The mechanism of insulin signaling on browning of white preadipocytes remains unclear. Human and mouse primary subcutaneous white preadipocytes  were induced to transdifferentiate into beige adipocytes under conditions of intact or blocked insulin signaling, respectively. Level of phosphoinositide-3-kinase (PI3K) after induction of beige adipocytes under conditions of normal insulin signaling, phosphorylated protein kinase B (pAKT), peroxisome proliferator-activated receptor γ coactivator-1 alpha (PGC-1α), zinc-fifinger transcriptional factor PRD1-BF1-RIZ1 homologous domain-containing protein 16 (PRDM16), uncoupling protein 1 (UCP1), peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein beta (C/EBPβ) were significantly increased. Conversely, when insulin signaling is incompletely inhibited, the expression of the thermogenic and adipogenic factors is significantly reduced, with obvious impairment of adipogenesis. However, phosphorylation level of adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK) and expression level of sirtuin type 1 (SIRT1) had increased. These white preadipocytes from different donors showed similar dynamic change in morphology and molecular levels during the browning. The present data indicate that insulin signaling plays a important role in regulation of browning of hsASCs and msASCs through PI3K-AKT-UCP1 signaling pathway. The insulin-AMPK-SIRT1 pathway was also involved in the adipocytes browning, while its effect is limited. Brown adipocytes (BACs) consumes energy in the form of heat production and is used as a defence mechanism against hypothermia, especially in infants, and it also contributes to metabolic homoeostasis , that prThese phenomena may suggest that intact insulin signalling not only regulates the adipogenesis of white preadipocytes and maintains mature WACs phenotype , but alsInsulin, dexamethasone (Dex), 3-isobutyl-1-methylxanthine (IBMX), collagenase type I, and linsitinib (OSI-906) were purchased from Sigma-Aldrich (USA). Dimethyl Sulphoxide (DMSO) was purchased from Merck . TRizol, Dulbecco’s modified Eagle’s-F12 medium (DMEM/F12), foetal bovine serum (FBS), and an antibiotic mixture (penicillin- streptomycin) were purchased from GIBCO-BRL .2 flasks, called F0 cells. Based on the fact that mesenchymal stromal cells (MSCs) takes longer to attach to the bottom of a dish than fibroblasts and are more sensitive to trypsin, a strategy of differential adherence and incomplete digestion is used to gradually improve the purity of MSCs. Briefly, when the F0 cells grow to 70% confluence, the old culture medium is disgarded and the cells are rinsed with DMEM/F12, followed by adding 1 ml 1% of trypsin to digest the cells at 37C for 1–2 minutes. After 50% of the cells start to float, 2 ml of CGM is gently added, cell suspension is collected, and cell pellets are collected after centrifugation for 1 minute at 800 rpm. The cells were reseeded into 25 cm2 flask, and labelled as F1 generation. When F1 cells were confluent to 70%, the purification steps were repeated as described above. After final purification the F4 or F5 cells were seeded onto 12-well dishes. These cells were used to induce differentiation to brown-like adipocytes.The cells used in this study were primary human subcutaneous preadipocytes (hsASCs) cryopreserved in our laboratory. They were isolated from subcutaneous WAT of plastic surgery healthy recipients, and were digested in 0.1% (v/v) collagenase type I solution , at 37°C water bath with constant agitation at 100 rpm for 60 minutes. The cell pellets were then resuspended in a cell growth medium , 100 U/ml penicillin and 100 µg/ml streptomycin), filtered through a 100 µm strainer, and were seeded into 25 cmdb/+, db/+) C57BL mice were purchased from the Jackson Laboratory , and were inbred to obtain homogeneous Leprdb/db (db/db) mice. Genotypes of these mice were identified by PCR. In view of spontaneous obesity in db/db mice over 4 weeks of age, the genotype can also be confirmed by obese phenotype. C57BL wild type (WT) mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd  as a lean control. All mice were kept and bred in a temperature-controlled room (22 ± 2°C) on a 12:12-hour light–dark cycle in specific pathogen-free animal facilities with a standard diet and water ad libitum from 22 d of age. Each time, six to eight week-old two genotypes of male mice  were used to isolate subcutaneous primary preadipocytes for experiments, using a collagenase type I digestion method that has been described earlier, while digestion time was shortened to 30 minutes. Mouse adipose-derived stem cells (ASCs) were also purified by differential adhesion and an incomplete digestion method similar to the isolation and purification of hsASCs. The WT and obese mice subcutaneous preadipocytes were named as WT msASCs and obese msASCs, respectively.Leptin receptor spontaneous heterogeneous point mutate (Lepr4 cells/cm2 were cultured in 12-well plates. After the cells were post-confluent (D0), they were exposed to CBAM , and the date was recorded as D0. Two days later, the medium was replaced with a beige adipogenic maintenance medium , and were further cultured for 5 d. Moreover, parts of the WT msASCs were exposed at 26°C from D6 for 1 d, from D5 for 2 d, and from D4 for 3 d in the beige adipogenic media , 1 μM T3, 250 μM indomycin and 10 mg/mL transferin, from Sigma-Aldrich, USA). The date was recorded as differentiation day 0 (D0). Three days later, the medium was replaced with a differentiation maintain medium  and 10 mg/mL transferrin, from Sigma-Aldrich, USA) for 9 days. Moreover, to investigate if low-temperature exposure influences beige adipocyte formation, part of the hsASCs were exposed at 26°C from D8 or D10 for 4 or 2 d, respectively, until D12 ). The meic media ).FigureThe hsASCs and msASCs were set up into two groups. One group was induced for beige adipogenesis without inhibition of insulin signalling at 37°C. Another group was induced for beige adipogenesis accompanied with different concentrations of OSI-906 from D0 to incompletely block both insulin and insulin-like growth factor 1 (IGF-1) signallings.n ≥ 7) was determined according to the Lowry method, the data was indicated as an adipogenic differentiation ratio using percentages of appropriate Ctrl (+).Lipid formation in beige adipocyte was identified by Nile red staining. To quantify intracellular lipid accumulation, the cells were fixed with formalin and were stained with Oil Red O (ORO) solution (60% saturated in isopropanol). Intracellular ORO was extracted using isopropanol, and light absorbance by the extracted dye was measured . The content of samples at several time points , db/db) under insulin signalling inhibition and intact state. We found that adipogenesis rate had decreased from 78 ± 4.50% to 5 ± 1.36% after inhibition of insulin signalling in WT msASCs , which T msASCs ). Obese T msASCs , CBAM) wT msASCs , CBAM+OST msASCs ) and proT msASCs  levels wIt should be noted that when the inhibition rates of insulin signalling in hsASCs and msASCs were 87 ± 2.49% and 91 ± 1.23%, respectively, other browning related molecules such as SIRT1 and pAMPK were significantly increased as shown in To verify the effect of low temperature on browning of hsASCs and WT msASCs, we comparatively analysed the differences in cell morphology, and expression patterns of browning markers under 37°C and 26°C situations during browning. Low temperature exposure reduced the rate of adipogenesis, i.e. smaller lipid droplets and less cells of beige differentiation. This tendency of dynamic change was positively correlated with the number of days of low-temperature exposure in the hsASCs ) and theThe formation of both WACs and BACs is a physiological phenomenon in the body that induces differentiation of preadipocytes into their corresponding mature adipocytes ,4,8,10. Insulin through its receptor signal promotes lipids, carbohydrates, and proteins into macromolecules that are involved in inducing nutrient storage in the body, cell growth, and cell differentiation ,17,18,22Evidence indicates that irisin, PPARγ, myostatin, FGF21, cold temperatures, and β-epinephrine agonist are involved in white adipocyte browning ,19,20,24Pharmacological and genetic interventions of PRDM16 in mature adipocytes lower the phosphorylation level of AKT, while PPARγ agonist promotes expression of PRDM16 in subcutaneous WAT ,30,35,36+ levels, promotes the differentiation of brown preadipocytes, and causes white preadipocyte browning [Based on the fact that a few numbers of white preadipocytes are still browning, we speculate that it is due to the insulin signalling being not completely blocked, has remained partially function, or other browning regulatory molecules are involved, such as insulin signalling independent and UCP-1 independent pathways ,39. Morebrowning ,37. In t− derived brown adipocytes have reduced lipogenic gene expression in BAT and increased WACs browning [Overall, the insulin signalling positively regulates browning of white preadipocytes in human and mice, which not only promotes the process of adipogenesis, but also promotes the transcription and translation levels of brown molecules PRDM16, PGC-1α, and UCP1 ,30,35,36browning ,46, favoIt has also been reported that cold exposure can increases proliferation, differentiation, and UCP1 activity, but also promotes the browning of WACs or adipocytes directly by sensimg temperature to activate thermogenesis ,13,14,47We provide evidence that the browning of human and mouse white primary preadipocytes was confirmed at the cell morphology, gene, and protein expression level using our modified method. Moreover, the results suggested that insulin signalling plays an important regulatory role in the process via PI3K-AKT-UCP1 signalling pathway. Dysfunction of the insulin signalling can significantly reduce white primary preadipocyte transdifferentiation into beige adipocyte. The effect of insulin on the process is independent of mammal species (humans and mice) of the cell donors, and mouse phenotype (lean and obese). In addition, the AMPK-SIRT1 signalling pathway is also involved in beige adipogenesis, although its ability to promote browning of white preadipocyte is limited. Moreover, cold acclimation reduces lipid production and accumulation in hsASCs and WT msASCs, while it promotes thermogenesis during beige formation . Other m


To investigate the association between the level of glycosylated hemoglobin (HbA1c) and preoperative deep vein thrombosis (DVT) and that between diabetes mellitus (DM) and preoperative DVT in patient undergoing total joint arthroplasty (TJA).A total of 1386 patients were enrolled. We created the receiver operating characteristic (ROC) curve of HbA1c, and based on the cutoff value, patients were divided into two groups. Risk factors were subsequently examined. Chi-square test or Fisher’s exact test was adopted for enumeration data. The results were expressed in percentages (%), and DVT-related variates were analyzed. We included the variates that were statistically significant in the univariate analysis in the multivariate binary logistic regression analysis and calculated the adjusted odds ratio (OR) and 95% confidence interval (95% CI).P = 0.005; 95% CI [1.20–2.80]), 2.22 , 2.47 , 2.03 ; 1.85 ; and 2.86 times , respectively. And multivariate logistic regression revealed that the risk of preoperative DVT in TJA patients with HbA1c ≥ 6.15%, HbA1c between 7 and 7.9%, HbA1c ≥ 8%, DM patients, female patients, and major surgery in the last 12 months increased by 1.77 ; 2.10 ; 2.50 ; 2.01 ; 1.80 ; and 3.04 times , respectively.Preoperative DVT was 100 cases (7.22%) and DM in 301 cases (21.7%). We determined the cutoff value of HbA1c of 6.15% using the ROC curve as the area under the curve (AUC) was 0.548. Univariate logistic regression revealed that the risk of preoperative DVT in TJA patients with HbA1c ≥ 6.15%, HbA1c between 7 and 7.9%, HbA1c ≥ 8%, DM, female, and major surgery in the last 12 months increased by 1.84 (We conclude that HbA1c ≥ 6.15%, DM, female and major surgery in the last 12 months are the independent risk factors for preoperative DVT in patients undergoing TJA. And patients with a higher HbA1c level are at an increased risk of preoperative DVT.Trial registration: ChiCRT2100054844. More TJAs of the hip (THA) and the knee (TKA) are anticipated to be performed in the next decade . VTE, a According to the latest global figures, 381.8 million adults (8.3%) are affected by DM, and this number is expected to rise to 591.9 million (8.8%) by 2035 . The gloThe impact of preoperative DM and elevated HbA1c levels on complications from TJA have all been the focus of most previous studies. Currently, there are no studies to verify the association between the level of glycosylated hemoglobin (HbA1c) and preoperative deep vein thrombosis (DVT), and that between diabetes mellitus (DM) and preoperative DVT in patient undergoing TJA. Therefore, we used data from our hospital to retrospectively analyze their relationships.Inclusion criteria were patients who underwent TJA in our hospital between January 1, 2017, and December 31, 2021, and had HbA1c records before surgery; a total of 1474 TJA patients were enrolled.Exclusion criteria: (1) a history of VTE (5 cases); (2) use of anticoagulation medications : atrial fibrillation (10 cases), coronary heart disease (CHD) patients with installed stents and anticoagulant therapy (15 cases); (3) thrombophilic genetic disorders (0 cases); (4) joint infection: knee joint (12 cases), hip joint (5 cases); (5) tuberculosis of the joint: knee joint (6 cases), hip joint (8 cases); (6) tumors of the joints: knee joint (15 cases), hip joint (4 cases); (7) under the age of 20 (8 cases); (8) pregnant (0 cases). Finally, 1387 patients with TJA were enrolled.We created the receiver operating characteristic (ROC) curve of HbA1c, and based on the cutoff value, patients were divided into two groups. The risk factors for DVT before TJA were subsequently examined. Based on their deep vein ultrasound results, patients were divided into two groups: DVT group and non-DVT group. High-risk factors for DVT before TJA were subsequently analyzed. Then, we used multivariate binary logistic regression analysis to verify. This study has been approved by Medical Research and Ethics Review  and registered in the WHO International Clinical Trials Registration (ChiCRT2100054844).We collected clinical data by accessing the electronic medical records and surgical anesthesia information systems. All patients received pulse Doppler ultrasound with Philips IE33 GE Vivid 9, C5-1 linear probes at a frequency of 5–10 Hz in their lower extremities and were co-diagnosed by two experienced sonographers. The positive criteria for DVT included venous incompressibility, intravascular filling defect, and the absence of a Doppler signal. We also collected DVT formation sites: distal thrombus ; proximal thrombus ; mixed thrombus . Patients’ general information included: name, admission number, height, weight, BMI (body mass index), age, gender, and preoperative diagnosis. Their medical records included: preoperative hypertension, DM, CHD, chronic obstructive pulmonary disease (COPD), chronic bronchitis, rheumatoid arthritis (RA), osteoarthritis (OA), cerebral infarction, cancer, renal failure, use of corticosteroids, preoperative smoking, alcohol consumption, major surgery  in 12 mP < 0.05 was considered statistically significant.We used SPSS 26.0 for statistical analyses. Specifically, we determined the cutoff value for HbA1c by establishing the ROC curve for the DVT and HbA1c levels in patients undergoing TJA. The patients were then divided into two groups based on the cutoff value: those with HbA1c levels higher than the cutoff value and those with HbA1c levels lower than the cutoff value. And risk factors were subsequently examined. Chi-square test or Fisher’s exact test was adopted for enumeration data. The results were expressed in percentages (%), and DVT-related variates were analyzed. The variates that were statistically significant in the univariate analysis were included in the multivariate binary logistic regression analysis, and the adjusted odds ratio (OR) and 95% CI were calculated for the evaluation of the association between TJA, DM, HbA1c, and preoperative DVT. A total of 1386 patients who underwent TJA in our hospital and had preoperative HbA1c level records were enrolled in this retrospective study. The mean age of all TJA patients was 64.24 ± 0.3 years, 71.24 ± 0.80 years in the DVT group, and 63.7 ± 0.32 years in the non-DVT group  . According to the American Diabetes Association, DM is defined as HbA1c ≥ 6.5% and prediabetes is HbA1c between 5.7 and 6.4% ), 2.22 , 2.47 , 2.03 ; 1.85 ; and 2.86 times , respectively ; 2.10 ; 2.50 ; 2.01 ; 1.80 ; and 3.04 times , respectively.Considering the multicollinearity of classification of HbA1c at 6.15%, grade of HbA1c, and DM, we conducted a binary logistic regression analysis on these variables separately with gender and the history of major surgery in the last 12 months Fig. . And mulJiao X et al. proposed that the incidence of DVT in early stage following TKA was closely associated with DM and proportional to HbA1c levels . HoweverHbA1c reflects the average plasma glucose concentration over a period and is recommended for blood glucose control in those who have DM . Due to Nagashima M et al. proposed the cutoff value of preoperative HbA1c of 5.9% and suggested that patients with an HbA1c ≥ 5.9% before TKA may require careful perioperative glycemic control . StrykerCapozzi JD et al. reported that one-third of patients undergoing TJA had undiagnosed dysglycemia detected on routine screening . Three hMraovic B et al. reported a 3.2-fold increase in the risk of PE among patients with DM compared with patients without DM . Jiao X Among all patients enrolled in the present study were osteoarthritis (OA). OA and the metabolic syndrome are recognized as the low-grade inflammatory condition with elevations in systemic inflammatory mediators such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) . A proin2 and that of male patients was 23.79 ± 0.15 kg/m2, P < 0.01. Female patients had higher preoperative BMIs, and the difference was statistically significant. High BMI is often associated with poor hemodynamics and may induce the formation of DVT ) was statistically significant, future studies with bigger sample size are needed to further verify the critical value.In this study, the correlation with DVT in TJA patients was explored by using materials such as preoperative medical history, preoperative laboratory examinations, and preoperative auxiliary examinations. However, this study has certain limitations. As a retrospective study, some data are incomplete. Moreover, although the critical value of HbA1c with preoperative DVT of 6.15% and the AUC of 0.548 (Our study found that the independent risk factors for preoperative DVT in patients undergoing TJA were HbA1c ≥ 6.15%, DM, female, and having undergone major surgery in the last 12 months. Patients undergoing TJA who had a higher level of HbA1c should be screened for DVT before surgery because they had an increased risk of preoperative DVT.


Citrus spp., exploring the potential implication of SIRT2. Naringenin (NAR), hesperetin (HSP), naringin (NRG), and neohesperidin (NHP) inhibited SIRT2 activity in the isolated recombinant enzyme, and more, the combination between NAR and HSP. In monocytic leukemic THP-1 cells, only NAR and HSP induced antiproliferative effects, altering the cell cycle. These effects may be ascribed to SIRT2 inhibition since these flavonoids reduced its gene expression and hampered the deacetylation of p53, known sirtuin substrate, and contextually modulated the expression of the downstream cell cycle regulators p21 and cyclin E1. Additionally, these two flavanones proved to interact with the SIRT2 inhibitory site, as shown by docking simulations. Our results suggest that both NAR and HSP may act as anti-leukemic agents, alone and in combination, via targeting the SIRT2/p53/p21/cyclin E1 pathway, thus encouraging deeper investigations. Acute myeloid leukemia (AML) represents the most alarming hematological disease for adults. Several genetic modifications are known to be pivotal in AML; however, SIRT2 over-expression has attracted the scientific community’s attention as an unfavorable prognostic marker. The plant kingdom is a treasure trove of bioactive principles, with flavonoids standing out among the others. On this line, the aim of this study was to investigate the anti-leukemic properties of the main flavanones of  Acute myeloid leukemia (AML) is the most common hematologic neoplasm in adults, characterized by an accumulation of abnormal myeloblasts, most frequently in the bone marrow, leading to its failure and, eventually, death . The inc+-dependent enzymes, are the focus of scientific community’s attention owing to the fact that they regulate a large number of cellular pathways implied in cellular aging and age-associated diseases, among which cancer [The acetylation state of histones regulates epigenetic processes by affecting transcription factor access to DNA, thus determining gene expression levels. Histone deacetylases (HDACs) are implied in this mechanism, which in turn increases DNA/histone complex compaction ,5. Amongh cancer . In parth cancer . Therefore, compounds able to inhibit SIRT2 activity are thought to be novel therapeutical approaches to ameliorate conditions such as leukemias, among which AML.Plant kingdom provides an uncountable number of active principles capable of modulating, blocking, or enhancing several cellular pathways, involved in a wide plethora of physio-pathological conditions. Flavonoids stand out among the others for their anti-infective , neuroprCitrus spp., namely naringenin (NAR), hesperetin (HSP), naringin (NRG) and neohesperidin  were tested according to the manufacturer’s guidelines. SIRT2 inhibitor SirReal2  was used as positive control. The fluorescence was read after 30 min using a FLUOstar Omega Plate Reader  at 350–360 nm excitation wavelength and 450–465 nm emission wavelength.SIRT2 activity was determined using SIRT2 Direct Fluorescent Screening assay kit (n. 700280) purchased from Cayman Chemical Company  . For celThe effects of the combination between increasing concentrations of HSP and NAR (50–400 µM) was assessed in terms of SIRT2 activity on the recombinant isolated enzyme, combined at different ratios, following the checkboard method. In particular, pharmacological interaction models of Loewe additivity and Bliss independence have been developed, and a recent protocol has been employed to measure the Zero Interaction Potency (ZIP) . Resultsv/v heat-inactivated fetal bovine serum (FBS), L-glutamine (2 mM), HEPES (10 mM), sodium pyruvate (1 mM), glucose (2.5 g/L), 2-mercaptoethanol (0.05 mM), penicillin (100 IU/mL) and streptomycin (100 µg/mL), at 37 °C in a humified 5% CO2 atmosphere. Medium was renewed every 2 days and split performed when cells reached maximum density (1 × 106 cells/mL). Human peripheral blood mononuclear cells (PBMCs) were originally obtained from ATCC and cultured using supplemented RPMI 1640 medium (10% v/v FBS), at 37 °C in a 5% CO2 environment. Each reagent for cell culture was from Gibco .The human leukemia monocytic THP-1 cell line was originally obtained from ATCC . The cells were grown in RPMI 1640 medium supplemented with 10% 4 cells/well in 200 µL. Cells were treated with the flavanones NAR, NRG, HSP, NHP (50–400 µM). For vehicle control, we added the same amount of DMSO present in the highest concentration of the flavanones tested to assess whether no toxic effect was induced by the solvent. The plates were then incubated for 24, 48 and 72 h at 37 °C, prior adding 20 µL of resazurin  0.01% w/v solution to each well and kept for further 3 h. Fluorescence was measured on a microplate reader POLARstar Omega (BMG Labtech) with an excitation wavelength of 544 nm and an emission one of 590 nm.The evaluation of the antiproliferative activity was assessed by resazurin assay, as an index of mitochondrial functionality, and propidium iodide (PI) staining to detect dead cells . For the6 cells/well) were treated with NAR, NRG, HSP, NHP (100–400 µM) in 6-well plates, which were incubated at 37 °C for 72 h. Subsequently, cells were harvested, washed twice and resuspended in 100 µL of PBS plus 10 µL of PI solution . Stained cells were incubated for 30 min at room temperature in the dark and fluorescence of at least 10,000 events was analyzed by a Novocyte 2000 cytofluorimeter  with FL-2 channel.For PI staining, cells (1 × 105 cells/well) and treated with the flavanones NAR and HSP (100–400 µM) or with their combination at concentrations of 100 µM for 24, 48 and 72 h. Then, cells were collected, centrifuged, washed with PBS, and fixed in 70% ice-cold ethanol while gently vortexed. After at least 2 h at 4 °C, cells were centrifugated, washed twice with cold PBS, and resuspended in 250 μL of PBS together with 5 μL of RNase A  at 37 °C for 1 h. After incubation, 10 µL of PI (1 mg/mL) were added to samples, and immediately acquired by cytofluorimeter. Three independent sets of at least 10,000 events were collected for each condition.The ability of flavanones to interfere with progression of cell cycle was evaluated by flow cytometry . BrieflyTo assess the levels of acetylated p53 in THP-1 cells treated for 24 h with NAR and HSP  and their combination (100/100 µM), or with SIRT2 inhibitors SirReal2 (10 µM) and nicotinamide  , a comme6 cells/dish with fresh medium (untreated cells), NAR and HSP , as well as their combination (100/100 µM) at 37 °C for 12 and 24 h. Then, total RNA from untreated and treated cells was extracted using TRIzol reagent , according to the manufacturer’s instructions. Afterward, 2 µg of total RNA was reverse transcribed into cDNA using the High-Capacity cDNA Archive Kit , as previously described [−∆∆CT relative quantification method against β-actin (ACTB), used as the housekeeping control. The values are expressed as n-fold change compared to untreated cells. When the value was less than 1, it was converted into its inverse to report downregulated genes.THP-1 cells were plated in 100 mm Petri dishes at a density of 1 × 10escribed . QuantitDocking studies were performed by AutoDock4 suite , using tp-values lower or equal to 0.05 were considered statistically significant.The assays were carried out in different replicates, as described above. Data obtained were expressed as mean ± standard error of the means (SEM). Statistical evaluation of results was performed using one-way analysis of variance (ANOVA), depending on the assay. Multiple comparisons of the means of the groups were performed by the post hoc Student–Newman–Keuls test . The cell-free model consisting of the isolated recombinant enzyme. In this setting, each flavanone was able to inhibit SIRT2 activity, despite to a different extent  at different ratios in the isolated enzyme. The obtained results have been processed to investigate the presence of antagonism or synergism. A clear antagonism was observed between the pairs NAR-NHP, NAR-NRG, NHP-NRG, and NRG-HSP, whereas a weak synergistic interaction was detected between NHP and HSP (data not shown). Unlike the other combinations, the flavanones NAR and HSP, which individually elicited the strongest inhibitory effect on SIRT2 enzymatic activity, showed a great synergism in counteracting SIRT2 activity . Indeed,To investigate whether the studied flavanones possessed anti-leukemic activity, the effect of NAR and HSP, along with their glycosidic counterparts NRG and NHP, was assessed in a human monocytic leukemia THP-1 cell line, which represents a preclinical model of the M5 subtype of AML. In these cells, both NRG and NHP hampered viability at 72 h by no more than 20% at the highest concentration tested (400 µM). On the contrary, the aglycones NAR and HSP significantly reduced cell proliferation already at 24 h  and up to 64.0 ± 2.3% and 66.0 ± 2.4% at 72 h, respectively A.In order to assess the potential cytotoxicity of the flavanones under study, we repeated the resazurin assay in normal human PBMCs, testing the same timings and concentrations. Noteworthy, none of these caused any inhibitory effects on PBMCs growth, if not after exposure to the flavanones NAR and HSP at the concentration of 400 µM for 72 h, where a slight but not significant reduction in cell viability was observed B.At this point, we have thus identified non-cytotoxic concentrations employed in the following experiments.The PI staining corroborated the outcome observed with the resazurin assay . ConsistGiven these results, further in vitro experiments were performed considering only NAR and HSP.With the aim of comprehending the mode of death elicited by NAR, HSP, and their combination, their influence on the cell cycle progression of THP-1 cells was evaluated. After 24 h of treatment, just a slight modulation of cell cycle progression was observed with 400 µM of NAR and HSP (data not shown). After 48 h of treatment, 100 µM of NAR did not elicit any modification of the ratio among cell cycle phases respect to the control, whereas 200 µM and even more 400 µM increased the percentage of cells in S phase. Similarly, 100 µM of HSP did not alter the cell cycle at 48 h, while 200 µM increased the cell population in S and G2/M phases. The accumulation of cells in the S phase was also recorded with HSP at 400 µM concentration. Moreover, the combination between NAR and HSP at a molar ratio of 1:1 (100 µM) displayed a stronger effect than the two flavanones alone A. ConsisOf note, cells populating the sub-G0/G1 phase (hypodiploid cells), a known sign of apoptosis, are relevant after treatment with HSP , NAR (400 µM), and their combination (100/100 µM) at both 48 and 72 h .As a direct in vitro effect of SIRT2 activity modulation, the levels of acetylated p53 protein were evaluated by means of an ELISA. In detail, treatment with both NAR and HSP 100 µM for 24 h did not alter levels of acetylated p53 with respect to control cells . On the Finally, to appropriately compare the abovementioned results, we used a specific  and a non-specific SIRT2 inhibitor  as positive controls. In comparison with what occurs in untreated cells, the incubation of THP-1 cells with both inhibitors significantly increased the degree of p53 acetylation by 1.23- and 1.67-fold, respectively. This suggests that NAR and HSP behave as synthetic SIRT2 inhibitors.Besides inhibiting SIRT2 enzymatic activity, NAR and HSP were also able to modulate its gene expression. Indeed, the treatment with both HSP 200 µM and NAR 400 µM for 12 h significantly reduced SIRT2 mRNA levels by 1.25-fold down compared to controls, as well as their equimolar combination of 100 µM concentration, which brought no effects for single flavanones. Of note, the highest tested concentration of HSP (400 µM) significantly lowered SIRT2 mRNA expression after 12 h . In detail, compared to controls, treatment with NAR 200 µM for 12 h increased p21 mRNA levels in THP-1 cells by 1.7-fold and with HSP 200 µM by 1.5-fold. For the concentrations of 400 µM of NAR and HSP, a similar increase in p21 levels  was recorded after 12 h. Likewise, this growing trend was maintained after 24 h of treatment with NAR (2.7-fold vs. CTRL) and HSP (2.2-fold vs. CTRL) 200 µM, whereas it was even higher with NAR 400 µM, which significantly raised p21 gene expression by 4.0-fold, and HSP 400 µM by 4.9-fold respect to controls. On this line, after 24 h of incubation, the equimolar combination (100 µM) between the two flavanones increased p21 mRNA levels by 2.1-fold respect control, similarly to HSP 200 µM C.Contrariwise, cyclin E1 expression was significantly hampered by NAR and HSP already after 12 h of treatment. Indeed, NAR reduced mRNA levels of cyclin E1 by 1.25-, 1.62- and 2.49-fold down at 100, 200, and 400 µM, respectively, whereas HSP lowered cyclin E1 mRNA quantity by 1.70- and 2.04-fold down at 200 and 400 µM, respectively. Their combination decreased by 1.52-fold down cyclin E1 expression after 12 h of incubation. After 24 h of treatment, NAR and HSP significantly reduced cyclin E1 gene expression at 100 µM , 200 µM , and 400 µM . Their association (100/100 µM) brought a result like that of the 200 µM of both NAR and HSP alone D, while To investigate the molecular interactions between the flavanones NAR and HSP with SIRT2, we performed docking simulations using AutoDock4 suite , employiThe computational studies suggested that all studied compounds had a similar network of interactions as SirReal2 in the lipophilic pocket of SIRT2, forming crucial contacts within active site residues. The inspection of docking results suggested that the 2-(4-hydroxyphenyl)-substituent of NAR, taken as an example, forms crucial π-T-shaped interaction with Phe131, whereas the chromen-4-one moiety occupies the hydrophobic region close to Tyr139 and Phe190 residues. Moreover, our molecular simulation revealed that NAR, such as HSP, made two hydrogen bond interactions with oxygen atoms of Ile118 and Ala135 backbone. Compared to SirReal2, NAR loses the crucial π-π stacking interaction within the selectivity pocket of SIRT2. This could explain the minor activity of the flavanones .Citrus fruits, and, due to their presence, these are endowed with well-acknowledged protective activities exploited to defend human health [Citrus fruits and their derivatives have been long investigated for their role in this nefarious disease [The main source of dietary flavonoids is n health ,31,32, an health ,34. Rega disease ,36,37, a disease .Citrus spp. Therefore, the first step was to assess the effect of NAR, HSP, and their glycosidic counterparts, NRG and NHP, on the recombinant isolated SIRT2 enzyme. Interestingly, all flavanones were able to inhibit the deacetylase activity of SIRT2, despite to different extent, thus being the first to report these effects.In this work, we started from the assumption that quercetin-derived compounds have been demonstrated to possess SIRT2 inhibition activity , and, giPreviously, we demonstrated how natural products combined may elicit stronger effects than when alone . On thisIn the light of the inhibitory effects of the selected flavanones on the SIRT2 recombinant enzyme and that SIRT2 expression was increased in the M5 subtype of AML patients , we deciPrevious reports suggested the capability of many flavonoids as anti-leukemic agents via targeting multiple pathways ,41. In oGiven these results, we investigated which type of cell death was elicited by NAR and HSP by assessing whether these compounds can interfere with the progression of THP-1 cells during the cell cycle. This cellular process is governed by checkpoints that are carefully followed by normal cells; nevertheless, if genetic aberrations or abnormalities occur, cells will enter a state of cell cycle arrest . In our To verify the sirtuin inhibition by flavanones in vitro, we evaluated the level of p53 acetylation, the main cellular target of SIRT2 deacetylase activity . InteresSince the enzyme modulation can be achieved by either affecting the activity or the gene expression, we wondered whether the lowered SIRT2 activity could be originated by a decrease in its mRNA levels, in addition to the inhibition of its enzymatic activity. Therefore, when we quantified the total SIRT2 gene levels, we observed an early modulation (12 h) of mRNA expression at the highest tested concentration (400 µM) of NAR and HSP, as well as with their combination at equimolar ratio (100 µM), an effect that was totally abolished at 24 h of treatment, except for HSP 400 µM. This outcome may suggest that cancer cells respond to SIRT2 inhibition with an increase in its gene expression in order to restore its functionality, which is crucial for the survival of AML cells. Notably, HSP 400 µM steadily maintained the suppression of SIRT2 expression, suggesting its potentiality in AML.During the cell cycle, p53 directly binds DNA to promote the transcription of several factors, among which p21 is the pivotal one . The imm+-binding capacity as well as N- and C-terminal extensions. The catalytic core contains a variant of the Rossmann fold and a small domain consisting of helical and zinc-binding modules. The abovementioned domains are separated by a large lipophilic area that represents a wide groove containing an active site where deacetylation of substrates occurs. The catalytic groove accommodates various inhibitors from natural and synthetic sources [Finally, to understand how NAR and HSP interact with SIRT2, we investigated the putative binding mechanism of these two flavanones by computational techniques. SIRT2 structure was fully deciphered in 2001 by Finnin and co-workers  who reve sources ,50. The  sources  revealedCitrus fruits, can exert anti-leukemic effects on the human leukemic monocytic cell line THP-1 through growth inhibition via the arrest of cell cycle progression. Interestingly, these mechanisms appear to be linked to the inhibition of SIRT2, a known proliferation marker in relapsing AML, which makes flavonoids attractive candidates for the management of this pathology. In addition, we showed that NAR and HSP can reciprocally either enhance their antiproliferative effects, thus providing further new possibilities for studying the combined anti-leukemic activity of natural products.Hematological malignancies continue to represent a significant challenge, being frequently depicted as incurable diseases. Therefore, urgent development of novel therapeutic agents is needed to overcome the failure of standard therapies and to improve the patients’ survival rate. Some plant-derived products, such as flavonoids, have gained great interest due to their pharmacological potential. Among these, we demonstrated that NAR and HSP, the two most common flavanones of


Aspergillus sclerotiorum PSU-RSPG178, were found to exhibit inhibitory effects against 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase and oxidative stress in an in vitro assay. Whether or not the known pyrrolidine asperidine B and the recently isolated piperidine asperidine C have lipid-lowering effects remains unknown. Thus, this study aimed to investigate the hypocholesterolemic effects of asperidines B and C and identify the mechanisms involved in using in vitro, ex vivo, and in vivo models. The results show that both compounds interfered with cholesterol micelle formation by increasing bile acid binding capacity, similar to the action of the bile acid sequestrant drug cholestyramine. However, only asperidine B, but not asperidine C, was found to inhibit cholesterol uptake in Caco-2 cells by up-regulating LXRα without changing cholesterol transporter NPC1L1 protein expression. Likewise, reduced cholesterol absorption via asperidine-B-mediated activation of LXRα was also observed in isolated rat jejunal loops. Asperidine B consistently decreases plasma cholesterol absorption, similar to the effect of ezetimibe in rats. Therefore, asperidine B, the pyrrolidine derivative, has therapeutic potential to be developed into a type of cholesterol absorption inhibitor for the treatment of hypercholesterolemia.Isolated secondary metabolites asperidine B (preussin) and asperidine C, produced by the soil-derived fungus  An imbalance of lipid levels, including hypercholesterolemia- and hypertriglyceridemia-induced obesity, has been known to become a major risk factor for several diseases, including type 2 diabetes mellitus, cardiovascular disease, hypertension, cancer, and non-alcoholic fatty liver disease ,2,3. TheStudies have previously reported that a lack of intestinal NPC1L1 resulted in a reduction in plasma cholesterol and low-density lipoprotein cholesterol (LDL-C) and improved fatty liver in high-cholesterol-diet-fed mice, while intestinal NPC1L1 expression increased in cholesterol-depleted-diet-fed porcine ,8. FurthA. sclerotiorum PSU-RSPG178 have shown antimalarial and anti-mycobacterial activity -cholesterol mixed micelle transport in a concentration-dependent manner when compared with the control. Notably, at higher concentrations of asperidine B (75 and 100 μM), there was a similar degree of inhibition to that elicited by 100 μM ezetimibe, an NPC1L1 inhibitor. On the other hand, asperidine C had no effect on [3H]-cholesterol mixed micelle transport , but not a recently isolated piperidine derivative, asperidine C, in lowering cholesterol by inhibiting cholesterol absorption in in vitro, ex vivo, and in vivo models. This pyrrolidine interference with bile acid binding capacity results in reduced cholesterol micellar formation and absorption. Asperidine B also exhibits a high potency, similar to that of the prescribed drug, ezetimibe, mediated by LXRα activation, which could subsequently interfere with NPC1L1 function. Since cholesterol homeostasis depends upon two major sources—receipt from the diet and de novo cholesterol synthesis—the former is approximately proportional to intestinal cholesterol absorption by contributing roughly 50% of total plasma cholesterol . SeveralAt present, ezetimibe is known to be the first and only prescribed lipid-lowering drug for the inhibition of dietary cholesterol absorption, which is crucial for statin-intolerant patients . It has Azadirachta indica leaf extract is also reflected in cholesterol absorption -cholesterol, specific activity 49 Ci/mmol, was obtained from Perkin Elmer . A complete protease inhibitor cocktail was purchased from Merck . All other chemicals with high purity were obtained from commercial sources.Asperidines B and C  used in this study were isolated from the soil-derived fungus 2 gas. The sample was reconstituted by sonication in phosphate buffered saline (PBS) and passed through 0.22 µm syringe filters. Asperidine B or C at 100 μM was then added into the mixed micelle solution and incubated for 3 h at 37 °C. The size of the micellar complex was subsequently measured using a particle size analyzer . Similarly, to determine the micellar cholesterol, 10 mM unlabeled cholesterol, 1 mM sodium taurocholate, and 0.6 mM phosphatidylcholine were mixed in PBS. The mixed micelle solution was incubated in the presence or absence of 100 μM asperidines B or C for 3 h at 37 °C. The micelles were filtered through a 0.22 µm membrane to separate precipitated micelles from intermicellar complex. The filtrated micellar cholesterol was measured using a commercial colorimetric cholesterol assay . Furthermore, we investigated the interference of bile acid binding capacity by asperidines B or C and compared it with that of cholestyramine, a bile acid sequestrant drug. Briefly, asperidines B or C at 100 μM was incubated with either 2 mM taurocholic acid, glycodeoxycholic acid, or taurodeoxycholic acid at pH 7.0, 37 °C for 2 h. The bile acid mixture was centrifuged at 10,000 rpm for 10 min, and the supernatant was filtered through a 0.22 µm membrane filter to separate bound and free bile acids. The filtrated free bile acid was mixed with a reaction mixture containing 0.133 M tris buffer at pH 9.5, 1 M hydrazine hydrate, 7.7 mM nicotinamide adenine dinucleotide (NAD), and 1 unitmL/3α-hydroxysteroid dehydrogenase. The reaction mixture was further incubated at 37 °C for 2 h. The rate of thio-NADH formation was measured at a wavelength of 340 nm and calculated as a percentage of the control.As previously described , the eff3H]-cholesterol, 2 µM unlabeled cholesterol, 0.1 mM phosphatidylcholine, and 4 mM sodium taurocholate. The mixture solution was evaporated with N2 gas and reconstituted by sonicating in serum-free DMEM/F12 for 1 h. The cholesterol mixed micelle solution was subsequently filtered through a 0.22 µm syringe filter prior to use in the cholesterol transport study.A cholesterol mixed micelle solution was prepared for the cholesterol transport study by mixing 1 µCi/mL [3H]-cholesterol, 1 µM unlabeled cholesterol, 50 µM phosphatidyl-choline, and 2 mM sodium taurocholate was mixed and evaporated with N2 gas and stored at −20 °C until use. The cholesterol micelles were freshly prepared by dissolving the lipid film in PBS and sonicating for 1 h. The cholesterol micelle solution was subsequently filtered through a 0.22 µm syringe filter and maintained at 37 °C before use.For the in vivo cholesterol absorption experiment, the micelle solution containing 10 µCi/mL -cholesterol mixed micelles in the presence or absence of 12.5–100 µM asperidines B and C for 3 h. The medium was removed, and the cells were washed three times with ice-cold PBS. The cells were subsequently dissolved in 1 N NaOH and neutralized by 1 N HCl. The radioactivity of cholesterol transport was measured using liquid scintillation counters . The intracellular [3H]-cholesterol micelle radioactivity was calculated as fmol/mg protein/min.Differentiated Caco-2 cells were washed three times with PBS (pH 7.4) and incubated with 1 μCi/mL -cholesterol mixed micelles in the presence or absence of asperidine B, asperidine C, or ezetimibe. The jejunal loop was then incubated in PBS buffer for 30 min at room temperature. The PBS buffer was also collected after 10, 20, and 30 min to ensure leakage of the [3H]-cholesterol throughout incubation. To identify mechanisms involved with the NPC1L1 function, LXRα activator (GW3965) 1 µM or LXRα inhibitor (SR9238) 10 µM were replaced either on their own or combined with a tested compound. At the end of the experiment, the jejunal loop was cut horizontally and the epithelial tissue was removed. The jejunal epithelium was weighed, dissolved in 1 N NaOH, and neutralized by using 1 N HCl. The intracellular [3H]-cholesterol radioactivity was measured using liquid scintillation counters , and calculated as fmol/mg protein/min.Male Wistar rats weighing 300–350 g were obtained from Nomura Siam International . The animal facilities and protocols were approved by the Laboratory Animal Care and Use Committee at the Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand (protocol no: 17/2564 and 03/2565). All the rats were housed in a room maintained at 25 ± 1 °C on a 12:12 h dark–light cycle and acclimated for 1 week with free access to chow and water. To address the effect of asperidines B and C on cholesterol transport using isolated jejunal loops as previously described [3H]-cholesterol level was measured at 0 min as a baseline. Subsequently, 10 µCi/mL [3H]-cholesterol micelles containing either the tested compound or ezetimibe were orally administered, and the blood samples were collected at 4, 8, 12, 24, 28, 32, and 48 h from the tail vein. Plasma radioactivity was analyzed by liquid scintillation spectroscopy .To further determine actual cholesterol absorption in an in vivo model, the animals fasted for 12–16 h. The blood [p < 0.05.Data were expressed as mean ± SEM. Statistical differences were assessed using one-way ANOVA followed by Tukey’s post hoc test. Statistical analyses were conducted using the statistical package for IBM SPSS Statistical Software version 23 . Differences were considered to be significant when This study demonstrates the promising and high-efficacy cholesterol-lowering action of a pyrrolidine derivative, asperidine B (preussin), by reducing intestinal cholesterol absorption, mediated by the activation of LXRα and interference with bile acid binding capacity. Therefore, asperidine B (preussin) containing a pyrrolidine core has great potential to be developed as a cholesterol absorption inhibitor for the treatment of hypercholesterolemia. The identification of molecular targets and understanding the mechanisms of action of asperidine B (preussin) warrants further investigation.


Paralichthys olivaceus), as the main marine aquaculture fish, has high economic value, mainly distributed in China, Korea, and Japan. At present, the viruses that are extremely harmful to Japanese flounder are Lymphocytes disease virus (LCDV), Hirame rhabdovirus (HIRRV), Viral hemorrhagic septicemia virus (VHSV), et al., were difficult to control in the event of a large-scale epidemic, causing significant economic losses to the Japanese flounder culture industry. Fish cell lines are an important research tool with the advantages of low assay conditions, accurate control of assay results, and ease of operation, and thus have been widely used in virology research. We established a new cell line Japanese flounder spleen (JFSP) from the spleen tissue of the Japanese flounder (P. olivaceus). We explored the optimal growth conditions, chromosome morphology, and number and transfection experiment of JFSP cells in vitro. The susceptibility of JFSP cells to different viruses, including Bohle virus (BIV), Viral hemorrhagic septicemia virus (VHSV), Hirame rhabdovirus (HIRRV), Infectious hematopoietic necrosis virus (IHNV), and Lymphocystis disease virus (LCDV) and the expression changes of immune-related genes after virus infection were detected. The established JFSP cell line has enriched the fish cell resource bank and can be used as an excellent tool for genetic manipulation, studying host-virus interactions, and the development of potential vaccines.In recent years, with the continuous development of the mariculture industry, the scale of mariculture has gradually expanded, and the problem of mariculture diseases is prominent. Among them, viral diseases and bacterial diseases are the main concerns. Japanese flounder  was established and characterized in this study. The JFSP cells grew rapidly at 29 °C, and the optimum fetal bovine serum concentration in the L-15 medium was 15%. Cells were subcultured for more than 80 passages. The JFSP cells have a diploid chromosome number of 2n = 68, which differs from the chromosome number of normal diploid Japanese flounder. The established cells were susceptible to Bohle virus (BIV), Viral hemorrhagic septicemia virus (VHSV), Hirame rhabdovirus (HIRRV), Infectious hematopoietic necrosis virus (IHNV), and Lymphocystis disease virus (LCDV), as evidenced by varying degrees of cytopathic effects (CPE). Replication of the virus in JFSP cells was confirmed by qRT-PCR and transmission electron microscopy. In addition, the expression of four immune-related genes, TRAF3, IL-1β, TNF-α, and TLR2, was differentially altered following viral infection. The results indicated that the cells underwent an antiviral immune response. JFSP cell line is an ideal tool in vitro for virology. The use of fish cell lines to study the immune genes and immune mechanism of fish and to clarify the immune mechanism of fish has important theoretical significance and practical application value for the fundamental prevention and treatment of fish diseases.A new cell line Japanese flounder spleen (JFSP) derived from the spleen of Japanese flounder ( Paralichthys olivaceus), as the main marine aquaculture fish, has high economic value, mainly distributed in China, Korea, and Japan. Diseases caused by viruses such as lymphocytes disease virus (LCDV) [Viral diseases cause massive death, resulting in huge economic losses for the aquaculture industry worldwide. Japanese flounder (s (LCDV) , hirame s (LCDV) , viral hs (LCDV) ,4, chinos (LCDV)  et al. ws (LCDV) ,7,8,9.Oncorhynchus mykiss) [in vitro to the cellular level has led to the rapid development of marine fish cell lines in the last decade. In Japanese flounder, the cell lines, including the brain [The fish cell line is widely used for fish virus isolation and research. Since the first fish cell line was established from the gonad of rainbow trout ( mykiss) , there h mykiss) ,12. Prev mykiss) ,14. Howehe brain ,16, fin he brain , embryo he brain , gill [1he brain , skin [2he brain , spleen he brain , and kidhe brain , have beAs an important hematopoietic and immune organ of fish, the spleen plays an irreplaceable role in specific and non-specific immunity of fish. This study aims to establish a new cell line, JFSP, derived from the spleen of Japanese flounder. Moreover, the study objective is to determine the optimal growth conditions, chromosome number, and transfection efficiency of JFSP. The JFSP cell susceptibility to different viruses from fish and amphibians will be assessed for the first time in this study. Additionally, the study will explore the immune-related gene expression patterns of JFSP cells upon viral infection.2 cell culture flasks and cultured at 23 °C. A 5 mL complete culture medium containing L-15 medium, 15% fetal bovine serum , 8% Penicillin-Streptomycin-Amphotericin B Solution (100×) , 1% Japanese flounder serum, and Japanese flounder embryo extract, 2 ng/mL fibroblast growth factor  and Leukemia Inhibitory Factor  was added to the culture flasks. One-half of the medium was changed every four days for three weeks. Observation of cell apposition and changes in cell morphology by inverted fluorescence microscope .Healthy Japanese flounder  was obtained from the Beidaihe Central Experiment Station, Chinese Academy of Fishery Sciences. The fish was narcotized with MS-222, exsanguinated with a syringe, and soaked with 75% alcohol. Spleen tissue was dissected in sterile Petri dishes and washed four times in Leibovitz’s L-15  medium containing 8% Penicillin-Streptomycin-Amphotericin B Solution (100×) , and then immersed in Penicillin-Streptomycin-Amphotericin B Solution (100×) for 5 min. After washing, the minced tissues were dispersed into single cells using 0.25% trypsin solution for 30 min. The tissue fragments were inoculated into 25 cmThe old medium was removed, and the cell sheets were washed with Floating lotion containing L-15 medium, 8% Penicillin-Streptomycin-Amphotericin B Solution (100×)  after the primary cell cultures had grown to confluence, and 90% of the culture flask bottom was covered with a monolayer of cells. The confluent cells were dispersed with 1 mL of 0.25% trypsin solution, subcultured at a ratio of 1:2, and maintained in an L-15 growth medium containing 15% FBS. The subcultured cells were passaged every 3–6 d. After the initial 10 subcultures, the growth medium was changed to L-15 with 10% FBS and reduced levels of antibiotics 1% Penicillin-Streptomycin-Amphotericin B Solution (100×) .P. olivaceus) spleen (JFSP) cells were trypsinized, centrifuged, and then resuspended in an ice-cold storage medium , and 70% L-15) at 2 × 106 cells/mL for cryopreservation at every five passages. A 2 mL cell suspension was dispensed into 2 mL cryovials and then kept at 4 °C for 30 min, −20 °C for 1 h, and −80 °C overnight. They were then transferred to liquid nitrogen (−196 °C) for prolonged storage.Japanese flounder  according to the manufacturer’s instructions. The partial fragment sequence of the 18s rRNA gene was amplified using specific primers forward (5′-CCTGAGAAACGGCTACCACAT-3′) and reverse (5′-ATCCCGAGGTCCAACTACGAG-3′) (Accession Number: EF126037) designed according to the sequence available in the National Center for Biotechnology Information (NCBI) database. The 20 μL PCR reaction mixture contained 10 μL of the PrimeSTAR Max Premix (2×) , 1 μL of each primer, 1 μL of the extracted genomic DNA, and 7 μL of the nuclease-free water. The optimum conditions for PCR include initial denaturation at 98 °C for 3 min, 35 cycles at 98 °C for 10 s, 60 °C for 5 s, and final elongation at 72 °C for 3 s. PCR products were analyzed on 1% agarose gel and sequenced using Sanger’s sequencing method.3 cells per well in 96-well plates with different FBS concentrations , temperatures , and culture medium , then incubated for seven days to measure cell growth rate. Using the Cell Proliferation Kit  and following the steps in the manufacturer’s instructions, the cell growth rate was estimated every 24 h after seeding.The effect of different FBS concentrations, temperatures, and culture medium on cell growth was examined in passage 55. JFSP cells were seeded at a density of 2 × 10g rpm for 5 min. The cells were resuspended in 1 mL of 0.075 mol/L KCl solution at 37 °C for 40 min. Then it was fixed three times at 4 °C with Carnoy’s solution  for 15 min. After centrifugation, the cells were suspended in 0.5 mL fixative solution, dripped onto −20 °C pre-cooled slides, and then immediately fire-roasted with the aim of dispersing the chromosomes. After staining with Giemsa solution  according to the instructions, chromosomes were observed and counted under a light microscope.Chromosome analysis was performed using JFSP cells at subculture 65. The passage cells were cultured at 4 °C for 30 min before being transferred to a carbon dioxide cell incubator . Colchicine with the final concentration of 0.5 or 1 μg/mL  was added and incubated in the incubator for 5 h. Cells were digested with trypsin for 1 min and centrifuged at 1500× TM 3000 Instruction manual , Opti-MEM  diluted Lipo3000 reagent is used to mix well as reaction solution 1. The pEGFP-N1 premixture was prepared by diluting pEGFP-N1 with Opti-MEM, and the reaction liquid 2 was prepared by adding P3000. Then the reaction solution 1 and 2 were prepared into a plasmid-liposome complex according to the ratio of 1:1. After incubating for 15 min at room temperature, it was added to the cells. Finally, the mixture was added to cells, cultured at 23 °C for 2 d, and observed under a microscope.Cells were inoculated into 12-well plates one day in advance until cell confluence reached 70 to 90% for subcellular localization experiments. According to LipofectamineJFSP cells susceptibility to Bohle virus (BIV), VHSV, HIRRV, infectious hematopoietic necrosis virus (IHNV), and LCDV were investigated. JFSP cells were seeded in 6-well plates and inoculated with 200 μL of virus inoculum for 1 h. The virus was removed and washed twice with L-15, then the infected cells were incubated with L-15 medium containing 3% FBS at 17 °C and subsequently observed for the cytopathic effect (CPE).2 cell culture flasks one day before. Until the cells reached 80% confluence, the cells were infected with 1 mL of virus solution and adsorbed for 1 h. The virus solution was removed, then washed twice and incubated at 17 °C for 3–4 d to observe CPE. The cells were collected when the CPE reached 70%; the medium was discarded, and the cells were fixed in room temperature electron microscope fixative  for 5 min and collected with a cell scraper , centrifuged at 2000× g rpm for 5 min. After discarding the supernatant, a new electron microscope fixative  was added, fixed at room temperature for 30 min, and stored at 4 °C. The cells were prepared for TEM analysis.JFSP cells at passage 65 were passaged in 75 cm−1 to 10−7 as a standard curve to count the number of copies according to the Cycle number Ct value. Viral loads of BIV, VHSV, HIRRV, IHNV, or LCDV were determined on a q225 qRT-PCR platform  using TB Green Premix Ex Taq II  and primers  to estimate the replication efficiency of the virus. The viral DNA/RNA was extracted using the TIANamp Virus DNA/RNA Kit , the total RNA concentration was determined, and 1 ug was reverse transcribed into cDNA. The remaining sample was stored at −80 °C. 0.5 μL of the sample was used as a template for qRT-PCR experiments. Three replicates were set up, and the uninfected group was used as a negative control group. The virus plasmid DNA was diluted from 10 primers . The ampTRAF3 and IL-1β, TNF-α, and TLR2, were examined in JFSP cells using TB Green Premium Ex Taq II  on a q225 qRT-PCR platform  to analyze the immune response to BIV, HIRRV, VHSV, IHNV, and LCDV infection. The primer pairs are listed in −ΔΔCt method [Cell samples were collected from JFSP cells infected with the BIV, HIRRV, VHSV, IHNV, and LCDV for 36 h p.i., cellular RNA was extracted using the trizol method, and the concentration was measured and reverse transcribed into cDNA. The expression levels of the immune genes t method .Primary cells migrating from spleen tissue fragments were observed on the fifth day and forming a monolayer within one month at 23 °C A. In theP. olivaceus, indicating the originating of the established cell line  medium containing 15% FBS, with rapid growth at 29 °C A. JFSP cThe chromosomal information of JFSP cells was analyzed at passage 65. The chromosome morphologies were mostly telocentric or submetacentric based on 137 metaphase plates A. The ceTM 3000 (Invitrogen) at passages 40 and 80. The expression of GFP in the JFSP cell line was observed by fluorescence microscope. It was found that the fluorescence expression could be detected at 12 h. Approximately 30% of the cells displayed green fluorescence after 24 h of transfection  (p < 0.01) (10 VHSV RNA copies/μg at 3 d p.i. (p < 0.01) (10 HIRRV RNA copies/μg at 3 d p.i. (p < 0.01) (10 IHNV RNA copies/μg at 3 d p.i. (p < 0.05) (10 LCDV RNA copies/μg at 3 d p.i. (p < 0.05)  A. Post-i < 0.01) B. The HI < 0.01) C, while  < 0.05) D, and LC < 0.05) F. The reJFSP cells inoculated with the BIV virus revealed an abundance of hexagonal, vesicular membrane virus particles with a diagonal diameter of 144 ± 8 nm A in ultrIL-1β, TRAF3, TNF-α, and TLR2 expression levels in JFSP cells after virus infection. TRAF3 upregulation showed significant differences after 36 h of BIV infection (p < 0.001). TRAF3 expression was significantly upregulated after HIRRV and VHSV infection (p < 0.05 or 0.01) but significantly downregulated after IHNV infection (p < 0.01) and downregulated after LCDV infection but not significantly different (p > 0.05) (IL-1β expression level was significantly upregulated to peak levels 36 h after VHSV infection (p < 0.05). BIV and HIRRV infections showed highly significant (p < 0.01), and IHNV infection showed extremely significant differences (p < 0.001), but infection with LCDV showed no significant difference from the control group (p > 0.05) (TNF-α upregulation showed very significant differences after BIV and HIRRV-infected cells after 36 h (p < 0.01). The expression was significantly upregulated (p < 0.05) after VHSV infection. In contrast, TNF-α expression was extremely significantly downregulated after IHNV infection (p < 0.001) but downregulated after LCDV infection, not significantly (p > 0.05)   A. The IL > 0.05) B. TNF-α  > 0.05) C. The TLr 0.001) D.Paralichthys olivaceus). The optimum growth condition for JFSP cells was in L-15 medium with 15% FBS at 29 °C. Up to now, the JFSP cell line has been subcultured for more than 80 passages and cryopreserved at different passages. PCR analysis and sequence analysis showed 100% sequence similarity of 18s rRNA between JFSP and P. olivaceus.In this study, we established and characterized a JFSP cell line from the spleen tissue of Japanese flounder  [Oreochromis mossambicus) [Cell line susceptibility and species specificity are essential for conducting studies on viral pathogen isolation, characterization, and propagation. However, it is clear that not every cell line can be used to identify all viruses. Different cell lines have different susceptibilities to viruses, so it is important to establish susceptible cell lines for different viruses. Several studies have been published on using Japanese flounder cell lines for virus isolation, including the gill cell FG, embryonic cell line HINAE, and kidney cell line for HIRRV isolation ,22. The carifer)  and Tilaambicus) . HoweverTNF-α, IL-1β, and IL6. Studies have revealed that fish infected with VHSV or IHNV have high IL-1β levels [IL-1β is significantly upregulated after BIV, VHSV, HIRRV, and IHNV infections. TLR2 is a pattern recognition receptor [TLR2 was downregulated after VHSV, BIV, HIRRV, HIRRV, and LCDV infections. TNF-α is a tumor necrosis factor, and TRAF3 acts as a tumor necrosis factor receptor that binds to TNF and participates in apoptosis and inflammatory responses together with antiviral functions. Studies showed that TNF-α expression was downregulated after LCDV infection in fish [TNF-α and TRAF3 were significantly downregulated after IHNV infection. LCDV infection showed a downregulation trend but not significantly, presumably due to the short duration of viral stimulation. In contrast, TNF-α was significantly upregulated after BIV, VHSV, and HIRRV infections. The study of the regulation of immune-related cytokine expression contributes to understanding cellular immunity levels and improves cell ability to resist resistance to pathogenic microorganisms.Viral infections predispose host cells to damage and necrosis, leading to innate immune response initiation by host pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs)  and chanβ levels ,35,36. Treceptor ,38 that  in fish . In thisIn summary, we established a cell line JFSP from Japanese flounder spleen tissue that displayed susceptibility to fish viruses BIV, VHSV, HIRRV, IHNV, and LCDV. These characteristics make JFSP cells an excellent tool for studying exogenous gene expression, gene function validation, and host-virus interactions.


The p21-activated kinase 4 (PAK4) is a member of the PAKs family. It is overexpressed in multiple tumor tissues. Pharmacological inhibition of PAK4 attenuates proliferation, migration, and invasion of cancer cells. Recent studies revealed that inhibition of PAK4 sensitizes immunotherapy which has been extensively exploited as a new strategy to treat cancer. In the past few years, a large number of PAK4 inhibitors have been reported. Of note, the allosteric inhibitor KPT-9274 has been tested in phase Ⅰ clinic trials. Herein, we provide an update on recent research progress on the PAK4 mediated signaling pathway and highlight the development of the PAK4 small molecular inhibitors in recent 5 years. Meanwhile, challenges, limitations, and future developmental directions will be discussed as well. AccumulDue to the homology of protein structure and sequence, PAKs are divided into group Ⅰ (PAK1-3) and group Ⅱ (PAK4-6) . All isoin vivo , an N-terminal p21-binding domain (PBD), a pseudosubstrate sequence (PS) or an autoinhibitory domain (AID), and a basic residue cluster for membrane targeting  Radu et. The preon state . Cdc42 won state . The binon state . The N-ton state . Moreoveon state .G93A mice, a high level of PAK4 in the spinal neurons was found to block degeneration of motor neuron, thus prolonging survival rates , and blocking of PAK4 induces reduction of Bad phosphorylation and upregulation of tumor-suppressive miRNAs in PDAC (β (CEBPB) activation, resulting in upregulation of CLDN4. Blocking of PAK4 decreased the activation of PAK4/CEBPB/CLDN4 and subsequently suppressed migration and invasion of breast cancer  . In norm50 values of 27 and 830 nm respectively in the biochemical and the proliferation assay against A549 cells. Mechanism study confirms that 7 inhibits phosphorylation of PAK4 and its downstream proteins LIMK1 and Cofilin, and mitigates cancer cell migration and invasion. Further optimization provided the analogue 8   (max and AUC(0−∞) after oral administration  , a scaff.790 μm) , leading.790 μm)  exhibiteoline 10  is anothof 60 nm . Furtherof 60 nm , showing 3.1 μm) . The cry 3.1 μm) . It is o 3.1 μm)  was then 3.1 μm) . It was stration . In HCT-stration .50 value of 8.7 μm. The co-crystal structure of PAK4 in complex with 13 shows that the imidazopyridine and pyrazole rings form sufficient hydrogen-bonding interactions in the hinge loop interaction  , showingeraction . 3-Aminoeraction  is anotheraction . At conceraction . Compouneraction  bearing of 15 μm . It showf 4.3 μm , and redf 4.3 μm . Phenantf 4.3 μm  is a modf 420 nm . In the pound 17  bearing ectively . It inhi50 value of 21.36 μm and has no significant binding to PAK1, PAK5, and PAK6. It suppressed the invasion of human gastric cancer SGC7901 cells and alleviated metastasis related SCG10 phosphorylation through downregulation of the PAK4/LIMK1/cofilin and the downstream protein SCG10 signaling pathways. Meanwhile, Cheng and co-workers  cells with IC50 values of approximately 10 µm. Mechanism studies confirmed that this compound is an ATP competitive inhibitor and interacts with Asp 444 in the PAK4 kinase domain, Moreover, compound 20 significantly suppressed the growth of ESCC PDX model, thus is worthy of further investigation as an effective PAK4 inhibitor.Through structure-based virtual screening, 6-hydroxy-2-mercapto-3-phenylpyrimidin-4(3H)-one 18 SUP-106,  was iden-workers  recently-workers  conducte50 against PAK4   . PF-3758 0.24 nm . In HCT- 0.24 nm . Recent  0.24 nm . Furtherr tissue , indicatUnfortunately, clinical treatment with PF-3758309 showed no objective response . In dogs+, EpCAM+)  with Everolimus (2.5 mg/kg) produced a statistically significant suppression in tumor growth, a scenario not observed for single agent of Everolimus (Pancreatic carcinoma (PCC) is one of the most malignant tumors with high lethality worldwide and its drug treatment remains an unmet medical need . Recent  EpCAM+) . In BON- EpCAM+) . In addierolimus . Mechanierolimus .In the meantime, PAKs also play important roles in leukemic stem cells and several resistant cancer cells . KPT-927In addition, more combinations of KPT-9274 with drugs of various mechanisms were investigated and synergetic effects were generally achieved. For example, administration of KPT-9274 with the DNA-damage agent bendamustine afforded a synergetic antitumor effect higher than each drug in the Waldenstrom macroglobulinemia (WM) BCWM-1 xenograft model . In the Recently, KPT-9274 was also reported to have therapeutical potential for triple negative breast cancer (TNBC). PAK4 is overexpressed in TNBC cells and the levels of PAK4, phosphorylated PAK4 and downstream phosphorylated β-catenin as well as phosphorylated cofilin were remarkedly reduced after treatment with KPT-9274 for 72 h in SUM159 TNBC cells . Among t2/M arrest and apoptosis. In the meantime, c-Myc and Cyclin D1, the two RCC oncogenic proteins, were decreased drastically, and the NAD levels were also reduced due to its inhibition of NAMPT  has been reported as well . Treatmeof NAMPT . Furtherof NAMPT .p = 0.0007 vs. 0.01)  . Meanwhis. 0.01) . Taken tAt present, there are three phase Ⅰ clinical trials of KPT-9274 in progress for solid tumors, Non-Hodgkin’s lymphoma, and acute myeloid leukemia , respectively. Among them, “NCT02702492” was terminated in 2021, but the detailed results have not been disclosed yet. In addition, the other two clinical trials are still in the process of patient recruiting.Since the first recognition in 1994, the PAK family has gained a continuously increasing interest both in the biological function and their roles in disease modulation. Culminating evidences have shown that the aberrant PAK4 signaling is critically implicated in nearly all hallmarks of cancer and overexpression of its mRNA or proteins has been observed in a large number of diverse human tumors, including those highly malignant ones. Genetic knockout or pharmacological inhibition have postulated that PAK4 is an extremely attractive drug target and many PAK4 inhibitors either as ATP competitive or as allosteric inhibitors bearing multiple chemotypes have been developed and tested as a first line or as adjuvant cancer treatment. Unfortunately, the majority of current reported PAK4 inhibitors suffer from poor selectivity, insufficient antitumor efficacy, and unfavorable drug-like properties. Therefore, by far, there are only two PAK4 inhibitors  entering into clinical trials. PF-3758309 is the first PAK inhibitor tested in the clinical trial, but was terminated due to its poor PAK4 selectively and adverse events together with pharmacokinetic issues observed from patients during phase I study. The emergence of the allosteric inhibitor KPT-9274 draws a new prospect for PAK4 inhibitors. KPT-9274 not only has high selectivity for PAK4 with an allosteric mechanism and also is a potent inhibitor targeting NAMPT, an enzyme important for tumor development and progression as well. In addition, PAK4 was found to play an active role in excluding tumor-specific T cells from the tumor microenvironment, and the PAK4 inhibitor KPT-9274 has showed synergic effects to increase tumor-specific T cell infiltration and hence sensitize non-responsive or resistant tumors to PD-1 blockade therapy. Therefore, KPT-9274 has great potentials either as monotherapy or as combinations with immunotherapy to treat both immune inflamed and poorly inflamed/resistant tumors. However, the fate of KPT-9274 and other PAK4 inhibitors has to wait for the overall outcomes of the final clinical trials on human patients.In addition, it has to be alert that although the mRNA and protein levels of PAK4 are generally observed in cancer cells, the precise mechanism underlying this increase and involvement of PAK4 in the proliferation and transformation of cancer cells are not completely understood. Some studies show that modulation of mTOR and the subsequent WNT/β-catenin pathway might be a critical mechanism for PAK4 in cell proliferative, metabolism, and immune cell infiltration, however, PAK4 certainly has many other functions and many other pathways can be involved in the WNT/β-catenin signaling. Therefore, much effort is need to further investigate the fundamental biological mechanisms PAK4 underlying, which will be critically important for patient stratification and biomarker selectivity for clinical trials.


As metabolomics increasingly finds its way from basic science into applied and regulatory environments, analytical demands on nontargeted mass spectrometric detection methods continue to rise. In addition to improved chemical comprehensiveness, current developments aim at enhanced robustness and repeatability to allow long-term, inter-study, and meta-analyses. Comprehensive metabolomics relies on electrospray ionization (ESI) as the most versatile ionization technique, and recent liquid chromatography-high resolution mass spectrometry (LC-HRMS) instrumentation continues to overcome technical limitations that have hindered the adoption of ESI for applications in the past. Still, developing and standardizing nontargeted ESI methods and instrumental setups remains costly in terms of time and required chemicals, as large panels of metabolite standards are needed to reflect biochemical diversity. In this paper, we investigated in how far a nontargeted pilot experiment, consisting only of a few measurements of a test sample dilution series and comprehensive statistical analysis, can replace conventional targeted evaluation procedures. To examine this potential, two instrumental ESI ion source setups were compared, reflecting a common scenario in practical method development. Two types of feature evaluations were performed, (a) summary statistics solely involving feature intensity values, and (b) analyses additionally including chemical interpretation. Results were compared in detail to a targeted evaluation of a large metabolite standard panel. We reflect on the advantages and shortcomings of both strategies in the context of current harmonization initiatives in the metabolomics field. Metabolomics, a bioanalytical approach originally devised to study metabolic changes in biological model organisms, is now widely used in more applied fields of clinical diagnostics and pharmaceutical research ,2. ActivBioanalysis in metabolomics makes use of two major technologies, nuclear magnetic resonance (NMR) and hyphenated mass spectrometry . NMR provides structure elucidation and quantitative capabilities for biomatrices in situ but requires relatively large sample amounts. GC-MS combines efficient chromatographic separation with sensitive detection but is limited to small volatile compounds or compounds made volatile by derivatization. LC-MS, by contrast, can detect larger metabolites natively due to the use of soft electrospray ionization (ESI), coupled directly to the LC effluent. Polar to medium polar molecules usually ionize well under ESI , with anCommercial LC-MS instruments and ESI ion sources, in particular, are continually refined to enhance operational robustness and sensitivity, addressing many of the above-named needs in nontargeted analysis. Technical enhancements apply to sprayer configuration and arrangement of ion optics (reviewed in ). Also, The goal of the present study was to investigate if a defined nontargeted test experiment can efficiently guide and support method development and evaluation of different instrumental setups. A minimal experiment was devised consisting of a few measurements of a test sample and extensive data analysis of the feature space, ensuring practicality and transferability. Targeted testing, comprising a large commercial metabolite library, was carried out for comparison, representing a common but time-consuming and costly procedure. Both strategies were exemplarily employed for evaluating two instrumental setups for their performance in nontargeted metabolomics: (1) LC-HRMS using a standard ESI interface as reference (REF); and (2) LC-HRMS equipped with a high-temperature “IonBooster” interface representing an alternative setup (ALT) with potentially improved performance. Results showed that similar conclusions about instrumental performance could be drawn from both strategies, suggesting that feature-based evaluation procedures can contribute to much-needed accelerated method development in nontargeted analysis.m/z] ratio and retention time [RT] pairs; looked significantly more populated  than in the REF setup when HILIC was used and 2.3-fold higher when RPC was used  of these compounds, we obtained positive fold-changes for the ALT setup similar to the nontargeted evaluation  in the more sensitive setup. Comparing samples at lower concentrations, by contrast, did not use the full ionization capacity of the less sensitive setup. We, therefore, investigated if a dilution series of the biosample could be used to avoid this problem. Eight sequential one-in-four dilutions of the original sample were prepared  and submitted to LC-HRMS analysis. Feature intensities were then analyzed for ‘robust’ fold-changes considering all concentration levels, as outlined in was used B,C. A towas used , we condaluation . While tfindMAIN ; B: 75% acetonitrile + 10 mM NH4COOH, pH 3.0 [HCOOH]) and a 75 mm × 2.1 mm × 1.7 µm BEH Amide column . The gradient was programmed as follows: 0 min 1% A, 0.3 min 1%, 5.3 min 65%, 5.31 min 99%, 5.7 min 99%, 5.71 min 1%, 7.5 min 1%. A flow rate of 0.8 mL min−1 was used, 0.5 mL min−1 of which were discarded post-column using a T piece to remain within the optimal working range of the standard ion source. The second method employed the reversed-phase (RPC) mode, using a 75 mm × 2.1 mm × 1.8 µm HSS-T3 column  and H2O/acetonitrile each with 0.1% formic acid as mobile phases. Gradient conditions were: 0 min 99% A, 0.3 min 99%, 5.5 min 50%, 5.6 min 1%, 6.5 min 1%, 6.6 min 99%, 7.5 min 99%. Injection volume was 5 µL for the biological sample, 0.2 µL and 1 µL for metabolite standards in positive mode, and 1 µL and 5 µL for metabolite standards in negative mode.Two chromatography methods were used. A hydrophilic liquid interaction chromatography (HILIC) method was established using a buffered solvent system  mode used MSE-like alternating low (8 eV) and high (40–100 eV) collision energies. Ion source temperatures and gas flows were chosen according to the manufacturer’s recommendations for intermediate LC flow rates (0.3 mL min−1). For ESI(+) mode, these were as follows (IonBooster settings in brackets): capillary voltage 4500 V (1000 V), endplate offset −500 V (−400 V), nebulizer pressure 2.5 bar (4.1 bar), dry heater 200 °C (200 °C), dry gas [N2] 8 L min−1. For ESI(-) mode, settings were identical except for capillary voltage . Settings only applicable to IonBooster were charging voltage (300 V) and vaporizer temperature (350 °C).QqTOF analyses were carried out using a scan rate of 8 sMass calibration was performed against an ion series produced by sodium formate, spiked into the LC effluent at the end of each chromatographic run.−1, while the LC flow rate was 0.3 mL min−1 as described. Changes in signal intensities were monitored based on extracted base peak chromatograms (BPC) of the 17 target masses ([M+H]+ ± 1 mDa). As a blank reference, pure mobile phase A was used.An intermediate dilution of the biological sample  without addition of internal standards was analyzed using the above HILIC method. Pharmaceutical mix 17, diluted 1:200 in acetonitrile/methanol 3:1, was infused post-column using a syringe pump and T piece. The infusion flow rate was 60 µL minhttps://proteowizard.sourceforge.io/tools.shtml) accessed on 22 June 2021. Peak detection and alignment over samples were performed in R [Raw Bruker (.d) files were calibrated in the vendor software  and exported to mzML files using msconvert . Alignments were verified by overlaid BPCs of QC compounds. For all subsequent evaluations , featureIons of the same compound, including molecular peaks, isotopes, adducts, fragments, etc., were grouped into pseudospectra using an iterative procedure. First, peaks with similar retention times were combined into time groups using the hierarchical clustering function in R. In each time group, the combination of adducts explaining the greatest proportion of intensity was identified with findMAIN , and allDeconvoluted spectra  were bat2-fold changes (here: 1.5–8). If one such stretch reached or exceeded a defined minimum length (here: 3), 2 linear regression models were fit to the intensity values using standard R functionality (function ‘lm’), the first one to the longest identified stretch (s1) and a second one to the stretch spanning the first intensity value (highest concentration) to the center intensity value of s1. Slopes were extracted from both models , and their difference (Δa) was calculated and returned by the function. In case no stretch reached or exceeded the minimum length, a “missing value” was returned, and the feature was not considered in the overall analysis. After applying the function to all features, Δa values were averaged over 5 s retention time windows and visualized as shown.An R function was implemented to analyze ion suppression effects based on feature intensities. Intensity values of a feature were arranged in order of increasing sample concentration and log2-transformed. These values were analyzed for consecutive stretches exhibiting defined positive logVenn diagrams were produced using the VennDiagram R package.


The study of gender-related peculiarities of vertebral pain syndrome course in order age patients appears to be highly relevant.Study population included 46 female patients and 38 male patients in the age between 60 and 75 years old; mean age was 67,4±6,6 years old.Pain syndrome intensity was assessed using the visual analogue scale (VAS), vegetative disfunction was assessed using A.M. Wayne Questionnaire; the Toronto Alexithymia ScaleThe conducted comparative study showed that the male patients perceived the pain syndrome as more intense as compared to the female patients in lumbar spine: 4,5 ±0,8 vs 3,6±0,5 scores  and in thoracic spine: 4,1 ±1,0 vs. 3,4±1,0 . On the other hand, in vegetative dysfunction assessment, the male patients demonstrated generally lower score: 43,3±7,5 scores vs. 59,6+10,3 in female patients, р <0,001. The results of correlation analysis pf interrelations between alexithymia and pain intensity revealed the differences between the study groups in emotion recognition accuracy : female patients showed lower scores  as compared to the male patients .Therefore, the vertebral pain syndrome tends to be more pronounced in older age male patients as compared to the similar population of older age female patients. Therefore, vertebral pain syndrome correction requires multidisciplinary approach, including psychotherapeutic support.


PADI1 and PADI3) and corneodesmosome degradation, changes that are consistent with increased corneocyte retention. Several overarching principles governing epidermal lipid expression were also noted. For example, there was a strong negative correlation between the expression of 18-carbon and 22-carbon sphingoid base ceramides. Disease-specific alterations in epidermal lipid gene expression and their corresponding alterations to the epidermal lipidome were characterized. Lipid biomarkers with diagnostic utility for inflammatory and precancerous conditions were identified, and a 2-analyte diagnostic model of psoriasis was constructed using a step-forward algorithm. Finally, gene coexpression analysis revealed a strong connection between lipid and immune gene expression. This work highlights (a) mechanisms by which the epidermis is uniquely adapted for the specific environmental insults encountered at different body surfaces and (b) how inflammation-associated alterations in gene expression affect the epidermal lipidome.The epidermis is the outermost layer of skin. Here, we used targeted lipid profiling to characterize the biogeographic alterations of human epidermal lipids across 12 anatomically distinct body sites, and we used single-cell RNA-Seq to compare keratinocyte gene expression at acral and nonacral sites. We demonstrate that acral skin has low expression of EOS acyl-ceramides and the genes involved in their synthesis, as well as low expression of genes involved in filaggrin and keratin citrullination ( Combined, these lipids form a permeability barrier that limits transepidermal water loss (The stratum corneum (SC) is the outermost layer of the epidermis. It is composed of terminally differentiated and enucleated keratinocytes that reside within a lipid matrix, like bricks and mortar. The lipid matrix is a heterogenous mixture of free fatty acids (FAs), cholesterol, and ceramides  characterize a variety of disease-specific alterations in epidermal lipid expression; (g) identify and validate disease-specific diagnostic lipid biomarkers; and (h) demonstrate how disease-associated cytokines can alter lipid gene expression, likely resulting in the characteristic changes in epidermal lipid expression that are associated with the same cytokines.Here, we (a) identify transcriptional alterations that likely account for the site-specific structural changes of the human epidermis; (b) construct a detailed biogeographic map of epidermal lipid expression and make predictions as to how compositional changes to epidermal lipids can alter the structure of the SC; (c) uncover the general rules governing correlative relationships among different lipid structures; (d) identify potentially novel clusters of coregulated lipid genes; (e) link lipid gene expression to the expression of autoinflammatory mediators, especially –95, 3.1 × 10–85, and 1.0 × 10–81, respectively). Specifically, the expression of the NH ceramide Cer(t20:1[6OH]/26:0) was relatively high within the epidermis of the AB, AC, AX, and PF and relatively low within the epidermis of the AL, CK, DH, GB, and PH. In contrast, the AH ceramide Cer(t18:1[6OH]/22:0[2OH]) had the opposite expression pattern, with relative high expression in the epidermis of AL, CK, GB, and PH , with no underlying skin or major medical conditions. Each individual had 12 different anatomic sites sampled . A targeted mass spectrometry approach  was then, and PH . These l, and PH . The lacNext, to better visualize the biogeographic variation in epidermal lipid expression, we constructed (a) pie charts from representative highly abundant lipids and (b) a cluster dendrogram to organize anatomic sites by their lipid similarities. For each location, 2 pie charts were constructed: one for representative ceramides and the second for common unsaturated FAs and cholesterol, where 100% equals the combined total for the listed lipids . Lipids selected for inclusion in the pie charts had (a) variable expression by anatomic location and (b) were abundant enough to be visualized in the pie chart after exclusion of saturated FAs. This analysis revealed that GB, AL, and CK had a high relative abundance of cholesterol sulfate (Chol-SO4) . In contWhen lipid similarities were presented hierarchically as a cluster dendrogram, the high degree of similarity between CK, AL, and GB; UB, AC, and VF; AX, AB, and PF; and DH and PLE was clearly evident . Of thesTo investigate how epidermal ceramides of acral skin were synthesized, we next performed RNA-Seq to search for differential expression in lipid-associated metabolic genes that could account for the noted differences in the epidermal ceramides. Differential gene expression analysis demonstrated that 45 out of a set of 83 genes (54%) related to lipid metabolism were significantly differentially expressed in palm versus trunk skin (FDR < 0.05) . A princSPTLC2 is significantly upregulated only in granular layer keratinocytes and not in spinous or basal layer keratinocytes. In contrast, CERS4 expression is increased across all keratinocyte layers in acral skin. Diagrams of how these alterations in lipid-associated genes should alter epidermal PH ceramide expression are presented in ABHD5, was strongly downregulated in acral skin  need to be degraded. Desmosomes hold together keratinocytes in the epidermis. At the outermost layer of the stratum granulosum, corneodesmosomes are formed from the addition of corneodesmosin to the extracellular portion of the desmosomes. The resulting electron-dense structures are integrated into the cornified cell envelopes. Desquamation occurs when the corneodesmosomes are fully degraded. Since acral skin is hallmarked by corneocyte retention, we searched for alterations in the acral skin transcriptome that would delay corneocyte desquamation. Single-cell RNA-Seq revealed that acral skin granular layer keratinocytes have low expression of corneodesmosome-degrading and KLK7 . In contegulated , but thealm skin . Also, tinocytes , while tegulated . Numerouegulated . These aKRT10 in acral skin   . Furtherof KRT10 . Thus, iatoderma , 17. Sinnd KRT78 , B and Cic KRT77 . Finallyll lines . Other dn = 37); nonlesional psoriasis (PN) (n = 16); lesional AD (n = 10); nonlesional atopic dermatitis (AN) (n = 9); AK (n = 10); SK (n = 9), TI (n = 3); and healthy control (NN) (n = 20) skin , seborrheic keratoses (SK), and tinea corporis (TI) can be diagnosed by their clinical appearance, especially by their nearly pathognomonic epidermal changes. We, thus, hypothesized that the characteristic epidermal features  associated with these diseases should have corresponding molecular signatures in the epidermal lipidome. To investigate this possibility, we performed targeted mass spectrometry to profile epidermal lipids in lesional psoriasis skin (PP) (20) skin .–25; –22, 1.6 × 10–23, 7.7 × 10–26, 6.7 × 10–26, respectively)  in lesional skin of psoriasis, AD, and TI (FDR = 1.7 × 10ctively) .P = 1.4 × 10–7)  .To visualize the major lipid compositional differences between psoriasis, AD, and healthy controls, we constructed 3 different types of pie charts that displayed (a) the relative abundance of the different lipid classes, (b) the relative abundance of select FAs and Chol-SO4, and (c) the relative abundance of select ceramides. In all the above pie charts, 100% equals the combined total of only the listed lipids — not every epidermal lipid . ClearlyTo better appreciate alterations in lower abundance lipids, we next constructed heatmaps displaying the relative expression of all 351 monitored lipids in AD and psoriasis compared with healthy controls . SK and To further visualize the lipid similarities between diagnostic groups, a cluster dendrogram was constructed . The resn = 27], PSO [n = 28], control [n = 38]) to search for transcriptome alterations that could account for these changes  curves for each of them. This analysis revealed that the NH ceramide Cer(t18:1[6OH]/30:0) was an accurate single analyte classifier of psoriasis  . Specifin = 37, PP) against all other diagnostic groups . This model was composed of 2 lipid analytes, NH ceramide Cer(t18:1[6OH]/30:0) and AS ceramide Cer(d16:1/28:0[2OH]),(5.45*[NH ceramide Cer(t18:1(6OH)/30:0)]-1.74*[AS ceramide Cer(d16:1/28:0[2OH])]-12.16)  . Interro]-12.16) .–7, 1.3 × 10–5, 1.3 × 10–5, respectively) , Cer(t20:1[6OH]/24:0[2OH]), Cer(t19:1[6OH]/24:0[2OH]) (FDR = 9.29 × 10ctively) .After characterizing the biogeography of epidermal lipid expression and its disease-associated alterations, we next sought to determine how the expression of individual lipids related to one another. For this, we first pooled data from each diagnostic group and then calculated lipid correlation coefficients for every possible lipid-lipid comparison, 123,201 in total . This idr2, where r represents the Pearson’s correlation coefficient of each lipid-lipid comparison. From the resulting image, a variety of strong intra- and intersubclass lipid-lipid correlations were apparent. The t-SNE plot also clearly demonstrated that there are groups of lipids from within each subclass that have similar correlation patterns, but not all lipids from within the same subclass behave similarly  dimensionality reduction strategy, using the pairwise distance formula, 1 – imilarly .r = 0.99, FDR = 2.5 × 10–86)       . Likewis× 10–49)  and AH c× 10–44) . Negativ× 10–46) , and AH × 10–45) . NS(C18)× 10–34) .r = 0.95, FDR = 2.9 × 10–55)   and NP ceramide Cer(t22:0/26:0)   and NDS ceramide Cer(d18:0/20:0)   . Likewis× 10–49) . FA 24:1× 10–49) — e.g., F× 10–22) . In cont× 10–32) . In cont× 10–32) , and it × 10–32) . Finally× 10–32) .r = 0.80, FDR = 3.7 × 10–22)      ceramides — e.g., Chol-SO4 positively correlates with NS ceramide Cer(d18:1/23:0) (× 10–22) . In cont× 10–22)  — e.g., × 10–19) . Finally× 10–26) . They al× 10–45) . However× 10–33) . As a ge× 10–33) .ELOVL and CERS lipid-gene family members in 50 primary keratinocyte cell lines. This analysis revealed that the ELOVL and CERS genes formed 2 clusters based on their strong correlative relationships, the ELOVL4 cluster  and the ELOVL6 cluster   and various genes known to be involved in inflammation . The strong link between ELOVL4 and IL36B, CDK7, CHMP2B, and S100A13 was also evident in RNA-Seq data sets of psoriasis lesional skin , rs62407622, located within the ELOVL4 gene that was associated with a strong reduction in ELOVL4, both in primary keratinocytes  and the barrier genes  were all decreased in the ELOVL4lo keratinocytes homozygous for rs62407622. In contrast, keratinocytes homozygous for rs62407622 had increased expression of S100A13 and TLR3  . A 2D pland IL18 . A scatt 2 genes . Several  . These gLike ceramides, free FAs are also abundant in the epidermal lipidome, especially saturated FAs. Albeit less abundant, unsaturated FAs also make important contributions to the structure of the lipid matrix or “mortar.” This is of particular relevance because we found that unsaturated FAs had a high degree of biogeographic variation . For exaSPTLC2 was upregulated and SPTLC3 was downregulated in acral skin granular layer keratinocytes  ceramides but increased expression of NS Cer(d18:1/18:0)   catalyzed the rate-limiting step in sphingolipid biosynthesis, with SPTLC2 favoring generation of 18-carbon sphingoid bases and SPTLC3 favoring generation of 16-carbon sphingoid bases; SPTLC3 also supported synthesis of longer carbon sphingolipids when present within SPTLC1-SPTLC3-SPTSSB. Single-cell RNA-Seq revealed that inocytes . This exinocytes . The preinocytes . CERS4 sinocytes . Also, tral skin  was consral skin , as ABHDynthesis , 20. Fur:1/18:0) . Thereforal skin  might beThe biogeographic alterations in epidermal lipids just described will undoubtedly manifest as physical changes to the lipid “mortar” located between the corneocyte “bricks.” For example, the lipid envelope is a monolayer of FAs and acyl-ceramides esterified to the cornified envelope –24. It iPADI1 and PADI3) were strongly downregulated in acral skin  were upregulated in granular layer acral keratinocytes  are highly upregulated in granular layer acral keratinocytes , an ichthyosis that is hallmarked by thick hyperkeratotic skin. Interestingly, KRT1-associated EHK has an accompanying palmoplantar keratoderma that is generally absent in KRT10-associated EHK. Palmoplantar keratoderma also happens to be the clinical manifestation of KRT9 deficiency  lipid-lipid correlations, which followed specific rules . For exaHowever, some structurally dissimilar lipids were also highly correlative. A prime example of this is Chol-SO4, whose expression positively correlated with NS ceramide Cer(d18:1/23:0) (and similar ceramides) and negatively correlated with NH ceramide Cer(t20:1[6OH]/27:0) (and similar ceramides) . AnotherELOVL1, ELOVL3, ELOVL4, ELOVL7, CERS3) and   . Interesne genes . Immune d MAP3K8 . It is linocytes . Thus, tinocytes . This isatoderma , a psoristaphylococcus colonization in AD with AS, ADS, NS, and NDS ceramide abundance  increased the expression of SPTLC2 , which mSPTLC2 . LikewisFinally, we explored lipid profiling as a diagnostic tool with real-world clinical applications. The shortage of dermatologists outside of major cities and the ease and low cost of tape stripping compared with skin biopsy makes lipid profiling an attractive diagnostic tool. To this end, we identified 77 lipid analytes with utility as diagnostic biomarkers . We alsoAlthough we focused on dermatologic diseases, expanding our biomarker research endeavors to applications outside of dermatology is clearly possible. Serum ceramides are being investigated as potential predictive biomarkers for aging, diabetes, and cardiovascular disease –34. GiveAll participating subjects were adults recruited from the Department of Dermatology at the UCD. Patients met respective clinical criteria for a diagnosis of psoriasis, AD, AK, SK, or TI. All diagnoses were made by a board-certified dermatologist. The diagnosis of TI was confirmed with potassium hydroxide (KOH) microscopy. Patients were not on any topical or systemic therapy at the time of sampling. For psoriasis and AD samples, lesional and nonlesional anatomically matched paired samples were taken to control for body location. Individuals without underlying dermatologic conditions were used as healthy controls. Normal healthy subjects for acral and nonacral skin sites were recruited from the Department of Dermatology at the University of Michigan. Groups were balanced for sex.All transcriptomic data described in this manuscript have been deposited in SRA under the BioProject IDs PRJNA858182, PRJNA858056, and PRJNA742431. All other data and material generated as a part of this work will be made available upon request.For full statistical information, see The acral biopsy study was approved by the IRB of the University of Michigan. Informed consent was obtained from all participating patients.AAM and STL are cofirst authors. Order of authorship was determined by seniority. Conceptualization was contributed by FP and EM. Methodology was contributed by EM, AAM, STL, AT, YX, AIM, SMW, IEA, JW, YI, FTL, BA, LCT, JEG, and CBL. Investigation was contributed by AAM, STL, CA, AT, AIM, SMW, IEA, FP, ACB, YI, A Kumar, RU, JMK, EAW, CM, MYC, HX, A Kirane, GL, LCT, CBL, JEG, and EM. Project administration was contributed by EM. Supervision was contributed by EM, AAM, and SMW. Review and editing of the manuscript was contributed by all authors.