Hyperbolic relationship between insulin secretion and sensitivity on oral glucose tolerance test

The utility of the disposition index as a measure of beta-cell compensatory capacity rests on the established hyperbolic relationship between its component insulin secretion and sensitivity measures as derived from the intravenous glucose tolerance test (IVGTT). If one is to derive an analogous measure of beta-cell compensation from the oral glucose tolerance test (OGTT), it is thus necessary to first establish the existence of this hyperbolic relationship between OGTT-based measures of insulin secretion and insulin sensitivity. In this context, we tested five OGTT-based measures of secretion (insulinogenic index, Stumvoll first phase, Stumvoll second phase, ratio of total area-under-the-insulin-curve to area-under-the-glucose-curve (AUC(ins/gluc)), and incremental AUC(ins/gluc)) with two measures of sensitivity (Matsuda index and 1/Homeostasis Model of Assessment for insulin resistance (HOMA-IR)). Using a model of log(secretion measure) = constant + beta x log(sensitivity measure), a hyperbolic relationship can be established if beta is approximately equal to -1, with 95% confidence interval (CI) excluding 0. In 277 women with normal glucose tolerance (NGT), the pairing of total AUC(ins/gluc) and Matsuda index was the only combination that satisfied these criteria (beta = -0.99, 95% CI (-1.66, -0.33)). This pairing also satisfied hyperbolic criteria in 53 women with impaired glucose tolerance (IGT) (beta = -1.02, (-1.72, -0.32)). In a separate data set, this pairing yielded distinct hyperbolae for NGT (n = 245) (beta = -0.99, (-1.67, -0.32)), IGT (n = 116) (beta = -1.18, (-1.84, -0.53)), and diabetes (n = 43) (beta = -1.37, (-2.46, -0.29)). Moreover, the product of AUC(ins/gluc) and Matsuda index progressively decreased from NGT (212) to IGT (193) to diabetes (104) (P < 0.001), consistent with declining beta-cell function. In summary, a hyperbolic relationship can be demonstrated between OGTT-derived AUC(ins/gluc) and Matsuda index across a range of glucose tolerance. Based on these findings, the product of these two indices emerges as a potential OGTT-based measure of beta-cell function.     

Versican V1 proteolysis in human aorta in vivo occurs at the Glu441-Ala442 bond, a site that is cleaved by recombinant ADAMTS-1 and ADAMTS-4

Mature human aorta contains a 70-kDa versican fragment, which reacts with a neoepitope antiserum to the C-terminal peptide sequence DPEAAE. This protein therefore appears to represent the G1 domain of versican V1 (G1-DPEAAE(441)), which has been generated in vivo by proteolytic cleavage at the Glu(441)-Ala(442) bond, within the sequence DPEAAE(441)-A(442)RRGQ. Because the equivalent aggrecan product (G1-NITEGE(341)) and brevican product (G1-EAVESE(395)) are generated by ADAMTS-mediated cleavage of the respective proteoglycans, we tested the capacity of recombinant ADAMTS-1 and ADAMTS-4 to cleave versican at Glu(441)-Ala(442). Both enzymes cleaved a recombinant versican substrate and native human versican at the Glu(441)-Ala(442) bond and the mature form of ADAMTS-4 was detected by Western analysis of extracts of aortic intima. We conclude that versican V1 proteolysis in vivo can be catalyzed by one or more members of the ADAMTS family of metalloproteinases.     

The Full-Length Isoform of Human Papillomavirus 16 E6 and Its Splice Variant E6* Bind to Different Sites on the Procaspase 8 Death Effector Domain

Human papillomavirus 16 is a causative agent of most cases of cervical cancer and has also been implicated in the development of some head and neck cancers. The early viral E6 gene codes for two alternatively spliced isoforms, E6_large and E6*. We have previously demonstrated the differential effects of E6_large and E6* binding on the expression and stability of procaspase 8, a key mediator of the apoptotic pathway. Additionally, we have reported that E6 binds to the FADD death effector domain (DED) at a novel E6 binding domain. Sequence similarities between the FADD and procaspase 8 DEDs suggested a specific region for E6_large/procaspase 8 binding, which was subsequently confirmed by mutational analysis as well as by the ability of peptides capable of blocking E6/FADD binding to also block E6_large/caspase 8 binding. However, the binding of the smaller isoform, E6*, to procaspase 8 occurs at a different region, as deletion and point mutations that disrupt E6_large/caspase 8 DED binding do not disrupt E6*/caspase 8 DED binding. In addition, peptide inhibitors that can block E6_large/procaspase 8 binding do not affect the binding of E6* to procaspase 8. These results demonstrate that the residues that mediate E6*/procaspase 8 DED binding localize to a different region on the protein and employ a separate binding motif. This provides a molecular explanation for our initial findings that the two E6 isoforms affect procaspase 8 stability in an opposing manner.The relationship between viruses and cancers is reflected in the observation that viral infections account for approximately 10 to 15% of the cancer burden worldwide (6, 60). This makes viral infections one of the preventable risk factors of cancer. Viruses are associated with several human malignancies, including hepatitis B and C virus-associated hepatocellular carcinomas (48), Epstein-Barr virus-associated nasopharyngeal carcinomas and lymphomas (36), and human T-cell leukemia virus-associated adult T-cell leukemia (8, 28). Although there is a correlation between infection and the onset of cancer, the frequency of infection supersedes the incidence of cancer inception, suggesting that the presence of the virus alone is not sufficient to trigger carcinogenesis. Progression from viral infection to tumor development therefore requires additional environmental and cellular factors in addition to the expression and activity of virus-encoded proteins (40).High-risk strains of human papillomavirus (HPV) (high-risk HPV [HR-HPV]) such as HPV16 and HPV18 have been implicated in most cases of cervical cancer and also in a subset of head and neck cancers (24, 26, 39). Infection with oncogenic strains of HPV represents up to 75% of all infections. Furthermore, 1/10 of all deaths among women worldwide can be attributed to HR-HPV-related cancers (44, 45). The key players in promoting cell transformation and immortalization following HPV infection are the viral early proteins E6 and E7. These proteins are well known for their ability to interact with the tumor suppressor p53 or members of the retinoblastoma family of proteins including pRb, p107, and p130, respectively (3, 17, 41). In addition to p53, HR-HPV E6 (HR-E6) binds to a number of cellular proteins involved in various aspects of cell proliferation and virus survival (reviewed in references 34 and 53). Our laboratory has reported that E6 binds to key mediators of the apoptotic pathway including tumor necrosis factor (TNF) R1 (22), the FADD death effector domain (DED) (21), and the procaspase 8 DED (20) and, in doing so, impedes apoptosis from taking place.As noted above, HR-E6 binds to TNF R1, blocking the adaptor molecule TRADD from binding to the membrane receptor. Similarly, the binding of HR-E6 to the FADD DED, a molecule common to the TNF-, Fas L-, and TRAIL-mediated extrinsic pathways of apoptosis, leads to the accelerated degradation of FADD and thereby inhibits the binding of additional downstream molecules necessary for programmed cell death. Additionally, we have reported that HR-E6 binds to procaspase 8, another molecule common to all three receptor-mediated pathways. The importance of procaspase 8 can be demonstrated by the many proteins produced by viruses to either inactivate or inhibit this apoptotic mediator in order to evade clearance by the host immune response. Such proteins include the herpes simplex virus R1 subunit that interferes with caspase 8 activation (31); the molluscum contagiosum virus MC159 protein that binds to the DEDs of both FADD and procaspase 8, thereby inhibiting their interaction (25); the human herpesvirus 8 FLICE protein that obstructs procaspase 8 cleavage and prevents its activation (4); and the cowpox virus serpin CrmA, which, along with the human cytomegalovirus UL136 proteins, inhibits caspase 8 activation (50, 56). In a like manner, HR-HPV16 produces the early protein E6 that binds to procaspase 8. Interestingly, however, we have found that the two splice products of the E6 gene, E6_large, a protein of about 16 kDa, and E6*, a protein less than half the size of E6_large, bind to and affect procaspase 8 stability differentially. While the large isoform accelerates the degradation of procaspase 8, leading to its destabilization, the short isoform leads to the stabilization of protein expression and an increase in activity. These observations suggest that the bindings of these two E6 isoforms have different functional consequences and may well localize to different regions on procaspase 8.We have previously identified a novel E6 binding site on the FADD DED (54). Based on sequence comparisons between the DEDs of FADD and procaspase 8, we proposed that the binding motif that mediates oncoprotein binding to both proteins would be similar. To test this possibility, we performed a series of mutational and peptide competitor-based experiments and discovered that the motifs on caspase 8 and on FADD that mediate binding between E6 and its cellular partner are indeed similar. Interestingly, however, the motif by which E6* binds to procaspase 8 is located in another region of the protein. These findings provide a molecular explanation for our previously reported observations concerning the differential effects of the binding of each isoform to the procaspase 8 DED. These findings also demonstrate the ability of peptide inhibitors to successfully impair E6 binding to its cellular targets and contribute to the discovery of therapeutic agents that are effective against cervical cancer.     

Protein kinase D regulates RhoA activity via rhotekin phosphorylation

The members of the protein kinase D (PKD) family of serine/threonine kinases are major targets for tumor-promoting phorbol esters, G protein-coupled receptors, and activated protein kinase C isoforms (PKCs). The expanding list of cellular processes in which PKDs exert their function via phosphorylation of various substrates include proliferation, apoptosis, migration, angiogenesis, and vesicle trafficking. Therefore, identification of novel PKD substrates is necessary to understand the profound role of this kinase family in signal transduction. Here, we show that rhotekin, an effector of RhoA GTPase, is a novel substrate of PKD. We identified Ser-435 in rhotekin as the potential site targeted by PKD in vivo. Expression of a phosphomimetic S435E rhotekin mutant resulted in an increase of endogenous active RhoA GTPase levels. Phosphorylation of rhotekin by PKD2 modulates the anchoring of the RhoA in the plasma membrane. Consequently, the S435E rhotekin mutant displayed enhanced stress fiber formation when expressed in serum-starved fibroblasts. Our data thus identify a novel role of PKD as a regulator of RhoA activity and actin stress fiber formation through phosphorylation of rhotekin.     

Flash Infrared Annealing for Antisolvent‐Free Highly Efficient Perovskite Solar Cells

Organic–inorganic perovskites have demonstrated an impressive potential for the design of the next generation of solar cells. Perovskite solar cells (PSCs) are currently considered for scaling up and commercialization. Many of the lab‐scale preparation methods are however difficult to scale up or are environmentally unfriendly. The highest efficient PSCs are currently prepared using the antisolvent method, which utilizes a significant amount of an organic solvent to induce perovskite crystallization in a thin film. An antisolvent‐free method is developed in this work using flash infrared annealing (FIRA) to prepare methylammonium lead iodide (MAPbI₃) PSCs with a record stabilized power conversion efficiency of 18.3%. With an irradiation time of fewer than 2 s, FIRA enables the coating of glass and plastic substrates with pinhole‐free perovskite films that exhibit micrometer‐size crystalline domains. This work discusses the FIRA‐induced crystallization mechanism and unveils the main parameters controlling the film morphology. The replacement of the antisolvent method and the larger crystalline domains resulting from flash annealing make FIRA a highly promising method for the scale‐up of PSC manufacture.     

Mammalian RNAi: a practical guide

Silencing of gene expression by RNA interference (RNAi) has become a powerful tool for the functional annotation of the Caenorhabditis elegans and Drosophila melanogaster genomes. Recent advances in the design and delivery of targeting molecules now permit efficient and highly specific gene silencing in mammalian systems as well. RNAi offers a simple, fast, and cost-effective alternative to existing gene targeting technologies both in cell-based and in vivo settings. Synthetic small interfering RNA (siRNA) and retroviral short hairpin RNA (shRNA) libraries targeting thousands of human and mouse genes are publicly available for high-throughput genetic screens, and knockdown animals can be rapidly generated by lentivirus-mediated transgenesis. RNAi also holds great promise as a novel therapeutic approach. This review provides insight into the current gene silencing techniques in mammalian systems.     

Inhibition of the amino‐acid transporter LAT1 demonstrates anti‐neoplastic activity in medulloblastoma

Most cases of medulloblastoma (MB) occur in young children. While the overall survival rate can be relatively high, current treatments combining surgery, chemo‐ and radiotherapy are very destructive for patient development and quality of life. Moreover, aggressive forms and recurrences of MB cannot be controlled by classical therapies. Therefore, new therapeutic approaches yielding good efficacy and low toxicity for healthy tissues are required to improve patient outcome. Cancer cells sustain their proliferation by optimizing their nutrient uptake capacities. The L‐type amino acid transporter 1 (LAT1) is an essential amino acid carrier overexpressed in aggressive human cancers that was described as a potential therapeutic target. In this study, we investigated the therapeutic potential of JPH203, a LAT1‐specific pharmacological inhibitor, on two independent MB cell lines belonging to subgroups 3 (HD‐MB03) and Shh (DAOY). We show that while displaying low toxicity towards normal cerebral cells, JPH203 disrupts AA homeostasis, mTORC1 activity, proliferation and survival in MB cells. Moreover, we demonstrate that a long‐term treatment with JPH203 does not lead to resistance in MB cells. Therefore, this study suggests that targeting LAT1 with JPH203 is a promising therapeutic approach for MB treatment.