Prospective evaluation of 68 Ga-NODAGA-RGD PET-CT in patients of carcinoma thyroid with thyroglobulin elevated negative radioiodine scintigraphy (TENIS) with a head-to-head comparison with FDG-PET/CT.

BACKGROUND AND AIM: This study aimed to examine the expression of RGD binding integrins in patients of elevated serum thyroglobulin (Tg) level with negative radioiodine scintigraphy (TENIS) employing 68 Ga-NODAGA-RGD PET-CT. MATERIAL AND METHODS: This was a prospective study involving 30 proven cases of TENIS with histopathological diagnosis of differentiated thyroid carcinoma post-surgery. In addition to observing the lesional concentration on 68 Ga-NODAGA-RGD PET-CT, a 4-point visual grading system (grade I-IV), was undertaken to estimate the degree of radiotracer avidity, for potential of theranostics. RESULTS: On 18 F-FDG-PET/CT, the uptake was seen in 182 lesions out of a total of 200 (91%). 68 Ga-NODAGA-RGD PET-CT showed expression in a total of 110/200 (55%) lesions. On patient-specific analysis, 68 Ga-NODAGA-RGD PET-CT was positive for the disease in 21/30 patients (70%) and negative in 9/30 (30%) patients. The overall patient-specific sensitivity and specificity of 68 Ga-NODAGA-RGDPET-CT were 75% and 100%, respectively. 18 F-FDG PET-CT was positive for the disease in 26/30 patients (86.66%) and negative in 4/30 (13.33%) patients. The overall patient-specific sensitivity and specificity of 18 F-FDG-PET/CT were 92.86% and 100%, respectively. The 4-point visual grading system revealed 14/200 (7%) lesions demonstrating Grade I uptake, 49/200 (24.5%) lesions grade II uptake, 17/200 (8.5%) lesions grade III uptake and 40/200 (20%) lesions grade IV uptake. CONCLUSION: The results suggested that RGD-binding integrin is expressed in a sizeable fraction of metastatic lesions of TENIS cases, albeit demonstrating a varying degree of uptake. Out of the soft tissue, lung, and bone lesions, metastatic bone lesions showed more RGD affinity than other sites. The patients with substantial RGD uptake on a 4-point visual grading system may be potential targets for RGD-based therapy.

Rare Presentations of Differentiated Thyroid Cancer Exposing Dr Jekyll and Mr Hyde Nature of an Otherwise Indolent Disease: Case Series.

ABSTRACT: Differentiated thyroid carcinoma (DTC) usually manifests as an indolent cancer with good prognosis. However, rarely uncommon sites of metastatic involvement can worsen the prognosis and require aggressive therapeutic approach. Here in, we describe 5 patients (3 women and 2 men) harboring rare sites of metastatic involvement from DTC including the adrenals, colon, kidneys, urinary bladder, brachial plexus, and superior vena cava with contiguous right atrial involvement. The awareness of such rare sites of involvement from DTC is imperative for treating clinicians to plan individualistic approach in management including multiprong therapies for better patient care.

A high-content flow cytometry and dual CRISPR-Cas9 based platform to quantify genetic interactions.

Probing epistasis between two genes can be a critical first step in identifying the molecular players in a cellular pathway. The advent of CRISPR-Cas mediated genetic screen has enabled studying of these genetic interactions at a genomic scale. However, when combining depletion of two genes using CRISPR Cas9, reduced targeting efficiencies due to competition for Cas loading and recombination in the cloning step have emerged as key challenges. Moreover, given conventional CRISPR screens typically involve comparison between the initial and final time point, it is difficult to parse the time kinetics with which a perturbed genetic interaction impacts viability, and it also becomes challenging to assess epistasis with essential genes. Here, we discuss a high-throughput flow-based approach to study genetic interactions. By utilizing two different Cas9 orthologs and monitoring viability at multiple time points, this approach helps to effectively mitigate the limitations of Cas9 competition and enables assessment of genetic interactions with both essential and non-essential genes at a high temporal resolution.

Exogenous application of nanocarrier-mediated double-stranded RNA manipulates physiological traits and defence response against bacterial diseases.

Stability and delivery are major challenges associated with exogenous double-stranded RNA (dsRNA) application into plants. We report the encapsulation and delivery of dsRNA in cationic poly-aspartic acid-derived polymer (CPP6) into plant cells. CPP6 stabilizes the dsRNAs during long exposure at varied temperatures and pH, and protects against RNase A degradation. CPP6 helps dsRNA uptake through roots or foliar spray and facilitates systemic movement to induce endogenous gene silencing. The fluorescence of Arabidopsis GFP-overexpressing transgenic plants was significantly reduced after infiltration with gfp-dsRNA-CPP6 by silencing of the transgene compared to plants treated only with gfp-dsRNA. The plant endogenous genes flowering locus T (FT) and phytochrome interacting factor 4 (PIF4) were downregulated by a foliar spray of ft-dsRNA-CPP6 and pif4-dsRNA-CPP6 in Arabidopsis, with delayed flowering and enhanced biomass. The rice PDS gene targeted by pds-dsRNA-CPP6 through root uptake was effectively silenced and plants showed a dwarf and albino phenotype. The NaCl-induced OsbZIP23 was targeted through root uptake of bzip23-dsRNA-CPP6 and showed reduced transcripts and seedling growth compared to treatment with naked dsRNA. The negative regulators of plant defence SDIR1 and SWEET14 were targeted through foliar spray to provide durable resistance against bacterial leaf blight disease caused by Xanthomonas oryzae pv. oryzae (Xoo). Overall, the study demonstrates that transient silencing of plant endogenous genes using polymer-encapsulated dsRNA provides prolonged and durable resistance against Xoo, which could be a promising tool for crop protection and for sustaining productivity.

Temozolomide and flavonoids against glioma: from absorption and metabolism to exosomal delivery.

Patients with glioblastoma multiforme and anaplastic astrocytoma are treated with temozolomide. Although it has been demonstrated that temozolomide increases GBM patient survival, it has also been connected to negative immune-related adverse effects. Numerous research investigations have shown that flavonoids have strong antioxidant and chemo-preventive effects. Consequently, it might lessen chemotherapeutic medicines' side effects while also increasing therapeutic effectiveness. The need for creating innovative, secure, and efficient drug carriers for cancer therapy has increased over time. Recent research indicates that exosomes have enormous potential to serve as carriers and cutting-edge drug delivery systems to the target cell. In recent years, researchers have been paying considerable attention to exosomes because of their favorable biodistribution, biocompatibility, and low immunogenicity. In the present review, the mechanistic information of the anti-glioblastoma effects of temozolomide and flavonoids coupled with their exosomal delivery to the targeted cell has been discussed. In addition, we discuss the safety aspects of temozolomide and flavonoids against glioma. The in-depth information of temozolomide and flavonoids action via exosomal delivery can unravel novel strategies to target Glioma.

68 Ga-PSMA-11 PET/CT and 64 CuCl 2 PET/CT Help in Identifying Rare Metastatic Site of Penile Shaft in a Patient of Carcinoma Prostate.

ABSTRACT: A 71-year-old man, presenting with complaints of burning sensation and pain during urination, finally diagnosed with prostate carcinoma. Ultrasonography of the abdomen and pelvis revealed prostatomegaly. Serum PSA level was elevated, and TRUS-guided biopsy demonstrated acinar adenocarcinoma (Gleason score: 5 + 4 = 9). 68 Ga-PSMA-11 PET/CT for initial staging showed PSMA-avid enlarged prostate, pelvic lymphadenopathy, and focal PSMA uptake in the left side of the shaft of the penis. The patient also underwent a 64 CuCl 2 PET/CT, which demonstrated similar findings of enlarged prostate and adenopathy with focally increased tracer uptake in the shaft of the penis coinciding with the lesion observed on 68 Ga-PSMA-11 PET/CT, thereby detecting a rare metastatic site from carcinoma prostate.

Recent advances and future directions in etiopathogenesis and mechanisms of reactive oxygen species in cancer treatment.

A class of exceptionally bioactive molecules known as reactive oxygen species (ROS) have been widely studied in the context of cancer. They play a significant role in the etiopathogenesis for cancer. Implication of ROS in cancer biology is an evolving area, considering the recent advances; insights into their generation, role of genomic and epigenetic regulators for ROS, earlier thought to be a chemical process, with interrelations with cell death pathways- Apoptosis, ferroptosis, necroptosis and autophagy has been explored for newer targets that shift the balance of ROS towards cancer cell death. ROS are signal transducers that induce angiogenesis, invasion, cell migration, and proliferation at low to moderate concentrations and are considered normal by-products of a range of biological activities. Although ROS is known to exist in the oncology domain since time immemorial, its excessive quantities are known to damage organelles, membranes, lipids, proteins, and nucleic acids, resulting in cell death. In the last two decades, numerous studies have demonstrated immunotherapies and other anticancer treatments that modulate ROS levels have promising in vitro and in vivo effects. This review also explores recent targets for therapeutic interventions in cancer that are based on ROS generation or inhibition to disrupt the cell oxidative stress balance. Examples include-metabolic targets, targeted therapy with biomarkers, natural extracts and nutraceuticals and targets developed in the area of nano medicine. In this review, we present the molecular pathways which can be used to create therapy plans that target cancer by regulating ROS levels, particularly current developments and potential prospects for the effective implementation of ROS-mediated therapies in clinical settings. The recent advances in complex interaction with apoptosis especially ferroptosis and its role in epigenomics and modifications are a new paradigm, to just mechanical action of ROS, as highlighted in this review. Their inhibition by nutraceuticals and natural extracts has been a scientific challenging avenue that is explored. Also, the inhibition of generation of ROS by inhibitors, immune modulators and inhibitors of apoptosis and ferroptosis is explored in this review.

68 Ga-PSMA-11 PET/CT Imaging in Brain Gliomas and Its Correlation With Clinicopathological Prognostic Parameters.

BACKGROUND: Gliomas are the most common primary central nervous system tumors, of which the malignant gliomas account for 60%-75%. The primary and secondary brain malignancies are highly treatment resistant, and their marked angiogenesis attracts interest as a potential therapeutic target. The grade of gliomas, Ki-67 index, and IDH mutation status are among the major prognostic markers in gliomas. Prostate-specific membrane antigen (PSMA) is a zinc-dependent peptidase that is not only expressed in prostate cancer cells but also in the tumor neovasculature. The initial PSMA PET studies in central nervous system tumors using 68 Ga-HBED-CC-PSMA ( 68 Ga-PSMA-11) PET tracer confirmed selective target expression in gliomas of different grades, with higher expression in high-grade glioma compared with low-grade glioma. AIMS AND OBJECTIVES: The aim of the present study was to correlate and compare the 68 Ga-PSMA-11 and 18 F-FDG uptake in brain tumors with their clinicopathological prognostic parameters, so as to study their prognostic implications. In addition, the study also aimed to identify patients who are likely to benefit from potential PSMA-targeted therapies. PATIENTS AND METHODS: This ongoing prospective study was approved by the institutional scientific and medical ethics committee. The patients with primary or recurrent glioma lesions on MRI underwent regional brain PET/CT scanning with 68 Ga-PSMA-11 and 18 F-FDG. The final histopathology of the brain lesions (glioma grade), Ki-67 index, and IDH mutation status were compared with SUV max values of the 68 Ga-PSMA-11 and 18 F-FDG PET/CT. RESULTS: A total of 15 patients (13 males and 2 females; age range, 21-73 years; median age, 58 years) were included in this study analysis. Among the 15 patients, 10 were treatment naive and 2 were patients with recurrent glioma. Three patients turned out to be WHO grade I-II, 6 belonged to grade III, and 6 grade IV (glioblastoma multiforme) on final histopathology. The 68 Ga-PSMA-11 PET/CT showed tracer uptake in all high-grade gliomas with good tumor-to-background ratio. It was PSMA nonavid in 2/3 low-grade gliomas, and it showed low-grade uptake in 1/3 patients. PSMA expression (as evaluated by SUV max values) was significantly higher in higher-grade tumors, those with IDH mutation wildtype status, and higher Ki-67 indices. FDG PET SUV max also showed significant correlation with these prognostic parameters. CONCLUSIONS: In these preliminary results, PSMA PET appears to be an important tool in the evaluation and prognosis of gliomas. PSMA-directed theranostics can be explored as a personalized approach in gliomas with high PSMA uptake. However, with the limitation of small sample size, larger clinical trials are warranted to draw conclusive evidence regarding the same.

Childhood health and educational disadvantage are associated with adult multimorbidity in the global south: findings from a cross-sectional analysis of nationally representative surveys in India and Brazil.

INTRODUCTION: Multimorbidity has emerged as a major healthcare challenge in low/middle-income countries (LMICs) such as India and Brazil. Life course epidemiology suggests that adverse events in early life contribute to an individual's later health in adulthood. However, little is known about the influence of early life health and social factors on the development of multimorbidity in adulthood in LMICs. We aimed to explore the association of adult multimorbidity with childhood health and social disadvantages among two LMICs, India and Brazil. METHODS: We conducted a secondary data analysis of older adults aged ≥50 years using nationally representative surveys from Longitudinal Ageing Study in India, 2017-2018 (n=51 481) and 'Estudo Longitudinal da Saude e Bem-Estar dos Idosos Brasileirous', 2015-2016 (n=8730). We estimated the prevalence of multimorbidity along with 95% CI as a measure of uncertainty for all weighted proportions. Log link in generalised linear model was used to assess the association between childhood health and disadvantages with multimorbidity, reported as adjusted prevalence ratio (APR). RESULTS: The prevalence of multimorbidity was 25.53% and 55.24% in India and Brazil, respectively. Participants who perceived their childhood health as poor and missed school for a month or more due to illness had the highest level of multimorbidity across both countries. After adjusting for age and gender, a significant association between adult multimorbidity and poor self-rated childhood health (APR: (India: 1.38, 1.16 to 1.65) and (Brazil: 1.19, 1.09 to 1.30)); and missed school for a month due to illness (AOR: (India: 1.73, 1.49 to 2.01) and (Brazil: 1.16, 1.08 to 1.25)) was observed. CONCLUSION: Early life health, educational and economic disadvantages are associated with adult multimorbidity and appear to contribute to the later course of life. A life course approach to the prevention of multimorbidity in adulthood in LMICs may be useful in health programmes and policies.

Angiogenesis Imaging of Adrenocortical Carcinoma with Ga-68-NODAGA-RGD Positron Emission Tomography: Opening New Horizons in Multimodality Imaging from Theranostic Perspective.

A 53-year-old female, with a known case of adrenocortical carcinoma (ACC), underwent F-18 fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) for initial staging, which revealed FDG avid large left suprarenal mass contiguous with hypermetabolic tumor thrombus in the inferior vena cava (IVC) through the left renal vein. Thereafter, she underwent angiogenesis imaging using Ga-68-NODAGA-RGD PET/CT, which showed similar avid tracer uptake in both primary and IVC thrombus. Demonstration of RGD avidity in ACC in this case opens a new horizon for targeted radionuclide therapy (e.g., Lu-177 RGD) in selected patients, who may have limited therapeutic options.

Effect of Ga substitution with Al in ZSM-5 zeolite in methanethiol-to-hydrocarbon conversion.

The catalytic properties of conventional H-[Al]-ZSM-5 and gallium-substituted H-[Ga]-ZSM-5 were evaluated in the conversion of methanethiol to ethylene (CH3SH → 1/2C2H4 + H2S). Dimethyl sulfide (DMS), aromatics, and CH4 were formed as byproducts on the H-[Al]-ZSM-5 catalyst. The introduction of Ga into the ZSM-5 structure provided a high ethylene yield with relatively high selectivity for olefins. Based on the temperature-programmed desorption of NH3 and pyridine adsorption on zeolites, the strength of acid sites was decreased by introducing Ga into the ZSM-5 structure. Undesirable reactions seemed less likely to occur at weakly acidic sites. The suppression of the formation of dimethyl sulfide (CH3SH → 1/2C2H6S + 1/2H2S) and the sequential reaction of ethylene to produce aromatics provided a high yield of ethylene over H-[Ga]-ZSM-5.

Extracellular Matrix-Derived Damage-Associated Molecular Patterns (DAMP): Implications in Systemic Sclerosis and Fibrosis.

Damage-associated molecular patterns (DAMPs) are intracellular molecules released under cellular stress or recurring tissue injury, which serve as endogenous ligands for toll-like receptors (TLRs). Such DAMPs are either actively secreted by immune cells or passively released into the extracellular environment from damaged cells or generated as alternatively spliced mRNA variants of extracellular matrix (ECM) glycoproteins. When recognized by pattern recognition receptors (PRRs) such as TLRs, DAMPs trigger innate immune responses. Currently, the best-characterized PRRs include, in addition to TLRs, nucleotide-binding oligomerization domain-like receptors, RIG-I-like RNA helicases, C-type lectin receptors, and many more. Systemic sclerosis (SSc) is a chronic autoimmune condition characterized by inflammation and progressive fibrosis in multiple organs. Using an unbiased survey for SSc-associated DAMPs, we have identified the ECM glycoproteins fibronectin-containing extra domain A and tenascin C as the most highly upregulated in SSc skin and lung biopsies. These DAMPs activate TLR4 on resident stromal cells to elicit profibrotic responses and sustained myofibroblasts activation resulting in progressive fibrosis. This review summarizes the current understanding of the complex functional roles of DAMPs in the progression and failure of resolution of fibrosis in general, with a particular focus on SSc, and considers viable therapeutic approaches targeting DAMPs.

Pharmacological inhibition of TAK1 prevents and induces regression of experimental organ fibrosis.

Multiorgan fibrosis in systemic sclerosis (SSc) accounts for substantial mortality and lacks effective therapies. Lying at the crossroad of TGF-ß and TLR signaling, TGF-ß-activated kinase 1 (TAK1) might have a pathogenic role in SSc. We therefore sought to evaluate the TAK1 signaling axis in patients with SSc and to investigate pharmacological TAK1 blockade using a potentially novel drug-like selective TAK1 inhibitor, HS-276. Inhibiting TAK1 abrogated TGF-ß1 stimulation of collagen synthesis and myofibroblasts differentiation in healthy skin fibroblasts, and it ameliorated constitutive activation of SSc skin fibroblasts. Moreover, treatment with HS-276 prevented dermal and pulmonary fibrosis and reduced the expression of profibrotic mediators in bleomycin-treated mice. Importantly, initiating HS-276 treatment even after fibrosis was already established prevented its progression in affected organs. Together, these findings implicate TAK1 in the pathogenesis of SSc and identify targeted TAK1 inhibition using a small molecule as a potential strategy for the treatment of SSc and other fibrotic diseases.

Comparison of the molecular profiling of core biopsy with surgical specimens in breast cancers and the effect of neoadjuvant therapy on the same - A North Indian study.

Aims and Objectives: The purpose of this study was to determine the concordance of core needle biopsy (CNB) and surgical specimens for determining the molecular profiling and to observe the changes in the same after neoadjuvant chemotherapy. Materials and Methods: This was a cross-sectional study over a period of one year on 95 cases. Immunohistochemical (IHC) staining was done as per the staining protocol in a fully automated BioGenex Xmatrx staining machine. Results: On CNB, estrogen receptor (ER) positivity was seen in 58 out of 95 cases, comprising 61% of the total, and on mastectomy, it was positive in 43 (45%) cases. Progesterone receptor (PR) positivity was seen in 59 (62%) cases on CNB and 44 (46%) cases on mastectomy. Total 7 (7%) were human epidermal growth factor receptor 2 (HER2)/neu positive on CNB and 8 (8%) on mastectomy, respectively. There were 15 (15.7%) that showed discordant results after neoadjuvant therapy. Estrogen status changed from negative to positive in 1 (7%) case and positive to negative in 14 (93%) cases. Progesterone status changed from positive to negative in all 15 cases (100%). There was no change in the HER2/neu status. The agreement of hormone receptor status between CNB and subsequent mastectomy in the present study was found to be substantial (kappa value for ER, PR, and HER2neu as 0.608, 0.648, and 0.648, respectively. Conclusion: IHC is a cost-effective method to assess hormone receptor expression. This study shows that ER, PR, and HER2/neu expression in CNB should be reassessed in excision specimens for the better management of endocrine therapy.

Synthesis of 2,5-dimethylhexene by isobutene dimerization with H2S co-feeding.

This study investigates the effect of hydrogen sulfide (H2S) co-feeding on the synthesis of 2,5-dimethyl-1-hexene, 2,5-dimethyl-2-hexene, and 2,5-dimethylhexane (2,5-DMHs), useful compounds, using the dimerization of isobutene under mild pressure conditions. The dimerization of isobutene did not proceed in the absence of H2S, whereas the desired products of 2,5-DMHs were produced under H2S co-feeding conditions. The effect of reactor size on the dimerization reaction was then examined, and the optimal reactor was discussed. To enhance the yield of 2,5-DMHs, we changed the reaction conditions of the temperature, molar ratio of isobutene to H2S (iso-C4[double bond, length as m-dash]/H2S) in the feed gas, and the total feed pressure. The optimum reaction condition was at 375 °C and 2/1 of iso-C4[double bond, length as m-dash]/H2S. The product of 2,5-DMHs monotonously increased by an increment of total pressure from 1.0 to 3.0 atm with a fixed iso-C4[double bond, length as m-dash]/H2S ratio at 2/1.

Characterization of the Sediment Bacterial Community Structure and Composition in Najafgarh Lake and Adjoining Dhansa Barrage.

This study was undertaken to assess the changes in the community structure, diversity, and composition of sediment bacteria in a shallow lake, Najafgarh Lake (NL), that receives untreated sewage effluent through drains connected to it. These changes were analyzed by comparing the sediment bacterial community structure of NL to the sediment bacterial community structure of Dhansa Barrage (DB), which receives no such effluents. 16S rRNA amplicon was used for bacterial community analysis. Water and sediment samples were also analyzed and compared revealing high conductivity, ammonia, nitrite content, and low dissolved oxygen in NL. The organic matter content is also higher in the sediments of NL. Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria are the predominant phyla in both sites and account for 91% of total bacterial abundance in DB and only 77% in the case of NL. Proteobacteria have the highest relative abundance, accounting for around 42% of the total bacterial population in the case of DB and Firmicutes has the highest relative abundance in Najafgarh at 30%. The diversity analysis found significant differences in the community structure at the two sites. The variation in the bacterial communities in the two wetlands is significantly associated with two water parameters (conductivity and temperature) and two sediment parameters (Sediment Nitrogen and Sediment Organic Matter). Correlation Analysis showed that high ammonia, nitrite, and conductance in NL resulted in bacterial communities shifting towards phyla abundant in degraded ecosystems like Acidobacteria, Choloroflexi, Caldiserica, Aminicenantes, Thaumarchaeota, and Planctomycetes.

Highly stable Fe/CeO2 catalyst for the reverse water gas shift reaction in the presence of H2S.

This study focused on evaluating the catalytic properties for the reverse water gas shift reaction (RWGS: CO2 + H2 → CO + H2O ΔH 0 = 42.1 kJ mol-1) in the presence of hydrogen sulfide (H2S) over a Fe/CeO2 catalyst, commercial Cu-Zn catalyst for the WGS reaction (MDC-7), and Co-Mo catalyst for hydrocarbon desulfurization. The Fe/CeO2 catalyst exhibited a relatively high catalytic activity to RWGS, compared to the commercial MDC-7 and Co-Mo catalysts. In addition, the Fe/CeO2 catalyst showed stable performance in the RWGS environment that contained high concentrations of H2S. The role of co-feeding H2S was investigated over the Fe/CeO2 catalyst by the temperature programmed reaction (TPR) of CO2 and H2 in the presence of H2S. The result of TPR indicated that the co-feeding H2S might enhance RWGS performance due to H2S acting as the hydrogen source to reduce CO2.

Salt-Induced Doping and Templating of Laser-Induced Graphene Supercapacitors.

The use of inexpensive and widely available CO2 lasers to selectively irradiate polymer films and form a graphene foam, termed laser-induced graphene (LIG), has incited significant research attention. The simple and rapid nature of the approach and the high conductivity and porosity of LIG have motivated its widespread application in electrochemical energy storage devices such as batteries and supercapacitors. However, nearly all high-performance LIG-based supercapacitors reported to date are prepared from costly, petroleum-based polyimide (Kapton, PI). Herein, we demonstrate that incorporating microparticles of inexpensive, non-toxic, and widely abundant sodium salts such as NaCl and Na2SO4 into poly(furfuryl alcohol) (PFA) resins enables the formation of high-performance LIG. The embedded particles aid in carbonization and act as a template for pore formation. While increasing both the carbon yield and surface area of the electrodes, the salt also dopes the LIG formed with S or Cl. The combination of these effects results in a two- to four-order-of-magnitude increase in device areal capacitance, from 8 µF/cm2 for PFA/no salt at 5 mV/s to up to 80 mF/cm2 for some PFA/20% Na2SO4 samples at 0.05 mA/cm2, significantly higher than that of PI-based devices and most other LIG precursors.

Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence.

Replication stress is a hallmark of several ovarian cancers. Replication stress can emerge from multiple sources, including double-strand breaks, transcription-replication conflicts, or amplified oncogenes, inevitably resulting in the generation of single-stranded DNA (ssDNA). Quantifying ssDNA, therefore, presents an opportunity to assess the level of replication stress in different cell types and under various DNA-damaging conditions or treatments. Emerging evidence also suggests that ssDNA can be a predictor of responses to chemotherapeutic drugs that target DNA repair. Here, we describe a detailed immunofluorescence-based methodology to quantify ssDNA. This methodology involves labeling the genome with a thymidine analog, followed by the antibody-based detection of the analog at the chromatin under non-denaturing conditions. Stretches of ssDNA can be visualized as foci under a fluorescence microscope. The number and intensity of the foci directly co-relate with the level of ssDNA present in the nucleus. We also describe an automated pipeline to quantify the ssDNA signal. The method is rapid and reproducible. Furthermore, the simplicity of this methodology makes it amenable to high-throughput applications such as drug and genetic screens.