Gastrinoma is a rare form of neuroendocrine neoplasm. The presence of a primary lymph node localization of gastrinoma is a much debated and controversial topic in the literature, as regards whether these cases represent metastatic disease from an as yet unidentified primary tumor, or the de novo occurrence of a gastrinoma in a lymph node.We report the case of a 24-year-old male with intense epigastric pain treated at the beginning with high dose proton pump inhibitors. Further workup with CT and subsequent laparotomy revealed a single peripancreatic lymph node. Histological examination highlighted a well-differentiated neuroendocrine tumor.This case underlines that the primitive lymph node gastrinoma is a distinct nosological entity with a precise location in the context of rare neuroendocrine tumors that should be considered when specific symptoms are associated with the identification of isolated lymph nodes, after excluding any possible primitive locations of neoplastic localization. Gastrinoma is a rare form of neuroendocrine neoplasm that ectopically secretes gastrin and gives rise to a clinical syndrome of peptic ulcer, first described by Zollinger and Ellison in 1955 . GastrinHelicobacter pylori infection was absent. Further workup with abdominal computed tomography (CT) revealed an ovoid lesion measuring 1.3 cm, slightly hypervascularized and exophytic, adjacent to the lateral margin of the head of the pancreas, in the space between the duodenal bulb and the inferior vena cava . Previously, for several months, the patient had experienced upper abdominal pain and gained partial benefit from the administration of high dose proton pump inhibitors (PPI). An upper gastrointestinal endoscopy  did not reveal specific erosions or inflammatory lesions of the gastric mucosa, and Meticulous surgical exploration of the operative field  demonstrated the lesion in the pancreatic head, with a diameter of about 2 cm. The nodule, approximately 2 cm in diameter with a smooth, regular surface, was excised and a frozen section submitted to pathology for extemporaneous intraoperative examination. Intraoperative frozen sections analysis disclosed a single lymph node measuring 1.5 cm in maximum diameter, almost completely replaced by a tumor whose appearance was consistent with a low-grade neuroendocrine tumor (NET-G1). After careful exploration of the gastrinoma triangle, no lesion other than the one identified preoperatively was found at surgical exploration and intraoperative ultrasound (IOUS); therefore, further procedures were suspended and the abdomen was closed. The definitive histology and immunohistochemistry analysis on paraffin-embedded sections confirmed a lymph nodule of a well-differentiated neuroendocrine tumor (WD - NET), with less than 3% of cells staining positive for Ki67 (MIB-1) and a number of mitoses < 2 for 10HPF, thereby classified in accordance with 2017 World Health Organization (WHO) classification [In the literature, the presence of lymph node primary gastrinomas is still controversial due to the uncommoness of the tumor . It is d


Sepsis is defined as dysregulated host response caused by systemic infection, leading to organ failure. It is a life-threatening condition, often requiring admission to an intensive care unit (ICU). The causative agents and processes involved are multifactorial but are characterized by an overarching inflammatory response, sharing elements in common with severe inflammatory response syndrome (SIRS) of non-infectious origin. Sepsis presents with a range of pathophysiological and genetic features which make clinical differentiation from SIRS very challenging. This may reflect a poor understanding of the key gene inter-activities and/or pathway associations underlying these disease processes. Improved understanding is critical for early differential recognition of sepsis and SIRS and to improve patient management and clinical outcomes. Judicious selection of gene biomarkers suitable for development of diagnostic tests/testing could make differentiation of sepsis and SIRS feasible. Here we describe a methodologic framework for the identification and validation of biomarkers in SIRS, sepsis and septic shock patients, using a 2-tier gene screening, artificial neural network (ANN) data mining technique, using previously published gene expression datasets. Eight key hub markers have been identified which may delineate distinct, core disease processes and which show potential for informing underlying immunological and pathological processes and thus patient stratification and treatment. These do not show sufficient fold change differences between the different disease states to be useful as primary diagnostic biomarkers, but are instrumental in identifying candidate pathways and other associated biomarkers for further exploration. The fouidentify a panel of gene expression profile biomarkers which distinguish sepsis patients from those who had clinical outcomes consistent with SIRS or the more severe septic shockinvestigate the immune-pathogenesis of these markers, primarily in sepsiscompare these profiles to those observed in resolved SIRSuncover the likely cell types associated with key identified hub markers.The combined data outputs from these objectives may provide valuable information for development of biomarkers for diagnostic purposes and provide valuable information on some of the key metabolic pathways and/or cell types involved in the underlying pathological processes.http://www.ebi.ac.uk/arrayexpress/) under accession number E-GEOD-9960 , showed no increased interaction between the hubs in any of the disease states except pediatric sepsis compared with healthy controls. This entity plays a role in transport of monocarboxylates including lactic acid and pyruvate. Lactate is postulated to increase PD-L1 expression on monocytes and macrophages which could contribute to immunosuppression . It assoFOSL2 expression is regulated by TGF-β1 and is a key transcriptional regulator which plays a role in invariant natural killer T-cell (iNKT) development . AlteratKLRK1  is a transmembrane protein involved in natural killer cell (NK cell) and CD8 T-cell activation –224. It Expression of this entity was reduced in pediatric SIRs and sepsis and this was maintained in septic shock and resolved SIRS. Its expression remained relatively unchanged in adult SIRS and sepsis. It is interesting that this marker is downregulated in these disease conditions and alongside many of the associated entities, appears to be inhibited. KLRK1 did not associate with any other hubs in any of the control or disease states. It showed association with CD2, IL2RB, and IL7R in adult SIRS and KLRG1 and TGFBR3 in adult sepsis, among others. It showed interaction with CD28 and TLR7 in pediatric SIRS and CD8A, KLRC3, GIMAP5, and GIMAP6 in resolved-SIRS. There is connection between KLRK1 and CD2, NLRC3 (a NOD-like receptor involved in innate immunity), CD247 (CD3-ξ chain), CD96 and other markers of T and NK function. Association with many of these markers was confirmed using similar entities analysis  e.g., CDh) subtype  is primarily a myosin light chain that may regulate muscle contraction by modulating the ATPase activity of myosin heads. However, various isoforms of this exist. The inducible form MLC-2C plays a role in monocyte/macrophage lineage development and platelet production –87. In tThis hub gene exhibited the greatest conservation of associated entities, compared with other hub entities, across all the control and disease interaction map analyses. However, it also did not associate with any other hubs in any of the control or disease states. In the pediatric control it associated with a number of entities including SELP, CMTM5, and TGF1l1, in pediatric SIRS with ITGB3 and ITGA2B, TREML1, and CMTM5. All of these are associated with MYL9 in pediatric sepsis and in pediatric septic shock, except for CMTM5 in the latter. CMTM5, SELP, and TREML1 were also observed in pediatric resolved SIRS, in addition to ALOX12 , which was also seen in pediatric sepsis. This latter entity and its reaction products have been shown to regulate platelet function . The proSELP (p-selectin) has been implicated in sepsis for some considerable time and may contribute to neutrophil recruitment and NET formation –250. AscThe profile of interactions for adult sepsis is quite different than that for pediatric sepsis, with association instead with platelet membrane glycoproteins GP5 and GP6, PLOD2, COL6A3 and PROS1, ITGA3, and ITGB3BP (integrin subunit beta 3 binding protein), inferring activation down a different pathway. GP5 (human platelet glycoprotein V) is a part of the Ib-V-IX system of surface glycoproteins that comprise the receptor for von Willebrand factor and mediate the adhesion of platelets to injured vascular surfaces in the arterial circulation –278. GP6Many of the above entities are components of platelet α-granules . Thus, pMany of the above pathway observations were also confirmed from the results of the pathway analysis. Entities in the blood coagulation pathway (P00011) were found to be overlapped in SIRS, sepsis, and septic shock. These pathway reactions are perhaps revealed in the profile of entities associated with MYL9. The integrin signaling pathway (P00034) was also found to be represented in all disease groups and the healthy controls. Tissue injury caused by infection and also by an inappropriate host response, can lead to multiple dysfunctions during sepsis progression.A two-tier gene screening using ANNs was conducted to identify a panel of markers and their relationships related to sepsis and SIRS. On the first-tier screening process, a panel of 48 candidate markers and 3 housekeeping genes were identified. Amongst these 48 candidate markers, 26 showed a consistent upregulation response in adult SIRS and the remaining 22 were upregulated in adult sepsis. The interaction between these genes were then analyzed in the second-tier screening process. Eight key hub genes were identified. Further investigation on pathogenesis activities of these hub genes in stratifying sepsis from SIRS, septic shock, resolved SIRS and bacteraemia were conducted.Our findings are summarized below:CD177 can be taken to imply a substantial infiltration of CD177 positive neutrophils in both SIRS and sepsisGPR84 appears to oppose and provide an inhibitory control for CD177 and this may be taken to mean diametrically opposing and competing pathways, either in the same cell type or subtypes, or in two different cell types. The cell-type assignation is unknown, and this requires further investigationTDRD9 may be involved in regulation of cell proliferation, adhesion and phagocytosis/autophagy, perhaps involved in pathogen clearance/killing. The cell type assignation is also unknown and requires further investigationFGF13 may be neutrophil-associated and shows some linkage with hub genes TDRD9 and PCOLCE2The pathogenic role or cell association of PCOLCE2 and SLC16A3 are unclear, but these may function in metabolic processes, perhaps associated with hypoxia and carboxylic acid metabolism and are closely linked to each other and TDRD9. They may also be linked to immunosuppressive cell activities, however this requires further investigationKLRK1 is most likely associated with NK cell activity and its reduction in expression can be taken to mean loss or down-regulation of NK cells/markers from the periphery. This could be due to inhibition, cell death or egress from the peripheryKLRK1 inhibition may be actioned through ITKThis process appears to be inhibited/driven by T-cells and/or APCs, which also appear to promote the activities of other key hub genes such as CD177, FGF13, and PCOLCE2The activity of MYL9 could be ascribed to platelet activation and blood clotting cascades with reasonable confidenceThe activity surrounding MYL9 is a key differentiator between SIRS and sepsis in both adults and children, with apparent inhibition of these activities/pathways in SIRS and activation in sepsisThis is consistent with the observation of intravascular coagulation being a key feature of disease pathology in sepsis and a major cause of observed organ damage.While the hub genes showed significant fold increases over controls, they did not show sufficient fold-change differences between SIRS and sepsis and lack discriminatory power to differentiate these two conditions. Although some of these hubs and associated gene interactions cannot yet be easily described in the context of this study, they may provide the basis for further investigation into gene regulatory networks and pathways and their specific involvement in disease development and progression. They may also provide a new source of other biomarkers for prospective investigations in differential diagnosis. A better understanding of the disease processes underpinning each condition, could also pave the way for improvements in patient management and care. The interaction analysis has provided additional information as to the likely source of a few of the hub markers e.g., CD177, FGF13, KLRK1, and MYL9, which could be ascribed to a cell type with reasonable confidence i.e., neutrophils, NK cells, and platelets. These cell types have been implicated previously in the immunopathology of sepsis and this may pave the way for new or improved targeted therapies. The remaining hub genes GPR84, TDRD9, PCOLCE2, and SLC16A3 could not be assigned to a cell type with the same degree of confidence and their cell-associated expression and role in underlying disease immunopathology requires additional study. Further work is in progress to investigate these biomarkers and associated pathways, to provide clarification on the likely basis of cell type-associated expression and identify diagnostically useful biomarkers.ArrayExpress repository using the following accession numbers: E-GEOD-9960, E-GEOD-28750, E-GEOD-13904, E-GEOD-6269.Publicly available datasets were analyzed in this study. These data are available in the Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.DT and KK conducted the data analysis. DT, KK, TS, and GB wrote and edited the paper.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.


Current treatment modalities for IDD include conservative and surgical strategies. Unfortunately, there is a significant number of patients in which conventional therapies fail with the result that these patients remain suffering from chronic pain and disability. Furthermore, none of the current therapies successfully address the underlying biological problem - the symptomatic degenerated disc. Both spinal fusion as well as total disc replacement devices reduce spinal motion and are associated with adjacent segment disease. Thus, there is an unmet need for novel and stage-adjusted therapies to combat IDD. Several new treatment options aiming to regenerate the IVD are currently under investigation. The most common approaches include tissue engineering, growth factor therapy, gene therapy, and cell-based treatments according to the stage of degeneration. Recently, the regenerative activity of small molecules  on IDD was demonstrated. However, small molecule-based therapy in IDD is still in its infancy due to limited knowledge about the mechanisms that control different cell signaling pathways of IVD homeostasis. Small molecules can act as anti-inflammatory, anti-apoptotic, anti-oxidative, and anabolic agents, which can prevent further degeneration of disc cells and enhance their regeneration. This review pursues to give a comprehensive overview of small molecules, focusing on low molecular weight organic compounds, and their potential utilization in patients with IDD based on recent  Globally, chronic low back pain (CLBP) symptoms occur in ~60-80% of people during their lifetime, which has a significant socioeconomic impact via reduced quality of life and work efficacy in vitro and in vivo animal and human studies showed cellular senescence as a critical mechanism in the progression of IVD aging, increased inflammation, elevated catabolism, and subsequently IDD IDD features extracellular matrix (ECM) degradation, accelerated cartilaginous and bone remodeling, the release of proinflammatory cytokines, altered spine biomechanics, angiogenesis, and neoinnervation, altogether causing CLBP and disability Most therapeutic options for IDD like analgesics, anti-inflammatory medications, and physical therapy are currently limited to symptomatic treatments, which only delay or mask the degradation process of the IVD. Surgical intervention is used as a last resort, with procedures such as total disc replacement or spinal fusion, which are associated with a substantial risk of intraoperative and postoperative complications in vitro, in vivo, and clinical studies Therefore, there is a strong demand to find new therapeutic agents (or utilize well-known drugs which were proven effective in treating other diseases) aiming to relieve discogenic pain and regenerate damaged IVDs through restoration of tissue homeostasis. In this regard, several small molecules have shown promising results as alternative therapeutic agents in Figure ) The IVD is the largest avascular structure in the human body that contains three main components; the soft mucoid NP core, the lamellar AF tissue that encloses the NP, and the CEPs which cover both top and bottom of the IVD .Inflammation is another factor that is thought to play an important role in the development of IDD Table  In addition, we will discuss new strategies and approaches which were used in recent studies and the future direction for using these molecules to regenerate IVD cells. In vitro, in vivo, and clinical studies related to the discussed small molecules are listed in Table  The effective in vitro concentrations of different small molecules for regeneration of disc cells are listed in In the context of this review, we focus on small molecules as low molecular weight  compounds, including synthetic or natural products Although detailed pathways and molecular interactions between discogenic pain, disc degeneration, and inflammation remain to be elucidated, some inflammatory cytokines and related pathways are known as potential targets for therapies in IDD in vitro studies, small molecules, including naringin, cannabidiol (CBD), epigallocatechin gallate (EGCG), curcumin, icariin, resveratrol, berberine, and tofacitinib showed an impact on the downregulation of IL-1 and TNF-α levels in IVD cells . According to previous literature, icariin, resveratrol, and EGCG can inhibit NF-kB and p38/MAPK signaling pathways, thereby modulating inflammatory responses and preventing the development of a degenerative cascade in vitroIn various et al. investigated the effects of tofacitinib in an inflammatory and degenerative bovine IVD organ culture model. Tofacitinib citrate (2.5 mg/mL) was added daily to the culture medium to simulate a systemic application of the drug. The results showed that tofacitinib could slow down the degenerative response and reduce inflammation in the organ culture model by selectively inhibiting the Janus kinase 3 (JAK3) pathway in vitro study showed that luteoloside, a flavonoid glycoside, could suppress inflammatory factors, such as TNF-α and IL-6, in IL-1β-primed NP cells through inhibition of the NF-κB signaling cascade. They demonstrated that luteoloside promoted the nuclear factor erythroid 2-related factor 2 (Nrf2) translocation to the nuclei; Nrf2 can act through activation of the Nrf2/HO-1 (heme oxygenase-1) signaling in NP cells and mitigate inflammation by suppressing the NF-κB signaling cascade and by anti-apoptotic function Lang et al. showed that pre-treatment with CBD suppressed the production of COX‑2 and inflammatory cytokines (i.e. IL-6 and IL‑1β) in NP cells et al. used the JAK antagonist tofacitinib for pre-treatment of rat AF cells and then cells were incubated with inflammatory cytokines such as IL-6. Tofacitinib significantly decreased the expression level of COX-2 and, subsequently, the production of PGE2 Figure 2).Cyclooxygenase-2 (COX-2), which regulates prostaglandin E2 (PGE2) synthesis, is another candidate for modulation of inflammatory responses in IVDs. Chen Figure 3). Recent studies showed that naringin and icariin could upregulate the cellular concentrations of anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) and downregulate the apoptotic effect of promoter proteins, including cleaved caspase 3 and BCL-associated X (Bax) to modulate the apoptotic rate of human NP derived cells et al. confirmed the protective effect of CBD on hydrogen peroxide-induced apoptosis. They identified that CBD increased cell viability and reduced apoptosis in NP cells after exposure to hydrogen peroxide by reducing the expression level of caspase 3 and promoting the Bcl-2 protein expression Therapeutic regulation of transduction pathways can modulate the programmed cell death (PCD) process, which is considered to play a significant role in IVD cell degeneration The effect of 17 beta-estradiol (E2) was also assessed on isolated NP cells from healthy rats and their intact IVDs, which were cultured with or without TNF-α. It was shown that in NP cells, E2 significantly increased matrix macromolecules expression, telomerase activity, and cell proliferation potential but attenuated senescence markers (p53 and p16). P53 plays a critical role in intrinsic pathways of apoptosis; therefore, the reduction of this marker could lead to a decrease in apoptosis Resveratrol is another small molecule which could induce anti-apoptotic genes  and simultaneously reduce the expression level of pro-apoptotic genes such as Bax or caspase 3. Moreover, when LY294002 was used as a strong inhibitor of PI3K/AKT, the anti-apoptotic effects of resveratrol in IL-1β-primed NP cells were attenuated The protective effect of berberine by inhibiting NF-κB activation has been shown on IL-1β stimulated human NP cells undergoing apoptosis Cutibacterium acnes can induce inflammatory response (IL-1β) and initiate IDD. The number of NLRP-positive cells significantly increased in C. acnes infected disc tissue, which suggested that pyroptosis activation may be induced by C. acnesPyroptosis is another form of programmed cell death which can lead to production of proinflammatory mediators Figure 3). For instance, the effect of naringin on oxidative stress-induced apoptosis was investigated in rat NP-derived mesenchymal stem cells (MSCs). The findings showed that naringin had protective effects against hydrogen peroxide induced NP cell apoptosis. The potential mechanism of naringin to alleviate apoptosis may be due to the activation of the ROS-mediated PI3K/AKT pathway et al. evaluated the anti-oxidative effect of EGCG on human IVD cells exposed to hydrogen peroxide. Their results demonstrated that survival of the treated disc cells by EGCG under severe oxidative stress was considerably enhanced in comparison to the control cells, which happened through activation of PI3K/AKT pathway and inhibition of cytochrome c release from mitochondria During IDD, usually excessive reactive oxygen species (ROS) are produced and released locally, suggesting a contribution of oxidative stress to the degeneration process and opening a new horizon regarding the pathogenesis of IDD. ROS, as active mediators, are involved in various cell signaling pathways and cell metabolisms, including matrix degradation, inflammation, apoptosis, autophagy, and senescence of IVD cells. Moreover, ROS can change the structure of matrix proteins in NP, AF, and CEP leading to the impairment of the IVD's mechanical function and acceleration of IVD degeneration processes The inhibitory effect of E2 on ROS generation was studied by several investigators. ROS/NF-κB pathway of rat NP cells is affected by the interaction of estrogen receptor and E2, which inhibits TNF-α-induced premature senescence et al. They found that icariin improved the viability and function of human NP derived stem cells by preserving the phenotype after thawing the cells in vitro. The protective effect of resveratrol is due to the stimulation of sirtuin 1 and the PI3K/AKT pathway which was activated in different settings in vitro et al. observed that the production of ROS under hydrogen peroxide exposure was down-regulated with berberine, which protected human NP cells against oxidative stress-induced apoptosis in vitro study showed that luteoloside could successfully suppress iNOS and modulate ROS production in IL-1β treated NP cells Cryopreservation can be used to allow the storage of cells over prolonged periods of time. While cryopreservation at -196°C would render IVD cells metabolically inactive, cells usually suffer insults during freeze-thawing such as the generation of ROS in vivo and preclinical evidence to support the efficiency of these molecules to retard the process of IDD. Further in vivo and clinical studies are required to develop effective anti-oxidative therapies for IVD regeneration.Although most of the studies showed an anti-oxidative effect of these small molecules on IVD cells, there is not enough Figure 2). Li et al. evaluated the influence of naringin on the growth of degenerative NP cells and its regenerative effects on protein and gene expression. Naringin treatment elevated the protein expression of collagen type II, aggrecan, and SOX6, and decreased the gene expression of MMP3 Several studies assessed the anabolic and anti-catabolic effect of small molecules on IVD cells and reported their beneficial impacts on IDD  percutaneously. The regeneration of discs was assessed by magnetic resonance imaging (MRI), real-time polymerase chain reaction (RT-PCR), and histological analysis. An increased aggrecan and decreased MMP13 gene expression in the treatment group compared to the carrier was observed, and increased matrix PG production was confirmed by histology Through an in vitro study, gefitinib at 10 µM was administered 30 minutes before the treatment of rat NP cells with 10 ng/ml TGF-α. After 48h of treatment with TGF-α, RNA and protein analyses were performed. It was concluded that gefitinib caused therapeutic inhibition of EGFR signaling thereby inhibiting IVD degeneration and enhancing IVD matrix synthesis in TGF-α treated NP cells in vitro studies showed that luteoloside and APO 866 (daporinad) increased the content of ECM-related proteins such as type II collagen and aggrecan and reduced the expression level of MMP13 and ADAMTS5 in IL-1β primed disc cells It has been shown that the expression level of anabolic genes in NP cells increased by metformin, while the expression of catabolic genes considerably decreased Several investigations have demonstrated that autophagy occurs in IVD cells in vitro studies. Shi et al. used autophagy markers (LC3 and Beclin-1) to assess the impact of APO866 on autophagy of NP cells as well as their apoptosis. The results showed that APO866 inhibited IL-1β-induced NP cell apoptosis by induction of autophagy. These findings showed the therapeutic potential of APO866 for IDD The impact of small molecules on autophagy was assessed in different In molecular signaling, the mammalian target of rapamycin (mTOR) acts as a negative regulator of autophagy. The mTOR is a serine/threonine kinase which regulates cellular activation such as cell growth and division, cell motility and cell survival. The mTOR exists in two distinct protein complexes including mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Protein kinase B (known as AKT), an essential pro-survival mediator by suppressing apoptosis, regulates mTORC1 and mTORC2 et al. observed a reduced number of senescent disc (NP and AF) cells and decreased SASP factors as well as an increase in cell proliferation after treatment with curcumin or o-vanillin. This effect was due to the selective induction of apoptosis in senescent cells without negative effects on the proliferating cells Senescent cells are non-dividing cells, which are still metabolically active. These cells have been shown to contribute to the catabolic shift in IVD tissue during degeneration by secreting the senescence-associated secretory phenotype (SASP) which contributes to a pro-inflammatory milieu et al. evaluated the effect of BMPs for regeneration of IVD both in vitro and ex vivo. Their results showed that BMP heterodimers could successfully upregulate the aggrecan and type II collagen gene expression, as well as glycosaminoglycan (GAG) synthesis of NP cells Bone morphogenetic proteins (BMPs such as BMP-2 and BMP-7) have shown promise in IVD regeneration. Li In IDD, increased Wnt signaling suppresses progenitor cell proliferation and induces apoptosis of NP cells in vivo studies using small molecules and targeting IDD and related degenerative processes have been published (Table 4). All these in vivo studies showed promising results of different small molecules on IDD; however, there is still limited evidence for a regenerative impact of small molecules on degenerated disc tissue. Most of the cited studies showed the preventive effects of the drugs on IDD progression. Only one study showed that EGCG could reach the MRI and histological scoring levels measured in the sham group in vivo investigations (Table 3) followed the animal cases up to 12 weeks, which is a very short-term follow-up and could explain such insignificant results To date, several in vivo investigations used standard methods to analyze the regenerative process or the inhibition of IDD progression, including X-ray, MRI, histopathology and immunohistochemistry (IHC) . However, these methods have some limitations to assess the beneficial effects of small molecules on IDD. For instance, MRI was used as a technique to analyze the disc height index (DHI) or the Pfirrmann grade in different treatment groups, whereby 3.0 Tesla (T) or lower (1.5T) clinical MRIs were frequently used (7 out of 10 in vivo studies on rodents). The 1.5T or 3.0T MRI and related image quality are not suitable for small animal imaging , which may lead to an irreproducible scoring by the radiologists Most of the Table 3, 16 out of 18 studies utilized rodent and lagomorph animal models (rat and rabbit) to explore the effect of small molecules on IDD regeneration or discogenic pain. However, significant limitations exist with using such animal models for IVD regeneration studies. For instance, in the rat tail models, the mechanism of disc injury is different due to different mechanical loading and persistence of notochordal cells which affect the regeneration processes. Furthermore, due to the smaller size of the disc, the diffusion of nutrition is different in comparison with the human disc. Another limitation is the lack of human-like longitudinal compression in the quadrupedal animal models et al. studied the therapeutic effect and the safety of celecoxib-loaded microspheres administered in a canine IDD model. This study showed that there was no evidence of adverse effects on MRI or macroscopic evaluation of IVDs. The diagnostic analysis of NP PG content revealed that site-targeted and sustained administration of CXB inhibited IDD. Local delivery of the COX-2 inhibitor showed decreased neuronal growth factor and PGE2 tissue levels, which led to an inhibition of the inflammation and alleviation of pain Table 3) is another limiting factor for the majority of in vivo studies, which can make it difficult to interpret the results due to high variations.Small laboratory mammals are the main animal models for IDD et al. and Lin et al. used this method to analyze the effect of EGCG and resveratrol on IVD regeneration and related discogenic pain in vivo studies, it is unknown whether the regeneration of the IVD is correlated with the resolution of discogenic pain.The behavioral assessment of discogenic pain  in animal models is still in its infancy et al. evaluated the presence of the MMP- and ADAMTS-cleaved aggrecan neoepitopes in vivo, using IHC. These neoepitopes produced by catabolic enzymes in the degeneration process could be used for early detection of IDD in vivo bioluminescence imaging. This idea might be developed in the future for early diagnosis of IDD and the results could be used for evaluation of the treatment responses in both animal models and clinical cases .In a rat tail static compression model, Yurube et al. conducted a retrospective cohort study to evaluate the use of statins in higher doses for patients suffering from a spinal degenerative joint disease (SDJD). They described an inversely proportional relationship between the dose of statins prescribed to patients suffering from hypercholesterolemia and the risk of SDJD occurrence in vivo and clinical studies, the application of biological treatment strategies for the regeneration of IVDs remains a largely undiscovered field which has great potential for further investigations.Until now, there have been no record of clinical trials  and only three clinical studies have been reported regarding the effect of small molecules on IVD regeneration: Five human patients with non-small cell lung cancer and IDD received gefitinib treatment over the past five years, which not only resulted in tumor regression but also ameliorated IDD in vitro studies have investigated the potential of small molecules to induce autophagy, for stimulating the natural process of IVD cells to adapt and regenerate in a stressful environment in vivo studies in vivo bioluminescence) should be developed to detect early IDD or to evaluate the prompt tissue responses to different small molecules in vivo.Small molecule drugs have shown promising therapeutic potential for the regeneration of IVD tissue and attenuation of the degenerative process. Nowadays, the focus in medicine is on individualized therapies and the factors aggravating the degenerative process in an individual patient need to be considered in more detail. Research towards therapies that induce the intrinsic regenerative potential of the IVD and CEP cells are of high interest due to the long-lasting effects and limited disturbance of the tissue homeostasis which happens with external manipulation. Several ex vivo and in vivo models. Small molecules have a huge therapeutic potential, as their application can be directed with controlled release formulas and in many cases, the evidence of their efficacy is already proven in the treatment of other diseases in the clinics in situ show promising results to resolve this problem in the future The promising results of the discussed investigations are only the foundation to answer the more complex questions arising from In vitro results have shown the beneficial effects of small molecules on regeneration of degenerated IVDs through attenuation of inflammation, oxidative stress, apoptosis, catabolism, and stimulation of anabolic processes via different signaling pathways. Similarly, most in vivo results also indicate beneficial effects of these small molecules on the amelioration of IDD in vivo evidence, due to a lack of representative animal models; the lack of an adequate delivery system for different small molecules; and the lack of patient stratification methods to identify the patients who would respond to and benefit from a small molecule-based therapy. The potential necessity of long-term treatment should also be emphasized to achieve a successful therapy from small molecule agents for regeneration of degenerated IVD. The avascular nature of the IVD may increase the duration of treatment, particularly for oral administration of small molecules which should first be absorbed by digestive tracts, delivered to the target site via the systemic circulation and finally reach the degenerated disc through diffusion.in vitro models. Considerations for establishing an appropriate animal model include factors like the absence of notochordal cells, size of IVD tissue, body mass relative to humans, mechanical compression forces upon the IVD, type of injury and ethics in vivo studies could be used to decrease the outcome variation and make it easier to draw conclusions To bridge the gap between rudimentary animal models and clinical studies, a more clinically relevant animal model should be used to better replicate the complexity of the human IVD and the pathology of IDD. The modeling of pain or inflammation, systemic response to degeneration and related treatment are only represented to a limited extent in the established in vivo imaging techniques, using equipment with higher sensitivity and resolution is also recommended. Recently, better MRI systems have become available for small animal models that are able to generate magnetic fields of 9.5T or even 21T, offering an excellent tool for small animal preclinical studies in vitro studies, followed by ex-vivo IDD organ culture models to confirm the results. Furthermore, new experimental techniques such as lab-on-a-chip that simulate the activities, mechanics and physiological response of entire organs and organ systems are being developed 126. Recently, a microfluidic disc-on-a-chip device has been developed that was tailored for laboratory small animal disc organs as a long-term ex-vivo organ culture platform. This device lays groundwork for future studies by simulating the chronic nature of IDD Behavioral pain assessment should be utilized to assess the effect of small molecules on IDD such as von Frey filament test, gait analysis, weight loading, and hot plate analysis. It should be highlighted that the collaboration of neuroscience, orthopedic, and biomedical sciences is a necessity to extend the current understanding of the complex and multifactorial pathophysiology of IDD and discogenic pain in vitro investigations. Moreover, it will be of importance to investigate other regenerative effects of different small molecules, such as the induction of NP-like cell differentiation in endogenous and exogenous stem or progenitor cells.In conclusion, small molecule therapy is an alternative treatment for conventional therapies and surgical approaches in discogenic pain. Effective regeneration of the degenerated IVD with small molecules has opened an important area of research in IVD regeneration in the last decade. However, it is of great importance to investigate the mechanism of action of small molecules in targeting different signaling pathways towards an effective therapy. Several of these small molecules are effective in inhibiting apoptosis, inflammation, oxidative stress, and senescence, which can prevent degeneration of the disc cells. They also showed anabolic and anti-catabolic effects (intrinsic regeneration) which are critical factors for the regeneration of damaged IVD. Further analysis, especially in large animal and advanced organ culture models and then clinical studies are needed to confirm the preliminary results obtained from Supplementary figures and tables.Click here for additional data file.


Chronic hypoxia is an essential component in many cardiac diseases. The heart consumes a substantial amount of energy and it is important to maintain the balance of energy supply and demand when oxygen is limited. Previous studies showed that the heart switches from fatty acid to glucose to maintain metabolic efficiency in the adaptation to chronic hypoxia. However, the underlying mechanism of this adaptive cardiac metabolism remains to be fully characterized. Moreover, how the altered cardiac metabolism affects the heart function in patients with chronic hypoxia has not been discussed in the current literature. In this review, we summarized new findings from animal and human studies to illustrate the mechanism underlying the adaptive cardiac metabolism under chronic hypoxia. Clinical focus is given to certain patients that are subject to the impact of chronic hypoxia, and potential treatment strategies that modulate cardiac metabolism and may improve the heart function in these patients are also summarized. The oxidation of ketones is comparable to glucose and pyruvate in terms of oxygen efficiency  to measure mitochondrial oxidation, results from different studies should be interpreted based on the specific methodology and specimen types  during this process , can also be affected under chronic hypoxia. CoQ10 is a vitamin-like organic component of ETC and is ubiquitously expressed in different tissues  and creatine kinase (CK). This energy buffer system catalyzes the reversible reaction: Phosphocreatine + ADP + Hin vivo measurement of phosphocreatine and ATP in high-land Sherpas  family. The biochemical properties and the detailed regulation of Hif-1α by oxygen have been summarized in a previous review  in the metabolic adaptation to chronic hypoxia should be mentioned. PKC isozymes are serine/threonine kinases that regulates the protective response against ischemic injury in the heart  and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) are significantly downregulated , sustained elevation of myocardial Hif-1α regulates the metabolic switch from fatty acid to glucose for ATP production since birth. However, when they enter puberty, the rise of insulin resistance attenuates myocardial expression of Hif-1α and reduces the capacity of the heart to use glucose, which disrupts the adaptive cardiac metabolic program, decreases ATP production and leads to cardiac dysfunction  and mitogen-activated protein kinase (MAPK) pathways , a direct inhibitor of PDK enzyme that serves to increase PDH activity. DCA has been shown to improve the coupling of glucose oxidation to glycolysis in hypertrophied hearts , an established antianginal drug that inhibit fatty acid metabolism through inhibition of mitochondrial 3-ketoacyl thiolase and enhance PDH activity to improve the metabolic efficiency in the myocardium (McCarthy et al., The third option is CPT1 inhibition, which could result in decreased mitochondrial uptake of long-chain fatty acid, reduced β-oxidation, and promotion of glucose oxidation. Although several synthetic CPT1 inhibitors, including oxfenicine, etormoxir and perhexiline, have been developed in the past decades, none of them have made into clinical practice to treat heart failure (Heggermont et al., In recent years, novel strategies are emerging to modulate cardiac metabolism by direct targeting of specific regulators. For instance, direct modulation of cardiac PDK4 activity could offer a potentially feasible strategy to enhance glucose metabolism in chronically hypoxic hearts. A recent study found that conditional cardiac deletion of PDK4 could result in an increase in PDH activity and consequently an increase in glucose relative to fatty-acid oxidation, which was associated with reduced cardiac fibrosis and improved left ventricular function in mice following myocardial infarction (Cardoso et al., As we had discussed above, insulin resistance and altered insulin signaling could impair the protective HIF-1α pathway in response against chronic hypoxia. Hence, another potentially feasible strategy to maintain the adaptive cardiac metabolism under chronic hypoxia is by improving cardiac insulin sensitivity. Pioglitazone is a potent insulin sensitizer that targets PPARγ signaling to decrease systemic and myocardial insulin resistance (Soccio et al., Modulation of CoQ10 biosynthesis, or direct supplementation of CoQ10, should be considered in patients with chronic hypoxia. Although the biological effects of CoQ10 have been reported in various cardiac diseases (Zozina et al., In response to chronic hypoxia, the hearts initiate transcriptional programs to increase reliance on carbohydrates over fatty acid for ATP production, with altered mitochondrial structure and function that improve metabolic efficiency and reduce ROS generation, as well as beneficial changes in glycogen storage and high energy phosphate system. Collectively, the adaptive cardiac program improves the regulation of the balance between energy demand and energy supply when oxygen is limited. Of note, this adaptive program is prone to be disrupted by systemic pathological factors, such as insulin resistance, and it can be complicated with other pathological factors concomitantly seen in patients with chronic hypoxia. The presence of these risk factors can pre-dispose to altered adaptive metabolism and cardiac dysfunction. Currently available pharmacological strategies are limited and none have been tested in patients with chronic hypoxia. Therefore, more investigational studies are needed to unravel the critical features and mechanism for maladaptive cardiac metabolism in these patients.ZS and YL contributed to the conception of the article. ZS drafted the first manuscript and YL contributed to the revision of the manuscript and graphical design. HZ contributed to the critical revision of the manuscript. HZ and YL obtained funds. All authors approved the submission of the article.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.


The characteristics of fetal membrane cells and their phenotypic adaptations to support pregnancy or promote parturition are defined by global patterns of gene expression controlled by chromatin structure. Heritable epigenetic chromatin modifications that include DNA methylation and covalent histone modifications establish chromatin regions permissive or exclusive of regulatory interactions defining the cell-specific scope and potential of gene activity. Non-coding RNAs acting at the transcriptional and post-transcriptional levels complement the system by robustly stabilizing gene expression patterns and contributing to ordered phenotype transitions. Here we review currently available information about epigenetic gene regulation in the amnion and the chorion laeve. In addition, we provide an overview of epigenetic phenomena in the decidua, which is the maternal tissue fused to the chorion membrane forming the anatomical and functional unit called choriodecidua. The relationship of gene expression with DNA (CpG) methylation, histone acetylation and methylation, micro RNAs, long non-coding RNAs and chromatin accessibility is discussed in the context of normal pregnancy, parturition and pregnancy complications. Data generated using clinical samples and cell culture models strongly suggests that epigenetic events are associated with the phenotypic transitions of fetal membrane cells during the establishment, maintenance and termination of pregnancy potentially driving and consolidating the changes as pregnancy progresses. Disease conditions and environmental factors may produce epigenetic footprints that indicate exposures and mediate adverse pregnancy outcomes. Although knowledge is expanding rapidly, fetal membrane epigenetics is still in an early stage of development necessitating further research to realize its remarkable basic and translational potential. Current consensus defines an epigenetic trait as “a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence.”  The fetaEpigenetic events establish, sustain and adjust chromatin structure in a cell-specific fashion through DNA methylation, post-translational histone modifications and regulatory non-coding RNAs. The resulting chromatin landscapes determine cell-specific gene expression patterns, which determine tissue phenotypes . Evidencin vitro under appropriate conditions  is the most thoroughly studied epigenetic chromatin modification. CpG methylation is traditionally considered to silence genes by promoting closed chromatin structure or recruitment of repressor complexes to gene regulatory regions . There ade novo” methylation at unmethylated CpG sites. DNMT3L is a catalytically inactive essential cofactor of DNMT3A and -3B. The characteristics of DNA methyltransferases have been extensively studied and reviewed in the literature . The reaction is catalyzed by the Ten-Eleven Translocation group of dioxygenases , which use molecular oxygen and 2-oxoglutarate as co-substrates. TETs can oxidize 5-hmC further to 5-formyl and 5-carboxyl cytosine, which are detected by the base excision DNA repair (BER) system eventually replacing 5meC with unmodified cytosine in the CpG motifs . The cenDNA methylation in the fetal membranes has been studied so far using traditional techniques that do not discriminate between 5meC and 5-hmC such as bisulfite conversion  or have p < 0.0001), with no overlap among the top 15 differentially methylated genes in the two comparisons. ITGA11) and trombospondin-2 (THBS2), were selected for verification using bisulfite pyrosequencing and real-time RT-PCR with independently collected samples. Both genes showed lower methylation and higher expression preterm than at term. Although this study used genome-wide discovery approaches to find previously discovered and expected relationships between gene methylation and expression, it has confirmed that CpG methylation in the amnion is dynamic and related to transcriptional activity at a subset of genes linked to tissue function.CpG methylation has been profiled genome-wide in the amnion using the Illumina Infinium HumanMethylation27k BeadChip (HM27k) array . The arrSIPA1L1 gene) with a robust cell type-specific methylation difference, which could be used as a marker to correct for the variable cell type composition in amnion tissue samples. Genome wide methylation analysis was conducted with an assay called HELP-tagging, which utilized the methylation sensitive restriction enzyme, HpaII. With this assay the authors surveyed the methylation state of over 545,000 CpG sites in normal versus pre-eclamptic amnion samples  and found 4,058 differentially methylated sites in 3,035 genes. Methylation of 123, 85, and 99 sites were influenced by systolic blood pressure, proteinuria grade and the combination of the two, respectively, in regression models. RNAseq with a subset of samples revealed that 41 genes were differentially expressed in pre-eclampsia; however, none of the differentially methylated sites were in the vicinity of the differentially expressed genes, indicating the complexity of 5meCpG-mediated gene regulation. Overall, the presence of pre-eclampsia-associated epigenetic “signatures” in the amnion is remarkable, because this tissue is not a prime player in the disease. It appears, however, that epigenetic plasticity makes the amnion a useful surrogate to report the effects of adverse intrauterine conditions on fetal tissues in general, which may impact on disease susceptibility later in life.Pre-eclampsia is a severe pregnancy complication characterized by hypertension, proteinuria and maternal inflammatory reactions. Its pathogenesis is unclear but abnormal placentation, angiogenic imbalance and endothelial dysfunction are well-documented attributes of the condition. DNA methylation has been studied in the early onset form (<34 weeks of gestation) of the disease using formaldehyde-fixed, paraffin-embedded full thickness fetal membrane samples that included amnion, chorion and attached decidua . CpG metMMP1 gene promoter in the amnion in normal pregnancies and in cases of preterm pre-labor rupture of the membranes (pPROM). Matrix metalloproteases (MMPs), such as MMP1, have a key role in breaking down the collagen matrix of the amnion contributing to membrane rupture at term and preterm birth. The MMP1 proximal promoter contains no CpG island, only 14 sporadic CpG sites, which are fairly highly methylated (≈50%) in amnion samples, as determined by clonal bisulfite sequencing. Treatment of primary cultures of amnion mesenchymal cells with the DNA-demethylating agent, 5-aza-3′-deoxycytidine (5-AZA), increased MMP1 expression and decreased the methylation of one CpG motif at 1,538 bases upstream of the transcription initiation site. Interestingly, the methylation level of this site was significantly lower in amnion samples from deliveries complicated by pPROM compared to normal term controls. This group also described a previously undetected polymorphism in the MMP1 promoter that generated a new CpG site as a minor allele. The presence of this CpG reduced MMP1 promoter activity in amnion mesenchymal cells and even more so when it was methylated. In vivo, this site was always methylated and, remarkably, was significantly protective of pPROM in an African American population. This study illustrates that epigenetic and genetic differences may combine forming complex patterns of regulation that can be understood by analyses involving both levels. In the case of MMP1, the effect of DNA methylation on gene activity interacted with the polymorphism of the promoter shaping the impact on fetal membrane integrity and the risk of pPROM.Methylation analysis of candidate genes having well-established functions is a straightforward approach to explore the role of DNA methylation in gene regulation with physiological relevance. TIMP1 proximal promoter contains no CpG island and the 25 CpG sites around the transcriptional start site (−275)–(+279) are highly methylated in the amnion of female fetuses compared to males, as shown by TIMP1 mRNA abundance was higher in female than in male amnions and mRNA expression did not change with spontaneous labor despite a significant decrease of promoter methylation. Lipopolysaccharide (LPS) treatment of amnion explants increased methylation without effect on expression. Pre-treatment with the DNA demethylating agent, 5-AZA , did not affect TIMP1 mRNA, or TIMP1 promoter methylation levels in male or female amnions but sensitized the tissues to respond to LPS with increased TIMP1 expression. These data suggest that TIMP1 promoter methylation is dynamic but not linked to the level of gene activity in the amnion. Responsiveness to LPS might be influenced by methylation possibly at remote sites in trans.TIMP1 is a protease inhibitor protein that interacts with MMPs to control extracellular matrix remodeling. The PTGS2, BMP2, NAMPT/PBEF, CXCL2), steroid receptor genes  and renin-angiotensin system components  were examined with the technique for promoter methylation density in amnion samples from early gestation (11–17 weeks) and after term delivery with or without labor. With the exception of KLK1 , the proximal promoters of these genes have relatively high CpG density overlapping with CpG islands. Methylation densities of these promoters showed bimodal distribution with either highly methylated or sparsely methylated copies. The distributions did not change with gestational age or with labor but varied between genes and among individuals. Furthermore, the distribution of highly versus sparsely methylated promoter copies did not correlate with expression levels but correlated significantly between individuals. This suggests that the methylation of these promoters was established in early pregnancy (before 11–17 weeks) in a gene-specific fashion under the influence of individual conditions and was maintained until after delivery. In agreement with this, the expression of the DNMTs, DNMT1 and -3a, were highest in early pregnancy and decreased by term. Interestingly, the low CpG KLK1 promoter exhibited intermediate methylation density in some of the samples and a loss of methylation at term, indicating dynamic methylation without a significant influence on expression level.Methylation of candidate genes in the amnion has been assessed in two further studies using the Methyl-Profiler PCR system . The MetCovalent post-translational modifications of histones, which include acetylation, methylation, phosphorylation, ubiquitination, and sumoylation, are pervasive throughout the chromatin and are organized in patterns characteristic of genomic features, such as promoters, enhancers, transcribed sequences and heterochromatin regions . They coPTGS2 gene, which encodes a key enzyme of prostaglandin biosynthesis  and at term  have not been published with amnion or chorion to inform about gestational changes or pathological alterations. Studies employing ChIP combined with PCR, however, have demonstrated the presence of histone modifications in the promoter regions of a few labor-associated genes in the amnion. Gene activating histone-3 and -4 acetylation  and histone-3, lysine-4 demethylation (H3K4me2) have been reported at the promoter of the ynthesis , 2011. Fncreased . Histone at term . The expex vivo preserves progesterone receptor expression in its non-laboring state . Micro-RNAs act as guides to direct protein complexes to mRNAs or non-coding RNAs by sequence recognition . DependiPLA2G4B mRNA, which encodes a phospholipase involved in prostaglandin biosynthesis. They have also demonstrated a marked down-regulation of Dicer, a key enzyme of miRNA biogenesis, with advancing pregnancy in agreement with a widespread reduction of miRNA levels. This finding suggests that the homeostatic role of miRNAs, which is to stabilize a transcriptome that maintains the pregnancy-supporting phenotype of the membranes, is weakened at term, thus facilitating the transition to a labor-promoting state. An even higher number of miRNAs (875 miRNAs included in the miRCURY Array v.11 from Exiqon) have been tested in isolated amnion tissues at term and after preterm labor  by fetal membranes by strict definition. It forms the maternal side of the maternal-fetal contact zone, however, and is fused with the chorion leave so intimately that it is practically impossible to separate them completely, even with sharp dissection  and estradiol (E2) or cAMP treatments of cultured endometrial stromal cells  sequencing. Collectively, these data suggest that DNA methylation dynamics is slight in the decidua and the involvement of CpG methylation in the decidualization process is subtle. Other epigenetic processes, such as histone modifications, may play a more predominant role.Differentiation of the endometrium to decidua involves the transformation of endometrial stromal cells to decidual cells , which hal cells . These sal cells . In spital cells , suggest7k array . The top in vivo . In a siin vivo and in vitro is associated with the decreasing expression of EZH2, which is the catalytic component of the histone methyltransferase complex, PRC2  choriodecidua samples leaving the contribution of the maternal (decidua) and the fetal (chorion laeve) components to be determined .Finally, there is considerable interest in defining the role of decidual non-coding-RNAs in the pathogenesis of early pregnancy loss , pre-eclThe information currently available about epigenetic events in the fetal membranes and the decidua establishes structural changes of the chromatin as an important contributor to gene regulation in these tissues throughout pregnancy and in labor. The major types of chromatin modifications, such as DNA (CpG) methylation and histone modifications, have been found and many of them located in genome-wide and candidate gene-focused studies. Alterations in modification levels and patterns have been detected in association with gestational age, labor status and pathological conditions. Long and short non-coding RNAs (miRNAs), which represent a separate, but connected, branch of epigenetic regulation, have also been described and studied in detail. •Most data about epigenetic events in the fetal membranes describe associations between chromatin modifications, non-coding RNAs and outcomes related to various physiological or pathological states of pregnancy. Associations do not prove causation; therefore, inferences about epigenetic involvement remain tentative. Evidence supporting functional roles can be generated by interventional studies that target components of the epigenetic machinery using inhibitors of chromatin modifying enzymes and specially designed epigenetic chemical probes . A few o•in vivo are subject to environmental exposures and lifestyle conditions such as smoking, diet, toxic substances and effects of social stress. Epigenetic mechanisms mediate the long term  effects of these exposures and conditions as determined in animal models (agouti locus in mice (Fetal membrane tissues l models . Furtherl models . “Metastl models , for exa in mice  and were in mice . Smoking in mice , but the•Epigenetic events in the chorion leave are much less studied than in the amnion and the decidua. The chorion leave constitutes the fetal side of the maternal-fetal contact zone and a role in establishing and maintaining the maternal tolerance of the fetus is implied by its location . Chorion•The amnion, chorion and decidua contain several types of cells, and experimental results with tissue samples represent a sum generated by heterogeneous cell mixtures that often vary in proportions. Chromatin modification patterns are cell specific, and epigenetic differences predominant in a particular cell type may be masked by the average. Analysis of isolated individual cell types or correction for cellular heterogeneity using cell-specific markers are approaches that can alleviate the problem of variable cell composition and increase the robustness of results. Importantly, the expanding suite of single cell epigenomic techniques  offers e•To identify chromatin loci where epigenetic alterations occur during pregnancy, labor and pathological conditions will require the generation and bioinformatic integration of genome-wide DNA methylation and histone modification datasets, as these modifications buttress the chromatin together and function in a combined fashion . Inclusi•Lastly, it is important to emphasize that advanced, genome-wide approaches and the more traditional analysis of individual candidate genes supplement each other and pursuing them in combination can result in optimal outcomes. Validation of key findings in genome-wide screens, and mechanistic studies in culture systems in general benefit from focusing on genes and regulatory regions pinpointed by genomic scale analyses. Emerging unexplored epigenetic mechanisms like non-cytosine DNA methylation  and protTZ conceptualized the work and drafted the manuscript. JP provided intellectual input, edited the manuscript drafts, and finalized the figure designs. Both 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.


Additionally, affected males had significantly higher partial pressure of carbon dioxide (pCO2) and total carbon dioxide (TCO2) than unaffected males. Wooden breast affected broilers also possessed a significantly heavier pectoralis major muscle and whole feathered wing compared to unaffected broilers. Blood gas disturbances characterized by high pCO2 and low pH are indicative of insufficient respiratory gas exchange, suggesting that wooden breast affected broilers have an elevated metabolic rate that may also be inadequately compensated due to cardiovascular deficiencies such as poor venous return or respiratory insufficiency. Lung tissues from 12 birds with extreme sO2 values were subsequently examined to assess whether lung pathology contributed to the observed blood gas disturbance. Comparison of lung morphology between affected and unaffected birds revealed no apparent differences that could contribute to decreased parabronchial gas exchange. However, an interesting finding was the detection of pulmonary phlebitis in one of the wooden breast-affected samples consistent with vascular changes observed in pectoralis major muscle exhibiting the wooden breast phenotype. Our results suggest that the effects of wooden breast are not limited to the pectoralis major muscle and further indicate the importance of research into metabolic changes associated with the myopathy.Wooden breast syndrome is a widespread and economically important myopathy and vasculopathy of fast growing, commercial broiler chickens, primarily affecting birds with high feed efficiency and large breast muscle yield. To investigate potential systemic physiological differences between birds affected and unaffected by wooden breast, a total of 103 market-age Cobb 500 broilers were sampled for 13 blood parameters and the relative weights of the pectoralis major muscle, pectoralis minor muscle, external oblique muscle, wing, heart, lungs, liver, and spleen. Blood analysis was performed on samples taken from the brachial vein of live birds and revealed significant differences in venous blood gases between affected and unaffected chickens. Chickens with wooden breast exhibited significantly higher potassium (K Modern commercial broiler chickens have undergone intensive selection for production traits such as high muscle yield, rapid growth, and high feed efficiency to meet consumer demand for low-cost lean chicken meat, specifically chicken breast meat. Such breeding strategies have produced remarkable results, nearly halving the time for birds to reach market weight while simultaneously increasing the breast muscle weight by about two-thirds since the 1950s . UnfortuOne such myopathy, commonly called wooden breast (WB), causes the pectoralis major muscle to become grossly pale, enlarged, and palpably firm, with visible signs of inflammation such as petechial hemorrhages and tissue edema . These mResearch on WB and related myopathies is focused largely on the pectoralis major muscle, with only minor attention paid to potential systemic disparities accompanying the condition. However, factors that predispose broilers to WB – growth rate, feed efficiency, and breast muscle yield – can broadly be categorized as relating to general metabolism and body form. To date, there has been no comprehensive comparison of muscle and organ weights beyond the pectoralis major muscle weight and abdominal fat percentage between WB affected and unaffected broilers. An analysis of body weight distribution in WB birds may aid in identifying systemic physiological predisposition to or pathophysiologic effects of the myopathy. Intensive selection for commercially valuable traits has been shown to underpin biological imbalances in meat-type chickens, such as insufficient cardiopulmonary capacity to accommodate sustained rapid growth, resulting in pulmonary hypertension syndrome, or ascites . It is k2) and increased total carbon dioxide (TCO2), bicarbonate (HCO3–), and base excess (BE). The same blood analysis conducted at 42 days of age produced similar results with regard to blood gas changes in WB affected birds, with the additional finding that packed cell volume  was significantly associated with WB severity (+) levels in affected birds in a study evaluating the venous blood of male broilers at 42 days of age.Microscopic characterizations of WB have also largely been limited to the pectoralis major muscle , althougseverity . LivingsThus, the objective of the current study was to further the systemic characterization of WB myopathy by comparing blood parameters, body weight distribution, and lung histology between affected and unaffected broilers.The University of Delaware Institutional Animal Care and Use Committee approved the animal protocol (48R-2015-0) followed for this scientific study. Euthanasia was performed by means of cervical dislocation, and all efforts were made to maximize bird welfare.This experiment was conducted in chicken houses located at the University of Delaware under environmental conditions simulating a commercial setting. As part of a genome-wide association study, a total of 542 Cobb 500 broilers from the same breeding population were raised in 4 chicken houses and provided free access to feed and water. At 47 days of age, 103 birds were selected for blood analysis based on manual palpation of the breast muscle. These birds were selected to achieve an approximately equal number exhibiting no palpable breast muscle firmness  and severe palpable firmness . Blood analysis using i-STAT requires sampling of blood from live birds. Therefore, bird selection was performed using breast muscle palpation of live birds while all statistical analyses utilized more accurate scoring of WB based on gross evaluation of the pectoralis major muscle at necropsy, as described below.Birds were euthanized by cervical dislocation at 52 or 53 days of age. During necropsy, the pectoralis major muscles were evaluated for gross lesions and palpable firmness associated with WB and each bird was assigned a WB score using a 0–4 scale; 0-Normal indicates the bird had no macroscopic signs of the myopathy, 1-Very Mild indicates 1% or less of the breast muscle was affected, 2-Mild indicates between 1% and 10% of the breast muscle was affected, 3-Moderate indicates between 10 and 50% of the breast muscle was affected, and a score of 4-Severe indicates that more than 50% was affected. This scoring system is the same one implemented by Gallus gallus has been demonstrated in previous studies (+), K+, ionized calcium (iCa), and glucose (Glu); hematologic parameters Hct and hemoglobin (Hb); and blood gas parameters including pH, partial pressure of carbon dioxide (pCO2), TCO2, HCO3–, BE, oxygen saturation (sO2), and pO2. After all measurements were completed, data was downloaded from the analyzer and consolidated for statistical analysis. Body weight at 47 days was also measured at this time.At 47 days of age, 1 ml of blood was drawn from the brachial wing vein of each bird using a 3 ml syringe with 23-gauge needle that had been prepared by aspirating and expelling a small volume of liquid heparin prior to blood collection. The blood was deposited immediately into a new i-STAT CG8 + cartridge inserted in the i-STAT 1 Analyzer  to perform rapid blood analysis. While designed for clinical use in humans, the i-STAT system’s performance in  studies . CG8 + tThe same 103 birds used for blood analyses were also used to evaluate body weight distribution. After euthanasia, the left pectoralis major muscle, left pectoralis minor muscle, left external oblique muscle, heart, lungs, liver, spleen, and whole feathered left wing, disarticulated at the shoulder, were dissected from each bird. The weight of each dissected body part was recorded, along with the body weight of the bird before necropsy.P ≤ 0.05 were considered significant for all tests.Statistical analyses were performed using JMP software . To improve statistical power, birds were grouped according to WB status: unaffected birds included those assigned a WB score of 0-Normal or 1-Very Mild and affected birds included those assigned a score of 2-Mild, 3-Moderate, or 4-Severe. All i-STAT measurements were analyzed using a mixed linear model with WB status, sex, WB-sex interaction, and body weight at 47 days as fixed effects and poultry house as a random effect. Body part weights were analyzed using a mixed linear model with WB status, sex, WB-sex interaction, and body weight at necropsy as fixed effects and poultry house as a random effect. Effects with 2 values and six birds with the highest sO2 values  were selected for microscopic examination of lung tissue based on blood gas values measured at 47 days of age. In the low sO2 group, 5 birds were classified as affected, with WB scores of 3-Moderate or 4-Severe, and 1 bird was classified as unaffected, with a WB score of 0-Normal. In the high sO2 group, all 6 birds were classified as unaffected, with a WB score of 0-Normal. From each of the selected birds, lung tissue from the cranial and caudal aspects from either the left or right lung were harvested and fixed by immersion in 10% neutral buffered formalin. Samples were processed routinely for staining with hematoxylin and eosin as described by Of the 103 broilers used in this study, six birds  with the lowest sO2 status (high or low), sex, or WB status.Lung tissue was examined microscopically by a veterinarian (Papah) and a certified veterinary anatomic pathologist (Brannick) for histopathologic lesions or other tissue changes which could affect systemic blood parameters, such as the presence of inflammation, fibrosis, lymphoid follicular hyperplasia (lymphocytic nodules), cartilaginous nodules, edema or hemorrhage in the gas exchange areas, thickening of parabronchial walls, and obstruction of parabronchi. Assessment of all slides was performed in a blinded fashion and later microscopic lesions were assessed for an association with sOThe effects of WB, sex, WB-sex interaction, and body weight on the weights of the dissected left pectoralis major muscle, left pectoralis minor muscle, left external oblique muscle, whole feathered left wing, heart, lungs, liver, and spleen are provided in The external oblique muscle showed an interesting effect of the WB-sex interaction, with affected males and unaffected females having the highest average weights of those muscles and unaffected males having the lowest weights. Similarly, the weight of the pectoralis minor muscle was highest in unaffected females and lowest in unaffected males. One potential explanation for the observed group means of the external oblique muscle is as an adaptive response to the size of the combined pectoralis major and pectoralis minor muscle. Avian respiration relies on movement of the sternum to allow expansion of the bellows-like air sacs during inhalation . The extCompared to females in the present study, males possessed larger wings and lungs, but a smaller liver after accounting for WB and body weight. There was no significant difference in the size of the pectoralis major muscle between males and females in our model. Body weight had a significant effect on the sizes of all body parts except the lungs, with larger birds possessing generally heavier body parts but relatively smaller lungs.+ and significantly lower pH, sO2, and pO2. Affected male birds also possessed significantly higher pCO2 and TCO2 values compared to unaffected male birds. Although affected female birds had pCO2 and TCO2 values higher than those of unaffected female birds, the effect was not significant potentially due to the smaller number of female birds sampled. These results are largely in accordance with previously published data of blood parameters measured at 42 days of age .Elevated pCOurbances . Respirahe lungs . As carbof HCO3– . Livings2 production (increased metabolism), a relative decrease in respiratory gas exchange (cardiopulmonary insufficiency), or both  pumps have been found to account for 40–50% of the resting metabolic rate in mouse skeletal muscle, or 12–15% of whole body resting oxygen consumption , or reduced respiratory musculature .2, as indicated by blood gas analysis in this study, and negatively affect gas exchange by slowing the flow of blood back to the gas exchange surfaces of the lungs. Impaired venous return from phlebitis may exacerbate what some characterize as existing vascular insufficiency in the pectoralis major of commercial broilers. At the cellular level, large breast muscles are produced by increasing the number, diameter, and length of muscle fibers  and has been previously reported in broiler chickens in association with ascites syndrome  observed in tissues from either high sOculature  and was syndrome . In the syndrome . This fiOther minor histopathologic findings included bronchus-associated lymphoid tissue (BALT) hyperplasia, occasionally with discrete nodular follicle formation, and extramedullary hematopoiesis (EMH). Generalized and nodular lymphoid hyperplasia likely indicate immune response to inhaled antigens or irritants and are not suspected to be related to WB. The airway associated lymphoid tissues did not appear to impinge upon or obstruct airway lumens. The physiologic process of EMH is common in tissues of young animals, but can sometimes occur to produce additional red blood cells in response to low tissue oxygenation. However, the limited extent and level of EMH in the lung tissues in the present study indicate that EMH is likely an incidental rather than clinically significant finding. Mild, limited EMH is a common finding in avian tissues, including lung, even in clinically normal birds. Artifactual changes from specimen collection and processing, such as acute hemorrhage with no tissue reaction and collapse of air spaces with no indication of true airway obstruction (atelectasis) were observed but disregarded for purposes of analysis.2 status were not associated with histopathologic changes in the lungs, suggesting that structural abnormalities or disease in the lungs are not likely contributors to the WB phenotype. The chondro-osseous metaplasia lesions observed in this study are likely sequelae of the WB syndrome rather than precipitating factors in the development of the disease. The finding of pulmonary phlebitis in one WB bird suggests that the phlebitis associated with WB is not restricted to the musculature but may be systemic in nature.Altogether, WB status, sex, or sOlipoprotein lipase (LPL), fatty acid binding protein 4 (FABP4), and adiponectin c1q and collagen domain containing (ADIPOQ), in the lung tissue of WB affected broilers  rather than its etiology and further studies are required to ascertain whether a blood gas disturbance is present during early stages of the disease.The findings of the present study indicate that WB is associated with blood gas disturbances characterized primarily by increased venous KThe datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by the University of Delaware Institutional Animal Care and Use Committee.BA conceptualized, designed, and supervised the study. JL performed the statistical analysis and wrote the original draft of the manuscript. EB, MP, and CL conducted histopathological examination of lung samples and EB wrote sections of the manuscript relevant to that examination. BA, JL, SV, EB, and MP contributed to manuscript revision. All authors read 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.


Maize Chlorotic Mottle Virus (MCMV) in combination with any of the potyviruses namely; maize dwarf mosaic virus (MDMV), sugarcane mosaic virus (SCMV) and tritimovirus wheat streak mosaic virus (WSMV). In Africa, the disease occurs due to combined infections of maize by MCMV and SCMV, leading to severe yield losses. Efforts to address the disease spread have been ongoing. Serological techniques including enzyme-linked immuno-sorbent assay (ELISA), polymerase chain reaction (PCR), genome-wide association (GWAS) mapping and next generation sequencing have been effectively used to detect and characterize MLN causative pathogens. Various management strategies have been adapted to control MLN including use of resistant varieties, phytosanitary measures and better cultural practices. This review looks at the current knowledge on MLN causative viruses, genetic architecture and molecular basis underlying their synergistic interactions. Lastly, some research gaps towards MLN management will be identified. The information gathered may be useful for developing strategies towards future MLN management and maize improvement in Africa.Maize lethal necrosis (MLN) disease is new to Africa. First report was in Kenya in 2012, since then the disease has rapidly spread to most parts of eastern and central Africa region including Tanzania, Burundi, DRC Congo, Rwanda, Uganda, Ethiopia and similar symptoms were observed in South Sudan. Elsewhere, the disease was caused by infection of  Zea mays L., 2n=2x=20) is a major staple food and source of income and livelihood for the majority of smallholder farming communities in sub-Saharan Africa  and any of SCMV, MDMV  or WSMV  , DRC Congo , MCMV-KS (Kansas) and MCMV-YN (Yunnan), and different unconfirmed strains have been suspected in some parts of Africa including Nigeria, Rwanda, Sao Tome and Principe, Tanzania, Togo, Zambia and Zimbabwe  and aphid  are the common vectors of MLN viruses in Africa and the viruses can survive on different host plants such as cassava, beans, maize, sorghum, onions, rice, peppers coriander, peas, various grasses, Bidens pilosa and Tithonia diversifolia , southern corn rootworm (Diabrotica undecimpunctata), western corn rootworm (Diabrotica virgifera), Systena frontalis, Diabortica longcornis and Oulema melanopa , the only reported arthropod vector of MCMV, is the most common insect that transmit MCMV in most maize growing areas in Africa  and they function both as genome and messenger RNA. The viruses have evolved the ability to use the metabolic machinery of the host cell so as to produce their own genetic materials that they can use for multiplication and translation processes necessary for their survival as indicated in Viral particle enters host cell through wounds made mechanically or by vectors on the cell wall or by deposition into an ovule by an infected pollen grain  through replication by RdRp and translation of ssRNA(+) by ribosomes must occur on the same genomic template and at the same time. However, to avoid encounter between replication and translation apparatuses as well as collision of the two molecules as they move along the same template during the synthesis processes, ssRNA(+) viruses have developed a mechanism whereby ribosomes complete translation of ssRNA(+) before the negative strand replication by RP can start on the same template  in the surroundings are common signs and indications of potential MLN infections.Common symptoms of MLN include higher intensity of chlorotic spots, stunted growth and yellowing of the suspected maize plant . Phenotypic observation of symptoms of diseases alone cannot reveal the exact cause(s). Cells obtained from young leaf of virus-infected plants contain inclusion bodies of fairly distinctive shapes and sizes that can be seen using advanced tools so as to reveal the genus of the virus. Another important technique in detecting plant viruses is bioassay, which could be further confirmed by electron microscopy, serology and molecular techniques. Particles of many viruses are not always easy to find under the electron microscope; however, even when such particles are revealed, proof that the particles are of the virus that causes the particular disease requires much additional work and time  involved. Also, symptoms caused by the MLN viruses may vary according to the age of the maize plant, variety involved, environmental conditions, strain of the MLN viruses, and different viruses may cause similar symptoms on the same plant. Sometimes the disease symptom could result from co-infection by more than one virus , is easy to apply and is commonly used for detection of MLN viruses , and nucleic acid hybridization with radio-labelled and non-radio-labelled probes, and DNA/RNA probes  and polyclonal antibodies (PABs). The technique is widely applicable because ELISA based on RT-PCR method has some shortfalls since the method is more specific to a particular species or strain of a virus  and wheat streak mosaic virus  and they also infect maize, sorghum, wheat, oat, rye, johnsongrass and most species of Gramineae.Viruses of MLN are so complex and can survive and develop different strains or isolates across a wide range of hosts. Maize chlorotic mottle virus colonizes maize plant as the only natural host; however, the virus can experimentally infect grasses in the family Plant viruses are composed of minute building blocks (particles) called virions that make the complete viable structure. Shape of the virions differs from one virus to another and that is the main reason why viruses differ in their physical appearance when observed under electronic microscope as indicated in The genome is about 4-5.4 kb in length and about 30 nm in diameter. Both 5′ and 3′ terminals are protected by untranslated regions (UTRs)  as described in in trans. In addition, ORF2 produces P111 (111 kDa) protein by translational read-through of UAG stop codon found at its N-terminus. P111 is implicated in viral replication with some functions in trans. The function of ORF3 is not well understood  protein is expressed from ORF5 when the UAG stop codon of ORF4 is suppressed. The protein plays key roles in systemic infection. A 25 kDa coat protein (CP) expressed from the 3' proximal, is involved in cell-to-cell movement of virus. It is assumed that MCMV can overcome host defense mechanisms (RNA silencing) by expressing viral suppression of RNA silencing (VSRs) through P31, P32 and P50  encodes a 32 kDa protein which is assumed to be involved in viral accumulation. ORF2 (P50) encodes a 50 kDa protein and is associated with viral replication as well as function Genome of SCMV is 9610 nt long including the untranslated regions (UTRs) at both 5′ and 3′ terminals, which functions as infectious genome as well as messenger RNA, as presented in 4E at it C-terminal which interacts with eukaryotic translation initiation factors (eIF4E and its isoform eIFiso4E) by binding to siRNA or miRNA of the host, resulting to VSRs and enhanced viral infectivity  is expressed by autoproteolytic cleavage using serine protease and is involved in virus replication by stimulating helper component protease (HC-Pro) during VSRs. HC-Pro is synthesized through cleavage by cysteine protease. It is a multifunctional viral component which acts both as protein and enzyme (proteinase), and contains N-terminal, Central and HC-Pro domains. The N-terminal domain enhances virus transmission by aphids. It allows binding of virions to the cuticle of the maxillary food canal and foregut of aphids, virulence genome amplification and accumulation of virus  is a viral coat protein with major roles in virus movement, genome amplification and vector transmission. It is synthesized by the C-terminal of the polyprotein and is involved in encapsidation of viral genome using its multifunctional subunits  which is a small opening connecting adjacent cells , and polerovirus Beet western yellows virus (family Luteoviridae) has been reported with increased concentrations of all the three viruses and severe symptoms and stunting in sugar beet  plays major role in synergistic interactions as well as in replication and movement in SCMV. During co-infection of MCMV and SCMV, HC-Pro could be prioritized as VSRs, leading to reduction in SCMV replication and movement. Therefore, SCMV concentration in the mixed infections remains constant and being maintained by viral proteins such as P1 and VPg which are not strong enhancers of replication and movement proteins. This might explain why SCMV concentration in mixed infection with MCMV is unable to increase as for the MCMV  and sweet potato chlorotic stunt virus , the sweet potato plants showed severe symptoms in leaves and stunting of the plants. Concentration of SPFMV was increased 600-fold while that of the non-potyvirus partner (SPCSV) remained the same as in singly infected plants. This was because SPCSV was involved in VSRs of the host. As such, amplification and movement of SPFMV were enhanced  and both share some similar functions Scheets, . P1 may h-type thioredoxin responsible for resistance at Scmv1 locus, is reported to be involved in suppression of SCMV RNA accumulation  is generated and the Calvin cycle pathways within the photosystem I are affected. As a result, production of chlorophyll is reduced and MLN symptoms are expressed by the plant  gene, involved in COnt Cann, . Ferredont Cann, . Althougnt Cann, .A number of quantitative trait loci (QTLs) related to resistance to viruses have been reported in maize and so far genetic of resistance to MDMV, SCMV and WSMV is most studied of all viral diseases of maize  have been confirmed important and provide strong resistance against SCMV in maize. Scmv1 is responsible for protection against early infection while Scmv2 protects against late SCMV infections, and the two loci segregate as dominant genes , sugarcane, common weeds , and wild grasses. MCMV incidence is exacerbated in continuous maize production fields . However, it should be noted that the two scales 1-5 and 1-9 are similar because: 1=1; 1.5=2; 2=3; 2.5=4; 3=5; 3.5=6; 4=7; 4.5=8; and 5=9.A quantitative scale of 1-9 introduced by Reddy and Singh (1984) is also widely used based on diseased tissues relative to the whole plant: 1=resistant ; 2=resistant to moderately resistant ; 3=moderately resistant (mild chlorotic streaks on emerging leaves); 4=moderately to moderate resistant (moderate chlorotic streaks on emerging new leaves); 5=moderate resistant (chlorotic streaks and mottling throughout plants); 6=moderate to moderately susceptible ; 7=moderately susceptible ; 8=moderately susceptible to susceptible ; and 9=susceptible (complete plant necrosis and dead plants)  in collaboration with Kenya Agriculture and Livestock Research Organization (KALRO) has established an advanced MLN screening facility in Naivasha, Kenya of which partners including national research institutions (NARIs) have been screening their respective maize germplasm for resistnce to MLN viruses. The MLN screening facility uses artificial inoculation in order to create optimal screening conditions for MLN while ensuring minimal risk of disease escape. Optimized procedures have been developed to ensure uniform, high MLN disease pressure at the screening site (Olsen et al., Vast majority of African maize lines are susceptible to MLN and since the emergence of MLN in Africa in 2012, not much has been done on breeding for resistance to the deadly disease at national levels. CIMMYT, in collaboration with KALRO and other partners, has developed some maize hybrids and inbred lines that are tolerant to MLN. These materials are available for adoption by national research institutions (Olsen et al., Maize lethal necrosis disease is a new challenge to maize production and is threatening food security in Arica. For the last 4 years, most of the research interventions on MLN in Africa have been concentrating on etiology, epidemiology, screening and characterization of germplasm for sources of resistance to the disease (Adams et al., Each MLN virus exists in different strains and isolates; however, it is not known which combination of isolates gives severe MLN. Information on mode of inheritance of resistance to MCMV is inadequate. Multiple QTLs are linked to resistance to MLN; however, number of genes involved in resistance to MLN is not well reported. MLN is caused by co-infections of MCMV and SCMV; nevertheless, information on which virus to infect first so as to cause severe MLN synergism is lacking. Similarly, transmission of MLN through seed requires further investigation. Although a number of maize lines tolerant to MLN have been identified, their utilization in commercial hybrid is inadequate.MLN is a complex disease of maize with severe yield losses in Africa, hence its control requires effective and robust research approaches. A short-term strategy would be accessing tolerant lines from CIMMYT for adaptation in national programs. Mid-term research strategy may involve introgression of the tolerant genes into the susceptible, but farmers preferred maize varieties. In the long run, the breeding approach should corporate host resistance and vector management where transgenic breeding techniques could be employed.