Structural and mechanistic insights into Quinolone Synthase to address its functional promiscuity.

Quinolone synthase from Aegle marmelos (AmQNS) is a type III polyketide synthase that yields therapeutically effective quinolone and acridone compounds. Addressing the structural and molecular underpinnings of AmQNS and its substrate interaction in terms of its high selectivity and specificity can aid in the development of numerous novel compounds. This paper presents a high-resolution AmQNS crystal structure and explains its mechanistic role in synthetic selectivity. Additionally, we provide a model framework to comprehend structural constraints on ketide insertion and postulate that AmQNS's steric and electrostatic selectivity plays a role in its ability to bind to various core substrates, resulting in its synthetic diversity. AmQNS prefers quinolone synthesis and can accommodate large substrates because of its wide active site entrance. However, our research suggests that acridone is exclusively synthesized in the presence of high malonyl-CoA concentrations. Potential implications of functionally relevant residue mutations were also investigated, which will assist in harnessing the benefits of mutations for targeted polyketide production. The pharmaceutical industry stands to gain from these findings as they expand the pool of potential drug candidates, and these methodologies can also be applied to additional promising enzymes.

Telomerase inhibitors induce mitochondrial oxidation and DNA damage-dependent cell death rescued by Bcl-2/Bcl-xL.

BACKGROUND: Reactivation of telomerase is a hallmark of cancer and the majority of cancers over-express telomerase. Telomerase-dependent telomere length maintenance confers immortality to cancer cells. However, telomere length-independent cell survival functions of telomerase also play a critical role in tumorigenesis. Multiple telomerase inhibitors have been developed as therapeutics and include anti-sense oligonucleotides, telomerase RNA component targeting agents, chemical inhibitors of telomerase, small molecule inhibitors of hTERT, and telomerase vaccine. In general, telomerase inhibitors affect cell proliferation and survival of cells depending on the telomere length reduction, culminating in replicative senescence or cell death by crisis. However, most telomerase inhibitors kill cancer cells prior to significant reduction in telomere length, suggesting telomere length independent role of telomerase in early telomere dysfunction-dependent cell death. METHODS: In this study, we explored the mechanism of cell death induced by three prominent telomerase inhibitors utilizing a series of genetically encoded sensor cells including redox and DNA damage sensor cells. RESULTS: We report that telomerase inhibitors induce early cell cycle inhibition, followed by redox alterations at cytosol and mitochondria. Massive mitochondrial oxidation and DNA damage induce classical cell death involving mitochondrial transmembrane potential loss and mitochondrial permeabilization. Real-time imaging of the progression of mitochondrial oxidation revealed that treated cells undergo a biphasic mitochondrial redox alteration during telomerase inhibition, emphasizing the potential role of telomerase in the redox regulation at mitochondria. Additionally, silencing of hTERT confirmed its predominant role in maintaining mitochondrial redox homeostasis. Interestingly, the study also demonstrated that anti-apoptotic Bcl-2 family proteins still confer protection against cell death induced by telomerase inhibitors. CONCLUSION: The study demonstrates that redox alterations and DNA damage contribute to early cell death by telomerase inhibitors and anti-apoptotic Bcl-2 family proteins confer protection from cell death by their ability to safeguard mitochondria from oxidation damage.

Impact of age at vaccination and cervical HPV infection status on binding and neutralizing antibody titers at 10 years after receiving single or higher doses of quadrivalent HPV vaccine.

Long-term follow-up of a cohort of unmarried girls who received one, two, or three doses of quadrivalent HPV vaccine, between 10 and 18 years of age, in an Indian multi-centric study allowed us to compare antibody responses between the younger and older age cohorts at 10-years post-vaccination, and study the impact of initiation of sexual activity and cervical HPV infections on antibody levels. Among the younger (10-14 years) recipients of a single dose, 97.7% and 98.2% had detectable binding antibody titers against HPV 16 and HPV 18 respectively at ten years post-vaccination. The proportions among those receiving a single dose at age 15-18 years were 92.3% and 94.2% against HPV 16 and HPV 18 respectively. Mean HPV 16 binding antibody titers were 2.1 folds (95%CI 1.4 to 3.3) higher in those vaccinated at ages 10-14 years, and 1.9 folds (95%CI 1.2 to 3.0) higher in those vaccinated at 15-18 years compared to mean titers seen in the unvaccinated women. Compared to previous timepoints of 36 or 48 months, binding antibodies against HPV 16 and neutralizing antibodies against both HPV 16 and HPV 18 were significantly higher at 10 years. This rise was more pronounced in participants vaccinated at 15-18 years. No association of marital status or cervical HPV infections was observed with the rise in titer. Durability of antibody response in single dose recipients correlated well with the high efficacy of a single dose against persistent HPV 16/18 infections irrespective of age at vaccination, as we reported earlier.

Impact of HPV vaccination on HPV-related oral infections.

BACKGROUND: Human papillomavirus (HPV) is one of the most common sexually transmitted infections worldwide. Although the efficacy of the HPV vaccine in preventing the development of cervical pre-malignant lesions has been well demonstrated, the efficacy of the HPV vaccine in preventing HPV infection in the upper respiratory tract has been poorly studied. METHODS: In the context of the IARC cohort study of two versus three doses of HPV vaccine in India, we compared the HPV type prevalence in the oral cavity of women vaccinated with three doses, two doses, or a single dose of quadrivalent HPV vaccine with that of unvaccinated women. A total of 997 oral samples, from 818 vaccinated women and 179 unvaccinated women, were collected at three study sites. All the participants were sexually active at the time of sample collection. RESULTS: The age-standardized proportion (ASP) of HPV16/18 infections was 2.0 % (95 % CI, 1.0-3.0 %) in vaccinated women and 4.2 % (95 % CI, 1.2-7.2 %) in unvaccinated women. HPV16 was detected in 3.5 % of single-dose recipients, 1.2 % of two-dose recipients (days 1 and 180), and 1.5 % of three-dose recipients (days 1, 60, and 180), whereas 3.3 % of the unvaccinated women tested positive for HPV16. The same trend was observed for HPV18. DISCUSSION: Our findings agree with those of previous studies on the efficacy of HPV vaccination in reducing oral HPV infections and provide indications that a single vaccine dose may be less efficient than two or three doses in preventing oral HPV infection.

Mutational landscape and in silico structure models of SARS-CoV-2 spike receptor binding domain reveal key molecular determinants for virus-host interaction.

BACKGROUND: SARS-CoV-2, the causative agent of COVID-19 pandemic is a RNA virus prone to mutations. Formation of a stable binding interface between the Receptor Binding Domain (RBD) of SARS-CoV-2 Spike (S) protein and Angiotensin-Converting Enzyme 2 (ACE2) of host is pivotal for viral entry. RBD has been shown to mutate frequently during pandemic. Although, a few mutations in RBD exhibit enhanced transmission rates leading to rise of new variants of concern, most RBD mutations show sustained ACE2 binding and virus infectivity. Yet, how all these mutations make the binding interface constantly favourable for virus remain enigmatic. This study aims to delineate molecular rearrangements in the binding interface of SARS-CoV-2 RBD mutants. RESULTS: Here, we have generated a mutational and structural landscape of SARS-CoV-2 RBD in first six months of the pandemic. We analyzed 31,403 SARS-CoV-2 genomes randomly across the globe, and identified 444 non-synonymous mutations in RBD that cause 49 distinct amino acid substitutions in contact and non-contact amino acid residues. Molecular phylogenetic analysis suggested independent emergence of RBD mutants. Structural mapping of these mutations on the SARS-CoV-2 Wuhan reference strain RBD and structural comparison with RBDs from bat-CoV, SARS-CoV, and pangolin-CoV, all bound to human or mouse ACE2, revealed several changes in the interfacial interactions in all three binding clusters. Interestingly, interactions mediated via N487 residue in cluster-I and Y449, G496, T500, G502 residues in cluster-III remained largely unchanged in all RBD mutants. Further analysis showed that these interactions are evolutionarily conserved in sarbecoviruses which use ACE2 for entry. Importantly, despite extensive changes in the interface, RBD-ACE2 stability and binding affinities were maintained in all the analyzed mutants. Taken together, these findings reveal how SARS-CoV-2 uses its RBD residues to constantly remodel the binding interface. CONCLUSION: Our study broadly signifies understanding virus-host binding interfaces and their alterations during pandemic. Our findings propose a possible interface remodelling mechanism used by SARS-CoV-2 to escape deleterious mutations. Future investigations will focus on functional validation of in-silico findings and on investigating interface remodelling mechanisms across sarbecoviruses. Thus, in long run, this study may provide novel clues to therapeutically target RBD-ACE2 interface for pan-sarbecovirus infections.

Vaccine efficacy against persistent human papillomavirus (HPV) 16/18 infection at 10 years after one, two, and three doses of quadrivalent HPV vaccine in girls in India: a multicentre, prospective, cohort study.

BACKGROUND: A randomised trial designed to compare three and two doses of quadrivalent human papillomavirus (HPV) vaccine in adolescent girls in India was converted to a cohort study after suspension of HPV vaccination in trials by the Indian Government. In this Article, the revised aim of the cohort study was to compare vaccine efficacy of single dose to that of three and two doses in protecting against persistent HPV 16 and 18 infection at 10 years post vaccination. METHODS: In the randomised trial, unmarried girls aged 10-18 years were recruited from nine centres across India and randomly assigned to either two doses or three doses of the quadrivalent HPV vaccine (Gardasil [Merck Sharp & Dohme, Whitehouse Station, NJ, USA]; 0·5 mL administered intramuscularly). After suspension of recruitment and vaccination, the study became a longitudinal, prospective cohort study by default, and participants were allocated to four cohorts on the basis of the number vaccine doses received per protocol: the two-dose cohort (received vaccine on days 1 and 180 or later), three-dose cohort (days 1, 60, and 180 or later), two-dose default cohort (days 1 and 60 or later), and the single-dose default cohort. Participants were followed up yearly. Cervical specimens were collected from participants 18 months after marriage or 6 months after first childbirth, whichever was earlier, to assess incident and persistent HPV infections. Married participants were screened for cervical cancer as they reached 25 years of age. Unvaccinated women age-matched to the married vaccinated participants were recruited to serve as controls. Vaccine efficacy against persistent HPV 16 and 18 infections (the primary endpoint) was analysed for single-dose recipients and compared with that in two-dose and three-dose recipients after adjusting for imbalance in the distribution of potential confounders between the unvaccinated and vaccinated cohorts. This trial is registered with ISRCTN, ISRCTN98283094, and ClinicalTrials.gov, NCT00923702. FINDINGS: Vaccinated participants were recruited between Sept 1, 2009, and April 8, 2010 (date of vaccination suspension), and followed up over a median duration of 9·0 years (IQR 8·2-9·6). 4348 participants had three doses, 4980 had two doses (0 and 6 months), and 4949 had a single dose. Vaccine efficacy against persistent HPV 16 and 18 infection among participants evaluable for the endpoint was 95·4% (95% CI 85·0-99·9) in the single-dose default cohort (2135 women assessed), 93·1% (77·3-99·8) in the two-dose cohort (1452 women assessed), and 93·3% (77·5-99·7) in three-dose recipients (1460 women assessed). INTERPRETATION: A single dose of HPV vaccine provides similar protection against persistent infection from HPV 16 and 18, the genotypes responsible for nearly 70% of cervical cancers, to that provided by two or three doses. FUNDING: Bill & Melinda Gates Foundation.

Cyclophilin A induces macrophage apoptosis and enhances atherosclerotic lesions in high-fat diet-fed hyperglycemic rabbits.

Macrophage apoptosis is a key contributor to the progression of atherosclerosis. Cyclophilin A, a monocyte secretory protein associated with the initiation of atherosclerosis has an inherent nuclease activity. This study reports the mechanism by which cyclophilin A causes apoptosis of macrophages and accelerates the progression of atherosclerosis. Aortic lesion formation and apoptosis were studied in New Zealand White rabbits (NZW) which were fed high-fat diet (HFD) for 12 weeks. Using monocytes and HFD-fed rabbits we demonstrate that cyclophilin A induces mitochondrial membrane potential loss and mitochondrial pore transition protein opening through caspase 3 activation. En face staining revealed a significant increase in the lesion area in HFD-fed rabbits. Levels of glucose, cholesterol and proinflammatory cytokines were higher in these animals compared to rabbits fed with a normal diet. In the aorta of HFD-fed rabbits, medial vascular smooth muscle cells were disorganized and there was a loss of integrity of the endothelium. An 8-fold increase was seen in the number of apoptotic cells in the lesion area of HFD-fed NZW rabbits which were associated with an elevation in plasma cyclophilin A levels. siRNA knockdown of cyclophilin A gene reduced activation of caspase 3 in macrophages. Treatment with cyclosporine A, an inhibitor of cyclophilin A, significantly attenuated apoptosis in macrophages. Our study indicates that inhibitors of proinflammatory cytokines such as cyclophilin A may arrest macrophage apoptosis and result in a regression of advanced atherosclerotic lesions.

Acquisition, prevalence and clearance of type-specific human papillomavirus infections in young sexually active Indian women: A community-based multicentric cohort study.

In context of the ongoing multi-centric HPV vaccine study in India, unvaccinated married women (N = 1484) aged 18-23 years were recruited in 2012-2015 as age-matched controls to the vaccinated women and followed up yearly. We assess type-specific prevalence, natural history and potential determinants of human papillomavirus (HPV) infection in these unvaccinated women. Cervical samples were collected yearly for at least four consecutive years. A Multiplex Type-Specific E7-Based polymerase chain reaction assay was used to detect 21 HPV types. HPV prevalence was 36.4% during 6 years. Most common HPV types were 16 (6.5%) and 31 (6.1%). Highest persistence were observed for HPV 35 (62.5%) and 52 (25%). New HPV acquisition rate was 5.6/1000 person-months of observation (PMO), highest for HPV 16 (1.1/1000 PMO). Type-specific clearance rates ranged between 2.9-5.5/100 PMO. HPV 16 and/or 18 infections were 41% (95% CI 4-63%) lower among women with 2-<3 years between marriage and first cervical sample collection compared to those with <2 years. HPV prevalence and acquisition rates in young Indian women were lower than their Western counterparts. HPV 16 infections being most common shows the importance and potential impact of HPV vaccination in India. Women with 2-3 years exposure had reduced risk possibly due to higher infections clearance.

Coordinated dysregulation of cancer progression by the HER family and p21-activated kinases.

Most epithelial cancer types are polygenic in nature and are driven by coordinated dysregulation of multiple regulatory pathways, genes, and protein modifications. The process of coordinated regulation of cancer promoting pathways in response to extrinsic and intrinsic signals facilitates the dysregulation of several pathways with complementary functions, contributing to the hallmarks of cancer. Dysregulation and hyperactivation of cell surface human epidermal growth factor receptors (HERs) and cytoskeleton remodeling by p21-activated kinases (PAKs) are two prominent interconnected aspects of oncogenesis. We briefly discuss the discoveries and significant advances in the area of coordinated regulation of HERs and PAKs in the development and progression of breast and other epithelial cancers. We also discuss how initial studies involving heregulin signaling via HER3-HER2 axis and HER2-overexpressing breast cancer cells not only discovered a mechanistic role of PAK1 in breast cancer pathobiology but also acted as a bridge in generating a broader cancer research interest in other PAK family members and cancer types and catalyzed establishing the role of PAKs in human cancer, at-large. In addition, growth factor stimulation of the PAK pathway also helped to recognize new facets of PAKs, connecting the PAK pathway to oncogenesis, nuclear signaling, gene expression, mitotic progression, DNA damage response, among other phenotypic responses, and shaped the field of PAK cancer research. Finally, we recount some of the current limitations of HER- and PAK-directed therapeutics in counteracting acquired therapeutic resistance and discuss how cancer's as a polygenic disease may be best targeted with a polygenic approach.

HER family in cancer progression: From discovery to 2020 and beyond.

The human epidermal growth factor receptor (HER) family of receptor tyrosine kinases (RTKs) are among the first layer of molecules that receive, interpret, and transduce signals leading to distinct cancer cell phenotypes. Since the discovery of the tooth-lid factor-later characterized as the epidermal growth factor (EGF)-and its high-affinity binding EGF receptor, HER kinases have emerged as one of the commonly upregulated or hyperactivated or mutated kinases in epithelial tumors, thus allowing HER1-3 family members to regulate several hallmarks of cancer development and progression. Each member of the HER family exhibits shared and unique structural features to engage multiple receptor activation modes, leading to a range of overlapping and distinct phenotypes. EGFR, the founding HER family member, provided the roadmap for the development of the cell surface RTK-directed targeted cancer therapy by serving as a prototype/precursor for the currently used HER-directed cancer drugs. We herein provide a brief account of the discoveries, defining moments, and historical context of the HER family and guidepost advances in basic, translational, and clinical research that solidified a prominent position of the HER family in cancer research and treatment. We also discuss the significance of HER3 pseudokinase in cancer biology; its unique structural features that drive transregulation among HER1-3, leading to a superior proximal signaling response; and potential role of HER3 as a shared effector of acquired therapeutic resistance against diverse oncology drugs. Finally, we also narrate some of the current drawbacks of HER-directed therapies and provide insights into postulated advances in HER biology with extensive implications of these therapies in cancer research and treatment.

RTKs in pathobiology of head and neck cancers.

Non-communicable diseases contribute to 71% of the deaths worldwide, of which cancers rank second after cardiovascular diseases. Among all the cancers, head and neck cancers (HNC) are consequential in augmenting the global cancer incidence as well as mortality. Receptor tyrosine kinases (RTKs) are emphatic for the matter that they serve as biomarkers aiding the analysis of tumor progression and metastasis as well as diagnosis, prognosis and therapeutic progression in the patients. The extensive researches on HNC have made significant furtherance in numerous targeted therapies, but for the escalating therapeutic resistance. This review explicates RTKs in HNC, their signaling pathways involved in tumorigenesis, metastasis and stemness induction, the association of non-coding RNAs with RTKs, an overview of RTK based therapy and associated resistance in HNC, as well as a sneak peek into the HPV positive HNC and its therapy. The review extrapolates the cardinal role of RTKs and RTK based therapy as superior to other existing therapeutic interventions for HNC.

Molecular Networking and Whole-Genome Analysis Aid Discovery of an Angucycline That Inactivates mTORC1/C2 and Induces Programmed Cell Death.

Rediscovery of known compounds and time consumed in identification, especially high molecular weight compounds with complex structure, have let down interest in drug discovery. In this study, whole-genome analysis of microbe and Global Natural Products Social (GNPS) molecular networking helped in initial understanding of possible compounds produced by the microbe. Genome data revealed 10 biosythethic gene clusters that encode for secondary metabolites with anticancer potential. NMR analysis of the pure compound revealed the presence of a four-ringed benz[a]anthracene, thus confirming angucycline; molecular networking further confirmed production of this class of compounds. The type II polyketide synthase gene identified in the microbial genome was matched with the urdamycin cluster by BLAST analysis. This information led to ease in identification of urdamycin E and a novel natural derivative, urdamycin V, purified from Streptomyces sp. OA293. Urdamycin E (Urd E) induced apoptosis and autophagy in cancer cell lines. Urd E exerted anticancer action through inactivation of the mTOR complex by preventing phosphorylation at Ser 2448 and Ser 2481 of mTORC1 and mTORC2, respectively. Significant reduction in phosphorylation of the major downstream regulators of both mTORC1 (p70s6k and 4e-bp1) and mTORC2 (Akt) were observed, thus further confirming complete inhibition of the mTOR pathway. Urd E presents itself as a novel mTOR inhibitor that employs a novel mechanism in mTOR pathway inhibition.

Epigenetic Dysregulation at the Crossroad of Women's Cancer.

An increasingly number of women of all age groups are affected by cancer, despite substantial progress in our understanding of cancer pathobiology, the underlying genomic alterations and signaling cascades, and cellular-environmental interactions. Though our understanding of women's cancer is far more complete than ever before, there is no comprehensive model to explain the reasons behind the increased incidents of certain reproductive cancer among older as well as younger women. It is generally suspected that environmental and life-style factors affecting hormonal and growth control pathways might help account for the rise of women's cancers in younger age, as well, via epigenetic mechanisms. Epigenetic regulators play an important role in orchestrating an orderly coordination of cellular signals in gene activity in response to upstream signaling and/or epigenetic modifiers present in a dynamic extracellular milieu. Here we will discuss the broad principles of epigenetic regulation of DNA methylation and demethylation, histone acetylation and deacetylation, and RNA methylation in women's cancers in the context of gene expression, hormonal action, and the EGFR family of cell surface receptor tyrosine kinases. We anticipate that a better understanding of the epigenetics of women's cancers may provide new regulatory leads and further fuel the development of new epigenetic biomarkers and therapeutic approaches.

Two-dose recommendation for Human Papillomavirus vaccine can be extended up to 18 years - updated evidence from Indian follow-up cohort study.

Earlier publication from the ongoing multi-centric study of the International Agency for Research on Cancer to evaluate less than three doses of the quadrivalent Human Papillomavirus (HPV) vaccine in India amongst unmarried girls demonstrated non-inferior total antibody titres, neutralizing antibody titres and antibody avidity in 2-dose recipients compared to 3-dose recipients at 15-18 years of age (Bhatla et al., 2018) [7]. The number of participants recruited at 15-18 years of age was 1515 and 1795 in the 3-dose and the 2-dose groups respectively. At a median follow-up of 7 years, incident HPV 16/18 infections were detected in 1.6% women receiving two doses and 0.8% women receiving three doses at 15-18 years. Frequency of incident infection was 7.0% in the age- and site-matched unvaccinated women (N = 1484). No persistent infection from HPV 16 was observed in the 2- or 3-dose recipients and one (0.2%) persistent HPV 18 infection was documented, each in the 3-dose and 2-dose cohorts. Among the unvaccinated women, the frequency of HPV 16/18 persistent infection was 1.7%. The protection offered by two doses of quadrivalent HPV vaccine against incident and persistent infections in recipients at 15-18 years is comparable to that seen in 3-dose recipients at 15-18 years.

Estrogen receptor-α regulation of microRNA-590 targets FAM171A1-a modifier of breast cancer invasiveness.

The pathobiology and aggressiveness of the triple negative breast cancer (TNBC) are influenced by genes that are preferentially expressed in TNBC cells. However, the nature of such genes with the role in invasiveness of TNBC cells is not fully understood. Here, we identified FAM171A1, member (A1) of the family with sequence similarity 171, as an overexpressed candidate gene in TNBC cells and tumors as compared to estrogen receptor-alpha (ERα) positive breast cancer. We found that the expression of FAM171A1 correlates well with the loss of ERα as well as its newly identified target miR590-5p in TNBC but not in ERα-positive cells. In addition, we report that ERα regulates FAM171A1 expression through a mechanism which involves ERα stimulation of miR590-5p expression via binding to its promoter, and in-turn, miR590-5p suppression of FAM171A1 expression. Further, we found that the levels of FAM171A1 correlate well with cancer cell aggressiveness as depletion or overexpression of FAM171A1 confers reduced or increased ability of TNBC cells to form mammospheres, respectively in accordance with the previous report of increased mammosphere formation potential of metastatic cells. In brief, results presented here have demonstrated that ERα regulation of FAM171A1 expression via miR590-5p explains the molecular basis of the noticed reduced levels of FAM171A1 in ER-positive breast cancer cells and that FAM171A1 is a preferably TNBC- overexpressed gene. Further, the noted loss of ERα-miR590-5p axis may upregulate the expression of FAM171A1 and consequently, resulting aggressiveness of TNBC cells. These findings suggest that FAM171A1 might represent a potentially novel therapeutic target for TNBC tumors.

A high-throughput real-time in vitro assay using mitochondrial targeted roGFP for screening of drugs targeting mitochondria.

Most toxic compounds including cancer drugs target mitochondria culminating in its permeabilization. Cancer drug-screening and toxicological testing of compounds require cost-effective and sensitive high-throughput methods to detect mitochondrial damage. Real-time methods for detection of mitochondrial damage are less toxic, allow kinetic measurements with good spatial resolution and are preferred over end-stage assays. Cancer cell lines stably expressing genetically encoded mitochondrial-targeted redox-GFP2 (mt-roGFP) were developed and validated for its suitability as a mitochondrial damage sensor. Diverse imaging platforms and flow-cytometry were utilized for ratiometric analysis of redox changes with known toxic and cancer drugs. Key events of cell death and mitochondrial damage were studied at single-cell level coupled with mt-roGFP. Cells stably expressing mt-roGFP and H2B-mCherry were developed for high-throughput screening (HTS) application. Most cancer drugs while inducing mitochondrial permeabilization trigger mitochondrial-oxidation that can be detected at single-cell level with mt-roGFP. The image-based assay using mt-roGFP outperformed other quantitative methods of apoptosis in ease of screening. Incorporation of H2B-mCherry ensures accurate and complete automated segmentation with excellent Z value. The results substantiate that most cancer drugs and known plant-derived antioxidants trigger cell-death through mitochondrial redox alterations with pronounced ratio change in the mt-roGFP probe. Real-time analysis of mitochondrial oxidation and mitochondrial permeabilization reveal a biphasic ratio change in dying cells, with an initial redox surge before mitochondrial permeabilization followed by a drastic increase in ratio after complete mitochondrial permeabilization. Overall, the results prove that mitochondrial oxidation is a reliable indicator of mitochondrial damage, which can be readily determined in live cells using mt-roGFP employing diverse imaging techniques. The assay described is highly sensitive, easy to adapt to HTS platforms and is a valuable resource for identifying cytotoxic agents that target mitochondria and also for dissecting cell signaling events relevant to redox biology.

Genome editing of oncogenes with ZFNs and TALENs: caveats in nuclease design.

BACKGROUND: Gene knockout technologies involving programmable nucleases have been used to create knockouts in several applications. Gene editing using Zinc-finger nucleases (ZFNs), Transcription activator like effectors (TALEs) and CRISPR/Cas systems has been used to create changes in the genome in order to make it non-functional. In the present study, we have looked into the possibility of using six fingered CompoZr ZFN pair to target the E6 gene of HPV 16 genome. METHODS: HPV 16+ve cell lines; SiHa and CaSki were used for experiments. CompoZr ZFNs targeting E6 gene were designed and constructed by Sigma-Aldrich. TALENs targeting E6 and E7 genes were made using TALEN assembly kit. Gene editing was monitored by T7E1 mismatch nuclease and Nuclease resistance assays. Levels of E6 and E7 were further analyzed by RT-PCR, western blot as well as immunoflourescence analyses. To check if there is any interference due to methylation, cell lines were treated with sodium butyrate, and Nocodazole. RESULTS: Although ZFN editing activity in yeast based MEL-I assay was high, it yielded very low activity in tumor cell lines; only 6% editing in CaSki and negligible activity in SiHa cell lines. Though editing efficiency was better in CaSki, no significant reduction in E6 protein levels was observed in immunocytochemical analysis. Further, in silico analysis of DNA binding prediction revealed that some of the ZFN modules bound to sequence that did not match the target sequence. Hence, alternate ZFN pairs for E6 and E7 were not synthesized since no further active sites could be identified by in silico analyses. Then we designed TALENs to target E6 and E7 gene. TALENs designed to target E7 gene led to reduction of E7 levels in CaSki and SiHa cervical cancer cell lines. However, TALEN designed to target E6 gene did not yield any editing activity. CONCLUSIONS: Our study highlights that designed nucleases intended to obtain bulk effect should have a reasonable editing activity which reflects phenotypically as well. Nucleases with low editing efficiency, intended for generation of knockout cell lines nucleases could be obtained by single cell cloning. This could serve as a criterion for designing ZFNs and TALENs.

Are two doses of human papillomavirus vaccine sufficient for girls aged 15-18 years? Results from a cohort study in India.

Extending two-dose recommendations of HPV vaccine to girls between 15 and 18 years will reduce program cost and improve compliance. Immunogenicity and vaccine targeted HPV infection outcomes were compared between 1795 girls aged 15-18 years receiving two (1-180 days) and 1515 girls of same age receiving three (1-60-180 days) doses. Immunogenicity outcomes in 15-18 year old two-dose recipients were also compared with the 10-14 year old three-dose (N = 2833) and two-dose (N = 3184) recipients. The 15-18 year old two-dose recipients had non-inferior L1-binding antibody titres at seven months against vaccine-targeted HPV types compared to three-dose recipients at 15-18 years and three-dose recipients at 10-14 years of age. Neutralizing antibody titres at 18 months in 15-18 year old two-dose recipients were non-inferior to same age three-dose recipients for all except HPV 18. The titres were inferior to those in the 10-14 year old three-dose recipients for all targeted types. Frequency of incident infections from vaccine-targeted HPV types in the 15-18 year old two-dose recipients was similar to the three dose recipients. None of the girls receiving two or three doses had persistent infection from vaccine-targeted types. These findings support that two doses of HPV vaccine can be extended to girls aged 15-18 years.

Metformin attenuates effects of cyclophilin A on macrophages, reduces lipid uptake and secretion of cytokines by repressing decreased AMPK activity.

Growing evidence implicates cyclophilin A secreted by vascular wall cells and monocytes as a key mediator in atherosclerosis. Cyclophilin A in addition to its proliferative effects, during hyperglycemic conditions, increases lipid uptake in macrophages by increasing scavenger receptors on the cell's surface. It also promotes macrophage migration across endothelial cells and conversion of macrophages into foam cells. Given the known effects of metformin in reducing vascular complications of diabetes, we investigated the effect of metformin on cyclophilin A action in macrophages. Using an ex vivo model of cultured macrophages isolated from patients with type 2 diabetes with and without coronary artery disease (CAD), we measured the effect of metformin on cyclophilin A expression, lipid accumulation, expression of scavenger receptors, plasma cytokine levels and AMP-activated protein kinase (AMPK) activity in macrophages. In addition, the effects of metformin on migration of monocytes, reactive oxygen species (ROS) formation, lipid uptake in the presence of cyclophilin A inhibitors and comparison with pioglitazone were studied using THP-1 monocytes. Metformin reduced cyclophilin A expression in human monocyte-derived macrophages. Metformin also decreased the effects of cyclophilin A on macrophages such as oxidized low-density lipoprotein (oxLDL) uptake, scavenger receptor expression, ROS formation and secretion of inflammatory cytokines in high-glucose conditions. Metformin reversed cyclophilin A-induced decrease in AMPK-1α activity in macrophages. These effects of metformin were similar to those of cyclophilin A inhibitors. Metformin can thus function as a suppressor of pro-inflammatory effects of cyclophilin A in high-glucose conditions by attenuating its expression and repressing cyclophilin A-induced decrease in AMPK-1α activity in macrophages.

TALEN based HPV-E7 editing triggers necrotic cell death in cervical cancer cells.

Human Papillomavirus E7 and E6 oncoproteins have been considered as suitable candidate anti-viral targets since they cause malignant conversion in cervical cancers. Transcription Activator-Like Effector Nucleases (TALENs) are recent editing tools to knockout genes by inducing double stranded breaks at specific sites in the genome. In here, we have designed specific TALENs to target E7 and analyzed their efficiency in inducing cell death in cervical cancer cells. We found that designed TALENs could yield about 10-12% editing activity as observed from T7E1 and nuclease resistance assays. Down-regulation of E7 and E6 was further evident at the transcript as well as proteins levels indicating that the selected TALENs were effective. TALEN-mediated E7 editing led to cell death as ascertained by cell cycle and Annexin V assays. Annexin profiling suggested that cell death could be due to necrosis as observed by upregulation of necrotic markers such as LDH A, Rip-1, and Cyclophilin A. Necrosis appears to be a better therapeutic response as it could further activate pro-inflammatory cytokines to attract immune cells to eliminate HPV-integrated cells and therefore TALEN editing strategy has the potential to be a promising tool as an adjuvant therapy in cervical cancer along with surgery.