High Molecular Weight DNA Fragments Research Articles

Potential of Seaweed Extract on Growth, Physiological, Cytological and Biochemical Parameters of Wheat (Triticum aestivum L.) Seedlings

Synthetic fertilizers are widely employed in agriculture relative to biofertilizers. Long-term use affects soil fertility, plant vigor, human and livestock health, and the soil microbial community. Seaweeds were employed as biofertilizers in sustainable agriculture to overcome this issue and boost the agricultural productivity. Therefore, the current study investigates the effect of wheat seeds priming with the aqueous extracts of Ulva linza or Corallina officinalis seaweeds at various concentrations (5, 10, 15, 20, and 30%). Wheat grains were primed with seaweed extracts and grown in petri dishes, and the germination rate, growth attributes, physiological parameters, cytological traits, and molecular analyses were evaluated. The physico-chemical characterization revealed that U. linza possessed higher levels of ash, soluble proteins, and sugars, as well as essential macronutrients and amino acids, than C. officinalis. Seedling growth parameters, physiological characteristics, and mitotic index all showed a substantial increase in low concentrations of both extracts when compared to the control. Higher concentrations, on the other hand, exhibited an inhibitory effect as well as certain chromosomal abnormalities. In the treated seedlings, new proteins were expressed, which could be owing to the activity of bioactive components in the extracts. As compared to untreated seedlings, PCR-RAPD analysis utilizing five markers revealed that both seaweed extracts triggered low and high molecular weight DNA fragments, as well as fragments specific to each extract. Seaweed extracts could be employed as an environmentally friendly and soil-safe alternative to typical chemical fertilizers.

Whole human blood DNA degradation associated with artificial ultraviolet and solar radiations as a function of exposure time

Laboratory investigations were conducted to evaluate the effect of ultraviolet radiation components and solar radiation exposure as a function of time on the degradation of whole human blood DNA from the standpoint of forensic analysis. Ten μL of whole human male blood samples were exposed to UV-A, UV-B, UV-C, and solar radiation at 20 min intervals up to 120 min. Allele frequencies of 16 short tandem repeat (STR) markers were monitored by employing current forensic typing DNA techniques. The STR markers were grouped into high, medium, and low molecular weight categories. Results revealed that even 20 min exposure to 4.89 eV UV-C photons (ʎ = 254 nm) with radiation intensity of 1200 μW/cm2 would degrade whole human male blood DNA samples significantly, making them unfit for human identification due to the breakdown of high molecular weight STRs. Exposure of blood samples to 4.11 eV UV-B photons (ʎ = 302 nm) with radiation intensity of 900 μW/cm2 resulted in complete degradation of high molecular weight STRs after 60 min. Partial breakdown of medium and low molecular weight STRs started after 80 min exposure. The degradation index (DI) values appear to show that the degradation of the DNA template molecule was relatively less in the low molecular weight DNA fragments as compared with high molecular weight DNA fragments. This finding indicates that genetic profiles obtained from whole human male blood exposed to this radiation for 60 min will give inconclusive results. Samples exposed up to 120 min to 3.40 eV UV-A photons (ʎ = 365 nm) and 3.10–3.94 eV photons of solar radiation did not appear to produce appreciable degradation in any of three molecular weight STRs in the whole human blood DNA samples.

A Multiplex PCR-Based Assay for Detection of Plasma Cell Free DNA Integrity as a Marker of Bladder Cancer

Background: Cell free DNA (cfDNA) analysis in patient plasma has been suggested for screening of different cancers. Healthy cells may shed more degraded DNA than non-apoptotic tumor cells. Objectives: Our goal was to check whether DNA fragments extracted from patient’s plasma with bladder cancer (BC) had higher integrity than DNA purified from plasma of healthy controls. Methods: cfDNA was extracted from the plasma of a healthy control group and patients with BC. The association between clinical status and length of DNA fragments was examined. Plasma cfDNA integrity was examined using multiplex PCR with specific primers producing progressively long PCR fragments (200, 400 & 800-bp). Length and concentration of PCR amplicons obtained from BC patients and healthy individuals were compared. Results: Using a multiplex PCR assay, the used p53 primers efficiently amplified the increasingly long fragments. High molecular weight DNA fragments (400 & 800-bp) in the plasma cfDNA are associated with the presence of cancer. Moreover, elevation of the 200-bp fragment concentration in the plasma cfDNA is significantly (p < 0.002) associated with the presence of cancer. Conclusions: The detected long DNA fragments in plasma cfDNA is associated with BC indicating that the multiplex PCR assay of plasma cfDNA integrity could be a useful future marker for the detection of BC.

A Multiplex PCR-Based Assay for Detection of Plasma Cell Free DNA Integrity as a Marker of Bladder Cancer

Background: Cell free DNA (cfDNA) analysis in patient plasma has been suggested for screening of different cancers. Healthy cells may shed more degraded DNA than non-apoptotic tumor cells. Objectives: Our goal was to check whether DNA fragments extracted from patient’s plasma with bladder cancer (BC) had higher integrity than DNA purified from plasma of healthy controls. Methods: cfDNA was extracted from the plasma of a healthy control group and patients with BC. The association between clinical status and length of DNA fragments was examined. Plasma cfDNA integrity was examined using multiplex PCR with specific primers producing progressively long PCR fragments (200, 400 & 800-bp). Length and concentration of PCR amplicons obtained from BC patients and healthy individuals were compared. Results: Using a multiplex PCR assay, the used p53 primers efficiently amplified the increasingly long fragments. High molecular weight DNA fragments (400 & 800-bp) in the plasma cfDNA are associated with the presence of cancer. Moreover, elevation of the 200-bp fragment concentration in the plasma cfDNA is significantly (p < 0.002) associated with the presence of cancer. Conclusions: The detected long DNA fragments in plasma cfDNA is associated with BC indicating that the multiplex PCR assay of plasma cfDNA integrity could be a useful future marker for the detection of BC.

An accurate algorithm for the detection of DNA fragments from dilution pool sequencing experiments.

The short read lengths of current high-throughput sequencing technologies limit the ability to recover long-range haplotype information. Dilution pool methods for preparing DNA sequencing libraries from high molecular weight DNA fragments enable the recovery of long DNA fragments from short sequence reads. These approaches require computational methods for identifying the DNA fragments using aligned sequence reads and assembling the fragments into long haplotypes. Although a number of computational methods have been developed for haplotype assembly, the problem of identifying DNA fragments from dilution pool sequence data has not received much attention. We formulate the problem of detecting DNA fragments from dilution pool sequencing experiments as a genome segmentation problem and develop an algorithm that uses dynamic programming to optimize a likelihood function derived from a generative model for the sequence reads. This algorithm uses an iterative approach to automatically infer the mean background read depth and the number of fragments in each pool. Using simulated data, we demonstrate that our method, FragmentCut, has 25-30% greater sensitivity compared with an HMM based method for fragment detection and can also detect overlapping fragments. On a whole-genome human fosmid pool dataset, the haplotypes assembled using the fragments identified by FragmentCut had greater N50 length, 16.2% lower switch error rate and 35.8% lower mismatch error rate compared with two existing methods. We further demonstrate the greater accuracy of our method using two additional dilution pool datasets. FragmentCut is available from https://bansal-lab.github.io/software/FragmentCut. vibansal@ucsd.edu. Supplementary data are available at Bioinformatics online.

A Fosmid Pool-Based Next Generation Sequencing Approach to Haplotype-Resolve Whole Genomes.

Haplotype resolution of human genomes is essential to describe and interpret genetic variation and its impact on biology and disease. Our approach to haplotyping relies on converting genomic DNA into a fosmid library, which represents the entire diploid genome as a collection of haploid DNA clones of ~40 kb in size. These can be partitioned into pools such that the probability that the same pool contains both parental haplotypes is reduced to ~1 %. This is the key principle of this method, allowing entire pools of fosmids to be massively parallel sequenced, yielding haploid sequence output. Here, we present a detailed protocol for fosmid pool-based next generation sequencing to haplotype-resolve whole genomes including the following steps: (1) generation of high molecular weight DNA fragments of ~40 kb in size from genomic DNA; (2) fosmid cloning and partitioning into 96-well plates; (3) barcoded sequencing library preparation from fosmid pools for next generation sequencing; and (4) computational analysis of fosmid sequences and assembly into contiguous haploid sequences.This method can be used in combination with, but also without, whole genome shotgun sequencing to extensively resolve heterozygous SNPs and structural variants within genomic regions, resulting in haploid contigs of several hundred kb up to several Mb. This method has a broad range of applications including population and ancestry genetics, the clinical interpretation of mutations in personal genomes, the analysis of cancer genomes and highly complex disease gene regions such as MHC. Moreover, haplotype-resolved genome sequencing allows description and interpretation of the diploid nature of genome biology, for example through the analysis of haploid gene forms and allele-specific phenomena. Application of this method has enabled the production of most of the molecular haplotype-resolved genomes reported to date.

Abstract 5232: Changes in plasma circulating DNA composition during focal ablative therapy for liver cancer

Abstract Circulating cell-free DNA (cfDNA) may potentially serve as a diagnostic or therapeutic biomarker. We tested our established methodologies for cfDNA profiling on blood samples obtained during focal ablative treatments for liver cancer, given that such interventions may cause abrupt spillage of circulating tumor DNA (ctDNA) into the bloodstream. Following written informed consent, serial blood samples from 13 patients with hepatocellular carcinoma (HCC) were drawn into cfDNA-optimized collection tubes before treatment, 2-3 hours post-treatment, and a few weeks after. Nine patients underwent radiofrequency ablation and 4 patients received transarterial chemoembolization. The cfDNA concentration was assessed by quantitative PCR of Alu-amplicons (Alu-101 and Alu-226) on 13 pre-treatment samples, 12 immediate post-treatment samples and 8 follow-up samples. Aberrant cfDNA methylation at genes GSTP1 and RASSF1 was assessed by pyrosequencing to investigate tumor-specific cfDNA. Mean cfDNA levels of patients with HCC increased by an average of 3.6-fold (up to 9.3-fold) from baseline immediately after treatment (p = 0.0019). Levels measured 1-15 weeks after treatment returned to on average 87% of the baseline. Each specimen contained both high molecular weight DNA (consistent with abrupt release of DNA from disrupted cells) and smaller apoptotic cfDNA fragments that ranged in size of 160-180 bp. DNA integrity (Alu-226/Alu-101) was similar before treatment, immediately after, and in the follow-up samples. Compared to the paired leukocyte DNA methylation levels, higher methylation levels at RASSF1 and/or GSTP1 were observed in all 13 pre-treatment cfDNA samples, supporting at least partial tumor origin of the isolated cfDNA. RASSF1 methylation moderately correlated with GSTP1 methylation (r = 0.55, p&amp;lt;0.001). Excluding one patient with exceptionally high RASSF1 and GSTP1 methylation levels, the averaged amount of RASSF1/GSTP1-methylated ctDNA correlated with the tumor size prior to the procedure (r = 0.72, p = 0.008), while no association was found between the AFP levels and tumor burden. The characteristics of cfDNA in cancer patients may change significantly following therapeutic interventions. We have established an improved workflow for cfDNA quantification and characterization in plasma that can be applied to the peri-treatment setting and are in the process of evaluating ctDNA analysis protocols for EDTA blood as well. These preliminary observations in a limited number of specimens support the potential of quantifying ctDNA as a marker of tumor cell destruction. Preliminary observation of a correlation between aberrantly-methylated cfDNA levels and HCC tumor size suggests that pre-treatment cfDNA profiling may inform on tumor burden. Further research will be needed to validate the clinical significance of the quantitative and epigenetic changes in cfDNA samples from patients with HCC. Citation Format: Maarit I. Tiirikainen, Karolina Peplowska, Min-Ae Song, Kyle Miyazaki, Linda L. Wong, Sandi A. Kwee. Changes in plasma circulating DNA composition during focal ablative therapy for liver cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5232. doi:10.1158/1538-7445.AM2015-5232

Pulsed-field gel electrophoresis for Leuconostoc mesenteroides and L. pseudomesenteroides.

Pulsed-field gel electrophoresis (PFGE) is a technique using alternating electric fields to help the separation of high molecular weight DNA fragments with a high resolution. This method consists of the digestion of bacterial chromosomal DNA with rare-cutting restriction enzymes and of applying an alternating electrical current between spatially distinct pairs of electrodes. DNA molecules migrate at different speeds according to the size of the fragment. This method is considered as the "gold standard" for genotyping, genetic fingerprinting, epidemiological studies, genome size estimation, and studying radiation-induced DNA damage and repair.

Pulsed field gel electrophoresis for dairy propionibacteria.

Pulsed field gel electrophoresis (PFGE) is a technique using alternating electric fields to migrate high molecular weight DNA fragments with a high resolution. This method consists of the digestion of bacterial chromosomal DNA with rare-cutting restriction enzymes and in applying an alternating electrical current between spatially distinct pairs of electrodes. DNA molecules migrate at different speeds according to the size of the fragments. Among other things, this technique is considered as the "gold standard" for genotyping, genetic fingerprinting, epidemiological studies, genome size estimation, and studying radiation-induced DNA damage and repair. This chapter describes a PFGE method that can be used to differentiate dairy propionibacteria.

Synthesis of Nanoparticles Using Euphorbia prostrata Extract Reveals a Shift from Apoptosis to G0/G1 Arrest in Leishmania donovani

The aim of the present investigation was to synthesize silver (Ag) and titanium dioxide (TiO2) nanoparticles (NPs) using the aqueous leaves extract of Euphorbia prostrata as antileishmanial agents and to explore the mechanism of induced cell death. In vitro antileishmanial activity of synthesized NPs was tested against promastigotes of Leishmania donovani by alamar Blue® cell viability reagent and propidium iodide uptake assay. The effective leishmanicidal activity of synthesized Ag NPs was further confirmed by cell cycle progression, externalized phosphatidylserine, DNA fragmentation assay, reactive oxygen species (ROS) level, intracellular non-protein thiols and transmission electron microscopy (TEM) of the treated parasites. TEM analysis of the synthesized Ag NPs and TiO2 NPs showed spherical shape with an average size of 12.82 ± 2.50 and 83.22 ± 1.50 nm, respectively. Ag NPs was found to be the most active agent against Leishmania parasites after 24 h exposure with IC50 value of 14.94 μg/mL. A significant increase in G0/ G1 phase of the cell cycle with subsequent decrease in S and G2/M phases was observed when compared to control and thus confirming the growth inhibitory effect of synthesized Ag NPs. Decreased ROS level was also observed which could be responsible for caspase independent shift from apoptosis (G0/G1 arrest) to massive necrosis. High molecular weight DNA fragmentation as a positive consequence of necrotic cell death was also visualized. In the present study, the unique trypanothione/trypanothione reductase (TR) system of Leishmania cells was significantly inhibited by synthesized Ag NPs was reported. The green synthesized Ag NPs may provide promising leads for the development of cost effective and safer alternative treatment against visceral leishmaniasis.

Abstract 1179: Nelarabine front-line therapy for adult T-lymphoblastic leukemia/lymphoma (T-LBL/ALL): preliminary results of a single center experience.

Abstract Background. Precursor T cell LBL/ALL occurs most frequently in late childhood, adolescence, and young adulthood, with a 2:1 male predominance; it comprises 15 and 25 percent of childhood and adult ALL, respectively, and 2 percent of adult non-Hodgkin lymphoma. Nelarabine is an anticancer prodrug of arabinofuranosylguanine (ara-G); it inhibits DNA synthesis and leads to high molecular weight DNA fragmentation and cell death. Nelarabine has showed relevant efficacy in phase II clinical trials, both in pediatric and in adult LBL/ALLpopulations. Design and Methods. We report clinical outcome results of 9 newly diagnosed and younger than 60 years T-ALL patients (median age 29 years, range 22-45 years, 3/6 M/F, 8 T-ALL, 1 T-LBL) treated according to pediatric-like adapted schedule. Cytogentics data and molecular biologic features will be provided on site. Induction cycle included Vincristine, daunoblastine, L-asparaginase and Prednisone. After induction, all the patients received consolidation therapy with cyclophosphamyde, L-asparaginase, Cytarabine and 6-Mercaptopurine. Subsequently all the patients receveid Nelarabine 1500 mg/sqm (days 1-3-5 every 21) for two cycles. All the patients shared the same strategy for intensification, which consisted in allogenic stem cell transplantation, if available, or additional courses of consolidation chemotherapy. Durations of complete remission (CR) and overall survival (OS) were estimated according to the Kaplan-Meier method. The CR duration was dated from start of CR to first evidence of disease recurrence. Results. After a single induction course, 9/9 patients obtained a CR (100%). Eight patients underwent an allogenic bone marrow transplantation. After a median follow-up of 24 months, 7/9 patients (78%) are alive in CR. The median CR duration and OS were 13.4 and 24.4 months, respectively. Neurological toxicity of grade 3 has not been reported. We did not observe grade 3-4 haematological toxicity. Conclusion. Nelarabine is a promising drug, which induces a remarkable complete remission rate at the expense of a very low and manageable toxicity. Acknowledgments. Work supported by European LeukemiaNet, AIRC, AIL, PRIN, University of Bologna and BolognAIL. Citation Format: Chiara Sartor, Mariachiara Abbenante, Cristina Papayannidis, Ilaria Iacobucci, Stefania Paolini, Emanuela Ottaviani, Annalisa Lonetti, Sarah Parisi, Anna Ferrari, Viviana Guadagnuolo, Sandro Grilli, Nicoletta Testoni, Michele Cavo, Giovanni Martinelli. Nelarabine front-line therapy for adult T-lymphoblastic leukemia/lymphoma (T-LBL/ALL): preliminary results of a single center experience. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1179. doi:10.1158/1538-7445.AM2013-1179

Hypoxic stress triggers a programmed cell death pathway to induce vascular cavity formation in Pisum sativum roots.

Flooding at warm temperatures induces hypoxic stress in Pisum sativum seedling roots. In response, some undifferentiated cells in the primary root vascular cylinder start degenerating and form a longitudinal vascular cavity. Changes in cellular morphology and cell wall ultrastructure detected previously in the late stages of cavity formation suggest possible involvement of programmed cell death (PCD). In this study, cytological events occurring in the early stages of cavity formation were investigated. Systematic DNA fragmentation, a feature of many PCD pathways, was detected in the cavity-forming roots after 3 h of flooding in situ by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay and in isolated total DNA by gel electrophoresis. High molecular weight DNA fragments of about 20-30 kb were detected by pulse-field gel electrophoresis, but no low-molecular weight internucleosomal DNA fragments were detected by conventional gel electrophoresis. Release of mitochondrial cytochrome c protein into the cytosol, an integral part of mitochondria-dependent PCD pathways, was detected in the cavity-forming roots within 2 h of flooding by fluorescence microscopy of immunolabeled cytochrome c in situ and in isolated mitochondrial and cytosolic protein fractions by western blotting. DNA fragmentation and cytochrome c release remained confined to the undifferentiated cells in center of the root vascular cylinders, even after 24 h of flooding, while outer vascular cylinder cells and cortical cells maintained cellular integrity and normal activity. These findings confirm that hypoxia-induced vascular cavity formation in P. sativum roots involves PCD, and provides a chronological model of cytological events involved in this rare and understudied PCD system.

A simplified, non-invasive fecal-based DNA integrity assay and iFOBT for colorectal cancer detection

Neoplasia cells exfoliated from colorectal epithelium have dysfunctional apoptotic mechanisms, thus it is possible to identify high-molecular weight DNA fragments in feces. This prospective single-center study was performed to evaluate the sensitivity and specificity of fecal-based DNA integrity versus immunological fecal occult blood test (iFOBT) and calprotectin for colorectal cancer (CRC) and adenoma detection. Feces were collected from 204 subjects and DNA integrity was quantified by quantitative-denaturing high performance liquid chromatography (QdHPLC). Calprotectin and iFOBT were assessed using commercial kits. The diagnostic performance was calculated by receiver operating characteristic (ROC) curves analysis. A total of 192 fecal specimens were analyzed and 12 samples were excluded due to DNA degradation. We found long DNA (L-DNA) occurrence in feces with a sensitivity of 86% (n = 24/28) and a specificity of 81% for CRC detection. To minimize false-positive cases of the developed test, area under the curve of ROC was evaluated such that the specificity was increased to 92% with decreased sensitivity to 79%, p = 0.0001 for CRC detection. iFOBT was positive in 51% (n = 14/27) while calprotectin was positive in 75% (n = 18/27). The combination of iFOBT and L-DNA identified a greater number of CRC cases with a sensitivity of 89% and a specificity of 95%, p < 0.001. The combination also improved the sensitivity of polyps, particularly high-grade dysplasia and advanced adenoma (33%, p = 0.0015) as opposed to a single evaluation assay (17-21%). This study illustrates the usefulness of fecal DNA integrity assay by QdHPLC as a non-invasive, easy-to-perform, and reproducible method with a high level of sensitivity in detecting individuals with colorectal neoplasia. Combination of iFOBT and L-DNA improves the sensitivity for CRC and adenoma detection.

Methadone induces CAD degradation and AIF-mediated necrotic-like cell death in neuroblastoma cells

Methadone ( d, l-methadone hydrochloride) is a full-opioid agonist, originally developed as a substitution for heroin or other opiates abusers. Nowadays methadone is also being applied as long-lasting analgesics in cancer, and it is proposed as a promising agent for leukemia therapy. Previously, we have demonstrated that high concentrations of methadone (0.5 mM) induced necrotic-like cell death in SH-SY5Y cells. The pathway involved is caspase-independent but involves impairment of mitochondrial ATP synthesis and mitochondrial cytochrome c release. However, the downstream mitochondrial pathways remained unclear. Here, we studied the participation of apoptosis inducing factor (AIF) in methadone-induced cell death. Methadone resulted in a translocation of AIF from mitochondria to the nucleus. Translocation was inhibited by cyclosporine A, but not by lack of Bax protein. Therefore the effect seems mediated by the formation of the mitochondrial transition pore, but is apparently independent of Bax. Furthermore, methadone-treated SH-SY5Y nuclei show characteristics that are typical for stage I nuclear condensation. Methadone did not induce degradation of DNA into oligonucleosomal fragments or into high molecular weight DNA fragments. Absence of DNA fragmentation coincided with a considerable decrease in the levels of the caspase-actived endonuclase DNase and its chaperone-inhibitor ICAD. In conclusion, our results provide mechanistic insights into the molecular mechanisms that underlie methadone-induced cell death. This knowledge may prove useful to develop novel strategies to prevent toxic side-effects of methadone thereby sustaining its use as therapeutical agent against tumors.

A Highly Toxic Cellular Prion Protein Induces a Novel, Nonapoptotic Form of Neuronal Death

Several different deletions within the N-terminal tail of the prion protein (PrP) induce massive neuronal death when expressed in transgenic mice. This toxicity is dose-dependently suppressed by coexpression of full-length PrP, suggesting that it results from subversion of a normal physiological activity of cellular PrP. We performed a combined biochemical and morphological analysis of Tg(DeltaCR) mice, which express PrP carrying a 21-aa deletion (residues 105-125) within a highly conserved region of the protein. Death of cerebellar granule neurons in Tg(DeltaCR) mice is not accompanied by activation of either caspase-3 or caspase-8 or by increased levels of the autophagy marker, LC3-II. In electron micrographs, degenerating granule neurons displayed a unique morphology characterized by heterogeneous condensation of the nuclear matrix without formation of discrete chromatin masses typical of neuronal apoptosis. Our data demonstrate that perturbations in PrP functional activity induce a novel, nonapoptotic, nonautophagic form of neuronal death whose morphological features are reminiscent of those associated with excitotoxic stress.

DNA fingerprints of sheep using an M13 probe

The bacteriophage M13 DNA was used to detect hypervariable minisatellites in several families of Booroola sheep as well as Merino and Suffolk sheep. Digestion of sheep DNA gave rise to three to eight fragments with different restriction enzymes demonstrating considerable polymorphism between the different breeds. The length of informative DNA fragments varied in size from 6 to 20kb. The DNA fingerprints generated were individual specific and allowed for differentiation between closely related animals. The pattern obtained with sheep DNA was different from that observed with humans and other vertebrates in the proportion of high molecular weight DNA fragments present. Pedigree analysis of DNA patterns of dams and their offspring for several sets of twins and triplets showed a clear distinction between individuals and failed to reveal the presence of monozygosity.

Nelarabine

Nelarabine is an anticancer prodrug of arabinofuranosylguanine (ara-G), which is metabolized in cells to the cytotoxic metabolite ara-G triphosphate (ara-GTP). Ara-GTP competes with deoxyguanosine triphosphate for incorporation into DNA. Once incorporated, it inhibits DNA synthesis and leads to high molecular weight DNA fragmentation and cell death. In paediatric and adult patients with T-cell acute lymphoblastic leukaemia or T-cell lymphoblastic lymphoma, nelarabine induced a complete response, with or without complete haematological recovery, in approximately one-fifth of patients who had not responded to, or had relapsed following treatment with, two or more prior chemotherapy regimens. The median overall survival time was 13.1 and 20.6 weeks in paediatric and adult patients, with corresponding 1-year survival rates of 14% and 29%. Treatment-emergent adverse events were common, but non-haematological events were mostly of mild or moderate severity. Neurological events, which may be severe and irreversible, were the most likely adverse events to limit treatment.

The RNA binding protein RNPS1 alleviates ASF/SF2 depletion-induced genomic instability

Formation of transcription-induced R-loops poses a critical threat to genomic integrity throughout evolution. We have recently shown that the SR protein ASF/SF2 prevents R-loop formation in vertebrates by cotranscriptionally binding to nascent mRNA precursors to prevent their reassociation with template DNA. Here, we identify another RNA binding protein, RNPS1, that when overexpressed strongly suppresses the high molecular weight (HMW) DNA fragmentation, hypermutation, and G2 cell cycle arrest phenotypes of ASF/SF2-depleted cells. Furthermore, ablation of RNPS1 by RNA interference in HeLa cells leads to accumulation of HMW DNA fragments. As ASF/SF2 depletion does not influence RNPS1 expression, and RNPS1 cannot compensate for ASF/SF2 function in splicing, our data suggest that RNPS1 is able to function together with ASF/SF2 to form RNP complexes on nascent transcripts, and thereby prevent formation of transcriptional R-loops.

Impairment of the mitochondrial respiratory enzyme activity triggers sequential activation of apoptosis‐inducing factor‐dependent and caspase‐dependent signaling pathways to induce apoptosis after spinal cord injury

The mitochondrion participates in caspase-independent or caspase-dependent apoptotic pathways through the release of apoptosis-inducing factor or cytochrome c. Whether both mitochondrial apoptotic cascades are triggered in the injured spinal cord remains unknown. Here, we demonstrated that neurons, astrocytes and microglia in spinal segments proximal to a complete spinal cord transection underwent two phases of apoptotic cell death. The early phase of high-molecular weight (HMW) DNA fragmentation was associated with nuclear translocation of apoptosis-inducing factor, reduction in mitochondrial respiratory chain enzyme activity and decrease in cellular ATP concentration. The delayed phase of low-molecular weight (LMW) DNA fragmentation was accompanied by cytosolic release of cytochrome c, activation of caspases 9 and 3, and resumption of mitochondrial respiratory functions and ATP contents. Microinfusion of coenzyme Q(10), an electron carrier in mitochondrial respiratory chain, into the epicenter of the transected spinal cord attenuated both phases of induced apoptosis, and reversed the elicited mitochondrial dysfunction, bioenergetic failure, and activation of apoptosis-inducing factor, cytochrome c, or caspases 9 and 3. We conclude that mitochondrial dysfunction after spinal cord transection represents the initiating cellular events that trigger the sequential activation of apoptosis-inducing factor-dependent and caspase-dependent signaling cascades, leading to apoptotic cell death in the injured spinal cord.

Dynamic Role of Postsynaptic Caspase-3 and BIRC4 in Zebra Finch Song-Response Habituation

Activation of the protease caspase-3 is commonly thought to cause apoptotic cell death. Here, we show that caspase-3 activity is regulated at postsynaptic sites in brain following stimuli associated with memory (neural activation and subsequent response habituation) instead of cell death. In the zebra finch auditory forebrain, the concentration of caspase-3 active sites increases briefly within minutes after exposure to tape-recorded birdsong. With confocal and immunoelectron microscopy, we localize the activated enzyme to dendritic spines. The activated caspase-3 protein is present even in unstimulated brain but bound to an endogenous inhibitor, BIRC4 (xIAP), suggesting a mechanism for rapid release and sequestering at specific synaptic sites. Caspase-3 activity is necessary to consolidate a persistent physiological trace of the song stimulus, as demonstrated using pharmacological interference and the zenk gene habituation assay. Thus, the brain appears to have adapted a core component of cell death machinery to serve a unique role in learning and memory.