N = 97) had a median (range) age of 62 years (38–85), 5 (2–14) prior therapy lines, and 85% were double refractory. The overall response rate (ORR) was 4.3, 20.0, 29.2, and 24.0% with isatuximab 3 mg/kg Q2W, 10 mg/kg Q2W/Q4W, 10 mg/kg Q2W, and 20 mg/kg QW/Q2W, respectively. At doses ≥10 mg/kg, median progression-free survival and overall survival were 4.6 and 18.7 months, respectively, and the ORR was 40.9% (9/22) in patients with high-risk cytogenetics. CD38 receptor density was similar in responders and non-responders. The most common non-hematologic adverse events  were nausea (34.0%), fatigue (32.0%), and upper respiratory tract infections (28.9%). Infusion reactions  occurred in 51.5% of patients. In conclusion, isatuximab is active and generally well tolerated in heavily pretreated RRMM, with greatest efficacy at doses ≥10 mg/kg.A Phase 2 dose-finding study evaluated isatuximab, an anti-CD38 monoclonal antibody, in relapsed/refractory multiple myeloma . Patients with ≥3 prior lines or refractory to both immunomodulatory drugs and proteasome inhibitors  were randomized to isatuximab 3 mg/kg every 2 weeks (Q2W), 10 mg/kg Q2W(2 cycles)/Q4W, or 10 mg/kg Q2W. A fourth arm evaluated 20 mg/kg QW(1 cycle)/Q2W. Patients ( The introduction of proteasome inhibitors (PIs) and immunomodulatory agents (IMiDs) has improved clinical outcomes in patients with multiple myeloma (MM) , and 5-yCD38 is a transmembrane glycoprotein that has multiple functions as both a receptor and an enzyme. It is uniformly, and often highly, expressed on malignant plasma cells, making it an attractive target for the treatment of MM , 11. IsaIn a Phase 1 dose escalation study of single agent isatuximab in patients with RRMM who had previously received an IMiD and a PI, the overall response rate (ORR) was 24% (15/63 patients) at doses 10–20 mg/kg, including one complete response (CR) [This Phase 2, open-label, randomized, international, multicenter study (NCT01084252) was conducted across 17 study centers . The protocol was approved by institutional review boards/ethics committees and patients provided written informed consent to participate. The study was conducted according to the Declaration of Helsinki and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use Good Clinical Practice.Adult patients (≥18 years) with MM were eligible to participate if their disease was refractory to both an IMiD and a PI, or if they had received three or more prior lines of therapy that included an IMiD and PI. All patients must also have received an alkylating agent, have had measurable disease, and obtained a minimal response or better to at least one prior line of therapy, according to International Myeloma Working Group (IMWG) Uniform Response Criteria . Other kPatients were randomized in a 1:1:1 ratio to one of three dosing regimens, all administered in 28-day cycles. Arm 1 received isatuximab 3 mg/kg every 2 weeks (Q2W), Arm 2 received isatuximab 10 mg/kg Q2W, and Arm 3 received isatuximab 10 mg/kg Q2W for two cycles followed by 10 mg/kg every 4 weeks . Randomization was carried out via an Interactive Voice Response System/Interactive Web Response System and was stratified by prior treatment with pomalidomide and/or carfilzomib. As a protocol amendment, based on pharmacokinetic (PK) analysis of Phase 1 data, a fourth arm was added for which patients received isatuximab 20 mg/kg once a week (QW) during the first 28-day cycle and Q2W thereafter. Intrapatient dose escalation (not to exceed 20 mg/kg on the Q2W schedule) was permitted at the discretion of the study sponsor and treating physician for patients with progressive disease if the original dose regimen was tolerated for at least one cycle.Isatuximab was administered at an initial infusion rate of 175 mg/h, which could be increased in the absence of infusion reactions (IRs) up to a maximum of 400 mg/h. Prophylaxis was given 15–30 min prior to isatuximab infusion to reduce the risk and severity of IRs, consisting of diphenhydramine 25–50 mg IV, methylprednisolone 100 mg IV, ranitidine 50 mg IV , and acetaminophen (paracetamol) 650–1000 mg PO.Patients continued treatment until disease progression, unacceptable toxicity, or another reason for discontinuation. At the end of treatment, those with disease progression were followed every 3 months until death or study cut-off date and those without disease progression were followed monthly until disease progression, initiation of another anticancer therapy, death, or study cut-off date.The primary efficacy endpoint was ORR, which included stringent CR, CR, very good partial response (VGPR), or partial response (PR). Responses were assessed by the Independent Adjudication Committee (IAC) using the IMWG Uniform Response Criteria , and werExploratory endpoints included analysis of Fc gamma receptor polymorphism from baseline blood samples and CD38 receptor density from baseline bone marrow aspirates to evaluate correlations with clinical response.Disease assessment was on Day 1 of Cycle 2, and Day 1 of every subsequent cycle thereafter. Safety was evaluated based on physical examination, laboratory tests, and adverse event (AE) reporting, which was graded by the National Cancer Institute Common Terminology Criteria for Adverse Events v4.03. High-risk cytogenetics, defined as t(4:14) and/or del(17p), were assessed at a central laboratory using fluorescent in situ hybridization, or local assessment when central data were not available. Blood concentrations of isatuximab were analyzed for population PK using non-linear mixed effects modeling.A selection design was used to maximize the probability of selecting the best of four isatuximab doses using ORR as the endpoint . Target Analyses of efficacy and safety were based on the all treated population, which comprised all patients who received at least one full or partial administration of isatuximab. For the primary endpoint of ORR, results are summarized as descriptive statistics.Duration of response, OS, and PFS were analyzed using the Kaplan–Meier method with estimated median and 95% confidence interval (CI). The exploratory analysis of Fc gamma receptor polymorphism used logistic regression analysis. All other analyses are summarized with descriptive statistics.The study started on July 2, 2014, with a part 1 data cut-off of December 9, 2016; 12 months after the first dose of the last patient. A total of 97 patients were treated with isatuximab in the dose-finding section of the study.Patient demographics, disease characteristics, and treatment history are presented by dose group in Table Patient disposition is shown in Fig. The ORR by IAC assessment for all dose groups was 19.6% (19/97 patients), including 11.3% (11/97 patients) with a VGPR and 8.2% (8/97) with a PR; no CR were observed. The ORR based on investigator assessment was similar at 21.6% (21/97 patients). The ORR by dose  and 66/97 (68.0%) patients had a grade ≥3 TEAE. The most common any grade TEAEs (≥20% of patients), grade ≥3 TEAEs (≥5% of patients), and hematologic abnormalities are presented in Table Besides IRs, drug-related TEAEs reported in ≥5% of patients consisted of chills (16.5%), nausea (15.5%), dyspnea (12.4%), chest discomfort (11.3%), flushing (11.3%), cough (8.2%), headache (7.2%), vomiting (6.2%), and wheezing (5.2%). Except for dyspnea, which occurred at grade ≥3 in 1 patient (1.0%), none of these drug-related TEAEs were reported at grade >2.The most frequently reported grade ≥3 TEAEs were hematologic abnormalities and pneumonia Table . Grade 3Five AEs (5.2%) led to withdrawal from the study, of which two were IRs: one grade 4 IR (anaphylactic reaction and bronchospasm), one grade 3 IR, one sudden death (not considered related to isatuximab), one grade 3 platelet count decrease, and one grade 5 atrial fibrillation (not considered related to isatuximab). There were eight (8.2%) deaths during the treatment period: three due to AEs , and five due to progressive disease, none of which were considered related to treatment.None of the 94 evaluable patients were positive for antidrug antibodies at baseline or after isatuximab administration.Of the 60 patients evaluable for CD38 receptor density at baseline, median CD38 receptor density was 151,317 molecules/cell  in 15 responders and 143,408 molecules/cell  in 45 non-responders. At active doses ≥10 mg/kg, the ORR for patients with CD38 receptor density above or below the threshold value of 150,000 molecules/cell was 33.3% (8/24 patients) and 27.3% (6/22 patients), respectively.The prevalence of FcγRIIIA (CD16) cytogenetic variants was 10.7% (10/93 patients) FcγRIIIA V/V, 39.8% (37/93) F/F, and 49.5% (46/93) F/V. The highest ORR  was observed in the V/V variant population, compared with 8.1% (3/37) for F/F and 19.6% (9/46) for F/V.Cmax) at Cycle 1 Day 1 (6.9-fold increase), whereas area under the curve (AUC) at Week 1 (AUC 1W) increased in a greater than dose-proportional manner (8.5-fold increase). When the dose was increased 2-fold, from 10 to 20 mg/kg, there was a dose-proportional increase in Cmax and AUC 1W (both 1.8-fold increase) . For the 20 mg/kg QW/Q2W schedule, accumulation ratios at Cycle 2 were 2.2 for Cmax and 3.3 for Ctrough, and were similar at Cycle 6 , indicating that steady state is reached by Cycle 2 for this administration schedule .The efficacy of isatuximab is important given the prior treatment burden of patients in this study, comprising a median five previous lines of therapy and 85% dual refractoriness to IMiDs and PIs. It is well recognized that responses diminish with successive treatment regimens , and reaResponses to isatuximab were consistently demonstrated in all high-risk groups analyzed, including those with more than three prior lines of therapy, double and quadruple refractory disease, ≥70 years of age, and at least one high-risk cytogenetic abnormality. Indeed, the ORR was higher in older and high-risk cytogenetic patients than the overall population, although small subgroup numbers warrant caution when interpreting the data. The efficacy of isatuximab in these hard-to-treat patients is promising, especially given real-world data showing reduced response rates and OS in older patients compared with their younger counterparts –21 and rn = 106), patients had a median five prior lines of therapy (range 2–14) and 95% were double refractory to a PI and IMiD. The ORR (primary endpoint) was 29.2% , median PFS was 3.7 months and median OS was 17.5 months [The results of this study with isatuximab monotherapy compare favorably with those from studies supporting approved single agents in RRMM. In the Phase 2 SIRIUS study of daratumumab, patients had received an alkylating agent and at least three prior lines of therapy, including a PI and IMiD, or were double refractory to the last PI and IMiD received. In the daratumumab 16 mg/kg arm , without incurring any increased risk of IRs.A previous study found an association between receptor density and response to daratumumab, reflecting an observed link between CD38 expression levels on patient MM cells and induced cell death by daratumumab via ADCC and CDC , 29. In Isatuximab induces both Fc-dependent and Fc-independent pathways to kill CD38-expressing tumor cells , 13. Of Cmax and Ctrough between Cycle 2 and Cycle 6 were observed with both 10 mg/kg regimens. Furthermore, PK data from a Phase 1 study of isatuximab in patients with RRMM or other CD38-positive hematologic malignancies indicate that, at 10 mg/kg Q2W, Cycle 1 plasma trough levels of isatuximab are consistently above the lowest pharmacological active dose in mouse tumor models (10 μg/ml), and at dose 20 mg/kg Q2W, Cycle 2 trough levels are above the concentration needed for tumor eradication (129 μg/ml) [This study confirms the findings from a Phase 1 study of single agent isatuximab in heavily pretreated patients with RRMM , where the ORR was 24% at doses ≥10 mg/kg . There w9 μg/ml) . NotablyAs patients were not randomized to the 20 mg/kg QW/Q2W group, direct comparisons cannot be made between this and the other study arms. It is also noteworthy that detection of the therapeutic monoclonal antibody may obscure whether a patient with an IgG-kappa M-spike has attained a complete hematologic remission. A CR per IMWG criteria requires the absence of M-protein by immunofixation, while a VGPR allows M-protein detectable by immunofixation but not on electrophoresis , 33. HigPart 2 of this study is ongoing to evaluate isatuximab 20 mg/kg as monotherapy or in combination with dexamethasone in patients with RRMM (NCT01084252). Isatuximab is also being investigated in combination regimens –36 and PIn conclusion, isatuximab demonstrated single agent activity in heavily pretreated patients with RRMM, including high-risk patients, and these responses were more durable at doses ≥10 mg/kg. Isatuximab was generally well tolerated. The time burden of infusions could be safely reduced following the first infusion and most IRs were mild-to-moderate in severity and rarely occurred after the first infusion. Isatuximab is a significant new treatment option for patients with RRMM and studies are ongoing for use both as a single agent and in combination with other treatment regimens.Supplemental Figure legendsSupplemental Figure 1Supplemental Figure 2


RSVH occurred in 534/14 504 (3.7%) infants; equating to a rate of 5.65 per 100 patient-seasons, with the rate in individual wGA groups dependent upon exposure time (P = 0.032). Most RSVHs (60.1%) occurred in December–January. Median age at RSVH was 88 days (interquartile range (IQR): 54–159). Respiratory support was required by 82.0% of infants: oxygen in 70.4% (median 4 (IQR: 2–6) days); non-invasive ventilation in 19.3% (median 3 (IQR: 2–5) days); and mechanical ventilation in 10.2% (median 5 (IQR: 3–7) days). Intensive care unit admission was required by 17.9% of infants (median 6 days (IQR: 2–8) days). Median overall hospital length of stay (LOS) was 5 (IQR: 3–8) days. Hospital resource use was similar across wGA groups except for overall LOS, which was shortest in those born 35 wGA . Strategies to reduce the burden of RSVH in otherwise healthy 32–35 wGA infants are indicated.To provide comprehensive information on the epidemiology and burden of respiratory syncytial virus hospitalisation (RSVH) in preterm infants, a pooled analysis was undertaken of seven multicentre, prospective, observational studies from across the Northern Hemisphere (2000–2014). Data from all 32 RSVHs aountries . Certainountries –6.An important group that has generated much debate comprises those children born 32–35 weeks' gestational age (wGA), in terms of their level of risk for RSVH compared to those born at lower and higher wGA and the cost-effective deployment of prophylaxis with palivizumab . This isper 100 patient-seasons. The median hospital length of stay (LOS) was 6 days, and 22% required intensive care unit (ICU) admission for a median of 8 days. Supplemental oxygen support was required by 70.4% of infants for a median of 5 days and 12.7% required mechanical ventilation for a median of 5 days [A number of prospective studies have assessed the burden of RSVH in children born 32–35 wGA within Northern Hemisphere countries –19. To pf 5 days .Herein we analysed those born at 32 wGA, stratified results across individual wGA, and assessed those born and hospitalised outside of the RSV season.0) to 35 weeks and 6 days (356)) were identified via a systematic search of PubMed and EMBASE from January 1998 to January 2015. Study selection criteria included a multicentre, observational, prospective design; inclusion of >1000 preterm infants; and laboratory-confirmed RSV infection in all subjects -based assays), who were hospitalised with an LRTI. Studies where >15% of infants received palivizumab prophylaxis were excluded  and only one study (most recent) from an individual country was included (to limit data to one representative study from each country). The overall aim was to include large, high-quality, cross-sectional studies that, when combined, provide an overview of the burden of RSV generalisable across the Northern Hemisphere.The selection of studies comprising the pooled dataset has been described previously . BrieflyFactors Linked to Respiratory Syncytial Virus Infection Requiring Hospitalization in Premature Infants Study  [Pediatric Investigators Collaborative Network on Infections in Canada  [Preterm Risk Estimation Measure for RSV Hospitalization in Ireland  [Italian National Birth Cohort  [RSV Respiratory Events among Preterm Infants Outcomes and Risk Tracking  [Predictors Associated with RSV HOspitalization in Nonprophylaxed, Premature Infants  [The original review comprised seven studies spanning 2000–2014) [00–2014 [, Spain) ; RISK St Canada) ; RSV PreIreland) ; Italian, Italy) ; RSV ResRT, USA) ; and Preational) .0–356 wGA infants (≤1 year) were included in this analysis except: those with a comorbidity associated with an increased risk of RSVH ; those who had received palivizumab prophylaxis; and those with an RSV-negative respiratory-related hospitalisation. Gestational age was determined within each study based on standard criteria, namely ultrasonographic dating and/or the time between the first day of the last menstrual period and delivery date.All 32The individual datasets from the seven studies were previously shown to be homogeneous when analysed in 33–35 wGA infants born and hospitalised during the RSV season . Predicaper 100 patient-seasons, with the latter adjusted for length of exposure during the RSV season for the full cohort . SeasonThe following outcomes related to RSVHs were assessed: requirement for and duration of oxygen support; requirement for and duration of other non-invasive respiratory support/ventilation ; requirement for and duration of mechanical ventilation ; admission to and LOS in ICU; and overall hospital LOS.P-value <0.05 was considered statistically significant.LOS and duration of respiratory support required were expressed as a median with interquartile range (IQR). Length of follow-up was presented as a mean plus standard deviation (SD). RSVH incidence rates were compared using the Kruskal–Wallis test and Spearman (non-parametric) correlation. Differences in hospital resource use by wGA were assessed using the Kruskal–Wallis test. Analyses of partial seasonal exposure in relation to RSVH across gestational age groups were carried out using Cox regression. Analyses were performed using SPSS for Windows version 15.0 , Microsoft Access SQL  and/or Microsoft Access/Excel VBScript (Microsoft). A 0 and 356 wGA, for which year-round data were available for 9020 (n = 9686) there were: 1046/9686 (10.8%) infants born at 32 wGA, 2235/9686 (23.1%) at 33 wGA, 3646/9686 (37.6%) at 34 wGA and 2759/9686 (28.5%) at 35 wGA.In total, the pooled analysis included 14 504 infants born between 32for 9020 . For theper 100 patient-seasons was 5.65. December and January accounted for over half of RSVHs (60.1%), with 94.1% of RSVHs occurring between November and March . Overall, 42.3% of RSVHs (157/371) occurred in infants born outside the RSV season.Of the 14 504 infants, 534 had a confirmed RSVH, representing an incidence of 3.7%. The corresponding rate nd March ; across Median age at RSVH was 88 (IQR: 54–159) days, with approximately half of RSVHs  occurring in infants <3 months old and four-fifths occurring in infants ≤6 months old . For those born within and outside the RSV season, the median age at RSVH was 62 (IQR: 40–96) days and 143 (IQR: 94–188) days, respectively.χ2: P = 0.09; Spearman: P = 0.10). However, when converted to a rate per 100 patient-seasons, the difference between wGA groups was significant (Spearman: P = 0.03): 35 wGA 5.66; 34 wGA 6.14; 33 wGA 7.29; and 32 wGA 7.84. The length of seasonal exposure to RSV also increased with increasing wGA (Spearman: P = 0.003). Cox regression analysis showed an upward trend in mean seasonal exposure rates amongst RSVH cases between 32 wGA (0.638) and 35 wGA (0.707) and the Wald statistic for seasonal exposure as a predictor of change in hazard for RSVH was highly significant (P < 0.001) in every group. The proportion of RSVHs outside the RSV season did not differ significantly with wGA, albeit there was a rising rate with increasing wGA . Median age at RSVH did not significantly differ across wGA groups (Spearman: P = 0.21), although infants born at 32 wGA (118 (IQR: 64–195) days) seemed to have an extended period of risk compared to those born at ³33 wGA (33 wGA: 82 (IQR: 55–153) days; 34 wGA: 91 (IQR: 51–154) days; 35 wGA: 89 (IQR: 47–159)).RSVH incidence tended to increase with decreasing wGA at birth, although this did not reach statistical significance: 4.0% for those born 35 wGA; 4.0% for 34 wGA; 4.7% for 33 wGA; and 5.0% for 32 wGA  required some form of oxygen therapy or respiratory support during their RSVH; 70.4% required supplemental oxygen, 19.3% non-invasive ventilation and 10.2% mechanical ventilation . SupplemP = 1.000; Spearman: P = 0.06). None of the other hospital resource variables differed significantly between 32, 33, 34 and 35 wGA infants except the median duration of overall hospital LOS, which was lower (P < 0.001) for those born 35 wGA (3 (IQR: 2–6) days) after a progressive increase in stay from 32 to 34 wGA (32: 4 (3–7) days; 33: 5 (3–8) days; 34: 6 (4–9) days).The percentages of infants requiring any form of respiratory support were highest in those born most premature, although results were not significantly different across wGA groups: 59.6% (31/52) for those born 32 wGA; 50.5% (53/105) for 33 wGA; 44.1% (64/145) for 34 wGA; and 43.6% (48/110) for 35 wGA  of all RSVHs occurring in December and January. While the asserted start and end dates of the RSV season varied by country (and sometimes within the country) and year-to-year, the main at-risk period spanned from November through March .This pooled analysis of 14 504 infants provides a comprehensive evaluation of the epidemiology and burden of RSVH in 32–35 wGA infants in the Northern Hemisphere. The overall incidence of RSVH was 3.7% and the overall rate P = 0.10), the rate per 100 patient-seasons did significantly increase with decreasing wGA . Other studies, such as those from Denmark [n = 964, 3.9%) were greater than full-term infants  3.2, 95% CI 1.4–7.1), but equal to <32 wGA infants  [P = 0.003; Cox: P < 0.001).While RSVH incidence did not significantly differ with wGA  . Our stuP = 0.21), although infants born 32 wGA appeared to have a longer at-risk period (median 118 days) than those born 33–35 wGA (82–91 days). Similar results were reported in the US SENTINEL 1 study, where the median age at RSVH was 3 months for 29–32 wGA infants and 2 months for 33–35 wGA infants [vs. 3.9%; P = 0.25), possibly reflecting younger chronological age in the former balanced against the increased duration of exposure in the latter.The median age at RSVH was approximately 3 months (88 days), with half (53%) of all RSVHs occurring in those <3 months of age and four-fifths in those ≤6 months of age (82%). The median age at RSVH did not differ significantly across wGA . The costs associated with an RSVH episode in preterm infants have been estimated at a median $14 000–77 000 in the USA [RSVHs were associated with considerable resource use, with 82% of infants requiring some form of respiratory support, 10% mechanical ventilation for a median of 5 days and 18% ICU admission for a median of 6 days. Studies from the USA, including SENTINEL 1, have reported higher ICU admission rates of 40–50% in similar wGA infants , 27, alb5–6 days , respect5–6 days , 29. In  the USA , 25 and  the USA . After R the USA , 32.P = 0.001) and duration of hospitalisation (non-parametric Levene test: P < 0.001) [The main limitations of our pooled analysis were related to some salient differences in methodology/design of the included studies. Most notably, only four of seven studies included 32 wGA infants, although data were available on >1000 such infants. Other differences included the fact that the PICNIC study enrolled only infants born and hospitalised during the RSV season (+1 month) and variable lengths of follow-up . Heterog< 0.001) . The stu< 0.001) , 18, 19.< 0.001) . In the < 0.001) , a quartOur study provides comprehensive data on the epidemiology and burden of RSVH in 32–35 wGA infants in the Northern Hemisphere that can help inform healthcare policy on the deployment of preventive strategies, such as palivizumab. The use of a country-specific or customisable risk factor scoring tool to identify the highest risk infants  should b


Fenofibrate, a commonly used antilipidemic drug, has been verified to exert hepatic protective effects in other cells and animal models. The purpose of this study was to identify the function of fenofibrate on mouse hepatic IR injury and discuss the possible mechanisms. A segmental (70%) hepatic warm ischemia model was established in Balb/c mice. Serum and liver tissue samples were collected for detecting pathological changes at 2, 8, and 24 h after reperfusion, while fenofibrate  was injected intraperitoneally 1 hour prior to surgery. Compared to the IR group, pretreatment of FF could reduce the inflammatory response and inhibit apoptosis and autophagy. Furthermore, fenofibrate can activate PPAR- Ischemia/reperfusion (I/R) injury is a major concern during surgical procedures such as liver resection, trauma, and transplantation, which can lead to liver injury or even failure for its inevitable interruption and subsequent restoration of circulation , 2. Hepaα (PPARα) agonist, the nuclear receptor superfamily member, which was discovered in 1990 [β-oxidation of fatty acids, inflammation, oxidative stress, and even tumorigenesis and cancer progression [Fenofibrate belongs to the group of fibrate drugs, which are generally used in the treatment of dyslipidemia and combined hyperlipidemia patients . Also, f in 1990 . As an agression . Severalgression –14. But α, and they together participate in mechanisms like inflammation, apoptosis, autophagy, and oxidative stress that are involved in IR. There are reasons to believe fenofibrate may function through activating the AMPK/PPAR-α pathway.AMPK, adenosine monophosphate–activated protein kinase, a key sensor of cellular nutrient supply and energy status, plays crucial roles in regulating cellular growth and metabolism and is related to processes such as autophagy in eukaryotes . It was α- and AMPK-dependent manner.Herein, we evaluated the value of fenofibrate in hepatocellular protection during hepatic ischemia/reperfusion injury. The present study investigated whether and how fenofibrate administration would affect liver functions and its underlying mechanisms in a well-established murine IR model based on our previous research . We hypoα, IL-6, Bax, Bcl-2, Beclin-1, P62, LC3, Casepase-9, Caspase-3, and Nrf-2 were provided by Proteintech . The TNF-α and IL-1β antibodies were from Abcam . The AMPK and p-AMPK antibodies were from CST . The TdT-mediated dUTP nick end labeling (TUNEL) apoptosis assay kit was from Roche .Fenofibrate was purchased from Kingmorn Life Science  and suspended with 10% DMSO. ALT and AST regent kits were obtained from Jiancheng Bioengineering Institute . The enzyme-linked immunosorbent assay (ELISA) kits were acquired from eBioscience . The RNA polymerase chain reaction (PCR) kit was from Takara Biotechnology . The antibodies for PPAR-Male Balb/c mice weighing 23 ± 2 g and aged 6–8 weeks old were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. . The mice were group-housed in standard plastic cages at ambient temperature (23 ± 1°C) and 55% humidity with a 12 h light-dark cycle, having access to food and water ad libitum. All animal experiments were carried out in accordance with the National Institutes of Health Guidelines and with approval of the Animal Care and Use Committee of Shanghai Tongji University.n = 6); (b) sham group (n = 18); (c) I/R without any pretreatment (n = 18); (d) I/R with pretreatment of FF (50 mg/kg); and (e) I/R with pretreatment of FF (100 mg/kg). Fenofibrate was administered by intravenous route 1 h before surgery. A total of six mice were selected to sacrifice from each group  at 2, 8, and 24 hours after reperfusion to obtain blood and tissue samples.78 mice were randomly assigned to one of five groups as follows: (a) normal control group (Blood samples were collected and then placed at 4°C for 5 hours. Serum was separated by centrifugation of the blood at 4600 × g (4°C) for 10 minutes. Serum was collected, aliquoted, and stored at −80°C until the biochemical analysis. A portion of the median and left liver lobes was quickly isolated and preserved in 4% paraformaldehyde solution for at least 24 hours at 4°C for histopathological assessment while the remaining liver tissue was collected, snap frozen with liquid nitrogen, and stored at −80°C for the subsequent experiments.A model of segmental (70%) hepatic warm IR was used, as previously described . Food waThe hepatoduodenal ligament was dissected, and a microvascular atraumatic clamp was placed on the portal pedicle to the median and left lobe of the liver for partial hepatic ischemia. After 45 minutes, the clamp was removed to initiate liver reperfusion and the wound was sewn with 4–0 silk. Sham groups were subjected to the same procedure but without vascular occlusion. A constant warm body temperature was needed to be maintained during the procedure.β and TNF-α was determined using ELISA kits, following the manufacturer's instructions.Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured with commercially available colorimetric assay kits as described by the manufacturers of Jiancheng Bioengineering Institute. The amount of IL-1μm thick sections and stained with hematoxylin and eosin (H&E). Inflammation and tissue damage were confirmed with a light microscope.The prepared liver specimens were dehydrated with ethanol and embedded in paraffin. Samples were sliced into 5 μm polyvinylidene fluoride membranes. Nonspecific binding sites were blocked with PBS containing 5% nonfat milk for at least 1 h at room temperature, and the membranes were incubated overnight at 4°C with the following primary antibodies and dilutions: β-actin , TNF-α , IL-1β , IL-6 , LC3 (1 : 500), Beclin-1 (1 : 500), Bcl-2 , Bax , caspase3 , caspase9 , P62 , PPAR-α (1 : 500), AMPK , p-AMPK , and Nrf-2 . The next day, membranes were washed three times for 10 min each using PBST (PBS containing 0.1% Tween 20) and then incubated with secondary antibodies at 1 : 2000 for 1 h at room temperature protected from light. Membranes were then washed 3 times with PBST and scanned with the Odyssey two-color infrared laser imaging system . The gray values were quantified using ImageJ analysis software.Total protein of liver tissue was extracted from liver tissues stored in liquid nitrogen with radioimmunoprecipitation assay (RIPA) lysis buffer mixed with protease inhibitors (PI) and phenylmethyl-sulfonyl fluoride (PMSF). Protein concentration was quantified using the bicinchoninic acid protein assay kit . Equal amounts of total protein were separated on 7.5–12.5% SDS-polyacrylamide gels and then transferred onto 0.22 α, anti-IL-1β, anti-Bcl-2, anti-Bax, anti-Beclin-1, anti-PPAR-α, and anti-Nrf-2 , anti-LC3, and p-AMPK (1 : 100), followed by incubation in secondary antibody (1: 50) for 1 h at 37°C. A diaminobenzidine (DAB) kit was used to analyze antibody binding under a light microscope. The stained area was measured by using Image-Pro Plus software (version 6.0).Paraffin-embedded liver sections were dewaxed in xylene and dehydrated with gradient alcohol. Antigen retrieval was performed by citrate buffer and incubated in a 95°C water bath for 20 min. To block endogenous peroxidases, the sections were incubated with 3% hydrogen peroxide for 10 min at room temperature. Sections were washed with PBS three times and then treated with 5% bovine serum albumin (BSA) for 20 min to block nonspecific proteins. Next, the liver specimens were incubated overnight at 4°C with the following primary antibodies and dilutions: anti-TNF-β-actin.Total RNA was extracted from stored frozen liver specimens using TRIzol reagent . RNA was reverse-transcribed into cDNA with a Reverse-Transcription Kit . Gene expression was measured using SYBR Premix Ex Taq , and cDNA was quantified with the 7900HT Fast Real-Time PCR System . Oligonucleotide primer sequences are listed in μm sections were then digested with 20 μg/ml proteinase K for 30 min. After washing 4 times with PBS, the sections were added with TUNEL reaction mixture. Finally, the positive areas were observed by the light microscope.The hepatocyte apoptosis was determined by TUNEL assay. Followed by dewaxing and rehydrating, the prepared 5 t-test and one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. P < 0.05 was considered statistically significant. Statistical analyses were performed, and the graphic charts were plotted by GraphPad Prism 6 software.Experimental data were presented as the mean ± standard deviation (SD), and experiments were repeated at least three times. Statistical differences between groups were analyzed by Student's The levels of biomarkers of hepatic function, ALT and AST, in serum were significantly elevated in the IR group at 3 time points . This inα, IL-6, and IL-1β, the proinflammatory cytokines, were detected to explore the effect of FF on inflammation in terms of serology, protein levels, and gene transcription. As shown by ELISA results, the expression of TNF-α and IL-1β in serum was significantly higher than that in the sham group and peaked at 8 hours after reperfusion  performed obviously  showed a clear difference between the IR group and IR+FF  groups, which may indicate that fenofibrate could activate the phosphorylation of AMPK. These conclusions could be confirmed by PCR, western blotting, and IHC Figures . As it iHepatic IR injury is a clinically relevant processes that occurs in liver resection, trauma, and transplantation, which is the paradoxical damage increasing upon reperfusion of ischemic organs . Hence, For the above purpose, we used a well-established Balb/c mice model of hepatic IR injury. Ischemia-reperfusion process resulted in elevated serum ALT and AST levels, both of which are indicators of early acute hepatic damage, while fenofibrate pretreatment decreased liver enzyme activities, and this protective effect was dose-related. These results were further validated by the findings of pathological changes. The necrotic area and massive inflammatory cell infiltration indicated that FF could reduce the severity of liver injury caused by hepatic IR.β, and TNF-α [α, IL-6, and IL-1β. And the favorable anti-inflammatory profiles are potentiated in the high-dose group (100 mg/kg).The physiological and pathophysiological processes involved in hepatic IR injury are complicated. During the initial phase, the oxidative phosphorylation levels of hepatocytes decrease due to oxygen deficiency, thus affecting the generation of adenosine triphosphate (ATP). The levels of mitochondrial reactive oxygen species (ROS) production in hepatocytes are out of control under abnormal circumstances such as ATP depletion . Kupffernd TNF-α . Neutropnd TNF-α . Besidesnd TNF-α . In the α plays a pivotal role in various signaling pathways according to previous studies, which could result in extrinsic hepatocyte apoptosis in HIR injury [The proinflammatory cytokine TNF-R injury . MoreoveR injury . The proR injury . Bcl-2, R injury . In our There are profound interactions between autophagy and apoptosis: they can mutually reinforce and inhibit in many physical activities , 30. Bclα is a key transcription factor that mediates the nucleus–mitochondrial interactions to regulate inflammation, lipid metabolism, and mitochondrial functions in various tissues and cells, and it has become a main target in NFALD [α activation increases the expression of sirtuin-1 (SIRT1), which inhibits NF-κB, depending on the AMPK pathway and thus reducing inflammation [α and IFN-γ, but the levels had increased in PPARα knockout mice [α activation could decrease IR-induced liver, heart, and brain injury by suppressing inflammation, apoptosis, and lipid peroxidation [α can promote the expression of Bcl-2 and subsequently inactivate apoptosis [α can also regulate hepatocyte autophagy. Evidence showed that PPAR-α activation attenuated the immune response and protect liver from acute failure through autophagic activation [α, can regulate the expression of PPARα to affect autophagy [α to assess whether it also took part in IR injury and found that PPAR-α expression was upregulated in fenofibrate preconditioning groups.Next, we need to explore the mechanisms of fenofibrate on how to attenuate the injury of IR. PPAR-in NFALD , 37. PPAammation , 39. Deaout mice . It was xidation , 42. Furpoptosis . An expepoptosis . PPAR-α tivation . A studyutophagy . HoweverκB levels [AMPK is a serine/threonine kinase that can be activated by ATP depletion, antioxidant interference, and ROS production . In hepaB levels . In addiB levels . As mentB levels . Nrf2, wB levels . AccumulB levels , 59. We α, and PPAR-α, helps modulate hepatic lipid metabolism for the improvement of liver injury [α [α pathway [α [α expression by promoting PPAR-α transcriptional activity, while treating with compound C, an AMPK inhibitor, could reverse the expression of PPAR-α, which illustrated that PPAR-α activation was in an AMPK-dependent way [α was increased. In the meantime, the expression of apoptosis- and autophagy-related proteins was consistent with our idea. We concluded that fenofibrate protected the liver from damage by activating the AMPK/PPAR-α pathway and accordingly inhibited apoptosis and autophagy.It has been reported that AMPK, the activation of which is a trigger to downstream mediators such as SIRT-1, PGC-1r injury . It is wnjury [α . AdipoRo pathway . Liu et thway [α . Furtherdent way , 61, 63.α via the phosphorylation of AMPK, which could alleviate apoptosis and autophagy in hepatic IR Thus, our study showed that fenofibrate can activate PPAR-α pathway.Taken together, fenofibrate alleviated hepatic ischemia/reperfusion injury in mice effectively. Fenofibrate reduced the release of inflammatory cytokines and inhibited hepatocyte apoptosis, autophagy, and ROS. And this protective effect is partly based on the activation of the AMPK/PPAR-


Pseudocerastes and Eristicophis (Viperidae: Viperinae) are known as the desert vipers due to their association with the arid environments of the Middle East. These species have received limited research attention and little is known about their venom or ecology. In this study, a comprehensive analysis of desert viper venoms was conducted by visualising the venom proteomes via gel electrophoresis and assessing the crude venoms for their cytotoxic, haemotoxic, and neurotoxic properties. Plasmas sourced from human, toad, and chicken were used as models to assess possible prey-linked venom activity. The venoms demonstrated substantial divergence in composition and bioactivity across all experiments. Pseudocerastes urarachnoides venom activated human coagulation factors X and prothrombin and demonstrated potent procoagulant activity in human, toad, and chicken plasmas, in stark contrast to the potent neurotoxic venom of P. fieldi. The venom of E. macmahonii also induced coagulation, though this did not appear to be via the activation of factor X or prothrombin. The coagulant properties of P. fieldi and P. persicus venoms varied among plasmas, demonstrating strong anticoagulant activity in the amphibian and human plasmas but no significant effect in that of bird. This is conjectured to reflect prey-specific toxin activity, though further ecological studies are required to confirm any dietary associations. This study reinforces the notion that phylogenetic relatedness of snakes cannot readily predict venom protein composition or function. The significant venom variation between these species raises serious concerns regarding antivenom paraspecificity. Future assessment of antivenom is crucial.Snakes of the genera  Pseudocerastes , the spider-tailed viper Pseudocerastes urarachnoides (Viperidae: Viperinae)  from western Iran . getation ,24, and getation ,10,24,25chnoides ,15. The E. macmahonii venom possesses procoagulant properties  B; mid-cu1450.00] C), thougpersicus 9 ± 3.7 sP. urarachnoides venom was very strong, with clotting times comparable to those achieved by Sri Lankan Russell’s viper venom (Daboia russelii) (13.5 s)—a species infamous for its rapid procoagulant activity  also retained its procoagulant activity with decreasing venom concentrations. At its mid-curve clotting point, 0.4 ± 0.0 µg/mL venom induced a clot in 26 ± 0.5 s—a doubling in clot time despite a 50-fold decrease in venom concentration. While there is virtually no data available regarding snakebite envenoming by P. urarachnoides, these findings indicate that a bite by this species is likely to result in clinically relevant coagulopathy.In contrast, the procoagulant activity of vity see . The extE. macmahonii venom possessed procoagulant activity, it was significantly weaker than that of P. urarachnoides (Tukey’s: p < 0.0001). Furthermore, the venom’s procoagulant toxins appeared to have a relatively low affinity for their plasma targets as clotting times declined quickly with decreasing venom concentrations . For example, despite that E. macmahonii venom exhibited a significantly faster maximal clotting speed compared to that of P. persicus (unpaired t-test: t (4) = 20.79, p < 0.00001), there was no significant difference between their mid-curve clot times  = 1.273, p = 0.2720). Accordingly, the potency of E. macmahonii venom  was lower than that of P. persicus , though this difference was not significant (unpaired t-test: t (4) = 2.069, p = 0.1074). Overall, the procoagulant activity of E. macmahonii venom could best be described as moderate. These results also suggest that the posited low levels of procoagulant toxins within P. persicus venom may have a high affinity for plasma components. Such coagulant activity could therefore be an important consideration surrounding P. persicus envenoming as it may contribute to coagulopathy, even at low venom doses.While Pseudocerastes urarachnoides venom readily cleaved both prothrombin and FX . Baseline parameters were established by measuring the spontaneous clotting of plasmas without venom. To assess influence of the venoms on clot dynamics, data were normalised to these controls (spontaneous control = 0%) and potential maxima (100%). Three parameters are presented: “split point,” the time interval of clot initiation (minutes); “angle,” the rate of clot formation (degrees); and “maximum amplitude,” the elasticity (and thus strength) of the resultant clot (millimetres). Statistical analyses compared venom-induced clotting values with the spontaneous controls, unless otherwise stated.Factor X is highly conserved between the taxa used as models, and this was reflected by human FXa activating clotting in each plasma sample .Human thrombin, however, was unable to clot toad plasma see , which iE. macmahonii did not induce coagulation in toad plasma. Post hoc testing indicated that the venom had a weak to moderate anticoagulant effect, slowing down clot initiation speed by only 13 ± 2.6% and slowing clot formation rate by 65 ± 13.7%. Clot strength was unaffected (unpaired t-test: t (4), p = 0.5823). The venom did, however, demonstrate rapid procoagulant activity in both chicken and human plasmas . The venom’s effect on chicken plasma was slightly greater than on human (Unpaired t-test: t (6) = 2.471, p = 0.0484). While the venom elevated the rate of clot formation in both plasmas, these increases were not significant  = 1.850, p = 0.1138. Human: 11 ± 21.3%; unpaired t-test: t (6) = 0.5963, p = 0.5728). The venom’s effects on clot strength were minimal  = 2.954, p = 0.0255. Human: −13 ± 11.6%; unpaired t-test: t (4) = 1.473, p = 0.2147).The venom of E. macmahonii venom in toad plasma suggests that, overall, the venom is not specialised for acting on toad physiology, at least in the case of coagulant activity. Despite limited information on the feeding ecology of E. macmahonii and a small number of amphibian species being sympatric with this viper, niche partitioning may make it unlikely that toads form an important dietary item, as amphibians are rare in the shifting, sandy dunes it occupies. Eristicophis macmahonii is said to occasionally consume rodents [E. macmahonii—a generalist that includes large proportions of ectotherms such as lizards and arthropods—is predominantly haemotoxic. In contrast, that of P. fieldi—a generalist that appears to prefer birds and rodents—is dominated by neurotoxins. Such “ectothermic/endothermic prey” inferences are likely a major over-simplification and would benefit from greater incorporation of individual species’ ecologies.The lack of strong activity by the  rodents ,8,18,34, rodents ,36,37. I rodents ,8,18,34. rodents ,39, thesPseudocerastes urarachnoides venom possessed rapid, taxonomically “broad-spectrum” procoagulant activity, significantly increasing both clot initiation speed and clot formation rate in each plasma tested  or greater than (P. fieldi) the spontaneous clotting control in both formation rate and strength. Thus, although clot formation was slightly delayed, clotting function of chicken plasma was fundamentally uncompromised by these two venoms. Given that these species are known to consume a high proportion of avian prey [2s that dominate P. fieldi’s venom. Notably, the venoms completely inhibited clotting of toad plasma  the substantial divergences in the bioactivities of these species’ venoms, despite that all members of the clade superficially share many similarities in their ecology; and ii) the lack of phylogenetic patterns associated with these activities, despite that there is typically similarity between the venom profiles of closely related snakes.P. fieldi is paired with a seemingly specialised proteome and venom activity , while the dietary generalism of P. persicus and E. macmahonii is paired with diverse venom biochemistry. In contrast, the diverse venom proteome of P. urarachnoides is paired with a specialised diet and venom activity . These findings superficially deviate from suppositions that indicate dietary generalism favours toxinological diversity [P. persicus venom shares greater proteomic and functional similarity to P. fieldi than to P. urarachnoides with whom it is sister. Furthermore, the venom proteomes of P. urarachnoides and E. macmahonii, which diverged around 16–18 Mya, most closely resemble that of typical vipers and thus represent the posited plesiotypic condition of the clade [P. urarachnoides and P. fieldi venoms present an apotypic state—P. fieldi more so if both venom function and proteome are considered. However, P. urarachnoides arguably presents the most derived predatory characters, should venom function be considered in parallel with morphological traits.Overall, this clade of closely related vipers presents an unusual example of venom variation. The dietary generalism of iversity  and evadhe clade ; yet, fu2s and procoagulant toxins within the clade, the common ancestor of this group may have possessed a venom most resembling that of E. macmahonii. Unfortunately, the lack of consistent patterns between venom proteome and bioactivity in relation to these species’ phylogenetic relationships or feeding ecologies renders any trends of venom evolution obscure, begging the question as to what may be influencing their divergence. Venom variation is commonly linked with populational or age-driven differences in prey availability or endothermic/ectothermic prey groupings [Given the conjectured shared (though highly variable) expression of neurotoxic PLAroupings ,39,44. Hroupings ,45. The roupings . These fP. persicus—the venom of which possesses the weakest activity of these species (according to human-oriented in vitro models). Future assessment of antivenom cross-reactivity is therefore crucial.Regardless of evolutionary drivers, this study reinforces the notion that phylogenetic relatedness of snakes cannot readily predict venom protein composition or activity, even where overlaps in ecology are evident. This considerable venom variation raises serious concerns regarding the paraspecificity of the Razi polyvalent antivenom, which is specific for Eristicophis macmahonii venom was collected from 3 adult male snakes from the Nushki district (30.12° N 67.01° E) of Balochistan, Pakistan by SAA. Pseudocerastes fieldi and P. persicus venoms were purchased from Latoxan. Pseudocerastes urarachnoides were collected under the approval of NIMAD # 942485 by PG and BF. Venoms were immediately flash frozen in liquid nitrogen and were subsequently lyophilised and stored at −80 °C until use. Samples were pooled to account for any potential variation in toxin expression between individuals. The lyophilised venoms were reconstituted to a working stock concentration of 1 mg/mL in 50% deionised H2O (Milli-Q) and 50% glycerol (Sigma-Aldrich) to prevent freezing and thus reduce enzyme degradation associated with freeze-thaw cycles. Working stock aliquots were stored at −20 °C and used for all subsequent analyses.All venom work was undertaken under the University of Queensland IBSC approval #IBC134BSBS2015. Human plasma was sourced from the Australian Red Cross  under human ethics approval number 2016000256. Two batches of pooled human plasma  were further combined and aliquoted. The aliquots were flash frozen in liquid nitrogen and stored at −80 °C. Immediately prior to use for experimentation, aliquots were defrosted by immersing in a water bath for 5 min at 37 °C. Clotting time parameters were established for the plasma by measuring clot formation (in triplicate) in the presence and absence of an activator: 46–48 s (s) and 300–500 s, respectively . To check for plasma degradation and ensure the clotting parameters of the plasma aliquots were consistent over time, positive and negative controls were conducted prior to each experimental assay; namely, clotting time was measured in the presence and absence of an activator and compared to pre-established parameters. Chicken and toad bloods were extracted from anaesthetised animals by a trained veterinarian under the University of Queensland Animal Ethics approval SBS/019/14/ARC. Blood was citrated (3.2%) and spun at 15,000 relative centrifugal force (RCF) for 15 min, supernatant (plasma) extracted and aliquoted. The aliquots were flash frozen in liquid nitrogen and stored at −80 °C until use. Immediately prior to use for experimentation, aliquots were defrosted by immersing in a water bath for 5 min at 37 °C.2O (Milli-Q).One mm 12% SDS-PAGE gels were prepared using the following recipe for resolving gel layer: 3.3 mL deionised H2O, 2.5 mL 1.5 M Tris-HCl buffer pH 8.8 , 100 µL 10% SDS , 4 mL 30% acrylamide mix , 100 µL 10% APS , 4 µL TEMED ; and stacking gel layer: 1.4 mL deionised H2O, 250 µL 0.5 M Tris-HCl buffer pH 6.8, 20 μL 10% SDS , 330 mL 30% acrylamide mix , 20 μL 10% APS , 2 µL TEMED . 10× gel running buffer was prepared using the following recipe: 250 mM Tris , 1.92 M glycine , 1% SDS , pH 8.3. For the 1D gels, venom samples were prepared with and without 5% 2-mercaptoethanol , and were boiled at 100 °C for 5 min to denature proteins. Twenty µg venom samples were loaded into the gels and were run in 1× gel running buffer at room temperature for 20 min at 90 V  and then 120 V until the dye front approached the bottom of the gel. Gels were then stained with colloidal coomassie brilliant blue G250 , 3% orthophosphoric acid , 170 g/L ammonium sulphate , 1 g/L coomassie blue G250 , and de-stained in deionised HFor 2D gels, 300 µg venom samples were loaded onto immobilised pH gradient (IPG) strips  and incubated overnight at room temperature. Isoelectric focussing was conducted using a PROTEAN i12 IEF CELL (Bio-Rad Lab) under the following conditions: 50 µA current; 100 V for 1 h, 500 V for 1 h, 1000 V for 1 h, and 8000 V until 98,400 V/h. To reduce and alkylate the samples, IPG strips were incubated in a reducing equilibration buffer  for 10 min, followed by further incubated for 20 min in an alkylating equilibration buffer . The strips were then rinsed with SDS-PAGE running buffer and positioned on top of 12% polyacrylamide gels using 0.5% agarose. Gels were run at 4 °C with a current of 10 mA/per gel for 20 min followed by 20 mA/per gel for the rest of the run until the bromophenol dye front was within 0.5 cm of the base of the gel. After the run, gels were briefly washed with water and stained with 0.2% colloidal Coomassie brilliant blue G250 overnight.Visible spots were picked from the gel, washed with 100 µL 50 mM ABC twice and destained with 50 mM ABC/50% acetonitrile (ACN), and then incubated with 100% ACN until opaque. Twenty μL of trypsin solution (20 μg trypsin in 100 μL 1 mM HCl and 900 µL of 40 mM ammonium bicarbonate (ABC)) was diluted in 380 μL 40 mM ABC, then 30 µL was added to gel spots and incubated overnight at 37 °C. Subsequently, spots were removed from solution and peptides eluted by adding 1% trifluoroacetic acid (TFA), incubating for 20 min, transferring solution and then repeating with 5% ACN/1% TFA. Solutions were centrifuged at 16,000 RCF for 10 min and supernatant extracted. Solutions were then ziptipped by wetting tip  with 100% ACN, equilibrating with 5% ACN/0.1% TFA, loading sample, washing tip with 5% ACN/0.1% TFA, eluting sample with 60% ACN/0.1% TFA, dehydrating sample and then and resuspending in 0.1% formic acid.Peptides extracted from in-gel tryptic digestion were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Agilent Zorbax stable bond C18 column  on a Shimadzu Nexera UHPLC system  coupled to an AB SCIEX 5600 Triple TOF mass spectrometer . The chromatographic elution was achieved at a flow rate of 400 µL/min via a gradient of 1 to 40% solvent  over 4 min. The MS/MS spectra were acquired at a rate of 20 scans per second with a cycle time of 2.3 s, and the resultant 20 most intense ions were obtained and searched against UniProt databanks using Protein Pilot v4.0 software .2-95% humidified environment. A minimum of three replicates were conducted per treatment. Cell death was visualised via light microscopy using Zeiss Axiovert 200 with a Plan- Apochromat 63 × 1.4 numerical aperture after 24h of treatment with Pseudocerastes or Eristicophis venoms at 10 µg/mL.Pancreatic tumour (PaTu-T) cells, melanoma patient derived cells (MM96L), and human neonatal foreskin fibroblasts (NFF) were selected as representatives of both healthy and unhealthy cells from different organs to account for any variation in cell types. Cells were maintained in RPMI medium supplemented with 1% penicillin-streptomycin and 10% foetal bovine serum (FBS). Cells were trypsinised (0.25% trypsin) and seeded 24 h prior to the experiments in 96-well flat bottom plate at a density of 5000 and 3000 cells/well for NFF and MM96L cells, respectively, and at 80% confluence for PaTu-T cells. Plates were incubated at 37 °C in a 5% COGallus gallus) were euthanized with CO2 between 4–10 days after hatching . Replicates were run using tissues of different ages to account for differences in effects related to tissue development. Chick biventer cervicis nerve-muscle were dissected and preparations were mounted under 1 g tension in 5 mL organ baths containing physiological salt solution  at 34 °C and bubbled with carbogen . Electrodes were placed around the tendon of the biventer muscle. Electrical stimulation of the motor nerve  using a Grass S88 stimulator  evoked indirect twitches. Selective stimulation of the nerve was confirmed by the abolition of twitches with d-tubocurarine (10 µM), a nicotinic acetylcholine receptor (nAChR) competitive antagonist. Tissues were then washed repeatedly with physiological salt solution to restore twitch responses to nerve stimulation. The stimulation was ceased, and the contractile responses to acetylcholine (1 mM ACh for 30 s), carbachol (20 µM CCh for 60 s), and potassium chloride  were obtained and recorded. The organ bath was then washed, and electrical stimulation was resumed and maintained for 30 min to allow the preparation to equilibrate. Venom (10 µg/mL) was added to the organ bath and the twitch height was recorded until twitch response was abolished. If twitch response did not cease, measurements were stopped at 60 min. The stimulator was turned off again and the bath was washed. Contractile responses to ACh, CCh, and KCl were obtained again to compare with responses prior to venom addition. The twitch responses to electrical stimulation and contractile responses to agonists  were measured using a Grass FT03 force displacement transducer  and recorded on a PowerLab system . At least four replicates were run for each venom.Male chicks . Venom working stock was manually diluted to 0.1 mg/mL in Owren Koller (OK) Buffer (Stago Cat# 00360) and loaded into the analyser. All subsequent dilutions, steps, and measurements were automated. Venom-induced clotting times were measured over a 9-point dilution series . Assay conditions were as follows: 50 µL venom  was incubated with 50 μL of CaCl2 , 50 µL phospholipid , and 25 μL of OK Buffer for 120 s at 37 °C. 75 µL of human plasma was then added and time until clot formation measured by the analyser using a viscosity-based detection system. Each dilution series was repeated in triplicate. To measure toxin co-factor dependency, the above assay was repeated in the presence and absence of calcium and/or phospholipid. Conditions were identical except for 50 µL OK buffer being used in place of the absent co-factor in order to maintain a consistent final volume (250 μL). Plasma negative controls were conducted under identical conditions except for 50 μL of blank solution  substituting the venom. Positive controls were conducted by incubating 50 μL Kaolin  with 50 µL plasma for 120 s at 37 °C. 50 μL CaCl2  was then added and time until clot formation measured.Coagulant activity was assessed by incubating the venoms with human plasma and measuring time until clot formation. The coagulation analyses were performed using a Stago STA-R Maxwww.graphpad.com).To measure the activation of the coagulation factor Factor X (FX) by the venom, 10 µL venom  was plated in at least triplicate on a 384 well plate along with 10 µL FX  (0.1 µg dry weight) and 10 µL phospholipid  Buffer). To account for direct substrate cleavage by the venom, 10 µL venom  was plated in triplicate on a 384-well plate along with 10 µL phospholipid and 10 µL buffer  to substitute the FX for later subtraction. Ten µL (0.1 µg dry weight) of FXa  in at least triplicate was used as a positive control. Negative control consisted of 20 µL dilution buffer and 10 µL phospholipid. Activity was measured by adding 70 µL quenched Fluorogenic Peptide Substrate Cat#ES002  . Fluorescence was monitored (excitation at 390 nm and emission at 460 nm) at 37 °C over 400 min or until activity ceased. To measure prothrombin activation, test conditions were identical except prothrombin  was used in place of FX, thrombin  in place of FXa, and Fluorogenic Peptide Substrate Cat#ES011 (measured at 390 nm/460 nm) in place of substrate Cat#ES002. Data were normalised against no reaction (0%) and the positive control (100%) using GraphPad Prism version 8.0.0 for iOS . This machinery consists of a stationary pin suspended by a torsion wire into the cup. The cup oscillates, and as the clot develops the changes in the viscoelasticity of the fluid are detected by the pin and wire, which act as a torque transducer and generates a “trace” output. For the experiments, 189 µL of human, chicken, or toad plasma was combined with 72 μL CaCl2 (25mM stock solution Stago Cat# 00367 STA), 72 μL phospholipid  buffer), and 20 μL OK buffer in an assay cup . In the final step, 7 µL venom (1 mg/mL in 1:1 glycerol:deionised H2O) was added, pipette mixed, and then the machine was immediately started. Tests were run at 37 °C and measured for 30 min. Negative and positive controls followed identical methods, except 7 µL of 1:1 glycerol:deionised H2O (negative control) and 7 µL thrombin, FXa , and/or kaolin  (positive controls) replaced the 7 µL venom in the final step. As these plasmas had low levels of platelets, values attained in these assays are not comparative to in vivo coagulation. For example, clot strength is compromised by the lack of fibrin-binding platelets, which makes assessment of the venoms’ influence on such parameters difficult. However, blood factors that are upstream in the coagulation cascade, such as FXII, are known to spontaneously convert into their active form. This initiates the coagulation cascade despite the absence of an activator, and the resulting “spontaneous” clot is structured according to the native proportions of blood factors in the platelet-poor plasma used in in vitro assays. The venom data were therefore normalised against the spontaneous clotting control data  to assess the venoms’ influence on in vitro coagulation cascade kinetics.The effects of venoms on clot dynamics of plasma were measured using a Thromboelastographwww.graphpad.com). Venom neurotoxicity data were normalised against the controls and tested for normality using Shapiro-Wilk, Kolmogorov Smirnov, and Q-Q plots. Contractile heights at 60 min were compared via one-way ANOVA with Tukey’s post hoc and paired t-tests (control vs. venom) using Prism default settings with 95% confidence interval. To analyse the data obtained in the Stago coagulation analyses, dose-response plasma coagulation curves were normalised against maximal response of the venom’s clotting time (100%) and blank plasma control spontaneous clotting time (0%). EC50s were then calculated using the non-linear regression (log)dose vs. response model—variable slope with Shapiro–Wilk, Kolmogorov Smirnov, and Q-Q plot diagnostic tests for normality and confidence intervals set to 95%. Clotting times were compared using one-way ANOVA  with Tukey’s post hoc analysis or unpaired T-tests using Prism default settings with a 99% confidence interval. Thromboelastography data were tested for normality using Shapiro-Wilk, Kolmogorov Smirnov, and Q-Q plot diagnostic tests. The data were normalised against the spontaneous clotting control data for each plasma . As positive controls (thrombin and FXa) were of human origin, they could not be used to obtain maximal values of clotting parameters against which to normalise venom-induced clot data. “Potential maxima” values for clot maximum amplitude and angle for chicken and toad plasma were therefore obtained and estimated from previous studies that characterised the clotting parameters of alike animal plasmas under comparable methodologies [Data were graphed and analysed using GraphPad Prism version 8.0.0 for iOS (GraphPad Software, San Diego, California, USA, dologies ,48,49. V


Crithmum maritimum, significantly inhibited cell growth in HCC cells. By 1H-NMR spectroscopy, here we show that Crithmum maritimum ethyl acetate extract counteracts the Warburg effect, by reducing intracellular lactate, inhibits protein anabolism, by decreasing amino acid level, and affects membrane biosynthesis by lowering choline and phosphocholine. Also, we observed an effect on lipid homeostasis, with a reduction in triglycerides, cholesterol, monounsaturated fatty acids (MUFA), and diunsaturated fatty acids (DUFA), and an increase in polyunsaturated fatty acids (PUFA). Taken together, these data demonstrate that Crithmum maritimum-induced cytostasis is exerted through a multi-effect action, targeting key metabolic processes in HCC cells. Overall, our findings highlight the role of Crithmum maritimum as a promising tool for the prevention and the improvement of the therapeutic options for HCC and other types of tumours.Hepatocellular carcinoma (HCC) is nowadays the sixth cause of tumour-related deceases worldwide, estimated to become the third in Western countries by 2030. New drugs for HCC treatment still have many adverse effects. Several lines of evidence indicate that plant metabolites offer concrete opportunities for developing new therapeutic strategies for many diseases, including cancer. We previously reported that ethyl acetate extract of a spontaneous edible plant harvested in Apulia,  This trend finds the basis in the increased prevalence of metabolic diseases, such as metabolic syndrome (MetS), diabetes, obesity, and non-alcoholic fatty liver disease (NAFLD)2. The therapy of HCC is mainly based upon surgery and pharmacology employing tyrosine-kinase inhibitors, such as sorafenib, regorafenib, and levantinib2, often in combination with immunotherapeutic agents, for instance, pembrolizumab and nivolumab3. Such pharmacological approaches result in many unfavourable consequences and are not tolerated by patients in the long range5. Thus, innovative and effective therapeutic strategies, with fewer side effects, are extremely needed for HCC.Hepatocellular carcinoma (HCC) is currently the sixth principal cause of tumour-related deceases worldwide and it is expected to grow into the third one in the Western world by 2030, regardless of the reduced incidence of chronic hepatitis infections7, including cancer8. We previously demonstrated that ethyl acetate extract of a spontaneous edible plant harvested in Apulia, Crithmum maritimum L., exerted a significant inhibitory effect on cell growth in HCC cells by affecting cell cycle regulation and apoptosis9. Metabolomics offers a trustable and effective tool to investigate the behaviour of biological systems by analysing their metabolic fingerprint10. Many applications have been described so far in the characterisation of the metabolic profile of plants12, as well as in the assessment of changes of the metabolic signature after a selected treatment, such as plant-derived medicines and conventional drugs13, or plant-derived metabolites14. Here, we aimed at further characterising the effect of Crithmum maritimum in HCC cells by using a metabolomic approach. We, therefore, characterised the effect of the ethyl acetate extract of Crithmum maritimum on the metabolic profile in two HCC cell lines, Huh7 and HepG2, by analysing, with 1NMR-spectroscopy, cell pellets (aqueous and lipid fractions) and cell culture media. Moreover, we analysed the whole plant powder and the four extracts to determine their metabolite composition. Our results show that ethyl acetate extract of Crithmum maritimum substantially changes the metabolic profile in both HCC cell lines. In particular, in the aqueous fraction, we observed a decrease in intracellular lactate, many amino acids, choline, and phosphocholine. Interestingly, the decrease of intracellular lactate in the aqueous fraction was paralleled by the increase in cell culture media, where an increase of glucose was also measured. Notably, we also found that ethyl acetate extract of Crithmum maritimum modulates lipid homeostasis, by decreasing triglycerides, cholesterol, and monounsaturated fatty acids (MUFA) and di-unsaturated fatty acids (DUFA), and increasing polyunsaturated fatty acids (PUFA). Moreover, the metabolite composition of the whole plant powder and the ethyl acetate extract offers interesting insights for a better understanding of the broad effect shown by Crithmum maritimum. Overall, our findings show for the first time the multitarget action of Crithmum maritimum extract in inhibiting HCC cell growth as well as the reversion of the pathologic metabolic profile of HCC cells towards normal patterns.Plant-derived products and extracts are gaining consideration for therapeutic purposes, and noteworthy effects have been described concerning many pathologiesCrithmum maritimum lyophilized powder, as described in the Materials and Methods section. The workflow from plant lyophilized powder preparation to 1H-NMR analysis is summarised in Fig. 1H-NMR experiments on cell extracts and cell culture media. To corroborate the effect of Crithmum maritimum in other tumour histotypes, we carried out the same experiment in a non-HCC cell line, namely HeLa, and observed a similar antiproliferative effect  and 1.01 (CH3-25) were assigned to β-sitosterol, as also confirmed by the carbons at 12.2 ppm and 19.1 ppm, respectively. The 1H resonance at 0.70 ppm was identified as stigmasterol17, while the smaller signal observed at 0.55 ppm was assigned to a sterol molecule. Crithmum maritimum and halophytes in general are known to be important sources of phytosterols18.Then, we characterised by tes Fig. . These ites Fig. . Characttes Fig. a. The prH3) of all fatty acid chains were observed at 0.88 ppm. A more de-shielded signal at 0.98 ppm was assigned to ω3-fatty acids methyl groups. Broad signals in the range 1.26–1.39, 1.59–1.67, and 2.25–2.41 ppm were detected and identified as characteristic for methylene (n-CH2), β-methylene, and α-methylene (-CH2COO-) protons adjacent to the carbonyl group of all fatty acids. The unsaturated fatty acids were identified by the diagnostic resonances of allylic (–CH2CH=CH–) and olefinic (–CH=CH–) protons at 2–2.10 and 5.29–5.45 ppm respectively. The presence of polyunsaturated fatty acids such as linoleic (C18:2 ω6) and linolenic (C18:3 ω3) was assessed by the presence of bis-allylic protons (–CH=CHCH2CH=CH–) at 2.74–2.82 ppm. Unsaturated fatty acids are highly represented in wild plants and a significant amount of ω3 and ω6 series was previously reported in Crithmum maritimum leaves20. Several signals characterized the region between 3.5 and 4.6 ppm  protons was identified at 5.28 ppm. A doublet at 4.90 ppm (J 3.78 Hz) coupled with a proton signal at 3.80 and a 13C at 99 ppm in the HSQC spectrum was assigned to the anomeric proton typical of glycodiacylglycerols sugar moiety. Digalactosyldiacylglycerol is known to be the major glycerolipids of plastids in plants17.Terminal methyl groups , 5.22 CH(1), and 4.90 CH(3), together with the spin system at 0.88 (–CH3), 1.26–1.39 (n-CH2–), 5.29–5.45 (–CH=CH–) were assigned to falcarindiol. Interestingly, these resonances were not observed in the 1H-NMR spectra of the other three fractions  and 7.60 ppm (J = 15 Hz) are compatible with caffeoyl moieties of hydroxycinnamic acids, such as chlorogenic acid, reported to be major phenolic compounds in Crithmum maritimum aerial parts25. Interestingly, strongly coupled doublets at 7.0 and 6.78 ppm were not observed in the methanol extract . Cells were treated for 48 h with the extract at the concentration of 0.5 μM, which was used in our previous work. Cell culture media were collected for further analyses. Then, cells were harvested and the pellet obtained after centrifugation was used for the analysis performed on both aqueous and lipid fractions.We next evaluated by resolved NMR spectra were used to accurately identify and assign metabolites according to literature data32. Then, a multivariate analysis (MVA) was performed. In a first step, a general overview for both the HepG2 and Huh7 cell lines was obtained by unsupervised PCA  analyses, while supervised OPLS-DA models were used to elucidate the most reliable class-discriminating variables that were highly diagnostic for CM and DMSO group separation. OPLS-DA models , clearly evidenced differences in the concentrations of polar metabolites of the two cell lines for the treated compared with untreated samples . For metabolites defined in the 1H-NMR spectra by multiple patterns of signals, the non-overlapping signals were chosen, and relevant data obtained were expressed as normalised integrals for the discriminating metabolites  and cell culture media, 1D and 2D COSY, HSQC, HMBC, and J-les Fig.  online. 2X = 0.89, R2Y = 0.80, Q2 = 0.25 and R2X = 0.78, R2Y = 0.94, Q2 = 0.65. Data evidence a specific trend in the level of metabolites in Crithmum maritimum-treated samples when compared with the vehicle (DMSO). In HepG2 cell culture media only glycine, lactate, and valine were increased in Crithmum maritimum-treated samples. On the other hand, in Huh7 samples, relatively higher levels of alanine, formate, glycine, glutamate, lactate, lysine, methionine, methylhistidine, pyroglutamate, phenylalanine, tyrosine, and valine were detected. Relevant metabolites found by MVA were evaluated by the integration of the unbiased signal in the 1H-NMR spectra, and t-test analysis was used to determine statistical significance. Results, expressed as normalized integrals for the discriminating metabolites, are reported in Figs. Similarly, the corresponding OPLS-DA models were obtained for cell culture media samples Fig. a,b. BothCrithmum maritimum-treated HepG2 and Huh7 cells compared with vehicle (DMSO) samples. Non-polar extracts were characterized by the presence of esterified lipids  and minor components, such as sterols  and phospholipids 3 groups of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC). As shown by the OPLS-DA models . A moderate relative higher content of fatty acids  was observed in Crithmum maritimum-treated extracts of HepG2 cells if compared to vehicle (DMSO). Indeed, relatively lower levels of cholesterol , SFA, DUFA, TAGs, and phospholipids  were detected in Crithmum maritimum-treated HepG2 cells. A similar trend was observed in Huh7 cells, except for PUFA ω-3. Relevant metabolites found by MVA were evaluated by the integration of the unbiased signal in the 1H-NMR spectra, and the t-test accounted for the significance of variation. Results, expressed as normalized integrals for the discriminating metabolites, are reported in Fig. Finally, we analysed the lipid fraction in 33. Several plant-derived compounds are under evaluation as potential anti-cancer drugs34, and some are being tested in clinical trials35. Scientific evidence supports the view that beneficial effect on health lies in the complex blend of compounds characteristic of each plant, rather than to the action of a single or few molecules37. Indeed, the anticancer effect of a mixture of compounds from desert plant extracts has been described in HCC cells38. Also, some reports demonstrated the effectiveness of the ethyl acetate extract of three traditional plants on cervical cancer39 and HCC41.Studying the effects of plants for the prevention and cure of different diseases is a growing area of interest for the scientific communityCrithmum maritimum ethyl acetate extract exerts an evident cytostatic effect in HCC cell lines9. In this study, we extended the analysis using a metabolomic approach to obtain additional insights for a better understanding of the mechanism of action of this plant extract. Our results clearly show that the metabolite signature notably changed in both cell lines upon treatment with the Crithmum maritimum ethyl acetate extract. This offers an interesting framework, which outlines a multi-target effect on key metabolites 46, can be explained by the increased demand for membrane phospholipids due to the accelerated proliferative rate in cancer cells.We have previously shown that tes Fig. c that arCrithmum maritimum ethyl acetate extract, we found an important decrease of saturated fatty acids (SFAs), triglycerides (TAGs), total cholesterol , and mono- and di-unsaturated fatty acids (MUFA and DUFA). Moreover, we observed a significant decrease in free cholesterol and an increase in ω-6 PUFA in both cell lines. ω-3 PUFA were increased in HepG2 cells and slightly decreased instead in Huh7. Recent studies have underlined the role of lipid homeostasis for tumour development and progression. In a study performed in 20 patients, the level of PUFA was significantly lower compared to healthy liver tissue47. More recently, cholesterol was also found to be essential for cancer cell metabolism and was proposed as a promising therapeutic target48. We also observed a slight difference in terms of response to the Crithmum maritimum ethyl acetate extract between the two employed cell lines, which may be due to their different genetic background which accounts for HCC heterogeneity49. Indeed, our study shows that the multi-target action of Crithmum maritimum ethyl acetate extract is effective upon this heterogeneity. This supports the idea of employing a blend of plant-derived bioactive compounds against cancer. This view is further corroborated by the observation that the effect of Crithmum maritimum is still maintained upon extracellular acidic pH conditions. Moreover, since we observed that Crithmum maritimum is also effective in a non-HCC cell line, further investigations should be extended to other malignancies.As for the lipid fraction, upon treatment with Crithmum maritimum ethyl acetate extract normalise the metabolic profile of HCC cells by modulating key metabolic processes. Such pathways were reported to contribute to the tumorigenic phenotype of HCC. As depicted in Fig. Crithmum maritimum in HCC cells.Overall, our data demonstrate that Crithmum maritimum-mediated cytostasis and pave the way for further investigations on the multiple mechanisms by which this plant exerts such effect. This is in line with the need of developing systems pharmacology approaches for improving the treatment of tumours. Plant secondary metabolites have evolved for defence purposes against different categories of aggressors. For this reason, these metabolites are thought to interact with a plethora of targets in biological systems. The synergistic action of many phytochemicals may provide effective tools in disease management, including cancer51. In the case of Crithmum maritimum, the synergy of several secondary metabolites, such as phytosterols, glycerolipids, polyacetylenes, hydroxycinnamic acids, and hydroxybenzoic acid derivatives accounts for the observed cytostatic effect in HCC. A systems pharmacology approach using Crithmum maritimum will offer concrete opportunities for developing new effective therapeutic opportunities for HCC by integrating established drug protocols with plant-derived formulations.Our findings identify a metabolic basis for All of the solvents employed for the extraction procedure and analytical determinations were of analytical grade and HPLC grade. Hexane was purchased from Honeywell, ethyl acetate, methanol, and ethanol from Sigma-Aldrich. Dimethyl sulfoxide (DMSO) was purchased from Corning [cat. # 25-950-CQC].1H-NMR analyses were of analytical grade. CDCl3, CD3OD (99.8 atom%D), TMS (0.03% v/v) were purchased from Armar Chemicals .All chemical reagents used for Crithmum maritimum L. was harvested on the Apulian coast in the Bari metropolitan area (Bari S. Spirito). The extraction procedure was performed as previously described9. Briefly, after the whole plant was desiccated at 60 °C, the powder was obtained by mechanical trituration. The average yield of extraction obtained was ≈ 96% fresh/dried plant material. The powder was then subjected to the first extraction with hexane, by adding 150 mL of hexane to 150 mg plant powder in a conical flask and leaving the mixture under gentle orbital shaking for 48 h. Then, after filtration, the extract was let to evaporate at room temperature under a chemical hood. The extraction protocol described above was then repeated for ethyl acetate, methanol, and ethanol. Obtained extracts were then solubilised in cell culture-grade DMSO. The concentration of the extract was determined by high-pressure liquid chromatography (HPLC) as previously described9.2).Huh7 and HepG2 cell lines were purchased from JCRB cell bank . HeLa cell line was purchased from ATCC cell bank , supplemented with 1X MEM-Nonessential Amino Acids [Corning cat. # 25-025-CIR], 20 mM Hepes Buffer [Aurogene cat. # AU-L0180-500], 1X Antibiotic–Antimycotic solution [Corning cat. # 30-004-CI] and 10% Foetal Bovine Serum (FBS) [Corning cat. # 35-079-CV]. Cells were grown under standard culturing conditions 55.Cell samples: NMR samples were obtained from Huh7 and HepG2 cell pellets and cell culture medium (CCM). Cell extracts were prepared according to a modified Bligh and Dyer two-step methodCrithmum m. extract: to obtain a satisfactory dissolution of polar and non-polar analytes, the ethyl acetate extract was resuspended in a 1:1 CD3OD:CDCl3  mixture. 600 μL of the mixture was transferred into a 5-mm NMR tube and immediately subjected to NMR analysis.For 1H Jres, 1H COSY, 1H-13C HSQC, and HMBC) and by comparison with published data57.For each aqueous sample a 1D sequence with pre-saturation and composite pulse for selection (zgcppr Bruker standard pulse sequence) was acquired, with 256 transients, 16 dummy scans, 5 s relaxation delay, size of fid of 64 K data points, a spectral width of 12,019.230 Hz (20.0276 ppm) and an acquisition time of 2.73 s, and with chemical shift referencing to trimethylsilyl propionic-2,2,3,3-d 4 acid sodium salt (TSP) signal (δ = 0.00 ppm). The resulting FIDs were multiplied by an exponential weighting function corresponding to a line broadening of 0.3 Hz before Fourier transformation, automated phasing, and baseline correction. For each lipid extract a one-dimensional experiment (zg Bruker pulse program) was run with 128 scans, 64 K time domain, spectral width 20.0276 ppm , 5 s delay, p1 8 µs and 2.73 s acquisition time. All spectra were referenced to the tetramethylsilane (TMS) signal (δ = 0.00 ppm). Metabolites were assigned based on 2D NMR spectra analysis , in a spectral window of 20.0276 ppm , 128 scans, and a 90° pulse of 7.55 µs, 64 K time domain, spectral width 20.0276 ppm , 5 s delay, and 2.73 s acquisition time. After the acquisition, the standard FID processing procedures were carried out, by using TopSpin 3.5 , such as the Fourier transform , the phase and baseline correction, and 0.3 Hz line broadening. All the 1H-NMR spectra were calibrated to the internal standard TMS (δ = 0.00 ppm). Characterization of metabolites was performed by the analysis of two-dimensional homo- and heteronuclear NMR spectra  and by comparison with published data58.1H observation, equipped with a z-axis gradient coil and automatic tuning-matching (ATM). Experiments were acquired at 300 K in automation mode after loading individual samples on a Bruker Automatic Sample Changer, interfaced with the software IconNMR (Bruker).All measurements were performed on a Bruker Avance III 600 Ascend NMR spectrometer  operating at 600.13 MHz for , 1H-NMR spectra—regions between 7.75–6.75, 3.48–3.25, and 1.70–1.44 ppm—were excluded before analysis due to the residual peaks of solvents . For each of the resulting data sets, a matrix was obtained, made of the bucketed 1H-NMR spectra values (columns) measured for each sample (rows). The mean centred and Pareto scaling procedures were applied to the data before multivariate statistical analyses, to attenuate the effect of dominant variables and noise while amplifying weak signals to the largest possible60.To account for variations of the overall concentrations of samples, NMR spectra were processed using Topspin 3.6.1 and Amix 3.9.13 software  for simultaneous visual inspection and the successive bucketing process. NMR spectra were segmented in rectangular buckets of fixed 0.04 ppm width and integrated. For aqueous cell fractions, spectral regions between 5.00–4.50 ppm were discarded because of the residual peak of water signal, while for CCM NMR spectra, signals of Hepes and DMSO and its affected neighbouring regions between 3.87–3.77 and 3.20–2.55 ppm were discarded. For lipid extract1H-NMR spectroscopy, metabolites of interest were quantified by analysing the integrals of selected distinctive unbiased NMR signals. Integral values for metabolite quantitative analysis were referred to as the internal standard . Results, represented as mean intensities and standard error mean of the selected NMR signals, were validated by the univariate t-test63. Levels of statistical significance were at least at p-values < 0.05 with a 95% confidence level. The univariate analysis made on the integral area of metabolites between groups was performed using MeTPA MetaboAnalyst software64.By 2O 10% v/v to obtain a 1 mg/mL solution. Before Crystal violet addition, cells were fixed in 4% paraformaldehyde. Colour elution was performed with 10% acetic acid and absorbance was measured at λ = 595 nm using an iMark plate reader [Bio-Rad cat. #168–1135].End-point proliferation was assessed by crystal violet staining 72 h after plant extract addition. Crystal violet [Sigma-Aldrich cat. #C3886] was diluted in EtOH/H1H-NMR, relevant metabolites detected by MVA were evaluated by the integration of the unbiased signal in the 1H-NMR spectra, and statistical significance, set at p < 0.05, was assessed by t-test.Concerning Cell proliferation data were analysed by Two-way ANOVA, followed by Dunnett’s multiple comparisons test. For data not normally distributed, the Kruskal–Wallis test followed by Dunn’s multiple comparisons test was employed.Supplementary Information


GPR50) in HCC is overexpressed and that GPR50 knockdown may downregulate cancer cell progression through attenuation of the Notch signaling pathway. GPR50 knockdown was found to reduce HCC progression by inactivating Notch signaling in a ligand-independent manner through a disintegrin and metalloproteinase metallopeptidase domain 17 (ADAM17), a proteolytic enzyme that cleaves the Notch receptor, which was corroborated by GPR50 overexpression in hepatocytes. GPR50 silencing also downregulated transcription and translation of ADAM17 through the AKT/specificity protein-1 (SP1) signaling axis. Notably, GPR50 was found to directly interact with ADAM17. Overall, we demonstrate a novel GPR50-mediated regulation of the ADAM17-Notch signaling pathway, which can provide insights into HCC progression and prognosis and development of Notch-based HCC treatment strategies.Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide, and it is thus critical to identify novel molecular biomarkers of HCC prognosis and elucidate the molecular mechanisms underlying HCC progression. Here, we show that G-protein-coupled receptor 50 (  Cho and colleagues demonstrate that orphan GPR50 can function in HCC in a ligand-independent manner. GPR50 can promote the HCC progression via the ligand-independent ADAM17-mediated Notch signaling pathway, which can provide insights into HCC progression and prognosis and development of Notch-based HCC treatment strategies. GPCRs are important proteins in drug development and are reported to be key targets for more than 30% of drugs on the market.,GPR50 can act as a tumor suppressor in breast cancer (BRC);,GPR50 in cancer progression.GPR50, another member of orphan GPCRs, exhibits high sequence similarity with the melatonin receptors MT1 and MT2; however, melatonin is not a GPR50 ligand.GPR50 in HCC progression and prognosis. As GPR50 was described as a tumor suppressor in breast cancer, we examined whether GPR50 plays an oncogenic or a tumor-suppressor role in HCC. We found that GPR50 is overexpressed in HCC and that GPR50 knockdown can suppress HCC progression by downregulating the Notch signaling pathway. Our findings also indicate that GPR50 forms a novel molecular complex with a disintegrin and metalloproteinase (ADAM) metallopeptidase domain 17 (ADAM17) and regulates ADAM17 activity, activating the Notch signaling pathway in HCC in a ligand-independent manner. This pathway is also partially regulated by GPR50-mediated ADAM17 transcription via the noncanonical AKT/specificity protein 1 (SP1) axis. Thus, our results support the potential of targeting HCC via the GPR50/ADAM17/Notch signaling pathway.In this study, we aimed to uncover the role of https://www.oncomine.org/resource/login.html) to examine the expression status of GPR50 in various cancers, we found dysregulated GPR50 expression (Wooster cell line dataset) that was especially enhanced in BRC, cervical (CEC), esophagus (ESC), liver (HCC), and lung (LUC) cancers  datasets. The GEO data showed that GPR50 expression was significantly upregulated in liver cancers  and downregulated in breast, cervical, esophagus, and lung cancers (GPR50 expression in various cancer patients using The Cancer Genome Atlas (TCGA) database via the SurvExpress web. Among the indicated cancers, high GPR50 expression exhibited a significant (p = 0.0118), poor prognostic role in HCC, whereas a nonsignificant prognostic role was found for other cancers, including breast, cervical, esophagus, and lung cancers  and western blot analyses and found that GPR50 expression was clearly overexpressed in the HCC cell lines  in two HCC cell lines: HepG2 and SNU475. GPR50 knockdown was confirmed (approximately 95%) via RT-PCR and western blot analysis  and 2, as well as delta-like ligand 1 (DLL1), 3, and 4, was not significantly altered in shGPR50-HepG2 and SNU475 cells compared with their normal counterparts /mitogen-activated protein kinase (MAPK), and p38/MAPK signaling pathways, as previous studies reported that transcription of ADAM17 can be regulated through these pathways.GPR50 knockdown (GPR50 suppression (GPR50-knockdown cells with the AKT inhibitor LY294002 (20 μM).GPR50-knockdown cells compared with control cells -rich sequence that was depicted to bind various transcription factors, including SP1,,GPR50 silencing regulates SP1 expression. We examined the mRNA expression levels of all SP family genes  and found that SP1 expression was significantly downregulated upon GPR50 suppression  treatment of ADAM17-overexpressed SNU475 cells reduced ADAM17 activity, which was marked in shGPR50-SNU475 cells  and/or the Notch signaling (γ-secretase) inhibitor DAPT (20 μM).reatment H. Moreovreatment I, whereaG2 cells J and 7K.,,,,An orphan GPCR (GPR50) was shown to regulate bipolar-affective disorder, lipid metabolism, thermogenesis, adipogenesis, and neuronal development,GPR50 knockdown attenuates cell proliferation, sphere formation, migration, and drug resistance of HCC cells, whereas overexpression of GPR50 upregulates cancer properties in hepatocytes  components, and shedding of membrane proteins ; fertilization; development; inflammation; asthma; and neurodegenerative diseases, such as Alzheimer’s disease.,,,Consistent with our results, a number of studies depicted that ligand-independent Notch activation is mediated by ADAM17.,We further demonstrated that GPR50 can regulate ADAM17 transcription and translation through the AKT/SP1 signaling axis, which is in agreement with previous studies that ADAM17 can be transcriptionally regulated via the AKT, ERK/MAPK, and p38/MAPK signaling pathways.In conclusion, we demonstrated how the orphan receptor GPR50 regulates the ligand-independent activation of Notch signaling through GPR50-mediated modulation of ADAM17 activity in HCC . A signaGPR50 gene in various cancers were retrieved from the Oncomine database (https://www.oncomine.org/resource/login.html).,https://www.ncbi.nlm.nih.gov/geo/).The expected expression levels of the http://bioinformatica.mty.itesm.mx:8080/Biomatec/SurvivaX.jsp),GPR50 mRNA expression levels and clinical outcomes. This database contains more than 39,000 samples and 225 datasets covering tumors from more than 26 different tissues. With the use of this platform, survival plots were generated for GPR50 in specific cancer types using TCGA data. We further performed an integrative analysis of GPR50 and clinical characteristics using cBioPortal, an open-access resource found at http://www.cbioportal.org/,,With the use of a web-based tool for survival analysis, SurvExpress  and were authenticated using short tandem repeat (STR) profiling.The human HCC cell lines  and the normal hepatocyte cell line MIHA (ATCC) were cultured in DMEM  or RPMI 1640 , supplemented with 10% heat-inactivated fetal bovine serum , 100 U/mL penicillin , and 100 mg/mL streptomycin . Cells were cultured at 37°C in a humidified atmosphere of 5% COGPR50 knockdown (shGPR50-1 and -2); the sequences are listed in BamHI and EcoRI restriction enzyme sites, according to the manufacturer’s instructions. Briefly, two oligonucleotides were annealed at 95°C for 2 min in a heat block with annealing buffer, and then the samples were left to cool down to room temperature. The annealed, double-stranded DNA was then ligated into a lentiviral vector using the T4 DNA ligase enzyme . Sequences of the newly constructed plasmids were confirmed by sequencing analysis.Sense and antisense oligonucleotides were synthesized for control (scramble) or GPR50, the pGEM-T Easy vector (Promega) was used to clone the complete GPR50 coding sequence using the primers listed in XbaI and EcoRI restriction enzymes and the primers listed in For overexpression of JAG1, DLL1, NICD, and ADAM17, cells were incubated overnight to a cell density of 2 × 105 cells per well in a 24-well culture plate. The cells were then transfected with the expression vectors for hemagglutinin (HA)-JAG1, GFP-DLL1, FLAG-NICD , and FLAG-ADAM17 For overexpression of 8 IU/mL viral particles for stable knockdown in the indicated cells. All experiments were started 72 h postinfection.To generate the lentivirus, the Rev response element (RRE)/REV lentivirus expressing systemGAPDH) was used as a loading control, as described previously.GAPDH was used as an internal control, as described previously.Total RNA was extracted using an Easy-Blue RNA Extraction Kit , and the purified total RNA (2 μg) was reverse transcribed into cDNA using a cDNA synthesis kit (Promega), according to the manufacturer’s instructions. For PCR analysis, 1 μL of synthesized cDNA, specific forward and reverse primers, and r-Taq Plus Master Mix  were mixed and analyzed by thermocycler PCR, after which, the PCR products were analyzed by agarose gel electrophoresis. DNA was stained via ethidium bromide (EtBr), observed under UV light, and imaged. The images were then analyzed in Photoshop CS6 , and the relative expression fold changes were measured using ImageJ; the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase . Protein samples (40–60 μg) were then loaded onto either 10% or 12% SDS-PAGE gels and electrophoresed, after which, the proteins were transferred to nitrocellulose blotting membranes. The membranes were blocked with 5% skim milk for 1 h and subsequently incubated with primary antibodies against GPR50 , NANOG , OCT4 , ABCG2 , phospho-p38 , NICD , FLAG , PI3K , AKT , phospho-AKT , ERK , phospho-ERK , p38 , GSK3β , phospho-GSK3β , HES1 , actin , and α-tubulin  at 4°C overnight. The membranes were then washed three times at 10-min intervals with Tris-buffered saline and Tween 20  buffer and incubated with secondary antibodies, including anti-mouse (SC-2005), -goat (SC-2020), or -rabbit (SC-2004) immunoglobulins (Igs) tagged with horseradish peroxidase (HRP) at room temperature for 1 h. Next, after washing with TBS-T buffer for 30 min, the immunoreactive proteins were visualized using an enhanced chemiluminescence (ECL) detection kit , as previously described.,95Total cell lysates were extracted from the indicated cells using lysis buffer. The cell lysates were then incubated at 4°C for 15 min, vortexed every 2–3 min, and centrifuged at 16,600 × 4 cells/well) were seeded onto 12-well plates. Cells were counted, starting from 24 h up until day 5 using a trypan blue kit. For cell viability assays, cells were seeded onto 96-well plates, and at the indicated time points, EZ-cytox WST-1 reagent  was added at a ratio of 1:10 and incubated at 37°C in a humidified atmosphere of 5% CO2 for ∼4 h. Afterward, the relative absorbance was measured at 450 nm using a fluorescence microplate reader, as previously described.,95For cell proliferation analysis, control (scramble-transduced), GPR50-knockdown, or GPR50-overexpressing cells  for 3 h, after which, the cells were scratched with a 200-μL pipette tip. The indicated wound areas in the dishes were marked, and photos were taken every 12 h. The pictures were analyzed in ImageJ, and the wound closure percentage (%) was determined, as previously described.5 cells) were seeded onto noncoated Petri dishes with sphere-forming medium containing serum-free DMEM/F12 media with B27 supplement, 20 ng/mL EGF (Sigma-Aldrich), 10 μg/mL insulin (Sigma-Aldrich), and 1% bovine serum albumin (Sigma-Aldrich). After 6 days, colonies were gently collected into conical tubes  and stained with crystal violet (Sigma-Aldrich). Finally, the colonies were disassociated using 0.25% trypsin-EDTA , after which, the disassociated cells were counted and presented as the percent (%) of sphere-forming cells, as previously described.,The indicated cells  of surviving cells, as previously described.For drug-resistance analysis, 1 × 105 cells/well) were seeded onto 12-well plates and transiently transfected with 1 μg of either HES1 or HES5 luciferase plasmid using HyliMax transfection reagent .For the luciferase assay, the indicated cells (1 × 107 cells were harvested in 1 mL of ice-cold PBS (pH 7.2–7.4) with protease inhibitor and stored at −20°C overnight. After two freeze-thaw cycles, the cell lysates were centrifuged for 5 min at 5,000 × g at 2°C–8°C, and then the supernatant was collected and stored at −20°C until future use, after measurement of the protein concentrations in the supernatant. Approximately 15 mg of protein was mixed with substrate for 15–30 min, and the optical density (OD) of each well was determined within 5 min using a microplate reader .ADAM17 activity assay was performed using an ADAM17 Activity Assay Kit , according to the manufacturer’s instructions.g for 1 min. The pellets were then washed thrice with ice-cold cell lysis buffer, after which, the immunoprecipitated proteins were analyzed by western blotting, as described above.To analyze protein interactions, a coIP assay was performed with the indicated samples. Briefly, 400 μg of cell lysate was pretreated with 30 μL Protein A/G Agarose beads (Santa Cruz Biotechnology) to remove nonspecific IgG. The supernatant was then collected in new tubes and incubated overnight with 3–4 μg primary antibodies (anti-GPR50 or anti-ADAM17) and rabbit IgG on an agitator at 4°C. Subsequently, Protein A/G Agarose was added, and the mixture was incubated for an additional 3 h and spun down at 4,000 × All experiments were performed three times, and the data are presented as the mean ± standard deviation (SD). For statistical analysis, an unpaired t test was performed between two groups (control versus treated), and p values <0.05 were considered statistically significant.All data referenced in the manuscript can be downloaded from websites indicated in the Materials and Methods section.S.K.S. conceived of and participated in the study design, performed the experiments, analyzed the data, and wrote the manuscript. H.Y.C., G.-M.Y., P.K.B., K.K., G.-H.K., and M.G. performed some experiments and analyzed the data. S.-G.C. designed the study, reorganized the data, and wrote and edited the manuscript. All authors reviewed and approved the manuscript.The authors declare no competing interests.