Interaction between Heparin and Fibronectin: Using Quartz Crystal Microbalance with Dissipation, Immunochemistry and Isothermal Titration Calorimetry

The adsorption behavior of heparin and fibronectin was studied by quartz crystal microbalance with dissipation(QCM-D), and the interaction between heparin and fibronectin was evaluated using immunochemistry and isothermal titration calorimetry(ITC) measurement. The results showed that there was competitive adsorption between heparin and fibronectin, and the preadsorption of fibronectin could prevent subsequent heparin adsorption to some extent, and the adsorbed Hep/Fn complex on the surface was in a rigid form. The bioactivity of heparin and fibronectin could be affected by the bulk concentration of each, and both heparin and fibronectin in Hep/Fn complex formed under p H 4 condition displayed larger bioactivity than that formed under p H 7 condition. Moreover, the fibronectin showed more exposed cell-binding sites at the p H value lower than physiological condition. The results of ITC further suggested that the interaction between heparin and fibronectin under p H 4 was stronger than under p H 7, and the complex was also more stable. The study brings forth the detailed interaction between heparin and fibronectin, which will be helpful for better understanding the interaction mechanism of the two biomolecules. The results may be potentially useful for the development of new generation of cardiovascular biomaterials.

The Establishment of Immunochemistry Test Based on a Synthetic Peptide Antibody for the Detection of a Porcine Circovirus-Like Virus P1

Recently, a novel porcine circovirus-like virus P1 with a circular DNA genome of 0.648 kb was identified. P1 antigen was detected both in vitro and in vivo by synthetic peptide-derived polyclonal antibody-based immunochemistry. The designed peptides were synthesized by solid-phase technique, purified by high performance liquid chromatography, coupled to Keyhole limpet hemocyanin, and injected into rabbits to prepare polyclonal antibody. The emergence of positive cells revealed that synthetic peptide could elicit antibodies against P1 and viral protein could be synthesized. The polyclonal peptide antibodies described here was successfully applied to immunochemical staining and proved helpful in diagnosing P1.

Relationship between DNA ploidy, expression of ki-67 antigen and gastric cancer metastasis

AIM To evaluate the relationship between the expression of Ki 67 antigen and the pathobiological behaviours of gastric cancers especially their distant metastases. METHODS Fifty six specimens of gastric cancer routinely fixed in formalin and embedded in paraffin (FFEP) were studied by immunohistochemical method. RESULTS Expression of Ki 67 antigen was significantly related to the distant metastases to liver, ovary and adrenal gland ( P <0 005), but not related to the histological type, growth pattern, depth of invasion, histological differentiation and the metastases to local lymph nodes ( P >0 05). Furthermore, the Ki 67 antigen expression was significantly related to the DNA aneuploidy pattern, which is closely related to poor prognosis ( P <0 05). CONCLUSION Overexpression of Ki 67 can be used as an objective marker of the proliferative activity for predicting prognosis of gastric cancer and metastatic potential to distant organs.

Fos expression incatecholaminergic medullary neu-rons induced by chemical stimulation of stomach projecting to the paraventricular nucleus of hypothalamus in rats

AIM To determine whether medullary catecholaminergic neurons expressing Fos induced by chemical stimulation of the stomach project to the paraventricular nucleus of hypothalamus (PVH) in rats. METHODS A triple labeling method of horseradish peroxidase (HRP) retrograde tracing combined with Fos (ABC method) and tyrosin hydroxylase (TH) (PAP method) immunohistochemical stainings was used in the present study. RESULTS Seven kinds of labeled neurons were found in the nucleus tractus solitarii (NTS), the ventrolateral medulla (VLM) and the reticular formation of the medulla (RF): Fos like immunoreactive (LI) neurons, TH LI neurons and HRP retrogradely single labeled neurons, Fos/HRP, Fos/TH and HRP/TH double labeled neurons, and Fos/HRP/TH triple labeled neurons. CONCLUSION Ascending projections from the NTS, VLM and RF to the PVH might be involved in the transmitting process of the visceral noxious stimulation.

BMPR2 and HIF1-α overexpression in resected osteosarcoma correlates with distant metastasis and patient survival

Objective： Bone morphogenetic protein receptor 2（BMPR2） and hypoxia-inducible factor 1-α（HIF1-α） existed abnormal expression in several types of cancer. However, their expressions and related roles in osteosarcoma are largely unknown.Methods：To investigate the clinical significance of BMPR2 and HIF1-αin osteosarcoma,we analyzed their expression levels in 103 osteosarcoma specimens by immunochemistry.Meanwhile,we conducted a follow-up to examine the metastatic behavior and overall survival（OS）of osteosarcoma patients.Results：Among 103 tissues,61 cases had BMPR2-positive expression and 57 cases had HIF1-αpositive expression.A significant correlation was noticed between BMPR2 and HIF1-αexpression in osteosarcoma specimens（P=0.035）.Receiver-operating characteristic（ROC）curves were calculated to investigate the predictive value of the two markers in tumor metastasis.By means of univariate and multivariate analysis,BMPR2 and HIF1-αexpression,as well as higher tumor grade,were identified as significant risk factors for OS in patients with osteosarcoma.Kaplan-Meier survival analysis revealed that the patients with BMPR2 and HIF1-αpositive expression had worse OS compared with patients with BMPR2-negative or HIF1-α-negative staining.Conclusions：It can be concluded that BMPR2 and HIF1-αexpression is highly correlated with metastatic behavior in patients with osteosarcoma and can serve as predictive markers for metastasis and OS of these patients.

ALTERATION OF G1-CYCLINS (D1 AND E) IN TRANSITIONAL CELL CARCINOMA OF HUMAN URINARY BLADDER WITH INFECTION OF HPV-18

Objective: This study was designed to investigate differential pattern of G1-cyclins (D1 and E) in transitional cell carcinoma (TCC) of human urinary bladder with or without human papillomavirus-18 (HPV-18) infection. Methods: Immunohistochemistry method was used in the detection of the expression of G1-cyclins in 57 cases of TCC (7 normal bladders as control), and HPV-18 DNA was found in 29 cases by polymerases chain reaction (PCR). Results: Cyclin D1 expression was found in 41 of 57 (71.93%) TCCs and it was reverse associated with HPV (x 2=8.21, P<0.05). And cyclin D1 expression was found in 16 of 29 (55.17%) in HPV-18 infection group and 25 of 28 (89.29%) in non-HPV infection group. Cyclin E expression was detected in 36 of 57 (63.16%) and the association between the cyclin E expression and HPV infection was not found (x2=0.52, P>0.05). Cyclin E expression was found in 17 of 29 (56.82%) in HPV-18 infection group and 19 of 28 (67.86%) in non-HPV infection group. There was obvious difference in the cyclin D1 and cyclin E expression between the TCC and normal tissue (x 2=7.46, P<0.05; x 2=7.45, P<0.05, respectively). Conclusion: These data demonstrated that HPV infection altered the control of G1 cell cycle. And changes of G1 cell cycle regulatory proteins, either by interaction of cellular protein with viral oncoproteins or by changes in the cellular proteins themselves, may be critical for carcinogenesis of TCC of urinary bladder.

Changes in N-methyl-D-aspartate receptor 2A subunit expression caused by binocular form deprivation and chondroitin sulfate proteoglycan degradation

Light deprivation is known to induce a significant decrease in the percentage of N-methyi-D- aspartate receptor 2A subunit （NR2A）-expressing neurons during development. The purpose of this study was to investigate the effects of binocular form deprivation （BFD） and chondroitin sulfate proteoglycan （CSPG） degradation on NR2A expression via an immunohistochemical study, around the end of a critical developmental period. The results show that the positive staining of NR2A in the normal rat visual cortex increases gradually from postnatal 3-5 weeks （P 〈 0.05）, but the changes from 5 weeks to 7 weeks were not significant. The positive staining of NR2A following BFD in the rat visual cortex slightly increased from postnatal 3-7 weeks （P 〉 0.05）. The positive staining of NR2A in the CSPG-treated group was insignificant compared with the BFD group at the same time point from 4 weeks to 7 weeks （P 〉 0.05）. Thus, the effect of BFD on NR2A expression in the rat visual cortex was similar to that of CSPG degradation around the end of the critical developmental period.

Cost-effective screening using a two-antibody panel for detecting mismatch repair deficiency in sporadic colorectal cancer

BACKGROUND The microsatellite instability(MSI)test and immunohistochemistry(IHC)are widely used to screen DNA mismatch repair(MMR)deficiency in sporadic colorectal cancer(CRC).For IHC,a two-antibody panel of MLH1 and MSH2 or four-antibody panel of MLH1,MSH2,PMS2,and MSH6 are used.In general,MSI is known as a more accurate screening test than IHC.AIM To compare two-and four-antibody panels of IHC in terms of accuracy and cost benefit on the basis of MSI testing for detecting MMR deficiency.METHODS We retrospectively analyzed patients with CRC who underwent curative surgery between 2015 and 2017 at a tertiary referral center.Both IHC with four antibodies and MSI tests were routinely performed.The sensitivity and specificity of a fourand two types of two-antibody panels(PMS2/MSH6 and MLH1/MSH2)were compared on the basis of MSI testing for detecting MMR deficiency.RESULTS High-frequency MSI was found in 5.5%(n=193)of the patients(n=3486).The sensitivities of the four-and two types of two-antibody panels were 97.4%,92.2%,and 87.6%,respectively.The specificities of the three types of panels did not differ significantly(99.6%for the four-antibody and PMS2/MSH6 panels,99.7%for the MLH1/MSH2 panel).Based on Cohen's kappa statistic(κ),four-and twoantibody panels were in almost perfect agreement with the MSI test(κ>0.9).The costs of the MSI test and the four-and two-antibody panels of IHC were approximately$200,$160,and$80,respectively.CONCLUSION Considering the cost of the four-antibody panel IHC compared to that of the twoantibody panel IHC,a two-antibody panel of PMS2/MSH6 might be the best choice in terms of balancing cost-effectiveness and accuracy.

The Distribution of the 5-hydroxytryptamin(5-HT) Immunoreactive Cells in the Pancreas of Guinea-pig

Immunocytochemical localization of 5-hydroxytryptamine (5-HT) containing cells of the guinea-pig pancreas was studied by means of PAP-glucose oxidase-DAB-nickel (PAP-GDN) and PAPstaining methods on paraffin sections. In this study we demonstrated the presence of 5-HTimmunoreactive cells in the endocrine (islets) and exocrine pancreas of the guinea-ping. Cells exhib-iting 54-HT immunoreactivity were found in the center and the periphery of the pancreatic islets. The5-HT immunoreactive granules were evenly distributed throughout the cytoplasm, but was faint inthe nuclei. Cells of this type were also distributed in the exocrine pancreas. Grouped or single or single cellsoccurred intercalatedly between acinar cells, as well as in the epithlium of ducts and in the connectivetissues near the ducts or acini.

Mucinous adenocarcinoma of the buttock associated with hidradenitis:A case report

BACKGROUND Mucinous adenocarcinomas of the buttock are rare and have an uncertain etiology and natural course.They are usually related to chronic anal fistulas,hidradenitis suppurativa,or Crohn's disease.Here,we report a case of mucinous adenocarcinoma associated with hidradenitis and contradictory immunochemistry results.CASE SUMMARY A 62-year-old man complained of recurrent abscesses of the buttock for 3 years.He had several scars and nodules in bilateral buttocks,with purulent discharge.The skin lesions did not appear to originate from the anus.The patient was diagnosed with recurrent abscesses due to hidradenitis suppurativa at the first visit.He showed purulent and subsequent mucin discharge in the first operation and was diagnosed with mucinous adenocarcinoma.Several examinations were performed to determine disease origin and staging.There were no significant findings or evidence of anal fistulas.Hence,he underwent wide local excision and V-Y advancement flap in the second operation.The final diagnosis was mucinous adenocarcinoma without any evidence of anal fistulas.Additional immunochemistry test results were negative for cytokeratin(CK)7 and positive for CK20 and CDX2,with a colorectal origin.A pathologist suggested that the disease originated from a chronic anal fistula.The patient has remained free of recurrence for 24 mo.CONCLUSION Although the patient with mucinous adenocarcinoma showed an atypical course,immunochemistry helped detect the disease origin.

Determination of Calcineurin Inhibitors in Dried Blood Spots from Kidney Transplant Recipients

Background: Determination of cyclosporine A (CsA) and tacrolimus (Tac) in dried blood spots (DBS) could enable drug monitoring in transplanted patients without the necessity of having to take venous blood samples. Therefore, we have developed a method for quantitative determination of calcineurin inhibitors (CNI) by liquid-chromatography-tandem mass spectrometry (LCMS). Methods: In a study with 68 kidney transplant recipients (KTR, 34 CsA, 34 Tac), we tested the clinical application of LCMS monitoring in DBS in comparison to LCMS in whole blood. Results: The measuring range is proven for 27.33 to 1345 ng/ml for CsA and for 1.63 to 39.7 ng/ml for Tac. The requirements for clinical chemical analyses for precision and accuracy are complied with. Stability is documented for a period of 14 days. The study showed the following deviations from LCMS in whole blood for determination of CsA and Tac in DBS after introducing a correction factor by the haematocrit (Hct) value (CsA trough level: mean = 4.7%, ±1.96 standard deviation (SD) -52.1% to 61.4%, N = 96;CsA peak level: mean = 7.3%, ±1.96 SD -39.7% to 54.4%, N = 95;Tac trough level: mean = -0.5%, ±1.96 SD -76.4% to 75.3%, N = 88;Tac peak level: mean = 3.9%, ±1.96 SD -80.1% to 88.7%, N = 92). Conclusions: Our data show comparable results with the reference method by means of LCMS in whole blood. Therefore, DBS of KTR for determination of CNI levels could be transported on filter cards by mail to the respective laboratory resistant to breakage and the hazard of infection.

Does Muller Cell Differentiation Occur Prior to the Emergence of Synapses in Embryonic Turtle Retina？

MOiler cells are the main glial cells in the retina, and are related to plexiform layer activity. Recent studies have demonstrated that Muller cells are involved in the synaptic conservation, plasticity, development and metabolism of glutamate. During turtle retinal development, layers, cells and synapses appear at different times. The aim of this research is to study the emergence of Muller cells during embryonic development and their relationship with the synaptogenesis. The authors used retinas from Trachemys scripta elegans embryos at stages S14, 18, 20, 23, and 26. Some retinas were processed with immunocytochemistry in order to detect the presence of glutamine synthetase in Muller cells, which was used as a marker of these ceils. Other retinas from the same stages were processed for ultrastructural studies. Samples were observed in confocal and transmission electron microscopes, respectively. The present results show that glutamine synthetase expression in MOiler cells occurs at S18, before the emergence of the retinal layers and the early synapses.

Quantitative measurement of p53-p53 antibody interactions by quartz crystal microbalance: A modelsystem for immunochemical calibration

We are developing methods to quantify antibody interactions that include a quartz crystal microbalance (QCM) system to measure, on a molecular basis, the interaction of p53 and anti-p53 antibodies. Previously, as a model system, we developed a measurement device consisting of p53 protein (human wild type), characterized by mass spectroscopy and immobilized at various concentrations on a glass slide. The device is designed as a control to be used with immunohistochemical (IHC) assays that incorporate commercially available anti-p53 antibodies and probes. In the current study, p53 protein is covalently immobilized on a silicon dioxide-coated quartz crystal and the resonance frequency shift is followed in-situ. The functionalized sensor is then incubated with the anti-p53 antibody, which provides a direct gravimetric measure of the antibody-antigen binding. This previously described method allows the comparison of the surface immobilized protein concentrations with estimates obtained by fluorescence measurement. The p53 functionalized QCM system offers an independent measure of surface immobilized protein concentration and is essential in development of our IHC calibration prototypes. These results provide a benchmark for comparing antibody systems that may be used in developing other molecular diagnostic assays such as immunocytochemical analysis and the detection of biomarker proteins in blood and urine.

Identification and characterization of Argonaute gene family and meiosis-enriched Argonaute during sporogenesis in maize

Argonaute（AGO） proteins play a key role in regulation of gene expression through small RNA‐directed RNA cleavage and translational repression, and are essential for multiple developmental processes. In the present study, 17 AGO genes of maize（Zea mays L., ZmAGOs） were identified using a Hidden Markov Model and validated by rapid amplification of cDNA ends assay. Subsequently, quantitative PCR revealed that expressions of these genes were higher in reproductive than in vegetative tissues. AGOs presented five temporal and spatial expression patterns, which were likely modulated by DNA methylation, 50‐untranslated exons and microRNA‐mediated feedback loops. Intriguingly, ZmAGO18 b was highly expressed in tassels during meiosis. Furthermore, in situ hybridization and immunofluorescence showed that ZmAResearchGO18b was enriched in the tapetum and germ cells in meiotic anthers. We hypothesized that ZmAGOs are highly expressed in reproductive tissues, and that ZmAGO18 b is a tapetum and germ cell‐specific member of the AGO family in maize.

COMPARATIVE ANALYSIS OF ISOELECTRO FOCUSING PATTERN AND IMMUNOCHEM ICAL CHARACTERS OF RuBPcase OF LEAVES BETWEEN HYBRID PLANTS OF Spinacia oleracea L.+ Nicotiana tabacum L. AND THEIR PARENT PLANTS

Ribulose-1, 5-bisphosphate carboxylase/oxygenase (EC 4, 1, 1, 39, abbreviation: RuBPcase)is a bifunction enzyme. Because of its properties in genetic function and structure, RuBPcase has been intensively studied. In the absence of certain effective genetic markers for identifying hybrids, the analysis of the isoelectrofocusing pattern of RuBPcase has become a standard procedure to verify the nature of the hybrid plants.