Polygonum perfoliatum L. (PPL) is an herbal medicine with anti-lipid peroxidation and anti-inflammatory properties. However, detailed hepatoprotective effects of PPL against NAFLD and its underlying mechanisms are not fully understood. Here, we found that PPL protects against high fat diet (HFD)-induced hepatic steatosis, lipid peroxidation, and glucose-lipid metabolism dysfunction in NAFLD mice. We therefore performed a label-free quantitative proteomic profiling analysis to determine the effect of PPL treatment on liver tissue proteomics and identified that activated PPARs/CPT1A/CPT2-mediated hepatic fatty acid β-oxidation (FAO) process was significantly altered. In vitro treatment of hepatocytes with PPL confirmed this altered process and FAO inhibitor etomoxir (ETO) attenuated the lipid-lowering activity of PPL in hepatocytes. Ultra-high-performance liquid chromatography/Q Exactive-HFX (UPLC/QE-HFX) was used to determine the material basis of anti-NAFLD activity of PPL. Our results have demonstrated the efficacy and potential mechanisms of PPL as an effective pharmacological therapy of NAFLD.Non-alcoholic fatty liver disease (NAFLD) is a predominant contributor to end-stage liver disease in the forthcoming decades.  NAFLD is currently one of the most prevalent chronic liver diseases, accounting for approximately a quarter of the global epidemic, and its prevalence will continue to increase in the foreseeable future . NAFLD hvia CPT1 and CPT2 located in both outer and inner mitochondrial membranes, respectively. In addition, acyl-CoA is converted to acetyl coenzyme A (acetyl-CoA) by multiple dehydrogenases, which undergoes repeated tricarboxylic acid (TCA) cycle and ultimately generate large amounts of energy , phlegm and dampness retention are the main causes of hyperlipidemia and NAFLD. PPL belongs to the Chinese herbal medicine, which has the effects of clearing heat and detoxification, dispelling phlegm, and relieving dampness, and is widely distributed in many regions of China . TherefoIn this study, we demonstrated the therapeutic effects of PPL on NAFLD progression based on an HFD-induced NAFLD model and characterized the protein expression profile, signaling pathway activation and histopathological changes. Our findings indicated that PPL might be a promising therapeutic agent for NAFLD through activation of FAO.PPL was purchased from Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine . Voucher samples were deposited in the Central Laboratory of Shuguang Hospital. Serum biochemical indexes were measured using a full-automatic biochemical analyzer. MDA  and SOD  kits were purchased from Elabscience . Liver TC  and TG  levels were determined using APPLYGEN assay kits . Primary antibodies included rabbit anti-PPARγ , rabbit anti-PPARα , mouse anti-CPT1A , rabbit anti-CPT2 , rabbit anti-SREBP2 , mouse anti-HMGCR , rabbit anti-CYP7A1 , and actin .To obtain an extract of PPL, 200 g PPL was soaked in 2 L distilled water at room temperature (RT) for 1 h and then heat to reflux for another 1 h. The extract was filtered, and the residue was subjected to a secondary extraction. The extracted mixture was concentrated under reduced pressure and further lyophilized to a fine powder to obtain 20.52 g lyophilized powder with a final yield of 10.26%.Male C57BL/6 mice were purchased from Jiesijie Research Institute  and divided into two groups. Mice in Group 1 were orally administered with PPL extract , and blood was collected from the orbit 1 h after administration. Mice in Group 2 were given saline and the serum was collected as a control group. Both serum samples were inactivated by heating at 56°C for 30 min. The protein precipitation was removed by adding twice the volume of acetonitrile  and the supernatant was transferred into a rotary evaporator  for concentration. The pure water was re-dissolved and desalted by a LC-C18 solid phase extraction column , and transferred into a vacuum centrifugal concentrator . The concentrate was weighed, filtered, and sterilized by re-dissolving in pure water and stored at −80 °C until subsequent experiments.Total proteins were extracted from mice liver tissues sampled from HFD and HFD + PPL groups (n = 3) with 300 µL lysis buffer supplemented with 1 mM Phenylmethanesulfonyl fluoride (PMSF). Samples were centrifuged at 15,000 g for 15 min to remove insoluble particles, and then the precipitate was again excluded. After adding 5 times the volume of cold acetone and precipitating overnight at −20°C, the precipitate was collected by centrifugation at 12,000 g for 15 min at 4 °C. The precipitation was dried at RT for 3 min and then dissolved in SDS-lysis buffer for 2 h. Sample supernatant was collected and centrifuged again to remove the precipitate. The protein concentration was determined according to BCA method and samples were aliquoted to be stored at −80 °C. Base on BCA assay results, 100 μg of protein was extracted for filter-aided sample preparation (FASP) digestion. IAA was added to a final concentration of 50 mM and incubated at RT for 40 min in a dark place, followed by another 1 h incubation at 60 °C. The solution was discarded after centrifugation on a filter at 4 °C for 25 min. After adding 100 µl of 300 mM triethyl ammonium bicarbonate (TEAB), the solution was centrifuged twice for 20 min each time. After washing, TEAB and trypsin were added to the filtration unit and digested overnight for 12 h at 37°C. Finally, EAB was eluted and lyophilized. The raw data was imported into Maxquant for label-free quantitative analysis with a search engine Andromeda.Mass spectrometric analysis was performed on a Q Exactive HFX mass spectrometer using a UPLC system with a BEH Amide column . 25 mmol/L acetate of ammonium and 25 mmol/L hydroxide of ammonium in water (pH = 9.85) A) and acetonitrile B) were respectively used. The auto-sampler was set to 4 °C and the injection volume was 3 L. To obtain MS/MS spectra in information-dependent acquisition mode (IDA), a QE HFX mass spectrometer was utilized. In IDA mode, the acquisition software performed continuous evaluation of the full scan MS spectrum. Accordingly, the electrospray lonization (ESI) source conditions were as follows: 15 Arb sheath gas flow rate, 15 Arb aux gas flow rate, 350 °C capillary temperature, 60,000 full MS/MS resolution, 10/15/60 collision energy in normalized collisional energy (NCE) mode, and 3.6 (positive) or −3.2 (negative) spray voltage. The raw data was converted from XML to mzXML using ProteoWizard, and then the peak data was extracted in R using an in-house program developed by XCMS to align and integrate it. A cut-off of 0.3 was determined in metabolite annotation using an internal MS2 database.ad libitum access to standardized food and tip-filtered water. Male C57BL/6 mice were given HFD  for 13 weeks to establish a NAFLD pathological model. Mice treated with normal chow diet (NCD) were defined as the control group. Mice fed with HFD + PPL  were defined as the treatment group. Mice treated with physiological saline and PPL (400 mg/kg) were used to verify the toxicity of PPL. All experimental procedures were authorized according to the standards in the Guide for the Care and Use of Laboratory Animals. All animal experiments were approved by the Institutional Animal Care and Use Committee of Shuguang Hospital, Shanghai Chinese traditional medicine University.Male C57BL/6 mice were purchased from Jiesijie Research Institute . Six-week-old male mice (weight: 20 ± 2 g) were maintained in a standard environment at 22°C–24°C with a 12:12 h light-dark cycle, humidity (65% ± 5%) and via oral gavage for another 6 weeks. Mice were fasted overnight, drank water freely, and anesthetized prior to specimen collection. The whole body, liver, and adipose tissues were sampled from each group of mice, weighed, and photographed for documentation. Specimens were divided into three parts, and either frozen in liquid nitrogen for proteomic studies, or stored at −80°C for biochemical analysis, or fixed in paraformaldehyde  for further morphological and histological analysis of the liver.Thirty-eight C57BL/6 male mice were randomly divided into 6 groups: 1) Normal chow diet (NCD) group (n = 6); 2) NCD + PPL (400 mg/kg) group (n = 6); 3) High fat diet (HFD) group (n = 7); 4) HFD + PPL (100 mg/kg) group (n = 6); 5) HFD + PPL (200 mg/kg) group (n = 6); 6) HFD + PPL (400 mg/kg) group (n = 7). PPL was dissolved in saline. Both NCD and HFD groups were simultaneously given saline during PPL treatment in HFD + PPL group. Mice were continuously fed HFD for 13 weeks to induce NAFLD model and treated with PPL or saline Blood specimens were allowed to stand at RT for 30 min and then centrifuged at 4°C for 15 min at 3,000 rpm to obtain serum samples. High-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), serum triglycerides (TG), total cholesterols (TC), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were determined using kits  according to the manufacturer’s instructions.Mice liver samples were fixed with 4% paraformaldehyde. White adipose tissues (WAT) and brown adipose tissue (BAT) were fixed with adipose tissue-specific fixed fluid. The liver and adipose tissue were sectioned into 5-µm sections and stained with hematoxylin and eosin (HE) solutions according to standard procedures. Slides were observed under an inverted microscope . Adipocyte diameter and area of white and brown adipocytes were measured using ImageJ software .2O. Finally, slides were observed under an inverted microscope.For liver sections staining, frozen liver sections were performed at optimal cutting temperature, rewarmed at RT, rinsed in distilled water, and stained with Oil Red O stain solution  for 6–10 min. After staining, sections were soaked in 60% isopropyl alcohol and washed with distilled water. Sections were sealed with glycerin gelatin. For cell staining, cells were harvested and fixed with 4% paraformaldehyde, washed twice, oil red O stained for 6–10 min, examined for lipid droplets. After that, samples were rinsed in 60% isopropyl alcohol and washed three times with ddHFor mice liver staining, liver tissue sections were frozen for 30 min and immersed in PBS for 10 min, and then soaked in BODIPY 493/503  for 30 min, protecting from light during staining. After washing with PBS three times, slides were counter-stained with DAPI . For cell staining, hepatocytes were rinsed three times with PBS and fixed in 4% paraformaldehyde for 15 min. After washing, samples were stained with BODIPY 493/503 for 30 min, and then counter-stained with DAPI for 5 min. Images were observed under an inverted microscope and fluorescence was quantified using an ImageJ software.ad libitum. Fasting blood glucose levels (0 min) were measured before intraperitoneal injection of glucose (1 gkg−1 body weight), and blood was taken from the tail tip to record blood glucose levels . The insulin tolerance test (ITT) was performed 3 days after the GTT test. Mice were given a 6 h fast (07:00–13:00) with free access to water, followed by an intraperitoneal injection of insulin (0.75 gkg−1 body weight). Blood glucose concentrations  were recorded from the tail tip.For glucose tolerance test (GTT), mice were fasted overnight for 12 h (20:00–08:00) and drank water 6 cells per well) at 37°C in a 5% CO2 atmosphere in DMEM/F12 medium  supplemented with 1% penicillin/streptomycin plus 10% FBS. The following day, both hepatocytes were washed twice and replaced with new culture medium. Blank serum metabolites, PPL metabolites (5.10 μg/ml) and 50 μM PA plus 200 μM OA were added 24 h before cell harvesting. Cells were subsequently collected for further analysis.Mouse primary hepatocytes were isolated from C57BL/6J male mice according to a two-step perfusion method as previously described . Briefly6 cells per well). After overnight incubation, cells were co-incubated with PPL metabolites  and PA/OA for another 24 h. Finally, cells were collected and homogenized in lysis-buffer to determine TG content according to instructions of APPLYGEN Assay Kit.AML12 cells and mice primary hepatocytes were inoculated in 6-well plates (2×103 cells/well). Cell viability was determined using a CCK-8 assay kit . Each well was incubated with 100 μl of medium and 10 μl of CCK-8 reagent for no more than 4 h. The absorbance of each well was determined at 450 nm.Cells were inoculated in a 96-well plate . RT-qPCR was performed using a ChamQTM Universal SYBR® qPCR Master Mix . The relative expression levels of the genes were determined by SYBR-green-based RT-qPCR. The results were analyzed using the 2−ΔΔCT method and normalized toβ-actin values. Primer sequences are listed in Total RNA was extracted from mice liver tissues or cells using TRIzol reagent . A total of 2 μg RNA was reversely transcribed using a HiScriptTotal proteins were extracted from mice liver tissues or cells homogenized in RIPA Lysis Buffer  supplanted with a protease inhibitor cocktail . BCA Protein Assay Reagent  was used for protein concentration quantification. Approximately 40 μg of total protein samples were loaded to 10% SDS-PAGE, followed by an electrophoretic transfer to a nitrocellulose membrane. After that, the membrane was blocked with 5% BSA  for at RT for 1 h, and incubated at 4°C overnight against primary antibodies. After washing, membranes were incubated with secondary antibodies for 1 h. Finally, an Enhanced chemiluminescence Western blot kit  was used to visualize the target protein.The pathological and morphological changes of tissue sections were assessed at ×200 magnification. The histopathological grading criteria scores of liver tissue sections include periportal-bridging necrosis, intralobular degeneration, portal inflammation, and fibrosis . The hist-tests and among groups using one-way analysis of variance (ANOVA) tests. p < 0.05 was considered statistically significant . If a statistically significant change was found (p < 0.05), a post hoc comparison was performed using Bonferroni correction.Data were expressed as the mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism software (v 8.4.2). Data were compared between groups using Student’s The results showed that PPL treatment significantly reversed body weight gain , but no IL-6 and MCP-1. Accordingly, PPL significantly increased gene expression levels of browning markers such as Tmem26, CD137 and Cited1 (PGC1α) and significantly inhibited PPARγ activation, while simultaneously promoting PPARα expression in BAT  in HFD and HFD + PPL groups, according to ratio analysis of protein expression levels. Of these, 191 were upregulated DEPs (FC ≥ 1.5) and 58 were downregulated DEPs (FC ≤ 0.667) (We further used a label-free quantitative proteomic profiling to identify the role of PPL at the hepatic tissue proteomic level to explore the key protein and molecular mechanisms underlying PPL protection against NAFLD. The protein expression profiles of liver tissues from mice in HFD and HFD + PPL (400 mg/kg) groups were subjected to by proteomic analysis, and a total of 2,693 plausible proteins were identified. A total of 249 differentially expressed proteins (DEPs) were identified by ≤ 0.667) . KEGG pa≤ 0.667) . Metabol≤ 0.667) , with PP≤ 0.667) . In addiACADVL), Acyl-CoA dehydrogenase long chain (ACADL), Acyl-CoA dehydrogenase medium chain (ACADM), Acyl-CoA dehydrogenase short chain (ACADS), Acyl-CoA dehydrogenase short/branched chain (ACADSB), which were responsible for facilitating the FAO dehydrogenation reaction. As shown in ACADVL, ACADL, ACADM, ACADS and ACADSB, compared to those detected from the model group. In terms of glucose metabolism, PPL reversed the expression of gluconeogenesis-related genes (such as G6PC and PCK1) while increased the level of glucose transporter gene (Glut2). In addition, PPL inhibited the expression of cholesterol synthesis-related proteins (SREBP2 and HMGCR) and increased the expression level of cholesterol metabolism-related protein (CYP7A1)  . Taken tIn vitro analysis of cellular steatosis characterized by lipid accumulation was performed in AML12 cells by incubating cells with OA plus PA. The CCK-8 assay results showed no cytotoxic effects of PPL on AML12 cells at concentrations below 100 μg/ml  + ETO (50 μM) did not affect the cell viability of hepatocytes (ETO is a fatty acid oxidation inhibitor that inhibits the activity of CPT1A enzyme through binding to its catalytic site and thus has been widely used to validate the molecular function of the hypolipidemic activity of PPL metabolites atocytes . Accordiatocytes . Consistatocytes . These rTo explore the material basis of PPL for ameliorating HFD-induced NAFLD, UPLC/QE-HFX analysis was used. The results showed that total 29 compounds were identified from PPL metabolites . Of thesObesity is one of the main causes of NAFLD worldwide, accompanied by hyperlipidemia, hypertension, and hyperglycemia, and its pathological roots lie in the imbalance between caloric intake and energy expenditure . Excess In recent years, many studies have shown that numerous plant metabolites or monomers therein can exert different combinations of biological activities to ameliorate weight gain as well as reverse hepatic steatosis . PreviouTo investigate the biology functions of PPL blocking high fat-treated obesity and NAFLD, proteomic analysis was conducted in this study. Compared to the HFD group, we found that FAO was the most affected biological process in mice liver tissues from PPL group. Among the FAO-related proteins, CPT1A and CPT2 were found to be simultaneously involved in PPAR signaling pathway, fatty acid metabolism, and fatty acid degradation, suggesting that the pair of isozymes located in the inner and outer mitochondrial membranes functions a key role in the inhibition of NAFLD. In addition, ACADVL was also detected, which encodes a dehydrogenase that catalyzes the first step in physiological β-oxidation of fatty acid to generate Enoyl-CoA . Accordiin vivo.Mitochondrial FAO is the primary pathway of fatty acid catabolism and plays a crucial role in the regulation of daily energy homeostasis . AccordiIn this study, flavonoid components were found to be probably the main components of anti-NAFLD in PPL. Among them, kaempferol, genistein, quercetin and phloretin can improve NAFLD through PPAR signaling pathway . Both bain vitro, we therefore hypothesized that PPL could play a similar role in PA/OA-stimulated hepatocyte models. Subsequently, immortalized hepatocytes, AML12 cells, which retain representative hepatocyte characteristics, were used as a suitable cell line in this study. The results showed that PPL significantly reversed the elevated TG levels in PA/OA-stimulated AML12 cells, presumably associated with the upregulation of CPT1A, CPT2 and PPARα protein expression levels. In addition, elevated FAO, further inhibited PPARγ protein expression to block lipid storage. However, the FAO inhibitor ETO significantly reversed the hypolipidemic effect delivered from PPL metabolites in PA/OA-treated hepatocytes and replicated a similar phenotype in primary mice hepatocytes. All these results suggested that the hypolipidemic effect of PPL might be related to the promotion of mitochondrial β-oxidation, implying FAO might be an attractive pharmacological mechanism for explaining PPL treatment against NAFLD.Previous results have shown that various flavonoids in PPL are able to reduce lipid accumulation In summary, our results demonstrated that PPL, a traditional Chinese medicine, could effectively ameliorate HFD-induced hyperlipidemia, abnormal liver function, lipid peroxidation, as well as hepatic steatosis. This efficacy was achieved by promoting mitochondrial FAO through PPARs/CPT1A/CPT2 pathway. Furthermore, flavonoids might be the major active ingredients in PPL. Our findings have indicated a prospect for the development of PPL and its flavonoids in clinical treatment of NAFLD.


Fall is a Southerner's reward for having survived the summer.James Farmer–Ochsner Journal contains four original research articles, one innovative program report, a quality improvement article, and five case reports and clinical observations which serve as the principal elements for this edition. This issue also contains the third quarterly Health, Medicine, and Society column, this time authored by Deborah F. Grimes, the Ochsner Health Chief Diversity Officer.The Fall 2023 issue of the The original research includes an article by Pettus, Gavlinski, Beermann, et al on “Implementation of and Barriers to Optimizing Postpartum Care by Resident and Attending Physicians” followed by “Prognostic Factors of Morbimortality in Patients Treated for Xanthogranulomatous Pyelonephritis” authored by Garcia-Chairez, Robles-Torres, Rios-Palacios, et al. Next, please find work by Price-Haywood and Burton detailing “Racial Differences in Strength of Associations Between Colorectal Cancer Screening, Area Deprivation, Demographics, and Clinical Characteristics” followed by “Is It All About the Form? Norm- vs Criterion-Referenced Ratings and Faculty Inter-Rater Reliability” authored by Scielzo, Abdelfattah, and Ryder.An innovative program description is offered by a group of Ochsner physicians and trainees including Fixler, Oliaro, Frieden, et al who report on a very timely experience with “Alert to Action: Implementing Artificial Intelligence–Driven Clinical Decision Support Tools for Sepsis.” Our quality improvement article this edition comes from Riopelle, Kozmenko, Wyche, et al looking at “Lower Double-Wall Puncture Rate During Ultrasound-Guided Internal Jugular Vein Cannulation Using Sharper, Narrower-Gauge, and/or Length-Optimized Needles: A 6-Year Quality Improvement Clinical Series in Adult Patients.”Case reports and clinical observations include “Carcinosarcoma of the Esophagus–A Diagnostic Challenge” authored by Jain, Varshney, Aggarwal, et al, followed by “Syncope and Cardiac Arrhythmias Caused by a Paratracheal Mass” authored by Clayton, Eckholdt, Samra, et al. Jain, Selvakumar, Varshney, et al contributed “Gangliocytic Paraganglioma of the Duodenum: A Masquerader,” and Kajy, Rechenberg, Kerndt, and Wolschleger discuss “Cardiac Tamponade Secondary to Hemorrhagic Pericardial Effusion: A Complication of STEMI.” Rounding out this section is “Massive Renal Cyst Displacing Intra-Abdominal Structures” by Mohammed, Zarm, Velez, and Mohamed.the, hottest summers temperature-wise since weather records were first kept. The promise of cooler temperatures is not far away, and Southerners will certainly welcome them as a reward for surviving this summer.Summer 2023 in the Deep South will certainly go down in the record books as one of the, if not Ochsner Journal accomplished a major milestone this summer: for the first time ever, the Journal has been assigned a journal impact factor (JIF) in Clarivate Analytics Journal Citation Reports. For 2022, the Ochsner Journal JIF is 1.2. In addition to a solid JIF, the Ochsner Journal offers several publication benefits: full-text availability at PubMed, Web of Science, Google Scholar, and other databases; open-access publication with absolutely no article processing charges or other publication fees ; and CC BY copyright that vests copyright with the authors, not with the Journal.Ochsner Journal has been a pioneer in nonpredatory, open-access publishing. Congratulations to Managing Editor Kathleen McFadden, the editorial board, our reviewers, and all who have trusted their work to our review and publication for making this distinction possible.Since its inception in 1999, the


Centre for Evidence in Transplantation have developed the Transplant Trial Watch. The Transplant Trial Watch is a monthly overview of 10 new randomised controlled trials (RCTs) and systematic reviews. This page of Transplant International offers commentaries on methodological issues and clinical implications on two articles of particular interest from the CET Transplant Trial Watch monthly selection. For all high quality evidence in solid organ transplantation, visit the Transplant Library: www.transplantlibrary.com.To keep the transplantation community informed about recently published level 1 evidence in organ transplantation ESOT and the RANDOMISED CONTROLLED TRIAL 1Transplantation Outcomes with Donor Hearts after Circulatory Death.by Schroder, J. N., et al. New England Journal of Medicine 2023; 388(23): 2121–2131.The aim of this study was to investigate posttransplant outcomes of hearts obtained from donation after circulatory death (DCD) versus donation after brain death (DBD) donors.Participants were randomised to receive a heart from either a DCD or DBD donor.297 adult candidates for heart transplantation were randomised, out of which 180 underwent transplantation.The primary efficacy outcome was patient survival adjusted for prespecified donor and recipient risk factors. The secondary efficacy outcome was the donor-heart utilization rate.1 year posttransplantation.ex-vivo perfusion) or DBD organ, whichever came first. 297 wait-listed patients were randomised, of whom 180 were transplanted in the study – 90 with DBD organs, and 90 with DCD organs. At 6 months post-transplant, there was no difference in risk-adjusted survival or other clinical outcomes between the two groups. This is a very well-designed study. Studies that alter with organ allocation are challenging as they must not disadvantage patients by reducing the chances of an organ offer. By allowing patients in the DCD arm to receive a DBD organ if allocated, the investigators overcome this. At least in the short term, outcomes from DCD hearts assessed ex-vivo appear equivalent, and have the potential to increase the rate of transplantation – within the study, 67% patients randomised to the DCD cohort were transplanted compared to 39% in the DBD cohort.This multicentre study randomised patients on the heart transplant waiting list to waiting for a standard, DBD organ; or to a DCD organ  to conventional liver transplantation (CLT).The technique being tested is IFLT compared with CLT. IFLT is a complex technique in which during DBD donation the perfusion cannulas of a Liver Assist can be placed in the donor liver prior to cessation of donor circulation. The arterial canula placed via the splenic artery, portal vein via and vein graft and the outflow canula into the infra-hepatic cava. The perfusion can then seamlessly be transferred from donor circulation to NMP, the liver is then procured and continued NMP until implantation. The supra-hepatic caval (piggyback), portal vein and hepatic arterial anastomoses are then performed in the recipient while NMP continues, and once completed the NMP cannulas are removed, and hepatic perfusion transferred from NMP to recipient without interruption of perfusion.65 adult whole liver-only transplant recipients.The primary endpoint was early allograft dysfunction (EAD) within 7 days as defined by the Olthoff criteria. The secondary endpoints included primary non-function, post-reperfusion syndrome, biliary complications, post-reperfusion lactate, post-transplant LFTs, patient and graft survival at 1, 6, and 12 months, ITU stay and overall hospital stay.12 months.n = 32 and the CLT n = 33, of these 2 (6%) in the IFLT experience EAD and 8 (24%) in the CLT (p = 0.044) which was the primary endpoint. In some of the secondary endpoints they found significant improvement with IFLT: peak ALT and ASK at 7 days, total bilirubin, post-op lactate positive perfusate microbial culture and non-anastomotic strictures at 12 months. When scrutinising these strictures, there were 2 in IFLT (one mild and one moderate) and 9 in CLT (five mild and four moderate) none of which required intervention. The marked reduction in post-reperfusion syndrome is important 3 (9%) in IFLT and 21 (64%) in CLT given the risk of post-reperfusion cardiac arrest. They found no significant differences in primary non-function, over-all hospital stay, anastomotic stenosis (though the rate was higher in IFLT) and, graft and patient survival. They present an impressive success given the complexity of the procedure, however this is its key limitation. Despite the improvement in EAD, strictures and post-reperfusion syndrome there was no measurable benefit in patient or graft survival within the first year and none of the strictures require intervention. It was done in a set of low risk DBD donors, a cohort in which similar benefits have been seen with NMP alone. There are technical limitations, it was performed with a liver assist device which is not transportable, thus donor and recipient must be in the same location. The technique is of interest and a great technical achievement, but a study of larger numbers with a wider range of DBD donors and longer-term follow-up is required.This small unblinded randomised trial was conducted in a single high volume transplant centre in China by the group who have been pioneering the ischaemia-free liver transplant technique since its first publication in 2018. Images and videos of their technique have been included in their 3 publications on their reports and protocols. The IFLT cohort was 3.Modified intention-to-treat analysis.Yes.ChiCTR1900021158.Non-industry funded.in situ normothermic perfusion. The liver was then procured and moved to the reservoir of the Liver Assist for ex situ normothermic machine perfusion and moved to the recipient locality for transplant. For the liver implantation to the recipient, the anastomoses of the inferior vena cava, portal vein, and hepatic artery were performed under continuous in situ normothermic machine perfusion. Machine perfusion was discontinued after the donor liver had been revascularized. Then the biliary tract was reconstructed.This is a very interesting randomised controlled trial in liver transplantation that has the potential to significantly change practice and improve transplant outcomes. 68 liver transplant recipients from donation after brain death were randomised to standard treatment or for an “Ischemia-Free Liver Transplant” (IFLT). The trial was conducted at a single hospital in China. The study was adequately randomised, but the clinical team could not be blinded to the intervention, understandably. For the intervention group, the Liver Assist device  was used to establish There was therefore zero cold ischemic time for the IFLT group. Mean cold ischaemic time in the standard care group was approximately 7 h, and mean normothermic perfusion time in the IFLT group was approximately 7 h.The primary outcome was Early Allograft Dysfunction (EAD) and this was significantly reduced by IFLT (6% versus 24%), as were peak ALT, AST and bilirubin levels. Post-reperfusion syndrome was dramatically reduced, from 64% to 9%. Non-anastomotic biliary strictures were also significantly reduced (8% versus 36%), although this was recorded as seen on protocol MRCP.This clinical trial has shown a dramatic reduction in the ischemia reperfusion injury of transplant livers through the novel use of technology to remove the cold ischemic phase of the organ preservation period. The donor liver is kept warm and perfused all through the process of procurement from the donor body, preservation outside the body, and during the implant into the recipient up until the moment of reperfusion with the recipient’s blood. The technique clearly improved early transplant function. The reduction in non-anastomotic strictures was largely asymptomatic, so it remains to be seen if this technique can significantly reduce the risk of symptomatic strictures in higher risk livers.


In Group B (n = 35), specific tension was applied to the grafts with the use of a tensioner device  Graft Tensioning System). A total of 60 N was applied to the semitendinosus, and 40 N was applied to the gracilis. Clinical outcomes were assessed at 6, 12, and 24 months. Results: There were no significant differences between the baseline demographic and clinical data among the patients of the two groups . The patients were followed up for a minimum of 24 months (mean ± SD). There were no significant differences in the side-to-side anterior knee laxity, the IKDC, the Lysholm Knee, and the Tegner Activity Scale scores for up to 24 months after operation. The pivot shift test was negative in all cases, and no graft failure was reported at a 2-year follow-up. Conclusion: No significant differences were found with respect to postoperative anterior knee laxity, clinical outcomes, activity level, and patient satisfaction between the application of manual tension and the use of the graft-tensioning system during tibial fixation while performing anatomic single-bundle ACL reconstruction with a quadruple hamstring tendon graft. Further high-quality clinical studies are required to elucidate whether device-assisted tension is superior to manual tension.The optimal initial graft tension during ACL reconstruction is still a matter of debate. Manual tension is commonly applied to the graft during tibial fixation. However, this has been associated with a greater graft failure rate than that associated with device-assisted tensioning. This study aims to compare the clinical outcomes between the application of manual tension and the use of the ConMed Linvatec SE™ Graft Tensioning System during graft fixation while performing anatomic single-bundle ACL reconstruction. Methods: A prospective comparative study was conducted between September 2015 and May 2017. Sixty-four patients  with isolated ACL injuries (and who would be subjected to ACL reconstruction with a quadruple hamstring tendon graft) were divided into two groups. In Group A ( Anterior cruciate ligament (ACL) reconstruction with a patellar tendon or hamstring autograft is currently the most common surgical technique for managing ACL ruptures. ACL reconstruction techniques using autografts have shown satisfactory results. Implantation techniques, graft fixation, and tensioning, along with postoperative rehabilitation, have demonstrated significant improvement ,2. DespiAn important variable affecting the outcome of ACL reconstruction is the in vivo tensioning of the graft at the time of fixation ,4. The iCurrently, the most common method for applying graft tension is manual tensioning, where a maximum one-handed pull on the graft is applied during tibial fixation, with an estimated tension of 41–60 N. Manual tensioning is an important step in the surgical procedure to ensure that the reconstructed ACL graft is appropriately tensioned. It involves applying a specific amount of tension to the graft to mimic the natural tension and length of the original ACL. The goal is to achieve optimal graft tension that allows for proper knee stability and a functional range of motion. Manual tension is the preferred tensioning method for 80% of Australian surgeons because of its easiness . HoweverSeveral factors influence the graft-tensioning process. The choice of graft can impact the tensioning requirements. Different grafts  have varying biomechanical properties and degrees of elasticity, thus affecting the ideal tensioning range . IndividWhile manual tensioning is simple, it causes a greater degree of variability in the amount of tension applied compared to device-assisted tensioning . PreviouThe value of this study is that it offers information and data to assess whether the use of graft-tensioning systems provides any additional benefit for the patients in comparison to the application of manual tension during ACL reconstruction. Specifically, the aim of this study is to compare the clinical outcomes between the application of manual tension and the use of the ConMed Linvatec SE™ Graft Tensioning System during graft fixation while performing anatomic single-bundle ACL reconstruction with a quadruple hamstring tendon graft. The current study hypothesizes that applying a tensioning device may be associated with greater postoperative knee stability and better functional outcomes compared to applying manual tension.A prospective, comparative study was conducted at our institution between September 2015 and May 2017. The study was approved by the corresponding institutional review board before initiation. All patients provided informed consent after a full explanation of the study protocol. Every participant of the study was skeletally mature and had been suffering from unilateral ACL deficiency at the time of surgery. The diagnosis of ACL tear was based on patients’ history, clinical examination, and magnetic resonance imaging (MRI) of the knee. Exclusion criteria were meniscal injuries, bony avulsions, multi-ligamentous injuries, chondral lesions, and osteoarthritis. Moreover, patients that had undergone any form of previous knee operation were excluded from the study.n = 29), common tension was applied manually to both grafts. In Group B (n = 35), a specific level of tension was applied to the grafts with the use of a tensioner device [ConMed Linvatec SE™  Graft Tensioning System]. A total of 60 N was applied to the semitendinosus, and 40 N was applied to the gracilis tendon.Patients’ demographic data, such as gender, age at the time of operation, height, and weight, were recorded. Bone mass indices (BMIs) were also calculated. The time interval between injury and operation and the width of the femoral and tibial tunnels were also recorded. Sixty-four patients with isolated ACL injuries who were to be subjected to ACL reconstruction with a quadruple hamstring tendon graft were divided into two groups. In Group A ). The angle of tibial fixation was 15°. The knee’s range of motion was determined. At this stage, the graft was tensioned via repeated cycling (20 cycles). A wire was passed through the tibial tunnel beside the graft. In Group A, graft tensioning was achieved through free one-hand pull force. In Group B, the graft was fixed using the ConMed Linvatec SE™ Graft Tensioning System, applying 60 N to the semitendinosus and 40 N to the gracilis. A tibial interference screw was placed centrally by separating the bundles of the graft and placing the bundles on the entire surface of the bone tunnel. The manual tension applied by the first author, using a dynamometer, was measured for 5 patients, and mean tension was calculated at 55.3 N.The knee joint was thoroughly irrigated to remove any debris or blood. The incisions were closed with staples, and sterile dressings were applied. Patients were placed in a hinged knee brace for postoperative protection and stability. Postoperatively, all patients followed the same rehabilitation program. No running was allowed until four months after surgery. Return to full sports activity was generally permitted at nine months.The first author conducted the patients’ evaluations. A KT-1000 arthrometer  was used to measure anterior tibial translation at maximum manual pull, which is expressed as the difference between the injured and uninjured knee in millimeters (mm). The anterior tibial translation was measured preoperatively, intraoperatively under general anesthesia, and postoperatively at 0, 3, and 6 months following ACL reconstruction. The International Knee Documentation Committee Subjective Knee Form (IKDC), the Lysholm Knee Score, and the Tegner Activity Scale questionnaires  were used to assess functional outcomes ,19. All t-test for independent samples or the Mann–Whitney test in case the data did not follow a normal distribution. All statistical analyses were performed with the statistical package SPSS, version 17.00 . All tests were two-sided. A p-value of <0.05 was set as the level defining a statistically significant difference.The values of the variables are presented using the number of participants (N), the mean values (M), and the standard deviations (SD). Frequencies (n) and the corresponding percentages (%) were used for categorical variables. The normality of distribution of the measurements was assessed using the Kolmogorov–Smirnov test. Comparisons of the variables at any given time between the two groups were performed using the The sample size was initially 111 patients subjected to ACL reconstruction. Preoperatively, 30 patients were excluded due to meniscal injuries, and 2 patients were excluded due to collateral ligament injuries or chondral lesions. After intraoperative inspection, 14 more patients were excluded due to meniscal injuries, and 1 patient was rejected due to a chondral lesion. Finally, 64 patients were included in the study.2 (range 22.37–26.07 kg/m2). As shown in The study cohort consisted of 64 patients (50 men-14 women), with a mean age of 29.3 years (range 14–45 years). The mean weight was 72.4 kg (range 61–88 kg), the mean height was 173.1 cm (range 162–185 cm), and the mean BMI was 24.14 kg/mp > 0.05).p = 0.172 and p = 0.450, respectively). No graft failures were reported at the 2-year follow-up.The present study compared the clinical outcomes of applying manual tension and using a tensioner device during graft fixation upon performing ACL reconstruction. The most important finding was that manual or device-assisted graft tensioning did not result in any statistically significant differences concerning the postoperative objective anteroposterior knee laxity, the IKDC score, the Lysholm knee score, and the Tegner Activity Scale score after a 2-year follow-up. No cases of graft laxity or knee instability were detected in either group.The findings of this study agree with those reported in a randomized controlled trial by Grunau et al., who found no differences in anterior knee laxity, IKDC score, and quality of life between the manual tensioning of single-bundle hamstring ACL grafts and device-assisted tensioning among 127 ACL reconstructions . On the The clinical outcome of arthroscopically assisted ACL reconstruction is affected by the initial graft tension at a certain degree of knee flexion . Under-tThe ideal tension of an ACL graft is still a matter of controversy. In a randomized controlled trial, the application of 90 N of tension lead to a better clinical outcome in patellar tendon grafts compared to 45 N of tension . BiomechThe technique of applying manual tension is widely preferred while performing graft fixation in ACL reconstruction as it is a simple method that is easy to perform. Some surgeons prefer to fix the graft at a certain level of tension using a manual tensioner ,32. HoweThe present study has certain limitations. Firstly, the selection of patients was not randomized; however, the baseline demographic and clinical data were not different among the patients of the two groups. Secondly, we supposed that the tension applied manually was similar in all cases in Group A, but we did not repeat the measurement for all patients. According to Cunningham et al., most surgeons apply a “sufficient magnitude” of initial tension, ranging from 40 to 90 N, to a graft at full extension or a slightly flexed position . Lastly,No significant difference was found regarding postoperative anterior knee laxity, clinical outcomes, activity level, and patient satisfaction between the application of manual tension and the use of the ConMed Linvatec SE™ Graft Tensioning System during graft fixation while performing an anatomic single-bundle ACL reconstruction with a quadruple hamstring tendon graft. Further high-quality clinical studies are required to elucidate whether device-assisted tension is superior to manual tension.


Skin Health and Disease, the words of the late former German chancellor Helmut Schmidt reverberate in my thoughts. Loosely translated, he once quipped that ‘If you have visions, you need to see a doctor.’Sitting in my office in Lübeck on a late Spring Friday afternoon, charting the future editorial direction of Skin Health and Disease, the first new journal from the British Association of Dermatologists in over half a century, started as a vision. The Society recognised the importance and potential of a fully open access Dermatology journal and, under the leadership of George Millington and his editorial team, have delivered an international, multidisciplinary journal covering all aspects of dermatology from basic science to translational and clinical research. The journal has published 10 issues to date, including two special editions centred on psychodermatologyJoking aside, having a vision is crucial to organise and plan our direction of travel. As I take over at the helm, is it already mission accomplished?Well, we've come a long way in the last 3 years but there's still plenty of work ahead. Although the journal is indexed and searchable on PubMed Central, we are applying to be indexed on Medline. We have also applied to be indexed on Web of Science, which will allow us to gain our first impact factor next year. However, we recognise that impact factor is not the only measure of the success of journals. We are accessible worldwide and the publication reporting new onset vitiligo following COVID‐19 infection was downloaded 5281 times. In 2022, we had 77 820 downloads in total.With new article categories, including Scientific/Research letters and Images in Dermatology and a clear social media presence we're striving to attract even more high‐quality submissions, hallmarked with robust methodology, scientific validity and reliability. In addition, we've developed a competitive pricing plan, with APC discounts available on several article types and waivers and discounts to corresponding authors based in low‐ and lower middle‐income countries.Aiming to improve our recognition both nationally and internationally, as a key member of the British Association of Dermatologists' journal family, we want to secure our place as the ‘go to’ open access journal for interdisciplinary and multi‐professional dermatology research. In fact, we can now announce our third special issue which will focus on skin biology and ageing, meeting our commitment to focus on skin health, and its maintenance, throughout the life cycle.The task of turning our vision into reality has really only just begun and I'm looking forward to the journey.Ewan A. Langan: Writing–original draft (Lead); Writing–review & editing (Lead).The author declares no conflicts of interest.This article received no specific grant from any funding agency in the public, commercial, or not‐for‐profit sectors.Not applicable.


As expected, pasta samples showed the highest value of SDS/av starch compared to couscous and bread. Fusilli and cavatelli samples presented the highest SDS/av starch ratio , then came spaghetti 49.39 ± 2.83% and penne 45.93 ± 1.19%, while couscous presented the lowest value of SDS/av starch (2.64 ± 0.50%), followed by bread (11.78 ± 2.63%). Our study confirmed that the pasta making process efficiently mediates an increase in SDS/Av starch content, which has been specifically quantified above 40%, therefore strongly related to a lowered glycemic response in vivo. Our results strengthened the concept that pasta is a good source of SDS, which makes it useful for glycemic control. The rate at which starch is digested in the human intestine elicits different glycemic responses and reflects the glycemic index (GI) of foods. In vitro measurement of starch digestibility can reflect the GI of food. Differences in starch digestibility among four durum wheat pasta samples, couscous, and bread were evaluated to better describe the role of the pasta making process in affecting starch digestibility. Statistical differences in RDS (rapidly digestible starch), SDS (slowly digestible starch), and RS (resistant starch) of products were found ( The rate at which starch present in foods is digested and absorbed within the gastrointestinal tract elicits different glycemic responses, which are usually quantified to calculate the Glycemic Index of foods (GI) . In factAmong cereals, pasta represents an important staple food, contributing to the intake of complex carbohydrates. Compared to other carbohydrate-rich foods, pasta stimulates a lower post-prandial glycemic response, representing an important low GI element of the Mediterranean diet ,11. ThisBecause of this, different starchy products, made starting from the same raw materials but different processes, may have different GI values. This is the case for two other starchy wheat-based foods commonly consumed within the Mediterranean area, such as couscous and bread ,14. In fTherefore, the aim of this study was to investigate the effect of pasta production on the SDS content in comparison with other semolina-based products, such as couscous and bread; moreover, different shapes (both short and long) of wheat-based pasta  were analyzed for better understanding the role of different pasta process in affecting starch digestibility.v/v), mixing gently, and letting it stand for 5 min before the analysis. Bread was baked using the ingredients and following the procedure described in In the present study the starch digestibility of 4 durum wheat pasta samples, one sample of bread and one sample of couscous were analysed; in order to avoid any bias deriving from using a different type of ingredients, all the samples were prepared with the same type of semolina. Couscous and semolina for bread preparation were industrially manufactured by Barilla G. e R. F.lli S.p.A and commercially available in Europe. Couscous was prepared by pouring boiling water on couscous  were analysed in order to evaluate the influence of pasta production and pasta shape on starch digestibility. Penne, fusilli, and cavatelli were short pastas, while spaghetti was a long pasta. To reduce the variation due to different grain harvests and to limit industrial and technological variabilities, the pasta samples were obtained the same year of production from a single factory. After industrial production, all pasta samples were stored as per package instruction. Therefore, once received, the samples were kept at room temperature in a cool and dry place. Each pasta sample was cooked in unsalted boiling water (70 g of pasta in 1 L of boiling water), according to the package instruction. The preparation instructions for all test foods are shown in The percentage of SDS, RDS, and RS of tested foods were measured according to the method proposed by Englyst and colleagues . Briefly20), and 120 min of incubation (G120),was analysed using an automatic glucose analyser . Therefore, rapidly available glucose (RAG), slowly available glucose (SAG), RDS, and SDS were calculated as follows:Glucose released after 20 min of incubation  with Tukey HSD as a post hoc test. Statistical analysis was performed using SPSS software .p < 0.05). As expected, bread showed the highest value of RDS (33.20 ± 0.79 g/100 g), followed by couscous (30.40 ± 0.58 g/100 g). Pasta samples showed a significantly lower values from bread and couscous, with cavatelli having 11.54 ± 0.81 g/100 g of RDS, fusilli having 12.01 ± 1.56 g/100 g of RDS, followed by spaghetti having 14.64 ± 0.69 g/100 g of RDS, and penne having 15.50 ± 0.97 g/100 g of RDS (p < 0.05). Considering the SDS content, pasta samples did not show any statistical difference among the different shapes analysed (p > 0.05), with SDS ranging from 13.52 ± 1.29 g/100 g for cavatelli, to 15.07 ± 0.16 g/100 g for fusilli, while bread and couscous presented a significantly lower content of SDS than the pasta samples . As expected, RS represents only a minor fraction in these products. After comparing results, bread showed the highest content of RS (1.06 ± 0.05 g/100 g), followed by spaghetti and fusilli, cavatelli, and penne, which presented similar RS content, and couscous had the lowest value.The values of RDS, SDS, and RS (g/100 g product “as eaten”) for each product are reported in p < 0.05). The pasta samples showed a higher value of SDS/av starch compared to couscous and bread. Fusilli and cavatelli samples showed the highest ratios of SDS/av starch  and the lowest ratios of RDS/av starch . Spaghetti showed intermediate ratios of both SDS/av starch (49.39 ± 2.83%) and RDS/av starch (50.61 ± 2.83%), followed by penne sample that had the lowest ratio of SDS/av starch (45.93 ± 1.19%) and the highest ratio of RDS/av starch (54.06 ± 1.19%), among pasta samples. Couscous had the lowest value of SDS/av starch (2.64 ± 0.50%), followed by bread (11.78 ± 2.63%). Considering the RDS/av starch content, bread and couscous showed the highest value .The % contribution of RDS and SDS as available starch (considered as 100%) is graphically reported in In the present study, the effect of pasta making process on the quality of starch (express as SDS/av starch) has been analysed. Pasta resulted in a mean 39% larger increase in SDS/av starch than bread, and this was 49% higher than couscous. Results obtained confirm that the technological process behind the production of pasta may be responsible for the greater level of SDS, resulting in a significant reduction in rate of starch digestion compared to bread and couscous. Considering that all the analysed pastas, independently from the shape, showed values of SDS/av starch above 40%, they resulted all potentially eligible for bearing the approved health claim on the lowered post-prandial glycemic response in vivo.In 2011, European Food Safety Authority (EFSA) confirmed that the rate of starch digestibility assessed in vitro in cereal products can be considered a good parameter to predict the in vivo effect on post-prandial glycemic responses. In particular, a cause–effect relationship has been established between the consumption of cereal products that have at least 40% of available starch as SDS and reduced post-prandial glycemic responses . More reDespite the positive characteristics described for pasta, a reduction in consumption of this food during the years has been reported. One of the main reasons behind this dietary habit is probably due to the overflowing fake news about carbohydrate-rich foods consumption and gain weight, even though several scientific contributions clearly debunk this myth . Among cThe present study presents some strengths and limitations worth highlighting. The first strength is linked to the samples analysed, all specifically produced starting from the same semolina to avoid any bias deriving from different wheat cultivars, batches, and milling processes used. Moreover, bread was produced without the addition of other ingredients interfering with the SDS and GI of products, such as fats , in order to study only the effect on SDS induced by the different technological process applied. Limitations are certainly linked to the absence of a complete characterization of other technological and molecular parameters that could better describe this in vitro behaviour. Lastly, as already well described in a previous work published , in vivoThe present work supports the evidence that pasta making process is an efficient method to enhance the content of SDS on available starch. Moreover, pasta showed a good content of SDS and a relevant SDS/Av starch ratio, which has been specifically quantified for these samples above 40%, and, therefore, it potentially mediates a reduction in post-prandial glycemia, as described in the scientific opinion on SDS published by EFSA. On the contrary, other starchy foods commonly consumed as alternative to pasta, such as bread and couscous, made with the same durum wheat semolina, presented a scarce content of SDS/av starch, therefore reflecting their higher glycemic response in vivo. Our results strengthened the concept that pasta, despite the size and shape, is a good source of SDS, which makes it useful for the glycemic control associated with several health benefits, including reduced insulin demand, improved blood glucose control, and reduced blood lipid levels. Additionally, the scientific literature has highlighted that the consumption of pasta is involved in decreasing body weight, resulting in optimal carbohydrate-based foods, which is also the case for obese or overweigh individuals. These are important elements for scientifically supporting the consumption of pasta products, considering the recommended portion size and consumption frequency, as they may play an important role in the promotion of health and prevention and management of several chronic diseases.


Plasmodium falciparum, Trypanosoma cruzi, and Dirofilaria repens. Bacteriophages offer advantages such as being immunogenic carriers, low production costs, and customization options, making them a promising alternative to traditional vaccines. The purpose of this study has been to highlight an approach that encompasses the entire process from epitope identification to vaccine production using a single technique, without requiring additional manipulation. Unlike conventional methods, phage display demonstrates exceptional efficiency and speed, which could provide significant advantages in critical scenarios such as pandemics.Traditional vaccines use inactivated or weakened forms of pathogens which could have side effects and inadequate immune responses. To overcome these challenges, phage display has emerged as a valuable tool for identifying specific epitopes that could be used in vaccines. This review emphasizes the direct connection between epitope identification and vaccine development, filling a crucial gap in the field. This technique allows vaccines to be engineered to effectively stimulate the immune system by presenting carefully selected epitopes. Phage display involves screening libraries of random peptides or gene/genome fragments using serum samples from infected, convalescent, or vaccinated individuals. This method has been used to identify epitopes from various pathogens including SARS-CoV-2, Mycobacterium tuberculosis, hepatitis viruses, H5N1, HIV-1, Human T-lymphotropic virus 1,  Epitopes are crucial components in the development of effective vaccines against infectious pathogens. These regions on the antigen’s surface engage with antibodies, B-cell and T-cell receptors, and serve as the portions of a pathogen that the immune system identifies and attacks. By identifying and selecting the most potent epitopes, it becomes feasible to design vaccines that trigger a strong immune response against the pathogen, while minimizing harm to the host ,2.Mycobacterium tuberculosis. These epitopes not only hold value in the realm of Tuberculosis (TB) serodiagnosis but also play a crucial role in the development of a potential TB vaccine , and M2 ectodomain .In another study, sera from individuals immunized with the H5N1 A/Indonesia/05/2005 (clade 2.1) VLP vaccine, which was produced in Sf9 insect cells, were used. The study aimed to identify epitopes of the influenza virus hemagglutinin (HA1/HA2) and neuraminidase (NA) using a GFPDL library. The purified antibodies derived from these sera exhibited superior recognition of the oligomeric form of HA1 compared to its monomeric form. Moreover, these antibodies demonstrated NA-inhibiting activity, attributed to their binding to a C-terminal epitope in close proximity to the sialic acid binding site .In a study conducted by Scala et al. (1999), mimotopes were isolated from phage display libraries utilizing sera obtained from HIV-infected patients. Among the selected mimotopes, there were ones that accurately mimicked HIV epitopes generated during infection, specifically regions of gp120 C2 and gp41. These mimotopes were not only recognized by sera from SHIV-infected monkeys but also stimulated the production of neutralizing antibodies when administered to immunized mice .Similarly, mimotopes of HIV-1 were identified from M13 phage libraries by employing sera from individuals infected with HIV-1 subtype B. These particular mimotopes effectively mimicked the V3 crown epitope of HIV-1, resulting in the production of antibodies that bound to V3 peptides .In another notable study conducted by Palacios-Rodríguez et al. (2007), CxxKxxC sequences were identified from a C7C phage display library using IgG obtained from HIV-1-infected patients. These sequences exhibited a resemblance to the immunodominant epitope CSGKLIC found in the envelope gp41 glycoprotein. When administered as a mixture in mice, these sequences induced higher titers of antibodies against the HIV-1 epitope .Machado et al. (2022), conducted a study aimed at identifying mimotopes of Human T-lymphotropic virus 1 (HTLV-1) epitopes. To accomplish this, they utilized a pool of IgG from HTLV-1 patients to screen a phage display library. The primary objective was to discover specific peptide sequences that could mimic critical regions of HTLV-1 antigens. This approach provided them access to a diverse repertoire of potential mimotopes through a random 12-mer peptide library. Consequently, they were able to screen and select peptides that closely resembled the targeted epitopes, displaying a high degree of similarity. Notably, the chosen mimotopes closely resembled epitopes found in the envelope protein (gp46), Tax protein, and HTLV-1 protease, demonstrating promising characteristics in terms of their potential immunological relevance. These mimotopes could effectively serve as surrogate antigens, triggering immune responses similar to those elicited by the authentic HTLV-1 antigens .Plasmodium falciparum-infected erythrocyte surface antigens, a study employed a phage display library. Antibodies that were extracted from a pool of sera from malaria-infected individuals using P. falciparum-infected erythrocyte were used to screen the phage display library. After multiple rounds of panning several phage clones were selected. Among the 23 randomly chosen sequenced clones, 13 unique sequences were identified, and 3 of these sequences showed similarity to membrane proteins known to exist on P. falciparum-infected erythrocyte. Interestingly, most of the phage clones (7 out of 8) selected after the 4th panning exhibited specific binding to IgG from malaria-infected individuals. This specific binding was confirmed using sera from 24 malaria-infected individuals and 11 from uninfected individuals. Notably, one phage clone was most frequently detected after the 4th panning, demonstrating stronger binding to IgG in all malaria-infected individuals samples compared to any serum from uninfected individuals. Moreover, a rabbit antiserum raised against the peptide expressed by this clone specifically recognized the surface of P. falciparum-infected erythrocyte, leading to their hemolysis [To identify mimotopes of emolysis .Trypanosoma cruzi enables the identification of thousands of antigens recognized by serum samples from patients with Chagas disease. Previous studies have shown a correlation between levels of anti-T. cruzi antibodies in patients and the persistence of the parasite, as well as eventual disease resolution. Certain T. cruzi antigens have been proposed as indicators of therapeutic efficacy. Therefore, the antibody response map generated through genome phage display holds significant potential in identifying and improving new biomarkers for Chagas disease. While four of the selected epitopes identified in this study demonstrated a noticeable correlation with the corresponding IgG pools, none of them proved to be specific markers for disease status. However, the study successfully validated at least five epitopes that are novel and not found in the existing database. The genomic phage platform presented in this study is a reproducible and effective tool for the rapid simultaneous identification of epitopes and antigens, not only in Chagas disease but also in emerging/reemerging acute pathogens on a global scale [Teixeira et al. (2021) developed an integrated platform for genome phage display of the eukaryotic parasite al scale .Dirofilaria repens. This innovative approach facilitated the discovery of highly immunoreactive peptides that can serve as novel diagnostic markers for D. repens infections [Through the utilization of phage display technology and a 12-mer peptide library, Pękacz et al., (2022) successfully identified epitopes that exhibited a high reactivity with IgG from sera of dogs infected with fections .Vaccine development traditionally involves using inactivated or attenuated forms of pathogens or their components to stimulate an immune response. However, these approaches can have drawbacks, including side effects or inadequate immune responses. To overcome these challenges, researchers have turned to phage display technology, which enables the identification of specific epitopes that can selectively target the pathogen, reducing the risk of side effects. By selecting peptide sequences that bind to a particular target, phage display facilitates the design of vaccines that effectively elicit an immune response against the target.In this innovative approach, phage particles displaying the chosen epitopes are administered to patients, prompting their immune systems to recognize the epitopes as foreign and mount an immune response against them. This response can prove effective in combating the target, whether it be a virus or cancer cell.Phage-based vaccines offer numerous advantages over traditional vaccines. They have the capacity to elicit both humoral and cellular immune responses, which are crucial for comprehensive protection against pathogens. Additionally, these vaccines are relatively easy to produce and cost-effective , making However, there are still challenges that need to be addressed to optimize the selection and delivery of epitopes in phage-based vaccines, ensuring their maximum effectiveness in generating a protective immune response. It is crucial to continue exploring methods that enhance the stability and immunogenicity of selected epitopes, while also developing strategies for the efficient delivery and presentation of phage particles to target cells. By overcoming these challenges, phage display technology holds immense promise for the development of highly targeted and effective vaccines against a wide range of diseases, including infectious diseases and cancers.The identification and selection of epitopes play a crucial role in the development of vaccines, diagnostic tests, and therapeutic antibodies. Various techniques are available for this purpose, including X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, peptide arrays, site-directed mutagenesis, bioinformatics and computational modeling, and phage display. The choice of technique depends on the specific application and the type of epitope or mimotope under investigation. Among these techniques, phage display stands out due to its simplicity in epitope selection and screening. Moreover, the resulting phagosomes can be used as vaccines without requiring additional manipulation.To successfully select epitopes or mimotopes using phage display, several important steps must be carefully executed. Firstly, a phage display library is needed to facilitate the selection of target epitopes. These libraries can either be constructed in-house or obtained as pre-made libraries, such as those provided by New England Biolabs. There are two main types of libraries commonly used: random phage display peptide libraries (RPL) and gene or genome fragment libraries (GFL). Each type of library has its own advantages and disadvantages, and the choice depends on factors such as the specific research question, the nature of the target protein, and the available resources.Obtaining the appropriate source of antibodies is another critical aspect when selecting epitopes for the library. These antibodies can be derived from patients who are recovering from an infectious disease or individuals who have been immunized with a vaccine. It’s important to note that these antibodies need to undergo purification using protein A/G affinity chromatography or antigen-based chromatography, if the antigen is available, to ensure that selection is not biased against other components present in the samples. However, it should be acknowledged that serum samples typically contain polyclonal antibodies, which necessitates a screening assay following the selection process.Before commencing the selection or biopanning process, two protocols must be prepared and optimized. The first protocol involves biopanning the library, wherein a pool of phage clones recognized by the antibodies is selected. The second protocol is employed to identify the most promising hits or binders of interest within this pool, usually through a phage ELISA or an inhibition assay.In addition to the steps outlined in the biopanning process, it is essential to highlight the importance of performing a subtractive selection using antibodies from negative samples. This critical step aims to eliminate non-specific binders in the library and clones that may bind to the constant part of the antibodies. To mitigate any potential interference during the panning process, it is recommended to add these antibodies to the phage library. One approach to achieving this is by incubating the serum sample on a surface coated with helper phages  or the original phage without an insert  This effectively removes phage-specific antibodies before further utilization.After obtaining the selected hits, it is important to optimize them if needed, possibly through techniques like site-directed mutagenesis. Subsequently, a thorough characterization of the hits is necessary to assess their antibody binding capability, their potential to elicit an immune response, and the effectiveness of that immune response.The epitopes of a pathogen identified through phage display have diverse applications, including the development of diagnostic tests, understanding pathogen-host interactions, and the creation of therapeutics such as monoclonal antibodies, small molecule inhibitors, and vaccines.Moreover, numerous studies have employed phage display techniques in the context of infectious diseases, using two key components: a phage display library and antibodies from samples taken from infected, convalescent, or vaccinated individuals. For instance, in the case of hepatitis A virus (HAV), mimotopes were identified using antibodies from hepatitis A patients. One of the identified phages exhibited the ability to hinder the attachment of anti-HAV antibodies, eliciting a specific immune response in mice. Similarly, phage display libraries were utilized to discover mimotopes of the hepatitis B virus (HBV) surface antigen (HBsAg), leading to the generation of highly specific antibodies in vaccinated mice. Furthermore, mimotopes of the human hepatitis C virus (HCV) core protein were identified using serum from an HCV-infected patient. Comparable studies were conducted for hepatitis E virus (HEV), H5N1 influenza, HIV, and Human T-lymphotropic virus 1 (HTLV-1), resulting in the identification of mimotopes and the induction of immune responses. These studies exemplify the immense potential of phage display libraries in identifying specific epitopes for vaccine development, designed to be safe and effective in preventing the associated diseases.The development of vaccines has traditionally relied on the use of inactivated or attenuated forms of pathogens. However, these methods have their limitations, such as side effects and insufficient immune responses. To address these challenges, phage display has emerged as a valuable tool for identifying specific epitopes that can selectively target pathogens, mitigating the risk of side effects associated with using whole pathogens. By harnessing the power of phage display, vaccines can be engineered to effectively stimulate the immune system by presenting carefully chosen epitopes.It is worth noting that phage-based vaccines are not currently available in the market. Nonetheless, research and development in this area have shown promising results. For instance, bacteriophage vaccines based on M13 have demonstrated safety in both humans  and animAnother significant advantage of phage-based vaccines is their relatively low production cost. This makes them particularly suitable for regions with limited resources that are frequently affected by infectious diseases. These regions often face challenges in accessing and affording conventional vaccines. By developing and producing their own phage-based vaccines, these regions can gain more control over their healthcare systems and contribute to improved disease prevention and public health outcomes.Additionally, phage-based vaccines offer the flexibility of customization. Researchers can select and optimize specific epitopes from pathogenic organisms, tailoring the vaccine to target the most relevant components for an effective immune response. This precision in epitope selection can lead to vaccines that are highly specific and potent in combating infectious diseases.However, it is important to note that further research and development are needed to address certain challenges associated with phage-based vaccines. These challenges include improving the stability of phage particles, enhancing immunogenicity, and ensuring efficient delivery to target cells. Researchers are actively exploring various strategies to optimize these aspects and maximize the potential of phage display technology in vaccine development.This comprehensive review illuminates the unique perspective of phage display, establishing a direct connection between epitope identification and vaccine development to address a specific gap in the field. In


Arteries can stiffen via different mechanisms due to the distending effects of blood pressure, the extracellular (ECM) and vascular smooth muscle cells (VSMC). This short review discusses how these simple models can be applied to the complex biomechanics of arteries to gain physiological insight into why an individual’s arteries are stiff and identify new therapeutic strategies. In the Multi-Ethnic Study of Atherosclerosis, the important question of whether arteries stiffen with aging due to load-dependent or structural stiffening was investigated. Structural stiffening was consistently observed with aging, but load-dependent stiffening was highly variable. Importantly, the high load-dependent stiffness was associated with future cardiovascular disease events, but structural stiffness was not. Clinical studies in older, hypertensive adults surprisingly show that decreasing vascular smooth muscle tone can cause clinically significant increases in arterial stiffness. To understand this paradox, the author developed a model simple enough for clinical data but with biologically relevant extracellular matrix (ECM) and vascular smooth muscle cell (VSMC) stiffness parameters. The effect of VSMC tone on arterial stiffness depends on the ECM–VSMC stiffness ratio. Future research is needed to develop a framework that incorporates both the blood pressure dependence of arterial stiffness and the VSMC–ECM interaction on hemodynamics. This could result in personalized arterial stiffness treatments and improved CVD outcomes. The subtitle of this review is “Learning to De-Stiffen Arteries” because our results have so far only shown that we can acutely make arteries stiffer. We are optimistic though that the findings and the analytic techniques covered here will be one of the many steps along the path of the arterial stiffness research community learning how to de-stiffen arteries. Hypertension is a major cause of renal, neural, and cardiovascular disease (CVD), putting the many individuals with poor blood pressure control at high risk for end-organ damage and death . ArteriaArteries can stiffen via different mechanisms and without understanding why an individual’s arteries are stiff, the ability to personalize treatment based on arterial stiffness is limited. Mathematical models of arterial biomechanics are a useful tool for understanding the blood pressure dependence of arterial stiffness in addition to factors such as extracellular matrix (ECM) vs. vascular smooth muscle cell (VSMC) mechanics , 6. HoweThis short review discusses how these simple models can be applied to the complex biomechanics of arteries to gain physiological insight into why an individual’s arteries are stiff and identify new therapeutic strategies. The subtitle of this review is “Learning to De-Stiffen Arteries” because we have so far only shown that we can acutely make arteries stiffer. We are optimistic though that the findings and the analytic techniques covered here will be one of the many steps along the path of the arterial stiffness research community learning how to de-stiffen arteries.Using carotid artery ultrasound data from the Multi-Ethnic Study (MESA) , myself First, a traditional stiffness metric  is calculated at the individual’s actual blood pressure to represent total arterial stiffness. Second, a single exponential model is fitted to the measured pressure–diameter data using Eq. PWVLD) would be calculatedLast, load-dependent stiffness is calculated as the difference between total stiffness and structural stiffness. For example, load-dependent pulse wave velocity , we found that structural stiffening was consistently observed with aging but that load-dependent stiffening was highly variable  [As a follow-up analysis, we looked at the association between load-dependent vs structural stiffening with CVD events and incident hypertension in MESA in 5873 participants over an average of 14.3 year of participant follow-up . Surprisllow-up) . Structuk is the VSMC activation (k = 0 equates to fully relaxed VSMCs), kref is a reference VSMC activation, and Historically, increased large elastic artery stiffness has been believed to be primarily due to two main extracellular matrix (ECM)-related mechanisms: (1) load-dependent stiffening due to higher blood pressure and (2) structural stiffening representative of changes intrinsic to the artery. However, there is an increasing amount of data showing that arterial stiffness can acutely be modified independently of blood pressure in humans , 18, while tones . After uincreasing smooth muscle tone can have a stronger or equivalent effect on improving carotid artery mechanics compared with decreasing blood pressure [This work has been highlighted in editorials as an important first step toward understanding the role of VSMCs in arterial dysfunction  and raispressure , althougVSMC mechanics may also contribute to hypertensive response to exercise (excessively increased blood pressure with exercise), which is associated with left ventricular hypertrophy and higher rates of CVD . ExercisRm) is associated with incident heart failure events [Z) in the circulation [PWV is pulse wave velocity, and A is artery cross-sectional area. In each individual, Rm could be elevated due to remodeling of the central arteries, remodeling of the peripheral arteries, the non-linear effect of elevated blood pressure changing arterial stiffness and thus impedance, or a combination of these effects. Simple 1D models of hemodynamics, such as a T-tube model [The cellular and molecular mechanisms underlying changes in the blood pressure dependence of stiffness and the interplay of VSMC and ECM on arterial stiffness are undoubtedly important and should be a focus of future research. It will be equally important, however, to integrate the effect of these changes in central and conduit artery stiffness into the hemodynamics of the whole cardiovascular system. Central–peripheral arterial interactions can be quantified through wave reflections using either time or frequency domain approaches . Wave ree events . From 1Dculation . Impedanbe model  for the be model  for the be model  could beIt may also be possible to understand the impact of VSMC–ECM interactions on central and peripheral arterial mechanics by measuring pressure-flow relationships before and after administration of a vasoactive agent. This analysis could be performed by incorporating the simple exponential smooth muscle mechanics model as a constitutive relationship into 1D hemodynamics models. This analytical approach to assess VSMC–ECM mechanical interactions in the central vs. peripheral vasculature holds great promise for holistically understanding circulatory physiology but would be dependent on future research identifying what experimental data need to be collected to uniquely identify all parameters and if this data could be collected non-invasively in humans.Arteries can stiffen via different mechanisms and simple models can be applied to complex arterial mechanics to gain physiological insight into why an individual’s arteries are stiff and identify new therapeutic strategies. In the Multi-Ethnic Study of Atherosclerosis, the important question of whether arteries stiffen with aging due to load-dependent or structural stiffening was investigated. Structural stiffening was consistently observed with aging, but load-dependent stiffening was highly variable. Importantly, the high load-dependent stiffness was associated with future CVD events, but structural stiffness was not. Clinical studies in older, hypertensive adults surprisingly show that decreasing VSMC tone can cause clinically significant increases in arterial stiffness. To understand this paradox, the author developed a model simple enough for clinical data but with biologically relevant ECM and VSMC parameters. The effect of VSMC tone on arterial stiffness depends on the ECM–VSMC stiffness ratio. Future research is needed to develop a framework that incorporates both the blood pressure dependence of arterial stiffness and the VSMC–ECM interaction on hemodynamics. This could result in personalized arterial stiffness treatments and improved CVD outcomes.