Effect of avian influenza virus subtype H9N2 on the expression of complement-associated genes in chicken erythrocytes.

1. The H9N2 subtype avian influenza virus can infect both chickens and humans. Previous studies have reported a role for erythrocytes in immunity. However, the role of H9N2 against chicken erythrocytes and the presence of complement-related genes in erythrocytes has not been studied. This research investigated the effect of H9N2 on complement-associated gene expression in chicken erythrocytes.2. The expression of complement-associated genes (C1s, C1q, C2, C3, C3ar1, C4, C4a, C5, C5ar1, C7, CD93 and CFD) was detected by reverse transcription-polymerase chain reaction (RT-PCR). Quantitative Real-Time PCR (qRT-PCR) was used to analyse the differential expression of complement-associated genes in chicken erythrocytes at 0 h, 2 h, 6 h and 10 h after the interaction between H9N2 virus and chicken erythrocytes in vitro and 3, 7 and 14 d after H9N2 virus nasal infection of chicks.3. Expression levels of C1q, C4, C1s, C2, C3, C5, C7 and CD93 were significantly up-regulated at 2 h and significantly down-regulated at 10 h. Gene expression levels of C1q, C3ar1, C4a, CFD and C5ar1 were seen to be different at each time point. The expression levels of C1q, C4, C1s, C2, C3, C5, C7, CFD, C3ar1, C4a and C5ar1 were significantly up-regulated at 7 d and the gene expression of levels of C3, CD93 and C5ar1 were seen to be different at each time point.4. The results confirmed that all the complement-associated genes were expressed in chicken erythrocytes and showed the H9N2 virus interaction with chicken erythrocytes and subsequent regulation of chicken erythrocyte complement-associated genes expression. This study reported, for the first time, the relationship between H9N2 and complement system of chicken erythrocytes, which will provide a foundation for further research into the prevention and control of H9N2 infection.

[Diagnostic value of platelet associated biomarkers in chronic periprosthetic joint infection].

Objective: To evaluate the diagnostic value of platelet count(PC),PC to mean platelet volume(MPV) ratio(PC/MPV) and plateletcrit(PCT) in chronic periprosthetic joint infection(PJI). Method: The medical records of 159 patients who underwent hip or knee revisions at Department of Joint Surgery,Affiliated Hospital of Qingdao University from August 2013 to June 2019 were retrospectively reviewed. There were 51 patients(26 knees and 25 hips) in the PJI group,which included 28 males and 23 females,aged (68.0±11.8)years (range:32 to 84 years)with a body mass index(BMI)of (26.1±3.6) kg/m².There were 116 patients(19 knees and 97 hips) in the aseptic loosening(AL) group,including 67 males and 49 females,aged (70.3±8.9)years(range:49 to 89 years)with a BMI of (25.0±3.6)kg/m².The plasma C-reactive protein(CRP),erythrocyte sedimentation rate(ESR),PC,MPV,PC/MPV and PCT levels of the two groups were recorded and analyzed. Receiver operating characteristic curve was used to calculate the sensitivity and specificity of each biomarker,expect for MPV,and the diagnostic value of each biomarker was compared according to the area under the curve(AUC).Independent-sample t test or Mann-Whitney U test were used for comparison between groups. Result: Compared with AL group,AJI group had significantly higher levels of CRP,ESR,PC,PC/MPV and PCT(all P<0.05),but lower level of MPV (P<0.05).The AUCs for CRP,ESR,PC,PC/MPV and PCT were 0.820, 0.829, 0.689, 0.668 and 0.676,respectively. Based on the Youden index,the optimal predictive cutoff for CRP was 11.12 mg/L,with a sensitivity of 74.4% and a specificity of 87.1%.The optimal predictive cutoff for ESR was 17.60 mm/1 h,with a sensitivity of 81.4% and a specificity of 75.3%.The optimal predictive cutoff for PC was 243.00×109/L,with a sensitivity of 60.6% and a specificity of 71.8%.The optimal predictive cutoff for PC/MPV was 24.95,the sensitivity was 58.1% and the specificity was 74.1%.And the optimal predictive cutoff for PCT was 0.24%,with a sensitivity of 69.8% and a specificity of 63.5%. Conclusion: PC,PC to MPV ratio and PCT were of limited value to diagnose PJI.

[Clinical analysis of IgG4-related lung disease].

Objective: To improve the understanding and treatment of IgG4-related lung disease (IgG4-RLD) by analyzing the clinical characteristics of patients. Methods: A total of 13 patients with IgG4-related lung disease (IgG4-RLD) diagnosed by pathology at Zhongshan Hospital affiliated to Fudan University during December 2007 to December 2017 were included. The clinical characteristics, chest CT, pathological features, serum IgG4 levels, therapy and prognosis of these 13 patients were analyzed. Results: The 13 patients with IgG4-RLD included 8 men and 5 women, with an average age of (51.1±14.8) years. The clinical manifestations were varied, 10 patients showed respiratory symptoms, mainly including cough (n=9), expectoration (n=6), hemoptysis (n=6), chest tightness (n=2) and breathless (n=2). Multiple organs were involved in 12 cases. Chest CT showed solid lung nodules (n=9), ground glass shadows (n=1), alveolar-interstitial infiltration (n=3), bronchovascular lesions (n=3), and the solid lung nodules was predominant. Enlargement of lymph nodes in the mediastinal and bilateral hilum of the lung were present in 10 cases. Serum IgG4 levels were elevated in all the 13 cases, with an average concentration of (7.92 ±13.98)g/L. Quite amount of lymphocytes, plasma cell infiltration and fibrosis were common pathologic findings. Immunohistochemical staining showed a large number of IgG4 positive plasma cells infiltration with IgG4 positive plasma cells count 10-320/HP in 13 cases. The ratio of IgG4 positive plasma cells to IgG positive plasma cells was higher than 40%. Eight patients received glucocorticoid therapy alone, five received glucocorticoids with immunosuppressant therapy. After treatment, 10 patients were with remission, and disease progressed in 3 patients, but no death. Conclusions: The clinical manifestations and imaging features of IgG4-RLD are diverse, and lesions can often involve multiple organs. The diagnosis could be made based on pathological features and IgG4 serum levels. Glucocorticoid can be used as the first choice for the treatment of IgG4-RLD.

Direct manipulation of metallic nanosheets by shear force microscopy.

Micro/nanomanipulation is a rapidly growing technology and holds promising applications in various fields, including photonic/electronic devices, chemical/biosensors etc. In this work, we present that shear force microscopy (ShFM) can be exploited to manipulate metallic nanosheets besides imaging. The manipulation is realized via controlling the shear force sensor probe position and shear force magnitude based on our homemade ShFM system under an optical microscopy for in situ observation. The main feature of the ShFM system is usage of a piezoelectric bimorph sensor, which has the ability of self-excitation and detection. Moreover, the shear force magnitude as a function of the spring constant of the sensor and setpoint is obtained, which indicates that operation modes can be switched between imaging and manipulation through designing the spring constant before experiment and changing the setpoint during manipulation process, respectively. We believe that this alternative manipulation technique could be used to assemble other nanostructures with different shapes, sizes and compositions for new properties and wider applications.

Quercetin alleviates pulmonary angiogenesis in a rat model of hepatopulmonary syndrome.

Quercetin shows protective effects against hepatopulmonary syndrome (HPS), as demonstrated in a rat model. However, whether these effects involve pulmonary vascular angiogenesis in HPS remains unclear. Therefore, this study aimed to assess the effect of quercetin on pulmonary vascular angiogenesis and explore the underlying mechanisms. Male Sprague-Dawley rats weighing 200-250 g underwent sham operation or common bile duct ligation (CBDL). Two weeks after surgery, HIF-1α and NFκB levels were assessed in rat lung tissue by immunohistochemistry and western blot. Then, CBDL and sham-operated rats were further divided into 2 subgroups each to receive intraperitoneal administration of quercetin (50 mg/kg daily) or 0.2% Tween for two weeks: Sham (Sham+Tween; n=8), CBDL (CBDL+Tween; n=8), Q (Sham+quercetin; n=8), and CBDL+Q (CBDL+quercetin; n=8). After treatment, lung tissue specimens were assessed for protein (immunohistochemistry and western blot) and/or gene expression (quantitative real-time PCR) levels of relevant disease markers, including VEGFA, VEGFR2, Akt/p-Akt, HIF-1α, vWf, and IκB/p-IκB. Finally, arterial blood was analyzed for alveolar arterial oxygen pressure gradient (AaPO2). Two weeks after CBDL, HIF-1α expression in the lung decreased, but was gradually restored at four weeks. Treatment with quercetin did not significantly alter HIF-1α levels, but did reduce AaPO2 as well as lung tissue NF-κB activity, VEGFA gene and protein levels, Akt activity, and angiogenesis. Although hypoxia is an important feature in HPS, our findings suggest that HIF-1α was not the main cause for the VEGFA increase. Interestingly, quercetin inhibited pulmonary vascular angiogenesis in rats with HPS, with involvement of Akt/NF-κB and VEGFA/VEGFR-2 pathways.

Quercetin alleviates pulmonary angiogenesis in a rat model of hepatopulmonary syndrome

Quercetin shows protective effects against hepatopulmonary syndrome (HPS), as demonstrated in a rat model. However, whether these effects involve pulmonary vascular angiogenesis in HPS remains unclear. Therefore, this study aimed to assess the effect of quercetin on pulmonary vascular angiogenesis and explore the underlying mechanisms. Male Sprague-Dawley rats weighing 200-250 g underwent sham operation or common bile duct ligation (CBDL). Two weeks after surgery, HIF-1α and NFκB levels were assessed in rat lung tissue by immunohistochemistry and western blot. Then, CBDL and sham-operated rats were further divided into 2 subgroups each to receive intraperitoneal administration of quercetin (50 mg/kg daily) or 0.2% Tween for two weeks: Sham (Sham+Tween; n=8), CBDL (CBDL+Tween; n=8), Q (Sham+quercetin; n=8), and CBDL+Q (CBDL+quercetin; n=8). After treatment, lung tissue specimens were assessed for protein (immunohistochemistry and western blot) and/or gene expression (quantitative real-time PCR) levels of relevant disease markers, including VEGFA, VEGFR2, Akt/p-Akt, HIF-1α, vWf, and IκB/p-IκB. Finally, arterial blood was analyzed for alveolar arterial oxygen pressure gradient (AaPO2). Two weeks after CBDL, HIF-1α expression in the lung decreased, but was gradually restored at four weeks. Treatment with quercetin did not significantly alter HIF-1α levels, but did reduce AaPO2 as well as lung tissue NF-κB activity, VEGFA gene and protein levels, Akt activity, and angiogenesis. Although hypoxia is an important feature in HPS, our findings suggest that HIF-1α was not the main cause for the VEGFA increase. Interestingly, quercetin inhibited pulmonary vascular angiogenesis in rats with HPS, with involvement of Akt/NF-κB and VEGFA/VEGFR-2 pathways.

Inhibition of dipeptidyl peptidase IV protects tacrolimus-induced kidney injury.

Accumulating evidence shows that a gut-released hormone, the glucagon-like peptide-1 (GLP-1), has not only a glucose-lowering effect but also a renoprotective effect against kidney injury. In this study, we investigated whether a dipeptidyl peptidase (DPP) IV inhibitor has a protective effect against tacrolimus-induced renal injury. Rats were treated with tacrolimus (1.5 mg/kg, subcutaneously) and the DPP IV inhibitor MK0626 (10 or 20 mg/kg, oral gavage) for 4 weeks. MK0626 treatment attenuated tacrolimus-induced renal dysfunction, tubulointerstitial fibrosis, and arteriolopathy. Moreover, these improvements were accompanied by a reduction in oxidative stress and apoptosis. MK0626 treatment increased the blood level of GLP-1 and the level of its receptor in tissue sections but did not alter the levels of other DPP IV substrates, such as neuropeptide Y and the stromal cell-derived factor-1. These data suggest that DPP IV inhibition has an important role in the renoprotection against tacrolimus-induced nephrotoxicity via antioxidative and antiapoptotic effects and preservation of the GLP-1 system.

MiR-207/352 regulate lysosomal-associated membrane proteins and enzymes following ischemic stroke.

The role of microRNAs (miRNAs) in lysosome-mediated neuronal death and survival following ischemic stroke remains unknown. Herein, using miRNA and mRNA gene expression profiling microarrays, we identified the differentially expressed 24 miRNAs and 494 genes in the cortical peri-infarct area, respectively. Integrating the miRNA targets and mRNA expression profiles, we found 47 genes of miRNA targets, including lysosomal-associated membrane protein 2 (LAMP2), Hexb, Bcl2, etc. MiR-207 and miR-352 were mainly downregulated after ischemic stroke, followed by a slight return to baseline during post-middle cerebral artery occlusion (MCAO) 1d to 7d. Furthermore, the luciferase reporter assay demonstrated that LAMP2 and Hexb were the direct targets of miR-207 and miR-352, respectively. After lateral ventricle injection with miR-207 agonist mimics, the neurological deficit scores and infarct volumes were attenuated, and the structure of mitochondria ridges was improved. In addition, miR-207 mimics could reduce the number of cellular lysosome and autophagosome, whereas increase the number of autophagic vacuoles, indicating miR-207 might affect the latter part of lysosomal-autophagy pathway and mitochondria-induced apoptosis. These results suggested that miR-207 and miR-352 were involved in lysosomal pathway for mediating ischemic injury and spontaneous recovery. MiR-207 mimics as potential target drugs could protect against autophagic cell death after ischemic stroke.

Fractional iron solubility of aerosol particles enhanced by biomass burning and ship emission in Shanghai, East China.

In terms of understanding Fe mobilization from aerosol particles in East China, the PM2.5 particles were collected in spring at Shanghai. Combined with the backtrajectory analysis, the PM2.5/PM10 and Ca/Al ratios, a serious dust-storm episode (DSE) during the sampling was identified. The single-particle analysis showed that the major iron-bearing class is the aluminosilicate dust during DSE, while the Fe-bearing aerosols are dominated by coal fly ash, followed by a minority of iron oxides during the non-dust storm days (NDS). Chemical analyses of samples showed that the fractional Fe solubility (%FeS) is much higher during NDS than that during DSE, and a strong inverse relationship of R(2)=0.967 between %FeS and total atmospheric iron loading were found, suggested that total Fe (FeT) is not controlling soluble Fe (FeS) during the sampling. Furthermore, no relationship between FeS and any of acidic species was established, suggesting that acidic process on aerosol surfaces are not involved in the trend of iron solubility. It was thus proposed that the source-dependent composition of aerosol particles is a primary determinant for %FeS. Specially, the Al/Fe ratio is poorly correlated (R(2)=0.113) with %FeS, while the apparent relationship between %FeS and the calculated KBB(+)/Fe ratio (R(2)=0.888) and the V/Fe ratio (R(2)=0.736) were observed, reflecting that %FeS could be controlled by both biomass burning and oil ash from ship emission, rather than mineral particles and coal fly ash, although the latter two are the main contributors to the atmospheric Fe loading during the sampling. Such information can be useful improving our understanding on iron solubility on East China, which may further correlate with iron bioavailability to the ocean, as well as human health effects associated with exposure to fine Fe-rich particles in densely populated metropolis in China.

Measurement of gain characteristics of semiconductor lasers by amplified spontaneous emissions from dual facets.

In this letter, we describe a novel gain measurement approach for semiconductor edge-emitting lasers, with which TE and TM gain spectra can be easily obtained by collecting the amplified spontaneous emissions at dual facets of the device. An unstrained and continuously-operated GaAs/AlGaAs single quantum well laser strip is used to illustrate this method. The measured gain spectra are compared with theoretical gain curves to analyze the gain polarization characteristics and the relevant subband structure in the valence band of the well using the measured gain spectra.

RIP140 is associated with subclinical inflammation in type 2 diabetic patients.

AIMS: To evaluate the expression level of RIP140 (receptor interaction protein 140) and its correlation with inflammatory cytokine production and free fatty acids (FFAs) in type 2 diabetes. METHODS: Plasma and peripheral blood mononuclear cells (PBMCs) were collected from 24 diabetic and 30 healthy individuals. The levels of FFAs, TC, TG, HDL-C, LDL-C, FIN, and FBG were measured. The insulin resistance index was calculated using the homeostasis model assessment (HOMA). Additionally, PBMCs from control group were cultured alone or with 500 µmol/L palmitic acid (PA). Levels of RIP140 TNF-α, and IL-6 in PBMCs were analyzed using real-time RT-PCR, Western blots and ELISA. The relationship between RIP140 and other variables was performed using SPSS 11.5 software. RESULTS: TG, LDL-C, FIN, FBG, HOMA, and HDL-C were significantly different between diabetic patients and the control group. Levels of RIP140, TNF-α, and IL-6 were higher in the diabetic group compared to control. RIP140 expression was positively correlated with FFAs, HDL-c, TNF-α, IL-6, FIN, FBG, and HOMA. Finally, 500 µmol/L PA treatment increased RIP140 expression and the secretion of inflammatory cytokines in cultured control PBMCs. CONCLUSIONS: Increased RIP140 level may be closely associated with inflammation and disorder of lipid and glucose metabolism in diabetic patients.

Clinical significance of the ratio between FOXP3 positive regulatory T cell and interleukin-17 secreting cell in renal allograft biopsies with acute T-cell-mediated rejection.

The aim of this study is to investigate the clinical significance of the ratio between interleukin-17 (IL-17) secreting cell and FOXP3-positive regulatory T cell (FOXP3(+) Treg) infiltration in renal allograft tissues with acute T-cell-mediated rejection (ATCMR). Fifty-six patients with biopsy-proven ATCMR were included. Infiltration of FOXP3(+) Treg and IL-17-secreting cells was evaluated with immunostaining for FOXP3 or IL-17 on the biopsy specimens, and the patients were divided into the FOXP3 high group (Log FOXP3/IL-17 > 0·45) or the IL-17 high group (Log FOXP3/IL-17 < 0·45). We compared the allograft function, severity of tissue injury, and clinical outcome between the two groups. In the IL-17 high group, allograft function was significantly decreased compared with the FOXP3 high group (P < 0·05). The severity of interstitial and tubular injury in the IL-17 high group was higher than the FOXP3 high group (P < 0·05). The proportions of steroid-resistant rejection, incomplete recovery and recurrent ATCMR were higher in the IL-17 high group than in the FOXP3 high group (all indicators, P < 0·05). The IL-17 high group showed lower 1-year (54% versus 90%, P < 0·05) and 5-year (38% versus 85%, P < 0·05) allograft survival rates compared with the FOXP3 high group. Multivariate analysis revealed that the FOXP3/IL-17 ratio was a significant predictor for allograft outcome. The FOXP3/IL-17 ratio is a useful indicator for representing the severity of tissue injury, allograft dysfunction and for predicting the clinical outcome of ATCMR.

Duration of Ross River viraemia in a mouse model--implications for transfusion transmission.

BACKGROUND AND OBJECTIVES: There is little data on the duration of viraemia following infection with Ross River virus (RRV), the most common cause of arbovirus disease in Australia. In particular, no accurate estimate exists for the duration of pre-symptomatic RRV infection, which is important in assessing the potential for transfusion transmission. MATERIALS AND METHODS: We used an established mouse model of RRV infection involving adult Swiss outbred mice to measure viraemia following infection. Applying our experimental data to a published probabilistic model for estimating the risk of dengue transmission by transfused blood, we derived comparable risk estimates for RRV. RESULTS: Ross River virus RNA was measured using highly sensitive real-time PCR in serum samples to determine the duration of asymptomatic viraemia, which typically lasted 5 days, but extended to 9 days in some mice. Assuming the potential for transfusion transmission is proven, the risk of RRV transmission by blood during a 2004 outbreak in Cairns, Australia was retrospectively estimated as 1 in 13,542 (range from 1 in 4765 to 47,563). CONCLUSION: This study provides updated epidemiological data useful to underpin modelling to assess the potential risk of transfusion-transmitted RRV. Using an established model for dengue, the risk estimate for RRV transmission is comparable in the same geographical region. Should transfusion be proven as a route of transmission, this supports consideration of appropriate mitigation strategies to safeguard blood recipients.

Distinct roles of the anterior cingulate cortex in spinal and supraspinal bee venom-induced pain behaviors.

A wide variety of human and animal experiments suggest that the anterior cingulate cortex (ACC) is one of the key brain substrates subserving higher order processing of noxious information. However, no sufficient data are now available regarding the mediation by ACC of different levels of pain processing as well as its potential descending modulation of spinal nociception. Using the well-developed rat bee venom (BV) model, the present study evaluated the effect of lesions of bilateral ACC on two levels of spontaneous nociceptive behaviors (spinally-processed persistent paw flinching reflex and supraspinally-processed paw lifting/licking) and heat or mechanical hypersensitivity under the inflammatory pain state. In contrast to the sham lesion group (saline microinjection into the ACC), bilateral complete ACC chemical lesions (kainic acid microinjection into the ACC) significantly decreased the BV-induced paw lifting and licking behavior (less time spent by the animal in paw lifting/licking) but produced no influence upon spinally-processed spontaneous paw flinching reflex (no change in number of paw flinches following subcutaneous BV injection). Moreover, the bilateral ACC lesions relieved the BV-evoked primary thermal or mechanical hypersensitivity compared with the sham control group. However, incomplete lesions of bilateral ACC failed to affect the abovementioned pain-related behaviors. No effects were seen on basal pain sensitivity in either group of rats. Motor coordination, as measured by Rota-Rod treadmill test, was not impaired by bilateral ACC lesions. These results implicate that the ACC area of the brain plays differential roles in the mediation of different levels of spontaneous pain-related behaviors. The present study also provides additional evidence for the ACC-mediated descending facilitation of primary hyperalgesia (pain hypersensitivity) identified in the injured area under inflammatory pain state.

Spatial and temporal variations of eutrophication in Western Chaohu Lake, China.

Chaohu Lake is one of the five largest fresh lakes in China. Now it is one of the three most eutrophic lakes of China attracting more and more attention in the world-wide-concern. From Zhongmiao Temple to Qitouzui Cape, the lake is divided into two parts. The eutrophication state of the lake western part is more serious than that of the eastern part, mainly because the former is the final place of industrial and municipal wastewater from Hefei City, the capital of Anhui Province. Through six routine national sampling sites of the Western Chaohu Lake, we analyzed the yearly variation of eutrophication from 1984 to 2004, the monthly variation from 2001 to 2004 and the spatial distribution and variation of the six sampling sites in the year 1992, 1996, 2000 and 2004 with Trophic State Index (TSI). The main reasons for Chaohu Lake eutrophication and the spatial and temporal variations of eutrophication in Western Chaohu Lake were discussed.

Effects of bee venom peptidergic components on rat pain-related behaviors and inflammation.

To identify the active components of honeybee venom in production of inflammation and pain-related behaviors, five major peptidergic subfractions were separated, purified and identified from the whole honeybee venom. Among them, four active peptidergic components were characterized as apamin, mast-cell degranulating peptide (MCDP), phospholipase A(2) (PLA(2))-related peptide and melittin, respectively. All five subfractions were effective in production of local inflammatory responses (paw edema) in rats although the efficacies were different. Among the five identified subfractions, only MCDP, PLA(2)-related peptide and melittin were able to produce ongoing pain-related behaviors shown as paw flinches, while only apamin and melittin were potent to produce both thermal and mechanical hypersensitivity. As shown in our previous report, melittin was the most potent polypeptide in production of local inflammation as well as ongoing pain and hypersensitivity. To further explore the peripheral mechanisms underlying melittin-induced nociception and hypersensitivity, a single dose of capsazepine, a blocker of thermal nociceptor transient receptor potential vanilloid receptor 1, was treated s.c. prior to or after melittin administration. The results showed that both pre- and post-treatment of capsazepine could significantly prevent and suppress the melittin-induced ongoing nociceptive responses and thermal hypersensitivity, but were without influencing mechanical hypersensitivity. The present results suggest that the naturally occurring peptidergic substances of the whole honeybee venom have various pharmacological potencies to produce local inflammation, nociception and pain hypersensitivity in mammals, and among the five identified reverse-phase high pressure liquid chromatography subfractions (four polypeptides), melittin, a polypeptide occupying over 50% of the whole honeybee venom, plays a central role in production of local inflammation, nociception and hyperalgesia or allodynia following the experimental honeybee's sting. Peripheral transient receptor potential vanilloid receptor 1 is likely to be involved in melittin-produced ongoing pain and heat hyperalgesia, but not mechanical hyperalgesia, in rats.

Development of a shear force scanning near-field fluorescence microscope for biological applications.

In this paper, a shear force scanning near-field fluorescence microscope combined with a confocal laser microspectrofluorometer is described. The shear force detection is realized based on a bimorph cantilever, which provides a very sensitive, reliable, and easy to use method to control the probe-sample distance during scanning. With the system, high-quality shear force imaging of various samples has been carried out. Furthermore, simultaneous shear force and near-field fluorescence imaging of biological cells has also been realized. As an example, we especially present the result on the distribution of P-glycoprotein in the plasma membrane of human small cell lung cancer cells, suggesting that the system would be a promising tool for biological applications.

Non-optical bimorph-based tapping-mode force sensing method for scanning near-field optical microscopy.

A non-optical bimorph-based tapping-mode force sensing method for tip-sample distance control in scanning near-field optical microscopy is developed. Tapping-mode force sensing is accomplished by use of a suitable piezoelectric bimorph cantilever, attaching an optical fibre tip to the extremity of the cantilever free end and fixing the guiding portion of the fibre to a stationary part near the tip to decouple it from the cantilever. This method is mainly characterized by the use of a bimorph, which carries out simultaneous excitation and detection of mechanical vibration at its resonance frequency owing to piezoelectric and anti-piezoelectric effects, resulting in simplicity, compactness, ease of implementation and lack of parasitic optical background. In conjugation with a commercially available SPM controller, tapping-mode images of various samples, such as gratings, human breast adenocarcinoma cells, red blood cells and a close-packed layer of 220-nm polystyrene spheres, have been obtained. Furthermore, topographic and near-field optical images of a layer of polystyrene spheres have also been taken simultaneously. The results suggest that the tapping-mode set-up described here is reliable and sensitive, and shows promise for biological applications.

[Cloning of the sugar related biosynthesis gene cluster from Streptomyces tenebrarius H6].

Streptomyces tenebrarius H6 produces a complex of aminoglycoside antibiotics, such as apramycin, tobramycin and kanamycin B etc. To study the apramycin biosynthetic genes the genomic library from the Streptomyces tenebrarius H6 was established using E. coli/Streptomyces shuttle vector pKC505 by in vitro packing. The probability of finding a specific gene from the library composed of 3,000 colonies was over 99.9%. According to the highly conserved sequence of the genes involved in 6-deoxyhexose biosynthesis, primers were designed and 0.6 kb fragment homologous to strE gene was obtained by PCR. 30 positive clones were found from the genomic library of S. tenebrarius H6 with the 0.6 kb fragment as a probe. Overlapped regions were localized by Southern hybridization and putative sugar related biosynthetic gene cluster was mapped by restriction enzyme digestions. An ORF of dTDP-glucose-4,6-dehydratase gene consisted of 1,132 bp, designated as aprE, was obtained and submitted to GenBank under the accession number of AF306787. A DNA sequence highly homologous to strL coding dTDP-4-dehydrorhamnose reductase was found linked with aprE gene.