The slow rate of neuronal regeneration that follows peripheral nerve repair results in poor recovery, particularly where reinnervation of muscles is delayed, leading to atrophy and permanent loss of function. There is a clear clinical need to develop drug treatments that can accelerate nerve regeneration safely, restoring connections before the target tissues deteriorate irreversibly. The identification that the Rho/Rho-associated kinase (ROCK) pathway acts to limit neuronal growth rate is a promising advancement towards the development of drugs. Targeting Rho or ROCK directly can act to suppress the activity of this pathway; however, the pathway can also be modulated through the activation of upstream receptors; one of particular interest being peroxisome proliferator-activated receptor gamma (PPAR-γ). The connection between the PPAR-γ receptor and the Rho/ROCK pathway is the suppression of the conversion of inactive guanosine diphosphate (GDP)-Rho to active guanosine triphosphate GTP-Rho, resulting in the suppression of Rho/ROCK activity. PPAR-γ is known for its role in cellular metabolism that leads to cell growth and differentiation. However, more recently there has been a growing interest in targeting PPAR-γ in peripheral nerve injury (PNI). The localisation and expression of PPAR-γ in neural cells following a PNI has been reported and further in vitro and in vivo studies have shown that delivering PPAR-γ agonists following injury promotes nerve regeneration, leading to improvements in functional recovery. This review explores the potential of repurposing PPAR-γ agonists to treat PNI and their prospective translation to the clinic. Nerve damage resulting from a peripheral nerve injury (PNI) can be highly debilitating to a patient causing life-long loss or disturbance to end-organ function . The mosDespite advancements made in PNI research, effective treatment acting to improve functional recovery remains challenging . The curDrug treatments that promote axonal regeneration with correlating functional recovery could provide an alternative treatment. Current drug therapies tend to focus on the resulting symptoms from a PNI such as neuropathic pain, inflammation, and weakness without modifying the condition itself . ApproprUnlike the central nervous system (CNS) the peripheral nervous system (PNS) does have some innate regenerative capacity, however, the rate of this regeneration is remarkably slow (~1 mm/day) ,9. This This review explores how the Rho/ROCK pathway could be modulated in PNI, in particular via the peroxisome proliferator-activated receptor gamma (PPAR-γ). Articles that have studied the use of drugs or small molecules that target PPAR-γ are reviewed, with a particular focus on where these have been used to promote regeneration or functional recovery following PNI. The 86 articles included were published between 1995 and 2021. Articles that studied PPAR-γ in optic nerve, neuropathic pain, neuroprotection, inflammation or neurodegenerative disease were excluded. The study of PPAR-γ in other nervous system disorders is important; however, this review focuses specifically on peripheral nerve regeneration and functional recovery.Studies have demonstrated the effect of signaling pathway modulation on nerve regeneration  16,17].,17.16,17In brief, activation of the GTPase Rho (or RhoA) to its GTP-bound form with the help of downstream effector kinase ROCK leads to stiffening of the actin cytoskeleton, this initialises changes in the signaling of many downstream effectors, which in turn inhibits axonal elongation and mediates growth cone collapse  16,20,2,220,21. Rho, together with Rac and Cdc42, belongs to the family of small GTPases, which have a role in cellular motility and cytokinesis ,23. ModuRho GTPases can be viewed as molecular switches whose activity depends upon regulatory proteins specific to each family member; these include guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs). Inhibitors targeting these proteins would provide a basis to control neuronal growth following PNI, however, a lack of specific tool compounds has hampered progress ,29. Rho The Rho-associated kinase (ROCK) is the most extensively studied downstream effector in the Rho pathway. ROCK is a serine/threonine protein kinase belonging to the AGC family ,31. Two The Rho/ROCK pathway  can alsoStudies have clearly demonstrated that neurons, particularly motor neurons, have an increased responsiveness to the Rho/ROCK pathway following PNI ,34 furthPPAR-γ is a member of the nuclear receptor family that heterodimerizes with retinoic acid-X receptors and is rendered transcriptionally active by binding to a specific DNA sequence element termed the PPAR response element ,39. ActiMore specifically, ligands bind to the ligand-binding pocket (LBP) of PPAR-γ modulating the activation function 2 (AF-2). AF-2 is a coactivator binding surface comprised of residues from the C-terminal helices (H) 12, H3 and H5 ,42. AgonThe role of PPAR-γ in nerve tissue remains unclear, however, immunohistochemical analysis has demonstrated that PPAR-γ can be found in Schwann cells of myelinated fibres and endothelial cells in rat peripheral nerves . This waA recent study has suggested that PPAR-γ activation has a role in controlling the phenotype of Schwann cells . FollowiTreatment with the PPAR-γ agonist, troglitazone in a primary rat hippocampal culture induced neurite outgrowth and increased axon length . This waPPAR-γ ligands can also be beneficial to improve neuro-inflammation and treat neuropathic pain ,52. PPARFurther studies have indicated that PPAR-γ activation has beneficial effects against oxidative stress, mitochondrial dysfunction, and apoptosis in several cell-based models for degenerative diseases such as Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and spinal cord injury ,59. PPARDrug discovery and development is expensive and time-consuming with a high risk of failure, therefore more companies and academic researchers are exploring drug repurposing as an alternative . This teAgonists for the PPAR-γ usually contain a lipophilic backbone and an acidic moiety, usually a carboxylate  and manyTo date promising results have been reported for treatment with ibuprofen and diclofenac treatment following PNI, for example Madura et al., 2011 demonstrated an improvement in functional recovery and remyelination of axons after 3 months following 3 weeks ibuprofen treatment in a rat tibial nerve transection model. Furthermore, treatment with diclofenac delivered to a transection of a rat sciatic nerve in an artery graft elicited improvement in functional recovery and axon regeneration .To the best of our knowledge other NSAIDs have yet to be investigated in PNI models; however, the relative affinities of a number of NSAIDs for PPAR-γ have been determined  and follAnother class of drugs that act upon PPAR-γ is the glitazones which are more commonly used as anti-diabetics. Pioglitazone and rosiglitazone have, however, been studied for use in PNI. In a mouse sciatic nerve crush injury model, three weeks of treatment with pioglitazone improved myelination , furtherNSAIDs have also demonstrated positive effects on regeneration in the CNS by inhibiting Rho activation . In bothDuan et al. reported that three days of treatment in vitro with ROCK inhibitors Y27632 and fasudil lead to tolerance of ROCK inhibition and attenuation of neurite outgrowth, but the tolerance was decreased with the cyclooxygenase-2 (COX-2) inhibitor NS398 . This to11 in rats [A preclinical study combining the treatment of the ROCK inhibitor fasudil and COX-2 inhibitor celecoxib demonstrated a synergistic effect by decreasing COX-2 and ROCK-II activation in a spinal cord injury site following a hemisection at the crest of T in rats . The loc in rats .To address this hypothesis another study was conducted using the anti-inflammatory drug methylprednisolone and the PPAR-γ agonist rosiglitazone, which also demonstrated a positive synergistic effect. The combined treatment of methylprednisolone and rosiglitazone following a laminectomy and compressive spinal cord injury in rats, had a pronounced effect on attenuation of inflammation and apoptosis. Increased functional recovery was observed when the two drugs were administered together in comparison to using either drug treatment alone .This review has focused on targeting PPAR-γ in the PNS, however, there are studies that have explored targeting PPAR-γ in the CNS ,77,78,79One recent study that is out of the inclusion criteria of this review but could have an influence on the study of PPAR-γ for CNS or PNS treatments is a randomised, placebo- controlled trial that found no correlation between ibuprofen dose and PPAR-γ gene expression during an emotion-related neural activation .To conclude, there is evidence in support of targeting PPAR-γ in peripheral nerve regeneration, which provides a platform for the development of pharmacological interventions. The repurposing of approved drugs is likely to be valuable in moving therapies rapidly towards the clinic. Although there have been many advancements made in the identification of targets for pharmacological agents to treat nerve injury, the field is still in its infancy. Only a few agents have been directly tested in humans to date for applications related to peripheral nerve function. Ibuprofen as well as other NSAIDs are routinely used for inflammation and pain and already have well-established safety and efficacy profiles . TherefoFuture work would need to address the additional issues associated with method and site of drug administration  to minim


Realimentation can compensate for weight loss from poor-quality feedstuffs or drought. Mature cows fluctuate in body weight throughout the year due to nutrient availability. The objective of this study was to determine whether cows that differ in weight gain during realimentation also differ in the abundance of transcripts for enzymes associated with energy utilization in skeletal muscle. Mature cows were subjected to feed restriction followed by ad libitum feed. Skeletal muscle transcriptome expression differences during the two feeding periods were determined from cows with greater (n = 6) and less (n = 6) weight gain during the ad libitum feeding period.Bonferroni < 0.05). These genes were over-represented in lysosome, aminoacyl-tRNA biosynthesis, and glutathione metabolism pathways. Validation of the expression of five of the genes was performed and four were confirmed. These data suggest that realimentation weight gain for all cows is partially controlled by protein turnover, but oxidative stress and cellular signaling pathways are also involved in the muscle tissue. This dataset provides insight into molecular mechanisms utilized by mature cows during realimentation after a period of low abundance feed.A total of 567 differentially expressed genes (408 up- and 159 down-regulated) were identified for the comparison of restriction and ad libitum periods (PThe online version contains supplementary material available at 10.1186/s13104-021-05757-8. Cattle producers are faced with the challenge of increasing production to feed the growing population, and over 2/3 of the budget is the feed to achieve production goals. Compensatory gain (CG) increases production while maintaining or even decreasing inputs and affects an animal’s lean tissue and fat deposition. Forage quality fluctuates with season; thus, the mature beef cow can experience periods of nutrient restriction followed by realimentation. This, along with the utilization of CG for improved feed efficiency, highlights the necessity to understand the mechanisms that allow for certain cattle to respond differently to nutritional shifts.Previous work investigated these mechanisms in the adipose tissue of mature cows  and discKeogh et al.  investig1 bulls from Angus and Hereford dams were mated to F1 cows from these matings to produce cross progeny. At 5 years, cows were not bred and moved to an individual feed intake facility with Calan Gates . Feed restriction and ad libitum feeding diets are described in [The samples and phenotypes presented in this paper were collected previously . CrossbrP < 0.01).Cows most divergent in body weight (BW) gain  during the realimentation period were selected and skeletal muscle samples were processed. This classification was based on realimentation to identify cows with divergent BW gain during realimentation following nutrient restriction, and represent variation in CG. Cows selected for high gain had higher body weights and feed intakes than cows selected for lower gain (P < 0.0005). Cows with greater gain displayed average Gain:Feed ratio of 0.12, compared to 0.08 for cows with lesser gain  was isolated from 50–100 mg of tissue with TriPure reagent , and quantified using a NanoDrop 8000 spectrophotometer . The 260/280 measurements were > 1.8 for all samples. Quality of total RNA was assessed on an Agilent 2100 Bioanalyzer and RNA integrity number (RIN) values were ≥ 7 for all samples.P of < 0.05 was obtained. Raw data files were deposited in the NCBI gene expression omnibus (GEO) as series record GSE94777.For microarray, 250 ng of total RNA was used with the Bovine 1.1ST array strips on the Affymetrix GeneAtlas System . Transformed data were analyzed using a repeated measures model where gain class was fitted as a fixed effect and restricted and ad libitum samples as time points. Gain class was tested on the animal error term while time and its interaction with gain class was tested on the residual. Genes were considered differentially expressed when a Bonferroni adjusted P-value < 0.05 between cows with high and low gain. However, none were significant after Bonferroni correction. After Bonferroni correction, the comparison of feed restriction and realimentation time points produced 567 differentially expressed genes  from the analysis comparing feed restriction to realimentation  were used. Real-time PCR was performed in triplicate for all samples and genes on a Bio-Rad CFX384 (BioRad) instrument using SsoAdvance SYBR Green master mix (Bio-Rad). The RT-qPCR reaction was performed according to the manufacturer’s instructions. Relative transcript abundance of each target gene was calculated using the 2−ΔΔCt method [Validation using real-time quantitative polymerase chain reaction (RT-qPCR) was performed on five of the DEG (t method  with theP < 0.05), with trends for aminoacyl-tRNA biosynthesis, glutathione metabolism, and renin-angiotensin system pathways (P < 0.1). Up-regulated genes were enriched for phototransduction (P < 0.05). All genes identified for the lysosome pathway were expressed in higher abundance during realimentation. Lysosomes are responsible for digesting macromolecules including proteins and organelles [Functional annotation of the 567 DEG identified in the feed restriction and realimentation analysis was performed in the database for annotation, visualization, and integrated discovery (DAVID) v6.8 . The genes over-represented were SHMT1, SHMT2, LAMTOR1, LAMTOR4, LOC614531 and RRAGA. All were down-regulated in the feed restricted animals, and all annotated genes have functions in amino acid synthesis or cellular responses to amino acid availability. The two LAMTOR genes and RRAGA encode proteins that are crucial to the activation of the mammalian target of rapamycin complex 1 cascade to promote cell growth in response to cell signals such as nutrient and amino acid levels [SHMT1 and SHMT2  between restriction and realimentation was evaluated using the PANTHER Classification System , 13 for d levels . The lisd levels , glycineXKR4 has been implicated in gain and feed intake in cattle [UCN is involved in mammalian appetite and stress response [CUX1, ETV1, EIF4EBP2, FOXJ3, and SOX30). These were all transcribed in higher abundance during realimentation in muscle. Genes with inflammatory functions were identified in the muscle tissue of cows transitioning from restriction to realimentation, including IL18BP, IL18RAP, IL34, IL36A, INHBE, and SELE. Genes IL34, IL36A, INHBE and SELE may be of particular importance, as they were detected in the adipose of these cows. These inflammatory response genes differ in function yet are important regulators of cellular response to stresses including inflammation, nutritional stress, and disease [We have previously evaluated the adipose from these same cows  and founn cattle  and UCN response , 24. Gen disease –31. The The nutritional challenges implemented here reflect those experienced by cattle grazing seasonal pastures and native ranges, which represents the majority of the U.S. cow herd. The objective of this discovery study was to investigate differences in the skeletal muscle transcriptome explaining the molecular responses in BW gain in mature cows during realimentation. While some pathways associated with protein turnover and energy metabolism were identified, many of the biological processes discovered were modulators of these pathways in some capacity, even if not directly identified as belonging to those biological processes.In summary, we identified common pathways among cows in the muscle tissue during realimentation, which included protein turnover, tRNA synthesis, and glutathione metabolism. These appear to be pathways that are critical for CG of cows during realimentation and provide insight into the underlying biological mechanisms. This study is the first to evaluate high versus low gaining mature cows for biological differences in the muscle tissue during feed restriction and realimentation. Further studies on larger groups of animals are necessary to better evaluate these genes as potential biological markers for CG.A limitation of this study was the small sampling size (n = 12). There were 121 cows in this study and we selected those most divergent in BW gain during realimentation. Obtaining clear differences between animals with high versus low gain phenotype while balancing the cost to perform the microarray experiments were critical factors for our sample size. Future aims include validation of some of the most highly and lowly expressed genes detected in this study in samples from cows biopsied in different years.Additional file 1: Table S1. Statistical analysis data of the expression of genes in the muscle tissue of mature cows for high versus low gain, feed restriction versus ad libitum feed time points, and the interaction between gain and time.Additional file 2: Fig. S1. Heatmap illustrating the changes in expression for the 30 most up-regulated and the 30 most down-regulated genes between the feed restriction diet and ad libitum feed.Additional file 3: Table S3. Comparison of microarray data with RT-qPCR data for five genes identified as differentially expressed in the muscle tissue of cows (n = 12) fed ad libitum after feed restriction.Additional file 4: Table S4. PANTHER biological processes identified as over-represented in the muscle tissue of mature cows between feed restriction and ad libitum periods.


Clinical relapses early after autologous stem cell transplantation portrays an inferior clinical outcome. Early relapse in this setting with extramedullary disease (EMD) of lung involvement in multiple myeloma is rare. To our knowledge, this is the first reported case of lymphangitic spread of myeloma with pulmonary parenchymal and pleural involvement occurring at first relapse. Multiple myeloma (MM) is characterized by malignant clonal proliferation of plasma cells and is known for relapsing disease course with a small cure fraction. Extramedullary disease (EMD) in MM exemplifies a clonally evolved population of myeloma cells that can survive independent of the bone marrow (BM) microenvironment and confers treatment resistance and adverse clinical outcome. Herein, we report a rare case of fulminant pulmonary lymphangitic relapse of myeloma with extramedullary pulmonary involvement as the first clinical presentation of relapse occurring within 90 days after an upfront autologous stem cell transplantation (ASCT).18F-FDG PET at diagnosis showed multiple focal lesions with no extramedullary disease. The patient proceeded with 30 Gy local radiation to thoracic spine and induction therapy with KRD for 4 cycles achieving a partial response (IMWG criteria), followed by high dose chemotherapy (HDC) and ASCT. ASCT did not further deepen the response and the patient attained partial response, hence the plan was to proceed with additional KRD consolidation followed by maintenance therapy. However, the patient presented with worsening shortness of breath (SOB) and cough approximately D + 90 after ASCT requiring hospitalization. A computed tomography (CT) at admission showed new bilateral lower lobe infiltrates with consolidation and innumerous pulmonary nodules  IGH-MAF translocation, TP53 deletion, and gain of MAFb (20q12) observed in 29–96% of plasma-cell-enriched cells analyzed. This is in contrast to the FISH studies at initial diagnosis which showed 1q21 gain, the gain of MYC, t  in addition to the original TP53 clone. Cytogenetics showed a complex tetraploid karyotype with (del)17P. Taken together, the clinical picture was consistent with a marrow relapse and extramedullary myelomatous lung involvement with lymphangitic spread and malignant pleural effusion. The patient became progressively hypoxic, requiring admission to the intensive care unit. She was started on methylprednisolone 1 gram daily for 3 days followed by bendamustine (90 mg/m2) × 2 days. Unfortunately, the MM was proved difficult to control, thus we proceeded with salvage KPACE with minimal symptomatic improvement. Given the incurable nature of disease and limited prognosis, the patient opted for hospice care.A 63-year-old female presented with R-ISS stage II IgG kappa MM after disease progression from monoclonal gammopathy of undetermined significance (MGUS). She had high-risk cytogenetics del17p (unknown clone size) on FISH with complex karyotype. She presented with back pain resulting from a thoracic vertebral paramedullary mass at T12 with no neurological compromise.  nodules . She was nodules . Video-a nodules . Bone ma3.The incidence of EMD in MM had increased from 6.5% in 2005 to 23.7% in 2014 based on a recent registry study, and this is ascribed to the improving survival of MM patients and routine integration of novel imaging techniques such as PET CT and MRI DWIBS in clinical practice [EMD correlates with a high-risk 70-gene expression profiling signature with enrichment of MF molecular subgroup and the PR molecular subgroup . In addiOur case is unique due to the clinical presentation of early fulminant relapse within 90 days of ASCT with lymphangitic and parenchymal involvement of the lung along with the pleura. The relapse pattern could be due to a hematogenous inoculum of plasma cells in the lung followed by perivascular lymphatic spread into pulmonary parenchyma. In addition, early relapse within the first 24 months of ASCT occurring in 35–38% of newly diagnosed multiple myeloma (NDMM) confers an inferior clinical outcome , 11.Our case highlights the very aggressive nature of the disease depicted by the presence of high-risk cytogenetics of (del)17p at initial diagnosis, suboptimal response to induction therapy, early relapse within 3 months of ASCT with additional high-risk cytogenetics including 1q21, and complex tetraploid karyotype with poor response to salvage therapy. Interestingly, in our case, there is a striking discordance in the flow cytometry expression of CD56 or neural cell adhesion molecule (NCAM) with BM being negative and extramedullary lung site positive perhaps suggesting a clonal evolution . Publish


Hantaviridae) usually occurs via inhalation of aerosols contaminated with viral particles derived from infected rodents and organ manifestation of infections is characterized by lung and kidney involvement. Orthohantaviruses found in Eurasia cause hemorrhagic fever with renal syndrome (HFRS) and New World orthohantaviruses cause hantavirus cardiopulmonary syndrome (HCPS). However, cases of infection with Old World orthohantaviruses with severe pulmonary manifestations have also been observed. Therefore, human airway cells may represent initial targets for orthohantavirus infection and may also play a role in the pathogenesis of infections with Eurasian orthohantaviruses.Transmission of all known pathogenic orthohantaviruses  in vitro. In addition, we examined the expression of orthohantaviral receptors in these cell types. To minimize donor-specific effects, cells from two different donors were tested for each cell type.3 was not detectable in these cell types. In addition, replication kinetics and viral release demonstrate enormous donor-specific variations.Productive infection with PUUV was observed for endothelial cells of the microvasculature and for the three tested epithelial cell types derived from different sites of the respiratory tract. Interestingly, infection and particle release were also detected in bronchial and bronchiolar epithelial cells although expression of the orthohantaviral receptor integrin βVβ3 surface expression on bronchial and small airway epithelial cells indicates an alternate mode of orthohantaviral entry in these cells that is independent from integrin β3.The human respiratory epithelium is among the first targets of orthohantaviral infection and may contribute to virus replication, dissemination and pathogenesis of HFRS-causing orthohantaviruses. Differences in initial pulmonary infection due to donor-specific factors may play a role in the observed broad variance of severity and symptoms of orthohantavirus disease in patients. The absence of detectable levels of integrin α Infections with orthohantaviruses, formerly named hantaviruses, cause two diseases: hemorrhagic fever with renal syndrome (HFRS) by Old World orthohantaviruses found in Eurasia and hantavirus cardiopulmonary syndrome (HCPS) by New World orthohantaviruses of the American continents , 2. HFRSThe clinical picture of infectious diseases depends on the tropism of the invading pathogen. Infection of target cells contributes to the clinical symptoms and is responsible for the dysfunction or even failure of the affected organ. Histopathological analysis of autopsy samples derived from HCPS patients infected with the New World orthohantavirus Sin Nombre (SNV) demonstrates that viral antigen was found in a number of organs such as lung, kidney, spleen, intestine, or lymph nodes . The preDetection of orthohantaviral genomes and antigen in endothelial and epithelial cells of various tissues indicates a broad range of organ tropism and may explain the extrarenal manifestations observed in Old World orthohantavirus infection. HFRS-cases complicated by acute respiratory distress syndrome caused by HTNV are described in China and cases with severe respiratory symptoms are reported in the context of PUUV infections in Europe , 18–22. Orthohantaviruses are transmitted via inhalation of aerosols containing virions excreted from infected host animals. Therefore, cells of the respiratory tract may represent initial targets for infection by inhaled particles. However, replication of Old World orthohantaviruses in lung cells is not investigated in detail so far. To analyze if pulmonary cells may serve as primary entry site in human infections with Old World orthohantavirus PUUV, we examined receptor expression and infection of human primary cells derived from different parts of the respiratory tract.2. Vero E6 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum.Primary human pulmonary microvascular endothelial cells (HPMEC), human small airway epithelial cells (HSAEpC), and human bronchial epithelial cells (HBEpC) were obtained from PromoCell (Germany). Human pulmonary alveolar epithelial cells (HPAEpiC) were purchased from ScienCell (USA). The cells were maintained in Endothelial Cell Growth Medium MV2, Small Airway Epithelial Growth Medium, Airway Epithelial Growth Medium , and Alveolar Epithelial Cell Medium (ScienCell). Only cells from passages two to six were used. Cells derived from two different donors were analyzed for every cell type. Each individual is indicated by a three digit numeral identity code: HPMEC: donor #011 and #016; HBEpC: donor #359 and #404; HSAEpC: donor #210 und #306; HPAEpiC: donor #144 and #167. Cells were seeded at 10,000 cells/cm5 IU/ml) was added at a multiplicity of infection (MOI) of 1 to primary pulmonary cells. After incubation for two hours at 37 °C, unbound virus was removed by triple washing with the appropriate medium. Infection was analyzed by immunofluorescence and Western blot analysis for orthohantaviral nucleocapsid protein (N protein). Infection kinetics were monitored by immunofluorescence staining of N protein and quantification of the percentage of positive cells. Experiments were performed in triplicates for each donor. Representative figures of day 6 post infection were shown in Figs. Puumala virus (PUUV) strain Vranica isolated from bank vole , 28 was To quantify the release of infectious particles from pulmonary cells, a first round of infection assay in Vero E6 cells was performed. Number of initially infected cells was quantified early after inoculation (48 h post inoculation) during the first replication cycle to avoid an overestimation of viral titers by second rounds of infection.Cell-free supernatants from infected human pulmonary cells were collected on day two, four and six after infection and 10 µl of supernatant were added to Vero E6 cells for two hours. Inoculum was removed by a triple washing with medium. After 48 h, the total number of initially infected Vero E6 cells was quantified by immunofluorescence staining of PUUV N protein. The total number of infected Vero E6 cells corresponds to the number of infectious units (IU) in 10 µl. Titers were expressed as IU per ml supernatant. Experiments were performed in triplicates. Representative images of Vero E6 monolayers inoculated with supernatant of day 6 after infection were taken and shown in Figs. Vβ3, clone LM609  (Millipore). Cell nuclei were stained with Hoechst 33342 (Invitrogen). Images were taken using an Axiocam 506 camera attached to an Axio Observer.D1 microscope (Zeiss). For Western blot analysis, the following primary antibodies were used: rabbit anti-PUUV N protein, rabbit anti-integrin β3  (Santa Cruz) and mouse anti-α-tubulin (Sigma). Equal loading was verified by the detection of tubulin on the same membrane. Detection was performed by using near infrared fluorescent dye (IRDye)-conjugated secondary antibodies and an Odyssey infrared imaging system (Li-Cor Biosciences).For immunofluorescence, cells grown on coverslips were fixed, permeabilized and stained with primary and appropriate fluorescently-labelled secondary antibodies. The following antibodies were used: mouse anti-CD31  (Dako), mouse anti-cytokeratin 18  (Millipore), mouse anti-PUUV nucleocapsid protein (Progen), and mouse anti-integrin αVβ3  antibody, clone LM609 (Millipore) or the corresponding isotype control. After one hour of incubation on ice, cells were washed and analyzed by flow cytometry with FACSCalibur (BD Pharmingen). Flow cytometry analysis of cell viability was monitored by using Via-Probe™ Cell Viability Solution (BD Pharmingen) according to manufacturer’s instructions.Cells were washed, scraped and stained with allophycocyanin (APC)-conjugated mouse anti-CD55  antibody (BD Pharmingen) and phycoerythrin (PE)-conjugated mouse anti-integrin αP values of < 0.05 were considered significant. *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001; ns: not significant.Data were analyzed using Prism 5.0 (GraphPad Software Inc.). Values of two groups were compared using two-tailed Student’s t-test. Vβ3 was observed by immunofluorescence  for their permissiveness and susceptibility to PUUV Fig. . The endnce Fig. A.Inoculation of HPMECs with PUUV resulted in productive infection as shown by the detection of N protein in cells, lysate and supernatant (SN) Fig. B. The reVβ3 heterodimer was not detectable. In contrast, expression of integrin αVβ3 was present in HPAEpiCs  that have no detectable integrin αVβ3 expression  that express integrin αThe respiratory tract is the first region of contact for many pathogens. The ability of the pathogen to enter and to replicate in certain pulmonary cell types may influence the clinical picture and disease severity as shown for influenza virus and coronaviruses , 32. HowCell culture infection studies in pulmonary cells also exist for orthohantaviruses. In vitro infection of human lung microvascular endothelial cells with SNV or HTNV in a 3D human lung tissue model results in the induction of chemokine expression and may contribute to an immune-mediated increase in vascular permeability . StudiesOur work with human primary pulmonary endothelial and epithelial cells demonstrates the permissiveness of human cells within different parts of the respiratory tract for the Old World orthohantavirus PUUV. Epithelial and endothelial cells of the lung support orthohantaviral replication as observed for the infection of different cells from the kidney, which represent epithelial  and endothelial  cell types , 45, 46.vβ3 or integrin β3 subunit. The absence of integrin β3 expression in normal human bronchial epithelium was also reported in several studies analyzing tissue samples or primary human bronchial epithelial cells [3 was described to serve as entry receptor for pathogenic orthohantaviruses by cell culture studies [3 expression was also observed [. describe that orthohantaviral entry in human airway epithelial cells depends on macropinocytosis and identified differences between the New World orthohantavirus ANDV and the Old World orthohantavirus HTNV [Interestingly, productive infection of bronchial and small airway epithelial cells occurred without detectable expression of integrin αal cells –49. Inte studies , 14. In  studies , 50–53. observed –56. Torrrus HTNV . As a coThe replication analysis revealed differences in the kinetics of PUUV infection and release between donors. Donor-specific variation in permissiveness was also described for the infection of human airway epithelial cells with H1N1 Influenza virus and Nipah virus , 59. TheBased on the detection of orthohantaviral antigen in lungs of infected patients and the results from in vitro cell culture studies, pulmonary cells may represent target cells of orthohantavirus infection. The release of infectious particles indicates a possible viral spread in lung tissue. Nevertheless, a person-to-person transmission is not described for orthohantaviruses with the exception of ANDV –70. The In summary, the replication of orthohantaviruses in pulmonary epithelial cells may play a pivotal role in the initial phase of transmission, virus dissemination, and pathogenesis of pulmonary symptoms in orthohantavirus disease. Differences between donors and orthohantavirus species in pulmonary infection should be investigated in more detail in future work.Orthohantaviruses are transmitted via inhalation of contaminated aerosols and infections are characterized by specific manifestations in kidney and/or lung. As shown for renal cells, pulmonary cells may represent target cells of PUUV infection. The infection of bronchial and bronchiolar cells occurs in the absence of detectable integrin β3 expression indicating an alternate mode of entry in pulmonary cells. Patient-specific differences in the initial pulmonary replication as observed in vitro for cells from different donors may contribute to the severity of the clinical course. Therefore, the detailed analysis of orthohantaviral replication cycle in pulmonary cells is of special interest and will provide helpful insights in the mechanisms of orthohantaviral initial infection and its impact on pathogenesis.


As one of the leading causes of bone fracture in postmenopausal women and in older men, osteoporosis worldwide is attracting more attention in recent decades. Osteoporosis is a common disease mainly resulting from an imbalance of bone formation and bone resorption. Pharmaceutically active compounds that both activate osteogenesis, while repressing osteoclastogenesis hold the potential of being therapeutic medications for osteoporosis treatment. In the present study, sesamin, a bioactive ingredient derived from the seed of Sesamum Indicum, was screened out from a bioactive compound library and shown to exhibit dual-regulating functions on these two processes. Sesamin was demonstrated to promote osteogenesis by upregulating Wnt/β-catenin, while repressing osteoclastogenesis via downregulating NF-κB signaling . Furthermore, DANCR was found to be the key regulator in sesamin-mediated bone formation and resorption . In an ovariectomy (OVX)-induced osteoporotic mouse model, sesamin could rescue OVX-induced bone loss and impairment. The increased serum level of DANCR caused by OVX was also downregulated upon sesamin treatment. In conclusion, our results demonstrate that sesamin plays a dual-functional role in both osteogenesis activation and osteoclastogenesis de-activation in a DANCR-dependent manner, suggesting that it may be a possible medication candidate for osteoporotic patients with elevated DNACR expression levels. Osteoporosis is a chronic bone disease with deteriorated bone tissue microstructure and reduced bone quality. Osteoporosis complications include fractures, chronic pain and secondary osteoarthritis, which may further increase the patients’ suffering and financial burden . CurrentPromotion of osteogenic differentiation and repression of osteoclast formation were revealed to be effective treatment methods for osteoporosis therapy. As reported before, Wnt/β-catenin and NF-κB signaling pathways play major roles in these two differentiation processes, respectively . In ostePrevious studies revealed that small molecules promoted osteogenesis by activating Wnt/β-catenin signaling, or repressed osteoclastognesis via deactivating NF-κB signaling activities. Li’s group reported that lithium released from lithium chloride/calcium phosphate cement could activate osteogenic differentiation and bone regeneration by activating Wnt/β-catenin signaling pathway, and further ameliorate osteoporosis progression . Zhao etA cell-based luciferase activity assay was applied to identify compounds that modulate osteogenesis and osteoclastogenesis activity simultaneously that could then be used in pharmacological proof of concept studies in vivo. To identify small molecules that could potentially stimulate osteoblast and repress osteoclast differentiation, concurrently, we designed a high-throughput screening system that allowed us to measure the luciferase activity of master regulators during osteogenesis and osteoclastogenesis. The TOPFlash vector reflects Wnt/β-catenin transcription activity, while NF-κB response element (RE) vector is responsive to NF-κB signaling activity. The luciferase activities of TOPFlash and NF-κB RE vectors upon treatment of small molecules were plotted, respectively A,B. The We further tested the ability of sesamin to promote BMSCs osteogenesis by activating the Wnt/β-catenin signaling pathway. The viability of mouse bone marrow MSCs treated with sesamin revealed no significant effects at concentration ranging from 1 to 10 μM A. The BMThe osteoclastogenesis of osteoclast progenitor cell line, RAW264.7 was induced with RANKL, with the co-treatment of sesamin. The cytotoxicity assay showed that sesamin had no effect on osteoclast precursor cells at concentrations from 0 to 10 μM A. SesamiMany lncRNAs have been demonstrated to serve as critical regulators in osteoporosis. In these studies, we propose that a ubiquitously expressed lncRNA in musculoskeletal tissues maybe a universal modulating factor and regulated by sesamin in osteoporosis. To identify the lncRNA targeted by sesamin , literature review was performed to screen out possible candidate lncRNAs that may regulate both osteoblast and osteoclast differentiation processes. We identified one lncRNA, DANCR which could exert both osteogenesis repressing and osteoclastogenesis promoting functions . Here, wWe also studied the mediating effect of DANCR on sesamin repressed osteoclastogenesis. DANCR expression in RAW264.7 cells was inhibited by sesamin treatment A. The IκWe next determined the efficacy of sesamin in osteoporosis in vivo. In the OVX-induced osteoporosis mouse model, two groups of mice were administrated with two different doses of sesamin daily  for two months. The femurs of the sacrificed mice were collected for analysis. Micro-CT analysis revealed that the altered cancellous bone microstructure at the distal site of the femur after OVX were significantly improved by sesamin treatment A. The boWe further performed histological and histomorphometrical analysis of the distal femur metaphysis. TRAP staining indicated that the osteoclast amount in the trabecular bone surfaces were reduced upon OVX surgery. Sesamin treatment increased the TRAP-positive staining area (red arrows) in the distal head of the femurs compared with the OVX group, at both low and high dosage. Immunostaining showed that OCN and OPN expression were reduced in the OVX group as compared to the Sham group, while their expression was partially enhanced in the Sesamin-L and Sesamin-H groups A. The voIn the present study, sesamin was found to promote osteogenesis by activating Wnt/β-catenin while repressing osteoclastogenesis via de-activating NF-κB signaling pathways. Further investigation of the underlying mechanisms demonstrated that DANCR was involved in sesamin mediated osteogenesis and osteoclastogenesis , respectively. Knockdown of DANCR could abolish the stimulating effect of sesamin on Wnt/β-catenin as well as its repressive effect on NF-κB signaling activities. Therefore, our study provides a new mechanism of sesamin in osteoporosis prevention and may represent a therapeutic candidate for osteoporotic patients.For osteoporosis treatment, several pharmacological medications could be used with the aim of increasing bone mass and strength by inhibiting bone resorption or promoting bone formation . HoweverIn recent years, the noncoding RNAs (ncRNAs) were revealed to be largely involved in the regulation of osteoporosis. Among them, long non-coding RNAs (lncRNAs) with a length of over 200 bases have recently been identified as novel regulators of osteoporotis related gene activity . The regOsteoporosis is a common disease mainly resulting from the imbalance of bone formation and bone resorption. In our study, we found that upon ovariectomy, the bone resorption in the trabecular bone of the mouse femur was enhanced while the bone formation was inhibited as revealed by a significant reduction of bone microstructure and reduced bone mechanical properties. The expression level of TRAP, a specific marker of bone resorption was increased, while OPN and OCN, the late-stage markers of osteoblastic differentiation and bone regeneration were deceased in OVX mice compared with Sham control. Sesamin treatment could effectively reverse this trend of OVX-induced bone loss. Previous studies also revealed a significant upregulation of DANCR in blood mononuclear cells (MNCs), and DANCR could promote the expression of IL-6 and TNFα expression and elevated the bone resorption activity of MNCs in postmenopausal women . In our In summary, our results show that the efficacy of sesamin in osteoporosis is through the regulation of DANCR expression and the downstream Wnt/β-catenin and NF-κB signaling pathways during osteogenesis and osteoclastogenesis, respectively. This study elucidates a new mechanism/role of DANCR in mediating the beneficial effects of sesamin in postmenopausal osteoporosis treatment and sheds light on developing sesamin as a novel agent for osteoporotic patients, especially for postmenopausal women with high serum DANCR levels. However, although sesamin is considered as a nutraceutical in foods and supplement products, intake of sesamin from daily foods could be not sufficient for anti-osteoporosis activity because of its poor solubility and chemical instability, which results in low bioavailability. Thereafter, the dosage of sesamin we applied for osteoporosis treatment is much higher than the clinical dosage of odanacatib and romosozumab. The physiochemical properties of this natural molecule will be studied as to optimize its delivery capability and to expand its application in clinical practice.Selleck natural product library was purchased from Selleck Chemicals . This collection contains 143 active compounds of nature product selected by a team of medicinal chemists and pharmacists for their high structural and pharmacological diversity, high cell permeability and effective bioavailability and reliable safety. The compounds were supplied as DMSO solution at the concentration of 10 mM.2 incubator at 37 °C.The human embryonic kidney 293 (HEK293) cells and mouse mononuclear macrophage-like cell line RAW 264.7 were obtained from ATCC and was cultured with Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin/neomycin (PSN). The mouse bone marrow mesenchymal stem cells (BMSCs) were isolated and kept in our laboratory as previously described . The BMSThe DANCR small interfere RNAs (si-DANCR) and control siRNAs (si-NC) were synthesized by GenePharma . Wnt/β-catenin response element (RE) luciferase reporter vector TOPFlash, NF-κB response element (RE) luciferase reporter vector and Renilla luciferase control reporter vectors were purchased from Upstate Cell Signaling . The siRNA as well as plasmids were transfected using Lipofectamine 3000  following the manufacturer’s instructions.A high-throughput luciferase activity screening method was applied to screen out the putative natural products which regulate promoter luciferase activities. The HEK293 cells were seeded in 24-well plates at the density of 5000 cells/well. When the cells were 80% confluent, they were co-transfected with Renilla vector together with TOPFlash or NF-κB RE reporter vector, respectively. The cells were then treated with chemicals from Selleck small product chemical library at the final concentration of 10 μM. After one-day treatment, the cells were harvested and applied to dual-luciferase assay according to the instructions of dual-luciferase assay reagent  with some modifications. In brief, cell lysate was placed into a PerkinElmer VictorTM X2 2030 multilateral reader  to measure the firefly luciferase activity as well as the renilla luciferase activity. The ratio of firefly luciferase to renilla activity in each sample was revealed as a measurement of the normalized luciferase activity. Triplicated experiments were performed.3/well) or RAW246.7 cells (5 × 104/well) were seeded in 96-well plate and cultured overnight. The cells were then treated with various concentrations of sesamin for 72 h. After that, 10 μL of 0.5 mg/mL MTT was applied into each well and incubate at 37 °C for additional 4 h. After incubation, the supernatant was discarded and 100 μL DMSO was applied for solubilization of formazan crystals. The light absorbance at 570 nm was measured with a Spectramax Gemini dual-scanning microplate reader .The effect of sesamin on the viability of BMSCs and RAW246.7 cells were measured by using 3--2,5-diphenyltetrazolium bromide (MTT) assay. In brief, BMSCs  with the addition of sesamin . The ALP activity assay was performed 7 days post-osteogenesis induction following the previous protocol [To induce the BMSCs osteogenesis, the mouse BMSCs were seeded in 12-well plates. When reached 90% confluency, the osteogenesis induction was applied to the cells by using StemProprotocol . The cel4 cells/cm2. RANKL (50 ng/mL) and sesamin  or DMSO were applied to the cells for the osteoclastogenesis induction. After 5-day induction, the cells were fixed in 4% paraformaldehyde for 10 min and stained with Tartrate-resistant acid phosphatase (TRAP) activity kit according to the manufacturer’s instructions . The TRAP-positive multinucleated (nuclei > 3) cells were scored as osteoclasts under a light microscope.Raw 264.7 cells were seeded in 24-well plate at the density of 5 × 10n = 24) or sham  surgery. OVX surgery was performed by removing the bilateral ovaries as described previously [All animal experiments were approved by the Ethics Committee of Chinese University of Hong Kong and performed in accordance with the Code of Ethics of the World Medical Association. Thirty-two C57BL/6 mice  were subjected to either ovariectomy  as described before [To analyze the microstructure of distal femoral metaphysis, the high-resolution micro-computed tomography (μCT) 40  with a voltage of 70 kV and 10.5 μm isotropic resolution was used. The gray-scale images were employed to perform three-dimensional (3D) reconstruction of the mineralized tissues using a fixed threshold , Toluidine blue O staining  and IHC staining according to manufacturer’s instruction. For IHC staining, the sections were incubated with primary antibodies including rabbit anti-OCN  and rabbit anti-OPN  overnight at 4 °C. The horseradish peroxidase-streptavidin system  was used for signal detection which followed by counterstaining with hematoxylin.To process the un-decalcified femur specimens, the femurs were fixed by 10% buffered formalin overnight and embedded in methyl methacrylate. The samples were sectioned at 7-μm thickness by RM2155 hard tissue microtome . The sections were stained by the von Kossa/nuclear fast red method as well as Masson-Goldner trichrome staining technique following the previous protocol . The uns−ΔΔCq method and normalized by using GAPDH expression level. The primers used were listed in Total RNA from tissue culture was extracted with TRIzol™ reagent . RNA from mice serum was extracted using TRIzol LS Reagen . The cDNA was reversely transcribed by using PrimeScript RT Master Mix  according to the manufacturer’s protocols. Real-time PCR was performed using Power SYBR Green PCR Mix  on the QuantStudio 7 Flex Real-time PCR system . The target gene expression level was calculated by the 2Total protein of harvested cells was lysed by RIPA buffer  which supplemented with protease inhibitor cocktail . Collection of cytoplasmic and nuclear protein was performed using NEPER Nuclear and Cytoplasmic Extraction Reagents  according to the manufacturer’s protocol. Whole cell extracts, cytoplasmic fractions or nuclear extract protein samples were electrophoresed on the SDS-PAGE gel and electroblotted onto a PVDF membrane as described in previous studies . The PVDt-test for parametric data and by Mann–Whitney U test for non-parametric data analysis. The analysis was performed with GraphPad Prism 8 . p < 0.05 was revealed as significant difference.All data were presented as mean ± standard deviation. Experiments were repeated independently at least three times, and representative data are shown. The statistical significance between two groups was calculated by unpaired, two-tailed Student’s


Extracorporeal membrane oxygenation (ECMO) has been well described as a viable option for patients in need of temporary supplemental oxygenation when ventilator capabilities have failed to augment a patient’s condition. Less described is the potential use of ECMO for lung protection in the setting of gigantic bullae despite initially adequate oxygenation.We describe how the early incorporation of ECMO in a patient with coronavirus disease 2019 and necrotizing pneumonia complicated by multiple large and gigantic bullae led to a favorable outcome.The decision to start ECMO early, despite room for ventilator oxygenation adjustments, may have helped to prevent potential, significant complications such as tension pneumothorax while on positive pressure, thus potentially optimizing the outcome in this patient. Venovenous extracorporeal membrane oxygenation (VV-ECMO) is a known treatment strategy that can be used for refractory respiratory failure by running the patient’s deoxygenated blood through a membrane oxygenator before returning the blood to the area of the right atrium.6Indications for ECMO in the setting of severe acute respiratory distress syndrome (ARDS) secondary to COVID-19 infections have yet to be defined. A review of 10 case reports and three case series failed to outline any specific usefulness of ECMO in the setting of coronavirus infections with severe ARDS sequelae.72) as well as increasing positive end expiratory pressure (PEEP).10While ECMO hasn’t been shown to have specific benefits in COVID-19 infections, ECMO may be advantageous in severe but potentially reversible ARDS as well as surgical augmentation. However, ECMO as an option for lung protection in the setting of acute respiratory failure requiring mechanical ventilation with underlying gigantic pulmonary bullae has not yet been reported. It is already known that underlying bullae raise the risk for spontaneous pneumothorax.We discuss how early initiation of VV-ECMO was used to protect a patient with gigantic bullae requiring mechanical ventilation from developing an iatrogenic pneumothorax while on positive pressure.2 through bag-valve-mask, and a core temperature of 36.7° Celsius.A young adult patient presented to our emergency department (ED) via emergency medical services (EMS) as a medical code secondary to respiratory distress and altered mentation. The unidentified male with an estimated age between 20–30 was in respiratory extremis and unable to communicate with providers. Per EMS, when they arrived at the house they found the patient in the bathtub, minimally responsive with a pulse oximetry of 60%. The patient was immediately brought to the ED. Initial Glasgow Coma Scale was six. Vital signs showed a heart rate of 161 beats per minute, blood pressure of 116/75 millimeters of mercury (mm Hg), tachypnea at 34 breaths per minute (bpm), pulse oximetry 90% on 100% FiO2 of 100%. The patient was also started on broad-spectrum antibiotics and sedated with dexmedetomidine and propofol infusion. Post-intubation chest radiograph (CXR) showed bilateral interstitial infiltrates as well as a gigantic bleb in the right upper lobe , rate of 16, PEEP of 5 mm Hg, and FiOper lobe .Thoracic computed tomography obtained while within the ED showed a necrotizing multifocal pneumonia with lower lobe predominant bronchiectasis, giant right upper lobe bulla, and large right lower lobe bulla , 3.9/liter (L) (reference range: 4.5–11.0 109/L), lactic acid of 4.4 millimoles per liter (mmol/L) (0.5–2.2 mmol/L), and a positive COVID-19 screen. Initial arterial blood gas showed a pH of 7.155 (reference range: 7.35–7.45), partial pressure of carbon dioxide (PCO2) of 75.4 mm Hg (35–45 mm Hg), partial pressure of arterial oxygen (PaO2) 115.5 mm Hg (80–100 mm Hg), and bicarbonate (HCO3) 25.8 milliequivalents per liter (mEq/L) (reference range: 38–42 mEq/L).Initial laboratory evaluation showed an elevated white blood cell count of 48.0 × 102 and PEEP of five centimeters of water (cm H20). A STAT repeat CXR confirmed the absence of a pneumothorax, and bedside ultrasound showed a hyperdynamic left ventricle and flat inferior vena cava. The patient was bolused three liters of normal saline while central venous and arterial lines were placed, after which norepinephrine was initiated.A few hours into the patient’s stay, while still in the ED he developed hypotension and hypoxia to 88% on 100% FiOWhat do we already know about this clinical entity?Extracorporeal membrane oxygenation (ECMO) has been shown to be advantageous in severe but potentially reversable acute respiratory distress syndrome as well as surgical augmentation.What makes this presentation of disease reportable?ECMO has not yet been reported as an option for lung protection in the setting of respiratory failure requiring mechanical ventilation with underlying gigantic pulmonary bullae.What is the major learning point?Early initiation of venovenous ECMO in the setting of gigantic bullae, may have helped to avoid an iatrogenic pneumothorax while under positive pressure ventilation.How might this improve emergency medicine practice?May lead to early identification for the involvement of ECMO equipped facilities to expedite collaboration with critical consultants.20, rate of 24 bpm, 100% FiO2, and a PEEP of 5 cm H20. Repeat arterial blood gas showed a pH 7.19, PCO2 71.5 mm Hg, PaO2 84.7 mm Hg, and HCO3 of 27.2 mEq/L. The patient continued to desaturate down to 85% on 100% FiO2, so PEEP was increased from five to eight cmH20. Because of the patient’s gigantic bullae, worsening oxygenation and hypercapnia, and concerns for inevitable iatrogenic pneumothorax, the decision was made to consult the ECMO team. Of note, at this time the patient’s arterial oxygenation was not in a critical range (84.7 mm Hg), and in a typical situation this value alone would not be a typical indication for ECMO. The ECMO team agreed that due to the complicated nature of the patient’s disease process and underlying severity of lung injury he was an appropriate candidate for VV-ECMO. The patient was taken emergently to the operating room and received a right ventricular assist device, and VV-ECMO was initiated.He then developed high peak pressures. Due to the high pressures, the ventilator settings were transitioned to pressure control with a driving pressure of 18 cm HStaphylococcus aureus (MRSA). The patient received daily chest CXRs post-ECMO cannulation to tract placement and lung pathology. Post-extubation chest CXR the following day showed no new pathology; however, on the second morning post extubation he was found to have developed an asymptomatic spontaneous pneumothorax on routine chest CXR. The patient remained hemodynamically stable and did not require tube thoracostomy, potentially aiding to an outcome that would have been drastically different if he had still been on positive pressure ventilation.Post VV-ECMO initiation, the patient was extubated in under 24 hours and transitioned to 3L nasal cannula. Sedation was weaned and the patient quickly became fully alert and was able to follow commands and communicate. On hospital day 3 his blood cultures grew positive for methicillin-resistant The final diagnosis was established as acute hypoxic respiratory failure due to COVID-19 pneumonia with superimposed MRSA. The patient was discharged to his mother’s house with home health care and home oxygen on hospital day 29 on two liters nasal cannula.2. Increasing this patient’s PEEP to correct for hypoxia likely would have led to positive pressure iatrogenic pneumothorax, which would have further decompensated the patient’s already poor pulmonary baseline. This hypothesis is further supported by the presence of a spontaneous pneumothorax two days post extubation. By initiating ECMO early, mechanical ventilation was discontinued prior to the development of a spontaneous pneumothorax, thus avoiding significant complications and mortality associated with a pneumothorax under positive pressure.As demonstrated by this case, the early initiation of VV-ECMO in the setting of gigantic bullae helped to avoid an iatrogenic pneumothorax while under positive pressure ventilation, decreased the number of days on mechanical ventilation, maintained hemodynamic stability in the setting of spontaneous pneumothorax, and likely led to an overall decrease in intensive care unit stay. Very early on our patient developed signs of worsening oxygenation status despite 100% FiOOur report is not without limitations. This treatment strategy requires a facility equipped with ECMO capabilities. While this may not be generalizable to all health centers, it does provide a potential strategy whereby one may begin to engage early on with nearby ECMO-capable facilities for potential transfer. Additionally, because this report was a discussion of one patient there was the possibility of unseen variables that potentially helped impact personal outcome.Further studies into the use of ECMO to prevent significant complications associated with gigantic bullae and positive pressure ventilation may provide useful information regarding patient care. This information may aid in the earlier identification of disease, necessity of possible transfers to ECMO-equipped facilities, expedited involvement of critical consultants, and potential for favorable patient outcomes.


Diagnostic and treatment guidelines serve several important purposes with an overall aim to improve medical care. The 2021 American Heart Association (AHA)/American College of Cardiology (ACC) Chest Pain Guidelines . For theJournal of the American College of Cardiology [Circulation [Journal of Cardiovascular Magnetic Resonance. Reviewing the full Guidelines is necessary to get a detailed appreciation for how the various imaging modalities are ‘weighted’ in particular recommendations. Recommendation number from the full document is included for reference  patients, defined as those with a 30-day risk of death or major adverse cardiovascular event (MACE) < 1%, the 2021 AHA/ACC Chest Pain Guidelines provide a class 2a recommendation indicating that it is reasonable to discharge the patient home without hospitalization or urgent cardiac testing. This is one of the “Top 10 Take-Home Messages” of the updated guidelines.Reducing imaging indications will reduce imaging costs and the number of cardiac tests that confirm no evidence of significant CAD. While an initial strategy of no-testing is appropriate in these patients, testing remains an option for patients with persistent or worsening symptoms. From a CMR perspective, there is time to schedule appropriate patients in a non-acute setting rather than doing imaging as an emergency procedure.without known CAD (4.1.2.1.4). Stress imaging, including CMR, is given a class 2a recommendation for sequential testing after an inconclusive coronary computed tomography angiographic (CCTA) study (4.1.2.1.7). The 2021 AHA/ACC Chest Pain Guideline puts CMR on par with the other stress imaging modalities regardless of whether or not a patient can exercise or whether or not the electrocardiogram (ECG) is interpretable  along with CCTA, in patients with previously known non-obstructive CAD, and without any preference of one modality over the others. This recommendation also brings CMR to the same level as the other non-invasive imaging modalities. Of note, among those with known CAD, exercise testing without imaging is no longer considered an appropriate study.For intermediate risk patients with acute chest pain and There is a class 2a recommendation for CMR or echocardiography to establish alternative diagnoses once obstructive CAD has been excluded by CCTA or invasive coronary angiography (ICA). CMR has become well established for detecting other pathologies such is myocardial infarction with no obstructive coronary arteries (MINOCA) (4.2.3.1) or myocarditis which can present acutely in the absence of obstructive CAD (4.1.3.3). Stress CMR could also be used to establish the diagnosis of ischemia in patients with no obstructive coronary arteries (INOCA) or microvascular disease (5.2.2.3).The 2021 AHA/ACC Chest Pain Guidelines favor computed tomography (CT) as the first choice in the assessment of possible acute aortic syndromes given its faster speed and wider availability. CMR is given a class I recommendation as an alternative if CT is contraindicated or unavailable (4.2.1.2).In patients with possible myopericarditis (4.2.3.2), the new guidelines give CMR a class I recommendation for distinguishing myocarditis from other causes and for assessing myocardial and pericardial inflammation and fibrosis. These recommendations align with increasing community awareness of CMR as the test of choice for an indication often labeled MINOCA. (4.2.3.1).The 2021 AHA/ACC Chest Pain Guidelines also support a class 2a recommendation among patients with acute chest pain and known or suspected valvular heart disease if transthoracic echocardiography (TTE) or transesophageal echocardiography (TEE) are not technically adequate for assessing valvular heart disease 4.2.4.3). CMR has the ability to objectively quantify the severity of regurgitant heart lesions . Again, CMR is not differentiated from the other stress imaging modalities.In patients with known obstructive CAD who have stable chest pain despite optimal therapy, stress imaging receives a class I indication for diagnosing myocardial ischemia, estimating risk of MACE, and guiding therapeutic decision making (5.2.1.5). There is an additional new class 2a indication for both PET and CMR adding quantification of myocardial blood flow reserve (MBFR) to improve diagnostic accuracy and enhance risk stratification (5.2.1.8). This is particularly important given the growing availability of CMR techniques for performing absolute quantification of myocardial blood flow.In patients with known non-obstructive CAD, stress CMR received a Class 2a recommendation for assessing INOCA (5.2.2.3). Stress CMR with the addition of quantitative myocardial blood flow assessment is given a class 2a recommendation for the diagnosis of coronary microvascular dysfunction and for assessing MACE (5.2.3.3).“In a 55-year old male patient with atypical angina CCTA, single photon emission computed tomography (SPECT), PET and stress CMR can reliably rule-out anatomically significant CAD but stress ECG or stress echocardiography cannot (A). To assess the performance of imaging tests to detect functionally significant CAD ) in the same patient (B) one can see that PET and stress CMR results can both rule-out and rule-in significant CAD while CCTA can only confidently rule-out if a negative result is documented. ICA and SPECT are not recommended tests in this patient.”  cannot confidently rule-in functionally significant CAD and now advocates fractional flow reserve (FFR)-CT to determine functional significance of what is anatomically described as “obstructive CAD” (50–90% stenosis) and extending further to a 40% narrowing. A stress CMR may be more cost-effective than FFR-CT or PET in sequential testing. While, CCTA may be the best test to exclude anatomically significant CAD, PET and CMR can effectively rule-in and rule-out functionally significant CAD.The 2021 AHA/ACC Chest Pain Guidelines have 4 recommendations for FFR-CT in acute and chronic stable chest pain syndromes and FFR-CT was broadly described as an instrument for assessing likelihood of ischemia with established robustness for decision-making in lesions of 40–90% detected on CTA. FFR-CT was displayed in numerous flow charts to represent equivalent class 2a recommendations compared to any other functional imaging modality.However, the diagnostic and prognostic utilities of FFR-CT are not as robustly evidenced as any of the stress imaging modalities . FFR-CT does not improve the sensitivity of CT, and only modestly improves the specificity of identifying flow-limiting obstructive coronary artery lesions when compared with invasive FFR and functional testing –8. FFR-CFurthermore, there are practical implications regarding FFR-CT. FFR-CT is only feasible in a subset of CCTA cases that are relatively artifact-free and remains highly limited in patients with prior coronary artery stenting, extensive calcification, severe valvular heart disease, sequential luminal lesions or prior coronary artery bypass graft surgery. FFR-CT is currently only available by a single company . The CPT Category III code used to reimburse FFR-CT is reserved for emerging technologies. Finally, FFR-CT costs 3-times as much as a standard CCTA.The 2021 AHA/ACC Chest Pain Guidelines put all stress imaging modalities in the same “functional imaging” group for the purpose of comparison against CCTA, as shown in many flow charts and tables. This approach has little clinical basis but also misses critical attributes of different ischemia tests that may be relevant in the management of a vastly diverse patient spectrum. In the contemporary era, there is clear randomised trial evidence for the use of stress CMR in reducing unnecessary ICA referral or coronary revascularization rates, and thus improving patient care, health outcomes and healthcare resource utilization , 13.The largest randomized trial to date, PROMISE , prospectively evaluated the utility of an anatomic (CCTA) testing approach in comparison with functional testing (stress imaging and treadmill ECG testing) amongst 193 North American centers in over 10,000 patients . After aThe SCOT-HEART (Scottish COmputed Tomography of the HEART) trial conducted within the United Kingdom failed to meet its original primary endpoint, but did observe a late reduction in non-fatal myhocardial infarction (MI) . HoweverThe 2021 AHA/ACC Chest Pain Guidelines do not have clear definitions of “Obstructive CAD”, “Anatomically Significant CAD”, and “Functionally Significant CAD”. These three definitions overlap with each other and contribute to miscommunication among healthcare providers and confuse patients. In general, the 2021 AHA/ACC Chest Pain Guidelines do not systematically differentiate anatomic CAD and functional CAD. This may contribute to an overestimation of the utility of CCTA compared with stress imaging modalities.Invasive FFR has been shown in several multi-center trials to provide better outcomes than management by stenosis severity [“Obstructive CAD” has been used to describe a 50% or greater coronary artery diameter stenosis by quantitative coronary angiography (QCA) and is now widely used to describe CCTA findings. However, the majority of 50–70% stenoses are not severe enough to impair coronary flow reserve . Invasivseverity –19. ThusThe 2021 AHA/ACC Chest Pain Guidelines briefly mention women-specific considerations in text but do not make any formal recommendations that recognize appropriateness or level of evidence. CCTA, SPECT, and PET directly deliver radiation to the breasts. Now that zero ionizing radiation methodologies like CMR can perform as well as CCTA and PET, and are superior to stress echocardiography and SPECT, CMR seems the logical choice for stress perfusion imaging in women if local equipment and expertise is available.CMR has matured into a powerful diagnostic tool as evidenced by the wide range of clinical indications recognized in the 2021 AHA/ACC Chest Pain Guidelines and other international guidelines. Figure The CMR community should be proud of the hard work that provided the data leading to multiple Class I and Class 2a Recommendations, which are now finally more aligned with the 2014 ESC Guidelines on Revascularization in which stress CMR features in Class IA recommendations [CMR and PET appear to have superior diagnostic accuracy compared with SPECT and stress echocardiography. The relatively low specificity of CCTA for functionally significant CAD is a weakness. The reliance on FFR-CT to provide a computer-based substitute for a full physiological assessment of CAD may or may not be cost-effective over the long haul. CMR researchers should also continue to refine quantitative methods as the current Guidelines are the first to formally recognize the value of quantifying myocardial blood flow reserve by CMR. Studies focused on women will help highlight the role of stress CMR versus other modalities in the diagnosis of CAD as well as other causes of symptoms.


PPD symptoms partially mediated and aggravated the association between preterm birth and child mental disorders. Preterm birth, maternal PPD symptoms and child mental disorders are associated, calling for timely prevention interventions.Preterm birth has been linked with postpartum depressive (PPD) disorders and high symptom levels, but evidence remains conflicting and limited in quality. It remains unclear whether PPD symptoms of mothers with preterm babies were already elevated before childbirth, and whether PPD symptoms mediate/aggravate the effect of preterm birth on child mental disorders. We examined whether preterm birth associated with maternal PPD symptoms, depressive symptoms trajectories from antenatal to postpartum stage, and whether PPD symptoms mediated/aggravated associations between preterm birth and child mental disorders. Mothers of preterm (n = 125) and term-born (n = 3033) children of the Prediction and Prevention of Preeclampsia and Intrauterine Growth Restriction study reported depressive symptoms four times within 8 weeks before and twice within 12 months after childbirth. Child mental and behavioral disorder diagnoses until age 8.4–12.8 years came from medical register. Preterm birth associated with higher PPD symptoms (mean difference = 0.19 SD, 95% CI 0.01, 0.37,  It poses a risk for neurosensory impairments, and for physical, mental and behavioural disorders later in life5. Preterm birth also induces substantial challenges and distress among the parents of the preterm infants6. Hence, preterm birth has been implicated as a risk factor for maternal postpartum depression (PPD)7.Preterm birth (< 37 completed weeks of gestation) is the leading cause of perinatal morbidity and mortality8, we systematically assessed the quality of evidence of previous studies testing the association between preterm birth and maternal PPD D Tables  and ST2.D Tables , ST2. Of15, which suggests that PPD may be on a pathway between preterm birth and mental and behavioral disorders in children. One previous study has reported that maternal major depressive disorder (MDD) diagnosis between childbirth and child’s assessment in early childhood mediated the association between lower gestational age and child’s risk for anxiety disorders16. Another study reported that maternal PPD symptoms 9 months postpartum did not mediate the association between preterm birth and child’s cognitive function in early childhood17. However, we are not aware of studies that have tested whether maternal PPD symptoms mediate the associations between preterm birth and child mental and behavioral disorders. It also remains unknown if maternal PPD aggravates the association of preterm birth with these child mental health outcomes.Apart from the implicated association with preterm birth, maternal PPD has been also linked with the child’s higher risk for mental and behavioral disorders18) from the childbirth until 12 months postpartum. Further, to unravel if the depressive symptoms levels of women with a preterm baby were high already in the antenatal stage, we studied if preterm birth was associated with depressive symptoms trajectories from the antenatal to the postpartum stage. We also examined if these associations were independent of maternal mood disorders diagnosed before childbirth, and examined if maternal PPD symptoms mediated the association between preterm birth and mental and behavioral disorders in the children followed-up from birth until mid-childhood. Finally, we examined if the effects of preterm birth and maternal PPD on mental and behavioral disorders in the children were additive. We took into account important covariates and assessed whether maternal obstetric complications associated with the risk of preterm delivery, namely preeclampsia, placenta previa, chorionamnionitis, premature rupture of membranes and mode of delivery19, modified the associations of preterm birth with maternal PPD, depressive symptoms trajectories and child mental and behavioral disorders.We examined in a large pregnancy cohort of Finnish women and their children if preterm birth was associated with maternal PPD symptoms and probable clinical PPD  and 12% (n = 15) were born very or extremely preterm . Preterm born compared with term born children were more often boys. Mothers of preterm born compared with term born children more often had a pregnancy complicated by preeclampsia and premature rupture of membranes, they more often delivered via Caesarean section, especially urgent/emergency Caesarean section, and reported higher levels of antenatal depressive symptoms 8 weeks before childbirth  of the mothers in our sample the first PPD symptoms assessment occurred within the first 10 days after childbirth, a period known as the baby blues period27. Hence, since the vast majority of the mothers in our sample provided their first PPD assessment outside of baby blues period, and all second PPD assessments were way beyond this period, the baby blues period is unlikely to bias the associations. Young age of the children included in this study pertains to another limitation and precludes studying mental and behavioral disorders with peak age at onset beyond childhood, such as substance use disorders or schizophrenia-spectrum disorders28. Finally, our study is also limited by lacking data on paternal depressive symptoms, which may influence the associations. It has been previously demonstrated that in comparison to the fathers of term born children, fathers of preterm born children had elevated depressive symptoms 9 months after childbirth. These elevated paternal PPD symptoms, in turn, predicted child’s lower cognitive function in early childhood17. However, the symptoms did not mediate the associations between preterm birth and child’s cognitive function17.However, our study does not unravel the mechanisms underlying the association between preterm birth and maternal PPD symptoms, and mechanisms underlying their association with increased risk of mental and behavioral disorders in children. While data attrition in the child follow-up was null, our study suffers from selective dropout, as the women who participated in the postpartum follow-up and reported PPD symptoms were older, used substances less, and less often had been diagnosed with mood disorders than the women who did not. However, even selective dropout has been found to only marginally alter predictive modelsIn conclusion, our findings suggest that preterm birth and maternal PPD symptoms are associated. The continuity of depressive symptomatology from antenatal to postpartum period and its associations with preterm birth suggests that addressing depressive symptoms already in pregnancy may have beneficial effects on the risk of preterm birth, PPD and subsequently on child mental and behavioral disorders. Mediation and additive effects of preterm birth and maternal PPD on child’s higher risk for mental and behavioral disorders call for prevention interventions aimed at reducing PPD symptoms in women who gave preterm birth.29. In brief, it comprises 4777 mothers who gave birth to a singleton live-born child in Finland between 2006 and 2010. Recruitment took place at ten study hospitals in Southern/Eastern Finland at the first fetal ultrasound screening in early pregnancy. Three women have since withdrawn from the study. We excluded 6 mother–child dyads with unknown gestational age, 29 with missing Finnish Medical Birth Register (MBR) data, 178 with post-term birth , and 4 whom we did not have data on child mental or behavioral disorders from the Finnish Care Register for Health Care (HILMO). Of the 4559 women, 3158 (69.3%) completed questionnaires on depressive symptoms twice up to 12 months postpartum, and of them 125 (4.0%) gave preterm birth.The Prediction and Prevention of Preeclampsia and Intrauterine Growth Restriction (PREDO) study is described in detail elsewhereDiagnoses of any mental disorder from birth until 31st December 2018, when the children were 8.4–12.8  9.5–10.8) years of age, were available for all children of the 3158 women.Characteristics of the women who did provide the data on PPD and those who did not are shown in the Table +6 gestational weeks + days or less and term birth as birth between 37+0 and 41+6 gestational weeks + days.Gestational age was derived from hospital records and/or MBR. We defined preterm birth as birth at 3618 twice up to 12 months postpartum . The 20 CES-D questions were rated on a scale from none (0) to all of the time (3). Higher scores indicate more depressive symptoms during the past week. We defined PPD symptoms as the mean of the two postpartum CES-D scores and probable clinical PPD as mean CES-D ≥ 1618.The women completed the Center for Epidemiologic Studies Depression Scale (CES‐D)30, a timeframe which is captured by the four biweekly consecutive CES-D measurement points before childbirth.Using the CES-D, the women also rated prospectively biweekly their antenatal depressive symptoms up to 14 times starting from 12 to 14 gestational week until delivery for women who gave birth to a preterm baby and until 38–39 gestational weeks for women who gave birth to a full-term baby. Of these biweekly CES-D measurements we identified the four consecutive measurements before childbirth: we have previously shown in this sample that in women who gave birth to a preterm baby, depressive symptoms begin increasing approximately 8 weeks before childbirth31. We studied any mental and behavioral disorder (ICD-10: F00-F99) as the outcome.We identified diagnoses of mental and behavioral disorders from the HILMO from the birth of the child between 11/07/2006 and 07/24/2010 to 12/31/2018. The HILMO includes primary and subsidiary diagnoses of all inpatient hospital treatments and outpatient visits in specialized medical care coded using the International Statistical Classification of Diseases and Related Health Problems-10 (ICD-10). HILMO is a valid tool for researchThe following variables were included as covariates: maternal age at childbirth (years), smoking at any time during pregnancy (no/any smoking during pregnancy), mode of delivery , child's sex  and age (years) at first mental and behavioral disorder diagnosis with data extracted from hospital records, MBR and/or HILMO; maternal education level  levels 0 to 5)/tertiary (ISCED levels 6 to 8) and alcohol use  were self‐reported in early pregnancy. Smoking and alcohol use were combined into one variable representing smoking and/or alcohol use at any time during pregnancy (yes/no). Maternal mood disorder diagnoses before the childbirth were identified from HILMO . Effect modifiers of preterm birth on maternal and child outcomes included mode of delivery (defined as above), maternal preeclampsia , which was extracted from the hospital records, MBR and /or HILMO and verified by an expert jury comprising two medical doctors and a research nurse with expertise in obstetrics and gynecology, placenta previa (ICD-10 code O44), chorioamnionitis (ICD-10 code O41.1) and premature rupture of membranes (ICD-10 code O42), which were extracted from medical records, MBR and/or HILMO.Associations between preterm birth and maternal PPD symptoms levels were tested using linear regression and with probable clinical PPD using logistic regression.32, the optimal solution was based on (1) goodness-of-fit criteria , (2) reasonable distribution of participants across subgroups (at least 10% of the sample), (3) high certainty of classification identified by posterior probabilities, and (4) clear clinical characteristics of the participants within each of the subgroups. Using the optimal LCA solution, we then tested whether the women who gave birth to a preterm and term baby differed in their odds of belonging to groups differing in their depressive symptoms trajectories identified by the LCA.To identify trajectories of maternal depressive symptoms between antenatal and postpartum stage, we conducted latent class analysis (LCA) based on their four antenatal and two postpartum CES-D scores. We compared solutions with two to six latent classes. Based on criteria for the optimal number of classes described by Kongsted and Nielsen33. Before testing mediation, we ensured that the criteria for mediation were met, namely that the predictor, mediator and outcome variables were associated. Therefore, before proceeding to mediation analyses we also examined if preterm birth and maternal PPD symptoms were associated with mental and behavioral disorders in children by using Cox proportional hazards models. The proportionality assumptions were met .To study whether maternal PPD symptoms mediated the association between preterm birth and mental and behavioral disorders in the children, we used SPSS PROCESS macro with 5000 bootstrapped samplesTo study if mode of delivery, maternal preeclampsia, placenta previa, chorioamnionitis and premature rupture of membranes modified the effects of preterm birth on maternal PPD, depressive symptoms trajectories, and child mental and behavioral disorders, we included interaction terms between these variables and preterm birth into statistical models, followed by main effects of these variables.We then examined if preterm birth and maternal PPD symptoms had additive effects on the hazard of mental and behavioral disorders in children by applying Cox proportional hazards models. For this analysis, we compared the hazards of mental and behavioral disorders in the children who were born at term and whose mothers reported no probable clinical PPD (referent) with (a) children born at term and whose mothers reported probable clinical PPD, (b) preterm children whose mothers reported no probable clinical PPD and (c) preterm children whose mothers reported probable clinical PPD.We report the associations as unadjusted and adjusted for all covariates. Additionally, in examining the associations between preterm birth and PPD, we conducted sensitivity analyses in which we excluded women with a previous history of mood disorders diagnosed before childbirth. Statistical analyses were conducted by using SAS 9.4 , Stata 15  and SPSS-IBM . All methods were carried out in accordance with relevant guidelines and regulations.The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.


DNA methylation and demethylation at CpG islands is one of the main regulatory factors that allow cells to respond to different stimuli. These regulatory mechanisms help in developing tissue without affecting the genomic composition or undergoing selection. Liver and backfat play important roles in regulating lipid metabolism and control various pathways involved in reproductive performance, meat quality, and immunity. Genes inside these tissue store a plethora of information and an understanding of these genes is required to enhance tissue characteristics in the future generation.C7orf50, ACTB and MLC1 in backfat and TNNT3, SIX2, SDK1, CLSTN3, LTBP4, CFAP74, SLC22A23, FOXC1, GMDS, GSC, GATA4, SEMA5A and HOXA5 in the liver, were categorized as differentially-methylated. Subsequently, Motif analysis for DMRs was performed to understand the role of the methylated motif for tissue-specific differentiation. Gene ontology studies revealed association with collagen fibril organization, the Bone Morphogenetic Proteins (BMP) signaling pathway in backfat and cholesterol biosynthesis, bile acid and bile salt transport, and immunity-related pathways in methylated genes expressed in the liver.A total of 16 CpG islands were identified, and they were involved in differentially methylation regions (DMRs) as well as differentially expressed genes (DEGs) of liver and backfat tissue samples. The genes In this study, to understand the role of genes in the differentiation process, we have performed whole-genome bisulfite sequencing (WGBS) and RNA-seq analysis of Nanchukmacdon pigs. Methylation and motif analysis reveals the critical role of CpG islands and transcriptional factors binding site (TFBS) in guiding the differential patterns. Our findings could help in understanding how methylation of certain genes plays an important role and can be used as biomarkers to study tissue specific characteristics.The online version contains supplementary material available at 10.1186/s12864-021-08123-x. Pork is an important high-protein food consumed across the world and requires timely effort to monitor and sustain the quality of meat. Several molecular breeding programs are being run around the world to understand and fulfil future requirements with enhanced food quality which largely depends upon the taste and composition, and these factors ultimately shape the breeding program by the choice of meat , 2. The Despite having the same genome, an unknown mechanism is governing the gene expression, development, genome imprinting, diseases, and diversification, and has been involved in evolutionary changes in different tissues , 10. A sN=5+5) samples of backfat and liver respectively and integrated the analysis results to understand the tissue characteristics. The methylation pattern in the CpG island was further studied to determine the potential role in the hyper-methylated region with the respective expression pattern in the specific tissues. RNA-seq analysis abled us to analyze the differential expression patterns, as well as gene ontology studies, reveals the close association in different biological important pathways that were enriched in various tissue under methylated conditions. Along with this, we also aimed to conduct a de novo whole genome motif analysis to identify the methylated motif and the transcription factor binding sites in terms of overall changes of tissue and specific pathways.In the present work, we reported genes involved in tissue-specific changes at the methylation level and the role of gene expression in the regions. To this end, we performed WGBS and RNA-seq of (https://www.bioinformatics.babraham.ac.uk/projects/seqmonk/), a tool for visualization of high throughput mapped sequence data, we detected a sharp increase at the 2 kb upstream region of the TSS region and at downstream of TTS region. CpG methylation help in stabilizing chromatin structure as well as it also controls the regulation of related gene expression and these effects could overall responsible for stabilization of genome > 99.4 %. The overall methylation composition was inclined towards liver Fig. A and metome Fig. . A DMR sre Table  S. scrofaIn the present investigation, to understand the role of genes involved in tissue-specific diversification, we have presented a comprehensive view of comparative methylation pattern with differentially expressed genes amongst backfat and liver tissue in Nanchukmacdon Pig. Methylation analysis is one of the most promising methods recently evolved and used to accurately decipher the diversification in cross tissue differentiation patterns as well as to identify close relationships amongst different tissues. Studying these patterns will ultimately help us in identifying markers that specifically target breeds to enhance tissues of interest. Therefore, methylation analysis at CpG islands were performed by integrating WGBS with RNA-seq data from different tissues and profiled to identify potential genes and regions that are hyper-methylated and upregulated or down-regulated in backfat and liver in CpG islands which could be playing important roles in tissue-specific diversification.de novo methylated regions and found the rank 1 motif includes “TATA box” promoter sequence, which specifies to other molecules where transcription begins and strongly modulates cell- and tissue-specific RANKL expression and the osteoclastogenesis process [We performed a DMR analysis for  process . We obse process  . The pigs were euthanized with an anesthetic injection given into the ear vein with an overdose of Alfaxan (0.7 mg/kg) and bulk tissue (10 mg) was thereafter collected [In the present study, Nanchukmacdon pigs were grown in farm of National Institute of Animal Science located on Jeju island with close monitoring of their health. Five boars with average age of 26 months were randomly selected for effective size calculation to collect samples for WGBS, and RNA-seq analysis (ollected . Subsequollected . GenomicN=5) after RNA isolation of backfat and liver tissues using the TRIzol method following the prescribed protocol and previously described [gDNA from Nanchukmacdon pig backfat and liver was subjected to bisulfite conversion using the fragment size (250 bp±25 bp), and the sequencing libraries were constructed as previously described . Similarescribed . The seqsscrofa11.1 using Bismark [The WGBS data were analyzed as previously described to understand methylation patterns in the identified genes . Trim_ga Bismark  and the  Bismark –40. LogiS. scrofa genome (Sscrofa11.1) using Hisat2 [Similarly, we performed a RNA-seq analysis as it enables a comprehensive understanding of the expression pattern of tissue-specific changes in genes. With statistical advanced tools, we performed a quality check by FastQC to assess low-quality pair-end reads  and furtg Hisat2 , 44 follg Hisat2 . Finallyg Hisat2  was usedHyper-methylated regions were predicted with a cutoff of ±25 in DMRs in backfat and liver. We were interested in understanding the motif for these methylated regions in GC% of the CpG island found near to the transcription start site and these motif analysis was performed by findMotifsGenome.pl module of HOMER software at the default parameter . Rank-wiAfter identifying DEGs commonly found in backfat and liver, methylated regions with FDR ≤ 0.05 and log2FoldChange ≥±2 were compiled and submitted to DAVID v6.8  for funcSscrofa11.1 as the reference and selected a track for the CpG island. The identified island was used to extract DMRs located in the genomic coordinates and we extracted the region of interest that plays a crucial role in tissue diversification.Based on DMRs, we aimed to identify regions either inclined towards backfat or liver by comparing CpG island coordinates retrieved from the UCSC genome browser . A totalAdditional file 1Figure S1. Comparative methylation pattern of identified genes using SeqMonk.Additional file 2Figure S2. GO results for Biological process (BP), Molecular function (MF), Cellular compartment.Additional file 3File S1. Cytosine methylation report for backfat and liver.Additional file 4File S2. Differentially methylated as well as expressed gene list for backfat and liver.Additional file 5File S3. Gene ontology studies of identified genes in hypermethylation condition identified in backfat and liver tissue samples.Additional file 6Table S1. Motif output predicted results.