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Background: Haemaphysalis qinghaiensis is a tick species distributed only in China. Due to its ability to transmit a variety of pathogens, including species of the genera Anaplasma, Rickettsia, Babesia, and Theileria, it seriously endangers livestock husbandry. However, the microbial community of the midgut of H. qinghaiensis females collected from yaks and Tibetan sheep has not yet been characterized using metagenomic sequencing technology., Methods: Haemaphysalis qinghaiensis were collected from the skins of yaks and Tibetan sheep in Gansu Province, China. Genomic DNA was extracted from the midguts and midgut contents of fully engorged H. qinghaiensis females collected from the two hosts. Metagenomic sequencing technology was used to analyze the microbial community of the two groups., Results: Fifty-seven phyla, 483 genera, and 755 species were identified in the two groups of samples. The ticks from the two hosts harbored common and unique microorganisms. At the phylum level, the dominant common phyla were Proteobacteria, Firmicutes, and Mucoromycota. At the genus level, the dominant common genera were Anaplasma, Ehrlichia, and Pseudomonas. At the species level, bacteria including Anaplasma phagocytophilum, Ehrlichia minasensis, and Pseudomonas aeruginosa along with eukaryotes such as Synchytrium endobioticum and Rhizophagus irregularis, and viruses such as the orf virus, Alphadintovirus mayetiola, and Parasteatoda house spider adintovirus were detected in both groups. In addition, the midgut of H. qinghaiensis collected from yaks had unique microbial taxa including two phyla, eight genera, and 23 species. Unique microorganisms in the midgut of H. qinghaiensis collected from Tibetan sheep included two phyla, 14 genera, and 32 species. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that the functional genes of the microbiome of H. qinghaiensis were annotated to six pathways, and the metabolic pathways included 11 metabolic processes, in which the genes involved in carbohydrate metabolism were the most abundant, followed by the genes involved in lipid metabolism., Conclusions: These findings indicate that most of the microbial species in the collected H. qinghaiensis ticks were the same in both hosts, but there were also slight differences. The analytical data from this study have enhanced our understanding of the midgut microbial composition of H. qinghaiensis collected from different hosts. The database of H. qinghaiensis microbe constructed from this study will lay the foundation for predicting tick-borne diseases. Furthermore, a comprehensive understanding of tick microbiomes will be useful for understanding vector competency and interactions with ticks and midgut microorganisms., (© 2024. The Author(s).)

Haemaphysalis longicornis is a common tick species that carries several pathogens. There are few reports on the influence of different hosts on the structure of midgut microflora in H. longicornis . In this study, midgut contents of fully engorged female H. longicornis were collected from the surface of tiger ( Panthera tigris ) and deer ( Dama dama ). The bacterial genomic DNA of each sample was extracted, and the V3-V4 regions of the bacterial 16S rRNA were sequenced using the Illumina NovaSeq sequencing. The diversity of the bacterial community of the fully engorged female H. longicornis on the surface of tiger was higher than that of deer. In total, 8 phyla and 73 genera of bacteria annotations were detected in the two groups. At the phylum level, the bacterial phyla common to the two groups were Proteobacteria, Firmicutes, and Actinobacteriota. At the genus level, there were 20 common bacterial genera, among which the relative abundances of Coxiella , Morganella , Diplorickettsia , and Acinetobacter were high. The Morganella species was further identified to be Morganella morganii . The alpha diversity index indicated that the bacterial diversity of the tiger group was higher than that of the deer group. Bacteroidota, Patescibacteria, Desulfobacterota, Verrucomicrobiota, and Cyanobacteria were solely detected in the tiger group. A total of 52 bacterial genera were unique in the tiger group, while one bacterial genus was unique in the deer group. This study indicates that there are differences in the structure of the gut bacteria of the same tick species among different hosts. Further culture-based methods are needed to provide a more comprehensive understanding of the tick microbiota parasitizing different hosts.

Ticks are blood-sucking ectoparasites that secrete immunomodulatory substances in saliva to hosts during engorging. Cystatins, a tick salivary protein and natural inhibitor of Cathepsins, are attracting growing interest globally because of the immunosuppressive activities and the feasibility as an antigen for developing anti-tick vaccines. This review outlines the classification and the structure of tick Cystatins, and focuses on the anti-inflammatory effects and molecular mechanisms. Tick Cystatins can be divided into four families based on structures and cystatin 1 and cystatin 2 are the most abundant. They are injected into hosts during blood feeding and effectively mitigate the host inflammatory response. Mechanically, tick Cystatins exert anti-inflammatory properties through the inhibition of TLR-NF-κb, JAK-STAT and p38 MAPK signaling pathways. Further investigations are crucial to confirm the reduction of inflammation in other cell types like neutrophils and mast cells, and fully elucidate the underlying mechanism (like the structural mechanism) to make Cystatin a potential candidate for the development of novel anti-inflammation agents., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Human lice have always been a major public health concern due to their vector capacity for louse-borne infectious diseases, like trench fever, louse-borne relapsing fever, and epidemic fever, which are caused by Bartonella quintana, Borrelia recurrentis, and Rickettsia prowazekii, respectively. Those diseases are currently re-emerging in the regions of poor hygiene, social poverty, or wars with life-threatening consequences. These louse-borne diseases have also caused outbreaks among populations in jails and refugee camps. In addition, antibodies and DNAs to those pathogens have been steadily detected in homeless populations. Importantly, more bacterial pathogens have been detected in human lice, and some have been transmitted by human lice in laboratories. Here, we provide a comprehensive review and update on louse-borne infectious diseases/bacterial pathogens., Competing Interests: Declaration of competing interest We declare no competing interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Tick eggs contain all essential proteins for embryogenesis, and egg proteins are a potential reservoir of tick-protective antigens. However, the protein profile and dynamics during embryonic development remain unknown. This study aimed to depict the protein profile and dynamics in tick embryogenesis, further providing protein candidates for targeted interventions. Eggs from Haemaphysalis flava ticks were incubated at 28 °C and 85% relative humidity. On days 0 (newly laid eggs without incubation), 7, 14 and 21, eggs were collected, dewaxed and subject to protein extraction. Extracted proteins were digested by filter-aided sample preparation and analyzed by liquid chromatography-tandem mass spectrometry (LC/MS-MS). MS data were searched against an in-house H. flava protein database for tick-derived protein identification. Abundances of 40 selected high-confidence proteins were further quantified by LC-parallel reaction monitoring (PRM)/MS analysis throughout egg incubation. A total of 93 high-confidence proteins were identified in eggs on 0-day incubation. Identified proteins belonged to seven functional categories: transporters, enzymes, proteinase inhibitors, immunity-related proteins, cytoskeletal proteins, heat shock proteins and uncharacterized proteins. The enzyme category contained the most types of proteins. Neutrophil elastase inhibitors represented the most abundant proteins in terms of intensity-based absolute-protein-quantification. LC-PRM/MS revealed that the abundances of 20 proteins increased including enolase, calreticulin, actin, GAPDH et cetera, and the abundances of 11 proteins decreased including vitellogenins, neutrophil elastase inhibitor, carboxypeptidase Q, et cetera from 0- to 21-day incubation. This study provides the most comprehensive egg protein profile and dynamics during tick embryogenesis. Further investigations are needed to test the tick-control efficacy by targeting the egg proteins., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Tick saliva is a reservoir of bioactive proteins. Saliva protein compositions change dynamically during blood-feeding. Decipherment of protein profiles in different blood-feeding stages may bring deeper insight into tick feeding physiology and provide targets for immunologic control alternatives. However, having the infancy of tick genome sequencing, assembly, annotation, and limited knowledge of tick salivary proteins restrain the data interpretation. Here, we aimed to depict the saliva protein profile in partially- (PE) and fully-engorged (FE) Haemaphysalis flava ticks, with a special focus on the analysis of those uncharacterized proteins. Saliva was collected from PE and FE adult female H. flava ticks. Saliva proteins were analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS-MS). MS data were searched against an in-house salivary gland transcriptome library for identification of tick-derived proteins. Abundances of proteins were compared between PE and FE ticks. The uncharacterized proteins detected in saliva were further bioinformatically analyzed. In total, 614 proteins were identified including 94 host proteins and 520 tick-derived proteins. The 226 tick-derived high-confidence proteins were classified into 10 categories: transporters, enzymes, protease inhibitors, immunity-related proteins, lipocalins, glycine-rich proteins, muscle proteins, secreted proteins, uncharacterized proteins and others. A total of 98 proteins were shared in both PE and FE with 74 only in PE and 54 only in FE. Abundances of 24 shared proteins were significantly higher in PE. The profile of top 15 most abundant proteins was also different between PE and FE ticks. The 65 uncharacterized proteins detected in tick saliva were branched into subclusters 1 A, 1B, 2, 3 A, 3B and 3 C based on particular motifs like RGD, LRR, indicating their diverse predicted functions like anti-coagulation, regulation of innate immune, or other functions. This study provides and compares saliva proteomes of H. flava ticks in two feeding stages with special cluster analysis on the uncharacterized proteins. Further investigations are needed to confirm the roles of these uncharacterized proteins in ticks., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Ixodes hunanensis n. sp. (Acari: Ixodidae), is identified based on the morphological characteristics and molecular biological analyses of males and females ex hog badger, Arctonyx collaris Cuvier (Carnivora: Mustelidae) from China. Adults of this new species are similar to those of other species of the subgenus Pholeoixodes Schulze, 1942, from which they can be distinguished by the shape of basis capituli, development of cornua, size of porose areas, shape, and size of spurs on coxae and phylogenetic analyses of the cox1 and 16S rRNA sequences., (© 2022 Royal Entomological Society.)

Background: Haemaphysalis flava is a hematophagous ectoparasite that acquires the nutrition needed for development and reproduction by sucking blood and digesting the blood meal. During blood-sucking and blood-meal digestion, the prevention of blood coagulation is important for this tick. Previous studies have shown that heat shock cognate 70 (HSC70) protein has certain anticoagulant activities, but its immunogenicity remains unclear. Also, whether the mutation of individual bases of the TKD-like peptide of HSC70 through the overlap extension method can change its anticoagulant activities and immunogenicity remains to be investigated., Methods: The gene encoding the HSC70 protein was cloned from a complementary DNA library synthesized from H. flava. The coding gene of the TKD-like peptide of HSC70 was mutated into a TKD peptide coding gene (HSC70 TKD ) using the overlap extension method. Escherichia coli prokaryotic expression plasmids were constructed to obtain the recombinant proteins of HSC70 (rHSC70) and HSC70 TKD (rHSC70 TKD ). The purified rHSC70 and rHSC70 TKD were evaluated at different concentrations for anticoagulant activities using four in vitro clotting assays. Emulsifying recombinant proteins with complete and incomplete Freund's adjuvants were subcutaneously immunized in Sprague Dawley rats. The serum antibody titers and serum concentrations of interferon-gamma (IFN-γ) and interleukin-4 (IL-4) were detected using an indirect enzyme-linked immunosorbent assay to assess the immunogenicity of rHSC70 and rHSC70 TKD ., Results: The open reading frame of HSC70 was successfully amplified and found to have a length of 1958 bp. The gene encoding the TKD-like peptide of HSC70 was artificially mutated, with the 1373-position adenine (A) of the original sequence mutated into guanine (G), the 1385-position cytosine (C) mutated into G and the 1386-position G mutated into C. rHSC70 and rHSC70 TKD that fused with His-tag were obtained using the expression plasmids pET-28a-HSC70 and pET-28a-HSC70 TKD , respectively. rHSC70 and rHSC70 TKD prolonged the thrombin time (TT) and reduced the fibrinogen (FIB) content in the plasma, but did not affect the prothrombin time (PT) or activated partial thromboplastin time (APTT) when compared to the negative control. Interestingly, the ability of rHSC70 TKD to prolong the TT and reduce the FIB content in the plasma was better than that of rHSC70. The specific antibody titers of both rHSC70 and rHSC70 TKD in rat serum reached 1:124,000 14 days after the third immunization. The serum concentration of IFN-γ in the rHSC70 TKD group was higher than that in the rHSC70 group. The rHSC70 group has the highest serum concentration of IL-4, and the serum concentration of IL-4 in the rHSC70 TKD group was higher than that in the negative group., Conclusions: rHSC70 and rHSC70 TKD exhibited anticoagulant activities by prolonging the TT and reducing the FIB content in vitro. rHSC70 TKD had better anticoagulant activities than rHSC70. Both rHSC70 and rHSC70 TKD had good immunogenicity and induced humoral and cellular immunity., (© 2022. The Author(s).)

Background: Tick hemolymph bathes internal organs, acts as an exchange medium for nutrients and cellular metabolites, and offers protection against pathogens. Hemolymph is abundant in proteins. However, there has been limited integrated protein analysis in tick hemolymph thus far. Moreover, there are difficulties in differentiating tick-derived proteins from the host source. The aim of this study was to profile the tick/host protein components in the hemolymph of Haemaphysalis flava., Methods: Hemolymph from adult engorged H. flava females was collected by leg amputation from the Erinaceus europaeus host. Hemolymph proteins were extracted by a filter-aided sample preparation protocol, digested by trypsin, and assayed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). MS raw data were searched against the UniProt Erinaceidae database and H. flava protein database for host- and tick-derived protein identification. Protein abundance was further quantified by intensity-based absolute quantification (iBAQ)., Results: Proteins extracted from hemolymph unevenly varied in size with intense bands between 100 and 130 kDa. In total, 312 proteins were identified in the present study. Therein 40 proteins were identified to be host-derived proteins, of which 18 were high-confidence proteins. Top 10 abundant host-derived proteins included hemoglobin subunit-α and subunit-β, albumin, serotransferrin-like, ubiquitin-like, haptoglobin, α-1-antitrypsin-like protein, histone H2B, apolipoprotein A-I, and C3-β. In contrast, 169 were high-confidence tick-derived proteins. These proteins were classified into six categories based on reported functions in ticks, i.e., enzymes, enzyme inhibitors, transporters, immune-related proteins, muscle proteins, and heat shock proteins. The abundance of Vg, microplusin and α-2-macroglobulin was the highest among tick-derived proteins as indicated by iBAQ., Conclusions: Numerous tick- and host-derived proteins were identified in hemolymph. The protein profile of H. flava hemolymph revealed a sophisticated protein system in the physiological processes of anticoagulation, digestion of blood meal, and innate immunity. More investigations are needed to characterize tick-derived proteins in hemolymph., (© 2022. The Author(s).)

The full-length cDNA of two ferritins of Haemaphysalis flava were cloned after which recombinant Hf-FER1 and Hf-FER2 were expressed and their function was analyzed. In addition, RNA interference (RNAi) based on the injection of Hf-fer1 or Hf-fer2 dsRNA into fully engorged female ticks was performed. The cDNA encoding Hf-FER1 is 834 bp in length. It contains an iron-responsive element in the 5' untranslated region and encodes 174 amino acid residues. The full-length cDNA of Hf-FER2 contains 696 bp and encodes 199 amino acids, including a putative signal peptide sequence. Hf-FER1 and Hf-FER2 both have the ferroxidase iron center and the ferrihydrite nucleation center. The evolutionary relationship of Hf-FER1 and Hf-FER2 was established, and the predicted quaternary structures were assembled as typical spherical shells composed of 24 subunits which was demonstrated by nature PAGE. Real-time PCR showed that Hf-fer1 and Hf-fer2 were expressed in all developmental stages, with the highest expression in fully engorged females. The expression of Hf-fer1 and Hf-fer2 were relatively high in unfed larvae. Hf-fer1 was expressed in all tissues and was especially abundant in the salivary glands of fully engorged females. In contrast, the highest levels of Hf-fer2 were found in the midgut of fully engorged females, and no expression was found in the salivary glands of this life stage. Both recombinant Hf-FER1 and Hf-FER2 had iron-binding capabilities. Silencing of both Hf-fer1 and Hf-fer2 affected fecundity. Compared to the control, the percentage of ticks that laid eggs in the Hf-fer1 and Hf-fer2 RNAi groups was 73.3% and 66.7%, respectively. The silenced ticks that laid eggs had lower egg weight to body weight ratios, and the eggs had abnormal morphologies. The hatchability of eggs with normal morphology in the Hf-fer1 and Hf-fer2 silenced groups was 47.8% and 22.8%, respectively, which was significantly different from the control group (P < 0.005). These findings indicate that Hf-FER1 and Hf-FER2 play important roles in the iron storage of H. flava., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Background: Ticks, which are ectoparasites of animals, may carry multiple pathogens. The cattle tick Rhipicephalus microplus is an important bovine parasite in China. However, the midgut microbiome of R. microplus from China has not been characterized via metagenomic methods., Methods: Rhipicephalus microplus were collected from cattle in the city of Changsha in Hunan province, China. The DNA of the midgut contents was extracted from fully engorged adult female R. microplus. A DNA library was constructed and sequenced using an Illumina HiSeq sequencing platform. SOAPdenovo software was used to assemble and analyze the clean data. The latent class analysis algorithm applied to system classification by MEGAN software was used to annotate the information on the species' sequences. DIAMOND software was used to compare unigenes with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and functional annotation was carried out based on the results of the comparison., Results: The dominant phyla in the five samples were Firmicutes, Proteobacteria, and Actinobacteria. Streptococcus, Mycobacterium, Anaplasma, Enterococcus, Shigella, Lactobacillus, Brachyspira, Pseudomonas, Enterobacter, Bacillus, and Lactococcus were the dominant genera in the five samples. The endosymbiotic bacterium Wolbachia was also detected in all of the samples. Mycobacterium malmesburyense, Streptococcus pneumoniae, Anaplasma phagocytophilum, Enterococcus faecium, Shigella sonnei, Enterococcus faecalis, Lactobacillus casei, Brachyspira hampsonii, Pseudomonas syringae, Enterobacter cloacae, and Lactococcus garvieae were the dominant species in the five samples. In addition to these bacterial species, we also detected some eukaryotes, such as Rhizophagus irregularis, Enterospora canceri, Smittium culicis, Zancudomyces culisetae, Trachipleistophora hominis, and viruses such as orf virus, human endogenous retrovirus type W, enzootic nasal tumor virus of goats, bovine retrovirus CH15, and galidia endogenous retrovirus in all of the samples at the species level. The results of the annotated KEGG pathway predictions for the gene functions of the midgut microflora of R. microplus indicated genes involved in lipid and amino acid metabolism, infectious diseases (e.g., Streptococcus pneumonia infection, human granulocytic anaplasmosis, Shigella sonnei infection, Salmonella enterica infection, and pathogenic Escherichia coli infection), and cancer., Conclusions: Our study revealed that the midgut microbiome of R. microplus is not only composed of a large number of bacteria, but that a portion also comprises eukaryotes and viruses. The data presented here enhance our understanding of this tick's midgut microbiome and provide fundamental information for the control of ticks and tick-borne diseases., (© 2022. The Author(s).)

Argas persicus is an ectoparasite of poultry. The bacterial community structure and the pathogenic bacteria associated with different developmental stages of A. persicus have implications for control. Argas persicus were collected from chickens in the city of Jiuquan in Gansu, China. Bacterial DNA was extracted from the midgut contents of blood engorged larvae, nymphs and adult females. The V3-V4 hypervariable regions of 16S rRNA genes were sequenced using the IonS5™XL platform. Identification of Rickettsia spp. and detection of Coxiella burnetii were performed using PCR on target genes. The bacterial diversity within larvae was the highest and the bacterial diversity within nymphs was greater than that of adults. At different classification levels, seven bacterial phyla were common phyla, 27 genera were common genera, and 18 species were common species in the three samples. At the phylum level, Proteobacteria showed a marked predominance in all samples. Rickettsia, Stenotrophomonas, Spiroplasma, and Coxiella were the dominant bacteria at the genus level. The Rickettsia species in A. persicus was identified as Rickettsia hoogstraalii and the Coxiella species was identified as a Coxiella-like endosymbiont. Additionally, some bacterial species such as Pseudomonas geniculata, Sphingomonas koreensis, and Acinetobacter haemolyticus were reported here for the first time in A. persicus., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

In this study, the authors sequenced and characterized the complete mitochondrial (mt) genomes of two hard ticks of the genus Ixodes, I. nipponensis and Ixodes (Pholeoixodes) sp., which were 14 505 and 14 543 bp in length, respectively. Their mt genomes encoded 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNA genes and two ribosomal RNA genes, and have only one non-coding region. The gene order in their mt genomes was the same as that of other Ixodes spp. mt genomes. The average sequence identity, combined nucleotide diversity, non-synonymous/synonymous substitutions ratio analyses consistently demonstrated that cox1, rrnS, cox2, cox3 and cytb were the most conserved and atp8, nad6 and nad2 were the most variable genes across Ixodes mitogenomes. Phylogeny of the present Ixodes spp., and other selected hard tick species, based on concatenated amino acid sequences of PCGs, confirmed their position within the genus Ixodes and sub-family Ixodinae. The novel mt markers described herein will be useful for further studies of the population genetics, molecular epidemiology and systematics of hard ticks., (© 2021 The Royal Entomological Society.)

Aims: Adipocyte-secreted microvesicles (MVs)-derived microRNAs (miRNAs) are relevant to adipogenic and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in osteonecrosis of the femoral head (ONFH). Our aims are to investigate the mechanism of adipocyte-derived MVs-miR-148a in ONFH., Materials and Methods: Adipocyte-derived MVs were identified via transmission electron microscopy and specific markers expression. The adipogenic and osteogenic differentiation were investigated by Oil-Red O staining, alkaline phosphatase (ALP) activity, Alizarin Red S (ARS) staining and osteogenic or adipogenic factors levels. Genes and proteins expression were detected by using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The relationship between miR-148a and Wnt5a was tested via dual-luciferase reporter analysis. The adipogenic differentiation and osteogenic differentiation in methylprednisolone (MPS)-induced ONFH rat model were assessed via hematoxylin-eosin (HE) staining, and immunohistochemical staining of collagen I (COL I)., Key Findings: Adipocyte-derived MVs promoted adipogenic differentiation via increasing Oil-Red O staining positive cells, adiponectin (Adipoq), acid-binding protein 2 (aP2) and peroxisome proliferator-activated receptor γ (PPAR-γ) levels, and repressed osteogenic differentiation of BMSCs via decreasing ARS staining positive cells, ALP, Runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) levels. MiR-148a was present in adipocyte-derived MVs, and miR-148a knockdown inhibited adipogenic differentiation and promoted osteogenic differentiation. Furthermore, Wnt5a expression was regulated by miR-148a. MiR-148a overexpression facilitated adipogenic differentiation and suppressed osteogenic differentiation via regulating the Wnt5a/Ror2 pathway. Adipocyte-derived MVs promoted adipogenic differentiation and inhibited osteogenic differentiation in MPS-induced ONFH rat model., Significance: Adipocyte-derived MVs-miR-148a promoted adipogenic differentiation and suppressed osteogenic differentiation via targeting the Wnt5a/Ror2 pathway., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objectives: To compare the treatment effects of periodontal endoscope-assisted and traditional subgingival scaling on residual pockets., Methods: A total of 13 patients with periodontitis from Dept. of Periodontics, West China Hospital of Stomatology, Sichuan University were recruited. After 4-6 weeks of initial treatment, the residual pockets with a probing depth (PD) of ≥4 mm and attachment loss (AL) of ≥4 mm and bleeding on probing were examined with traditional (control group) and periodontal endoscope-assisted subgingival scaling (endoscopy group) in a randomly controlled split-mouth design. At baseline and 6 weeks and 3 months after treatment, plaque index (PLI), PD, AL, and bleeding index (BI) were measured. Differences in these clinical parameters within and between groups and patient-reported outcomes were compared., Results: A total of the 694 sites of 251 teeth were included in this trial. Both groups showed significant improvement in each periodontal parameters 6 weeks and 3 months after treatment ( P <0.001). For sites in a single-rooted tooth, sites with PD≥5 mm, and sites without vertical alveolar bone resorption and furcation involvement, the PD in endoscopy group was significantly lower than that in the control group at 6 weeks and 3 months after treatment ( P <0.05)., Conclusions: Periodontal endoscope-assisted subgingival scaling resulted in better effects than traditional subgingival scaling when the residual pockets were in a single-rooted tooth, with a PD of ≥5 mm but without vertical alveolar bone resorption and furcation involvement.

Ticks are hematophagous ectoparasites and cause a major public health threat worldwide. Development of anti-tick vaccines is regarded to be an optimal alternative for tick control. AV422, a unique protein in ticks, is secreted into hosts during blood-feeding, but its roles are not confirmed in Haemaphysalis flava ticks. We retrieved a gene fragment encoding AV422 from a transcriptome dataset of H. flava, and based on it, we reconstructed the full length of AV422 from H. flava (Hf-AV422) by rapid amplification of cDNA ends. Expression profiles of Hf-AV422 in whole ticks and organs of different engorgement levels were determined by qPCR. Then its opening reading frame (ORF) was expressed in Escherichia coli strain BL21 (DE3). The prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT) assays were conducted to test anticoagulant activities of the purified recombinant protein (rHf-AV422). The full length of AV422 was 1152 bp. Hf-AV422 showed to be conserved as indicated by multiple sequence alignment. Expression of Hf-AV422 was significantly higher in salivary glands and cuticles than in ovaries. Its expression in whole ticks decreased during engorgement with the highest levels in 1/4 engorged ticks. rHf-AV422 prolonged PT, APTT and TT when incubated with rabbit plasma. Our data demonstrated that Hf-AV422 is a conserved salivary protein with anticoagulant activity. Further studies are needed to test in detail its functional properties to ensure it an adequate antigen candidate for the development of broad-spectrum vaccines against ticks., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: The family Hoplopleuridae contains at least 183 species of blood-sucking lice, which widely parasitize both mice and rats. Fragmented mitochondrial (mt) genomes have been reported in two rat lice (Hoplopleura kitti and H. akanezumi) from this family, but some minichromosomes were unidentified in their mt genomes., Methods: We sequenced the mt genome of the rat louse Hoplopleura sp. with an Illumina platform and compared its mt genome organization with H. kitti and H. akanezumi., Results: Fragmented mt genome of the rat louse Hoplopleura sp. contains 37 genes which are on 12 circular mt minichromosomes. Each mt minichromosome is 1.8-2.7 kb long and contains 1-5 genes and one large non-coding region. The gene content and arrangement of mt minichromosomes of Hoplopleura sp. (n = 3) and H. kitti (n = 3) are different from those in H. akanezumi (n = 3). Phylogenetic analyses based on the deduced amino acid sequences of the eight protein-coding genes showed that the Hoplopleura sp. was more closely related to H. akanezumi than to H. kitti, and then they formed a monophyletic group., Conclusions: Comparison among the three rat lice revealed variation in the composition of mt minichromosomes within the genus Hoplopleura. Hoplopleura sp. is the first species from the family Hoplopleuridae for which a complete fragmented mt genome has been sequenced. The new data provide useful genetic markers for studying the population genetics, molecular systematics and phylogenetics of blood-sucking lice.

Melophagus ovinus is a type of ectoparasite infesting sheep. Data regarding the comprehensive bacterial community associated with the whole body and midgut of M. ovinus under different engorged statuses are required. Melophagus ovinus were collected from the city of Jiuquan, China. Bacterial DNA was extracted from the whole body and midgut of fully engorged female adults, or newly hatched and unfed adult female M. ovinus. The 16S rRNA gene V3-V4 hypervariable regions were sequenced using the IonS5™XL platform (Thermo Fisher Scientific, Waltham, MA, U.S.A.). The whole body bacterial diversity of the newly hatched, unfed adult females was greater compared with that of the other three samples. Proteobacteria was the dominant bacterial phylum in all of the samples. Of the 42 total bacterial genera present in all of the experimental samples, Arsenophonus, Bartonella and Wolbachia were the dominant genera. The relative abundance of Arsenophonus in midgut was greater than that in the whole body. The relative abundance of Bartonella in fully engorged adults was far greater than those in newly hatched, unfed adults. The relative abundance of Wolbachia was highest in the whole body of newly hatched, unfed adults. Seventeen bacterial species were identified in all experimental samples. Bartonella chomelii, Streptococcus hyointestinalis and Escherichia coli were the first species reported in M. ovinus., (© 2019 The Royal Entomological Society.)

The heat shock protein 70 (HSPA) family and their genes have been studied in ticks and are considered as possible antigen candidates for the development of anti-tick vaccines. However, knowledge about their members, structure and function in ticks is incomplete. Based on our transcriptomic data, the full length of four HSPA genes in Haemaphysalis flava (Acari: Ixodidae) was cloned via rapid amplification of cDNA ends. The open reading frame of HSPA2A, HSPA2B, HSPA5 and HSPA9 was 1920, 1911, 1983 and 2088 bp in length, respectively. Three family signatures and one localization motif were in the encoding proteins. HSPA2A and HSPA2B were predicted to be located at cytoplasm/nucleus, whereas HSPA5 and HSPA9 were at endoplasmic reticulum and mitochondria, respectively. In silico simulation demonstrated that those proteins had distinct numbers of α-helixes, extended strands and coils, and different antigenic epitopes. Expression of HSPA5 and HSPA9 in the salivary gland was significantly higher in partially-engorged female adult ticks than the fully-engorged (P < 0.01) as shown by a quantitative polymerase chain reaction. Our data indicated that H. flava ticks had at least four HSPA genes encoding proteins with different cellular locations, structures and expression profiles, suggesting their diverse roles in tick biology., (© 2019 The Royal Entomological Society.)

Amblyomma javanense is an important ectoparasite of Manis javanica, although the population genetics, molecular biology and systematics of A. javanense remain poorly understood. In the present study, the mitochondrial genome of A. javanense was sequenced using the Illumina HiSeq sequencing platform (Illumina, San Diego, CA, U.S.A.) and compared with the genomes of two closely related species: Amblyomma fimbriatum and Amblyomma americanum. The intraspecies and interspecies relationships of A. javanense and another 21 selected species were investigated by constructing a maximum-likelihood tree and a neighbour-joining tree. The mitochondrial genome of A. javanense was 14 780 bp in length and contained 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and two control regions. The results of the comparisons indicate that there is great similarity among these three species, and both trees indicate that A. javanense is a member of the Amblyomminae. The study of A. javanense of pangolins also indicates the premise and foundation of the relationship between the parasite and other species., (© 2019 The Royal Entomological Society.)

Dermacentor silvarum is widely distributed in northern China and transmits several pathogens that cause diseases in humans and domestic animals. We analysed the comprehensive bacterial community of the saliva and midgut from partially and fully engorged female adult D. silvarum. Dermacentor silvarum samples were collected from Guyuan, China. Bacterial DNA was extracted from the saliva and midgut contents of partially or fully engorged female adult D. silvarum. Sequencing of the V3-V4 hypervariable regions of the 16S rRNA genes was performed using the IonS5 TM XL platform. The bacterial diversity in saliva was higher than in the midgut. The bacterial diversity of saliva from fully engorged ticks was greater than in partially engorged tick saliva. The bacterial diversity in midguts from partially engorged ticks was greater than in fully engorged tick midguts. Proteobacteria was the most dominant bacterial phylum in all of the samples. Twenty-nine bacterial genera were detected in all of the samples. Rickettsia, Anaplasma, and Stenotrophomonas were the main genera. The symbionts Coxiella, Arsenophonus, and Wolbachia were also detected in all of the samples. Eight bacterial species were identified in all of the experimental samples. Anaplasma marginale was reported for the first time in D. silvarum.

The sheep ked Melophagus ovinus is mainly found in Europe, Northwestern Africa, and Asia. Although M. ovinus is an important ectoparasite of sheep in many countries, the population genetics, molecular biology, and systematics of this ectoparasite remain poorly understood. Herein, we determined the mitochondrial (mt) genome of M. ovinus from Gansu Province, China (MOG) and compared with that of M. ovinus Xinjiang Uygur Autonomous Region, China (MOX). The mt genome sequence (15,044 bp) of M. ovinus MOG was significantly shorter (529 bp) than M. ovinus MOX. Nucleotide sequence difference in the whole mt genome except for non-coding region was 0.37% between M. ovinus MOG and MOX. For the 13 protein-coding genes, comparison revealed sequence divergences at both the nucleotide (0-1.1%) and amino acid (0-0.59%) levels between M. ovinus MOG and MOX, respectively. Interestingly, the cox1 gene of M. ovinus MOX is predicted to employ unusual mt start codons AAA, which has not been predicted previously for any parasite genome. Phylogenetic analyses showed that M. ovinus (Hippoboscoidea) is related to the superfamilies Oestroidea + Muscoidea. Our results have also indicated the paraphylies of the four families (Anthomyiidae, Calliphoridae, Muscidae, and Oestridae) and two superfamilies (Oestroidea and Muscoidea). This mt genome of M. ovinus provides useful molecular markers for studies into the population genetics, molecular biology, and systematics of this ectoparasite.

In this article, the color of ghost imaging (GI) was studied theoretically and experimentally. The theoretical analysis and experimental data show that the color of GI with rotating ground glass plate and computational GI are the same as the light source. If multiwavelength source is used in these schemes, a full color image without distortion can be obtained. In contrast, the color of GI with spatial light modulator as well as that in a quantum system is a superimposed one, depending on the idle and object light beams, and following the principle of light color superposition. Correspondingly, a full color image can also be obtained under the condition of multiwavelength source, but with color distortion existing.

Protein glycosylation is one of the most important protein post-translational modifications that can affect life activities by endowing the protein with various structural and functional features. Saliva is an easy-to-obtain, noninvasive body fluid that contains components originating from serum, gingival crevicular fluid, and oropharyngeal mucosae. In recent years, understanding of saliva has been constantly updated with the developments in related research. Studies have shown that salivary proteins can be used as diagnostic markers for certain diseases, and changes of protein glycosylation in saliva are generally considered to be related to many diseases. In this review, salivary protein glycosylation and its relationship with systemic and oral diseases were discussed.

Background: Melophagus ovinus, one of the most common haematophagous ectoparasites of sheep, can cause anaemia and reductions in weight gain, wool growth and hide value. However, no information is available about the microfloral structure of the midgut of this ectoparasite. In the present study, we investigated the microbial community structure of the midgut contents of fully engorged female and male M. ovinus using Illumina HiSeq., Results: The phylum showing the highest abundance was Proteobacteria (99.9%). The dominant bacterial genera in females and males were Bartonella, Arsenophonus and Wolbachia. Some less abundant bacterial genera were also detected, including Enterobacter, Acinetobacter, Halomonas, Shewanella, Bacillus and Staphylococcus., Conclusions: Bartonella, Arsenophonus and Wolbachia were the dominant bacterial genera in the midgut of female and male M. ovinus. Although detected, Enterobacter, Acinetobacter, Halomonas, Shewanella, Bacillus and Staphylococcus showed low abundances. Importantly, this is the first report of the presence of Arsenophonus, Wolbachia, Enterobacter, Halomonas, Shewanella, Bacillus and Staphylococcus in the midgut of M. ovinus.

Newborns are highly susceptible to DNA virus infections, which may result from the characteristics of neonatal innate immune systems. Here we analyzed for the first time the development of innate immune sensing and signaling of intracellular DNA virus infection in human newborns and young children. Both mRNA and protein expression of cGAS, an intracellular DNA sensor, were shown to be significantly reduced in neonatal peripheral blood mononuclear cells (PBMCs). In addition, cGAS expression in neonatal PBMCs could be induced upon herpes simplex virus type 1 (HSV-1) or interferon-α (IFNα) stimulation. Furthermore, production of the second messenger cGAMP and activation of the transcriptional factor IRF3 was severely decreased in neonatal cord blood mononuclear cells (CBMCs) or PBMCs compared with adults. In contrast, the downstream signaling STING-TBK1-IRF3 appeared to be functional in neonatal PBMCs, as demonstrated by the fact that IRF3 phosphorylation and IFNβ production in these cells could be activated by cGAMP. Intriguingly, decreased expression of cGAS in neonatal cells can be rescued by DNA demethylation, with concomitant enhancement in IFNβ induction by HSV-1. Thus, cGAS restoration or STING stimulation by small molecules during infancy might improve the age-dependent susceptibility to DNA virus infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Analysis of the diverse interactions of multiple signaling pathways is an emerging challenge in the era of networking pharmacology. To reveal imbalanced signaling pathways and pharmacological mechanisms involved in ischemic process, we designed systemic experiments from top-down to bottom-up for investigating the variations of multiple pathways in mouse hippocampal cells. A total of 711 focal cerebral ischemia-reperfused animals (504 mice and 207 rats), induced by occlusion of the middle cerebral artery, were obtained to conduct 4 experiments. The mice were used to analyze the pharmacological effects of four single compounds, baicalin (BA), jasminoidin (JA), ursodeoxycholic acid (UA) and concha margaritifera (CM) and two combination therapies (BA+JA, and JA+UA). Moreover, the mouse models were also used for microarray and western blotting test. The rat models were used for infarction volume test, magnetic resonance imaging (MRI) test and neurological score analysis to validate the pharmacological effects in another species. The results of western blotting confirmed that the expression of the key proteins involved in the ischemiaactivated Wnt and nuclear factor κB (NF-κB) pathway was markedly altered. In addition, based on the screened gene expression profiles of ischemia hippocampus, a variety of altered genes contributed to the 9 stroke-related pathways based on literature review [Wnt, extracellular signal-regulated kinase (ERK), janus kinase (JAK), mitogen-activated protein kinase (MAPK), gonadotropin-releasing hormone (GnRH), calcium/calmodulin-dependent protein kinase (CaMK), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF)] in different groups. Thus, we believed that the 9 signaling pathways were significantly imbalanced in different groups. However, analysis of overlapping genes was insufficient to reveal the expression profiles of imbalanced pathways between or within various conditions treated with different compounds or compound mixtures. Therefore, global similarity index (GSI) is introduced to quantify the genotypic outcomes of gene expression profiles. Independent experiments in mice on the effects of infarction volume, neurologic deficit score and the results of MRI in rats showed that GSI was suitable for the spectral measurement of imbalance in those 9 biochemical pathways with a predictive accuracy of 81.0% as assessed by leave-one-out cross-validation.

Plant water sources were estimated by two or three compartment linear mixing models using hydrogen and oxygen isotope (deltaD and delta18O) values of different components such as plant xylem water, precipitation and river water as well as soil water on the Tibetan Plateau in the summer of 2005. Four dominant species (Quercus aquifolioides, Pinus tabulaeformis, Salix rehderiana and Nitraria tangutorum) in three typical ecosystems (forest, shrub and desert) were investigated in this study. Stable isotope ratios of the summer precipitations and the soil water presented variations in spatial and temporal scales. delta18O values of N. tangutorum xylem water were constant in the whole growth season and very similar to those of deep soil water. Water sources for all of the plants came from both precipitations and soil water. Plants switched rapidly among different water sources when environmental water conditions changed. Rainwater had different contributions to the plants, which was influenced by amounts of precipitation. The percentage of plant xylem water derived from rainwater rose with an increase in precipitation. Water sources for broad-leaved and coniferous species were different although they grew in the same environmental conditions. For example, the broad-leaved species Q. aquifolioides used mainly the water from deep soil, while 92.5% of xylem water of the coniferous species P. tabulaeformis was derived from rainwater during the growth season. The study will be helpful for us to fully understand responses of species on the Tibetan Plateau to changes in precipitation patterns, and to assess accurately changes of vegetation distribution in the future.

Recent studies confirmed that the new cell survival signal pathway of Insulin-PI3K-Akt exerted cyto-protective actions involving anti-apoptosis. This study was undertaken to investigate the potential neuroprotective effects of insulin in the pathogenesis of spinal cord injury (SCI) and evaluate its therapeutic effects in adult rats. SCI was produced by extradural compression using modified Allen's stall with damage energy of 40 g-cm force. One group of rats was subjected to SCI in combination with the administration of recombinant human insulin dissolved in 50% glucose solution at the dose of 1 IU/kg day, for 7 days. At the same time, another group of rats was subjected to SCI in combination with the administration of an equal volume of sterile saline solution. Functional recovery was evaluated using open-field walking, inclined plane tests, and motor evoked potentials (MEPs) during the first 14 days post-trauma. Levels of protein for B-cell lymphoma/leukemia-2 gene (Bcl-2), Caspase-3, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were quantified in the injured spinal cord by Western blot analysis. Neuronal apoptosis was detected by TUNEL, and spinal cord blood flow (SCBF) was measured by laser-Doppler flowmetry (LDF). Ultimately, the data established the effectiveness of insulin treatment in improving neurologic recovery, increasing the expression of anti-apoptotic bcl-2 proteins, inhibiting caspase-3 expression decreasing neuronal apoptosis, reducing the expression of proinflammatory cytokines iNOS and COX-2, and ameliorating microcirculation of injured spinal cord after moderate contusive SCI in rats. In sum, this study reported the beneficial effects of insulin in the treatment of SCI, with the suggestion that insulin should be considered as a potential therapeutic agent.

Objective: To investigate the effect of interleukin-6 (IL-6) on the apoptosis of annulus fibrosus (AF) cell induced by interleukin-1beta (IL-1beta)., Methods: Cultured AF cells were divided into 6 groups and treated with no drug, 10 ng/mL IL-6, 10 ng/mL IL-1beta, 10 ng/mL IL-1beta and Z-VAD-FMK (a caspase-9 inhibitor), 10 ng/mL IL-1beta and 10 ng/mL IL-6, 10 ng/mL IL-1beta and 100 ng/mL IL-6, respectively. After three days of culture, the apoptosis rate, the positive rates of caspase-3, -8, and -9 of AF cells were detected with flow cytometry., Results: The apoptosis rates of cells in group 1 to 6 were 2.67% +/- 1.08%, 2.71% +/- 0.53%, 20.37% +/- 1.57%, 11.34% +/- 0.67%, 18.17% +/- 0.74%, and 9.42% +/- 1.08%, respectively. There was no significant difference between group 1 and 2, while the apoptosis rates of group 4, 5, and 6 were significantly lower than group 3 (P = 0.001, P = 0.172, and P = 0.001, respectively). Positive rates of caspase-3 in group 5 (12.35% +/- 0.64%) and 6 (9.26% +/- 0.36%) were significantly lower than group 3 (17.14% +/- 0.72%; P = 0.001 and P < 0.001, respectively). And positive rates of caspase-9 in group 5 (15.13% +/- 1.45%) and 6 (10.17% +/- 2.50%) were significantly lower than group 3 (19.4% +/- 0.98% ; P = 0.014 and P = 0.004, respectively). But there was not obvious change of caspase-8 activity after IL-6 was added., Conclusion: IL-6 is capable of protecting AF cells from IL-1beta induced apoptosis in vitro. Mechanism of the protection is related with the inhibition of caspase-3 and -9 activities.

Objective: To evaluate the clinical effects of microvascular decompression in treating trigeminal neuralgia., Methods: Surgical experience and operative findings of 2643 cases of trigeminal neuralgia treated by microvascular decompression were analyzed retrospectively., Results: Two thousand four hundred and eighty-seven of 2643 cases were cured, 76 cases were ineffectiveness, 48 cases were effectiveness and 31 were ineffective. One patient died. Two thousand one hundred and thirty-six cases were followed up in 3-240 months, 1918 cases were cured, 85 cases were obviously effective, 39 cases were effective and 30 were ineffective. Sixty-four cases were pain relapsed and 37 cases were cured by second operation among them., Conclusion: The etiology of trigeminal neuralgia is that the unusual vascular oppress the root entry zone, and arachnoid membrane circling the nerve is thickened and sticked. To untie the arachnoid membrane and decompress the offending vascular is the effective methods in treating trigeminal neuralgia.

In comparison to cation (K+, Na+, and Ca2+) channels, much less is currently known about the functional role of anion (Cl-) channels in cardiovascular physiology and pathophysiology. Over the past 15 years, various types of Cl- currents have been recorded in cardiac cells from different species including humans. All cardiac Cl- channels described to date may be encoded by five different Cl- channel genes: the PKA- and PKC-activated cystic fibrosis tansmembrane conductance regulator (CFTR), the volume-regulated ClC-2 and ClC-3, and the Ca2+-activated CLCA or Bestrophin. Recent studies using multiple approaches to examine the functional role of Cl- channels in the context of health and disease have demonstrated that Cl- channels might contribute to: 1) arrhythmogenesis in myocardial injury; 2) cardiac ischemic preconditioning; and 3) the adaptive remodeling of the heart during myocardial hypertrophy and heart failure. Therefore, anion channels represent very attractive novel targets for therapeutic approaches to the treatment of heart diseases. Recent evidence suggests that Cl- channels, like cation channels, might function as a multiprotein complex or functional module. In the post-genome era, the emergence of functional proteomics has necessitated a new paradigm shift to the structural and functional assessment of integrated Cl- channel multiprotein complexes in the heart, which could provide new insight into our understanding of the underlying mechanisms responsible for heart disease and protection.

Objective: The aim of work was to evaluate the efficacy of microvascular decompression (MVD) in patients with trigeminal neuralgia (TN) associated with hypertension., Methods: Five hundred and thirty-eight cases of TN associated with hypertension treated with MVD were retrospectively analyzed, all the cases were treated with MVD in the zone of the ventrolateral medulla oblongata and inspected postoperative blood pressure., Results: Among 538 cases, 341 cases (63.4%) had cure result, 71 cases (13.2%) had obvious effectiveness, 53 cases (9.8%) had effectiveness, and 73 cases had no effectiveness. Four hundred and eighty-three cases have been followed 1.0 approximately 15.8 years, mean 6.3 years. At the time of their follow-up, 313 cases had cure result, 63 cases had obvious effectiveness, 42 cases had effectiveness and 65 cases had no effectiveness., Conclusions: It was pathogenesis of nedulla oblongata by abnormal vascular tab, long-term aching stimulation and emotional stress. It was an effective method to treat TN associated with hypertension with MVD in this zone.

Objective: To elucidate the effects of exogenous basic fibroblast growth factor (bFGF) on biological characteristics of rat osteoblasts cultured in vitro., Methods: The osteoblasts isolated from a Sprague-Dawley rat and cultured in vitro were treated with different concentrations of bFGF (5-50 ng/ml) respectively. At 24 hours after treatment, the proliferating cell nuclear antigen was measured with immunocytochemistry, alkaline phosphatase (ALP) activity was determined and the expression of transforming growth factor beta 1 (TGF-beta(1)) was detected to observe the effects of bFGF on growth and differentiation of osteoblasts., Results: bFGF (5-50 ng/ml) could obviously promote the growth of osteoblasts. The intracellular expression of TGF-beta(1) mRNA increased significantly, but the intracellular ALP content decreased., Conclusions: bFGF can obviously stimulate the proliferation of osteoblasts and promote the synthesis of TGF-beta(1), but cannot promote the differentiation of osteoblasts.

Background & Objective: Sellar region tumors are common intracranial tumor. This study was designed to investigate the methods of the removal of tumors in sellar region by pterional approach., Methods: A total of 119 cases with sellar region tumors were performed operation. Before the operation, the relationships between the tumors and their periphery were determined according to CT, MR and the cerebral angiography and then the tumors were removed by pterional approach microsurgery in 4 different spaces., Results: The number of the patients treated with total, subtotal, and partial resection were 72, 36, and 11, respectively; with 85 cases significantly improved, 24 getting better, 8 without changes and 2 dead. Follow-up half a year, 92 cases were survival in the half a year of follow-up, in which 61 cases were able to work with normal life, 15 could do light work, 9 could take care of themselves but 6 patients were disabled, and 1 died of heart disease., Conclusion: Pterional approach can be used to remove many kinds of tumors in sellar region and with better exploration, therefore, increase the total removal and cure rate of such tumors.

Objective: To investigate the effect of transforming growth factor-beta 1 (TGF-beta 1) gene transfer on the biological characteristics of osteoblasts., Methods: The expression of TGF-beta 1 in the transfected osteoblasts was detected by in situ hybridization and assay of TGF-beta 1 activity in the supernatant (mink lung epithelium cell growth-inhibition test). The effects of gene transfer and supernatant of the transfected osteoblasts on the proliferation and alkaline phosphatase(ALP) activity of osteoblasts were detected by 3H-TdR and MTT., Results: The results of in situ hybridization analysis suggested that the osteoblasts transfected by TGF-beta 1 gene could express TGF-beta 1 obviously. The complex medium, which was the mixture of serum-free DMEM and the activated supernatant according to 1:1, 1:2, 1:4, could inhibit growth of Mv-1-Lu evidently and the ratios of inhibition were 16.3%, 22.7%, 28.2% respectively. TGF-beta 1 gene transfer had no effect on the biological characteristics of osteoblasts, but the activated supernatant of transfected osteoblasts stimulated proliferation and inhibited ALP activity of osteoblasts., Conclusion: TGF-beta 1 gene transfer promotes the expression of TGF-beta 1 and the biological characteristics of transfected osteoblasts are stable, which is helpful for gene therapy of bone defects in vivo.

Objective: To investigate the effect of transforming growth factor (TGF) beta 1 gene transfection on the growth of mesenchymal stem cells(MSCs) and to evaluate a new biomimetic biodegradable polymer as scaffolds for applications in articular cartilage tissue engineering., Methods: Principles of tissue engineering were combined organically with principles of gene therapy to produce cultured periosteum-derived MSCs transduced with the full-length rat TGF-beta 1 cDNA in vitro. These cells were then seeded onto three-dimensional porous poly-DL-lactide scaffolds modified with poly-L-lysine that mimicked cell-binding domains found on natural extracellular matrix to promote specific cell adhesion. The adhesion, proliferation, and differentiation of the transfected MSCs were examined with scanning electron microscope within 2 weeks., Results: All cells adhered to the biomimetic matrices well, but more cartilage-like tissue was formed for TGF-beta 1 gene modified MSCs/scaffolds composites than for the control groups. Transfer of gene encoding TGF-beta 1 to MSCs promoted its proliferation and differentiation significantly., Conclusions: The TGF-beta 1 gene transduced MSCs/biomimetic matrix composites used in this study was the first attempt to apply the principles of molecular tissue engineering for articular cartilage repair. This new molecular tissue engineering approach could be of potential benefit to repair damaged articular cartilage, especially in osteoarthritis. The new biomimetic biodegradable polymer matrices modified with biomolecules not only have good structural compatibility, but also have better interfacial compatibility and bioactivity, and can be used as scaffolds for articular cartilage tissue engineering.

In rabbit atrial myocytes, depolarization of the membrane results in a rapidly activating transient outward current (I(to)) that then decays to a sustained level. The sustained current (Isus) remains constant for at least 5 s during continued depolarization. The present study was designed to identify the ionic mechanism underlying Isus with the use of whole cell voltage-clamp techniques. After exposure to 2 mM 4-aminopyridine (4-AP), the 4-AP-sensitive transient outward current (I(to1)) was abolished, but Isus was unaffected. Isus was not blocked by the K+ channel blockers tetraethylammonium chloride and Ba2+, was not changed by increasing superfusate K+ concentration, and was still present when K+ was replaced by Cs+ in both the superfusate and the pipette. Isus was significantly reduced by the Cl- transport blockers 4-acetamido-4'-isothiocyanatostilbene-2.2'-disulfonic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. The current-voltage relations of Isus showed outward rectification, and the reversal potential of Isus shifted with changes in the transmembrane Cl- gradient in the fashion expected for a Cl- current. We conclude that Isus in rabbit atrium is due to a noninactivating Cl- current which, unlike previously described cardiac Cl- currents, is manifest in the absence of exogenous stimulators of adenosine 3',5'-cyclic monophosphate formation, cytosolic Ca2+ transients, or cell swelling.