DHPLC法分析甘肃东乡族RBP4基因多态性与超重、肥胖、胰岛素抵抗的相关性

目的 探讨甘肃东乡族视黄醇结合蛋白4（RBP4）基因多态性与超重肥胖、胰岛素抵抗的关系。方法用DHPLC技术筛查东乡族113例正常体重者及107例超重肥胖者RBP4基因第五内含子＋7542位点的多态性。结果（1）DM组＋7542位点基因型和等位基因频率分布在体重正常和超重肥胖者之间无统计学差异。非DM组超重肥胖者TT基因型和T等位基因频率明显高于体重正常者。（2）在非DM人群中超重和体重正常组均发现TT基因型者的平均HOMA-IR值明显高于其他基因型者。结论甘肃东乡族RBP4基因多态性可能与超重肥胖、胰岛素抵抗相关。     

SLC30A8基因rs13266634C/T多态性与甘肃省东乡族及回族2型糖尿病的相关性研究

目的 探讨SLC30A8基因rs13266634C/T遗传多态性与甘肃东乡族及回族T2DM的关系。 方法 应用PCR–RFLP检测甘肃东乡族、回族T2DM（T2DM组）患者和健康对照者 （NC组）者SLC30A8的基因型。 结果 东乡族、回族T2DM组CC基因型（39.07% vs 40.80%）、C等位基因（61.80% vs 62.40%）频率均高于NC组（23.30% vs 26.00%;50.00% vs 51.60%）（P〈0.05）。 结论 SLC30A8基因多态性与甘肃东乡族和回族人群T2DM发病有关,C是其风险等位基因。     

PRKAA2基因rs2051040多态性与东乡族2型糖尿病、脂代谢及体质指数关系的研究

目的探讨PRKAA2基因rs2051040多态性与东乡族T2DM发病风险及脂代谢的关系。方法运用高效变性液相色谱技术检测218名东乡族人群中PRKAA2基因rs2051040多态性,其中T2DM患者(T2DM组)104例,健康者(NC组)114名。分析该多态性位点基因型及等位基因频率与T2DM的关系,比较不同基因型间生化指标的差异。结果 rs2051040多态性位点的基因型和等位基因频率两组间差异无统计学意义,但T2DM组rs2051040多态性与HDL-C及BMI相关。结论 rs2051040多态性位点与东乡族T2DM发生无相关性,但与T2DM患者血脂和BMI相关。     

我国东乡族Ghrelin基因Leu72Met多态性与2型糖尿病的关系

目的 明确Ghrelin基因Leu72Met多态性在中国甘肃省东乡族的分布并探讨其与2型糖尿病的关系.方法 用PCR-DHPLC技术和测序法检测甘肃省东乡族T2DM患者及正常对照者(NC组)Ghrelin基因Leu72Met多态性.结果 Leu72Met基因型和等位基因频率在T2DM组和NC组中的分布差异无统计学意义(P＞0.05);NC组CA+AA型的FIns、HOMA-IR明显高于CC型;T2DM组CC基因型的FPG、血肌酐(SCr)水平明显高于CA+AA型(P均＜0.05).结论 Ghrelin基因Leu72Met多态性可能参与胰岛素抵抗和2型糖尿病的发病机制.     

我国东乡族Ghrelin基因Leu7 2 Met多态性与2型糖尿病的关系

目的明确Ghrelin基因Leu72Met多态性在中国甘肃省东乡族的分布并探讨其与2型糖尿病的关系。方法用PCR-DHPLC技术和测序法检测甘肃省东乡族T2DM患者及正常对照者(NC组)Ghrelin基因Leu72Met多态性。结果 Leu72Met基因型和等位基因频率在T2DM组和NC组中的分布差异无统计学意义(P0.05);NC组cA+AA型的Fins、HOMA-IR明显高于CC型;T2DM组CC基因型的FPG、血肌酐(SCr)水平明显高于cA+AA型(P均0.05)。结论 Ghrelin基因Leu72Met多态性可能参与胰岛素抵抗和2型糖尿病的发病机制。     

乙酰辅酶A羧化酶B基因多态性与2型糖尿病的相关性研究

目的探讨乙酰辅酶A羧化酶B(ACACB)基因(rs2268388及rs2268393)多态性与中国昆明地区汉族人群T2DM的相关性。方法分别运用聚合酶链反应-限制性片段长度多态性(PCRRFLP)技术及Taqman探针技术,在昆明地区汉族人群中对184例T2DM患者和70名健康对照者(NC)的ACACB基因(rs2268388及rs2268393)多态性进行检测,并比较分析各组间基因型、等位基因频率、相关临床和生化指标。结果 T2DM组和NC组ACACB基因rs2268393各基因型和等位基因频率比较,差异无统计学意义(χ2=4.810,P=0.09;χ2=1.29,P=0.24);T2DM组ACACB基因rs2268388AA基因型和A等位基因频率高于NC组(χ2=10.469,P=0.005;χ2=4.71,P=0.007);T2DM组ACACB基因rs2268388及rs2268393位点AABB基因型分布频率高于NC组(χ2=10.526,P=0.001);AABB基因型是T2DM发生的危险因素(OR:1.44,95%CI:1.32~1.57);二分类Logistic回归分析表明,ACACB基因rs2268388AA基因型、高BMI及高WHR是T2DM发生的危险因素,HDL-C是T2DM发生的保护因素。结论在昆明地区汉族人群中,ACACB基因rs2268388AA基因型可能与T2DM发生相关。     

我国西部人群脂联素基因+45位点多态性与肥胖、胰岛素抵抗及2型糖尿病的相关性

目的探讨宁夏汉族人群脂联素基因＋45位核苷酸T／G多态性与肥胖、胰岛素抵抗（IR）及2型糖尿病（T2DM）的相关性。方法采用聚合酶链式反应-限制性内切酶长度多态性技术，对100例T2DM患者和101例正常对照（NC）者脂联素基因＋45位点进行基因分型；并计算BMI和HOMA-IR。结果（1）T2DM组GG基因型频率明显高于NC组（P〈0．01），G等位基因频率明显高于NC组（P〈0．01）。（2）在T2DM组中，GG＋TG基因型的BMI、HOMA-IR大于TT基因型（P〈0．01）。在NC组中，各基因型间BMI、HOMA-IR的差异无统计学意义。在T2DM组中，而BMII〉25组的GG＋TG基因型频率高于BMI〈25组（P〈0．01），G等位基因频率也高于BMI〈25组（P〈0．01）。结论脂联素基因＋45位核苷酸T／G多态性与肥胖、IR及T2DM相关。     

β_2肾上腺素能受体基因R16G多态与哈萨克族男性肥胖的相关性

目的探讨β2肾上腺素能受体基因R16G多态在哈萨克族人群肥胖发病中所起的作用。方法应用Taqman技术检测331例哈萨克族正常体重者,380例超重者以及232例肥胖者中ADRB2基因R16G多态性,观察各基因型和等位基因频率在不同BMI水平的分布及其与超重肥胖的关系。结果哈萨克族正常体重组、超重组与肥胖组组间基因型和等位基因频率的分布无统计学差异,但突变型纯合子基因型频率在哈萨克族超重组及肥胖组有增高趋势;将哈萨克族人群按性别分层并用logistic回归模型校正年龄、血压之后,显示男性组间基因型频率分布存在统计学差异(χ2=8.707,P=0.013),携带突变型纯合子(AA基因型)的个体患肥胖的风险为GG基因型的2.18倍。结论ADRB2基因R16G多态与哈萨克族男性肥胖相关,AA基因型可能是哈萨克族男性肥胖的风险因子。     

β2肾上腺素能受体基因R16G多态与哈萨克族男性肥胖的相关性

目的 探讨β2肾上腺素能受体基因R16G多态在哈萨克族人群肥胖发病中所起的作用.方法 应用Taqman技术检测331例哈萨克族正常体重者,380例超重者以及232例肥胖者中ADRB2基因R16G多态性,观察各基因型和等位基因频率在不同BMI水平的分布及其与超重肥胖的关系.结果 哈萨克族正常体重组、超重组与肥胖组组间基因型和等位基因频率的分布无统计学差异,但突变型纯合子基因型频率在哈萨克族超重组及肥胖组有增高趋势;将哈萨克族人群按性别分层并用logistic回归模型校正年龄、血压之后,显示男性组间基因型频率分布存在统计学差异(χ2=8.707, P=0.013),携带突变型纯合子(AA基因型)的个体患肥胖的风险为GG基因型的2.18倍.结论 ADRB2基因R16G多态与哈萨克族男性肥胖相关,AA基因型可能是哈萨克族男性肥胖的风险因子.     

昆明汉族2型糖尿病肾病与AT1R基因A1166C多态性关系

运用限制性片段长度多态性聚合酶链反应(PCR-RFLP)检测184例T2DM患者,其中110例为DN者[DN( )组]、74例为无肾病者[DN(-)组]及56例正常对照组(NC组)的AT1R基因多态性.结果 (1)昆明汉族正常人群中AT1R基因型AA型86%,AC型14%,CC型未检出;A等位基因频率93%,C等位基因频率7%.(2)T2DM组与NC组A1R基因T的A1166C多态基因型频率和等位基因频率分布均无差异(P＞0.05).(3)DN( )与DN(-)中AT1R基因的A1166C多态基因型频率和等位基因频率分布均无差异(P＞0.05).结论 (1)昆明地区汉族正常人群AT1R基因多态性频率分布具有一定的地区特征.(2)昆明汉族T2DM及2型DN与AT1R基因A1166C多态性无关.     

Combined targeting of HER2 and VEGFR2 for effective treatment of HER2-amplified breast cancer brain metastases

Brain metastases are a serious obstacle in the treatment of patients with human epidermal growth factor receptor-2 (HER2)–amplified breast cancer. Although extracranial disease is controlled with HER2 inhibitors in the majority of patients, brain metastases often develop. Because these brain metastases do not respond to therapy, they are frequently the reason for treatment failure. We developed a mouse model of HER2-amplified breast cancer brain metastasis using an orthotopic xenograft of BT474 cells. As seen in patients, the HER2 inhibitors trastuzumab and lapatinib controlled tumor progression in the breast but failed to contain tumor growth in the brain. We observed that the combination of a HER2 inhibitor with an anti–VEGF receptor-2 (VEGFR2) antibody significantly slows tumor growth in the brain, resulting in a striking survival benefit. This benefit appears largely due to an enhanced antiangiogenic effect: Combination therapy reduced both the total and functional microvascular density in the brain xenografts. In addition, the combination therapy led to a marked increase in necrosis of the brain lesions. Moreover, we observed even better antitumor activity after combining both trastuzumab and lapatinib with the anti-VEGFR2 antibody. This triple-drug combination prolonged the median overall survival fivefold compared with the control-treated group and twofold compared with either two-drug regimen. These findings support the clinical development of this three-drug regimen for the treatment of HER2-amplified breast cancer brain metastases.     

SARS-CoV-2 spike engagement of ACE2 primes S2′ site cleavage and fusion initiation

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has resulted in tremendous loss worldwide. Although viral spike (S) protein binding of angiotensin-converting enzyme 2 (ACE2) has been established, the functional consequences of the initial receptor binding and the stepwise fusion process are not clear. By utilizing a cell–cell fusion system, in complement with a pseudoviral infection model, we found that the spike engagement of ACE2 primed the generation of S2′ fragments in target cells, a key proteolytic event coupled with spike-mediated membrane fusion. Mutagenesis of an S2′ cleavage site at the arginine (R) 815, but not an S2 cleavage site at arginine 685, was sufficient to prevent subsequent syncytia formation and infection in a variety of cell lines and primary cells isolated from human ACE2 knock-in mice. The requirement for S2′ cleavage at the R815 site was also broadly shared by other SARS-CoV-2 spike variants, such as the Alpha, Beta, and Delta variants of concern. Thus, our study highlights an essential role for host receptor engagement and the key residue of spike for proteolytic activation, and uncovers a targetable mechanism for host cell infection by SARS-CoV-2.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has exceeded 240 million cases across the globe, but the molecular mechanisms of viral infection and host pathogenesis remain elusive. The SARS-CoV-2 spike (S) glycoprotein is a class I fusion protein decorated on the viral lipid envelope and is a key determinant of viral entry (1). The SARS-CoV-2 spike monomer contains two fragments: The amino terminus S1 subunit contains a receptor binding domain (RBD) (2–5), which recognizes the host receptor angiotensin-converting enzyme 2 (ACE2) for initial docking, while the carboxyl terminus S2 subunit catalyzes the fusion of viral and cell membranes (6, 7), enabling the subsequent release of viral RNA genome and downstream replication within the infected cells (8). Although many studies have captured the stationary phases of spike binding to human ACE2 (9–11), key molecular and cellular processes downstream of receptor recognition have not been explored.Spike can be proteolytically processed (12). SARS-CoV-2 spike encodes a polybasic cleavage site at its S1/S2 junction, and is posttranslationally processed by the endopeptidase furin (13, 14); cleaved S1 and S2 subunits remain noncovalently attached and fusion competent (15). Furin-cleaved S1 also exposes a C-terminal motif recognized by the host receptor neuropilin-1 (NRP1) (16, 17), which can facilitate SARS-CoV-2 entry. Although spike protein is autoprocessed, additional proteolytic cleavage event within the S2 subunit is proposed to be responsible for the subsequent membrane fusion (18, 19). This cleavage can be mediated at the plasma membrane by the type II transmembrane serine proteases (TMPRSS2) (20–23), or processed by the lysosomal cathepsins during the endocytosis of viral particles (24). Secreted tissue proteases, such as elastase and trypsin, can also facilitate this cleavage event and promote infection (25). As a result, this proteolytic event within the S2 subunit induces the release of a highly conserved hydrophobic region, known as the fusion peptide (18), which subsequently anchors the target host cell membrane (6, 26). A conformational reconfiguration within the S2 subunit then pulls the viral and host membranes into close proximity, allowing lipid membranes to fuse (7, 27–29). The unilateral change of the S2 subunit is of the utmost importance during viral entry, but molecular events regulating the spike processing and activation have not been demonstrated.Cells infected with SARS-CoV-2 drive the fusion with adjacent ACE2-expressing cells, producing morphologically distinct multinuclear giant cells, also known as syncytia (2, 30, 31). Spike-mediated syncytia have been reported in the postmortem lung samples of severe COVID-19 patients (32, 33). Apart from virus to cell transmission, spike-driven syncytia formation may provide an additional route for cell–cell transmission of SARS-CoV-2. Here, by using a cell–cell fusion system, in complement with a pseudoviral particle infection model, we study the functional and molecular requirements of spike activation. Through analyzing the prefusion and postfusion spike protein products, we show that proteolytic cleavage at the S2′ site is triggered by human cell receptor recognition in a range of immortalized cell lines and humanized primary cells. Generation of the S2′ fragment specifically requires spike recognition of functional host ACE2 and is conserved in the several variants of concern. We highlight that arginine 815, but not arginine residues at the S1/S2 cleavage site, is indispensable for the S2′ cleavage and syncytia formation in wild type (WT), as well as the more infectious Alpha, Beta, and Delta spike variants. Hence, these data highlight that both receptor recognition and proteolytic event at the S2′ site are functionally important for spike-mediated membrane fusion and SARS-CoV-2 infection.     

Intestinal myofibroblast-specific Tpl2-Cox-2-PGE2 pathway links innate sensing to epithelial homeostasis

Tumor progression locus-2 (Tpl2) kinase is a major inflammatory mediator in immune cell types recently found to be genetically associated with inflammatory bowel diseases (IBDs). Here we show that Tpl2 may exert a dominant homeostatic rather than inflammatory function in the intestine mediated specifically by subepithelial intestinal myofibroblasts (IMFs). Mice with complete or IMF-specific Tpl2 ablation are highly susceptible to epithelial injury-induced colitis showing impaired compensatory proliferation in crypts and extensive ulcerations without significant changes in inflammatory responses. Following epithelial injury, IMFs sense innate or inflammatory signals and activate, via Tpl2, the cyclooxygenase-2 (Cox-2)-prostaglandin E₂ (PGE₂) pathway, which we show here to be essential for the epithelial homeostatic response. Exogenous PGE₂ administration rescues mice with complete or IMF-specific Tpl2 ablation from defects in crypt function and susceptibility to colitis. We also show that Tpl2 expression is decreased in IMFs isolated from the inflamed ileum of IBD patients indicating that Tpl2 function in IMFs may be highly relevant to human disease. The IMF-mediated mechanism we propose also involves the IBD-associated genes IL1R1, MAPK1, and the PGE₂ receptor-encoding PTGER4. Our results establish a previously unidentified myofibroblast-specific innate pathway that regulates intestinal homeostasis and may underlie IBD susceptibility in humans.Inflammatory bowel diseases (IBDs), encompassing Crohn’s disease and ulcerative colitis, are chronic inflammatory disorders of the gastrointestinal tract that develop from a complex and largely unknown etiology (1). Recent genome-wide association studies (GWAS) for IBD showed significant association with SNP rs1042058 in mitogen-activated protein kinase kinase kinase 8 (MAP3K8) gene (2) which encodes tumor progression locus-2 (Tpl2) kinase (SI Appendix, Fig. S1 A and B). Molecularly, Tpl2 is known to bind to NF-ĸB1-p105 in a stabilized but inactive form (3) and, when activated, is released to phosphorylate the major immediate targets MEK1/2 activating mainly the ERK pathway and downstream inflammatory mediators (3). The NFKB1 gene is also genetically associated with IBD in GWAS (2), highlighting the relevance of this pathway to human disease. Tpl2^−/− mice show defective TNF expression in response to LPS (4), high susceptibility to Listeria due to defective IL-1β expression (5), and impaired Th1 responses (6). Tpl2 also mediates pathogenesis in the TNF-driven and T-cell–mediated Tnf^ΔARE mouse model of Crohn’s-like IBD, indicating a potential pathogenic relevance of this pathway in specific contexts (7). Due to these proinflammatory functions, established mainly in cells of hematopoietic origin, Tpl2 is considered to be an appealing pharmacological target for the treatment of IBD and other inflammatory diseases (3, 8).The genetic association of MAP3K8 with IBD, however, may indicate unknown homeostatic functions of Tpl2 in the intestine. GWAS results provide insights into the complexity underlying MAP3K8 association because along with NFKB1, an additional 25 genes functionally related to Tpl2 are genetically associated with IBD (2). These functional interactions can be clustered in a wide spectrum of cell types and potential mechanisms (SI Appendix, Fig. S2 A and B and Table S1). Indeed, Tpl2 functions in many different cell types downstream of receptors such as CD40 (9), CD3/CD28 (10), TNFRI (11), IL-1R (11), NOD2 (5), or TLR4 (4). Notably, these pathways may have opposing roles in intestinal homeostasis and IBD; for example, contrasting homeostatic and epithelial cell death-promoting roles have been suggested for TLR4 and TNFRI, respectively (12, 13). A cell-specific approach is therefore required for detailed mechanistic understanding of the role of Tpl2 in IBD and effective therapeutic design.Here, studying the role of Tpl2 in intestinal homeostasis and IBD pathogenesis in a cell-specific manner, we identify a myofibroblast-specific Tpl2-Cox-2-PGE₂ pathway which plays a homeostatic role in the gut by promoting the compensatory proliferative response of the epithelium upon injury. This mechanism highlights the dominant role of IMFs in regulating epithelial homeostasis and also provides a possible explanation for the MAP3K8 association with IBD (see Fig. 8).Open in a separate windowFig. 8.Schematic representation of the mechanism proposed in the present study and the relevant genes which are genetically associated with IBD pathogenesis in humans. Following epithelial injury IMFs sense penetrating bacteria and the inflammatory milieu generated to activate a Tpl2-ERK-Cox-2-PGE₂–mediated homeostatic pathway and promote compensatory epithelial proliferation in the crypts. Indicated are genes genetically associated with IBD in recent GWAS (human symbols in parentheses) (2). These genes include MAP3K8 encoding Tpl2 kinase, NFKB1 encoding NF-κB1 (p105) which physically interacts with Tpl2 regulating its function and preventing its degradation (3), MAPK1 encoding ERK2, IL1R1 encoding IL-1 receptor, and the PTGER4 gene encoding the PGE₂ receptor EP4.     

Structural basis for the high Ca2+ affinity of the ubiquitous SERCA2b Ca2+ pump

Sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA) Ca²⁺ transporters pump cytosolic Ca²⁺ into the endoplasmic reticulum, maintaining a Ca²⁺ gradient that controls vital cell functions ranging from proliferation to death. To meet the physiological demand of the cell, SERCA activity is regulated by adjusting the affinity for Ca²⁺ ions. Of all SERCA isoforms, the housekeeping SERCA2b isoform displays the highest Ca²⁺ affinity because of a unique C-terminal extension (2b-tail). Here, an extensive structure–function analysis of SERCA2b mutants and SERCA1a2b chimera revealed how the 2b-tail controls Ca²⁺ affinity. Its transmembrane (TM) segment (TM11) and luminal extension functionally cooperate and interact with TM7/TM10 and luminal loops of SERCA2b, respectively. This stabilizes the Ca²⁺-bound E1 conformation and alters Ca²⁺-transport kinetics, which provides the rationale for the higher apparent Ca²⁺ affinity. Based on our NMR structure of TM11 and guided by mutagenesis results, a structural model was developed for SERCA2b that supports the proposed 2b-tail mechanism and is reminiscent of the interaction between the α- and β-subunits of Na⁺,K⁺-ATPase. The 2b-tail interaction site may represent a novel target to increase the Ca²⁺ affinity of malfunctioning SERCA2a in the failing heart to improve contractility.     

Upregulation of annexin A2 in H(2)O(2)-induced premature senescence as evidenced by 2D-DIGE proteome analysis

Human diploid fibroblasts undergo premature senescence after treatment with sublethal concentration of H(2)O(2). We report the first proteomic study of microsomal proteins in the context of H(2)O(2)-induced premature senescence by using 2D-DIGE approach. Twelve different proteins with altered abundance at day 3 after treatment with H(2)O(2) were identified. Among them, we demonstrated a re-localization of annexin A2 in plasma membrane.     

Molecular basis of the interaction between the antiapoptotic Bcl-2 family proteins and the proapoptotic protein ASPP2

We have characterized the molecular basis of the interaction between ASPP2 and Bcl-2, which are key proteins in the apoptotic pathway. The C-terminal ankyrin repeats and SH3 domain of ASPP2 (ASPP2_Ank-SH3) mediate its interactions with the antiapoptotic protein Bcl-2. We used biophysical and computational methods to identify the interaction sites of Bcl-2 and its homologues with ASPP2. Using peptide array screening, we found that ASPP2_Ank-SH3 binds two homologous sites in all three Bcl proteins tested: (i) the conserved BH4 motif, and (ii) a binding site for proapoptotic regulators. Quantitative binding studies revealed that binding of ASPP2_Ank-SH3 to the Bcl-2 family members is selective at two levels: (i) interaction with Bcl-2-derived peptides is the tightest compared to peptides from the other family members, and (ii) within Bcl-2, binding of ASPP2_Ank-SH3 to the BH4 domain is tightest. Sequence alignment of the ASPP2-binding peptides combined with binding studies of mutated peptides revealed that two nonconserved positions where only Bcl-2 contains positively charged residues account for its tighter binding. The experimental binding results served as a basis for docking analysis, by which we modeled the complexes of ASPP2_Ank-SH3 with the full-length Bcl proteins. Using peptide arrays and quantitative binding studies, we found that Bcl-2 binds three loops in ASPP2_Ank-SH3 with similar affinity, in agreement with our predicted model. Based on our results, we propose a mechanism in which ASPP2 induces apoptosis by inhibiting functional sites of the antiapoptotic Bcl-2 proteins.     

Diels–Alder Cycloaddition with CO,CO2, SO2, or N2 Extrusion: A Powerful Tool for Material Chemistry

Phenyl, naphthyl, polyarylphenyl, coronene, and other aromatic and polyaromatic moieties primarily influence the final materials’ properties. One of the synthetic tools used to implement (hetero)aromatic moieties into final structures is Diels–Alder cycloaddition (DAC), typically combined with Scholl dehydrocondensation. Substituted 2-pyranones, 1,1-dioxothiophenes, and, especially, 1,3-cyclopentadienones are valuable substrates for [4 + 2] cycloaddition, leading to multisubstituted derivatives of benzene, naphthalene, and other aromatics. Cycloadditions of dienes can be carried out with extrusion of carbon dioxide, carbon oxide, or sulphur dioxide. When pyranones, dioxothiophenes, or cyclopentadienones and DA cycloaddition are aided with acetylenes including masked ones, conjugated or isolated diynes, or polyynes and arynes, aromatic systems are obtained. This review covers the development and the current state of knowledge regarding thermal DA cycloaddition of dienes mentioned above and dienophiles leading to (hetero)aromatics via CO, CO₂, or SO₂ extrusion. Particular attention was paid to the role that introduced aromatic moieties play in designing molecular structures with expected properties. Undoubtedly, the DAC variants described in this review, combined with other modern synthetic tools, constitute a convenient and efficient way of obtaining functionalized nanomaterials, continually showing the potential to impact materials sciences and new technologies in the nearest future.     

Local Ca(2+) signaling and EC coupling in heart: Ca(2+) sparks and the regulation of the [Ca(2+)](i) transient

The elementary event of Ca(2+) release in heart is the Ca(2+) spark. It occurs at a low rate during diastole, activated only by the low cytosolic [Ca(2+)](i). Synchronized activation of many sparks is due to the high local [Ca(2+)](i) in the region surrounding the sarcoplasmic reticulum (SR) Ca(2+) release channels and is responsible for the systolic [Ca(2+)](i) transient. The biophysical basis of this calcium signaling is discussed. Attention is placed on the local organization of the ryanodine receptors (SR Ca(2+) release channels, RyRs) and the other proteins that underlie and modulate excitation-contraction (EC) coupling. A brief review of specific elements that regulate SR Ca(2+) release (including SR lumenal Ca(2+) and coupled gating of RyRs) is presented. Finally integrative calcium signaling in heart is presented in the context of normal heart function and heart failure.