Unraveling the inner workings of neural circuits entails understanding the cellular origin and axonal pathfinding of various neuronal groups during development. In the embryonic hindbrain, different subtypes of dorsal interneurons (dINs) evolve along the dorsal-ventral (DV) axis of rhombomeres and are imperative for the assembly of central brainstem circuits. dINs are divided into two classes, class A and class B, each containing four neuronal subgroups (dA1-4 and dB1-4) that are born in well-defined DV positions. While all interneurons belonging to class A express the transcription factor Olig3 and become excitatory, all class B interneurons express the transcription factor Lbx1 but are diverse in their excitatory or inhibitory fate. Moreover, within every class, each interneuron subtype displays its own specification genes and axonal projection patterns which are required to govern the stage-by-stage assembly of their connectivity toward their target sites. Remarkably, despite the similar genetic landmark of each dINs subgroup along the anterior-posterior (AP) axis of the hindbrain, genetic fate maps of some dA/dB neuronal subtypes uncovered their contribution to different nuclei centers in relation to their rhombomeric origin. Thus, DV and AP positional information has to be orchestrated in each dA/dB subpopulation to form distinct neuronal circuits in the hindbrain. Over the span of several decades, different axonal routes have been well-documented to dynamically emerge and grow throughout the hindbrain DV and AP positions. Yet, the genetic link between these distinct axonal bundles and their neuronal origin is not fully clear. In this study, we reviewed the available data regarding the association between the specification of early-born dorsal interneuron subpopulations in the hindbrain and their axonal circuitry development and fate, as well as the present existing knowledge on molecular effectors underlying the process of axonal growth. The vertebrate central nervous system (CNS) is composed of a vast array of neuronal circuits that are assembled in a stepwise manner to give rise to the enormous diversity of cells and functions. Cell fate acquisition, neural cell migration, and axonal projections are all initiated in the developing neural tube and give rise to the neuronal networks of genetically defined neurons that are interconnected within the CNS, as well as with afferent/efferent connections with peripheral targets. It is only with the advent of molecular techniques of lineage tracing, designated gene mutations, and enhanced developmental analyses that have allowed the emerging understanding of the details of neural connectivity assembly. Although many studies uncovered genes that regulate different aspects of the multi-event process that spans from neural specification to circuit formation, there is still missing knowledge regarding how these complex mechanisms are orchestrated to give rise to functional networks, and what goes wrong in neurodevelopmental disorders.via multiple circuits that regulate vital functions such as breathing, fine-tuning movement, blood pressure adjustment, auditory and vestibular sensations and facial movement , along the anterior-posterior (AP) extent of the hindbrain anlage  Lumsden. A more In all vertebrates, hindbrain compartmentalization is controlled by combinatorial expression and activity of multiple families of transcription and signaling factors across each rhombomere, leading to the generation of distinct cell lineage compartments . The ideInvestigation of the neuronal patterns along the dorsal hindbrain has identified 6–8 progenitor domains that are born in designated positions in some or all rhombomeres . In contThe specification of the different dA/dB subclasses requires coordinated signaling cues that arise from the roof and floor plate (FP) , and provide positional information which leads to the birth of individual neuronal fates . The mecFollowing the differentiation of neurons, they start migrating to their final destinations in the mantle layer as well as to project axons that extend toward their target sites in a stepwise manner, under the control of guidance cues along their pathways . In the This review aims to cover the gap between the vast knowledge on hindbrain nuclei projections and their association to specific dA/dB INs along the hindbrain AP axis. We will not discuss r0, r1, and their contribution to the isthmus and cerebellum, and mainly concentrate on r2–r7/8, the rhombomeric units which are morphologically evident and coincide in the two rhombomere-classification systems in use . Moreove+ neurons in several nuclei subtypes in the auditory system, such as in the ventral and dorsal cochlear nuclei  and the superior olivary nuclei (SON), Atoh1+ neurons in the spinal, medial, and lateral vestibular nuclei  and in the vestibular nucleus X, Atoh1+ neurons in the main and descending sensory spinal trigeminal nuclei and Atoh1+ neurons which establish the various precerebellar nuclei (PCNs), which include the pontine gray nuclei (PGN), reticulotegmental nucleus, lateral reticular nucleus (LRN), and external cuneate nucleus (ECN)  . Interesus (ECN) . All of at birth . Despitevia Mossy Fiber (MF) tracts . However, only with the development of genetic tools that utilized Atho1-enhancer elements to label neuronal precursors in the dorsal RL, these trajectories could be attributed to the dA1 subpopulation  tracts . Further) tracts . Based opulation . Subtracd nuclei . As suchavian NL , while tavian NL .+ lineages on both sides of the hindbrain, the ability to trace unilateral projections at subsequent stages was compromised. In a series of studies in the chick hindbrain, we aimed at tracing the sequential axonal growth of dA1 INs at multiple stages, using unilateral electroporation of plasmids encoding dA1-specific enhancers upstream of Cre recombinase together with nuclear, cytoplasmic, or synaptic GFP reporters. Hindbrains were electroporated at stages when dA1/Atoh1+ cells are specified and their axons were traced for 2–16 days  progenitors that upon maturation express the transcription factors Lhx1/5, Forkhead box protein 2 (Foxp2), and Pou4f1 (+ neurons to the inferior olivary nucleus (ION), a major pre-cerebellar center that connects to the cerebellar Purkinje layer via climbing fiber (CF) axons  axons . Yet, fuThe axonal projection patterns of dA2 neurons have not yet been delineated. This is in contrast to the spinal cord, where genetic labeling of dI2 INs in the chick spinal cord uncovered their contralateral ascending or descending projections, according to the thoracic/sacral level, before turning laterally in the white matter toward the LF . Genetic+)   (Qian et+) . Previou+) . However+/Tlx3+/Ascl1+) neurons that emerge from r4–r7 contribute to the nucleus of the solitary tract (NTS), a major relay station for visceral sensory information regulating the activity of the cardiovascular, respiratory, vocalization, and digestive systems  nucleus, the dorsal motor nucleus of the vagus, dorsal regions of the tegmental nucleus, the cerebellar vermis, the paratrigeminal nucleus, the ventrolateral catecholaminergic column in the medulla, and the spinal trigeminal tract . Complex nucleus . Notablyvia the ipsi/contralateral ventral and lateral funiculi. These projections further branch in a complementary fashion to reach the dorsal/ventral horns of the spinal cord at different axial levels  . The A5 l levels . Finallyl levels .Altogether, dA3 INs exhibit complex fates and display multiple ascending and descending axonal routes , emphasi+/Olig3+/Mash+ progenitors that also express the bHLH factor Pancreas Specific Transcription factor 1a (Ptf1a)  and were suggested to depend on Ptf1a for their inhibitory neuronal fate   (Fedtsov (Ptf1a) . dA4 neu (Ptf1a) , like alnal fate . While tnal fate . So far,nal fate . Formati neurons . Intrigu+ neurons in wild type and mutated zebrafish/mouse embryos confirmed that Ptf1a+ ION neurons extend excitatory commissural projections that innervate Purkinje cells  (+/lbx1+) derivatives were shown to give rise to multiple GABAergic neuronal cell types such as the inhibitory stellate cells, cartwheel cells, and Golgi cells, as well as to a small glycinergic population within the VCN , three relay sensory hubs located in the medial column of the medulla and function to control eye, head and neck movements to maintain balance  . The DCNris (NA) . Within  the VCN . Moreove balance , as well balance . This we+ neuronal derivatives. For instance, the mammalian DCN/avian NA was found to form local medullary connections with the ipsilateral mammalian VCN/avian NM. This center projects to the nearby SON and the lateral lemniscal nuclei and extends projections through the ipsi and contralateral DF into the mammalian inferior colliculi/avian nucleus mesencephalicus lateralis pars dorsalis (MLD) in the anterior midbrain, an auditory center that transmits inputs to the medial geniculate body of the thalamus  to the midbrain Edinger–Westphal nuclei   . Other vy fibers  (Akert ey fibers . Albeit +/Lhx1/5+/Pax2+) dINs and demonstrate their multiple axonal projections that extended at subsequent time points  neurons to the RTN/parafacial respiratory group (pFRG) in the ventral medulla. This glutamatergic center relays modulatory input to the preBötC to control respiration rhythm by chemosensing CO2 levels in the blood , that descends along the spinal cord to connect to motor neurons of the extensor musculature of the limbs and the trunk . These LAltogether, the complex reality of several dB2 subgroups as recognized by their unique transcriptional profile and fates among different rhombomeres, require further fate map investigations to reveal their precise destination and axonal connection to different somatosensory brain circuits.+ progenitor domain and has a unique transcriptional profile of Lbx1/Tlx3/Lmx1b/Pou4f1/FoxP2  and the principal trigeminal nucleus (Pr5), that relay various sensory modalities including temperature, touch, and pain from the ipsilateral faces   . This nep (cVRG) . As suchreathing . Since W+ neurons project ipsi and contralateral axons that innervate somatic motoneurons of tibialis anterior and/or gastrocnemius  family, is expressed in PGN neurons in the ventral hindbrain. Zic1 was found to drive axon laterality choice to the ipsilateral, rather than the contralateral pontocerebellar tract, by inhibiting axonal midline crossing . PGN neuling cue . Future + group, require Robo/Slit signaling for axonal elongation from the dorsal rhombic lip toward the FP and for crossing the midline, which fails to occur in Robo3 mutant mice  family of transmembrane receptors and their soluble ligands Slit are chemorepulsive cues fundamental for commissural axonal guidance in the CNS . Mutatioant mice . Similarent mice . At varient mice . Interesent mice . These fent mice , 2015 isent mice , indicatent mice .Cell adhesion molecules (CAMs) are instructive for axonal pathfinding, elongation, and fasciculation . SeveralEph proteins, including the Eph receptors and their ligands ephrins, are fundamental for axonal guidance in the CNS . A serieMultiple pieces of evidence have shown the involvement of the chemoattractant molecule Netrin and its receptor Deleted in Colorectal Cancer (DCC) in hindbrain commissural axons . For insThe role of the neuropilin (Npn) receptors and their semaphorin (Sema) ligands in axonal growth is well known in various sensory and motor neural systems . In the in vivo identification of FMRP activity in developing axons in the hindbrain. Further studies are required to elucidate FMRP-downstream RNA targets in dA1 axons and to uncover whether fragile X patients suffer from axonal development deficits in the auditory brainstem.The RNA binding protein FMRP is broadly expressed in the CNS where it acts as a reversible repressor of specific mRNA translation . FunctioThe orderly and conserved development of the segmented hindbrain in vertebrates, together with its fundamental roles to transmit and process sensory-motor orders that arrive from the surroundings, or the spinal cord and higher brain centers, makes it a fascinating CNS domain. Yet, how it is set to produce diverse neurons that are programmed to assemble dedicated neuronal circuits that execute a wealth of physiological actions, is only partially clear. The ground plan to develop 8 dINs subclasses along the longitudinal axis of the hindbrain which have to respond to antagonistic roof plate and floor plate DV cues along with rhombomere-specific AP positional information is unique. Many important studies along the years have initially mapped definite DV and AP positions in the hindbrain that drive specific axonal tracts and neuronal clustering which in turn give rise to different brainstem nuclei. With the generation of transgenic and mutant animal models, the genetic code of all individual dA/dB subtypes has been decoded, enabling investigation of the developmental program, circuit formation, and fate of dA1-4/dB1-4 neuronal subgroups. Yet, many challenges remain to fully correlate the identity of specific dINs in the early embryo with brainstem nuclei and projections at advanced stages, to identify the mechanism by which each dA/dB subgroup, which displays a uniform genetic identity along the AP axis, can eventually give rise to different types of neuronal cell types with distinct axonal connections, target sites, and functions, and to uncover how multiple attractive and repulsive axonal cues are orchestrated to guide the step-by-step assembly of multiple axonal projections that arise from individual and/or neighboring dINs in the developing hindbrain.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.DH, AK, YW, and DS-D participated in writing and revising the manuscript and preparing the figures and contributed to the data presented in the manuscript. All authors contributed to the article and approved the submitted version.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.


Maternal immune activation (MIA) is a known risk factor for schizophrenia (SCZ) and autism spectrum disorder (ASD) and is often modelled in animal studies in order to study the effect of prenatal infection on brain function including behaviour and gene expression. Although the effect of MIA on gene expression are highly heterogeneous, combining data from multiple gene expression studies in a robust method may shed light on the true underlying biological effects caused by MIA and this could inform studies of SCZ and ASD. This study combined four RNA-seq and microarray datasets in an overlap analysis and ranked meta-analysis in order to investigate genes, pathways and cell types dysregulated in the MIA mouse models. Genes linked to SCZ and ASD and crucial in neurodevelopmental processes including neural tube folding, regulation of cellular stress and neuronal/glial cell differentiation were among the most consistently dysregulated in these ranked analyses. Gene ontologies including K+ ion channel function, neuron and glial cell differentiation, synaptic structure, axonal outgrowth, cilia function and lipid metabolism were also strongly implicated. Single-cell analysis identified excitatory and inhibitory cell types in the cortex, hippocampus and striatum that may be affected by MIA and are also enriched for genes associated with SCZ, ASD and cognitive phenotypes. This points to the cellular location of molecular mechanisms that may be consistent between the MIA model and neurodevelopmental disease, improving our understanding of its utility to study prenatal infection as an environmental stressor. Prenatal infection is among the most well-studied environmental risk factors for abnormal neurodevelopment , present+ and Ca2+) [Many studies of MIA models include analysis of gene expression to investigate the effect of infection on molecular genetic function within the brains of offspring. Technologies for transcriptome-wide analysis including RNA sequencing (RNA-seq) and microarrays facilitate the detection of gene expression changes between experimental treatment groups and controls. These techniques provide insights into the effect of MIA on brain gene expression. Individual studies find MIA to elicit several changes to the expression of genes involved in ion channel signalling (notably Knd Ca2+) , myelin nd Ca2+) ,10, neurnd Ca2+) , synapsend Ca2+)  and nuclnd Ca2+) .Investigating expression changes for individual genes alone can lack context. Most biological processes are influenced by multiple genes. For this reason, Gene Ontology (GO) analysis can be usefully performed on sets of differentially expressed genes (DEGs) in order to find biological pathways, cellular compartments and molecular functions that may be disrupted as a result of MIA. In combination with specific genetic and ontological changes, investigating vulnerable cell types can also crucially inform on the biological impact of gene expression changes. Large single cell transcriptomics studies employing single cell RNA-seq (scRNA-seq) ,12 now pOur aim was to combine and meta-analyse gene expression data from multiple studies of MIA models to identify genes that showed consistent changes in expression across those studies. Based on these data, we next identified biological processes and individual cell types associated with these genes. We further investigated if these genes, processes and cell types were enriched for risk genes reported in human studies of neurodevelopmental phenotypes. Our rationale for doing so was that significant overlap between human studies and the MIA model could point to cell types and molecular mechanisms that are vulnerable to genetic variation and environmental stress, and may underpin the cognitive and social changes common to both.https://pubmed.ncbi.nlm.nih.gov (accessed on 7 January 2021)) identified 33 original research papers reporting on MIA models up until 31 December 2020. Of these, twelve used Poly(I:C) as the stimulating agent in mouse and the number was further reduced to nine when only considering those studies that included transcriptome-wide analysis of gene expression using either RNA-seq or microarray. Three studies were excluded based on their experimental parameters; analysis of foetal brain tissue rather than adult brain [A comprehensive literature search using NCBI PubMed ) [p-value  of < 0.05. Where necessary, the biomaRt 2.40.5 [Sets of DEGs were subjected to Gene Ontology (GO) statistical overrepresentation tests using ConsensusPathDB -treated animals compared to controls, regardless of magnitude). Once genes were separated into upregulated and downregulated lists, they were ordered individually by significance and each gene was given a ranking (the most significant gene was ranked first). The rankings for each were then arithmetically summed or multiplied to produce sum rank and product rank, respectively. The top 20 genes in each of the 4 lists were subjected to a literature search for links to SCZ, ASD and/or neurodevelopment. The top 5% of each list were subjected to GO analysis.https://ctg.cncr.nl/software/MAGMA/aux_files/NCBI37.3.zip (accessed on 27 January 2021)) and 1000 Genomes European Panel reference (https://ctg.cncr.nl/software/MAGMA/ref_data/g1000_eur.zip (accessed on 27 January 2021)) files, the latter which MAGMA uses to account for linkage disequilibrium (LD) between SNPs. Second, MAGMA generated p-values for individual gene reflecting their level of association with a phenotype. Thirdly, using each gene’s association result, MAGMA tested each gene-set for enrichment of genes associated with SCZ, ASD, IQ or EA.GWAS summary stats for SCZ  , ASD (18Gene-sets were tested for enrichment of de novo mutations found in published exome sequencing studies of SCZ 3394 trios) ,29,30,3194 trios p-values less than Bonferroni-adjusted significance thresholds for the human data were then compared to those cell types from the MIA analysis.Data from two separate scRNA-seq studies on the mouse brain (565 cell types ) and nern = 39 in total) compared to the mPFC study  data. The one consistent factor across the four datasets was that more DEGs were significantly upregulated than downregulated in treatment versus control groups.cellular retinoic acid binding protein 1 (Crabp1) and dopamine receptor D3 (Drd3) and only Crabp1’s change in expression was in the same direction for all three studies where it was downregulated in treated animals compared to controls. The D3 dopamine receptor plays a role in clinical antipsychotic response [n = 72), NA and mPFC (n = 63), and AM and NAc (n = 12). response . Crabp1 response ,38,39. OThese three sets of DEGs that overlapped between studies were subjected to GO analysis. Several GO terms relevant to neurodevelopment were found to be enriched for overlapping DEGs: neuron differentiation, nervous system development, glial cell differentiation, ensheathment of neurons, myelination, synaptic structure and function, and dopamine receptor signalling. Separate meta-analyses of upregulated  and downregulated genes were performed next. Nine hundred and forty-seven genes were consistently upregulated, and 428 genes were consistently downregulated in all four datasets. After ranking within each dataset, ranks were then combined across the datasets by two methods, a product of the ranks and a sum of the ranks (from here on termed product rank and sum rank). There was a high degree of consistency between the product rank and the sum rank for the downregulated genes where eleven genes were in the top twenty positions for both methods . There w+ channel structure and function, synaptic structure, neuron projection and vesicle transport. The full list of significantly enriched GO terms for these gene-sets are in Cck, Slc4a7, and Kcnn1  are members of the enriched neuron projection (GO:0043005) ontology. Furthermore, Cck and Slc17a5 (positions 1 and 15) are members of the enriched presynapse (GO:0098793) ontology. As discussed below, many of the individual highest-ranking genes also have ties to SCZ/ASD including Cck (position 1 in summed rank) and Szt2, Fgf10, Serinc2 and Crhbp , as well as many having ties to crucial neurodevelopmental processes.To see what biological processes were enriched for the highest-ranking genes, GO analysis was performed on the top 5% by rank of these upregulated and downregulated genes. Enriched GO terms for upregulated genes in the top 5% for sum rank included KNine gene-sets were included in this analysis; the four sets of DEGs from the individual studies , the three sets of DEGs that overlapped between AM and mPFC, NAc and mPFC, and AM and NAc, and lastly the two set of genes that were consistently upregulated and consistently downregulated across all four studies from the meta-analysis. Each gene-set was analysed in two independent datasets of scRNA-seq gene expression data from the mouse brain  . Despite this, as gene expression patterns are very often strongly correlated across brain regions, we still proceeded to initially identify genes that had consistent changes in expression across those studies. Although analysis of the FC data shows an apparently diminished biological response compared to the other datasets, the original study found behavioural effects consistent with ASD as well as transcriptomic changes involving glutamatergic neurotransmission, mTOR signalling and Kal study . Among tFgf10, Cck, Crhbp, Igfbp7, Napepld) and one downregulated gene (Zeb1) among the top 20 genes were found to have links to SCZ. Higher concentration of Fgf10 product is associated with increased positive and negative syndrome scale (PANSS) total score in humans [Cck gene has a neuropeptide product in which the human orthologue is found to have abnormal levels of mRNA in the entorhinal cortex of schizophrenia patients [CCK gene product (CCK-AR) may also be associated with positive symptoms of schizophrenia, particularly auditory hallucinations [CRHBP is a gene involved in the HPA-axis found to have links with suicide attempt [IGFBP7 gene product was found to be significantly lower in patients with schizophrenia compared to controls [NAPEPLD contains a polymorphism associated with SCZ in a Han Chinese sample [ZEB1 was implicated in human schizophrenia in a GWAS region-based analysis that analysed signals from independent 100 kb regions [When the datasets were meta-analysed, five upregulated genes  and three upregulated genes  among the top 20 genes have possible links to ASD. Of the downregulated genes, each has a link to ASD or autism-like behaviour through deletion. Loss of Adcy5 in mouse dorsal striatum lead to autism-like behaviour [DOCK10 following an autosomal recessive inheritance pattern was attributed as being causal of ASD [GLI3 was associated with causing subclinical autism in the father of a child with ASD and Greig cephalopolysyndactyly syndrome [SZT2 mutation was reported to be causal of a syndrome incorporating autistic features as well as intellectual disability, epilepsy and developmental delay [Serinc2 is a proposed ASD candidate following a chromosomal array [Maf has variants associated with Aymé-Gripp syndrome, which includes ASD as a characteristic [Three downregulated genes  may be involved in macrocephaly, developmental delay, pharmacoresistant epilepsy and intellectual disability phenotypes [Serinc2 [Kcnn1 [Rybp, Scube3 and Ifrd1  gene products are found in high levels in the developing neural tube, with Rybp playing a crucial role in mouse neural tube closure in early development [Rybp, Slc4a7, Fgf10, Zeb1, Hmgn2 and Gli3 play a role in neuronal differentiation [Fgf10 gene also plays a role in axonal regeneration and recovery [Rem2 has a function in forming glutamate synapses and dendrite outgrowth [Rgmb plays a role in axonal regeneration [Fgf10 provides protection against ischemia-driven oxidative stress [Syt10 and Slc4a7 act to protect neurons from excitotoxic injury [Many of the top genes have associations with relevant disorders and important neurodevelopmental processes. The in mouse  and mutaenotypes ,61,62,63[Serinc2 ,65 and K2 [Kcnn1  are stroelopment ,68,69. Gntiation ,73,74,75recovery  as well recovery . Anotherutgrowth , while Rneration . Some ofe stress , while Sc injury ,81. Cck, Slc4a7, Slc17a5, Napepld and Kcnn1 may be interfering with the ability to maintain normal synaptic structure and function and leading to aberrant neurotransmission and ultimately, behaviour.Gene ontology analysis of the highest-ranking genes revealed enrichments for genes involved in ontologies of synaptic structure, neuron projection and vesicle transport, all of which are crucial to the proper function and maintenance of synapses. Synapse function is implicated in schizophrenia in studies based on both post-mortem analysis of human brains  and gene+ NAc DEGs, all seven genes  that are members of the enriched “ensheathment of neurons” GO term and four out fo the five gene members  of the enriched “glial cell differentiation” GO term are highly expressed in the enriched cortical and hippocampal oligodendrocytes from the Saunders data. All of these genes, with the exception of Mal, are also among the top 5% of genes specifically expressed in the enriched MOL2 oligodendrocyte cell type in the Zeisel data. Three of the seven genes from the mPFC+ AM overlap  that are members of the “cilium” GO term are in the top 5% of genes specifically expressed in the enriched EPEN ependymal cell type in the Zeisel data. Finally, one of two DEGs from the mPFC+ AM overlap (Adcy5) is a member of the enriched “adenylate cyclase-activating dopamine receptor signalling pathway” GO term and this gene is highly expressed in dSPN and iSPN neurons among dopaminergic striatal cell types in the Saunders data, while Adcy5 is also in the top 1% of genes specifically expressed in both of the enriched dopaminergic striatal MSN cell types  from the Zeisel data. Although the enriched lipid metabolism GO terms that could be linked to a cell type, a disruption in lipid metabolism may have a link to human SCZ development. Recent evidence showed baseline serum lipid differences between individuals at clinical high risk for psychosis (CHR) and healthy control, as well as distinct serum lipid profiles between CHR individuals who went on to develop psychosis and those who did not after 5-year follow up [Using scRNA-seq data to identify the cell types that are enriched for DEGs was not performed in any of the original studies. This analysis provided some interesting insights. Firstly, several the DEGs that are driving the cell type enrichments are also driving the GO enrichments, indicating cell types where different biological process may be disrupted. For example for the mPFCollow up .Several of the cell types found to be enriched for DEGs from MIA studies are also enriched for genes associated with SCZ, ASD or cognitive phenotypes in human studies. These include excitatory glutamatergic neurons  in the cortex and CA1 principal cells, hippocampal interneurons and cells of the entorhinal cortex. Studies of SCZ implicate the hippocampus at the anatomical level, with meta-analysis finding reductions in hippocampal volume in both first-episode psychosis and chronic schizophrenia compared to healthy control . The entDespite these cell type links between the MIA transcriptomic data and human GWAS studies, there was no direct overlap between MIA DEGs and genes associated with SCZ, ASD, IQ and EA. Furthermore, no enrichments for rare de novo mutations contributing to SCZ or ASD were found among MIA DEGs. This result in the context of the cell type enrichment similarities suggests that the additional risk for SCZ caused by maternal infection may be mediated through a smaller set of genes regulating a small number of vulnerable cell types. Larger genome-wide investigations may not be able to effectively detect this signal in isolation. It is possible that other risk factors may induce similar cell-specific enrichments that, when integrated with MIA, would facilitate the detection of a more generalised risk spread across a larger set of genes. This suggests that while the same cell types could be affected by genetic variation in the human disorders and environmental stress in the MIA models, underpinning the cognitive and social behaviours common to both, it is not the same genes that are affected.There are several limitations to the approach used in this study. First, the low number of datasets make it difficult to generalise findings to future studies of MIA. Second, two datasets are from the same study, performed under identical conditions, leading to possible biases in the data. Third, although the need for all genes to be expressed in the same direction is a robust measure, it suffers from a general lack of power and misses out on many genes that may have been differentially expressed in the same direction in at least three studies. Fourth, expression fold changes were not taken into consideration when finding and ranking unidirectional genes. Finally, gene expression data are derived solely from male mice, and the confounding effects of sex on the outcome cannot be studied.In summary, this study combined four RNA-seq and microarray datasets in an overlap analysis and ranked meta-analysis in order to investigate genes, pathways and cell types dysregulated in MIA mouse models. Genes linked to SCZ and ASD and crucial in neurodevelopmental processes including neural tube folding, regulation of cellular stress and neuronal/glial cell differentiation were among the most consistently dysregulated in these ranked analyses. Gene ontologies including K+ ion channel function, neuron and glial cell differentiation, synaptic structure, axonal outgrowth, cilia function and lipid metabolism were enriched for DEGs. Single-cell analysis identified excitatory and inhibitory cell types in the cortex, hippocampus and striatum that may be affected by MIA and are also enriched for genes associated with SCZ, ASD and cognitive phenotypes, pointing to the location of molecular mechanisms that may be consistent between the MIA model and the neurodevelopmental diseases that the model is used to study. Understanding the link between genetics, environment, and this early neurodevelopment, which is facilitated by molecular analysis of MIA models, is essential for study of neurodevelopmental disorders.


Powerful bioinformatics tools have provided a wealth of novel miRNA–transcription factor networks crucial in controlling gene regulation. In this review, we focus on the biological functions of miRNAs targeting ZNF521, explaining the molecular mechanisms by which the dysregulation of this axis contributes to malignancy. ZNF521 is a stem cell-associated co-transcription factor implicated in the regulation of hematopoietic, neural, and mesenchymal stem cells. The aberrant expression of ZNF521 transcripts, frequently associated with miRNA deregulation, has been detected in several tumors including pancreatic, hepatocellular, gastric, bladder transitional cell carcinomas as well as in breast and ovarian cancers. miRNA expression profiling tools are currently identifying a multitude of miRNAs, involved together with oncogenes and TFs in the regulation of oncogenesis, including ZNF521, which may be candidates for diagnostic and prognostic biomarkers of cancer. Most of the miRNA targets are transcription factors (TFs), which are critical in trans-regulating gene networks in many cellular systems . TFs areThe miRNA–TF target regulation is orchestrated by a complex mechanism in which the expression of a miRNA can be regulated by TFs, and a gene encoding TFs can be inhibited by miRNAs  Figure .miRNAs and TFs act in a coordinated manner by forming two different autoregulatory feedback loops, the unilateral or reciprocal negative feedback loops and double-negative feedback loops, in which the expression of a component can be directly influenced by the presence or absence of the other . In the In this review, we discuss miRNAs targeting the transcription factor ZNF521 to clarify the current literature, largely based on bioinformatic analysis. The interaction between different miRNAs and the transcription factor ZNF521 is at the center of complex regulatory networks during growth and development as well as during tumorigenesis. Currently, the investigation of the molecular mechanisms underlying the relationship between specific miRNAs and the ZNF521 target gene could aid in the understanding of tumorigenesis and provide molecular targets for alternative therapeutic approaches. Zinc finger protein 521  is a stem cell-associated transcription co-factor composed of 30 zinc fingers organized in 5 domains and an N-terminal transcriptional repressor motif that binds to the components of the nuclear remodeling and histone deacetylase (NuRD) complex ,12. hZNFIn addition to the role for ZNF521 in cell fate determination in different types of progenitor cells, this TF also plays a crucial role in cancer malignancies. The overexpression of this protein sustains clonogenic growth, migration and tumorigenicity of medulloblastoma cells, and recently it was demonstrated to interact with GLI1 and GLI2 proteins stimulating the trans-activation of the Sonic Hedgehog pathway in medulloblastoma ,26. Zfp5In light of the remarkable multifunctional regulatory capabilities of ZNF521, in recent years, many studies have focused on identifying potential miRNAs able to target and modulate ZNF521 using novel bioinformatics platforms. The growing interest in miRNAs is linked to their ability to control the development and progression of tumors by targeting gene regulation at both the transcriptional and post-transcriptional levels, as well as transcription factors (TFs), such as ZNF521, which are considered important regulatory actors in solid and hematological malignancies. In addition to the role for ZNF521 in cell fate determination in different types of progenitor cells, this TF also plays a crucial role in cancer malignancies.Pancreatic cancers were studied by bioinformatic in silico analysis  to identCompared to pancreatic cancers, hepatocellular carcinomas (HCC) express high miR-802, which is up-regulated compared to adjacent, non-tumor tissues. In 2020, Yang et al.  demonstrIn addition, it was shown in HCC cells that ZNF521, acting as a co-transcriptional repressor, binding Runx2  and inhiIn 2014, Bai and colleagues performed an integrated analysis of miRNA and mRNA in HCC cells; they compared miRNA and mRNA expression profiling in HepG2 cells and normal liver HL-7702 cells using next-generation sequencing technology. Bioinformatics analysis with specific enrichment tools  allowed the identification of potentially dysregulated miRNAs correlated to target genes and their classification according to the biological processes and pathways in which they could be upregulated. In a complex network of mRNAs and miRNAs, ZNF521 seems to have a close relationship with miR-105 and miR-361-5p, although the molecular meaning was not addressed. ZNF521 was considered as a predicted target gene of these dysregulated miRNAs . However, it must be considered that a single miRNA can target and regulate many mRNAs and an individual mRNA may be regulated by multiple miRNAs, resulting in complex regulatory networks . FurtherThe cellular gene expression profile can be influenced by epigenetic events such as DNA methylation, histone modification, and miRNA-associated silencing during carcinogenesis. Human miRNA microarray analysis performed in eight HCC cell lines demonstrated that in Chromosome 19 the miRNA cluster (C19MC), which harbors 43 miRNA genes, was upregulated after combined treatment with DNA methylation and histone deacetylase inhibitors . Unlike The correlation between ZNF521 and miR-517a was also detected in studies focusing on low birth weight infants. miR-517a expression was highly expressed in the placentas of low birth weight newborns compared to normal birth weight newborns and its overexpression was implicated in the molecular mechanisms underlying the inhibition of trophoblast invasion. The 3′UTR sequence alignment and miRNA binding site prediction by TargetScan identified ZNF521 as a potential regulated gene targeted by miR-517a as well as other genes including SEMA3A and EIF4G3. Further studies are required to understand whether ZNF521 can be considered a downstream mRNA targeted by miR-517a to regulate trophoblast invasion .ZNF521 also plays a central role in gastric tumorigenesis and progression through regulation by miR-204-5p . MiR-204From an integrative model of miRNA and mRNA expression, it was found that ZNF521 was one of the genes which plays a protective role in the patients affected by breast invasive carcinoma (BRCA) who had undergone radiation therapy .The results showed that nine mRNAs  exerted a protective effect while five miRNAs  and eight mRNAs  were considered as high-risk genes .In ovarian endometriosis ZNF521 appears also in a complex integrated miRNA–TF–gene regulatory network , it is pZNF521 expression is influenced by miRNAs also in urological malignancies, including bladder cancer (BC). A microarray platform was used to investigate the profiles of miRNA expression in BC compared to normal bladder urothelium. ZNF521, together with TMCC1, TNIP1, VSTM2B, ISL1, emerged as target genes of miR-517a, the expression of which is significantly enhanced in bladder cancer ,59. miR-Idiopathic pulmonary fibrosis (IPF) is a severe scarring chronic lung disease characterized by a progressive and irreversible impairment in lung function . The molThe microRNA microarray dataset GSE32538 and the mRNA datasets GSE32537, GSE53845, and GSE10667 identified a role for ZNF521 in the biological processes of IPF. ZNF521 is differentially expressed in IPF and can be deregulated by specific miRNAs , which have been recently identified as regulators for genes in pulmonary fibrosis ,65.In addition, protein–gene interactions analyzed by the STRING web tool revealed the relationship among 67 common differentially expressed genes in IPF. This analysis identified nine hub nodes  and the proteins with the highest degree of interaction connected to them. In this network ZNF521, together with LEPREL1, TGFB3, COL15A1, COL17A1, and TNC, interacts with the hub node COL1A1 ,65.In lung epithelial cells and fibroblasts, these miRNAs are predicted to exert a profibrotic role targeting COL1 gene expression; for this reason, COL1 was reported as the first of the top nine nodes in the protein–protein interaction network of DEGs. The biological validation of miRNA-mediated regulatory networks among the DE-miRNAs and DEGs could be interesting to identify new potential biomarkers for the prediction of IPF progression .The mouse protein Zfp521 is highly expressed in neural stem cells playing a role in the ontogenesis and in the regulation of specialized functions in the central nervous system . Zfp521 The human protein ZNF521 was found to be a target gene for miR-381 in the striatum of macaques during acute-SIV infection and chronic administration. Within the neuroimmune-modulatory microRNA profile miR-381, whose expression was significantly increased, belongs to a cluster of five miRNAs  located close together on chromosome 7 in Rhesus and chromosome 14 in humans, in a region particularly enriched for the H3K227Ac histone mark and, therefore, with high transcriptional activity associated with Fos, Jun, FoxA1, Foxa2, Sp1 and GATA . FurtherThe miRNAs targeting ZNF521 in these different cancers and cellular systems have been reassumed in In this review we discuss the current knowledge concerning miRNAs targeting the transcription factor ZNF521, as yet not described in this context. We focused the attention on data from computational miRNA prediction tools identifying putative miRNAs targeting ZNF521 and cellular/molecular experimental models validating the bioinformatics data and clarifying the mechanisms by which miRNAs can also regulate ZNF521 expression in gene regulatory networks. ZNF521 is a stem cell-associated transcription co-factor with recognized regulatory functions in the immature compartment of the hematopoietic, mesenchymal and neural systems. However, it is still to be investigated whether ZNF521 targeting miRNAs plays a role in inducing the expansion of hematopoietic stem cells where this transcriptional factor is abundantly expressed ; this coTaken together, ZNF521 can be considered as a cancer-related gene which is aberrantly expressed in MLL-rearranged AML and involved in the induction of B-cell lymphomagenesis. ZNF521 expression is also deregulated in solid tumors including medulloblastoma cells with an increased tumorigenic potential as well as in other solid tumor types including hepatocellular carcinoma, gastric cancer cells, invasive bladder transitional cell carcinoma as well as breast and ovarian cancers. Integrated analysis of high-throughput systems revealed a close functional inverse relationship between the specific miRNAs and ZNF521 expression levels comparing cancers and controls. The dysregulation of the miRNA–ZNF521 axis may be crucial for the acquisition of cancer-initiating properties by stem cells and for the molecular changes occurring during cancer progression. Identifying new miRNAs targeting ZNF521 and understanding the molecular basis of their interaction will support the search for novel biomarkers or development of advanced therapeutic strategies.


Recent findings suggest that changes in human odors caused by malaria infection have significant potential as diagnostic biomarkers. However, uncertainty remains regarding the specificity of such biomarkers, particularly in populations where many different pathological conditions may elicit similar symptoms. We explored the ability of volatile biomarkers to predict malaria infection status in Kenyan schoolchildren exhibiting a range of malaria-like symptoms. Using genetic algorithm models to explore data from skin volatile collections, we were able to identify malaria infection with 100% accuracy among children with fever and 75% accuracy among children with other symptoms. While we observed characteristic changes in volatile patterns driven by symptomatology, our models also identified malaria-specific biomarkers with robust predictive capability even in the presence of other pathogens that elicit similar symptoms. However, a major challenge for the development of volatile disease biomarkers is posed by the inherent variability of human volatile emissions, which are highly labile and can be influenced by a wide range of genetic, physiological, and environmental factors3. In light of this variability, there is reason to speculate that volatile biomarkers might be of particular value in diagnosing diseases caused by insect-borne pathogens, which frequently manipulate the odors of their hosts in ways that influence vector behavior and might therefore be highly conserved6. Indeed, a number of recent studies on human malaria have identified characteristic changes in the volatile emissions from the skin and breath of infected individuals12. Yet, uncertainty remains about the physiological bases of these volatile changes and the extent to which they are uniquely caused by malaria or products of pathological processes that might be shared with other disease states. Addressing this uncertainty has important implications for understanding the diagnostic value of such biomarkers, particularly for use in human populations where numerous pathological conditions are widespread and may give rise to similar symptomatology, as is often the case for malaria endemic regions.The presence of disease can alter human odors, including volatile emissions from skin and breath. The potential diagnostic value of such changes in volatile chemistry has long been recognized, and volatile-based diagnostics are being actively explored for a number of diseases, including several types of cancer9 and breath12 volatiles, as well as the volatile emissions of rodent malaria hosts13, while others have characterized the emissions of Plasmodium cells grown in vitro16. Effects of infection on host odors have also been reported to influence vector behavior, with several studies reporting increased mosquito attraction to hosts harboring transmissible stages of Plasmodium parasite18 and at least one showing enhanced attraction to host odor profiles in which individual compounds were manipulated to mimic the relative up- or downregulation caused by infection13.A number of previous studies have documented malaria-induced changes in human skin19, suggesting that the pathogen may benefit from actively manipulating host volatiles20. To the extent that Plasmodium parasites, or other vector-borne pathogens, alter host odors in ways that consistently influence vector attraction, they may also generate unique patterns of effects on host volatile profiles—possibly tailored to the olfactory responses of particular vector species—distinct from more general changes in volatile emissions that arise as mere byproducts of pathology. The presence of such unique signatures of infection might, in turn, facilitate the identification of pathogen-specific biomarkers capable of reliably predicting infection status even in populations where numerous pathological conditions elicit similar symptoms.Enhanced vector recruitment during the transmissible stage of infection is hypothesized to facilitate malaria transmission7. Yet, while these characteristic changes were highly predictive of malaria infection, the specific processes by which the presence of malaria parasites alter human volatiles remain almost entirely unknown. One recent study reported that a malaria-derived isoprenoid increases the production of several monoterpenes and aldehydes in vitro21, but this does not account for the majority of volatile alterations observed by our and other studies12 suggesting that the underlying mechanisms are likely to be complex.The identification of such robust biomarkers is of particular interest for malaria, which frequently occurs in populations affected by numerous other diseases and ailments that elicit similar symptoms, including fever, headaches, diarrhea, vomiting, etc., and might therefore plausibly be expected to have somewhat similar effects on underlying physiological processes that also influence volatile emissions. In the case of malaria, disease progression often entails alternating asymptomatic and symptomatic phases that may recur indefinitely if not treated. In a previous study, we reported differential up and down regulation of specific volatile organic compounds in both symptomatic and asymptomatic schoolchildren in Kenya7. Specifically, we compared the skin volatile profiles of symptomatic schoolchildren who tested positive for malaria to those of children presenting similar  symptoms, but who tested negative for malaria. We used a genetic algorithm framework to predict malaria infection and identify volatile compounds associated with disease status. Our goal was thus to discover which volatile changes might be specific to malaria, and which might be caused by infection with other illnesses or conditions.To explore the extent to which changes in the odors of malaria infected humans are associated with the presence of symptoms, we analyzed the volatile profiles of symptomatic children, using volatile data previously collected from a large field trial in Western Kenya-F1,48 = 2.96, p = 0.022) and feet . These results indicate that the effect of malaria infection on volatile profiles is apparent even when directly compared to other conditions that produce similar symptoms.We first explored whether there was a clear signature of malaria infection among symptomatic children. In addition to examining differences in the volatile profiles of malaria-infected and uninfected children presenting any symptom , we made similar comparisons for non-exclusive subsets of children exhibiting the two most commonly observed symptoms in our dataset: fever and diarrhea. For each of these three symptom categories, discriminant analysis of principal components (DAPC) revealed clear separation in the overall volatile composition between individuals with and without malaria infection Fig. , S1, witWe next used a genetic algorithm predictive model trained to recognize malaria infection based on a subset of the data (70%) to predict malaria infection status in the remaining test set (30%). For children exhibiting any symptom, this model was able to predict malaria status with 75% accuracy Tables and 2. GiPlasmodium completes its lifecycle within the host, fever is typically associated with the rupture of mature schizont cells and the release of merozoites that then reinvade red blood cells22. The resulting intermittent cyclic fever may cause unique physiological effects compared to other ailments, and may also play a role in the downregulation of volatiles, including the compounds that drive the predictive accuracy of our model  and using either foot or arm volatiles Table . Several7. The overlap in compounds identified as important predictors of malaria infection and of symptoms in malaria-free children (but with different patterns of up and down regulation) is intriguing and may reflect the influences of different pathological conditions on the same underlying physiological processes.Comparing the volatile emissions of malaria-free children exhibiting any symptom to those without symptoms revealed significant downregulation of several compounds Fig. , with et25. In the case of lung cancer, a large-scale trial for diagnostic detection using VOCs is currently underway26. For many other diseases, however, significant challenges remain to be overcome prior to the implementation of large-scale trials, including the identification of a robust and reproducible set of candidate biomarkers3, which can be complicated by the absence of standardized methods for volatile collection and analysis27. In the case of malaria, several previous studies have reported successful prediction of infection status based on analysis of VOCs; however, there is considerable variation in the predictive compounds identified, likely owing to divergent methodologies across studies. Two compounds, hexanal and nonanal, have consistently been found informative with respect to with malaria status, each reported in three separate studies from skin and breath volatiles as well as in the prediction here of malaria status in symptomatic children , and the Kenya Medical Research Institute (SERU 391) and all experiments were performed in accordance with relevant guidelines and regulations. Before sample collection, the study and consent form were explained to parents/guardians and their written informed consent was obtained.This study was approved by The Pennsylvania State University , ETH Zürich (EK2015-Participant exclusion criteria included (1) receipt of antimalarial medication during the previous 2 wk; (2) chronic disease, such as HIV; (3) not signing (or having a parent sign) the consent form; and (4) refusal of malaria treatment.7. Symptoms were self-reported in an initial interview using a standardized questionnaire; symptoms indicative of malaria included fever, abdominal pain, rash, diarrhea, vomiting and body aches. For our analysis, we sorted children into the categories (1) any symptom (2) fever, (3) diarrhea and (4) asymptomatic. The any symptom category comprised children with fever, diarrhea, abdominal pain or vomiting; the fever and diarrhea categories were non-exclusive subsets of the any symptom category. Abdominal pain and vomiting were not analyzed independently due to low numbers of children with these symptoms. Malaria infection status was initially assessed via rapid diagnostic testing (SD Bioline), then confirmed by light microscopy and PCR. For the current study, children in the malaria infected category tested positive by both microscopy and PCR.Our analyses were performed on data derived from skin volatile samples (1 h collections from arms and feet) collected from students at 41 primary schools near Mbita Point, Kenya between 2013 and 2016The dataset used for this study comprised volatile profiles for 114 children. Once categorized into symptoms for the below analyses, numbers of children in each category were as follows: malaria infected children with any symptom = 29, fever = 18, diarrhea = 11. Malaria-free children with any symptom = 22, fever = 11, diarrhea = 9, asymptomatic = 16.7).Arm and foot volatiles were collected simultaneously for 1 h by enclosing the arm (from wrist to above the elbow) or foot (to below the knee) in a teflon bag, pushing filtered air through an entry port  and pulling it through an exit port  where it was collected on an adsorbent HaySepQ filter. Compounds trapped on filters were then eluted with 150ul dichloromethane and analyzed by GC–MS  with the Euclidean similarity on the scaled data43.Discriminant Analysis of Principal Components (DAPC)44. They have been used as a promising multivariate approach in the analysis of metabolomics datasets46. The algorithm works by evolving initial sets of variables (chromosomes) from a random population via cycles of replication, recombination and mutation of the fittest chromosomes. The iterations were repeated for 100 generations, with a population size of 50 candidate solutions for each model and crossover and mutation probabilities of 0.8 and 0.1, respectively. The performance of the fittest GA model was used to predict the samples in the validation set. To complement the predictive models, a differential analysis between infection statuses was performed using the limma R package47. The GA models were formulated with the caret R package48.We used a Genetic Algorithm (GA) to predict disease condition and to select one or more sets of compounds for each of the symptom subsets to be assessed as a potential biomarker for malaria. Each subset was randomly split into 70% for model training and 30% for validation. Using the training set, each model was trained five times using a repeated tenfold cross-validation. Genetic Algorithms are feature selection procedures that are conceptually based on the principle of evolution by natural selectionSupplementary information.


Heterotaxy is a rare syndrome associated with cardiac complexity, anatomic variability and high morbidity and mortality. It is often challenging to visualize and provide an accurate diagnosis of the cardiac anatomy prior to surgery with the use of conventional imaging techniques. We report a unique case demonstrating how the use of three-dimensional (3D) cardiac printed model allowed us to better understand the anatomical complexity and plan a tailored surgical approach for successful biventricular repair in a patient with heterotaxy syndrome. Heterotaxy syndrome is a rare disorder characterized by abnormal lateralization and isomerism of thoraco-abdominal organs across the left–right axis of the body ,2,3,4. IThe patient is a 5-month-old male who was diagnosed with multiple congenital anomalies including heterotaxy syndrome, polysplenia, intestinal malrotation, absent septum pellucidum, cleft lip and palate, small left kidney with hydronephrosis and right inguinal hernia at birth. His post-natal echocardiogram revealed mesocardia with apex to the left, midline liver, atrial situs ambiguous with a common atrium and ipsilateral pulmonary veins (left pulmonary veins draining into the left-sided atrium and right pulmonary veins draining into the right-sided atrium), D-looped ventricles, normally related great arteries , interrupted inferior vena cava (IVC) with hemiazygos continuation to the left superior vena cava (SVC) draining into the left and anterior aspect of the common atrium, hepatic veins draining to the common atrium, a partial AV canal, small anterior muscular ventricular septal defect (VSD), and a moderate sized patent ductus arteriosus (PDA) . GeneticAt three and a half months of age, the patient underwent elective cleft lip and palate repair where he experienced acute hypoxemic respiratory failure and cardiorespiratory arrest, as well as multiple failed attempts at extubation. Cardiac computerized tomography angiography (CTA) confirmed the cardiac anatomy previously seen on post-natal echocardiogram . It alsoThe DICOM  files from CTA were imported into the segmentation software. Mimics Medical 23 software was used for 3D segmentation. The resulting 3D segmented virtual model was converted to a STL (Stereolithography or Standard Tessellation Language) file that is compatible with 3D printing. The heart model design was then printed using Stratasys J750 printer to generate the flexible cardiac model. Loose powder present at the end of the building process was vacuumed off to reveal the final product. Upon examination of the cardiac model, the surgical decision was to perform a complex biventricular surgical repair. At the age of 5 months, the patient underwent partial AV canal repair with closure of mitral valve cleft. A modified Warden procedure was performed. As previously planned on the 3D model, a large systemic venous baffle was created to drain systemic venous circulation into the right atrium. The base of LSVC, left-sided atrial appendage and right-sided atrial appendage were fashioned to form the posterior wall of the baffle. The joint of the LSVC to the left atrium was closed with a pericardial patch. A 30-degree CardioCel patch was used to form the anterior wall of the systemic venous baffle. With that, the LSVC and hemiazygos vein that carries the flow from the IVC was directed into the right atrium. The mitral cleft was closed with an interrupted 6-0 Prolene sutures. To create an atrial septum, a CardioCel bovine pericardial patch was used. The patch was started from the posterior and inferior portion of the left atrium and the suture line was carried out anteriorly. This method allowed us to move toward the space of tissue between the mitral valve and the tricuspid valve. Then, the same patch was turned to the superior portion to create an interatrial septum which was missing in this patient. At the end of the procedure, separation between the right atrium from the left atrium was obtained, incorporating the four pulmonary veins communicating with the mitral valve, and the hepatic veins together with blood coming from the superior vein baffle communicating with the tricuspid valve.Normal biventricular size and systolic function with no evidence of significant intracardiac shunt was obtained on cardiac MRI performed 5 weeks postoperatively. His postoperative course from the cardiac perspective remained uneventful until discharge . Figure Historically, heterotaxy patients have had poor surgical outcomes associated with high morbidity and mortality due to structural abnormalities of systemic and pulmonary venous connections, increased incidence of single ventricle physiology, pulmonary and aortic outflow obstruction, arrhythmias and infections associated with splenic dysfunction ,11,12,13In recent years, 3D printing has been increasingly used in the surgical field, especially for reconstructive surgeries in craniomaxillofacial ,17, orthIn addition, 3D printed heart models have helped reduce communication barriers between patients, their families, and physicians. It aids in the education of cardiac anatomy, pathology, surgical procedures, and post-operative progress ,29. ThesFrom extensive literature review, there are 14 reports of successful biventricular repair using 3D printed cardiac models in complex congenital heart disease . Two priAgarwal A. discussed the use of 3D cardiac modeling to explore the possibility of biventricular repair in a 4-year-old patient with heterotaxy syndrome as well as its importance in the planning of her complex cardiac surgery . ValverdThe remaining reports discussed biventricular repair in various other complex cardiac anatomy, and the benefits of 3D cardiac modeling in visualizing complex cardiac anatomy which helped simplify decisions related to surgical options. Riesenkampff et al. produced 11 cardiac models of patients with complex anatomy to investigate the possibility of biventricular repair. There was a unanimous agreement among physicians that these models improved their understanding of the pathology, and helped streamline discussions regarding surgical options for the patients . ValverdAs evident from the table and previous reported cases, 3D-printed models have helped in understanding the anatomy, spatial relationships of important and adjoining structures, deciding between surgical options, as well as surgical simulations and for educational purposes.Despite the benefits of 3D printing, the high costs involved in the initial set-up can prevent widespread use of the technology ,33,34,35However, its ability to provide tactile experience, valuable information for pre-surgical planning, and improve patient communication proves that it has the potential to be a useful tool in the future of congenital heart surgery . This case highlights the use of a 3D-printed model in helping plan a unique surgical approach to achieve a successful biventricular repair in a patient with heterotaxy syndrome. 3D printing is proving to be an integral tool in understanding the anatomical complexity, planning the surgical approach, and achieving successful outcomes for patients with complex congenital heart defects.


Pattern recognition receptors (PRRs) play a central role in the inflammation that ensues following microbial infection by their recognition of molecular patterns present in invading microorganisms but also following tissue damage by recognising molecules released during disease states. Such receptors are expressed in a variety of cells and in various compartments of these cells. PRR binding of molecular patterns results in an intracellular signalling cascade and the eventual activation of transcription factors and the release of cytokines, chemokines, and vasoactive molecules. PRRs and their accessory molecules are subject to tight regulation in these cells so as to not overreact or react in unnecessary circumstances. They are also key to reacting to infection and in stimulating the immune system when needed. Therefore, targeting PRRs offers a potential therapeutic approach for chronic inflammatory disease, infections and as vaccine adjuvants. In this review, the current knowledge on anti-viral PRRs and their signalling pathways is reviewed. Finally, compounds that target PRRs and that have been tested in clinical trials for chronic infections and as adjuvants in vaccine trials are discussed. Infection as well as tissue injury/stress can activate inflammatory responses. Inflammation is widely recognised by the cardinal symptoms of fever, redness, oedema, pain, and loss of function. Inflammation is necessary to help with the removal of the source of infection, to help protect the infected tissue(s), and to restore homeostasis. Both immune and non-immune cells produce cytokines, chemokines, and vasoactive peptides. These molecules attract immune cells such as neutrophils that are normally restricted to the vasculature, allowing them to enter into the inflamed or infected tissue ,2,3. VirViruses can be categorised as RNA viruses, DNA viruses or retroviruses (which can have RNA or DNA in some cases). They can also be categorised by whether their nucleic acid is positive or negative sense. In addition, replication of these viruses also generates intermediate molecules such as ssRNA and/or dsRNA ,8. TheseThe first PRRs to be discovered were Toll-like receptors. They are most highly expressed in immune cells, but some are also expressed in non-immune cells such as epithelial cells. Basal expression levels vary across the body ,13. TLRsOther receptor families such as RLRs, NLRs and DNA sensors such as absent in melanoma 2 (AIM2) and cyclic GMP-AMP synthase (cGAS) can recognise a range of microbial nucleic acids in the cytosol . This stIn humans, there are 10 TLR family members that are membrane-bound receptors located intracellularly and on the cell surface . Ligand tlr3 deficient mice, which are susceptible to lethal viral infection [The intracellular TLR, TLR3 was identified as recognising polyinosinic-polycytidylic acid (poly(I:C)), a synthetic analogue of double stranded RNA (dsRNA) . TLR3 isnfection . TLR7 renfection ,22.Expression of TLR7 is relatively high in plasmacytoid dendritic cells (pDCs). TLR8 also recognises ssRNA and is similar phylogenetically to TLR7. Interestingly, no release of interferon or cytokines occurs in the absence of TLR7 following ssRNA stimulation but immune responses are normal in TLR8 deficient mice ,22. It mTLR recognition of RNA and DNA is limited to the endosomal compartment of immune cells. Other PRRs have evolved for sensing nucleic acids in different cellular locations and cell types to detect viruses at different stages of their life cycle . The secThe third category of PRRs are cytosolic receptors that recognise microbial DNA during infection. DNA is mostly contained within the nucleus or mitochondria in mammalian cells. During infection, viruses inject DNA into the cytosol of cells. DNA sensors include absent in melanoma 2 (AIM2) and cyclic GMP-AMP (cGAMP) synthase (cGAS) among others. This then activates an endoplasmic reticulum-bound adaptor called stimulator of interferon genes (STING). STING undergoes a conformational change and inducing the production of type I interferon via the activation of TBK1 and IRF3 . All of PRRs and their signalling pathways could be pharmacologically targeted at the receptor level, the downstream kinases, or the protein–protein interactions between adaptor molecules. This review will highlight the receptors specifically as targets rather than downstream events. To date, PRR agonists, antagonists and antagonistic antibodies have been designed. Clinical trials have evaluated these molecules as adjuvants for vaccines and cancer as well as for infections, acute and chronic inflammatory diseases, and neurological conditions. This review will focus on those tested for anti-virals and adjuvants for vaccines. Reviews focusing on PRRs as targets in cancer and other inflammatory diseases can be found elsewhere ,38.Despite vaccination programmes being in place in many countries, there is a still an unmet clinical need to treat hepatitis B and C viruses, human immunodeficiency virus (HIV), human papillomavirus (HPV), herpes simplex virus (HSV) and more recently coronavirus (SARS-CoV-2) infections. These infections are associated with chronic disease and are lethal in many cases. Stimulating anti-viral pathways by using agonists of PRRs may help reduce the viral load and hence the associated disease. Hepatitis B virus (HBV) is a non-cytopathic DNA virus that is a common cause of liver disease. The virus generates intermediates of closed circular DNA in the nucleus of hepatocytes which have a long half-life and that get transcribed . StimulaThe TLR7 agonists tested to date in phase I trials include GS-9620, RO7020531 and TQ-A3334 were all shown to be safe and well tolerated in hepatitis B patients and healthy volunteers ,41,42,43Chronic hepatitis C viral (HCV) infection can lead to liver cirrhosis and carcinoma . SimilarHIV infection affects millions worldwide and can become latent even in people who have been successfully treated with highly active anti-retroviral therapy (HAART). Viral particles can re-emerge later when therapy is interrupted. Using TLR agonists could be useful as latency reverting agents . Given tHuman papillomavirus (HPV) infection is associated with the presentation of anogenital warts as well as cervical and anal cancers. Like many viral infections mentioned so far, imiquimod has been tested in trials for HPV. Administered to HPV patients with warts as a cream (5%) caused decreases in viral load as measured by HPV DNA as well as increases in IFN, TNF and IL12 mRNA . HIV patHerpes simplex virus II infection is also known to lead to genital warts, and it can establish persistent infection by evading immune responses . TopicalFor many years, vaccines have failed in trials due to a lack of efficacy and/or durability and often because immune responses were not strong enough to antigens. The use of adjuvants has helped resolve this but there was still a limited number of these molecules that were safe. PRR adjuvants have recently been test in trials due to their ability to bridge the innate and adaptive immune systems . There hDespite success with antiretroviral drug therapy, there is still a need for vaccines for HIV/AIDS. Many TLR agonists have been tested with various types of HIV vaccines. Poly ICLC, a TLR3 agonist has been combined with DCVax (a fusion protein consisting of an antibody to the dendritic cell receptor CD205 and the HIV gag p24 protein). This was tested in a phase I trial using healthy volunteers, but no results were available at the time of writing. The TLR4 agonist MPL and the NOD2 agonist muramyl dipeptide (MDP) were combined with the recombinant protein gp120 subunit vaccine in an initial phase I trial and showed neutralising antibody activity . The TLRHuman papillomavirus vaccines have had success in recent years. Several trials have been designed to investigate the efficacy and safety of combining these with TLR agonists including MPL (TLR4) and imiquimod (TLR7). Cervarix, a vaccine against HPV16/18 strains uses an adjuvant called AS04, containing MPL and aluminium salt. In a phase III trial of over 2000 adolescent girls aged 10–14, the mean titers of antibodies to both strains were shown to be maintained at higher levels up to 10 years later in those vaccinated with the AS04 adjuvant ,96. TLR7Herpes simplex virus 2 is regarded as a primary causative agent of genital herpes. Prophylactic vaccination is proposed to help reduce sexual transmission. An investigational HSV vaccine (glycoprotein D subunit vaccine) has been tested with some TLR agonists including MPL (as AS04) and the TLR9 agonist CpG1018. It was reported that the vaccine had an acceptable safety profile, it was well tolerated and immunogenic, as determined by geometric mean concentration of anti-gD2 antibodies. However, there was no control group to determine the effect of imiquimod .Influenza infections cause thousands of deaths annually and mutation makes it difficult to control. Vaccination is key to reducing mortality. An intranasal quadrivalent flu vaccine FluMist was combined with the TLR3 agonist rintatolimod (Ampligen) and tested in a phase I/II trial. Despite being reported to be well tolerated, improving antibody titers, and demonstrating cross-reactivity to strains of avian flu, the trial was terminated . RecentlFinally, imiquimod was given topically prior to an intradermal trivalent influenza vaccine and found to be safe, well tolerated. It also significantly increased, prolonged, and expedited the immunogenicity of the vaccine ,107. A pIn summary, agonists of ant-viral TLRs  seem to hold the most promise as both anti-viral drugs and as vaccine adjuvants. As many of the PRR signalling pathways culminate in NF-κB and IRF activation , targeti


Bone infection has always been the focus of orthopedic research. Mesenchymal stem cells (MSCs) are the natural progenitors of osteoblasts, and the process of osteogenesis is triggered in response to different signals from the extracellular matrix. MSCs exert important functions including secretion and immune regulation and also play a key role in bone regeneration. The biological behavior of MSCs in acute and chronic inflammation, especially the transformation between acute inflammation and chronic inflammation, has aroused great interest among researchers. This paper reviews the recent literature and summarizes the behavior and biological characteristics of MSCs in acute and chronic inflammation to stimulate further research on MSCs and treatment of bone diseases. Staphylococcus aureus and Staphylococcus epidermidis are responsible in most cases in vitroBone infection has always been the focus of orthopedic research. Bone infection is a serious complication in orthopedics and often leads to severe bone destruction and nonunion. When acute infection is not effectively controlled, it may become chronic, which often leads to disastrous consequences. Osteomyelitis is a typical bone infection, and it often requires prolonged antibiotic treatment and surgical intervention. Osteomyelitis is caused by bacterial pathogens, and During the process of bone regeneration, inflammatory factors can activate and mobilize endogenous MSCs, which migrate from their niche to the damaged site A literature search was conducted with PubMed, EMBASE and Web of Science databases. The following terms: , were used to search eligible studies from 2002 to 2020, with 80 relevant English articles included in this review.It is know from previous studies that in the process of regulating the osteogenic differentiation of MSCs, signaling pathways such as mitogen-activated protein kinases (MAPK) MAPK is a conserved family of enzymes that can transmit and spread external stimuli, and coordinate cell responses through phosphorylation cascades to adapt to environmental changes In addition, the phosphorylated p38MAPK activates mitogen and stress-activated protein kinases 1 (MSK 1) and mitogen and stress-activated protein kinases 2 (MSK 2), which can phosphorylate the NF-κB complex transactivating Ser276 of the p65 subunit, thereby enhancing the NF-κB signaling in vivo and in vitro. It activates SMAD1/5/8 and several mitogen-activated protein kinases (MAPK), which ultimately leads to the increase in the runt-related transcription factor 2 (RUNX2) and the core binding factor A1 (CBFA1) BMPs are members of the transforming growth factor β (TGF-β) superfamily The SMAD protein family can be divided into 3 categories, the receptor-activated SMAD (R-SMAD), the common pathway SMAD (Co-SMAD) and the inhibitory SMAD (I-SMAD) The Wnt/β-catenin signaling pathway regulates the osteogenic differentiation, bone formation and bone metabolism disorders of bone marrow mesenchymal stem cells Ward et al. suggested that the up-regulation of podoplanin in the infected state is associated with the increased migration ability of MSCs MSCs will recruit and exert biological effects under the action of cytokines and chemokines in acute inflammation, and this process also allows the organism to heal from infections and tissue damage. Infection or trauma to tissues is usually detected by receptors largely located on the surface of innate immune cells, which are called pattern recognition receptors (PRRs). These PRRs then identify the pathogen-related molecular patterns or damage-associated molecular patterns and trigger the inflammatory response. Acute inflammation peaks at 24 to 48 h after injury and is complete after about 7 days Xu et al. showed that the purinergic receptor p2x and ligand gated ion channel, 7 (P2X7) receptors are also key molecules that regulate the osteogenesis of MSCs in acute inflammatory conditions In the presence of acute infection, the acute inflammation response plays an important role in bone regeneration, and it is a crucial first step for bone healing. By contrast, the absence and inhibition of an acute inflammatory response may lead to impaired bone healing In the process of inflammatory response, the same cytokine will have different regulatory effects on the osteogenic differentiation of MSCs under different concentration and action time conditions. Studies have shown that low concentration of TNF-α (1 ng/ml) promotes the osteogenic differentiation of MSCs in the short term (1 d and 3 d) and increases the mRNA expression of BMP-2 and SMAD 1, but inhibits the osteogenic differentiation of MSCs at long term (7 d). However, whether long-term or short-term, high concentrations of TNF-α inhibited the osteogenic differentiation of MSCs and the expression of SMAD 1, but resulted in high expression of BMP-2 Very similarly, studies have shown that IL-10 has concentration-dependent, dual roles in the osteogenesis of MSCs through P38MAPK and NF-kB signaling pathways. Under low-dose conditions (0.1-1 ng/ml), it can promote osteogenesis. But when the concentration of IL-10 exceeds 10 ng/ml, it will inhibit osteogenesis Evans et al included 24 cases of 36 high-energy penetrating injuries, of which 13 (36%) had heterotopic ossification. After debridement, the tissue fragments and local exudate were tested for inflammatory factors and found that IL-6, IL-10 and monocyte chemoattractant protein-1 are significantly related to the occurrence of the heterotopic ossification Among the ossification caused by inflammation, heterotopic ossification is a very special type of decease, which we will separately describe in detail. Heterotopic ossification (HO) often occurs after severe trauma or burn In vitro, MSCs can suppress the proliferation of T cells induced by mitogens and alloantigens. Although the exact mechanisms underlying the immunosuppressive effects of MSCs are still unclear, most evidence indicates that soluble factors are involved. These factors include prostaglandin E2 (PGE2), indoleamine 2,3-dioxygenase (IDO), hepatocyte growth factor (HGF), IFN-γ, and TGF-β1. MSCs can also alter the balance of Th1/Th2 cells Moretta et al. showed that resting unstimulated MSCs are capable of inhibiting NK cell proliferation, cytokine release, and cytotoxicity, via PGE2 and IDO In acute infections, MSCs mainly exert immunosuppressive effects and, under certain conditions, may also exhibit proinflammatory effects. MSCs induce a complex regulation of a variety of immune cells in acute infection and interact with immune cells through proinflammatory and immunosuppressive regulatory directions. MSCs have immunomodulatory functions, which are mainly achieved by interacting with different immune cell subpopulations Munir et al. believe that in chronic inflammation, MSCs residing in tissues will acquire a proinflammatory phenotype opposite to that of normal MSCs and will then produce proinflammatory factors In chronic inflammation, the immune regulatory function and osteogenic differentiation ability of MSCs are altered, which eventually leads to the persistence of inflammation, bone destruction, and nonunion. The inflammatory response is an essential and critical step in the healing process of acute bone infections; however, when the pathogen is difficult to remove, it will turn into chronic infection.  Saeedi et al. used MSCs primed by modified LPS to treat sepsis, and, in this context, MSCs presented strong antibacterial properties Yuan et al. used MSCs to reduce methicillin-resistant Staphylococcus aureus infection in rat models Because of the effective differentiation potential of MSCs and their immunoregulatory properties during the inflammatory response, MSCs have become highly regarded in tissue engineering, wound repair, immunotherapy, and infection treatment. Many studies have proposed MSCs as a treatment for infection.In an acute inflammation response, MSCs will first migrate to the site of infection and then regulate their differentiation and immunological effects via the stimulation of inflammatory factors. When an acute infection shifts to a chronic inflammation, the immune regulatory function and osteogenic differentiation ability of MSCs will be changed, which will eventually lead to the persistence of inflammation, bone destruction, and nonunion. The differentiation and immune regulation ability of MSCs under infection is bidirectionally regulated through multiple pathways. The transformation between acute inflammation and chronic inflammation has become a crucial point of tissue repair. The behavior of MSCs in acute and chronic inflammation states is worthy of further study, which may reveal the key involvement of MSCs in the whole process of infection.


CCN proteins also participate in osteoclastogenesis and myogenesis. In adult life, CCN proteins serve as mechanosensory proteins in the musculoskeletal system providing a steady response to environmental stimuli and participating in fracture healing. Substantial evidence also supports the involvement of CCN proteins in inflammatory pathologies, such as osteoarthritis and rheumatoid arthritis, as well as in cancers affecting the musculoskeletal system and bone metastasis. These matricellular proteins indeed show involvement in inflammation and cancer, thus representing intriguing therapeutic targets. This review discusses the current understanding of CCN proteins in the musculoskeletal system as well as the controversies and challenges associated with their multiple and complex roles, and it aims to link the dispersed knowledge in an effort to stimulate and guide readers to an area that the writers consider to have significant impact and relevant potentialities.The acronym for the CCN family was recently revised to represent “cellular communication network”. These six, small, cysteine-enriched and evolutionarily conserved proteins are secreted matricellular proteins, that convey and modulate intercellular communication by interacting with structural proteins, signalling factors and cell surface receptors. Their role in the development and physiology of musculoskeletal system, constituted by connective tissues where cells are interspersed in the cellular matrix, has been broadly studied. Previous research has highlighted a crucial balance of CCN proteins in mesenchymal stem cell commitment and a pivotal role for CCN1, CCN2 and their  CCN proteins were first discovered in the late 1980s and early 1990s as small proteins (30–40 kDa) enriched in cysteine content  and characterized by a tetramodular structure. The first reports described them as growth factors due to their ability to control adhesion, proliferation, migration, apoptosis, and extracellular matrix deposition in diverse cell types . As resCCN gene family members are conserved among vertebrates in term of amino acid sequences, protein structures and genetic loci. Bioinformatic analysis revealed an expansion from the sole homologue found in amphioxus, cestode, oyster, and fruit fly to the 4 homologs in lamprey, 5 in ascidian and 9 in zebrafish. Six members belong to this family in mammals, such as mice and humans    and administration of anti-CCN1 monoclonal antibodies, or drugs (rosiglitazone) in collagen-induced (CIA) murine models of RA invariably led to an amelioration of RA symptoms with decreased inflammatory scores and monocyte infiltration, decreased production of proinflammatory cytokines such as IL-6 and IL1β and increased production of antinflammatory IL10, decreased MMP activity and osteoclastogenesis with reduced RORc, TNFα, IL17 and sRANKL levels, and ultimately reduced osteoclast populations  and prostate cancer (85%) and myeloma (95%)  or bone (CCN2) invasiveness (Kim et al. CCN2, as observed in breast cancer, CCN3 and CCN4 levels are up regulated in prostate cancer and bone-metastatic cells or corresponding bone metastasis (Dankner et al. CCN1 is up regulated in multiple myeloma tissues as well as in patients’ sera (Johnson et al. CCN1 and CCN2 play pivotal stimulating roles in development, physiology, pathology, and malignancy of the musculoskeletal system. CCN4 has a minor role in all aspects whereas CCN3 counteracts the actions of CCN2. The roles of CCN5 and CCN6 in the musculoskeletal system remain poorly studied and unclear. Efforts should be made to perform a more coordinated study of the effects of these family members as a team rather than single entities in the physiology of both osteoclasts and skeletal muscle cells as well as degenerative diseases and cancer. Regenerative approaches could also benefit from considering the integrated role of CCN family members in fine-tuning the function of diverse tissues and cell types in the musculoskeletal system. Lessons learned from the proinflammatory role of CCN1 and CCN2 in rheumatoid arthritis should prompt studies on the ability of CCN family members to affect the tumour microenvironment and immune evasion. Differential expression between osteosarcomas/bone metastasis and surrounding normal bone or between low- and high-grade chondrosarcomas offers a rationale for developing targeted therapeutics, at least for young adult patients. Last, given the involvement of CCN2 in bone metastasis regardless of the of primary tumour site, its level in biological fluid could provide a precocious marker of skeleton related events.


Sepsis-induced myocardial dysfunction considerably increases mortality risk in patients with sepsis. Previous studies from our group have shown that sepsis alters the expression of structural proteins in cardiac cells, resulting in cardiomyocyte degeneration and impaired communication between cardiac cells. Caveolin-3 (CAV3) is a structural protein present in caveolae, located in the membrane of cardiac muscle cells, which regulates physiological processes such as calcium homeostasis. In sepsis, there is a disruption of calcium homeostasis, which increases the concentration of intracellular calcium, which can lead to the activation of potent cellular enzymes/proteases which cause severe cellular injury and death. The purpose of the present study was to test the hypotheses that sepsis induces CAV3 overexpression in the heart, and the regulation of L-type calcium channels directly relates to the regulation of CAV3 expression. Severe sepsis increases the expression of CAV3 in the heart, as immunostaining in our study showed CAV3 presence in the cardiomyocyte membrane and cytoplasm, in comparison with our control groups (without sepsis) that showed CAV3 presence predominantly in the plasma membrane. The administration of verapamil, an L-type calcium channel inhibitor, resulted in a decrease in mortality rates of septic mice. This effect was accompanied by a reduction in the expression of CAV3 and attenuation of cardiac lesions in septic mice treated with verapamil. Our results indicate that CAV3 has a vital role in cardiac dysfunction development in sepsis and that the regulation of L-type calcium channels may be related to its expression. Sepsis is a potentially fatal organ dysfunction, characterized by an unregulated response of a host to an infection . DespitePrevious results from our research group demonstrated that structural changes in cardiac cells elucidate the physiopathology of SIMD . During Dystrophin proteins act as critical components of the dystrophin-glycoprotein complex (DGC), establishing connections between the intracellular cardiac contractile machinery and the extracellular matrix. Furthermore, DGC performs three essential basic functions: stabilization of the membrane during contraction cycles, transduction of contractile force, and the organization of membrane specializations . In addi2+ channels (LTCCs) i, reducing the contraction i in cardiomyocytes exposed to the serum of septic mice i in cultured neonatal cardiomyocytes treated with septic animal serum [2+]i involve failures in the channels that regulate the entry of Ca2+, microruptures in the plasma membrane, and the excessive release of Ca2+ by the sarcoplasmic reticulum i activates proteases inside the cell, such as calpain. In sepsis, calpain expression in cardiomyocytes increases, with a concomitant reduction in dystrophin-glycoprotein complex (DGC) proteins [The increase in [Caproteins . The disproteins , 39. Theproteins . Experimproteins . Consistproteins .κB activation and thereby contributes to the mechanism of microvascular permeability in sepsis [A study using the lung of septic mice (induced by the CLP model) demonstrated a reduction of CAV1, which was reported as a host cytoprotective factor to regulate the number of available caveolae that can be used by pathogens as an escape mechanism from lysosomal degradation . Surprisn sepsis .The cause of CAV3 overexpression in sepsis is still unknown. Studies that induced overexpression of CAV3 in transgenic mice showed severe cardiomyocyte degeneration with reduced cardiac function, in addition to skeletal muscle damage and negative regulation of DGC; such findings are similar to those found in DMD , 45. How2+ waves in cells absent from CAV3, and this also occurred when the interaction of CAV3 with G protein was interrupted [2+-dependent calcineurin-NFAT pathway [There are strong indications that CAV3 regulates calcium homeostasis in cardiac cells, an important relationship in maintaining cellular physiology. One study showed the absence of slow Caerrupted . On the  pathway .Our results indicate that sepsis leads to increased expression of CAV3 in the heart, and the treatment with verapamil can directly or indirectly modulate its expression resulting in a reduction of mortality rates and cardiac injuries.


Skeletal class II and III malocclusions are craniofacial disorders that negatively impact people’s quality of life worldwide. Unfortunately, the growth patterns of skeletal malocclusions and their clinical correction prognoses are difficult to predict largely due to lack of knowledge of their precise etiology. Inspired by the strong inheritance pattern of a specific type of skeletal malocclusion, previous genome-wide association studies (GWAS) were reanalyzed, resulting in the identification of 19 skeletal class II malocclusion-associated and 53 skeletal class III malocclusion-associated genes. Functional enrichment of these genes created a signal pathway atlas in which most of the genes were associated with bone and cartilage growth and development, as expected, while some were characterized by functions related to skeletal muscle maturation and construction. Interestingly, several genes and enriched pathways are involved in both skeletal class II and III malocclusions, indicating the key regulatory effects of these genes and pathways in craniofacial development. There is no doubt that further investigation is necessary to validate these recognized genes’ and pathways’ specific function(s) related to maxillary and mandibular development. In summary, this systematic review provides initial insight on developing novel gene-based treatment strategies for skeletal malocclusions and paves the path for precision medicine where dental care providers can make an accurate prediction of the craniofacial growth of an individual patient based on his/her genetic profile. Among the three dimensions of craniofacial structure, the sagittal dimension is the primary focus and most crucial plane in orthodontic diagnosis and treatment planning . SkeletaTo date, orthognathic surgical correction is still the last-resort treatment of severe skeletal class II and III malocclusions to achieve balanced anatomic and functional jaw relationships and improve facial appearance . HoweverSince a strong inheritance pattern of a specific type of skeletal malocclusion running in families has been observed in various races ,26, therThis systematic review followed the 2020 Preferred Reporting Items for Systemic Reviews and Meta-Analyses (PRISMA) guidelines , and thep-value and a false discovery rate (FDR) less than 0.05 were recorded, and the top 20 pathways with the lowest FDR values were further discussed. Pathway functional annotations were based on PubMed focusing on bone, cartilage, osteoblasts, osteoclasts, and osteocytes. Functions of genes not recognized in the Reactome database were annotated by the Uniprot database ,,33,FGFR2nd MMP13 ,58,59 weNFATc1 is a skeletal class III malocclusion-associated gene  [Downregulated by FGFR2 , the “PI2 (BMP2)  while de2 (BMP2) . These rFGFR2 SNPs are located in introns  gene in mouse osteoblasts led to a striking decrease in cancellous bone volume, connectivity, and trabecular number, accompanied by an increase in trabecular spacing, while the rate of mineralization of osteoid was significantly decreased [“Insulin receptor signaling cascade”, which was identified by pathway enrichment of the genes associated with skeletal class II and class III malocclusions , is knowecreased . This coecreased .Both insulin receptor substrate 1 (IRS1) and insulin receptor substrate 2 (IRS2) are involved in the signal transduction from IGF1R to its downstream mediator RAC-alpha serine/threonine-protein kinase (Akt1) . PreviouBecause they are both connected to the PI3K→Akt axis, a complex interaction exists between FGF-related signal transduction and the “Insulin receptor signaling cascade” ,109,112.runt-related transcription factor 2 (RUNX2) promotes the activity of FGFR2 and FGFR3, leading to the proliferation of pre-osteoblastic cells [RUNX2 promotes the differentiation of secondary chondrocytes and the formation of the cartilage of mandibular condyles [RUNX2 gene may more frequently lead to mandibular retrognathism and thus induce skeletal class II malocclusion. This hypothesis is supported by previous animals studies that have shown that RUNX2-deficiency in mice resulted in a lack of mandibular condylar cartilage and mandibular bone [RUNX2 gene was identified as the seed for three skeletal class II malocclusion-associated enriched pathways, as expected  was found to be more highly expressed in the masseter muscle of patients with skeletal class III malocclusion than in patients with skeletal class II malocclusion [lysine acetyltransferase 6B (KAT6B) in the masseter muscle of skeletal class III malocclusion patients than in the same tissue type belonging to skeletal class II malocclusion patients, thereby indicating that KAT6B may be associated with mandibular prognathism [cclusion . In addignathism ,52. CleaIt is important to note that GWAS can only indicate the association between identified genes and a targeted clinical condition, while functional validation at the molecular level is a prerequisite for forming any conclusions. As stated above, current knowledge of the functions of the skeletal class II and III-associated genes in the musculoskeletal system is extensive. However, the specific impacts of these genes on the craniofacial region are largely unknown. We hope that the current review and the conducted pathway enrichment can provide clues about the potential relationships among the identified genes and identify which genes and pathways can be prioritized for functional validation in future studies. We would also like to emphasize that the current review focused on the sagittal dimension, which is a crucial dimension in orthodontic diagnosis and treatment planning. Because craniofacial structure develops three-dimensionally, further evaluation of the genes involved in the vertical and transverse growth and development of the craniofacial structure is also needed.By reviewing and reanalyzing available human studies, 19 genes were found to be associated with skeletal class II malocclusion and 53 genes with skeletal class III malocclusion. Using the Reactome database, most of these genes were enriched in pathways related to bone and cartilage regulation and growth, as expected. Interestingly, multiple muscle-related genes and pathways have also been identified, which aligns with functional matrix theory in that the muscles surrounding the jaws may also contribute to the development of these two types of malocclusions. In addition, several SNPs were associated with both skeletal class II and III malocclusions, with the majority of these SNPs being located in the introns of their respective genes. It is possible that these SNPs result in a more sensitive isoform of the respective genes in response to factors associated with the surrounding microenvironment, such as mechanotransductive stimulation and/or pre- and post-translational regulations that might include epigenetic modification and miRNA-level interaction. Although multiple confirmation studies must be conducted, we hope our current study can provide a relatively panoramic view of the current understanding of skeletal class II and III malocclusions at the genetic level and thus potentially help dental care providers make more accurate craniofacial sagittal growth predictions and treatment strategy selections based on the patients’ genetic profiles in the future.