Streptococcus pneumoniae is a leading cause of pneumonia and one of the most common causes of death globally. The impact of S. pneumoniae on host molecular processes that lead to detrimental pulmonary consequences is not fully understood. Here, we show that S. pneumoniae induces toxic DNA double-strand breaks (DSBs) in human alveolar epithelial cells, as indicated by ataxia telangiectasia mutated kinase (ATM)-dependent phosphorylation of histone H2AX and colocalization with p53-binding protein (53BP1). Furthermore, results show that DNA damage occurs in a bacterial contact-independent fashion and that Streptococcus pyruvate oxidase (SpxB), which enables synthesis of H 2 O 2 , plays a critical role in inducing DSBs. The extent of DNA damage correlates with the extent of apoptosis, and DNA damage precedes apoptosis, which is consistent with the time required for execution of apoptosis. Furthermore, addition of catalase, which neutralizes H 2 O 2 , greatly suppresses S. pneumoniae-induced DNA damage and apoptosis. Importantly, S. pneumoniae induces DSBs in the lungs of animals with acute pneumonia, and H 2 O 2 production by S. pneumoniae in vivo contributes to its genotoxicity and virulence. One of the major DSBs repair pathways is nonhomologous end joining for which Ku70/80 is essential for repair. We find that deficiency of Ku80 causes an increase in the levels of DSBs and apoptosis, underscoring the importance of DNA repair in preventing S. pneumoniaeinduced genotoxicity. Taken together, this study shows that S. pneumoniae-induced damage to the host cell genome exacerbates its toxicity and pathogenesis, making DNA repair a potentially important susceptibility factor in people who suffer from pneumonia.DNA damage | Streptococcus pneumoniae | hydrogen peroxide | γH2AX | Ku80

Injury-and ischemia-induced angiogenesis is critical for tissue repair and requires nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS). We present evidence that NO induces angiogenesis by modulating the level of the angiogenesis inhibitor thrombospondin 2 (TSP2). TSP2 levels were higher than WT in eNOS KO tissues in hind-limb ischemia and cutaneous wounds. In vitro studies confirmed that NO represses TSP2 promoter activity. Moreover, double-eNOS/TSP2 KO mice were generated and found to rescue the phenotype of eNOS KO mice. Studies in mice with knock-in constitutively active or inactive eNOS on the Akt-1 KO background showed that eNOS activity correlates with TSP2 levels. Our observations of NO-mediated regulation of angiogenesis via the suppression of TSP2 expression provide a description of improved eNOS KO phenotype by means other than restoring NO signaling.wound healing | extracellular matrix | Akt | matrix metalloproteinases E ndothelial nitric oxide synthase (eNOS) and its bioactive product nitric oxide (NO) are well-established proangiogenic molecules. Endothelial-derived NO is crucial for maintenance of proper vasodilatory tone and regulation of an antiproliferative and antiapoptotic state for endothelial cells (ECs) and has essential roles in physiological angiogenesis (1-3). Pharmacological inhibition or genetic disruption of eNOS limited angiogenesis during tissue repair, resulting in delayed wound closure (2, 4). Addition of NO donors to wounds enhanced angiogenesis and accelerated healing (5-7). eNOS KO mice recovered poorly from hind-limb ischemia as a consequence of decreased angiogenesis (3,8). These mice also displayed accelerated atherosclerosis, neointimal thickening postinjury, and hypertension (1, 9). Taken together, these observations highlight the ability of eNOS-derived NO to influence vascular function.Thrombospondins (TSPs) are a small family of antiangiogenic matricellular proteins (10). TSPs enhance clearance of matrix metalloproteinase (MMP)-2 and MMP-9 (11-13) and interact with cell-surface receptors, including α v β 3 , very low density lipoprotein receptor (VLDLR), CD36, and CD47, to inhibit angiogenesis (14). Further, ultrastructural studies demonstrated that TSP2 influences ECM assembly (15, 16). TSP2 KO mice displayed improved recovery of blood flow following ischemia (17), altered foreign body response (18,19), and accelerated wound healing (16,20,21). In contrast, TSP1 KO mice displayed delayed healing because of insufficient stimulation of inflammation (13). Consistent with these observations, the expression of TSP1 and TSP2 in tissue repair was associated with the inflammatory and repair phases, respectively (13, 16). Recently, several studies linked components of the Akt-eNOS cascade with TSPs. Specifically, TSP1 has been described to blunt the ability of NO to activate soluble guanyl cyclase (sGC) (22, 23) through interactions with CD36 and CD47 during ischemia. TSP1 has also been described to diminish eNOS activity by blocking phosphorylation at S117...

Influenza viruses account for significant morbidity worldwide. Inflammatory responses, including excessive generation of reactive oxygen and nitrogen species (RONS), mediate lung injury in severe Influenza infections. However, the molecular basis of inflammation-induced lung damage is not fully understood. Here, we studied influenza H1N1 infected cells in vitro, as well as H1N1 infected mice, and we monitored molecular and cellular responses over the course of two weeks in vivo. We show that influenza induces DNA damage both when cells are directly exposed to virus in vitro (measured using the comet assay) and also when cells are exposed to virus in vivo (estimated via γH2AX foci). We show that DNA damage, as well as responses to DNA damage, persist in vivo until long after virus has been cleared, at times when there are inflammation associated RONS (measured by xanthine oxidase activity and oxidative products). The frequency of lung epithelial and immune cells with increased γH2AX foci is elevated in vivo, especially for dividing cells (Ki-67 positive) exposed to oxidative stress during tissue regeneration. Additionally, we observed a significant increase in apoptotic cells as well as increased levels of DSB repair proteins Ku70, Ku86 and Rad51 during the regenerative phase. In conclusion, results show that influenza induces DNA both in vitro and in vivo, and that DNA damage responses are activated, raising the possibility that DNA repair capacity may be a determining factor for tissue recovery and disease outcome.

With pharmaceutical companies shrinking their research departments and exiting out of efforts related to unprofitable diseases, society has become increasingly dependent on academic institutions to perform drug discovery and early-stage translational research. Academic drug discovery and translational research programs assist in shepherding promising therapeutic opportunities through the so-called valley of death in the hope that a successful new drug will result in saved lives, improved health, economic growth, and financial return. We have interviewed directors of 16 such academic programs in the United States and found that these programs and the projects therein face numerous challenges in reaching the clinic, including limited funding, lack of know-how, and lack of a regional drug development ecosystem. If these issues can be addressed through novel industry partnerships, the revision of government policies, and expanded programs in translational education, more effective new therapies are more likely to reach patients in need. Expected final online publication date for the Annual Review of Pharmacology and Toxicology Volume 59 is January 6, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

It is well established that inflammation leads to the creation of potent DNA damaging chemicals, including reactive oxygen and nitrogen species. Nitric oxide can react with glutathione to create S-nitrosoglutathione (GSNO), which can in turn lead to S-nitrosated proteins. Of particular interest is the impact of GSNO on the function of DNA repair enzymes. The base excision repair (BER) pathway can be initiated by the alkyl-adenine DNA glycosylase (AAG), a monofunctional glycosylase that removes methylated bases. After base removal, an abasic site is formed, which then gets cleaved by AP endonuclease and processed by downstream BER enzymes. Interestingly, using the Fluorescence-based Multiplexed Host Cell Reactivation Assay (FM-HCR), we show that GSNO actually enhances AAG activity, which is consistent with the literature. This raised the possibility that there might be imbalanced BER when cells are challenged with a methylating agent. To further explore this possibility, we confirmed that GSNO can cause AP endonuclease to translocate from the nucleus to the cytoplasm, which might further exacerbate imbalanced BER by increasing the levels of AP sites. Analysis of abasic sites indeed shows GSNO induces an increase in the level of AP sites. Furthermore, analysis of DNA damage using the CometChip (a higher throughput version of the comet assay) shows an increase in the levels of BER intermediates. Finally, we found that GSNO exposure is associated with an increase in methylation-induced cytotoxicity. Taken together, these studies support a model wherein GSNO increases BER initiation while processing of AP sites is decreased, leading to a toxic increase in BER intermediates. This model is also supported by additional studies performed in our laboratory showing that inflammation in vivo leads to increased large-scale sequence rearrangements. Taken together, this work provides new evidence that inflammatory chemicals can drive cytotoxicity and mutagenesis via BER imbalance.

DNA damage and alterations in global DNA methylation status are associated with multiple human diseases and are frequently correlated with clinically relevant information. Therefore, assessing DNA damage and epigenetic modifications, including DNA methylation, is critical for predicting human exposure risk of pharmacological and biological agents. We previously developed a higher-throughput platform for the single cell gel electrophoresis (comet) assay, CometChip, to assess DNA damage and genotoxic potential. Here, we utilized the methylation-dependent endonuclease, McrBC, to develop a modified alkaline comet assay, “EpiComet”, which allows single platform evaluation of genotoxicity and global DNA methylation [5-methylcytosine (5-mC)] status of single cell populations under user-defined conditions. Further, we leveraged the CometChip platform to create an EpiComet-Chip system capable of performing quantification across simultaneous exposure protocols to enable unprecedented speed and simplicity. This system detected global methylation alterations in response to exposures which included chemotherapeutic and environmental agents. Using EpiComet-Chip on 63 matched samples; we correctly identified single sample hypermethylation (≥1.5-fold) at 87% (20/23), hypomethylation (≥1.25-fold) at 100% (9/9), with a 4% (2/54) false negative rate (FNR) and 10% (4/40) false positive rate (FPR). Using a more stringent threshold to define hypermethylation (≥1.75-fold) allowed us to correctly identify 94% of hypermethylation (17/18), but increased our FPR to 16% (7/45). The successful application of this novel technology will aid hazard identification and risk characterization of FDA-regulated products, while providing utility for investigating epigenetic modes of action of agents in target organs, since the assay is amenable to cultured cells or nucleated cells from any tissue.

Ionising radiation causes various types of DNA damages including double strand breaks (DSBs). DSBs are often recognized by DNA repair protein ATM which forms gamma-H2AX foci at the site of the DSBs that can be visualized using immunohistochemistry. However most of such experiments are of low throughput in terms of imaging and image analysis techniques. Most of the studies still use manual counting or classification. Hence they are limited to counting a low number of foci per cell ( 5 foci per nucleus) as the quantification process is extremely labour intensive. Therefore we have developed a high throughput instrumentation and computational pipeline specialized for gamma-H2AX foci quantification. A population of cells with highly clustered foci inside nuclei were imaged, in 3D with submicron resolution, using an in-house developed high throughput image cytometer. Imaging speeds as high as 800 cells/second in 3D were achieved by using HiLo wide-field depth resolved imaging and a remote z-scanning technique. Then the number of foci per cell nucleus were quantified using a 3D extended maxima transform based algorithm. Our results suggests that while most of the other 2D imaging and manual quantification studies can count only up to about 5 foci per nucleus our method is capable of counting more than 100. Moreover we show that 3D analysis is significantly superior compared to the 2D techniques.Keywords: image cytometry, foci counting , gamma-h2ax, image analysis Various agents, such as ionizing radiation and pollutants, can cause cancer by inducing DNA damage which can lead to sequence rearrangements in cells. Of many types of DNA damages, one of the most genotoxic is the double strand break (DSB). DSBs are recognized by DNA repair proteins, including ATM, which phosphorylates H2AX at serine 129 to form gamma-H2AX. Phosphorylation of H2AX stretches for tens of kb from the breakpoint, leading to a structural change that can be visualized by immunohistochemistry.1 These foci can be easily detected by secondary antibodies that fluoresce. Thus, fluorescence based detection of the gamma-H2AX marker of DNA damage has been used broadly to study DNA damage and repair, and in particular, to study DSBs. 2Quantification of the frequency of repair foci can be done using different criteria. The most widely used is by classifying cells as positives and negatives using a threshold for the number of foci inside the cell nucleus. This threshold is usually kept at very low values such as 5 foci per nucleus. However quantifying the number of foci per cell, rather than the number of cells with a focus frequency higher than a specified number, provides a much more accurate representation of the DSB event frequency. It is not feasible for scientists to count the number of foci per cell, since there can be over a dozen foci per cell (often overlapping), and robust experimental designs require upwards of 100 cells per condition. Therefore, what is needed is an automated counting algorithm that can function in 3D. In addition to the ...

Ionising radiation causes various types of DNA damages including double strand breaks (DSBs). DSBs are often recognized by DNA repair protein ATM which forms gamma-H2AX foci at the site of the DSBs that can be visualized using immunohistochemistry. However most of such experiments are of low throughput in terms of imaging and image analysis techniques. Most of the studies still use manual counting or classification. Hence they are limited to counting a low number of foci per cell ( 5 foci per nucleus) as the quantification process is extremely labour intensive. Therefore we have developed a high throughput instrumentation and computational pipeline specialized for gamma-H2AX foci quantification. A population of cells with highly clustered foci inside nuclei were imaged, in 3D with submicron resolution, using an in-house developed high throughput image cytometer. Imaging speeds as high as 800 cells/second in 3D were achieved by using HiLo wide-field depth resolved imaging and a remote z-scanning technique. Then the number of foci per cell nucleus were quantified using a 3D extended maxima transform based algorithm. Our results suggests that while most of the other 2D imaging and manual quantification studies can count only up to about 5 foci per nucleus our method is capable of counting more than 100. Moreover we show that 3D analysis is significantly superior compared to the 2D techniques.Keywords: image cytometry, foci counting , gamma-h2ax, image analysis Various agents, such as ionizing radiation and pollutants, can cause cancer by inducing DNA damage which can lead to sequence rearrangements in cells. Of many types of DNA damages, one of the most genotoxic is the double strand break (DSB). DSBs are recognized by DNA repair proteins, including ATM, which phosphorylates H2AX at serine 129 to form gamma-H2AX. Phosphorylation of H2AX stretches for tens of kb from the breakpoint, leading to a structural change that can be visualized by immunohistochemistry.1 These foci can be easily detected by secondary antibodies that fluoresce. Thus, fluorescence based detection of the gamma-H2AX marker of DNA damage has been used broadly to study DNA damage and repair, and in particular, to study DSBs. 2Quantification of the frequency of repair foci can be done using different criteria. The most widely used is by classifying cells as positives and negatives using a threshold for the number of foci inside the cell nucleus. This threshold is usually kept at very low values such as 5 foci per nucleus. However quantifying the number of foci per cell, rather than the number of cells with a focus frequency higher than a specified number, provides a much more accurate representation of the DSB event frequency. It is not feasible for scientists to count the number of foci per cell, since there can be over a dozen foci per cell (often overlapping), and robust experimental designs require upwards of 100 cells per condition. Therefore, what is needed is an automated counting algorithm that can function in 3D. In addition to the ...