Regulating pri/pre-microRNA up/down expressed in cancer proliferation, angiogenesis and metastasis using selected potent triterpenoids.

MicroRNAs (miRNAs) play a crucial role in cancer progression by selectively inducing translational degradation of messenger RNA (mRNA) via sequence-specific interactions with the 3'-untranslated region (3'-UTR). The potential targeting of miRNA has been recognized as a significant avenue for investigating the biological progression of diverse cancer types. Consequently, targeting of pri-miRNA and pre-miRNA by phytochemicals emerges as a viable strategy in the realm of anticancer therapies. Among phytochemicals, triterpenoids have garnered significant recognition for their chemotherapeutic and chemopreventive capabilities in combating multiple cancers. To date, there is a dearth of literature about the molecular interactions between triterpenoids and miRNAs. The primary objective of this investigation is to discern the potential triterpenoids that can function as modulators for specific miRNAs, namely pri-miRNA-19b-2, pre-miR21, microRNA 20b, pri-miRNA-208a, pri-miRNA-378a, pri-miRNA-320b-2, and pri-miRNA-300, achieved through the use of in silico investigations. The study primarily focused on performing drug-likeness, computer-aided toxicity, and pharmacokinetic prediction studies for triterpenoids. Furthermore, molecular docking and simulation techniques were employed to investigate these compounds. The triterpenoids studied were shown to have drug-likeness characteristics, although asiatic acid, lupeol, and pristimerin were able to pass all toxicity tests. Among the triterpenoids that underwent docking, pristimerin had a significant binding energy of -10.9 kcal/mol during its interaction with pri-miR-378a. The stable interaction between the pristimerin and miRNA complex was demonstrated by molecular dynamics simulation. As a result, pristimerin has the potential to act as a modulator of carcinogenic miRNAs, making it a promising candidate for cancer prevention and treatment due to its tailored modulation of miRNA activity.

Exploration of selected monoterpenes as potential TRPC channel family modulator in lung cancer, an in-silico upshot.

Lung cancer is still the most frequent cause of cancer-related death, accounting for nearly two million cases yearly. As cancer is a multifactorial disease, developing novel molecular therapeutics that can simultaneously target multiple associated cellular processes has become necessary. Ion channels are diverse regulators of cancer-related processes such as abnormal proliferation, invasion, migration, tumor progression, inhibition of apoptosis, and chemoresistance. Among the various families of ion channels, the transient receptor potential canonical channel family steps out in the context of lung cancer, as several members have been postulated as prognostic markers for lung cancer. Phytochemicals have been found to have health benefits in the treatment of a variety of diseases and disorders. Among phytochemicals, monoterpenes are effective in treating both the early and late stages of cancer. The molecular docking interaction analysis was conducted to evaluate the binding potential of selected monoterpenes with TRPC3, TRPC4, TRPC5, and TRPC6 involved in different phases of carcinogenesis. Amongst the selected monoterpenes, thymoquinone exhibited the highest binding energy of -6.7 kcal/mol against the TRPC4 channel, and all amino acid binding residues were similar to those of the known inhibitor for TRPC4. In addition, molecular-dynamic simulation results parameters, such as RMSD, RMSF, and Rg, indicated that thymoquinone did not impact the protein compactness and exhibited stability during the interaction. The average interaction energy between thymoquinone and TRPC4 protein was -26.85 kJ/mol. In-silico Drug-likeness and ADMET profiling indicated that thymoquinone is a druggable candidate with minimal toxicity. We propose further investigation and evaluation of thymoquinone for lead optimization and drug development.Communicated by Ramaswamy H. Sarma.

Thymoquinone exhibited the highest BE −6.7 kcal/mol against the TRPC4 channel.Thymoquinone passed all drug-likeness parameters.Thymoquinone showed 99.38% of intestinal absorption in ADMET analysis.MD confirms thymoquinone forms stable molecular interaction with TRPC4.

Corrigendum: The molecular basis of differential host responses to avian influenza viruses in avian species with differing susceptibility.

[This corrects the article DOI: 10.3389/fcimb.2023.1067993.].

An Interaction Network Driven Approach for Identifying Cervical, Endometrial, Vulvar Carcinomic Biomarkers and Their Multi-targeted Inhibitory Agents from Few Widely Available Medicinal Plants.

Differently expressed genes (DEGs) across cervical (CC), endometrial (EC), and vulvar carcinoma (VC) may serve as potential biomarkers for these progressive tumor conditions. In this study, DEGs of cervical (CC), endometrial (EC), and vulvar carcinoma (VC) were identified by microarray analysis. The interaction network between the identified 124 DEGs was constructed and analyzed to identify the hub genes and genes with high stress centrality. DEGs, namely, CDK1 and MMP9, were found to show highest degree and highest stress centrality respectively from the gene interaction network of 124 nodes and 1171 edges. DEG CDK1 is found to be overlapping in both cervical and endometrial carcinomic conditions while DEG MMP9 is found in vulvar carcinomic condition. Further, as it is studied that many phytochemicals play an important role as medicinal drugs, we have identified phytochemicals from few widely available medicinal plants and performed comprehensive computational study to identify a multi-targeted phytochemical against the identified DEGs, which are crucially responsible for the progression of these carcinomic conditions. Virtual screening of the phytochemicals against the target DEG protein structures with PDB IDs 4Y72 and 1GKC resulted in identifying the multi-targeted phytochemical against both the proteins. The molecular docking and dynamics simulation studies reveal that luteolin can act as a multi-targeted agent. Thus, the interactional and structural insights of luteolin toward the DEG proteins signify that it can be further explored as a multi-targeted agent against the cervical, endometrial, and vulvar carcinoma.

The molecular basis of differential host responses to avian influenza viruses in avian species with differing susceptibility.

Introduction: Highly pathogenic avian influenza (HPAI) viruses, such as H5N1, continue to pose a serious threat to animal agriculture, wildlife and to public health. Controlling and mitigating this disease in domestic birds requires a better understanding of what makes some species highly susceptible (such as turkey and chicken) while others are highly resistant (such as pigeon and goose). Susceptibility to H5N1 varies both with species and strain; for example, species that are tolerant of most H5N1 strains, such as crows and ducks, have shown high mortality to emerging strains in recent years. Therefore, in this study we aimed to examine and compare the response of these six species, to low pathogenic avian influenza (H9N2) and two strains of H5N1 with differing virulence (clade 2.2 and clade 2.3.2.1) to determine how susceptible and tolerant species respond to HPAI challenge. Methods: Birds were challenged in infection trials and samples (brain, ileum and lung) were collected at three time points post infection. The transcriptomic response of birds was examined using a comparative approach, revealing several important discoveries. Results: We found that susceptible birds had high viral loads and strong neuro-inflammatory response in the brain, which may explain the neurological symptoms and high mortality rates exhibited following H5N1 infection. We discovered differential regulation of genes associated with nerve function in the lung and ileum, with stronger differential regulation in resistant species. This has intriguing implications for the transmission of the virus to the central nervous system (CNS) and may also indicate neuro-immune involvement at the mucosal surfaces. Additionally, we identified delayed timing of the immune response in ducks and crows following infection with the more deadly H5N1 strain, which may account for the higher mortality in these species caused by this strain. Lastly, we identified candidate genes with potential roles in susceptibility/resistance which provide excellent targets for future research. Discussion: This study has helped elucidate the responses underlying susceptibility to H5N1 influenza in avian species, which will be critical in developing sustainable strategies for future control of HPAI in domestic poultry.

A hypothetical model of multi-layered cost-effective wastewater treatment plant integrating microbial fuel cell and nanofiltration technology: A comprehensive review on wastewater treatment and sustainable remediation.

Wastewater management has emerged as an uprising concern that demands immediate attention from environmentalists worldwide. Indiscriminate and irrational release of industrial and poultry wastes, sewage, pharmaceuticals, mining, pesticides, fertilizers, dyes and radioactive wastes, contribute immensely to water pollution. This has led to the aggravation of critical health concerns as evident from the uprising trends of antimicrobial resistance, and the presence of xenobiotics and pollutant traces in humans and animals due to the process of biomagnification. Therefore, the development of reliable, affordable and sustainable technologies for the supply of fresh water is the need of the hour. Conventional wastewater treatment often involves physical, chemical, and biological processes to remove solids from the effluent, including colloids, organic matter, nutrients, and soluble pollutants (metals, organics). Synthetic biology has been explored in recent years, incorporating both biological and engineering concepts to refine existing wastewater treatment technologies. In addition to outlining the benefits and drawbacks of the current technologies, this review addresses novel wastewater treatment techniques, especially those using dedicated rational design and engineering of organisms and their constituent parts. Furthermore, the review hypothesizes designing a multi-bedded wastewater treatment plant that is highly cost-efficient, sustainable and requires easy installation and handling. The novel setup envisages removing all the major wastewater pollutants, providing water fit for household, irrigation and storage purposes.

Neolamarckia cadamba (Roxb.) Bosser (Rubiaceae) extracts: promising prospects for anticancer and antibacterial potential through in vitro and in silico studies.

Neolamarckia cadamba is an Indian traditional medicinal plant having various therapeutic potentials. In the present study, we did solvent-based extraction of Neolamarckia cadamba leaves. The extracted samples were screened against liver cancer cell line (HepG2) and bacteria (Escherichia coli). MTT cytotoxic assay was performed for in vitro analysis of extracted samples against the HepG2 cell lines and the normal human prostate PNT2 cell line. Chloroform extract of Neolamarckia cadamba leaves showed better activity with IC50 value 69 µg/ml. DH5α strain of Escherichia coli (E. coli) was cultured in Luria Bertani (LB) broth media and minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) were calculated. Solvent extract chloroform showed better activity in MTT analysis and antibacterial screening and it was taken for characterization of phytocomposition by Fourier transform infrared (FTIR) and gas chromatography mass spectrometry (GC-MS). The identified phytoconstituents were docked with potential targets of liver cancer and E. coli. The phytochemical 1-(5-Hydroxy-6-hydroxymethyl-tetrahydropyran-2-yl)-5-methyl-1H-pyrimidine-2,4-dione shows highest docking score against the targets PDGFRA (PDB ID: 6JOL) and Beta-ketoacyl synthase 1(PDB ID: 1FJ4) and their stability was further confirmed by molecular dynamics simulation studies.

Exploring the mechanism of action of podophyllotoxin derivatives through molecular docking, molecular dynamics simulation and MM/PBSA studies.

The chemical structure of a compound directly affects its biological activity, as different functional groups can change a compound's activity. With this in mind, the current study aims to predict the likely mechanism of action of several podophyllotoxin derivatives whose biological activities have already been documented. The interactions of the derivatives of podophyllotoxin with tubulin (PDB ID: 6NNG) and topoisomerase II (PDB ID: 3QX3), the two recognised targets of podophyllotoxin, were examined using molecular docking experiments. According to the molecular docking result, tubulin, and the investigated variants of podophyllotoxin interact more effectively than topoisomerase. The greatest docking score of the compounds was -12.200 against tubulin and -4.511 against topoisomerase, indicating that tubulin is the target of these drugs. Further to ascertain the strength of the interaction between the best-docked derivatives and the target protein, additional molecular dynamics investigations were also incorporated. With tubulin, the derivatives engage steadily, while with topoisomerase, the ligands shift from the protein's initial binding site to its DNA binding site. MMPBSA analysis was used to examine the stability of their relationship.Communicated by Ramaswamy H. Sarma.

Identification of SARS-CoV-2 inhibitors through phylogenetics and drug repurposing.

The novel coronavirus that has affected the whole world is declared a pandemic by the World Health Organization. Since the emergence of this virus, researchers worldwide have searched for potential antivirals against it. Being an RNA virus, it shows a high rate of mutability and variability in its genome. In the present study, all the reported SARS-CoV-2 genomes isolated from diverse regions of the world available in the GISAID database have been considered for phylogenetic analysis. The strain identified at the root is subjected to phylogenetic analysis with genomes of other known human viruses obtained from NCBI for identifying the nearest viral neighbor. Furthermore, the phylogenetic relationship between various human viruses was used to repurpose the known antiviral drugs towards coronavirus using in silico docking approach. The phylogeny reveals the link of the COVID virus with adenovirus. The known drugs against adenovirus are considered in the present study for drug repurposing through molecular docking analysis. The reference inhibitors of the respective targets were also considered in the docking study. The protein targets, namely protease, endoribonuclease, methyltransferase, phosphatase, and spike protein, are considered for screening with the known drug of adenovirus. Ribavirin, known to treat adenoviral infection, shows the best docking score, suggesting its use as a repurposed drug to treat SARS-CoV-2. Furthermore, the potency of the ribavirin drug is analyzed using molecular dynamics studies. Supplementary Information: The online version contains supplementary material available at 10.1007/s11224-022-02019-6.

Microbiome Diversity in Vaginal Fluid and Sensitivity Patterns in Preterm Premature Rupture of Membrane Cases.

INTRODUCTION: Preterm premature rupture of membranes (PPROM) is spontaneous rupture of the fetal membranes before 37 completed weeks and before the onset of labor. PPROM occurs in 3% of all pregnancies and is responsible for approximately one-third of all preterm deliveries. It leads to increase in perinatal morbidity and mortality. AIM: The present study aimed to characterize the microbiome of vaginal fluid, which will be helpful in the selection of empiric antimicrobial therapy. MATERIALS AND METHOD: A prospective observational study was conducted in the Department of Obstetrics and Gynaecology, Pradyumna Bal Memorial Hospital, Kalinga Institute of Medical Sciences (KIMS), Bhubaneswar during the period of October 2019 to June 2021 to characterize the microorganisms in the vaginal fluid and their antimicrobial sensitivity patterns found in antenatal women presenting with PPROM. A total of 160 antenatal women diagnosed with PPROM, gestational age between 28 weeks to 36 weeks and 6 days were included in the study. High vaginal swabs were collected for microbial culture and sensitivity. RESULTS: Out of 160 samples, the growth of organisms was observed in 134 (85.09%) samples. Out of them, 133 were monomicrobial, one was polymicrobial. Common isolated infections included Enterococcus faecalis (17.39%), followed by Staphylococcus aureus (14.29%), Escherichia coli (11.18%), and Staphylococcus haemolyticus (6.21%). Most of them were sensitive to ampicillin followed by linezolid and vancomycin. S. aureus was most sensitive to linezolid followed by gentamicin and vancomycin. Most isolates were multidrug-resistant. CONCLUSION: The empirical antimicrobial treatment started for PPROM management should be based on the established changing microbiological pattern and sensitivities with due consideration of geographical and demographic variations.

Whole-genome comparative analysis reveals genetic mechanisms of disease resistance and heat tolerance of tropical Bos indicus cattle breeds.

Bos indicus cattle breeds have been naturally selected for thousands of years for disease resistance and thermo-tolerance. However, the genetic mechanisms underlying these specific inherited characteristics must be elucidated. Hence, in this study, a whole-genome comparative analysis of the Bos indicus cattle breeds Kangayam, Tharparkar, Sahiwal, Red Sindhi, and Hariana of the Indian subcontinent was conducted. Genetic variant identification analysis revealed 155 851 012 SNPs and 10 062 805 InDels in the mapped reads across all Bos indicus cattle breeds. The functional annotation of 17 252 genes that comprised both SNPs and InDels, with high functional impact on proteins, was carried out. The functional annotation results revealed the pathways involved in the innate immune response, including toll-like receptors, retinoic acid-inducible gene I-like receptors, NOD-like receptors, Jak-STAT signaling pathways, and non-synonymous variants in the candidate immune genes. We also identified several pathways involved in the heat shock response, hair and skin properties, oxidative stress response, osmotic stress response, thermal sweating, feed intake, metabolism, and non-synonymous variants in the candidate thermo-tolerant genes. These pathways and genes directly or indirectly contribute to the disease resistance and thermo-tolerance adaptations of Bos indicus cattle breeds.

Proteomic analysis of differential expression of lung proteins in response to highly pathogenic avian influenza virus infection in chickens.

Elucidation of the molecular pathogenesis underlying virus-host interactions is important for the development of new diagnostic and therapeutic strategies against highly pathogenic avian influenza (HPAI) virus infection in chickens. However, the pathogenesis of HPAI virus in chickens is not completely understood. To identify the intracellular signaling pathways and critical host proteins associated with influenza pathogenesis, we analyzed the lung proteome of a chicken infected with HPAI H5N1 virus (A/duck/India/02CA10/2011/Agartala). Mass spectrometry data sets were searched against the chicken UniProt reference database. At the local false discovery rate level of 5%, a total of 3313 proteins with the presence of at least one unique peptide were identified in the chicken lung proteome datasets. Differential expression analysis of these proteins showed that 247 and 1754 proteins were downregulated at 12 h and 48 h postinfection, respectively. We observed expression of proteins of the predominant signaling pathways, including Toll-like receptors (TLRs), retinoic acid-inducible gene I-like receptors (RLRs), NOD-like receptors (NLRs), and JAK-STAT signaling. Activation of these pathways is associated with the cytokine storm effect and thus may be the cause of the severity of HPAI H5N1 infection in chickens. We also observed the expression of myeloid differentiation primary response protein (MyD88), inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB), interleukin 1 receptor associated kinase 4 (IRAK4), RELA proto-oncogene NF-κB subunit (RELA), and mitochondrial antiviral signaling protein (MAVS), which are involved in critical signaling pathways, as well as other, less-commonly identified proteins such as hepatocyte nuclear factor 4 alpha (HNF4A), ELAV-like RNA binding protein 1 (ELAVL1), fibronectin 1 (FN1), COP9 signalosome subunit 5 (COPS5), cullin 1 (CUL1), breast cancer type 1 susceptibility protein (BRCA1), and the FYN proto-oncogene Src family tyrosine kinase (FYN) as main hub proteins that might play important roles in influenza pathogenesis in chickens. In summary, we identified the signaling pathways and the proteomic determinants associated with disease pathogenesis in chickens infected with HPAI H5N1 virus.

Identification of hub genes in common cancers of women in India and targeting for the search of anticancer agent from Punica granatum phytoconstituent using interaction network analysis and virtual screening.

Cancer is one of the most dreadful diseases across the globe, with the advancement in this field a great advent has been achieved in treating cancer by various therapies like chemotherapy, radiation therapy, hormone therapy, gene therapy, and many more but also the most serious concern associated with the available treatments are the toxicities or the side effects linked to them, apart from this the treatment of many malignancies are still not available, because of these such issues, tremendous research is still going on in the whole world to find a better and more potent treatment option for cancer. Cancer develops due to the synergistic effects of both genetic and epigenetic factors. The mutations that change the normal functioning of the genes are responsible for cancer. Various genes are associated with cancers; many genes are commonly found to be mutated in diverse cancer types. In the present work, the genetic co-relation among the top five common cancers in Indian women has tried to be established, after that the identification of the hub gene was carried out with the use of CytoHubba module of Cytoscape. The hub gene product signaling pathway was then targeted for molecular docking with phytoconstituents of Punica granatum while the stability of the docked protein and ligand complex was validated through Molecular Dynamics Simulation.Communicated by Ramaswamy H. Sarma.

First complete genome characterization of duck plague virus from India.

In this study, we report the complete genome sequencing of the Duck plague virus from India for the first time. The sequencing was done on the MinION nanopore sequencer from Oxford Nanopore Technologies. The closest relative is the European strain 2085v, with 99.98 and 99.8% identity at the amino acid and nucleotide level respectively. Moreover, 72 out of 77 ORFs are completely conserved between the 2 strains. The high similarity with the European strain over the only three other pathogenic strains reported from China points to the circulation of European strain in India. The fly pathways of migratory birds and co-habitation with native species being a probable reason. More complete genome data from diverse sampling locations are needed to characterize the genomic features, develop diagnostics, vaccines, and understand the evolution of the virus.

Evolutionary Signatures Governing the Codon Usage Bias in Coronaviruses and Their Implications for Viruses Infecting Various Bat Species.

Many viruses that cause serious diseases in humans and animals, including the betacoronaviruses (beta-CoVs), such as SARS-CoV, MERS-CoV, and the recently identified SARS-CoV-2, have natural reservoirs in bats. Because these viruses rely entirely on the host cellular machinery for survival, their evolution is likely to be guided by the link between the codon usage of the virus and that of its host. As a result, specific cellular microenvironments of the diverse hosts and/or host tissues imprint peculiar molecular signatures in virus genomes. Our study is aimed at deciphering some of these signatures. Using a variety of genetic methods we demonstrated that trends in codon usage across chiroptera-hosted CoVs are collaboratively driven by geographically different host-species and temporal-spatial distribution. We not only found that chiroptera-hosted CoVs are the ancestors of SARS-CoV-2, but we also revealed that SARS-CoV-2 has the codon usage characteristics similar to those seen in CoVs infecting the Rhinolophus sp. Surprisingly, the envelope gene of beta-CoVs infecting Rhinolophus sp., including SARS-CoV-2, had extremely high CpG levels, which appears to be an evolutionarily conserved trait. The dissection of the furin cleavage site of various CoVs infecting hosts revealed host-specific preferences for arginine codons; however, arginine is encoded by a wider variety of synonymous codons in the murine CoV (MHV-A59) furin cleavage site. Our findings also highlight the latent diversity of CoVs in mammals that has yet to be fully explored.

SARS-CoV-2 Delta Variant among Asiatic Lions, India.

In May 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was detected in Asiatic lions in a zoological park in India. Sequence and phylogenetic analyses showed the SARS-CoV-2 strains were the B.1.617.2 (Delta) variant. To reduce transmission of variants of concern, surveillance of SARS-CoV-2 in wild animal populations should be increased.

Identification and molecular characterization of H9N2 viruses carrying multiple mammalian adaptation markers in resident birds in central-western wetlands in India.

We report here a targeted risk-based study to investigate the presence of influenza A viruses at the migratory-wild-domestic bird interface across the major wetlands of central India's Maharashtra state during the winter migration season. The H9N2 viruses have been isolated and confirmed in 3.86% (33/854) of the fecal samples of resident birds. To investigate the genetic pools of H9N2 circulating in resident birds, we sequenced two isolates of H9N2 from distant wetlands. Sequence and phylogenetic analyses have shown that these viruses are triple reassortants, with HA, NA, NP, and M genes belonging to G1 sub-lineage (A/quail/Hong Kong/G1/1997), PB2, PB1, and NS genes originating from the prototype Eurasian lineage (A/mallard/France/090360/2009) and PA gene deriving from Y439/Korean-like (A/duck/Hong Kong/Y439/97) sub-lineage. It was confirmed not only that four of their gene segments had a high genetic association with the zoonotic H9N2 virus, A/Human/India/TCM2581/2019, but also that they had many molecular markers associated with mammalian adaptation and enhanced virulence in mammals including the unique multiple basic amino acids, KSKR↓GLF at the HA cleavage site, and analog N-and O-glycosylation patterns on HA with that of the zoonotic H9N2 virus. Furthermore, future experiments would be to characterize these isolates biologically to address the public health concern. Importantly, due to the identification of these viruses at a strategic geographical location in India (a major stop-over point in the Central Asian flyway), these novel viruses also pose a possible threat to be exported to other regions via migratory/resident birds. Consequently, systematic investigation and active monitoring are a prerequisite for identifying and preventing the spread of viruses of zoonotic potential by enforcing strict biosecurity measures.

A Life Course Study on Traumatic Brain Injury and Physical and Emotional Trauma in Foster Children.

Foster children are exposed to high levels of abuse, violence, and other adverse events throughout their childhood and adolescent years. Forms of brain injury, notably traumatic brain injury (TBI), are understudied in the foster child population. This study aimed to explore different forms of brain injury and their cognitive, behavioral, and psychological/emotional effects on current and former foster children using a life course perspective. A thematic analysis with a life course perspective was used to examine semi-structured, open-ended interviews conducted with current and previous foster children between the ages of 16 and 29 years. The study included 47 participants: 25 males (53%) and 22 females (47%) with an average age of 21 years and an average of 11.2 years of education. Of 47 current and previous foster children between the ages of 16 and 29, two-thirds had sustained one or more TBIs. Through a thematic analysis, four overarching and inter-related themes emerged from the data: frequent TBI, normalization (of abuse, violence, injury, and neglect), emotional trauma, and dangerous coping methods such as alcohol use in 94% and recreational drug use in 81%. Normalization of adverse events, emotional trauma, and the use of dangerous coping methods occurred in 66%, 81%, and 49% of participants, respectively, and are the cumulative toxic long-term effects of early negative life experiences and repeated forms of brain injury. Early and continued exposure to TBI, abuse, violence, and/or neglect with continued maladaptive behaviors suggests that the participants may have experienced changes in brain structure and function over their lives that provided the milieu for continued vulnerability to personal and future injury to future generations. These behavioral and perceptual changes point to a toxic combination of injuries that result in continued vulnerability to repeated injury through contextual exposure to risks and maladaptive normalization, emotional trauma, and risky coping styles.