Background and Objectives: Caesarean scar pregnancy (CSP) refers to placental implantation on or in the scar of a previous caesarean section and represents a potentially life-threatening condition. CSP is considered a diagnostic challenge in obstetrics, with the diagnosis relying mainly on transvaginal ultrasound (TVS) and the management depending upon case presentation and available healthcare infrastructures. Case Presentation: We present a case of 34-year-old G3P2 with a history of two-previous caesarean sections referred to the outpatient gynaecology clinic of our Department at the 7th week (7/40) of gestation with abnormal early pregnancy TVS findings, illustrating the gestational sac attached to the caesarean scar and a foetal pole with evidence of foetal cardiac activity. We discuss the outcome of an alternative combined medical and surgical approach we followed as well as an updated review of the current literature. Conclusions: The ideal management of CSP requires tertiary centers, equipment availability and experienced healthcare professionals capable of dealing with any possible complication, as well as individualized treatment based on each case presentation. Caesarean Scar Pregnancy (CSP) represents one of the rarest forms of endometrial pregnancy ,2. CesarA 34-year-old gravida 3 para 2 (G3P2) woman with a history of two previous caesarean sections was referred by a physician to the early pregnancy outpatient clinic of the University Hospital of Larisa during the 7th week (7/40) of gestation due to abnormal transvaginal ultrasound (TVS) findings, illustrating the gestational sac attached to the caesarean scar and a fetal pole with evidence of fetal cardiac activity . She had2) was given [Initially, after informing the patient accordingly and obtaining written consent, an unsuccessful attempt for pregnancy termination by injecting potassium chloride (KCl) in the gestational sac by ultrasonographic guidance was performed ,7,8,9. Oas given ,11. Sequas given . The iniAt that time the medical team considered the possibility of a second dose of methotrexate. However, surgical intervention was favored, and one day later (8th day) decision for a diagnostic hysteroscopy and suction of the scar pregnancy was taken. In particular, suction curettage and foley bulb induction approach was chosen and the patient received counselling and consented accordingly prior to the procedure . The labApproximately, two hours prior to the diagnostic hysteroscopy the patient received 800 μg of misoprostol sublingually. Under general anesthesia and ultrasonographic guidance, dilatation of the cervix with Hegars’ was performed and a Karman’s cannula number 4 was inserted to access the sac level via the cervix, followed by suction. Subsequently, a 16 French foley catheter was placed on the sac’s level and the balloon was inflated by saline until the bleeding was stopped, while simultaneously, 10 IU of oxytocin were administered intravenously. The patient recovered with hemodynamical stability and no evidence of intraoperative complications. Twenty-four hours postoperatively the β-hCG levels reached 39,531 mIU/mL. Thereafter, the foley catheter was removed.The patient was discharged from the hospital at postoperative day 2 and one week later histopathological assessment was completed, reporting on products of conception that correspond to a 1st trimester pregnancy and no presence of trophoblastic disease. The β-hCG serum levels were undetectable on the day 24 after surgical intervention .The first case of a CSP was reported in medical bibliography in 1978 in a G2P1 23 years old South African Zulu woman . As resuThe actual mechanism generating this condition remains uncertain. A variety of theories have been proposed so far including: (a) the endogenous migration of the gestational sac through either a wedge defect in the lower uterine segment or a microscopic fistula within the scar ,15; (b) It has to be underlined that scar pregnancy represents a different pathology compared to that of an intrauterine pregnancy with placenta accreta. In cases with placenta accreta formation, the products of conception are primarily settled in the uterine cavity and the leading cause of fluctuating degrees of invasion of the myometrium by trophoblastic tissues is the absence of decidua basalis . In scarTwo different types of scar pregnancies have been identified. Type I is believed to be caused by implantation in the scar tissue of the previous caesarean section with expansion towards the cervico-isthmic space or the uterine cavity ,23. In tThe primary and optimal CSP’s diagnostic modality remains transvaginal ultrasonography in the 1st or early 2nd trimester, which provides high resolution; however, color Doppler in combination to grayscale evaluation is also recommended, allowing detailed visualization of the placental site implantation as well as definition of fetal and extraembryonic structures . The TypThe ultrasonographic diagnostic findings suggestive of CSP may also include: (1) an empty endometrial and endocervical cavity; (2), a nested gestational sac and placenta, on/in the scar; (3) a triangular ≤8/40 weeks), rounded or oval shaped gestational sac (≥8/40 weeks) filling the scar “niche” ; (4) a thin (1–3 mm) or absent layer of myometrium between the urinary bladder and the gestational sac; 5) a distinct or rich vascular pattern around the area of the scar, and (6) an embryonic or fetal pole, yolk sac, or both with presence or absence of fetal cardiac activity. Bulging or ballooning of the lower uterine segment in the midline sagittal transabdominal view has also been considered to be supportive of CSP diagnosis [0 weeks,  a distinIn order to assure the maximal benefit from primary diagnosis and treatment, all gestating individuals with a history of previous caesarean sections are advised to be appointed for a first trimester scan at Early Pregnancy Assessment Clinic (EPAC) after a positive pregnancy test. The examination of choice remains transvaginal ultrasonography (TVS), which might be combined with a transabdominal scan in cases where a panoramic view is required, and additional three-dimensional Power Doppler can confirm ultrasonographic impression. In equivocal cases, magnetic resonance imaging (MRI) may corroborate the initial ultrasonographic diagnosis ,27.The available treatment modalities comprise of expectant management , medical management with methotrexate administration and surgical intervention; the choice of treatment method is mainly dependent on the case presentation and the clinical symptoms . The avaSummarizing operative treatment approaches, besides the hysteroscopic, laparoscopic or laparotomic surgical excision, vacuum aspiration and suction, as in our case, can also be used to remove the scar ,29. The Management of CSP in the first and early second trimesters should preferably be undertaken in a center of excellence where a variety of treatment options and blood bank services are available. All hemodynamically unstable patients should undergo immediate surgical intervention, principally with a minimally invasive approach. For patients with hemodynamical stability, management options include either medical or surgical termination of pregnancy or even continuation of the pregnancy in special circumstances. In CSPs individuals with a fetal demise, expectant management could be offered in combination with medical or surgical treatment ,31. ExpeCSP should be treated without delay following diagnosis, and a swift decision for termination should be made because of the increased risk for bleeding in case the pregnancy continues to grow . The priOnly a few cases of laparoscopic treatment of CSP have been reported. Recently, Kathopoulis et al. shared their experience with laparoscopic management of two cases utilizing different operative techniques. While the authors conclude that the laparoscopic approach appears to be a safe and effective technique for the management of CSP, applied either as a primary intervention or after failure of medical management, they consider laparoscopic removal of CSP as mandatory when the scar gestation is growing towards the bladder and abdominal cavity (type II CSP) . LaparosA recent meta-analysis by Wu et al. of 32 articles, which were reviewed systematically, including a population of 3380 individuals having CSP history, investigated the reproductive outcomes of women as well as how treatments approaches used might affect subsequent pregnancy outcomes. The authors concluded that these individuals are at higher risk for ectopic pregnancy (16.6%), with recurrent CSP rates and risk for spontaneous miscarriages in cured CSP cases being significantly increased in subsequent pregnancies; therefore, contraception should be recommended in those patients who do not wishing future fertility ,40. BaseCaesarean scar pregnancy is a rare obstetrical condition, which may result in the woman being in a life-threatening situation such as uterine rupture and massive haemorrhage, possibly leading to maternal death. This situation represents a diagnostic challenge in obstetrics and gynaecology clinical practice, and management should be timely while careful decisions made as soon as possible. Clinicians should rely on transvaginal ultrasonography as the primary diagnostic modality. Women should have access to all appropriate management options for CSP. If local facilities do not provide all options in non-life-threatening situations, then clear referral pathways should exist to allow them to access appropriate care. Ideal CSP management requires specialized centers and blood bank services, appropriate equipment and experienced healthcare professionals who can deal with possible complications. Frequently, management needs to be individualized, as in our case, where decision for combined treatment approaches was made in the concept of personalized medicine and the ideal management of such a life-threatening condition.


There has been a growing appreciation recently that mutagenic processes can be studied through the lenses of mutational signatures, which represent characteristic mutation patterns attributed to individual mutagens. However, the causal links between mutagens and observed mutation patterns as well as other types of interactions between mutagenic processes and molecular pathways are not fully understood, limiting the utility of mutational signatures.GeneSigNet that constructs an influence network among genes and mutational signatures. The approach leverages sparse partial correlation among other statistical techniques to uncover dominant influence relations between the activities of network nodes.To gain insights into these relationships, we developed a network-based method, named GeneSigNet to cancer data sets, we uncovered important relations between mutational signatures and several cellular processes that can shed light on cancer-related processes. Our results are consistent with previous findings, such as the impact of homologous recombination deficiency on clustered APOBEC mutations in breast cancer. The network identified by GeneSigNet also suggest an interaction between APOBEC hypermutation and activation of regulatory T Cells (Tregs), as well as a relation between APOBEC mutations and changes in DNA conformation. GeneSigNet also exposed a possible link between the SBS8 signature of unknown etiology and the Nucleotide Excision Repair (NER) pathway.Applying GeneSigNet provides a new and powerful method to reveal the relation between mutational signatures and gene expression. The GeneSigNet method was implemented in python, and installable package, source codes and the data sets used for and generated during this study are available at the Github site https://github.com/ncbi/GeneSigNet.The online version contains supplementary material available at 10.1186/s13073-023-01162-x. This provides an opportunity to leverage such signatures for studying interactions between mutagenic processes and other cellular process.Traditionally, research in cancer genomics has been focused on the identification of cancer driving mutations that confer a growth advantage to cancer cells. However, since cancer often emerges as a byproduct of various mutagenic processes such as UV light or a faulty DNA repair mechanism, cancer genomes also accumulate numerous mutations with seemingly no direct roles in carcinogenesis. Different mutagenic processes often lead to distinct patterns of somatic mutations called Starting from the pioneering work of Alexandrov et al. , severalThere are several know examples where deficiencies in the activities of some genes such as MUTYH , ERCC2 , MSH6 6 have beeThere are also many examples of the reverse relation. For instance, tobacco smoking is not only mutagenic but it is also believed to activate the immune response . SpecifiFinally, mutagenic processes themselves have been known to interact with each other. For example, homologous recombination deficiency (HRD) is often accompanied by mutational signatures related to APOBEC activity , 14.Applying a clustering-based method to genome-wide expression and mutational signature data, Kim et al. identified coherently expressed groups of genes associated with specific combinations of mutational signatures, providing interesting insights regarding these signatures . HoweverGeneSigNet (Gene and Signature Influence Network Model), aiming to uncover interactions among mutagenic and cellular processes, and to provide insights into mutagenic processes underlying individual signatures. Utilizing gene expression and mutational signature data from cancer patients, GeneSigNet constructs a Gene-Signature Network (GSN) that represents asymmetric dependencies among two types of node entities — genes and MutStates. MutState nodes are in one-to-one correspondence with mutational signatures and each MutState represents an abstract (directly unobserved) cell state associated with the corresponding mutational signature  also builds on partial correlation relations, which computes a sparse asymmetric weight matrix representing directed influences among nodes in the network. The sparsity constraints ensure that only a small fraction of influential edges have nonzero weights while many entries of such inferred matrix are set to zero and one-directional .In contrast, the statistical higher moments were used as indicators of the direction of dependency between two variables  under thGeneSigNet method builds on the last two approaches. First, a sparse partial correlation technique (SPCS) is used to obtain an initial sparse weighted directed network. We opted to infer a sparse network in order to focus on strongest trends that are more likely to suggest mechanistic explanations. This initial network contains bidirectional edges. Next, where applicable, a novel partial higher moment strategy is used to resolve (orient) bidirectional edges. We note that GeneSigNet does not attempt to construct a fully resolved oriented graph, leaving many edges as bidirectional. We found that this approach compares favorably to the previously proposed techniques that can be used for this task.The GeneSigNet to two cancer datasets, breast cancer and lung adenocarcinoma, for which sufficient numbers of patient samples with gene expression data are available and the interactions of mutational signatures are partially known. For the network reconstruction, we utilized genes in two GO categories: metabolism and immunity. It is increasingly appreciated that cellular metabolic regulation can lead to DNA damage and can impact DNA repair, stimulating interest in the understanding of the cross-talk between DNA-damage and metabolism [We applied tabolism . In additabolism . TherefoGeneSigNet model are overall consistent with current knowledge, but also include several interesting novel findings. In particular, the model suggests a causative relation from the homologous recombination deficiency signature (SBS3) to a clustered APOBEC mutation signature, and also linked Signature 8 (SBS8) to nucleotide excision repair (NER) pathway. The latter connection is consistent with the recent findings based on an experimental study in mouse [GeneSigNet identified a novel relation between APOBEC hypermutation and the activation of regulatory T cells which presents an important implication in immunotherapy, and captured a relation of APOBEC signature (SBS2) with DNA conformation changes among other findings. Taken together, our results demonstrate that GeneSigNet provides novel and important insights.The relations inferred by the in mouse . In addiGeneSigNet is a construction of a Gene-Signature Network (GSN) consisting of two types of nodes: nodes corresponding to genes, and nodes corresponding to mutational signatures  effectuating a mutational signature. The activity of this state is measured as the exposure of the corresponding mutational signature in the given patient. Statistical relations on the activities of genes and MutStates are likely to shed light on the genes and pathways associated with, and potentially contributing to, the level of the exposure of the corresponding mutational signature. To this end, GeneSigNet infers directed edges between both types of nodes, utilizing patient-specific gene expression (for genes) and exposures of mutational signatures (for MutStates) as described below  constructing a preliminary directed network using a sparse partial correlation selection (SPCS), and (ii) revising the initial network by testing whether the orientation of bidirectional edges can be decided using a partial higher moment strategy adapted to a network setting. The workflow of the GeneSigNet method is shown in Fig. GeneSigNet constructs an initial weighted-directed graph using a sparse partial correlation selection (SPCS)  Fig. B. Specifmentclass2pt{minimGeneSigNet refines the initial network by reducing the number of bidirectional edges remaining after the first step. The idea is an adaptation of the basic bivariate higher moment strategy to multivariate analysis. Specifically, for any two nodes having potential effects on each other in the initial network , we first utilize the partial correlation technique to remove confounding effects due to the presence of the other partial higher moment strategy.In the second step, the pair . Under tWe note that the method relies on simplifying assumptions which might not be fully satisfied in real biologic relations. In particular, the assumption that all confounding effects come from the activities of the remaining Figure GeneSigNet is provided in Additional file Finally, alternate scaling , a matriThe normalized gene expression data for 266 breast cancer (BRCA) patients were downloaded from Table S7 in . Gene exSigMa [SigMa divided all mutations into two groups, close-by Clustered and Dispersed mutations, and assigned each of these mutations to one of 12 COSMIC v2 signatures [SigMa as described above, signature phenotype profiles 1D, 2C/D, 3C/D, 5D, 8C/D, and 13C/D that had exposure levels of at least 10% within each group were selected for further analysis (the numbering refers to the COSMIC signature index and C/D denotes signatures attributed to clustered and dispersed mutations). Examining their correlation patterns among patients, some of the signatures were grouped as follows: Signatures 3C/D and 8D were combined into DSB (double-stranded DNA break repair) related signatures, and Signatures 2C and 13C/D into APOBEC related signatures. The remaining signatures are treated separately, resulting in Signature 1, 2D, 5, APOBEC, DSB. A log transformation was consequently performed on exposures of each signature to make its distribution shape closer to a bell curve of normality.For mutational signatures, somatic mutation data were downloaded from the ICGC data portal . The 3,4SigMa . SigMa dgnatures  which weFurthermore, we included binary information of homologous recombination deficiency as an additional variable in the analysis. The binary alteration information was obtained by aggregating functional inactivation information for BRCA1/BRCA2 and 16 other HR genes as provided in Supplementary Tables 4a and 4b of Davies et al. . The posThe expression data (RNA-seq) of the lung adenocarcinoma (LUAD) from The Cancer Genome Atlas (TCGA) project were downloaded from the Genomic Data Commons Data Portal on 2020-06-05 . NormaliThe TCGA LUAD exome mutation spectra were downloaded from Synapse  and decohttps://github.com/ncbi/GeneSigNet [We analyzed 466 tumor samples that had both gene expression and mutational signature exposure data available. We analyzed 2433 genes belonging to the DNA metabolic process and immune system process in GO terms (genes that are not expressed in at least 10% of the samples were omitted). The gene expression and mutational signature exposure data were combined to form an input data matrix. All the data is available at the Github site neSigNet .The numbers of recovered edges and average degrees for each of the two cancer networks are summarized in Additional file GeneSigNet infers a directed network representing information flow between genes’ expression and signatures’ exposure. We compared the performance of GeneSigNet to four competing approaches which, similarly to GeneSigNet, strive to predict influence flow between network nodes. We compare GeneSigNet to the following key approaches: (1) the independent component analysis-based method LiNGAM [d LiNGAM , (2) thed LiNGAM  previousd LiNGAM , (3) thed LiNGAM  and (4) d LiNGAM . Additiod LiNGAM  for the d LiNGAM , 40, 41.d LiNGAM , this alTo generate data, we implemented the data simulation schema provided in LiNGAM . This apGeneSigNet, GeneNet, LiNGAM, SPCS and ElasticNet construct a single network that is the output of the method-specific optimization procedure, thus their performances are represented by single points in Fig. GeneSigNet had the highest precision in predicting the direction of the influence. The F-score , was also the highest for GeneSigNet although only slightly better than the second best method, GeneNet. In addition, we also evaluated the methods on their ability to recover the values of the lower triangular matrix used to simulate the data. This measure was proposed in [GeneSigNet’ ability to reconstruct the input matrix was equal or better relative to other methods, suggesting that GeneSigNet indeed captured dominating influences using a smaller number of edges.The performances of the methods are summarized in Fig. posed in  and measGeneSigNet had the highest precision. Surprisingly, GeneNet and the regression tree methods resulted in worse than random performance in the evaluation on the BRCA data. Unfortunately, in the case of real data, the weights of the reference edges are unknown so we do not have a measure corresponding to the correlation function used on simulated data.We also compared the performance of the methods on breast cancer (BRCA) and lung cancer (LUAD) data sets , APOBEC-D (SBS2 corresponding to disperse APOBEC mutations), DSB (SBS3 and clustered SBS8), SBS5, and SBS8D (dispersed SBS8). In addition to gene expressions and exposures of mutational signatures, we included a node indicating the binary status of homologous recombination deficiency (HRD) as it is assumed to lead to specific patterns of mutational signatures in BRCA [GeneSigNet to construct a GSN for genes, mutational signatures, and HRD status, and to find relations between these features.We utilized breast cancer (BRCA) data collection obtained from ICGC which includes 266 cancer samples providing both whole-genome sequencing data and gene expression data  plays an important role in the double-strand break (DSB) repair mechanism and that HR deficiency is associated with the DSB signature [GeneSigNet identified the known negative impact of BRCA1 expression on the DSB signature which is also consistent with the role of BRCA1 in HRD [GeneSigNet captured the impact of HRD on chromosome separation, reflecting the role of homologous recombination in maintaining genomic stability [APOBEC-D with telomere maintenance, consistent with the well recognized role of APOBEC mutagenesis in replication [Many relations uncovered with ignature . Indeed,1 in HRD . Furthertability , 48, andlication , 50.Fig.GeneSigNet method to uncover non-obvious relationships.Interestingly, our method linked SBS8 to the nucleotide excision repair (NER) pathway Fig. . The etiPrevious studies speculated that APOBEC related mutational signatures can arise in multiple different scenarios. First, double-strand breaks (DSB) created by the homologous recombination deficiency (HRD) provide mutational opportunities for APOBEC enzymes to act on the ssDNA regions, resulting in clustered APOBEC mutations , 51, 52.GeneSigNet supports a positive influence of the DSB on APOBEC-C MutState, consistent with the assumption that double-strand breaks provide an opportunity for APOBEC mutations. Additionally, our analysis reveals that the expression level of the APOBEC3B enzyme is associated with the strength of the DSB signature. Indeed, a previous study proposed that APOBEC3 proteins are recruited to DSB sites to participate in the DSB repair process [APOBEC-C strength by two different mechanisms: (i) increased mutation opportunity due to ssDNA created by DSB and (ii) increased mutation probability due to increased APOBEC3B expression. Note that increased APOBEC expression would also increase APOBEC mutations in the “fog” regions proposed in [Focusing on the interactions of APOBEC signatures with the other MutStates and genes, we observe that  process . Thus, Dposed in .APOBEC-D is positively influenced by APOBEC-C activity, without direct relation to DSB. In fact, GeneSigNet inferred a negative influence from HR status to APOBEC-D MutState, confirming that different mutagenic processes are involved in clustered and dispersed APOBEC mutations  revealed significant enrichment in positive regulation of regulatory T cells (Tregs) differentiation  allowing them to benefit from immunotherapy . In partWe next analyzed lung adenocarcinoma (LUAD) data using 466 cancer samples from the TCGA project. The exposure levels of 6 COSMIC mutational signatures  present in the exome sequencing data were integrated with the RNAseq expression data of 2433 genes belonging to the DNA metabolic and immune system processes in Gene Ontology terms to uncover influence between signatures and genes  with the expressions of APOBEC3B and APOBEC3A enzymes, and also identified the association of SBS13 with pyrimidine related catabolic processes, potentially reflecting the fact that SBS13 involves a pyrimidine to pyrimidine mutation  contributing to this GO term. This suggests a relation between DNA stress mediated by TOP1 and APOBEC activity.As discussed earlier, APOBEC can only act on single-stranded DNA (ssDNA). Interestingly, one of the GO terms associated with SBS2 MutState identified by nge Fig. . DNA geoGeneSigNet, a new network-based approach that infers the relation between gene expression and the strength of mutation patterns (signature exposures) allowing us to uncover the relations between signatures and processes involved in DNA repair and immune response among other cellular processes. Recognizing the limitations of the previous approaches, GeneSigNet relies on a construction of a sparse directed network. For each node (gene or MutState), it selects a sparse set of incoming edges representing dominating incoming effects so that their combination explains the activity of the node. Aiming to capture the most dominant influences, the method utilizes sparse partial correlation coefficients.Elucidating the nature of mutagenic processes and their interactions with cellular processes is of fundamental importance for understating cancer etiology and guiding cancer therapy. Here, we propose In this study we focused on the relations that can be inferred based on genes’ expressions and exposures of mutational signatures. It is possible to include other features as well. We leveraged this flexibility of our method to include for breast cancer germline perturbations that are known to predispose homologous recombination deficiency (HD).The construction of a sparse network utilized by our approach has both advantages and disadvantages. Sparse networks are easier to interpret and more likely to suggest mechanistic insights. However, by restricting edge set, they might lose some information. In our previous studies , 69 we hGeneSigNet are consistent with the current knowledge, boosting the confidence in the method’s applicability. In addition, GeneSigNet provided several new biological insights concerning the relation between mutagenic processes and other cellular processes. For example, the uncovered relation between APOBEC hypermutation and activation of regulatory T cell can have an important implication in immunotherapy.For the biological results, overall the relations discovered by GeneSigNet, for uncovering potentially causal relations between gene expression and mutagenic processes represented by mutational signatures. The main idea of the approach is to construct an influence network representing dependency flow between genes and mutational signatures. We opted to infer a sparse network in order to focus on the strongest trends that are more likely to suggest mechanistic explanations. We note that focusing on a sparse set of edges reduces the power of GO enrichment analysis and requires more specific biological knowledge for interpreting the results. Yet, this potential disadvantage is compensated by the compelling mechanistic insights provided by the method.In this work, we report a computational method, named Additional file 1. Supplemental Information includes Sections S1, S1.1, S1.2, S1.3, S1.4, and S1.5: detailed descriptions of the GeneSigNet, and data simulation schema for validation, Table S1: Supplemental table providing the topological information on the inferred networks, Figs. S1, S2, S3, and S4: Supplemental figures for visualizing the performance of the GeneSigNet method and the extended subnetworks covering the mutational signatures and their direct neighbors. (PDF)Additional file 2. Tables S2 (BRCA), and S3 (LUAD) : Supplemental tables providing the genes enriched in up and down streams of mutational signatures and their interaction weights in the gene signature networks of BRCA and LUAD. (Excel)Additional file 3. Tables S4 (BRCA) and S5 (LUAD): Supplemental tables providing the GO terms enriched for causal and affected genes of mutational signatures in the gene signature networks of BRCA and LUAD. (Excel)


Given its central role in cell metabolism, mTOR is involved in several biological processes. Among those, mTOR is a driver of senescence, a process that might contribute to the establishment of chronic lung disease because the characteristic irreversible inhibition of cell proliferation, associated to the acquisition of a pro-inflammatory senescence-associated secretory phenotype (SASP) supports the loss of lung parenchyma. The deregulation of mTORC1 is a hallmark of lymphangioleiomyomatosis (LAM), a rare pulmonary disease predominantly affecting women which causes cystic remodeling of the lung and progressive loss of lung function. LAM cells have senescent features and secrete SASP components, such as growth factors and pro-inflammatory molecules, like cancer cells. Using LAM as a paradigm of chronic and metastatic lung disease, here we review the published data that point out the role of dysregulated lipid metabolism in LAM pathogenesis. We will discuss lipids’ role in the development and progression of the disease, to hypothesize novel LAM biomarkers and to propose the pharmacological regulation of lipids metabolism as an innovative approach for the treatment of the disease.A chronic inflammatory condition characterizes various lung diseases. Interestingly, a great contribution to inflammation is made by altered lipids metabolism, that can be caused by the deregulation of the mammalian target of rapamycin complex-1 (mTORC1) activity. There is evidence that one of mTOR downstream effectors, the sterol regulatory element-binding protein (SREBP), regulates the transcription of enzymes involved in the  Insig1/2Δ/Δ mice causes neutral lipid accumulation in type II cells and in the alveoli, resulting in pulmonary inflammation and airspace remodeling  is a rare progressive lung disease characterized by lung cystic destruction, and the progressive loss of lung function . LAM occignaling . mTOR haignaling . Recentlignaling .The minireview describes the current state of knowledge of emerging role of lipids in LAM considering their contribution in inflammation and in the pathogenesis and progression of chronic lung diseases.Lung inflammatory diseases are tightly linked with aberrant SREBP activity in respiratory epithelial cells and with dyslipidemia. SREBP2-mediated lipid metabolism promotion correlates with COVID-19)-induced cytokine storm . The inc-induced in vitro generated models demonstrated the presence of the proinflammatory interleukin-6 (IL-6), CXC chemokine ligand 8 (IL-8), and monocyte chemoattractant protein 1 (2), which is synthesized from the arachidonic acid, is expressed at higher levels in COPD fibroblasts compared to healthy controls and is responsible for the autocrine and paracrine spreading of senescence in neighboring cells, as well as of the induction of inflammatory response through the up-regulation of its receptors EP2 and EP4 in a cyclooxygenase 2 -dependent reactive oxygen species response  to maintain their status and to communicate within their microenvironment. SASP factors, both released as soluble molecules or within extracellular vesicles (EVs), have strong biological activities on neighboring cells, including the promotion of inflammation. Indeed, the study of SASP in all rotein 1 . Even ifrotein 1 . Of noterotein 1 . The conrotein 1 . Moreoverotein 1 . Of noterotein 1 . Senescerotein 1  and it hrotein 1 . For exaresponse . Notablytigation . Additiotigation . Finallytigation .TSC1 or TSC2 tumor suppressor genes that causes the hyperactivation of Rheb with an increase of mTORC1 and adenosine 5′-monophosphate activated protein kinase (AMPK) activity  primarily affects women of childbearing age. LAM cells are smooth muscle-like cells bearing a mutation in activity , 40. AMPactivity . mTORC1 activity . Moreoveactivity . As a coynthesis , 43. Furynthesis .The proliferation of LAM cells in the lung parenchyma is associated with diffuse cystic lesions and their infiltration into the lymphatic walls causes lymphatic abnormalities leading to damage or obstruction of lymphatic vessels . LymphanThe discovery of the genetic basis of LAM cells led to consider the inhibition of mTOR hyperactivation as a therapeutic approach. Rapamycin indeed inhibits LAM cell proliferation resulting in the stabilization of the lung function, in the reduction of lymphatic abnormalities, and of the angiomyolipoma volumes, with no efficacy in the regression of the existing pulmonary lesions. So far, the treatment with rapamycin is the only approved for LAM .After being classified as an interstitial lung disease, LAM has been recently reconsidered as a low-grade, destructive, metastasizing neoplasm for several features as the invasive LAM cell properties, the recurrency in donor allografts of patients who have undergone lung transplantation, and the metabolic reprogramming. However, LAM differs from other neoplasms for the bilateral and symmetrical disruption of the lung without a clear primary tumor site and dominant mass lesions .We recently demonstrated that LAM/TSC cells, derived by chylous thorax of a LAM/TSC patient, have senescent features dependent from mTOR hyperactivation and the capability to induce senescence in neighboring cells . As demoFurthermore, mTOR role in LAM senescence can be supported by the senolitic effect of rapamycin and the relapse following the suspension of rapamycin treatment caused by an irreversible senescent state, called geroconversion, sustained by mTOR hyperactivation .in vitro model of CF by reducing ceramide accumulation with Myriocin, the inhibitor of the rate-limiting step in sphingolipid biosynthesis  cells have a metabolic signature of increased fatty acid uptake and synthesis, a characteristic in common with cancer cells , making –/– mice have a strong positivity to the ganglioside D3 (GD3), whose expression is a characteristic that LAM cells maintain also after multiple passages in vitro , consistently with the hypothesis that tuberin loss might enhance several phospholipid and neutral lipid species. The treatment with the mTOR inhibitors rapamycin or torin1, or the down-regulation of SREBP1 does not suppress LPCs, while the down-regulation of specific PLAs decreases selectively the proliferation of Tsc2–/– MEFs compared with Tsc2+/+ MEFs  reduced the growth and induced apoptosis on tuberin-deficient cells derived from LAM patient. Interestingly, the tuberin induced-expression in these cells prevents the effects of MAFP treatment  were found in the plasma of women with LAM compared with healthy controls, suggesting novel potential biomarkers . LPCs ar+/+ MEFs . Among Preatment .–/– MEFs are sensitive to the treatment with rintaserin in combination with chloroquine, while the impact of those two drugs on Tsc2+/+ MEFs is minimal  and [18F]fluoroacetate (FACE) to detect tuberin-deficient cells in solid tumors and to monitor the response to rapamycin through dynamic PET (18F]FCH uptake, but not [18F]FACE uptake in tuberin-deficient xenografts was rapamycin-sensitive and in vitro study on tuberin-deficient cells indicated that this difference might be due to an accumulation of these two compounds in two different cells compartment. In fact, [18F]FCH is mainly incorporated into lipids, while [18F]FACE enters into mitochondria and can be used as marker of mitochondrial activity, which is not suppressed by rapamycin, confirming a specific metabolic feature of tuberin-deficiency in LAM cells (Finally, the altered lipid metabolism in LAM cells can be exploited to identify novel metabolic imaging biomarkers for the disease. Indeed, the previously reported enhancing in PC levels and neutral lipids in LAM cells  allowed amic PET . [18F]FCAM cells .Regulation of precise lipid species plays a critical role in senescence and senescence-mediated inflammation associated to chronic pulmonary diseases. Aberrant activity of SREBP and its upstream regulator, mTOR, is highly involved in lipotoxicity that induces inflammation and fibrosis in several lung diseases including LAM . The eviAll authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.


Nature, Delaunay and colleagues demonstrate that tRNA modifications in mitochondria have the potential to affect the synthesis of mitochondrial proteins involved in oxidative phosphorylation (OXPHOS), which drives the invasive spread of cancer cells. The inhibition of one particular RNA-modifying enzyme was sufficient to prevent the cancer cell invasion and dissemination.1In a recent study published in Glycolysis and OXPHOS are two main metabolic pathways that support cancer cell development and progression. While primary tumors preferentially utilize glucose as a source for their energetic requirement , less is known about metabolic plasticity required for dissemination of cancer cells from primary tumor sites. Metastasis, which is a multistep process enabling the tumor cells to spread from primary tumors to adjacent and distinct tissues, is responsible for approximately 90% of cancer-related deaths. The metastatic development requires cancer cells to leave the primary tumors and to metabolically adopt to changing microenvironment. Figure 2 however the exact underlying mechanisms and the respective roles of OXPHOS and glycolysis during metastatic transition are still unknown. Some previous studies reported that metformin, a widely used anti-diabetic drug approved for the treatment of patients with type 2 diabetes, promotes anticancer effects.3 Although several mechanism have been proposed for its anti-tumoral activity, the dominant effects of metformin are generally mediated by inhibiting mitochondrial electron transport chain (ETC) complex I.Mitochondria, which continually undergo the highly dynamic processes of mitogenesis and mitophagy, are crucial for OXPHOS, and cancer cells are characterized by mitochondrial metabolic plasticity and heterogeneity. It is plausible to speculate that some mitochondrial metabolites might be implicated in driving oncogenesis. Mitochondria were previously proposed to be involved in both aspects of metastasis, migration, and invasion of cancer cells,Nature, Delaunay and colleagues show that the specific modifications 5-methylcytosine (m5C) and its derivative 5-formylcytosine (f5C) at position 34 in mitochondrial tRNAMet are required for the development of CD36-driven metastasis. The functional aspects of modifications in mitochondrial tRNAs are poorly understood. So far, 18 different types of post-transcriptional modifications introduced by tRNA-modifying enzymes have been found in 22 human mitochondrial tRNAs.4 It is known that all 22 tRNA species translate essential proteins belonging to respiratory chain complexes. The data from Delaunay et al. suggest that the m5C modification of mitochondrial tRNAs is an important prerequisite for metabolic remodeling of metastasis-initiating cells that are dependent on mitochondrial protein synthetic machinery and mitochondrial respiration. The loss of a specific mitochondrial enzyme, NOP2/Sun RNA methyltransferase (NSUN3) led to metabolic switch associated with an elevated glycolysis, an upregulation of glucose transporter 1 (GLUT1), and a limited OXPHOS, as well as a slight reduction in most tricarboxylic acid (TCA) metabolites. As a result, the invasive spread of tumor cells was strongly decreased. Interestingly, the growth of primary tumors that primarily utilize glycolysis was not affected by the loss of m5C modification. The authors conclude that site-specific mitochondrial tRNA modifications likely serve as a “sensor of cellular energy requirements”, which might be a novel therapeutic target for modifying metabolic plasticity needed for metastatic tumors. Moreover, the NSUN3 function as potential clinical biomarker for metastases was examined in patients with head and neck cancer with high expression of CD36. Indeed, a high cellular expression of the enzyme NSUN3 and increased m5C levels were highly predictive for lymph node metastases.The capacity of mitochondrial tRNAs to modify tumor development and metastasis has not been elucidated yet. In a recent publication in 5C formation in mitochondrial tRNAs by being able to suppress cancer cell invasion and metastatic spread. Although this novel concept suggests that specific antibiotics may be therapeutically exploited to inhibit progress of metastatic cancer, we might need a deeper understanding of such findings, as a global change of patients‘ microbiome is expected following the treatment with antibiotics. Several studies have demonstrated that antibiotic-induced disturbances in the composition of intestinal microbiota modulate T cell-mediated anti-cancer immunity.5 It is possible that alterations in microbiome in the gut (or even within the microbiome-harboring tumors) caused by antibiotic treatment may also impact on metastatic behavior of tumors.To gain insights into potential therapeutic aspects, the authors treated both in vitro-derived tumoroids and tumor-bearing mice with several classes of antibiotics. Of note, only antibiotics such as doxycycline, chloramphenicol, and linezolid, which are able to inhibit mitochondrial protein synthesis without affecting protein translation in the cytoplasm, reduced the number of invading leading cells in tumoroids and metastases in vivo. Thus, the pharmacological inhibition of mitochondrial translation fully recapitulated the functional loss of mMet, known as m5C was crucial for the development of metastasis. Both, the blockade of the specific enzyme, NSUN3, responsible for m5C modification and treatment of mice with antibiotics targeting mitochondrial protein synthesis strongly decreased the invasive spread of cancer cells from the primary tumors. Thus, modifications of mitochondrial tRNAs regulate the metabolic plasticity in metastasis by supporting mitochondrial respiration of invasive cancer cells located at the periphery of primary tumors. The metabolic feature of such invasive cancer cells performing high degree of OXPHOS may be used in the future for the prevention of metastases. Although this finding represents an innovative work contributing to the therapeutic exploration of how mitochondrial RNA modifications could be targeted for cancer treatment, various aspects have remained unclarified. Since the majority of normal tissue cells rely on OXPHOS, the OXPHOS-dependent metabolic reprogramming might have various adverse effects. The function of some immune cells such NK cells and CTLs that recognize tumor cells can be impaired by inhibiting protein synthesis in mitochondria. Moreover, for some cancer patients, the high OXPHOS is associated with higher sensitivity to chemotherapy and better prognosis . As high-level of f5C modification in mitochondrial tRNAs is present in various tissues, future work should shed light on the role of this modification and the corresponding tRNA-modifying enzyme ALKBH1 in cancer progression. Finally, the treatment with mitochondria-specific antibiotics would introduce global perturbations in gut microbiota composition, which might lead to suppression of anti-tumor immunity. Although a better understanding of mitochondrial RNA modifications may open new therapeutic opportunities, additional studies will be required to define their therapeutic potential for cancer treatment.In summary, Delaunay and colleagues identified the specific mitochondrial RNA modifications that are associated with invasive spread of cancer cells and poor prognosis of neck and head cancer. The specific modification of mitochondrial tRNA


Psychiatric disorders are often distinguished from neurological disorders in that the former do not have characteristic lesions or findings from cerebrospinal fluid, electroencephalograms (EEGs), or brain imaging, and furthermore do not have commonly recognized convergent mechanisms. Psychiatric disorders commonly involve clinical diagnosis of phenotypic behavioral disturbances of mood and psychosis, often with a poorly understood contribution of environmental factors. As such, psychiatric disease has been challenging to model preclinically for mechanistic understanding and pharmaceutical development. This review compares commonly used animal paradigms of preclinical testing with evolving techniques of induced pluripotent cell culture with a focus on emerging three-dimensional models. Advances in complexity of 3D cultures, recapitulating electrical activity in utero, and disease modeling of psychosis, mood, and environmentally induced disorders are reviewed. Insights from these rapidly expanding technologies are discussed as they pertain to the utility of human organoid and other models in finding novel research directions, validating pharmaceutical action, and recapitulating human disease. Psychiatric diseases have been notoriously difficult to study from a basic science perspective, and unfortunately, consolidated mechanisms of mood, psychotic, and environmentally induced disorders at the most fundamental level have eluded researchers for centuries. These obstacles to discovery come from both technical and ethical perspectives. From a technical viewpoint, understanding psychiatric disorders relative to neurological diseases is difficult as brain ‘lesions,’ or signals in cerebrospinal fluid (CSF), electrophysiological, or imaging studies are not as robust and apparent in disorders we consider psychiatric in nature. Ethically speaking, preclinical experimentation of in vivo and in vitro central nervous systems (CNSs) involves unique sets of moral implications, briefly discussed below.It is worth noting that several disorders with known genotype lead to cognitive and behavioral phenotypes that may fall under the umbrella of psychiatry. In this review we will focus primarily on neurodevelopmental and acquired psychiatric disorders without a definitive genetic cause, as these present a particularly distinct and under-reviewed challenge in creating preclinical models.The ethical challenges in studying psychiatric disorders preclinically lie in several realms and are not solved by the review of advanced culture methods described herein. The chief modalities currently involved in preclinical testing involve testing on animals, typically primates and rodents, with burgeoning contributions from in vitro human cell platforms Fig. . ExperimWhile we use animals as surrogates for testing disorders of higher-order cognition, consciousness, and behavior, as animals are unable to provide consent, one must use a principle of comparative beneficence to justify animal testing. As animals display complex behaviors that necessitate some degree of higher-order thinking and cognition that is generally considered to be related to cerebral complexity, one may also expect a degree of subjective suffering in research animals whose experience likely resides somewhere on a continuum of consciousness. Researchers already assume a degree of consciousness with ongoing and historical experiments, as an example rodents have been used as surrogates for testing in consciousness studies, including with thalamic lesions . Other aThe ethics landscape of human organoid research is an emerging topic beyond the scope of the present article, and while several viewpoints have been published in recent years, there exists no consensus framework on best practices or broadly adopted research restrictions –9. A recAn alternative or perhaps a complement to preclinical animal testing that has been emerging for greater than a century is the study of neuronal cultures in controlled laboratory settings. The cultures were studied initially in explant tissue surviving ex vivo, and this field has now developed into sophisticated analyses of complex human stem cell-based neural networks growing in three dimensions (3D). At the present relatively advanced stage, these networks have limited ability for efferent output and cannot yet display phenotypic behavior, a hallmark of psychiatric disease. However, significant advancements in cell culture techniques toward surrogacy for in vivo human neurodevelopment warrant discussion as an alternative validated preclinical complement to the paradigm of animal models.Beyond preclinical testing modalities, most aspects of psychiatric illness, including etiologies, remain controversial. Mechanistic origins described from polygenic to environmental contributions as well as neurodevelopmental alterations in utero through adolescence and adult life have all been suggested and researched extensively. The myriad of potential etiologies may be investigated by several means, and in the case of commonly cited and complex polygenic origin, it has been argued that 3D cell models would have utility in decoding the impact of genetic variants on pathological brain development leading to mental illness .Moving forward in this review, we will begin with a comparison of animal models for psychiatric research, followed by an overview of the development of advanced in vitro cell cultures, and then succeeded by subsections of relevant psychiatric models. This work builds on prior reviews of cell culture technology, including modeling of the neurotypical CNS and its development with its unique set of complexities, described extensively in prior work –21 and iAs animal models, in particular rodent models, have been used extensively in a preclinical context to characterize genetic and neural mechanisms of psychiatric disorders and to facilitate new treatments , a limitRecent advances in murine models have correlated hallucination-like precepts in mice, through ketamine administration, and optogenetically boosting striatum dopamine signaling, which responded in an expected way to haloperidol administration . As noteCurrently, available rodent animal models are used frequently for basic drug screening and investigations in neuroscience, however they are still time-consuming, costly, and do not study the molecular and cellular effects of these drugs on the human brain . While tFocusing first on murine models concerning inflammation, recent analysis has found that murine model transcriptional responses to inflammation correlate poorly with humans, particularly found when comparing murine models of trauma, burns, and endotoxemia, which did not correlate with highly consistent genomic response to these insults in human patients . This seFurther comparing species, rodents are social creatures with some aspects that humans would find familiar including forms of auditory communication (with ultrasonic squeaks) relating to purported positive/negative affect . HoweverBeyond the macro-scale species differences, on the level closer to preclinical studies of neural cell biology, there exist several differences in cell development in the CNS. The neocortex germinal layer in murine outer radial glial cells populates a distinct neural layer in the developing cortex, the subventricular zone (SVZ) , whereasExpanding on biochemical and cellular differences between rodent model species and humans, the cell cycle is estimated to operate 2–3 times faster in mice than in humans , 56, witOn the level of neurotransmitters there similar exists several human-specific changes when compared with murine studies. Dopaminergic neurons contain substantially higher levels of dopamine in human neurons compared to murine neurons  as well 2A receptors in the striatum [2A receptors have species-dependent differences in dissociation kinetics with serotonergic psychedelics, with a prominent amino acid substitution in the orthosteric binding pocket [The target of neurotransmitters, cell surface receptors, is another area of species’ difference, notably there are substantial differences in terms of the species-specific chemical neuroanatomy of 5-HTstriatum . Focusing pocket .When examining higher-level cortical electrical organization in species, there are noted differences including human hippocampal theta waves showing decreases in amplitude, frequency, and duration from rodent equivalents , 69. As Over one hundred and ten years have passed since Ross Granville Harrison made the advance of observing explanted neural explants from frogs in hanging drop culture, greatly propelling the ability to examine neurons ex vivo , 71. In The ability to differentiate neurons from fibroblast samples through induced pluripotency has particularly risen to prominence since the turn of the millennium and furthermore the expansions of cultures of cells into 3D have dramatically accelerated in recent years . While rIn the effort to expand in vitro cell cultures to 3D, non-planar hiPSC cultures have been in development since they were differentiated in the form of cortical neuro-epithelia in 2008. In likely the earliest report of brain organoids there were notable differences in the neuroepithelial structure between human and murine embryonic stem cell-derived cortical tissues, with a continuous versus polarized circular neuroepithelia, respectively, found after extended culture . In 2012As different research groups use individual protocols and have diverse discoveries, a common critique and noted limitation of current organoid technology has been batch-to-batch variability. This finding is improved by directed or guided differentiation toward specific brain regions, as opposed to unguided protocols, however, it remains an important consideration. Human cell-derived brain organoid models have been validated by multiple groups, and it has been demonstrated that reliable CNS tissue can be generated ex embryo in a reproducible and developmentally constrained manner that transcends individual organoids, experimental batch, genetic background, and sex . Further2+ transients in response to glutamate [Broadly, organoids technology has advanced in efforts toward understanding neural circuitry and neurotransmitters, notably pertinent to understanding psychiatry and psychotropic drugs. Organoids exhibit spontaneous Calutamate , and glulutamate . Functiolutamate , includilutamate .To advance the complexity of neural systems modeled in vitro, several groups have published techniques where two or more brain regions are fused in a coculture system or in an assembly (“assembloids”). Techniques for combining 3D cultures of neural cells have been in development since at least 2013, where neurospheroids in millimeter-sized cubic molds were differentiated separately into hippocampal and cortical neuron cultures and then combined in coculture with their connections visualized . Notable® coated organoids leading to primitive vascularization of the organoids in the form of cluster of differentiation 31 (CD31)—positive cells forming tubular channels [Other advancements include adding astrocytes or microglia in coculture to help with synaptogenesis, leading to increased electrophysiological maturity over conventionally generated methods . Furtherchannels . Other tchannels . A functchannels .The development of inhibitory neurons in human 3D neural culture is another active area of research, and GABAergic mediation of glutamatergic activity is important for synchronized burst activity in older brain organoid cultures . OrganoiTechnical engineering efforts in organoids have likewise developed as hiPSCs can be encapsulated in a bioink in a defined, scalable, and versatile platform and bioprinted in a 3D manner . The 3D Schizophrenia is polygenic, hypothesized neurodevelopmental disorder, with a yet unknown molecular origin, although with a well-established theory of overactive dopaminergic signaling in the midbrain , 105. MoAs schizophrenia is a disease of unknowns, there are a multitude of different paths to take toward mechanistic study, especially with in vitro experiments, and in the next section we will review some known features of schizophrenia and emerging discoveries with advanced cell modeling approaches. It has been suggested that there is a developmental or perhaps senescent cascade in schizophrenia as there is a mysterious gray and white matter reduction in schizophrenia patients , 107. AlSchizophrenia has been modeled using hiPSCs since 2011, when idiopathic schizophrenic patient samples were used in 2D culture with neuronal phenotypes identified including diminished neuronal connectivity with decreased neurite numbers, postsynaptic density protein (PSD95)-protein levels (excitatory synapse marker), and decreased glutamate receptor expression. Further, there was altered expression in cAMP and Wnt signaling pathways and these phenotypes were rescued with added loxapine, an antipsychotic, to culture media . Also, hMoving forward with the relevance of DISC1 to schizophrenia modeling, previous studies have shown that both DISC1 and NudE Neurodevelopment Protein 1 (NDEL1) can regulate the cell cycle by interacting with glycogen synthase kinase 3 beta  and LIS1As discussed, an area of active research in schizophrenia is altered neurodevelopment. A further finding in schizophrenia patient-derived organoids was disruption of the developmental strata, including increased Ki67+ neural progenitor cells (NPCs) in the intermediate zone and atypically placed deep subcortical neurons, accompanied by significant loss of T-box, brain 1 (TBR1) positive pioneer neurons from the top cortical layer. Overall abnormal subcortical neurogenesis was consistent with transcriptome studies of NPCs from schizophrenia hiPSCs. Also apparent was visibly diminished different pan-neuronal expressing fibers and random directionality of calretinin-positive neurites . GlutamaFurther investigating the exhibitory and inhibitory developmental cascade, glutamatergic and GABAergic neurons in independently differentiated forebrain subdomains were then combined in coculture recapitulating the saltatory migration of interneurons. To develop this assembly these sub-brain regions modeled as human cortical spheroids and human subpallium spheroids were placed in a conical tube and fused together after 3 days. This migration was reminiscent of interneuron migration in the fetal forebrain . Given tAdditional efforts to determine higher-level physical brain phenotype changes in schizophrenia return to previous studies of DISC1. The group involved had previously made an isogenic hiPSC model which showed in 2D culture that the balanced translocation of a loss of long DISC1 isoforms had a reduction in the production of T-box brain protein 2 (TBR2)—positive NPCs and an increase in baseline Wnt signaling . The groWhether or not DISC1 leads to clinically significant brain phenotypic changes is controversial as patients with DISC1 disruption do not have gross cortical structural defects, thus the phenotype may be amplified due to inherent qualities of the organoid system, including lack of support systems (microglia and neurovasculature). However, there are animal models with a reduction in cortical volume, enlarged ventricles, and thinning of the cortex with DISC1 disruption –138. In Critical to the modeling of downstream dopamine dysregulation in schizophrenia, a human midbrain dopaminergic system was developed which optimized tyrosine-hydroxylase expressing dopaminergic neurons. It was also shown that toxin-induced dopaminergic neuronal cell death was preserved in midbrain organoid models. The authors note this could replace the ‘highly used and ethically compromised’ 6OH-Da rodent model, with ‘great potential for drug screening’ .Further research questions on schizophrenia regard the contribution of neuroglia to pathophysiology. There is a significant increase in microglia density in patients with schizophrenia as compared with matched controls, and it is unknown if this is an environmental or genetic component as stress leads to elevated microglial activity in hippocampal regions . Other fMicroglia are centrally acting neural immune cells with ‘trained innate immunity’ with research leading to a possible role in schizophrenia . To modeFurther work has used schizophrenia patient-derived organoids to identify differences from controls in progenitor and differentiated neurons, discovering two novel gene growth and transcription factors, Pleiotrophin and BRN2, respectively. These factors were downregulated at the mRNA level to the peptide level within progenitors and rescue experiments could restore reduced neuron number in schizophrenia patient-derived organoids . Interes+ channel function and increased frequency of GABA transmission. Glutamate transmission appeared unchanged between groups and the phenotype is suggested to reflect an alteration in synaptic E/I balance. The cognitive tests of schizophrenia patients correlated to spontaneous excitatory postsynaptic transmission amplitude, and the number of Na+ peaks showed association with positive symptoms of schizophrenia [In a recent study, the bench to bedside approach was utilized as hiPSC-derived neurons were differentiated from fibroblasts in schizophrenia patients and controls, and electrical activity compared to patient performance on a variety of cognitive tasks. It was found that schizophrenia patient-derived neurons had altered Naophrenia .The question of heritability in schizophrenia as touched on previously is curious as twin concordance is estimated to be 50% . PatientDespite decades of research, most psychiatric disorders remain without consensus etiologies, however, they are generally theorized to be dependent on the action of neurotransmitters. The diverse receptor interactions of known psychotropic medications indicate that this is a clinically relevant hypothesis. While most of the organoid literature focuses on glutamatergic/GABAergic signaling, organoids have been developed to replicate dopaminergic and serotonergic signaling and have led to valuable insights into the mechanism and pharmacokinetics of antidepressants. In this section, we will explore human cell-based models which have shown promise in elucidating the origin of mood states and effects of clinical treatment.In one study of well-characterized MDD patients, hiPSCs were generated from selective serotonin reuptake inhibitor (SSRI)-remitters and SSRI-non-remitters, and serotonergic neurotransmission was examined in differentiated forebrain neurons. It was found that non-remitter patient-derived neurons displayed serotonin-induced hyperactivity downstream of upregulated serotonergic receptors, not seen in healthy or remitter samples. The authors indicated postsynaptic forebrain hyperactivity downstream of SSRI treatment could play a role in treatment resistance .In a demonstration of the advantage of advanced engineering of organoids, human choroid plexus organoids that created cysts of CSF-like fluid were used to test transport across the internal fluid of the organoids. Bupropion was found to cross into the organoid internal fluid and reached baseline levels similar to what is found in vivo .Another study used a class of antidepressants known as monoamine oxidase (MAO) inhibitors (MAOI). The study used tranylcypromine , a nonselective and irreversible MAOI, and tested the effect of exposure of 0–10 μM 2-CPA on human cerebral organoids. They found that 2-CPA impaired neural growth with a noted decrease in expression of Ki-67, and a cleaved caspase 3 expression increase. Global decrease of lysine-specific demethylase 1a (LSD1) and an increase in histone H3 lysine K4 (H3K4) methylation was also observed. Intriguingly LSD1 has sequence homology with MAO , and theDifferentiated neurons have been studied for a variety of mood disorders, including bipolar disorder, an important mechanistic distinction from unipolar depression as a bipolar diathesis indicates an improved response to mood stabilizers relative to antidepressants . In one 2+ imaging, hyperactive action potential firing found in hiPSCs of patients with bipolar disorder was selectively reversed with lithium, although only in patients who were classified as lithium responders [In further bipolar disease research in a 2D hiPSC model, hippocampal dentate gyrus-like neurons derived from bipolar patients detected mitochondrial abnormalities as compared to controls. In addition, using patch clamp and somatic Casponders .In a brain organoid model with cells derived from bipolar disorder patients from a research group that published a similar methodology with schizophrenia patients , genome-As psychiatrists treat all patients with behavioral disturbance, the etiologies are not limited to mood and psychotic disorders thought to be polygenic or neurodevelopmental in origin. Psychiatrists also treat individuals who suffer from substance use disorders, as well as cognitive disorders, including from exposure to environmental insults, notably prenatal exposure to alcohol and other substances. These disorders incur a high cost to the individual and society, and several labs have developed techniques for the in vitro modeling of pathogenesis and treatment of these conditions, demonstrating the utility of 3D cell culture techniques for propagating insight into environmentally-induced disorders.Autism studies are discussed in depth in other reviews concerning 3D in vitro models of neurodevelopmental disorders , 168–176Brain organoids that replicated first-trimester development were exposed to 50 mM of ethanol, which led to premature neural differentiation, more pronounced with prolonged exposure. This ‘hyperdifferentiation’ of glutamatergic neurons with conserved differentiation of GABAergic neurons was speculated to change E/I balance. Other changes included expression changes of neurogenesis-related genes in the Hippo pathway, a pathway which regulates brain size, notably including roof plate-specific spondin-2 (RSPO2), known to be involved in craniofacial morphogenesis. Cell death was also induced by ethanol exposure .In another model system, a novel alginate hollow fiber system for preparing brain organoids was used , and orgThe developmental consequences of prenatal nicotine exposure is an evolving and complex topic. However, there is a demonstrated association between prenatal nicotine exposure and altered brain structure and function as well a correlation with behavioral issues, notably ADHD, in exposed offspring . EffortsPrenatal cocaine exposure is another concerning neurodevelopmental insult, also studied in a 3D organoid model. In the model, it was found that cytochrome P450 3A5 (CYP3A5) mediated the generation of reactive oxygen species with cocaine exposure, as well as inhibition of neocortical progenitor cell proliferation, induction of premature neuronal differentiation, and interruption of neuronal tissue development. Knockdown of CP3A5 reversed these findings, suggesting CYP3A5 as a target for therapeutics designed to rescue harmful neurodevelopmental effects of in utero cocaine exposure . Similar2A, 5-HT2C, downregulating nuclear factors of activated T-cells (NFAT) and kappa B (NF- ΚB). Treated organoids exhibited downregulation of metabotropic glutamate receptor 5 (mGLuR5), which has a role in several drugs of abuse; notably mice lacking mGLuR5 do not self-administer cocaine. 5-MeO-DMT also caused significant upregulation of ephrin-B2 (EFNB2), and ephrin type-B receptor (EPHB), and other secondary messengers in dendritic spine formation [Another area of research into psychoactive substances concerns 5-methoxy-N,N-dimethyltryptamine (5-MeO DMT), an ‘entheogenic serotonin-like molecule,’ and structural analog of serotonin and melatonin. This substance is reportedly associated with cognitive gain and antidepressant effects. In studies using human cerebral organoid models, anti-inflammatory effects were found through downregulation through toll-like receptor and 5-HTormation .In experiments not limited to exogenous psychoactive substances, numerous studies have sought to determine how risk factors in the supportive developmental environment may lead to downstream consequences affecting nervous system functioning. In this section, we will review a selection of other factors relevant to psychiatric disease and cognition, from toxic to protective molecules, trace elements, and hypoxic conditions.Vincristine is a chemotherapeutic with the potential for a high degree of neurotoxicity, albeit with a controversial mechanism for damage to exposed neural tissue . In an oA contrasting utility of organoid models was shown through testing human brain organoids with minocycline, a tetracycline antibiotic, which is also under investigation for treatment of early psychosis due to its theorized neuroprotective qualities, and its high degree of lipophilicity and thus ability to cross the blood-brain barrier. This compound was tested in a model of neonatal hypoxia, and morphological atrophic changes and hypoxic stress gene expression changes were rescued by minocycline in this model .2 modeled mid-gestation human cortex in conditions of hypoxic encephalopathy of prematurity and second-trimester placental insufficiency. Several notable changes were found, notably TBR2-positive progenitors in the SVZ were particularly affected by oxygen deprivation. This response was thought to involve the unfolded protein response (UPR), a response pathway to endoplasmic reticulum stress. It was found that the UPR modulator, integrated stress response inhibitor, (ISRIB) which is BBB permeable, prevented the hypoxia-related TBR2 defects [Hypoxia as a condition is highly relevant to preclinical disease testing, as prenatal hypoxia correlates with postnatal morphological changes in brain structures involved in learning and memory and impaired development of cognitive functions . In one  defects .2 for 24 h, and 28 days after differentiation, and it was found that the progenitor population of ORG had immediate and prolonged apoptosis. As the ORG are more prominent specifically in primates, (Fig. In an additional work, organoids were cultured in a hypoxic chamber at 3% OOther miscellaneous relevant developmental factors include specific micronutrients or trace elements, as little is known about the in vivo developmental composition of the developing human CNS, although metal metabolism is known to be disrupted in neurodegenerative and neuropsychiatric disorders. Studies detailing elemental composition in organoids showed distinct levels of phosphorus, potassium and sulfur. It was also shown that trace elements change distribution inside organoids during differentiation. Potassium was seen to be first concentrating at the periphery then became evenly distributed as differentiation continued. Zn was more homogenous and then became peripheral at 45 days. Glutamate occupied the outer portion of organoids, interesting because Zn is present at high levels in glutamatergic synapses and Zn dependent metalloproteinases facilitate neural migration and outgrowth. These metal concentrations are clinically relevant as developmental deficiencies in Zn nutrition are associated with memory defects and maternal Fe deficiency may be a risk for schizophrenia in offspring , 199.Building on over a century of lab culture of neural tissue, human brain organoid models have increased in complexity in phenotypes and relevance to human development and circuitry, particularly in the past decade. Although these models do not have the benefit of behavioral outputs found in animal models, they are increasingly relevant to psychiatric diseases of uncertain etiology, whether polygenic, neurodevelopment, or acquired. Moreover, organoid models are likely to be increasingly relevant to translational researchers, including in searching for novel therapeutic compounds. Disease models will similarly be useful for psychiatrists seeking to further understand the complex neurochemistry of commonly treated diseases as they portend to known kinetic actions of neurotransmitters and upstream contributions of neurodevelopmental and environmental interactions. Areas of ongoing improvement include, but are not limited to, increasing recapitulation of the complex interaction between developmental regions of the CNS, and further maturation and sophisticated analysis of electrical signals, including those produced from stimuli.Human organoid studies nevertheless face several limitations, related to many features including indistinct batch reliability, and limited recapitulation toward maturing in utero development. Limited batch reproducibility in unguided organoids necessitates directed patterning to increase subtype specificity, and while assemblies of multiple organoids can recreate aspects of CNS subtype interactions, they remain limited in reiterating the full scope of CNS development. Perfusable vasculature also remains to be solved with in vitro models and the BBB remains a difficult to recapitulate phenomenon. As these technologies develop, it will also be important to increase the ability to record efferent responses to stimuli, as well as increase the ability for sensory input, although these will necessitate a continued discussion of best practice ethical principles.


In ruminants, most of the metabolic diseases  occur within the first month of lactation. For producers, these disorders represent enormous economic losses due to reductions in milk yield, increased risk of culling and mortality, and increased incidence and duration of common postpartum diseases .Liver is an essential organ, that plays an important role in preserving and regulating the metabolism of lipids, glucose, proteins in the body as well as energy metabolism. On the other hand, adipose tissue is a primary site for energy storage in the form of neutral triacylglycerol and controls systemic energy balance by regulating lipid mobilization and distribution in the body. Adipose tissue acts as an endocrine organ that produces numerous bioactive factors, such as TNF-α, IL-6, leptin, adiponectin and resistin, which communicate with other organs and affect metabolic homeostasis. In ruminants, a crosstalk between adipose tissue and liver is critical to keep animal's metabolic homeostasis. Thus, understanding liver and adipose tissue functions are pivotal in the context of preventing metabolic dysfunction of ruminants. Therefore, this Research Topic “Functions of liver and adipose tissue in metabolic disorder diseases of ruminants” aimed at compiling scientific reports focused on the alterations of liver and adipose tissue functions in the occurrence of metabolic disorder diseases of ruminants, the molecular mechanism of liver and adipose tissue dysfunction in ruminants, the crosstalk between liver and adipose tissue in the control of metabolic homeostasis of ruminants, and the potential strategy to detect and treat liver damage and adipose tissue dysfunction in the development and progress of ruminant metabolic dysfunction.Xue et al. assessed hepatic injury and described its metabolic mechanism in ruminants fed diets with different dietary energy levels. They used 25 Yunnan semi-fine wool sheep and fed them with five different dietary metabolic energy levels: low energy, medium–low energy, medium energy, medium–high energy, and high energy. They concluded that both high and low dietary energy levels caused hepatic injury and their data on liver tissue metabonomic analysis showed that hepatic injury might be caused by altered metabolism and lipid accumulation induced by lipid mobilization.This Research Topic accepted seven articles covering the aforementioned aspects. Deng et al. determined the relationship between negative energy balance, insulin resistance and inflammation in dairy cows and assessed the role of non-esterified fatty acids in the nuclear factor kappa beta (NF-κB) inflammatory and insulin signaling pathways through Toll-like receptor 4 (TLR4). For that, primary calf hepatocytes were cultured and added different concentrations of NEFAs to assess the mRNA and protein levels of inflammatory and insulin signaling pathways. They reported that high-dose NEFAs (2.4 mM) can activate the TLR4/NF-κB inflammatory signaling pathway and reduce the sensitivity of the insulin pathway through the TLR4/PI3K/AKT metabolic axis.Transition dairy cows are often in negative energy balance leading to lipid mobilization and high serum β-hydroxybutyrate (BHBA) and non-esterified fatty acid (NEFAs) levels, which can later induce ketosis and fatty liver in dairy cows. Mohsin et al. characterized plasma levels of GH, β-hydroxybutyrate acid (BHBA) and non-esterified fatty acid (NEFA) and glucose in ketotic cows and healthy control (CON) cows; to measure the liver function test indices in ketotic and healthy CON cows. Their study concluded that during postpartum, higher plasma GH levels in SCK cows might involve the initiation of body adipose tissue mobilization, resulting in hyperketonemia. In the same line, Yu et al. determined whether the enhanced transcription factor EB (TFEB) transcriptional activity contributes to lipolysis of adipose tissue in SCK cows and explored the possibility of establishing a therapeutic strategy by using TFEB as a target to control lipolysis. Interestingly, their findings indicated that enhanced transcriptional activity of TFEB may contribute to lipolysis of adipose tissue in dairy cows with SCK. The regulation of TFEB activity may be an effective therapeutic strategy for controlling overt lipolysis in ketotic cows.It has been suggested that during early lactation and the transition period, higher plasma growth hormone (GH) levels in subclinical ketosis (SCK) might involve the initiation of body adipose tissues mobilization, resulting in metabolic disorders in ruminants particularly hyperketonemia. Yang et al. performed a study to determine if TTO alleviates palmitic acid (PA)-induced lipid accumulation in bovine hepatocytes. Hepatocytes isolated from mid-lactating Holstein cows were used for this purpose. In summary, they showed that TTO exerts anti-inflammatory effects and acts to promote lipid homeostasis in bovine hepatocytes. These effects were associated with inhibition of NF-κB signaling and SREBP1c expression. Their data suggests that TTO treatment may be a promising therapeutic approach to imbalanced lipid homeostasis, inflammation, and endoplasmic reticulum stress in dairy cows shortly before and after calving.Essential oils extracted from plants contain secondary metabolites that exhibit anti-inflammatory activity and tea tree oil (TTO) has been shown to play an important role in lipid metabolism, as it ameliorates inflammatory responses. Thus, Reichelt et al. investigated the suitability of the fetal bovine hepatocyte-derived cell line BFH12 as a model for hepatosteatosis which is a common metabolic disorder of dairy cows, especially during early lactation. They found that BFH12 may be a useful in vitro model to study bovine hepatosteatosis and its underlying molecular mechanisms.Stiensmeier and Schmicke elucidated if the active form of thyroid hormones (T3) may have an additive and direct effect on hepatic GHR mRNA expression in vitro in bovine primary hepatocytes. With that study, they confirmed that T3 has a stimulatory effect on GHR1A mRNA expression at least at in vitro levels.Lastly, in a brief research report, In summary, results from the above-mentioned studies have improved our understanding on the functions of liver and adipose tissue in metabolic disorder diseases of ruminants. Despite all existing literature related to this topic. Many efforts are still needed to elucidate way for preventing metabolic problems during the first weeks after parturition in dairy ruminants, especially in cattle.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Acute kidney injury (AKI) is still a headache for clinicians and scientists as a possible reason for increased death among intensive care unit (ICU) patients after invasive cardiac surgery. Furthermore, the diagnostic process for AKI using conventional biomarkers is not sufficient to ensure early warning of this condition because of the morbid influence of non-renal factors that definitively delay the time for the prognosis. These imposed limitations have led to significant amounts of research targeted towards identifying novel biomarkers for AKI with a sustained degree of sensitivity and specificity. Here, we reviewed previous studies conducted on the Klotho, CYR61 and YKL-40 biomarkers in relation to AKI.  Review of the literature conducted in the Institute of Clinical Chemistry & Biochemistry, Ljubljana University Medical Center, Slovenia.  The literature was searched in PubMed and the Cochrane Library. From the database of this specialty, we selected 17 references that matched our context for detailed analysis and further investigation.  The studies reviewed showed notable differences in their results relating to the diagnostic impact of Klotho, CYR61 and YKL-40 on early prediction of AKI.  The results regarding the Klotho, CYR61 and YKL-40 biomarkers showed markedly equivocal performance in the previous studies and did not fulfill the expectations that these factors would form valid possible biomarkers for AKI. AKIN used two values of SCr within two days instead of baseline SCr, regardless of glomerular filtration rate (GFR) changes. According to AKIN, stage 3 AKI was confirmed when the duration of increased SCr levels did not exceed 48 h and the patient required renal replacement therapy (RRT).The failure of both RIFLE and AKIN to fulfill precise prognostic stratification of AKI severity and to provide a unified definition of AKI was the reason for establishing the Kidney Disease Improving Global Outcomes (KDIGO) guidelines. These novel criteria suggested that AKI should be defined by SCr levels that reached 26.4 µmol/l within 48 h or increased to a level 1.5 times higher than the baseline level within 7 days, which provides a sufficient timeframe for AKI diagnosis.,,,,The AKI incidence rate worldwide has remained imprecise because of the small number of case report studies, gaps in the data collected from patients and differences in definitions of AKI between developed and developing countries.Klotho (KL) is a novel phosphatonin encoded by the anti-aging KL gene located on chromosome 13q12 as an inactive single-pass transmembrane protein.,The pathological importance of Klotho emerged through studies on animal models for AKI that had previously undergone ischemic reperfusion injury (IRI) or unilateral urethral obstruction (UUO). Thus, a transient reduction in renal Klotho mRNA expression was shown in response to renal tubular injury.,CYR61 is a cysteine-rich matricellular protein encoded by the CYR61 gene located on chromosome 1p22.3. It is intercalated with various integrins and heparin sulfate proteoglycans and is associated with extracellular matrix formation, cell adhesion, proliferation, differentiation, angiogenesis, apoptosis and inflammation due to its biochemical features, which resemble Wnt-1 proto-oncogene, and its number of growth factors.E. coli , and from human patients with sepsis. They showed similar quantitative increases in comparison with controls without AKI.Chitinase-3-like protein 1 (CHI3L1) or YKL-40 is a 40 kDa glycoproteinThe objective of the current review was to focus on the suitability and validity of Klotho, CYR61 and YKL-40 as ideal predictive biomarkers for acute kidney injury.We conducted a comprehensive systematic search by using the main known databases: PubMed, SCOPUS, SciELO, Lilacs, ScienceDirect and Google Scholar. The MeSH search terms included: (''Klotho and Acute Kidney Injury''), (''CYR61 and Acute Kidney Injury'') AND (Chitinase-3-like protein 1 and Acute Kidney Injury''). The search strategy was designed for the PubMed database and was altered as needed for use in other databases. Our last search was finished in January 2016. References were written in the English language. The inclusion criterion was that all research articles, review articles and observational studies included needed to match our context, i.e. "the propensity of CYR61, Klotho and YKL-40 to be novel biomarkers for AKI". Additionally, we excluded papers that investigated these biomarkers in relation to chronic kidney disease (CKD) and other diseases as well as AKI.,,,,,,,,,,,,,,,,Our search revealed a total of 2917 references. From the title and abstract, while omitting review articles, case reports and similar results, the number of papers was reduced to 17, which included seven relating to the biomarker YKL-40, three relating to CYR61 and seven relating to Klotho . BrieflyIn this review article, we discuss the propensity of some novel biomarkers for early detection of AKI. Traditional biomarkers have been proven to be unable to satisfactorily distinguish AKI during the first 24 hours before kidney function is disrupted. This is certain to delay the diagnostic process and gives rise to the possibility that the patient's condition will worsen. Despite the paucity of studies on biomarkers and AKI (for reasons mentioned earlier), we conducted a comprehensive review of the literature encompassing all papers relating to our context, focusing on all the results.,,,Recent papers have inferred that reduced levels of Klotho correlated with emergence of soft tissue calcifications, cardiovascular diseases, senescence, cancers, chronic hypertension, osteoporosis, renal failure, diabetes mellitus, oxidative stress and uremic parathyroid hyperplasia.Sugiura et al. indicated that renal Klotho levels in rats started to fall on the first day and completely returned to normal within 10 days.A clinical study on urinary Klotho levels found that these were lower in AKI patients than in healthy individuals and recommended that this should be a candidate biomarker for AKI.Studies on Klotho were found to exhibit a variety of problematic issues: almost all the studies related to animal models rather than humans, with a narrow scale; there were unexplained variations between comparable studies; the mechanism of Klotho in AKI remains unknown, the behavior of Klotho in animal models differed from its behavior in humans; there was a lack of knowledge of ideal Klotho timing and normal cutoff ranges; and the urinary and plasma levels of Klotho were not indicative for renal Klotho, which might suggest that confounding factors and discrepancies in laboratory methodologies were present.,,According to Vaidya and Muramatsu et al., CYR-61 was rapidly stimulated in the proximal renal tubules and was excreted in urine within 3-6 h after bilateral renal ischemic injury in rats. Its peak was within 6-9 h and it declined after 24 h.Other previous papers investigating CYR61 expression found that it was induced by several growth factors, exposure to UV irradiation,The diagnostic capacity of urinary CYR61 as a biomarker might be blocked through: 1) its poor specificity, since it is normally abundant under both physiological and pathological conditions; 2) its rapid decline over time in spite of AKI progression; 3) the insensitivity of the immunoblotting technique used in quantification in urine; and 4) the fact that most studies were conducted on animal models because of difficulty in obtaining samples from human patients without prolonged routine registry for clinical trials in accordance with the World Health Organization (WHO) requirements and without prior patient approval.,Hall et al. showed that increased levels of urinary YKL-40 of up to 5 ng/ml were moderately correlated with AKI progression and/or mortality among patients. Moreover, apparent increases in YKL-40 levels in urine were observed in cases of kidney transplantation among patients hospitalized within 24 hours of developing AKI.,Another study by Schmidt and Malyszko et al. reported that urinary YKL-40 was better than serum YKL-40 levels for distinguishing between delayed graft function and slow or immediate graft function, within 3 days after kidney transplantation. Delayed graft function produces greater severity of ischemic kidney injury, while the damage from other types tends to become repaired.The results regarding the Klotho, CYR61 and YKL-40 biomarkers showed markedly equivocal performance in the previous studies and did not fulfill the expectations that these factors would form valid possible biomarkers for AKI.


Transverse myelitis (TM) is characterized by acute development of motor, sensory and autonomic dysfunctions due to horizontally diffused inflammation in one or more segments of the spinal cord in the absence of a compressive lesion. The not well-known inflammation process induces demyelination resulting in neurological dysfunction.In this case report we used a functional Near-Infrared Spectroscopy (fNIRS) technique to evaluate changes in the peri-spinal vascular response induced by a peripheral median nerve electrical stimulation in a patient with chronic transverse myelitis (TM). fNIRS showed drastically reduced signal amplitude in the peri-spinal vascular response, compared to that obtained from a healthy control group throughout most of the C7-T1 and T10-L2 spinal cord segments.The potential use of this relatively non-invasive fNIRS technology support the potential clinical application of this method for functional test of the spinal cord through the assessment of the spinal neurovascular response. Transverse myelitis (TM) is characterized by acute motor, sensory and autonomic dysfunctions due to the horizontally diffused inflammation in one or more segments of the spinal cord in the absence of a compressive lesion . The infCurrently, MRI or computed tomography assessment of the spinal cord is mainly structural, however, they provide little information on spinal function . At the 2) and deoxyhemoglobin (Hb), which is directly correlated with neuronal activity ) and deoxyhemoglobin (Δ [Hb]) and lower (T10 and L2) spinal cord, which were selected due to the cervical and lumbar sensory enlargement of the spinal cord  applied to the skin using electroconductive gel, over the nerve at the wrist medial midline. An array of 4 light sources and 4 photodetectors totaling 8 NIRS channels with a source-detector optodes distance of 4.5 cm were used to record the concentration changes of oxyhemoglobin  designed for brain fNIRS and based on a continuous wave technology was modified to record spinal NVRs. The system uses time multiplexed method to combine sources and detectors without interference, with dual wavelengths of 760 nm and 850 nm i.e., under and above hemoglobin spectral isosbestic point within < 1 pW sensitivity.A commercially available fNIRS device   was relatively normal Table , left spThe results reported here strongly suggest the ability of the non-invasive fNIRS assessment of the peri-spinal NVR to detect the functional alteration in the TM patient. The regional blood flow in a neuronal population is tight- and directly associated to the neuronal metabolic rate and activity, process known as neuro-vascular coupling , 11, 14 Despite that the BMI for the patient was higher than that for healthy controls, Valenzuela et al., 2021 11) did not find statistical association between subjects’s BMI and spinal NVR parameters such as amplitude, rise time or duration, thus, contribution of the BMI to the spinal NVR in this case should be marginal [ did not These results strongly support the application of the new fNIRS to assess the spinal NRV in TM and its potential use on functional assessment of the human spinal cord, and opens a wide range of possibilities of examining other spinal inflammatory diseases, edema from different etiology, pharmacological effects, and ischemic lesions at the spinal cord in the same way that affects the brain .