Features of thromboelastogram in populations exposed to or transferring from high altitude.

Background: Thromboelastogram (TEG) is an effective indicator that monitors the dynamic changes of blood coagulation in real-time. It still remains controversial about the performance and influence of coagulation at high altitude. The present study intends to describe comprehensively the clinical features of TEG in populations exposed to or transferring from high altitude. Methods: Two groups were recruited in the present study. Group A included young males who worked at high-altitude (4888 m or 5418 m) areas for some time, while Group B included young males who had recently returned from high-altitude (4888 m or 5418 m) areas. Medical examinations were performed using portable devices. Spearman's test was used to evaluate the correlations between thromboelastogram (TEG) variables and other variables. Logistic regression analysis was used to analyze the factors affecting various abnormal TEG variables. Results: A total of 51 adult males were included in the two groups. Significantly increased reaction time (R) and decreased maximum amplitude (MA) were found in group B (P < 0.05). No significant differences were observed in the comparisons of K and angle between the two groups. Various TEG variables were identified to be correlated with different coagulation and biochemical variables. Logistic regression analysis demonstrated that abnormal R was independently associated with direct bilirubin, and abnormal K was independently associated with the platelet count in Group A (P < 0.05). However, none of the factors were independently associated with abnormal TEG variables in Group B. Conclusion: Populations exposed to or transferring from high altitudes are characterized by different TEG characteristics. Our findings give a comprehensive description of the complex interaction between TEG indexes, coagulation dynamics, and hematological parameters, which can help guide the development of appropriate medical approaches tailored to the unique needs of these populations.

Short-Chain Fatty Acids Promote Immunotherapy by Modulating Immune Regulatory Property in B Cells.

The dysfunction of regulatory B cells (Breg) may result in immune inflammation such as allergic rhinitis (AR); the underlying mechanism is not fully understood yet. Short-chain fatty acids, such as propionic acid (PA), have immune regulatory functions. This study is aimed at testing a hypothesis that modulates PA production alleviating airway allergy through maintaining Breg functions. B cells were isolated from the blood obtained from AR patients and healthy control (HC) subjects. The stabilization of IL-10 mRNA in B cells was tested with RT-qPCR. An AR mouse model was developed to test the role of PA in stabilizing the IL-10 expression in B cells. We found that the serum PA levels were negatively correlated with the serum Th2 cytokine levels in AR patients. Serum PA levels were positively associated with peripheral CD5+ B cell frequency in AR patients; the CD5+ B cells were also IL-10+. The spontaneous IL-10 mRNA decay was observed in B cells, which was prevented by the presence of PA through activating GPR43. PA counteracted the effects of Tristetraprolin (TTP) on inducing IL-10 mRNA decay in B cells through the AKT/T-bet/granzyme B pathway. Administration of Yupinfeng San, a Chinese traditional medical formula, or indole-3-PA, induced PA production by intestinal bacteria to stabilize the IL-10 expression in B cells, which promoted the allergen specific immunotherapy, and efficiently alleviated experimental AR. In summary, the data show that CD5+ B cells produce IL-10. The serum lower PA levels are associated with the lower frequency of CD5+ B cells in AR patients. Administration with Yupinfeng San or indole-3-PA can improve Breg functions and alleviate experimental AR.

TaWRKY74 participates copper tolerance through regulation of TaGST1 expression and GSH content in wheat.

Glutathione S-transferase (GST) is the key enzyme in glutathione (GSH) synthesis, and plays a crucial role in copper (Cu) detoxification. Nonetheless, its regulatory mechanisms remain largely unclear. In this study, we identified a Cu-induced glutathione S-transferase 1 (TaGST1) gene in wheat. Yeast one-hybrid (Y1H) screened out TaWRKY74, which was one member from the WRKY transcription factor family. The bindings between TaGST1 promoter and TaWRKY74 were further verified by using another Y1H and luciferase assays. Expression of TaWRKY74 was induced more than 30-folds by Cu stress. Functions of TaWRKY74 were tested by using transiently silence methods. In transiently TaWRKY74-silenced wheat plants, TaWRKY74 and TaGST1 expression, GST activity, and GSH content was significantly inhibited by 25.68%, 19.88%, 27.66%, and 12.68% in shoots, and 53.81%, 52.11%, 23.47%, and 17.11% in roots, respectively. However, contents of hydrogen peroxide, malondialdehyde, or Cu were significantly increased by 2.58%, 12.45%, or 37.74% in shoots, and 25.24%, 53.84%, and 103.99% in roots, respectively. Notably, exogenous application of GSH reversed the adverse effects of transiently TaWRKY74-silenced wheat plants during Cu stress. Taken together, our results suggesting that TaWRKY74 regulated TaGST1 expression and affected GSH accumulation under Cu stress, and could be useful to ameliorate Cu toxicity for crop food safety.

[Structural Characteristics of Microbial Communities in the Sediments of the Niyang River in Tibet].

The Niyang River is an important tributary of the Yarlong Zangbo River, which is also an important water source in the Gongbujiangda and Linzhi areas of Tibet. In this study, water and sediment samples from 18 sites in the Niyang River were collected. The physical and chemical properties, concentrations of 12 heavy metals, and the microbial community in the sediments were determined. The microbial community structures in the sediments collected in 2017 and 2018 were highly similar at the phylum and genus levels. The most dominant type of bacteria in the sediment of the Niyang River was Proteobacteria. Other dominant bacteria included Bacteroidetes, Acidbacteria, and Actinobacteria. At the genus level, the abundance of Flavobacterium was high. Other conditional pathogenic bacteria, such as Aeromonas and Acinetobacter, were detected. Cluster analysis found that there were certain spatial differences among the upstream, midstream, and downstream microbial communities. The microbial community of sediments in the power station reservoir area was unique. Correlation analysis showed that the temperature, dissolved oxygen, electrical conductance, Cr, Zn, Sr, and Ba in the sediments had high correlation with abundance of specific microbes at the phylum level. Redundancy analysis suggested that total nitrogen, total phosphorus, dissolved oxygen, Cr, Sr, Ba, and Mn were the main influencing factors of microbial community structure in the sediments of the Niyang River. The results of this study provide data support for understanding the spatial and temporal distribution of the microbial community in sediments of the Niyang River and identifying their environmental impact factors.

CREB1-induced lncRNA LEF1-AS1 contributes to colorectal cancer progression via the miR-489/DIAPH1 axis.

Long non-coding RNAs (lncRNAs) have been identified as new regulatory factors in tumor progression. Lymphoid enhancer-binding factor 1 antisense RNA 1 (LEF1-AS1) was a recently identified lncRNA. This research aimed to investigate the roles and mechanisms of LEF1-AS1 in colorectal cancer (CRC). We firstly showed that LEF1-AS1 expression was upregulated in human CRC tissues and cell lines. LEF1-AS1 upregulation was demonstrated to be induced by CREB1. Clinical study revealed that high LEF1-AS1 expression was positively associated with histological grade, lymph nodes metastasis, and decreased survivals of CRC patients. Functionally, down-regulation of LEF1-AS1 using si-LEF1-AS1 decreased cell growth, migration and invasion, as well as increased apoptosis in CRC cells. Mechanically, LEF1-AS1 functioned as competing endogenous RNA (ceRNA) for miR-489 to positively recover DIAPH1, thus playing an oncogenic role in CRC pathogenesis. Overall, our observations identified a novel CRC-related lncRNA LEF1-AS1 and discovered a critical role for this lncRNA as a ceRNA in CRC pathogenesis, suggesting that it may serve as a novel biomarker for prognosis and act as a therapeutic target for CRC treatment.

[Ecological pattern of urban forest landscape of Ji'nan City, China]. / æµŽå—åŸŽå¸‚æ£®æž—æ™¯è§‚ç”Ÿæ€æ ¼å±€.

The ecological pattern research on urban forest landscape is the foundation and premise of urban forest system planning and urban ecological construction, as well as an important way of optimizing urban spatial structure, giving full play to urban forest ecological function and creating ecological and livable environment. Ecological pattern of urban forest landscape in built-up area of Ji'nan city was quantitatively analyzed and the corresponding optimization countermeasures were put forward through quantitative analysis and gradient analysis of the overall landscape ecological pattern by RS and GIS technology with 10 landscape indices at patch level and landscape level. The results showed that urban forest coverage rate in built-up areas was 15.8%. At the landscape level, the overall landscape ecological pattern of urban forest showed following patterns: relatively complete types of urban forest landscape patches, greater degree of landscape fragmentation, different area of various types of urban forest, large major patches, and highly connected same patch types. At the level of patch type, the ecological public welfare forest was dominated by large patches. The shape of scenic recreational forest was regular and dominated by large and medium patches. The shape of road forest and subsidiary forest was complex and dominated by small patches. The production and management forest was lacking. At the level of patch type, landscape recreational forest was the dominant type, ecological public welfare forest patch area was the largest, road forest and ancillary forest fragmentation was large. At the level of landscape, urban central forest landscape fragmentation was large, human interference was serious, and landscape shape was complex. Based on the analysis of the ecological pattern of urban forest landscape, we proposed the construction scheme of Ji'nan urban forest ecological network, which was "one ring, two networks, three four axes, and multi-point chessboard". Such scheme would help strengthen the connectivity of various types of urban forests and exert overall ecological benefits of large-scale ecosystems.

The relationship between human papillomavirus and penile cancer over the past decade: a systematic review and meta-analysis.

Human papillomavirus (HPV) infection appears to play an important role in the development of penile cancer (PeCa), but their relationship remains unclear. Therefore, we performed a systematic review and meta-analysis to elucidate their relationship. We systematically searched Embase, PubMed, Cochrane Library, and Web of Science for case-control studies and cross-sectional studies using polymerase chain reaction (PCR) technology on formalin-fixed paraffin-embedded (FFPE) or paraffin-embedded (PE) PeCa tissues to detect HPV (published between January 1, 2007, and December 29, 2017; no language restrictions). Twenty-two studies were identified, and 1664 cases were available for analysis. The combined HPV infectious risk of PeCa is 51.0% (95% confidence interval [CI]: 43.0%-60.0%). The three most common subtypes of HPV were HPV16 (28.5%), HPV18 (2.3%), and HPV6 (2.3%). The virus was relevantly associated with basaloid (85.5%, 95% CI: 77.2%-93.8%) and warty (50.0%, 95% CI: 35.2%-64.8%) carcinomas. The invasiveness of PeCa was not associated with HPV (χ[2] = 0.181, df = 1, P < 0.671). HPV infection in PeCa tended to be moderately differentiated (54.4%, 95% CI: 47.7%-61.1%). This study found that almost half of PeCa patients are associated with HPV. The most commonly associated genotype is HPV16, but several other genotypes were also detected. In addition to types 6 and 11, other single low-risk HPV infections have been found to contribute to PeCa to a lesser degree. HPV-positive tumors tend to exhibit warty and/or basaloid features, corresponding to a moderate histological grade. The role of HPV in PeCa should be revisited to provide evidence for the development of PeCa in the presence of HPV infection.

[Research progress on pathogenesis of ischemic stroke and traditional Chinese medicine commonly used for treatment of ischemic stroke].

Stroke has become the second leading cause of death in the world, and the most common type is the ischemic stroke. Due to its rapid onset and complex conditions, ischemic stroke is a major neurological disorder that causes disability. Ischemic stroke mainly results from atherosclerosis, and the pathogenesis of ischemic stroke mainly includes energy metabolism disorders in the brain, the toxicity of excitatory amino acids, oxidative/nitrification stress, inflammatory response, apoptosis, and autophagy. With the characteristics of multi-component and multi-target, traditional Chinese medicine could be used to treat ischemic stroke at different stages. This article summarized the latest research progress on the pathogenesis of ischemic stroke and commonly used traditional Chinese medicine for treatment of ischemic stroke in order to provide references for the further research and clinical treatment of ischemic stroke.

Network Intervention, a Method to Address Complex Therapeutic Strategies.

Objective: Network-based approaches emerged as powerful tools for studying complex diseases. Our intention in this article was to raise awareness of the benefits of new therapeutic strategy in biological networks context and provide an introduction to this topic. Methods: This article will discuss the rational for network intervention, and outline some of the important aspects of deciphering targets activities in the network and future embodiments of network intervention. We also present examples of network intervention based on the strategies these approaches use. Results: Network intervention seeks for target combinations to perturb a specific subset of nodes in disease networks to inhibit the bypass mechanisms at systems level. Experimental results derived from our studies are discussed, with conclusions that lead to future research directions. A simple diagram is designed to give a way to find the minimum number of external input required for a network intervention based on the graph theory and get the analytical value of the least input. Conclusion: Creating network intervention that addresses blindness and unthinking action in this way could, therefore, provide more benefit than multi-target therapy. We hope that this article will give readers an appreciation for a new therapeutic strategy that has been proposed for improving clinical benefit by adopting network-based approaches as well as insight into their properties.

Systems Pharmacology Dissection of Traditional Chinese Medicine Wen-Dan Decoction for Treatment of Cardiovascular Diseases.

Cardiovascular diseases (CVDs) have been recognized as first killer of human health. The underlying mechanisms of CVDs are extremely complicated and not fully revealed, leading to a challenge for CVDs treatment in modern medicine. Traditional Chinese medicine (TCM) characterized by multiple compounds and targets has shown its marked effects on CVDs therapy. However, system-level understanding of the molecular mechanisms is still ambiguous. In this study, a system pharmacology approach was developed to reveal the underlying molecular mechanisms of a clinically effective herb formula (Wen-Dan Decoction) in treating CVDs. 127 potential active compounds and their corresponding 283 direct targets were identified in Wen-Dan Decoction. The networks among active compounds, targets, and diseases were built to reveal the pharmacological mechanisms of Wen-Dan Decoction. A "CVDs pathway" consisted of several regulatory modules participating in therapeutic effects of Wen-Dan Decoction in CVDs. All the data demonstrates that Wen-Dan Decoction has multiscale beneficial activity in CVDs treatment, which provides a new way for uncovering the molecular mechanisms and new evidence for clinical application of Wen-Dan Decoction in cardiovascular disease.

Function of the ERFL1a Transcription Factor in Wheat Responses to Water Deficiency.

The APETALA2/ethylene response factor (AP2/ERF) superfamily is involved in the responses of plants to biotic and abiotic stresses; however, the functions and mechanisms of some members of this family in plants are unclear. In our previous study, expression of TaERFL1a, a member of the AP2/ERF family, was remarkably induced in wheat seedlings suffering freezing stress. In this study, we show that its expression was rapidly upregulated in response to salt, cold, and water deficiency, suggesting roles in the responses to abiotic stresses. Further, transient barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) resulted in significantly reduced tolerance to 20% PEG6000-stimulated water deficiency. Subcellular localization and transcriptional activation assays separately showed that TaERFL1a was targeted to the nucleus and possessed transcriptional activation activity. Yeast two-hybrid library screening identified six interacting proteins, and of these, the interactions between TaERFL1a and TaSGT1, and TaERFL1a and TaDAD2 proteins were further confirmed by yeast co-transformation and bimolecular fluorescent complementation (BiFC). Collectively, our results suggest that TaERFL1a is a stress-responsive transcription factor, which could be functionally related to proteins involved in the abiotic stress responses of plants.

Monocarboxylate transporters MCT1 and MCT4 are independent prognostic biomarkers for the survival of patients with clear cell renal cell carcinoma and those receiving therapy targeting angiogenesis.

BACKGROUND: Prognostic biomarkers for patients with clear cell renal cell carcinoma (ccRCC), particularly those receiving therapy targeting angiogenesis, are not well established. In this study, we examined the correlations of monocarboxylate transporter 1 (MCT1) and MCT4, 2 critical transporters for glycolytic metabolism, with various clinicopathological parameters as well as survival of patients with ccRCC and those treated with vascular endothelial growth factor receptor (VEGFR) inhibitors. METHODS: A cohort of 150 ccRCC patients were recruited into this study. All patients underwent radical or partial nephrectomy as the first-line treatment, and 38 received targeted therapy (sorafenib or sunitinib) after the surgery. Expression levels of MCT1, MCT4, and CD34 were examined by immunohistochemistry. Correlations between MCT1 or MCT4 expression and different clinicopathological parameters or patient survival were analyzed among all as well as patients receiving targeted therapy. RESULTS: MCT1 or MCT4 expression did not significantly correlate with sex, age, tumor diameter, microvascular density, tumor staging, pathological Furmann grade, or MSKCC (P>0.05). High expression of either MCT1 or MCT4 significantly correlated with reduced overall survival (OS) and progression-free survival (PFS) among the total cohort of ccRCC patients. For patients receiving targeted therapy, high expression of either MCT1 or MCT4 significantly correlated with reduced PFS, but not OS. Both conditions were independent prognostic biomarkers for reduced PFS among all patients or those receiving targeted therapy. CONCLUSION: MCT1 and MCT4 are prognostic biomarkers for patients with ccRCC or those receiving targeted therapy. High expression of these 2 proteins predicts reduced PFS in these patients.

Engineering a lipase B from Candida antactica with efficient perhydrolysis performance by eliminating its hydrolase activity.

A Ser105Ala mutant of the lipase B from Candida antarctica enables 'perhydrolase-only' reactions. At the example of the chemoenzymatic Baeyer-Villiger oxidation of cyclohexanone, we demonstrate that with this mutant selective oxidation can be achieved in deep eutectic solvent while essentially eliminating the undesired hydrolysis reaction of the product.

Targeting of MCT1 and PFKFB3 influences cell proliferation and apoptosis in bladder cancer by altering the tumor microenvironment.

Phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3) and monocarboxylate transporter 1 (MCT1) play important roles in tumor endothelial cells (ECs) and several biological processes. The present study was conducted to study the effects of PFKFB3 and MCT1 on cell proliferation and apoptosis in the tumor microenvironment by co-culture of HUVECs and T24, a bladder cancer (BC) cell line, using a microfluidic device. Immunofluorescence assay showed that HUVEC activity was significantly enhanced under co-culture with T24 cells, according to the stronger fluorescence intensity of CD31 and CD105 than that in the signal­cultured cells. Quercetin treatment inhibited MCT1 expression but did not affect PFKFB3 expression. Knockdown of MCT1 or/and PFKFB3 increased the apoptosis rate of the HUVECs under single-culture and co-culture situations by staining with calcein and propidium iodide. Meanwhile, cell proliferation and lactic concentration were significantly decreased after the blocking of MCT1 or/and PFKFB3, as compared with that in the control group. No obvious differences in the effects on apoptosis, proliferation and lactic concentration were found between cells treated with quercetin and siMCT1. Thus, we concluded that the targeting of MCT1 and PFKFB3 regulated cell proliferation and apoptosis in BC cells by altering the tumor microenvironment, and quercetin exhibited a potential antitumor effect by targeting MCT1.

Aqueous two-phase system based on natural quaternary ammonium compounds for the extraction of proteins.

Aqueous two-phase systems, based on the use of natural quaternary ammonium compounds, were developed to establish a benign biotechnological route for efficient protein separation. In this study, aqueous two-phase systems of two natural resources betaine and choline with polyethyleneglycol (PEG400/600) or inorganic salts (K2 HPO4 /K3 PO4 ) were formed. It was shown that in the K2 HPO4 -containing aqueous two-phase system, hydrophobic interactions were an important driving force of protein partitioning, while protein size played a vital role in aqueous two-phase systems that contained polyethylene glycol. An extraction efficiency of more than 90% for bovine serum albumin in the betaine/K2 HPO4 aqueous two-phase system can be obtained, and this betaine-based aqueous two-phase system provided a gentle and stable environment for the protein. In addition, after investigation of the cluster phenomenon in the betaine/K2 HPO4 aqueous two-phase systems, it was suggested that this phenomenon also played a significant role for protein extraction in this system. The development of aqueous two-phase systems based on natural quaternary ammonium compounds not only provided an effective and greener method of aqueous two-phase system to meet the requirements of green chemistry but also may help to solve the mystery of the compartmentalization of biomolecules in cells.

Active-State Structures of a Small Heat-Shock Protein Revealed a Molecular Switch for Chaperone Function.

Small heat-shock proteins (sHsps) maintain cellular homeostasis by binding to denatured client proteins to prevent aggregation. Numerous studies indicate that the N-terminal domain (NTD) of sHsps is responsible for binding to client proteins, but the binding mechanism and chaperone activity regulation remain elusive. Here, we report the crystal structures of the wild-type and mutants of an sHsp from Sulfolobus solfataricus representing the inactive and active state of this protein, respectively. All three structures reveal well-defined NTD, but their conformations are remarkably different. The mutant NTDs show disrupted helices presenting a reformed hydrophobic surface compatible with recognizing client proteins. Our functional data show that mutating key hydrophobic residues in this region drastically altered the chaperone activity of this sHsp. These data suggest a new model in which a molecular switch located in NTD facilitates conformational changes for client protein binding.

Enzymatic selective synthesis of 1,3-DAG based on deep eutectic solvent acting as substrate and solvent.

In this study, enzymatic selective esterification of oleic acid with glycerol based on deep eutectic solvent acting as substrate and solvent was studied. As choline chloride (ChCl) or betaine can effectively change the chemical reaction characteristics of glycerol when they are mixed with a certain molar ratio of glycerol, several factors crucial to the lipase catalytic esterification of glycerol with oleic acid was investigated. Results showed that, betaine had more moderate effects than ChCl on the lipase, and water content had an important influence of the esterification and the enzyme selectivity. Significant changes of the glyceride compositions and enzyme selectivity were found in ChCl adding system compared with pure glycerol system; optimum accumulation of DAG especially 1,3-DAG because of the eutectic effect of ChCl was found in this system. Furthermore, in a model 1,3-DAG esterification synthesis system catalyzed by Novozym 435, high content (42.9 mol%) of the 1,3-DAG could be obtained in ChCl adding system within 1 h.

Bladder Tumor Heterogeneity: The Impact on Clinical Treatment.

Bladder cancer relapse and treatment failure in most patients have often been attributed to chemoresistance in tumor cells and metastasis. Emerging evidence indicates that tumor heterogeneity may play an equally important role and extends to virtually all measurable properties of cancer cells. Although the idea of tumor heterogeneity is not new, little attention has been paid to applying it to understand and control bladder cancer progression. With the development of biotechnology, such as Gene sequencing, recent advances in understanding its generation model, original basis, consequent problems, and derived therapies provide great potential for tumor heterogeneity to be considered a new insight in the treatment of bladder cancers.

Warburg effect or reverse Warburg effect? A review of cancer metabolism.

Cancer is a major threat to human health. A considerable amount of research has focused on elucidating the nature of cancer from its pathogenesis to treatment and prevention. Tumor cell metabolism has been considered a hallmark of cancer. Cancer cells differ from normal cells through unlimited cell division, and show a greater need for energy for their rapid growth and duplication. Research on glycometabolism, as the key point of energy metabolism, has played a unique role. In the 1920s, Warburg found that cancer cells prefer to produce adenosine triphosphate (ATP) by glycolysis, which is a less efficient pathway compared to oxidative phosphorylation. This striking discovery, called 'the Warburg effect', has influenced and guided the study of the mechanism and treatment of tumors for generations, but its causal relationship with cancer progression is still unclear. Some studies have now shown contradicting evidence and a new hypothesis, the reverse Warburg effect, has been put forward, in which cancer cells produce most of their ATP via glycolysis, even under aerobic conditions. In this review we discuss the new points concerning the energy metabolism of a tumor, as well as the current facts and perspectives.

Learning curve for hand-assisted laparoscopic D2 radical gastrectomy.

AIM: To describe the learning curves of hand-assisted laparoscopic D2 radical gastrectomy (HALG) for the treatment of gastric cancer. METHODS: The HALG surgical procedure consists of three stages: surgery under direct vision via the port for hand assistance, hand-assisted laparoscopic surgery, and gastrointestinal tract reconstruction. According to the order of the date of surgery, patients were divided into 6 groups (A-F) with 20 cases in each group. All surgeries were performed by the same group of surgeons. We performed a comprehensive and in-depth retrospective comparative analysis of the clinical data of all patients, with the clinical data including general patient information and intraoperative and postoperative observation indicators. RESULTS: There were no differences in the basic information among the patient groups (P > 0.05). The operative time of the hand-assisted surgery stage in group A was 8-10 min longer than the other groups, with the difference being statistically significant (P = 0.01). There were no differences in total operative time between the groups (P = 0.30). Postoperative intestinal function recovery time in group A was longer than that of other groups (P = 0.02). Lengths of hospital stay and surgical quality indicators (such as intraoperative blood loss, numbers of detected lymph nodes, intraoperative side injury, postoperative complications, reoperation rate, and readmission rate 30 d after surgery) were not significantly different among the groups. CONCLUSION: HALG is a surgical procedure that can be easily mastered, with a learning curve closely related to the operative time of the hand-assisted laparoscopic surgery stage.