Rabies is a disease of antiquity and has a history spanning millennia ever since the first interactions between humans and dogs. The alarming fatalities caused by this disease have triggered rabies prevention strategies since the first century BC. There have been numerous attempts over the past 100 years to develop rabies vaccineswith the goal of preventing rabies in both humans and animals. Thepre-Pasteurian vaccinologists, paved the way for the actual history of rabies vaccines with the development of first generation vaccines. Further improvements for less reactive and more immunogenic vaccines have led to the expansion of embryo vaccines, tissue culture vaccines, cell culture vaccines, modified live vaccines, inactivated vaccines, and adjuvanted vaccines. The adventof recombinant technology and reverse genetics have given insight into the rabies viral genome and facilitated genome manipulations, which in turn led to the emergence of next-generation rabies vaccines, such as recombinant vaccines, viral vector vaccines, genetically modified vaccines, and nucleic acid vaccines. These vaccines were very helpful in overcoming the drawbacks of conventional rabies vaccines with increased immunogenicity and clinical efficacies. The path traversed in the development of rabies vaccines from Pasteur to the modern era vaccines, though, faced numerous challenges;these pioneering works have formed the cornerstone for the generation of thecurrent successful vaccines to prevent rabies. In the future, advancements in the scientific technologies and research focus will definitely lay the path for much more sophisticated vaccine candidates for rabies elimination. Rabies has a historical importance ever since the beginning of human and dog relationships nearly 40,000 years ago. The Mesopotamian records reveal the existence of a very hazardous ‘mad dog’ disease, which reveals the interaction of dogs with a most deadly rabies virus . Rabies,Rhabdoviridae. It is a bullet shaped virus, holding a single-stranded, negative-sense RNA genome of about 12 kb, which encodes five major structural proteins from 3′ to 5′ viz., nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase (L)  and show remarkable potential for addressing a variety diseases . Thisis Generally, adequate levels of virus-neutralizing antibodies are directed against the rabies virus glycoprotein, which in turn, correlates with rabies protection. The glycoprotein gene can be easily cloned into the proper expression vectors using the techniques of recombinant DNA technology, which facilitates the efficient in vivo expression of glycoproteins and resulted in the production of rabies DNA vaccines. Since 1994, DNA vaccines have been suggested as a more affordable and effective method for rabies prophylaxis, and theirviability has been shown in a variety of animal models, including companion animals . The gly+ and CD8+ T lymphocytes in inoculated mice and protect them from lethal intracerebral challenge infection [RNA vaccines are the simplest nucleic acid vaccines and emerged as a potentialmorepromising alternative platform for vaccine development. RNA vaccines have a number of advantages over DNA vaccines. RNA vaccines are directly translated in the cytoplasm, eliminating the need for transport into the nucleus and resulting in fast antigen expression with no possible risk of integration with the host genome. It is a known fact that RNA is a powerful adjuvant that increases the host immune response by communicating through pattern recognition receptors, such asthe Toll-like receptors or the retinoic acid-inducible gene I-like receptors . The abinfection . Similarnfection . A self-nfection . HoweverProtein vaccines stimulate the immune system by using peptides or proteins as antigens. Protein antigens can be produced naturally from the pathogen that causes the infectious disease, either in their entire form or as derived split-products. Protein subunit vaccines can also be created as heterologous proteins in recombinant systems, including recombinant bacteria, yeast, insect cells, or mammalian cells, as an alternative to natural sources . The RABApart from protein vaccines, a number of antigenic epitopes of the RABV G protein were identified, and synthetic peptides mimicking these G protein epitopes have been produced and used as vaccines. Animals inoculated with a synthetic peptide encapsulating the rabies virus’s G5 antigenic region produced antibodies with strong avidity but low neutralization potential . PeptideViral vector vaccines were developed by cloning the antigen of interest into a heterologous virus that will serve as a carrier molecule to transfer the foreign gene of interest within the host cells, thus leading to its subsequent expression . The firSeveral researchers targeted different viral vectors for the expression of rabies viral glycoprotein. The recombinant adeno-associated viruses (AAVs) with RVG vaccinations enhanced the protective levels of Rabies Virus Neutralizing Antibodies (RVNAs), with single intramuscular vaccination . SeveralAnimal vaccination is an effective way of protecting public health through the reduction of zoonotic pathogens. The long history of rabies vaccine development has landed with many potent vaccines for parenteral vaccination strategies. However, the vaccination of wildlife or free-roaming domestic animals, such asstray dogs, through parenteral vaccination remains a challenge. Hence the need for an alternative vaccination strategy for the eradication of rabies has led to the development of oral rabies vaccines (ORVs). The oral vaccines are given orally using a bait designed with the vaccine suspension-sealed sachet encased in a palatable bait material, such as animal intestine, chicken head, egg, and dog food-based materials, depending on the target species. Upon consumption, the chewing motion causes the sachet to be perforated, releasing the vaccination suspension into the mouth. Here, the vaccine is mostly absorbed by the palatine tonsils where, following minimal replication at the site of entry, it triggers a protective immune response . CurrentThe development of modified live rabies virus oral vaccines started way back in 1935, derived from the Street Alabama Dufferin (SAD) rabies virus strain. This parental SAD strain was isolated from the salivary glands of a rabid dog in the USA. Subsequent serial passages of the SAD strain in unnatural hosts, such asmice, chick embryos, and non-neural cell lines, includinghamster kidney and pig kidney cells, with further heat stabilization, has resulted in thehighly attenuated SAD-Bern, Evelyn Rokitnicki Abelseth (ERA), and SAD-B19 strains. These strains constitute the first-generation ORVs, which werethe keystone for controlling wildlife rabies in Europe and havebeen widely used ORVs across the globe . FurtherThe success of field trails in generating immune responses in free-roaming dogs whenusing SPBN GASGAS was appreciable in certain developing countries. Using oral bait with SPBN GASGAS supplied in Thailand for 2444 dogs, which were not accessible for parenteral vaccination, about 65.6% of the roaming dogs at these locations were effectively immunized orally . An evalThe development of vector-based vaccines helped to overcome the risks associated with MLVs. These viral vector-based rabies vaccines are more promising for both veterinary and human use ,130,131.Currently, two commercially licensed VBVs are available for controlling wildlife rabies viz., RABORAL V-RG, which uses recombinant vaccinia virus vector , and ONRThe details of the current research in the development of different vaccines and advantages and disadvantages of different vaccines are summarized in The ability of a vaccine to stimulate the immune response is also dependent on the route of inoculation of the vaccine. Intradermal vaccination delivers antigens into the space between the epidermis and the dermis. This space is an anatomically favorable site for immune stimulation, as the dermis and epidermis layers of the skin are abundant sources of antigen-presenting cells, such as Langerhans cells, dermal dendritic cells, and dermal macrophages, which are known to take part in vaccine-induced immune responses, and skin is seen as a desirable vaccination target. The dermis’ extensive network of blood capillaries and lymphatic arteries also makes it easier for leukocytes and dendritic cells to travel from the skin to the secondary lymphoid organs. Most rabies vaccinations administer an increased amount of antigens via the intramuscular (IM) route. Hence, its usage is restricted in many under-developed nations due to the price of these antigens and occasionally their scarcity. Reduced dosages  administered via the intradermal (ID) route areable to elicit immunological reactions similar to those brought on by the standard dose administered via the IM route .In the United States, pre-exposure immunization with the ID method of rabies vaccines was first authorized in 1986, which was followed by the development of a cost-effective multi-site intradermal (ID) immunization approach. The intradermal administration of contemporary rabies vaccines for post-exposure prophylaxis was advised by the WHO Expert Committee in 1991 . AccordiRABV typically infects the nervous system (NS). Yet, in the early stages of infection, virus particles are “injected” into the muscles and skin, and before the virus enters the nervous system, it might cause an immunological reaction in the periphery . Once RA+ T lymphocytes. Though it is typically believed that cytotoxic T cells are more crucial than antibodies to remove virus infection from tissues, rabies is an exception. Moreover, CD8+ T cell activation results in a pathogenic reaction that is clinically linked to paralysis. Based on the high risk of generating a potent, harmful CD8 response in the nervous system, this knowledge should generally prevent the use of live vaccines, such as DNA vaccines or recombinant viruses, as post-exposure immunizations [+ T cells. Because post-exposure immunizations set up an immune response in peripheral secondary organs, they are likely to provide protection. Activated lymphocytes, CD4- producing plasmocytes, secrete antibodies thatgain entry into the nervous tissue parenchyma and neutralize the virus [The most crucial function of rabies vaccination is to induce a persistent antibodyresponse through the activation of CD4izations . Yet, duhe virus .Thousands of people die from rabies each year, with the majority of those deaths occurring in Asia and Africa. However, rabies can be prevented throughvaccination if post-exposure prophylaxis (PEP) is administered promptly and effectively. Rabies exposure is roughly divided into three categories: I, II, and III, depending on the nature of the probable rabid animal interactions. Any exposures that are found to provide a rabies risk necessitate PEP, which involves vaccination, the thoroughwashing and disinfection of all bite and scratch wounds, local wound infiltration with RIG , and the prompt local treatment of all bite and scratch wounds . The priRabies Immune Globulin (RIG) is either a polyclonal or monoclonal antibody that specifically targets the G protein epitope, which wasfound to be very effective at neutralizing RABV prior to the virus entering the central nervous system (CNS) . This wiA class of double-stranded RNA molecules of about 21–23 bp, known as siRNA, also referred to as short interfering RNA or silencing RNA, repress the expression of particular genes by degrading mRNA following transcription via the RNAi pathway. The siRNA-based gene silencing of target genes has become an option for antiviral defense against many illnesses and disorders. Research has focused on plasmids encoding siRNA or viral vector-based systems of siRNA targeting specific RABV genes, which were constructed and tested for anti-rabies efficacies ,171. EspThe recent advancements in rabies vaccine developments have generatedpromising tools for preventing and eradicating rabies. However, there are certain areas to be focused on in the near future. The current development ofthe newly invented vaccines is quite appreciable; however, the development of novel vaccine delivery systems remains a vital areas of study, which will help to formulate effective strategic measures to eradicate this illness. Failure of the rabies vaccines due to insufficient vaccine potency as a result of exposure to unfavorable temperatures during storage andshipping critically necessitates research on the developmentof potential rabies vaccines that can be stored and transported outsidethe usual cold chain, which will definitely revolutionize vaccine distribution by increasing the effectiveness, efficiency, and affordability ofvaccine delivery. Recent research on sugar-matrix thermostabilization (SMT) technology for viral-vectored vaccines will be a novel platform in the future for eliminating theneed for the cold-chain storage of vaccines. Further exploration for novel adjuvants  will definitely be a hopeful option in improving the potency of inactivated anti-rabies vaccines. Application opportunities for the creation of multivalent vaccines arepromising. The development of successful bivalent viral vector-based vaccines against rabies and canine distemper will have potential to control both diseases with a single vaccine candidate, especially in developing countries. Moreover, future vaccine development should focus oncreating a multivalent vaccine with broad-spectrum protection efficacy considering other members of Lyssavirus, which will be very effectual in curtailing this deadliest disease. Although the quality and availability of rabies vaccines and RIGs produced from cell cultures have progressively improved, there has consistently been modest interest in creating antiviral therapy, which warrants research on potential therapeutic interventions for clinical application, through potential antiviral medications. More research on rabies vaccines using small interfering RNA (siRNA) in the natural host or the development bi-specific antibody (BsAb)-based therapies for rabies virus holds more promising options in the therapeutic aspects of rabies. Moreover, improving post-exposure vaccination in animals and redefining the regimen of vaccination forvarious types of vaccines remain the need of the hour. The further development of novel andimproved immunogenic oral bait vaccines in target hosts will be much appreciated in controllingrabies, especially in the free-roaming dog population in developing countries through mass dog vaccination programs.The saga in the development of anti-rabies vaccines and the path traversed in the development of rabies vaccines from Pasteur to the modern era of immunization has faced many ups and downs. However, still, these pioneering works have laid a strong foundation for the successful development of vaccines to prevent human deaths and curtail canine rabies at the moment and thus holds much appreciation. Moreover, the roadmap ahead in vaccine development with advanced scientific technologies to manipulate the rabies viral genome and novel vaccine carriers will definitely provideoutstanding developments in vaccine research in the near future. However, the immediate need of countries endemic for dog-mediated rabies is to employ mass dog vaccination using the parenteral vaccines for domestic dogs and accessible community dogs, complemented by ORVs for free-ranging dogs that are difficult to be caught. This combined approach could make the Global Strategic Plan for the global elimination of dog-mediated human rabies deaths by 2030 a reality.


Cardiometabolic diseases encompass a range of interrelated conditions that arise from underlying metabolic perturbations precipitated by genetic, environmental, and lifestyle factors. While obesity, dyslipidaemia, smoking, and insulin resistance are major risk factors for cardiometabolic diseases, individuals still present in the absence of such traditional risk factors, making it difficult to determine those at greatest risk of disease. Thus, it is crucial to elucidate the genetic, environmental, and molecular underpinnings to better understand, diagnose, and treat cardiometabolic diseases. Much of this information can be garnered using systems genetics, which takes population-based approaches to investigate how genetic variance contributes to complex traits. Despite the important advances made by human genome-wide association studies (GWAS) in this space, corroboration of these findings has been hampered by limitations including the inability to control environmental influence, limited access to pertinent metabolic tissues, and often, poor classification of diseases or phenotypes. A complementary approach to human GWAS is the utilisation of model systems such as genetically diverse mouse panels to study natural genetic and phenotypic variation in a controlled environment. Here, we review mouse genetic reference panels and the opportunities they provide for the study of cardiometabolic diseases and related traits. We discuss how the post-GWAS era has prompted a shift in focus from discovery of novel genetic variants to understanding gene function. Finally, we highlight key advantages and challenges of integrating complementary genetic and multi-omics data from human and mouse populations to advance biological discovery. Cardiometabolic diseases encompass a range of interrelated conditions that develop from underlying metabolic perturbations precipitated by genetic, environmental, and lifestyle factors. A significant burden of disease in developed countries is attributable to these conditions including coronary artery disease (CAD), heart failure (HF), obesity, type 2 diabetes (T2D), and non-alcoholic fatty liver disease (NAFLD) . TraditiIn this review, we provide an overview of mouse genetic reference panels and the opportunities they provide for the study of cardiometabolic diseases and relevant traits, with particular emphasis on the Hybrid Mouse Diversity Panel (HMDP). We discuss how systems genetics research has shifted focus in the post-genome-wide assocation study (GWAS) era from discovery of novel genetic variants, to understanding gene function. In doing so, we highlight the substantial contributions that systems geneticists have made to advance the field , while distal associations are referred to as trans (i.e. acting via distant genomic architecture). In the context of germline associations to molecular traits such as expression, DNA plays a regulatory role in the production of RNA and consequent protein, and therefore, co-mapping of a cis-eQTL with a given trait infers a potential causal relationship, although experimental validation is usually required for confirmation of such findings.Over 20 years ago, the Human Genome Project generated the first detailed annotation of the ~3 billion base pair human DNA sequence . This seHuman GWAS have uncovered many genes that contribute to complex diseases including NAFLD , CAD De, and obeIt is becoming increasingly evident that the major proportion of complex conditions, including cardiometabolic diseases, are influenced by hundreds to thousands of subtle genetic variants in combination , indicatSeveral large-scale human population studies have been undertaken to generate resources comprising clinical, molecular, genetic, and omics-derived data. A non-exhaustive list of relevant resources is provided in Dissection of the genetic component of disease is, however, further complicated by the variable influence of environmental factors on differing genetic backgrounds, known as gene-by-environment (G×E) interactions. Even traits that are highly heritable and penetrable, such as obesity, are amenable to environmental influence and indeed genetic background , which hCaenorhabditis elegans) (Drosophila melanogaster)  of model organisms including worms (elegans) , flies (ogaster) , mice (Bogaster) , and ratogaster)  are a coogaster) . Panels ogaster) , Collaboogaster) , Diversiogaster) , Um-Het3ogaster) , ILSXISSogaster) , and HMDogaster) . Geneticogaster) . Alternamplexity . This brmplexity . Importamplexity  is compamplexity .With the ongoing development of high-throughput sequencing technologies, mouse genomes can be readily sequenced at high fidelity, and through integration with complementary omics data, regulatory loci can be identified with high confidence. Whole genome sequencing is available for all BXD lines and many CC strains, while SNP array data is available for most inbred mouse panels . Since tex vivo genetic screen  F1 males  program and has since been utilised by the National Institute on Ageing (NIA) Interventions Testing Program (ITP), which aims to identify non-invasive interventions that extend lifespan and promote healthy ageing . The breF1 males . Thereforiations . The mairiations . Het3 miriations . Other sriations . Finallyriations .The HMDP consists of 100+ genetically diverse inbred strains of mice that have undergone extensive genotyping . The panThe HMDP has been studied in a basal setting and following genetic, pharmacological, and lifestyle interventions, to mimic features of cardiometabolic diseases or healthy interventions as described below. For example, to explore the genetic regulation of the hepatic and plasma lipidome, Parker and colleagues integrated genetic data with liver proteomics and lipidomics, as well as plasma lipidomics from 107 HMDP strains, in the absence of intervention . ThroughCETP) transgene  diet for 16 weeks  was identified across the Ath-HMDP in both the aorta and liver. This gene is found in the same locus as APOE and APOC1 and was speculated to partly mediate the effect of rs2075650  algorithm, this network was shown to be regulated by seven key driver genes, or ‘hub genes’. Such genes are central to the regulation of entire gene networks and thus would be obvious candidates for therapeutic intervention due to the widespread impact that would likely result from their manipulation. Indeed, independent small interfering RNA knockdown of four key driver genes activated other genes within the network and modulated THP-1 foam cell formation, validating their involvement in the regulation of atherosclerosis.Polygenic diseases are often influenced by the interaction of gene networks within and between multiple tissues. Network modelling approaches can assist in elucidating the genetic circuits within one or multiple tissues that drive biological or pathological processes. One such approach is weighted gene co-expression network analysis (WGCNA), a global expression analysis tool developed by SNPs in genes that regulate lipid and glucose pathways can contribute to atherosclerosis susceptibility . To expltrans-eQTL regulated by NADH:ubiquinone oxidoreductase core subunit V2 (Ndufv2) that controlled the expression of 89 genes, of which approximately one third were OXPHOS genes. Subsequent validation experiments confirmed a causal role for Ndufv2 in the sex-specific regulation of obesity via alterations in mitochondrial function.In a separate study, Chella Krishnan and colleagues demonstrated that adipose mitochondrial oxidative phosphorylation (OXPHOS) genes were elevated in female mice across the HF/HS-HMDP in a tissue-specific and sexually dimorphic manner. This effect was conserved across the human STARNET and GTEx cohorts . An assoin vivo, including plasma insulin and glucose levels following fasting, or in response to an oral glucose tolerance test  was identified and experimentally validated as a novel regulator of hepatic and plasma TG and diglyceride (DG) abundance . These f and T2D . These s and T2D . They coThe above studies performed ‘look-up’ type integration, with specific outcomes being tested for association. However, more non-directed approaches may provide broader utility in identifying novel, evolutionarily conserved drivers of complex disease. Indeed, in a recent study, Li and colleagues performed WGCNA using liver expression data obtained from the HMDP and DO mouse populations to construct mouse liver co-expression modules . The enrMAFF/Maff and ATF3/Atf3 to orchestrate a liver network enriched in genes linked to atherosclerosis. Bioinformatic analysis of liver gene expression data from STARNET and multiple HMDP cohorts indicated a potential role for MAFF/Maff and ATF3/Atf3 in the context-specific regulation of lipid and lipoprotein metabolism via the low-density lipoprotein receptor (LDLR/Ldlr). These findings were validated in silico and in vitro, suggesting that MAFF/Maff and ATF3/Atf3 regulate lipid metabolism and atherosclerosis in an inflammation-dependent manner.With regard to non-directed integration of data obtained from human and mouse populations, von Scheidt and colleagues comprehensively explored the literature and catalogued all the genes from experimental mouse models that significantly altered lesion size or plaque stability (827 genes from over 9000 publications) and compared them with orthologous human GWAS-derived genes for CAD (244 genes from 169 significant and suggestive GWAS loci). They demonstrated that only 46 orthologous GWAS-derived genes had been studied in mice, 45 of which significantly impacted atherosclerosis . Furthercis-eQTLs, including those for phosphatase and actin regulator 2 (Phactr2) and eukaryotic translation initiation factor 3 subunit H (Eif3h), the gene expression of which significantly correlated with liver fibrosis. In subsequent analyses, mouse hepatic gene expression data was compared to gene expression in liver explants of 68 individuals undergoing bariatric surgery, including a subset of individuals with histologically diagnosed NASH  that are likely to be causally associated with a given SNP. However, if phenotypic components themselves are tightly correlated, as is often the case for functionally related genes, proteins, and lipid species, this can inflate mediation statistics and increase the rate of false positives. Despite advances in statistical network modelling approaches such as WGCNA or Bayesian networking and its derivative KDA algorithm, these approaches are prone to false positive relationships due to inherent limitations in the modelling of complex and dynamic biological systems, or simply the result of bystander genes. Integrative approaches that combine the advantages of both human and mouse datasets to assign priority to the most promising candidate genes can help reduce the rate of false positive associations. However, this does not obviate the requirement for validation experiments to establish causality and confirm directionality. Further mechanistic insights can also be obtained either in the absence of tissue crosstalk using in vitro experimentation, or in the presence of multi-cellular and multi-organ crosstalk using in vivo studies, which are more representative of a biological system.An important aspect of systems genetics is the validation of candidate targets to establish causality. Indeed, statistical causal inference techniques such as in vivo , in mouse liver cells. This led to the identification of several high-confidence, cross-species conserved, cholesterol regulating genes, such as sestrin1 (Sesn1), which was functionally characterised to play a role in cholesterol biosynthesis using in vivo and in vitro systems. This is an elegant example of how systematic, data-driven validation studies can be used to assign priority to candidate genes and provide insight into the molecular mechanism of a gene under investigation.With thousands of loci having been identified for complex diseases, a remaining challenge is the prioritisation of high-confidence genes with therapeutic potential. For example, as mentioned above, Li and colleagues integrated mouse liver gene expression networks with data from a human lipid GWAS. This led to the identification of 25 novel genes implicated in cholesterol metabolism . Using aAlthough the ability of human GWAS to consistently identify the causal gene(s) within a given locus for complex traits has been impeded by numerous logistical challenges, such methodologies have pioneered the gene discovery revolution and have been the springboard to contemporary genetic analytical approaches. As a complementary approach to GWAS in humans, GRPs in model organisms such as mice have also been vastly informative. As stand-alone methods, both human and mouse GWAS have been successful in their own right at identifying causal genetic variants, however their integration stands to elevate their individual successes. We have highlighted here the many potential advantages of comparing, overlaying, and integrating complementary datasets between mice and humans to improve these outcomes. Indeed, more extensive integration of data from human and mouse populations can be leveraged to maximise biological discovery and clinical translation. However, more work is necessary to advance this knowledge towards prognostic, diagnostic, and therapeutic outcomes.GeneNetwork.org (Starnet.mssm.edu (CoffeeProt.com (Pheweb.org/metsim-metab) (Metabolomics.baker.edu.au)  , or Inst.edu.au)  would beWith the systems genetics field now at a major crossroads on how to best leverage contemporary methodologies including PGS and Mendelian randomisation, several questions remain that will ultimately influence the direction taken in years to come. The full potential of cross-species data integration remains to be seen, with the field continuing to evolve with regard to greater depth of sample analyses, larger cohort numbers, and high-throughput analytical approaches being developed. So, what is needed from existing infrastructure to convince both clinical and discovery scientists that genetic information is a powerful complementary tool to guide discovery and therapeutic translation? Do we need to integrate genetic information from multiple ethnicities, in larger numbers and continue to push the boundaries in computational science? Although identifying disease-associated variants is undoubtedly important, should we place more emphasis on overlaying multiple biological layers such as the abundance of genes, proteins, and other intermediate metabolites to improve our confidence in the most likely causal gene(s) and underlying biology? What does full integration of human and mouse data look like and how far should this be pursued? Can we perform cross-species meta-analysis to generate more precise and powerful comparisons? Can we develop an approach to comprehensively define functional orthology across species to facilitate biological interpretation between humans and mice? Whatever the answers may be, the field must work through these challenges in a systematic and collaborative manner. Undoubtedly, these approaches, combined with addressing the abovementioned limitations, are likely to provide unique opportunities for discovery and clinical translation to improve human health and pave the way for a new direction for the field of systems genetics, greatly facilitating progress towards precision medicine.


Post-traumatic segmental renal infarction is an extremely rare event, especially in case of minor blunt abdominal trauma. While major trauma guidelines are well established, several problems account for the adequate management of minor trauma. Herein, we report a case of minor blunt abdominal trauma determining traumatic thrombosis of the apical renal artery and segmental renal infarction, firstly diagnosed by CEUS in emergency care setting. Renal injury accounts for approximately 1–5% of all trauma admissions and as many as 10% of patients who sustain abdominal trauma . HoweverA 21-year-old male patient referred to our Emergency department with a history of minor blunt abdominal trauma sustained after falling off a bunk bed. The patient complained of pain localized to the left lumbar and hypochondriac region. No external wound or ecchymosis was noted at physical examination.The patient was hemodynamically stable . Laboratory tests were unremarkable , no macro or microhematuria was detected.Because of the absence of any criteria for major abdominal trauma  and no risk factors , 9, the After microbubbles were administered intravenously, a cortico medullary anechoic band-like area was immediately detected at the upper pole of the left kidney and after 32 s the absence of enhancement was persistent and homogenous. At 45 s, the upper pole remained avascular (Figs. E-FAST is a useful bedside tool for ruling in pneumothorax, pericardial effusion, and intra-abdominal free fluid in the trauma setting. However, FAST examination is an imperfect test. Indeed, its sensitivity is extremely variable between 41 and 95%, and has not to be used to rule out intra-abdominal injuries –15. ThusPatients with minor trauma are usually managed with FAST examination and segmental x rays, depending on clinical symptoms. In case of negative e-FAST, and with no clinical or laboratory alterations during the observation period, patients are usually dismissed. However, even in cases of negative FAST examinations, in patients where a clinical or laboratory alterations may lead to the persistence of any suspicion of misdiagnosed conditions, repeated laboratory analysis, and serial FAST examination are required. At serial ultrasound examination, additional ultrasound view and B-mode examination of parenchyma are performed to increase ultrasound sensitivity , 16. HowWe reported an interesting case of a multimodality approach to minor blunt abdominal trauma in a young patient. Particularly, our case underlines the CEUS's role in minor trauma. CEUS can be extremely useful in screening patients requiring second-level imaging, reporting no alterations at the E-FAST examinations. It may represent a problem-solving imaging method and a speeding-up modality for the prompt management of patients.Particularly, although more studies are needed to confirm these results, CEUS examination may be proposed as first-line imaging in patients with a negative FAST examination reporting focused symptoms after minor blunt abdominal trauma. Indeed, the diagnostic value of CEUS has been showed to be higher than the conventional ultrasound examination . NotablyRenal trauma is responsible for 30.8% of acute renal infarction, meanwhile, post-traumatic segmental renal infarction is an extremely rare event. Segmental renal infarction has been reported secondary to cardiogenic and systemic conditions or iatrogenic causes, traumatic segmental renal infarction secondary to a thrombosed segmental artery is an exceptional event .The rate of thrombosis of the renal artery after renal trauma is estimated around 2% , and isoAvascular lesions, such as infarcts, are confidently identified at CEUS examination; infarcts appear on CEUS as wedge-shaped areas showing no enhancement. In our case, the avascular lesion presented a band-like morphology. The band-like appearance of the infarct was due to a segmental apical artery according to pre-segmental anatomical branching of renal arteries, the knowledge of segmental and pre-segmental renal artery branching is important to correctly identify vascular lesions, which may present different wedge-shaped or band-like morphologies, depending on the arterial distribution.In our case, we were not able to identify the cortical rim signs, probably due to the cute and traumatic event. However, MDCT with intravenous contrast confirmed the infarcted area on the upper pole of the left kidney, identifying the renal pre-segmental artery branching and the traumatic thrombosis of the apical artery.Our case demonstrated the potential central role that CEUS examination may have in the management of minor blunt trauma in clinically symptomatic patients with a negative FAST examination. However, more studies are needed to confirm the role of CEUS in these emergency situations. Post-traumatic Segmental Renal Infarction due to Renal Apical Artery Thrombosis is an exceptional event and the knowledge of renal artery segmentation is important to characterize the avascular region. In our case, CEUS correctly identified the renal infarct, and justified the second-level imaging with MDCT with intravenous contrast that accurately demonstrated the renal artery branching and apical artery thrombosis. Hence, CEUS examination may represent a first-line diagnostic tool to improve the correct selection of patients needing second line examinations, improving the diagnostic sensibility of clinically symptomatic patients showing no alterations at FAST examination. However, more studies are needed to confirm our results, and to clarify the potential role of CEUS.


Processes that damage the optic nerve, including elevated intraocular pressure, trauma, ischemia, and compression, often cause visual loss for which there is no current treatment. It has long been believed that patients who experience damage to the optic nerve will never regain useful vision because the nerve cannot regenerate or repair itself. This belief is based on three assumptions: 1) a mammalian retinal ganglion cell (RGC) cannot be prevented from dying once its cell body or its axon has been injured; (2) an injured mammalian RGC whose axon has degenerated cannot be induced to extend a new axon; and (3) even if an injured mammalian RGC could be induced to regenerate, the regenerating axon cannot be directed toward its correct target in the central nervous system (CNS) [ a mammalThe optic nerve, similar to most pathways in the mature central nervous system, cannot regenerate if injured, and within days, RGCs begin to die. Research over the past two decades has identified several strategies to enable RGCs to regenerate axons the entire length of the optic nerve, in some cases leading to modest reinnervation of di- and mesencephalic visual relay centers. A review by Wong and Benowitz  primarilEpigenetic factors are known to influence tissue development, functionality, and their response to pathophysiology. In their review, Ashok et al.  focus onThe goal of neuroprotection in optic neuropathies is to prevent loss of RGCs and to preserve their function. The ideal time window for initiating neuroprotective treatments should be the preclinical period at which RGCs start losing their functional integrity before dying. In their review, Porciatti et al.  discuss One of the most interesting therapies for a variety of acute optic neuropathies is erythropoietin (EPO), which has been shown to have neuroprotective properties in extra-hematopoietic tissues, especially the retina. It is postulated that EPO may interact with its heterodimer receptor (EPOR/βcR) to exert its anti-apoptosis, anti-inflammatory, and anti-oxidation effects in preventing RGC death through different intracellular signaling pathways. In their review, Lai et al.  summarizPrimary open angle glaucoma (POAG), a chronic optic neuropathy, remains the leading cause of irreversible blindness worldwide. It is driven in part by the pro-fibrotic cytokine transforming growth factor beta (TGF-β) and leads to extracellular matrix remodeling at the lamina cribrosa of the optic nerve head. Despite an array of medical and surgical treatments targeting the only known modifiable risk factor, raised intraocular pressure, many patients still progress and develop significant visual field loss and eventual blindness. The search for alternative treatment strategies targeting the underlying fibrotic transformation in the optic nerve head and trabecular meshwork in glaucoma is ongoing. MicroRNAs are small non-coding RNAs known to regulate post-transcriptional gene expression. Extensive research has been undertaken to uncover the complex role of miRNAs in gene expression and miRNA dysregulation in fibrotic diseases. MiR-29 is a family of miRNAs which are strongly anti-fibrotic in their effects on the TGF-β signaling pathway and the regulation of extracellular matrix production and deposition. In their review, Smyth et al.  discuss A reduction in intraocular pressure remains the only proven treatment for POAG, but it does not prevent further neurodegeneration. In their review, Strickland et al.  discuss Optic neuritis is an inflammatory condition involving the optic nerve and is the most common acute optic neuropathy in young adults. Optic neuritis can be idiopathic or represent an early manifestation of demyelinating diseases, mostly multiple sclerosis (MS), at least in the Western hemisphere. Other causes include antibody-driven optic neuritis associated with neuromyelitis optica spectrum disorder (NMOSD), myelin oligodendrocyte glycoprotein antibody disease (MOGAD), chronic/relapsing inflammatory optic neuropathy , sarcoidosis, and a variety of infectious causes such as Lyme disease, Cat Scratch disease, syphilis, and tuberculosis. Appropriate and timely diagnosis is essential to rapidly decide on the appropriate treatment, maximize visual recovery, and minimize recurrences. Saitakis and Chwalisz  review tAs noted in the review by Saitakis and Chwalisz , one of Non-arteritic anterior ischemic optic neuropathy (NAION) is the most common cause of sudden optic nerve (ON)-related vision loss in humans. Fortunately, there are several animal models of the condition. In particular, the rodent NAION model (rNAION) closely resembles clinical NAION in its pathophysiological changes and physiological responses and enables analyses of the specific responses to sudden ischemic axonopathy and of the effectiveness of potential treatments. However, there are anatomic and genetic differences between human and rodent optic nerves, and the inducing factors for the human disease and the model are different. These variables can result in marked differences in lesion development between the two species, as well as differences in the possible responses to various treatments. Bernstein et al.  discuss Leber hereditary optic neuropathy (LHON) is the most common primary mitochondrial DNA disorder. It is characterized by bilateral severe central subacute vision loss due to specific loss of RGCs and their axons. Historically, treatment options have been quite limited, but ongoing clinical trials show promise, with significant advances being made in the testing of free radical scavengers and gene therapy. Spiegel and Sadun  summarizThe activation of G-protein-coupled receptor 110 (GPR110) has been shown to stimulate neurite extension in developing neurons and after axon injury in adult mice. In the first paper in this issue, Kwon et al.  report tToomey et al.  remind uSome animal species have the potential for partial or complete regeneration of neural tissue. Sugitani et al.  point ouPioglitazone (PGZ) is a drug that selectively stimulates the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-γ) and to a lesser extent PPAR-α. It modulates the transcription of the genes involved in the control of glucose and lipid metabolism in various tissues. Sun et al.  assessedThe most common mutation causing LHON is at site 11778 and is transmitted  to all maternal lineages. However, not everyone harboring the 11778 mutation develops LHON and men are much more often affected than woman. Nuclear modifier genes have been presumed to affect the penetrance of LHON, but conventional genetic methods have failed to clarify these issues. Cheng et al.  performeAzithromycin is an antibiotic that also has been shown to be neuroprotective in some studies. Zloto et al.  assess tAnother drug that has been shown to exhibit RGC protection is granulocyte colony-stimulating factor (GCSF); however, the mechanisms by which this occurs are unclear. To investigate the mechanisms involved in RGC protection by GCSF, Tsai et al.  examinedThe AQP4 autoantibodies found in most patients with NMOSD are believed to cross the blood–brain barrier, target astrocytes, activate complement, and eventually lead to astrocyte destruction, demyelination, and axonal damage. However, it is still not clear what the primary pathological event is. Zveik et al.  hypothesFinally, we are grateful to all the invited researchers who contributed to this Special Issue dealing with optic nerve regeneration and repair. Hopefully, these contributions will have both new and lasting impacts on our quest to restore vision to those who suffer from both acute and chronic optic nerve damage.


Movement control is among the top brain-computer interface (BCI) application scenarios for potential end-users. Movement control could involve moving a computer cursor on a screen, controlling a robotic arm, or reanimating one's own limbs. Current approaches vary in numerous factors, including signal modality [ranging from single unit spiking activity up to scalp-based electroencephalographic (EEG) recordings], control strategies, decoding algorithms, artifact mitigation, and feedback modalities . In spite of this diversity, there are shared key challenges that must be solved in order to deliver dependable assistive technology that will meet the needs of end-users. In addition to safety, availability, and portability, a dependable BCI has to eliminate or alleviate time-consuming system (re-)calibration processes, be independent of artifacts introduced by body movements or external stimulations, and generalize to various movement tasks with different dynamics.The goal of this Research Topic was to share recent advances among diverse approaches and, thereby, facilitate exchange between groups and research directions. We aimed for a scope ranging from original neuroscientific studies investigating neural correlates of goal-directed movements to neural engineering applications toward dependable BCIs for movement control.In this Research Topic we collected four original scientific papers which shed light to specific aspects of the quest to develop dependable BCI technology. All of the articles use non-implanted neural recording technologies. Three manuscripts use EEG recordings of brain activity, while the fourth manuscript uses surface electromyography (sEMG) recordings of muscle activity during a large variety on hand movements.BCIs rely on an understanding of the neural correlates of movement at the level of the brain, however in order to restore natural movement, it is also important to understand the peripheral activity of these movements, i.e., the EMG activity, to be able to further connect movement generation with movement execution in all levels.Peng et al. proposed a method combining feature selection and Ensemble Extreme Learning Machine (EELM) to improve the recognition performance based on sEMG signals. The authors compared the suggested methods with classical ensemble classifiers on a data set with 52 different hand movements from 10 able-bodied participants. Ultimately, sEMG could be used to classify these different movements with >75% accuracy.To enhance accuracy and stability of gesture recognition based on sEMG, Zhou et al. conducted a study to evaluate whether neurofeedback training based on up-regulation of the alpha band relative power had an effect on motor imagery BCI performance. A general finding was that such alpha neurofeedback training could successfully help study participants increase their alpha rhythm power and improve their motor imagery BCI performance. Also, on an individual level, participants with increased alpha power had a larger performance improvement.Many BCIs rely on motor imagery to generate consistent patterns of neural activity that can be used as a control signal, however imagery performance can vary widely from person-to-person. In earlier studies, it was found that resting-state alpha-band power, also described as mu-band power, is positively correlated with motor imagery-based BCI performance. Plucknett et al. and Srisrisawang and Müller-Putz studied feature representations to aid subsequent decoding pipelines with regard to performance increases and interpretation.The next two papers in the collection, Plucknett et al. investigated how various combinations of feature extraction and metric learning algorithms affected feature representations in a self-paced, binary center-out movement task. As a supervised dimensionality reduction technique, the goal in metric learning is to project data in a low-dimensional space that maintains discriminative information. In their study with a publicly available EEG dataset comprising two subjects, they report that the considered metric learning algorithms did not improve classification performance but were useful to identify the importance of EEG channels encoding discriminative information. Their findings confirmed the importance of low-frequency (<5Hz) EEG activity in movement discrimination tasks.Srisrisawang and Müller-Putz, they tackle the problem of co-linearity due to the higher number of dimensions in the source space by two folds: (i) they selected signals in predefined regions of interest (ROIs) and (ii) they applied dimensionality reduction techniques like computing the mean (Mean), principal component analysis (PCA), and locality preserving projections (LPP) in pre-defined ROIs. Also the effect of using individual head models compared to a template model was investigated. Statistical tests revealed no significant differences between the source-space and sensor-space approaches when decoding reach velocities. Similarly, there was no significant difference between subject-specific and template head models. While no significant differences in performance were noted for correlation-based decoding of velocity, further investigations are still necessary when it comes to other decoding approaches such as a classification task.The majority of EEG BCI studies for hand trajectory decoding rely on low frequency activity using features in sensor space, rather than source space which estimates the activity at locations in the brain from the sensor activity using inverse modeling techniques. In the study of Peng et al.) and EEG  and one EEG-based neurofeedback training . Despite the progress made in these and other works, further research is required toward establishing dependable BCIs for movement control.All articles in this collection addressed important issues concerning BCIs for movement control. Three papers investigated feature extraction and classification methods from EMG (All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


The ability to model physiological systems through 3D neural in-vitro systems may enable new treatments for various diseases while lowering the need for challenging animal and human testing. Creating such an environment, and even more impactful, one that mimics human brain tissue under mechanical stimulation, would be extremely useful to study a range of human-specific biological processes and conditions related to brain trauma. One approach is to use human cerebral organoids (hCOs) in-vitro models. hCOs recreate key cytoarchitectural features of the human brain, distinguishing themselves from more traditional 2D cultures and organ-on-a-chip models, as well as in-vivo animal models. Here, we propose a novel approach to emulate mild and moderate traumatic brain injury (TBI) using hCOs that undergo strain rates indicative of TBI. We subjected the hCOs to mild (2 s TBI is incurred to the brain due to a blow, foreign object penetrating the skull, or acceleration-deceleration forces2. Most injuries occur during collision sports, motor vehicle accidents, and military combat. There are mitigation strategies like wearing proper headgear and seat belts to reduce the risk of head injuries; nevertheless, people are still susceptible to TBI as these countermeasures do not completely inhibit the mechanical impact on the brain. Research shows traumatic brain injury is linked to symptoms like memory loss, headache, fatigue, and photosensitivity4. Additionally, TBI presents a risk factor for Alzheimer’s disease and neurodegenerative syndromes, including Chronic Traumatic Encephalitis (CTE)5. Mostly, TBI sequelae are not curable but can only be partially addressed and symptomatically treated with pharmaceutical-, physical-, cognitive-, speech-, psychological-, occupational-, and behavioral therapy10. Methods to quantify TBI-related changes in gene expression remain elusive, and there are no treatments to reverse any damage to the brain after injury.Traumatic brain injury (TBI) hospitalized 223,135 people in 2019 and caused the death of 64,362 people in 2020 in the United States12, atherosclerosis14, and pulmonary fibrosis16. Evidence shows that TBI can trigger similar inflammatory responses in the brain19. However, investigations of potential impact of TBI on gene expression, epigenetic modifications, and other processes that can influence the functioning of genes and cellular pathways over an extended period following the injury are nascent. Most studies are in mice and rats20 and human testing of TBI is primarily limited to postmortem studies or clinical imaging22. The population with the highest risk of TBI  demonstrate similar clinical course and outcomes, including TBI-induced structural brain abnormalities and post-TBI psychiatric complications28. Thus, the high prevalence of TBI and TBI-induced clinical complications warrant the need to characterize the genomic landscape and develop a suitable in-vitro model for this disease.The mechanical stimulation of cells plays a continuous role in the development, growth, remodeling, and maintenance of biological systems. The mechanotransduction response of cells can trigger a range of chronic diseases, including osteoarthritis33. These approaches have provided tremendous benefits that complement mice models with many enhanced benefits. The most immediate benefit is that these organoids are composed of human tissue, likely having a better representation for a 3D arrangement for human disease models. Further, organoids provide an advantage in genomic editing, allowing for the creation of specific mutations to mimic specific diseases where the genomic insult is known. They can also introduce corrections to the genome to restore wild-type function where hCOs have been created directly from patients with specific diseases. Finally, hCOs allow us to model more complex diseases by introducing genetic mutations into these human cerebral models, understanding disease initiation and progression better, and making better treatment decisions that mouse models could not achieve35.Organoids produced from iPSCs (induced pluripotent stem cells) or hESCs (human embryonic stem cells), have been proven effective in modeling various diseases, including microcephaly, autism, schizophrenia, and cancer36. Our work takes a different path by focusing on only human-specific models for TBI research. Similarly, Shoemaker et al. made significant contributions to the field by utilizing fluorescence and absorption techniques to visualize trauma-related differences in human cortical spheroids37. Their work shed light on certain aspects of TBI pathology. Our study employed RNA sequencing as a powerful tool to identify and analyze gene products associated with different TBI conditions.Ramirez et al. conducted a groundbreaking study that merits recognition, as they were the first to utilize cerebral organoids for modeling traumatic brain injury (TBI). Their innovative approach using penetrative techniques and slow compression methods on human cerebral organoids and mouse models provided valuable insights into TBI mechanismsOur work highlights the ability to capture and analyze gene expression patterns at different TBI stimulation profiles. While previous TBI hCO research observed TBI effects through fluorescence imaging, our approach enables a deeper understanding of the underlying molecular mechanisms involved. By leveraging different techniques and focusing on human-specific models, our research offers a distinct perspective that complements and expands upon their findings.We propose a novel approach to emulate mild and moderate TBI on human cerebral organoids by uniaxially compressing them with relevant strain rates and observing how TBI influences neuron signaling and gene expression in the human cerebral organoids Fig. . We asseWe developed an integrated approach to mechanically stimulate human cerebral organoids. Our method combines multi-cellular systems within a more neural mimetic approach to better understand the neural response to mechanical stimulation similar to TBI. We used our custom-designed mechanical stimulation system through a highly controlled voice coil actuator to stimulate the organoids, varying the strain rates to analyze different mechanical signatures related to TBI. In this, we collected the organoids and analyzed them using RNA sequencing to understand the genetic responses of our multi-cellular systems. Moreover, we examined real-time cellular calcium response to understand how cells responded to a known mechanotransduction calcium signaling pathway. This approach also allowed us to compare the genetic response signatures to other well-known neural and mechanically related findings, further enhancing our understanding of how mechanical loading affects neural-related systems.33. We achieved fully formed organoids to model the human brain starting with human embryonic stem cells, which we cultured in a 4-week period, to develop embryoid bodies, neural ectoderm, and cerebral organoid tissue  approach44 and found in trauma45. Interestingly, calcium is a significant factor in the execution of this process46. The NAD biosynthesis pathway acts as a critical regulator of physiologic homeostasis in the face of infection, circadian disorder, and inflammation47. The interleukin 2 (IL-2) pathway has had increasing evidence of its involvement in trauma. For example, there is evidence of high levels of IL-2 receptor in trauma48; however the trauma was burn patients. More specifically, in TBI patients, IL-2 has been shown to activate regulatory T cells to suppress neuronal inflammation and, therefore, may act as a means of trying to alleviate the symptoms associated with trauma49. To explore the particular genes that are associated with mild and moderate mechanotransduction of hCOs, we examined the differential expression of hCOs under mild and moderate mechanotransduction. We ran an unsupervised analysis of all three conditions of hCOs that were studied and visualized in a heatmap where the gene expression for control, mild and moderate hCOs show distinct gene activation  promotes cell death, and its activation has been associated with inflammation51. FOXP3 (Forkhead Box P3) has been implicated in Immunodysregulation53 with its primary functions include DNA-binding transcription factor activity and sequence-specific DNA binding.Building on our analysis from RNA sequencing, we explored the gene ontology as it related to hCO mechanotransduction Fig. A. ConsisTo mimic TBI injury on human cerebral organoids (hCOs) and view calcium signaling after injury, we used our Mechanical Excitation Testbed Fig. A. The TBOur research focuses on using human cerebral organoids, which are human-specific 3D brain models, to address significant challenges in treating traumatic brain injury. Current-day treatment for TBI includes rest, over-the-counter pain relievers, anti-seizure drugs, diuretics, coma-inducing drugs, and surgery—all depending on the degree of brain injury. After these initial treatments, rehabilitation is the next step for treatment. Some patients improve with these methods, and others do not. This treatment method is the extent of clinical practice.The physical consequences of traumatic brain injury are more readily apparent than nonphysical ones, which motivated our research to investigate the extent of gene expression changes following such injuries. Our study has revealed previously concealed genetic expression effects that can impact gene functioning and cellular pathways, as evidenced by our genetic and cellular mechanical stimulation experiments. Specifically, using our human-specific model, we conducted genetic analyses that demonstrated specific genetic products based on the severity of the injury.Furthermore, our investigations demonstrated an increasingly pronounced calcium signaling response in neuronal cells as we applied greater mechanical stimulation, indicating a heightened mechanotransduction reaction under escalating mechanical insults. Utilizing human cerebral organoids (hCOs) provided a physiologically relevant model specific to humans, enabling us to gain valuable insights into the neuronal response to traumatic brain injuries.Notably, the genes expressed under increasing mechanical insult have been associated with neurodegenerative diseases like Hashimoto’s Encephalopathy, genes that regulate cell death and inflammation, and immune dysregulation, all of which can negatively affect patients with TBI. These findings highlight the relevance and potential clinical implications of our research.Through our work, we identified genetic products that arise from TBI. This critical step enables us to pave the way for personalized treatments that focus on the specific patient and can precisely target essential gene products and biological pathways, ultimately halting or potentially reversing the progression of the TBI-induced disease. By leveraging drugs already in production, our research holds promise for developing effective therapies that can address the newly identified genetic products derived from TBI.56, postmortem examinations of human subjects58, and studies involving living human subjects60. Some previous research has explored the versatility of disease modeling using organoids to model Microcephaly29 and the Zika virus61. Additionally, TBI has been studied for TBI37. These studies have provided valuable insights into TBI’s phenotypic cellular effects, with one particular TBI study utilizing mouse models in conjunction with the organoid models. However, the previous studies on TBI did not investigate the differences in calcium or genetic products between mild and moderate traumatic brain injuries.Previous research on traumatic brain injury (TBI) These studies employed various methodologies, including animal modelsHuman cerebral organoids present a remarkable opportunity to bridge the gap between studies conducted on animal models and direct human experimentation by providing a personalized, rapid, and robust laboratory-constructed model. By leveraging this innovative approach, we aim to advance the understanding of traumatic brain injury (TBI) and its disease onset.62. Briefly, we plated hESCs reaching 80% confluency in low attachment 96-well plates. From this, embryoid bodies formed. We fed them every other day for 5 days with a 50 We cultured human embryonic stem cells (hESCs) using a modified version of the Linkous protocol63.For the compression of organoids, we used the Mechanical Excitation Testbed (MET). The MET comprises a voice coil actuator, a z-axis positioner, and a precision end effector, giving us access to a wide range of frequencies to simulate TBI and precisely compress the organoid with controlled strain rates and strain for a controlled number of pulses. This system allows us to recreate a more realistic 3D simulation environment for the hCOs. Additional details on the MET can be found in Bobo 2020 Fig. B63.64. However, since we are using cerebral organoids, we want to understand the mechanical conditions to mimic these injuries. We control the strain imposed on the organoid and the strain rate with the VCA and microcontroller, and we use these to mimic the mechanics of the brain under dynamic loading , which is a cell-permeant CaTo better understand neural identity, activity, and 3D organization of cerebral organoids as a response to mechanics through calcium releaseWe did RNA sequencing on the organoids, which included differential gene expression analysis and functional analysis to observe how TBI influenced the genetic expression in the cerebral organoids. After obtaining the raw data, we obtained an error rate distribution, GC-content distribution, and Data Filtering for a quality check. Next, we examined a mapping of the genes to the brain reference genome. Then, we split between the gene expression quantification, where we received correlation analysis-quality contract check on samples between samples. Finally, we implemented differential expression analysis and functional analysis focusing on enrichment analysis in Gene Ontology.71, which takes the sum of the gray values of all the pixels in our 512 To observe neuron signaling in the human cerebral organoids, we measured the intensity of the Fluo-4 AM dyed hCOs with a Zeiss Spinning Disk Confocal Microscope (Axio Observer Z1 System, Supplementary Information.


Drosophila and higher organisms. Consequently, the fruit fly is an excellent in vivo model for identifying the genes and mechanisms involved in muscle development. Moreover, there is growing evidence indicating that specific conserved genes and signaling pathways govern the formation of tissues that connect the muscles to the skeleton. In this review, we present an overview of the different stages of tendon development, from the specification of tendon progenitors to the assembly of a stable myotendinous junction across three different myogenic contexts in Drosophila: larval, flight and leg muscle development. We underline the different aspects of tendon cell specification and differentiation in embryo and during metamorphosis that result into tendon morphological and functional diversity.Myogenesis is a developmental process that is largely conserved in both  Drosophila melanogaster is an established model used to better understand the cellular and molecular events in the conserved myogenesis process /Egr-like transcription factor  with specialized tendon-like cells . Both tendon-like cells and muscles, but also other tissues such as fat body, secrete extracellular matrix proteins that form the equivalent of a Myo-Tendinous Junction (MTJ) present in vertebrates. As a holometabolous flying insect,  jumping . Accordidon cell . The powdon cell  and tenddon cell . Whereasn factor , subsequHere we review existing knowledge on the development of tendons that connect with larval and insect flight and leg muscles, in addition to recent findings that emphasize reciprocal interactions between developing tendons and muscles.Drosophila, all tendon cells are characterized by the expression of stripe (sr), which is the earliest known marker of tendon cell specification  within the epidermis that can attract muscle fibers  and Wingless (Wg) signaling pathways (sr. In the most anterior row (n°1), sr expression is induced through Ci-mediated Hh signaling in the Rho+ domain  that are present on the surface of the wing and leg discs, respectively . Wing ansr . Althougsr is expressed in five clusters that are confined to the notum region, which gives rise to the thoracic dorsal body wall , a LIM-homeodomain protein  that create the notum prepattern (achaete-scute complex (ac-sc), which encodes transcription factors that regulate the development of sensory bristles  and thisbe (ths), two secreted ligands for the FGF receptor Heartless (Htl), is not restricted to specific tendon cells and is not directly expressed by tendon cells, their broad expression in the ectoderm appears to globally direct myotube leading edges toward their attachment sites  inhibits its sr mRNA nuclear export leading to its degradation, whereas How (S) stabilizes it and favors srA isoform splicing leading to the upregulation of SrA protein . Dei and Sr regulate the expression of cell adhesion molecules required for the establishment of the MTJ .sr (see above). However, only a few studies have examined the cellular and molecular mechanisms that control the later development of flight and leg muscle tendons. In the wing disc, Broad-complex transcription factors, upon 20-hydroxyecdysone regulation, are potential regulators of tendon cell terminal differentiation to ensure the correct attachment of flight muscles . However, the reduction in sr expression precludes the elongation of the tendon, suggesting that leg disc sr is required to trigger a differentiation program that allows the collective migration of these cells   . Moreovemuscles) . Consequithelium . Strikinithelium . Moreoveithelium . These omultiple edematous wing (mew/αPS1), inflated (if/αPS2) and myospheroid (mys/βPS). αPS and 1βPS subunits first heterodimerize at the tendon membrane, whereas the αPS2 and βPS link together slightly later on the muscle side to consolidate the MTJ (slowdown (slow) mutants. Slow is secreted by tendon cells and when mutated, Tsp accumulates prematurely at the growing end of the muscle leading to the formation of a weak muscle-tendon junction  that connect to the actin cytoskeleton. These molecules were identified primarily by studying the MTJ of larval muscles, whose role was thoroughly reviewed by  the MTJ . One of  the MTJ . The Tspjunction . Moreovejunction . Thus, ijunction , Kon con the MTJ .As mentioned above, other molecules have been found to participate in the MTJ and have been already listed by dpy or qsm mutated alleles display cuticle depressions at flight muscle attachment sites (Drosophila Rho-kinase (DRok) to regulate elastic properties of the actin cytoskeleton to maintain the shape of the epithelium and polarity during the muscle compaction process , the tendon cell must also bind to the cuticle (chitin-rich exoskeleton). As in vertebrate, tendons are at the intersection between smooth contractile tissue (muscle) and rigid tissue (skeleton or exoskeleton). During myogenesis, mechanical tension is required for myofibril sarcomere organization and formation . Howevernt sites . This prnt sites . Within nt sites . Chas acnt sites . It has nt sites . Accordi process . Interes process .Drosophila model shows a variety of muscle attachments that reflect the diversity of its somatic musculature. Drosophila tendons differ in several aspects: single cell or monolayer cells that ensure larval and flight muscle attachments are morphologically distinct from tube-shape tendons of the leg, different combinations of signaling pathways are required to specify the tendon cell fate in these different systems and specific transcription factors are differentially expressed in different tendons. Therefore, Drosophila are of great interest to uncover the developmental program of each tendon type. For instance, pioneer work has identified Sr/EGR as a central regulator of tendon cell fate, however more recent studies have identified a few new conserved factors that contribute to underlying specification and differentiation of particular tendons, thereby participating in tendon diversity. With the rapid development of high-throughput sequencing technology, comparative transcriptomic analysis would enhance our knowledge of the molecular mechanisms underlying tendon diversity during development. Lastly, tendon diversity may also result into differential composition of ECM between tendon types. In vertebrates, Collagen proteins are the major components of the MTJ (Drosophila muscle-tendon adhesion is less well-documented (Tendons are part of connective tissues precursors that are recognized as an important source of extrinsic cues that regulate skeletal muscle differentiation, growth, and patterning. Despite their critical importance, the understanding of connective tissue development lags behind that of other musculoskeletal system components in vertebrates. As described in this review, the  the MTJ  whereas cumented . It woulFinally, tendons and muscle connective tissues in general are associated with major clinical challenges, including tendon scarring and muscle dystrophies. A greater understanding of connective tissue cell differentiation and how they interconnect with muscles and the skeleton is critical to address mechanisms that contribute to pathologies.