A Bispecific Antibody That Targets the Membrane-Proximal Region of Mesothelin and Retains High Anticancer Activity in the Presence of Shed Mesothelin.

Mesothelin (MSLN) is a cell-surface protein that is expressed in many cancers, which makes it a popular target for Ab-based cancer therapy. However, MSLN is shed from cancer cells at high levels via proteases that cleave at its membrane-proximal C-terminal region. Shed MSLN accumulates in patients' fluids and tumors and can block Ab-based MSLN-targeting drugs from killing cancer cells. A previously established mAb, 15B6, binds MSLN at its protease-sensitive C-terminal region and does not bind shed MSLN. Moreover, 15B6 variable fragment (Fv)-derived chimeric antigen receptor T cells are not inhibited by shed MSLN and kill tumors in mice more effectively than mAb SS1 Fv-derived chimeric antigen receptor T cells, which bind an epitope retained in shed MSLN. In this study, we have established 15B6 Fv-derived MSLN × CD3 bispecific antibodies (BsAb) that target MSLN-expressing cancers. We identified our lead candidate BsAb 5 after screening multiple 15B6-derived BsAb formats in vitro for cytotoxic activity. BsAb 5 activates T cells to kill various cancer cell lines in a MSLN-specific manner. MSLN 296-591 His, a recombinant protein mimicking shed MSLN, does not inhibit 15B6-derived BsAb 5 but completely inhibits humanized SS1-derived BsAb 7. Furthermore, BsAb 5 inhibits and delays tumor growth and is not inhibited by MSLN 296-585 His in mice. Our findings indicate that by targeting the protease-sensitive region of MSLN, BsAb 5 has high MSLN-specific anticancer activity that is not inhibited by shed MSLN. BsAb 5 may be a promising immunotherapy candidate for MSLN-expressing cancers.

A bispecific antibody that targets the membrane-proximal region of mesothelin and retains high anticancer activity in the presence of shed mesothelin.

Mesothelin (MSLN) is a cell-surface protein that is expressed on many cancers, which makes it a popular target for antibody-based cancer therapy. However, MSLN is shed from cancer cells at high levels via proteases that cleave at its membrane-proximal C-terminal region. Shed MSLN accumulates in patient fluids and tumors and can block antibody-based MSLN-targeting drugs from killing cancer cells. A previously established monoclonal antibody (mAb), 15B6, binds MSLN at its protease-sensitive C-terminal region and does not bind shed MSLN. 15B6 variable fragment (Fv)-derived chimeric antigen receptor (CAR) T cells are not inhibited by shed MSLN and kill tumors in mice more effectively than mAb SS1 Fv-derived CAR T cells, which bind an epitope retained in shed MSLN. Here, we have established 15B6 Fv-derived MSLN x CD3 bispecific antibodies (BsAbs) that target MSLN-expressing cancers. We identified our lead candidate, BsAb 5, after screening multiple 15B6-derived BsAb formats in vitro for cytotoxic activity. BsAb 5 activates T cells to kill various cancer cell lines in a MSLN-specific manner. MSLN 296-591 His, a recombinant protein mimicking shed MSLN, does not inhibit 15B6-derived BsAb 5 but completely inhibits humanized SS1-derived BsAb 7. Furthermore, BsAb 5 inhibits and delays tumor growth and is not inhibited by MSLN 296-585 His in mice. Our findings indicate that by targeting the protease-sensitive region of MSLN, BsAb 5 has high MSLN-specific anticancer activity that is not inhibited by shed MSLN. BsAb 5 may be a promising immunotherapy candidate for MSLN-expressing cancers.

Allicin extracted from Allium sativum shows potent anti-cancer and antioxidant properties in zebrafish.

Garlic (Allium sativum) is an important flavouring component in Indian cuisine. Allicin, a sulphur containing compound, is the most abundant component of garlic and has been widely studied for its antimicrobial and antioxidant properties. It is also known to play a role in the regulation of blood pressure and cholesterol levels. Despite the known health benefits associated with allicin, systematic studies on its anti-cancer properties using animal models are very limited. This study aimed to develop a simple method for the extraction of allicin from fresh garlic, study the stability of the extracted compound at various temperatures, and evaluate the antioxidant, anti-proliferative, pro-apoptotic and anti-angiogenic properties in zebrafish. A five-month stability study indicated that allicin remains significantly stable at temperatures 4 °C and below but shows extensive degradation if stored at room temperature. The in vivo studies in zebrafish using a combination of mutants and transgenic lines demonstrated the antioxidant, anti-proliferative, apoptotic and anti-angiogenic properties of allicin. The study highlights the importance of natural bioactive compounds as potential anti-cancer agents that can be studied further.

Assays with Patient-Derived Organoids to Evaluate the Impact of Microbial Infection on Base Excision Repair (BER) Enzymes.

Microbes play an important role in regulating cellular responses and the induction of chronic diseases. Infection and chronic inflammation can cause DNA damage, and the accumulation of mutations leads to cancer development. The well-known examples of cancer-associated microbes are Helicobacter pylori in gastric cancer and Fusobacterium nucleatum (Fn), Bacteroides fragilis, and E.coli NC101 in colorectal cancer (CRC). These carcinopathogens modify the expressions of the base excision repair enzymes and cause DNA damage. This chapter will show how Fn can initiate CRC through the downregulation of a critical enzyme of the base excision repair (BER) pathway that subsequently causes accumulation of DNA damage. We used the stem cell-based organoid model and enteroid-derived monolayer (EDM) from the murine and human colon to assess the impact of infection on the expression of BER enzymes on the transcriptional and translational levels and to develop other functional assays. For example, we used this EDM model to assess the inflammatory response, DNA damage response, and physiological responses, where we correlated the level of these parameters to BER enzyme levels.

Investigation of anti-proliferative and anti-angiogenic properties of Parkia javanica bark and fruit extracts in zebrafish.

The use of herbal products as traditional medicines has been a practice in India for centuries. Due to high ethnic diversity, the pool of herbal medicines is enormous, and they are often preferred over modern medicines in certain parts of the country. Cancer is one of the major non-communicable diseases affecting people worldwide. Despite considerable research, cancer is a disease that is still not understood completely, and there have been constant efforts towards the identification of novel drugs or approaches in cancer management. Parkia javanica, an important medicinal plant and a rich source of flavonoids and terpenoids, is widely studied for its antioxidant and anti-inflammatory activities. Traditionally, the fruit and bark extracts of P. javanica find use as home remedy for dysentery and piles in NE India. Moreover, the fruits are consumed by the people of North-East (NE) India as vegetables, either in steamed or cooked form. In this study, crude extracts of P. javanica fruit and bark were obtained, the sub-lethal dose was determined and were then analyzed for anti-proliferative and anti-angiogenic properties using a battery of assays in zebrafish embryos. The sub-lethal concentration 50 (LC50) was found to be 28.66 mg/L and 346.66 mg/L for bark and fruit extract respectively, indicating a decreased toxicity of the fruit extract compared to that of the bark. The anti-proliferative and anti-angiogenic properties were more pronounced for the fruit extract compared to the bark extract. Although preliminary, the results of the study suggest that P. javanica fruits possess potent anti-angiogenic and anti-proliferative properties, which can be further studied for the isolation of active phytochemicals for use as therapeutic agents.

Multi-year seabed environmental baseline in deep-sea offshore oil prospective areas established using microbial biodiversity.

Microorganisms are the ocean's first responders to marine pollution events, yet baseline studies rarely focus on microbial communities. Temporal and spatial microbial biodiversity baselines were established using bacterial 16S rRNA gene amplicon sequencing of seafloor sediments in a deep-water oil prospective area along the Scotian Slope off Canada's east coast sampled during 2015-2018. Bacterial diversity was generally similar in space and time, with members of the family Woeseiaceae detected consistently in >1 % relative abundance, similar to seabed sediments in other parts of the world. Anomalous biodiversity results at one site featured lower Woeseiaceae as well as higher levels of bacterial groups specifically associated with cold seeps such as Aminicenantes. This was unexpected given that site selection was based on sediment geochemistry not revealing any petroleum hydrocarbons in these locations. This finding highlights the sensitivity and specificity of microbial DNA sequencing in environmental monitoring. Microbiome assessments like this one represent an important strategy for incorporating microbial biodiversity as a new and useful metric for establishing robust environmental baselines that are necessary for understanding ecosystem responses to marine pollution.

Poly (A)-specific ribonuclease deficiency impacts oogenesis in zebrafish.

Poly (A)-specific ribonuclease (PARN) is the most important 3'-5'exonuclease involved in the process of deadenylation, the removal of poly (A) tails of mRNAs. Although PARN is primarily known for its role in mRNA stability, recent studies suggest several other functions of PARN including a role in telomere biology, non-coding RNA maturation, trimming of miRNAs, ribosome biogenesis and TP53 function. Moreover, PARN expression is de-regulated in many cancers, including solid tumours and hematopoietic malignancies. To better understand the in vivo role of PARN, we used a zebrafish model to study the physiological consequences of Parn loss-of-function. Exon 19 of the gene, which partially codes for the RNA binding domain of the protein, was targeted for CRISPR-Cas9-directed genome editing. Contrary to the expectations, no developmental defects were observed in the zebrafish with a parn nonsense mutation. Intriguingly, the parn null mutants were viable and fertile, but turned out to only develop into males. Histological analysis of the gonads in the mutants and their wild type siblings revealed a defective maturation of gonadal cells in the parn null mutants. The results of this study highlight yet another emerging function of Parn, i.e., its role in oogenesis.

Regulation of DNA damage response by trimeric G-proteins.

Upon sensing DNA double-strand breaks (DSBs), eukaryotic cells either die or repair DSBs via one of the two competing pathways, i.e., non-homologous end-joining (NHEJ) or homologous recombination (HR). We show that cell fate after DSBs hinges on GIV/Girdin, a guanine nucleotide-exchange modulator of heterotrimeric Giα•ßγ protein. GIV suppresses HR by binding and sequestering BRCA1, a key coordinator of multiple steps within the HR pathway, away from DSBs; it does so using a C-terminal motif that binds BRCA1's BRCT-modules via both phospho-dependent and -independent mechanisms. Using another non-overlapping C-terminal motif GIV binds and activates Gi and enhances the "free" Gßγ→PI-3-kinase→Akt pathway, which promotes survival and is known to suppress HR, favor NHEJ. Absence of GIV, or loss of either of its C-terminal motifs enhanced cell death upon genotoxic stress. Because GIV selectively binds other BRCT-containing proteins suggests that G-proteins may fine-tune sensing, repair, and survival after diverse types of DNA damage.

Molecular etiology of defective nuclear and mitochondrial ribosome biogenesis: Clinical phenotypes and therapy.

Ribosomopathies are rare congenital disorders associated with defective ribosome biogenesis due to pathogenic variations in genes that encode proteins related to ribosome function and biogenesis. Defects in ribosome biogenesis result in a nucleolar stress response involving the TP53 tumor suppressor protein and impaired protein synthesis leading to a deregulated translational output. Despite the accepted notion that ribosomes are omnipresent and essential for all cells, most ribosomopathies show tissue-specific phenotypes affecting blood cells, hair, spleen, or skin. On the other hand, defects in mitochondrial ribosome biogenesis are associated with a range of clinical manifestations affecting more than one organ. Intriguingly, the deregulated ribosomal function is also a feature in several human malignancies with a selective upregulation or downregulation of specific ribosome components. Here, we highlight the clinical conditions associated with defective ribosome biogenesis in the nucleus and mitochondria with a description of the affected genes and the implicated pathways, along with a note on the treatment strategies currently available for these disorders.

Wild-type and cancer-prone zebrafish exhibit distinct gut microbial diversity and differential anti-inflammatory response upon infection.

The study investigated the gut microbial diversity and the role of gut-associated microorganisms in modulating the immune responses in normal (wild-type) and TP53M214K (cancer-prone) zebrafish. Biochemical tests, genus/species-specific PCR, and 16S rDNA sequencing were performed to characterize the bacteria isolated from the gut of wild-type (WT) and cancer-prone zebrafish. Gut microbiome analysis revealed greater diversity but reduced bacterial load in wild-type zebrafish compared with cancer-prone zebrafish, which had lesser diversity but higher bacterial load. Interestingly, the gut in cancer-prone fish showed selective colonization by opportunistic pathogens. The bacterial isolates showed resistance to antibiotics such as tetracycline, nalidixic acid, and ciprofloxacin. Gnotobiotic zebrafish embryos were established, and mono-colonization with the isolated bacteria was done to examine the expression of anti-inflammatory genes using real-time PCR. Variable expression of IL10 and IL4 was observed in germ-free (GF) wild-type embryos when mono-colonized with Staphylococcus sciuri and Vibrio cholerae. In contrast, germ-free TP53 mutant embryos showed a consistent downregulation of both the anti-inflammatory genes. Thus, a better immune response in WT embryos against S. sciuri or V. cholerae infection than in cancer-prone fish was observed, suggesting that genetic predisposition could contribute to disabling the immune system against infection.

Effect of NaCl, high iron, iron chelator and antibiotics on growth, virulence gene expression and drug susceptibility in non-typhoidal Salmonella: an in vitro fitness study.

Salmonella is one among the most versatile and resilient enteric pathogens that is known to have developed various survival strategies within the host system. The ability of the bacteria to circumvent the physiological parameters as well as dodge the antimicrobial stress environment within the host is one of the most crucial steps in establishing an infection. With an alarming rise in multi-drug resistant serovars of non-typhoidal Salmonella and lack of vaccine for combatting the infections, behaviour of the bacteria in the presence of host physiological conditions (NaCl, high and low iron) and antibiotics will help in understanding the survival strategies as well as mechanisms of resistance. Two multi-drug resistant and two sensitive serovars of Salmonella Weltevreden and Salmonella Newport isolated from poultry and seafood were used for growth kinetics and virulence gene expression study. The results obtained revealed that despite similar resistance pattern, effect of individual class of antibiotics on the growth of serovars varied. On the contrary, no significant difference was observed in growth pattern on exposure to these in vitro experimental conditions. Nevertheless, coupling these conditions with antibiotics drastically reduced the minimum inhibitory concentration (MIC) of antibiotics in resistant strains. A first of its kind study that draws attention on the significant effect of antibiotics and physiological conditions on MIC between resistant and sensitive non-typhoidal Salmonella serovars and expression of virulence genes from Salmonella pathogenicity island (SPI) 1 and 2 (invA, hilC, fliC2, sseA and ssrB).

Detection of clinically-relevant EGFR variations in de novo small cell lung carcinoma by droplet digital PCR.

Targeted therapy that utilizes tyrosine kinase inhibitors (TKIs), specific to epidermal growth factor receptors (EGFR) has changed the landscape of treatment of non-small cell lung cancer (NSCLC). The success or failure of this approach depends on presence of certain variations in the tyrosine kinase domain of EGFR gene. Generally, patients diagnosed with Small cell lung cancer (SCLC) are considered ineligible for TKI therapy owing to the absence of EGFR variations. . However, there is evidence of these variations being detected in SCLCs, both in de-novo and in transformed SCLCs (TKI-treated adenocarcinomas). Despite the presence of clinically-relevant EGFR variations in SCLCs, the response to TKIs has been inconsistent.  Liquid biopsy is a well-established approach in lung cancer management with proven diagnostic, prognostic and predictive applications. It relies on detection of circulating tumor-derived nucleic acids present in plasma of the patient. In this study, a liquid biopsy approach was utilized to screen 118 consecutive lung cancer patients for four clinically-relevant variations in EGFR gene, which included three activating/sensitizing variations (Ex18 G719S, Ex19del E746-A750 and Ex21 L858R) and one acquired/resistance (Ex20 T790M, de novo) variation by droplet digital PCR, the most advanced third generation PCR technique. As expected, clinically-relevant EGFR variations were found in majority of the non-small cell lung cancer cases. However, among the handful of small cell lung cancer samples screened, sensitizing variations (Ex18 G719S and Ex21 L858R) were seen in almost all of them. Interestingly, Ex20 T790M variation was not detected in any of the cases screened.  The results of our study indicate that EGFR variations are present in SCLCs and highly sensitive liquid biopsy techniques like ddPCR can be effectively utilized for this purpose of screening EGFR variations in such samples.

Highly active CAR T cells that bind to a juxtamembrane region of mesothelin and are not blocked by shed mesothelin.

SignificanceMesothelin (MSLN) is a cell-surface protein that is a popular target for antibody-based therapies. We have identified shed MSLN as a major obstacle to successful antibody therapies and prepared a monoclonal antibody that inhibits shedding and makes very active CAR T cells whose activity is not blocked by shed MSLN and merits further preclinical development.

Kinase-deficient BTK mutants confer ibrutinib resistance through activation of the kinase HCK.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib irreversibly binds BTK at Cys481, inhibiting its kinase activity and thus blocking transduction of B cell receptor (BCR) signaling. Although ibrutinib is durably effective in patients with B cell malignancies, many patients still develop ibrutinib-resistant disease. Resistance can arise because of mutations at the ibrutinib-binding site in BTK. Here, we characterized the mechanism by which two BTK mutations, C481F and C481Y, may lead to ibrutinib resistance. Both mutants lacked detectable kinase activity in in vitro kinase assays. Structural modeling suggested that bulky Phe and Tyr side chains at position 481 sterically hinder access to the ATP-binding pocket in BTK, contributing to loss of kinase activity. Nonetheless, BCR signaling still propagated through BTK C481F and C481Y mutants to downstream effectors, the phospholipase PLCγ2 and the transcription factor NF-κB. This maintenance of BCR signaling was partially achieved through the physical recruitment and kinase-independent activation of hematopoietic cell kinase (HCK). Upon BCR activation, BTK C481F or C481Y was phosphorylated by Src family kinases at Tyr551, which then bound to the SH2 domain of HCK. Modeling suggested that this binding disrupted an intramolecular autoinhibitory interaction in HCK. Activated HCK subsequently phosphorylated PLCγ2, which propagated BCR signaling and promoted clonogenic cell proliferation. This kinase-independent mechanism could inform therapeutic approaches to CLL bearing either the C481F or C481Y BTK mutants.

PARN Knockdown in Cell Lines Results in Differential and Cell-Specific Alterations in the Expression of Cancer-Associated mRNAs.

Ribonucleases (RNases) is the collective term used for the group of enzymes that are involved in mRNA degradation. The shortening of the poly (A) tail through deadenylation is the preferred mechanism of degradation of most eukaryotic mRNAs and poly (A)-specific ribonuclease (PARN) is the most important player in deadenylation.  Besides its primarily role in mRNA stability, PARN is also involved in several non-conventional functions. It is conceivable that a decreased RNase activity can alter the stability of cancer-associated mRNAs and this alteration may be differential in cells of different origin. METHODS: The effects of siRNA-mediated knockdown of PARN on the post-transcriptional expression of 16 oncogenes and 18 tumor suppressor genes in cells derived from different lineages (NCI-H460 and NCI-H522; lung cancer) and (HEK-293; kidney) were investigated. Further, the effects of PARN depletion on proliferation and death of the lung cancer cells were investigated. RESULTS: Quantitative real time PCR analysis revealed an cell-specific alteration in the expression of the target onco and tumor suppressor genes upon PARN depletion, differently, for cells derived from different lineages. The tumor suppressor genes showed a consistent pattern of down regulation upon PARN depletion in all the three cell types tested. In contrast, the expression of oncogenes was not consistent; while some oncogenes showed overexpression in HEK 293 cells, the majority of them were downregulated in the lung cancer cells. Further, PARN depletion did not alter the proliferation of lung cancer cells, which was in contrast to previous reports. CONCLUSION: The results of this study reveal that PARN deficiency leads to an altered stability of cancer-associated mRNA, distinctly, in cells of different lineages. Despite previous reports suggesting a potential therapeutic role of PARN in cancer, our results suggest that PARN may not be an important biomarker, particularly in lung cancer.

Non-Invasive Saliva-based Detection of Gene Mutations in Oral Cancer Patients by Oral Rub and Rinse Technique.

BACKGROUND: Oral Squamous Cell Carcinoma (OSCC) is the most widely reported cancer worldwide. Liquid biopsy, a method that relies on identification of tumor-associated cells and/or cell free nucleic acids from body fluids is becoming increasingly popular in cancer diagnostics. The aim of the study was to evaluate the feasibility of Oral Rub and Rinse (ORR) technique in determining the genetic changes in common biomarkers of oral cancer such as TP53 using DNA obtained from saliva of oral cancer patients. METHODS: A total of 15 oral cancer patients were recruited in the study and pre-surgical saliva samples were collected using the ORR technique. Tissue samples included in the study were obtained during the surgical excision of the cancerous oral lesion. Genomic DNA was isolated from the salivary cell plug and the tissues and the TP53 gene was amplified by PCR. The PCR products of all the exons of TP53 (Exons 2 to 11) were electrophoresed on agarose gel, purified and sequenced by Sanger method. The obtained sequences were compared with the reference sequence of TP53 gene. STATISTICAL ANALYSIS USED: Descriptive statistics were used and reported as frequency and percentage. RESULTS: Capillary sequencing of TP53 gene from tissue DNA revealed the presence of codon 72 c.215C>G (p.Pro72Arg) polymorphism in 10 patients (67%) and a heterozygous mutation at codon 172 c.514 G>T (p.Val172Phe) in 2 patients (13%). Among the 10 samples that showed codon 72 polymorphism, matched salivary DNA was available for 6 samples and 4 out of these showed same genetic change at codon 72. Similarly, of the 3 samples that showed codon 172 mutation, matched salivary DNA was available for 1 sample and the mutation status was identical. CONCLUSION: The results suggest a potential for clinical applications of ORR technique as an alternative to invasive tissue biopsy for detection of genetic changes in candidate biomarkers in oral cancer.

Poly (A)-specific ribonuclease (PARN): More than just "mRNA stock clearing".

In eukaryotic cells, the balance between the synthesis and the degradation decides the steady-state levels of messenger RNAs (mRNA). The removal of adenosine residues from the poly(A) tail, called deadenylation, is the first and the most crucial step in the process of mRNA degradation. Poly (A)-specific ribonuclease (PARN) is one such enzyme that catalyses the process of deadenylation. Although PARN has been primarily known as the regulator of the mRNA stability, recent evidence clearly suggests several other functions of PARN, including a role in embryogenesis, oocyte maturation, cell-cycle progression, telomere biology, non-coding RNA maturation and ribosome biogenesis. Also, deregulated PARN activity is shown to be a hallmark of specific disease conditions. Pathogenic variants in the PARN gene have been observed in various cancers and inherited bone marrow failure syndromes. The focus in this review is to highlight the emerging functions of PARN, particularly in the context of human diseases.

Transcription coupled base excision repair in mammalian cells: So little is known and so much to uncover.

Oxidized bases in the genome has been implicated in various human pathologies, including cancer, aging and neurological diseases. Their repair is initiated with excision by DNA glycosylases (DGs) in the base excision repair (BER) pathway. Among the five oxidized base-specific human DGs, OGG1 and NTH1 preferentially excise oxidized purines and pyrimidines, respectively, while NEILs remove both oxidized purines and pyrimidines. However, little is known about why cells possess multiple DGs with overlapping substrate specificities. Studies of the past decades revealed that some DGs are involved in repair of oxidized DNA base lesions in the actively transcribed regions. Preferential removal of lesions from the transcribed strands of active genes, called transcription-coupled repair (TCR), was discovered as a distinct sub-pathway of nucleotide excision repair; however, such repair of oxidized DNA bases had not been established until our recent demonstration of NEIL2's role in TC-BER of the nuclear genome. We have shown that NEIL2 forms a distinct transcriptionally active, repair proficient complex. More importantly, we for the first time reconstituted TC-BER using purified components. These studies are important for characterizing critical requirement for the process. However, because NEIL2 cannot remove all types of oxidized bases, it is unlikely to be the only DNA glycosylase involved in TC-BER. Hence, we postulate TC-BER process to be universally involved in maintaining the functional integrity of active genes, especially in post-mitotic, non-growing cells. We further postulate that abnormal bases (e.g., uracil), and alkylated and other small DNA base adducts are also repaired via TC-BER. In this review, we have provided an overview of the various aspects of TC-BER in mammalian cells with the hope of generating significant interest of many researchers in the field. Further studies aimed at better understanding the mechanistic aspects of TC-BER could help elucidate the linkage of TC-BER deficiency to various human pathologies.

TP53 codon 72 polymorphism and type 2 diabetes: a case-control study in South Indian population.

TP53 functions primarily as a tumor suppressor, controlling a myriad of signalling pathways that prevent a cell from undergoing malignant transformation. This tumor suppressive function requires an activation and stabilization of TP53 in response to cell stressors. However, besides its cancer-preventive functions, TP53 is also known to be involved in diverse cellular processes including metabolism, reproduction, stem cell renewal and development. Indeed, several lines of evidence strongly suggest that TP53 plays crucial role in diabetes. A number of studies have evaluated the association of genetic alterations (single nucleotide variations) in TP53 gene with the development of diabetes. However, the results have not been consistent. The aim of this study was to evaluate whether the C/G polymorphism at codon 72 (Pro72/Arg72), located in exon 4 of TP53, is associated with type 2 diabetes in South Indian population. A total of 74 type 2 diabetic patients and 54 non-diabetic subjects were screened. None of the three genotypes, namely C/C (Pro/Pro), C/G (Pro/Arg), and G/G (Arg/Arg) was found to be significantly associated with type 2 diabetes in our study group. The findings of this study indicate that TP53 codon 72 polymorphism is not associated with increased risk of type 2 diabetes in South Indian population. Further studies with a large cohort size would be necessary to corroborate the observations of this study. Nevertheless, this study represents the first genetic analysis of TP53 variants in South Indian type 2 diabetic patients.

Quantitative analysis of cell-free DNA by droplet digital PCR reveals the presence of EGFR mutations in non-malignant lung pathologies.

Tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) are effectively used in treatment of non-small cell lung cancer (NSCLC). Mutation profile of tyrosine kinase domain of EGFR determines the eligibility of the patients for tyrosine kinase inhibitor (TKI) therapy. Liquid biopsy, which relies on circulating tumor-derived nucleic acids, has emerged as an effective tool in lung cancer management with proven diagnostic, prognostic and predictive applications. We screened 100 subjects, suspected to have lung malignancy, for four hotspot mutations including three activating (G719S, Ex19del E746-A750 and L858R) and one acquired (T790M, de novo) in EGFR gene by droplet digital PCR (ddPCR). While 97 subjects were subsequently confirmed to have lung malignancy based on histo/cytopathological studies, three cases turned out to be non-malignant lung pathologies that were completely cured by antibiotic therapy. Intriguingly, ddPCR revealed the presence of EGFR mutations in these non-malignant subjects. Two cases showed the presence of G719S and T790M mutations respectively and another had compound mutations (T790M and L858R). The detection of EGFR mutations in non-malignant pulmonary conditions opens up a new area of research.