Gene expression specificity is a core aspect of transcriptional regulation—genes are expressed in specific temporal and spatial patterns to promote distinct developmental and environmental responses. Transcriptomic analyses have elucidated many of these gene expression profiles but have mostly focused on genes that encode for proteins. Far less studied is the role of other transcriptional products, such as long intergenic noncoding RNAs (lincRNAs), in gene expression. In animal systems, lincRNAs have been shown to affect chromatin structure, mRNA splicing and stability, and translation . In planPlant Physiology, In this issue of The authors went on to classify the lincRNAs by their expression profiles, using tissue- and condition-specific transcriptomic data. The lincRNAs grouped in distinct clusters with specific expression profiles and in fact were more specific in their expression patterns than protein-coding genes. The root-expressed cluster showed the most specificity, suggesting that lincRNAs may play an important role in transcriptional regulation of root development . The autUsing a variety of genomic datasets, Liu et al. identified thousands of Arabidopsis lincRNAs and determined their evolutionary conservation and selection signatures, tissue-specific expression profiles, chromatin status, and potential regulation by TFs. By focusing on specificity in lincRNA transcriptional profiles, the authors were able to contextualize their annotations and generate hypotheses as to the function of these lincRNAs. This dataset can serve as a resource for researchers interested in targeting lincRNA loci for CRISPR-based disruption, allowing rapid and potentially high-throughput functional analysis of lincRNAs’ role in development. Recent advancements in tissue-specific and inducible CRISPR systems can expand the complex genetics that can be revealed through such precision editing approaches . The autThe authors used an innovative approach to determine relationships between TF-binding and lincRNA expression and found that many evolutionarily constrained lincRNAs were regulated by known gene regulatory networks. Interestingly, lincRNA promoters did not contain the conserved sequences that are often hallmarks of TF binding, suggesting that, unlike for protein-coding genes, regulation of these loci may not rely on sequence conservation. Further research could analyze the mechanism by which TFs regulate lincRNA expression, which may rely on chromatin accessibility or positional information . These g


In situ amplification and spread within the tumor mass are the key benefits of such replication-competent viruses. Oncolytic virotherapy is divided into two main groups, according to tumor specificity: naturally oncolytic viruses to replicate in human cancer cells and gene-modified viruses engineered to accomplish selective oncolysis  cancer models to investigate the efficacy of oncolytic viruses; 2) OVs as cancer immunotherapeutic agents; 3) novel viral platforms; 4) combination therapies; and 5) methods to develop OV quantification.Carter et al.) used stable organoid cell cultures derived from breast cancer tissue to develop a protocol to study the effects of oncolytic viruses. They used an established three-dimensional organoid model derived from tissue from 10 patients with primary breast cancer.Carter and colleagues  and a vaccinia virus (GLV) genetically engineered to express different transgenes. The most significant oncolytic effects were observed with oncolytic viruses expressing a suicide gene combined with the prodrug 5-FC. Therefore, the Salman et al.) established 3D coculture spheres in vitro consisting of two types of cells: the first type is derived from the breast cancer cell line (MCF-7 or AMJ13), and the second type is normal adipose tissue-derived mesenchymal stem cells. The floater culture plate was used to culture the cells so they could form spheroids, which were transferred to a special scaffold dish. The newly formed 3D culture spheroids were used to assess the oncolytic activity of the Newcastle disease virus AMHA1 strain by labeling the Newcastle disease virus (NDV) with a fluorescent PKH67 linker to track virus infection. They found that introducing chemical fluorescent dye into NDV particles is an effective strategy to identify virus particles in the infected co-culture spheroid model. Their results revealed that the oncolytic strain of NDV AMHA1 replicates effectively in cancer cells but not in normal cells in the 3D coculture system. This combined model of 3D coculture plus fluorescent tracking indicates that the NDV AMHA1 strain is selective and effective in antitumor virotherapy.Salman, Al-Shammari and colleagues  evaluated the systemic delivery of HSV1716 in multiple mouse models of breast cancer. They found a direct relationship between virus tolerability and mouse strains. They tested the C57/B16, FVB, and Balb/c strains and found different responses. Intravenous administration of OV induces a lethal side effect in Balb/c mice, while C57/B16 is the most tolerant. These differences in response to OV in mouse strains may produce confusing results, which are mainly due to the interaction between OV and the immune system of these different strains. Eventually, this leads to a decrease in predictive value and low clinical efficacy. Howard and associates treated Balb/c mice with immunomodulators before injecting OV to decrease side effects. However, they could not estimate whether the immunomodulators increased virus tolerability. However, this work presented data to support therapeutic modulation of immune subsets with the aim of promoting a pro-inflammatory reaction, particularly by increasing CD8 + T cell levels that combat tumor growth. Finally, they are stating that the inconsistencies found in mouse models will help to have a larger picture, making it applicable, possibly to heterogenic human populations, which will lead to the development of translational oncolytic virotherapy.Howard and his associates  reviewed possible immunotherapies that can be combined with oncolytic viruses for the treatment of Melanoma according to its molecular characteristics. They explored molecular changes in Melanoma that can be targeted taking into consideration that melanoma has a high mutation rate, leading to the appearance of tumor-specific antigens (TSA) and infiltration of lymphocytes, facilitating the utilization of therapeutic technologies that elicit novel or reinstate preexisting responses from the immune system. Immune checkpoint inhibitors are one of these innovative therapeutic technologies for melanoma treatment that could be combined with oncolytic virotherapy for synergistic action. They propose that a virus’s oncolytic action is due to virus replication and activation of innate and adaptive antiviral immune responses. This may be just as important, if not more so, than viral replication.Cerqueira and colleagues  VC2-OVA that expresses a fragment of ovalbumin (OVA) as a fusion protein with the virion capsid protein VP26. They evaluated the efficacy of VC2-OVA to work as a vector capable of stimulating specific antitumor immunity in a syngeneic murine melanoma model. The administration of VC2-OVA through therapeutic vaccination resulted in a notable decrease in the presence of tumor cells in the lungs of mice that were intravenously exposed to B16cOVA cells. Furthermore, the administration of VC2-OVA resulted in strong preventive antitumor activity and prolonged the survival of mice that were intradermally implanted with B16cOVA tumors compared to mice inoculated with a control virus. Their findings demonstrate the efficacy of VC2 as an oncolytic virotherapy, showing promise for its potential application as a combined oncolytic virotherapy and personalized vaccine in the treatment of human and animal malignancies.A research work by Kaufman et al., they described the progress made in advancing Talimogene laherparepvec (T-VEC) to the most proper melanoma patients, expansion to patients with non-melanoma cancers, and clinical trial results of T-VEC combination studies. T-VEC is a modified oncolytic herpes Simplex virus type 1 (HSV-1) that encodes granulocyte-macrophage colony stimulating factor (GM-CSF). T-VEC is adapted for selective replication in melanoma cells, and GM-CSF was expressed to augment host antitumor immunity. T-VEC is indicated for the local treatment of recurrent melanoma after primary surgery and is the first-in-class oncolytic virus to achieve FDA approval in 2015. Its tumor cell selective replication was improved by careful deletion of the two viral infected cell protein (ICP) 34.5 genes, which encodes the neurovirulence factor. Furthermore, deletion of ICP47 is believed to promote antitumor immunity as it facilitates MHCI loading of tumor-associated antigens. It showed very promising clinical outcomes. Additionally, it appeared that T-VEC could be used in combination with or sequentially to checkpoint blockade, without influence on therapeutic responses. T-VEC may be an important consideration for older patients with melanoma who may not be able to tolerate other systemic options.In the review by Kaufman et al., there is evidence indicating the existence of specific patient subsets within the melanoma population who may exhibit a higher likelihood of seeing therapeutic benefits from T-VEC treatment. Mostly, patients with head and neck melanoma appeared to have higher response rates. Despite the higher mutation load due to Sun exposure being postulated, no demonstration of this was offered. Other potentially interesting settings are allotransplanted patients who cannot receive potent immunotherapy, such as immune checkpoint blockade, due to the risk of rejection of allograft, as well as patients with early-stage I-II melanoma patients, such as neoadjuvant treatment. As far as other cancers are concerned, T-VEC showed ex vivo preclinical activity, and then clinical trials have been planned. In general, accessible tumors for intratumoral injection have been a priority, and this has included head and neck squamous cell carcinoma, soft tissue sarcoma, and breast cancer. Other studies have been conducted to evaluate T-VEC in pancreatic cancer, hepatocellular carcinoma, and non-melanoma skin cancers. The combination with CTLA4 blockers seems promising, but further investigation is needed. It should be noted that some data support the role of elements of the antiviral interferon signaling machinery in tumor cells as possible predictive biomarkers of oncolytic virus (OV) activity and merit further clinical investigation .According to the findings reported by Cristi et al.), described genetic modifications that were done to OVs to improve the killing ability of tumor cells directly, to dismantle the tumor microenvironment, or to alter tumor cell signaling and enhance antitumor immunity. Although many OVs have progressed to human clinical trials, their performance as monotherapy has not been as successful as expected. Importantly, recent literature suggests that the oncolytic potential of these viruses can be further increased by genetically modifying the viruses.A review article .These advances are particularly important to increase virus spread and reduce metastasis, as demonstrated in animal models. The extracellular matrix (ECM) in a tumor does not have the same characteristics as that in normal tissues. In the tumor, the ECM is more rigid, abundant and dense. Because of this, tumor ECM acts as a barrier for therapeutic agents such as OV. At the same time, the barrier impairs oxygen and nutrients supply, activating apoptosis and senescence. Thus, ECM is a candidate cancer therapeutic target. Since at the onset of metastasis, during the invasion process, remodeling of the ECM is mainly done by metalloproteases (MMPs), both adenovirus- and vaccinia-based OVs have been genetically modified to exploit the natural functions of MMPs and enhance virus dissemination. Other attempts have been made by adding relaxin, hyaluronidase, or exonucleases. Overall, it resulted in increased virus spread and reduced tumor growth, including metastases in some cases  showed that Ad-Apoptin-hTERTp-E1a (Ad-VT), a bispecific oncolytic adenovirus, can effectively induce cell death of breast cancer cells and has a better effect when used in combination with chemotherapy drugs (1–2). Ad-VT has no cytotoxicity in normal cells, with the advantage of specifically inducing tumor cell apoptosis, through the expression of the apoptin protein . On the contrary, MDR1 and BCRP levels remain unchanged. Furthermore, since Ad-VT can induce cell death through either apoptosis or autophagy, the authors also explored the second mechanism. They found that MRP1 expression was significantly affected by autophagy inhibition, while it was not modified by apoptosis inhibitors. Therefore, they concluded that Ad-VT restored sensitivity to anthracyclines through autophagy induction. Clearly, the effect was shown to be mediated by mTOR inhibition; When the authors analyzed the expression of the mTOR protein after adding an autophagy inhibitor, they found that inhibition of autophagy significantly increased mTOR activation and that treatment of MCF-7/ADR cells with 60 MOI Ad-VT treatment reversed the effects caused by autophagy inhibitors. This effect was likely mediated by the AMPK pathway and more specifically by the AMPK-mTOR-eIF4F signaling axis. Traditionally, the relationship between autophagy and drug resistance has been divided into two distinct mechanisms and their related effects: one is associated with its protective mechanism against tumor drug resistance, and the other is related to autophagy-induced cell death, which increases tumor sensitivity to apoptosis. In their study, Li et al. could effectively highlight both effects in MCF-7/ADR treated with Ad-VT. It would certainly be interesting to assess whether similar effects can be induced in other types of cancer cells.Recently, Li and colleagues ( protein . In theiLundstrom) on genetically engineered alphavirus vectors, which have been evaluated for prophylactic and therapeutic use for a broad range of cancer indications in various animal models and in several clinical. Although, based on numerous vaccine studies, it has not been possible to demonstrate superiority of any alphavirus system with respect to immune responses or therapeutic efficacy. In most cases, robust immune responses have been obtained, including humoral and cellular responses. Th1-biased immunogenicity confirmed the potential of alphavirus-based cancer vaccine. The possibility to include alphavirus-based delivery of cytotoxic genes, antitumor genes, immunostimulatory genes, apoptosis induced by alphaviruses, and RNA interference in the form of short interfering RNAs and microRNAs expands the possibilities of therapeutic interventions. Moreover, alphavirus vectors can be applied as recombinant viral particles, including replication-deficient, replication-proficient, and oncolytic viruses, as well as RNA replicons and DNA replicons. It has been demonstrated that the stability of RNA and its resistance against degradation can be improved by RNA encapsulation in lipid nanoparticles. Several studies have also confirmed that due to the presence of alphavirus replicons, both RNA replicons and DNA replicons can induce the same immune response at 100 to 1,000 times lower doses compared to synthetic mRNA and conventional DNA plasmids, respectively. Although alphaviruses have shown good safety and efficacy in various animal models, transfer to humans has often generated disappointingly weak immune responses in clinical trials. Several issues such as targeting, delivery, dose optimization, and potential combination therapy need to be addressed.Another review article  can be classified as either lytic or nonlytic, as it selectively infects cells that possess a compromised interferon system. This characteristic enhances the safety profile of NDV when utilized as a vaccine. Incorporation of foreign genes is not necessary for NDV to exhibit a potent anticancer impact and maintain persistent expression of foreign genes. The integration of NDV viral therapy with conventional and emerging tumor treatment modalities has been documented and holds significant potential for widespread implementation. However, many inquiries about NDV therapy, similar to other oncolytic viruses (OV), persist without definitive answers. These unsolved questions encompass the practical methodologies for administering NDV therapy, optimal genetic engineering approaches, the therapeutic sequence for immune checkpoint inhibitors, and the most effective combination partners for NDV therapy. At present, there is a lack of established consensus on the most effective and appropriate approach for patients to utilize the virus, both in terms of methodology and timing. The presence of a tumor microenvironmental barrier and the cytoplasmic matrix in solid tumors may limit and suppress virus entry and multiplication, hence reducing its oncolytic efficacy. The presence of an excessive number of foreign genes may have an impact on the replication process of Newcastle Disease Virus (NDV). Furthermore, the NDV purification process requires extensive measures to achieve a clinical-grade virus product. Integrating NDV therapy with conventional and alternative medicines has the potential to emerge as an innovative approach in the field of cancer treatment. The potential increase in the anticancer effect can be achieved by integrating NDV viral therapy with existing immunotherapy, using the immunomodulatory impact of NDV. Consequently, NDV will emerge as a promising candidate for tumor therapy in the near future.Corbett et al.) discussed one such promising oncolytic virus called the Seneca Valley Virus (SVV-001) and its therapeutic implications. SVV development has seen seismic evolution over the past decade and now boasts of being the only OV with a practically applicable biomarker for viral tropism. We discuss relevant preclinical and clinical data involving SVV and how bioselecting for TEM8/ANTXR1, a negative tumor prognosticator, can lead to first of its kind biomarker-driven oncolytic viral cancer therapy. The initial discovery of SVV-001 revealed its specificity for neuroendocrine tumors, highlighting its remarkable capacity to revolutionize the field of neuroendocrine neoplasm therapies. This novel treatment has shown the ability to induce a substantial tumor response, even in cases where immunotherapy was previously believed to be ineffective. However, initial investigations were limited due to the absence of a biomarker that could be used to identify patients who were susceptible to severe viral vasculitis (SVV). The discovery of TEM8/ANTXR1 as a receptor for SVV-001, a therapeutic agent that can be administered through intratumoral injections, in a patient population with a high prevalence of biomarkers, and in conjunction with dual checkpoint blockade to enhance treatment responses, established the foundation for future clinical trials utilizing SVV-001 with a more targeted and strategic approach. The treatment paradigm under consideration was originally designed to address neuroendocrine neoplasms. However, recent advances in understanding the plasticity of neuroendocrine transformation in various solid tumor types, as well as studies revealing widespread upregulation of TEM8/ANTXR1, indicate that SVV-001 may have the capability to target numerous other tumor types that exhibit high resistance to therapy and are associated with high mortality rates. Gaining a greater understanding of the specific immune tumor microenvironment associated with upregulation of TEM8/ANTXR1 in high-grade neuroendocrine carcinoma, well differentiated neuroendocrine tumors, and related tumor types is crucial for effectively using SVV-001 as a therapeutic approach for these conditions. Furthermore, this understanding is essential for the advancement of novel agents that can be used in conjunction with SVV-001.In the next review , a specific alpha-glucosidase inhibitor, to induce glucose deficit combined with oncolytic Newcastle disease virus (NDV) to enhance antitumor action. In this study, a murine model of breast cancer was used, in which mammary adenocarcinoma tumor cells (AN3) were subjected to treatment with ACA, NDV, and a combined administration of both compounds. The research includes an investigation of various parameters, including antitumor efficacy, relative body weight, glucose level, hexokinase level (HK-1) determined by enzyme-linked immunosorbent assay (ELISA), glycolysis product (pyruvate), total adenosine triphosphate (ATP), oxidative stress markers (reactive oxygen species and reduced glutathione), and apoptosis assessed by immunohistochemistry. The findings demonstrated an important level of antitumor efficacy after the administration of combination therapy. The observed antitumor activity was associated with a drop in body weight and glucose levels, downregulation of HK-1, inhibition of glycolysis products such as pyruvate and total ATP, activation of oxidative stress characterized by an increase in reactive oxygen species (ROS) and a decrease in reduced glutathione, as well as the appearance of apoptotic cell death. The results suggest a novel approach to combating breast cancer by targeting glycolysis suppression, glucose deprivation, oxidative stress, and apoptosis, with potential therapeutic applications.In research work by In summary, the findings of this investigation provide solid evidence in favor of the innovative hypothesis that ACA triggers glucose restriction, while virotherapy acts synergistically to improve metabolic oxidative stress and induce apoptosis. This study presents novel findings that indicate that ACA-induced glucose restriction acts in synergy with oncolytic NDV, which demonstrates a promising therapeutic approach that targets the glycolysis pathway for enhanced efficacy and safety. The integration of many therapeutic approaches into this unique treatment modality demonstrates a strong potential for application in clinical therapy.Green-Tripp et al.). Oncolytic adenoviruses (OAds) have been genetically modified to exhibit the ability to specifically induce lysis, elimination, and activation of host antitumor immune responses, while protecting normal cells from injury. The common modifications observed involve the removal of certain components within the early gene products, such as the E1B55 KDa protein and particular segments of the E1A protein. Alternatively, the introduction of tumor-specific promoters can also be employed as a modification strategy. The efficacy of oncolytic adenoviruses (OAds) in the treatment of several types of adenocarcinomas in patients with breast cancer (BC) has not been adequately evaluated in clinical trials. Preclinical research showed effectiveness in breast cancer cell lines, namely, triple negative breast cancer cells, using innovative adenoviral mutants that showed encouraging results. In this review, Green-Tripp et al. examined the results described for the most promising oncolytic adenoviruses (OAds) in preclinical investigations and clinical trials, both as standalone treatments and in combination with established conventional therapies or emerging therapeutic approaches. The present focus of research involves investigating the efficacy of combining OAds with small molecule medications that target the epidermal growth factor receptor (EGFR), androgen receptor (AR), and DNA damage repair through new PARP inhibitors. These combinations have been shown to exhibit improved efficacy. The co-administration of Olaparib, a PARP inhibitor, with oncolytic adenoviruses (OAds) demonstrated a significant and significant anti-neoplastic response. The most encouraging results have been observed to date when oncolytic adenoviruses (OAds) are used in combination with antibodies targeting immunological checkpoints or expressing cytokines derived from the viral backbone. Although multiple clinical trials and preclinical research have provided evidence of the safety and efficacy of cancer-selective oncolytic adenoviruses (OAds), additional advances are required to effectively eradicate metastatic lesions, enhance immune activation, and promote intratumoral viral dissemination.The next article on this Research Topic described that the latest discoveries related to oncolytic adenoviruses (OAds) have the potential to offer a novel approach to improve the outcomes of individuals affected by triple negative breast cancer (TNBC) and other forms of breast cancer (BC)  concluding the presence of relatively good results of studies in the field of treatment of solid cancers such as gastric cancer using oncolytic viruses, it seems that these viruses can be used more widely in combination therapies to increase the efficiency and effectiveness of cancer treatment. Nevertheless, this therapeutic method has its difficulties and requires more studies. In peritoneal metastasis gastric cancer, virotherapy can limit peritoneal metastasis and tumor metastasis to the peritoneum in diverse ways, such as direct oncolysis of tumor cells, as well as inhibition of mechanisms and molecules involved in angiogenesis. Alternatively, inserting genes with antitumor function into the genome of oncolytic viruses for expression in virus-infected tumor cells can enhance therapeutic effect. Viruses seem to have a wide range of unknown functions, and due to their extraordinary capabilities, such as their ability to replicate in hypoxic conditions, which is one of the drawbacks of cancer therapy, shortly, they can be used to treat cancers to the maximum benefit performance.In the next review .Despite careful quality control, during high titer production, “wild-type-” like replication-competent adenovirus (RCA) contaminants can be generated through recombination events due to the DNA sequence similarity between OV and host cells It is obvious that oncolytic virotherapy is progressing in steady and wide steps toward being among conventional cancer therapies. Since the approval of more than one type of OV in clinical use and many in clinical trials, more research is expected to focus on increasing the efficacy to be used as a first-line therapy. The development of oncolytic viruses that target specific types of mutations and genetic alterations in cancer cells will help treat difficult tumors clinically and give more hope to cancer patients.


Neuroimaging studies suggest involvement of frontal, striatal, limbic and cerebellar regions in trichotillomania, an obsessive-compulsive related disorder. However, findings regarding the underlying neural circuitry remains limited and inconsistent. Graph theoretical analysis offers a way to identify structural brain networks in trichotillomania. T1-weighted MRI scans were acquired in adult females with trichotillomania (n = 23) and healthy controls (n = 16). Graph theoretical analysis was used to investigate structural networks as derived from cortical thickness and volumetric FreeSurfer output. Hubs, brain regions with highest connectivity in the global network, were identified, and group differences were determined. Regions with highest connectivity on a regional level were also determined. There were no differences in small-worldness or other network measures between groups. Hubs in the global network of trichotillomania patients included temporal, parietal, and occipital regions (at 2SD above mean network connectivity), as well as frontal and striatal regions (at 1SD above mean network connectivity). In contrast, in healthy controls hubs at 2SD represented different frontal, parietal and temporal regions, while at 1SD hubs were widespread. The inferior temporal gyrus, involved in object recognition as part of the ventral visual pathway, had significantly higher connectivity on a global and regional level in trichotillomania. The study included women only and sample size was limited. This study adds to the trichotillomania literature on structural brain network connectivity. Our study findings are consistent with previous studies that have implicated somatosensory, sensorimotor and frontal-striatal circuitry in trichotillomania, and partially overlap with structural connectivity findings in obsessive-compulsive disorder. Trichotillomania , one of the obsessive-compulsive and related disorders (OCRDs) in DSM-5, is associated with repetitive hair-pulling resulting in hair loss and repeated attempts to decrease or stop the behaviour. The condition is associated with significant distress and impairment in several life domains  connectivity that have been reported in several neuropsychiatric disorders may be associated with altered topology including in small-world organization, and in the balance between the integration and segregation of information  and the University of Cape Town (HREC Ref. 261/2007). Participants provided written informed consent. The study was conducted according to the ethical guidelines of the Declaration of Helsinki.The Structured Clinical Interview for Obsessive-Compulsive Spectrum Disorders (SCID-OCSD)  There were no group differences in network measures of segregation  or integration .Regarding global connectivity, in the TTM group hubs at 2SD were the left inferior temporal gyrus, parietal, and occipital regions, while hubs at 1SD additionally were in frontal and striatal regions . Local connectivity was significantly higher in controls compared to TTM in the right pericalcarine gyrus (p = 0.04), located below the cuneus in the occipital lobe.To our knowledge, this study is the first to use graph theoretical analysis to investigate structural brain networks in TTM. The main findings suggest 1) similar small-world properties and network measures including segregation and integration between patients and controls, 2) involvement of different hubs in the global network of TTM patients compared to controls, including inferior temporal, parietal and occipital hubs (at 2SD above mean network connectivity) and frontal and striatal hubs (at 1SD above mean network connectivity), and 3) a role for the inferior temporal gyrus on a global and regional level in TTM, involved in object recognition as part of the ventral visual pathway.Corresponding small-worldness and network measures in both TTM and controls suggest brain networks that generally have similar network arrangement. This is partly consistent with some studies in OCD, the key example of the obsessive-compulsive and related disorders (OCRDs), that found similar small-world properties compared to controls (Kim et al., Our findings indeed suggest a distributed set of regions operating as hubs in the global network in TTM. These include temporal, parietal, and occipital regions, as well as frontal and striatal regions. Hubs found across lobes in the TTM group broadly coincide with regions implicated in TTM using other imaging modalities, e.g. diffusion tensor imaging and resting state functional imaging (Slikboer et al., The current study findings also suggest involvement of frontal-striatal circuitry in TTM. Consistent with aspects of earlier studies Grachev, , we founHubs in our controls represented principal regions typically found in structural networks of healthy adults, which include those of the default mode network (Hagmann et al. This study had limitations. First, the study included women only, in line with epidemiological data suggesting that the majority of individuals with TTM are female (American Psychiatric Association, Our findings largely coincide with previous studies implicating somatosensory, sensorimotor and frontal-striatal circuitry in TTM. Our findings also partially overlap with structural connectivity findings in OCD, a key example of the OCRDs. Further study in larger samples is needed to differentiate structural and functional networks of this condition.


Rapid breakthroughs in nucleic acid nanotechnology have always driven the creation of nano-assemblies with programmable design, potent functionality, good biocompatibility, and remarkable biosafety during the last few decades. Researchers are constantly looking for more powerful techniques that provide enhanced accuracy with greater resolution. The self-assembly of rationally designed nanostructures is now possible because of bottom-up structural nucleic acid (DNA and RNA) nanotechnology, notably DNA origami. Because DNA origami nanostructures can be organized precisely with nanoscale accuracy, they serve as a solid foundation for the exact arrangement of other functional materials for use in a number of applications in structural biology, biophysics, renewable energy, photonics, electronics, medicine, etc. DNA origami facilitates the creation of next-generation drug vectors to help in the solving of the rising demand on disease detection and therapy, as well as other biomedicine-related strategies in the real world. These DNA nanostructures, generated using Watson–Crick base pairing, exhibit a wide variety of properties, including great adaptability, precise programmability, and exceptionally low cytotoxicity in vitro and in vivo. This paper summarizes the synthesis of DNA origami and the drug encapsulation ability of functionalized DNA origami nanostructures. Finally, the remaining obstacles and prospects for DNA origami nanostructures in biomedical sciences are also highlighted. Naked therapeutic materials that include small and biomolecular drugs have several inherent difficulties that impede them from executing their activities fully in the body. This includes lower solubility, as well as stability against chemical and enzymatic breakdown, difficulty to cross biologic barriers, undesired side effects, and toxicity ,2,3. SevTo combat numerous illnesses, many nucleic acid therapies and chemotherapeutic drugs have been designed. Nowadays, synthetic drug deliveries are problematic because of these current limiting factors, such as stability, targeted transportation, solubility, regulated release, systemic distribution, and membrane penetration. Considering the potential immunogenicity and cytotoxicity of such carriers, their broad applicability is restricted. Through nucleic acid alterations, flexible chemically modified DNA origami nanostructures can be constructed. Because of its rich responsiveness and programmable sequences, DNA has gained increasing interest in the development of nanomaterials with predictable nanostructures and customizable functionalities, which have shown significant prospects in drug delivery. On the one hand, through sequence designing, DNA sequences with responsiveness, molecular recognition, and therapeutic effectiveness may be readily incorporated into the framework of DNA nanostructures. Drug delivery is a prospective application for these chemically altered DNA origami nanostructures with their increased stability and connected functional moieties via chemical alterations. DNA origami can be used as a drug loading vehicle to treat several diseases, such as multidrug-resistant leukemia, breast cancer, etc. . The druDNA-based nanotechnology and, in particular, the DNA origami technique are progressively approaching real-world biological applications. Unfortunately, many of these applications are still restricted by the limited stability of DNA nanostructures in biological fluids. The presence of digestive enzymes and the low magnesium ion concentration of the DNA nanostructures might cause unwinding and structural collapse , limitinThis review will address the uses of DNA origami nanostructures in precision nanomedicine and evaluate their utility and practicality. The discussion will begin with an overview of DNA origami and some of the structures that may be constructed. After that, an explanation of the synthesis of DNA origami, the mechanism utilized to make DNA origami nanostructures, and recent applications of this technology for the targeted drug delivery will be provided. We will also discuss our prospective on the future of DNA-origami-based drug carriers. We believe that this review will not only rigorously explain current advancements in DNA origami, but will also inspire both scientists and beginners to promote the improvements in this fascinating area.The area of DNA nanotechnology has experienced a revolution with the introduction of DNA origami technology, which involves folding long viral “scaffold DNA strands” with chemically manufactured “staple DNA strands” into particular 2D and 3D structures . These b2 -9-Pentylcarbazole Diiodide (BMEPC) is a carbazole-derived photosensitizer. 56MESS is a platinum-based compound. Quercetin and luteolin belong to plant flavonoids. Actinomycin-D is a natural chromopeptide.After assembling hundreds of complementary DNA helper strands and M13mp18 phage DNA into DNA origami carriers, the anthracycline doxorubicin (DOX) drug was noncovalently intercalated into the carriers. A thorough analysis of the anticancer effects of DOX/origami in vivo was carried out  Figure .3 were divided into four groups . DOX and DOX/origami intravenous injections at dosages equivalent to 4 mg/kg of DOX each were administered every three days. As a control, 0.9% saline was administered in the same volume as bare origami (0.08 mg/kg/day). When compared to the saline-treated group, the nude mice treated with DOX and DOX/origami both showed tumor volume suppression effects. In addition, the DOX/origami therapy showed a considerably greater ratio of lowering tumor burden than the DOX-treated group. Using the empty DNA origami, there was no tumor inhibition. In summary, DOX containing DNA origami that was directed on a breast tumor in BALB/c mice showed excellent anticancer activity without any systemic harm -9-Pentylcarbazole Diiodide (BMEPC) is a component of photo dynamic treatment (PDT), a cancer therapy option that uses ultraviolet or visible light in combination with a photosensitizer and molecule oxygen. This mixture has the potential to create extremely reactive oxygen species, which may eventually result in the multiple processes that destroy tumor cells. BMEPC may be added to DNA origami, which tumor cells can absorb. When exposed to radiation, BMEPC can produce free radicals and cause apoptosis . This fiIn order to accomplish a multidrug combination therapy, a nano antibody that targets the inhibition of the EGFR , one of the tumor indicators, was mixed with the platinum-based medication 56MESS and intercalated into a tetrahedron DNA origami. It has been discovered that the DNA nanoplatform and nanobodies worked together to treat tumors with exceptional selectivity and without any discernible harm .Aclarubicin is an anti-cancer drug used to treat acute nonlymphocytic leukemia intercalated into DNA origami . When fiMitoxantrone is a drug that is loaded into the double strands of the folate-overhung tetrahedron DNA origami and utilized as an anticancer agent. The folate-overhung mitoxantrone tetrahedra DNA origami (about 25 nm) may target leukemic cells, travel through the nucleus membrane, trigger apoptosis, and improve the overall effectiveness of treating leukemic cells in vitro and in mice with leukemia  38]..38].Drug cisplatin is used to assess the cytotoxicity of DNA origami nanostructures on FaDu cells, as well as to cross-link the structures . After 48–72 h, cell viability from nanomolar doses of cisplatin-loaded DONs is reduced to 50%  39]..39].The anti-inflammatory, antioxidant, toxic, anti-cancer, and immunomodulatory properties of quercetin (flavonoids) demonstrate its potential therapeutic value. Despite its many positive effects on human health, quercetin has some drawbacks, including its hydrophobic nature, low bioavailability, poor solubility, and poor permeability. Quercetin is encapsulated in DNA origami to improve its solubility and absorption in order to overcome some of its drawbacks . A typicThe well-known antibiotic actinomycin-D, which is intercalated in DNA origami and has significant antibacterial and anticancer action, belongs to the actinomycin group. Its cytotoxic and antitumor effects are caused by a number of mechanisms, many of which are linked to DNA functioning, which inhibits the production of RNA and, in turn, protein synthesis. The two main mechanisms are intercalation to DNA and stabilization of topoisomerases I and II cleavable complexes with DNA, in which a polypeptide lactone ring occupies a position in the minor groove of the DNA helix, and a phenoxazone ring localizes between the GpC base pair sequence in DNA, or the drug penetrates to a location in the DNA structure where topoisomerase binds with DNA, respectively. In addition, it has been hypothesized that actinomycin D’s sluggish dissociation from DNA complexes, photodynamic activity, free radical generation, and other biochemical impacts of activity may be significant determinants of this drug’s biological action .Since CpG patterns are much more common in microbial DNA than in vertebrate genomes, the immune system recognizes them as an indication of pathogen invasion. These sequences are identified by Toll-like receptor 9 (TLR9) when they are unmethylated, which can severely activate the innate and adaptive immune systems. As a result, CpG oligodeoxynucleotides are a strong option for use as an adjuvant in immunotherapy vaccines. The fact that native CpG dinucleotides are very vulnerable to nuclease degradation poses a problem for their application, hence stabilizing modifications, such as phosphorothioate (PTO)-modified backbones, are investigated. Due to the fact that PTO-CpGs are not as effective as adjuvants and may harm organs or lymphoid tissue, DNA origami is being investigated as a possible nanocarrier of CpG sequences for immunotherapies ,44. By hIn a recent study, the “Square block” DNA origami platform was introduced to investigate the significance of the spacing of CpG oligonucleotides that bind Toll-like receptors and so serve as danger signals for dendritic cells. It has been demonstrated that when CpG is separated at 3.5 nm, square blocks promote Th1 immune polarization in vivo and in vitro tumor-treatment models. This DNA origami vaccine improves DC activation, NK cell activation, antigen cross-presentation, Th1-polarized CD4 activation, and CD8 T cell activation. This research introduced a DNA-origami-based cancer vaccine that delivers antigen and CpG immune adjuvant with appropriate spacing for Th1 immune polarization .Octahedral DNA origami used as a nano-vehicle for the delivery of siRNAs. Although photothermal treatment and chemotherapy are frequently employed to treat cancer, their effectiveness is frequently constrained by multidrug resistance. Small interfering RNAs (siRNAs) have been widely used in cancer therapy to battle multidrug resistance to chemotherapeutic medicines and hyperthermia because of their capacity to decrease the expression of target genes. It is still extremely difficult to administer siRNAs and chemo-photothermal agents effectively in vivo. In this study, octahedral DNA origami frameworks (OctDOFs) are built as a nano-vehicle for precisely organizing and orchestrating the distribution of siRNAs in combinational cancer treatment. In order to effectively downregulate the connective tissue growth factor (CTGF) and heat shock protein 72 (HSP72) for the dual sensitization of cancer cells to chemotherapeutic medicines and hyperthermia, the stiff OctDOFs structure’s inner cavity sterically prevents RNase destruction and protein binding. The suggested OctDOFs demonstrated improved cytotoxicity and tumor suppression efficacy in vitro and in vivo by increasing chemo-photothermal therapeutic potency with siRNAs. A novel siRNA delivery platform for targeted medicine and combination treatment is created by this nano-vehicle . In a stDrug carriers must contain sufficient amounts of drug, which must be loaded and delivered in a regulated way. The cargo integration approach is determined by the drug’s characteristics, the kind of DNA origami, and the necessity to preserve drug activity after release. With continued advances in mass production and diverse ways for stabilizing DONs in physiological fluids, the possibility of DNA origami nanostructures for biological applications is being properly explored ,56. AlthThe endosomal compartment is where drug-encapsulated DNA origami is typically held after absorption, where it must either escape or release a loaded payload. Using electroporation, which involves rupturing the outer membrane with a powerful electrical field to let the origami structures pass through, it is possible to directly transport drug-encapsulated DNA origami into the cytoplasm ,66,67,68Many targeting ligands, as previously noted, can be linked to the DNA origami to enhance cellular absorption, drug transport, and administration. Triangle DNA origami is readily modified to integrate aptamers, a family of nucleic acids that may detect membrane receptors, trigger aptamer-receptor mediated endocytosis in HeLa cells, and dramatically increase the effectiveness of DOX administration . It has Less research has been conducted on the distribution of therapeutically carried DNA origami in vivo than has been performed in vitro; however, there has been some substantial in vivo DNA origami research performed. One of the earliest in vivo studies of DNA origami/ DOX dispersion was given by Q. Zhang et al. This research used triangle-shaped DNA origami nanostructures to provide remarkable therapeutic outcomes in vivo without systemic toxicity and a high passive tumor targeting accumulation of DOX in cancer regions . In addiTo explore their cell transport activities, DNA origami nanostructure carriers are frequently tagged with fluorescent dyes. Because such fluorescent dyes are observable under a microscope, they are perfect model molecules for drug carriers to transport into cells D. It hasMolecular self-assembly is widely employed in the areas of effective catalyst materials, biosensors, medicinal biomaterials, and molecular devices. DNA is a biomacromolecule made up of deoxyribonucleotide units, which serves as the transmitter of genetic data. DNA nanotechnology transcends DNA’s initial features as a molecule that stores and transfers genetic information from its biological environment by utilizing its distinctive base pairing and innate biocompatibility to construct structurally defined supramolecular frameworks. As DNA technology advances, the assembly process of DNA nanostructures is no longer confined to DNA hybridization but includes other biological interactions . Below, [A] Cost: One of the most difficult obstacles standing in the way of the practical uses of DNA origami as a medication delivery system is cost. At a synthesis scale of roughly 10 nmol, staple strands of a 7000 bp origami structure are commonly available for purchase for several hundred dollars. The true cost of individual DNA origami design would be significantly greater if other expenses, such as scaffold DNA, oligonucleotide functionalization, and origami purification, were taken into consideration. Therefore, it is vitally necessary to develop cost-effective scaffold and staple DNA synthesis techniques. Given that basic DNA tile structures have previously been generated in vivo, it may be possible to resolve this problem by producing DNA strands or perhaps whole origami structures in vivo .2+ and Na+) are necessary to neutralize the negative charge of the DNA backbone and maintain DNA origami structures because of the extraordinarily dense packing of DNA duplexes in these nanostructures. The amounts of cationic ions in typical physiological solutions (such PBS and medium) are insufficient to stabilize DNA origami constructs. The stability of DNA origami in fluids that simulate physiological circumstances has been tested in several experiments. Less dense things, such as wire-frame origami creations, have been discovered to be more stable in cation-depleted fluids [[B] In vitro and in vivo stability: Another problem that has to be solved is the stability of DNA origami in vitro and in vivo. High quantities of cationic ions  can help to minimize structural defects. Straightforward methods should also be developed to allow a convenient examination of structural quality.[G] Defects: DNA origami structures contain assembly defects, which can hinder heteroelement or therapeutic incorporation. Optimizing structural designs  and assembly conditions  sequences have been employed as a model cargo and have been covalently attached to DNA nanocarriers in order to elicit an immunological response. A Fab fragment, AuNPs, and active enzymes have all been reported to be contained inside a DNA origami nanostructure’s hollow. The cargo and enzymes were able to be more stable, catalytically active, and resistant to protease digestion because of these DNA origami nanostructures, according to the data ,67,92.[D] Cellular internalization: It has been demonstrated that DNA origami nanostructures with larger sizes and stronger compactness enable more effective internalization than structures with smaller compactness or isolated ssDNA . DNA ori[E] Therapeutic efficacy: The high loading, minimal cytotoxicity, perfect stability, and releasing capability of nanocarriers all contribute to high effectiveness in cancer therapy. Numerous studies have shown that DNA origami nanostructures improved anticancer functions and circumvented drug resistance. DOX-infused triangular and tubular DNA origami nanostructures, according to Jiang and colleagues, boosted the apoptosis of DOX-resistant breast cancer . It has [F] Photodynamic therapy: In photodynamic therapy (PDT), cancer cells are killed by combining light with photosensitizers. There are several photosensitizers for PDT, including silicon phthalocyanine Pc 4, aminolevulinic acid, and porphyrins. Some medicines, however, have drawbacks such as slow absorption, quick clearance, and poor solubility, which therefore, lead to insufficient therapeutic effectiveness. Additionally, DNA origami nanostructures have been applied in PDT as nanocarriers of photosensitizers .[G] Further investigations: To completely understand the stability issue with DNA origami constructs, more research is required. Although the effective cell entry of DNA origami structures has been experimentally confirmed, the precise endocytosis process has not yet been identified through rigorous mechanistic research. Another difficult obstacle is that every study has indicated that DNA origami constructs eventually make their way to lysosomes for digestion. DNA origami vehicles may be required to escape from the lysosome in order to facilitate an effective cargo release into the cytosol. Potential tactics include conjugating functional molecules onto DNA origami to promote lysosomal escape or utilizing targeted ligands to start absorption via a non-lysosomal route. Before any clinical drug-delivery applications, a deeper comprehension of the pharmacokinetics and pharmacodynamics of DNA origami constructs in vivo is also required .Because of its improved drug targeting and lower drug toxicity, nanomedicine has grown at an exponential rate. It makes use of interactions in which nanotechnological materials and biological systems interface with one another to improve delivery performance.The diversified DNA nanostructures have evolved into some of the most prominent self-assembly systems with a lengthy and notable history. Since it was first discovered, DNA origami has made incredible progress toward a variety of biological applications. This paper covers several approaches currently utilized for the construction of DNA origami nanostructures. The use of these DNA nanostructures with well-defined parameters for accurate control in the delivery of drug and gene therapy is also explored. Several possible applications of DNA origami have been presented, covering areas from nanomaterials to drug delivery, and they have demonstrated potential as strategies of interacting with cells. The cellular transport of DNA nanostructures is an emerging field of interest, which we cover in this review, concentrating largely on DNA origami structures. We pointed out several drugs that can be encapsulated with DNA origami and we concluded the prospectives and challenges of DNA origami from our point of view. While DNA-origami-based nanotechnology has great potential for precise nanomedicine, it is still in its early stages. Prior to clinical translation, certain critical obstacles must be addressed: (I) Unclear operating mechanism. Although DNA nanostructures have been explored for drug delivery, more research into the mechanics of transfection is desperately required since the real mechanism of uptake and how crucial parameters, such as size and shape, impact uptake are still unknown; (II) Further testing of safety profile. DNA, being a naturally biocompatible and biodegradable polymer, performs quite well in some types of cells and in mice. In these preliminary trials, there was no antibody reaction against DNA nanostructures. Nevertheless, considering the complexities of the human body, the impact of particle physicochemical characteristics on renal systems, and the doubtful but potentially harmful genome recombination, more research on DNA origami nanostructures in different types of organs is required before they can be used in clinical settings. The practical implementation of DNA origami nanostructures in vivo would be driven by additional mechanistic research on the fate of DNA nanostructures in vivo and the development of effective ways to reduce interruption from the physiological environment. Chemically altered DNA origami with many customizable sites has the ability to accurately co-load and effectively co-deliver a variety of therapeutic drugs via a protective barrier to improve nuclease resistance. We anticipate that in the near future, such chemically altered DNA origami nanostructures will be extensively utilized in the construction of drug carriers and we also be applicable to pre-clinical and clinical investigations. This type of strategically designed drug delivery method is important in reducing the systemic toxicity found in conventional administration and improving medication pharmacodynamics. This study encourages the advancement of innovative DNA-origami-based targeted drug delivery for the efficient and appropriate co-delivery of encapsulated cargoes in anti-cancer therapeutic applications, and also paving the way for the future medical applications of DNA origami nanostructures as prospective delivery platforms for tumor-targeted therapies. We believe that with further advancement in drug delivery systems and with the resolution of scalability difficulties, DNA-origami-based nanotechnology can bring a new notion into carrier systems and provide beneficial clinical results. Recent discoveries in the design and implementation of DNA origami nanostructures demonstrate that there is substantial potential for improvement, which should lead to significant applications for these nanostructures in material sciences and healthcare. These findings may contribute to the development and optimization of DNA origami nanostructures as drug carrier nanoplatforms for biomedical applications. This topic will undoubtedly attract multidisciplinary research efforts among chemists, biologists, doctors, and bioengineers, and interesting new discoveries will emerge.


The need for pre-clinical models to better predict the individual clinical outcome has driven the development of improved patient-derived models. Among variety of cancer models, patient derived xenografts (PDXs) and patient-derived organoids (PDOs) have shown promise to advance the field towards realizing the goals of precision cancer medicine –4. HowevThrough the collection of several insightful reviews, perspectives, and original research articles, this Research Topic aims to highlight recent progress and facilitate scintillating discussion to advance the preclinical research aligned with the goals of precision cancer medicine. We summarize here some of the key aspects covered in the various contributions, offering the readers a comprehensive understanding of this impactful area of research.Andus et al., report the establishment and characterization of three patient derived cell lines (PDCs) for non-small cell lung cancer (NSCLC) subtypes: adeno-, squamous cell, and pleomorphic carcinoma. PDCs of pleomorphic cell type carcinoma, for which the biological and drug response information are scarce, represent new preclinical models. Similarly, Driesch et al., report the establishment of a PDC as well as the first in-depth characterization of Medullary pancreatic carcinoma, a rare subtype of pancreatic ductal adenocarcinoma. These PDC models, with the detailed characterization of their molecular and morphological features and drug-sensitivity profile, represent valuable pre-clinical tools for advancement of precision cancer therapy.Cancer cell line models, for decades, have been extensively used in studies to gain valuable insights into tumor progression and drug responses. However, due to clonal selection and repeated passages under serum-containing medium for years, cell lines tend to have genetic drift from the original tumors . NeverthBeraud et al., report the development and characterization of five PDX models and their utility in evaluating therapeutic modalities such as androgen deprivation, PARP inhibitors and chemotherapy. These prostate cancer PDXs included hormone naïve, castrate resistant and neuroendocrine subtypes, and revealed a favorable response to PARP inhibitors in a BRCA2 mutant neuroendocrine model. Similarly, Yu et al. report the generation of a large panel of 171 PDX models, including 85 colorectal cancer (CRC), 21 esophageal, and 65 gastric cancer PDX models. The authors correlated the clinicopathological and molecular features of tumors with PDX take rates and demonstrated that tumor biomarkers and proliferation index could be validated in PDXs. Interestingly, cetuximab had a significantly lower overall tumor response rate in patients with RAS mutation. Screening of drug sensitivity in PDX models led the authors to identify two patients with K-Ras mutant tumors that responded to cetuximab, demonstrating the use of PDX models in patient case reports of CRC therapy. Two different incisive reviews by Liu et al. and Aaltonen et al., describe recent advances and knowledge gaps in lung cancer and neuroblastoma research, respectively, where patient derived models have been extensively used in co-clinical avatar studies for precision medicine.Models of prostate cancer (PCa) are also scarce, with nearly a dozen established cell lines and fewer organoid models , yet perFerrarini et al., performed a proof-of-concept study using lung adenocarcinoma PDXs and demonstrated plurimetabolic state in 9 of 14 PDXs analyzed. Expanding on the utility of PDXs in Neurooncology, Alcaniz et al., established and characterized 23 IDH-wt glioblastoma (GBM) PDXs, initially as heterotopic GBM PDXs, then propagated as brain orthotopic models, to assess drug resistance, and the impact of TME and blood brain barrier (BBB), which are key limitations to therapeutic response in orthotopic GBM models  signaling in limiting cholesterol metabolism which supports medulloblastoma growth. Despite the extensive utility of PDXs propagated in immune compromised animals and ex vivo in tissue culture, PDX models have limited applicability specially to study TME or tumor immune response. Furthermore, tumor take rates are variable, consequently only a fraction of any given tumor type can be timely established as PDXs for co-clinical avatar studies. Slower growth rate and clonal selection in the mouse background can also cause unpredictability, these challenges have been overlooked with their growing list of advantages in oncology.Beyond the evaluation of drug distribution and therapeutic responses in live animals, PDX models can serve in multiple ways. For example, readily available PDX samples can be leveraged to identify novel targets and biomarkers through exploratory studies, which otherwise may not be feasible due to paucity of patient tissue. M models . Deng etVerduin et al.) and a perspective article . The review collected data obtained from 60 published data spanning different tumor types. The authors concluded that PDOs have indeed the potential to predict response to treatment and have high translatability to the clinics. They highlighted the possibility to use PDOs in high-throughput studies to define targets, examine treatment response and further personalize these treatments. The authors especially highlight the discordance between in vitro vs. in vivo drug efficacy, that could be due to dose limiting toxicity in normal tissues. Therefore, evaluating drug sensitivity using normal tissue organoids in parallel with that in cancer organoids is a valuable addition to the organoid based co-clinical avatars in precision cancer medicine. Wang et al. specifically touched on the potential of PDOs as models in cancer immunotherapy. The use of complex organoids with enhanced heterogeneity of cell populations is an approach we must encourage to have the potential of studying the interaction between cancer cells and stroma, especially immune cells, at the basic and translational levels. Since PDOs lack vasculature, nervous system and drug metabolic pathways, this platform cannot be used for testing all drugs. Considering these limitations, Tan et al., adapted a strategy to use PDOs with available PDXs, established for the advanced gallbladder cancer, for initial rapid drug screening and in vivo drug efficacy validation, respectively, and allude to making these two models complementary. This approach can guide the individualized treatment strategies for cancer patients, when PDXs could be established in timely fashion.Patient-derived tumor organoids (PDOs) are cultures of cells resected tumors from an individual patient. PDOs can serve as models to understand patient-specific drug responses and to investigate cancer cell growth, and molecular analysis. The overall importance of PDOs has been nicely highlighted by a review article  using humanized mouse models. First, Tan et al, report that gallbladder tumor PDXs in humanized mice, generated with human peripheral blood mononuclear cells and treated with nivolumab, show immune cell death in tumors and that PD-L1 expression is not a direct indicator of the tumor suppressive activity of ICI, a finding that reflects the consensus on the unreliability of PD-L1 expression as a biomarker and the need for better biomarkers for response to ICI-based immunotherapy. Second, Marie et al., report the improvement of the traditional CRC PDX models by developing novel humanized CRC PDX models utilizing autologous (from same CRC patient) or allogeneic  PBMCs. Marie et al., elegantly demonstrated that while immune (HLA)-mismatched allogeneic models developed graft-vs-tumor effects limiting their utility, autologous humanized mice were proven useful, when used within a window-of-treatment, in detecting T-cell responses, myeloid cell infiltration and predicting responses to ICIs and potential combination therapies. The authors also demonstrate that modulation of immuno-suppressive cells, such as regulatory T cells within the TME can augment the efficacy of ICI agents in CRC. The use of newly available mouse strains with expression of human cytokines or those lacking mouse major histocompatibility proteins could potentially improve the support of engrafted immune cells and the therapeutic window in humanized animals.Cancer immunotherapy has revolutionized the field of oncology by prolonging survival and quality of life for cancer patients. However, a comprehensive Overall, the collection of articles in this Research Topic provides important additions to the arena of clinically relevant patient derived models to be utilized for drug testing and offers a roadmap to build on to achieve the goals of precision cancer medicine.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Here, we test (1) whether these scores contribute to prediction over-and-above family history, (2) the extent to which PGS prediction reflects inherited genetic variation N = 2483, 1565 monozygotic/918 dizygotic). Twins' parents were assessed for histories of substance use disorder. Twins were assessed for behavioral disinhibition at age 11 and substance use from ages 14 to 24. PGS prediction of substance use was examined using linear mixed-effects, within-twin pair, and structural equation models.PGSs for alcohol, cannabis, and nicotine use/use disorder were calculated for Minnesota Twin Family Study participants (Nearly all PGS measures were associated with multiple types of substance use independently of family history. However, most within-pair PGS prediction estimates were substantially smaller than the corresponding between-pair estimates, suggesting that prediction is driven in part by demography and indirect genetic effects of parents. Path analyses indicated the effects of both PGSs and family history on substance use were mediated via disinhibition in preadolescence.PGSs capturing risk of substance use and use disorder can be combined with family history measures to augment prediction of substance use outcomes. Results highlight indirect sources of genetic associations and preadolescent elevations in behavioral disinhibition as two routes through which these scores may relate to substance use. Substance use disorders (SUDs) are one of the most common and disabling behavioral health problems , PGSs also predict trait variance via demographic processes (i.e. population stratification and assortative mating)  tend to predict both the use of that and other substances (e.g. cannabis and nicotine) /932 DZ), 49.0% male]. To be eligible for the study, participants had to reside within a day's drive of Minneapolis, live with at least one biological parent, and have no physical or mental conditions that would interfere with completing a day-long, in-person assessment. Participants were recruited the year they turned 11 years old between 1977 and 1984 or 1988 and 1994. Families were representative of the area they were drawn from in terms of socioeconomic status, history of mental health treatment, and urban v. rural residence. Consistent with the demographics of Minnesota for the target birth years, 96% of participants reported non-Hispanic White race and ethnicity. Detailed overviews of the MTFS, twin samples, inclusion/exclusion criteria, and assessment procedures are provided in previous studies , a longitudinal study of same-sex twins , cannabis , and nicotine use  in adolescence and adulthood. Twins with higher scores on each PGS tended to be more disinhibited than twins with lower PGS . Family history of alcohol use disorder, cannabis use disorder, and nicotine dependence were also each associated with higher behavioral disinhibition (online Supplementary Table S4).Consistent with prior research, twins with greater behavioral disinhibition at age 11 tended to also report greater alcohol , Regular Smoking-PGS , family history of alcohol use disorder , and family history of nicotine dependence  remained significant, indicating that each measure was independently associated with substance use via elevated behavioral disinhibition. Conversely, the indirect paths from the Drinks Per Week-PGS, Problematic Alcohol Use-PGS, Lifetime Cannabis Use-PGS, and family history of cannabis use disorder to latent substance use via behavioral disinhibition were reduced to near-zero, likely due to the weaker associations between these measures and our latent disinhibition factor. Significant direct effects from the Problematic Alcohol-PGS, the Regular Smoking-PGS, and family history of nicotine dependence to substance use suggest that these measures additionally contribute to substance use via mechanisms that do not involve behavioral disinhibition  predicted the use of multiple types of substances, we estimated a latent substance use factor as our outcome in these mediation models. Results from individual-indicator models indicated significant indirect effects from all risk indicators (other than the Nicotine Dependence-PGS) to substance use between ages 14 and 24 via behavioral disinhibition at age 11 (online Supplementary Table S5). In the model including all nine indicators, only indirect paths for the Cannabis Use Disorder-PGS [hibition .Fig. 1.Results from this longitudinal study of substance use advance the understanding of recently developed PGSs capturing risk of substance use/SUDs in three ways. First, we showed that all six PGSs remained associated with at least one substance use index after adjusting for family history of the corresponding SUD, suggesting independent effects. Second, we contrasted within- and between-pair PGS effects to show that PGS prediction is substantially driven by the effects of demography and indirect genetic effects of parents. Third, longitudinal mediation analyses indicated that multiple PGS and family history measures contributed to substance use in adolescence and adulthood via elevated behavioral disinhibition in preadolescence, highlighting a possible developmental pathway that might partly explain the nonspecific associations commonly observed between these indicators and multiple types of substance use.Strengths of the current study include the multiple approaches to estimating risk and different types of substance use assessed, allowing us to examine the consistency of associations between these measures as well as test for independent effects. Our finding that all PGS and family history measures were associated with the use of multiple substances is in line with previous studies  is also substantially driven by these indirect processes  (Hicks et al.,


Screening for cancer is an important preventive strategy for the early detection of precancerous lesions and its management. There has been inadequate evidence on cervical cancer screening (CCS) practices among HIV-positive women in rural western Uganda. This study aimed to assess the prevalence and predictors of CCS among HIV-positive women, as well as knowledge and practices regarding cervical cancer screeningA cross-sectional, analytical study was conducted among HIV-positive women attending HIV care facilities located in rural settings of western Uganda. A validated and interview-based data collection form was used to capture statistics regarding demographics, HIV care, obstetric profile, health belief constructs, and knowledge and history of CCS from the participants. Bivariate and multivariate logistic regression analyses were used to correlate women’s characteristics and health beliefs toward CCS practices.The prevalence of CCS among HIV-positive women was found to be 39.1% (95%CI: 14.0–71.7). A multivariate logistic regression analysis showed that post-secondary education attainment , four years or more lapsing after being diagnosed as HIV-positive , having more than one child , antenatal care attendance , post-natal care attendance , and having good knowledge regarding CC  were positively associated with adherence to CCS among HIV-positive women in western Uganda. Health Belief Model (HBM) constructs like the perceived risk of developing CC , worries about developing CC , believing that CC leads to death , that screening assists in early identification  and treatment  of precancerous lesions, reducing the risk of mortality , and the reassurance provided by negative test results  were positively associated with adhering to CCS. A female doctor performing the screening  as well as offering a free screening service  were significantly associated with CCS. Meanwhile, screening being painful , expensive , time-consuming , embarrassing , and the fear of positive results  were found to have a significant negative association with adhering to CCS.Only one-third of HIV-positive women had undergone CCS. Variables including secondary education attainment, four years or more lapsing after being diagnosed as HIV-positive, having more than one child, antenatal care attendance, post-natal care attendance, and knowledge about CC were positively associated with CCS adherence. Educational programs should be geared towards the risk of CC, severity of cases, benefits of screening, and reducing barriers associated with screening, which can significantly improve cervical CCS among HIV-positive women. The study proposes the incorporation of free screening services and the inclusion of trained female staff in CC prevention policies to improve CCS. Globally, cervical cancer ranks as the fourth-most prevalent cancer among women, resulting in 311,365 fatalities in 2018  were significantly associated with cervical cancer screening practices. Variables including time since diagnosis of HIV more than four years , disclosure of HIV status , parity of more than one , visited an ANC at least once , and visited a post-natal unit  were also significantly associated with cervical cancer screening practices among HIV-positive women. Knowledge regarding cervical cancer , screening , a facility that offers screening services , and patients who are able to afford screening for cervical cancer screening  were significantly associated with screening practices among HIV-positive women. The associations of participants’ demographics, clinical profile, obstetrics profile, and knowledge level towards cervical cancer screening practices among HIV-positive women are represented in Table Within the framework of the perceived susceptibility, severity, and seriousness construct of the HBM, variables such as the perception of a high risk of developing cervical cancer , experiencing a lot of worry about developing cervical cancer , and cervical cancer could result in death  were significantly linked to the cervical cancer screening practices among HIV-positive women. Perceived barriers like screening being painful , expensive , time-consuming , embarrassing , and the fear of positivity  were significantly and negatively associated with cervical cancer screening practices among HIV-positive women. The perceived benefits of cervical cancer screening, such as the identification of precancerous lesions , early treatment of precancerous lesions  the reduction in mortality risk , and relieving participants’ concerns when the test results are negative , were found to be strongly associated with the practice of screening among women living with HIV.Clues to action constructs like a female doctor performing screening  and offering a free screening service  were significantly associated with adherence to cervical cancer screening practices among HIV-positive women. Table HIV-positive women may experience an increased susceptibility to cervical cancer throughout their lifespan. The sequence of events begins with an elevated susceptibility to human papillomavirus (HPV) infection, followed by an accelerated transition from high-risk HPV infection to precancerous lesions, ultimately leading to the development of cervical cancer. Additionally, there is a diminished likelihood of regression of precancerous lesions and a heightened incidence of recurrence after treatment. The WHO advises that all HIV-positive women should have cervical cancer screening starting at age 25, and repeat the screening every 3 to 5 years . This isThe current study revealed that the prevalence of cervical cancer screening among HIV-positive women was found to be 39.1%. The prevalence of cervical cancer screening among HIV-positive women in rural western Uganda is comparatively lower when compared to studies conducted in high income countries (HICs) such as the United States (78.0%), Italy (91.0%), and Canada (82.0%) –24. The The findings of the multivariate logistic regression analysis revealed that educational status, length of time since diagnosis, parity, ANC and PNC visits, disclosure of HIV status, and knowledge of HIV-positive women were significantly associated with adherence to cervical cancer screening practices. The study shows that educated HIV-positive women  were positively associated with cervical cancer screening practices compared to uneducated women. This indicates that educated women have better healthcare-seeking behaviors, awareness about screening, and decision-making power than uneducated women. Similar findings are also observed in studies conducted in Italy, Korea, Taiwan, and Ethiopia , 28, 29.Women diagnosed with HIV four or more years ago, a parity of more than one, and attending ANC and PNC visits were more likely to undergo cervical screening. The primary reason for the practice of cervical cancer screening within this population is attributed to their regular and prolonged attendance at healthcare facilities for routine HIV care and treatment of HIV and other medical conditions. This facilitates the women in acquiring knowledge about cervical cancer and screening from healthcare professionals, which can enhance their cervical cancer screening practices. This is in line with the findings of the studies conducted in Kenya, northern Italy, and Ethiopia , 25, 31.Women who had more than one child and regular attendance at ANC and PNC visits were more likely associated with undertaking cervical screening practices. This may be due to women who had children visiting healthcare facilities regularly for perinatal care in addition to routine HIV care follow-up compared to nulliparous women. Consequently, such individuals may have the opportunity to receive information and guidance on utilizing screening services from healthcare professionals. This finding is similar with the result of a study conducted in northwest Ethiopia .Women who revealed their HIV status to their partner are significantly more likely to engage in cervical cancer screening practices compared to women who kept their HIV status hidden. This may be due to emotional support received from partners in caring for the HIV-positive women. A study conducted in central Uganda revealed that partners’ emotional support is strongly associated with cervical cancer screening among HIV-positive women . Hence, It is crucial for HIV-positive women to possess adequate knowledge regarding cervical cancer, screening practices, and the availability of screening facilities. This knowledge is essential in promoting timely and appropriate uptake of screening, which aids in the detection of precancerous lesions and in the prevention of cervical cancer. In the current study, women’s knowledge level was significantly associated with their adherence to cervical cancer screening practices, similar with studies conducted in urban Uganda and Laos Republic , 30. TheThe findings of the multivariate logistic regression analysis revealed that Health Belief Model (HBM) constructs, such as perceived susceptibility of cervical cancer, severity and seriousness of cervical cancer, benefits of screening, and cues to action of screening, exhibited a significant association with their inclination to cervical cancer screening practices among HIV-positive women. Meanwhile, perceived barriers like screening are painful, expensive, time-consuming procedures, as well as embarrassing; fears of positivity concerns are significantly associated with lower odds of cervical cancer screening practices among HIV-positive women.According to the perceived susceptibility construct, women who believe they might be at risk of developing CC and worry about getting CC are significantly associated with cervical cancer screening practices. This suggests that it is important to raise awareness among HIV-positive women about their increased susceptibility to developing cervical cancer in comparison to women without HIV. Ultimately, this has the potential to improve adherence to screening practices among HIV-positive women. While excessive worry about contracting cervical cancer can have a negative impact on women’s mental well-being, a reasonable level of concern is essential to motivate women to undergo cervical cancer screening. As with perceived susceptibility, the seriousness/severity of the CC can also play a vital role in the uptake of screening practices. Within the framework of the perceived seriousness construct, there was a significant correlation between HIV-positive women who believed that cervical cancer could lead to death and their adherence to screening practices. Previous studies conducted in LMICs and HICs showed a similar finding that perceived susceptibility and seriousness of cervical cancer predicted adherence to cervical cancer screening practices –36. One The other variable that was positively associated with adhering to screening practices was the perceived benefits, such as screening helps in the early identification and treatment of precancerous lesions, reduces the risk of mortality, and provides reassurance for negative test results, which were positively associated with CCS. Cervical precancers are not cancers; they are lesions that may progress to cancer if left untreated. Screening tests such as the HPV test, the Papanicolaou (PAP) test, and visual inspection with acetic acid (VIA) can be utilized to identify precancerous lesions. HIV-positive women who possess knowledge about the efficacy of screening tests in detecting precancerous lesions at an early stage are more inclined to participate in screening procedures actively. Moreover, the timely identification of precancerous lesions on the cervix can be effectively treated through cryotherapy, a method that involves the eradication of abnormal cells through freezing, or LEEP, a technique that employs hot wire to destroy the abnormal cells. HIV-positive women should receive comprehensive counseling regarding the potential advantages of screening for timely identification and treatment of precancerous cervical lesions, which can effectively mitigate the risk of developing cervical cancer. The susceptibility findings indicate that women exhibit significant worry regarding the potential development of cervical cancer in the future. If the results indicate the absence of precancerous lesions, it can alleviate worry related to the likelihood of developing cervical cancer. The current study’s findings of the benefits offered by cervical cancer screening are in line with those of studies conducted in Botswana and Ethiopia , 35.However, HBM barrier constructs such as screening being painful, expensive, time-consuming, embarrassing, as well as the fear of positive results were found to have a significant negative association with CCS practices among HIV-positive women. These findings suggest that there is a need to design educational interventions that prompt reductions in barriers and enhance knowledge about the benefits offered by cervical cancer screening for HIV-positive women.The findings regarding the cues to actions construct reveal that a female doctor performing screening and offering free CC screening services are positively associated with cervical cancer screening. Involving female doctors encourages screening among participants, and can be correlated with the low screening rates seen among women who experienced embarrassment about CC screening. In order to overcome this barrier and improve the adherence to screening practices among HIV-positive women, it is necessary to only employ female personnel for conducting cervical cancer screenings. Cervical cancer screening facility access with no cost can encourage women to participate in the screening program.The aforementioned findings illustrate the potential impact of HIV-positive women’s health beliefs on the cervical cancer screening decision-making process. The inclusion of findings from the HBM and the consideration of HIV-positive women’s characteristics that influence decision making can be taken into consideration when formulating cervical cancer screening policy and designing sensitization programs in HIV.The scope of the study was restricted to HIV care clinics situated in rural regions of western Uganda. There is a need to expand this research in various regions of Uganda, to develop and offer a nation-wide cervical cancer screening program among HIV-positive women. The findings of this study help in addressing issues related to poor cervical cancer screening practices among HIV-positive women. The study did not endeavor to investigate the healthcare factors that are linked to the inadequate implementation of cervical cancer screening among HIV-positive women. The qualitative research focused on healthcare factors influencing cervical cancer screening among high-risk populations is necessary to strengthen screening uptake. Due to the cross-sectional nature of the study design, the actual causality of poor cervical cancer screening is not well established. The study participants were attending HIV care facilities; they were exposed to information on cervical cancer and screening availability, which can limit the generalization of screening practice findings to the general population of women. The primary strength of this study lies in its focus on rural western Uganda and its investigation of the factors related to cervical cancer screening practices among women who are HIV-positive. The utilization of interviews as the primary methodological approach in this study design allows for a reduction in response bias in the obtained findings.The study concludes that only one-third of HIV-positive women had undergone cervical cancer screening. Variables, including secondary education attainment, four years or more lapsing after being diagnosed HIV positive, having more than one child, antenatal care attendance, post-natal care attendance, and knowledge about cervical cancer, were positively associated with cervical cancer screening. Determinants of cervical cancer screening provide insights for public health interventions to prevent cervical cancer. These interventions may encompass activities such as advocating for the importance of perinatal visits, where women can acquire knowledge about CCS; creating visual materials for participants with no literacy or primary education levels; encouraging women in the early stages of HIV infection to screen for cervical cancer; and enhancing awareness regarding cervical cancer, screening, and healthcare facilities that offer screening services by providing structured educational programs. The educational programs should be geared towards the risk of CC, the severity of cases, the benefits of screening, and reducing barriers associated with screening can significantly improve cervical cancer screening among HIV-positive pregnant women. This study proposes the incorporation of free screening services and the inclusion of trained female staff in CC prevention policies to improve CC screening.


A bidirectional causal relationship has been established between temporomandibular disorders (TMDs) and chronic headaches. Recent advances in the neurobiology of chronic pain offer a framework for understanding the comorbidity between these two conditions that might reside in the shared biomolecular mechanisms of peripheral and central sensitization. The initiation of these processes is inflammatory in nature and is most likely mediated by key molecules, including calcitonin gene-related peptide (CGRP). This scoping review proposes that CGRP-mediated neuroinflammation in the trigeminal ganglion may partly explain the biomolecular bidirectional link between TMDs and chronic headaches. Finally, clinical implications of this neuropathologic process are briefly discussed. Temporomandibular disorders (TMD) encompass a group of conditions involving the masticatory muscles, the temporomandibular joint (TMJ) and associated structures ,2. TMD aAmong the various signs and symptoms that individuals with TMD manifest, headache is a common manifestation. Migraine and tension-type headache constitute the majority of the primary headaches encountered in the general population, with a prevalence of approximately 12%  and 45–7Recent studies highlighted the comorbid presence of headache and TMD, hypothesizing that a relationship between the two might exist. In this regard, a recent systematic review and meta-analysis supported a positive association between pain-related TMDs, migraine and chronic tension-type headaches . Other sImportantly, the Orofacial Pain: Prospective Evaluation and Risk Assessment (OPPERA) study demonstrated that prior headaches were a significant risk factor for the development of first-onset TMD symptoms . After tcentral sensitization .,35.13,35Different migraine pathogenetic mechanisms have been explored and supported in the literature. Beside the classic vascular pathophysiology of migraine, current research suggests that vasodilation is an epiphenomenon, and that peripheral and central sensitization in the trigeminal nociceptive system may explain the migraine pain . This pa1B/1D receptors. As 5-HT1B receptor is localized on smooth muscle cells of cerebral, meningeal and coronary arteries, and 5-HT1D is mainly expressed in the trigeminal ganglion, these drugs result in a strong vasoconstriction of the cranial arteries [Ergotamine derivatives and triptans, drugs approved for the acute treatment of migraine , mainly arteries . They alarteries .1F receptor agonists (lasmiditan) are newly Food and Drug Administration (FDA)-approved drugs for the acute treatment of migraine [1F receptors are located on terminals and cell bodies of the trigeminal ganglion neurons, acting at the peripheral nervous system and central nervous system. It can modulate CGRP from trigeminal ganglion neurons by potentially blocking its release and inhibiting the development of central sensitization [1F does not induce vasodilation but rather causes vasoconstriction [Ditans, 5-HTmigraine . 5- HT1Ftization . Contrartriction .CGRP receptors are localized on smooth muscles cells of meningeal and cerebral blood vessels. As a result, the release of CGRP by the activated meningeal C-fibers causes blood vessels to dilate . Direct Topiramate inhibits nitric oxide and proton mediated CGRP secretion in a time- and concentration-dependent fashion from sensory trigeminal neurons .Botulinum toxin-A (BoNT) at doses between 150 Units and 195 Units, repeated every 12 weeks, is listed among the FDA-approved therapies for prevention of chronic migraine . Among tThe role of CGRP in migraine pathogenesis is evidenced by many of the current migraine pharmacological therapies, which act by directly or indirectly inhibiting CGRP activation. For instance:Nevertheless, as many as 40% of patients suffering from chronic migraine are considered non-responders to therapy, i.e., not achieving more than 50% reduction in monthly headache days . This maThe levels of CGRP in saliva, cerebrospinal fluid and peripheral blood are elevated during severe migraine attacks .Clinical improvement of migraine symptoms after triptan administration is accompanied by decreased CGRP levels . In addiSubjects with chronic migraine have higher levels of CGRP in peripheral blood samples than those with episodic migraine .Important experimental clinical findings supporting the relationship between migraine and CGRP include the following findings:TMD exhibits a significant comorbidity with migraine. Current research supports that CGRP may constitute one important molecular link between headaches and TMD. Such a connection is evidenced by a migraine animal model, where the masseter muscle was injected with a chemical irritant to trigger a peripheral inflammatory response . As a reSpecifically in the case of TMJ-related trauma, peripheral insult or repetitive strain to the masseter causes CGRP expression in the trigeminal ganglion. When CGRP is expressed in the ganglion, it acts in an autocrine and paracrine fashion by stimulating satellite microglial cells to release pro-inflammatory mediators, causing a ganglion-wide inflammatory cascade . In thisThe role of CGRP as a key biomolecular link in neuroinflammation responsible for TMD development and maintenance is highlighted in the following studies .https://clinicaltrials.gov/ct2/show/NCT05162027 and https://clinicaltrials.gov/ct2/show/NCT04884763?term=CGRP&cond=Temporomandibular+Disorder&draw=2&rank=2) (Accessed on February 23 2025).The majority of the available literature on the use of anti-CGRP therapy in TMD has been conducted in animal studies. Interestingly, there is evidence suggesting a potential association between comorbidity of headache, migraine and TMD, and dysfunctional CGRP signaling. Studies have indicated alterations in CGRP levels and CGRP receptor expression in individuals with both headache and TMD compared to those with either condition alone. Comorbidity of headache and TMD could potentially serve as an indicator for the likelihood of successful anti-CGRP therapy ,70. WhilCGRP receptors are widely distributed through the trigeminovascular system .The inflammatory response from peripheral injury stimulates the expression of CGRP in the trigeminal ganglion and central relay centers .Biomolecular CRGP release in the trigeminal ganglion stimulates the release of pro-inflammatory mediators via supporting cells in a paracrine fashion .The resulting “inflammatory soup” in the ganglion (a sterile ganglionitis) is permissive of cross-excitation of all branches of the trigeminal nerve .When these findings are combined with those supporting the role of CRGP as a biomolecular key in headaches, a framework to better understand the shared pathogenesis of headache and TMD comorbidity emerges. This relationship is supported by the following findings:Taken together, these findings suggest that cross-excitation secondary to CGRP-mediated trigeminal ganglionitis may explain the physiologic basis for the comorbidity of TMD and headaches .Direct jaw trauma;Sports injuries;Motor vehicle accidents;Whiplash associated injuries;Hyperextension injuries;Strain from repetitive or continuous muscle activation;Bruxism and other parafunctional behaviors.The proliferation of research on mild traumatic brain injury (mTBI) and post-traumatic headaches (PTH) in the past decade has increased our understanding of chronic pain syndromes. There are many types of trauma that can impact the head, neck and jaw, and lead to both headache and TMD including:Importantly, an acute jaw, head and neck injury of any type will result in a four-fold increase in the odds of subsequent painful TMD development . Trauma There are well described clinical and phenotypic overlaps between PTHs and both migraine  and TMDsNot surprisingly, CGRP is implicated in the development of PTH after mTBI . AccordiAnother recent study supporting the link between CGRP and PTH revealed how the injection of CGRP in a cohort of patients diagnosed with PTH resulted in the immediate development of migraine-like headache in 72% of them . This stThe studies reviewed so far suggest that an anti-neuroinflammatory treatment in the acute phase of an injury has the potential to encourage healing and prevent central sensitization, and, as a result, a chronic pain state. Alternatively, if a chronic pain condition is already established, the same treatment strategy can be used in an abortive fashion for episodic symptom flare-ups. In this scenario, pharmacologic agents that directly or indirectly antagonize CGRP are already being used clinically for different types of headache ,82. CurrTherefore, the optimal approach in clinical practice should involve an early identification of those individuals in the acute phase with a high likelihood of developing a chronic pain state through the promotion of risk stratification and preventive treatment approaches. At this stage, conservative, non-invasive and inexpensive approaches are advocated to encourage both healing of the injured tissues and reduction of the risk factors that drive delayed healing and chronic pain.prompt treatment to prevent central sensitization is clear. If central neuroplastic changes can be prevented, chronic pain will not develop. A multimodal approach, using non-invasive and complementary measures, provides the best chance of curbing the cycle of pain amplification and progression to pain chronicity. The first few weeks after an injury correspond well with the proposed physiological mechanisms that drive central sensitization, i.e. neuroinflammation, microglial activation and neuroplastic adaptation. This is also supported by clinical findings, such as those seen in the OPPERA study . Each inBeside pharmacological approaches, other non-pharmacological strategies can be adopted. This is the case, for example, of an anti-inflammatory minimal sugar diet with nutritional supplements that attenuate biomolecular CGRP expression ,89 or thIn cases of pain within the temporomandibular structures, the protective biomechanical splinting of the TMJ and jaw and head muscle complex is a non-invasive effective intervention. As noted, there is a large population of CGRP-positive neurons in the TMJ complex , and masticatory muscles, tendons and fascia ,102,103.The use of behavioral interventions (such as mindfulness strategies  and deepPhysical therapies and exercise are considered front-line therapy for acute head, neck or TMD pain. Early mobility of both the jaw and neck is important for the interruption of peripheral sensitization. Multiple studies have shown that jaw exercises facilitate pain-free mouth opening and reductions in disturbances to daily living . PhysicaHeadache and TMD pathology are linked via the trigeminal system. The frequent comorbidity and bidirectional relationship of these two conditions may be supported by biomolecular pathways such as CGRP-mediated cross-excitation in an inflamed trigeminal ganglion. As a result, this excitation may drive peripheral and central sensitization of the structures innervated by the trigeminal system including both TMD and headache syndromes such as migraine. In both the acute and chronic phase of these conditions, treatments that inhibit CGRP may represent a promising preventive treatment strategy to minimize delayed recovery and chronic pain. This also highlights the importance of multimodal treatment with conservative, non-pharmacologic measures during the acute period including dietary changes, oral device therapy, behavioral management and physical therapies such as exercise to reduce peripheral and central risk factors to prevent chronic pain.