[Homozygous CFAP65 mutation induces multiple morphological abnormalities of sperm flagella: A preliminary genetic study].

OBJECTIVE: To search for the possible pathogenic genes for multiple morphological anomalies of sperm flagella (MMAF). METHODS: We performed whole exome sequencing (WES) of a typical case of MMAF and analyzed its possible pathogenic genes. We examined the semen sample from the patient and identified the ultrastructural characteristics of the sperm flagella under the scanning electron and transmission electron microscopes, and analyzed the expression pattern of cilia and flagela-associated protein 65 (CFAP65) in spermatogenesis by immunofluorescence assay. RESULTS: The MMAF patient was found with a homozygous pathogenic mutation of the CFAP65 gene c.2675G>A(p.Trp892*). Scanning electron microscopy showed that the sperm of the patient had typical characteristics of MMAF, that is, without tails or with folded tails, curly tails, short tails or irregular tails. Transmission electron microscopy revealed the loss and disorder of the "9+2" structure in the sperm flagellum, with abnormal assembly of the fibrous sheath, accompanied by loss of central microtubules and dynamin arms. Cellular immunofluorescence assay suggested that the CFAP65 gene was expressed at all levels of mouse germ cells. CONCLUSIONS: The CFAP65 gene is involved in the assembly of the sperm flagellum structure, and its mutation can cause the phenotype of MMAF, leading to male infertility.

Digestive Enzyme Corona Formed in the Gastrointestinal Tract and Its Impact on Epithelial Cell Uptake of Nanoparticles.

The fate of intravenously injected nanoparticles (NPs) is significantly affected by nano-protein interaction and corona formation. However, such an interaction between NPs and digestive enzymes occurring in the gastrointestinal tract (GIT) and its impacts on epithelial cell uptake are little known. We synthesized the poly(3-hydroxybutyrate- co-3-hydroxyhexanoate)-based cationic NPs (CNPs) and investigated the CNP-digestive enzyme interaction and its effect on the cellular uptake. The formation of enzyme corona was confirmed by size/zeta potential analysis, morphology, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and enzyme quantification. The cellular uptake of CNPs by Caco-2 cells was significantly reduced upon the formation of enzyme corona. Our findings demonstrate the digestive enzyme corona formation and its inhibited effect on the epithelial cell uptake of CNPs for the first time. Understanding the enzyme corona could offer a new insight into the fate of nanomedicines in the GIT, and this understanding would be highly beneficial for guiding future nanomedicine designs.

TCAB1: a potential target for diagnosis and therapy of head and neck carcinomas.

BACKGROUND: WRAP53, including α, ß and γ isoforms, plays an important role not only in the stability of p53 mRNA, but also in the assembly and trafficking of the telomerase holoenzyme. It has been considered an oncogene and is thought to promote the survival of cancer cells. The aim of this study was to detect the role of TCAB1 (except WRAP53α) in the occurrence and development of head and neck carcinomas. METHODS: Immunohistochemistry was used to detect the TCAB1 expression in clinical specimen sections and performed western blotting to check the TCAB1 expression levels in cell lines. TCAB1 was depleted using shRNA lentivirus and the knockdown efficiency was assessed using q-PCR and Western blotting. We performed CCK-8 assays and flow cytometry to check the cell proliferation potential and used the trans-well assay to test the invasion ability in vitro. Xenografts were used to detect the tumor formation potential in vivo. Moreover, we performed cDNA microarray to investigate the candidate factors involved in this process. RESULTS: We observed a notable overexpression of TCAB1 in head and neck carcinoma clinical specimens as well as in carcinoma cell lines. Knockdown of TCAB1 decreased the cellular proliferation potential and invasion ability in vitro. cDNA microarray analysis suggested the possible involvement of several pathways and factors associated with tumorigenesis and carcinoma development in the TCAB1-mediated regulation of cancers. Furthermore, the xenograft assay confirmed that the depletion of TCAB1 would inhibit tumor formation in nude mice. The immunohistochemistry results of the mice tumor tissue sections revealed that the cells in shTCAB1 xenografts showed decreased proliferation potential and increased apoptotic trend, meanwhile, the angiogenesis was inhibited in the smaller tumors form shTCAB1 cells. CONCLUSIONS: Our study demonstrated that depletion of TCAB1 decreased cellular proliferation and invasion potential both in vitro and in vivo. The data indicated that TCAB1 might facilitate the occurrence and development of head and neck carcinomas. In future, TCAB1 might be useful as a prognostic biomarker or a potential target for the diagnosis and therapy of head and neck carcinomas.

Effects of combined exogenous dextranase and sodium fluoride on Streptococcus mutans 25175 monospecies biofilms.

PURPOSE: To investigate the effects of exogenous dextranase and sodium fluoride on a S. mutans monospecies biofilm. METHODS: S. mutans 25175 was grown in tryptone soya broth medium, and biofilm was formed on glass slides with 1.0% sucrose. Exogenous dextranase and sodium fluoride were added alone or together. The biofilm morphology was analyzed by confocal laser scanning microscopy. The effects of the drug on the adhesion and exopolysaccharide production by the biofilms were evaluated by scintillation counting and the anthrone method, respectively. RESULTS: In this study, we found that the structure of initial biofilm and mature biofilm were partly altered by dextranase and high concentrations of sodium fluoride separately. However, dextranase combined with a low concentration of sodium fluoride could clearly destroy the typical tree-like structure of the biofilm, and led to less bacterial adhesion than when the dextranase or fluoride were used alone (P < 0.05). The amounts of soluble and insoluble exopolysaccharide were significantly reduced by combining dextranase with a low concentration of sodium fluoride, much more than when they were used alone (P < 0.05). These data indicate that dextranase and a low concentration of sodium fluoride may have synergistic effects against S. mutans biofilm and suggest the application of a low concentration of sodium fluoride in anticaries treatment.

Drug-free and non-crosslinked chitosan scaffolds with efficient antibacterial activity against both Gram-negative and Gram-positive bacteria.

Treatment of oral pathogens is important for both oral and systemic health. The antimicrobial activity of chitosan (CS)-based scaffolds either loading antibiotics or compositing with other agents are well documented. However, the intrinsic antibacterial activity of CS scaffolds alone has never been reported. Herein, we fabricated the non-crosslinked CS scaffold and investigated its antibacterial activity against typical oral pathogens, Gram-negative Porphyromonas gingivalis and Gram-positive Streptococcus mutans. We found both pathogens were completely killed by 1 mg CS scaffolds at 6 h, due largely to the CS-induced time-dependent bacteria clustering. Interestingly, ß-glycerophosphate crosslinked scaffolds showed no antibacterial activity. In conclusion, the bactericidal activity of CS scaffolds alone is reported for the first time. Together with the biodegradability, physical stability, biocompatibility and great antibacterial activity, the non-crosslinked CS scaffolds may have great potentials not only in treating oral diseases but also in wound healing and tissue engineering.

Understanding the sheet size-antibacterial activity relationship of graphene oxide and the nano-bio interaction-based physical mechanisms.

The antibiotics-independent antimicrobial activity of graphene oxide (GO) is of great importance since antibiotic therapy is facing great challenges from drug resistance. However, the relations of GO size with its antimicrobial activity and how the size regulates the antibacterial mechanisms are still unknown. Herein, we fabricated four GO suspensions with different sizes and demonstrated the parabolic relationship between GO size and its antibacterial activity against the Gram-positive cariogenic bacterium Streptococcus mutans. More interestingly, we found out how GO size regulated the nano-bio interaction-based physical antibacterial mechanisms. Increasing the size reduced the cutting effect but enhanced the cell entrapment effect, and vice versa. In conclusion, GO size affects its edge density and lateral dimension, further regulates its physical antibacterial mechanisms in different orientations and ultimately determines its activity. These findings provide a deep understanding of GO antibacterial property and may guide the design and development of GO for clinical use.

[Expression of human VIGILIN N terminus and observation of subcellular localization].

OBJECTIVE: To express human VIGILIN N terminus gene in E. coli, prepare the polyclonal antibody and study the subcell localization of human VIGILIN. METHODS: The N-terminal sequence of human VIGILIN was amplified by PCR and cloned into the polyclonal site of pGEX-4T-2 expression vector. The fusion protein was expressed under induction of IPTG in E. coli BL21. The extracted GST fusion protein was purified by GSTrap-FF affinity chromatography. The polyclonal antibody against human VIGILIN N was prepared by immunizing New Zealand white rabbits using the purified fusion protein as immunogen and analyzed the titer and specificity of the antiserum by ELISA and Western blot. Through immunofluorescence staining, the distribution of VIGILIN in cell was observed. RESULTS: Expression of the GST fusion protein was induced with 1 mmol/L IPTG at 28 C for 3 hours. The antibody titer was 1:16000. Western blot analysis demonstrated that the polyclonal antibody can recognize VIGILIN specifically. VIGILIN present in both the cytoplasm and the nucleus. Its distribution in the nucleus concentrated on the inner layer of nuclear membrane and the region close prominent area of DAPI staining. CONCLUSION: The human VIGILIN fusion protein was successfully expressed. The polyclonal antibody against human VIGILIN was generated and was further applied to the study of distribution of VIGILIN in cells. This study will provide a substantial base for further clarification of the quality and function of VIGILIN.

[Therapeutic effects of hTR-siRNA adenovirus on human cervical cancer xenografts in nude mice].

OBJECTIVE: To study the anti-tumor effects and its mechanism of hTR-siRNA adenovirus on human cervical cancer in vivo. METHODS: The in vivo model of human cervical cancer was established by the subcutaneous inoculation of HeLa cells at the right armpit of BALB/c nu/nu mice. After successful implantation, the mice were randomized into four groups, in which the mice were intratumorally injected with 0.1 mL of Ad-hTR-siRNA (10(13) pfu/L), or Ad-NT-siRNA (10(13) pfu/L), or Cisplatin (1.20 g/L), or serum-free DMEM alone respectively. These treatments were given once every 3 days for 6 times. After the last injection, the mice were observed for 7 days continuously and sacrificed at the end. The tumors were harvasted, weighed, and sectioned. The TUNEL assay was used to assess the apoptosis of these tumor cells. RESULTS: Tumors-implanted were established successfully by 100% with HeLa cells. As compared with Ad-NT-siRNA, Ad-hTR-siRNA could slow down tumor growth, decrease tumor volume (45.48%) and tumor weight (34.68%), as well as promote the apoptosis and necrosis of tumor cells. The TUNEL positive cells were about 11.8%. But the anti-tumor activity of Ad-hTR-siRNA didn't catch on Cisplatin's. CONCLUSION: This study indicated that the hTR-siRNA adenovirus could suppress cervical cancer-xenografted growth in vivo and induce tumor cell apoptosis or necrosis.

Antibiofilm effect of drug-free and cationic poly(D,L-lactide-co-glycolide) nanoparticles via nano-bacteria interactions.

AIM: Recently, nano-bio interactions and their biomedical impacts have drawn much attention, but nano-bacteria interaction and its function are unknown. Herein, we aim to synthesize drug-free and cationic nanoparticles (CNPs) and investigate CNP-bacteria interaction and its antibiofilm effect. MATERIALS & METHODS: The bioactivity of CNPs against Streptococcus mutans was examined by colony-forming units counting and scanning electron microscopy. CNP-bacteria interaction force was measured by atomic force microscopy. RESULTS: CNPs (217.7 nm, 14.7 mv) showed a concentration-dependent activity against bacteria. Particularly, CNPs at 200 µg/ml completely inhibited planktonic bacterial growth and biofilm formation, and disrupted ∼70% mature biofilm. CNP-bacteria interaction force was up to 184 nN. CONCLUSION: CNPs have great potentials for convenient local use for prevention and treatment of bacteria-related oral diseases.

Organic composite-mediated surface coating of human acellular bone matrix with strontium.

Acellular bone matrix (ACBM) provides an osteoconductive scaffold for bone repair, but its osteoinductivity is poor. Strontium (Sr) improves the osteoinductivity of bone implants. In this study, we developed an organic composite-mediated strontium coating strategy for ACBM scaffolds by using the ion chelating ability of carboxymethyl cellulose (CMC) and the surface adhesion ability of dopamine (DOPA). The organic coating composite, termed the CMC-DOPA-Sr composite, was synthesized under a mild condition, and its chemical structure and strontium ion chelating ability were then determined. After surface decoration, the physicochemical properties of the strontium-coated ACBM (ACBM-Sr) scaffolds were characterized, and their biocompatibility and osteoinductivity were determined in vitro and in vivo. The results showed that the CMC-DOPA-Sr composite facilitated strontium coating on the surface of ACBM scaffolds. The ACBM-Sr scaffolds possessed a sustained strontium ion release profile, exhibited good cytocompatibility, and enhanced the osteogenic differentiation of mesenchymal stem cells in vitro. Furthermore, the ACBM-Sr scaffolds showed good histocompatibility after subcutaneous implantation in nude mice. Taken together, this study provided a simple and mild strategy to realize strontium coating for ACBM scaffolds, which resulted in good biocompatibility and improved osteoinductivity.

Concentration-dependent protein adsorption at the nano-bio interfaces of polymeric nanoparticles and serum proteins.

AIM: A comprehensive understanding of nanoparticle (NP)-protein interaction (protein corona formation) is required. So far, many factors influencing this interaction have been investigated, like size and ζ potential. However, NPs exposure concentration has always been ignored. Herein, we aim to disclose the correlation of NPs exposure concentration with protein adsorption. MATERIALS & METHODS: Four polymeric NPs systems possessing similar sizes (230 ± 20 nm) but varied ζ potentials (-30 ∼ +40 mv) were prepared. Physicochemical properties and protein adsorption upon NP-protein interaction were characterized. RESULTS: Protein adsorption capacity and adsorbed protein types were NPs concentration-dependent. CONCLUSION: Considering the critical impacts of protein adsorption on NPs delivery, our work could be an urgent warning about the possible risks of dosage adjustment of nanoformulations.

[Construction of a reverse-subtracted cDNA library for differentially expressed genes in rat liver of prothrombotic state].

OBJECTIVE: To inquire into the mechanism of prothrombotic state (PTS) and the roles of liver therein by constructing a reverse-subtracted cDNA library for differentially expressed genes in rat liver of PTS. METHODS: The reverse-subtracted cDNA library for differentially expressed genes in rat liver of PTS was constructed by suppression subtractive hybridization. The rat model of PTS was induced by a high-carbohydrate diet. Poly A+ mRNAs were isolated from PTS and control rats, and cDNAs were synthesized from the mRNAs. After digestion by means of Ras I, cDNAs 400-600 bp in size were obtained. For suppression subtractive hybridization, cDNAs from PTS rat were used as Driver and the cDNAs from control rat as Tester. The Tester was divided into two parts and ligated to adaptor 1 and adaptor 2R respectively. After two times of subtractive hybridization and two times of nested PCR, the products of the last PCR amplification were inserted into T/A plasmid vectors to transform the Escherichia coli JM109 cells. The transformed cells were incubated at 37 degrees C overnight on a LB agar plate containing ampicillin (50 micrograms/ml), IPTG and X-gal. The colonies were counted. RESULTS: 78% of the colonies were white and the reverse-subtracted cDNA library for differentially expressed genes in rat liver of prothrombotic state was successfully constructed. CONCLUSION: Prothrombotic state caused by malfunction of homeostasis and fibrinolysis is an important risk factor of cardiovascular disease. Liver plays important roles in the development of PTS, for the majority of the factors in the coagulation as well as fibrinolytic cascades are generated by the liver and secreted into the bloodstream. The reverse-subtracted cDNA library for differentially expressed genes in rat liver of PTS, successfully constructed in the present study, provides an efficient way to further investigate the mechanism of PTS and the relevant liver functions.

Preformed albumin corona, a protective coating for nanoparticles based drug delivery system.

The non-specific interaction between nanoparticles (NPs) and plasma proteins occurs immediately after NPs enter the blood, resulting in the formation of the protein corona that thereafter replaces the original NPs and becomes what the organs and cells really see. Consequently, the in vivo fate of NPs and the biological responses to the NPs are changed. This is one substantial reason for the two main problems of the NPs based drug delivery system, i.e. nanotoxicity and rapid clearance of NPs from the blood after intravenous injection. Here, we demonstrate the successful application of the preformed albumin corona in inhibiting the plasma proteins adsorption and decreasing the complement activation, and ultimately in prolonging the blood circulation time and reducing the toxicity of the polymeric PHBHHx NPs. Since the interaction of proteins with various nano-materials and/or -particles is ubiquitous, pre-forming albumin corona has a great potential to be a versatile strategy for optimizing the NPs based drug delivery system.

The poor osteoinductive capability of human acellular bone matrix.

Demineralized bone matrix (DBM) has extensive clinical use for bone regeneration because of its osteoinductive and osteoconductive aptitude. It is suggested that the demineralization process in bone matrix preparation is influential in maintaining osteoinductivity; however, relevant investigations, especially into the osteoinductivity of acellular bone matrix, are not often performed. This study addressed the osteoinductive capability of human acellular cancellous bone matrix (ACBM) after subcutaneous implantation in a rat model. The growth and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) seeded in this material were also studied. Without the demineralization process, the ACBM we obtained had an interconnected porous network and the micropores in the surface were clearly exposed. After the ACBM was subcutaneously implanted for 4 months, new osteoid formation was noted but not typical mature bone formation. rBM-MSCs grew well in the ACBM and kept a steady morphology after continuous culture for 28 days. However, no mineralized nodule formation was detected and the expression levels of genes encoding osteogenic markers were significantly decreased. These results demonstrated that human ACBM possess the structural features of native bone and poor osteoinductivity; nonetheless this material helped to preserve the undifferentiated phenotype of rBM-MSCs. Such insights may further broaden our understanding of the application of ACBM for bone regeneration and the creation of stem cell niches.

Crocin inhibits proliferation and nucleic acid synthesis and induces apoptosis in the human tongue squamous cell carcinoma cell line Tca8113.

BACKGROUND: Cancer chemoprevention is a proven effective strategy for oral squamous cell carcinoma (OSCC). The present study was designed to investigate the effects of crocin, a potential chemopreventive agent, on growth and DNA and RNA content in a human tongue squamous cell carcinoma cell line, Tca8113. METHODS: Tca8113 cells were treated with crocin for 24, 48, 72, and 96 h at concentrations of 0.1, 0.2, 0.4, and 0.8 mM. Tumor cell viability was investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. In addition, Tca8113 cells were treated with 0.4 mM crocin and cytotoxic effects as an inducer of apoptosis were analyzed using flow cytometry. Furthermore, acridine orange (AO) staining and observation using laser scanning confocal microscopy (LSCM) were used to determine the effects of the drug on nucleic acid synthesis. RESULTS: Crocin decreased Tca8113 cell viability and growth remarkably at 24, 48, 72, and 96 h, in a concentration-dependent manner (P<0.05). In addition, 0.4 mM crocin significantly induced both early and late apoptosis of Tca8113 cells. Moreover, the cellular DNA and RNA content was significantly downregulated by 0.4 mM crocin compared with the negative control (P<0.01). CONCLUSIONS: Our observations support the feasibility of applying crocin as a chemoprophylactic agent and treatment for OSCCs.

[Effects of different concentrations of ethylenediamine tetraacetic acid paste on removing root canal smear].

OBJECTIVE: To evaluate the effect of different concentrations of ethylenediamine tetraacetic acid (EDTA) paste on the removing root canal smear layers and the degrees of erosion on the surface of the root canal walls at the different portions of canal. METHODS: Sixty human teeth with single root were instrumented using step-back technique, then were divided into six groups and treated with different concentrations EDTA paste and NaCLO solution. Group A: 0.9% saline; group B: 5.25% NaC10+5% EDTA; group C: 5.25% NaC10+10% EDTA; group D: 5.25% NaC10+15% EDTA; group E: 5.25% NaC10+17% EDTA; group F: 5.25% NaClO+20% EDTA. Then the teeth were split, the root canals with different treatments were examined with scanning electron microscope at the coronal, middle and apical thirds for smear layers removal and the degrees of erosion. RESULTS: The effect of EDTA paste on removing smear layers at the coronal, middle thirds of the root canal increased with the concentrations. 15% EDTA paste could remove the smear layers at the coronal and middle thirds of the root canal, but the effect on apical third was invalid, and no erosion could be found in the root canal wall. 17% and 20% EDTA paste produced the erosion to the root canal wall. CONCLUSION: The concentrations of the EDTA paste can influence the effect of the removing smear layers, and the concentration beyond 17% would produce the erosion to root canal wall.

[Effect of astragalus membranaceus on the proliferation, osteogenic capacity and structure of periodontal ligament cells in vitro].

OBJECTIVE: To investigate the effect of Astragalus membranaceus (APS) on the proliferation, osteogenic capacity and structure of periodontal ligament cells (PDLCs) in vitro. METHODS: PDLCs were cultured in vitro with APS of 0.08, 0.1, 0.2, 0.4 mg x mL(-1). Methyl thiazolyl tetrazolium (MTr), alkaline phosphatase (ALP) and cell structure were detected to determine the proliferation and differentiation of PDLCs proliferation and differentiation. RESULTS: When the APS was 0.2 mg x mL(-1), the absorbance of MTT and ALP exhibit significantly increased as compared to the control (P < 0.05). The cells cultured in vitro with APS of 0.2 mg x mL(-1) had the normal structure. CONCLUSION: APS with proper concentration in short-term culture may promote the proliferation and differentiation of PDLCs.

[The relationship in growth between A.actinomycetemcomitans and cariogenic bacteria in vitro].

PURPOSE: To investigate the correlation in growth between A.actinomycetemcomitans and cariogenic bacteria of S.mutans, L.acidophilus A.naeslundii A.viscosus in vitro. METHODS: Using improvement agar diffusion method and dual-species (A.actinomycetemcomitans with each of cariogenic bacteria) incubation in BHI broth, we studied the relationship between A.actinomycetemcomitans and 4 kinds cariogenic bacteria in growth. The characteristic and percentage of A.actinomycetemcomitans in dual-species biofilms were detected with SEM and CFU(colony forming units).The data were analyzed for significance using an independent two-sample t test with SPSS 10.0 software package. RESULTS: The agar diffusion essay showed that A.actinomycetemcomitans had no effect on the growth of S.mutans, L.acidophilus A.naeslundii and A.viscosus. While these 4 kinds of cariogenic bacteria could inhibit the growth of A.actinomycetemcomitans. The ratio of A.actinomycetemcomitans in the dual-species suspension decreased gradually.In dual-species BHI broth, the proportion of A.actinomycetemcomitans cultured alone ahead of 12 hours decreased as well, which came to 0 at 24-hour in S.mutans and L.acidophilus groups. There was significant difference between different time groups(P<0.05).The single A.actinomycetemcomitans was failed to form three-dimensional biofilm and just a small quantity of cells accumulated and adhered. On the contrary, the 4 kinds of single cariogenic bacteria could form mature biofilms respectively by themselves at 48-hour,of which the appearance of biofilms had no apparent change to that of single cariogenic bacteria after mixing A.actinomycetemcomitans. CFU of dual-species biofilms indicated that the percentage of A.actinomycetemcomitans in biofilms decreased gradually by changing medium per 12 hours. There were significant difference among the three time spots(P<0.05). CONCLUSION: The study shows that S.mutans, L.acidophilus A.naeslundii and A.viscosus could inhibit the growth of A.actinomycetemcomitans in vitro.