A multicenter single-arm trial of neoadjuvant pyrotinib and trastuzumab plus chemotherapy for HER2-positive breast cancer.

The objective of this multicenter, single-arm trial (ChiCTR1900022293) was to explore the efficacy and safety of neoadjuvant therapy with epirubicin, cyclophosphamide, and pyrotinib followed by docetaxel, trastuzumab, and pyrotinib (ECPy-THPy) in the treatment of patients with stage II-III HER2-positive breast cancer. The present study enrolled patients with stage II-III HER2-positive breast cancer. Epirubicin and cyclophosphamide were administrated for four 21-day cycles, followed by four cycles of docetaxel and trastuzumab. Pyrotinib was taken orally once per day throughout the treatment period. The primary endpoint was total pathological complete response (tpCR, ypT0/is ypN0) rate in the modified intention-to-treat (mITT) population. In total, 175 patients were included. The tpCR rate was 68.6% (95% CI, 60.7-75.8%), while the objective response rate was 89.1%. In the post-hoc subgroup analysis, no association between clinical characteristics and the tpCR rate was observed. The most common grade ≥3 adverse events were diarrhea (54.3%), followed by white blood cell count decreased (5.1%), and neutrophil count decreased (4.6%). In conclusion, the neoadjuvant regimen with ECPy-THPy showed promising pathological response and clinical benefits with an acceptable safety profile in patients with stage II-III HER2-positive breast cancer.

The loss of function GBA1 c.231C > G mutation associated with Parkinson disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease characterized by bradykinesia, rigidity, and tremor. However, familial PD caused by single-gene mutations remain relatively rare. Herein, we described a Chinese family affected by PD, which associated with a missense heterozygous glucocerebrosidase 1 (GBA1) mutation (c.231C > G). Clinical data on the proband and her family members were collected. Brain MRI showed no difference between affected and unaffected family members. Whole-exome sequencing (WES) was performed to identify the pathogenic mutation. WES revealed that the proband carried a missense mutation (c.231C > G) in GBA1 gene, which was considered to be associated with PD in this family. Sanger sequencing and co-segregation analyses were used to validate the mutation. Bioinformatics analysis indicated that the mutation was predicted to be damaging. In vitro functional analyses were performed to investigated the mutant gene. A decrease in mRNA and protein expression was observed in HEK293T cells transfected with mutant plasmids. The GBA1 c.231C > G mutation caused a decreased GBA1 concentration and enzyme activity. In conclusion, a loss of function mutation (c.231C > G) in GBA1 was identified in a Chinese PD family and was confirmed to be pathogenic through functional studies. This study help the family members understand the disease progression and provide a new example for studying the pathogenesis of GBA1-associated Parkinson disease.

[Analysis of clinical characteristics and prognostic risk factors in patients with Stanford type B aortic dissection complicated with hypertension].

OBJECTIVE: To investigate the clinical characteristics as well as short-term and long-term prognostic factors of patients with Stanford type B aortic dissection (TBAD) with hypertension. METHODS: Patients with TBAD who received thoracic endovascular aortic repair (TEVAR) admitted to Xiangyang Central Hospital from January 2014 to December 2018 were enrolled. The baseline data of patients admitted to the hospital were collected through the case management system, including gender, age, underlying diseases (hypertension, diabetes, coronary heart disease), smoking history, drinking history, duration of pain, vital signs at admission [heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP)], laboratory results [white blood cell count (WBC), platelet count (PLT), neutrophil/lymphocyte ratio (NLR), serum creatinine (SCr), C-reactive protein (CRP), D-dimer, ascending aorta diameter], etc. The clinical characteristics of TBAD patients with hypertension were analyzed. Logistic regression model and Cox proportional risk model were used to analyze the impact of hypertension on the short-term and long-term all-cause deaths after TEVAR in TBAD patients. RESULTS: Among 227 TBAD patients, 160 cases (70.5%) were complicated with hypertension, while 67 cases (29.5%) were not. The average age, the proportion of diabetes and coronary heart disease, and the level of SBP, DBP and SCr at admission of TBAD patients with hypertension were higher than those of TBAD without hypertension [age (years old): 53.1±11.9 vs. 42.8±14.1, combined with diabetes: 8.8% vs. 1.5%, combined with coronary heart disease: 6.3% vs. 0%, SBP (mmHg, 1 mmHg = 0.133 kPa): 147.9±18.1 vs. 136.9±15.2, DBP (mmHg): 93.9±11.9 vs. 89.1±13.8, SCr (µmol/L): 97.8±25.4 vs. 89.8±23.6, all P < 0.05]. The short-term mortality of TBAD with hypertension group was significantly higher than that of TBAD without hypertension group [6.3% (10/160) vs. 0% (0/67), χ2 = 4.386, P = 0.036]. 227 patients with TBAD were followed up for 3-66 months, with a median follow-up time of 25 months. There was no significant difference in long-term mortality between TBAD patients with and without hypertensive during discharge follow-up [13.1% (21/160) vs. 9.0% (6/67), χ2 = 0.784, P = 0.376]. Further multivariate Logistic regression analysis and Cox regression analysis did not indicate that hypertension was an independent risk factor for short-term and long-term death in TBAD patients [odds ratio (OR) and 95% confidence interval (95%CI) were 13.477 (0.541-330.215), 1.012 (0.990-1.035), both P > 0.05]. Age and HR were independent risk factors for the short-term mortality of TBAD patients [OR and 95%CI were 15.287 (1.051-226.415), 0.026 (0.002-0.840), both P < 0.05]. Age, PLT and D-dimer were independent risk factors for the long-term mortality of TBAD patients [OR and 95%CI were 1.808 (1.205-2.711), 0.555 (0.333-0.924), 1.482 (1.035-2.122), respectively, all P < 0.05]. CONCLUSIONS: The TBAD patients with hypertension have older age, high rates of diabetes or coronary heart disease. However, hypertension is not an independent risk factor for short-term and long-term mortality in TBAD patients.

A novel biosensor for the ultrasensitive detection of the lncRNA biomarker MALAT1 in non-small cell lung cancer.

Long non-coding RNAs (lncRNAs) have been proposed as diagnostic biomarkers for the screening of non-small cell lung cancer and monitoring disease progression. Accordingly, new, rapid, and cost-effective lncRNA biosensors that can be used clinically are urgently needed. Herein, a novel effective and ultrasensitive electrochemical biosensor was developed based on a gold nanocage coupled with an amidated multi-walled carbon nanotube (Au NCs/MWCNT-NH2)-decorated screen-printed carbon electrode (SPCE). Because of its large surface area, superior conductivity, and excellent biocompatibility, this SPCE Au NCs/MWCNT-NH2 lncRNA biosensor showed a wide linear range (10-7-10-14 M) and low limit of detection limit (42.8 fM) coupled with satisfactory selectivity and stability. Compared to traditional RT-PCR, the proposed method exhibits acceptable stability, good selectivity, is simpler to operate, has faster detection, and uses less costly raw materials. In summary, this biosensor may be a powerful tool for detecting lncRNAs for efficient clinical prognosis and cancer diagnosis.

CRISPR/Cas9 cleavage triggered ESDR for circulating tumor DNA detection based on a 3D graphene/AuPtPd nanoflower biosensor.

Circulating tumor DNA (ctDNA) plays an important role in the early diagnosis and prognosis of several cancers and is a credible biomarker for predicting the response to therapy. Additionally, the fact that the strategy used to detect ctDNA is non-invasive also adds to the advantages of using ctDNA for predicting disease diagnosis and prognosis. However, low abundance in peripheral blood and the high background of wild-type DNA impair the precise and specific measurement of ctDNA. In this study, we developed a novel 3D GR/AuPtPd nanoflower sensing platform based on CRISPR/Cas9 cleavage-triggered entropy-driven strand displacement reaction (ESDR) for the effective detection of ctDNA. Low levels of ctDNA could be detected using this method as the ESDR amplification does require complicated operation procedures and stringent reaction conditions. By combining the advantages of the site-specific cleavage by "gene magic scissors," Cas9/sgRNA, with those of the rapid amplification kinetics of entropy-driven strand displacement, our method resulted in amplification efficiency as well as high specificity for discriminating single-nucleotide mismatches. The 3D GR/AuPtPd nanoflower-based electrochemical biosensor displayed high specificity and worthy performance in assays with human serum. Therefore, this pioneered method provides a new paradigm for efficient ctDNA detection and shows great potential for use in clinical and diagnostic applications.

Inhibition of mitochondrial respiration by tigecycline selectively targets thyroid carcinoma and increases chemosensitivity.

The role of mitochondria in cancer and mitochondria-targeted therapy has been gaining attention for its effectiveness and selectivity between cancer and normal cells. In line with this notion, our work demonstrates that inducing mitochondrial dysfunction by tigecycline, a FDA-approved antibiotic, selectively targets thyroid cancer and enhances chemosensitivity. We found that tigecycline inhibited proliferation and induced apoptosis in a panel of thyroid cancer cell lines. Consistently, tigecycline inhibited thyroid cancer growth in mice without causing significant toxicity. The combination of tigecycline with paclitaxel achieved greater efficacy than paclitaxel alone in vitro and in vivo. Mechanistically, tigecycline inhibited mitochondrial respiration and ATP reduction through decreasing mitochondrial membrane potential and inhibiting mitochondrial translation, leading to oxidative stress and damage. In contrast, tigecycline was ineffective in mitochondrial respiration-deficient cells, confirming that tigecycline acts on thyroid cancer via inhibiting mitochondrial respiration. Interestingly, although tigecycline inhibited mitochondrial respiration in both thyroid cancer and normal thyroid cells in a similar manner, tigecycline was more effective in thyroid cancer than normal thyroid cells, suggesting that thyroid cancer cells are more dependent on mitochondrial functions than normal thyroid cells. This was supported by our observations that thyroid cancer cells had higher level of mitochondrial biogenesis compared to normal thyroid cells. Our work is the first to demonstrate that the combination of chemotherapy with tigecycline is a potential sensitizing strategy for thyroid cancer treatment. Our findings also highlight the higher dependence of thyroid cancer cells on mitochondrial functions than normal thyroid cells.

Mechanisms of cannabinoid CB2 receptor-mediated reduction of dopamine neuronal excitability in mouse ventral tegmental area.

BACKGROUND: We have recently reported that activation of cannabinoid type 2 receptors (CB2Rs) reduces dopamine (DA) neuron excitability in mouse ventral tegmental area (VTA). Here, we elucidate the underlying mechanisms. METHODS: Patch-clamp recordings were performed in mouse VTA slices and dissociated single VTA DA neurons. FINDINGS: Using cell-attached recording in VTA slices, bath-application of CB2R agonists (JWH133 or five other CB2R agonists) significantly reduced VTA DA neuron action potential (AP) firing rate. Under the patch-clamp whole-cell recording model, JWH133 (10 µM) mildly reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) but not miniature inhibitory postsynaptic currents (mIPSCs). JWH133 also did not alter evoked EPSCs or IPSCs. In freshly dissociated VTA DA neurons, JWH133 reduced AP firing rate, delayed AP initiation and enhanced AP after-hyperpolarization. In voltage-clamp recordings, JWH133 (1 µM) enhanced M-type K+ currents and this effect was absent in CB2-/- mice and abolished by co-administration of a selective CB2R antagonist (10 µM, AM630). CB2R-mediated inhibition in VTA DA neuron firing can be mimicked by M-current opener (10 µM retigabine) and blocked by M-current blocker (30 µM XE991). In addition, enhancement of neuronal cAMP by forskolin (10 µM) reduced M-current and increased DA neuron firing rate. Finally, pharmacological block of synaptic transmission by NBQX (10 µM), D-APV (50 µM) and picrotoxin (100 µM) in VTA slices failed to prevent CB2R-mediated inhibition, while intracellular infusion of guanosine 5'-O-2-thiodiphosphate (600 µM, GDP-ß-S) through recording electrode to block postsynaptic G-protein function prevented JWH133-induced reduction in AP firing. INTERPRETATION: Our results suggest that CB2Rs modulate VTA DA neuron excitability mainly through an intrinsic mechanism, including a CB2R-mediated reduction of intracellular cAMP, and in turn enhancement of M-type K+ currents. FUND: This research was supported by the Barrow Neuroscience Foundation, the BNI-BMS Seed Fund, and CNSF (81771437).

Cocaine Directly Inhibits α6-Containing Nicotinic Acetylcholine Receptors in Human SH-EP1 Cells and Mouse VTA DA Neurons.

Alpha6-containing nicotinic acetylcholine receptors are primarily found in neurons of the midbrain dopaminergic (DA) system, suggesting these receptors are potentially involved in drug reward and dependence. Here, we report a novel effect that cocaine directly inhibits α6N/α3Cß2ß3-nAChR (α6*-nAChRs) function. Human α6*-nAChRs were heterologously expressed within cells of the SH-EP1 cell line for functional characterization. Mechanically dissociated DA neurons from mouse ventral tegmental area (VTA) were used as a model of presynaptic α6*-nAChR activation since this method preserves terminal boutons. Patch-clamp recordings in whole-cell configuration were used to measure α6*-nAChR function as well as evaluate the effects of cocaine. In SH-EP1 cells containing heterologously expressed human α6*-nAChRs, cocaine inhibits nicotine-induced inward currents in a concentration-dependent manner with an IC50 value of 30 µM. Interestingly, in the presence of 30 µM cocaine, the maximal current response of the nicotine concentration-response curve is reduced without changing nicotine's EC50 value, suggesting a noncompetitive mechanism. Furthermore, analysis of whole-cell current kinetics demonstrated that cocaine slows nAChR channel activation but accelerates whole-cell current decay time. Our findings demonstrate that cocaine-induced inhibition occurs solely with bath application, but not during intracellular administration, and this inhibition is not use-dependent. Additionally, in Xenopus oocytes, cocaine inhibits both α6N/α3Cß2ß3-nAChRs and α6M211L/α3ICß2ß3-nCAhRs similarly, suggesting that cocaine may not act on the α3 transmembrane domain of chimeric α6N/α3Cß2ß3-nAChR. In mechanically isolated VTA DA neurons, cocaine abolishes α6*-nAChR-mediated enhancement of spontaneous inhibitory postsynaptic currents (sIPSCs). Collectively, these studies provide the first evidence that cocaine directly inhibits the function of both heterologously and naturally expressed α6*-nAChRs. These findings suggest that α6*-nAChRs may provide a novel pharmacological target mediating the effects of cocaine and may underlie a novel mechanism of cocaine reward and dependence.

Alpha6-containing nicotinic acetylcholine receptor is a highly sensitive target of alcohol.

Alcohol use disorder (AUD) is a serious public health problem that results in tremendous social, legal and medical costs to society. Unlike other addictive drugs, there is no specific molecular target for ethanol (EtOH). Here, we report a novel molecular target that mediates EtOH effects at concentrations below those that cause legally-defined inebriation. Using patch-clamp recording of human α6*-nicotinic acetylcholine receptor (α6*-nAChR) function when heterologously expressed in SH-EP1 human epithelial cells, we found that 0.1-5 mM EtOH significantly enhances α6*-nAChR-mediated currents with effects that are dependent on both EtOH and nicotine concentrations. EtOH exposure increased both whole-cell current rising slope and decay constants. This EtOH modulation was selective for α6*-nAChRs since it did not affect α3ß4-, α4ß2-, or α7-nAChRs. In addition, 5 mM EtOH also increased the frequency and amplitude of dopaminergic neuron transients in mouse brain nucleus accumbens slices, that were blocked by the α6*-nAChR antagonist, α-conotoxin MII, suggesting a role for native α6*-nAChRs in low-dose EtOH effects. Collectively, our data suggest that α6*-nAChRs are sensitive targets mediating low-dose EtOH effects through a positive allosteric mechanism, which provides new insight into mechanisms involved in pharmacologically-relevant alcohol effects contributing to AUD.

Heterogeneity of cannabinoid ligand-induced modulations in intracellular Ca2+ signals of mouse pancreatic acinar cells in vitro.

We recently reported that a CB2R agonist, GW405833 (GW), reduced both the ACh-induced Ca2+ oscillations and the L-arginine-induced Ca2+ signal enhancement in mouse pancreatic acinar cells, suggesting that GW-induced inhibition may prevent the pathogenesis of acute pancreatitis. In this study, we aim to evaluate the effects of other cannabinoid ligands on Ca2+ signaling in acinar cells. Patch-clamp whole-cell recordings were applied to measure ACh-induced intracellular Ca2+ oscillations in pancreatic acinar cells acutely dissociated from wild-type (WT), CB1R knockout (KO), and CB2R KO mice, and the pharmacological effects of various cannabinoid ligands on the Ca2+ oscillations were examined. We found that all the 8 CB2R agonists tested inhibited ACh-induced Ca2+ oscillations. Among them, GW, JWH133, and GP1a caused potent inhibition with IC50 values of 5.0, 6.7, and 1.2 µmol/L, respectively. In CB2R KO mice or in the presence of a CB2R antagonist (AM630), the inhibitory effects of these 3 CB2R agonists were abolished, suggesting that they acted through the CB2Rs. The CB1R agonist ACEA also induced inhibition of Ca2+ oscillations that existed in CB1R KO mice and in the presence of a CB1R antagonist (AM251), suggesting a non-CB1R effect. In WT, CB1R KO, and CB2R KO mice, a nonselective CBR agonist, WIN55,212-2, inhibited Ca2+ oscillations, which was not mediated by CB1Rs or CB2Rs. The endogenous cannabinoid substance, 2-arachidonoylglycerol (2-AG), did not show an inhibitory effect on Ca2+ oscillations. In conclusion, CB2R agonists play critical roles in modulating Ca2+ signals in mouse pancreatic acinar cells, while other cannabinoid ligands modulate Ca2+ oscillations in a heterogeneous manner through a CB receptor or non-CB-receptor mechanism.

Pharmacological and functional comparisons of α6/α3ß2ß3-nAChRs and α4ß2-nAChRs heterologously expressed in the human epithelial SH-EP1 cell line.

Neuronal nicotinic acetylcholine receptors containing α6 subunits (α6*-nAChRs) show highly restricted distribution in midbrain neurons associated with pleasure, reward, and mood control, suggesting an important impact of α6*-nAChRs in modulating mesolimbic functions. However, the function and pharmacology of α6*-nAChRs remain poorly understood because of the lack of selective agonists for α6*-nAChRs and the challenging heterologous expression of functional α6*-nAChRs in mammalian cell lines. In particular, the α6 subunit is commonly co-expressed with α4*-nAChRs in the midbrain, which masks α6*-nAChR (without α4) function and pharmacology. In this study, we systematically profiled the pharmacology and function of α6*-nAChRs and compared these properties with those of α4ß2 nAChRs expressed in the same cell line. Heterologously expressed human α6/α3 chimeric subunits (α6 N-terminal domain joined with α3 trans-membrane domains and intracellular loops) with ß2 and ß3 subunits in the human SH-EP1 cell line (α6*-nAChRs) were used. Patch-clamp whole-cell recordings were performed to measure these receptor-mediated currents. Functionally, the heterologously expressed α6*-nAChRs exhibited excellent function and showed distinct nicotine-induced current responses, such as kinetics, inward rectification and recovery from desensitization, compared with α4ß2-nAChRs. Pharmacologically, α6*-nAChR was highly sensitive to the α6 subunit-selective antagonist α-conotoxin MII but had lower sensitivity to mecamylamine and dihydro-ß-erythroidine. Nicotine and acetylcholine were found to be full agonists for α6*-nAChRs, whereas epibatidine and cytisine were determined to be partial agonists. Heterologously expressed α6*-nAChRs exhibited pharmacology and function distinct from those of α4ß2-nAChRs, suggesting that α6*-nAChRs may mediate different cholinergic signals. Our α6*-nAChR expression system can be used as an excellent cell model for future investigations of α6*-nAChR function and pharmacology.

Hippocampal synaptic and neural network deficits in young mice carrying the human APOE4 gene.

INTRODUCTION: Apolipoprotein E4 (APOE4) is a major genetic risk factor for late-onset sporadic Alzheimer disease. Emerging evidence demonstrates a hippocampus-associated learning and memory deficit in aged APOE4 human carriers and also in aged mice carrying human APOE4 gene. This suggests that either exogenous APOE4 or endogenous APOE4 alters the cognitive profile and hippocampal structure and function. However, little is known regarding how Apoe4 modulates hippocampal dendritic morphology, synaptic function, and neural network activity in young mice. AIM: In this study, we compared hippocampal dendritic and spine morphology and synaptic function of young (4 months) mice with transgenic expression of the human APOE4 and APOE3 genes. METHODS: Hippocampal dendritic and spine morphology and synaptic function were assessed by neuronal imaging and electrophysiological approaches. RESULTS: Morphology results showed that shortened dendritic length and reduced spine density occurred at hippocampal CA1 neurons in Apoe4 mice compared to Apoe3 mice. Electrophysiological results demonstrated that in the hippocampal CA3-CA1 synapses of young Apoe4 mice, basic synaptic transmission, and paired-pulse facilitation were enhanced but long-term potentiation and carbachol-induced hippocampal theta oscillations were impaired compared to young Apoe3 mice. However, both Apoe genotypes responded similarly to persistent stimulations (4, 10, and 40 Hz for 4 seconds). CONCLUSION: Our results suggest significant alterations in hippocampal dendritic structure and synaptic function in Apoe4 mice, even at an early age.

Brain cannabinoid receptor 2: expression, function and modulation.

Cannabis sativa (marijuana) is a fibrous flowering plant that produces an abundant variety of molecules, some with psychoactive effects. At least 4% of the world's adult population uses cannabis annually, making it one of the most frequently used illicit drugs in the world. The psychoactive effects of cannabis are mediated primarily through cannabinoid receptor (CBR) subtypes. The prevailing view is that CB1Rs are mainly expressed in the central neurons, whereas CB2Rs are predominantly expressed in peripheral immune cells. However, this traditional view has been challenged by emerging strong evidence that shows CB2Rs are moderately expressed and function in specific brain areas. New evidence has demonstrated that brain CB2Rs modulate animal drug-seeking behaviors, suggesting that these receptors may exist in brain regions that regulate drug addiction. Recently, we further confirmed that functional CB2Rs are expressed in mouse ventral tegmental area (VTA) dopamine (DA) neurons and that the activation of VTA CB2Rs reduces neuronal excitability and cocaine-seeking behavior. In addition, CB2R-mediated modulation of hippocampal CA3 neuronal excitability and network synchronization has been reported. Here, we briefly summarize recent lines of evidence showing how CB2Rs modulate function and pathophysiology in the CNS.

Cannabinoid receptor subtype 2 (CB2R) agonist, GW405833 reduces agonist-induced Ca(2+) oscillations in mouse pancreatic acinar cells.

Emerging evidence demonstrates that the blockade of intracellular Ca(2+) signals may protect pancreatic acinar cells against Ca(2+) overload, intracellular protease activation, and necrosis. The activation of cannabinoid receptor subtype 2 (CB2R) prevents acinar cell pathogenesis in animal models of acute pancreatitis. However, whether CB2Rs modulate intracellular Ca(2+) signals in pancreatic acinar cells is largely unknown. We evaluated the roles of CB2R agonist, GW405833 (GW) in agonist-induced Ca(2+) oscillations in pancreatic acinar cells using multiple experimental approaches with acute dissociated pancreatic acinar cells prepared from wild type, CB1R-knockout (KO), and CB2R-KO mice. Immunohistochemical labeling revealed that CB2R protein was expressed in mouse pancreatic acinar cells. Electrophysiological experiments showed that activation of CB2Rs by GW reduced acetylcholine (ACh)-, but not cholecystokinin (CCK)-induced Ca(2+) oscillations in a concentration-dependent manner; this inhibition was prevented by a selective CB2R antagonist, AM630, or was absent in CB2R-KO but not CB1R-KO mice. In addition, GW eliminated L-arginine-induced enhancement of Ca(2+) oscillations, pancreatic amylase, and pulmonary myeloperoxidase. Collectively, we provide novel evidence that activation of CB2Rs eliminates ACh-induced Ca(2+) oscillations and L-arginine-induced enhancement of Ca(2+) signaling in mouse pancreatic acinar cells, which suggests a potential cellular mechanism of CB2R-mediated protection in acute pancreatitis.

Netrin-1 with stem cells promote angiogenesis in limb ischemic rats.

BACKGROUND: Recent findings have elucidated that netrin-1 has ability of promoting angiogenesis besides the functions in nervous system. Autologous mesenchymal stem cells (MSCs) transplantation is now proved to be an effective method to treat peripheral arterial disease. However there are still many patients who cannot complete full treatments. Therefore it is necessary to improve the effectiveness. This study estimated the curative effects in chronic limb ischemia when MSCs allied with netrin-1. MATERIALS AND METHODS: Thirty-six rats were made into chronic limb ischemia models. They were randomly assigned to four groups, netrin-1 + MSCs group (treated with netrin-1 and MSCs derived from peripheral blood), MSCs group (treated with MSCs individually), netrin-1 group (treated with netrin-1 individually), and control group (treated with saline). Measurements of murine behaviors, vascular endothelial growth factor expression, and capillary density in ischemia limb were performed on days 7, 14, and 28 after treatments; measurements of contraction force in ischemia limb was performed on day 28 after treatments to compare differences among the groups. RESULTS: Netrin-1 allied with MSCs significantly increased Tarlov score, vascular endothelial growth factor expression, capillary density, and muscular strength in ischemia limb. CONCLUSIONS: Netrin-1 allied with MSCs derived from peripheral blood significantly promoted angiogenesis in aged rats with chronic limb ischemia. It may be a promising method of treating peripheral arterial disease in the future.

Clinical analysis for the application of endoscopic ultrasonography in the diagnosis of patients with a retroperitoneal space-occupying lesion.

OBJECTIVE: To report our experience and to evaluate the application of endoscopic ultrasonography (EUS) in the qualitative diagnosis of retroperitoneal space-occupying lesions. METHODS: Twenty-six patients with retroperitoneal space-occupying lesions confirmed by CT or MRI were studied. All the patients underwent endoscopic ultrasonography guided fine needle aspiration (EUS-FNA) at the Department of Gastroenterology, Xiangyang Central Hospital, Hubei Province, China from August 2009 to August 2011. Different parameters were evaluated, such as complications of EUS-FNA, the ratio of definite pathological diagnosis, and the pathologic types of all the specimens. RESULTS: Of the 26 patients, 24 had definite pathological diagnosis; the ratio of histodiagnosis was 92.3%. There were no complications such as hemorrhage, infection, or injury to the abdominal viscera in the process of EUS-FNA. Eight patients had benign tumors, with a ratio of 33.3%, and 16 patients had malignant tumors, with a ratio of 66.7%. Two patients had no definite pathological diagnosis because of the shortness of tissue sample. Eight patients did not undergo operation due to the diagnosis of benign tumors. CONCLUSION: The EUS-FNA has the advantage of lower complications, and higher diagnostic ratio, which is valuable in the qualitative diagnosis of retroperitoneal space-occupying lesions.

The relationship among human papilloma virus infection, survivin, and p53 gene in lung squamous carcinoma tissue.

OBJECTIVE: To study the relationship between the infection of human papillomavirus (HPV) type 16, type 18, the expression of survivin, and the mutation of p53 gene in lung squamous carcinoma tissue for the research of pathogenesis of lung carcinoma. METHODS: This study was carried out at the Laboratory of Molecular Biology, Xiangfan Central Hospital of Hubei Province, China from September 2008 to May 2010. Forty-five specimens of lung squamous carcinoma tissue confirmed by histopathology were the excisional specimens taken by the Thoracic Surgery of Xiangfan Central Hospital. Normal tissue, closely adjacent to the fresh carcinoma specimens, was used as the control group for p53 gene mutation analysis. Sixteen surgical excisional specimens of benign lung disease were used as a control group of non-carcinomatous diseases. Human papillomavirus DNA were detected by polymerase chain reaction (PCR), and we used the PCR-single-strand conformation polymorphism-ethidium bromide (PCR-SSCP-EB) method to detect the mutations of the p53 gene. The expression of the survivin gene was detected by immunohistochemistry methods. RESULTS: Approximately 68.9% of 45 lung squamous carcinoma tissue had p53 gene mutations. The mutation rate of exon 5-8 p53 were 15.6%, 17.8%, 15.6% and 20%. Approximately 42.2% of lung squamous cell carcinoma samples were shown to be positive for HPV DNA expression and 62.2% were positive for survivin expression. There was an inverse correlation between the presence of HPV infections and mutations of p53 gene; and the mutations of p53 gene and expression of survivin had a positive relationship. CONCLUSION: Mutation of p53 gene and HPV infection may facilitate each other in the generation of lung squamous cell carcinoma. Abnormal expression of the survivin gene may take part in the onset and progression of lung squamous cell carcinoma.

Construction and expression of anti-CD62P/anti-GPIIb-IIIa diabody.

OBJECTIVE: To construct 3 expression plasmids for the targeted therapy of thrombosis: single chain variable fragment (scFv) of monoclonal antibody (mAb) 7E3 that can identify and bind platelet glycoprotein GPIIb-IIIa complex, scFv of mAb WAPS12.2 that can identify and bind P selectin (CD62P), a diabody that can identify and bind GPIIb-IIIa and CD62P simultaneously, and to investigate whether the vectors can express correctly. METHODS: This study was carried out at the Laboratory of Hunan Yuantai Biological Technology Co. Ltd, Hubei, China from September 2007 to May 2008. Total RNA of mAb 7E3 cells and WAPS12.2 cells were obtained. Reverse transcriptase polymerase chain reaction (PCR) was carried out to obtain the genes of variable regions of light and heavy chains of 7E3 and WAPS12.2. Target genes were named 7E3VL, 7E3VH, CD62PVL, and CD62PVH. The 7E3VL-7E3VH and CD62PVL-CD62PVH were obtained by PCR and connected with pET-22b(+). Products were named pET-scFv7E3 and pET-scFvCD62P. The 7E3VL-CD62PVH and CD62PVL-7E3VH were obtained by using overlap PCR and were ligated to pET-22b(+). The products were named pET-ED1 and pET-ED2. The PCR was performed by taking pET-ED2 as a template to obtain the complete operon gene and was ligated to pET-ED1. The product was named pET-7ECD. RESULTS: The identification by restriction endonuclease cleavage and DNA sequencing confirmed that the construction of these expression plasmids was successful. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot confirmed that these plasmids expressed correctly. CONCLUSION: The expression plasmids pET-scFv7E3, pET-scFvCD62P, and pET-7ECD were constructed and expressed successfully, and laid a good foundation for further research on target-oriented thrombolytic agents.

Construction of humanized carcinoembryonic antigen specific single chain variable fragment and mitomycin conjugate.

AIM: To construct a new target-oriented conjugate of humanized carcinoembryonic antigen (CEA) specific single chain variable fragment (scFv) and mitomycin (MMC) against colorectal cancer, and to investigate its influence on the growth and apoptosis of colorectal cancer cells. METHODS: The primer was designed according to the gene sequence described in reference 16, which respectively contains restriction enzyme cleavage sites BamHI and EcoRI in its upstream and downstream. PCR was performed with the plasmid as template containing genes of humanized anti-CEA scFv. The product was digested by BamHI and EcoRI, and connected to an expression vector which also has the restriction enzyme cleavage sites BamHI and EcoR. Expression of the reaction was induced by isopropy-beta-D-thiogalactoside (IPTG). Then the expression product was covalently coupled with MMC by dextran T-40. The immunoreactivity of the conjugate against colorectal cancer cells as well as CEA was measured by enzyme linked immunosorbent assay (ELISA). The inhibiting ratio of conjugate on the growth of colorectal cancer cells was also measured by ELISA. The effect of conjugate on the apoptosis of colorectal cancer cells was determined by flow cytometry (FCM). RESULTS: Restriction endonuclease cleavage and gene sequencing confirmed that the expression vector was successfully constructed. Sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) confirmed that this vector correctly expressed the fusion protein. ELISA confirmed that the conjugate had quite a strong immunoreactivity against colorectal cancer cells and CEA. The conjugate had inhibitory effects on colorectal cancer cells in a concentration-dependent manner and could induce apoptosis of colorectal cancer cells in a concentration-dependent manner. CONCLUSION: The CEA-scFv-MMC conjugate can be successfully constructed and is able to inhibit the growth and induce apoptosis of colorectal cancer cells.

Construction of adeno-associated virus coexpression system for human angiopoietin-1 and VEGF gene.

BACKGROUND: Ischemic disease is one of the leading causes of death in the world. In order to further study gene therapy for ischemic disease, we constructed a recombinant plasmid for co-expression of human angiopoietin-1 and vascular endothelial growth factor 165(VEGF165) gene in adeno-associated virus (AAV) gene delivery system. METHODS: Human angiopoietin 1 and VEGF165 gene were obtained using PCR. The upstream of angiopoietin 1 contained restriction enzyme site HindIII, and the downstream of angiopoietin 1 contained restriction enzyme site BamHI. The upstream of VEGF165 contained restriction enzyme site BglII, and the downstream of VEGF165 contained restriction enzyme site BamHI. Using the multiple cloning sites (MCS) in plasmid pZero++ such as BamHI, BglII, HindIII, NotI, XhoI, XbaI, SalI, BspHI, KspI and the corresponding MCS in plasmid pAAV-MCS, angiopoietin 1 and VEGF165 gene were subcloned into pAAV-MCS. RESULTS: DNA sequencing revealed that the PCR- amplified angiopoietin 1 and VEGF165 were consistent with NCBI Gene Bank. The recombinant plasmid was identified using PCR and digestion, which proved to be consistent with our hypothesis. In recombinant plasmid, angiopoietin1 and VEGF possessed a CMV promoter and polyA terminator system respectively, thus assuring co-expression of the two genes. CONCLUSION: Successful construction of AAV co-expression system for human angiopoietin 1 and VEGF165 gene will provide the foundation for gene therapy to cure severe ischemic disease.