Tropheryma whipplei, most often presents with the classically described signs of malabsorption due to gastrointestinal colonization. However, it can also have signs and symptoms that clinically overlap with rheumatic diseases, potentially resulting in misdiagnosis. Furthermore, treatment with modern potent biologic immunosuppressive agents and classic disease modifying anti-rheumatic drugs (DMARDs) can lead to serious exacerbation of undiagnosed infections. We present the case of a middle-aged woman with long term complaints of arthalgias, who was diagnosed with seronegative rheumatoid arthritis and subsequently treated for almost 7 years with such immunosuppressive therapies. The patient’s disease course included chronic diarrhea that abruptly intensified and culminated in fatal hypovolemic shock/sepsis. A diagnosis of WD was made by autopsy examination, wherein several organ systems were found to be heavily involved by Tropheryma whipplei organisms, and their identification was confirmed with histochemical and molecular evaluation. Notably, most bacterial organisms were located deeply in the submucosa/muscularis of affected organs, a practical reminder to practicing pathologists that challenges the classic histopathologic description of Whipple disease as an infiltration of predominantly lamina propria, and the potential for sampling bias in typically superficial endoscopic biopsies during routine procedures.Whipple’s Disease, a rare diagnosis caused by the slow-growing bacterium  Tropheryma whipplei (T. whipplei), a fastidious, bacilliform, phylogenetically gram-positive bacteria characteristically positive for periodic acid schiff histochemical stain and diastase resistant (PAS-D+).-Whipple’s disease (WD) is an exceedingly rare systemic infection caused by the bacterium -The manifestations of WD are non-specific and may mimic other chronic disseminated infections as well as autoimmune disease. Symptoms of WD include, diarrhea, abdominal pain, adenopathy, arthralgias, and wasting with long-standing untreated disease. A prodromal stage has been reported with arthralgias years before the manifestation of gastrointestinal symptoms/malabsorption.T. whipplei intracellularly in macrophages. PAS staining is not sensitive or specific for T. whipplei, so additional molecular tests are usually performed to confirm the diagnosis. These tests may include T. whipplei-specific immunohistochemical (IHC) stains or detection of amplicons by polymerase chain reaction (PCR) directly from tissue sections or body fluids.,,Due to its rarity, a diagnostic work-up for WD generally takes place after more common disorders with overlapping presentations are ruled out. In these cases, WD is classically diagnosed with tissue biopsy of the small intestine or enlarged lymph node followed by PAS-D stain to visualize ,Several organ systems can concurrently be involved by WD, which may account for its diversity and severity in presentation. Progressive central nervous system involvement is among the most serious complications, and is most commonly seen as a triad of dementia, supranuclear ophthalmoplegia and myoclonus.Herein, we present a case of WD mimicking seronegative rheumatoid arthritis in a middle-aged female treated with long term immunosuppressive therapy, culminating in severe diarrhea, hypovolemic shock and death.A 58-year-old female presented to the emergency department (ED) as a transfer patient from another institution with undifferentiated shock, right bundle branch block, and a history of chronic diffuse, watery, non-bloody diarrhea that markedly worsened for one day duration. The patient had just recently moved from another state; her outside medical records were unable to be obtained making the true chronicity of some of her symptoms challenging to determine - it appears most were present for approximately 7 years in a relapsing-remitting fashion. Initial vitals on admission to the ED included blood pressure of 60/40 mmHg, a heart rate of 116 BPM, and a temperature of 35.9 C. No cardiac valvular dysfunction was noted during the physical examination at this time. Her clinical history included years of persistent fever of unknown origin, migratory arthralgias, nausea, and nonspecific neurologic symptoms . Due to this clinical picture, a working diagnosis of seronegative rheumatoid arthritis (RA) was proposed several years prior. Her general symptoms had persisted and worsened since this time despite almost 7 years of therapy with a number of immunosuppressive and disease-modifying anti-rheumatic drugs (DMARDs), which included: tocilizumab (an interleukin-6 receptor antagonist), leflunomide (a pyrimidine synthesis inhibitor), anakinra (an interleukin 1 receptor antagonist), rituximab , hydroxychloroquine, and prednisone.Several imaging studies were performed to locate the cause of the patient’s hypovolemia. X-ray and computed tomography imaging showed probable chronic inflammatory changes of the cecum and ascending colon, mild edema throughout the mesentery and fluid in the peritoneum and paracolic gutters, several calcified mesenteric lymph nodes, pleural thickening and effusion. A PET scan showed hypermetabolic activity of the lower esophagus to the cardia and of several of the partially calcified lymph nodes and diffuse hepatic steatosis. No sources of internal hemorrhage were noted.At the ED, attempts were made to correct severe hypotension and metabolic disturbances, but they were refractory to both pressors and IV fluids. Because the etiology of her hypovolemic shock was unknown, she also received broad spectrum antibiotics (including vancomycin and piperacillin/tazobactam), and steroids in an attempt to broadly cover bacterial sepsis. Her various laboratory studies after admission included: a metabolic (lactic) acidosis reaching a low of pH 6.8, mild leukocytosis (13.3 k/uL ), critically high potassium , low total protein , critically low hemoglobin , severe coagulopathy , signs of multi-organ damage, and eventual complete liver failure , and total bilirubin 1.4 mg/dL (0.2-1.4 mg/dL). Blood cultures did not yield any growth . Despite multiple successful resuscitation attempts during the course of her hospitalization, her critical condition was refractory to treatment, and she was declared dead 2 days after admission.Autopsy revealed a well-nourished adult female (BMI: 22.1) with diffuse petechiae of the trunk and focal soft tissue hemorrhage beyond what would be expected for iatrogenic resuscitation efforts, suggesting a coagulopathic process. The joints appeared normal and were without bony deformity or other stigmata of arthritis. There was no palpable lymphadenopathy. The proximal small intestine and duodenum showed mildly hyperemic and granular mucosa. The sigmoid colon was, in contrast, mostly unremarkable with minimal but rather diffuse pale-tan tenacious, adherent mucus. The mesentery was involved by multiple 0.5 – 2.0 cm calcified nodules (thought to be fat necrosis by prior imaging). The adrenal glands were markedly atrophic (2-3 g each [RR: 7-12 g for both]) likely secondary to her prolonged steroid therapy. The liver demonstrated diffuse spongy parenchymal collapse and softening as well as a variegated and congested appearance suggestive of hypovolemic shock. The pericardium was diffusely adherent to both the heart and overlying mediastinal tissues and without effusion. The heart contained numerous yellow vegetations of the cardiac valves, predominantly on the pulmonic and mitral valves. Notably, there were severe adhesions involving part of the bilateral pleura, intestine, and spleen. The bilateral kidneys had coarse cortical pitting (presumably from chronic hypertension) and a hemorrhagic-appearing renal collecting system, possibly due to coagulopathy and complications of shock. The uterus and bilateral ovaries were surgically absent, but no pelvic adhesions were observed. No significant internal hemorrhage or other trauma was identified to help explain the hypovolemic shock.Microscopically, widely disseminated bacilliform organisms were identified within the cytoplasm of histiocytes in numerous organs: small and large bowel, mesenteric lymph nodes, pericardium, myocardium, cardiac valves, liver, spleen, and pleura  to 7. TAt low power the liver appeared uninvolved, but upon close inspection at higher power (400x) rare single blue-tinged macrophages were present in a distribution completely limited to the sinusoids .The lymph nodes lacked true, well-formed granulomas and were rather diffusely replaced by organism-laden histiocytes with associated calcifications .The myocardium was sparsely involved with rare macrophages percolating between myocytes, predominantly in an epicardial distribution that was more or less contiguous with the overlying dense involvement along the adherent pericardium .The involved cardiac valves demonstrated discrete and sharply demarcated aggregates of histiocytes (laden with organisms by PAS-D stain) in an immediately subendocardial distribution that formed plaque-like nodules . There wSplenic involvement was complicated by marked post-mortem autolysis and hemorrhage but could be appreciated particularly in the soft tissues surrounding the hilum.T. whipplei by PCR at ARUP laboratories .This case of ,,,,,WD is a diagnosis of exclusion that can be considered after excluding the other more common disorders associated with symptoms of fever, arthralgias, GI and neurological symptoms.,,T. whipplei.The broad spectrum of symptoms associated with WD including diarrhea, arthralgias, and central nervous system (CNS) symptoms raises a differential diagnosis that may include inflammatory bowel disease, connective tissue disease, and other, more common infections such as tuberculosis, AIDS, and chronic diarrheal organisms.T. whipplei infection. The active enteritis observed microscopically is not a common finding reported in WD, but in our case may be a manifestation of the severity of disease. These GI findings in total not only explain the cause of diarrhea but may also have further contributed to hypovolemic shock, due to the massive fluid and associated protein loss. Further exacerbating the hypovolemic shock, the valvular vegetations, and severe pericardial and pleural adhesions resulting in a restrictive pleuropericarditis that most likely led to further decreased cardiac output and overall cardio-pulmonary dysfunction during the increased demand of cardiac output due to hypovolemia. The pathologic findings of the liver parenchyma included diffuse centrilobular necrosis, which is consistent with the patient’s hypovolemic shock.Several of our autopsy findings help explain the extent of our patient’s involvement by WD and the pathogenesis of her hypovolemic shock. The chronic diarrhea is most likely due to chronic malabsorption, and protein-losing enteropathy from small bowel T. whipplei cannot be detected with traditional blood cultures if there is suspicion of septic shock, which explains why no infectious etiology was clinically detected in this case – and the ultimate reason for the request for autopsy. Laboratory culture for confirmation of WD is both technically challenging and not clinically useful due to the exceptionally slow growth dynamics of T. whipplei (replication time estimated at 18 days) and an inability of the organisms to synthesize certain amino acids that are usually supplemented by their environment/host.,,,The autopsy findings also highlight a particular challenge in diagnosing WD by superficial luminal endoscopic biopsy. The bacteria-laden macrophage infiltrates may lie deep to the mucosa , and could potentially go unsampled in superficial biopsies, even in cases with a heavy burden of disease. The distribution of organisms in the small bowel, even in this patient with widely disseminated disease, is most dense in the muscularis propria, which is not typically intentionally sampled on routine biopsies. While admittedly somewhat obscured by the degree of autolysis secondary to autopsy, the number of organisms present in the tips and lacteal of the small bowel villi is relatively sparse. Thus, even a thorough pathologist actively looking for evidence of WD, may not be provided with a sufficient and deep enough sample to make the diagnosis.The remainder of the autopsy findings support WD as the etiology of this patient’s constellation of long-term signs and symptoms. No vasculitis, mononuclear cell infiltrates, or other auto-immune processes were identified histologically; admittedly, their identification may have been obscured by the degree of disseminated disease in this case. In either scenario, the presence of these findings would not alter the cause of death nor would their absence rule out these diseases. In short, there is no evidence for rheumatologic disease, which also would not explain the diverse symptomatology and severity of this patient’s disease course.,-did not involve suspected rheumatologic etiologies nor immunosuppressive therapies at all.,WD disease misdiagnosed as inflammatory rheumatoid disease has been reported in numerous studies, but none such cases appear to have led to a fatal outcome. In a retrospective European cohort of 113 patients diagnosed with WD,T. whipplei does not lead to disease in all individuals,,,,,,-T. whipplei with the human immune system is beyond the reach of our current understanding, but may account for the diversity of disease presentation.Clearly there is no specific symptomatology for WD and its clinical overlap with inflammatory rheumatoid disease poses a challenge for a timely diagnosis by our clinical colleagues. Speculatively, this could be due to patient-specific immune interactions with bacterial antigens, or simply due a chronic inflammatory state and immune dysregulation resulting from the long-term dissemination of a slow-growing pathogen, such as in some disseminated mycobacterial infections. Several research findings support an immunogenetic predisposition to full-fledged manifestation of WD: namely, colonization by This case report highlights the challenge of diagnosing a rare disease with an atypical clinical presentation, but may help serve as a reminder to consider unusual infections in patients with a similar constellation of symptoms. It also underscores the relevance and importance of a thorough autopsy examination, even with today’s complex imaging and laboratory testing. We also would like to highlight the potential sampling discrepancies that may arise with superficial endoscopic biopsies given the distribution of disease (even in cases with a high infectious burden such as this). WD, although while rare, should be considered whenever there are unexplained GI symptoms or arthropathies, with or without cardiac/CNS symptoms, to help gather more accurate and current data about its prevalence in various populations.


This special Research Topic of Frontiers in Physiology collates 16  articles on the endothelium (the cell layer that lines blood vessels) and other components of the vascular system. Over the past decade, it has become increasingly clear that the vascular network is not a mere conduit for blood and the endothelium is not a “wallpaper” of the vascular network; rather both are dynamic entities that sense and respond to mechanical and chemical stimulation from the microenvironment. Both endothelial cells and vascular tissue  are equipped with complex molecular machinery that initiate and amplify signaling processes leading to physiological and pathophysiological changes. These changes range from vasodilation, vasoconstriction and angiogenesis to inflammation, leakage, injury, and vascular remodeling and form the basis of vascular health, disease and other etiologies.2+ channels), activation of mitogen-activated protein (MAP) kinases, induction of cellular adhesion molecules (CAMs) which lead either to activation of downstream inflammation moieties (such as the NLRP3 inflammasome) or to modification of proteins that facilitate endothelial cell function. Collectively, these changes can alter vascular cell-cell junctions, vessel structure, and function. For instance, endothelial cells of a donor organ as the interface between the transplanted organ (graft) and the immune cells of the host, serve as the first target for host (recipient) immune cells post-transplant. Kummer et al. review how the sensing of a “foreign” entity by the human leukocyte antigen (HLA) I and II-recognizing natural killer (NK) cells, macrophages, and T-cells lead these cells to attack the endothelial layer. The damaged endothelial cells produce chemokines, express CAMs and drive a pro-inflammatory microenvironment. Inflammatory cells  are recruited into this environment and transmigrate from the bloodstream across the endothelial monolayer into the vessel wall, causing vascular damage. Vascular damage initiates repair mechanisms whereby migration and continued proliferation of vascular smooth muscle cells occur in the vascular structures causing fibrous intimal hyperplasia, which ultimately leads to transplant failure. Thus, endothelial preservation, as proposed by Jungraithmayr can be a promising therapeutic strategy for the protection of the endothelium against ischemia and reperfusion (i.e. maneuvers associated with transplant). Therefore, various receptors and signaling pathways that are well-recognized to be activated upon transplantation  can be blocked in order to facilitate endothelial preservation against injury.Endothelial signaling initiated by various stimuli involve reactive oxygen species (ROS) induced signaling events, calcium influx  that cause caspase and interleukin (IL)-1β, IL-18 release, leading to inflammation induced cell death (pyroptosis). Thus, various pathologies or agents that drive NLRP3 activation can also lead to endothelial inflammation and injury. As reported by Pereira et al. in a murine model of diabetes, wild-type mice showed reduction in endothelium-dependent vasodilation, increased vascular ROS generation, caspase-1, and IL-1β activation. These changes were not observed in Nlrp3−/− mice, which appear to be protected. Paul et al. review the mechanisms underpinning the links between periodontal disease (PD) and diabetes and cardiovascular disease. ROS and oxidative stress may also play a role in amplification of bacterial driven inflammation by triggering pathological processes in distant organs. A two-way association between PD and other health conditions presumably arises via ROS and intermediate inflammation signaling. Thus, blocking these intermediate inflammation-signaling events  can facilitate endothelial preservation. Modification of proteins also serve as intermediate signals that can alter inflammatory mediators/response. Silva et al. show that in a murine model of sepsis , inflammation is reduced by acylation of proteins by O-linked N-acetylglucosamine (O-GlcNAcylation); indeed, increase in O-GlcNAc reduces systemic inflammation and cardiovascular dysfunction, indicating the therapeutic potential of this pathway.ROS production and calcium influx are pivotal cellular events that drive inflammation signaling. ROS production in endothelial cells is tightly regulated by certain receptors including the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). As reported by 2+) are prominent signaling molecules that participate in various physiological functions, such as vasodilation, cell migration, growth and apoptosis. In naïve cells, mitochondria sequester and release Ca2+ via mitochondrial calcium uniporter complexes. While oxidative stress or other deterrent stimuli, increase mitochondrial calcium overload leading to apoptotic cell death, the calcium uniporter complexes regulate prosurvival mechanisms such as autophagy for endothelial protection. Natarajan et al. report that with in vitro ischemia (achieved by oxygen-glucose deprivation) endothelial cells downregulate the mitochondrial calcium uniporter complex 1 (MCUR1), but that the resultant autophagic flux is insufficient to protect ischemic endothelial cells and an additional autophagic flux  is required for cell protection indicating that therapeutic strategies can involve targeting multiple mechanisms of endothelial protection.Like ROS, calcium ions  a pharmacologically active component of Cnidium monnieri (L.) Cusson and report that it alleviates hyperplasia in a rat model of balloon angioplasty by targeting the NF-κB and TGF-β1/Smad2 signaling pathways that play a role in facilitating VSMC proliferation. Vascularization also has implications in preeclampsia, as reviewed by Raguema et al. Abnormal placental vascularization presumably arises from chronic hypoxia due to reduced placental blood flow leading to fetal growth retardation and later to pre-eclampsia.How does the vasculature respond to inflammation, oxidative damage and injury? A major response to vascular injury is a hyperproliferative response of vascular smooth muscle cells (VSMC). This gives rise of hyperplasia which leads to vessel remodeling. This is detrimental specifically in the context of maneuvers such as percutaneous coronary intervention where balloon angioplasty to remove arterial blockage is followed by hyperplasia (VSMC proliferation) that reinstates the blockage (restenosis). Restenosis can potentially be alleviated by blockade of vascular signaling that drives hyperproliferation. Toward that goal, Baruah et al. review intrinsic defects within endothelial cells from the earliest developmental time points, the disruption of the blood-brain barrier (BBB) can be the cause for posttraumatic epilepsy and other similar etiologies. Under control conditions, the BBB is maintained via tight endothelial cell-cell junctions, but these can be loosened by inflammation and oxidative damage. These changes lead to vascular abnormalities and autonomously support the development of hyperexcitability and epileptiform activity. Thus, BBB protection by alleviation in inflammation or by repair of junctional proteins can be a potential therapeutic strategy in epilepsy control.Another response is the disruption of the endothelial cell barrier. Indeed, as Epsley et al. review the effect of inflammation on bone modeling, which is a continual process to renew the adult skeleton through the sequential growth and resorption processes of bone cells (osteoblasts and osteoclasts). Nuclear factor RANK, an osteoclast receptor, and its ligand RANKL, expressed on the surface of osteoblasts, result in coordinated control of bone remodeling. Inflammation skews this process toward resorption via the interaction of inflammatory mediators and their related peptides with osteoblasts and osteoclasts, as well as other immune cells, to alter the expression of RANK and RANKL.Vascular inflammation also affects cellular signaling of bone. Englund and Langham review the use of MRI for vascular parameters via dynamic quantification of blood flow and oxygenation under conditions of ischemia or exercise and showcase this application for detection of endothelial dysfunction in clinical (cardiovascular disease) and sub-clinical settings . Wehrli et al. review the ability of MRI to detect vascular function. When paired with endothelial cell signaling assays, these “MRI-biochemical pairing” methods highlight the fact that changes at the cellular level  translate into altered vascular function at the macro level. Thus, MRI measures of vascular function may ultimately be used to complement the standard clinical metrics of vascular function and provide additional insight into efficacy of drugs for improvement of vascular function. Similarly, as reviewed by Mayer et al. endothelial signaling at cellular and molecular level, that contribute to atherosclerosis can be detected by non-invasive monitoring of calcification, a complex process facilitated by endothelial signaling that leads to transition of endothelial cells to osteoblast-like cells. Mayer et al. review existing literature on 18F-fluorodeoxyglucose (FDG) and 18F-sodium fluoride (NaF) positron emission tomography (PET) in assessing atherosclerosis via microcalcification and characteristic plaque buildup. Since the initiating event of atherogenesis is inflammation, the pathogenesis manifests in increased metabolic activity, detectable by FDG. At a later stage, atherosclerotic plaques become calcified. Calcification entails incorporation of calcium hydroxy apatite into the vascular walls. Injection of NaF as a tracer leads to partial exchange of hydroxyl ions by fluoride, therefore allowing direct detection of plaque constituents. Pourfathi et al. review the role of PET and hyperpolarized 13C MRI imaging to detect the various stages of pulmonary inflammation. The most common PET tracer for metabolic imaging is FDG. A hallmark of inflammatory lung disease is increased glycolysis manifesting in enhanced FDG PET activity. An alternative strategy, which is at an early stage of development, is to quantify the formation of lactic acid by means of hyperpolarized carbon-13 spectroscopic imaging. The carbonyl carbons of lactic acid and its exogenously administered metabolic precursor (pyruvate) have characteristic chemical shifts allowing their distinction.Changes in endothelial cell signaling that transiently or chronically impair endothelial function, vascular reactivity, and oxygen metabolism can be evaluated non-invasively by quantitative magnetic resonance imaging (MRI). Fetter et al. can alter blood flow and improve vascular function) or inhibition/blockade of ROS or NLRP3 or inflammatory and metabolic pathways may be beneficial to restore vascular function.In conclusion, endothelial signaling has an impact on overall health. Under conditions where these signals lead to remodeling that affects homeostasis, interventions (such as employing respiratory techniques as reported by SC drafted the editorial. All authors edited 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.


N = 5836) and subjected them to various integrated analyses. Many of these genes were initially identified in non-human species, and we investigated their orthologs in three non-human species  for further analysis. We characterized experimentally determined protein-protein interaction networks (PPIN) involving each species’ genes from 9 known protein databases and studied the enriched biological pathways among the individually constructed PPINs. We observed three important signaling pathways: FoxO signaling, mTOR signaling, and autophagy to be common and highly enriched in all four species . Our study implies that the interaction of proteins involved in the mechanistic target of rapamycin (mTOR) signaling pathway is somewhat limited to each species or that a “rewiring” of specific networks has taken place over time. To corroborate our findings, we repeated our analysis in 43 different human tissues. We investigated conserved modules in various tissue-specific PPINs of the longevity-associated genes based upon their protein expression. This analysis also revealed mTOR signaling as shared biological processes across four different human tissue-specific PPINs for liver, heart, skeletal muscle, and adipose tissue. Further, we explored our results’ translational potential by assessing the protein interactions with all the reported drugs and compounds that have been experimentally verified to promote longevity in the three-comparator species. We observed that the target proteins of the FDA-approved drug rapamycin (a known inhibitor of mTOR) were conserved across all four species. Drugs like melatonin and metformin exhibited shared targets with rapamycin in the human PPIN. The detailed information about the curated gene list, cross-species orthologs, PPIN, and pathways was assembled in an interactive data visualization portal using RStudio’s Shiny framework (https://agingnetwork.shinyapps.io/frontiers/).Intensive research efforts have been undertaken to slow human aging and therefore potentially delay the onset of age-related diseases. These efforts have generated an enormous amount of high-throughput data covering different levels in the physiologic hierarchy, e.g., genetic, epigenetic, transcriptomic, proteomic, and metabolomic, etc. We gathered 15 independent sources of information about genes potentially involved in human longevity and lifespan ( The aging process is affected by a number of interconnected genetic, epigenetic, metabolic, and environmental factors; however, their exact degree of contribution is unclear as there is no universally accepted theory of aging  within wThe advent of high-throughput technologies has led to the generation of enormous amounts of large-scale data from various cellular levels ranging from genomics to proteomics across different tissues and species. The availability of such technologies provides an excellent opportunity for studying aging through an integrative systems biology approach . In thisC. elegans). We found three critical signaling pathways: FoxO signaling, mTOR signaling, and autophagy, to be highly enriched  and shared between humans and all three other species. We found evidence for rewiring of specific protein networks of the mTOR signaling pathway across the species we studied. We also exploring whether or not our findings could shed light on human tissue-specific PPINs by correlating the tested genes’ mRNA and protein levels across 43 different human tissues using publicly available data. We found that highly expressed proteins were less likely to be the hub proteins in human tissue-specific networks. To explore our findings’ translational potential, we retrieved all experimentally verified drugs and compounds reported for longevity in the non-human species we studied and evaluated the potential targets of the aforesaid drugs for each species, including humans, using available drug-protein interaction information by determining the positions of these targets within their respective species-specific PPINs. Together, our results could benefit future studies considering effective combination therapies for age-related diseases, drug repurposing, or the development of novel therapeutics for enhancing longevity in humans.In the present work, we studied genes that have been reported to influence human aging and longevity. We used a network approach to decipher these genes interactions with physical protein-protein interaction networks and further investigate their properties in three different, evolutionary distant, species  for further analyses  used in our study (Where the CES of a gene (G) was calculated based on the available evidence that a gene is associated with longevity or aging across the 15 available datasets  using “gene to gene”  ortholog mapping from the Ensembl database using the g:Profiler web tool . We alsoThe raw physical protein-protein interactions (PPIs) information for human and mice, flies, worms were obtained from the results of experiments reported in 9 curated databases  and processed with the Integrated Interactions Database (IID) M . We charIn addition, we also constructed a broader physical PPIN, also term as “local-PPIN” for the 1007 aging and longevity associated genes in humans from three available human protein interactome resources: IID , MIST H, and HuRthe normalized transcripts per million (NX-TPM) values for 987 and 1001 genes in 34 and 43 different human tissues, respectively, available from each database. The 1007 genes were ultimately evaluated for four different tissues  based on the primary cell type information in the HPA databases, with variations in protein expression level of the genes assigned as high, moderate, weak and negative as described in the available annotations within the HPA database.The mRNA (transcript) abundance of the 1007 genes were curated using two public domain databases: the Genotype-Tissue Expression (GTEx)  database1. We used gene and pathway annotations in two common data sources for this: the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Actual enrichment statistics and p-values were calculated by applying a two-sided hypergeometric test followed by a Bonferroni correction to the resulting p-values (p < 0.05) to identify enrich or overrepresented pathway and terms in the respective databases  Unique ID (D008136 and D000375) from BioLitMine database . Further2 : which includes interactive visualizations created using RStudio’s Shiny framework  to rank each gene based on its evidence for being implicated in longevity and aging across the 15 resources for 1007 genes that were listed in at least two of those resources. These genes, listed in Aging affects different species in different ways and this fact contributes to the variation in lifespan exhibited by different species. However, many studies have shown that modulating genes in non-human species can affect the lifespan of those species and therefore provide a rationale for studying the human orthologs of those genes in studies of human longevity and aging . Thus, iC. elegans genes (see C. elegans) organisms, which is expected likely a result of gene loss events, duplication  events, and evolutionary constraints acting on the species  human gene orthologs of mouse genes, 449 human orthologs of fruit fly genes, and 411 human orthologs of enes see . 331 gen species .APOE, TP53, WRN, and LMNA) exhibit an intermediary ortholog score across 57 different species  within the four species-specific PPINs  . The numic PPINs  which clC. elegans did not, likely due to poor orthology determination and complex evolutionary histories of the genes including gene gain or loss events, indicating that human and mouse genes have become involved in complex interactions and regulatory circuits  over time. A comparison of the highly connected nodes among the top ten ranked proteins over the networks clearly indicates that “hub-like” proteins  in humans potentially lose their hub-like properties in non-human species. However, certain proteins, like GRB2, HDAC1, H1F1A, and MAPK1, remain highly connected within the species-specific PPINs  of the genes within the PPINs was computed based on their first-order interacting partners in the individual PPINs. Further, “hub” genes in those networks were identified by comparing different network topology metrics associated with the genes  together with the ranking of the genes based on their CES values in individual PPINs . As expeic PPINs . The exiEmerging evidence indicates that alterations in cellular signaling during the course of aging is important in controlling the lifespan in different species . It has p-value ≤ 0.001  and Kyoto Encyclopedia of Genes and Genomes (KEGG) gene and pathway annotations. We found that three important KEGG signaling pathways, autophagy (KEGG:04140), FoxO signaling (KEGG:04068) and mTOR signaling (KEGG:04150), are common and highly enriched in all four species with an adjusted  ≤ 0.001 . Our res ≤ 0.001 . Such di ≤ 0.001 .N = 111) and did not find mTOR signaling among the 27 enriched KEGG pathways , FoxO signaling (KEGG:04068) and mTOR signaling (KEGG:04150), were due to the presence of shared genes within the species-specific PPINs, we performed an independent pathway enrichment analysis for the genes that were common in the PPINs of all the four species , heart , skeletal muscle (ACTA1) and adipose  are not hubs in the respective tissue networks  and orthologs (COG) information together with the network properties, we have highlighted a couple of genes, such as AKT1, LMNA, and MYC, as important candidates in the conserved local-PPIN for different tissue-specific networks likely implicated in human longevity and aging.In addition, we identified an overlapping module of 701 defined PPIs implicated 160 genes which remain conserved across all four tissue-specific local-PPINs in our study . A numbeWe explored the functionality of those 160 conserved genes in the inter-tissue local-PPIN in a pathway enrichment analysis and identified a total of 85 different KEGG pathways that are enriched above the defined threshold cut-off . We notiC. elegans)  . We founelegans) .We identified the potential targets of the aforesaid drugs for each species, including humans, using available drug-protein interaction information . Using eAPOE, APP, SIRT1, IGF1, and IL6 have the highest number of literature citations and are also present on the top of our selected gene-list and also exhibit high CES values, which validates our CES ranking to some degree. On the other hand, we did not include some genes, such as CRP (CES Rank: 3456), COMT (CES Rank: 3480), VDR (CES Rank: 1124), because they had low CES ranking, yet are potential candidate genes for longevity and aging based on the literature. Obviously, studies that consider different criteria and curation strategies for identifying genes associated with longevity and aging are needed, as well as studies that accommodate new genes found to be associated with longevity and aging.We manually curated fifteen key data resources related to aging and longevity and identified 4060 non-redundant genes. Further, we selected 1007 genes as potential candidates through data harmonization efforts and performed a number of complementary integrated analyses. To verify the selected longevity and aging-related gene-list we used in our study, we performed a gene to literature-based mapping and identified the significant contribution of each gene related to human longevity and aging. A total of 1483 genes were mapped with the available PubMed literature to verify their links with human aging and longevity . We founWe considered a study of the connections between genes shown to be associated with longevity and aging. These connections included those arising from their interactions in protein-protein interaction networks (PPINs), expression patterns across different tissues, as well as their evolutionary-mediated connections as orthologs across species. Our findings shed light on the remarkable inter-species and human inter-tissue differences in the protein interactions, particularly involved in mTOR signaling, a key process for nutrient sensing known to mediate longevity and aging. Ultimately, our work provides a paradigm for how researchers can study genes related to human lifespan and aging using systems biology and evolutionary analyses and could lead to potential targets for longevity-enhancing drugs.The original contributions presented in the study are included in the article/AP and NS conceived the study. AP performed the data curation and analysis. AR contributed to shiny app development. AP and AR wrote the manuscript. NS participated in revising it critically for important intellectual content and gave final approval of the version to be submitted. 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.


Purpose of review: To discuss and summarize recent findings in peripheral neuropathy (PN) related to connective tissue diseases (CTD) including its prevalence, clinical manifestations, pathogenesis, diagnosis and treatment. Recent findings: Although PN is a common complication in CTD and has been well studied, recent research has shown that PN is more diverse and frequent in different subtypes of CTD than was expected. The incidence of PN in Sjögren’s syndrome and rheumatoid arthritis (RA) varies according to different disease subtypes, and the pathogenesis of neuropathic pain in different subtypes of eosinophilic granulomatosis with polyangiitis (EGPA) may also differ. Neurogenic inflammation, autoantibody-mediated changes, ischemia of the vascular wall and metabolic mechanisms have been shown to contribute to the pathogenesis of PN in CTD. Moreover, allergic inflammation has been recently identified as a possible new mechanism producing peripheral neuropathic pain associated with MPO-ANCA negative EGPA patients. Glucocorticoids are routinely used to relieve pain caused by PN. However, these steroids may cause hyperalgesia, exacerbate neuropathic pain, and activate the early phase of pain induction and produce hyperalgesia. Recently, neuroactive steroids, such as progesterone, tetrahydroprogesterone and testosterone, have been shown to exert protective effects for several PN symptoms, and in particular neuropathic pain. Neuroactive steroids will be an interesting topic for future research into PN in CTD. Summary: It is essential for the diagnosis and treatment of PN in CTD to be updated. Timely diagnosis, appropriate treatments, and multidisciplinary care are essential to minimize morbidity and decrease the risk of permanent neurologic deficits. Further studies are needed to guide diagnosis and treatment.  Connective tissue diseases (CTD) are chronic inflammatory autoimmune diseases induced by antibodies or T-cell responses directed against self-antigens, which can affect all body systems, including the central nervous system (CNS) and peripheral nervous system (PNS) . When thA literature review of original articles, review articles and case reports was conducted using Pubmed, Embase and Cochrane databases from January 2015 to June 2021. The search terms were: peripheral neuropathy; systemic lupus erythematosus; Sjögren’s syndrome; systemic sclerosis; mixed connective tissue disease; dermatomyositis; polymyositis; systemic vasculitis; Behçet disease; rheumatoid arthritis; anti-TNF-α therapy; and neuroactive steroids.Findings of literature reviewClassificationPN can be classified into multiple descriptive types according to different criteria  5]. The. The5]. 2. Prevalence and clinical manifestationsPN is a general term for disease that involves structural or functional impairment of the PNS, with various causes such as infection, autoimmune disorders , systemic or metabolic disorders, after exposure to toxic compounds and during drug therapy . Table 1Axonal sensory polyneuropathy and sensorimotor polyneuropathy can be characterized by paresthesia and defects  in the distal part of the symmetrical limb, mainly affecting the distal end of the lower limbs, and may be accompanied by burning pain in the feet. In addition to the above manifestations, motor weakness may be present in sensorimotor polyneuropathy, which is usually mild and limited to the extensor muscles of the toes or feet .Small-fiber neuropathy (SFN) is an algetic esthesioneurosis that usually results in burning pain and arises in the early stage of several systemic diseases such as diabetes, amyloidosis and CTDs . The maiAs a heterogeneous group of neurological disorders, the reported prevalence and clinical manifestations of PN in CTD varies widely . As showAmong the studies that have investigated the prevalence of PN in autoimmune diseases in recent years, SLE was one of the major diseases studied. In 2019, Shaban et al. ,12,13,14Although Guillain–Barré Syndrome (GBS) is a rare manifestation of SLE, which falls under the category of PNS-SLE, it was not mentioned in these studies. It is, however, important to remember that GBS can be one of the main causes of morbidity and mortality in PN-SLE patients ,16.In conclusion, SLE patients are easily susceptible to PN, but neurodiagnostic analysis of PN-SLE varies widely. PN-SLE should be given greater recognition. Further studies should focus on the differences between individual subtypes of neuropathy to explore the different pathologies and guide diagnosis and treatment.SS is the second most common chronic autoimmune rheumatic disease, including primary Sjögren’s syndrome (pSS) and secondary Sjögren’s syndrome (sSS). We found six valid cross-sectional studies and one cohort study that discussed the prevalence and clinical features of PN in SS ,21,22,23In conclusion, PN events are common in SS. Further studies should focus on the differences in the incidence and clinical patterns of PN events between sSS and pSS. PN might help to establish new international classification criteria and clinical practice guidelines for pSS in the future.There have been few studies on PN-SSc in recent years, with varying sample sizes and definitions for PN events (some studies used questionnaires to define neuropathic pain). Raja et al.  and PaikOne small sample size cross-sectional study from India calculated the prevalence of PN in PAN . The preIn summary, among CTD, PNA has a high prevalence of PN. The mechanism is arteritis of the vasa nervorum, leading to ischemic neuropathy . MononeuIn 2019, Bischof et al.  carried PN in EGPA primarily affected the lower extremities, with peroneal nerve involvement being the most frequent and severe. The sensory neuropathy was distributed mostly asymmetrically in the distal portion of the limbs, while the main manifestations of motor neuropathy were foot drop and muscle weakness. AVV patients may also have symptoms of autonomic dysfunction which is independent of the disease duration and its severity . AtypicaMononeuritis multiplex and distal symmetrical polyneuropathy, mixed neuropathy and lower limbs involvement are predominant PN manifestations in MPA patients ,46. SymmPN in RA was mainly reported as small sample sizes in cross-sectional studies in the past 5 years. Kaeley et al.  reportedPrevious studies have shown that the types of PN-RA are pure sensory, distal axonal sensory-motor, mononeuritis multiplex and entrapment neuropathy . HoweverStudies on PN in GCA and TAK were mainly reported as individual cases during the past 5 years. A literature review by Bougea et al.  found thPN involvement of TAK is much rarer. Isolated cranial nerve palsies was reported to be due to the involvement of the internal carotid artery or its branches . VasculiIn summary, PN in systemic vasculitis also requires a consideration of non-vasculitic neuropathies factors, especially compression neuropathies.PNS involvement in BD is extremely rare, only isolated cases of distal symmetrical polyneuropathy and mononeuritis multiplex have been reported . A retroPrevious studies suggested that the prevalence of PN-MCTD is approximately 10% to 17% , among wPrevious studies showed a prevalence of 7.5% in DM/PM patients with polyneuropathy . In 2016IgG4-RD can affect a wide range of visceral nerves including those innervating the kidney, prostate gland, epicardium, abdominal aorta, retroperitoneum and mesentery. The nerves comprising Auerbach’s plexus of the intestinal wall were most extensively involved . This miJanus kinase (JAK) inhibitors have been used to treat RA and other CTDs, basically targeting different molecules in the same signal pathway . In a stPNS involvement in ankylosing spondylitis (AS) and psoriatic arthritis (Pisa) were mainly related to tumor necrosis factor-α (TNF-α) antagonists, such as infliximab, adalimumab and etanercept . In 2015In CTD, neurogenic inflammation, autoantibodies-mediated changes, ischemia of the vascular wall and metabolic mechanisms are believed to contribute to the pathogenesis of PN and to be predominant in different diseases. Despite significant advances in our understanding the pathogenesis, no single pathogenetic mechanism is thought to be responsible for PN in CTD. Next, we discuss the possible mechanisms underlying disease pathogenesis. An increase in proinflammatory cytokine concentrations has been found in patients with vascular neuropathy . NocicepThe sural nerve biopsy specimens of PN-EGPA patients with negative MPO-ANCA were mainly characterized by eosinophil infiltration , suggestThe most possible pathogenesis of vasculitis-related PN is inflammation of precapillary arteries in the nerves . DepositA process of molecular mimicry may act as the starting motif to target different specific antigens within the structure of a nerve . Nodes oIgG and IgM anticardiolipin antibodies were detected in the serum of CTD patients . The exiPNS involvement in CTD can also be caused by metabolic disorders secondary to aggressive therapy, multiorgan pathology and endocrine abnormalities. Metabolic disorders may induce a reaction of demyelinating neuropathy and axon dystrophy in severe cases .The diagnosis of PN-CTD is based on recognition of the clinical symptoms and is supported by nerve conduction studies and neuropathology. A careful review of the illness history, current medication and laboratory results will help to arrive at a correct diagnosis.NCS can reveal the asymmetric or multifocal nature of neuropathy. Electromyogram (EMG)/NCS can establish whether patients have sensory-motor neuropathy, sensory neuronopathy or motor neuronopathy. Moreover, EMG/NCS can be applied to distinct polyradiculopathy with mononeuritis multiplex, as both patterns have very similar diffuse, non-length dependent neuropathies. Importantly, EMG/NCS can also be used to identify whether the affected structure of nerves is the axon itself or demyelination. In addition, needle electromyography is very useful in estimating the disease course, the extent of the injury and the existence of a superimposed myopathy .In In SLE, Saigal et al.  reportedThe sural nerve is most frequently used for biopsy, which contributes to the determination of the nature and extent of PN . BischofIn contrast, skin biopsy is an emerging, minimally invasive method for differentiating between length-dependent and non-length-dependent small fiber neuropathy , could aSomatic and autonomic functional nervous evaluation of SFN involves the sympathetic and parasympathetic autonomic functions, which is realized by determining the psychophysical sensory thresholds  through quantitative sensory testing (QST), pain-related tests and recording of laser-evoked potentials (LEP), single axon recording utilizing microneurography and tests . On the ®) device [What is more, current diagnostic technologies for SFN are also composed of quantitative sensory testing with determination of warm and cold detection thresholds , recording of LEP and sympathetic skin responses (SSRs), and measurement of electrochemical skin conductance (ESC) utilizing Sudoscan device .Modern imaging methods allow the precise localization of peripheral nerve damage. Sonoelastography  and ultrDiffusion-weighted magnetic resonance neurography (DW-MRN) is another emerging technique that can exploit the greater water diffusion anisotropy in peripheral nerves for improved visualization . Based oThere are no clear guidelines for the treatment of PN-CTD. In particular, the importance of PN when deciding the treatment strategy is often underestimated. In 2010, the guidelines of the Society for Neurology of USA recommended (with evidence level B) vasculitic peripheral neuropathy shock therapy with glucocorticoids, possibly in combination with immunosuppression as basic therapy . Major tIn 2019, Bortoluzzi et al.  systematInduction treatments with glucocorticoids with or without immunosuppressants was indicated for active vasculitic neuropathy , and the2+ channels; testosterone exerted anti-nociceptive effects in neuropathic rats; 17β-estradiol caused pain attenuation and a decrease of neuropathy-induced gliosis after sciatic nerve ligature; moreover, it exerted protective effects on neuropathic pain via estrogen receptors by inhibiting microglia activation and the production of inflammatory mediators. These observations support the view that neuroactive steroids may provide an interesting topic for neuropathic pain research in the near future.Glucocorticoids are routinely used to relieve pain. However, as reported in experimental models of neuropathic pain, glucocorticoids may contribute to hyperalgesia, exacerbate neuropathic pain, activate the early phase of pain induction and induce hyperalgesia . TherefoPN is a well-known consequence of CT, with a complex spectrum of overlapping clinical manifestations of rheumatologic and non-rheumatologic disorders, such as infections, drug toxicity or metabolic disease. Therefore, it is still a clinical issue and a challenge to rheumatologists and neurologists alike. The reported prevalence and clinical manifestations of PN in CTD varies widely. Recent studies have shown that seropositive RA and sSS may have a higher incidence of PN than seronegative RA and pSS. Furthermore, allergic inflammation was an underlying mechanism in MPO-ANCA negative EGPA patients. Although the majority of PN patients are resolved or improved over time by induction treatments, no reliable prediction of the treatment response or accepted diagnostic tools are available. Given the recent insights in PN-CTD pathogenesis and a better understanding of its disease course, new therapeutic targets and clinical management may be approved to improve the outcome of PN involvement in CTD in the future. However, some limitations of this review should also be considered, such as the wide variation in methods used to report complications and the frequency of reporting as determined by studies with different sample sizes in multiple age groups. Prospective clinical trials are needed for early detection of all possible medication-related side effects. Long-term evaluation of patients is also important to control recurrence.The reported prevalence and clinical manifestation of PN in CTD varies widely;The main mechanism of PN is different for each CTD and the pathogenesis may also vary in different subtypes of the same disease;Neuroactive steroids may have protective effects against several PN features, especially neuropathic pain.Key messages:


Chicken is known to be the most common meat type involved in food mislabeling and adulteration. Establishing a method to authenticate chicken content precisely and identifying chicken breeds as declared in processed food is crucial for protecting consumers’ rights. Categorizing the authentication method into their respective omics disciplines, such as genomics, transcriptomics, proteomics, lipidomics, metabolomics, and glycomics, and the implementation of bioinformatics or chemometrics in data analysis can assist the researcher in improving the currently available techniques. Designing a vast range of instruments and analytical methods at the molecular level is vital for overcoming the technical drawback in discriminating chicken from other species and even within its breed. This review aims to provide insight and highlight previous and current approaches suitable for countering different circumstances in chicken authentication. Gallus gallus, commonly known as a chicken, is a widely consumed species, and the demand for it keeps hiking up to 92.7 million metric tons in 2018 [ in 2018 . Moreove in 2018 . TherefoVariation in chicken breeds genetic composition is one of the major factors determining growth rate, feed intake and feed efficiency in chicken farming ,6,7. MorSubsequently, authenticating chicken in food can be achieved by identifying the presence of several stable and relevant biomarkers at the molecular level. In this sense, multi-omics discipline analytical approaches can facilitate the detection and identification of the target biomarkers. Omics studies include the entire flow of the central dogma of molecular biology, which starts from the information stored in DNA (genome) and is expressed (transcriptome) in the form of proteins (proteome), where the proteins play a role in anabolism and catabolism (metabolome) . Beyond Omics can be predominantly interpreted as a combination of diverse technologies applied for the qualification or quantification of a distinct molecular level. Unfortunately, it is impossible to detect a group of compounds by merely using a single approach. Hence, different omics and technologies should be developed and applied wisely in different circumstances to overcome each weakness found in a particular approach. Similarly to other analytical food authentication, chicken authentication based on omics involves indirect authentication of targeted and non-targeted analytical strategies. Targeted analysis refers to the detection and precise quantification of a particular set of secondary biological markers . The non-targeted analysis involves the simultaneous detection of up to hundreds of unspecified targets (mainly in metabolites and lipids), yielding a fingerprint reflecting the chicken and breeds identity. Chicken fingerprinting analysis mostly involves high-end analytical instruments such as mass spectrometry (MS) and its derivatives. On the other hand, fingerprints could also comprise data compiled from other analytical approaches or a combination of other complementary analytical methods .Since chicken authentication covers a large scope of the discipline, this review aims to provide an overview of recent developments in omics-based analytical technologies to assess chicken in meat and meat products. This includes a brief discussion of the pros and cons of each approach, as well as remarkable innovation and application in overcoming issues faced in different food authentication circumstances.The continuous development and improvement of novel and existing omics approaches have enabled the identification and quantification of biomolecules at high resolutions . When thGenomics studies on the chicken are essential for exploring potential discriminative genetic markers to trace the chicken species and breeds in meat products. Genomics studies require the aids of bioinformatics tools to handle robust DNA sequence data extracted from the chicken genome. The emergence of next-generation sequencing (NGS) in this era facilitated DNA sequencing, replacing the previous gold standard―Sanger sequencing. Although NGS has significantly reduced the time required for DNA sequencing, NGS is still relatively costly at this time, and the penetration of this technology into the market will suppress the cost in its advent .In the search for genetic markers for distinguishing three chicken breeds , Dongyep et al.  performew/w). The sequences’ data were uploaded in SGS™ . The All-Species ID software was aligned against a curated built-in database to identify the origin of the meat species. However, this NGS tool only allowed a rough quantification of the adulteration level. A similar Ion Torrent NGS was also performed by Ribani et al. [In addition, Cottenet et al.  recentlyi et al.  on 12S, When it comes to chicken breed discrimination, different strategies can be applied. These included random amplified polymorphic DNA-PCR (RAPD-PCR), amplified fragment length polymorphism (AFLP), and PCR-RFLP in combination with bioinformatics analysis for breed discrimination. In brief, these approaches are associated with genomic fingerprints, and fingerprint differences between breeds are interpreted as genetic distances. RAPD is a technique that works by amplifying genomic DNA by using at least one short oligonucleotide primer in low stringency conditions resulting in multiple amplification products from loci distributed through the genome . The RAPEcoRI/MseI or EcoRI/TaqI, to differentiate slow-growing from fast-growing chicken strains.AFLP is another PCR-based DNA fingerprinting method adapted from the RFLP and RAPD approaches . AFLP ofc subunit I gene (≈650 bp) [DNA-barcoding is another approach for chicken species and breeds identification. DNA barcoding can be carried out in the form of conventional full-length mitochondrial cytochrome ≈650 bp) , but it ≈650 bp) . Instead≈650 bp) . In this≈650 bp)  compared≈650 bp)  had perfGenetic traceability describes the use of DNA as a component to authenticate the species origin of an organism and its products. According to the European Parliament Resolution of 14 January 2014, DNA testing is suggested as a standard procedure for animal species determination for the purpose of fraud detection and control . Hence, In most cases, mitochondrial-based DNA analysis is preferred due to the high number of mitochondria that can be found in a cell, and many mitochondrial DNA copies stay inside each mitochondrion. Mitochondrial DNA is a potential candidate for species DNA identification given its maternal inheritance, low recombination probability, and the availability of conserved sequences. These reasons explain why mitochondrial DNA can serve as a naturally amplified source of genetic variation. In fact, most poultry DNA identification involves avian mitochondrial genes rather than nuclear DNA as a quantification component . Despiteb, cytochrome c oxidase subunit (CO), D-loop, and NADH dehydrogenase (ND)  refer  are amonIn DNA-based approaches for meat species identification, conventional PCR is a common technique used to amplify a small amount of contaminant DNA presence in meat samples but lacks quantitative capabilities, sensitivity, and is time-consuming, while many derivatives of it such as real time-PCR, multiplex-PCR, PCR-restriction fragment length polymorphism, and digital droplet-PCR offer much more advantages compared to conventional PCR. Notwithstanding, species identification by PCR in meat samples is challenged by the presence of inhibitory substances in meat sample matrices, and the risk of amplification inhibition is higher when more than 50 ng DNA is assayed . Real-tiAdditionally, PCR can be performed in a multiplex format using species-specific primers to detect different species simultaneously in one reaction, which saves cost and time. On the other hand, PCR-RFLP only offers species DNA fingerprinting based on banding patterns yielded from restriction endonuclease digestion of PCR amplicons, which is qualitative but not quantitative. Since the accurate quantification of a minute amount of target DNA in a matrix of non-interested DNA is highly challenged, a recently developed PCR technology known as digital droplet PCR (ddPCR) is able to encounter this difficulty . ddPCR wInterestingly, nucleic acid sensor assay can also design based on PCR amplification to rapidly detect chicken adulteration. Xiao et al.  developeIn addition to substituting chicken into more valuable minced meat, the incidence of adulterating relatively cheaper chicken offal/internal organs into skeletal muscle meat is defined as another form of food fraud . To protTherefore, transcriptomics is suitable for identifying the presence of offal in minced meat by targeting the product of transcription. Micro RNAs (miRNAs) are a class of endogenous non-coding RNAs comprising about 21 nucleotides long. This biomolecule is abundant in number, small in size (stable), and tissue-specific . Owing tIn the recent past, Vishnuraj et al.  developeThe selected markers are further applied on field samples by qRT-PCR assay to validate feasibility. Fortunately, the result is satisfying, with high sensitivity of detection (limit of detection = 0.01 ng/µL). The study also proved that the stability of miRNAs was enough to withstand heat treatment and could be used as markers in heat-treated products as well. To summarise, the application of transcriptomics in identifying the adulteration of non-muscle tissue shows a promising end. This approach only involves the same equipment used in DNA techniques and offers highly reproducible and direct results without incorporating complicated statistical analysis. Further studies on the stability of the miRNAs are needed to reassure that this technique is still relevant for harsh processed chicken meat products.When it comes to proteins, it serves as the necessary nutrients for the human diet and explains why humans consume chicken. Incorporating proteomics in chicken authentication makes it possible to confront the drawbacks of the DNA-based approach, wherein extreme food processing conditions such as heat and chemical treatment can potentially result in DNA degradation yielding non-specific DNA fragments . ChickenI) and molecular weight (Mw) can be conducted using techniques such as two-dimensional electrophoresis and OFFGEL fractionation, wherein a more detailed separation of peptides and amino acids based on mass-to-charge (m/z) ratio requires mass spectrometry instruments. In most cases, a bottom-up or peptide-based approach is applied for potential marker screening, where a class of proteins from different species is digested using a proteolytic enzyme known as trypsin. The enzymatic digestion reaction will yield a number of peptide fragments. The peptides will further be subjected to mass spectrometry analysis for protein identification purposes. The protein can be eventually identified by comparing the MS experimental data to the calculated mass values from the peptide sequence database such as MASCOT.The discovery of versatile species-specific markers for species identification is the benchmark for food authentication. Proteomics analysis typically involves a series of separation steps followed by identifying the proteins through mass spectrometry. Preliminary separation of proteins based on their isoelectric point  is an example of ambient MS, and the application of this instrument in the discovery of stable peptides for species authentication is demonstrated by Montowska et al. ,94. ThisChou et al.  and RamiApart from advanced instrumentation, the immunological approach is also an alternative to meat speciation. Developing a biorecognition element detecting the species-specific meat antigen is crucial in this sense. For this purpose, Chen et al.  developeInterestingly, it is possible to differentiate chicken breeds using the proteomics approach. The need to characterize the chicken breeds is demanded in food authentication and is also helpful for establishing a strategy for chicken population conservation. Zanetti et al.  had perfUnlike other omics disciplines, metabolomics is a wide field comprising the identification and quantification of multiple metabolites produced from a biological system . MetabolA novel metabolomics approach for meat species identification is reported by Zhou et al. . This ap1H NMR to 2D qNMR, previous issues encountered in 1D 1H NMR, such as the lack of quantification ability and overlapping issues, can be addressed. Principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) are employed to study the metabolites’ differences. The study found that Korean native chicken strains contain higher amounts of flavour enhancing metabolites and lower amounts of free amino acids than the broiler. This study proves the potential of 1D 1H NMR complement with 2D qNMR in acquiring interactive and accurate information on chicken breeds metabolite differences, which outperformed traditional chromatographic analysis.On the other hand, the emergence of nuclear magnetic resonance (NMR) spectroscopy as a pivotal instrument in metabolomics study is also a method of choice in addition to mass spectrometry . RecentlIn the past 20 years, lipidomics emerged as a subdiscipline of metabolomics together with the enhancement in analytical instruments and chemometrics . Data geChicken fat may serve as a potential adulterant in canola oil , butter Regarding the instrumentation in studying chicken lipidomes, FTIR is the most popular instrument due to its ability as a fingerprint analytical technique. FTIR offers an advantage in detecting closely similar fatty acids profiles from two different species that surpass the GC . General13C) values of fatty acids [Other than FTIR-based approaches, the application of elemental analyzer-isotope ratio mass spectrometry (EA-IRMS) in the study of fatty acids, monoacylglycerol, and diacylglycerol (MAG and DAG) also shows remarkable performance in food authentication. The significant differences in carbon isotope ratio  spectroscopy is also demonstrated by Alfar et al.  to autheGallus gallus domesticus Brisson) by profiling the lipid composition of this chicken from different ages, genders, and different body parts. Furthermore, a lipidomics comparison was made on the Taihe black-boned silky fowl with crossbred black-boned silky fowl. The result indicates that it is feasible to distinguish Taihe from crossbred black-boned silky fowls based on 47 statistically significant lipid molecules using OPLS-DA and ANOVA analysis. This finding provides insight into the possibility of chicken breeds authentication based on the lipidomics approach.Mi et al.  reportedRecently, Hrbek et al.  developeN-glycans. Hydrophilic interaction ultra-performance liquid chromatography (HILIC-UPLC) is preferred as the separation technique for N-Glycan labelling and analysis to overcome no retention on the typical reverse phase as glycans are highly polar molecules. Targeted sample peaks are then analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), separating the glycans according to the molecular masses. A tandem mass spectrometry MS/MS is further carried out to enable the identification of N-glycans structure. In this case, principal component analysis (PCA) was employed to discriminate samples, whereas partial least squares (PLS) regression analysis was used to evaluate the adulteration ratio. Among the meat samples, the N-glycans profile exhibits the most significant number of peaks. What is noteworthy is that the poultry meat sample (chicken and duck) contains relatively more minor N-glycans structures than other mammal species. This study also successfully discriminated against mixed binary and ternary species samples quantitatively.The application of glycomics in meat authentication has just emerged during these five years . Shi et N-glycans between different species as a profile to authenticate chicken or other species. The genes encoding the glycoenzymes participating in the synthesis of N-glycans are responsible for this structural specificity. Several advantages can be concluded from the use of the glycomics approach in chicken or species authentication. The use of N-glycan profiles of meats can overcome the drawbacks of the genomics approach in which the harsh processing of meat  may potentially degrade DNA. Data processing in the glycomics approach is also much more straightforward than the spectroscopic approach, which is rapid and requires minimal sample preparation. The author also claimed that the proposed approach could be accomplished within four hours, which is generally considered prompt.The study above described the significant compositional variation in N-glycan profile differs significantly among chickens with different ages, health status, and chicken breeds.Regarding the downside of the Shi et al.  approachChicken and its breed authentication are becoming more critical as the global consumer demand for chicken and its related meat products keeps increasing. Moreover, the growth in consumer awareness towards the meat product packaging information genuineness also invokes muscle food traceability and security. The detection of chicken adulteration based on molecular approaches is highly credentialed, but it requires complicated sample extraction and pretreatment methods. The genomics-based approach has emerged together with Next-Generation Sequencing technologies in species origin identification for meat products. Although NGS is fast and becoming less costly in meat species authentication, these tools are only useful in identifying hypothesis-free species in a given sample. In terms of DNA-based approaches, since many potential genetic markers have been identified in previous research, future works can focus on incorporating DNA markers into the development of DNA hybridization type biosensor as performed by Skouridou et al. . FurtherWith the emergence in the transcriptomics discipline, the detection of chicken offal adulteration based on miRNA also precedes the genomics approach, which cannot achieve the same results. In proteomics, the advancement in mass spectrometry technologies has brought convenience to high-resolution peptide marker discovery. Moreover, the selected peptide markers correspond to the species that can be used in adulterant detection. Still, enzymatic pre-digestion of the meat protein using trypsin is inevitable for bottom-up proteomics, while only a more advanced mass spectrometer can bypass this step. From the author’s point of view, the discovered potential peptide marker can serve as a novel developed assay target in which biosensors can be designed according to the peptide marker wherein the protein matrix should be digested by the enzyme first. The discrimination of chicken breeds based on metabolomics can be achieved well using chromatographic-mass spectrometry and NMR spectroscopy. Future applications of these approaches in differentiating chicken from other species are anticipated. The non-targeted lipidomics studies use spectroscopic instruments, especially FTIR, which discriminate species based on functional group profiles. Recent attempts on chicken lipidomics study using LC-MS enable the discrimination of chicken breeds based on identifying lipid molecules profiles in detail and are potentially preferred as an approach for high-resolution lipidomics study.Regarding glycomics, more effort should be placed on validating species-specific glycans by involving a higher number of samples and different health statuses and ages of given species. Since we are at the edge of Industrial Revolution 4.0, the application of big data tools for data mining and interpretation will be more easily accessible, thus, aiding the leap in chicken authentication. Using the robust data obtained from the previous experiments, linking the knowledge from different omics approaches is essential for inventing the novel, portable, miniaturized, and automated sensing technologies. Rapid and convenient muscle food assessment that simultaneously enhance the accuracy and resolution of currently available technologies will be the future trends in this research area.


Multiple names within the literature refer to a clinical picture affecting infants and consisting of a large or fast growing head circumference with enlarged cortical subarachnoid spaces (CSAS) while cranial sutures are open. This myriad of terms demonstrates the confusion about the entity, that may even group together different etiological processes. In this review, we aim to shed light on this matter in an effort to restate the defining features of the clinical picture and sum the evidence and current understanding of its pathophysiology and related imaging findings.Extensive and updated review of the literature with special focus on defining features, clinical history with long term evaluation and pathophysiological process.Functional and molecular CSF studies as well as clinical evidence challenges the common pathophysiological theory based on non-functional arachnoid villi. Conversely, there is increasing evidence supporting cerebro-venous system abnormalities as the main pathophysiological factor. Additionally, long term cohorts studies show that it may have subtle but irreversible neurodevelopmental consequences.Subarachnomegaly is an age-related condition of the infancy with radiological enlargement of CSAS and often self limiting course. However, considering the evidence on pathophysiology as outlined herein and long term outcome reports, further research effort is needed to assess the consequences of venous outflow impairment and enlarged CSAS and how this relates to imaging findings and neurodevelopment test results later in life Many different terms used in literature refer to this entity include benign external hydrocephalus, benign enlargement of subarachnoid space, extra-ventricular hydrocephalus, subdural effusions of infancy, pseudo-hydrocephalus, benign extra-axial collection of infancy, benign subdural effusions in infants, subdural hygroma, pseudohydrocephalus, benign communicating hydrocephalus, and extra-ventricular obstructive hydrocephalus –3.The main clinical feature of the entity is the development of macrocephaly presenting with a mean age of 6–7 months at onset with radiological enlargement of the cortical subarachnoid spaces, termed subarachnomegaly . PreviouAnother important hallmark is that there is typically mild or no ventriculomegaly, and signs of classic obstructive hydrocephalus are absent . In thisMany papers refer to subarachnomegaly as a self-limiting and benign entity , 8 sinceIn summary, subarachnomegaly comprises macrocephaly as the main clinical finding and enlargement of subarachnoidal spaces as the radiologically defining feature. In this work, we review radiological findings, clinical course, and recent evidence regarding etiology that supports the hypothesis of venous congestion as the principal pathophysiological factor in developing subarachnomegaly.Macrocephaly > 95th percentile, at the expense of enlarged cortical subarachnoid space .Predominantly frontal subarachnomegaly with mild or no ventriculomegaly ,Manifestation in infants 6–18 months of age, predominantly when sutures are open , 12Exclusion of classic hydrocephalus and related pathologies e.g. tethered cord Exclusion of hypomagnesemia, mucopolysaccharidosis, achondroplasia, agenesis of corpus callosum, Sotos syndrome and hyperglutaremia The typical clinical presentation begins with the referral of an otherwise normal infant because of a fast-growing head with crossing percentiles. To consider macrocephaly, head circumference must be two standard deviations above the mean for age and sex, as described in a standard growth chart. Typically, head circumference at birth is normal , 14 or sThe initial radiological assessment reveals enlarged subarachnoid space and no or mild ventriculomegaly. Subarachnomegaly is the most common cause of macrocephaly in infancy , 17, 18.A wide corpus of papers relates subarachnomegaly with an increased risk of developing subdural hematoma. Fig.  It is coThe majority of cases with subarachnomegaly are followed and observed without intervention as the radiological widening of the subarachnoid space progressively subsides spontaneously within 1–2 years . Head ciThe study of a macrocephalic infant usually begins with an ultrasound through the anterior fontanel. An increased cortical subarachnoid space with normal or mildly enlarged ventricles is used as a diagnostic criterion. Subarachnomegaly is predominantly frontal and is assessed via three main measurements: sinocortical width (SCW), craniocortical width (CCW), and interhemispheric distance (IHD) Fig. . Normal Ultrasound may not be sufficient to exclude other pathologies or distinguish subdural collections from widened CSAS or cerebral atrophy. If possible, a CT scan should be avoided in this early phase of life. Therefore, MRI is used to complete the assessment or to address complications of a known case in the follow-up. Particularly, MRI has allowed distinguishing between subdural effusions or hematoma and enlargement of the cortical subarachnoid space, helping to define subarachnomegaly as a distinct entity . Subdural collections appear more hyperintense in T1, proton-density, or T2-weighted sequences and lack the flow void sign (corresponding to a vessel) that appears in subarachnoidal spaces . MoreoveDiffusion tensor imaging (DTI) provides quantitative information about the diffusion properties within white matter. Fractional anisotropy (FA) is a DTI parameter that measures the spatial differential restriction to diffusion. In classical hydrocephalic patients, FA is typically low. This reduction is thought to reflect microstructural changes such as demyelination or loss of axonal integrity as damage after sustained increase of intracranial pressure (ICP). Increased FA values, on the contrary, indicate a high restriction to diffusion. In a retrospective analysis of a cohort of 15 subarachnomegaly infants, FA values were increased with respect to healthy controls in the genu and splenium of the corpus callosum  normalizing during a mean follow-up of 14 months . This fap = 0.028) [p < 0.001). Despite using a different scoring system to describe the severity of venous outflow impairment as compared to Sainz et al. [p = 0.01) [MR-venography has also become very relevant in understanding subarachnomegalic infants. In a significant proportion of these patients, the cerebrovenous system is compromised. In a cohort of 17 patients, 15 (88%) presented with abnormal phlebographies of their main venous sinuses, ranging from hypoplastic to severe stenosis. Moreover, the number and degree of affected venous segments showed a significant positive correlation with the CSAS volume with a Pearson coefficient of 0.57 (= 0.028) . In thei= 0.028)  confirmez et al. , these a = 0.01) Reversible collapse of the sinuses has been encountered , 26, poiMany theories and different etiological factors have been described. The common theory for subarachnomegaly in infants suggests that it is produced by impairment of CSF absorption by immature arachnoid villi , 28. AloThe widening of the CSAS at the expense of a growing skull with open fontanelles and sutures would prevent an increase of intracranial pressure. However, studies of arachnoid granulations reveal that villi immaturation or vili absence is a usual finding among all infants regardless of their CSF condition . A primaFurthermore, the main CSF absorption pathway appears not to be through the arachnoid villi neither in adults  nor in iThe CSAS becomes particularly relevant in regard to CSF dynamics in those clinical settings where ventricular size remains unchanged or only mildly increased as in infants with subarachnomegaly. In the cohort of Sainz et al. , 5/17 chAny form of cranial venous outflow congestion, as those shown in the MR-venography of a majority of subarachnomegalic patients, may result in an increased sinus venous pressure (venous hypertension) that will lead to a post-capillary rise of pressure. This, in turn, will increase the pressure within the capillary bed impairing also CSF reabsorption from the interstitial space. The subsequent increase in ICP (increased venous and increased CSF pressure) will promote an accelerated skull growth, eased by open sutures and fontanelles, creating a disproportion between the skull volume and the normal brain volume. This additional space is of course taken by the expanding CSAS. Added to the above-cited studies in subarachnomegalic children, there is mounting evidence of a relation between the venous system and CSF dynamics. Hydrocephalic infants exhibit high-pressure measurements in the venous system . AchondrMoreover, direct changes in the jugular outflow, and hence, the pressure in the cerebral venous system, have been related to changes in the cortical subarachnoid space volume , 41. In As described above, MR venogram abnormalities have been observed in subarachnomegalic patient cohorts or in case reports as an apparent common finding , 24, 27.Some subarachnomegalic infants, between 12 and 21% in the abovementioned phlebography studies, however, present with no MR-venography abnormalities. In these cases, a pre-capillary hypertension of the capillary bed is hypothesized corresponding to a hyperemic form of subarachnomegaly. An increased arterial inflow, instead of a venous stenosis, would end up raising capillary bed pressure producing consequently the same clinical picture . There iRegardless of the initial cause for the increased venous sinus pressure, it will in turn affect CSF absorption by increasing the capillary bed pressure. Increase in ICP then forces the infant skull to expand accordingly, contributing to CSAS widening. An increased capillary bed pressure will produce, apart from increasing CSF reabsorption resistance, an increase in cerebral blood volume augmenting brain turgor. This prevents exorbitant ventricular expansion despite increased CSF pressures like in hydrocephalus, forcing ventricular CSF displacement to the CSAS. Consequently, ventricular volume will remain normal or only slightly increased, at least in an initial phase. CSF pressure in CSAS will increase accordingly and further compromise venous outflow, fostered in case of venous wall instability.Subarachnomegaly patients are largely followed without intervention. In the majority of cases, enlargement of CSAS subsides spontaneously without apparent consequences. However, as mentioned above, this may be because long-term effects may be subtle and overlooked or not investigated systematically at all. Treatment options such as acetazolamide or shunts have not been statistically compared to the conservative approach. In few cases, where neurological or developmental delay symptoms become evident, more aggressive treatment options have been used. Some authors suggest the use of acetazolamide therapy for several weeks , 45. RepCSF diversion with shunts can help to break the vicious cycle by reducing the CSF pressure to the level of the venous pressure or even below. In the case of a sinus wall instability being the cause of the venous stenosis, the decrease of the CSF pressure below the venous sinus pressure will allow the sinus to expand again, as reported previously , 26. In Since the CSF accumulation is mostly within the CSAS, a lumboperitoneal (LP) shunt with an overdrainage preventing mechanism seems to be, from a theoretical point of view, the best option, since it drains from the spinal subarachnoidal space, which is in direct communication with the CSAS. Furthermore, ventriculoperitoneal (VP) shunts seem to have a high rate of ventricular catheter obstruction and malfunction in these patients. The combination of high venous pressure and brain turgor drives ventricle collapse as soon as CSF is removed. In the authors’ experience, all of the few VP shunts that were implanted in subarachnomegalic infants, with proven venous sinus stenosis and developmental delay, ended up with ventricular collapse despite overdrainage prevention and, hence, converted to LP shunts. LP shunt has become our standard of care for subarachnomegalic infants with developmental delay or exorbitant head growth. A lumbar catheter obstruction or overdrainage symptoms have not been encountered so far (unpublished data).Quantitative assessments, to decide for more aggressive treatment, are lacking as a standard guideline. No imaging features or parameters have been demonstrated as clear predictors to stratify the risk of neurodevelopmental impact. Along these lines, recent advances on MR sequences may help to clarify the relationship between parameter changes and long-term outcomes in the absence of adverted neurodevelopmental symptoms. In stratifying radiological and clinical (including invasive) findings according to the developmental status, indications for treatment will be more clear and evidence-based.The majority of patients are considered as neurologically and developmentally normal on follow-up . HoweverConsidering this condition as benign has prevented invasive pressure recordings, close radiological follow-up, and regular developmental assessments as a standard of care for these patients. Therefore, ICP values and dynamics for these patients still remain largely unclear and must be investigated in relation to long-term outcomes and related radiological features. Other CSF and venous dynamic-related pathologies should also be ruled out in adulthood for these subarachnomegaly patients since they may share etiological factors with known clinical pictures that express later in life. Normal pressure hydrocephalus patients, for instance, exhibit larger head circumferences compared to control subjects . FurtherSubarachnomegaly is an age-related condition of the infancy with radiological enlargement of CSAS and often self-limiting course. There is increasing evidence that supports cerebrovenous system abnormalities as the main pathophysiological factor resembling an early-onset analog of pseudotumor cerebri encountered in older children and adults.Considering the evidence on pathophysiology as outlined herein and long-term outcome reports, long-term follow-up data investigations with larger cohorts are needed to assess the consequences of venous outflow impairment and enlarged CSAS and how this relates to imaging findings and neurodevelopment test results later in life.