piRNAs as Modulators of Disease Pathogenesis.

Advances in understanding disease pathogenesis correlates to modifications in gene expression within different tissues and organ systems. In depth knowledge about the dysregulation of gene expression profiles is fundamental to fully uncover mechanisms in disease development and changes in host homeostasis. The body of knowledge surrounding mammalian regulatory elements, specifically regulators of chromatin structure, transcriptional and translational activation, has considerably surged within the past decade. A set of key regulators whose function still needs to be fully elucidated are small non-coding RNAs (sncRNAs). Due to their broad range of unfolding functions in the regulation of gene expression during transcription and translation, sncRNAs are becoming vital to many cellular processes. Within the past decade, a novel class of sncRNAs called PIWI-interacting RNAs (piRNAs) have been implicated in various diseases, and understanding their complete function is of vital importance. Historically, piRNAs have been shown to be indispensable in germline integrity and stem cell development. Accumulating research evidence continue to reveal the many arms of piRNA function. Although piRNA function and biogenesis has been extensively studied in Drosophila, it is thought that they play similar roles in vertebrate species, including humans. Compounding evidence suggests that piRNAs encompass a wider functional range than small interfering RNAs (siRNAs) and microRNAs (miRNAs), which have been studied more in terms of cellular homeostasis and disease. This review aims to summarize contemporary knowledge regarding biogenesis, and homeostatic function of piRNAs and their emerging roles in the development of pathologies related to cardiomyopathies, cancer, and infectious diseases.

Trypanosoma cruzi Modulates PIWI-Interacting RNA Expression in Primary Human Cardiac Myocytes during the Early Phase of Infection.

Trypanosoma cruzi dysregulates the gene expression profile of primary human cardiomyocytes (PHCM) during the early phase of infection through a mechanism which remains to be elucidated. The role that small non-coding RNAs (sncRNA) including PIWI-interacting RNA (piRNA) play in regulating gene expression during the early phase of infection is unknown. To understand how T. cruzi dysregulate gene expression in the heart, we challenged PHCM with T. cruzi trypomastigotes and analyzed sncRNA, especially piRNA, by RNA-sequencing. The parasite induced significant differential expression of host piRNAs, which can target and regulate the genes which are important during the early infection phase. An average of 21,595,866 (88.40%) of clean reads mapped to the human reference genome. The parasite induced 217 unique piRNAs that were significantly differentially expressed (q ≥ 0.8). Of these differentially expressed piRNAs, 6 were known and 211 were novel piRNAs. In silico analysis showed that some of the dysregulated known and novel piRNAs could target and potentially regulate the expression of genes including NFATC2, FOS and TGF-ß1, reported to play important roles during T. cruzi infection. Further evaluation of the specific functions of the piRNAs in the regulation of gene expression during the early phase of infection will enhance our understanding of the molecular mechanism of T. cruzi pathogenesis. Our novel findings constitute the first report that T. cruzi can induce differential expression of piRNAs in PHCM, advancing our knowledge about the involvement of piRNAs in an infectious disease model, which can be exploited for biomarker and therapeutic development.

Thrombospondin-1 Plays an Essential Role in Yes-Associated Protein Nuclear Translocation during the Early Phase of Trypanosoma cruzi Infection in Heart Endothelial Cells.

The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease. This neglected tropical disease causes severe morbidity and mortality in endemic regions. About 30% of T. cruzi infected individuals will present with cardiac complications. Invasive trypomastigotes released from infected cells can be carried in the vascular endothelial system to infect neighboring and distant cells. During the process of cellular infection, the parasite induces host cells, to increase the levels of host thrombospondin-1 (TSP-1), to facilitate the process of infection. TSP-1 plays important roles in the functioning of vascular cells, including vascular endothelial cells with important implications in cardiovascular health. Many signal transduction pathways, including the yes-associated protein 1 (YAP)/transcriptional coactivator, with PDZ-binding motif (TAZ) signaling, which are upstream of TSP-1, have been linked to the pathophysiology of heart damage. The molecular mechanisms by which T. cruzi signals, and eventually infects, heart endothelial cells remain unknown. To evaluate the importance of TSP-1 expression in heart endothelial cells during the process of T. cruzi infection, we exposed heart endothelial cells prepared from Wild Type and TSP-1 Knockout mouse to invasive T. cruzi trypomastigotes at multiple time points, and evaluated changes in the hippo signaling cascade using immunoblotting and immunofluorescence assays. We found that the parasite turned off the hippo signaling pathway in TSP-1KO heart endothelial cells. The levels of SAV1 and MOB1A increased to a maximum of 2.70 ± 0.23 and 5.74 ± 1.45-fold at 3 and 6 h, respectively, in TSP-1KO mouse heart endothelial cells (MHEC), compared to WT MHEC, following a parasite challenge. This was accompanied by a significant continuous increase in the nuclear translocation of downstream effector molecule YAP, to a maximum mean nuclear fluorescence intensity of 10.14 ± 0.40 at 6 h, compared to wild type cells. Furthermore, we found that increased nuclear translocated YAP significantly colocalized with the transcription co-activator molecule pan-TEAD, with a maximum Pearson's correlation coefficient of 0.51 ± 0.06 at 6 h, compared to YAP-Pan-TEAD colocalization in the WT MHEC, which decreased significantly, with a minimum Pearson's correlation coefficient of 0.30 ± 0.01 at 6 h. Our data indicate that, during the early phase of infection, upregulated TSP-1 is essential for the regulation of the hippo signaling pathway. These studies advance our understanding of the molecular interactions occurring between heart endothelial cells and T. cruzi, in the presence and absence of TSP-1, providing insights into processes linked to parasite dissemination and pathogenesis.

Bivalent Approach for Homodimeric Estradiol Based Ligand: Synthesis and Evaluation for Targeted Theranosis of ER(+) Breast Carcinomas.

The synthesis of estradiol based bivalent ligand [(EST)2DT] is reported and its potential for targeted imaging and therapy of ER(+) tumors has been evaluated. For the purpose, ethinylestradiol was functionalized with an azidoethylamine moiety via click chemistry. The resultant derivative was reacted in a bivalent mode with DTPA-dianhydride to form the multicoordinate chelating agent, (EST)2DT which displayed capability to bind (99m)Tc. The radiolabeled complex, (99m)Tc-(EST)2DT was obtained in >99% radiochemical purity and 20-48 GBq/µmol of specific activity. RBA assay revealed ∼15% binding with estrogen receptor. Evaluation of ligand on ER(+)-cell line (MCF-7) suggested enhanced and ER-mediated uptake. In vivo assays displayed early tracer accumulation in MCF-7 xenografts with tumor to muscle ratio ∼6 in 2 h and negligible uptakes in nontargeted organs. MTT assay performed on ER(+) and ER(-) cell lines displayed selective inhibition of ER(+) cancer cell growth with IC50 = 14.3 µM which was comparable to tamoxifen. The anticancer activity of the ligand is possibly due to the increase in ERß and suppression of ERα protein levels in gene transcription. The studies reveal the potential of (EST)2DT as diagnostic imaging agent with the additional benefits in therapy.

Identification of gallic acid based glycoconjugates as a novel tubulin polymerization inhibitors.

A novel class of gallic acid based glycoconjugates were designed and synthesized as potential anticancer agents. Among all the compounds screened, compound 2a showed potent anticancer activity against breast cancer cells. The latter resulted in tubulin polymerization inhibition and induced G2/M cell cycle arrest, generation of reactive oxygen species, mitochondrial depolarization and subsequent apoptosis in breast cancer cells. In addition, ultraviolet-visible spectroscopy and fluorescence quenching studies of the compound with tubulin confirmed direct interaction of compounds with tubulin. Molecular modeling studies revealed that it binds at the colchicine binding site in tubulin. Further, 2a also exhibited potent in vivo anticancer activity in LA-7 syngeneic rat mammary tumor model. Current data projects its strong candidature to be developed as anticancer agent.

Leading pedestrian intervals - Yay or Nay? A Before-After evaluation of multiple conflict types using an enhanced Non-Stationary framework integrating quantile regression into Bayesian hierarchical extreme value analysis.

A pedestrian was estimated to be killed every 85 min and injured every 7 min on US roads in 2019. Targeted safety treatments are particularly required at urban intersections where pedestrians regularly conflict with turning vehicles. Leading Pedestrian Intervals (LPIs) are an innovative, low-cost treatment where the pedestrian and vehicle usage of the potential conflict area (a crosswalk) is staggered in time to give the pedestrians a head start of a few seconds and reduce the "element of surprise" for right-turning vehicles. The effectiveness of LPI treatment on pedestrian safety is mixed, and most importantly, its effect on vehicle-vehicle conflicts is unknown. This study investigates the before-after effects of LPI treatments on vehicle-pedestrian and vehicle-vehicle crash risk by applying traffic conflict techniques. In particular, this study has developed a quantile regression technique within the extreme value model to estimate and compare crash risks before and after the installation of the LPI treatment. The before-after traffic movement video data (504 h in total) were collected from three signalized intersections in the City of Bellevue, Washington. The recorded movements were analyzed using Microsoft's proprietary computer vision platform, Edge Video Service, and Advanced Mobility Analytics Group's cloud-based SMART SafetyTM platform to automatedly extract traffic conflicts by analyzing road user trajectories. The treatment effect was measured using a Bayesian hierarchical extreme value model with the peak-over threshold approach. For the extreme value model, a Bayesian quantile regression analysis was conducted to estimate the conflict thresholds corresponding to a high (95th) quantile. Odds ratios were estimated for both conflict types using untreated crossing as a control group. Results indicate that the LPI treatment reduces the crash risk of pedestrians as measured by the reduction in extreme vehicle-pedestrian conflicts by about 42%. The LPI treatment has also been found not to negatively affect rear-end conflicts along the approaches leading to the LPI-treated pedestrian crossing at the signalized intersections. The findings of this study further emphasize the effectiveness of video analytics in proactive safety evaluations of engineering treatments.

Trypanosoma cruzi dysregulates expression profile of piRNAs in primary human cardiac fibroblasts during early infection phase.

Trypanosoma cruzi, the etiological agent of Chagas Disease, causes severe morbidity, mortality, and economic burden worldwide. Though originally endemic to Central and South America, globalization has led to increased parasite presence in most industrialized countries. About 40% of infected individuals will develop cardiovascular, neurological, and/or gastrointestinal pathologies. Accumulating evidence suggests that the parasite induces alterations in host gene expression profiles in order to facilitate infection and pathogenesis. The role of regulatory gene expression machinery during T. cruzi infection, particularly small noncoding RNAs, has yet to be elucidated. In this study, we aim to evaluate dysregulation of a class of sncRNAs called piRNAs during early phase of T. cruzi infection in primary human cardiac fibroblasts by RNA-Seq. We subsequently performed in silico analysis to predict piRNA-mRNA interactions. We validated the expression of these selected piRNAs and their targets during early parasite infection phase by stem loop qPCR and qPCR, respectively. We found about 26,496,863 clean reads (92.72%) which mapped to the human reference genome. During parasite challenge, 441 unique piRNAs were differentially expressed. Of these differentially expressed piRNAs, 29 were known and 412 were novel. In silico analysis showed several of these piRNAs were computationally predicted to target and potentially regulate expression of genes including SMAD2, EGR1, ICAM1, CX3CL1, and CXCR2, which have been implicated in parasite infection, pathogenesis, and various cardiomyopathies. Further evaluation of the function of these individual piRNAs in gene regulation and expression will enhance our understanding of early molecular mechanisms contributing to infection and pathogenesis. Our findings here suggest that piRNAs play important roles in infectious disease pathogenesis and can serve as potential biomarkers and therapeutic targets.

Transferability of multivariate extreme value models for safety assessment by applying artificial intelligence-based video analytics.

Traffic conflict techniques represent the state-of-the-art for road safety assessments. However, the lack of research on transferability of conflict-based crash risk models, which refers to applying the developed crash risk estimation models to a set of external sites, can reduce their appeal for large-scale traffic safety evaluations. Therefore, this study investigates the transferability of multivariate peak-over threshold models for estimating crash frequency-by-severity. In particular, the study proposes two transferability approaches: (i) an uncalibrated approach involving a direct application of the uncalibrated base model to the target sites and (ii) a threshold calibration approach involving calibration of conflict thresholds of the conflict indicators. In the latter approach, the conflict thresholds of the Modified Time-To-Collision (MTTC) and Delta-V indicators were calibrated using local data from the target sites. Finally, the two transferability approaches were compared with a complete re-estimation approach where all the model parameters were estimated using local data. All three approaches were tested for a target set of signalized intersections in Southeast Queensland, Australia. Traffic movements at the target intersections were observed using video cameras for two days (12 h each day). The road user trajectories and rear-end conflicts were extracted using an automated artificial intelligence-based algorithm utilizing state-of-the-art Computer Vision methods. The base models developed in an earlier study were then transferred to the target sites using the two transferability approaches and the local data from the target sites. Results show that the threshold calibration approach provides the most accurate and precise predictions of crash frequency-by-severity for target sites. Thus, for peak-over threshold models, the threshold parameter is the most important, and its calibration improves the performance of the base models. The complete re-estimation of models for individual target sites yields inferior fits and less precise crash estimates than the two transferability approaches since they utilize fewer traffic conflict extremes in their development than the larger dataset utilized in base model development. Therefore, the study results can significantly advance the applicability of traffic conflict models for crash risk estimation at transport facilities.

Thrombospondin-1 expression and modulation of Wnt and hippo signaling pathways during the early phase of Trypanosoma cruzi infection of heart endothelial cells.

The protozoan parasite, Trypanosoma cruzi, causes severe morbidity and mortality in afflicted individuals. Approximately 30% of T. cruzi infected individuals present with cardiac pathology. The invasive forms of the parasite are carried in the vascular system to infect other cells of the body. During transportation, the molecular mechanisms by which the parasite signals and interact with host endothelial cells (EC) especially heart endothelium is currently unknown. The parasite increases host thrombospondin-1 (TSP1) expression and activates the Wnt/ß-catenin and hippo signaling pathways during the early phase of infection. The links between TSP1 and activation of the signaling pathways and their impact on parasite infectivity during the early phase of infection remain unknown. To elucidate the significance of TSP1 function in YAP/ß-catenin colocalization and how they impact parasite infectivity during the early phase of infection, we challenged mouse heart endothelial cells (MHEC) from wild type (WT) and TSP1 knockout mice with T. cruzi and evaluated Wnt signaling, YAP/ß-catenin crosstalk, and how they affect parasite infection. We found that in the absence of TSP1, the parasite induced the expression of Wnt-5a to a maximum at 2 h (1.73±0.13), P< 0.001 and enhanced the level of phosphorylated glycogen synthase kinase 3ß at the same time point (2.99±0.24), P<0.001. In WT MHEC, the levels of Wnt-5a were toned down and the level of p-GSK-3ß was lowest at 2 h (0.47±0.06), P< 0.01 compared to uninfected control. This was accompanied by a continuous significant increase in the nuclear colocalization of ß-catenin/YAP in TSP1 KO MHEC with a maximum Pearson correlation coefficient of (0.67±0.02), P< 0.05 at 6 h. In WT MHEC, the nuclear colocalization of ß-catenin/YAP remained steady and showed a reduction at 6 h (0.29±0.007), P< 0.05. These results indicate that TSP1 plays an important role in regulating ß-catenin/YAP colocalization during the early phase of T. cruzi infection. Importantly, dysregulation of this crosstalk by pre-incubation of WT MHEC with a ß-catenin inhibitor, endo-IWR 1, dramatically reduced the level of infection of WT MHEC. Parasite infectivity of inhibitor treated WT MHEC was similar to the level of infection of TSP1 KO MHEC. These results indicate that the ß-catenin pathway induced by the parasite and regulated by TSP1 during the early phase of T. cruzi infection is an important potential therapeutic target, which can be explored for the prophylactic prevention of T. cruzi infection.

Trypanosoma cruzi Dysregulates piRNAs Computationally Predicted to Target IL-6 Signaling Molecules During Early Infection of Primary Human Cardiac Fibroblasts.

Trypanosoma cruzi, the etiological agent of Chagas disease, is an intracellular protozoan parasite, which is now present in most industrialized countries. About 40% of T. cruzi infected individuals will develop severe, incurable cardiovascular, gastrointestinal, or neurological disorders. The molecular mechanisms by which T. cruzi induces cardiopathogenesis remain to be determined. Previous studies showed that increased IL-6 expression in T. cruzi patients was associated with disease severity. IL-6 signaling was suggested to induce pro-inflammatory and pro-fibrotic responses, however, the role of this pathway during early infection remains to be elucidated. We reported that T. cruzi can dysregulate the expression of host PIWI-interacting RNAs (piRNAs) during early infection. Here, we aim to evaluate the dysregulation of IL-6 signaling and the piRNAs computationally predicted to target IL-6 molecules during early T. cruzi infection of primary human cardiac fibroblasts (PHCF). Using in silico analysis, we predict that piR_004506, piR_001356, and piR_017716 target IL6 and SOCS3 genes, respectively. We validated the piRNAs and target gene expression in T. cruzi challenged PHCF. Secreted IL-6, soluble gp-130, and sIL-6R in condition media were measured using a cytokine array and western blot analysis was used to measure pathway activation. We created a network of piRNAs, target genes, and genes within one degree of biological interaction. Our analysis revealed an inverse relationship between piRNA expression and the target transcripts during early infection, denoting the IL-6 pathway targeting piRNAs can be developed as potential therapeutics to mitigate T. cruzi cardiomyopathies.

A systematic mapping review of surrogate safety assessment using traffic conflict techniques.

Safety assessment of road sections and networks have historically relied on police-reported crash data. These data have several noteworthy and significant shortcomings, including under-reporting, subjectivism, post hoc assessment of crash causes and contributing factors, limited behavioural information, and omitted potential important crash-related factors resulting in an omitted variable bias. Moreover, crashes are relatively rare events and require long observation periods to justify expenditures. The rarity of crashes leads to a moral dilemma-we must wait for sufficient crashes to accrue at a site-some involving injuries and even death-to then justify improvements to prevent crashes. The more quickly the profession can end its reliance on crashes to assess road safety, the better. Surrogate safety assessment methodologies, in contrast, are proactive in design, do not rely on crashes, and require shorter observation timeframes in which to formulate reliable safety assessments. Although surrogate safety assessment methodologies have been developed and assessed over the past 50 years, an overarching and unifying framework does not exist to date. A unifying framework will help to contextualize the role of various methodological developments and begin a productive discussion in the literature about how the various pieces do or should fit together to understand road user risk better. This paper aims to fill this gap by thoroughly mapping traffic conflicts and surrogate safety methodologies. A total of 549 studies were meticulously reviewed to achieve this aim of developing a unifying framework. The resulting framework provides a consolidated and up-to-date summary of surrogate safety assessment methodologies and conflict measures and metrics. Further work is needed to advance surrogate safety methodologies. Critical research needs to include identifying a comprehensive and reliable set of surrogate measures for risk assessment, establishing rigorous relationships between conflicts and crashes, developing ways to capture road user behaviours into surrogate-based safety assessment, and integrating crash severity measures into risk estimation.

Pyranocarbazole derivatives as potent anti-cancer agents triggering tubulin polymerization stabilization induced activation of caspase-dependent apoptosis and downregulation of Akt/mTOR in breast cancer cells.

A series of new pyranocarbazole derivatives were synthesized via semi-synthetic modification of koenimbine (1a) and koenidine (1b) isolated from the leaves of Murraya koenigii. Among all, compound 3bg displayed significant anti-cancer activity against MDA-MB-231, DU145 and PC3 cell lines with the IC50 values of 3.8, 7.6 and 5.8 µM, respectively. It was also observed that the halogenated-benzyl substitution at N-9 position, C-3 Methyl and C-7 methoxy group on carbazole motif are favoured for anti-cancer activity. The detailed investigation was carried out with compound 3bg and its SEDDS (self-emulsifying drug delivery systems) formulation 3bgF. The in vivo drug release behavior study showed that the formulation enhanced slow release and better bioavailability at a tumor site. Compound 3bg and its formulation (3bgF) significantly inhibited cell proliferation and colony formation, induced G2/M arrest, reduced cellular ROS generation and induced caspase-dependent apoptosis in MDA-MB-231 cells. 3bg also induced significant alteration of Bax/Bcl expression ratio suggesting involvement of mitochondrial apoptosis. Additionally, 3bg caused down-regulation of mTOR/Akt survival pathway. 3bg do not bind to DNA, but interacts with tubulin as observed with in silico molecular docking studies. This interaction results in stabilization of tubulin polymerization similar to paclitaxel as detected in cell-free assay. Oral administration of 3bgF for 30 days at dose rate of 10 and 20 mg/kg body weight significantly reduced tumor growth in syngenic rat LA-7 mammary tumor model. These results indicated that the pyranocarbazole natural product based N-substituted analogues can act as potential anti-cancer lead.

Anti-colon cancer activity of Murraya koenigii leaves is due to constituent murrayazoline and O-methylmurrayamine A induced mTOR/AKT downregulation and mitochondrial apoptosis.

In recent years, many alkaloids of plant origin have attracted great attention due to their diverse range of biological properties including anti-hyperglycemic, anti-oxidant, anti-inflammatory, anti-diabetic and anti-tumor activity. Herein, the pyranocarbazole alkaloids were isolated from leaves of Murraya koenigii and their anti-cancer potential was investigated in different cancer cell lines. Among all tested compounds, murrayazoline and O-methylmurrayamine A demonstrated potent anti-cancer activity against DLD-1 colon cancer cells with the IC50 values of 5.7µM and 17.9µM, respectively, without any non-specific cytotoxicity against non-cancer HEK-293 and HaCaT cells. Further, studies of pure compounds revealed that the anti-cancer activity of compounds corresponds with altered cellular morphology, cell cycle arrest in G2/M phase, reactive oxygen species level and mitochondrial membrane depolarization of colon cancer cells. In addition, these compounds activated caspase-3 protein and upregulated Bax/Bcl-2 protein expression ratio leading to induction of caspase-dependent apoptosis in DLD-1 cells. These event induced by carbazole alkaloids also coincides with downregulation of Akt/mTOR suggesting downstream targeting of cell survival pathway. Thus, our in vitro studies not only provided scientific basis of the use of M. koenigii leaves in the traditional Indian Ayurveda medicines, but also expands possibilities of medicinal uses of M. koenigii leaves against colon cancer. Particularly, these findings will help in further investigating murrayazoline and O-methylmurrayamine A or their improvised derivatives as new therapeutics for the treatment of colon cancer.

Synthesis of targeted dibenzo[b,f]thiepines and dibenzo[b,f]oxepines as potential lead molecules with promising anti-breast cancer activity.

A targeted library of substituted dibenzo[b,f]thiepines and dibenzo[b,f]oxepines (prototypes I, II and III), and structurally analogous to tamoxifen have been synthesized as a new class of anti-breast cancer agents. All the prototype molecules exhibited potential antiproliferative activity against ER + ve and ER-ve breast cancer cell lines. Dibenzo[b,f]thiepine prototypes were found to be more active. Of all the compound tested, 14b exhibited potent in-vitro antiproliferative activity at 1.33 µM and 5 µM concentration in MCF-7 and MDA-MB-231 cell lines and was devoid of any cytotoxicity in normal HEK cells even at 50 µM. Cell cycle analysis showed that the compound 14b inhibited cell proliferation due to G0/G1 arrest in MCF-7 cells. Annexin-V FITC and PI staining experiments confirmed that the cell inhibition was primarily due to apoptosis and not by necrosis, which was also supported by LDH release assay experiment. Molecular docking studies showed better binding interaction of the new dibenzo[b,f]thiepine analogue 14b with the estrogen receptor (ER) as compared to 4-hydroxy-tamoxifen and this enhanced binding might be responsible for its estrogen antagonistic activity that induces cell cycle arrest, apoptosis and inhibition of breast cancer cells.