Mycoplasma bovis have been more frequently detected in calves infected with BRD compared to those detected in clinically healthy calves. Moreover, the data presented herein revealed co-infections by combination multiple viral pathogens with bacterial pathogens are closely involved in the onset of BRD. Taken together, our study demonstrates multiplex RT-qPCR which can simultaneously analyze multiple pathogens, including viruses and bacteria, and is useful for the early detection of BRD.Bovine respiratory diseases (BRD) are associated with various predisposing factors, such as physical and physiological stress factors, and bacterial and viral pathogens. These stressors and viruses suppress immune defenses, leading to bacterial growth in the upper respiratory tract and invasion of pathogens into the lower respiratory tract. Therefore, continuous monitoring of the causative pathogens would contribute to the early detection of BRD. Nasal swabs and sera from 63 clinically healthy calves were continuously collected from seven farms in Iwate prefecture from 2019 to 2021. We attempted to monitor dynamics of BRD-associated pathogens by multiplex real-time RT-PCR (RT-qPCR) using their nasal swab samples. In addition, we attempted to monitor fluctuation of antibody titers against each BRD-associated pathogen by virus neutralization test (VNT) using their sera. In contrast, nasal swabs from 89 calves infected with BRD were collected from 28 farms in Iwate prefecture from 2019 to 2021. We attempted to analyze their nasal swab samples by multiplex RT-qPCR aim to detect BRD-associated pathogens that are dominant in this region. As a result, our analyses using samples from clinically healthy calves showed that positive results by multiplex RT-qPCR were closely related to a significant increase of antibody titers by VNT in bovine coronavirus (BCoV), bovine torovirus (BToV), and bovine respiratory syncytial virus (BRSV). In addition, our data exhibited that BCoV, BToV, BRSV, bovine parainfluenza virus 3, and  Mannheimia haemolytica (Mh), Pasteurella multocida (Pm), Mycoplasma bovis (Mb), and Histophilus somni (Hs) , wh, wh27], The reaction volume per one unit was 20 μL, including 5 μL TaqPath 1-step Multiplex Master Mix, 1 μL  of mixture of forward and reverse primers (900 nmol/L each) and probe (250 nmol/L) per pathogen, 4 μL of sample, and 7 μL RNase-free water. Multiplex RT-qPCR was conducted using an Applied Biosystems QuantStudio 5 Real-Time PCR System  under the following conditions: 25 °C for 2 min, 53 °C for 10 min, and 95 °C for 2 min, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s. All results were analyzed using the QuantStudio Design and Analysis Software (Version 1.5).In the analysis by multiplex RT-qPCR, the cut-off value for pathogen detection was set at Ct 40 or less . In addition, the multiplex RT-qPCR analysis was performed in duplicate to robustly evaluate the possibility of non-specific reaction of the sample with high Ct values. The average of the two Ct values was used as the results. In addition, a retest was performed if no Ct value was obtained on one side, and cases when no Ct value was obtained on one side in the retest were considered to be negative.The virus neutralization test (VNT) was performed according to previously reported methods ,29. Each50/0.1 mL per each virus) of these strains was added to well and incubated at 37 °C for 1 h. Thereafter, 100 µL of bovine fetal muscular cells (approximately 1.5 × 104 cells) originally produced in our laboratory was added into all wells and incubated at 37 °C in 5% CO2 for 7 days.For Nose strain (BVDV1), KZ-91-CP strain (BVDV2), Los Angeles strain (BHV1) and Fukuroi strain (BAdV7), 50 µL (200 TCID50/0.1 mL per each virus) of these strains was added to well and incubated at 37 °C for 1 h. Thereafter, 100 µL of HRT-18G cells (approximately 1.5 × 104 cells) was added into all of the wells and incubated at 37 °C in 5% CO2 for 7 days.For Kakegawa strain (BCoV) and Aichi strain (BToV), 50 µL (200 TCID50/0.1 mL per each virus) of these strains was added to well and incubated at 37 °C for 1 h. Thereafter, 100 µL of Vero cells (approximately 1.5 × 104 cells) was added into all wells and incubated at 37 °C in 5% CO2 for 7 days.For 52-163-13 strain (BRSV) and BN-1 strain (BPIV3), 50 µL (200 TCIDThe appearance of the cytopathogenic effect (CPE) was observed using a microscope . The VN titer for each serum sample was expressed as the reciprocal of the highest dilution that inhibited CPE.An antibody titer of less than 2-fold was considered to be negative. A significant increase was defined as a 4-fold or greater increase in an antibody titer compared to two consecutive titers. For calves treated with vaccines containing BVDVs, BAdV7, BRSV, BPIV3, and BHV1, positive results obtained in each pathogen with multiplex RT-qPCR using nasal swabs from the animals, including cohabiting calves, were considered as a significant increase by the field strain or vaccine; otherwise, it was considered to be an effect of the vaccine. Furthermore, because cross-reactivity was observed between BVDV1 and BVDV2 antibodies, the significant increase of BVDV2 antibody titer in BVDV1 vaccinated calves was considered to be due to the vaccine ,31. A flClinical samples were collected as a part of routine diagnostic procedures, including monitoring of other pathogens; hence, permission regarding animal ethics was not required.Throughout interviews with farmers and diagnosis by veterinarians with experiences and knowledge during the investigation period, we confirmed an onset of respiratory disease in seven calves  and the death of two calves (nos. B1 and G6) from three farms  . In addiA total of 319 PCR-specific genes from nine pathogens were detected using 232 nasal swab samples from 63 clinically healthy calves collected in Iwate prefecture from 2019 to 2021. The pathogen-specific genes detected by multiplex RT-qPCR were Pm , Mh , Hs , BCoV , Mb , BAdVs , BRSV , BToV , and BPIV3 . Specific genes were not detected in BVDV 1 and 2, BIDV, and BHV1 by multiplex RT-qPCR .The antibody titers measured by VNT against each pathogen, using 232 sera from 63 clinically healthy calves, are summarized in The results by multiplex RT-qPCR and the results by VNT against each virus, using 232 nasal swab samples and sera from 63 clinically healthy calves, are summarized in The number of samples with multiplex RT-qPCR-positive, significant increase in antibody titers, and vaccination were BVDVs (0), BCoV (0), BToV (0), BAdVs (1), BRSV (2), BPIV3 (0), and BHV1 (0), respectively. The number of samples with multiplex RT-qPCR- positive, significant increase in antibody titers, and no vaccination were BVDVs (0), BCoV (16), BToV (2), BAdVs (0), BRSV (2), BPIV3 (0), and BHV1 (0), respectively. The number of samples with multiplex RT-qPCR-positive, no significant increase in antibody titers, and antibody possession were BVDVs (0), BCoV (3), BToV (1), BAdVs (3), BRSV (0), BPIV3 (0), and BHV1 (0), respectively. The number of samples with multiplex RT-qPCR-positive, no significant increase in antibody titers, and antibody no possession were BVDVs (0), BCoV (0), BToV (0), BAdVs (1), BRSV (0), BPIV3 (0), and BHV1 (0), respectively. The number of samples with multiplex RT-qPCR positive and unknown significant increase in antibody titers (No collection of pre- or post-serum) were BVDVs (0), BCoV (11), BToV (2), BAdVs (4), BRSV (1), BPIV3 (2), and BHV1 (0), respectively.p < 0.05) in the detection rates of BCoV, BToV, BRSV, BPIV3, and Mb between the two groups. In contrast, there were no significant differences in the detection rates of BVDVs, BAdVs, BIDV, BHV1, Mh, Pm, and Hs , Mh , BCoV , BRSV 29/89, 32.6%), Mb , BPIV3 , BToV , Hs , BAdVs , BHV1 , BVDVs , and BIDV  . In cont.6%, Mb  simultaneously detected two to five pathogens . In contIn this study, seven calves  showed respiratory disease and nine calves  were suspected to have respiratory disease among 63 clinically healthy calves during the investigation period. The multiplex RT-qPCR results from before to after the onset of the disease and the significant increase in antibody titers after the onset of the disease in these 16 calves is summarized in Viral and bacterial pathogens play an important role in BRD onset. Therefore, early detection of these pathogens can reduce the damage caused by BRD ,34. HoweTo analyze the presence of BRD-associated pathogens in nasal swabs of clinically healthy calves and their relationship to real infection, we compared results between multiplex RT-qPCR and VNT against BVDV1, BVDV2, BCoV, BToV, BAdV, BRSV, BPIV3, and BHV1 using 232 nasal swab samples and 232 serum samples from 69 clinically healthy calves. Herein, we focused on the percentage of samples with a significant increase in antibody titers against BCoV, BToV, BRSV, and BPIV3 that were positive with multiplex RT-qPCR. Except for cases that were unknown by VNT, the pathogens that showed a higher percentage of VNT were BCoV , BToV , and BRSV . For BPIV3, there were no applicable samples. Therefore, this suggests that the positive results by multiplex RT-qPCR could be an indicator of real infection. In addition, these findings revealed that several calves with antibody titers of 4 ≤ were shown to be positive by multiplex RT-qPCR. We speculate that those individuals infected more viruses than range that can protect them from BCoV infections by their VN titers. In addition, the positive results might be also influenced by host health condition and co-infection by several pathogens other than the amount of virus exposed.We compared 12 BRD-related pathogens detected by multiplex RT-qPCR using 89 and 232 nasal swab samples collected from 89 calves with respiratory symptoms and 63 clinically healthy calves during 2019–2021, respectively, owing to finding pathogens that are indicators of BRD onset. As results, we revealed that BCoV, BToV, BRSV, BPIV3, and Mb have been more frequently detected in calves with respiratory symptoms compared to those detected in clinically healthy calves. In previous studies, BRSV, BPIV3, and Mb have been reported as major pathogens of BRD ,35. TherPm and Mh have been reported to be resident bacteria in the nasal cavity . In addiOur analysis found one farm (No.A) where Mh and Pm were never detected for three years. In addition, our data showed that few viruses associated with BRD were detected at the farm. The farm had a small number of cows with little introduction of cattle and no other dairy farms in the surrounding area. These facts suggest that reduction of stress by cattle movement and herd organization, and enhancement of biosecurity in cattle management might be effective for the prevention of BRD ,42,43.In this study, we confirmed that seven calves showed BRD symptoms  out of 63 clinically healthy calves throughout the interview with farmers and diagnosis by veterinarians with experience and knowledge. In addition, there were epidemic cases of respiratory disease on three farms  during the investigation period, and moreover nine calves  were suspected to have the disease. Multiplex RT-qPCR and VNT were performed using samples from these 16 calves collected before and after BRD onset. Our analysis revealed one or two pathogens (mainly Mh and Pm) before BRD onset. Subsequently, mixed infections with two to four pathogens of viruses (mainly BCoV and BRSV) and bacteria (mainly Mh and Pm) were detected at BRD onset. Moreover, the number of pathogens detected using a nasal swab sample from healthy and BRD-infected calves was 1.4 and 2.3 pathogens, respectively. The pathogenesis mechanism of BRD has been suggested to be based on stress, immunosuppression, and potentially bacterial pathogens in the nasal cavity. At BRD onset, there is a shift in this homeostatic balance in the upper respiratory tract, resulting in colonization in the lower respiratory tract . MoreoveIdentifying causative pathogens of BRD requires rapid testing for multiple pathogens, including viruses and bacteria. Our investigation using samples from 16 BRD-infected calves showed that at least BCoV was associated with respiratory symptoms in 12 calves  on three farms . Of these, six calves (D4-D9) from farm D were negative for BCoV antibodies before BRD onset and then were infected with BCoV at BRD onset.In conclusion, we offer the application of multiplex RT-qPCR as a tool to monitor BRD causative pathogens and to detect the risk before BRD is spread within the herds. Analyses by multiplex RT-qPCR using nasal swab samples from calves with respiratory symptoms and clinically healthy calves suggests that BCoV, BToV, BRSV, BPIV3, and Mb are the main causative pathogens of BRD. Moreover, our data revealed that positive results by multiplex RT-qPCR are closely correlated, with significant increases in antibody titers by VNT regarding BCoV, BToV, and BRSV. Therefore, these facts support the idea that continuous monitoring of BRD-associated pathogens in healthy calves using multiplex RT-qPCR would contribute to early detection of BRD. Furthermore, this study found, in one of mechanism of BRD, that Mh and Pm existed in the nasal cavity before BRD onset, and that multiple pathogens of viruses and bacteria, such as BCoV, BRSV, Mh, and Pm were infected there. Therefore, we hope this methodology will be widely spread and accumulate more data in the future.


The objective of our study was to assess the effect of rumen-protected niacin supplementation on the transcriptome of liver tissue in growing Angus × Simmental steers and heifers through RNA-seq analysis. Consequently, we wanted to assess the known role of niacin in the physiological processes of vasodilation, detoxification, and immune function in beef hepatic tissue. Normal weaned calves (~8 months old) were provided either a control diet or a diet supplemented with rumen-protected niacin (6 g/hd/d) for a 30-day period, followed by a liver biopsy. We observed a significant list of changes at the transcriptome level due to rumen-protected niacin supplementation. Several metabolic pathways revealed potential positive effects to the animal’s liver metabolism due to administration of rumen-protected niacin; for example, a decrease in lipolysis, apoptosis, inflammatory responses, atherosclerosis, oxidative stress, fibrosis, and vasodilation-related pathways. Therefore, results from our study showed that the liver transcriptional machinery switched several metabolic pathways to a condition that could potentially benefit the health status of animals supplemented with rumen-protected niacin. In conclusion, based on the results of our study, we can suggest the utilization of rumen-protected niacin supplementation as a nutritional strategy could improve the health status of growing beef cattle in different beef production stages, such as backgrounding operations or new arrivals to a feedlot. Enzymes that depend on niacin are numerous and represent an important factor in metabolism in all organisms. For example, NAD+ participates in approximately 400 reactions, whereas NADP+ in 30 different reactions , actin cytoskeleton  can be explained in a way based on the lipid metabolism regulation function of niacin that we have discussed earlier. Niacin can prevent atherosclerotic cardiovascular disease, and oxidative modification of low-density lipoprotein (oxLDL), which is the major atherogenic modification of low-density lipoprotein [FAK) and actin polymerization can be reduced by niacin in mice [FAK phosphorylation and actin polymerization is associated with the inhibition of cell migration [FAK signaling pathway in mice [HCA2) is involved in cellular inflammatory responses. The chemokine-induced migration of macrophages can be inhibited by the niacin-activated HCA2 [As for the other significant pathways involved in cellular processes, it is worth mentioning the downregulation of cell motility and cell community KEGG categories, which mainly rely on the downregulation of the “regulation of actin cytoskeleton” pathway and “focal adhesion” pathway. A recent article investigating niacin deficiency and genetic instability also reported alterations of the focal adhesion signaling pathway in niacin-deficient cells in humans . Focal aoprotein . A previ in mice . Furtherigration . In addi in mice . Finallyted HCA2 , which a+ ratio, was shown to block the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway which is activated via phosphorylation [JAK2 is activated by several cytokines and growth factors, including IFN-γ, IL-4, IL-6, IL-12, IL-13, growth hormone (GH), and leptin [Niacin, known to lower cytosolic NADH/NADrylation . The inhd leptin . Signal d leptin .Niacin mediates its anti-inflammatory effects via HCA2-dependent mechanisms in monocytes and macrophages , 54 by iYWHAB, logFC = 1.24, FC = 0.03) that belongs to the Hippo signaling pathway which is a critical regulator of liver size [YWHAB expression plays critical roles in the termination of liver regeneration by inhibiting cellular proliferation in male rats [YWHAB to control liver size. The decrease in organ size could be explained also by the lipolytic effect of niacin in liver adipose tissue content [AXIN1, logFC = - 2.19, FC = 0.03) and nucleoside diphosphate kinase 2  genes, which are known inhibitors of the β-catenin pathway, were inhibited. The antagonistic effect of NKD2 on canonical Wnt signaling is achieved by inhibiting the translocation of β-catenin into the nucleus [Axin2 [LLGL2 , and SCRIB  genes which is a typical reaction during pathological conditions.Rumen-protected niacin increased the expression of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta  and calcium/calmodulin dependent protein kinase IV , in combination with inhibition of cAMP/PKA/CREB pathway, which is a major regulator of hepatic tissue proliferation and apoptosis [2+ pathway, the antilipolytic effect of nicotinic acid was evidenced by the Gi-mediated inhibition of adenylate cyclase 1, which inhibits cAMP production, through cholinergic receptor muscarinic 1 (CHRM1) activation [The cAMP signaling pathway presented signs of nicotinic acid uptake and transport in hepatic tissue, which appears to be regulated by an intracellular Can humans . These spoptosis . Coincidtivation .NPR2, logFC = 1.92, FDR = 0.02) and regulator of G protein signaling 2 ; both attenuate the increment of hepatic vascular resistance [MEF2, logFC = -2.75, FDR = 0.02) was inhibited by RPN supplementation. This gene has a role in the activation of hepatic stellate cells (HSCs), which represents a final common pathway of the hepatic response to liver injury [The cGMP-PKG signaling pathway is mainly related to vascular contraction and relaxation. A vasodilator effect of RPN supplementation may have been observed during our study by the activation of natriuretic peptide receptor 2 , which is activated in a calcium- and IP3R-dependent manner by cAMP and glucagon in primary hepatic tissue and by glucagon and fasting in vivo (WNT/Ca2+ pathway) [NFATC4, logFC = -1.79, FDR = 0.02), which induces the expression of cytokine genes in T-cells, especially IL-2 or IL-4 [Our results suggested that RPN supplementation could potentially lead to hepatic glucose production pathway) . Further or IL-4 .MMP-14, logFC = -3.51, FDR = 0.01) is associated with the degradation of several adhesion molecules, including fibronectin [MMP14 participates in the remodeling of the extracellular matrix which could be harmful to the liver tissue by producing accumulation of extracellular matrix leading to scar tissue formation [TGF-β1, logFC = -5.32, FDR = 0.04) [TGF-β1 or oxidative stress mediator hydrogen peroxide (H2O2), a major physiological stimulator of liver fibrosis [Matrix metalloproteinase 14 (ronectin  which isormation . Genes t = 0.04) . This stfibrosis .MEGF8, logFC = -6.87, FDR = 0.01; MGRN1, logFC = -2.44, FDR = 0.01), which are connected to a key transmembrane protein involved in cell-cell communication system called smoothened (Smo). Smoothened is critical for embryonic development and adult tissue homeostasis in vertebrates [Healthy adult hepatic tissue do not express Hedgehog ligands . Hedgehotebrates .PSEN2, logFC = 1.51, FDR = 0.04), which is thought to regulate cell differentiation and survival [Deltex 2 , which is closely involved in cell growth, differentiation, apoptosis, as well as intracellular signal transduction by modulating the Notch signaling pathway [Our results also suggest that RPN supplementation activates presenilin-2  KEGG pathway. This was the clearest sign of the well-known vasodilator effect of niacin. Briefly, angiotensinogen , which causes vasoconstriction and regulates blood pressure was inhibited by RPN supplementation. Phospholipase C beta 2  catalyzes the hydrolysis of 1-phosphatidylinositol 4,5-bisphosphate into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). This enzyme regulates the function of the endothelial barrier through intracellular signaling downstream of G protein-coupled receptors [GPR109A  couples to a G(i) protein and inhibits adenylate cyclase activity  [AGT.Rumen-protected niacin supplementation produced an inhibition of the Phospholipase D (eceptors . IP3 pateceptors . Therefo = 0.02) , leadingNiacin influenced VEGF signaling pathway by causing an inhibition on cell migration through actin reorganization and cell proliferation through the MAPK signaling pathway. A previous study showed that niacin interferes with the signaling cascade of chemoattractants in macrophages. More specifically, niacin might be inhibiting chemoattractant receptor activation that triggers actin cytoskeleton reorganization to form lamellipodia at the leading edge of macrophages. These are clear signs of the inhibitory effect of niacin on chemoattractant-induced cell migration  that leaHIF1A). In our study, RPN supplementation caused an inhibition of inflammation  and increased activation of angiogenesis  observed in the HIF1 pathway. TEK receptor tyrosine kinase (TEK) is an inducible early response gene involved in hepatic tissue proliferation and liver regeneration [Oxygen-sensing pathways, including the NOTCH-, PI3K-AKneration .GADD45, logFC = -2.63, FDR = 0.02), glucose metabolism  and immunoregulation . In contrast, one of the autophagy-related genes was activated . Briefly, GADD45 overexpression has been implicated in stress signaling in response to physiological or environmental stressors, which results in cell cycle arrest, DNA repair, cell survival and senescence, or apoptosis [G6PD), plays an important role in the production of NADPH and restoring the intracellular redox state in the setting of increased oxidant stress [KLF2) in endothelial cells results in reduced liver damage in mice [GABARAP, which is responsible for the autophagy mechanism involving general membrane remodeling [Furthermore, the FOXO signaling pathway was downregulated, potentially inhibiting oxidative stress resistance and DNA repair , cell adhesion , protein synthesis , and cell survival  in response to RPN supplementation. Briefly, p21-activated kinases (PAKs) mediate extracellular signals and regulate cell motility and morphology, cytoskeletal remodeling, cell proliferation, and apoptosis [AKT functions in an anti-apoptotic manner by directly phosphorylating the pro-apoptotic Bcl-2-associated death promoter (BAD) [AKT in insulin-sensitive tissues, such as liver. Furthermore, AKT/mTOR signaling pathway activation has a close relation with inflammation. Phosphorylation of AKT or mTOR can activate NF-κB, leading to its nuclear translocation, producing inflammatory cytokines, such as INF-γ and TNF-α [HCAR2, activated by RPN supplementation, took place through inhibiting AKT/mTOR signaling pathway in mice [Our results indicate the downregulation of the ERBB signaling pathway (poptosis . In termer (BAD) . A priornd TNF-α . This dr in mice .RAP1 acts as a molecular switch that regulates the cell’s response  to external stimuli . Within the RAP1 metabolic pathway, RPN inhibited the expression of guanine nucleotide exchange factor , which plays an important role in angiogenesis. A previous study showed that niacin inhibits angiogenesis likely through cytoskeleton remodeling in humans [FARP2, logFC = -2.68, FDR = 0.02) has been inhibited by RPN. In addition, FARP2 has a role in the actin cytoskeleton rearrangement of endothelial cells. Furthermore, the binding of integrins to their extracellular ligands is assisted by actin, producing an integrin–actin linkage mediated by talin 1 [RAP1 pathway, leading to attenuation of collagen accumulation exerting potential antifibrotic properties of niacin [n humans . In our  talin 1 . Talin 1f niacin .PLCB2, logFC = 1.44, FDR = 0.02) and sphingosine-1-phosphate receptor 5  and the inhibition of cathepsin D , AKT, MAPK11/15  and, BCL2 Associated X, Apoptosis Regulator . PLCB2 regulates the function of the hepatic endothelial barrier and mediates intracellular signaling downstream of G protein-coupled receptors. In previous research, an increase in phospholipase C gamma 2 (PLCγ2) mRNA was detected in the late phase of rat liver regeneration [TNF-α alone cannot induce apoptosis in normal hepatic tissue, because TNF-α also activates antiapoptotic signal pathways. TNF-α induces S1P generation via sphingosine kinase 2 , which activates survival signals such as the PI3K/AKT pathway and protects human hepatic tissue from TNF-α-induced apoptosis [S1PR5 and the inactivation of TNF receptor superfamily member 1A , which provides instructions for making a tumor necrosis factor receptor 1 (TNFR1), and SPHK2 that encodes one of two sphingosine kinase isozymes that catalyze the phosphorylation of sphingosine into sphingosine 1-phosphate (S1P). S1P mediates many cellular processes, including migration, proliferation, and apoptosis. Cell death mediated by TNF-α employs ceramide as an important second messenger. Ceramide is further hydrolyzed by ceramidase to sphingosine, which subsequently is converted to S1P by SPHK2. The balance between intracellular concentrations of ceramide and S1P may be a critical factor in the determination of cell fate, and our results suggest that RPN supplementation drives this reaction to greater S1P production leading to cell survival. Although, little is known about the signaling pathways regulated by ceramide in hepatic tissue [The sphingolipid signaling pathway shows a clear inhibition of apoptosis through the activation of phospholipase C beta 2 , diacylglycerol kinase theta , monoglyceride lipase , and phosphatidylethanolamine-N-methyltransferase  genes. In particular, the PEMT downregulation is remarkable, considering that PEMT pathway is known to play a role in lipid metabolism by regulating VLDL secretion in mice [PRKAG1, logFC = -1.43, FDR = 0.04), which appeared in line with the role of niacin in lipolysis inhibition via reduction of cAMP [PKA) activation leads to a less phosphorylation and activation of the lipolytic enzymes [PPP2R5E, logFC = 1.34, FDR = 0.04) which is known to inhibit AMP kinase [ACACA, logFC = -2.42, FDR = 0.03) is in line within this context, considering its role in lipid metabolism [CYP2D14, logFC = -2.42, FDR = 0.03), cytochrome P450 family 11 subfamily A member 1  and cytochrome P450 2D14-like . The inhibition of P450 enzymes by nicotinic acid has been long recognized [NDUFA13, logFC = 2.12, FDR = 0.01; NDUFB1, logFC = 1.86, FDR = 0.005, and NDUFC1, logFC = 2.23, FDR = 0.004) in our experiment and it is compatible with the niacin effect on boosting mitochondrial biogenesis and respiratory chain activity [Niacin is known to inhibit lipolysis , in part in mice . Further of cAMP . Mechani enzymes . In thisP kinase . Also, ttabolism . Within cognized . Cytochrcognized , and oxicognized . This wacognized , is convcognized  but are cognized . The maractivity . The facMLYCD, logFC = -1.51, FDR = 0.04) and the upregulation of acyl-CoA synthetase l ong chain family member 4 , which are known to play an important role in the control of fatty acid oxidation [Collectively, our results suggested from a transcriptomic point of view that niacin reduces hepatic triglyceride synthesis and increases hepatic lipid oxidation . Furtherxidation , 110, weAPOE, logFC = - 4.93, FDR = 0.01), which is involved in many steps in lipid and lipoprotein homeostasis, for the triglyceride-rich lipoproteins and for HDL [It Is also noteworthy to highlight the downregulation of ‘Digestive System’ KEGG subcategory, mainly relying on the downregulation of ‘Cholesterol metabolism’, ‘Carbohydrate digestion and absorption’ and ‘Protein digestion and absorption’ pathways. In particular, the downregulation of ‘Cholesterol metabolism’ was compatible with known niacin effect on total cholesterol decreasing . Within  for HDL . High ex for HDL ; thus, i for HDL .UGT1A6, logFC = 1.25, FDR = 0.03) and 3-hydroxy-3-methylglutaryl-CoA reductase  genes. UDP-glucuronosyltransferases (UGTs) are a superfamily of enzymes that generally fulfill detoxification roles, catalyzing the glucuronidation of various exogenous  as well as endogenous compounds  [HMGCR was particularly intriguing because of its role as rate-limiting enzyme for cholesterol synthesis [AMPK inactivates HMGCR, with a consequent inhibiting effect on cholesterol synthesis [PRKAG1 detected in our experiment. Nevertheless, the possible inhibition effect of nicotinic acid on HMGCR activity has been recently proposed [HMGCR, a key enzyme in cholesterol synthesis, would require further investigations in the light of evidence indicating its role as lipid-lowering molecule [HMGCR, we detected the downregulation of 3β-hydroxysteroid-Δ24 reductase , the final enzyme of the cholesterol biosynthetic pathway [APOA4, which is known to be involved in cholesterol transport [LBR, logFC = 2.02, FDR = 0.004), whose protein exhibits sterol reductase activity essential for cholesterol biosynthesis [In this general context, the downregulation of ‘Bile secretion’ pathway seemed inconsistent with the increased biliary secretion traditionally associated with hypocholesterolaemic action of niacin . Howeveritamins)  to increitamins) . Whereasynthesis . Indeed,ynthesis . Thus, iproposed , thus thmolecule . Althoug pathway  and APOAransport . At the ynthesis .COL4A1 , COL6A1 , COL6A2 , COL18A1 , which suggested and confirmed the role of niacin as anti-fibrotic agent, as previously shown in human liver [The marked downregulation of ‘Protein digestion and absorption’ pathway was also intriguing. This mainly relied on the downregulation of several genes belonging to the collagen family group, such as an liver .AKT1, Rho Guanine nucleotide-exchange factor , TLN1 and the simultaneous upregulation of Prostaglandin-Endoperoxide Synthase 1 . Akt is a serine–threonine kinase that contributes to signaling and activation responses of platelets in mice [ARHGEF1 is known to be involved in platelet activity [TLN1 is required for platelet integrins activation [PTGS1 is noteworthy considering that prostaglandins induce vasodilation [TGFB1 downregulation was in line with the documented inhibitory effect of niacin on TGFB1 mRNA expression in hamster [The downregulation of the ‘Immune system’ category was also noteworthy. This result relied on marked downregulation of several pathways, such as ‘Platelet activation’, ‘Chemokine’, ‘Neutrophil extracellular trap formation’, ‘Th17 cell differentiation’ and ‘Fc gamma R-mediated phagocytosis’ pathways. Considering the effects of platelet activation on vasocontraction , our res in mice . Interesactivity . TLN1 istivation . Furtherdilation  and inhidilation . The dowdilation , 132. Fu hamster .TGFB1. In this regard, the positive interaction of TGFB1 with endothelin-1, a potent vasoconstrictor secreted by vascular endothelial cells, has been described [TGFB1 might indicate a vasodilation effect of niacin connected with the presumable decrease of endothelin levels. Also, the downregulation of ‘Vascular smooth muscle cells’ pathway was noteworthy, since this could be compatible with a vasodilation state. Within this pathway, we detected the downregulation of AGT, a precursor for angiotensin [In this context, the downregulation of ‘Relaxin signaling’ pathway was interesting. Although originally known to be present in pregnant individuals, current research identified other biological functions of relaxin in both males and females with physiological roles in vasodilation , as showescribed . Thus, wiotensin , which aiotensin .TNFRSF1A, which is known to be involved in ‘Adipocytokine signaling’ pathway, is in line with this idea. TNFRSF1A encodes for a protein called tumor necrosis factor receptor 1 (TNFR1) that, when attached to another protein called tumor necrosis factor (TNF), can trigger either inflammation or self-destruction of the cell (apoptosis).Overall, the downregulation of ‘Immune system’ KEGG category also supported the notion of a potential anti-inflammatory effect of niacin . The dowAdipocytokines can influence many bioactivities: notably inflammatory processes, glucose and lipid metabolism . AdiponeAKT1 downregulation [G6PD, which encodes for a key enzyme involved in the last step of gluconeogenic and glycogenolytic pathways, suggesting gluconeogenesis inhibition. It is well-known that insulin exerts control of gluconeogenesis by acting on the liver, but also by acting on other tissues [CREB3L3, logFC = - 1.82, FDR = 0.02) is noteworthy, since CREB is known to be a key activator of the hepatic gluconeogenic gene regulation program [The downregulation of ‘Insulin signaling’ pathway was consistent with gulation  and was gulation , 150. Ingulation . Neverth tissues . For exa tissues . This sc program . Further program , 154.THRA, logFC = - 2.46, FDR = 0.03). Furthermore, within this pathway we also detected the downregulation of Notch Receptor 1 . The lower expression of NOTCH1 due to niacin treatment was already reported in an in vitro study on niacin effect on vascular inflammation inhibition in cattle [Lastly the downregulation of ‘Thyroid hormone’ pathway was compatible with the decrease of thyroid hormone levels associated with niacin supplementation . In thisn cattle .Rumen-protected niacin supplementation on growing steers and heifers for 30 days after weaning presented a significant list of potential benefits observed at the liver transcriptomics level. Several metabolic pathways revealed positive effects of administration of rumen-protected niacin on beef calves after weaning. The most impacted pathways showed that rumen-protected niacin had a down-regulatory effect on the expression of genes related to lipolysis, apoptosis, inflammatory responses, atherosclerosis, oxidative stress, and fibrosis, and enhancing vasodilation. Therefore, results from our study could potentially promote supplementation of rumen-protected niacin on beef cattle backgrounding operations or new arrivals to a feedlot, especially during the acclimation period when health status is often compromised. Although, a performance test with a greater number of animals should be conducted in order to confirm these results. Finally, it is important to remark that our study seeks to bring light on the specific role of niacin in growing beef cattle, and caution must be exercised when translating our findings to other species or cattle breeds .S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.


Immunotherapy has made great strides in the treatment of lung cancer, but a significant proportion of patients still do not respond to treatment. Therefore, the identification of novel targets is crucial to improving the response to immunotherapy. The tumor microenvironment (TME) is a complex niche composed of diverse pro-tumor molecules and cell populations, making the function and mechanism of a unique cell subset difficult to understand. However, the advent of single-cell RNA sequencing (scRNA-seq) technology has made it possible to identify cellular markers and understand their potential functions and mechanisms in the TME. In this review, we highlight recent advances emerging from scRNA-seq studies in lung cancer, with a particular focus on stromal cells. We elucidate the cellular developmental trajectory, phenotypic remodeling, and cell interactions during tumor progression. Our review proposes predictive biomarkers and novel targets for lung cancer immunotherapy based on cellular markers identified through scRNA-seq. The identification of novel targets could help improve the response to immunotherapy. The use of scRNA-seq technology could provide new strategies to understand the TME and develop personalized immunotherapy for lung cancer patients. ReceDue to the heterogeneity and plasticity of stromal cells, identifying and defining distinct cell subsets is a significant challenge. With the booming development of high-dimensional profiling techniques, single-cell RNA sequencing (scRNA-seq) has emerged as a valuable tool to characterize the gene expression, compositional differences, and functional states of individual stromal cells. In addition, cell lineages, the dynamics of cell fate and differentiation dynamics, and crosstalk of stromal cell subsets in the TME can be revealed by analyzing of T-cell receptor (TCR) repertoires in scRNA-seq. These studies contribute to a better understanding of the potential roles of stromal cells in both tumor progression and resistance to ICB therapy , 8. In t2The TME plays a significant role in tumor progression and therapeutic response in lung cancer , 10. The2.1Tumor-infiltrating lymphocytes (TILs) play a critical role in executing anti-tumor immune responses and mediating responses to ICB treatments. The heterogeneity and plasticity of TILs have been revealed by scRNA-seq analysis, providing new strategies for cancer immunotherapy.2.1.1+ T cells, including naive, memory, effector, effector memory T cells (Tems), tissue-resident memory T cells (Trms), and exhausted T cells (Texs)  or memory CD8+ T cells (characterized by high levels of ZNF683) are activated, subsequently proliferating and differentiating into effector CD8+ T cells  to recognize and kill tumor cells , as a transitional state, express GZMA, GZMB, perforin 1, NKG7  –19. The or cells , 14, 20. 1, NKG7 . The subhe lungs , 35.+CD8+ Tems , ZNF683+CD8+ , and LAYN+CD8+ (high-level PDCD1 and high-level ITGAE) , T cell activation (CD38), and high levels of PD-1 and TIM-3  is characterized by high expression levels of inhibitory receptor genes such as PDCD1, CTLA4, LAG-3, LAYN, TIGIT, and TIM3, and low expression levels of tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), and T-box transcription factor (TBX21)  , 26–29.  (TBX21) , 25. How2.1.2+CD8+ exhausted, LEF1+CD8+ naive, and CX3CR1+CD8+ effector subsets are located at the ends of different branches, with GZMK+CD8+ T cells and ZNF683+CD8+ T cells positioned in between them , and Tex CD4+ T cells. Naive CD4+ T cells are characterized by high expression of CCR7, TCF7, LEF1, and SELL; Tems CD4+ T cells are characterized by the GNLY+CD4+ T cluster expressing PRF1, GZMA, and GZMB and lower signatures of PDCD1, CTLA4, and HAVCR2; Tcms CD4+ T cells are defined as ANXA1+CD4+ T cells  and tumor necrosis factor (TNF), whereas tumor-resident Tregs have been shown to suppress the infiltration and anti-tumor activities of effector CD8+ T cells   , 20. Alt4+ Tregs , 38, 39.eg cells . The ela+IL2RA+CD4+ and CTLA4+CD4+ Tregs  and tumor necrosis factor receptor superfamily member 9 , which reflect an activated state  to TNFRSF9+ Treg (activated) cells. Moreover, scRNA-seq analysis has revealed a transitional phenotype, Th17-like CD4+ T cells, characterized by the high expression of KLRB1, which is a transitional state in the conversion of naive CD4+ T cells to Tregs  and memory follicular B cells (CD20+CD27+IGHD−). Similar to the subset of naive follicular B cells, another cohort identified naive-like B cell clusters in NSCLC with high levels of CD19, CD22, MS4A1 (CD20), TCL1A, and CD83  or M2-like (anti-inflammatory) subsets based on their distinct activation factors and effector functions . However+, SPP1+, and TREM2+ subsets  enriched in non-cancerous tissues. Unlike NLRP3+ macrophages, LYVE1+ macrophages have the potential to develop into C1Q+ TAM and reprogram the TME , SPP1, triggering receptor expressed on myeloid cells-2 (TREM2), NLRP3, LYVE1 –52. Prot subsets , 52, 53.g cancer –57. In aer types , 51. TREc subset , 49. Add the TME . Althoug+ and effector CD8+ T cells increases and the expression of PD-1 decreases in TRM-exhausted lesions, which in turn impairs the survival and growth of early tumor cells  and CX3CR1, both of which are involved in promoting lymphocyte cytotoxic functions. In contrast, the CD16− NK cluster upregulates tissue-resident markers such as ITGA1, ITGAE, and ZNF683  signaling genes , which are believed to play a role in T cells recruitment. Moreover, these cells were located in the TLSs, suggesting they may have an antigen presentation role. Promoting the differentiation of CAFs into the ADH1B+ subtype may improve the anti-tumor immune effect  and ACKR1+ tumor EC (ACKR1+SELP+IL1R1+) in early-stage lung cancer that radiologically manifests as part-solid nodules , EDN1+CCL2+ ECs, decreased. It is known that EDN1+CCL2+ ECs overexpress genes related to lymphocyte activation and homing ability, such as BIRC3, CCL2, CD44, and ICAM1. Collectively, tumor ECs are remodeled to decrease their activities of lymphocyte cell activation and homing and increase their ability of angiogenesis and matrix remodeling, contributing to tumor immune tolerance.As important stromal cells in the TME, ECs are associated with angiogenic functions and play an essential role in tumor initiation and progression . The nomRY1+ ECs , anotherogenesis , and ano nodules . Despite nodules , 90. In  nodules , 19. In 3The advent of scRNA-seq technology has provided new insights into the complex cellular interactions and signaling pathways within the TME, thereby aiding in the identification of potential immunotherapy targets. Several tools, such as “cellphone DB” and iTALK have been established to infer putative cell-to-cell interactions based on ligand-receptor signaling from high-resolution scRNA-seq data , 91–97. 3.1Increasing evidence suggests that cancer cells modify their immunogenic properties and remodel adjacent stromal cells to create an immunosuppressive microenvironment that favors their survival and proliferation , 48, 98.Recent scRNA-seq studies have revealed that tumor cells regulate the expression of relevant carcinogenic molecules which contribute to immune escape , 100. CDTumor cells orchestrate stromal cells to favor immunosuppressive environment reconstitution, including TAMs, CAFs, and ECs , 105. Pr3.2+ T-cells  to exhausted CD8 T-cells . Interesolerance . Anotherolerance . Furtherolerance . In addiolerance , 19. Lym4Although scRNA-seq has been a valuable tool in the study of lung cancer, it has its limitations in providing spatial information, which is critical for a comprehensively understanding the progression and intercellular interactions. To overcome these limitations, spatial transcriptomics (ST) has emerged as a cutting-edge technology that seamlessly integrates with scRNA-seq, enabling the study of gene expression patterns within their spatial context. ST can map gene expression to specific locations within tissue, enabling the study of gene expression patterns within their spatial context. In the context of lung cancer, this technique has demonstrated great potential for gaining a deeper understanding of the heterogeneity of the tumor microenvironment, revealing the interactions between cell subpopulations behind spatial organization patterns , 108–111+ T cells than inside or around tumors , a heterotopic structure that aggregates T and B lymphocytes, have been associated with increased local immune reactions; however, traditional research methods have limited our understanding of this structure. Recent studies utilizing ST have further investigated the structure of TLSs and identified that CXCR5 and CD79 are markers associated with TLSs. CXCR5 expression on B cells is a characteristic of subpopulations migrating to TLSs. TLSs are often located outside tumor nests, with higher densities of B cells and CD4d tumors . Additioructures . Interesis lower .Therefore, the integration of single-cell transcriptomics with ST strategies holds great potential to improve the accuracy of the TME characterization and to advance the development of novel diagnostic and therapeutic approaches. However, despite its promising applications, the current knowledge on spatial transcriptomics in lung cancer is limited. Thus, further experimental researches are required to complement the existing evidence and to elucidate the full potential of this innovative approach.Collectively, we have uncovered a complex cellular interplay in the TME, including the inhibition of T cell activation, recruitment of immunosuppressive cells, CAFs activation, angiogenesis, and extracellular matrix remodeling. We also elucidated the dynamic changes of stromal subsets during tumor response, thereby providing a theoretical basis for revealing novel clinical immunotherapeutic targets Figure 25Several immunotherapies such as anti-PD-1/PD-L1 and CTLA4 have recently achieved clinical benefits , 112–1145.1+CD8+ T cells have been associated with durable responses to ICB therapy in lung cancer, while LAYN+CD8+ T cells have been found to be exhausted and contributed to non-response to immunotherapy. Moreover, thymocyte selection-associated high mobility group box protein (TOX), which promotes exhaustion of tumor-infiltrating CD8+ T cells within the tumor, can be used to stratify patients during anti-tumor therapy, including anti-PD-1 immunotherapy , which was composed of PDCD1rophages . It was g cancer . These fg cancer Figure 36In conclusion, this review illustrates the complex biological picture of lung cancer and illuminates the dynamics of cellular and molecular networks during tumor development. To aid future research into treatment methods, we propose novel potential targets. Combination therapy targeting inhibitory molecules and blocking specific pathways may restore innate immunity, reshape the immune microenvironment, enhance the immune response and the therapeutic efficacy of immunotherapy .However, there are still some issues that need to be resolved. First, there is no uniform naming principle for cell subsets with the same function by different researchers owing to differences in methods and tissue types. Therefore, a relatively standard naming norm is needed in the future. Second, although scRNA-seq faithfully replicates global gene expression profiles, it is uncertain whether the expression levels of the transcripts truly reflect the functions of the subpopulation. Moreover, the interactions of stromal cells predicted based on ligand and receptor gene expression also need to be further verified. Hence, combining proteomics and spatial transcriptomics technologies is necessary to jointly study the functions of different subsets and cell-cell interactions in the TME. Third, on account of tumor heterogeneity, including lung cancer types (lung squamous cell carcinoma and lung adenocarcinoma), tumor sites (primary and metastatic tumors), and different populations, the classification and functions of cell subsets in the TME are heterogeneous. Therefore, finding critical subpopulations with similar functions and specific targets will assist in achieving individualized precision immunotherapy for lung cancer. Finally, due to their crucial role in immune checkpoint therapy, most current studies have focused on T cells, with limited attention given to other stromal cells in TME. In addition, although the researches on T cells are intensive, it remains to be explored whether targeting specific subsets can produce immune responses in a wide range of patients or whether immunotherapy resistance will occur during the treatment. ScRNA-seq technology is expected to be applied to explore the changes in the TME after immunotherapy and to reveal in-depth mechanisms of drug resistance. Despite these challenges, we believe that immune molecular typing based on the TME can better identify immune-responsive populations and provide a basis for precise lung cancer treatment.QZ conceived the idea, designed, and supervised the topic. QX, WP and SZ drafted the manuscript and designed the figures and tables. XW, QX, LY, ZW, XX and PZ revised the manuscript. All authors contributed to the article and approved the submitted version.


HNRNPH2 cause an X-linked neurodevelopmental disorder with features that include developmental delay, motor function deficits, and seizures. More than 90% of patients with hnRNPH2 have a missense mutation within or adjacent to the nuclear localization signal (NLS) of hnRNPH2. Here, we report that hnRNPH2 NLS mutations caused reduced interaction with the nuclear transport receptor Kapβ2 and resulted in modest cytoplasmic accumulation of hnRNPH2. We generated 2 knockin mouse models with human-equivalent mutations in Hnrnph2 as well as Hnrnph2-KO mice. Knockin mice recapitulated clinical features of the human disorder, including reduced survival in male mice, impaired motor and cognitive functions, and increased susceptibility to audiogenic seizures. In contrast, 2 independent lines of Hnrnph2-KO mice showed no detectable phenotypes. Notably, KO mice had upregulated expression of Hnrnph1, a paralog of Hnrnph2, whereas knockin mice failed to upregulate Hnrnph1. Thus, genetic compensation by Hnrnph1 may counteract the loss of hnRNPH2. These findings suggest that HNRNPH2-related disorder may be driven by a toxic gain of function or a complex loss of HNRNPH2 function with impaired compensation by HNRNPH1. The knockin mice described here are an important resource for preclinical studies to assess the therapeutic benefit of gene replacement or knockdown of mutant hnRNPH2.Mutations in   HNRNPH2 were identified in 2016 in 6 unrelated individuals as a novel cause of an X-linked neurodevelopmental disorder, the features of which include developmental delay, intellectual disability, autism spectrum disorder, tone abnormalities, and seizure (OMIM 300986) (HNRNPH2 associated with a range of overlapping phenotypes (De novo pathogenic variants in ure OMIM 00986 (3– 300986) . Althougenotypes –7.HNRNPH2-related neurodevelopmental disorder have a nonsynonymous single nucleotide variant within or adjacent to the nuclear localization signal (NLS) of hnRNPH2, with the 2 most common missense variants, R206W and R206Q, located within the NLS. Additional variants outside the NLS of hnRNPH2 have been reported in children with similar symptoms. Two of these, at residues 114 and 188, are recurrent, suggesting a potential pathogenic effect  family of proteins, which govern various aspects of nucleic acid metabolism, including transcription, RNA processing, alternative splicing, mRNA trafficking, mRNA stability, and translation . hnRNPH2HNRNPH2 remain largely unexamined. One recent in vitro study showed deficiencies in nucleocytoplasmic shuttling of hnRNPH2 with NLS mutations , as well as alterations in splicing associated with hnRNPH2 R114W , which induced the assembly of cytoplasmic stress granules, as marked by eIF3η staining . Importantly, despite mutation-dependent redistribution to the cytoplasm, the majority of hnRNPH2 was still found in the nucleus. Indeed, even with the mutation (P209L) that caused the greatest amount of cytoplasmic accumulation, we estimated that approximately 75% of hnRNPH2 was found in the nucleus. In contrast, the 2 non–PY-NLS variants did not show cytoplasmic accumulation of hnRNPH2 . The remaining 2 variants alter the amino acid sequence in RRM3 (D340V) or the C-terminal low complexity domain (A371Cfs*24), respectively. All hnRNPH2 variants within the PY-NLS showed accumulation of hnRNPH2 protein in the cytoplasm, although the levels of accumulation varied  resulted in an approximately 90% decrease in Kapβ2 protein levels , and the PY-NLS motif is perfectly conserved between the two species (Hnrnph2 (Hnrnph2em1(IMPC)J/MmJax, The Jackson Laboratory). These two KO lines differ in two respects: first, in contrast to our KO line, which was driven by an indel with consequent nonsense-mediated decay, the KOMP-KO line was generated by a 1,451 bp deletion in exon 4, which was predicted to result in a truncated, nonfunctional transcript. Second, the background strain of our Hnrnph2-mutant and -KO lines is C57BL/6J, whereas the KOMP KO is on a C57BL/6NJ background. Thus, we selected 2 distinct disease-associated Hnrnph2 knockin mouse lines (R206W and P209L) and 2 distinct Hnrnph2 KO lines for in-depth phenotypic analysis. Importantly, extensive phenotypic analysis of our indel-based KO line and the KOMP-KO line yielded equivalent results. For ease of presentation, we include data from our own indel-based KO line (hereafter referred to as KO) in all subsequent figures alongside data from our knockin lines, whereas all results from the KOMP-KO line are compiled in Like human  species . To inve, suggesting partial embryonic lethality of male mice bearing the P209L mutation . We did not observe any changes in hnRNPH2 R206W or KO mice , the number of mice with pathologically confirmed hydrocephalus suggested an increased incidence in hnRNPH2 P209L and R206W mutant mice, but not in KO mice, compared with WT controls . The cautermates . Neithertermates . Notablytermates  revealed KO mice . We noteant mice .To characterize developmental abnormalities in the central nervous system of the mutant mice, we performed a systematic histological analysis of hemizygous male mutant or KO mice and their WT littermates. H&E staining revealed no gross abnormalities in the brains of hnRNPH2 P209L, R206W, or KO male mice , apart fP209L/YHnrnph2 mice. Compared with neurons cultured from littermate control mice, neurons cultured from P209L/YHnrnph2 mice had a dramatic reduction in the dendritic arbor .In humans, pathogenic variants in pairment ; thus, wpairment , 28, we controls . When abcontrols . In addicontrols . Scores P209L/YHnrnph2, R206W/YHnrnph2, and P209L/XHnrnph2 mice compared with WT littermates, whereas all other mutant or KO male mice and female mice did not differ significantly from controls  without clinical seizures . The frequent falls of the mutant mice off the maze were unexpected and may be related to increased anxiety levels and/or impaired motor function. For mice that completed the 5-minute test, we found no significant difference in the total distance traveled, suggesting similar locomotor activity  fell from the maze as compared with controls (1 of 19)  between termates . Althougtermates . Togethethe task . During days 2–4 . Percentdays 2–4 . Given tdays 2–4 , 37, it training  . In the adult cortex, Hnrnph1 mRNA levels were significantly increased in male Hnrnph2-KO mice but not in P209L or R206W mutant male mice , we did not detect any increase in HNRNPH1 transcript levels. However, when we reached approximately 60% KD of HNRNPH2 (18 nM RNAi), we detected an increase of approximately 15% in HNRNPH1 transcript levels. At approximately 88% and approximately 98% KD of HNRNPH2 (24 and 30 nM RNAi), we found approximately 25% and approximately 55% increases of HNRNPH1 transcript levels, respectively  but were less affected in mutant-expressing neurons . We further noted that the severity of gene expression changes in mutant-expressing neurons was inversely correlated with the levels of HNRNPH1 upregulation, with R206W (99% of WT expression levels) being the most severe and R206Q (128% of WT expression levels) being the least severe  bearing hnRNPH2 R206W, R206Q, or P209L mutations or KO of pression , A and Bpression . Interespression . Howeverpression . This obt severe . The gent severe , C–F. Mot severe , G and HHnrnph2-KO mice (CTNNA2 (OMIM: 618174), TNPO2 (OMIM: 619556), ASH1L (OMIM: 617796), and SHANK1 (OMIM: 209850)  . Similarsplicing , G and HHNRNPH2 and its close paralog HNRNPH1 provide one such example, wherein similar mutations  cause syndromes with overlapping symptoms  in which disease phenotypes are absent in KO animals but are faithfully recapitulated in animals expressing disease mutations. However, our results are also consistent with an alternative disease mechanism in which mutations in HNRNPH2 directly cause a loss of hnRNPH2 function, but the persistence of significant hnRNPH2 protein in the nucleus results in a failure to induce compensatory HNRNPH1 expression. Indeed, HNRNPH1 has an expression pattern that is nearly identical to that of HNRNPH2 with respect to brain region and cell type, and it encodes a highly similar protein to hnRNPH2. Interestingly, whereas the expression of HNRNPH1 decreases as development proceeds, the expression of HNRNPH2 persists, such that cells become progressively more dependent upon hnRNPH2. In this context, our findings from RNA-Seq analyses — namely, that KO of HNRNPH2/Hnrnph2 and knockin of pathogenic mutations alter gene expression into the same direction, in which knockin mutations induce more severe transcriptome changes than KO and KO, but not knockin, of HNRNPH2/Hnrnph2 consistently leads to upregulation of HNRNPH1/Hnrnph1 — suggest that upregulation of Hnrnph1 is responsible for rescuing KO animals from the consequences of the loss of hnRNPH2 function. In contrast, the introduction of disease mutations in Hnrnph2 in mice is not accompanied by significant upregulation of Hnrnph1. As such, disease-causing mutations in HNRNPH2 thwart the physiological mechanism that would otherwise compensate for the loss of hnRNPH2 protein function.This pattern of phenotypes in physiological models — no apparent phenotypic consequence in KO mice, robust recapitulation of disease features in knockin mice — suggests two possible mechanisms as drivers of disease. The first possibility is a toxic gain of function, a mechanism with precedents in several common neurological diseases . Indeed, genetic compensation between Sample preparation and data processing of RNA-Seq are described in All animal experiments were reviewed and approved by the IACUC of St. Jude Children’s Research Hospital.RNA-Seq data were deposited to GEO under accession GSE226527, which includes GSE226525 (human) and GSE226526 (mouse).AK, XY, KO, AG, BJWT, HN, and FY designed and performed laboratory experiments. AK, KO, AG, BJWT, JM, AFC, YDW, TP, HS, SSZ, YMC, JPT, and HJK analyzed data. AK, JPT, and HJK drafted and revised the manuscript. JPT and HJK supervised the overall study.


The 22q11.2 deletion syndrome is a multisystemic disorder characterized by a marked variability of phenotypic features, making the diagnosis challenging for clinicians. The wide spectrum of clinical manifestations includes congenital heart defects—most frequently conotruncal cardiac anomalies—thymic hypoplasia and predominating cellular immune deficiency, laryngeal developmental defects, midline anomalies with cleft palate and velar insufficiency, structural airway defects, facial dysmorphism, parathyroid and thyroid gland hormonal dysfunctions, speech delay, developmental delay, and neurocognitive and psychiatric disorders. Significant progress has been made in understanding the complex molecular genetic etiology of 22q11.2 deletion syndrome underpinning the heterogeneity of clinical manifestations. The deletion is caused by chromosomal rearrangements in meiosis and is mediated by non-allelic homologous recombination events between low copy repeats or segmental duplications in the 22q11.2 region. A range of genetic modifiers and environmental factors, as well as the impact of hemizygosity on the remaining allele, contribute to the intricate genotype-phenotype relationships. This comprehensive review has been aimed at highlighting the molecular genetic background of 22q11.2 deletion syndrome in correlation with a clinical multidisciplinary approach. The chromosome 22q11.2 deletion syndrome (22q11.2 DS), also known as DiGeorge syndrome (DGS) or velocardiofacial syndrome (VCFS), is a genetic condition resulting from the impaired development of structures originating from the third and fourth pharyngeal pouches in the germinal stage. The clinical features of the syndrome include hypoparathyroidism and hypocalcemia, thymic hypoplasia, conotruncal heart defects, facial dysmorphism, and palatoschisis. The complex phenotype of children affected with 22q11.2 DS may show considerable intersubject variability, and the expanded clinical manifestations comprise craniofacial, neurological, cognitive, behavioral, ocular, speech and hearing, musculoskeletal, and internal organs, as well as airway, gastrointestinal or renal abnormalities [The incidence of 22q11.2 DS has been estimated to range from 1:3000 ,5,6. TheHowever, there is a paucity of reports on the clinical course of 22q11.2 DS in pediatric patients, and the data are scattered, thereby making them difficult to apply in the all-embracing pediatric practice. In this review, we aimed to establish a comprehensive molecular genetic and clinical approach to 22q11.2 DS addressed to pediatricians and specialists in other fields of medicine who encounter children affected with this syndrome and provide in-depth clinical care. We also sought to determine what is known and what is new in the multidisciplinary genetic and clinical diagnostic process and prophylactic measures, as well as therapeutic modalities in 22q11.2 DS.GP1BB variant) and cerebral dysgenesis (polymicrogyria), neuropathy, ichthyosis, and palmoplantar keratoderma (CEDNIK) syndrome  [Several clinical disorders make up 22q11.2 DS, such as velocardiofacial  and DiGeorge  syndromes, a group of disorders described by the acronym CATCH22 . The nomenclature may be confusing due to the remarkable heterogeneity of clinical phenotype and marked intersubject variability of the presenting symptomatology in 22q11.2 DS . A varievariant) .TBX1, proline dehydrogenase PRODH, catechol-O-methyltransferase COMT, cell division cycle 45 CDC45, GP1BB, SNAP29, and DiGeorge critical regions DGCR2, DGCR6, DGCR6L, and DGCR8. A proximal 1.25 Mb deletion comprising LCR22A to LCR22B affects about 5% of individuals with 22q11.2 DS, whereas 2% of them show a deletion spanning the LCR22A to LCR22C region, and the next 5% have a smaller, atypical, nested deletion extending from LCR22B or LCR22C to LCR22D. Distal deletions, flanked by LCR22D-E and LCR22D-F, have been reported less frequently [DGCR6, DGCR6L, and DGCR8 gene alleles and mi-RNAs, such as miR-185, miR-4716, miR-3618, miR-1286, miR-1306, and miR-6816. Several miRs—miR-96-5, miR-451a, and miR-17-92—target TBX1, thereby influencing its expression and, consequently, the clinical phenotype [The deletion types and sizes in 22q11.2 DS show a high degree of variability due to several low-copy-number repeat sequences  flanking the deleted region. Most patients (about 90%) show a 2.54 Mb heterozygous deletion comprising four repeats extending from LCR22A to LCR22D and involving approximately 40 genes . The disequently ,16. The henotype . The 22qhenotype .The de novo occurrence of the syndrome is observable in about 90–95% of affected individuals in all populations, yet autosomal dominant inheritance has also been ascertained in 6–28% of subjects . With itWith its average frequency estimated to be 1:4000 live births and 1:1000 in unselected fetuses , 22q.11.Craniofacial dysmorphism in 22q11.2 DS is characterized by a multiplicity of facial phenotypic features. Some of these facial characteristics are not easily recognized clinically in children as they are mild in nature, and age is an important determinant of facial morphology. These features include an elongated face, hypertelorism, wide nasal bridge, hooded eyelids, upslanted palpebral fissures, epicanthus, long nose with a bulbous tip, narrow alar base, short philtrum, small mouth, micrognathia, and low-set, posteriorly rotated, small ears. The retrognathic posture of the mandible, inward displacement of the lower part of the face and the ocular region, and an enlarged cranial base angle have been identified as the uppermost dysmorphology features in the cephalometric and spatially dense 3-dimensional analysis . High prAmong oropharyngeal developmental disorders, palatoschisis is a frequently observable phenotypic feature, occurring in the form of open cleft, submucosal cleft, palatopharyngeal disproportion, and cleft lingula ,41. VeloDevelopmental disorders of the upper airways in 22q11.2 DS include congenital laryngeal anomalies. Laryngeal atresia due to a glottic web or partial laryngeal stenosis, laryngomalacia, laryngeal cleft, and vocal fold abnormalities are characteristic parts of the syndromic features ,44. The CRKL encoding for a cytoplasmic adaptor protein (CRK-like proto-oncogene) involved in growth factor signaling [Congenital heart disease has been recognized in approximately 60–80% of children affected with 22q11.2 DS. The most commonly occurring subset of cardiac anomaly is a conotruncal defect, such as tetralogy of Fallot, pulmonary atresia with ventricular septal defect, truncus arteriosus, interrupted aortic arch type B, conoventricular and/or atrial septal defects, and aortic arch anomalies . The anaignaling . Moreoveignaling .The complex and variable phenotypes related to 22q11.2 DS also include mild to moderate intellectual disability, which occurs in approximately one-third of affected pediatric patients . In thesNoteworthy, 22q11.2 DS is one of the rare examples of a cytogenetic abnormality occurring in conjunction with a psychiatric disease, schizophrenia, autism spectrum disorders (ASD) with some level of social-communication impairment, and attention deficit/hyperreactivity disorder (ADHD), which occur in as many as 20% of affected children and adolescents ,59. The TBX1 gene in the formation of cardiac outflow structure and positioning of the thyroid gland [Endocrine manifestations are hallmarks of 22q11.2 DS and primarily comprise hypoparathyroidism, thyroid dysfunction, and growth retardation ,67. Hypoid gland . Growth id gland . Whereasid gland  or adrenid gland , are conid gland , hypertrid gland , and obeid gland , have beid gland .The summary of multisystemic clinical phenotypic features and their estimated frequency in patients with 22q11.2 deletion syndrome, based on literature reviews, is shown in TBX1 hemizygosity, other genes, such as CRKL in the affected 22q11.2 region, may also have a gene dosing, modifying effect on the phenotypic expression of the syndrome. CRKL is expressed in neural crest-derived tissues and involves thymic development. The effect of compound heterozygosity for TBX1 and CRKL deletion on clinical features and thymus development is additive [Immunodeficiency is a key feature of 22q11.2 DS and is secondary to thymic aplasia or hypoplasia with subsequent impaired thymocyte development. The third and fourth pharyngeal pouches are a common embryonic precursor for the thymus, parathyroid glands, and conotruncal regions of the heart. In 22q11.2 DS, maldevelopment of these organs is due to impaired migration of the neural crest cell into pouch ectoderm . In the additive .Consequently, a wide spectrum of T-cell alterations is seen in 22q11.2 DS, ranging from near normal to near completely immunodeficient. Mild T cell immunodeficiency may be found in children with apparently hypoplastic thymus because ectopic retropharyngeal thymic tissue may be preserved ,91. FurtReferring to anatomical maldevelopment and dysfunction in generating T cells, the humoral immunodeficiency and B cell abnormalities in children with 22q11.2 DS are secondary to T cell deficits . Low immTBX1. Rare DNA variants in transcriptional regulators involved in retinoic acid signaling, NCOR2 and EP300, were found to be associated with parameters of the immune functions, such as immunoglobulin levels, lymphocyte response to antigens and mitogens, and flow cytometric lymphocyte compartment. Retinoic acid plays an important role in maintaining immune homeostasis by enhancing the differentiation of regulatory T cells, modulating epithelial and mucosal immune responses, and regulating proinflammatory cytokine activity. Hence, genetic modifiers contributing to the individual’s genetic background and modulating variable penetrance may influence the immune response in 22q11.2 DS [Immune dysregulation in 22q11.2 DS has been foremostly ascribed to T-cell lymphopenia and deficiency in CD3+CD4+CD25++, FOXP3+ regulatory T cells, which play a crucial role in maintaining immune homeostasis and self-tolerance. Reduced thymic output related to an increased naive T cell subset, and subsequent T regulatory cell activation, control the expansion of the T cell compartment. A reduced number of regulatory T cells, with their activated phenotype and loss of suppressive capacity in children with 22q11.2 DS, are key features of deregulated T cell homeostasis . Beyond q11.2 DS ,104.Autoimmune disorders have been described in as many as 23% of pediatric patients with the syndrome , manifesThe peripheral homeostatic expansion of T cells driven by low thymic output and T cell lymphopenia may contribute to Th2-skewed lymphocyte phenotype and atopic manifestations, such as eczema and asthma . In thesThe thymus dysfunction and immunophenotypic abnormalities within the T cell compartments in 22q11.2 DS make affected children susceptible to cancerogenic viruses, such as Epstein-Barr virus (EBV) and human papilloma virus (HPV), that might be linked to an increased risk of malignant transformation. It has also been postulated that chronic immune activation of peripherally expanded T cell population in dysfunctional cellular immunity may underpin the predisposition to developing lymphoproliferative disorders and lymphomagenesis , as T ceTBX1 penetrance and related phenotypic expression is shown in The chromosomal 22q11.2 DS is a clinically highly variable microdeletion syndrome with differently expressed phenotypes, with wide interfamilial and intrafamilial variability in patients sharing the same genetic underpinnings . This isTBX1 gene during development of the heart, thymus, and parathyroid glands, as well as during formation of the palate, teeth, and craniofacial features, there has been growing evidence that TBX1 is a candidate gene for 22q11.2 DS [The diagnosis of 22q11.2 DS has been traditionally based on the recognition of clinical features and cytogenetic testing using the fluorescence in situ hybridization (FISH) technique. FISH is perceived as the golden standard genetic testing method to confirm the diagnosis of microdeletion syndromes. However, poor clinical accuracy, the low confirmatory rate in the screening of suspected microdeletion syndromes, and failure to detect other than the targeted microdeletion are the major drawbacks of this method. An important limitation of this method is a failure to identify atypical and nested deletions because it can recognize deletions in the proximal part of the critical region, including the typical LCR22A-D deletion. FISH is admittedly inexpensive, yet still a highly labor-intensive and time-consuming procedure . Importaq11.2 DS ,130. Theq11.2 DS .TBX1 gene within the LCR22A-B region and CRKL within the LCR22C-D region, has been elaborated [Newborn screening (NBS) for severe combined immunodeficiency (SCID) is identifying a subset of infants with 22q11.2 DS due to T cell lymphopenia and low TREC numbers, and, hence, it is not a universally reliable detecting method. Therefore, direct NBS for 22q11.2 DS to recognize affected neonates using the genomic approach with multiplex qPCR assay, targeted to the aborated .Whereas postnatal phenotypes have been widely characterized and categorized, prenatal diagnosis of 22q11.2 DS remains challenging due to a low rate of inheritance (10% of cases) and mild unrecognized parental features. Fetal ultrasound imaging may provide information on findings characteristic for 22q11.2 DS, such as polyhydramnios, hypoplasia or aplasia of the fetal thymus, central nervous system anomalies, such as asymmetric ventriculomegaly and dilated cavum septum pellucidum, and, rarely, skeletal anomalies, among others, such as bilateral talipes and anomalous vertebrae .The golden standard method for detecting 22q11.2 microdeletions remains first trimester screening by chromosomal microarray (CMA), which is performed in invasively obtained prenatal samples, such as chorionic villi and amniotic fluid. An important technical advance in prenatal noninvasive screening for 22q11.2 is cell-free DNA testing . Cell-frThe marked phenotypic variability of 22q11.2 DS is accompanied by a wide scope of immune deficits, ranging from mild to moderate T cell lymphopenia in the partial form of DiGeorge syndrome (pDGS) to profound combined T and B cell immunodeficiency in the complete form (cDGS). In the first case, hypoplastic ectopic thymus or microscopic thymic rests are found and successful spontaneous immunocorrection has been reported, whereas the latter case is characterized by complete athymia . In thosActive immunization in children with 22q11.2 DS requires optimizing to provide vaccination coverage against vaccine-preventable infections. Live vaccine practices with Bacille Calmette-Guerin (BCG), and vaccines against measles-mumps-rubella (MMR), varicella (VAR), and an intranasal live attenuated influenza vaccine (LAIV), are contraindicated in children with this syndrome as T cell lymphopenia makes them susceptible to adverse effects following live immunization (AEFLI). Furthermore, in those children who, due to hypogammaglobulinemia, receive immunoglobulin replacement therapy either intravenously or subcutaneously, live vaccines are inactivated by administered antibodies and thereby are contraindicated. Inactivated vaccines can be safely administered to immunodeficient patients as they do not pose the risk of an uncontrolled spreading of vaccine microorganisms in the patient’s body, and they are not inactivated by supplemented immunoglobulins. However, the immune response to vaccines with antigen-specific antibodies and memory B cell generation may be significantly reduced .9/L, a cytotoxic T cell count above 0.2 × 109/L, and a normal response to mitogen [However, many individuals with 22q11.2 DS with mild to moderate immunosuppression receive live viral MMR and varicella vaccines despite the known diagnosis and tolerate them well, without serious adverse effects ,150. Giv mitogen .Reports on nationwide studies capturing patients with 22q11.2 DS ,145,152


Sphingolipids (SLs) are structural, bioactive molecules with several key cellular roles, whereas 1,25-dihydroxyvitamin D D), the active form of vitamin D, is considered the major regulator of calcium homeostasis, although it also exerts other extraskeletal effects. Many studies reported the physiological connection between vitamin D and SLs, highlighting not only the effects of vitamin D on SL metabolism and signaling but also the influence of SLs on vitamin D levels and function, thus strongly suggesting a crosstalk between these molecules. After a brief description of 1,25(OH)D and SL metabolism, this review aims to discuss the preclinical and clinical evidence on the crosstalk between SLs and 1,25(OH)D, with a special focus on cardiometabolic diseases. Vitamin D is a well-known key factor in bone health, as its active form, 1,25-dihydroxyvitamin D D), affects intestinal absorption of calcium and phosphate, both of which are essential elements for proper bone mineralization ,4. In paHowever, some effects are too fast to be attributed to changes in gene expression but rather are to be linked to the membrane-VDR (mVDR) and appear to involve MARRS , thus affecting several signal transduction pathways . NoteworMoreover, different studies demonstrated a significant association between low levels of circulating 25(OH)D and an increased prevalence and extent of ischemic heart disease, as well as risk of CV events, including mortality . In partBased on epidemiological and preclinical studies, there is further evidence of an association between hypovitaminosis D and cerebrovascular diseases, including ischemic stroke . HoweverInterestingly, recent advances in vitamin D biology raised the complexity of this scenario by providing mechanistic alternatives to the different, and sometimes opposite, effects of vitamin D. Indeed, the identification of new, alternative pathways of vitamin D activation led to the discovery of additional biologically active metabolites that may interact not only with VDRs but also with other nuclear receptors, opening up a new area of research that requires further investigation to better understand the pleiotropic effects of vitamin D . In partSphingolipids (SLs) are structural elements of the cellular membrane and key components of ceramides (Cer), a group of bioactive molecules characterized by the presence of sphingoid bases in the hydrophobic region of their molecular structure, while phosphate groups, sugar residues, and/or hydroxyl groups build up the hydrophilic region. Several studies attribute to Cer a wide range of functions, thus linking them to several important physiological pathways  based on their specific structure  . Many stInterestingly, a growing body of scientific evidence suggests that targeting SLs may benefit inflammation and cardiometabolic diseases, thus representing a promising and novel therapeutic option to be developed and investigated . In thisHowever, despite the encouraging results available, there are still challenges and limitations to overcome  before SLs can be introduced in clinical practice as biomarkers for diagnosis, management, and therapeutic targets for CM diseases ,24,25.Due to the high complexity of this class of molecules, also taking into account their multiple and sometimes opposite biological functions, SL pathophysiology definitely represents a scientific challenge; however, current knowledge needs to be further deepened in order to be able to target specific sphingolipids without affecting other species, thus eliciting collateral adverse effects .Given the importance of vitamin D and SLs in cardiometabolic settings, it is worth highlighting that vitamin D, alongside many other actions , is also involved in SL metabolism regulation. In addition, Cer act as mediators of 1,25(OH)D on cell differentiation, thereby playing a significant role in the mechanism of action of 1,25(OH)D, suggesting a crosstalk between these molecular entities and common effectors  ,28,29.Hence, this review aims to outline vitamin D metabolism and discuss the link between vitamin D and SLs in preclinical and clinical investigations, as well as current pitfalls and potential future opportunities in this field. Moreover, considering that recent scientific results have shown that vitamin D3 can bind directly to VDRs with a much higher affinity than 1,25(OH)D when bound to vitamin-D-binding protein (VDBP) ,16, thisFrom a biochemical standpoint, vitamin D is a biologically inactive prohormone that needs hydroxylation reactions to exert its properties efficiently. This fat-soluble secosteroid exists in two main forms, vitamin D2  and vitamin D3 , chemically different in the side chain; indeed, unlike vitamin D3, vitamin D2 carries a double bond between C22 and C23 and a methyl group at C24  30,31,3,331,32. Nevertheless, dietary foods provide only a small percentage of the body’s vitamin D requirements, while the skin, particularly when exposed to sunlight, is the main vitamin D3 source, providing at least 80% of the vitamin D demand in humans ,34. In tUpon prolonged exposure to UV or solar radiation, a physiological control mechanism kicks in, and through the inhibition of vitamin D synthesis, PreD3 is converted to inactive metabolites  rather than vitamin D3. In this way, negative effects arising from excessive vitamin D synthesis are avoided .Once produced in the skin, vitamin D3 is released into the circulation and transported in serum through binding to VDBP. As a fat-soluble vitamin, a percentage of the newly synthesized vitamin D is also stored in the adipose tissue. Right after its synthesis, vitamin D has no biological activity but rather needs to be metabolized through two subsequent hydroxylation reactions, which lead to the active hormone 1,25(OH)D .The first reaction is a 25-hydroxylation, which takes place mainly in the liver: here, in the microsomal P450 fraction of hepatocytes, the enzyme CYP2R1 25-hydroxylates both vitamin D2 and vitamin D3, yielding 25-hydroxyvitamin D (25(OH)D). Although CYP2R1 is considered the major enzyme responsible for this conversion, other enzymes have shown the same activity, thus contributing to the levels of 25(OH)D ,35. Amon25(OH)D is the major circulating form of vitamin D and is, therefore, considered the main parameter to look at in order to obtain the best estimation of the body’s vitamin D pool. However, a second reaction is needed to obtain active vitamin D, namely 1,25(OH)D, with paracrine and autocrine functions. This second step is catalyzed by the enzyme 1-alpha-hydroxylase CYP27B1, mainly expressed in renal cells and, in a lower amount, in several other tissues, including keratinocytes, bone, placenta, and immune cells ,35,36. CThe known half-life of 25(OH)D is around 15–20 days, and most of the circulating vitamin D is transported by VDBP (85%) and albumin (15%) to target tissues , where it exerts its physiological function ,32.SLs are synthesized via three main pathways, briefly discussed below:trans-double bond in position C4–C5 trans of dhCer, thus forming the correspondent ceramide  mainly through four reactions. In the first reaction, serine palmitoyltransferase (SPT) condenses palmitoyl-CoA and serine into 3-ketosphinganine (3-KS); then, through a second reaction carried on by a 3-keto-dihydrosphingosine reductase (3-KDSR), 3-KS is converted into sphinganine (DHSph), which is then further acetylated to dihydroceramide (dhCer) through different isoforms of ceramide synthase (CerS). CerS indeed promotes the reaction between acyl-CoA (made up of different chain lengths) and the molecule of sphinganine. Finally, a dihydroceramide desaturase introduces a ceramide  24,26].,26.transSphingomyelinase pathway: In this pathway, the hydrolysis of sphingomyelin (SM), carried out by sphingomyelinases (SMases), leads to the production of ceramides and phosphocholine. SMases are enzymes differently expressed in the various tissues according to their pH and structural characteristics.Salvage pathway: It takes place in acidic subcellular compartments, like endosomes and lysosomes. Through the SMases enzyme, SM is metabolized to sphingosine (Sph), which is then recycled to produce Cer through the action of CerS ,26.Many studies have already reported vitamin D effects on SL metabolism and signaling, while others have demonstrated the role of SL on vitamin D levels and function, thus strongly suggesting a crosstalk between these pathways .2) gene expression levels, a receptor that negatively regulates S1P [1–5) mRNA expression levels were measured in monocytes isolated from healthy controls and type 2 diabetes patients, mRNA levels of S1P1 and S1P2 (linked to inflammation and insulin resistance) were downregulated by calcitriol in monocytes from healthy subjects as well as type 2 diabetes patients, while S1P3 and S1P4 gene expression (both involved in the synthesis of cytokines) was induced; moreover, while SPHK1 was upregulated by calcitriol in both groups, SPHK2 gene expression only increased in type 2 diabetes subjects [The first evidence that 1,25(OH)D modulates phospholipid metabolism at the intestinal, renal, bone, and parathyroid levels dates back to the 1980s . Accordiates S1P . Indeed,subjects .3 gene expression has been reported in human breast cancer cells following calcitriol administration, suggesting cell-type-specific responses [In contrast to these data, a reduction in S1Pesponses . On the esponses ,44,45. Iesponses . Other desponses .In the cardiometabolic (CM) field, recent data reported that vitamin D supplementation improves left ventricular dysfunction in a mouse model of diet-induced type 2 diabetes (10 weeks); at the 25th week, unsupplemented mice had increased myocardial levels of two lipotoxic species, Cer and diacylglycerol (DAG), which correlated with adverse effects at the cardiomyocyte level , which were normalized by vitamin D3 supplementation . As prevInterestingly, vitamin D affects the lipid composition (including SL) of adipocyte-derived extracellular vesicles, playing key roles in energy homeostasis . 1,25 . MoreoveFurthermore, it should be considered that SL, in particular S1P, and 1,25(OH)D, can share common effectors , while their synergistic effects are still unclear and deserve further investigations.Interestingly, studies showed that lysosomal ceramides, as well as vitamin D administration, modulate medial calcification in the arterial wall of smooth-muscle-cell-specific Smpd1 transgenic mice, suggesting that these factors might interact in the changes that contribute to the development of medial arterial calcification and arterial stiffness .Only a few human studies have assessed the association between vitamin D deficiency and its interaction with SL metabolism and signaling in metabolic and cardiometabolic diseases. In particular, vitamin D deficiency was associated with several metabolic complications and diseases, including hypertension, obesity, insulin resistance, and diabetes ,60. It iIn patients with type 2 diabetes, plasma levels of Cer, Sph, DHSph, and their phosphate derivatives were measured at the beginning of the study, after 6 months of vitamin D supplementation, and after another 6 months of follow-up. Results showed that both at baseline and 6 months after follow-up, there were no differences in plasma SL species between placebo and patients. However, after 6 months of supplementation, vitamin D significantly enhanced plasma levels of C18 dhCer (N-stearoyl-sphinganine (d18:0/18:0)) and C18 Cer (N-stearoyl-sphingosine (d18:1/18:0)) in patients with type 2 diabetes compared with the placebo group. Other dhCer and Cer remained, instead, unchanged after 6 months of vitamin D supplementation, as unchanged were also the plasma levels of sphingosine, DHSph, and their phosphate derivatives (S1P and DhS1P) . These rThe role of vitamin D supplementation in 70 overweight/obese African Americans was recently investigated in order to determine whether its supplementation altered circulating long-chain ceramides and their metabolites. For 16 weeks, subjects were randomly assigned to one of four groups: 600, 2000, or 4000 IU/day vitamin D3 supplements or placebo. In this study, serum levels of N-stearoyl-sphingosine (Cer d18:1/18:0) and stearoyl sphingomyelin (Cer d18:1/18:0) significantly increased after vitamin D3 supplementation in a dose–response manner, while other metabolites such as N-palmitoyl-sphingosine (Cer d18:1/16:0), N-palmitoyl-sphinganine (Cer d18:0/16:0), Sph, S1P and Cer d18:1/16:0 remained unchanged. Moreover, changes in N-stearoyl-sphinganine (Cer d18:0/18:0), Sph, and S1P were positively associated with changes in hemoglobin A1c, while Cer d18:1/18:0 and Cer d18:0/18:0 were associated with changes in BMI .A proteomic study on vitamin-D-deficient Saudi Arabian subjects identified several proteins  that are differentially expressed in obese versus lean people . Using mIn another cross-sectional, retrospective study, based on data from 277 participants randomly selected from a larger cohort of 1820 subjects, lipidomic profiling was investigated in relation to vitamin D status and dyslipidemia. This analysis highlighted, on the one hand, higher SM levels in subjects with both physiological levels of vitamin D and dyslipidemia, while in vitamin-D-deficient patients either with or without dyslipidemia, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were highly altered. Furthermore, levels of SL and ceramide-related metabolites in normo- and dyslipidemic subjects with vitamin sufficiency and deficiency were examined in the same study, revealing higher levels of N-stearoyl-sphingosine (Cer d18:1/18:0) in dyslipidemic and vitamin-D-deficient subjects .Taken together, these results show a significant alteration of the metabolomic profiles in dyslipidemic and vitamin-D-deficient individuals when compared with control groups, an alteration that reflects changes in key physiological pathways and that involves several classes of biological functional metabolites, including Cer, lysophospholipids, phosphatidylcholines, phosphatidylethanol amines, and SM.A more recent study investigated sphingolipid levels in the red blood cell membrane, as well as their relationship with some well-established biomarkers of cardiovascular health, including vitamin D status in healthy women  ,73. The The crosstalk between vitamin D and SLs has also been hypothesized for the nervous system . As in tHowever, although changes in the vitamin D and SL signaling pathways, as well as in blood levels, have been observed in patients with neurodegenerative diseases, few studies have actually demonstrated a crosstalk between vitamin D and SLs . InteresVitamin D affects multiple biological systems, playing a key role in the prevention and regulation of cardiometabolic diseases , which are largely the same effectors targeted by SLs. Not surprisingly, available data clearly support the existence of a crosstalk between the SL and vitamin D pathways : indeed,


Ferroptosis, a newly recognized form of nonapoptotic regulated cell death, is characterized by iron-dependent lipid peroxidation. Biological processes, such as iron metabolism, lipid peroxidation, and amino acid metabolism, are involved in the process of ferroptosis. However, the related molecular mechanism of ferroptosis has not yet been completely clarified, and specific and sensitive biomarkers for ferroptosis need to be explored. Recently, studies have revealed that ferroptosis probably causes or exacerbates the progress of cardiovascular diseases, and could be the potential therapeutic target for cardiovascular diseases. In this review, we summarize the molecular mechanisms regulating ferroptosis, inducers or inhibitors of ferroptosis, and the current progresses of ferroptosis in cardiovascular diseases. Furthermore, we discuss the emerging challenges and future perspectives, which may provide novel insights into the treatment of cardiovascular diseases. TherMultiple types of cell death, including apoptosis, necrosis, and pyroptosis, are associated with the pathogenesis of CVDs . AccumulIn this review, we focus on effects of ferroptosis and related mechanisms in the occurrence and development of CVDs, and highlight the effective strategy for targeting ferroptosis in CVDs.2.Ferroptosis is a complicated process that is moderated by a series of signaling and molecules. The occurrence and execution of ferroptosis contain three main events, iron dysregulation, GSH depletion, and lipid membrane oxidation . The reg2.1.+) and ferric (Fe3+) iron  transports Fe2+ into the iron pool (LIP) through SLC11A2  and ferritin heavy chain 1 (FTH1), or enters into the blood by ferroportin-1 (FPN) and lipocalin-2 (LCN-2)  iron . In gene3+) iron , 9. Six- SLC11A2 , 11. The (LCN-2) , 13.FPN, the sole exporter in charge of the efflux of surplus iron, is degraded after being bound to hepcidin . Accordi2+ concentration in extracellular space, and catalyzes the Fenton reaction to participate in the production of ROS, inducing lipid peroxidation and eventually ferroptosis  are more prone to lipid peroxidation under oxidative stress , 21. CelPUFAs can be acylated by acyl-CoA synthetase long-chain family member 4 (ACSL4) to form PUFA acyl-coenzyme A (PUFA-CoA), and then reacts with lysophosphatidylcholine acyltransferase 3 (LPCAT3) to form PUFA-PE. PUFA-PE is subsequently oxidized by lipoxygenases (LOXs) to generates PUFAs-OOH , 23. PerSeveral reports have also shown that LOXs, the 15-LOX-1 isoform, especially are a key regulator of lipid peroxidation of ferroptosis , 26. Sup2.3.−), a membrane amino acid transporter, which consists of the catalytic subunit solute carrier family 7 member 11 (SLC7A11) and the solute carrier family 3 member 2 (SLC3A2)  . System the cell . Inhibittabolism .In addition, it is widely accepted that GPX4 is another key point in the amino acid metabolic pathway. GPX4 belongs to the selenoprotein family that uses GSH as a reducing agent and turns GSH into oxidized glutathione (GSSG) . In the 3.− and GPX4, have been implicated in governing ferroptosis -Nuclear factor E2-related factor 2/antioxidant response element (Keap1-Nrf2-ARE) pathway is the main survival pathway in cells, which protect cells from oxidative and electrophilic insults . Nrf2 isAnother cellular signaling pathway is the ferroptosis suppressor protein 1 (FSP1)–coenzyme Q10 (CoQ10)–nicotinamide adenine dinucleotide phosphate (NADPH) pathway, which is associated with GPX4 and glutathione (GSH) to prevent lipid peroxidation and ferroptosis independently . TogetheGTP cyclohydrolase-1 (GCH1)-tetrahydrobiopterin/dihydrobiopterin (BH4/BH2)-phospholipid axis has been shown to be a primary signaling pathway of ferroptosis resistance through upregulation of CoQ10, therefore suppressing oxidative damage and ferroptosis . GCH1 ov4.Ferroptosis plays a central role in CVDs, and regulating the levels of some key molecules related to ferroptosis may be helpful in treating CVDs . Several4.1.− or altering intracellular iron levels. Many ferroptosis inducers has been found, which can be divided into four classes, class I-IV. Class I ferroptosis inducers inhibit cystine uptake through the system Xc−, such as erastin and sorafenib. Erastin binds to SLC7A5, which is one of six light chains that bind SLC3A2 to import cystine, decreasing cellular glutathione, and inducing ferroptosis . Class II inducers do not deplete GSH, but bind to and inactivate GPX4, resulting in the formation of ROS . Class I4.2.2+, thereby preventing ferroptosis  and dexrazoxane (DXZ), could deplete the cellular iron pool, and promote the excretion of excess Feroptosis , 45. Ferroptosis . Besidesroptosis . Reducedroptosis .5.Multiple cell death types are involved in the complicated mechanisms of CVDs. Recently, ferroptosis has been proven to play a central role in CVDs . The ass5.1.Acute myocardial infarction (AMI) is one of the most common high-risk CVDs . Timely + in endoplasmic reticulum (ER), triggering ferroptosis after H/R in cardiomyocytes  characterized with arterial wall lipid deposition is the pathological basis for many vascular disorders. AS has been shown to be associated with inflammation, deposition of lipid peroxides and iron . Ferroptclerosis . Inhibitclerosis . Endotheclerosis . Iron ovclerosis . In humaclerosis . Moreoveclerosis . Hepcidiclerosis .5.3.5.3.1.+ complex in mitochondria, causing mitochondria-dependent ferroptosis  level, causing increased lipid peroxidation and ferroptosis (Doxorubicin (DOX), a chemotherapeutic drug, can cause cardiotoxicity termed doxorubicin-induced cardiomyopathy (DIC). Ferroptosis, a new type of cell death form, has been implicated in DIC mice. Mitochondria-dependent ferroptosis is involved in the pathogenesis of DIC . DOX dowroptosis . Both inroptosis , 71. In roptosis .in vitro and in vivo  is a potent antitumor drug, and its clinical use is limited for severe cardiotoxic effects. ROS and iron homeostasis dependent ferroptosis play a significant role in 5-Fluorouracil-induced cardiotoxicity in H9c2 cardiomyocytes and mice . Resvera in vivo . Trastuz in vivo . Mitopha in vivo .5.3.3.Sepsis-induced cardiac injury may result in lethal consequences, and cardiomyocyte apoptosis, ferroptosis, and inflammation participate in the progress of sepsis-induced cardiomyopathy (SIC). Ferroptosis plays critical role in lipopolysaccharide (LPS)-induced cardiac injury. ICA69 upregulates of STING level, which further results intracellular lipid peroxidation, eventually aggravating ferroptosis and septic heart injury . Dex all5.3.4.−/GSH/GPX4 axis (Diabetic cardiomyopathy (DCM), a major complication of type 2 diabetes, is a leading cause of heart failure and death in advanced diabetes. Ferroptosis also engages in the pathogenesis of DCM. A recent study has reported that ferroptosis is involved diabetes-induced endothelial dysfunction by activation of the p53-xCT-GSH axis . AstragaPX4 axis .5.4.Heart failure (HF) is a complex clinical syndrome with high mortality and morbidity , 101. Li5.5.Atrial fibrillation (AF) is the most prevalent type of cardiac arrhythmia with higher morbidity and mortality, due to stroke, heart failure and other complications . Ferropt5.6.Hypertension is a major risk factor for numerous CVDs, and leads to pathological cardiac hypertrophy and eventually heart failure . Elabela6.in vitro cell cultures, ex vivo models, and animal studies. The clinical studies relevant to ferroptosis in cardiovascular diseases are urgently needed. Currently, many chemical compounds have been found to affect the process of ferroptosis, including dysregulation of iron metabolism, lipid peroxidation, and abnormal amino acid metabolism. However, selectivity and side-effects of these compounds remain to be examined. It still faces great challenges in translating these compounds as future pharmacological drugs. In addition to ferroptosis, other forms of cell death, such as apoptosis, regulated necrosis and pyroptosis, have also been involved in the pathogenesis of CVDs. For examples, apoptosis and necroptosis are detected in diabetic mouse hearts at different ages (Ferroptosis is a newly-identified iron-dependent form of cell death. Recent studies have suggested that ferroptosis could be a feasible therapeutic target for several cardiovascular diseases. However, the molecular mechanism of ferroptosis in cardiovascular diseases has not been fully understood. Reliable and sensitive biomarkers for ferroptosis need to be explored. Most investigations are confined to ent ages . Palmitient ages . HoweverIn conclusion, further studies are needed to identify specific biomarkers and central regulators of ferroptosis, providing solid evidence and basis for further exploration in clinical trials. Furthermore, it is important to clarify the different temporal stages of ferroptosis and other cell death forms in CVDs.