Association of Neutrophil-Lymphocyte Ratio and Red Blood Cell Distribution Width with Poor Outcome in Pediatric Cardiac Surgery - A Retrospective Observational Study.

BACKGROUND: Neutrophil-lymphocyte ratio (NLR) is a valuable indicator for evaluating inflammatory response and red blood cell distribution width (RBDW), a routinely available biomarker of likely erythropoietic dysfunction, which may be associated with adverse outcomes after cardiac surgery. This study aimed to investigate the association between these two readily available haematological parameters, with the poor outcomes in paediatric patients undergoing cardiac surgery. METHODS: A comprehensive review of medical records for paediatric patients who underwent cardiac surgery at our tertiary care centre between April 2022 and June 2023 was carried out. RBDW and NLR values were collected from complete blood count reports obtained on admission to the ICU. Demographic data, surgical details, and postoperative complications were also recorded. A receiver operating characteristic (ROC) curve and multivariable logistic regression were applied to identify the prognosis performance of preoperative NLR and RBDW for poor outcomes. RESULTS: The study included 219 patients meeting the inclusion criteria of which a total of 90 (41%) children experienced at least one of the poor outcomes. Preoperative NLR (AUC=0.88, 95%CI 0.36-0.70, cut off- 4.2) and RBDW (AUC=0.88, 95%CI 0.39-0.73, cut off- 18.5%) showed prognostic significance in the perioperative period. CONCLUSION: This retrospective observational study highlights a significant association between elevated Red Blood Cell Distribution Width (RBDW) and Neutrophil Lymphocyte Ratio (NLR) values and poor outcomes in paediatric patients undergoing cardiac surgery. These readily available haematological parameters could serve as potential prognostic indicators for identifying patients at risk of poor outcomes.

2,2-Diphenethyl Isothiocyanate Enhances Topoisomerase Inhibitor-Induced Cell Death and Suppresses Multi-Drug Resistance 1 in Breast Cancer Cells.

We previously reported that phenethyl isothiocyanate (PEITC), a dietary-related compound, can rescue mutant p53. A structure-activity relationships study showed that the synthetic analog 2,2-diphenylethyl isothiocyanate (DPEITC) is a more potent inducer of apoptosis than natural or synthetic ITCs. Here, we showed that DPEITC inhibited the growth of triple-negative breast cancer cells (MDA-MB-231, MDA-MB-468, and Hs578T) expressing "hotspot" p53 mutants, structural (p53R280K, p53R273H) or contact (p53V157F), at IC50 values significantly lower than PEITC. DPEITC inhibited the growth of HER2+ (p53R175H SK-BR-3, p53R175H AU565) and Luminal A (p53L194F T47D) breast cancer (BC) cells harboring a p53 structural mutant. DPEITC induced apoptosis, irrespective of BC subtypes, by rescuing p53 mutants. Accordingly, the rescued p53 mutants induced apoptosis by activating canonical WT p53 targets and delaying the cell cycle. DPEITC acted synergistically with doxorubicin and camptothecin to inhibit proliferation and induce apoptosis. Under these conditions, DPEITC delayed BC cells in the G1 phase, activated p53 canonical targets, and enhanced pS1981-ATM. DPEITC reduced the expression of MDR1 and ETS1. These findings are the first report of synergism between a synthetic ITC and a chemotherapy drug via mutant p53 rescue. Furthermore, our data demonstrate that ITCs suppress the expression of cellular proteins that play a role in chemoresistance.

The linker domain of the initiator DnaA contributes to its ATP binding and membrane association in E. coli chromosomal replication.

DnaA, the initiator of Escherichia coli chromosomal replication, has in its adenosine triphosphatase (ATPase) domain residues required for adenosine 5'-triphosphate (ATP) binding and membrane attachment. Here, we show that D118Q substitution in the DnaA linker domain, a domain known to be without major regulatory functions, influences ATP binding of DnaA and replication initiation in vivo. Although initiation defective by itself, overexpression of DnaA(D118Q) caused overinitiation of replication in dnaA46ts cells and prevented cell growth. The growth defect was rescued by overexpressing the initiation inhibitor, SeqA, indicating that the growth inhibition resulted from overinitiation. Small deletions within the linker showed another unexpected phenotype: cellular growth without requiring normal levels of anionic membrane lipids, a property found in DnaA mutated in its ATPase domain. The deleted proteins were defective in association with anionic membrane vesicles. These results show that changes in the linker domain can alter DnaA functions similarly to the previously shown changes in the ATPase domain.

Adamantyl Isothiocyanates as Mutant p53 Rescuing Agents and Their Structure-Activity Relationships.

Mutant p53 rescue by small molecules is a promising therapeutic strategy. In this structure-activity relationship study, we examined a series of adamantyl isothiocyanates (Ad-ITCs) to discover novel agents as therapeutics by targeting mutant p53. We demonstrated that the alkyl chain connecting adamantane and ITC is a crucial determinant for Ad-ITC inhibitory potency. Ad-ITC 6 with the longest chain between ITC and adamantane displayed the maximum growth inhibition in p53R280K, p53R273H, or p53R306Stop mutant cells. Ad-ITC 6 acted in a mutant p53-dependent manner. It rescued p53R280K and p53R273H mutants, thereby resulting in upregulating canonical wild-type (WT) p53 targets and phosphorylating ATM. Ad-ISeC 14 with selenium showed a significantly enhanced inhibitory potency, without affecting its ability to rescue mutant p53. Ad-ITCs selectively depleted mutant p53, but not the WT, and this activity correlates with their inhibitory potencies. These data suggest that Ad-ITCs may serve as novel promising leads for the p53-targeted drug development.

Pregnancy and Its Successful Outcome in a Patient with Multiple Myeloma.

Multiple myeloma is a B-cell neoplastic disorder and represents 1% of all cancers and 13% of hematological malignancies. It is predominantly a disease of elderly, and less than 3% of all cases are below the age of 40 years. We present the case of a 29-year-old lady with multiple myeloma who had spontaneous conception during maintenance therapy and subsequently a successful pregnancy outcome. She gave birth to a healthy female infant through normal vaginal delivery and subsequently could remain off therapy for 5 years. Since the patient had a history of abortions and stillbirth, it was a precious pregnancy and we could successfully salvage both the mother and the baby. Young female patients of myeloma who are in remission can be encouraged to start a family during their reproductive years with the support of a comprehensive care team of hematologists/oncologists and obstetricians.

p53 mutant-type in human prostate cancer cells determines the sensitivity to phenethyl isothiocyanate induced growth inhibition.

BACKGROUND: We reported previously that phenethyl isothiocyanate (PEITC), a dietary compound, can reactivate p53R175H mutant in vitro and in SK-BR-3 (p53R175H) breast xenograft model resulting in tumor inhibition. Because of the diversity of human cancers with p53 mutations, these findings raise important questions whether this mechanism operates in different cancer types with same or different p53 mutations. In this study, we investigated whether PEITC recuses mutant p53 in prostate cancer cells harboring different types of p53 mutants, structural and contact, in vitro and in vivo. METHODS: Cell proliferation, cell apoptosis and cell cycle arrest assays were performed to examine the effects of PEITC on prostate cancer cell lines with p53 mutation(s), wild-type p53, p53 null or normal prostate cells in vitro. Western blot analysis was used to monitor the expression levels of p53 protein, activation of ATM and upregulation of canonical p53 targets. Immunoprecipitation, subcellular protein fraction and qRT-PCR was performed to determine change in conformation and restoration of transactivation functions/ inhibition of gain-of-function (GOF) activities to p53 mutant(s). Mice xenograft models were established to evaluate the antitumor efficacy of PEITC and PEITC-induced reactivation of p53 mutant(s) in vivo. Immunohistochemistry of xenograft tumor tissues was performed to determine effects of PEITC on expression of Ki67 and mutant p53 in vivo. RESULTS: We demonstrated that PEITC inhibits the growth of prostate cancer cells with different "hotspot" p53 mutations (structural and contact), however, preferentially towards structural mutants. PEITC inhibits proliferation and induces apoptosis by rescuing mutant p53 in p53R248W contact (VCaP) and p53R175H structural (LAPC-4) mutant cells with differential potency. We further showed that PEITC inhibits the growth of DU145 cells that co-express p53P223L (structural) and p53V274F (contact) mutants by targeting p53P223L mutant selectively, but not p53V274F. The mutant p53 restored by PEITC induces apoptosis in DU145 cells by activating canonical p53 targets, delaying cells in G1 phase and phosphorylating ATM. Importantly, PEITC reactivated p53R175H and p53P223L/V274F mutants in LAPC-4 and DU145 prostate xenograft models, respectively, resulting in significant tumor inhibition. CONCLUSION: Our studies provide the first evidence that PEITC's anti-cancer activity is cancer cell type-independent, but p53 mutant-type dependent.

Case Report: Paragonimiasis Presenting with Pericardial Tamponade.

We report an unusual case of paragonimiasis in a Nepali patient presenting with massive pericardial effusion and pericardial tamponade. The patient reported neither the consumption of crabs or crayfish nor the consumption of wild animal meat, which are the usual sources of infection. It is suspected that the source of infection was instead the ingestion of raw live slugs as part of a traditional medicine treatment.

An endogenous DNA adduct as a prognostic biomarker for hepatocarcinogenesis and its prevention by Theaphenon E in mice.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide, mainly because of its poor prognosis. A valid mechanism-based prognostic biomarker is urgently needed. γ-hydroxy-1,N2 -propanodeoxyguanosine (γ-OHPdG) is an endogenously formed mutagenic DNA adduct derived from lipid peroxidation. We examined the relationship of γ-OHPdG with hepatocarcinogenesis in two animal models and its potential role as a prognostic biomarker for recurrence in HCC patients. Bioassays were conducted in xeroderma pigmentosum group A knockout mice and diethylnitrosamine-injected mice, both prone to HCC development. γ-OHPdG levels in the livers of these animals were determined. The effects of antioxidant treatments on γ-OHPdG and hepatocarcinogenesis were examined. Using two independent sets of HCC specimens from patients, we examined the relationship between γ-OHPdG and survival or recurrence-free survival. γ-OHPdG levels in liver DNA showed an age-dependent increase and consistently correlated with HCC development in all three animal models. Theaphenon E treatment significantly decreased γ-OHPdG levels in the liver DNA of xeroderma pigmentosum group A knockout mice and remarkably reduced HCC incidence in these mice to 14% from 100% in the controls. It also effectively inhibited HCC development in the diethylnitrosamine-injected mice. Using clinical samples from two groups of patients, our study revealed that higher levels of γ-OHPdG are strongly associated with low survival (P < 0.0001) and low recurrence-free survival (P = 0.007). CONCLUSION: These results support γ-OHPdG as a mechanism-based, biologically relevant biomarker for predicting the risk of HCC and its recurrence. (Hepatology 2018;67:159-170).

Biochemical and cell biological assays to identify and characterize DNA helicase inhibitors.

The growing number of DNA helicases implicated in hereditary disorders and cancer indicates that this particular class of enzymes plays key roles in genomic stability and cellular homeostasis. Indeed, a large body of work has provided molecular and cellular evidence that helicases act upon a variety of nucleic acid substrates and interact with numerous proteins to enact their functions in replication, DNA repair, recombination, and transcription. Understanding how helicases operate in unique and overlapping pathways is a great challenge to researchers. In this review, we describe a series of experimental approaches and methodologies to identify and characterize DNA helicase inhibitors which collectively provide an alternative and useful strategy to explore their biological significance in cell-based systems. These procedures were used in the discovery of biologically active compounds that inhibited the DNA unwinding function catalyzed by the human WRN helicase-nuclease defective in the premature aging disorder Werner syndrome. We describe in vitro and in vivo experimental approaches to characterize helicase inhibitors with WRN as the model, anticipating that these approaches may be extrapolated to other DNA helicases, particularly those implicated in DNA repair and/or the replication stress response.

A Click Chemistry Approach to Identify Protein Targets of Cancer Chemopreventive Phenethyl Isothiocyanate.

Here we report the identification of protein targets of chemopreventive phenethyl isothiocyanate (PEITC) via "click" chemistry in the A549 human lung cancer cell line, using a novel alkyne-tagged PEITC which was also found to show potent in vitro anticancer activity.

Targeting an Achilles' heel of cancer with a WRN helicase inhibitor.

Our recently published work suggests that DNA helicases such as the Werner syndrome helicase (WRN) represent a novel class of proteins to target for anticancer therapy. Specifically, pharmacological inhibition of WRN helicase activity in human cells defective in the Fanconi anemia (FA) pathway of interstrand cross-link (ICL) repair are sensitized to the DNA cross-linking agent and chemotherapy drug mitomycin C (MMC) by the WRN helicase inhibitor NSC 617145. (1) The mechanistic basis for the synergistic interaction between NSC 617145 and MMC is discussed in this paper and extrapolated to potential implications for genetic or chemically induced synthetic lethality provoked by cellular exposure to the WRN helicase inhibitor under the context of relevant DNA repair deficiencies associated with cancers or induced by small-molecule inhibitors. Experimental data are presented showing that small-molecule inhibition of WRN helicase elevates sensitivity to MMC-induced stress in human cells that are deficient in both FANCD2 and DNA protein kinase catalytic subunit (DNA-PKcs). These findings suggest a model in which drug-mediated inhibition of WRN helicase activity exacerbates the deleterious effects of MMC-induced DNA damage when both the FA and NHEJ pathways are defective. We conclude with a perspective for the FA pathway and synthetic lethality and implications for DNA repair helicase inhibitors that can be developed for anticancer strategies.

Werner syndrome helicase has a critical role in DNA damage responses in the absence of a functional fanconi anemia pathway.

Werner syndrome is genetically linked to mutations in WRN that encodes a DNA helicase-nuclease believed to operate at stalled replication forks. Using a newly identified small-molecule inhibitor of WRN helicase (NSC 617145), we investigated the role of WRN in the interstrand cross-link (ICL) response in cells derived from patients with Fanconi anemia, a hereditary disorder characterized by bone marrow failure and cancer. In FA-D2(-/-) cells, NSC 617145 acted synergistically with very low concentrations of mitomycin C to inhibit proliferation in a WRN-dependent manner and induce double-strand breaks (DSB) and chromosomal abnormalities. Under these conditions, ataxia-telangiectasia mutated activation and accumulation of DNA-dependent protein kinase, catalytic subunit pS2056 foci suggested an increased number of DSBs processed by nonhomologous end-joining (NHEJ). Rad51 foci were also elevated in FA-D2(-/-) cells exposed to NSC 617145 and mitomycin C, suggesting that WRN helicase inhibition interferes with later steps of homologous recombination at ICL-induced DSBs. Thus, when the Fanconi anemia pathway is defective, WRN helicase inhibition perturbs the normal ICL response, leading to NHEJ activation. Potential implication for treatment of Fanconi anemia-deficient tumors by their sensitization to DNA cross-linking agents is discussed.

Correlation of gallstone characteristics with mucosal changes in gall bladder.

BACKGROUND AND AIM: Gallstones are known to produce diverse histopathological changes in the gall bladder. Our aim was to correlate various gallstone characteristics (number, size, weight, volume and morphological type) with the type of mucosal response in gall bladder (inflammation, hyperplasia, metaplasia and carcinoma). METHODS: The study was conducted on 330 open cholecystectomy specimens with complete gallstones. The stones were assessed for various parameters i.e. number, size, weight, volume and morphological type. For microscopy, sections were obtained from the fundus, body and neck of the gallbladder. Additional sections were taken from abnormal looking areas. RESULTS: Out of the 330 cases, 194 (59%) had mixed stones, 84 (25%) combined, 30 (9%) pigment and 22 (7%) had cholesterol stones. Number of stones varied from a single calculus in 131 (39.6%) cases, double in 29 (8.8%) and multiple in the remaining 170 (51.6%) cases. Cholecystitis, hyperplasia, metaplasia and carcinoma were more commonly seen with mixed and multiple stones. The average weight of calculi in cholecystitis was 2.551 gm, in hyperplasia 3.619 gm, metaplasia4.549 gm and 17.96 gm in cases with carcinoma. Similarly, average volume of the stone(s) was 2.664 ml in cholecystitis, 3.742 ml in hyperplasia, 4.532 ml in metaplasia and 19.178 ml in carcinoma. The average calculus size (2.147 cm) was found to be maximum in cases with carcinoma, followed by hyperplasia (1.187 cm), metaplasia (1.145 cm) and cholecystitis (1.136 cm). CONCLUSION: As the weight, volume and size of the stone increases the changes in the gall bladder mucosa changes from cholecystitis, hyperplasia, metaplasia, dysplasia, to carcinoma.

Inhibition of helicase activity by a small molecule impairs Werner syndrome helicase (WRN) function in the cellular response to DNA damage or replication stress.

Modulation of DNA repair proteins by small molecules has attracted great interest. An in vitro helicase activity screen was used to identify molecules that modulate DNA unwinding by Werner syndrome helicase (WRN), mutated in the premature aging disorder Werner syndrome. A small molecule from the National Cancer Institute Diversity Set designated NSC 19630 [1-(propoxymethyl)-maleimide] was identified that inhibited WRN helicase activity but did not affect other DNA helicases [Bloom syndrome (BLM), Fanconi anemia group J (FANCJ), RECQ1, RecQ, UvrD, or DnaB). Exposure of human cells to NSC 19630 dramatically impaired growth and proliferation, induced apoptosis in a WRN-dependent manner, and resulted in elevated γ-H2AX and proliferating cell nuclear antigen (PCNA) foci. NSC 19630 exposure led to delayed S-phase progression, consistent with the accumulation of stalled replication forks, and to DNA damage in a WRN-dependent manner. Exposure to NSC 19630 sensitized cancer cells to the G-quadruplex-binding compound telomestatin or a poly(ADP ribose) polymerase (PARP) inhibitor. Sublethal dosage of NSC 19630 and the chemotherapy drug topotecan acted synergistically to inhibit cell proliferation and induce DNA damage. The use of this WRN helicase inhibitor molecule may provide insight into the importance of WRN-mediated pathway(s) important for DNA repair and the replicational stress response.

Molecular analyses of DNA helicases involved in the replicational stress response.

The importance of helicases in nucleic acid metabolism and human disease has raised the bar for understanding how these unique enzymes function to perform their biological roles at the molecular level. Here we will describe experimental procedures and strategies to investigate the functions of helicases. These functional assays have been used to study DNA helicases important for the maintenance of genomic stability and genetically linked to age-related diseases and cancer. We will focus on the description of fluorometric helicase assays, protein displacement assays, and methods to characterize helicase activity on alternate DNA structures (triplex and quadruplex) used by our laboratory. The procedures to study these helicase functions are described in step-by-step detail to enable researchers interested in nucleic acid metabolism and related fields to apply these techniques to their own research questions.

Hitting the bull's eye: novel directed cancer therapy through helicase-targeted synthetic lethality.

Designing strategies for anti-cancer therapy have posed a significant challenge. One approach has been to inhibit specific DNA repair proteins and their respective pathways to enhance chemotherapy and radiation therapy used to treat cancer patients. Synthetic lethality represents an approach that exploits pre-existing DNA repair deficiencies in certain tumors to develop inhibitors of DNA repair pathways that compensate for the tumor-associated repair deficiency. Since helicases play critical roles in the DNA damage response and DNA repair, particularly in actively dividing and replicating cells, it is proposed that the identification and characterization of synthetic lethal relationships of DNA helicases will be of value in developing improved anti-cancer treatment strategies. In this review, we discuss this hypothesis and current evidence for synthetic lethal interactions of eukaryotic DNA helicases in model systems.

Role of N-terminal hydrophobic region in modulating the subcellular localization and enzyme activity of the bisphosphate nucleotidase from Debaryomyces hansenii.

3', 5'-Bisphosphate nucleotidase is a ubiquitous enzyme that converts 3'-phosphoadenosine-5'-phosphate to adenosine-5'-phosphate and inorganic phosphate. These enzymes are highly sensitive to sodium and lithium and, thus, perform a crucial rate-limiting metabolic step during salt stress in yeast. Recently, we have identified a bisphosphate nucleotidase gene (DHAL2) from the halotolerant yeast Debaryomyces hansenii. One of the unique features of Dhal2p is that it contains an N-terminal 54-amino-acid-residue hydrophobic extension. In this study, we have shown that Dhal2p exists as a cytosolic as well as a membrane-bound form and that salt stress markedly influences the accumulation of the latter form in the cell. We have demonstrated that the N-terminal hydrophobic region was necessary for the synthesis of the membrane-bound isoform. It appeared that an alternative translation initiation was the major mechanism for the synthesis of these two forms. Moreover, the two forms exhibit significant differences in their substrate specificity. Unlike the cytosolic form, the membrane-bound form showed very high activity against inositol-1,4-bisphosphate. Thus, the present study for the first time reports the existence of multiple forms of a bisphosphate nucleotidase in any organism.

Molecular cloning and biochemical characterization of a 3'(2'),5'-bisphosphate nucleotidase from Debaryomyces hansenii.

The enzyme 3'(2'),5'-bisphosphate nucleotidase catalyses a reaction that converts 3'-phosphoadenosine-5'-phosphate (PAP) to adenosine-5'-phosphate (AMP) and inorganic phosphate (Pi). The enzyme from Saccharomyces cerevisiae is highly sensitive to sodium and lithium and is thus considered to be the in vivo target of salt toxicity in yeast. In S. cerevisiae, the HAL2 gene encodes this enzyme. We have cloned a homologous gene, DHAL2, from the halotolerant yeast Debaryomyces hansenii. DNA sequencing of this clone revealed a 1260 bp open reading frame (ORF) that putatively encoded a protein of 420 amino acid residues. S. cerevisiae transformed with DHAL2 gene displayed higher halotolerance. Biochemical studies showed that recombinant Dhal2p could efficiently utilize PAP (K(m)17 microM) and PAPS (K(m)48 microM) as substrate. Moreover, we present evidence that, in comparison to other homologues from yeast, Dhal2p displays significantly higher resistance towards lithium and sodium ions.