A Clinical Conundrum with Diagnostic and Therapeutic Challenge: a Tale of Two Disorders in One Case.

Living organisms are exposed to exogenous and endogenous agents that affect genomic integrity by creating DNA double strand breaks (DSBs). These breaks are repaired by DNA repair proteins to maintain homeostasis. Defects in DNA repair pathways also affect lymphocyte development and maturation, as DSB sites are critical intermediates for rearrangements required for V(D)J recombination. Recent classifications for inborn errors of immunity (IEIs) have listed DNA repair defect genes in a separate group, which suggests the importance of these genes for adaptive and innate immunity. We report an interesting case of a young female (index P1) with mutations in two different genes, DCLRE1C and FANCA, involved in DNA repair pathways. She presented with clinical manifestations attributed to both defects. With the advent of NGS, more than one defect is increasingly identified in patients with IEIs. Familial segregation studies and appropriate functional assays help ascertain the pathogenicity of these mutations and provide appropriate management and genetic counseling.

Establishment of ex vivo calibration curve for X-ray induced "dicentric + ring" and micronuclei in human peripheral lymphocytes for biodosimetry during radiological emergencies, and validation with dose blinded samples.

In the modern developing society, application of radiation has increased extensively. With significant improvement in the radiation protection practices, exposure to human could be minimized substantially, but cannot be avoided completely. Assessment of exposure is essential for regulatory decision and medical management as applicable. Until now, cytogenetic changes have served as surrogate marker of radiation exposure and have been extensively employed for biological dose estimation of various planned and unplanned exposures. Dicentric Chromosomal Aberration (DCA) is radiation specific and is considered as gold standard, micronucleus is not very specific to radiation and is considered as an alternative method for biodosimetry. In this study dose response curves were generated for X-ray induced "dicentric + ring" and micronuclei, in lymphocytes of three healthy volunteers [2 females (age 22, 23 years) and 1 male (24 year)]. The blood samples were irradiated with X-ray using LINAC (energy 6 MV, dose rate 6 Gy/min), in the dose range of 0-5Gy. Irradiated blood samples were cultured and processed to harvest metaphases, as per standard procedures recommended by International Atomic Energy Agency. Pooled data obtained from all the three volunteers, were in agreement with Poisson distribution for "dicentric + ring", however over dispersion was observed for micronuclei. Data ("dicentric + ring" and micronuclei) were fitted by linear quadratic model of the expression Y[bond, double bond]C + αD + ßD2 using Dose Estimate software, version 5.2. The data fit has resulted in linear coefficient α = 0.0006 (±0.0068) "dicentric + ring" cell-1 Gy-1 and quadratic coefficient ß = 0.0619 (±0.0043) "dicentric + ring" cell-1 Gy-2 for "dicentric + ring" and linear coefficient α = 0.0459 ± (0.0038) micronuclei cell-1 Gy-1 and quadratic coefficient ß = 0.0185 ± (0.0010) micronuclei cell-1 Gy-2 for micronuclei, respectively. Background frequencies for "dicentric + ring" and micronuclei were 0.0006 ± 0.0004 and 0.0077 ± 0.0012 cell-1, respectively. Established curves were validated, by reconstructing the doses of 8 dose blinded samples (4 by DCA and 4 by CBMN) using coefficients generated here. Estimated doses were within the variation of 0.9-16% for "dicentric + ring" and 21.7-31.2% for micronuclei respectively. These established curves have potential to be employed for biodosimetry of occupational, clinical and accidental exposures, for initial triage and medical management.

Amphiphilic interaction-mediated ordering of nanoparticles in Pickering emulsion droplets.

For various industrial processes, the stabilization of an oil phase is crucial and demands a proper balance of complex interactions in an emulsion system. In Pickering emulsions, this is achieved by introducing nanoparticles, which become organized at the oil-water interface. The influence of interparticle interactions towards the formation of a stable emulsion and the ordering of the stabilizing nanoparticles is intriguing and needs attention. In this work, the role of amphiphilic interactions between hydrophilic silica nanoparticles and the Pluronic F127 tri-block co-polymer towards the spontaneous formation of a fairly stable Pickering emulsion has been studied using small-angle X-ray scattering. Unlike the usual random arrangements of the nanoparticles in a conventional Pickering emulsion, we observed highly organized silica nanoparticles at the oil-water interface. The established standard raspberry structural model of the Pickering emulsion fails to explain such strong ordering as observed in the present case. A plausible formation mechanism of the present Pickering emulsion with a high on-surface silica correlation is elucidated on the basis of the combined interactions of the block co-polymer and silica particles. A computer model is developed to elucidate the effects of size and distribution of the surface-decorating nanoparticles and their positional correlation.

Establishment of in vitro Calibration Curve for 60Co-γ-rays Induced Phospho-53BP1 Foci, Rapid Biodosimetry and Initial Triage, and Comparative Evaluations With γH2AX and Cytogenetic Assays.

A rapid and reliable method for biodosimetry of populations exposed to ionizing radiation in the event of an incident or accident is crucial for initial triage and medical attention. DNA-double strand breaks (DSBs) are indicative of radiation exposure, and DSB-repair proteins (53BP1, γH2AX, ATM, etc.) are considered sensitive markers of DSB quantification. Phospho-53BP1 and γH2AX immunofluorescence technique serves as a sensitive, reliable, and reproducible tool for the detection and quantification of DSB-repair proteins, which can be used for biological dose estimations. In this study, dose-response curves were generated for 60Co-γ-rays induced phospho-53 Binding Protein 1 (phospho-53BP1) foci at 1, 2, 4, 8, 16, and 24 h, post-irradiation for a dose range of 0.05-4 Gy using fluorescence microscopy. Following ISO recommendations, minimum detection limits (MDLs) were estimated to be 16, 18, 25, 40, 50, and 75 mGy for dose-response curves generated at 1, 2, 4, 8, 16, and 24 h post-irradiation. Colocalization and correlation of phospho-53BP1 and γH2AX were also measured in irradiated peripheral blood lymphocytes (PBLs) to gain dual confirmation. Comparative evaluation of the established curve was made by γH2AX-immunofluorescence, dicentric chromosome assay (DCA), and reciprocal translocation (RT) assays by reconstructing the dose of 6 dose-blinded samples. Coefficients of respective in-house established dose-response curves were employed to reconstruct the blind doses. Estimated doses were within the variation of 4.124%. For lower doses (0.052 Gy), phospho-53BP1 and γH2AX assays gave closer estimates with the variation of -4.1 to + 9% in comparison to cytogenetic assays, where variations were -8.5 to 24%. For higher doses (3 and 4 Gy), both the cytogenetic and immunofluorescence (phospho-53BP1 and γH2AX), assays gave comparable close estimates, with -11.3 to + 14.3% and -10.3 to -13.7%, variations, respectively.

Protocol for one-step selective lysis of red blood cells and platelets with long-term preservation of white blood cells (human) at ambient temperature.

Current protocols for storage of white blood cells (WBCs) rely on constant refrigeration. The protocol described below explains the preparation of a fixative combination saline (FCS) formulation, which allows fixation of human WBCs and lysis of red blood cells and platelets (at ambient temperature, 4-35oC) in whole blood samples in one step. FCS can be used for storing and transporting blood at ambient temperatures for up to 4 months, without altering the nuclear morphology and genomic integrity of WBCs.

Establishment and multiparametric-cytogenetic validation of 60Co-gamma-ray induced, phospho-gamma-H2AX calibration curve for rapid biodosimetry and triage management during radiological emergencies.

Exposure to ionizing radiation is unavoidable to our modern developing society as its applications are widespread and increasing with societal development. The exposures may be planned as in medical applications or may be unplanned as in occupational work and radiological emergencies. Dose quantification of planned and unplanned exposures is essential to make crucial decisions for management of such exposures. This study aims to establish ex-vivo dose-response curve for 60Co-gamma-ray induced gamma-H2AX-foci by immunofluorescence using microscopy and flowcytometry with human lymphocytes. This technique has the potential to serve as a rapid tool for dose estimation and triage application during small to large scale radiological emergencies and clinical exposures. Response curves were generated for the dose range 0-4 Gy (at 1, 2, 4, 8, 16, 24, 48, 72 and 96 h of incubation after irradiation) with microscopy and 0-8 Gy (at 2, 4, 8, 16 and 24 h of incubation after irradiation) with flow cytometry. These curves can be applied for dose reconstruction when post exposure sampling is delayed up to 96 h. In order to evaluate Minimum Detection Limit (MDL) of the assay, variation of background frequency of gamma-H2AX-foci was measured in 12 volunteers. To understand the application window of the assay, gamma-H2AX foci decay kinetics has been studied up to 96 h with microscopy and response curves were generated from 1 to 96 hours post exposure. Gamma-H2AX fluorescence intensity decay kinetics was also studied up to 96 h with flow cytometry and response curves were generated from 2 to 24 hours post irradiation. Established curves were validated with dose blinded samples and also compared with standard cytogenetic assays. An inter-comparison of dose estimates was made among gamma-H2AX assay, dicentric aberrations and reciprocal translocations for application window in various dose ranges and time of blood collection after exposures.

Integrated transcriptomic and proteomic analysis of microplasts derived from macrophage-conditioned medium-treated MCF-7 breast cancer cells.

Microplasts are large extracellular vesicles originating from migratory, invasive, and metastatic cancer cells. Here, to gain insight into the role of microplasts in cancer progression, we performed a proteomic and transcriptomic characterization of microplasts isolated from MCF-7 breast cancer cells treated with macrophage-conditioned medium. These cells were found to be viable, highly migratory, and metabolically active, indicating that microplasts derived from these cells are not apoptotic bodies. Transcriptomic/proteomic analyses identified 10273 mRNAs and 821 proteins in microplasts. Interestingly, 377 microplast mRNAs coded for corresponding microplast proteins. Microplast mRNAs and proteins were mainly associated with binding and catalytic activities. Microplasts showed enrichment of mRNAs involved in transcription regulation and proteins involved in processes such as cell-cell adhesion and translation. Pathway analysis showed enrichment of ribosomes and carbon metabolism. These results suggest a close resemblance between microplasts and parent cells, with mRNA and protein cargo relevant in intercellular signaling.

Differential killing and radio-modifying effects of iodoacetate in mammalian normal and cancer cells.

To explore possible applications of iodoacetate (IA), a glycolytic inhibitor, in cancer treatment, we screened its cytotoxicity and radioprotective/sensitizing efficacy in three different mammalian cell lines; A549 (human lung carcinoma), MCF7 (human mammary cancer), a non-cancerous CHO (Chinese hamster ovary) cells and human lymphocytes. Experiments were carried out using IA concentrations ranging from 0.01 to 2.5 µg/ml, with or without 60Coγ-radiation. In the outcomes, IA was found to exhibit higher toxicity in the cancer cells, whereas it was non-toxic/marginally toxic to the non-cancerous cells. Considerably higher glucose uptake in both cancer cells lines was observed indicating higher rates of glycolysis. IA significantly inhibited glycolysis as reflected by GAPDH activity inhibition. Radiomodifying effects of IA were found to be concentration dependent in both cancerous and non-cancerous cells. The response in non-cancerous was found to be biphasic: at lower concentrations, it offered significant radioprotection; however, the protection decreased with increasing concentration. Moreover, at the highest tested concentration, marginal radiosensitization was also observed (as indicated by clonogenic assay). In both cancer cells, IA offered significant amount of radiosensitization which was considerably high at higher concentrations. Further experiments were carried out to estimate the Dose Modification Factor (DMF) to quantify and compare relative radiosensitization by IA in cancer and normal cell lines. The DMF was calculated for three different concentrations of IA, 0.5, 1, and 1.5 µg/ml, and corresponding values were found to be 1.26, 1.43, and 1.89 for A549 cancer cells, whereas for normal CHO cells, it was 1.13, 1.13, and 1.24. In conclusion, differential killing and radiosensitizing effects of IA suggest that it may have potential use as a anticancer agent and radiosensitizer in cancer therapy.

Standardization of CalyculinA induced PCC assay and its advantages over Okadaic acid PCC assay in Biodosimetry applications.

OBJECTIVE: In the present study an attempt was made to estimate coefficients of dose response curves for PCC aberrations induced by CalyculinA and Okadaic acid, using 60Co gamma radiation and 8 MeV pulsed electron beam for biodosimetry application. MATERIALS AND METHODS: The modified method outlined by Puig et al. 2013 was used to conduct Calyculin A and Okadaic acid induced PCC assay in human blood lymphocytes.Chemical treatment was given for the last 1 h of a 48 h culture. The study was carried out in the dose range 2.5 to 20 Gy using 60Co gamma rays and 8 MeV pulsed electron beam. RESULTS AND CONCLUSIONS: Results show a linear dose dependent increase with a slope of 0.047 ± 0.001 from Calycalin A PCC and 0.048 ± 0.002 form Okadaic acid PCC. The slope of the fragments curve was 0.327 ± 0.006 from Calyculin A and 0.328 ± 0.006 from Okadaic acid PCC. Further, dose calibration studies were carried out for 8 MeV electron using Calyculin A PCC assay and the obtained slope from ring yield was 0.054 ± 0.002 and 0.427 ± 0.009 from fragment yield.

Cyto-genotoxicity assessment of potential radioprotector, 3,3'-diselenodipropionic acid (DSePA) in Chinese Hamster Ovary (CHO) cells and human peripheral blood lymphocytes.

Our previous study showed that 3,3'-diselenodipropionic acid (DSePA), a simple, stable, and water-soluble organoselenium exhibiting glutathione peroxidase (GPx)-like activity offered good radioprotection under in vitro and in vivo conditions. Herein, we investigated the anti-genotoxic effect of DSePA in model cellular systems such as Chinese Hamster Ovary (CHO) cell line and human peripheral lymphocytes after exposure to γ-radiation. The measurements on the induction of γ-H2AX foci and micronuclei frequency in the cell nuclei indicated that pretreatment with DSePA significantly prevented the radiation induced DNA damage or genotoxicity and subsequent cytotoxicity without exerting its own toxicity. The maximum protective effect of DSePA was seen at a pre-treatment concentration of 3 µg/ml. The mechanistic investigations in CHO cells revealed that DSePA pretreatment prevented the radiation induced ROS generation, lipid peroxidation and subsequent apoptosis in these cells. Further, it was seen to augment the mRNA expressions of GPx2 significantly and GPx4 marginally without causing much change in the total GPx activity after radiation exposure. These results suggested the roles of GPx2 and GPx4 in DSePA mediated radioprotection. In conclusion our results confirm the nongenotoxic nature of the DSePA and validate its radioprotective efficacy and mechanisms of action in model cellular systems.