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Aim: Aggravated neuronal loss, caused mainly by neuronal apoptosis, is observed in the brain of patients with Alzheimer's disease (AD) and animal models of AD. A truncated form of Dual‐specific and tyrosine phosphorylation‐regulated protein kinase 1A (Dyrk1A) plays a vital role in AD pathogenesis. Downregulation of anti‐apoptotic Bcl‐xL is tightly correlated with neuronal loss in AD. However, the molecular regulation of neuronal apoptosis and Bcl‐x expression by Dyrk1A in AD remains largely elusive. Here, we aimed to explore the role and molecular mechanism of Dyrk1A in apoptosis. Methods: Cell Counting Kit‐8 (CCK8), flow cytometry, and TdT‐mediated dUTP Nick‐End Labeling (TUNEL) were used to check apoptosis. The cells, transfected with Dyrk1A or/and ASF with Bcl‐x minigene, were used to assay Bcl‐x expression by RT‐PCR and Western blots. Co‐immunoprecipitation, autoradiography, and immunofluorescence were conducted to check the interaction of ASF and Dyrk1A. Gene set enrichment analysis (GSEA) of apoptosis‐related genes was performed in mice overexpressing Dyrk1A (TgDyrk1A) and AD model 5xFAD mice. Results: Dyrk1A promoted Bcl‐xS expression and apoptosis. Splicing factor ASF promoted Bcl‐x exon 2b inclusion, leading to increased Bcl‐xL expression. Dyrk1A suppressed ASF‐mediated Bcl‐x exon 2b inclusion via phosphorylation. The C‐terminus deletion of Dyrk1A facilitated its binding and kinase activity to ASF. Moreover, Dyrk1a1–483 further suppressed the ASF‐mediated Bcl‐x exon 2b inclusion and aggravated apoptosis. The truncated Dyrk1A, increased Bcl‐xS, and enrichment of apoptosis‐related genes was observed in the brain of 5xFAD mice. Conclusions: We speculate that increased Dyrk1A and truncated Dyrk1A may aggravate neuronal apoptosis by decreasing the ratio of Bcl‐xL/Bcl‐xS via phosphorylating ASF in AD. [ABSTRACT FROM AUTHOR]

Abstract Bcl-x pre-mRNA splicing serves as a typical example to study the impact of alternative splicing in the modulation of cell death. Dysregulation of Bcl-x apoptotic isoforms caused by precarious equilibrium splicing is implicated in genesis and development of multiple human diseases, especially cancers. Exploring the mechanism of Bcl-x splicing and regulation has provided insight into the development of drugs that could contribute to sensitivity of cancer cells to death. On this basis, we review the multiple splicing patterns and structural characteristics of Bcl-x. Additionally, we outline the cis-regulatory elements, trans-acting factors as well as epigenetic modifications involved in the splicing regulation of Bcl-x. Furthermore, this review highlights aberrant splicing of Bcl-x involved in apoptosis evade, autophagy, metastasis, and therapy resistance of various cancer cells. Last, emphasis is given to the clinical role of targeting Bcl-x splicing correction in human cancer based on the splice-switching oligonucleotides, small molecular modulators and BH3 mimetics. Thus, it is highlighting significance of aberrant splicing isoforms of Bcl-x as targets for cancer therapy.

Bcl-x pre-mRNA splicing serves as a typical example to study the impact of alternative splicing in the modulation of cell death. Dysregulation of Bcl-x apoptotic isoforms caused by precarious equilibrium splicing is implicated in genesis and development of multiple human diseases, especially cancers. Exploring the mechanism of Bcl-x splicing and regulation has provided insight into the development of drugs that could contribute to sensitivity of cancer cells to death. On this basis, we review the multiple splicing patterns and structural characteristics of Bcl-x. Additionally, we outline the cis-regulatory elements, trans-acting factors as well as epigenetic modifications involved in the splicing regulation of Bcl-x. Furthermore, this review highlights aberrant splicing of Bcl-x involved in apoptosis evade, autophagy, metastasis, and therapy resistance of various cancer cells. Last, emphasis is given to the clinical role of targeting Bcl-x splicing correction in human cancer based on the splice-switching oligonucleotides, small molecular modulators and BH3 mimetics. Thus, it is highlighting significance of aberrant splicing isoforms of Bcl-x as targets for cancer therapy. [ABSTRACT FROM AUTHOR]

Alternative splicing is deregulated in cancer and alternatively spliced products can be linked to cancer hallmarks. Targeting alternative splicing could offer novel effective cancer treatments. We investigated the effects of the crude extract of a South African medicinal plant, Cotyledon orbiculata, on cell survival of colon (HCT116) and esophageal (OE33 and KYSE70) cancer cell lines. Using RNASeq, we discovered that the extract interfered with mRNA regulatory pathways. The extract caused hnRNPA2B1 to splice from the hnRNPB1 to the hnRNPA2 isoform, resulting in a switch in the BCL2L1 gene from Bcl-xL to Bcl-xS causing activation of caspase-3-cleavage and apoptosis. Similar splicing effects were induced by the known anti-cancer splicing modulator pladienolide B. Knockdown of hnRNPB1 using siRNA resulted in decreased cell viability and increased caspase-3-cleavage, and over-expression of hnRNPB1 prevented the effect of C. orbiculata extract on apoptosis and cell survival. The effect of the hnRNPA2/B1 splicing switch by the C. orbiculata extract increased hnRNPA2B1 binding to Bcl-xl/s, BCL2, MDM2, cMYC, CD44, CDK6, and cJUN mRNA. These findings suggest that apoptosis in HCT116, OE33, and KYSE cancer cells is controlled by switched splicing of hnRNPA2B1 and BCL2L1, providing evidence that hnRNPB1 regulates apoptosis. Inhibiting this splicing could have therapeutic potential for colon and esophageal cancers. Targeting hnRNPA2B1 splicing in colon cancer regulates splicing of BCL2L1 to induce apoptosis. This approach could be a useful therapeutic strategy to induce apoptosis and restrain cancer cell proliferation and tumor progression. Here, we found that the extract of Cotyledon orbiculata, a South African medicinal plant, had an anti-proliferative effect in cancer cells, mediated by apoptosis induced by alternative splicing of hnRNPA2B1 and BCL2L1.

Abstract Background Homoharringtonine (HHT) is a natural alkaloid with potent antitumor activity, but its precise mechanism of action is still poorly understood. Methods We examined the effect of HHT on alternative splicing of Bcl-x and Caspase 9 in various cells using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The mechanism of HHT-affected alternative splicing in these cells was investigated by treatment with protein phosphatase inhibitors and overexpression of a protein phosphatase. Results Treatment with HHT downregulated the levels of anti-apoptotic Bcl-xL and Caspase 9b mRNA with a concomitant increase in the mRNA levels of pro-apoptotic Bcl-xS and Caspase 9a in a dose- and time-dependent manner. Calyculin A, an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), significantly inhibited the effects of HHT on the alternative splicing of Bcl-x and Caspase 9, in contrast to okadaic acid, a specific inhibitor of PP2A. Overexpression of PP1 resulted in a decrease in the ratio of Bcl-xL/xS and an increase in the ratio of Caspase 9a/9b. Moreover, the effects of HHT on Bcl-x and Caspase 9 splicing were enhanced in response to PP1 overexpression. These results suggest that HHT-induced alternative splicing of Bcl-x and Caspase 9 is dependent on PP1 activation. In addition, overexpression of PP1 could induce apoptosis and sensitize MCF7 cells to apoptosis induced by HHT. Conclusion Homoharringtonine regulates the alternative splicing of Bcl-x and Caspase 9 through a PP1-dependent mechanism. Our study reveals a novel mechanism underlying the antitumor activities of HHT.

Background: Verrucous carcinoma (VC) should be considered a distinct clinicopathologic entity different from the more common oral squamous cell carcinoma (OSCC) because of its unique biological behavior. Best way to understand the behavior of these carcinomas is to study them by means of molecular methods, especially in tumor progression tests and Bcl-X is an important antiapoptotic member of the Bcl-2 family and is one of the newest and most useful markers to determine the aggressiveness of many carcinomas. The relationship between this Bcl-X protein and carcinomatous behavior toward it is not studied extensively, which we attempted to evaluate using immunohistochemical analysis in selected carcinomas of the head and neck region. Method: We studied Bcl-X protein expression in sections of thirty OSCC and ten VC samples and correlated this with tumor differentiation. Results: There was a significant difference in cytoplasmic staining of Bcl-X expression with statistical analysis (P < 0.005) for VC and OSCC when compared as a group. No significance was seen among the different histological grades of OSCC and when compared with VC individually. Conclusion: The significant result between OSCC and VC suggests that their biologic course is comparable and can be helpful in differentiating them with each other for establishment of a better treatment protocol.

Alternative splicing is deregulated in cancer and alternatively spliced products can be linked to cancer hallmarks. Targeting alternative splicing could offer novel effective cancer treatments. We investigated the effects of the crude extract of a South African medicinal plant, Cotyledon orbiculata , on cell survival of colon (HCT116) and esophageal (OE33 and KYSE70) cancer cell lines. Using RNASeq, we discovered that the extract interfered with mRNA regulatory pathways. The extract caused hnRNPA2B1 to splice from the hnRNPB1 to the hnRNPA2 isoform, resulting in a switch in the BCL2L1 gene from Bcl-xL to Bcl-xS causing activation of caspase-3-cleavage and apoptosis. Similar splicing effects were induced by the known anti-cancer splicing modulator pladienolide B. Knockdown of hnRNPB1 using siRNA resulted in decreased cell viability and increased caspase-3-cleavage, and over-expression of hnRNPB1 prevented the effect of C. orbiculata extract on apoptosis and cell survival. The effect of the hnRNPA2/B1 splicing switch by the C. orbiculata extract increased hnRNPA2B1 binding to Bcl-xl/s, BCL2, MDM2, cMYC, CD44, CDK6, and cJUN mRNA. These findings suggest that apoptosis in HCT116, OE33, and KYSE cancer cells is controlled by switched splicing of hnRNPA2B1 and BCL2L1, providing evidence that hnRNPB1 regulates apoptosis. Inhibiting this splicing could have therapeutic potential for colon and esophageal cancers. Targeting hnRNPA2B1 splicing in colon cancer regulates splicing of BCL2L1 to induce apoptosis. This approach could be a useful therapeutic strategy to induce apoptosis and restrain cancer cell proliferation and tumor progression. Here, we found that the extract of Cotyledon orbiculata , a South African medicinal plant, had an anti-proliferative effect in cancer cells, mediated by apoptosis induced by alternative splicing of hnRNPA2B1 and BCL2L1. [ABSTRACT FROM AUTHOR]

Summary: Imatinib mesylate (IM) resistance has become a major clinical problem for chronic myeloid leukaemia (CML). It is known that Bcl‐x splicing is deregulated and is involved in multiple malignant cancer initiation and chemotherapy resistance, including CML. The aim of the present study was to correct the abnormal splicing of Bcl‐x in CML and investigate the subsequent malignant phenotype changes, especially response to IM. The aberrant Bcl‐x splicing in CML cells was effectively restored using vivo‐Morpholino Antisense Oligomer (vMO). CCK‐8 cell viability assay and flow cytometry showed that restoring of Bcl‐x splicing increases IM‐induced growth inhibition and apoptosis of K562 cells. Moreover, a more significant similar phenomenon was observed in imatinib‐resistant CML cell lines K562/G01. Finally, establishment of CML xenograft model had also proved that correcting Bcl‐x splicing in vivo can also enhance the anti‐tumor effect of IM. Our findings suggest that vMO co‐operating with IM can effectively increase the sensitivity of CML cells to IM both in vitro and in vivo, and Bcl‐x splicing could become good candidates for chemotherapy‐sensitized target in IM‐resistant CML. [ABSTRACT FROM AUTHOR]

Apoptosis plays a vital role in cell homeostasis during development and disease. Bcl-x, a member of the Bcl-2 family of proteins, is a mitochondrial transmembrane protein that functions to regulate the intrinsic apoptosis pathway. An alternative splicing (AS) event in exon 2 of Bcl-x results in two isoforms of Bcl-x with antagonistic effects on cell survival: Bcl-xL (long isoform), which is anti-apoptotic, and Bcl-xS (short isoform), which is pro-apoptotic. Bcl-xL is the most abundant Bcl-x protein and functions to inhibit apoptosis by a number of different mechanisms including inhibition of Bax. In contrast, Bcl-xS can directly bind to and inhibit the anti-apoptotic Bcl-xL and Bcl-2 proteins, resulting in the release of the pro-apoptotic Bak. There are multiple splice factors and signaling pathways that influence the Bcl-xL/Bcl-xS splicing ratio, including serine/arginine-rich (SR) proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs), transcription factors, and cytokines. Dysregulation of the AS of Bcl-x has been implicated in cancer and diabetes. In cancer, the upregulation of Bcl-xL expression in tumor cells can result in resistance to chemotherapeutic agents. On the other hand, dysregulation of Bcl-x AS to promote Bcl-xS expression has been shown to be detrimental to pancreatic β-cells in diabetes, resulting in β-cell apoptosis. Therefore, manipulation of the splice factor, transcription factor, and signaling pathways that modulate this splicing event is fast emerging as a therapeutic avenue in the treatment of cancer and diabetes.

Background: Homoharringtonine (HHT) is a natural alkaloid with potent antitumor activity, but its precise mechanism of action is still poorly understood. Methods: We examined the effect of HHT on alternative splicing of Bcl-x and Caspase 9 in various cells using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The mechanism of HHT-affected alternative splicing in these cells was investigated by treatment with protein phosphatase inhibitors and overexpression of a protein phosphatase. Results: Treatment with HHT downregulated the levels of anti-apoptotic Bcl-xL and Caspase 9b mRNA with a concomitant increase in the mRNA levels of pro-apoptotic Bcl-xS and Caspase 9a in a dose- and time-dependent manner. Calyculin A, an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), significantly inhibited the effects of HHT on the alternative splicing of Bcl-x and Caspase 9, in contrast to okadaic acid, a specific inhibitor of PP2A. Overexpression of PP1 resulted in a decrease in the ratio of Bcl-xL/xS and an increase in the ratio of Caspase 9a/9b. Moreover, the effects of HHT on Bcl-x and Caspase 9 splicing were enhanced in response to PP1 overexpression. These results suggest that HHT-induced alternative splicing of Bcl-x and Caspase 9 is dependent on PP1 activation. In addition, overexpression of PP1 could induce apoptosis and sensitize MCF7 cells to apoptosis induced by HHT. Conclusion: Homoharringtonine regulates the alternative splicing of Bcl-x and Caspase 9 through a PP1-dependent mechanism. Our study reveals a novel mechanism underlying the antitumor activities of HHT. [ABSTRACT FROM AUTHOR]

Background: Verrucous carcinoma (VC) should be considered a distinct clinicopathologic entity different from the more common oral squamous cell carcinoma (OSCC) because of its unique biological behavior. Best way to understand the behavior of these carcinomas is to study them by means of molecular methods, especially in tumor progression tests and Bcl-X is an important antiapoptotic member of the Bcl-2 family and is one of the newest and most useful markers to determine the aggressiveness of many carcinomas. The relationship between this Bcl-X protein and carcinomatous behavior toward it is not studied extensively, which we attempted to evaluate using immunohistochemical analysis in selected carcinomas of the head and neck region.Method: We studied Bcl-X protein expression in sections of thirty OSCC and ten VC samples and correlated this with tumor differentiation.Results: There was a significant difference in cytoplasmic staining of Bcl-X expression with statistical analysis (P < 0.005) for VC and OSCC when compared as a group. No significance was seen among the different histological grades of OSCC and when compared with VC individually.Conclusion: The significant result between OSCC and VC suggests that their biologic course is comparable and can be helpful in differentiating them with each other for establishment of a better treatment protocol. [ABSTRACT FROM AUTHOR]

Apoptotic endonuclease EndoG plays a key role in the alternative splicing of mRNA of human TERT telomerase catalytic subunit. The aim of this work was to test the ability of EndoG to induce alternative splicing of mRNA of other genes and in other organisms. To determine new mRNA splice-variants, EndoG overexpression was induced in human, mouse and rat CD4+-T-lymphocytes followed by sequencing of total RNA of these cells. Sequencing results showed that besides TERT, EndoG induced alternative splicing of deoxyribonuclease I (DNase I), caspase-2 (Casp-2) and BCL-x. The expression level of EndoG strongly correlated with mRNA splicing-variants of TERT, DNase I, Casp-2, and BCL-x in intact CD4+-T cells of healthy donors as well as different lines of mice and rats. EndoG overexpression induced down-regulation of fulllength mRNAs of TERT, DNase I, Casp-2, and BCL-x and up-regulation of their short-length mRNAs. Alternative splicing of studied mRNAs resulted in down-regulation of enzymatic activity of proteins in vitro and in vivo. The results of this work confirm the ability of endonuclease EndoG to induce alternative splicing of several mRNAs in human, mice and rats. [ABSTRACT FROM AUTHOR]

Protein arginine N-methyltransferase 2 (PRMT2) functions in JAK-STAT and Wnt/β-catenin signaling pathways, serves as a nuclear receptor-dependent transcriptional co-activator, and represses NF-κB and E2F1 transcription factor activities to promote apoptosis. We have previously demonstrated that PRMT2 interacts with PRMT1 and increases its activity. Here, we reveal associations using proteomics between the PRMT2 SH3 domain and splicing factors including Src-associated in mitosis 68 kDa protein (SAM68), a PRMT1 substrate and trans-acting factor that mediates BCL-X alternative splicing. We determined that PRMT2 interacts with SAM68 in cells and regulates its subcellular localization via the SH3 domain of PRMT2, prompting us to investigate the potential role of PRMT2 in BCL-X alternative splicing. We found that the expression of the full-length, wildtype form of PRMT2 promotes an increase in the BCL-X(L)/BCL-X(s) ratio in TNF-α or LPS stimulated cells. These results indicate that active PRMT2 may play a role during inflammation in alternative splicing regulation. [ABSTRACT FROM AUTHOR]

Objectives: Since its recognition as a physiologic process associated with tumor, among molecular mechanisms involved in tumor progression, defects in regulation of apoptosis have generated an accelerating volume of research that has sought to elucidate the role of programed cell death in pathogenesis and treatment of various tumors. Therefore, this study was performed to understand better the diverse biological profile of epithelial odontogenic tumors with the help of immunohistochemical expression of Bcl-X protein. Materials and Methods: We studied Bcl-X protein expression in 45 cases of epithelial odontogenic tumors which included 15 cases each of ameloblastomas, keratocystic odontogenic tumor (KCOT) and adenomatoid odontogenic tumor (AOT) and correlated the expression with their growth pattern. Results: Cytoplasmic staining of Bcl-X revealed overexpression in ameloblastoma when compared to KCOT and AOT. Percentage of positive cells showed a statistically significant difference, P = 0.007 between ameloblastoma and KCOT, whereas P < 0.001 between ameloblastoma and AOT. However, no significance was observed between KCOT and AOT (P = 0.132). Conclusion: The present study supports the fact that epithelial odontogenic tumors show diverse growth profiles. An increased Bcl-X expression was seen in ameloblastoma compared to KCOT and least expression in case of AOT which could be indicative of more aggressive biological behavior and increased cell survival activity of ameloblastoma than KCOT and AOT. This signifies the diagnostic relevance of this biomarker and also could be a possible regulator of the proliferative compartment by contributing in tumor progression and cytodifferentiation of epithelial odontogenic tumors. [ABSTRACT FROM AUTHOR]

The maternal and paternal genomes play different roles in mammalian brains as a result of genomic imprinting, an epigenetic regulation leading to differential expression of the parental alleles of some genes. Here we investigate genomic imprinting in the cerebellum using a newly developed Bayesian statistical model that provides unprecedented transcript-level resolution. We uncover 160 imprinted transcripts, including 41 novel and independently validated imprinted genes. Strikingly, many genes exhibit parentally biased—rather than monoallelic—expression, with different magnitudes according to age, organ, and brain region. Developmental changes in parental bias and overall gene expression are strongly correlated, suggesting combined roles in regulating gene dosage. Finally, brain-specific deletion of the paternal, but not maternal, allele of the paternally-biased Bcl-x, (Bcl2l1) results in loss of specific neuron types, supporting the functional significance of parental biases. These findings reveal the remarkable complexity of genomic imprinting, with important implications for understanding the normal and diseased brain.

Bcl-2 family proteins are known to competitively regulate Ca; however, the specific inter-organelle signaling pathways and related cellular functions are not fully elucidated. In this study, a portion of Bcl-x was detected at the ER-mitochondrion interface or MAM ( mitochondria- associated ER membrane) in association with type 3 inositol 1,4,5-trisphosphate receptors (IPR3); an association facilitated by the BH4 and transmembrane domains of Bcl-x. Moreover, increasing Bcl-x expression enhanced transient mitochondrial Ca levels upon ER Ca depletion induced by short-term, non-apoptotic incubation with thapsigargin (Tg), while concomitantly reducing cytosolic Ca release. These mitochondrial changes appear to be IPR3-dependent and resulted in decreased NAD/NADH ratios and higher electron transport chain oxidase activity. Interestingly, extended Tg exposure stimulated ER stress, but not apoptosis, and further enhanced TCA cycling. Indeed, confocal analysis indicated that Bcl-x translocated to the MAM and increased its interaction with IPR3 following extended Tg treatment. Thus, the MAM is a critical cell-signaling junction whereby Bcl-x dynamically interacts with IPR3 to coordinate mitochondrial Ca transfer and alters cellular metabolism in order to increase the cells' bioenergetic capacity, particularly during periods of stress. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) has biphasic effects on regulating osteoblast survival and death. This study was aimed to evaluate the effects of NO pretreatment on hydrogen peroxide (HP)-induced insults of rat osteoblasts and the possible mechanisms. Exposure of osteoblasts prepared from rat calvarias to HP significantly increased intracellular reactive oxygen species levels, decreased alkaline phosphatase activity and cell survival, and ultimately induced cell apoptosis. However, NO pretreatment lowered HP-induced oxidative stress and apoptotic insults. In parallel, HP increased Bax levels and its translocation from the cytoplasm to mitochondria. NO pretreatment caused significant attenuations in HP-induced modulations in Bax synthesis and translocation. In contrast, pretreatment with NO enhanced levels and translocation of antiapoptotic Bcl-X protein in rat osteoblasts. RNA analyses further revealed that HP inhibited Bcl-X mRNA expression without affecting Bax mRNA levels. In comparison, NO induced Bcl-X mRNA production and alleviated HP-caused inhibition of this mRNA expression. As to the mechanism, HP suppressed RNA and protein levels of transcription factor GATA-5 in rat osteoblasts. Pretreatment with NO induced GATA-5 mRNA and protein expressions and simultaneously attenuated HP-induced inhibition of this gene's expression. Consequently, GATA-5 knockdown using RNA interference inhibited Bcl-X mRNA expression and concurrently lowered NO's protection against HP-induced apoptotic insults. Therefore, this study showed that NO can protect rat osteoblasts from HP-induced apoptotic insults. The protective mechanisms are mediated by GATA-5-mediated transcriptional induction of Bcl- X gene, and translocational modulation of Bcl-X and Bax proteins. [ABSTRACT FROM AUTHOR]

Context: Tobacco smoking generates a tremendous amount of free radicals that induce oxidative stress (OS) in diabetics (pancreatic islet cells are defective).Salacia oblongaWall. (Celastraceae) is a proven antioxidant and antidiabetic plant whose mechanism of action is yet to be explored. Objective: The present study focuses on the protective ability ofS. oblongain tobacco smoke-induced oxidatively stressed pancreatic β-cell line. Materials and methods: The RINm5f cell line was exposed to tobacco smoke concentrate (TSC) (0.5–10%, 24 h), plant extract (1–75 µg/ml, 3 h), and their combinations. Cell viability was determined through MTT assay. Microscopic analysis was carried out in unstained and nonyl acridine orange-stained cells. The effect of toxic doses of TSC on DNA integrity was analyzed through DNA fragmentation assay. The TSC-induced nitric oxide generation was determined spectrophototmetrically. The expression of anti-apoptotic protein Bcl-X under the above treatment conditions was carried out through RT-PCR. Results: The LD50dose for TSC was found to be 1% TSC.Salacia oblongaextracts (10 and 15 µg/ml) were found to be optimum safe doses that significantly increased cell viability and decreased the nitric oxide production in TSC-treated cells. Pre-treatment with plant extract suppressed apoptosis through probable increase in the expression of anti-apoptotic protein Bcl-X in TSC-treated cells. Thus, the overall efficiency of plant extract in recovering cellular damage was proven. Discussion and conclusion: The results suggest that TSC-induced cellular alterations are related to rise in nitric oxide and Bcl-X mRNA expression and propose thatS. oblongamay confer significant cytoprotection against OS-mediated injury in β-cells. [ABSTRACT FROM AUTHOR]

Epidermal growth factor (EGF) has been suggested to play a key role in the maintenance of epithelial cell survival during lactation. Previously, we demonstrated that EGF dependent activation of PI3K pathway prevents apoptosis in confluent murine HC11 cells cultured under low nutrient conditions. The EGF protective effect is associated with increased levels of the antiapoptotic protein Bcl-XL. Here, we identify the EGF-dependent mechanism involved in cell survival that converges in the regulation of bcl-X expression by activated CREB. EGF induces Bcl-XL expression through activation of a unique bcl-X promoter, the P1; being not only the PI3K/AKT signaling pathway but also the increase in cAMP levels and the concomitant PKA/CREB activation necessary for both bcl-XL upregulation and apoptosis avoidance. Results presented in this work suggest the existence of a novel connection between the EGF receptor and the adenylate cyclase that would have an impact in preventing apoptosis under low nutrient conditions. [ABSTRACT FROM AUTHOR]

Kaposi's sarcoma (KS) is the most common tumor affecting AIDS patients with over 20% of these patients afflicted by this disease. Previous studies have demonstrated that KS tumor cells predominantly express the prosurvival protein Bcl-XL compared with Bcl-2. In the current study, we have used an adenoviral vector that expresses Bcl-XS, a functional inhibitor of Bcl-XL, to study the significance of Bcl-XL expression in the KS cell line (SLK) or KS primary cultures. The results demonstrate that 75% to 80% of SLK or KS primary cells were killed by the Bcl-XS containing adenovirus whereas KS cells infected with control adenovirus showed no significant cell death or growth inhibition. Overexpression of Bcl-XL, but not Bcl-2, in SILK cells attenuated apoptosis induced by adenovirus Bcl-XS. Immunoprecipitation experiments revealed that adenoviral Bcl-XS associated with Bcl-XL, but not with Bcl-2. Mutational analysis showed that the α2 helical region of Bcl-XS containing the BH3 motif was critical for killing activity and interaction with Bcl-XL. These results suggest that Bcl-XS is a direct killer and Bcl-XL may act by interacting with and sequestering Bcl-XS. These studies also suggest that targeting Bcl-XL may be of therapeutic benefit for the treatment of tumors that are characterized by inappropriate expression of Bcl-XL.

Purpose: Sorafenib is a multikinase inhibitor that targets the MAPK pathway and is currently used for the treatment of hepatocellular and renal carcinoma. Recently, it has been shown that sorafenib is also cytotoxic to multiple myeloma (MM) cells. Here, we have further analyzed the mechanism of sorafenib-induced death in MM cells. Methods: Cell death induced by sorafenib in MM cell lines and in plasma cells from MM patients was evaluated by analysis of gene expression by RT-MLPA and quantitative PCR, protein levels and functionality by Western blot and flow cytometry and gene silencing with siRNA. Results: Cell death was characterized by phosphatidylserine exposure, ΔΨ loss, cytochrome c release and caspase activation, hallmarks of apoptosis. DL at 24 h ranged from 6 to 10 µM. Ex vivo treatment with 20 µM sorafenib induced apoptosis in around 80 % myeloma cells from six multiple myeloma patients. Sorafenib induced caspase-dependent degradation of Bcl-x and Mcl-1 proteins, destabilizing the mitochondria and speeding up the development of apoptosis. Sorafenib treatment increased levels of Puma at mRNA and protein level and gene silencing with siRNA confirmed a relevant role for Puma in the induction of apoptosis. Co-treatment with the pan-caspase inhibitor Z-VAD-fmk prevented cell death to a variable degree depending on the cell line. In RPMI 8226 cells, Z-VAD-fmk prevented most of sorafenib-induced death. However, death in MM.1S was only prevented by co-incubation with both Z-VAD-fmk and the RIP1K inhibitor necrostatin-1, indicating that under conditions of inefficient caspase activation, sorafenib induces death by necroptosis. Conclusion: Our results demonstrate a key role for Puma in the triggering of sorafenib-induced apoptosis and that this drug can also induce death by necroptosis in multiple myeloma cells. [ABSTRACT FROM AUTHOR]

Bcl-2 and Bcl-XL belong to a family of proteins overexpressed in a variety of human cancers which inhibit apoptosis in response to a number of stimuli including chemotherapeutic agents and ionizing radiation. To better understand the role of these polypeptides in modulating the response of cancer cells to ionizing radiation we used cell lines that were engineered to overexpress the two polypeptides. Although Bcl-2 and Bcl-XL overexpression resulted in inhibition of radiation induced apoptosis, it did not result in enhanced clonogenic survival. Consistent with this was the observation that Bcl-2 and Bcl-XL protected cells from DNA fragmentation, loss of mitochondrial membrane potential, and caspase activation for up to 72 hours after irradiation. Beyond 72 hours, there was a rapid loss in the ability of Bcl-2 and Bcl-XL to inhibit these markers of apoptosis. When Bcl-XL was analyzed at 72 hours after irradiation and beyond, a rapid accumulation of a 16-kDa form of Bcl-XL was observed. To test the hypothesis that cleavage of the 29-kDa form of Bcl-XL by caspases to a 16-kDa polypeptide results in its inability to inhibit apoptosis beyond 72 hours, we constructed a cell line that overexpressed a caspase-resistant form of Bcl-XL (Bcl-XL-Δloop). Cells overexpressing Bcl-XL-Δloop were resistant to apoptosis beyond 72 hours after irradiation and did not contain the 16-kDa form at these time points. In addition, Bcl-XL-Δloop overexpression resulted in enhanced clonogenic survival compared with control or Bcl-XL overexpressing cells. These results provide a molecular basis for the observation that expression of Bcl-2 or Bcl-XL is not a prognostic marker of tumor response to cancer therapy.

Alternative splicing ( AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL- X mRNA. This, in turn, results in the selection of the proximal 5′ splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL- XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL- XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival. [ABSTRACT FROM AUTHOR]

The Bcl-2 family of proteins are well-characterized regulators of the intrinsic apoptotic pathway. Proteins within this family can be classified as either prosurvival or prodeath members and the balance between them present at the mitochondrial membrane is what determines if the cell lives or dies. Specific interactions among Bcl-2 family proteins play a crucial role in regulating programmed cell death. Structural studies have established a conserved interaction pattern among Bcl-2 family members. This interaction is mediated by the binding of the hydrophobic face of the amphipathic α-helical BH3 domain into a conserved hydrophobic groove on the prosurvival partners. It has been reported that an increase in the helical content of BH3 mimetic peptides considerably improves the binding affinity. In this context, this work states for designing peptides derived from the BH3 domain of the proapoptotic protein Bak by substitution of some non-interacting residues by the helical inducing residue Aib. Different synthetic peptides preserving BakBH3 relevant interactions were proposed and simulated presenting a better predicted binding energy and higher helical content than the wild type Bak peptide. [ABSTRACT FROM AUTHOR]

Here we report the H, C and N resonance assignments of free Bcl-x and of Bcl-x in complex with an azobenzene-modified peptide derived from the BH3 domain of the pro-apoptotic Bak. The spectra suggest predominantly folded proteins; chemical shift difference analysis provides a detailed view of the reorganization occurring on peptide binding. [ABSTRACT FROM AUTHOR]

Anti-apoptotic Bcl-2-family members not only neutralize pro-apoptotic proteins but also directly regulate intracellular Ca signaling from the endoplasmic reticulum (ER), critically controlling cellular health, survival, and death initiation. Furthermore, distinct Bcl-2-family members may selectively regulate inositol 1,4,5-trisphosphate receptor (IPR): Bcl-2 likely acts as an endogenous inhibitor of the IPR, preventing pro-apoptotic Ca transients, while Bcl-X likely acts as an endogenous IPR-sensitizing protein promoting pro-survival Ca oscillations. Furthermore, distinct functional domains in Bcl-2 and Bcl-X may underlie the divergence in IPR regulation. The Bcl-2 homology (BH) 4 domain, which targets the central modulatory domain of the IPR, is likely to be Bcl-2's determining factor. In contrast, the hydrophobic cleft targets the C-terminal Ca-channel tail and might be more crucial for Bcl-X's function. Furthermore, one amino acid critically different in the sequence of Bcl-2's and Bcl-X's BH4 domains underpins their selective effect on Ca signaling and distinct biological properties of Bcl-2 versus Bcl-X. This difference is evolutionary conserved across five classes of vertebrates and may represent a fundamental divergence in their biological function. Moreover, these insights open novel avenues to selectively suppress malignant Bcl-2 function in cancer cells by targeting its BH4 domain, while maintaining essential Bcl-X functions in normal cells. Thus, IPR-derived molecules that mimic the BH4 domain's binding site on the IPR may function synergistically with BH3-mimetic molecules selectivity suppressing Bcl-2's proto-oncogenic activity. Finally, a more general role for the BH4 domain on IPRs, rather than solely anti-apoptotic, may not be excluded as part of a complex network of molecular interactions. [ABSTRACT FROM AUTHOR]

Cattle persistently infected with a noncytopathic Bovine viral diarrhea virus (BVDV) are at risk of developing fatal “mucosal disease” (MD). The authors investigated the role of various apoptosis pathways in the pathogenesis of lesions in animals suffering from MD. Therefore, they compared the expression of caspase-3, caspase-8, caspase-9, and Bcl-2L1 (Bcl-x) in tissues of 6 BVDV-free control animals, 7 persistently infected (PI) animals that showed no signs of MD (non-MD PI animals), and 11 animals with MD and correlated the staining with the localization of mucosal lesions. Caspase-3 and -9 staining were markedly stronger in MD cases and were associated with mucosal lesions, even though non-MD PI animals and negative controls also expressed caspase-9. Conversely, caspase-8 was not elevated in any of the animals analyzed. Interestingly, Bcl-x also colocalized with mucosal lesions in the MD cases. However, Bcl-x was similarly expressed in tissues from all 3 groups, and thus, its role in apoptosis needs to be clarified. This study clearly illustrates ex vivo that the activation of the intrinsic, but not the extrinsic, apoptosis pathway is a key element in the pathogenesis of MD lesions observed in cattle persistently infected with BVDV. However, whether direct induction of apoptosis in infected cells or indirect effects induced by the virus are responsible for the lesions observed remains to be established. [ABSTRACT FROM PUBLISHER]

Mesenchymal Stem Cells (MSCs) have high therapeutic value for regenerative medicine and tissue engineering due to their differentiation potential and non-immunogenic characteristics. They are also considered as an effective in vivo delivery agent because of their ability to migrate to the site of injury. A major roadblock in their use for cell-based therapies is their rareness in vivo. Therefore, it is important to obtain increased number of functional MSCs in vitro in order to have adequate numbers for therapeutic regiments. We aimed to investigate the role of estrogen and its mechanism in obtaining more MSCs. MSCs were isolated from female and ovariectomized rats and cultured in the presence and absence of 10 M estrogen. In the presence of estrogen, not only their CFU-F activity increased but also apoptotic rate decreased as shown by TUNEL staining leading to obtain more MSCs. Also the number of the cells in the colonies increased upon estrogen treatment. To reveal the mechanism of this effect, we focused on Bcl-2 family of proteins. Our immunoblotting experiments combined with knockdown studies suggested a critical role for anti-apoptotic Bcl-x and Bcl-2. Estrogen treatment up regulated the expression Bcl-x and Bcl-2. When we knocked down the expression of bcl-x and bcl-2, MSCs lacking these genes showed an increase in the apoptotic rate in contrast to normal MSCs following estrogen treatment. Therefore, estrogen treatment will be of great advantage for cell-based therapies in order to get more functional MSCs and may provide opportunities to develop new strategies for debilitating diseases. [ABSTRACT FROM AUTHOR]

Abstract: Alternative splicing plays an important role in the control of apoptosis. A number of genes related to apoptosis undergo alternative splicing. Among them, the apoptotic regulator Bcl-x produces two major isoforms, Bcl-xL and Bcl-xS, through the alternative splicing of exon 2 in its pre-mRNA. These isoforms have antagonistic function in apoptotic pathway; Bcl-xL is pro-apoptotic, while Bcl-xS is anti-apoptotic. The balanced ratio of two isoforms is important for cell survival. However, regulatory mechanisms of Bcl-x splicing remain poorly understood. Using a mini-gene system, we have found that a 105nt exonic region (E3b) located within exon 3 affects exon 2 splicing in the Bcl-x gene. Further deletion and mutagenesis studies demonstrate that this 105nt sequence contains various functional elements which promote skipping of exon 2b. One of these elements forms a stem-loop structure that stimulates skipping of exon 2b. Furthermore our results prove that the stem-loop structure functions as an enhancer in general pre-mRNA splicing. We conclude that we have identified a cis-regulatory element in exon 3 that affects splicing of exon 2 in the Bcl-x gene. This element could be potentially targeted to alter the ratio of Bcl-xL and Bcl-xS for treatment of tumors through an apoptotic pathway. [Copyright &y& Elsevier]

It has been reported that exposure to infrasound causes cardiac dysfunction. Allowing for the key role of apoptosis in the pathogenesis of cardiovascular diseases, the objective of this study was to investigate the apoptotic effects of infrasound. Cardiac myocytes cultured from neonatal rats were exposed to infrasound of 5 Hz at 130 dB. The apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Also, the expression levels of a series of apoptosis-related proteins were detected. As a result, infrasound induced apoptosis of cultured rat cardiac myocytes in a time-dependant manner. The expression of proapoptotic proteins such as Bax, caspase-3, caspase-8, caspase-9, and FAS was significantly up-regulated, with concomitant down-regulated expression of antiapoptotic proteins such as Bcl-x, and the inhibitory apoptosis proteins family proteins including XIAP, cIAP-1, and cIAP-2. The expression of poly (ADP-ribose) polymerase and β-catenin, which are the substrate proteins of caspase-3, was significantly decreased. In conclusion, infrasound is an apoptotic inducer of cardiac myocytes. [ABSTRACT FROM AUTHOR]

Plasminogen Kringle 5(K5) is a proteolytic fragment of plasminogen, which displays potent anti-angiogenic activities. K5 has been shown to induce apoptosis in proliferating endothelial cells; however the exact mechanism has not been well explored. The present study was designed to elucidate the possible molecular mechanism of K5-induced endothelial cell apoptosis. Our results showed that K5 inhibited basic fibroblast growth factors activated in human umbilical vein endothelial cells (HUVECs), indicating proliferation in a dose-dependent manner and induced endothelial cell death via apoptosis. K5 exposure activated caspase 7, 8 and 9. These results suggested that both the intrinsic mitochondrial apoptosis pathway and extrinsic pathway might be involved in K5-induced apoptosis. K5 reduced mitochondrial membrane potential (MMP) of HUVECs, demonstrating mitochondrial depolarization in HUVECs. K5 increased the ratio of Bak to Bcl-x on mitochondria, decreased the ratio in cytosol, and had no effect on the total amounts of these proteins. K5 also did not effect on Bax/Bcl-2 distribution. K5 increased the ratio of Bak to Bcl-x on mitochondrial that resulted in mitochondrial depolarization, cytochrome c release and consequently the cleavage of caspase 9. These results suggested that K5 induces endothelial cell apoptosis at least in part via activating mitochondrial apoptosis pathway. The regulation of K5 on Bak and Bcl-x distribution may play an important role in endothelial cell apoptosis. These results provide further insight into the anti-angiogenesis roles of K5 in angiogenesis-related ocular diseases and solid tumors. [ABSTRACT FROM AUTHOR]

Abstract: To persist, a virus must co-exist with the host that it infects, thus allowing the virus to survive and to subvert the programmed cell death of the host. In this study, we investigated whether the intrinsic pathway of the apoptotic process is suppressed in a previously reported macrophage cell line persistently infected with respiratory syncytial virus (RSV). To this end, after using staurosporine to induce apoptosis, we determined cell viability and the degree of annexin staining and DNA fragmentation between infected and mock-infected cells. RSV persistence leads to a subversion of apoptosis; whereas in mock-infected macrophages, apoptosis was evident. The cellular apoptotic pathway involve was searched by determining the activities of caspases and the expression of anti-apoptotic proteins. Although caspases-3 and -9 were expressed, their activities were altered; the activity of caspase-3 was reduced and that of caspase-9 could not be detected. Expression of anti-apoptotic proteins Bcl-2, Bcl-X, and XIAP was enhanced, with Bcl-X and XIAP being regulated post-transcriptionally; the induction of the anti-apoptotic factors and the reduced caspases activities might account for the subversion of apoptosis. The data implies that in our viral persistence model an anti-apoptotic program is induced relating alterations of caspases-3 and -9 activity and expression of anti-apoptotic proteins, suggesting that the intrinsic pathway is suppressed. These findings are of importance for understanding the intracellular genes involved in subversion of apoptosis by RSV persistence in macrophages. [Copyright &y& Elsevier]

Nasal natural killer (NK)-cell lymphoma was resistant to various antitumor agents. Although high expression of p-glycoprotein has been reported, other molecular mechanism of the chemo-resistance is largely unknown. Activation of STAT3 and expression of major apoptosis-related proteins Bcl-2, Bcl-x, and Mcl-1 were analyzed by immunohistochemistry. Effects of STAT3 inhibitor AG490 on NK-YS cell line were analyzed by Western blotting and flow cytometric apoptosis assay. STAT3 was activated in six of the nine nasal NK-cell lymphomas (67%). In contrast, STAT3 activation was detected in 35% of diffuse large B-cell lymphoma (DLBCL) and in 10% of follicular lymphoma (FL). Frequent activation of STAT3 was significantly correlated with Mcl-1 expression in nasal NK-cell lymphoma, i.e., Mcl-1 was positive in five of six STAT3-active cases and negative in all three STAT3-inactive ones. In DLBCL, not only six out of seven STAT3-active cases (86%) but also eight out of thirteen STAT3-inactive cases (62%) were positive for Mcl-1 expression. Latent membrane protein-1 was positive in four nasal NK-cell lymphomas, among which three cases showed intermediate STAT3 activation. Inhibition of STAT3 activation by JAK inhibitor AG490 decreased Mcl-1 expression and induced apoptosis in STAT3-active NK-YS cells. Serum starvation rather increased the Mcl-1 level in NK-YS cells, and this effect was also canceled by AG490. These results suggest that activation of STAT3-Mcl-1 axis may play a role in the chemotherapy resistance of nasal NK-cell lymphoma. The pathway may be one of the future therapeutic targets of this intractable disease. [ABSTRACT FROM AUTHOR]

Elevated cell death in human preimplantation embryos is one of the cellular events compromising pregnancy rates after assisted reproductive technology treatments. We therefore explored the molecular pathways regulating cell death at the blastocyst stage in human embryos cultured in vitro. Owing to limited availability of human embryos, these pathways were further characterized in mouse blastocysts. Gene expression studies revealed a positive correlation between the cell death index and the expression of Bcl-x transcript. Cell death activation in human blastocysts was accompanied by changes in Bcl-x splicing, favoring production of Bcl-xS, an activator of cell death. Expression of Bcl-xS was detected in a subset of human blastocysts that show particular clustering in dying and/or dead cells. Altering the Bcl-xL/ Bcl-xS ratio in mouse embryos, in antisense experiments, confirmed that upregulation of Bcl-xS, with concomitant downregulation of Bcl-xL, compromised developmental potential and committed a subset of cells to undergoing cell death. This was accompanied by increased accumulation of reactive oxygen species levels without any impact on mtDNA content. In addition, altered Bcl-x splicing in favor of Bcl-xS was stimulated by culture in HTF medium or by addition of excessive glucose, leading to compromised embryo development. Thus, we conclude that inappropriate culture conditions affect Bcl-x isoform expression, contributing to compromised preimplantation embryo development. [ABSTRACT FROM AUTHOR]

Abstract: Bcl-x, a potent regulator of cellular decisions of life and death, has multiple survival-enhancing activities that rely on distinct protein regions. Evidence suggests that depending on the local environment and the binding of protein or peptide partners, Bcl-x can take on several conformations that expose different protein regions. However, biological occurrence of conformational forms has been very difficult to study, because structure determination techniques use large quantities of protein, purified under conditions that change Bcl-x conformation. We show here that standard 2D isoelectric focusing techniques can be used to distinguish conformationally distinct forms of Bcl-x in cell lysates. Conformational isoelectric forms were manipulated through the use of detergents and buffers of differing pH. Our data indicate that post-translational modifications are not needed for or associated with conformational changes, distinguishing the dominant isoelectric forms of Bcl-x. We found that Bcl-x conformational isoelectric forms have preferred subcellular localization patterns. Moreover, conformational forms are differently regulated in certain locations during cytokine starvation of IL-3-dependent cells. Therefore, we provide evidence that 2DIEF can be used to view biologically distinct conformational differences in Bcl-x on minute quantities of unpurified protein from cells or lysates. [Copyright &y& Elsevier]

Abstract: The antipsychotic drug haloperidol is still used to treat psychosis and “agitation”, often with devastating consequences, particularly in geriatric and pre-demented patients. Cytotoxicity induced by haloperidol has been associated with induction of Bcl-XS, a pro-apoptotic member of the Bcl-2 family, as well as with modulation of the Akt pro-survival pathway. Using preneuronal PC12 and primary neuronal cultures, we show that haloperidol inactivates Akt. This induces the dephosphorylation of serine residues in Bcl-XS and promotes its association with the mitochondrial voltage-dependent anion channel (VDAC), as well as with cytochrome c- and caspase-3-dependent events. These events are sensitive to expression of constitutively active Akt. Mutation of Serine106 (Ser106), which is flanked by a putative Akt motif, hinders the association of the Bcl-XS protein with Akt, but promotes its association with VDAC. The dephosphorylation mimic, Bcl-XS(Ser106Ala), induces caspase-dependent PC12 and neuronal cell apoptosis. In contrast, Bcl-XS(Ser106Ala) induces a significant loss of VDAC expression, and cytochrome c- and caspase-independent toxicity in the non-neuronal HEK293A cells. We link haloperidol and Akt to Bcl-XS-sensitive toxicity via cell line-dependent mitochondrial events centering on VDAC. This clearly mitigates the chronic use of haloperidol in neuropsychiatric populations, but supports its use as a potential acute therapeutic in cancer, where apoptosis is desirable. [Copyright &y& Elsevier]

Objectives: Nasal natural killer (NK)/T-cell lymphoma is characterized by chemo-resistance, angiodestruction, and aggressive tumor progression. Few studies exist on molecular characteristics of this disease entity. Methods: Expression levels of major apoptosis-related proteins Bcl-2, Bcl-x, Mcl-1, Bax, and a proliferative marker Ki-67 were analyzed in 11 nasal NK/T-cell lymphoma cases by immunohistochemical methods. Nine cases were of NK-cell lineage and two cases were of T-cell lineage. For comparison, 12 follicular lymphoma (FL) cases and 16 diffuse large B-cell lymphoma (DLBCL) cases were also studied. Results and conclusions: Bax expression was low in all nasal NK-cell lymphoma cases, which constitute the major population of nasal NK/T-cell lymphoma. Bax expression in nasal NK-cell lymphoma was similar to FL and significantly lower compared with DLBCL. Bcl-2 expression was significantly lower in nasal NK/T-cell lymphoma compared with that of FL and DLBCL. Bcl-x expression was high in all three lymphomas. Two distinct Mcl-1 expression groups existed for nasal NK/T-cell lymphoma (6.2 ± 5.2% and 59.1 ± 12.3%, 95% CI). Ki-67 expression was high in nasal NK/T-cell lymphoma, and worse prognostic groups tended to express higher levels of Ki-67. The results suggest a combination of impaired apoptosis and aggressive proliferation in nasal NK/T-cell lymphoma, and may provide explanations for its poor prognosis. [ABSTRACT FROM AUTHOR]