In situ isolation of nuclei or nuclear proteins from adherent cells: a simple, effective method with less cytoplasmic contamination

BACKGROUND: Isolation of nuclei or nuclear proteins is a prerequisite for western blot, nuclear proteome profiling, and other evaluations of nuclear proteins. Here, we developed a simple method for in situ isolation of nuclei or nuclear proteins by in situ removing the extranuclear part of adherent cells via a classical nonionic detergent triton X-100. RESULTS: First, the feasibility of our method was confirmed by confocal microscopy, atomic force microscopy, scanning electron microscopy, dynamic light scattering, immunofluorescence imaging, and time-lapse dynamic observation. Next, the optimal concentration range (approximately 0.1-1% for ~ 10 min) of triton X-100 and the optimal treatment time (< 30 min) of 0.1-1% Triton X-100 for our method were determined via western blotting of eight extra-/ intra-nuclear proteins. Subsequently, the effectiveness, sensitivity, and cytoplasmic contamination of our method were tested by investigating the levels of phosphorylated p65 (a NF-κB subunit) in the nuclei of endothelial or tumor cells treated with/without lipopolysaccharide (LPS) via western blotting and by comparing with a commercial nuclear protein extraction kit (a classical detergent-based method). The data show that compared with the commercial kit our method obtained a higher yield of total nuclear proteins, a higher pP65 level in both control and LPS groups, and much lower content of GAPDH (as a reference for cytoplasmic contamination) in nuclei. CONCLUSIONS: The in situ isolation of nuclei or nuclear proteins from adherent cells in this study is a simple, effective method with less cytoplasmic contamination. This method/strategy has the potential of improving the quality of downstream evaluations including western blotting and proteomic profiling.

Fibrolipomatous hamartoma of the median nerve: a case report and literature review / Hamartoma fibrolipomatoso del nervio mediano: reporte de un caso y revisión de la literatura

Abstract Fibrolipomatous hamartoma (FLH) of the nerve, also known as lipomatosis of the nerve, neurofibrillary lipomatous lesion, or intraneural lipoma, is a rare benign soft tissue tumor which mainly occurs in the nerves of the upper limb, especially in the median nerve. In April 2021, a 30-year-old male patient was secondly admitted to our hospital and underwent his third surgery, due to the recurrence of a mass and pain in the right palm, noticeable swelling and numbness of the right index and ring fingers, and limited flexion and extension activities of the right ring finger. He first visited our hospital in December 2017 due to a mass and pain in the right palm and swelling and numbness of the right index and ring fingers. When the clinician asked for the patient medical history, his parents stated that his right middle finger was swollen after birth. When the patient was ten years old; he was diagnosed with "macrodactyly" at the local county hospital, not in our hospital, and subsequently, the middle finger was amputated at the metacarpophalangeal joint level at the local county hospital. The postoperative pathological examination was not performed at that time, which was the first surgery the patient received. FLH is clinically rare, and its exact epidemiology and etiology are poorly understood. FLH is highly suspected in cases where a painless mass is present in the wrist, combined with macrodactyly. Magnetic resonance imaging and pathological examination are helpful in clarifying the diagnosis. Although FLH is a benign tumor, an individual treatment plan is the best choice according to the severity of the patient's symptoms. Therefore, further exploration and understanding of this disease by clinicians radiologists, and pathologists is necessary.

Resumen El hamartoma fibrolipomatoso (FLH) del nervio, también conocido como lipomatosis del nervio, lesión neurofibrilar lipomatosa, o lipointraneural, es un tumor benigno de tejido blando poco frecuente, que se presenta principalmente en los nervios del miembro superior, especialmente en el nervio mediano. En abril de 2021, un paciente masculino de 30 años fue ingresado por segunda vez en nuestro hospital y sometido a su tercera cirugía debido a la recurrencia de una masa y dolor en la palma derecha, evidente hinchazón y entumecimiento de los dedos índice y anular derecho y limitadas actividades de flexión y extensión del dedo anular derecho. En diciembre de 2017, visitó por primera vez nuestro hospital debido a una masa y dolor en la palma derecha, y a la hinchazón y entumecimiento de los dedos índice y anular derecho. Cuando el clínico preguntó la historia clínica del paciente, sus padres declararon que su dedo medio derecho estaba hinchado después del nacimiento, y cuando el paciente tenía 10 años, fue diagnosticado con "macrodactilia" en el hospital local del condado, no en nuestro hospital Posteriormente, el dedo medio fue amputado a nivel de la articulación metacarpofalángica en el hospital comarcal local, pero no se realizó la patología postoperatoria en ese momento, siendo ésta la primera cirugía a la cual se sometió el paciente. La FLH es clínicamente rara, y su epidemiología y etiología exactas no se entienden bien. En los casos que presentan una masa indolora en la muñeca, combinada con macrodactilia, se sospecha de FLH. La resonancia magnética y la patología son útiles para aclarar el diagnóstico. Aunque la FLH es un tumor benigno, el plan de tratamiento individual es la mejor opción de acuerdo con la gravedad de los síntomas del paciente. Por lo tanto, es necesaria una mayor exploración y comprensión de esta enfermedad por parte de médicos, radiólogos y patólogos.

Surveillance results of diarrhea cases in Minhang District, Shanghai during 2014-2016 / 上海预防医学

Objective To investigate the etiology and epidemiological characteristics of diarrhea in Minhang District of Shanghai from 2014 to 2016, so as to provide scientific basis to monitor diarrhea disease and to control cluster epidemics. Methods The diarrhea cases in monitoring hospitals from 2014 to 2016 were analyzed by epidemiological methods.Stool specimens were collected using systematic sampling for etiological examination.All specimens were tested for cholera, Shigella, salmonella, Vibrio parahaemolyticus, rotavirus, norovirus and enteric adenovirus. Results Totally 11 742 cases of diarrhea were reported in Minhang District of Shanghai from 2014 to 2016.The majority of the patients were young and middle aged adults.885 stool samples were detected for pathogen, 454 cases (51.30%) were pathogen positive including 57 cases of bacterial and virus co-infection.The detection rates of bacteria and viruses were respectively 10.36%(92/454)and 34.46%(305/454).Three kinds of bacteria and five kinds of viruses were detected, that were mainly Vibrio parahaemolyticus (n=72), salmonella (n=19), norovirus (n=195) and rotavirus A (n=71).The positive rate of pathogens reached the peak from January to March.There was a significant difference in pathogen positive rate between different seasons, while no difference between different years, gender and age groups.The epidemic peaks of different pathogens were different, with bacteria in summer and virus in winter and spring. Conclusion The positive rate of pathogens that caused diarrhea in Minhang District shows a significant seasonal peak.Norovirus, Vibrio parahaemolyticus and rotavirus A are the three main pathogens.It is necessary to take effective measures for diarrhea disease control according to the epidemiological characteristics.

Simultaneous Determination of Furan and Vinyl Acetate in Vapor Phase of Mainstream Cigarette Smoke by GC-MS

ABSTRACT A simple and sensitive method for simultaneous determination of furan and vinyl acetate (VA) in vapor phase of mainstream cigarette smoke with cold trap and gas chromatography-mass spectrometry (GC-MS) was developed. A Cambridge filter pad (CFP) was placed in front of the impingers of smoking machine to remove the particle phase from cigarette smoke. Furan and VA in vapor phase of mainstream cigarette smoke were collected in two impingers connected in series by filled with methanol at -78°C. The solutions were added with deuterium-labeled furan-d4 and VA-d6 as internal standards and analyzed by GC-MS. The results showed that the calibration curves for furan and VA were linear (r2 > 0.9995) over the studied concentration range. The intra- and inter-day precision values for furan and VA were <7.07% and <9.62%, respectively. The extraction recoveries of furan and VA were in the range of 94.5-97.7% and 92.3-94.9%, respectively. Moreover, the limits of detection for furan and VA were 0.028 µg mL-1 and 1.3 ng mL-1, respectively. The validated method has been successfully applied to determine the emissions of furan and VA in the vapor phase of mainstream cigarette smoke under International Organization for Standardization (ISO) and Canadian Intense (CI) smoking regimen.

HPLC determination of the content and dissolution of isophenylcyclopentylamine hydrochloride capsules / 国际药学研究杂志

Objective To establish a method for the determination of the content and dissolution of isophenylcyclopentyl?amine hydrochloride capsules. Methods The HPLC analysis was performed on a Diamonsil C18 column(150 mm×4.6 mm,5μm). A mixture of 0.02 mol/L KH2PO4 solution containing 0.1%triethylamine with pH adjusted to 3.0 by phosphoric acid-methanol-acetonitrile (30:35:35)was used as the mobile phase with the flow rate at 1.0 ml/min. The detection wavelength was 224 nm. Dissolution was de?termined by the basket method,using the 500 ml of 0.1 mol/L hydrochloric acid solution,pH 4.5 acetic acid buffer,water and pH 6.8 phosphoric acid buffer as dissolution media under the 50,75 and 100 r/min rotation speeds to select the dissolution condition. Re?sults This method had high specificity. The linear range for the quantitative determination was 20.74-155.58 μg/ml(r=1.0000), and the average recovery was 100.1%. The linear range for the determination of dissolution was 2.08-24.90μg/ml(r=0.9998),with the average recovery of 98.9%. The method of dissolution tests was established:0.1 mol/L hydrochloric acid solution was used as disso?lution medium and rotation speed was 50 r/min. The determined content and dissolution of three batches of capsules fulfilled the re?quirements. Conclusion The method is simple,accurate and reproducible for the determination of the content and dissolution of iso?phenylcyclopentylamine hydrochloride capsules.

Echocardiographic diagnosis of infracardiac total anomalous pulmonary venous connection / 北京大学学报(医学版)

Objective:To investigate the clinical usefulness of echocardiography in the diagnosis of infracardiac total anomalous pulmonary venous connection (ITAPVC) in neonates and infants.Methods:Retrospective analysis on 8 patients with ITAPVC was performed using echocardiography between April 2006 and December 2016.There were 4 boys and 4 girls with a mean age of 79.8 days (ranging from 15 to 195 days).A combined scanning via parasternal,subcostal and apical acoustic windows had been employed to diagnose ITAPVC and to trace the course and site of the anomalous pulmonary venous drainage,and to confirm the direction of the inter-atrial shunt and enlargement of right atrium and right ventricle.Results:Of the 8 patients who received echocardiography,ITAPVC was diagnosed in 7 patients.Misdiagnosis by echocardiography was encountered in one patient.The diagnosis by echocardiography was compatible with the operative findings in 5 patients receiving surgery and with the results of multislice computed tomography in 6 patients.The diagnostic accuracy rate of ITAPVC was 87.5％.The indirect signs obtained from echocardiogram was coexistence of a small malformed,triangle-shaped left atrium and right to left shunting at atrial level with dilatation and tortuousness of portal vein or hepatic vein and abundant blood flow in liver.The direct signs was total pulmonary veins unconnected with left atrium,whose confluence joining into vertical vein drained right-inferiorly to portal vein or hepatic vein through diaphragm.Three parallel vessels including vertical vein,abdominal aorta and inferior vena cava arranged anteriorly,left-posteriorly and right-posteriorly with the opposite flow directions of inferior vena cava and the other two were found on sub-costal view.Sites of the drainage to the infra-diaphragm veins located portal vein in 8 patients.Stenosis of site of vertical vein connecting to portal vein or hepatic veins occurred in 3 patients.Conclusion:Echocardiography has significant value in the diagnosis of pediatric ITAPVC and is capable of providing important structural and hemodynamic information for preoperative assessment of surgery.With multiple windows and multiple sections,ITAPVC could be diagnosed accurately by echocardiography.However,it is necessary to differentiate ITAPVC with intrahepatic portosystemic venous shunts or hepatic arteriovenous fistula.

Progress on the role of Toll-like receptors in Candida albicans infections / 浙江大学学报·医学版

Toll like receptors (TLRs) are expressed mainly on innate immunocytes such as dendritic cells and macrophages, and may have the potential to recognize and bind to pathogen-associated molecular patterns (PAMPs) from Candida albicans, thereby triggering the downstream signals. The genetic polymorphism of TLRs is associated with susceptibility to Candida albicans. The activation of TLRs by PAMPs from Candida albicans can induce the production of proinflammatory cytokines that play key roles in the anti-infection of Candida albicans. However, in order to evade the immune response of host,Candida albicans can also change its bacterial phase. Understanding of the interaction between TLRs and Candida albicans will provide novel evidence to further clarify the mechanisms of anti-fungal immune response.

The construction of HCV multi-CTL epitopes dendritic cell vaccine and its effect of stimulating T cell response in vitro / 临床肝胆病杂志

@#Objective To construct dendritic cell(DC) vaccine expressing hepatitis C virus(HCV) multi-cytotoxic T lymphocyte(CTL) epitopes,which can stimulate T-cell response in vitro,providing certain information for immunization experiments in vivo for the next step.Methods DCs were infected with recombinant defective adenoviruses expressing two HCV CTL epitopes with tagged green fluorescent protein(GFP).The rate of infection was detected by the fluorescence microscope or flow cytometry;the expression of multiple CTL epitopes in DC was proved by RT-PCR and Western Blot.The cell surface markers of DC such as CD80,CD83,CD86 and HLA-DR were identified by flow cytometry.T cell proliferation effect promoted by DC was observed by cell counting kit-8(CCK-8).IL-12p70 in DC culture supernatant and IFN-γ in T cell supernatant were detected by Enzyme-Linked Immuno Sorbent Assay(ELISA).HCV specific CTL activity was measured by LDH release assay.Results Recombinant multi-CTL epitopes and GFP were successfully expressed in DC.Adenovirus can promote DC maturation,the percentages of CD80,CD83,CD86 and HLA-DR were(71.19±3.29)%,(81.21±5.07)%,(91.23±4.24)%,(97.95±5.31)% respectively.Infected DC promotes homologous T cell proliferation and the stimulation index was 6.806±0.247 when DC:T was 1:10.The secretion of IL-12 and IFN-γ was also increased to(193.83±6.25) pg/ml and(111.14±2.09) pg/ml respectively.The CTL stimulated by infected DC could specifically kill Huh-7.5 cells transfected with FL-J6/JFH transcripts.The cytotoxicity was 35.99% when the rate of effector to target was 100:1.Conclusion The multi-CTL epitopes recombinant adenovirus can effectively infect DC in vitro,promoting the T cell immune response,laying the foundation for developing anti-HCV DC vaccine.

Mitochondrial ultrastructure & release of proteins during liver regeneration.

BACKGROUND & OBJECTIVES: It has been reported that some proteins are released from mitochondria during liver regeneration after partial hepatectomy (PH), but the relationship between proteins release and mitochondrial permeability transition (MPT) remains unclear. We undertook this study to demonstrate the changes of mitochondrial ultrastructure and proteins release during liver regeneration and to determine the relationship between proteins release and MPT in liver regeneration in rats. METHODS: After PH and administration of cyclosporin-A (CsA, a specific inhibitor of MPT), ultrastructural morphology of mitochondria in the remnant liver were determined by electron microscopy. Catalytic activity of mitochondrial and cytosolic proteins including aspartate aminotransferase (AST) and glutamic acid dehydrogenase (GDH) was measured. RESULTS: The liver mitochondria at 24 and 72 h were quite variable in morphology and ultrastructure. The enzyme activities of AST and GDH in cytosol released from mitochondrial matrix changed significantly at 24 and 72 h. CsA can inhibit the permeability of mitochondria partly at the same time. INTERPRETATION & CONCLUSIONS: The changes of mitochondria in ultrastructure reflected the feature of MPT, and the changes of enzymes activities released from mitochondrial matrix were consistent with those of mitochondrial ultrastructure. CsA can inhibit these changes to some extent. There was a close relationship of MPT with mitochondrial ultrastructure and proteins release during liver regeneration.

Therapeutic effect of autologous cytokine-induced killer cells on patients with liver cirrhosis caused by HBV infection / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To observe the therapeutic effect of autologous cytokine-induced killer cells (CIK) on HBV DNA positive patients with liver cirrhosis.</p><p><b>METHODS</b>HBV DNA positive 33 patients with cirrhosis were treated with CIK. Before and after cultured in vitro and post-treatment, CD3+, CD3+CD4+, CD3+CD8+, CD3+CD56+ cells, mDC and pDC were detected by flow cytometry. The indexes of virus and liver function were compared between pre- and post-treatment.</p><p><b>RESULTS</b>CD3+, CD3+CD8+ cells and CD3+CD56+ cells were higher after cultured in vitro and after transfused back than those before culture (91.5 +/- 10.3, 74.4 +/- 9.9 vs. 67.9 +/- 12.8; 60.9 +/- 15.5, 37.3 +/- 15.1 vs. 27.9 +/- 10.9; 18.4 +/- 11.7, 14.5 +/- 7.5 vs. 10.6 +/- 7.1). The percentages of mDC and pDC also increased after-treatment vs. pre-treatment (0.54 +/- 0.18 vs. 0.70 +/- 0.29; 0.26 +/- 0.13 vs. 0.41 +/- 0.25). HBV DNA became undetectable in 12 patients and decrease exceeded 100 times in 4 patients after treatment. HBeAg became undetectable in 10 of 14 patients who were HBeAg positive pretreatment patients, among them 2 patients had HBeAb sero conversion. The liver function was improved after treatment. All patients tolerated the treatment.</p><p><b>CONCLUSION</b>CIK treatment can increase immune effector cells and has some antiviral effect and is safe.</p>

Growth inhibition of human hepatocellular carcinoma xenograft in nude mice by combined treatment with human cytokine-induced killer cells and chemotherapy / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To compare the inhibitory effects of cytokine-induced killer (CIK) cells alone, chemotherapeutic drug alone, and CIK cells combined with chemotherapeutic drug on the growth of hepatocellular carcinoma (HCC) cells transplanted in nude mice.</p><p><b>METHODS</b>Peripheral blood mononuclear cells (PBMC) collected from five healthy donors by blood cell separator were incubated in vitro to induce CIK cells in the presence of interferon-gamma (IFN-gamma), IL-2 and anti-CD3 monoclonal antibody (mAb). The phenotype of CIK cells was characterized by flow cytometric analysis. BEL-7402 HCC cells were inoculated subcutaneously to nude mice. On day 5, at the inoculation site were injected normal saline (group 1), CIK cells (3 x 10(7) and 6 x 10(7), group 2 and 3), mitomycin-C (MMC 80 microg in 0.2 ml, group 4), and CIK cells combined with MMC (group 5), respectively.</p><p><b>RESULTS</b>The percentage of CD3(+), CD3(+)CD8(+), CD3(+)CD56(+), CD25(+) cells increased from 64.0%, 28.0%, 7.8%, and 9.1% to 94.7%, 67.7%, 61.3%, and 84.0% respectively after cytokine induction. The percentage of CD3(+) and CD3(+)CD8(+) cells remained at high levels during incubation period, but that of CD25(+) and CD3(+)CD56(+) cells peaked respectively on day 7 and 13 and then declined. During the 90-day observation, the tumor formation rates were 100%, 70.0%, 80.0%, 70.0% and 66.7%; and the mouse survival rates were 10.0%, 60.0%, 40.0%, 50.0% and 75.0%, respectively from group 1 to group 5. Compared to the other groups, in the combined therapy group of mice, not only the tumor grew slowly and but also showed more marked tissue necrosis.</p><p><b>CONCLUSION</b>The growth inhibitory effect on human HCC transplanted in nude mice of combined CIK cells and MMC treatment is more potent than that of CIK cells or MMC alone.</p>

Single nucleotide polymorphism loci of HIV-1 coreceptor CCR5 gene in Chinese Han people / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the single nucleotide polymorphism(SNP) loci of HIV-1 coreceptor CCR5 gene in Chinese Han people.</p><p><b>METHODS</b>The coding region of CCR5 was amplified using 2 pairs of primers and the PCR products of all 42 healthy subjects were sequenced by 4 different primers. The results of sequencing were analyzed by DNAstar in search of SNP loci.</p><p><b>RESULTS</b>Six SNP loci were discovered in the coding region of CCR5, among them four SNPs, i.e. 184A-->G, 503G-->T, 688G-->A and 999G-->T, cause amino acids changes and two SNPs are nonsense mutations. One cytosine deletion at the 894nt results in frame shift mutation and prematured termination. 184A-->G, 503G-->T and 999G-->T were found in Chinese Han people for the first time. The allelic frequencies of mutant 184G, 503T and 999T alleles were 1.1%, 21.1% and 10.0% in healthy Hans, respectively. The population distribution of G503T markedly deviated from Hardy-Weinberg equilibrium.</p><p><b>CONCLUSION</b>The SNP loci in the coding region of CCR5 in Chinese Han people has its own characteristics, which is not consistent with those of Japanese and obviously different from those of Caucasian and African.</p>

Polymorphism of CXCR4 coding region of human immunodeficiency virus-1 in Chinese Han people / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the polymorphism of human immunodeficiency virus (HIV)-1 coreceptor CXCR4 in Chinese Han ethnic group for AIDS prevention and treatment.</p><p><b>METHODS</b>Totally 48 individuals were enrolled into the study. CXCR4 (cDNA No-AF147204) was cloned by PCR amplification using 2 pairs of primers, then sequenced using sequencing primers. The results of the same sequencing primers were analyzed by DNAstar software to find and identify single nucleotide polymorphism (SNP) sites.</p><p><b>RESULTS</b>Totally 7 SNPs were found in the coding region of CXCR4, among them 3 were synonymous mutation (C-->T at loci 129, 426 and 968), 3 were missense mutation (C-->T at locus 38, A-->T at locus 90, and A-->C at locus 712) and 1 was stop mutation (C-->T at 106, which converted the codon for glutamic acid into stop codon).</p><p><b>CONCLUSIONS</b>The polymorphism of CXCR4 coding region in Chinese Han is probably different from that of the other ethnic groups. Six of the 7 SNPs were discovered for the first time. Their influences on AIDS progression are worthy of studying.</p>

Distribution of HIV-1 coreceptor CX3CR1 allelic polymorphisms in general population, HIV-1 high-risk group and HIV-1 carriers of Chinese indigenous Han and Uygur people / 中华流行病学杂志

<p><b>OBJECTIVE</b>To investigate the V249I and T280M allelic polymorphisms of human immunodeficiency virus (HIV) coreceptor CX3CR1 in HIV-1 infected and uninfected population of Chinese indigenous Han and Uygur people and to probe the association between I249-M280 haplotype and HIV-1 susceptibility as well as AIDS progression.</p><p><b>METHODS</b>Genomic DNA of 223 Uygur subjects and 316 Han subjects were purified from PBMC. I249 and M280 allelic frequencies were identified by polymerase chain reaction (PCR)/nest polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. All data were tested by chi(2) or u statistics analysis.</p><p><b>RESULTS</b>Allelic frequencies of I249 and M280 were 16.1% and 13.3% in Uygur people, and 3.3% and 2.4% in Han people. No obvious difference existed between three groups of either ethnic group. However the allelic frequencies of HIV infected population were higher than those of general population, and those of general population higher than those of HIV-1 high-risk group. There was a strong linkage between I249 and M280 (P almost zero).</p><p><b>CONCLUSIONS</b>I249 mutation was the sine qua non of M280 mutation, and most I249 alleles were accompanied by M280. The frequency of I249-M280 haplotype in Uygur population (13.3%) was adjacent to Caucasian people (15.8%), and that of I249-T280 haplotype (2.8%) was obviously lower than Caucasian people (12.5%); while both of them in Han people were much lower (0.9% and 2.4%). I249-M280 haplotype could accelerate AIDS progression according to Faure et al, while might be associated with HIV-1 susceptibility.</p>