of LTDC in pediatric and adult cases through a retrospective observational analysis between 2008 and 2018. Data included patients' demographics, main complaint, preoperative investigations, surgical management, and recurrences. Seventeen patients were included: 8 pediatric and 9 adult patients. The most common presenting symptom was foreign body sensation (35.3%). In all, 50% (4/8) of the children had respiratory problems, while the most common symptom in adults was difficulty swallowing (8/9). Five patients were of recurrent LTDC; 3 referred patients were suspected of having recurrent epiglottic cysts. The total misdiagnosis rate was 35.3% (6/17): 14.3% (1/7) in children and 55.6% (5/9) in adults. Fiber optic laryngoscopic examination revealed that LTDCs mostly occurred at the base of the tongue (53.3%) and vallecula epiglottica (33.3%). Ultrasound examination revealed low to anechoic masses on the root of the tongue; 50% were regular in shape and 50% were irregular. All pediatric patients had regular masses (100%), but most adults had irregular masses (85.7%). In total, 76.5% of the patients underwent the Sistrunk procedure, and 23.5% underwent marsupialization alone. The mean follow-up length was 37.5 ±â€Š32.8 months. All patients were well at follow-up. In conclusion, direct laryngoscopy and ultrasound examination are essential for diagnosis as LTDCs can be confused with vallecular cysts. Surgical treatment such as marsupialization or the Sistrunk operation must be performed thoroughly.

DNA methylome profiling at single-base resolution through bisulfite sequencing of 5mC-immunoprecipitated DNA.

BACKGROUND: Detection of DNA methylome at single-base resolution is a significant challenge but promises to shed considerable light on human disease etiology. Current technologies could not detect DNA methylation genome-wide at single-base resolution with small amount of sequencing data and could not avoid detecting the methylation of repetitive elements which are considered as "junk DNA". METHODS: In this study, we have developed a novel DNA methylome profiling technology named MB-seq with its ability to identify genome-wide 5mC and quantify DNA methylation levels by introduced an assistant adapter AluI-linker This linker can be ligated to sonicated DNA and then be digested after the bisulfite treatment and amplification, which has no effect of MeDIP enrichment. Because many researchers are interested in investigating the methylation of functional regions such as promoters and gene bodies, we have also developed a novel alternative method named MRB-seq, which can be used to investigate the DNA methylation of functional regions by removing the repeats with Cot-1 DNA. RESULTS: In this study, we have developed MB-seq, a novel DNA methylome profiling technology combining MeDIP-seq with bisulfite conversion, which can precisely detect the 5mC sites and determine their DNA methylation level at single-base resolution in a cost-effective way. In addition, we have developed a new alternative method, MRB-seq (MeDIP-repetitive elements removal-bisulfite sequencing), which interrogates 5mCs in functional regions by depleting nearly half of repeat fragments enriched by MeDIP. Comparing MB-seq and MRB-seq to whole-genome BS-seq using the same batch of DNA from YH peripheral blood mononuclear cells. We found that the sequencing data of MB-seq and MRB-seq almost reaches saturation after generating 7-8 Gbp data, whereas BS-seq requires about 100 Gbp data to achieve the same effect. In comparison to MeDIP-seq and BS-seq, MB-seq offers several key advantages, including single-base resolution, discriminating the methylated sites within a CpG and non-CpG pattern and overcoming the false positive of MeDIP-seq due to the non-specific binding of 5-methylcytidine antibody to genomic fragments. CONCLUSION: Our novel developed method MB-seq can accelerate the decoding process of DNA methylation mechanism in human diseases because it requires 7-8 Gbp data to measure human methylome with enough coverage and sequencing depth, affording it a direct and practical application in the study of multiple samples. In addition, we have also provided a novel alternative MRB-seq method, which removes most repetitive sequences and allows researchers to genome-wide characterize DNA methylation of functional regions.

Preparation of Nano Au and Pt Alloy Microspheres Decorated with Reduced Graphene Oxide for Nonenzymatic Hydrogen Peroxide Sensing.

The flourish of nanotechnology has brought new vitality to the research and development of electrochemical sensing materials. In this work, we successfully synthesized Nano Au and Pt alloy microspheres decorated with reduced graphene oxide (RGO/nAPAMSs) by a simple, facile, and eco-friendly one-step reduction strategy for the fabrication of highly sensitive nonenzymatic H2O2 sensing interfaces. Energy-dispersive X-ray spectroscopy mapping (EDX mapping), energy-dispersive X-ray spectroscopy analysis (EDX), transmission electron microscopy (TEM), Fourier transform infrared spectrum (FT-IR), and X-ray diffraction spectrum (XRD) were employed to characterize RGO/nAPAMSs from a microscopic perspective. The results of cyclic voltammetry and chronoamperometry exhibited excellent electrochemical behaviors toward H2O2, with a rapid response time within 5 s, remarkable sensitivity of 1117.0 µA mM-1 cm-2, wide linear range of 0.005 to 4.0 mM and lower detection limit of 0.008 µM (S/N = 3), which provide RGO/nAPAMS not only a promising prospect for the quantitative detection of H2O2 but also a potential application in other fields of sensors. Moreover, further analysis showed the principles of the superior H2O2 sensing performance of RGO/nAPAMSs. This discovery provides a significant contribution to future study in nonenzymatic H2O2 sensing based on Nano Pt, Nano Au noble metal electrocatalysts.

Computer-Aided Design and Three-Dimensional-Printed Surgical Templates for Second-Stage Mandibular Reconstruction.

It is extremely difficult in clinical practice to accurately reset the physiologic positions of the mandibular condyle and ramus because of ongoing bone remodeling, muscle stretching, occlusal disorders, and other factors; this makes it difficult to obtain a good shape for a reconstructed mandible, as well as a good condylar position. The present study aimed to investigate a standardized method for mandibular reconstruction in cases of obsolete mandibular defects, using a 3-dimensional (3D)-printed surgical template enhanced by computer-aided design. We collected computed tomography data preoperatively to computerize the physiologic positions of the mandibular condyle and ramus on the sagittal, axial, and coronal planes. Surgical simulation and 3D-printed template preparation were then conducted to assist in the implementation of actual surgery. Postoperative review was performed to assess the repositioning of the condyle, where patients were found to regain a satisfactory condyle position. Reconstruction error was ±0.56 mm, fulfilling the preoperative design. No complications and discomfort were reported. Overall, the combined use of computer-aided design and 3D-printed surgical templates can standardize mandibular reconstruction in cases of obsolete mandibular body defects.

A Rapidly Self-Healing Host-Guest Supramolecular Hydrogel with High Mechanical Strength and Excellent Biocompatibility.

It is still a challenge to achieve both excellent mechanical strength and biocompatibility in hydrogels. In this study, we exploited two interactions to form a novel biocompatible, slicing-resistant, and self-healing hydrogel. The first was molecular host-guest recognition between a host (isocyanatoethyl acrylate modified ß-cyclodextrin) and a guest (2-(2-(2-(2-(adamantyl-1-oxy)ethoxy)ethoxy)ethoxy)ethanol acrylate) to form "three-arm" host-guest supramolecules (HGSMs), and the second was covalent bonding between HGSMs (achieved by UV-initiated polymerization) to form strong cross-links in the hydrogel. The host-guest interaction enabled the hydrogel to rapidly self-heal. When it was cut, fresh surfaces were formed with dangling host and guest molecules (due to the breaking of host-guest recognition), which rapidly recognized each other again to heal the hydrogel by recombination of the cut surfaces. The smart hydrogels hold promise for use as biomaterials for soft-tissue repair.

Transcriptome analysis of Ruditapes philippinarum hepatopancreas provides insights into immune signaling pathways under Vibrio anguillarum infection.

The Manila clam, Ruditapes philippinarum, is one of the most economically important aquatic clams that are harvested on a large scale by the mariculture industry in China. However, increasing reports of bacterial pathogenic diseases have had a negative effect on the aquaculture industry of R. philippinarum. In the present study, the two transcriptome libraries of untreated (termed H) and challenged Vibrio anguillarum (termed HV) hepatopancreas were constructed and sequenced from Manila clam using an Illumina-based paired-end sequencing platform. In total, 75,302,886 and 66,578,976 high-quality clean reads were assembled from 101,080,746 and 99,673,538 raw data points from the two transcriptome libraries described above, respectively. Furthermore, 156,116 unigenes were generated from 210,685 transcripts, with an N50 length of 1125 bp, and from the annotated SwissProt, NR, NT, KO, GO, KOG and KEGG databases. Moreover, a total of 4071 differentially expressed unigenes (HV vs H) were detected, including 903 up-regulated and 3168 down-regulated genes. Among these differentially expressed unigenes, 226 unigenes were annotated using KEGG annotation in 16 immune-related signaling pathways, including Toll-like receptor, NF-kappa B, MAPK, NOD-like receptor, RIG-I-like receptor, and the TNF and chemokine signaling pathways. Finally, 20,341 simple sequence repeats (SSRs) and 214,430 potential single nucleotide polymorphisms (SNPs) were detected from the H and HV transcriptome libraries. In conclusion, these studies identified many candidate immune-related genes and signaling pathways and conducted a comparative analysis of the differentially expressed unigenes from Manila clam hepatopancreas in response to V. anguillarum stimulation. These data laid the foundation for studying the innate immune systems and defense mechanisms in R. philippinarum.

The TLR13-MyD88-NF-κB signalling pathway of Cyclina sinensis plays vital roles in innate immune responses.

Toll-like receptors, the best known pattern recognition receptors, play important roles in recognizing non-self molecules and binding pathogen-associated molecular patterns in the innate immune system. In the present research, the cDNA and protein characterization of the TLR signalling pathway genes including IRAK4, TRAK6 and IKKα (named CsIRAK4, CsTRAF6 and CsIKKα, respectively) with the typical motifs from Cyclina sinensis showed significant similarity with their homologues from other shellfish. Furthermore, the mRNA transcripts of these three genes are ubiquitously expressed in all tissues tested and are dominantly expressed in C. sinensis haemocytes (P < 0.05). Moreover, IRAK4, TRAK6 and IKKα cDNA expression levels were all up-regulated after injection with Vibrio anguillarum, Micrococcus luteus and poly I:C (P < 0.01) as shown by quantitative real-time PCR, indicating that they were involved in responding to pathogenic stimulation. We explored the function of the TLR13-MyD88-NF-κB signalling pathway in the innate immune responses of C. sinensis by RNA interference and immune challenges. The results suggested the mRNA expression patterns of CsMyD88, CsIRAK4, CsTRAF6, CsIKKα, CsIκB, CsNF-κB, CsC-LYZ and CsAMP were all down-regulated (P < 0.01) in normal and stimulated C. sinensis haemocytes, revealing the involvement of the TLR13-MyD88-NF-κB signalling pathway in innate immunity by positively adjusting internal signalling factors and immune-related genes. In summary, a TLR13-MyD88-NF-κB signalling pathway exists and plays vital roles in innate immune responses in C. sinensis. These findings collectively lay the foundation for studying the functional characterization of internal signalling factors and establishing a regulatory network for the TLR signalling pathway in molluscs.

Identification and functional characterization of three TLR signaling pathway genes in Cyclina sinensis.

Toll-like receptors (TLRs) are an ancient family of pattern recognition receptors that play a critical role in initiating and activating the innate immune system. In this study, we identified two TLR genes (CsTLR4 and CsTLR13) and the MyD88 (CsMyD88) gene using a transcriptome library from Cyclina sinensis. The sequence features and mRNA expression profiles of the genes were characterized, and their functions in the immune response were investigated to validate the TLR signaling pathway and its potential role in immune defense. The expression patterns of CsTLR4, CsTLR13 and CsMyD88 were detected in all the tissues examined from healthy clams and were primarily expressed in the hemocytes (P < 0.05), as shown by real-time PCR. Upon challenge with Vibrio anguillarum and Micrococcus luteus, they were significantly increased in hemocytes (P < 0.01), whereas only CsTLR13 and CsMyD88 were up-regulated (P < 0.01) by poly (I:C) challenge. In addition, the mRNA expression level of CsC-LYZ and CsAMP was down-regulated at 72 h (P < 0.01) after injection with CsMyD88 RNAi. These findings might be valuable for understanding the innate immune signaling pathways of C. sinensis and enabling future studies on host-pathogen interactions.

Molecular cloning, characterization and functional analysis of a heat shock protein 70 gene in Cyclina sinensis.

Heat shock protein 70 (HSP70) is an important member of the heat shock protein superfamily and is involved in protecting organisms against various stressors. In the present study, we used RACE to clone a full-length Cyclina sinensis HSP70 cDNA termed CsHSP70. The full length of the CsHSP70 cDNA was 2308 bp, with a 5' untranslated region (UTR) of 42 bp, a 3' UTR of 268 bp, and an open reading frame (ORF) of 1998 bp encoding a polypeptide of 655 amino acids with an estimated molecular mass of 72.75 kDa and an estimated isoelectric point of 5.48. Quantitative real-time PCR was employed to analyze the tissue distribution and temporal expression of the CsHSP70 gene after bacterial challenge and cadmium (Cd) exposure. The CsHSP70 mRNA transcript was expressed ubiquitously in five examined tissues, with the highest expression in hemocytes (P < 0.05) and with the lowest expression in the hepatopancreas. Furthermore, the expression level of CsHSP70 in hemocytes at 3 h after Vibrio anguillarum challenge was extremely significantly up-regulated (P < 0.01). Moreover, the CsHSP70 transcript was up-regulated significantly following exposure to a safe Cd concentration (0.1 mg/L). Finally, after the CsHSP70 gene was silenced by RNA interference, the expression of the CsTLR13 and CsMyD88 genes were extremely significantly decreased (P < 0.01). The results indicated that CsHSP70 could play an important role in mediating the environmental stress and immune responses, and regulating TLR signaling pathway in C. sinensis.

Identification and expression analysis of IκB and NF-κB genes from Cyclina sinensis.

With the increasing economic importance of Cyclina sinensis aquaculture, interest in its defense mechanisms against pathogenic infection has grown in recent years. Inhibitor of nuclear factor-kappaB (IκB) and nuclear factor-kappaB (NF-κB) are proteins with central roles in many important physiological and pathological processes, such as innate immune responses. In this study, we identified CsIκB and CsNF-κB genes from a C. sinensis transcriptome library. In healthy adult clams, CsIκB and CsNF-κB genes were widely expressed in various tissues and highly expressed in hemocytes. Further, the expression levels of these genes were significantly increased in hemocytes challenged by Vibrio anguillarum, Micrococcus luteus and poly I:C. Inhibition of CsMyD88 expression by RNAi technology significantly altered the mRNA expression patterns of CsIκB and CsNF-κB as measured using quantitative real-time PCR. These results collectively indicated that the NF-κB signaling pathway, including CsIκB and CsNF-κB genes, might be involved in early innate immune responses and may be regulated by a MyD88-dependent signaling pathway in C. sinensis.

PacBio full-length transcriptome profiling of insect mitochondrial gene expression.

In this study, we sequenced the first full-length insect transcriptome using the Erthesina fullo Thunberg based on the PacBio platform. We constructed the first quantitative transcription map of animal mitochondrial genomes and built a straightforward and concise methodology to investigate mitochondrial gene transcription, RNA processing, mRNA maturation and several other related topics. Most of the results were consistent with the previous studies, while to the best of our knowledge some findings were reported for the first time in this study. The new findings included the high levels of mitochondrial gene expression, the 3' polyadenylation and possible 5' m(7)G caps of rRNAs, the isoform diversity of 12S rRNA, the polycistronic transcripts and natural antisense transcripts of mitochondrial genes et al. These findings could challenge and enrich fundamental concepts of mitochondrial gene transcription and RNA processing, particularly of the rRNA primary (sequence) structure. The methodology constructed in this study can also be used to study gene expression or RNA processing of nuclear genomes.

A Kazal-type serine proteinase inhibitor from Cyclina sinensis is involved in immune response and signal pathway initiation.

Serine protease inhibitors (SPIs) are an important group of protease inhibitors involved in a variety of biological processes. In the present study, a Kazal-type serine protease inhibitor homolog gene (designated as CsKPI) was identified from a Cyclina sinensis cDNA library. The open reading frame consists of 456 bp and encodes a protein of 151 amino acid residues with a theoretical molecular mass of 16.85 kDa and an isoelectric point of 5.74. Furthermore, using quantitative real-time PCR, we focused on the expression patterns of CsKPI found in tissues and on the stimulation of this gene's expression by bacteria. The results show that a higher-level mRNA expression of CsKPI was detected in hemocytes (P < 0.05) and was significantly upregulated at 3 h (P < 0.01) upon receiving bacterial challenges with Vibrio anguillarum. In addition, after the CsKPI gene was silenced by RNA interference, the expression of the CsTLR2 and CsMyD88 genes was extremely significantly decreased (P < 0.01) in C. sinensis. Finally, the recombinant CsKPI (rCsKPI) protein was purified and shown to exhibit less inhibitory activity than C-lyz against V. anguillarum in vitro. Hence, we propose that CsKPI plays an important role in the innate immunity and mediates TLR2 and MyD88-dependent pathway initiation in C. sinensis.