[Consistency of ALK Ventana-D5F3 immunohistochemistry interpretation in lung adenocarcinoma among Chinese histopathologists].

Objective: To understand the consistency of ALK Ventana-D5F3 immunohistochemistry (IHC) interpretation in Chinese lung adenocarcinoma among histopathologists from different hospitals, and to recommend solution for the problems found during the interpretation of ALK IHC in real world, with the aim of the precise selection of patients who can benefit from ALK targeted therapy. Methods: This was a multicenter and retrospective study. A total of 109 lung adenocarcinoma cases with ALK Ventana-D5F3 IHC staining were collected from 31 lung cancer centers in RATICAL research group from January to June in 2018. All cases were scanned into digital imaging with Ventana iSCANcoreo Digital Slide Scanning System and scored by 31 histopathologists from different centers according to ALK binary (positive or negative) interpretation based on its manufacturer's protocol. The cases with high inconsistency rate were further analyzed using FISH/RT-PCR/NGS. Results: There were 49 ALK positive cases and 60 ALK negative cases, confirmed by re-evaluation by the specialist panel. Two cases (No. 2302 and No.2701) scored as positive by local hospitals were rescored as negative, and were confirmed to be negative by RT-PCR/FISH/NGS. The false interpretation rate of these two cases was 58.1% (18/31) and 48.4% (15/31), respectively. Six out of 31 (19.4%) pathologists got 100% accuracy. The minimum consistency between every two pathologists was 75.8%.At least one pathologist gave negative judgement (false negative) or positive judgement (false positive) in the 49 positive or 60 negative cases, accounted for 26.5% (13/49), 41.7% (25/60), respectively, with at least one uncertainty interpretation accounted for 31.2% (34/109). Conclusion: There are certain heterogeneities and misclassifications in the real world interpretation of ALK-D5F3 IHC test, which need to be guided by the oncoming expert consensus based on the real world data.

[Diagnostic significance of lymph node core needle biopsy for lymphoproliferative disease: a clinicopathologic study of 1 013 cases].

Objective: To study the clinicopathologic features of lymphoproliferative disease by lymph node core needle biopsy(CNB)and to evaluate the diagnostic significance of CNB for lymphoproliferative disease. Methods: The annual distribution, entity constitute, clinical finding, gross feature, morphologic change, affiliate study and repeat biopsy diagnosis of 1 013 cases of lymph node CNB diagnosed at West China Hospital of Sichuan University from January 2009 to December 2015 were investigated. Results: (1) Proportion of lymph node CNB in total amount of biopsy specimens increased from 0.2% in 2009 to 0.8% in 2015.(2) The study cohort included 471 lymphomas, 12 atypical lymphoid hyperplasia (ALH), 136 suspected lymphomas, 372 benign lesions, and 22 cases of descriptive diagnoses. The most common types were diffuse large B cell lymphoma and T-lymphoblastic lymphoma. (3) Majority of patients were adolescents and children younger than 20 years or the elderly older than 60 years. 53.1% CNB tumor specimen consisted of ≥4 tissue cores and 40.5% were >2 cm in length. (4) 104 CNB cases with previous history of excision biopsy was included 45 carcinomas(no metastatic carcinoma was found), 32 lymphomas for treatment observation.1/14 suspicious lymphomas, 1/1 ALH and 3/22 cases benign lesions were diagnosed as lymphoma by repeat biopsy respectively. (5) 217 CNB cases were diagnosed as lymphoma by subsequent CNB (70), or subsequent excision biopsy (147) including 78.5%(73/93) suspected lymphomas, 5/7 ALH and 32.3%(20/62)benign lesions. Conclusions: Lymph node CNB has certain clinical indications, although limited for the diagnosis of lymphoproliferative disorders. Suspected lymphomas and ALH diagnosed by CNB should be followed by repeat tissue biopsy. For the benign lesions by CNB it does not rule out additional biopsy to further investigate the lesion.

Preparation of carotenoid extracts and nanoemulsions from Lycium barbarum L. and their effects on growth of HT-29 colon cancer cells.

Lycium barbarum L., a traditional Chinese herb widely used in Asian countries, has been demonstrated to be protective against chronic diseases such as age-related macular degeneration. The objectives of this study were to determine the carotenoid content in L. barbarum by high-performance liquid chromatography-mass spectrometry, followed by preparation of a carotenoid nanoemulsion to evaluate the mechanism of inhibition on HT-29 colon cancer cells. The highest extraction yield of carotenoids was attained by employing a solvent system of hexane-ethanol-acetone (1:1:1, v/v/v). Nine carotenoids, including neoxanthin (4.47 µg g-1), all-trans-zeaxanthin and its cis-isomers (1666.3 µg g-1), all-trans-ß-cryptoxanthin (51.69 µg g-1), all-trans-ß-carotene and its cis-isomers (20.11 µg g-1), were separated within 45 min and quantified using a YMC C30 column and a gradient mobile phase of methanol-water (9:1, v/v) (A) and methylene chloride (B). A highly stable carotenoid nanoemulsion composed of CapryolTM 90, Transcutol®HP, Tween 80 and deionized water was prepared with a mean particle size of 15.1 nm. Characterization of zeaxanthin standard, blank nanoemulsion, carotenoid extract and carotenoid nanoemulsion by differential scanning calorimetry curves and Fourier transform infrared spectra revealed a good dispersion of zeaxanthin-dominated carotenoid extract with no significant chemical change after incorporation into nanoemulsion. The in vitro release kinetic study showed a higher release profile at pH 5.2 than at physiological pH 7.4, suggesting a rapid release of carotenoids in the acidic environment (pH 4.5-6.5) characteristic of tumors. Both the carotenoid nanoemulsion and the extract were effective at inhibiting growth of HT-29 colon cancer cells, with an IC50 of 4.5 and 4.9 µg ml-1, respectively. Also, both treatments could up-regulate p53 and p21 expression and down-regulate CDK2, CDK1, cyclin A and cyclin B expression and arrest the cell cycle at G2/M. The study may form a basis for further exploration of L. barbarum nanoemulsion in cancer treatment.

[Application of immunohistochemistry for p16 and GATA3 and molecular HPV typing in diagnosis of secondary bladder involvement by cervical carcinoma].

Objective: To investigate the expression of p16 and GATA3 and the detection of human papillomavirus (HPV) in secondary bladder involvement by cervical carcinomas. Methods: Sixteen cases of cervical carcinoma with bladder involvement diagnosed from December 2008 to March 2016 were collected and evaluated by light microscopy, immunohistochemistry for p16 and GATA3 detection and PCR-reverse dot blot for molecular typing of HPV. Results: The age of the patients ranged from 25 to 76 years with median of 52 years. Morphologically, 14 cases(14/16) showed tumor nests infiltrating lamina propria or muscle bundles of the bladder. By immunohistochemistry, 15 cases (15/16) were found to be diffusely and strongly positive for p16, and 1 showed patchy staining pattern. Seven cases (7/7) of corresponding original cervical cancers were also diffusely and strongly positive for p16. GATA3 staining was negative in 13 cases (13/16), and focal weak to moderate positivity was detected in 3 cases.Three cases (3/7) of corresponding original cervical cancers showed focal weak to moderate positivity of GATA3. Fifteen cases (15/16) showed concordant high risk HPV-positivity, including HPV16 in 8 cases and HPV31 in one case. Five cases showed co-infection of HPV16 and HPV18. One case showed co-infection with HPV18 and HPV45. Conclusion: Differential diagnosis by p16 or GATA3 alone is of limited value. Combination of immunohistochemistry for p16 and GATA3 and molecular typing for HPV detection are useful to distinguish primary bladder carcinoma from the secondary involvement by cervical carcinoma.

Hepatitis B virus X protein activates human hepatic stellate cells through upregulating TGFß1.

We investigated the effects of the hepatitis B virus X gene (HBV X) on the activation of human hepatic stellate cells (HSCs) and the possible mechanisms underlying the pathway. Recombinant plasmid pHBV-X-IRES2-EGFP was constructed and transfected into HL-7702 cells using a lipid-mediated method. Transfected cells were screened by G418, which detected stable expression of the X gene by reverse transcription (RT)-PCR and Western blot analysis, and named L02/x. Cells not subjected to G418-selection were analyzed to confirm the transient expression of the X gene and named L02/48x. Subsequently, L02/x and L02/48x, together with non-HBx-expressing cells, were co-cultured with HSCs in a non-contact transwell system. After 36 h of co-culture, the proliferation and migration of HSCs was detected using different cell counting methods. Finally, the mRNA and protein levels of α-SMA, Col I, and TGFß1 in HSCs were detected by real-time PCR and western blot analysis. RT-PCR and Western blot analysis showed that L02/x and L02/48x cells can express HBV X gene mRNA and protein. Additionally, HSCs co-cultured with L02/x or L02/48x cells showed significantly higher proliferation and migration levels than control groups. Real-time PCR and Western blot analysis showed that the mRNA and protein expressions of α-SMA, Col I, and TGFß1 in HSCs co-cultured with HBx-expressing liver cells were higher than those in control groups. HBx protein activated HSCs in vitro, leading to increased proliferation and migration of HSCs and upregulation of α-SMA and Col I. The TGFß1 gene may be involved in this pathway.

[Distribution of mosquito species and associated viruses in Hami City of Xinjiang Uygur Autonomous Region from 2019 to 2020].

OBJECTIVE: To investigate the distribution of mosquito species and their associated viruses, and identify Culex pipiens subspecies in Hami City, Xinjiang Uygur Autonomous Region. METHODS: Mosquitoes were captured using mosquito trapping lamps method in Yizhou District, Yiwu County, and Balikun County of Hami City in mi-July, 2019 and 2020. The species and subspecies of all captured mosquitoes were characterized. In addition, the flavivirus, alphavirus, bunyavirus, Japanese encephalitis virus, Liaoning virus, Tahyna virus, tick-borne encephalitis virus and West Nile virus were detected using reverse-transcription PCR assay in captured mosquitoes. RESULTS: A total of 1 496 mosquitoes were captured from Yizhou District, Yiwu County, and Balikun County of Hami City, belonging to 3 genus and 3 species. Cx. pipiens was the dominant mosquito species (986 mosquitoes, 65.91%), followed by Aedes caspius (457 mosquitoes, 30.55%), while Culiseta alaskaensis had the lowest number (53 mosquitoes, 3.54%). All captured Cx. pipiens mosquitoes were identified as Cx. pipiens pipiens based on the terminalia of male mosquitoes. RT-PCR assay tested negative for flavivirus, alphavirus, bunyavirus, Japanese encephalitis virus, Liaoning virus, Tahyna virus, tick-borne encephalitis or West Nile virus in captured Cx. pipiens mosquitoes. CONCLUSIONS: There were 3 species of mosquitoes in Hami City from 2019 to 2020, including Cx. pipiens, Ae. Caspius and C. alaskaensis, with Cx. pipiens as the dominant mosquito species, and all captured Cx. pipiens mosquitoes were Cx. pipiens pipiens; however, no arboviruses were detected.

[Correlation analysis on meteorological factors regarding the incidence of hand, foot and mouth disease in Xinjiang Uygur Autonomous Region, 2011-2018].

Objective: To study the correlation between meteorological factors and the incidence of hand, foot and mouth disease (HFMD) in Xinjiang Uygur Autonomous Region (Xinjiang) so as to provide scientific evidence for the early warning, prediction, prevention and control of HFMD. Methods: Data on HFMD surveillance and related population was collected from the China Information System for Disease Control and Prevention from 2011 to 2018. Meteorological data was obtained from http://www.tianqihoubao.com. Correlation analysis on meteorological factors and the incidence of HFMD in Xinjiang was conducted, using the Excel 2007, SPSS 17.0, and Spatial Distribution Map by ArcGIS 10.2 software. Results: HFMD usually occurred between April and July. Numbers of patients reached the top in May and June. Temperature was positively correlated with the incidence of HFMD (r=0.370, P<0.01) while precipitation was positively correlated with the incidence of HFMD (r=0.747, P<0.01). The temperature threshold appeared as 5 ℃-35 ℃ for the incidence of HFMD. Interval period was one month between the peak of both the incidence of HFMD and the precipitation. A power function relationship (y=0.009 4x(2.332 9), R(2)=0.898 9) was noticed between the precipitation and the incidence of HFMD. Conclusions: The incidence of HFMD was closely related to the meteorological factors including temperature and precipitation in Xinjiang during 2011-2018. Our findings have provided evidence for the development of early warning system on HFMD in Xinjiang.

[Survey of the first case of Zika virus disease in the mainland of China].

OBJECTIVE: To analyze epidemiological characteristics of the first laboratory confirmed case of Zika virus disease in the mainland China, and provide evidence for the prevention and control of the spread of Zika virus disease. METHODS: Epidemiological survey was conducted for the first suspect case of Zika virus disease in China, and medical observation was conducted for the close contacts. The nucleic acid detection of Zika virus was conducted with Real-time RT-PCR by using blood and urine samples collected from the cases. RESULTS: The first case of Zika virus disease was confirmed. The case was cured and discharged on 18(th) day after the onset. The blood sample on 10(th) day after the onset and urine samples on 11-13(th) days after the onset were all positive for Zika virus. The case had the history of mosquito bites in the endemic country before the onset. The close contacts showed no Zika virus disease-like symptoms during the medical observation period. CONCLUSION: The case reported by Jiangxi province was the first imported case of Zika virus disease in the mainland of China. The infection was related with the mosquito bites in Venezuela where Zika virus disease in endemic.

The solution structure of a cytotoxic ribonuclease from the oocytes of Rana catesbeiana (bullfrog).

RC-RNase is a pyrimidine-guanine sequence-specific ribonuclease and a lectin possessing potent cell cytotoxicity. It was isolated from the oocytes of Rana catesbeiana (bull frog). From analysis of an extensive set of 1H homonuclear 2D NMR spectra we have completed the resonance assignments. Determination of the three-dimensional structure was carried out with the program X-PLOR using a total of 951 restraints including 814 NMR-derived distances, 61 torsion angles, and 76 hydrogen bond restraints. In the resultant family of 15 best structures, selected from a total of 150 calculated structures, the root-mean-square deviation from the average structure for the backbone heavy-atoms involved in well-defined secondary structure is 0.48 A, while that for all backbone heavy-atoms is 0.91 A. The structure of RC-RNase consists of three alpha-helices and two triple-stranded anti-parallel beta-sheets and folds in a kidney-shape, very similar to the X-ray crystal structure of a homolo gous protein, onconase isolated from Rana pipiens. We have also investigated the interaction between RC-RNase and two inhibitors, cytidylyl(2'-->5')guanosine (2',5'-CpG) and 2'-deoxycytidylyl(3'-->5')-2'-deoxyguanosine (3',5'-dCpdG). Based on the ligand-induced chemical shift changes in RC-RNase and the NOE cross-peaks between RC-RNase and the inhibitors, the key residues involved in protein-inhibitor interaction have been identified. The inhibitors were found to bind in a "retro-binding" mode, with the guanine base bonded to the B1 subsite. The His103 residue was found to occupy the B state with the imidazole ring pointing away from the active site. The structure coordinates and the NMR restraints have been deposited in the Brookhaven Protein Data Bank (1bc4 and 1bc4mr, respectively).

Restoration of erectile function with intracavernous injections of endothelial progenitor cells after bilateral cavernous nerve injury in rats.

Endothelial progenitor cells (EPCs) are bone marrow-derived endothelial cells capable of circulating, proliferating, and differentiating into mature endothelial cells. Circulating EPCs can be directly recruited to some extent at sites of injury, and their administration could accelerate repair or endothelialization of the damaged tissue. We investigated the effects of intracavernous injections of EPCs into the corpora cavernosa of rats with erectile dysfunction (ED) caused by bilateral cavernous nerve (CN) injury. Overall, 24 male Sprague-Dawley rats were randomized into three groups: sham surgery, vehicle-only, or EPC treatment. Rats in the EPC treatment and vehicle-only groups were subjected to bilateral CN injury before injection of EPCs or vehicle, respectively, into the corpora cavernosa. Four weeks after surgery, erectile function was assessed by measuring maximum intracavernosal pressure (ICP), change in ICP, area under the ICP curve, and ratio of change in ICP and mean arterial pressure (MAP; ΔICP/MAP). Penile tissue was histomorphometrically analyzed for the expression of neural nitric oxide synthase (nNOS), neurofilament-1 (NF-1), von Willebrand factor (vWF), endothelial NOS (eNOS), and smooth muscle cell content. Maximum ICP and all other functional parameters of erectile function were significantly reduced in the vehicle-only group vs. the sham and EPC treatment groups (all p < 0.001). Smooth muscle cell content was decreased in the vehicle-only vs. the sham and EPC treatment groups (both p < 0.01). Expressions of vWF and eNOS in the dorsal artery were significantly higher in the EPC treatment than the vehicle-only group (p < 0.05). In conclusion, EPC treatment restored erectile function in a rat model of bilateral CN injury through recruitment of EPCs toward the dorsal artery and preservation of smooth muscle cells in the corpus cavernosum. These findings elucidate the therapeutic potential of EPCs for treating ED in humans.

Gibberellic acid stabilises microtubules in maize suspension cells to cold and stimulates acetylation of alpha-tubulin.

Gibberellic acid is known to stabilise microtubules in plant organs against depolymerisation. We have now devised a simplified cell system for studying this. Pretreatment of a maize cell suspension with gibberellic acid for just 3 h stabilised protoplast microtubules against depolymerisation on ice. In other eukaryotes, acetylation of alpha-tubulin is known to correlate with microtubule stabilisation but this is not established in plants. By isolating the polymeric tubulin fraction from maize cytoskeletons and immunoblotting with the antibody 6-11B-1, we have demonstrated that gibberellic acid stimulates the acetylation of alpha-tubulin. This is the first demonstrated link between microtubule stabilisation and tubulin acetylation in higher plants.

The solution structure of the active domain of CAP18--a lipopolysaccharide binding protein from rabbit leukocytes.

We have employed the circular dichroism (CD) technique to characterize the solution structure of CAP18(106-137), a lipopolysaccharide (LPS) binding, antimicrobial protein, and its interaction with lipid A. Our results revealed that CAP18(106-137) may exist in at least three lipid A concentration-dependent, primarily helix conformations. The 'model' structure of CAP18(106-137) in 30% (v/v) TFE, determined by nuclear magnetic resonance (NMR) technique, was found to be a complete and very rigid helix. In this conformation, the cationic and hydrophobic groups of CAP18(106-137) are separated into patches and stripes in such a way that it can favorably interact with lipid A through either coulombic interaction with the diphosphoryl groups or hydrophobic interaction with the fatty acyl chains.

Homocysteine thiolactone induces apoptotic DNA damage mediated by increased intracellular hydrogen peroxide and caspase 3 activation in HL-60 cells.

The cytotoxicity of homocysteine derivatives on chromosomal damage in somatic cells is not well established. The present study used reactive homocysteine derivative of homocysteine thiolactone (Hcy) to investigate its causal effect on apoptotic DNA injury in human promyeloid HL-60 cells. Our results demonstrated that Hcy induced cell death and features of apoptosis including increased phosphotidylserine exposure on the membrane surface, increased apoptotic cells with hypoploid DNA contents, and internucleosomal DNA fragmentation, all of which occurred in a time- and concentration-dependent manner. Hcy treatment also significantly increased intracellular reactive oxygen species H2O2, which coincided with the elimination of caspase 3 proenzyme levels and increased caspase 3 activity at the time of the appearance of apoptotic DNA fragmentation. Preincubation of Hcy-treated HL-60 cells with catalase completely scavenged intracellular H2O2, thus inhibiting caspase 3 activity and protecting cells from apoptotic DNA damage. In contrast, superoxide dismutase failed to inhibit Hcy-induced DNA damage. Taken together, these results demonstrate that Hcy exerted its genotoxic effects on HL-60 cells through an apoptotic pathway, which is mediated by the activation of caspase 3 activity induced by an increase in intracellular hydrogen peroxide.

Folate deficiency-induced oxidative stress and apoptosis are mediated via homocysteine-dependent overproduction of hydrogen peroxide and enhanced activation of NF-kappaB in human Hep G2 cells.

Folate coenzymes are critical for de novo synthesis of purine and thymidine, and for interconversion of amino acids. Folate deficiency inhibits cellular proliferation, disturbs cell cycling, causes genetic damage and eventually results in cell death. Previously, we demonstrated that the demise of human hepatoma Hep G2 cells mediated by folate deficiency proceeded via a p53-independent apoptosis, and the perturbation of intracellular calcium homeostasis was also shown to be involved. To further delineate the mechanism associated with this observed phenomenon, Hep G2 cells were cultivated in the control or folate-deficient media (control media lacking folate, glycine, thymidine and hypoxanthine) for 4 weeks. At the end of this cultivation period, we found that TBARS (an index of lipid peroxidation) concentrations in the folate-deficient cells were drastically increased as compared to the control cells (0.04 vs 0.01 nmole/10(6) cells), indicating that a severe oxidative stress of the former cells had occurred. This phenomenon was also shown to coincide with the ability of these folate-deficient cells to elaborate increased amounts of H2O2 as compared to its folate-supplemented cells (2.87 vs 0.98 nmole/10(5) cells/h). Furthermore, the accelerated production of H2O2 by the folate-deficient cells was also closely correlated with the elevated homocysteine concentrations released in the culture medium (15.37 +/- 2.4 vs 3.58 +/- 2.4 micromole/L; P< 0.001). Finally, we demonstrated that folate deficiency was indeed capable of activating a redox-sensitive transcription factor, NF-kappaB, which is crucial in the control of a reactive oxygen species-mediated apoptosis. In summary, we show that folate deficiency-induced apoptosis is proceeded via the enhanced activation of NF-kappaB, which is the resulting form of the homocysteine-mediated overproduction of hydrogen peroxide.

Retinopathy, neuropathy and nephropathy in non-insulin-dependent diabetic patients.

In 1978, an epidemiological survey for adult diabetes was conducted in Taipei City. A total of 219 Chinese non-insulin-dependent diabetic patients were discovered and 217 of them were examined for retinopathy, neuropathy and nephropathy. Among the 110 men and 107 women studied, 63.1% were free of complications and the prevalences for retinopathy, nephropathy and neuropathy were 24.0%, 12.9% and 23.5%, respectively. The clinical and biochemical data of the patients were compared. For those with and those without complications, the diabetic duration (8.2 +/- 6.7 vs 4.1 +/- 2.7, years), percentage of insulin treatment (8.8% +/- 0.7%), percentage of hypertension (42.5% vs 26.3%), and the fasting plasma glucose (182.8 +/- 63.6 vs 135.0 +/- 44.6, mg/dl) were significantly different. Diabetic duration and glycemic control consistently correlated with retinopathy, nephropathy and neuropathy. Hypertension and insulin treatment were also associated positively with the complications. The more complications the diabetic patients had, the poorer the glycemic control, the longer the diabetic duration, a higher percentage of insulin treatment and hypertension were found.

Dose-dependent folic acid and memantine treatments promote synergistic or additive protection against Aß(25-35) peptide-induced apoptosis in SH-SY5Y cells mediated by mitochondria stress-associated death signals.

Increased dietary folic acid (FA) is associated with reduced risks of Alzheimer's disease (AD). The AD drug memantine (Mn) has had limited therapeutic effects for the treatment of patients with moderate to severe AD. This study investigated whether and the underlying mechanisms by which the combination of Mn and FA may have synergistic or additive effects in protecting against amyloid-ß(25-35) peptide (Aß)-induced neurocytotoxicity. Aß treatment of human neuroblastoma SH-SY5Y cells significantly induced a 6-fold increase of apoptotic cells compared with the Aß-untreated group. Preincubation of Aß-exposed cells with FA (500 µM) or Mn (20 µM) caused a 22% and 10% reduction of apoptotic cells, respectively, whereas the combo-treatments at such doses synergistically alleviated Aß-induced apoptosis by 60% (P<0.05). The apoptotic protection by the combo-treatments coincided with attenuating Aß-elicited mitochondrial (mt) membrane depolarization and abolishing Aß-induced mt cytochrome c release to the cytosol. Increased levels of FA at 1000 µM in combination with 20 µM Mn exerted an additive protection against Aß(25-35)-induced-apoptosis as compared to the isolate Mn group (P<0.05). The combo-treatments reversed Aß-elicited mt membrane depolarization, attenuated Aß-elicited mt cytochrome c release to the cytosol, and diminished Aß-promoted superoxide generation. The apoptotic-protection by such combo-treatments was partially abolished by carbonyl cyanide 3-chlorophenylhydrazone (mt membrane potential uncoupler) and sodium azide (mt cytochrome c oxidase inhibitor). Taken together, the data demonstrated that dose-dependent FA and Mn synergistically or additively protected SH-SY5Y cells against Aß-induced apoptosis, which was partially, if not completely, mediated by mt stress-associated death signals.