In the study of cancer, omics technologies are supporting the transition from traditional clinical approaches to precision medicine. Intra-tumoral heterogeneity (ITH) is detectable within a single tumor in which cancer cell subpopulations with different genome features coexist in a patient in different tumor areas or may evolve/differ over time. Colorectal carcinoma (CRC) is characterized by heterogeneous features involving genomic, epigenomic, and transcriptomic alterations. The study of ITH is a promising new frontier to lay the foundation towards successful CRC diagnosis and treatment. Genome and transcriptome sequencing together with editing technologies are revolutionizing biomedical research, representing the most promising tools for overcoming unmet clinical and research challenges. Rapid advances in both bulk and single-cell next-generation sequencing (NGS) are identifying primary and metastatic intratumoral genomic and transcriptional heterogeneity. They provide critical insight in the origin and spatiotemporal evolution of genomic clones responsible for early and late therapeutic resistance and relapse. Single-cell technologies can be used to define subpopulations within a known cell type by searching for differential gene expression within the cell population of interest and/or effectively isolating signal from rare cell populations that would not be detectable by other methods. Each single-cell sequencing analysis is driven by clustering of cells based on their differentially expressed genes. Genes that drive clustering can be used as unique markers for a specific cell population. In this review we analyzed, starting from published data, the possible achievement of a transition from clinical CRC research to precision medicine with an emphasis on new single-cell based techniques; at the same time, we focused on all approaches and issues related to this promising technology. This transition might enable noninvasive screening for early diagnosis, individualized prediction of therapeutic response, and discovery of additional novel drug targets. Genome and transcriptome sequencing and editing technologies, supplemented with machine learning, are setting the stage for the transition from traditional to precision medicine ,5,6,7,8.As inter-tumor heterogeneity is characterized by variability in patients with the same histologic type ,19, thisIntra-tumoral heterogeneity (ITH) is detectable: subpopulations of cancer cells differ in genome features and tumor areas and/or may evolve/differentiate over time ,21,22. TColorectal carcinoma (CRC) is a leading mortality cause worldwide ,11 and iCRC treatment has been standardized based on clinicopathological and genetic features (KRAS/NRAS/BRAF mutation and Microsatellite instability (MSI) status), as well as based on tumor staging. Characterization of multiple samples from the same patient proved to be a significant ITH indicator between different areas of the same tumor  as well as comparing the primary tumor and a subsequent local or distant recurrence  .The ability of next-generation sequencing (NGS) both at whole genome and single-cell levels to identify disease-associated variants and tumor features triggered a renewed interest on the effectiveness of biomedical and oncology research ,2,18,30.Although over the years conventional research has improved, as well as outcomes in CRC patients through diagnosis standardization, staging, and multimodal treatment, important critical and clinical issues remain unresolved ,7,8,9,32Recent considerations of dynamic clonal evolution , spatiotBased on a review of published data, we aimed at discussing the possible achievement of a transition from CRC clinical research to precision medicine with a special emphasis on new single-cell-based techniques, focusing on all approaches and issues related to these technologies. This transition may provide feasible non-invasive screening procedures for early diagnosis, individualized prediction of therapeutic response and discovery of additional novel drug targets.Proper analysis and extensive use of the large amount of data generated from single scNGS experiments are very challenging and require experienced personnel. A full understanding of the experimental and computational pathways starting from the wet lab to the sophisticated computer analysis of data is needed. Attention must be given to quality control measures for determining which individual cells to include for further examination, data normalization methods, clustering, and visualization for dimensional reduction of data into a two-dimensional graph.As far as the experimental design is involved, no less significant are the costs that vary from EUR 1–2 to a few cents per cell. The price is highly dependent on the number of cells sequenced, the desired sequencing depth, and the sequencing platform used.Regardless of cell separation method and labeling of mRNA molecules, all approaches rely on similar computational pipelines for transcriptional profiling. Some concepts are applicable to the majority of single-cell sequencing platforms that use DNA barcodes as an approach to link mRNA transcripts to a single-cell source. Single-cell technologies can be used to define subpopulations within a known cell type by seeking differential gene expression within the cell population of interest; at the same time, they can effectively isolate signal from rare cell populations that would not be detectable by other methods. Each individual cell analysis is driven by clustering of cells based on their differentially expressed genes. The genes driving the clustering can be utilized as unique markers for a specific cell population.There are several high-throughput single-cell sequencing platforms on the market at the moment: the most widely used and cost-effective are Fluidigm C1, DropSeq and Chromium 10X ,41. ThesThe various cell capture methods have to consider several parameters that differ from one method to the other and affect the final sequencing results. The main parameters are the number of starting cells , the method of cell separation  and the efficiency of cell capture .The C1 system isolates single cells into individual reaction chambers in the Fluidigm integrated fluidic circuit (IFC). The optically clear IFC enables the operator to automatically stain captured cells and examine them by microscopy for viability, surface markers or reporter genes. Cell lysing, reverse transcription, and cDNA amplification are performed on the C1 Single-Cell Auto Prep IFC using a SMARTer Ultra Low RNA Kit for cDNA synthesis ,47,48,49Droplet-based single-cell gene expression approaches, including DropSeq and the 10X platform, use microfluidic chips to isolate single cells along with individual microspheres embedded in oil droplets using a microfluidic so that each droplet contains a single cell ,51. The scRNA-seq data analysis poses several unique computational challenges that need the adaptation of existing workflows, as well as the development and application of new analytical strategies . Many anSingle-cell experiments can be considered as thousands of separate experiments, so it is essential to apply the right quality control (QC) metrics to decide which individual data sets are valid . For exaA common QC metric is the number of mitochondrial gene transcripts: excessive numbers of mitochondrial transcripts indicate cellular stress. In normal tissues, cells with excessive mitochondrial gene expression are not included in the analysis . HoweverAn important point in analyzing single-cell data is normalization to eliminate batch effects if multiple sequencing runs are to be compared. These batch effects can be caused by a non-avoidable number of technical variations given by different experimental sessions . In addition, for bulk RNA sequencing, data normalization involves comparing multiple batches of biological material; however, in sequencing individual cells that are not all the same type, normalization parameters are required to maintain cell-to-cell variability. A common way to normalize sequencing data is based on comparison with housekeeping genes ,63. BaseThe next analytic step is to use a clustering algorithm to determine which cells are closely related. The most widely used is the principal component analysis (PCA) , which uIntratumoral heterogeneity is a crucial factor in tumor biology, response to therapies and patient survival ,70. Due As single-cell sequencing technologies ensure a complete, unbiased analysis of cellular diversity within tumor masses, they can be used to explore the measurement somatic mutation rates, the clonal evolution of cell tumor lineages, and gain insights into chemotherapeutic drug response ,72. WholCurrently, several studies on single cells genomics and transcriptomics analysis ,73,74 haDai et al. generated a molecular census of tumor tissue cell types of a single CRC patient alongside with a clustering analysis to define gene expression at single-cell level. A total of 2824 cells were identified and classified into five distinct cell clusters. Each cluster was characterized by different cell markers: cluster 2 prevalently contained genes related to the major histocompatibility complex, while the remaining 4 possessed cell markers related to themselves. Gene Ontology term analysis demonstrates that cluster 1 genes were responsible for biological processes including ATP synthesis, cellular respiration, and energy derivation. Cluster 3 and 4 genes mainly supported cells by providing energy, generating extracellular matrix. Cluster 2 and 5 genes highlighted immunity functions including immune response, regulation of lymphocyte, leukocyte, and T-cell activation. Although the results of Dai et al. were obtained by a single CRC patient, they help us understand how different activated and quiescent, abnormal cellular subpopulations contribute to the initiation, maintenance, and progression of CRC disease . These dLi et al. performed an scRNA-seq analysis on 11 primary CRCs and matched normal mucosa to their microenvironments. They developed a method for single-cell transcriptome analysis defined reference component analysis (RCA) based on an algorithm that improves clustering accuracy.de novo. These reference data allow to identify a strong enrichment of stem/TA-like cells. Two distinct types of cancer-associated fibroblasts (CAFs), and epithelial–mesenchymal transition-related genes were found to be upregulated in the tumoral CAF subpopulation. CRC defined as single type in bulk transcriptomics, might be divided into subgroups with different survival probability rates by using single-cell signatures [Seven major cell types both in normal mucosa and CRC were isolated as well as epithelial cells, fibroblasts, endothelial cells, B cells, T cells, mast cells and myeloid cells. By using RCA, nine epithelial clusters and seven epithelial cell subtypes in human normal mucosa were isolated gnatures .A recent study characterized the individual cell response of CRC cell lines to genotoxic 5-fluorouracil (5FU)-induced DNA damage using a scRNA-seq approach. After 5FU treatment, the apparently single population CRC cells assume three distinctive transcriptome profiles, corresponding to diversified cell-fate responses: apoptosis, cell-cycle checkpoint, and stress resistance. Based on the group-specific expression gene patterns mediating DNA damage responses, it can be inferred how individual cells shape their transcriptome in response to DNA damage involving recurrence and chemoresistance. This might represent one of the most important challenges in current cancer treatment . The ideMetastasis is a complex biological process in which tumor cells move from the primary organ site and spread to distant organs through blood circulation . Variousfirst hit” mutation in APC that subsequently gave rise to both the primary and metastatic tumors. Data from this study revealed a late-dissemination model of metastasis in both CRC patients and provided unprecedented insight into metastasis at single-cell genomic resolution [A general difficulty in understanding metastatic lineages depends on the large intra-tumor heterogeneity at primary and metastatic sites. Leung et al. developed a highly multiplexed single-cell DNA sequencing approach to dissect the clonal evolution during the metastatic process. They studied two CRC patients with matching liver metastases. They observed monoclonal seeding in the first patient: a single clone acquired a large number of mutations before migrating to the liver to establish the second tumor site. In the second patient, they observed polyclonal seeding: two independent clones seeded metastases to the liver after migrating from the primary tumor lineage at different time points. Single-cell data also revealed a striking independent tumor lineage that did not metastasize, and early progenitor clones with the “solution . ActuallTang et al. characterized the evolutionary pattern of metastatic CRC (mCRC) by analyzing bulk and single-cell whole-exome sequencing (scWES) data of primary and metastatic tumors from seven CRC patients. They proved that genomic profile could be better explained by using scWES than through bulk sequencing. Rare mutations highlighted by scWES were undetectable in bulk data. Several subclones have been identified in both primary and metastatic tumor cells in MSI CRC patients. Although the individual cells of each subclone share a substantial number of mutations, few subclone-specific single nucleotide variants (SNVs) could characterize different cell clones with low mutation frequencies in the entire population of tumor cells.In MSS CRC patients, tumor cells were divided into two major cell populations from primary and metastatic lesions, that shared most SNVs and involved genes associated with CRC progression, such as TP53 and APC. Primary tumor cell populations were rich in AXIN3 and RASGRF1 genes mutation, known to be associated with tumor proliferation and invasion. In addition, 24 non-synonymous SNVs specific to metastatic cells in DNAH3, TBC1D4, CMYA5, MYO18A, PLEKHA7, and SLC19A3 genes have been identified, validating their functions in cell migration capacity .Another comparison of scWES versus bulk whole-exome sequencing (bulk WES) on two CRC patients with tumor and adenomatous polyps, showed that both had monoclonal origin and shared partial mutations in the same signaling pathways; however, each showed a specific spectrum of heterogeneous somatic mutations. Adenoma and cancer further developed intratumor heterogeneity accumulating non-random somatic mutations specifically in GPCR, PI3K-Akt and FGFR signaling pathways. New driver mutations were identified that developed during the evolution of both adenoma and cancer: on one hand OR1B1  was related to adenoma evolution; on the other hand, LAMA1  and ADCY3  had a role in CRC evolution. ScWES shows causality of mutations in certain pathways that would not be detected by bulk tumor sequencing. Furthermore, it can potentially establish whether specific mutations are mutually exclusive or occur sequentially in the same subclone of cells .de novo methylation or demethylation during metastasis were observed. As well as providing important information about the molecular alterations that occur during CRC progression and metastasis, multicellular sequencing showed that DNA methylation levels are consistent within lineages but can differ substantially between clones [To examine the genome, transcriptome, and methylome within CRC primary tumors and metastases, Bian et al. used a single-cell triple homology sequencing (scTrio-seq) technique . The scTn clones .To summarize, shedding light on the main mechanisms behind the development of metastasis based on the analysis of gene expression patterns at single-cell resolution should lead to tailoring individualized cancer treatment.To this end, the study of CRC heterogeneity through identification of tumor cells subpopulations and analysis of their features by single-cell omics technologies is crucial for the comprehension of the role of these cells and might lead to identify potential new targets for clinical treatment.Application of omics technologies on other types of cancers is opening a way to verify results also in diagnosis and treatment of other tumoral diseases, including CRC; this is the case of breast cancer (BC) thoroughly studied through single-cell omics technologies. For instance, Pinkney et al. adopted scRNA-seq to analyze the heterogeneity of lncRNA expression in vivo using Triple Negative BC (TNBC) xenografts; at the same time, they tried to assess whether lncRNA expression is sufficient to define cellular subpopulations. These authors observed that even if most lncRNAs are detectable at low levels in TNBC xenografts, a subpopulation of cells could not be defined. They showed highly heterogeneous expression patterns including global expression and subpopulation-specific expression; in addition, a hybrid pattern of lncRNAs was expressed in several but not all subpopulations .LncRNAs have been progressively identified as the main group of oncology targets acting as drivers in cancer, and are also being studied as clinical biomarkers . LncRNAsZhang et al. performed a single-cell RNA- and ATAC-sequencing to examine the immune cell dynamics in advanced TNBC patients treated with paclitaxel or paclitaxel plus atezolizumab (anti-PD-L1). High levels of baseline CXCL13+ T cells linked to macrophage proinflammatory features might predict responses to a drug combination. In patients responsive to drug combination, an increase of lymphoid tissue inducer cells, follicular B cells, CXCL13+ T cells, and type 1 dendritic cells was detected. The latter decreased after paclitaxel monotherapy . These dImmune checkpoint blockade (ICB) targeting PD-1/PD-L1 signaling axis and its use has achieved significant responses in cancer patients, although the mechanisms underlying ICB resistance have not been fully understood ,92. ThusIn-depth knowledge of the cells of interest is crucial to properly manage genomic data and make decisions of clinical impact based on standardized measurements and accurate and reproducible quality controls. The use of scRNA-seq provides one of the most innovative methods for addressing biological and medical questions concerning the underlying processes of various developmental, physiological, and disease systems. However, new programs and implementations of scRNA-seq methodologies have been started in recent years but further advances in both technology and specific approaches to use them are certainly warranted.The deployment of a number of processes will make it possible to extend the analysis of scRNA-seq studies not only on fresh material, but also on cryopreserved and fixed tissue samples aiming at introducing this technique into the clinical practice. Volume reduction and diffusion of techniques based primarily on microfluidics platforms should reduce costs at the same time leading to a standardized and simplified use of different devices.However, one of the current challenges is the creation of standardized collections and data catalogues from single cells due to the fact that the number of samples used so far in studies is small. Such analysis, in fact, requires a minimum/sufficient number of cells to ensure that all cell types are represented. Only a bioinformatician with experience in single-cell sequencing will be able to generate analyses that can be used to make meaningful biological inferences by choosing appropriate cutoffs for applied algorithms and avoiding misleading results. Currently, there are limited standardization protocols and guidelines on standards .Furthermore, development of single-cell gene expression maps for all tissues will be necessary, as it occurred in bulk transcriptomics evolution. Many studies, in fact, will benefit from these easily accessible archives that reduce the costs of comparison and replication in normal tissues; at the same time, significant advances in bioinformatics and computational methods thanks to data sharing are expected.Thus, the new challenge will be represented by the use of a true inter-omic and multidisciplinary approach that will lead to a comprehensive examination of individual cells; this will be achieved by characterizing the genome, epigenome, proteome and metabolome while simultaneously examining the tumor microenvironment, its immunological characteristics and the impact of pharmacogenomics; in addition, a clear picture of tumor development will be given, together with cancer evolution and interactions. It will be crucial to address genetic changes in the early stages of tumorigenesis deployment and how transcriptional subpopulations evolve into malignancy in later stages of tumor progression.The robustness of NGS systems in exploring heterogeneity, at genome and transcriptome scale, will validate ITH variability and might determine the discovery of novel targeted drugs; predictive biomarkers for individualized drug-oriented therapies might also be developed. Pharmacogenomic profiling might predict response to chemotherapy by correlating it with immune cell regulatory values that affect CRC survival mechanisms. Future CRC studies employing comparison of primary, metastatic tumor ITH and liquid biopsies might offer elucidating suggestions on the origins and evolution of genomic subclones responsible for drug resistance and recurrence.CRC is extensively marked by phenomena of inter- and intra-tumor heterogeneity, spatial and temporal differences regarding phenotypic and genotypic aspects, influencing recurrence and therapeutic response and having a strong poor impact on CRC patient’s outcome.Until now, genomic and transcriptome analyses on bulk tumor cell populations have helped to explain tumor heterogeneity and also allowed to classify them into subgroups with distinct molecular, morphological, and clinical features . The appMetastatic progression is linked to the majority of CRC-related deaths . In pati


Dysregulated immune response arising in the periphery can induce depressive symptoms through neuroimmune interactions. Inflammatory oral pathology can be a potent inducer of chronic neuroimmune response relevant to depression. We aimed to synthesize available evidence for the association between inflammatory periodontal diseases (IPD) and major depression (MD) in relation to a broad range of biomarkers.Medline, Embase, PsycInfo, Cochrane Library, Web of Science and Scopus databases were searched from inception until January 27, 2022. Search terms included subject headings and synonyms for inflammatory periodontal disease and depression. Studies that reported data on both depression and inflammatory periodontal disease as categories along with measurement of a biomarker were considered. Two reviewers independently selected the articles for inclusion, extracted data and assessed the quality of each study. The protocol for this study was registered with PROSPERO, CRD42021215524.Twenty-eight studies were included in the final review-eleven cross-sectional studies, seven case-control studies, and six prospective cohort studies conducted in humans; the remaining four were experimental animal studies. Eighteen studies including all animal studies reported a positive association between depression and periodontal disease; one study reported a negative association and another nine studies found no such associations. Twenty studies reported mixed associations between IPD and biomarkers . Biomarkers related to depression were gingival crevicular fluid cortisol, interleukin 6 (IL-6), Il-1β, immunoglobulin G against Bacterioides forsythus; root canal lipopolysaccharides; blood IL-6, IL-1β, cortisol, advanced oxidation protein products, nitric oxide metabolites, lipid hydroperoxides and trapping antioxidant parameter; whereas five studies found no associations between depression and a biomarker. Although animal studies showed interaction of immune, inflammatory and neurotrophic biomarkers in the relationship between depression and periodontal disease, human studies showed mixed findings. In most studies, there were risks of bias due to the sample selection and assessment protocol. Study heterogeneity and limited number of comparable studies reporting on shared biomarkers precluded a meta-analysis.Immune-inflammatory contribution to depression was evident in the context of inflammatory periodontal diseases, but whether biomarkers mediate the associations between IPD and MD needs to be tested through methodologically rigorous studies aiming specifically at this hypothesis. •Inflammatory signals from the periphery induces brain neuroimmune response.•Periodontal inflammation is a potential source of neuroinflammation in depression.•We did a systematic review on biomarkers shared by periodontitis and depression.•Biomarkers relevant to immune changes in depression was also found in periodontitis.•Studies should directly test biomarker roles in periodontitis–depression relations. However, recent findings suggest that pathological processes involved in these conditions have far-reaching consequences. In particular, the field of psychoneuroimmunology has established that inflammatory signals arising in the periphery can reach brain areas responsible for mood and behavioral regulation . In thisConsiderable amount of data suggests an increased occurrence of mental health issues in persons with inflammatory oral diseases, and vice versa . A 12-yeAltered levels of immune-inflammatory markers including pro-inflammatory cytokines, oxidative and nitrosative stress markers, neurotoxic metabolites of tryptophan degradation and reduced neurotrophic levels are detected in a considerable proportion of individuals with depression . A recenPorphyromonas gingivalis) through release of bacterial proteins such as lipopolysaccharides that not only activate systemic low-grade inflammation but also downregulate neurotrophic factor maturation in the brain  as a plausible source of such inflammation in depression. Firstly, IPD involves dysbiosis of the oral microbial community in favor of inflammation-provoking pathogens . Two authors screened the title and abstract of 1557 reports independently using Rayyan-a web-based screening solution  . Recordssolution .Inclusion criteria: (a) original peer-reviewed studies conducted in vivo, in humans or animal models, (b) either sex, (c) any age, (d) any human or animal samples, (e) examined both inflammatory periodontal disease and depression, and (e) reported on at least one biomarker. A broad definition of biomarkers was used as suggested by the US National Institutes of Health . Exclusi2.2Data were independently extracted by all authors in pairs using a standardized form. A third reviewer crosschecked the extraction sheet for each study for consistency and any discordance was resolved through discussion among the authors.We included studies under an umbrella of depression for any categories the authors defined as satisfying depression diagnosis or having adequate symptom load for depression. We operationalized IPD with any criteria for periodontal inflammation presented by the authors, but included studies that reported values of at least one of the three parameters, i.e, bleeding on probing (BOP), pocket probing depth (PPD) and clinical attachment level (CAL) necessary for a periodontal diagnosis.We closely evaluated the data against study design, study population and settings for possible meta-analysis of biomarkers reported in comparable studies. As a priori, at least three methodologically comparable studies reporting the same biomarkers would provide basis for further meta-analysis.2.3https://www.nhlbi.nih.gov/health-topics/study-quality-assessment-tools, and the tools contain 14 criteria  or 12 criteria (case-control studies) assessing potential selection, information and measurement biases or confounding factors, rated as yes, no, not reported, cannot determine, or not applicable. One point was given for yes, and 0 point for the other responses. Two authors graded each study independently, and the grading was subsequently discussed to reach consensus. Risk of bias matrices are presented as supplementary material  levels . SimilarTwenty of the included studies on human subjects reported on biomarkers related to periodontal disease, fiftteen of which found an association between the measured biomarkers and periodontitis. Most studies reported cortisol levels measured in saliva, GCF, serum or urine to be positively correlated with periodontal disease . HoweverStudies reporting on the GCF and salivary levels of cortisol, MMP -8, MMP-9 showed mixed associations with depression , 2007. K3.5In animal studies, several of the analyzed biomarkers were related to co-occurrence of periodontitis and depression. Bacterioides forsythus (Bf) was associated with periodontal disease only in subjects with higher depression scores (Systemic and urinary cortisol were associated with elevated depression scores in patients with acute necrotizing ulcerative gingivitis (ANUG) . One stun scores . Increasn scores . One stun scores . Patientn scores . In the n scores , there wOther studies did not find common biomarkers to explain the link between depression and IPD. 4We examined available literature for the association between depression and periodontitis putatively mediated or related to shared biomarkers. Out of 28 included studies, fourteen human and four animal studies found a positive association between IPD and MD while the rest reported no such associations. A previous systematic review on the relationship between periodontitis and depression selected 15 relevant studies out of which only six concluded on the positive association between these conditions , pointinIn the current review, animal studies  support The possibility of inflammation and depression being related was out of the question a few decades ago . The emeNevertheless, the questions raised are still a matter of debate: is the inflammation primary or does depression promote inflammation? One prospective study on young individuals did indicate that depression episodes increased CRP levels and as such may be considered an inflammatory promoter . More evBacteria are the major culprits in the development of periodontits, but the tissue response to this aggression results in the secretion and interaction of a plethora of inflammatory mediators contributing to the pathogenesis of periodontal disease . KnowingAs such, this may explain why the study by  did not Several other methodological limitations of these evaluated studies should be named. First, the majority of studies are observational. It is known that these designs come with the great weakness of not being able to protect against confounding. While most of them used statistical techniques to adjust for possible confounders, there may be residual unknown or unrecognized factors that remain in a heterogeneous adult study population . This isAnother critical aspect for the analyzed studies is the big age-span of included subjects. Even though the quality of studies increases if a statistical adjustment is included, other possible unknown age-related confounding factors, particularly multimorbidities associated with immune inflammatory alterations, may influence the outcome of an investigation.The conflicting results between the investigations may also be explained by the differences between the different levels of periodontal disease severity and the high variation of used clinical parameters. It is also the big variability in studied biomarkers along with testing methodology and limited number of studies for shared biomarkers using the same biological sample that hampered the meta-analysis of existing literature.In conclusion, there is a substantial data supporting associations between periodontal disease and depression and inflammatory contribution to each disorder. However, limited evidence is available on the role of biomarkers in the possible causal pathway between depression and periodontal disease. This is generated by the high heterogeneity among the type of study, the included populations, the assay methods, and the evaluated biomarkers. Future randomized control and prospective studies with standardized clinical and biological assessments are required to establish causality between periodontal inflammation and depression and further analyze the role of biomarkers in linking these diseases.This study was funded through internal resources at the affiliated institutions.None.


Female sex workers are highly mobile, which may influence their risk of experiencing physical and sexual violence. However, there remains a paucity of research, particularly longitudinal, from Sub-Saharan Africa exploring mobility and gender-based violence among female sex workers. To address this gap, this study examined the longitudinal relationship between work-related mobility and recent experience of physical or sexual gender-based violence from a client or partner among female sex workers in Iringa, Tanzania. A secondary data analysis was conducted using baseline and 18-month follow-up data from Project Shikamana, a community empowerment-based combination HIV prevention intervention. Responses from 387 female sex workers aged 18 years and older participating in both baseline and follow-up were analyzed. Unadjusted and adjusted Poisson regression models with robust variance estimations, accounting for clustering of female sex workers’ responses over time, were fit. Final models adjusted for socio-demographic characteristics and aspects of participants’ living situations and work environments. Recent physical or sexual violence from a client or partner was common . Twenty-six percent of female sex workers at baseline, and 11% at follow-up, had recently traveled outside of Iringa for sex work. In the final adjusted longitudinal model, female sex workers recently mobile for sex work had a 25% increased risk of any recent experience of physical or sexual gender-based violence when compared with their non-mobile counterparts . Interventions must identify ways–such as mobile support services, linkages and referrals to health and other social services while traveling, or the use of mobile or digital technology–to address mobile female sex workers’ unique needs while traveling. Future quantitative and qualitative research is needed to understand the context of female sex workers’ mobility and how and why mobility influences risk environments and experiences of gender-based violence. Female sex workers (FSWs) experience increased risk of physical and sexual violence globally ,2. Such The socio-structural environment in which FSWs live and work influences their risk of experiencing violence. Criminalization of sex work, gender inequities, inequitable power relations, economic constraints, and population mobility intersect to shape the contexts in which FSWs work and experience violence ,11–15. PWhile substantial research has highlighted the increased risk for GBV among refugee and displaced women and girls –24, lessTo fill these gaps, this article examines the longitudinal relationship between work-related mobility among FSWs and GBV in Iringa, Tanzania, where sex work remains criminalized . In 2015This study was approved by the Johns Hopkins Bloomberg School of Public Health (JHSPH) institutional review board (IRB) (Ref #: 00007065) as well as the Muhimbili University of Health and Applied Sciences (MUHAS) IRB and National Institute of Medical Research (NIMR) IRB in Tanzania (Ref #: 1593). Oral informed consent was received from all participants. Oral informed consent was used given that the study worked with individuals with historically marginalized identities at the intersection of HIV- and sex work-related stigma. The study opted for oral consent because documenting their signature created additional possible risks related to participants’ confidentiality. This decision was informed by prior experience conducting the informed consent process for HIV prevention studies among people who sell sex and in the cultural context of Iringa. The informed consent procedures for this study were designed to maximize understanding of potential risks to participants. Forms were translated into local languages, and content read aloud to participants. Understanding was ensured by asking participants to summarize the study and explain the reasons why they wanted to participate. Individuals were also provided with a copy of their consent form if they wanted it as well as information on how to contact the study staff to report adverse events associated with their participation in the research. This oral consent procedure was approved by the JHSPH IRB as well as MUHAS and NIMR in Tanzania as part of the approval of the study.This study draws on data collected from FSWs in Iringa, Tanzania, situated in the Southern Highlands of Tanzania. The Tanzania-Zambian (Tan-Zam) highway passes through Iringa, making mobility–be it by truckers traveling or residents–fundamental to life in this region –33. AgriAccording to the 2016–2017 Tanzania HIV Impact Survey, HIV prevalence is higher in Iringa (11.3%) among adults 15 years and older than nationally (4.7%) . GovernmTo examine the longitudinal relationship between sex work-related mobility and experience of GBV, this study drew on baseline and 18-month follow-up data collected as part of Project Shikamana. As described elsewhere ,40, ProjAfter a mapping of sex work venues in each community, time-location sampling was used to recruit both HIV-positive and HIV-negative FSWs for the baseline survey. Eligible FSWs were at least 18 years old and reported that they had exchanged sex for money, based on self-report, within the past month. Oral informed consent was received from all participants before beginning data collection.Four-hundred ninety-six (496) FSWs were recruited at baseline, with 387 also participating in the 18-month follow-up survey. The final sample analyzed here included all FSWs participating in both baseline and follow-up surveys (n = 387).The outcome of interest was any recent experience of physical or sexual GBV from any sexual partner, defined as any self-reported experience of either physical or sexual violence within the past six months from a new or regular client or non-paying partner. Any recent experience of physical or sexual GBV from a sexual partner at baseline was assessed based on responses to nine questions following the WHO and work by Decker and colleagues ,32,43. AConfounding and correlate variables considered for final models were both time variant and time invariant and included relevant socio-demographic characteristics and aspects of FSWs’ living situation and work environments.Time-variant covariates included in final adjusted models were marital/cohabitation status, average monthly income, alcohol consumption, recent drug use, average number of clients per week, and perceived financial security. Marital/cohabitation status was defined as currently married or living with a sex partner vs. not. Average monthly income was defined as self-reported average monthly income, including sex work, over the last six months and dichotomized at 120,000 Tsh  to compare higher vs. lower average monthly income. Alcohol consumption was measured using the AUDIT-3 short-form  and was Time invariant covariates measured at baseline included participants’ age , HIV status (HIV-infected at baseline vs. not), community (intervention vs. control community), participants’ ethnic group (Hehe vs. other), and educational attainment (secondary school or higher vs. less than secondary school).2 tests. To examine bivariate associations between sex work-related mobility as well as other covariates and the outcome of interest, Pearson’s χ2 tests were conducted to examine associations at baseline and at follow-up. As recent experience of physical or sexual GBV was commonly reported among study participants, unadjusted Poisson regression models were fit using generalized estimating equations (GEE) with robust variance estimations and an exchangeable correlation structure to examine the longitudinal relationship with sex work-related mobility as well as with other variables of interest. The use of robust Poisson regression was proposed as an alternative to logistic regression for common outcomes [Descriptive analyses examined recent experience of physical or sexual GBV, sex work-related mobility, and other covariates of interest at baseline and/or follow-up. Key baseline socio-demographic characteristics were compared between those individuals participating at baseline and follow-up (n = 387) and those not participating in the follow-up survey (n = 109) to examine potential effects of loss to follow-up using Pearson’s χoutcomes . Longituoutcomes .It was hypothesized that age was a potential effect modifier of the relationship between sex work-related mobility and recent experience of physical or sexual GBV. An interaction term between the explanatory variable of interest and this socio-demographic characteristic was included in bivariate robust Poisson regression models to examine this hypothesis. There was no significant evidence of effect modification at the bivariate level (p>0.05), and the interaction term was dropped in final multivariate models presented here.An adjusted robust Poisson regression model first included relevant factors related to FSWs’ socio-demographic characteristics, living situation, and work environments. A manual backward elimination method from this initial full robust Poisson model was conducted. Wald tests were used to assess overall contribution of variables to the final model. Variables were removed if they did not significantly contribute to the model assessed based on p>0.2. Following this step, retained covariates were included in the final model examining the longitudinal relationship between sex work-related mobility and recent experience of physical or sexual GBV. As part of a sensitivity analysis, the final model suggested following this manual backward selection approach was compared to models suggested using forward and backward stepwise selection with a cut-off of p = 0.2. Model fit was assessed using Cui’s quasilikelihood under the independence model criterion (QIC), an adaptation of Akaike’s information criterion (AIC) for use with GEE analyses, to identify the best group of covariates for the final model . The finThe final model adjusted for study visit (follow-up vs. baseline), community, HIV status at baseline, age, educational attainment at baseline, ethnic group, marital/cohabitation status, average number of clients per week, perceived financial security, alcohol consumption, and recent drug use. All coefficients were exponentiated and reflect either unadjusted or adjusted incidence rate ratios (IRR).There were no significant differences in the outcome or primary explanatory variables of interest at baseline between the final sample (n = 387) and those who did not participate in the follow-up survey (n = 109). Those lost to follow-up tended to be younger, not living with HIV, and not currently married or living with a sex partner.At baseline and follow-up, mobility for sex work was more common among FSWs reporting recent physical or sexual violence from a client or partner than among those with no recent experience of violence . Age, educational attainment, and average monthly income were marginally significantly associated with recent experience of physical or sexual GBV in the final adjusted model . ParticiIn this sample of FSWs from Iringa, Tanzania, FSWs with recent mobility for sex work had a higher risk of recent physical or sexual GBV when compared to those with no recent mobility for sex work after adjusting for socio-demographic characteristics and aspects of their living situations and work environments. Other aspects of FSWs’ living situations and work environments, such as FSWs’ self-reported average number of clients per week, hazardous alcohol consumption, and recent drug use, also had positive associations with any recent experience of physical or sexual GBV. This study, consistent with previous analyses conducted in Vancouver, Canada , strengtWhile addressing the factors that put FSWs at greater risk of GBV is increasingly a priority for public health interventions, these findings suggest that there is a need to acknowledge the ways in which mobile FSWs, particularly those mobile for the purposes of sex work, may experience unique risk environments that increase their risk for and experiences of GBV. Participatory involvement of mobile FSWs in the conceptualization and design of interventions would not only strengthen engagement, but also address the unique needs that mobile FSWs have while traveling. This could include, for example, mobile GBV services or the improved integration of GBV services into other mobile health services or outlets providing services to FSWs. These services could address both prevention and response and include social support, case management, and referrals. Lessons could be learned from those working in other settings at the intersection of substance use, sex work, and mobility , for howFuture research should build on these findings to understand the complex mechanisms through which mobility for work influences risk of GBV. A qualitative investigation, for example, of the drivers of mobility for work in Tanzania would illuminate how mobility for work influences FSWs’ risk environments. Quantitative studies could also build on the findings presented here to explore those socio-structural factors–including factors such as alcohol consumption, drug use, client interactions, or other aspects of FSWs’ risk environments–that may mediate the relationship between work-related mobility and recent experiences of GBV. In addition, while there was no significant evidence of effect modification by age, mobility and recent experiences of GBV were more common among select age groups of FSWs, and further quantitative research is needed to understand these dynamics including whether or through what pathways age and mobility influence FSWs’ risk of GBV. Studies have previously looked at the intersection of mobility and GBV and its effect on other health outcomes like depression  or HIV iThis study reinforces the connection between mobility and gender inequality, made manifest in instances of GBV , in our This study extends previous cross-sectional analyses  to examiSecond, this study is limited by its measures of GBV and FSWs’ mobility. Measures of GBV at baseline and follow-up limited analyses to any recent experience of GBV from a client or partner. Future research should ensure consistent measurement of GBV across time points, and may need to include GBV from other perpetrators, such as police or community members. In addition, there remains a need for future research to understand the nuanced relationships between mobility and GBV. Recent work in Zimbabwe has begun to identify typologies of mobility based on destination, duration, and other aspects of FSWs’ mobility . Future Finally, findings presented here may not be representative of the experiences of all FSWs in Tanzania. Analyses were conducted using baseline and follow-up data from Project Shikamana, a community-randomized trial conducted in two communities in Iringa. While Project Shikamana included activities focused on violence prevention, there were no significant reductions in violence found in adjusted models, suggesting the need for more comprehensive approaches that address the socio-structural factors that influence GBV in this context. The socio-structural environment and context in communities where this study was conducted may differ from other communities in Iringa and elsewhere in Tanzania, given their proximity to the Tan-Zam highway  that mayDrawing on baseline and follow-up data from a community randomized controlled trial in Iringa, Tanzania, this study examined the longitudinal relationship between recent mobility for the purposes of sex work and experiences of GBV from a client or partner. Evidence of a significant increased risk of violence among mobile FSWs as compared to their non-mobile counterparts suggests that efforts to reduce GBV among FSWs must acknowledge the unique needs of those FSWs who are mobile for sex work. Public health programs must identify ways–using digital technologies, for example–to engage mobile FSWs. Further understanding of the typologies of FSW mobility that integrate a qualitative understanding of FSWs’ trajectories with quantitative understandings of not only reason for mobility but also factors related to destination, duration, and frequency will help elucidate the pathways through which mobility influences FSWs’ risk environments and, ultimately, their experiences of violence. An integration of mobility into how we understand FSWs’ risk environments, building on the strong body of literature exploring the intersections between migration and gender, is a critical first step for those working with such mobile populations.


Older adults are at increased risk for COVID-19 illness, hospitalization, and mortality. Essential health care workers became the backbone of their care during the pandemic, and their experiences are worthy of discussion. This symposium will highlight the emotional impact of COVID-19 on health care workers, and their scope of practice in various health settings. Using data from a cross-sectional survey, presenter one will describe concerns and coping strategies among nursing home social workers during COVID-19. Next, presenter two will present findings on the emotional health and wellbeing of home care workers. Then, presenter three will describe the experience of the VA’s strike teams sent into 82 Florida nursing homes most impacted by COVID. Presenter four will describe findings from a qualitative study on emotional distress experienced by hospital-based medical directors, nurse practitioners, and other health care workers. Finally, presenter five will describe findings from a scope of practice among health care social workers study, with an emphasis on how policies in their respective settings impacted their work. Taken together, the presenters’ findings in this symposium have implications for practice and policy recommendations to improve the experience of health care workers during pandemics.


Opioid treatment programs (OTPs) are the primary source of medication-assisted treatment (MAT) for many individuals with opioid use disorder, including poor and uninsured patients and those involved in the criminal justice (CJ) system. Substance use treatment services that are tailored to the unique needs of patients often produce better outcomes, but little national research has addressed characteristics associated with whether OTPs offer services specifically tailored to community members involved in the CJ system. Medicaid expansion under the Affordable Care Act has broadly strengthened MAT services, but the role of expansion in supporting MAT services that are specifically tailored towards CJ-involved populations remains unknown. Moreover, it is unknown whether the availability of tailored services varies between Medicaid expansion states.n = 1679) and whether they offered services specifically tailored for CJ-involved patients. We used logistic regression to model the association between OTPs offering tailored services and state Medicaid expansion status, adjusted for state-level opioid overdose and community supervision rates.We used the 2019 National Survey of Substance Abuse Treatment Services to identify OTPs in the US (p < 0.0001). The predicted probability of offering tailored services varied by state; probability estimates for all expansion states were above the national mean, and estimates for all non-expansion states were below the national mean.Nationally, only a quarter of OTPs offered services tailored to CJ populations, and the majority of OTPs (73%) were located in Medicaid expansion states. Compared to OTPs in non-expansion states, OTPs in expansion states demonstrated nearly double the odds of offering tailored services (adjusted odds ratio = 1.90, 95% confidence interval = 1.41–2.57, Our findings reiterate the role of Medicaid in promoting the adoption of comprehensive OTP services for CJ-involved populations. However, the proportion of OTPs that offered tailored services was relatively low, pointing to the need to continually strengthen Medicaid services and coverage. Opioid use disorders (OUD) and preventable overdose-related deaths continue to drive an ongoing national epidemic. Rates of opioid misuse have risen consistently through 2019, with the majority of overdose-related deaths attributed to fentanyl, including fentanyl mixed with heroin, other opioids, and stimulants , but treThere are disproportionate burdens of OUD, treatment barriers, and poor treatment outcomes among individuals in the community who are involved in the CJ system. The majority of CJ-involved individuals are not incarcerated; more than four million Americans are on probation, parole, or are otherwise experiencing some form of correctional supervision by a court . Rates oGiven the persistent barriers to initiating and maintaining MAT, there is a need for OTPs to better tailor services to meet the needs of CJ-involved clients. OTPs offer a reliable resource for those who would otherwise not attend private office-based treatment, but additional resources and funding for OTPs could improve maintenance on MAT and other health outcomes. Several strategies could be incorporated into OTPs to tailor services for CJ clients, including connection to MAT immediately upon release  and expaState Medicaid expansions under the Affordable Care Act have broadly increased access to substance use treatment services among poor and marginalized populations, including CJ-involved populations who have historically been un- and under-insured , 22, 23.While Medicaid expansions have generally benefited the most vulnerable populations, to our knowledge no research has looked at differences in OTP CJ-tailored services between Medicaid expansion and non-expansion states. We hypothesized that OTPs in expansion states would be more likely than OTPs in non-expansion states to offer tailored services to CJ populations. In addition, we investigated differences between individual states within each of the two expansion groups.n = 1679). In short, N-SSATS is planned, directed, and maintained jointly by the Center for Behavioral Health Statistics and Quality, SAMHSA, and the U.S. Department of Health and Human Services. The dataset provides annual facility-level administrative data on the state, organization, structure, and a range of clinical and ancillary services provided by both public and private substance use treatment facilities, including OTPs. No client-level information is collected or included in the dataset. In 2019, the survey response rate of facilities that provided treatment for substance use disorders was 91%. Representatives from each facility responded to a 37-item questionnaire, via secure web-based survey, a paper questionnaire by mail, or a telephone interview. Further details about the survey design, data collection, and the dataset are available through SAMHSA  = 8.99), with the lowest and highest overdose rates identified in Nebraska  and West Virginia , respectively. The mean community supervision rate was 1767.4 per 100,000 (SD = 986.0), with the lowest and highest community supervision rates identified in New Hampshire  and Georgia , respectively.A minority of OTPs offered programs or groups specifically tailored towards CJ-involved clients . We identified no statistically significant association between our confounders (state-level overdose and community supervision rates) and our outcome. The national predicted probability of OTPs offering tailored services to CJ-involved clients was 0.26; notably, point estimates for every expansion state were above the national estimate and point estimates for every non-expansion state were below the national estimate (Fig. Holding state opioid overdose and community supervision rates constant, the odds of offering CJ-specific programs were significantly higher within OTPs in expansion states, compared to OTPs in non-expansion states (adjusted odds ratio = 1.90, 95% confidence interval = 1.41–2.57, Our study demonstrates that Medicaid expansion is clearly and positively associated with OTPs offering services and programs that are specifically tailored to CJ-involved MAT clients. The probability of offering tailored services to CJ populations was higher than the national average in all OTPs in expansion states, and lower than the national average in all OTPs in non-expansion states. This finding is in line with previous work reporting that CJ populations have benefited widely from expanded Medicaid services –41. GeneMedicaid expansion is an important factor in identifying state characteristics associated with OTP services. However, Khatri and colleagues  recentlyOTPs in expansion states may have access to more funding and resources that facilitate the implementation of additional tailored services. Our results also complement previous findings that OTPs in Medicaid expansion states are more likely than OTPs in non-expansion states to offer integrated and comprehensive mental health services . Our anaSome researchers have recently conceptualized what CJ-tailored services may look like on a broader scale. For example, Brinkley-Rubinstein and colleagues  recentlyWhile this is the first study to identify national trends in tailored CJ services within OTPs, our definition of programs and services specifically tailored to CJ-involved MAT clients is limited. N-SSATS does not provide detailed information about specific characteristics of tailored programs, nor does it provide demographic characteristics of individual treatment clients. A fuller and more in-depth understanding of tailoring services to vulnerable groups is needed in order to better inform health policy and to optimize clinical MAT practices. Future research could fill this gap by identifying specific characteristics of programs tailored for CJ-involved clients that are the most effective in engaging these clients and improving their outcomes.Our study only utilized 1 year of N-SSATS data. As a cross-sectional analysis of 2019 data, we are limited to demonstrating associations and are unable to identify a causal pathway between Medicaid expansion and changes in OTP services. Future research may address temporal changes in OTP services and differences by Medicaid expansion status, especially as several states have yet to opt into Medicaid expansion and the opioid and mass incarceration crises are not abating.Individuals involved in the CJ system make up a sizable portion of the overall MAT clientele in OTPs. OTPs in Medicaid expansion states are significantly more likely than OTPs in non-expansion states to offer programs specifically tailored to the needs of CJ-involved clients, reiterating the role of Medicaid in promoting the adoption of comprehensive services that provide more support to CJ-involved populations. However, tailored services remained low across all 50 states, with no state having more than 40% of its OTPs offering such programs. While promoting the adoption of Medicaid expansion is critical, this research also point to the need to continually strengthen MAT services in all states, including expansion states, with a focus on developing new services and programs that specifically target populations with the highest need.


Chronic graft-versus-host disease (cGvHD) is an immune mediated late complication of allogeneic hematopoietic stem cell transplantation . Discovery of adequate biomarkers could identify high-risk patients and provide an effective pre-emptive intervention or early modification of therapeutic strategy, thus reducing prevalence and severity of the disease among long-term survivors of alloHSCT. Inflammation, endothelial injury, and endothelial dysfunction are involved in cGvHD development. Altered levels of acute phase reactants have shown a strong correlation with the activity of several immune mediated disorders and are routinely used in clinical practice. Since elevated von Willebrand factor (VWF) and factor VIII (FVIII) levels have been described as acute phase reactants that may indicate endothelial dysfunction and inflammation in different settings, including chronic autoimmune diseases, they could serve as potential candidate biomarkers of cGvHD. In this review we focused on reported data regarding VWF and FVIII as well as other markers of inflammation and endothelial dysfunction, evaluating their potential role in cGvHD. Chronic graft-versus-host disease (cGvHD) is an iatrogenic, immune mediated complication of allogeneic hematopoietic stem cell transplantation  with clinical features resembling aspects of several autoimmune disorders. With its high incidence of approximately 50%  and mortVon Willebrand factor and FVIII are widely known as coagulation factors that play an important role in hemostasis. Beside the critical role of VWF in “platelet plug” formation (bridging molecule for platelet adhesion and promoting platelet aggregation) at sites of vascular injury, it also serves as a carrier of FVIII, protecting it from proteolytic degradation and transporting it to sites of injury, thus enhancing fibrin formation . A detaiAlthough synthesis regulation of VWF and FVIII is yet to be completely elucidated, it seems that different endothelial cells produce each of them, with an additional production of VWF by megakaryocytes. While most of VWF is synthesized in endothelium of larger extrahepatic vessels, FVIII is synthesized mostly in hepatic sinusoidal endothelial cells. Interestingly, murine models have shown predominant distribution of VWF mRNA in lung and brain, indicating that VWF is differently expressed in different tissues . It shouWhile VWF is well-recognized as the carrier of FVIII in plasma and protector of its degradation, the possible impact of FVIII on VWF plasma levels is poorly resolved. Some studies described a synergistic role of FVIII and platelets on the cleavage of VWF multimers by a disintegrin and metalloproteinase with ADAMTS13  , while sThe clinical utility of VWF and FVIII as biomarkers has been questioned due to a possible influence by many extrinsic patient related parameters, such as age, body weight, ABO antigen status, diet, smoking, ethnicity, and even exercise , 19–21. Despite the fact that increase of VWF and FVIII can also be caused by hematologic malignancies , 101, chThe term acute-phase reactants (APRs) relates to all markers whose measured plasma concentrations increase or decrease by at least 25% during inflammation or tissue injury . Most ofEndothelial injury caused by inflammation, on the other hand, has not been extensively studied in cGvHD so far. However, an increase of vascular inflammation markers, such as endothelin-1, gelsolin, and anti-LG3 (autoantibodies against perlecan/LG3), was observed, confirming thus important role of endothelial injury and inflammation in the onset of cGvHD . Of notein vitro evidence that some of the endothelium targeting immunological effector cells are in fact endothelial-, not just allo-specific  and by the Croatian Science Foundation Project IP-2016-06-8046 entitled “New biomarkers for chronic Graft-versus-Host disease”.The opinions expressed here are those of the authors and do not represent the official position of the National Institutes of Health or the US Government.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plants employ a diversified array of defense activities when they encounter stress. Continuous activation of defense pathways that were induced by mutation or altered expression of disease resistance genes and mRNA surveillance mechanisms develop abnormal phenotypes. These plants show continuous defense genes’ expression, reduced growth, and also manifest tissue damage by apoptosis. These macroscopic abrasions appear even in the absence of the pathogen and can be attributed to a condition known as autoimmunity. The question is whether it is possible to develop an autoimmune mutant that does not fetch yield and growth penalty and provides enhanced protection against various biotic and abiotic stresses via secondary metabolic pathways’ engineering. This review is a discussion about the common stress-fighting mechanisms, how the concept of cross-tolerance instigates propitious or protective autoimmunity, and how it can be achieved by engineering secondary metabolic pathways. Plants face many constraints in the continuously changing environment and have evolutionarily gained the defensive mechanisms that are inevitable for their survival. Numerous efforts have been taken for a better understanding of plant adaptive responses to improve agricultural productivity and quality. When plants encounter particular stress, numerous genes are induced, which results in elevated levels of diverse secondary metabolites and proteins that would help plants to cope with the stress . These mS-transferase and non-enzymatic antioxidant systems, such as ascorbic acid, glutathione, tocopherol, carotenoids, and flavonoids . In. In62]. Rhizoctonia solani present in the soil and delayed appearance of disease symptoms. Studies are being conducted to evaluate the resistance offered by altered or overexpressed genes involved in cellular signaling, whose continuous and elevated expression leads to defense activation. One of the examples is calcium-dependent protein kinase (CDPK). CDPKs are cellular secondary messengers and principal mediators of downstream responses in plant immune and stress signaling [CDPK1 gene, overexpressed in tobacco, was found to be rendering salinity and drought stress tolerance [A. thaliana forged the plant cytotoxic to the fungal pathogen Botrytis cinerea, along with rendering improved growth and development [PDF1.2 gene involved in the jasmonate signaling pathway was found to be induced after infection with the pathogen. On the other hand, it becomes responsible for the activation of salicylic acid-associated genes such as PR1 even in the absence of the pathogen. The SA-dependent activation of PR genes involves NPR proteins that both positively and negatively regulate their expression. In Arabidopsis, the NPR1 is a positive regulator of SAR, which enhances SA-induced PR gene expression. Elucidating the function of NPR3 and NPR4 by mutation studies showed constitutive expression of PR1, which induced autoimmune responses [PUB13-disrupted plants resulted in spontaneous cell death, the accumulation of hydrogen peroxide and salicylic acid (SA), and elevated resistance to biotrophic pathogens but increased susceptibility to necrotrophic pathogens. It is evident that there is a possibility to develop autoimmune genotypes, which show constitutive defense response without growth and yield penalty.Apart from mutations and aberrations in NB-LRR, autoimmunity can also be developed by disruption or constitutive expression of genes involved in various cellular processes. Studies on induced constitutive gene expression were initiated by . Initialignaling . The ginolerance . Apart felopment . The PDFesponses . The sinApart from the continuous activation and impaired regulation of NLR proteins, we propose that certain mutations in the secondary metabolic pathway genes can induce autoimmunity and can also help plants to be resilient to multiple types of stress. The list of autoimmune phenotypes with a continuous expression of resistance-related genes is given in Flavonoids are proven to be among the protecting agents in plants during high light and UV stresses . The flanod gene influencers such as flavones and isoflavones [Even though the functions of many flavonoids are yet to be revealed, there is increasing evidence that they are inevitable factors for eliciting defense response in many plant species. Phenolic acids such as hydroxyl benzoic acid and hydroxyl cinnamic acid produced from the intermediate steps of the phenylpropanoid pathway are involved in attracting the beneficial microorganisms and establishing a symbiotic relationship with the plant roots . It is pflavones ,81. It iflavones . The antType III PKS superfamily of enzymes is the most important among the enzymes involved in secondary metabolic pathways. The active site of these homodimeric proteins iteratively participates in catalyzing the priming, extension, and cyclization reactions to produce a wide range of plant phenylpropanoids including flavonoids, chalcones, resveratrols, benzophenones, quinolones, phenylphenalenones, and curcuminoids . Type IIPetunia mutant RL01, which served as a recipient of the A1 gene of Zea mays, encoding dihydroquercetin 4-reductase, reducing dihydrokaempferol to provide intermediate for pelargonidin biosynthesis that develops orange flowers [Aegle marmelos [Emblica officinalis [Aegle marmelos induced improved growth, chlorophyll content, and photosynthetic efficiency. The CHS overexpressed transgenic plants showed an increased accumulation of secondary metabolites, such as flavonoids, phenols, and alkaloids that are accredited to their higher tolerance to drought and salinity stress. A similar result was obtained with E. officinalis CHS mutants. Mutants of E. officinalis CHS developed by exchange of single amino acids (F215S and F265V) were found to be utilizing p-coumaroyl–CoA as well as N-methylanthraniloyl–CoA as substrates and yield active products such as naringenin, 4-hydroxy 1-methyl 2(H) quinolone, and 1,3-dihydroxy-n-methyl acridone. Along with this finding, the mutants showed improved growth and disease resistance.Engineering of secondary metabolite pathway was initiated in  flowers . Later,  flowers . Studiesmarmelos  and Emblicinalis  to unveiApart from the genes involved in phenylpropanoid biosynthesis, several other transcription factors (TF) regulate the expression of key enzymes. Those TFs can be the targets for the enhanced production of flavonoids. In Arabidopsis, MYB12 TF was found to be upregulating the chalcone synthase (CHS) and flavone synthase (FS) genes, thereby accumulating flavonoids .Ustilago maydis [1300 were used for introducing a biopeptide BP100, a synthetic and cationic α-helical undecapeptide that exhibited a high degree of antibacterial activity against economically important plant-pathogenic bacteria with very low toxicity levels. Although the mutants exhibited antimicrobial activity, the fitness of the plants was compromised [Most of the plant metabolic pathway engineering to date have used the constitutive promoter to enhance and activate the expression of native genes or transgenes in plants regardless of the tissue or the organ. One such promoter widely used is CaMV 35S promoter from the cauliflower mosaic virus, owing to its stability and activity in several plant species . One suco maydis . Apart fo maydis . Moreovepromised .AtMYB12 in transgenic Arabidopsis enhanced the production of flavonoids and showed a significant increase in ABA, proline, peroxidase (POD), and superoxide dismutase (SOD) activities, thereby improving the salt and drought tolerance of the plants [2O2 and malonaldehyde (MDA) was also observed in the above study, which also indicated that tissue damage and necrosis were prevented by reducing the ROS content since ROS  is considered to play the main role in hypersensitive responses [PR1 and PR2.Overexpression of e plants . Signifiesponses . In addiesponses . This drComplex mechanisms that evolved in plants help them to defend against both single and combination of stresses. It is indeed important to understand the complex defense mechanisms and signaling pathways for minimizing the stress effects on growth and reproduction, thereby improving their life span. It is evident that the studies on plant immunity against various combinations of stresses have revealed the complex nature of the signaling cross-talk. The information is still limited, even though the research continuously evolves due to the astounding progress in high throughput sequencing and well-developed bioinformatics technologies for genomic studies, proteomics, metabolomics, and whole-transcriptome profiling. Recently, the studies demonstrated that ROS synthesis, hormone signaling, MAPK pathways, and various transcription factors (TFs) can act as positive or negative regulators of the plant immune responses upon reception of biotic and abiotic stress stimuli, and are having non-specific, crucial roles in eliciting an immune response. The interaction between hormone-responsive and secondary metabolite biosynthetic genes is yet to be elucidated, which will allow for developing novel strategies to withstand the combination of both biotic and abiotic stresses encountered in field conditions.Therefore, it is very important to understand and accelerate the development of stress-resistant crops via precise genome editing techniques, such as transcription activator-like effector nucleases (TALENs) and clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 systems, and to promote the flourishment of plant biotechnology for the sustenance and welfare of living beings.


Excessive alcohol intake is a direct cause of alcoholic liver disease (ALD). ALD usually manifests as fatty liver in the initial stage and then develops into alcoholic hepatitis (ASH), fibrosis and cirrhosis. Severe alcoholism induces extensive hepatocyte death, liver failure, and even hepatocellular carcinoma (HCC). Currently, there are few effective clinical means to treat ALD, except for abstinence. Natural compounds are a class of compounds extracted from herbs with an explicit chemical structure. Several natural compounds, such as silymarin, quercetin, hesperidin, and berberine, have been shown to have curative effects on ALD without side effects. In this review, we pay particular attention to natural compounds and developing clinical drugs based on natural compounds for ALD, with the aim of providing a potential treatment for ALD. Alcohol liver disease (ALD), a liver disease caused by long-term heavy alcohol intake, is the leading cause of chronic liver disease (CLD), especially in Europe and America . With ecFew specific medicines for ALD treatment exist. The development of effective and safer ALD treatments is an unmet clinical need . In ChinALD is always accompanied by hepatic steatosis induced by the imbalance of lipid metabolism originating from excess alcohol . CD36/FA+) in hepatocytes, which in turn inhibits the tricarboxylic acid (TCA) cycle, interferes with β-oxidation of FAs, and results in excessive accumulation of FAs /oxidised nicotinamide adenine dinucleotide , an enzyme found in both the endoplasmic reticulum (ER) and mitochondria of hepatocytes, could metabolise alcohol to acetaldehyde in the presence of oxygen . AcetaldThe liver, a vital organ for alcohol metabolism, can be damaged by by-products of alcohol decomposition, such as reactive oxygen species (ROS) . CYP2E1 Acetaldehyde is linked to the pathway by which alcohol causes oxidative stress in the liver. Acetaldehyde is highly reactive and toxic, and causes structural and functional damages to organelles such as mitochondria . The funAlcohol-induced sensitisation of Kupffer cells (KCs) to lipopolysaccharide (LPS) is considered a hallmark of inflammation and ALD. The level of circulating LPS in ALD patients is significantly elevated and increases with disease severity . The secThe polarisation of KCs is also essential for the progression of inflammation . PolarisAutophagy, a cellular self-protection program, is responsible for maintaining cell homeostasis by routing cytoplasmic macromolecules or damaged organelles to lysosomes for degradation . Selectiin vitro and in vivo, Tatsukawa et al. showed that TG2 is a vital cross-linking enzyme in alcohol-induced hepatocyte apoptosis , and apoptosis , and the expression of regenerating family member 3 gamma (Reg3γ) was inhibited. Prebiotic fructooligosaccharide (FOS) treatment effectively upregulated the expression of Reg3γ, reduced intestinal bacterial overgrowth, and improved hepatic inflammation in ALD mice  translocation in ALD patients and ALD experimental models . Under nThe possible mechanism linking intestinal microbes and intestine-liver axis to ALD is depicted in Glucocorticoids are always used to treat severe alcoholic hepatitis (SAH) and acute alcoholic hepatitis (AAH). The potential therapeutic mechanism of glucocorticoids is to suppress inflammation by reducing immune and pro-inflammatory cytokine responses . HoweverPentoxifylline (PTX) is an oral phosphodiesterase inhibitor that can suppress inflammation by reducing serum TNF-α levels . PTX canMetadoxine, a drug used to treat acute alcoholism, could accelerate ethanol excretion and lower the level of ethanol in the blood . HoweverPTX alone has poor efficacy and is always combined with glucocorticoids in treating acute alcoholic hepatitis or severe alcoholic hepatitis, which is the critical stage of ALD. However, glucocorticoids have significant side effects and are not suitable for long-term treatment. Metadoxine is commonly used clinically for acute alcoholism because it promotes alcohol excretion. However, all of them indirectly improved liver damage caused by long-term drinking by improving alcohol metabolism. Metadoxine is not a specific treatment for ALD per se.Increasing evidence indicates that natural compounds can effectively inhibit ALD progression. Natural compounds have been demonstrated to be effective towards ALD by engaging in a variety of pathways, including improvement of lipid metabolism, suppression of oxidative stress and inflammation, and reduction of programmed cell death (PCD). Natural compounds affect cellular functions broadly with few side effects, and can be administered at different stages of ALD. Curcumin, the main active ingredient in turmeric, has been shown to reduce lipid accumulation in hepatocytes and subsequent steatosis by regulating the NF-E2–related factor 2 (Nrf2)-farnesoid X receptor (FXR) pathway . Some scDihydroquercetin, one of the most common dihydroflavonoids, is widely found in milk thistles and onions. Zhang et al. proposed that dihydroquercetin improves alcoholic liver steatosis by regulating the activation of the SIRT1-AMPK pathway . DihydroTanshinone IIA is superabundant in the root of Salvia miltiorrhiza BUNGE. Yin et al. found that tanshinone IIA can inhibit the synthesis of FAs and stimulate the oxidation of FAs, improving ALD . TanshinSilymarin, a flavonoid extracted from Silybum marianum, has been introduced as a hepatoprotective agent. Silymarin mainly repairs hepatic injury by alleviating lipid peroxidation and oxidative stress . Zhang, Isoorientin was confirmed to be abundant in buckwheat. In an animal experiment, Chen et al. discovered that isoorientin effectively increases the activities of the antioxidant SOD and GSH-Px and the expression of alpha-SMA, thereby suppressing alcohol-induced oxidative stress and protecting from liver fibrosis in Wistar rats . In addiOleanolic acid exists abundantly in fruits, vegetables, and herbs. Oleanolic acid has been proven to have promising pharmacological activities, such as hepatoprotective effects and antioxidant activities . The antAnthocyanins are a group of plant pigments widely present in flowers, fruits, and cereals . The heaBaicalin, an active ingredient extracted from Scutellaria baicalensis, is widely used in herbal preparations to treat ALD and inflammation. According to recent studies, baicalin plays a beneficial role in various invasive toxicant models by inhibiting inflammatory responses . AdditioPuerarin and sophoronol both increase the activity of ADH and ALDH, thereby promoting alcohol metabolism and reducing hepatotoxic damage caused by alcohol . In addiCatechin, a polyphenol antioxidant, inhibits activation of NF-κB in rats upon excessive alcohol uptake and prevents necrotizing inflammatory changes .in vivo and in vitro  activation and apoptosis from ethanol-induced toxicity in vitro .in vivo and in vitro  The clinical efficacy of most natural compounds is limited due to their low bioavailability. 2) It is difficult to define the most appropriate drug form and dosage without standardised pharmaceutical technology. Thus, the optimal liver protection effect cannot be realised. 3) Most compounds have not been subjected to double-blind, placebo-controlled clinical trials to evaluate their efficacy and mechanisms of action of natural compounds are still unclear because of scarce studies.Undoubtedly, natural compounds have enormous potentials to be developed as therapeutic drugs for ALD. However, in the field of ALD research, clinical trials are still needed for systematic reviews. Further research is needed on the potential role of natural compounds . We hope


Lymph node (LN) metastasis in gynecological malignancies represents the most important negative prognostic predictor . ConsideEscuin et al. showed the different expression profile of several circulating miRNAs in relation to the SLN status, suggesting the potential role of peripheral blood circulating nucleic acids as surrogate markers of LN metastases in early breast cancer patients.In breast cancer the presence of metastatic SLNs still necessitates the recommended procedure for axillary staging of early neoplasms . CurrentZhou et al., Siegenthaler et al.). Since the SLN utility and applications in vulvar cancer are still debated, Zhou et al. compared the safety of SLN biopsy (SLNB) with regional LN dissection (RLND) in patients with vulvar squamous cell cancer . Their findings indicate that SLNB is related to prolonged survival outcomes in patients with no metastatic or advanced-stage disease compared to RLND and no LN removed, irrespective of tumor size, surgery type, or invasion depth . A recent study has also established the clinical utility of ultrasound-guided fine-needle aspiration cytology (FNAC), stating that a positive result is sufficient to avoid an unnecessary SLN sampling, enabling the surgeon to perform a bilateral inguinofemoral lymphadenectomy  nanocolloid combined with an intraoperative injection of a blue dye. A novel, potentially interesting, SLN mapping technique has been proposed by Favre et al. in their randomized study, comparing early-stage patients undergoing SLN biopsy alone versus pelvic LN dissection, demonstrated no significant differences between the two groups in terms of overall survival and disease recurrence.Regarding SLN mapping in cervical cancer, the New ESGO/ESTRO/ESP guidelines incorporated SLN biopsy as an acceptable method of LN staging in early-stage cervical cancers, particularly in cases of small volume tumors . In the The gold-standard technique to process SLN in cervical cancer is the ultrastaging protocol , 7.This technique, requiring LNs serial sectioning and immunohistochemistry, is utilized in pathology laboratories to confirm the negative status of a LN and also detect small-volume metastases, ranging between 0.2 and 2 mm in size. Recently, OSNA protocol, based on a quantitative measurement of target mRNA in a metastatic LN, has been proposed as an efficient alternative method for the intra-operative assessment of SLN in cervical cancer patients , 9. To dZhai et al., in endometrial cancer SLN mapping has emerged as a reliable alternative to pelvic LN dissection. Several studies have demonstrated a high sensitivity and negative predictive value to detect nodal metastases leading to similar oncological outcomes between patients undergoing SLN and pelvic lymphadenectomy . Therefore, in 2020 National Comprehensive Cancer Network (NCCN) guidelines, SLN mapping has been recommended for staging purposes in EC patients . On the other hand, SLN biopsy demonstrated high sensitivity and negative predictive values in intermediate-risk EC patients .As illustrated in the review article by patients . In thisTherefore, according to available literature data, full lymphadenectomy could be avoided by performing SLNB in patients of low and intermediate risk while additional data on larger cohorts need to be collected in order to demonstrate the staging and prognostic benefits of SLN biopsy in high-risk EC.LN status is also a relevant prognostic factor in early ovarian carcinoma; however, para-aortic and pelvic lymphadenectomies carry a significant risk of intra- and post-operative morbidity , 12. SLNIn conclusion, SLN biopsy is an intraoperative procedure with potential for adequate staging with less treatment-related morbidity. It should be performed by a skilled multidisciplinary team, in oncology centers, preferably within the protection of clinical trials. Different methods of histopathological and molecular SLN assessment according to the different gynecological cancers have been proposed. This Research Topic, collecting several papers on this topic, discusses the need for standardization of pathological protocols, the molecular aspects of SLN evaluation in gynecological cancer, and the clinical benefits of this treatment option in routine practice.All authors contributed to the Research Topic editorial and performed the literature search. AS and GA drafted the manuscript. GZ critically revised the work. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


ABSTRACT IMPACT: If successful, this program can provide a scalable, patient-centered intervention to help patients taper off opioid medications in primary care settings. OBJECTIVES/GOALS: Tapering of chronic opioid therapy is often desirable but challenging in primary care and specialty clinics that lack behavioral health expertise. The objective of this pilot study is to determine the feasibility of testing a peer-delivered pain self-management program to assist primary care patients through an opioid taper. METHODS/STUDY POPULATION: To provide critical support to patients and providers during opioid medication tapering, we propose to conduct a 40 patient randomized controlled pilot of a 12-week telecare collaborative care program administered by a psychiatrist and peer recovery specialist team. The intervention will incorporate a validated positive psychology intervention for treating chronic pain. Additionally, participants will be invited to participate in semi-structured individual interviews to discuss their experience in the trial, what worked well, what could be improved, and potential strategies to bolster recruitment of additional patients in future studies. RESULTS/ANTICIPATED RESULTS: Our primary aim is to determine the effectiveness of our intervention in facilitating opioid medication weaning, with reduction in opioid dose as the primary outcome. Our secondary aims will be to assess pain outcomes, adherence to tapering, patient satisfaction, and barriers to adherence as described by patients. DISCUSSION/SIGNIFICANCE OF FINDINGS: This trial proposes a novel collaborative care approach for opioid weaning using proven, easy-to-deliver positive psychology tools for pain management that, if successful, could be implemented broadly in many clinics struggling to safely reduce opioid prescribing.