Abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease with few effective treatments. Iron, an essential trace element, influences cellular functional states through multiple pathways and has been implicated in the pathogenesis and development of AAA. Previous studies have associated iron deficiency (ID) with the phenotypic switching of vascular smooth muscle cells by affecting mitochondrial and endoplasmic reticulum function. In this study, we demonstrate the protective role of appropriate iron supplementation in the progression of AAA. Our data further showed that iron supplementation increases the abundance of regulatory T cells (Tregs) both in the peripheral immune system and in aortic tissues, contributing to its protective effect. Moreover, iron supplementation inhibits the expression of mitochondrial uncoupling protein 2 (UCP2) in Tregs, which in turn suppresses the phosphorylation of dynamin-related protein 1 (Drp1), thereby modulating the dynamic balance between mitochondrial fusion and fission. Our findings suggest that iron supplementation may serve as a potential intervention to delay and ameliorate the progression of AAA.

OBJECTIVE:The limited understanding of venous thromboembolism (VTE) has hindered the development of novel treatment approaches. This study aimed to identify critical factors underlying VTE. METHODS:The isobaric tags for relative and absolute quantitation (iTRAQ) method was applied to identify differentially expressed proteins in plasma samples from patients with VTE and healthy volunteers. Gene overexpression and knockdown techniques were used for cultured human umbilical vein endothelial cells (HUVECs) and VTE mouse models. Thrombosis and apoptosis related to the identified proteins were verified using qRT-PCR, western blot, and flow cytometry analyses. RESULTS:iTRAQ analysis revealed significant upregulation of keratin 1, pro-platelet basic protein, and hemopexin (HPX) in the plasma of VTE patients. HPX was highly expressed in both plasma and GSE19151 and GSE48000 datasets. qRT-PCR and western blot results for HPX overexpressing HUVECs and VTE mouse models revealed that HPX positively regulated the expression of platelet endothelium aggregation receptor 1 and von Willebrand factor, but negatively regulated the expression of serpin family D member 1 and alpha-2-macroglobulin. Flow cytometry revealed that HPX exhibited pro-apoptotic activity in HPX overexpressing HUVECs. Furthermore, HPX elevated cleaved caspase-3 expression and inhibited B-cell lymphoma-2 expression in HUVECs and VTE mouse models. CONCLUSION:HPX exhibits possible prothrombotic activity by regulating thrombosis - apoptosis-related proteins.

Hyperglycaemia is a key factor in the progression of diabetes complications. Dapagliflozin (DAPA), a new type of hypoglycaemic agent, has been shown to play an important role in anti-apoptotic, anti-inflammatory and antioxidant activities. Previous studies have demonstrated an endothelial protective effect of DAPA, but the underlying mechanism was still unclear. Autophagy is a homeostatic cellular mechanism that circulates unfolded proteins and damaged organelles through lysosomal dependent degradation. In this study, we aimed to investigate whether DAPA plays a protective role against high glucose (HG)-induced endothelial injury through regulating autophagy. The results showed that DAPA treatment resulted in increased cell viability. Additionally, DAPA treatment decreased interleukin (IL)-1β, IL-6, and tumour necrosis factor-α levels in endothelial cells subjected to HG conditions. We observed that HG inhibited autophagy, and DAPA increased the autophagy level by inhibiting the protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signalling pathway. Chloroquine reversed all of these beneficial effects as an autophagy inhibitor. In summary, the endothelial protective effect of DAPA in HG can be attributed in part to its role in activating of autophagy via the AKT/mTOR signalling pathway. Therefore, suggesting that the activation of autophagy by DAPA may be a novel target for the treatment of HG-induced endothelial cell injury.

Background: Association between depression and aneurysm has been implicated but the specific role of depression in aneurysm remains unclear. We aimed to comprehensively characterize the relation of major depressive disorder (MDD) with aneurysm by subtype. Methods: Harnessing summary statistics from genome-wide association studies (Ncase/Ncontrol = 7603/317,899 for aortic aneurysm; 7321/317,899 for thoracic aortic aneurysm; 3201/317,899 for abdominal aortic aneurysm; 1788/317,899 for cerebral aneurysm; and 246,363/561,190 for major depressive disorder), we estimated the genetic correlation between MDD and each of four aneurysm subtypes via LD Score Regression and tested the causality via various estimators under the bi-directional Mendelian randomization (MR) framework. Results: Positive genetic correlation of statistical significance, ranging between 0.15 (with thoracic aortic aneurysm, P = 0.005) and 0.25 (with abdominal aortic aneurysm, P = 0.001), was consistently observed for MDD with each aneurysm subtype. In the MR analysis of MDD as an exposure, genetic liability to MDD causally increased the risk of cerebral (odds ratio: 1.71; 95 % confidence interval: 1.26-2.34) but not aortic aneurysm. Replication analysis of an independent dataset (Ncase/Ncontrol = 6242/59,418) corroborated this signal. In contrast, causal effect was not evident for any neurysm subtype on susceptibility to MDD. Limitations: Aneurysm could have been underdiagnosed if asymptomatic, leading to an underestimated causal impact on MDD. Non-linearity of the causal effect was not tested due to the lack of individual-level data. Conclusions: Depression and aneurysm may share common pathomechanisms. Screening depressed population and improving the clinical management for depression may benefit the primary prevention of cerebral aneurysm.

Imatinib is a classical targeted drug to treat chronic myeloid leukemia (CML). However, it shows cardiotoxicity, which limits its clinical application. Long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) shows proapoptotic properties in human cells. This study is performed to investigate whether targeting MEG3 can attenuate imatinib-mediated cardiotoxicity to cardiomyocytes. In this work, H9c2 cells were divided into four groups: control group, hypoxia group, hypoxia + imatinib, and hypoxia + imatinib + MEG3 knockdown group. MEG3 and microRNA-129-5p (miR-129-5p) expression levels were detected by the quantitative real-time PCR (qRT-PCR). The viability and apoptosis of H9c2 cells were then evaluated by cell counting kit-8 (CCK-8), flow cytometry, and TUNEL assays. The targeting relationships between MEG3 and miR-129-5p, between miR-129-5p and high-mobility group box 1 (HMBG1), were validated by dual-luciferase reporter assay and RNA Immunoprecipitation (RIP) assay. The protein expression level of HMGB1 was detected by western blot. It was revealed that, Imatinib-inhibited cell viability and aggravated the apoptosis of H9c2 cells cultured in hypoxic condition, and MEG3 knockdown significantly counteracted this effect. MiR-129-5p was a downstream target of MEG3 and it directly targeted HMGB1, and knockdown of MEG3 inhibited HMGB1 expression in H9c2 cells. In conclusion, targeting MEG3 ameliorates imatinib-induced injury of cardiomyocytes via regulating miR-129-5p/HMGB1 axis.

颈动脉体瘤(carotid body tumor,CBT)是一种罕见的头颈部化学感受器肿瘤,发源于颈动脉分叉处颈动脉体的副神经节,紧邻或包绕颈动脉,手术切除是目前公认的有效治疗方法,但由于CBT病变位置特殊,结构复杂,瘤体血运丰富,手术切除难度大,风险高,可能出现偏瘫、死亡等严重并发症[1-2].本文旨在探讨手术治疗CBT的技术细节及其近期疗效,以期为临床治疗提供参考,现报道如下.

The gene expression profile of abdominal aortic aneurysm (AAA) neck is not fully understood. The etiology of AAA is considered to be related to atherosclerosis and the inflammatory response, involving congenital, genetic, metabolic, and other factors. The level of proprotein convertase subtilisin/kexin type 9 (PCSK9) is related to those of cholesterol, oxidized low-density lipoprotein, and triglycerides. PCSK9 inhibitors have significant effects on lowering LDL-cholesterol, reversing atherosclerotic plaques, and reducing the risk of cardiovascular events and have been approved by several lipid-lowering guidelines. This work was aimed to investigate the potential role of PCSK9 in the neck of AAA. We extracted the expression dataset (GSE47472) containing 14 AAA patients and 8 donors and single-cell RNAseq (scRNA-seq) data (GSE164678) of CaCl2-induced (AAA) samples from the Gene Expression Omnibus dataset. Through bioinformatics methods, we found that PCSK9 was up-regulated in the proximal neck of human AAA. In AAA, PCSK9 was mainly expressed in fibroblasts. Additionally, immune check-point PDCD1LG2 was also expressed higher in AAA neck than donor, while CTLA4, PDCD1, and SIGLEC15 were down-regulated in AAA neck. The expression of PCSK was correlated with PDCD1LG2, LAG3, and CTLA4 in AAA neck. Additionally, some ferroptosis-related genes were also down-regulated in AAA neck. PCSK9 was also correlated with ferroptosis-related genes in AAA neck. In conclusion, PCSK9 was highly expressed in AAA neck, and may exert its role through interacting with immune check-points and ferroptosis-related genes.

Pulmonary fibrosis (PF) is characterized by oxidative injury and excessive collagen synthesis in lung fibroblasts, causing impaired pulmonary function and chronic lung injury. Piceatannol, a dietary polyphenol, possesses vital pharmacological effects in metabolic disorders, cancers, cardiovascular disease and infectious disease; however, its role in PF is still not completely elucidated. Mice (8 to 10 weeks old) were administered bleomycin (BLM) intratracheally (2 U/kg) to establish an in vivo PF model. Murine primary lung fibroblasts were isolated and stimulated with TGF-β (10 ng/mL) for 48 h to induce its activation. Meanwhile, mice or primary lung fibroblasts were treated with different doses of piceatannol to observe its protective roles. Pulmonary function and arterial blood gas were detected to assess pulmonary physiological status. Collagen deposition and the mRNA levels of profibrotic genes were determined by H&E staining and RT-PCR. Meanwhile, the protein and mRNA markers, as well as end-product of oxidative stress were detected in vivo and in vitro. The results showed that pulmonary function was significantly impaired in BLM-induced mice, accompanied by elevated oxidative stress and excessive collagen synthesis. Piceatannol significantly improved pulmonary function and decreased oxidative injury as well as collagen synthesis in mice with PF. Mechanically, piceatannol treatment significantly inhibited the activation of JAK2/STAT3 signaling pathway in BLM-induced mice and TGF-β-induced lung fibroblasts. Additional findings also demonstrated that coumermycin A1 (C-A1), an agonist of JAK2, could abolish the effects of piceatannol on TGF-β-induced lung fibroblasts and reactivated the phosphorylation STAT3. Taken together, our study demonstrated that piceatannol could protect against oxidative injury and collagen synthesis during PF in a JAK2/STAT3 signaling pathway-dependent manner.

目的:研究二去水卫矛醇(DAG)对人非小细胞肺癌(NSCLC)H460细胞体内抗癌作用及其机制.方法:建立裸鼠人NSCLC H460细胞移植瘤模型,分为模型组、阳性对照组及DAG低、中、高剂量组(DAG-L组、DAG-M组、DAG-H组).采用TUNEL法检测肿瘤细胞凋亡,分别采用RT-qPCR法和Western blotting法检测肿瘤组织凋亡相关基因和蛋白表达.结果:与模型组比较,DAG各剂量组和阳性对照组瘤体体积和瘤重减小,抑瘤率均高于40％,肿瘤细胞凋亡率增高(均P<0.05);DAG-M组、DAG-H组和阳性对照组Bax、Caspase-3、Caspase-9、P53 mRNA相对表达量升高,DAG-L组Caspase-9 mRNA相对表达量升高,DAG各剂量组Bcl-2 mRNA和蛋白相对表达量均降低,Bax、P53蛋白表达量升高(均P<0.05).结论:DAG可抑制人NSCLC H460裸鼠皮下移植瘤的生长,其机制与诱导线粒体凋亡途径有关.

目的:建立三七皂苷R1、人参皂苷Rg1和人参皂苷Rb1在三七总皂苷(PNS)肠溶微丸中的含量测定方法,并考察其体外释放度,为其质量控制提供依据.方法:采用HPLC法,色谱柱为XB-C18(4.6 mm×250 mm,5μm),流动相为乙腈-水,梯度洗脱,流速为1.0 mL·min-1,柱温为30℃,检测波长为203 nm,加样量为20μL.按照2020年版《中华人民共和国药典》通则0931溶出度和释放度测定法,测定PNS在酸中和磷酸盐缓冲液中的释放度.结果:供试品中3种皂苷分离良好,线性关系良好;三七皂苷R1、人参皂苷Rg1和人参皂苷Rb1的线性范围分别为3.477～111.250、4.517～114.985、4.783～152.100μg·mL-1;检测限分别为0.584、0.631、0.565μg·mL-1,3批制剂中三七皂苷R1、人参皂苷Rg1和人参皂苷Rb1含量分别为10.40％、53.01％、32.49％,与PNS原料中的含量无明显差异,说明PNS主要有效成分的含量在制备过程中没有发生明显变化;三七皂苷R1、人参皂苷Rg1和人参皂苷Rb1在酸中释放量<10％,在pH值=6.8的磷酸盐缓冲液中45 min累积释放量>70％,说明3批制剂的体外释放度具有良好的稳定性和重现性.结论:该方法可以准确、快速的实现PNS肠溶微丸中多种皂苷类成分的定量测定,可用于PNS肠溶微丸的质量控制.

In this paper, we have carried out an experimental study to investigate the effects of different anesthetics on perioperative organ protection and postoperative cognitive function in patients undergoing cardiac valve replacement with cardiopulmonary bypass. To realize this idea, a total of 90 patients with single valve replacement under general anesthesia and hypothermic cardiopulmonary bypass from January 2020 to October 2021 were enrolled. These patients were assigned into three groups, with 30 cases in each group by the digital table method. Group A was anesthetized with sufentanil combined with dexmedetomidine. Group B was anesthetized with sufentanil combined with etomidate. Group C was anesthetized with sufentanil combined with propofol. Perioperative organ protection and postoperative cognitive function of the three groups were compared. At T0 time point, there was no significant difference in blood WBC, blood N, and CRP among groups A, B, and C (P > 0.05); At T4 and T5 time points, the indexes of blood WBC, blood N, and CRP in groups A, B, and C were higher compared to the T0 time point. At T4 and T5 time points, the indexes of blood WBC, blood N, and CRP in group A were significantly lower compared to group B and group C. Before treatment, there was no significant difference in ALT and AST among groups A, B, and C (P > 0.05). After treatment, the indexes of ALT and AST in group A were significantly lower compared to group B and group C at T4 and T5 time points (P < 0.05). Before treatment, there was no significant difference in urea and creatinine among groups A, B, and C (P > 0.05). After treatment, the urea and creatinine indexes of group A were significantly lower compared to group B and group C at T4 and T5 time points (P < 0.05). Before treatment, there was no significant difference in CK-MB and CTnl among groups A, B, and C (P > 0.05); After treatment, the indexes of CK-MB and CTnl in group A were significantly lower compared to group B and group C at T4 and T5 time points (P < 0.05). Before treatment, there was no significant difference in MOCA scores among groups A, B, and C (P > 0.05). After treatment, the MOCA scores of group A were significantly higher compared to group B and group C at T5 and T6 time points (P < 0.05). Sufentanil combined with dexmedetomidine for heart valve replacement under cardiopulmonary bypass can reduce the dosage of anesthetics during the operation and have a certain perioperative protective effect on important organs such as the heart, lung, liver, and kidney, which may be related to reducing intraoperative hemodynamic fluctuations and inhibiting inflammatory stress response.

目的 研究南蛇风的生药学特征.方法 采用原植物基原鉴定、性状鉴别、显微鉴别、TLC鉴别、常规检查等方法进行研究.结果 南蛇风为苏木科云实属植物喙荚云实的藤茎,其性状和显微鉴别特征均较明显,尤其是髄部特征;对南蛇风中的白桦脂酸成分建立了薄层色谱鉴别方法,水分测定值为7.5％～9.1％,总灰分为3.4％～4.5％,酸不溶性灰分为0.2％～0.3％,浸出物为3.6％～9.6％.结论 研究结果可为南蛇风的鉴定和质量标准的制定提供科学依据.

目的 研究p53对p125表达的影响,及其对人肝癌细胞SMMC-7721细胞增殖的影响.方法 用脂质体转染技术,将实验分为3组:对照组(转染空载体pGPU6/GFP/neo-shNC的7721-shNC细胞)、空白组(肝癌细胞SMMC-7721)、实验组(转染p53重组真核表达质粒pGPU6/GFP/neo-p53-shRNA的7721-p53-shRNA细胞).用蛋白免疫印迹法(Western Blotting)检测p53和p125蛋白表达水平,用实时荧光聚合酶链反应检测DNA聚合酶δ催化亚基基因1(POLD1)、视网膜母细胞瘤蛋白1(Rb1)、转录因子E2F、细胞周期依赖性蛋白激酶2(CDK2)及细胞周期素E(CylinE)基因的表达水平.结果 实验组、对照组和空白组的p53蛋白表达水平分别为0.22±0.03,0.55±0.07和0.53±0.06,p125蛋白分别为2.86±0.21,2.20±0.28和2.24±0.25,POLD1 mRNA分别为1.09±0.13,0.45±0.07和1.00±0,Rb1 mRNA分别为4.16±0.29,1.10±0.16和1.00±0,转录因子E2F mRNA分别为2.49±0.12,1.08±0.17和1.00±0,CDK2 mRNA分别为1.65±0.08,1.12±0.22和1.00±0,CyclinE mRNA分别为2.78±0.23,1.17±0.31和1.00±0,实验组的上述指标与对照组比较,差异均有统计学意义(均P<0.05).结论 通过RNA干扰技术反向验证了p53对p125表达的调控作用,其对肝癌细胞增殖具有抑制作用,可能是通过调控Rb1、转录因子E2F、CDK2和CyclinE等细胞周期因子基因表达而实现的.

Purpose of Review This review focuses on recent evidence examining the role gut microbiota play in coronary heart disease. It also provides a succinct overview of current and future therapies targeting the gut microbiota for coronary heart disease risk reduction. Recent Findings A consensus has been reached that differences exist in the gut microbiotas of patients with coronary heart disease. Studies have shown that the gut microbiota is associated with obesity, diabetes, dyslipidemia, and hypertension, which are risk factors for coronary heart disease. The gut microbiota is involved in mediating basic metabolic processes, such as cholesterol metabolism, uric acid metabolism, oxidative stress, and inflammatory reactions, through its metabolites, which can induce the development of atherosclerosis and coronary heart disease. Interfering with the composition of gut microbiota, supplementing probiotics, and fecal donation are active areas of research to potentially prevent and treat coronary heart disease. Summary Gut microbiota are causally associated with coronary heart disease. We analyzed the gut microbiota’s effects on risk factors for coronary heart disease and studied the effects of gut microbiota metabolites on coronary heart disease. Gut microbiota is a potential target for preventing and treating coronary heart disease.

目的 研究南蛇风的化学成分.方法 南蛇风85％乙醇提取物采用硅胶、薄层色谱、Sephadex LH-20进行分离纯化,根据理化性质及波谱数据鉴定所得化合物的结构.结果 从中分离得到11个化合物,分别鉴定为木栓酮(1)、正十三醇(2)、白桦脂酸(3)、bonducellin (4)、金色酰胺醇酯(5)、platanic acid (6)、白藜芦醇(7)、neocaesalpin L(8)、△～(5,22)豆甾醇-3-O-β-D-吡喃葡萄糖苷(9)、豆甾醇(10)、胡萝卜苷(11).结论 化合物2～3、5～6、9为首次从该属植物中分离得到,化合物1、7为首次从该植物中分离得到.

Objective:To evaluate the toxic effect of long-term intravenous injection of carboxylated singlewalled carbon nanotubes(c-SWNTs) on kidney in rats.Methods:Sixty-four rats were divided randomly into control group and c-SWNTs group,with 32cases in each group.Rats in the control group were injected with 5％ glucose solution via tail vein,and rats in c-SWNTs group were injected with c-SWNTS-glucose solution via tail vein.After 30d,60d,90d and 120d(90 dinjection+30 drecovery) of intervention,eight rats were selected from each group and killed after anesthesia.Ultrastructure of renal tissue was observed by transmission electron microscope(TEM).Hematoxylin-eosin(HE) staining and Masson staining were used to observe pathological changes of renal tissue.Western blotting was used to detect the expression of type Ⅲ collagen(Col Ⅲ) protein.Results:After 30 days of intervention,reduced glomerular volume,karyopyknosis and swollen mitochondria were observed in the c-SWNTs group.Locally thickened renal vessels walls,glomerular capillary loop adhered to Bowman's capsule, inflammatory cell infiltration in capillary loop and spread to periglomerular interstitium with the prolong of treatment time,obvious dilation of renal tubule lumen,and periglomerular and medulla interstitial fibrogenesis were found in the c-SWNTs group by HE staining and Masson staining.After 90 days,the rats in the c-SWNTs group had severest renal inflammation and fibrosis,and significantly higher protein level of ColⅢcompared with the control group(P<0.05).There was no significant difference in the protein expression of ColⅢafter 30days of recovery between control group and c-SWNTs group(P>0.05).Conclusion:Long-term intravenous injection of c-SWNTs might lead to renal cortex,renal medulla and renal vessels injuries,and reversible inflammatory response and renal fibrosis.

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) was closely involved in doxorubicin- (DOX-) induced cardiotoxicity. MicroRNA-200a (miR-200a) could target Keap1 mRNA and promote degradation of Keap1 mRNA, resulting in Nrf2 activation. However, the role of miR-200a in DOX-related cardiotoxicity remained unclear. Our study is aimed at investigating the effect of miR-200a on DOX-induced cardiotoxicity in mice. For cardiotropic expression, male mice received an injection of an adeno-associated virus 9 (AAV9) system carrying miR-200a or miR-scramble. Four weeks later, mice received a single intraperitoneal injection of DOX at 15 mg/kg. In our study, we found that miR-200a mRNA was the only microRNA that was significantly decreased in DOX-treated mice and H9c2 cells. miR-200a supplementation blocked whole-body wasting and heart atrophy caused by acute DOX injection, decreased the levels of cardiac troponin I and the N-terminal probrain natriuretic peptide, and improved cardiac and adult cardiomyocyte contractile function. Moreover, miR-200a reduced oxidative stress and cardiac apoptosis without affecting matrix metalloproteinase and inflammatory factors in mice with acute DOX injection. miR-200a also attenuated DOX-induced oxidative injury and cell loss in vitro. As expected, we found that miR-200a activated Nrf2 and Nrf2 deficiency abolished the protection provided by miR-200a supplementation in mice. miR-200a also provided cardiac benefits in a chronic model of DOX-induced cardiotoxicity. In conclusion, miR-200a protected against DOX-induced cardiotoxicity via activation of the Nrf2 signaling pathway. Our data suggest that miR-200a may represent a new cardioprotective strategy against DOX-induced cardiotoxicity.

目的:探索氯化两面针碱磷(NC)脂复合物(NC-PC)制备的最佳工艺条件.方法:以NC与磷脂的复合率为评价指标,分别对不同反应溶剂、反应时间、反应温度、药物反应浓度、药物磷脂投料比等因素进行考察,再通过综合的单因素试验和正交试验探究最佳的制备工艺.结果:最佳的制备条件:以甲醇为溶剂,NC的浓度为0.5 mg/mL,NC与磷脂的投料比为1∶3(mol/mo1),反应温度为40℃,反应时间为1.5h.此条件可使NC与磷脂的复合率达到85％以上.结论:所采用的复合物的制备工艺稳定可行,复合率高,为NC的制剂学研究提供了参考依据.

Patients with aortic dissection (AD) may present acute lung injury (ALI) that may affect the prognosis. In this study, we aim to investigate the roles and mechanism of IL-22 in the pathogenesis of AD complicated with ALI. Six hundred and twenty-one AD patients were included, and the incidence of ALI and pulmonary CT findings were analyzed. Mouse ALI model was established through AngII, and then IL-22 injection and AG490 were given. The pathological changes, infiltration of inflammatory cells, and expression of STAT3 were determined. For the in vitro experiment, cultivated pulmonary microvascular endothelial cells (PMVECs) were treated by angiotensin II (AngII), followed by treating with IL-22 and/or AG490. The expression and migration of STAT3 was determined. Flow cytometry was carried out to evaluate the apoptosis. IL-22 contributed to the expression of STAT3 in lung tissues and attenuation of ALI. IL-22 obviously inhibited the apoptosis of PMVECs mediated by AngII and downregulated the expression and intranuclear transmission of STAT3. Such phenomenon was completely inhibited upon administration of AG490, an inhibitor of JAK2. Our data showed IL-22 contributed to the inhibition of PMVEC apoptosis mediated by AngII through activating the JAK2/STAT3 signaling pathway, which may attenuate the ALI induced by AngII.