Synergistic Nanomedicine Delivering Topoisomerase I Toxin (SN-38) and Inhibitors of Polynucleotide Kinase 3'-Phosphatase (PNKP) for Enhanced Treatment of Colorectal Cancer.

Inhibitors of a DNA repair enzyme known as polynucleotide kinase 3'-phosphatase (PNKP) are expected to show synergistic cytotoxicity in combination with topoisomerase I (TOP1) inhibitors in cancer. In this study, the synergistic cytotoxicity of a novel inhibitor of PNKP, i.e., A83B4C63, with a potent TOP1 inhibitor, i.e., SN-38, against colorectal cancer cells was investigated. Polymeric micelles (PMs) for preferred tumor delivery of A83B4C63, developed through physical encapsulation of this compound in methoxy poly(ethylene oxide)-poly(α-benzyl carboxylate-ε-caprolactone) (mPEO-b-PBCL) micelles, were combined with SN-38 in free or PM form. The PM form of SN-38 was prepared through chemical conjugation of SN-38 to the functional end group of mPEO-b-PBCL and further assembly of mPEO-b-PBCL-SN-38 in water. Moreover, mixed micelles composed of mPEO-b-PBCL and mPEO-b-PBCL-SN-38 were used to co-load A83B4C63 and SN-38 in the same nanoformulation. The loading content (% w/w) of the SN-38 and A83B4C63 to mPEO-b-PBCL in the co-loaded formulation was 7.91 ± 0.66 and 16.13 ± 0.11% (w/w), respectively, compared to 15.67 ± 0.34 (% w/w) and 23.06 ± 0.63 (% w/w) for mPEO-b-PBCL micelles loading individual drugs. Notably, the average diameter of PMs co-encapsulating both SN-38 and A83B4C63 was larger than that of PMs encapsulating either of these compounds alone but still lower than 60 nm. The release of A83B4C63 from PMs co-encapsulating both drugs was 76.36 ± 1.41% within 24 h, which was significantly higher than that of A83B4C63-encapsulated micelles (42.70 ± 0.72%). In contrast, the release of SN-38 from PMs co-encapsulating both drugs was 44.15 ± 2.61% at 24 h, which was significantly lower than that of SN-38-conjugated PMs (74.16 ± 3.65%). Cytotoxicity evaluations by the MTS assay as analyzed by the Combenefit software suggested a clear synergy between PM/A83B4C63 (at a concentration range of 10-40 µM) and free SN-38 (at a concentration range of 0.001-1 µM). The synergistic cytotoxic concentration range for SN-38 was narrowed down to 0.1-1 or 0.01-1 µM when combined with PM/A83B4C63 at 10 or 20-40 µM, respectively. In general, PMs co-encapsulating A83B4C63 and SN-38 at drug concentrations within the synergistic range (10 µM for A83B4C63 and 0.05-1 µM for SN-38) showed slightly less enhancement of SN-38 anticancer activity than a combination of individual micelles, i.e., A83B4C63 PMs + SN-38 PMs at the same molar concentrations. This was attributed to the slower release of SN-38 from the SN-38 and A83B4C63 co-encapsulated PMs compared to PMs only encapsulating SN-38. Cotreatment of cells with TOP1 inhibitors and A83B4C63 formulation enhanced the expression level of γ-HA2X, cleaved PARP, caspase-3, and caspase-7 in most cases. This trend was more consistent and notable for PMs co-encapsulating both A83B4C63 and SN-38. The overall result from the study shows a synergy between PMs of SN-38 and A83B4C63 as a mixture of two PMs for individual drugs or PMs co-encapsulating both drugs.

A Sensitive Technique Unravels the Kinetics of Activation and Trans-Cleavage of CRISPR-Cas Systems.

Activation of the CRISPR-Cas13a system requires the formation of a crRNA-Cas13a ribonucleoprotein (RNP) complex and the binding of an RNA activator to the RNP. These two binding processes play a crucial role in the performance of the CRISPR-Cas13a system. However, the binding kinetics remain poorly understood, and a main challenge is the lack of a sensitive method for real-time measurements of the dynamically formed active CRISPR-Cas13a enzyme. We describe here a new method to study the binding kinetics and report the rate constants (kon and koff) and dissociation constant (Kd) for the binding between Cas13a and its activator. The method is able to unravel and quantify the kinetics of binding and cleavage separately, on the basis of measuring the real-time trans-cleavage rates of the CRISPR-Cas system and obtaining the real-time concentrations of the active CRISPR-Cas ternary complex. We further discovered that once activated, the Cas13a system operates at a wide range of temperatures (7-37 °C) with fast trans-cleavage kinetics. The new method and findings are important for diverse applications of the Cas13a system, such as the demonstrated quantification of microRNA at ambient temperatures (e.g., 25 °C).

Development of a DNAzyme Walker for the Detection of APE1 in Living Cancer Cells.

DNAzyme walker technology is a compelling option for bioanalytical and drug delivery applications. While nucleic acid and protein targets have been used to activate DNAzyme walkers, investigations into enzyme-triggered DNAzyme walkers in living cells are still in their early stages. The base excision repair (BER) pathway presents an array of enzymes that are overexpressed in cancer cells. Here, we introduce a DNAzyme walker system that sensitively and specifically detects the BER enzyme apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1). We constructed the DNAzyme walker on the surface of 20 nm-diameter gold nanoparticles. We achieved a detection limit of 160 fM of APE1 in a buffer and in whole cell lysate equivalent to the amount of APE1 in a single HeLa cell in a sample volume of 100 µL. Confocal imaging of the DNAzyme walking reveals a cytoplasmic distribution of APE1 in HeLa cells. Walking activity is tunable to exogenous Mn2+ concentrations and the uptake of the DNAzyme walker system does not require transfection assistance. We demonstrate the investigative potential of the DNAzyme walker for up-regulated or overactive enzyme biomarkers of the BER pathway in cancer cells.

Zika Virus Induces Mitotic Catastrophe in Human Neural Progenitors by Triggering Unscheduled Mitotic Entry in the Presence of DNA Damage While Functionally Depleting Nuclear PNKP.

Vertical transmission of Zika virus (ZIKV) leads with high frequency to congenital ZIKV syndrome (CZS), whose worst outcome is microcephaly. However, the mechanisms of congenital ZIKV neurodevelopmental pathologies, including direct cytotoxicity to neural progenitor cells (NPC), placental insufficiency, and immune responses, remain incompletely understood. At the cellular level, microcephaly typically results from death or insufficient proliferation of NPC or cortical neurons. NPC replicate fast, requiring efficient DNA damage responses to ensure genome stability. Like congenital ZIKV infection, mutations in the polynucleotide 5'-kinase 3'-phosphatase (PNKP) gene, which encodes a critical DNA damage repair enzyme, result in recessive syndromes often characterized by congenital microcephaly with seizures (MCSZ). We thus tested whether there were any links between ZIKV and PNKP. Here, we show that two PNKP phosphatase inhibitors or PNKP knockout inhibited ZIKV replication. PNKP relocalized from the nucleus to the cytoplasm in infected cells, colocalizing with the marker of ZIKV replication factories (RF) NS1 and resulting in functional nuclear PNKP depletion. Although infected NPC accumulated DNA damage, they failed to activate the DNA damage checkpoint kinases Chk1 and Chk2. ZIKV also induced activation of cytoplasmic CycA/CDK1 complexes, which trigger unscheduled mitotic entry. Inhibition of CDK1 activity inhibited ZIKV replication and the formation of RF, supporting a role of cytoplasmic CycA/CDK1 in RF morphogenesis. In brief, ZIKV infection induces mitotic catastrophe resulting from unscheduled mitotic entry in the presence of DNA damage. PNKP and CycA/CDK1 are thus host factors participating in ZIKV replication in NPC, and pathogenesis to neural progenitor cells. IMPORTANCE The 2015-2017 Zika virus (ZIKV) outbreak in Brazil and subsequent international epidemic revealed the strong association between ZIKV infection and congenital malformations, mostly neurodevelopmental defects up to microcephaly. The scale and global expansion of the epidemic, the new ZIKV outbreaks (Kerala state, India, 2021), and the potential burden of future ones pose a serious ongoing risk. However, the cellular and molecular mechanisms resulting in microcephaly remain incompletely understood. Here, we show that ZIKV infection of neuronal progenitor cells results in cytoplasmic sequestration of an essential DNA repair protein itself associated with microcephaly, with the consequent accumulation of DNA damage, together with an unscheduled activation of cytoplasmic CDK1/Cyclin A complexes in the presence of DNA damage. These alterations result in mitotic catastrophe of neuronal progenitors, which would lead to a depletion of cortical neurons during development.

Optimized Carbohydrate-Based Nanogel Formulation to Sensitize Hypoxic Tumors.

Solid tumors are often poorly vascularized, which impairs oxygen supply and drug delivery to the cells. This often leads to genetic and translational adaptations that promote tumor progression, invasion, metastasis, and resistance to conventional chemo-/radiotherapy and immunotherapy. A hypoxia-directed nanosensitizer formulation of a hypoxia-activated prodrug (HAP) was developed by encapsulating iodoazomycin arabinofuranoside (IAZA), a 2-nitroimidazole nucleoside-based HAP, in a functionally modified carbohydrate-based nanogel, facilitating delivery and accrual selectively in the hypoxic head and neck and prostate cancer cells. Although IAZA has been reported as a clinically validated hypoxia diagnostic agent, recent studies have pointed to its promising hypoxia-selective anti-tumor properties, which make IAZA an excellent candidate for further exploration as a multimodal theranostic of hypoxic tumors. The nanogels are composed of a galactose-based shell with an inner core of thermoresponsive (di(ethylene glycol) methyl ethyl methacrylate) (DEGMA). Optimization of the nanogels led to high IAZA-loading capacity (≅80-88%) and a slow time-controlled release over 50 h. Furthermore, nanoIAZA (encapsulated IAZA) displayed superior in vitro hypoxia-selective cytotoxicity and radiosensitization in comparison to free IAZA in the head and neck (FaDu) and prostate (PC3) cancer cell lines. The acute systemic toxicity profile of the nanogel (NG1) was studied in immunocompromised mice, indicating no signs of toxicity. Additionally, growth inhibition of subcutaneous FaDu xenograft tumors was observed with nanoIAZA, demonstrating that this nanoformulation offers a significant improvement in tumor regression and overall survival compared to the control.

Biodistribution and Activity of EGFR Targeted Polymeric Micelles Delivering a New Inhibitor of DNA Repair to Orthotopic Colorectal Cancer Xenografts with Metastasis.

The disruption of polynucleotide kinase/phosphatase (PNKP) in colorectal cancer (CRC) cells deficient in phosphatase and tensin homolog (PTEN) is expected to lead to the loss of cell viability by a process known as synthetic lethality. In previous studies, we have reported on the encapsulation of a novel inhibitor of PNKP, namely, A83B4C63, in polymeric micelles and its activity in slowing the growth of PTEN-deficient CRC cells as well as subcutaneous xenografts. In this study, to enhance drug delivery and specificity to CRC tumors, the surface of polymeric micelles carrying A83B4C63 was modified with GE11, a peptide targeting epidermal growth factor receptor (EGFR) overexpressed in about 70% of CRC tumors. Using molecular dynamics (MD) simulations, we assessed the binding site and affinity of GE11 for EGFR. The GE11-modified micelles, tagged with a near-infrared fluorophore, showed enhanced internalization by EGFR-overexpressing CRC cells in vitro and a trend toward increased primary tumor homing in an orthotopic CRC xenograft in vivo. In line with these observations, the GE11 modification of polymeric micelles was shown to positively contribute to the improved therapeutic activity of encapsulated A83B4C63 against HCT116-PTEN-/- cells in vitro and that of orthotopic CRC xenograft in vivo. In conclusion, our results provided proof of principle evidence for the potential benefit of EGFR targeted polymeric micellar formulations of A83B4C63 as monotherapeutics for aggressive and metastatic CRC tumors but at the same time highlighted the need for the development of EGFR ligands with improved physiological stability and EGFR binding.

Domain analysis of PNKP-XRCC1 interactions: Influence of genetic variants of XRCC1.

Polynucleotide kinase/phosphatase (PNKP) and X-ray repair cross-complementing 1 (XRCC1) are key proteins in the single-strand DNA break repair pathway. Phosphorylated XRCC1 stimulates PNKP by binding to its forkhead-associated (FHA) domain, whereas nonphosphorylated XRCC1 stimulates PNKP by interacting with the PNKP catalytic domain. Here, we have further studied the interactions between these two proteins, including two variants of XRCC1 (R194W and R280H) arising from single-nucleotide polymorphisms (SNPs) that have been associated with elevated cancer risk in some reports. We observed that interaction of the PNKP FHA domain with phosphorylated XRCC1 extends beyond the immediate, well-characterized phosphorylated region of XRCC1 (residues 515-526). We also found that an XRCC1 fragment, comprising residues 166-436, binds tightly to PNKP and DNA and efficiently activates PNKP's kinase activity. However, interaction of either of the SNP-derived variants of this fragment with PNKP was considerably weaker, and their stimulation of PNKP was severely reduced, although they still could bind DNA effectively. Laser microirradiation revealed reduced recruitment of PNKP to damaged DNA in cells expressing either XRCC1 variant compared with PNKP recruitment in cells expressing WT XRCC1 even though WT and variant XRCC1s were equally efficient at localizing to the damaged DNA. These findings suggest that the elevated risk of cancer associated with these XRCC1 SNPs reported in some studies may be due in part to the reduced ability of these XRCC1 variants to recruit PNKP to damaged DNA.

Synthesis and Analysis of 64Cu-Labeled GE11-Modified Polymeric Micellar Nanoparticles for EGFR-Targeted Molecular Imaging in a Colorectal Cancer Model.

Polymeric micellar nanoparticles represent versatile and biocompatible platforms for targeted drug delivery. However, tracking their biodistribution, stability, and clearance profile in vivo is challenging. The goal of this study was to prepare surface-modified micelles with peptide GE11 for targeting the epidermal growth factor receptor (EGFR). In vitro fluorescence studies demonstrated significantly higher internalization of GE11 micelles into EGFR-expressing HCT116 colon cancer cells versus EGFR-negative SW620 cells. Azo coupling chemistry of tyrosine residues in the peptide backbone with aryl diazonium salts was used to label the micelles with radionuclide 64Cu for positron emission tomography (PET) imaging. In vivo analysis of 64Cu-labeled micelles showed prolonged blood circulation and predominant hepatobiliary clearance. The biodistribution profile of EGFR-targeting GE11 micelles was compared with nontargeting HW12 micelles in HCT116 tumor-bearing mice. PET revealed increasing tumor-to-muscle ratios for both micelles over 48 h. Accumulation of GE11-containing micelles in HCT116 tumors was higher compared to HW12-decorated micelles. Our data suggest that the efficacy of image-guided therapies with micellar nanoparticles could be enhanced by active targeting, as demonstrated with cancer biomarker EGFR.

Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex.

Microhomology-mediated end joining (MMEJ), an error-prone pathway for DNA double-strand break (DSB) repair, is implicated in genomic rearrangement and oncogenic transformation; however, its contribution to repair of radiation-induced DSBs has not been characterized. We used recircularization of a linearized plasmid with 3΄-P-blocked termini, mimicking those at X-ray-induced strand breaks, to recapitulate DSB repair via MMEJ or nonhomologous end-joining (NHEJ). Sequence analysis of the circularized plasmids allowed measurement of relative activity of MMEJ versus NHEJ. While we predictably observed NHEJ to be the predominant pathway for DSB repair in our assay, MMEJ was significantly enhanced in preirradiated cells, independent of their radiation-induced arrest in the G2/M phase. MMEJ activation was dependent on XRCC1 phosphorylation by casein kinase 2 (CK2), enhancing XRCC1's interaction with the end resection enzymes MRE11 and CtIP. Both endonuclease and exonuclease activities of MRE11 were required for MMEJ, as has been observed for homology-directed DSB repair (HDR). Furthermore, the XRCC1 co-immunoprecipitate complex (IP) displayed MMEJ activity in vitro, which was significantly elevated after irradiation. Our studies thus suggest that radiation-mediated enhancement of MMEJ in cells surviving radiation therapy may contribute to their radioresistance and could be therapeutically targeted.

Structural and functional characterization of the PNKP-XRCC4-LigIV DNA repair complex.

Non-homologous end joining (NHEJ) repairs DNA double strand breaks in non-cycling eukaryotic cells. NHEJ relies on polynucleotide kinase/phosphatase (PNKP), which generates 5΄-phosphate/3΄-hydroxyl DNA termini that are critical for ligation by the NHEJ DNA ligase, LigIV. PNKP and LigIV require the NHEJ scaffolding protein, XRCC4. The PNKP FHA domain binds to the CK2-phosphorylated XRCC4 C-terminal tail, while LigIV uses its tandem BRCT repeats to bind the XRCC4 coiled-coil. Yet, the assembled PNKP-XRCC4-LigIV complex remains uncharacterized. Here, we report purification and characterization of a recombinant PNKP-XRCC4-LigIV complex. We show that the stable binding of PNKP in this complex requires XRCC4 phosphorylation and that only one PNKP protomer binds per XRCC4 dimer. Small angle X-ray scattering (SAXS) reveals a flexible multi-state complex that suggests that both the PNKP FHA and catalytic domains contact the XRCC4 coiled-coil and LigIV BRCT repeats. Hydrogen-deuterium exchange indicates protection of a surface on the PNKP phosphatase domain that may contact XRCC4-LigIV. A mutation on this surface (E326K) causes the hereditary neuro-developmental disorder, MCSZ. This mutation impairs PNKP recruitment to damaged DNA in human cells and provides a possible disease mechanism. Together, this work unveils multipoint contacts between PNKP and XRCC4-LigIV that regulate PNKP recruitment and activity within NHEJ.

RUNX3 Promotes the Tumorigenic Phenotype in KGN, a Human Granulosa Cell Tumor-Derived Cell Line.

Granulosa cell tumors of the ovary (GCT) are the predominant type of ovarian sex cord/stromal tumor. Although prognosis is generally favorable, the outcome for advanced and recurrent GCT is poor. A better understanding of the molecular pathogenesis of GCT is critical to developing effective therapeutic strategies. Here we have examined the potential role of the runt-related transcription factor RUNX3. There are only two GCT cell lines available. While RUNX3 is silenced in the GCT cell line KGN cells, it is highly expressed in another GCT cell line, COV434 cells. Re-expression of RUNX3 promotes proliferation, anchorage-independent growth, and motility in KGN cells in vitro and tumor formation in mice in vivo. Furthermore, expression of a dominant negative form of RUNX3 decreases proliferation of COV434 cells. To address a potential mechanism of action, we examined expression of cyclin D2 and the CDK inhibitor p27Kip1, two cell cycle regulators known to be critical determinants of GCT cell proliferation. We found that RUNX3 upregulates the expression of cyclin D2 at the mRNA and protein level, and decreases the level of the p27Kip1 protein, but not p27Kip1 mRNA. In conclusion, we demonstrate that RUNX proteins are expressed in GCT cell lines and human GCT specimens, albeit at variable levels, and RUNX3 may play an oncogenic role in a subset of GCTs.

The Rev1 interacting region (RIR) motif in the scaffold protein XRCC1 mediates a low-affinity interaction with polynucleotide kinase/phosphatase (PNKP) during DNA single-strand break repair.

The scaffold protein X-ray repair cross-complementing 1 (XRCC1) interacts with multiple enzymes involved in DNA base excision repair and single-strand break repair (SSBR) and is important for genetic integrity and normal neurological function. One of the most important interactions of XRCC1 is that with polynucleotide kinase/phosphatase (PNKP), a dual-function DNA kinase/phosphatase that processes damaged DNA termini and that, if mutated, results in ataxia with oculomotor apraxia 4 (AOA4) and microcephaly with early-onset seizures and developmental delay (MCSZ). XRCC1 and PNKP interact via a high-affinity phosphorylation-dependent interaction site in XRCC1 and a forkhead-associated domain in PNKP. Here, we identified using biochemical and biophysical approaches a second PNKP interaction site in XRCC1 that binds PNKP with lower affinity and independently of XRCC1 phosphorylation. However, this interaction nevertheless stimulated PNKP activity and promoted SSBR and cell survival. The low-affinity interaction site required the highly conserved Rev1-interacting region (RIR) motif in XRCC1 and included three critical and evolutionarily invariant phenylalanine residues. We propose a bipartite interaction model in which the previously identified high-affinity interaction acts as a molecular tether, holding XRCC1 and PNKP together and thereby promoting the low-affinity interaction identified here, which then stimulates PNKP directly.

Nanoencapsulation of Novel Inhibitors of PNKP for Selective Sensitization to Ionizing Radiation and Irinotecan and Induction of Synthetic Lethality.

There is increasing interest in developing and applying DNA repair inhibitors in cancer treatment to augment the efficacy of radiation and conventional genotoxic chemotherapy. However, targeting the inhibitor is required to avoid reducing the repair capacity of normal tissue. The aim of this study was to develop nanodelivery systems for the encapsulation of novel imidopiperidine-based inhibitors of the DNA 3'-phosphatase activity of polynucleotide kinase/phosphatase (PNKP), a DNA repair enzyme that plays a critical role in rejoining DNA single- and double-strand breaks. For this purpose, newly identified hit compounds with potent PNKP inhibitory activity, imidopiperidines A12B4C50 and A83B4C63 were encapsulated in polymeric micelles of different poly(ethylene oxide)- b-poly(ε-caprolactone) (PEO- b-PCL)-based structures. Our results showed efficient loading of A12B4C50 and A83B4C63 in PEO- b-PCLs with pendent carboxyl and benzyl carboxylate groups, respectively, and relatively slow release over 24 h. Both free and encapsulated inhibitors were able to sensitize HCT116 cells to radiation and the topoisomerase I poison, irinotecan. In addition, the encapsulated inhibitors were capable of inducing synthetic lethalilty in phosphatase and tensin homologue (PTEN)-deficient cells. We also established the validity of the peptide GE11 as a suitable ligand for active targeted delivery of nanoencapsulated drugs to colorectal cancer cells overexpressing epidermal growth factor receptor (EGFR). Our results show the potential of nanoencapsulated inhibitors of PNKP as either mono or combined therapeutic agents for colorectal cancer.

Characterization of DNA Substrate Binding to the Phosphatase Domain of the DNA Repair Enzyme Polynucleotide Kinase/Phosphatase.

Polynucleotide kinase/phosphatase (PNKP) is a DNA strand break repair enzyme that uses separate 5' kinase and 3' phosphatase active sites to convert damaged 5'-hydroxyl/3'-phosphate strand termini to ligatable 5'-phosphate/3'-hydroxyl ends. The phosphatase active site has received particular attention as a target of inhibition in cancer therapy development. The phosphatase domain dephosphorylates a range of single- and double-stranded substrates; however, structural studies have shown that the phosphatase catalytic cleft can bind only single-stranded substrates. Here we use a catalytically inactive but structurally intact phosphatase mutant to probe interactions between PNKP and a variety of single- and double-stranded DNA substrates using an electrophoretic mobility shift assay. This work indicates that the phosphatase domain binds 3'-phosphorylated single-stranded DNAs in a manner that is highly dependent on the presence of the 3'-phosphate. Double-stranded substrate binding, in contrast, is not as dependent on the 3'-phosphate. Experiments comparing blunt-end, 3'-overhanging, and frayed-end substrates indicate that the predicted loss of energy due to base pair disruption upon binding of the phosphatase active site is likely balanced by favorable interactions between the liberated complementary strand and PNKP. Comparison of the effects on substrate binding of mutations within the phosphatase active site cleft with mutations in surrounding positively charged surfaces suggests that the surrounding surfaces are important for binding to double-stranded substrates. We further show that while fluorescence polarization methods can detect specific binding of single-stranded DNAs with the phosphatase domain, this method does not detect specific interactions between the PNKP phosphatase and double-stranded substrates.

Prolonged withdrawal from cocaine self-administration affects prefrontal cortex- and basolateral amygdala-nucleus accumbens core circuits but not accumbens GABAergic local interneurons.

Withdrawal from extended-access cocaine self-administration leads to progressive intensification ('incubation') of cocaine craving. After prolonged withdrawal (1-2 months), when craving is high, expression of incubation depends on strengthening of excitatory inputs to medium spiny neurons (MSN) of the nucleus accumbens (NAc). These excitatory inputs interact with the intra-NAc GABAergic 'microcircuit', composed of MSN axon collaterals and GABAergic interneurons. Here, we investigated whether the increased glutamatergic neurotransmission observed after prolonged withdrawal is accompanied by altered GABAergic neurotransmission, focusing on NAc core. Rats self-administered cocaine or saline (6 hours/day) and then underwent >40 days of withdrawal. First, we investigated parvalbumin positive (PV+) interneurons, GABAergic fast-spiking interneurons that regulate MSN activity. Immunohistochemical studies revealed no significant change in PV signal intensity or the number of PV+ cells in cocaine rats versus saline controls. We then screened PV and other interneuron markers using immunoblotting. We detected no changes in levels of PV, calretinin, calbindin or neuronal nitric oxide synthase. Because expression of these markers is activity dependent, our results suggest no marked changes in interneuron activity. Finally, we utilized local field potential recording, which can detect GABA-mediated alterations at the circuit level, to investigate potential changes in two circuits implicated in cocaine craving: prelimbic prefrontal cortex to NAc core and basolateral amygdala to NAc core. We detected differential adaptations in these circuits, some of which may involve GABA. Overall, our results suggest that alterations in GABA transmission may accompany incubation of cocaine craving, but they are circuit specific and less pronounced than alterations in glutamate transmission.

DNA ligase III acts as a DNA strand break sensor in the cellular orchestration of DNA strand break repair.

In the current model of DNA SSBR, PARP1 is regarded as the sensor of single-strand breaks (SSBs). However, biochemical studies have implicated LIG3 as another possible SSB sensor. Using a laser micro-irradiation protocol that predominantly generates SSBs, we were able to demonstrate that PARP1 is dispensable for the accumulation of different single-strand break repair (SSBR) proteins at sites of DNA damage in live cells. Furthermore, we show in live cells for the first time that LIG3 plays a role in mediating the accumulation of the SSBR proteins XRCC1 and PNKP at sites of DNA damage. Importantly, the accumulation of LIG3 at sites of DNA damage did not require the BRCT domain-mediated interaction with XRCC1. We were able to show that the N-terminal ZnF domain of LIG3 plays a key role in the enzyme's SSB sensing function. Finally, we provide cellular evidence that LIG3 and not PARP1 acts as the sensor for DNA damage caused by the topoisomerase I inhibitor, irinotecan. Our results support the existence of a second damage-sensing mechanism in SSBR involving the detection of nicks in the genome by LIG3.

Characterization of Plasmodium falciparum ATP-dependent DNA helicase RuvB3.

BACKGROUND: Malaria is one of the most serious and widespread parasitic diseases affecting humans. Because of the spread of resistance in both parasites and the mosquito vectors to anti-malarial drugs and insecticides, controlling the spread of malaria is becoming difficult. Thus, identifying new drug targets is urgently needed. Helicases play key roles in a wide range of cellular activities involving DNA and RNA transactions, making them attractive anti-malarial drug targets. METHODS: ATP-dependent DNA helicase gene (PfRuvB3) of Plasmodium falciparum strain K1, a chloroquine and pyrimethamine-resistant strain, was inserted into pQE-TriSystem His-Strep 2 vector, heterologously expressed and affinity purified. Identity of recombinant PfRuvB3 was confirmed by western blotting coupled with tandem mass spectrometry. Helicase and ATPase activities were characterized as well as co-factors required for optimal function. RESULTS: Recombinant PfRuvB3 has molecular size of 59 kDa, showing both DNA helicase and ATPase activities. Its helicase activity is dependent on divalent cations (Cu2+, Mg2+, Ni+2 or Zn+2) and ATP or dATP but is inhibited by high NaCl concentration (>100 mM). PfPuvB3 is unable to act on blunt-ended duplex DNA, but manifests ATPase activity in the presence of either single- or double-stranded DNA. PfRuvB3.is inhibited by doxorubicin, daunorubicin and netropsin, known DNA helicase inhibitors. CONCLUSIONS: Purified recombinant PfRuvB3 contains both DNA helicase and ATPase activities. Differences in properties of RuvB between the malaria parasite obtained from the study and human host provide an avenue leading to the development of novel drugs targeting specifically the malaria form of RuvB family of DNA helicases.

Key Issues Related to Cryopreservation and Storage of Stem Cells and Cancer Stem Cells: Protecting Biological Integrity.

Cryopreservation and biobanking of stem cells are becoming increasingly important as stem cell technology and application attract the interest of industry, academic research, healthcare and patient organisations. Stem cell are already being used in the treatment of some diseases and it is anticipated that stem cell therapy will play a central role in future medicine. Similarly, the discovery of both hematopoietic and solid tumor stem cells and their clinical relevance have profoundly altered paradigms for cancer research as the cancer stem cells are considered promising new targets against cancer. Consequently, long-term cryopreservation and banking of normal and malignant stem cells is crucial and will inevitably become a routine procedure that requires highly regulated and safe methods of specimen storage. There is, however, an increasing amount of evidence showing contradictory results on the impact of cryopreservation and thawing of stem cells, including extensive physical and biological stresses, apoptosis and necrosis, mitochondrial injuries, changes to basal respiration and ATP production, cellular structural damage, telomere shortening and cellular senescence, and DNA damage and oxidative stress. Notably, cell surface proteins that play a major role in stem cell fate and are used as the biomarkers of stem cells are more vulnerable to cold stress than other proteins. There are also data supporting the alteration in some biological features and genetic integrity at the molecular level of the post-thawed stem cells. This article reviews the current and future challenges of cryopreservation of stem cells and stresses the need for further rigorous research on the methodologies for freezing and utilizing cancer stem cells following long-term storage.

Tidying up loose ends: the role of polynucleotide kinase/phosphatase in DNA strand break repair.

The termini of DNA strand breaks induced by internal and external factors often require processing before missing nucleotides can be replaced by DNA polymerases and the strands rejoined by DNA ligases. Polynucleotide kinase/phosphatase (PNKP) serves a crucial role in the repair of DNA strand breaks by catalyzing the restoration of 5'-phosphate and 3'-hydroxyl termini. It participates in several DNA repair pathways through interactions with other DNA repair proteins, notably XRCC1 and XRCC4. Recent studies have highlighted the physiological importance of PNKP in maintaining the genomic stability of normal tissues, particularly developing neural cells, as well as enhancing the resistance of cancer cells to genotoxic therapeutic agents.

Biobanking in the Twenty-First Century: Driving Population Metrics into Biobanking Quality.

Biospecimens are the essential substrates for human biomarker research. Across the globe, biobanks have developed the facilities and mechanisms to collect, process, store and distribute those substrates to researchers. However, despite some notable successes, less than one hundred of the tens of thousands of purported biomarkers have been independently validated. We propose the need for a new paradigm in biobanking; simply pursuing larger numbers of participants, larger networks of biobanks and higher sample integrity will not, in itself, transform the success rate or efficiency of biomarker research. We propose that biobanks must embrace the intrinsic observational nature of biospecimens and furnish the recipients of biospecimens with the population metrics (descriptive statistics) that can facilitate the scientific rigor that is mandated in other areas of observational research. In addition, we discuss the value of population-based ascertainment and recruitment and the importance of the timing of biospecimen collections. Any assessment of biospecimen quality must go beyond the sample itself and consider both the patient/participant selection and the most appropriate and informative timing for specimen collection, particularly prior to any treatment intervention in diseased populations. The examples and rationales that we present are based largely on cancer-related collections because the feasibility of population metrics is greatly assisted by the comprehensive registries that are more common for cancer than other chronic diseases. Changing the biobanking paradigm from tacitly 'experimental' to explicitly 'observational' represents a profound but urgent methodological shift that will influence the establishment, management, reporting and impact of biobanks in the twenty-first century.