Due to the low solubility of thalidomide in its present form of administration, coupled with its lack of specificity for targeted delivery and controlled drug release, it is an urgent need to find potent delivery methods that can significantly enhance its solubility, optimize the desired site of drug action, and mitigate its toxicity. In this study, the isolated exosomes were incubated with synthetic liposomes to form hybrid exosomes (HEs) that carried THD (HE-THD) with uniform size distribution. The results demonstrated that HE-THD could significantly abrogate the expansion and proliferation of Tregs induced by TNF, and this might result from blocking TNF-TNFR2 interaction. By encapsulating THD in hybrid exosomes, our drug delivery system successfully increased the solubility of THD, laying a foundation for future in vivo experiments that validate the antitumor activity of HE-THD by reducing the Treg frequency within the tumor microenvironment.Thalidomide (THD), a synthetic derivative of glutamic acid, was initially used as a sedative and antiemetic until the 1960s, when it was found to cause devastating teratogenic effects. However, subsequent studies have clearly demonstrated the anti-inflammatory, anti-angiogenic, and immunomodulatory properties of thalidomide, thus providing a rationale for its current use in the treatment of various autoimmune diseases and cancers. Our group found that thalidomide can suppress the regulatory T cells (Tregs), a minor subset of CD4 T+ Tregs, and the data were interpreted to mean that TNFR2, one of the two receptors (TNFR1 and TNFR2) for orchestrating the biological functions of TNF, is necessary to functionally activate Tregs that help tumors escape from host immune surveillance [Thalidomide (THD), a synthetic derivative of glutamic acid , was orieillance .Exosomes (EXOs) are small extracellular vesicles (EVs) with a diameter of 30–150 nm that are derived from endosomal multivesicular compartments and secreted to the extracellular environment when they fuse with the plasma membrane . ExosomeHowever, due to the low yield of exosomes, the quantity of available exosomes for use in application is constrained . In ordeC57BL/6 mice (Ly5.2) and Balb/c mice  were purchased from The Jackson Laboratory (JAX)  and maintained in the Animal Facility of University of Macau under specific pathogen-free conditions. The animal study protocol was approved by the Animal Research Ethics Committee of University of Macau.v/v) Fetal Bovine Serum (FBS)  and 1% penicillin and streptomycin , #15140148,at 37 °C in a 5% CO2 environment.Mouse macrophage cell line raw 264.7 were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) and supplemented with 10%  and cholesterol (#C6231) was purchased from Macklin . Mouse monoclonal antibody CD63, Alix, TSG101 (51), and beta-actin were purchased from. All other reagents and chemicals were of analytical grade. Anti-mouse antibodies (Abs) were obtained from BD Biosciences  including FITC anti-mouse TCRβ (H57-597), PerCP-Cy5.5 anti-mouse CD4 (RM4-5), and PE anti-mouse CD120b . APC anti-mouse/rat Foxp3 (FJK-16s) and Foxp3 Staining Set (FJK-16s) were obtained from eBioscience. The Recombinant Mouse TNF (rmTNF) and rmIL-2 were purchased from BD Pharmingen . CellTrace™ Violet were purchased from Thermofisher Scientific . CD4 (L3T4) MicroBeads mouse isolation kit (#130-097-054) were purchased from Miltenyl . Thalidomide (T144) and lenalidomide were from Sigma-Aldrich  Pomalidomide was from Selleck Chemicals .+ T cells from lymphocytes. A CFSE assay was used to label MACS-purified CD4+ cells. In a U-shaped 96-well plate, the cells (3 × 105 cells/well) were stimulated in the presence of IL-2, with or without TNF for 72 h. Thalidomide, pomalidomide, and lenalidomide at the desired concentrations were added. FACS was used to analyze the proportion and peoliferation of Tregs in CD4 T cells and TNFR2 expression by Tregs.C57BL/6 mice were used to harvest lymphocytes from spleens and lymph nodes . Mouse CD4 (L3T4) Microbeads and MS (Miltenyi Biotec) columns were used to isolate CD4TM violet-labeled lymphocytes from lymph nodes and spleen were planted in U-shaped 96-well plate and stimulated in the presence of IL-2, IL-2 + TNF, and IL-2 + TNF + HE-THD at the desired concentration for 72 h. FACS was used to analyze the expansion and proliferation of Tregs in CD4 T cells and surface TNFR2 expression by Tregs.CellTraceCD4 T cells were incubated with diluted PE anti-mouse CD120b antibodies after blocking FcR. For Foxp3 detection, anti-mouse Foxp3 (FJk-16S) staining kit was used to fix (16 h) and permeabilize the cells before blocking and labeling appropriated diluted antibodies. Acquisition was performed by BD FACSCanto II. FlowJo software was used to analyze the data. Samples were analyzed by BD Fortessa. Data were analyzed using FlowJo software.2 environment to harvest exosomes. The media were collected and centrifuged at 300× g for 10 min to remove cell and cell fragments. Then, the supernatant was collected and centrifuged at 2000× g for 30 min to eliminate larger vesicles. Then, the supernatant was concentrated through Amicon® Ultra-15 Centrifugal Filter Unit at 3000× g. The obtained supernatant was centrifuged at 10,000× g for 30 min at 4 °C to remove debris. Finally, it was high-speed centrifuged at 110,000× g, 2 h) to precipitate exosomes.Exosomes were harvested from raw 264.7 cells using differential centrifugation techniques. Raw 264.7 cells were cultured in an exosome-free DMEM medium for 48 h at 37 °C in a 5% COLiposomes are synthesized via simple membrane hydration and membrane extrusion as described by Pitchaimani et al. . BrieflyHybrid exosomes (HE) were synthesized via simple thin film hydration followed by a membrane extrusion method. Previously isolated exosomes were used to hydrate the dry lipid layer. A total of 200 µg protein equivalent to the exosomes was added to 1000 µg of lipid film in a final volume of 3 mL. It was then vortexed and sonicated  for the proper mixing. Thus, formed multilamellar HE solution was extruded through 400- and 200-nm polycarbonate membrane filter, respectively, to get nano-sized unilamellar HE.The hydrodynamic dimensions and surface charges of liposomes and HEs were characterized using dynamic light scattering tests . Exosomes and HE were characterized via SDS-PAGE analysis and Western blotting. In SDS-PAGE analysis, exosomes and HEs were mixed with a sample loading buffer (1:5) to yield a final protein concentration of 20 µg/mL. The mixture was incubated at 99 °C for 10 min, and 20 µL of each sample was loaded into protein gel wells. The gel was stained with Coomassie bright blue and imaged with a Bio-Rad imager.For Western blotting, after SDS-PAGE was completed, the gel was transferred to the PVDF membrane via a Western blot  for protDrug loading was carried out via the physical encapsulation technique. Because of its low solubility, the powder of THD is initially dissolved in an organic solvent with phospholipids and cholesterol. Thalidomide, phospholipids, and cholesterol are weighed according to a suitable weight ratio. Preferably, 1 mg thalidomide, 30 mg lecithin, and 50 mg cholesterol are placed in a 50-mL round bottom flask, and an organic solvent with 5 mL of ethanol is added to dissolve the above substances, then rotated and evaporated under reduced pressure for 40 min at 50 °C water bath temperature to remove the organic solvent; thus, a uniform transparent film is formed on the bottle wall. Add 3 mL of PBS with or without exosome, shake up, then place in 37 °C of oscillators and shake at a constant speed for 30 min to form the thalidomide phospholipid complex. Thalidomide exosome-liposome nano-preparation was vortexed and bath sonicated for 15 min before extruding through a 200-nm polycarbonate filter. The thalidomide-loaded HEs were purified by using a 10-k molecular weight cut-off (MWCO) amicon filter, with excess unloaded thalidomide being washed away with PBS. The amount of thalidomide in the wash was quantified using HPLC. The thalidomide loading efficiency and percentage drug content with respect to the weight of the carrier were quantified by calculating thalidomide in both filtrate and HE.DOX loading efficiency was calculated using the following equation:Drug release study was carried out in physiological  conditions. A total of 2 mL of 1 mg/mL of thalidomide encapsulated in HEs was dialyzed using 3.5 kDa dialysis membrane bag in 100 mL of drug release media kept at 37 °C while constantly stirring (80 RPM). A total of 500 µL of the release media was observed at regular intervals and replaced by fresh media. The amount of thalidomide in the release media was calculated using HPLC. For the control study, thalidomide encapsulated liposome was prepared, and its release characteristic was studied following the same protocol.Drug release was quantified by using the following equation:p < 0.05 was considered significant.All data were presented as means ± SEMs. T test was used to compare two groups of data, and one-way ANOVA was used to compare more than two groups of data via GraphPad Prism vision 9.0 , and + T cells were stimulated with IL-2 (10 ng/mL) with or without TNF (20 ng/mL), and co-cultured with THD, LEN, and POM at two different concentrations along with IL-2 plus TNF for a duration of 3 days. As shown in + T cells, which confirms our previous studies [p < 0.01). Additionally, we found that LEN and POM similarly abrogated Treg proliferation induced by TNF, with the proportion of inhibition being up to 11.88% and 12.87%, respectively (p < 0.05). These results highlight the ability of thalidomide and its analogs to prevent the TNF-mediated elevation of Treg cells. To begin with, we investigated the impact of thalidomide (THD) and its analogs, lenalidomide (LEN) and pomalidomide (POM), on Treg cells induced by TNF. In order to do so, we conducted an experiment in which purified CD4 studies ,26. Trea+ T cells cultural system we mentioned above, and CFSE labeling was used for cell proliferation analysis. As shown in p < 0.001). In line with our above findings, treatment with THD effectively suppressed TNF-induced Treg proliferation, with a proportion of inhibition ranging from 20.41% to 31.12% (p < 0.05). Similarly, LEN was also observed to abrogate Treg proliferative expansion induced by TNF, with a proportion of inhibition of 30.61% (p < 0.05). POM inhibited the proliferation of Treg cells induced by TNF in a dose dependent manner, with a range of inhibition from 26.02% to 43.88% (p < 0.01). These results suggest that THD and its analogs can effectively suppress TNF-mediated Treg proliferation and expansion.We then investigated whether THD and its analogs could affect the proliferative expansion of Tregs induced by TNF either in vitro. To this end, we use the same purified CD4p < 0.05~0.01). These findings suggest that THD has a promoting effect on the differentiation of Th1 and Th17 cells in vitro.We conducted further investigations to understand the effect of THD on the differentiation of Th subsets in vitro. Our results demonstrated that THD at a concentration of 20 μg/mL significantly promoted the generation of IFNγ-producing Th1 cells from 10.15% to 17.59% . FurtherExosomes (EXOs) were obtained from the murine macrophage cells raw 264.7 cultural medium using ultracentrifugation as described in the method. Liposomes were prepared using thin-film hydration technology with a molar ratio of lecithin to cholesterol = 3:5. The harvested exosomes were hybridized with the synthetic liposomes by extruding through polycarbonate membranes in 400- and 200-nm sizes. The ratio of liposome to exosome was optimized to 5:1. Exosomes were quantified based on protein content, while liposomes were quantified based on lipid mass.The size, surface properties, and protein content of liposomes, EXOs, and HEs were characterized. The hydrodynamic size of the liposomes and exosomes determined via a dynamic laser scattering (DLS) method were 138.2 ± 29.3 nm and 136.1 ± 27.47 nm with zeta potentials of −32 ± 2 mV and 13 ± 1 mV, respectively A,C. WherProtein characterization was used to further confirm the hybridization . As showNext, we loaded THD into HE using the membrane extrusion-mediated drug loading physical method and studied its drug loading and release kinetics to explore the potential application of HE-encapsulated thalidomide (HE-THD) in drug delivery application. Under the premise of the good stability of HEs, we further studied the pharmacokinetics of HEs at a normal physiological pH , as depicted in p < 0.01). Meanwhile, the proliferation of Treg cells induced by TNF also became inhibited by HE-THD, with the proportion of inhibition being 45.2% (p < 0.001).To investigate whether HE-encapsulated thalidomide has the same effect on the regulatory T cells as single small molecule compound thalidomide, lymphocytes from the lymph nodes of the spleen were labeled using the CellTrace violet method and stimulated with IL-2 (10 ng/mL) with or without TNF (20 ng/mL), and HE-THD (50 μM) was co-cultured with IL-2 plus TNF for 3 days. As shown in p < 0.01). Therefore, HE-THD has the in vitro activity in the inhibition of surface TNFR2 expression on Tregs via TNF stimulation. All in all, HE-THD has the capacity to downregulate the TNFR2 expression on Tregs surface and block the interaction between TNF-TNFR2, thus decreasing the proportion and proliferation of Tregs.Next, we try to figure out the mechanism of the function of HE-THD on regulatory T cells. We previously reported that TNF treatment preferentially promotes cell surface expression of TNF receptor type II (TNFR2) on Tregs . Since s+ and CD8+ T cells and that co-stimulation with T cell receptors results in synergistic effects that increase IL-2-mediated T cell proliferation. However, another study suggested that PBMCs that are treated with THD strongly induced IL-2 production but prevented the TNFR2 expression of T cell surfaces and the subsequent secretion of soluble TNFR2 (sTNFR2) during co-stimulation with αCD3 [Since the first synthesis of thalidomide (THD) in 1954, there has been a burst of information about the multiple bioactivities of this synthetic compound. Initial emphasis was on its sedative and antiemetic effects for morning sickness, but the horrific role of THD was later realized when it was discovered to be teratogenic, with over 10,000 infants born with congenital abnormalities. Subsequent studies established that THD exhibits anti-inflammatory, anti-angiogenic, and immunomodulatory effects and potentially works by inhibiting the inflammation-induced TNF production, and eventually became a drug approved by the FDA that modified the disease of ENL. THD and its analogs lenalidomide (LEN) and pomalidomide (POM) are now classified as immunomodulatory drugs (IMiDs) for the treatment of several inflammatory diseases and cancers . They alith αCD3 ,33.+ Treg subset, could be a promising strategy that either works alone or in combination to evoke effective cancer immunotherapy. It was reported that the combination therapy of THD and fludarabine is effective in the treatment of patients with chronic lymphocytic leukemia (CLL). This treatment can reduce the number of CLL and Treg cells simultaneously [+Tregs and improved tumor immunity remains to be established, its clinical outcome should not be discounted. Due to the low solubility of THD in its present form of administration, there is an urgent need to find potent delivery methods to substantially increase its solubility, improve its bioavailability, as well as reduce its toxicity.Although there continues to be uncertainty about the specific mechanism of THD, our data confirmed that THD and its analogs can significantly abrogate the expansion and proliferation of Tregs induced by TNF, and this probably resulted from blocking TNF–TNFR2 interaction. Treg infiltration into the TME is one of the critical features of the tumor immune-evasion mechanism. Several features of Tregs, such as the increased production of suppressive cytokines , upregulation of immune checkpoints and inhibitory receptors , direct cytotoxicity (perforin/granzyme-mediated), disturbance of T effector cell activity (IL-2 consumption), and induction of tolerogenic DCs, support tumor cell growth and create a niche that provides resistance to conventional chemotherapy and even immunotherapy . Therefoaneously . Therefo+ Tregs. We observed that HE-THD significantly abrogated the expansion and proliferation of Tregs induced by TNF (Exosomes (~30–150 nm diameter), a subtype of EVs naturally secreted from cells, have excellent biocompatibility due to the presence of membrane proteins such as tetraspanin and fibronectin, which allow them to act as intercellular messengers with a typical lipid bilayer structure . The prod by TNF , while id by TNF . We did d by TNF . Our in d by TNF . These fGiven their unique endogenous origin, exosomes enable a very stable delivery of biomolecules in body fluids. However, the extremely low yield of exosomes has impeded their widespread use for therapeutic purposes. To solve this problem, we hybridized exosomes derived from raw 264.7 cells with synthetic liposome to generate HEs with sizes around 122.6 ± 33.59 nm. . The met


These functional changes are; however, fully reversible even following prolonged/chronic transient exposure to TNF. Notably, antagonizing TNF signaling in transplantation recipients enhances donor HSC reconstitution. Our findings provide molecular and functional insight into HSC regulation, with implications for both acute and chronic inflammatory conditions.While tumor necrosis factor (TNF) is a critical mediator of appropriate immune response and tissue repair, its misregulation is linked to cancer, autoimmunity, bone marrow failure, and aging. Understanding the context-dependent roles of TNF is essential for elucidating normal and pathogenic conditions and to guide clinical therapy advancements. Prior studies suggested that TNF restricts the self-renewal capacity of hematopoietic stem cells (HSCs), but its long-term effect on HSCs remains unclear. Here, we demonstrate that   •Temporary TNF exposure results in reversible HSC repopulation capacity reductions•TNF-induced repopulation impairments coincide with altered quiescence status•TNF signaling inhibition enhances HSC reconstitution in transplantation recipients Biological sciences; Cell biology; Molecular biology; Stem cells research Furthermore, TNF signals through two distinct receptors, TNFR-p55 and TNFR-p75 that diverge in their affinities to soluble and membrane-bound TNF and their mediated signaling transduction.in vivo assays to assess the impact of transient short and prolonged TNF exposure on HSCs. We focused primarily on the capacity of HSCs to recuperate functional properties following the disruption of TNF exposure, a notion of direct clinical relevance in patients suffering from diseases involving TNF.TNF levels are elevated in several inflammatory conditions, including inflammatory bowel disease,in vivo TNF administration. For this, we intravenously injected mice with a single dose of TNF (2 μg/mouse). On the following day, we monitored the cellularity of different blood cell types in peripheral blood (PB) (We began to assess the acute hematopoietic responses following ood (PB) A and of ood (PB) D.Figure + T cells (2.5-fold) and a distinct redistribution within the myeloid cell compartment. While eosinophils and monocytes drastically declined, the numbers of neutrophils increased  cellularity, with a stronger effect on platelet (PLT) counts A. Investncreased A, thereb,−Sca+Kit+CD48−CD150+ phenotype, ,+ HSCs confirmed that TNF did not negatively influence on HSC numbers and, if anything, rather associated with their slight expansion , granulocyte-macrophage progenitors , megakaryocyte progenitors , and erythroid progenitors  (−Sca+Kit+ (LSK) fraction, which reside developmentally in between HSCs and lineage-committed progenitors, were unaltered, other myelo-erythroid progenitors were also reduced  D and S2C reduced B.1 and S/G2/M phases .2M checkpoint.in vivo TNF stimulation resulted in a striking enrichment of the proliferation-associated pathways  every third day from seven days prior to irradiation/transplantation until five days post transplantation  prior to transplantation could therefore increase HSC reconstitution.,in vivo administration of the clinically widely used TNF inhibitor adalimumab to irradiated transplantation recipients increased the donor contribution from transplanted HSCs. Although the endogenous myeloid and B cell contribution was virtually absent in both groups and the consequences to these cells could therefore not be assessed, the endogenous T cell contribution was significantly reduced in the adalimumab-treated group. This finding could potentially be advantageous for reducing graft rejection, although allograft situations also exist where the presence of TNF might be beneficial for reconstitution and induction of transplantation tolerance.TNF increases in response to irradiation, a procedure that is frequently utilized for lympho- and myeloablation in the preparation for HSC transplantation in both clinical and research settings. It has been previously suggested that irradiation-induced TNF causes production of reactive oxygen species (ROS) and that treatment with the antioxidant in vivo-administered TNF by altering their proliferation and repopulation activity. The transient exit of HSCs from quiescence that follows TNF exposure is accompanied by an impaired repopulation capacity that is fully restored upon return to quiescence, even after chronic TNF exposure. This notion is reassuring, given the central role of TNF upregulation in many infections and (auto-)inflammatory diseases. It also holds potential for the advancement of clinical therapies aimed at increasing donor reconstitution following transplantation.Collectively, our findings clarify and elaborate on our understanding of the regulatory roles of TNF on HSCs. We have shown that HSCs rapidly respond to ,One limitation with our study is that the levels of TNF exposure to HSCs may be higher than what is physiologically relevant. However, the optimal level of exogenously administered TNF required to achieve physiologically relevant levels in the BM, and how this relates to human physiology, remain unclear. In this study, we intravenously injected 2 μg of TNF to mice, a concentration that allows for comparison with previous key studies.alexandra.rundberg_nilsson@med.lu.se).Further information and requests for resources and reagents should be directed to the lead contact, Alexandra Rundberg Nilsson  intravenously. Controls were administered a volume-equivalent 0.9% saline solution.rd day totaling 5 doses per mouse, during a period from 7 days before to 5 days after lethal irradiation and transplantation. Volume-equivalent saline was administered to control mice.Adalimumab  was administered intraperitoneally at 50 μg/dose, every 3,PB and BM cells were isolated and prepared as previously described.The cell cycle status of HSCs was determined using the PE and FITC Mouse Anti-Ki-67 Set and the Cytofix/Cytoperm Solution Kit  according to the manufacturer’s instructions. Briefly, lineage-depleted BM was stained with antibodies used to define HSCs as described above. After staining, the pelleted cells were fixed and permeabilized by resuspension in BD Fix/Perm™ buffer for 20 minutes at 4°C. Cells were then washed twice with BD Perm/Wash buffer and stained with anti-Ki67 or IgG1 isotype control for 30 minutes, after which cells were washed twice and resuspended in 10 μg/mL 7-Aminoactinomycin D (7-AAD) diluted in Wash/Perm solution over night before analysis.HSC proliferation history was analyzed by intravenously injecting mice with 1 mg/4g body weight freshly prepared EZ-Link® Sulfo-NHS-LC-LC-Biotin. 2 days later, the mice were administered 2 μg TNF or saline intravenously and were subsequently euthanized 2 days following the TNF/saline administration to analyze proliferation history by biotin retention. BM cells were stained for HSC markers and streptavidin-BV605 was used to identify the biotin retention.All transplantations were carried out competitively with FACS purified HSCs as previously described.GSE115403 data set,,The Unpaired two-tailed student’s t-tests were applied to calculate statistical significance between groups in flow cytometry experiments using Graphpad Prism. The statistical details can be found in the corresponding figure legends. p-values <0.05 were considered significant. Mice used for treatment and transplantation were randomized into groups and was not blinded. Transplanted mice with donor reconstitution that did not exceed 1% in all evaluated lineages and/or within the HSC pool were considered unsuccessful and excluded from analysis. In accordance with these criteria, the following mice were excluded from analysis: 1) one TNF-treated mouse related to


Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous clinical syndrome with multiple underlying mechanisms and comorbidities that leads to a variety of clinical phenotypes. The identification and characterization of these phenotypes are essential for better understanding the precise pathophysiology of HFpEF, identifying appropriate treatment strategies, and improving patient outcomes. Despite accumulating data showing the potentiality of artificial intelligence (AI)-based phenotyping using clinical, biomarker, and imaging information from multiple dimensions in HFpEF management, contemporary guidelines and consensus do not incorporate these in daily practice. In the future, further studies are required to authenticate and substantiate these findings in order to establish a more standardized approach for clinical implementation. Heart failure with preserved ejection fraction (HFpEF) is diagnosed as heart failure with a left ventricular ejection fraction (LVEF) of ≥50% and elevated left ventricular filling pressures at rest or during exercise after careful exclusion of conditions that may mimic HFpEF ,2. HFpEFThere is currently little evidence supporting the effectiveness of conventional therapies utilized for HFpEF to reduce mortality rates, such as empagliflozin in the EMPEROR-Preserved trial and dapagliflozin in the DELIVER trial. However, emerging research suggests that treatment should be tailored to the specific comorbidities present in each patient . Some ofSeveral studies have examined the relationship between HFpEF and clinical entities and comorbidities. For example, in the TOPCAT trial, it was discovered that patients with HFpEF who had been previously hospitalized for heart failure exhibited a greater likelihood of experiencing cardiovascular death, heart failure hospitalization, or aborted cardiac arrest than their counterparts who did not have a history of hospitalization . The utiSince the amount of healthcare data generated on a daily basis is overwhelming for a contemporary doctor, far surpassing the computational capacity of the human brain , machineOne method to subtype HFpEF is based on the presence of underlying clinical entities or comorbidities that contribute to the development and progression of the disease, known as clinical phenotyping A 8]. Th. Th8]. THwang et al. explored the implications of coronary artery disease (CAD) in HFpEF. The authors suggested that HFpEF patients with CAD have distinct clinical and pathophysiological characteristics compared with those without CAD. CAD may play a significant role in the progression of HFpEF, and a better understanding of this relationship could lead to the development of more effective treatments. This study provides insights into the clinical entity and underlying comorbidity of HFpEF, and it highlights the importance of considering these factors when defining and classifying HFpEF phenotypes .Shah et al. identified distinct subgroups of HFpEF patients based on their comorbidities, including obesity-related comorbidities, metabolic syndrome, and pulmonary hypertension. This study showed that these subtypes had distinct clinical, biochemical, and imaging profiles, suggesting that they may represent different disease processes. The authors proposed that this phenomapping model-based clustering approach could be used to tailor treatments to individual patients and to improve outcomes in HFpEF  Figure B.Borlaug et al. investigated the associations between specific clinical entities, comorbidities, and HFpEF subtypes. Their study included 344 patients with HFpEF who were classified into four subtypes, based on the clinical entities or comorbidities present, as follows: (1) obesity-related, (2) hypertension-related, (3) diabetes-related, and (4) idiopathic. According to this research, the HFpEF subtype associated with obesity showed a greater prevalence of metabolic risk factors and more severe diastolic dysfunction compared to the other subtypes. The hypertension-related HFpEF subtype had a higher prevalence of left ventricular hypertrophy, concentric remodeling, and worse systolic function. The diabetes-related HFpEF subtype had a higher prevalence of diabetic nephropathy, worse systolic function, and more severe diastolic dysfunction. The idiopathic HFpEF subtype had a lower prevalence of comorbidities and less severe diastolic dysfunction than the other subtypes .Uijl et al. proposed a five-cluster model, labeled Cluster 1 and Clusters 2–5, among 6909 HFpEF from the Swedish Heart Failure Registry (SwedeHF) and externally validated this in 2153 patients from the Chronic Heart Failure ESC-guideline based Cardiology practice Quality project (CHECK-HF) registry, as shown in Cardiac imaging and measurement of the cardiac structure are critical aspects in diagnosing HFpEF, as the symptoms of this condition can be nonspecific. Non-invasive measures of cardiac structure and function can assist in improving diagnostic accuracy and differentiating the sub-phenotypes of HFpEF. Additionally, imaging can exclude alternative diagnoses that mimic HFpEF, such as hypertrophic cardiomyopathy, primary valvular heart disease, cardiac amyloidosis, and pericardial disease. While two-dimensional (2-D) transthoracic echocardiography is the most commonly used imaging modality, advanced imaging techniques, including cardiac magnetic resonance imaging and 2-D speckle tracking echocardiography, are used to identify distinct HFpEF phenotypes based on left ventricular structure and function ,31,32,35Several studies have investigated the use of echocardiography to identify the imaging-based phenotypes of HFpEF. Diastolic dysfunction, a hallmark feature of HFpEF, has been well adopted in the initial classification of HFpEF . One stuAnother study evaluated left atrial function using 2-D speckle tracking echocardiography and found that left atrial strain was significantly decreased in patients with HFpEF compared with that in controls. The study also found that left atrial strain was independently associated with a higher risk of adverse events, including hospitalization and mortality .Cardiac imaging and measures of cardiac structure and function are essential for diagnosing HFpEF, excluding alternative diagnoses, and identifying imaging-based phenotypes. Although 2-D echocardiography remains the most commonly used imaging modality, CMR and 3-D echocardiography offer more comprehensive information regarding cardiac structure and function. These imaging techniques may have important prognostic implications and help guide management strategies in patients with HFpEF.Personalized management of different HFpEF phenotypes using clustering targeting more specific molecular or pathological etiology driving underlying mechanisms has been proposed in several studies ,40,41,42Among the five clusters, patients with HFpEF in this group had a median age of 59 years and a relatively low burden of comorbidities, making them the youngest of the cohorts. The most common comorbidities in Cluster 1 were hypertension (46%) and obesity (42%). The principles of management for this group are to control blood pressure and reduce body weight. It is worth mentioning that cluster 1 includes patients who have recovered HFrEF, due to the higher percentage of implantable cardioverter-defibrillator or cardiac resynchronization therapy.In addition to the implantable devices, quite a few medications have demonstrated an established efficacy in previous HFpEF trials. Those drugs were renin–angiotensin–aldosterone system (RAAS) antagonists such as angioten-sin-converting enzyme inhibitors (ACEis), angiotensin II receptor blockers (ARBs), mineralocorticoid receptor antagonists (MRAs), and angiotensin receptors/neprilysin inhibitor (ARNi), which could be considered as first-line agents for the management of HFpEF. Lifestyle modifications were strongly suggested for this cluster. Significant improvements in quality of life and exercise tolerance were observed as a result of weight reduction, which was found to be safe. In addition to these benefits, weight loss in patients with HFpEF has been shown to have a positive impact on cardiac function and metabolic parameters, potentially leading to reduced doses of diuretics, antihypertensive agents, and diabetes medications.The individuals belonging to Cluster 2 were relatively older compared to those in Cluster 1, having a median age of 77 years. This cluster included patients with HFpEF characterized by AF without diabetes. Principles of management for this cluster align with the AF Better Care (ABC) pathway, including rate/rhythm control in AF management, as follows: (A) avoiding thromboembolic events with the use of anticoagulation. (B) better management of symptoms with personalized, symptom-directed decisions on rate or rhythm control. Rate control involves the use of beta-blockers/non-dihydropyridine (DHP) calcium channel blockers (CCBs) (diltiazem or verapamil)/digoxin; rhythm control involves the use of amiodarone/dronedarone or AF ablation. (C) Effective management of cardiovascular and coexisting conditions, including attention to psychological factors and lifestyle. Following the ABC pathway has been shown to lead to improved outcomes, including decreased risks of all-cause mortality, cardiovascular mortality, stroke, and hospitalization due to cardiovascular reasons. It is important to avoid excessive rate control in patients with both HFpEF and AF, as it may diminish their chronotropic reserve. In a trial comparing strict (<80 bpm) and lenient (<110 bpm) rate control in patients with AF, which may have included individuals with undiagnosed HFpEF, no significant differences in outcomes were observed.Among the five clusters, Cluster 3 patients were the oldest  with the highest N-terminal pro b-type natriuretic peptide (NT-proBNP) values. It was reasonable to eliminate any meaningful clinical phenotyping for this cluster, as it presented with an anticipated higher risk for an ominous outcome. Clinically, the principle of for the elderly is to reduce hospitalization rates and improve quality of life. Decongestion of diuretics has been shown to reduce hospitalization rates. In the TOPCAT trial, spironolactone was associated with a decrease in heart failure hospitalization rates compared with the placebo . This clCluster 4 was composed of patients who had a median age of 71 years and were identified as having diabetes but not AF. Serum glucose control is the mainstay of this cluster. SGLT2 inhibitors have emerged as a critical component of HFrEF therapy as they possess favorable pleiotropic effects on various body parts such as the kidney, liver, pancreas, blood vessels, and adipose tissue, apart from their primary role as an antidiabetic medication. The EMPEROR-Preserved trial was groundbreaking in the study of HFpEF as it compared the effects of empagliflozin with a placebo in patients with ejection fractions above 40%, irrespective of whether they had diabetes or not. The trial demonstrated a significant reduction in the risk of heart failure-related hospitalizations and cardiovascular mortality, as well as an improvement in renal outcomes.According to the DELIVER trial, dapagliflozin is superior to placebos in decreasing cardiovascular deaths and hospitalizations due to heart failure in patients with mildly reduced or preserved ejection fractions. Additionally, the study showed that dapagliflozin was effective in patients who previously had ejection fractions below 40% but later saw an increase to over 40%.Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) are also associated with positive cardiovascular effects. A recent meta-analysis involving 592 patients revealed that liraglutide was connected with significant enhancements in the left ventricular diastolic function.Cluster 5 was a union of Clusters 2 and 4. This cluster had a median age of 82 years, and its members had comorbidities of both diabetes and AF. In the DECLARE-TIMI 58 trial , dapagliflozin reduced the incidence of AF in patients with diabetes . EffortsObesity as a common etiology and co-morbidity for HFpEF has been shown to induce activated sympathetic system and RAAS  and further promote systemic inflammation ,46, whicElevated circulating levels of aldosterone, either directly from adipocytes or released from the adrenal gland in response to leptin through the adipokines-cell-signaling molecules secreted , along with the attenuated anti-aldosterone effects from natriuretic peptides due to an increased neprilysin activity in obesity, may potentiate  the deleImportantly, an activated leptin–aldosterone–neprilysin axis with sustained increases in aldosterone and neprilysin concentration may in turn accelerate the accumulation and inflammation of epicardial fat ,53. ReceIn summary, the identification and characterization of HFpEF phenotypes are important for guiding diagnosis, management, and research into novel treatment strategies. It was a prerequisite for us to identify that the presence of ischemic heart disease by itself induces prognostic implications. Given the complexity of HFpEF, a personalized approach to management that considers the underlying mechanisms and comorbidities in each patient is needed and might help to solve the puzzle of this challenging syndrome.


To provide a bibliographical-historical perspective and main interest in the field of myopia.  In this bibliographic study, the Web of Science Database was searched from 1999 to 2018. Recorded parameters included journal name, impact factor, year and language, number of authors, type and origin, methodology, number of subjects, funding, and topics. P = 0.034). The articles were published in 27 journals, with the majority in Investigative Ophthalmology, Vision Sciences (28%), and Ophthalmology (26%). Etiology, signs and symptoms, and treatment equally encompassed the topics. Papers addressing etiology, specifically genetic and environmental factors (P = 0.029), signs and symptoms (P = 0.001), and prevention, specifically public awareness , received significantly more citations. Treatment to decrease myopia progression was a much more common topic (68%) than refractive surgery (32%). Optical treatment was the most popular modality (39%). Half of the publications came from 3 countries: the United States (US), Australia, and Singapore. The highest ranked and cited papers came from the US (P = 0.028) and Singapore (P = 0.028).  Epidemiological assessments were the leading type of article (28%), and half of the papers were prospective studies. The number of citations for multicenter studies was significantly higher ( To our knowledge, this is the first report of the top-cited articles on myopia. There is a predominance of epidemiological assessments and multicenter studies originating from the US, Australia, and Singapore, assessing etiology, signs and symptoms, and prevention. These are more frequently cited, emphasizing the great interest in mapping the increase in the incidence of myopia in different countries, public health awareness, and myopia control. Myopia is a major public health concern, and its prevalence is increasing rapidly around the world . High myMyopia generally develops during the early to middle childhood years  and is aRefractive surgery is one of the most commonly performed ocular surgeries worldwide , 17, andRefractive surgery is one of the most frequently used ocular surgeries globally , 17, andThe purpose of this study was to discover the main topics investigated in the field of myopia, to provide a bibliographical-historical perspective of this major health problem, and to analyse the trends of interest in the medical community.The Web of Science database was searched by an experienced medical librarian for the keywords: “myopia,” “near sighted,” and “short sighted” in the title. The search for publications in peer-reviewed journals in all languages between 1999 and 2018 was conducted in May 2020. We chose 1999 as the first year included to provide a retrospective overview of the last 20 years, and 2018 was selected as the last year to allow enough time from when the article was published until it was cited. Papers were reviewed by 3 authors . Our main outcome measure was to characterize the 100 most cited papers on myopia. The secondary goal of this study was to provide a historical perspective on the most cited papers on myopia. Data recorded from each paper are presented in Supplementary p < 0.05 was considered significant. The SPSS software was used .Categorical variables were summarized according to their frequency and percentage. Continuous variables were also evaluated for the normal distribution. Since the absolute number of papers that originated from certain countries was small in number (2 or less), we merged them into a category termed “other” and performed logistic regression in order to determine the mean rank of each country. The correlations between the number of citations and the continuous, ordinal, and categorical variables were evaluated using Spearman's rank correlation coefficient, the Kruskal–Wallis test, and the Mann–Whitney test, respectively. The chi-square test was used to explore the association between the categorical variables; the false discovery rate method was used to correct for multiple comparisons; and the association between the total number of citations and the year of publication was evaluated using linear, quadratic, and cubic models. Statistical tests were two-sided, and n = 42), prevention (n = 30), signs and symptoms (n = 42), and treatment (n = 41). Twelve papers dealt with genetic etiology, 11 with environmental etiology, and 20 with both. Papers that assessed etiology (P=0.029) and prevention (P=0.005) and papers on signs and symptoms (P < 0.001) were significantly more frequently cited than papers that did not address these topics. Thirty papers focused on myopia prevention, 14 on public health awareness, four on myopia screening, and 12 on both public health awareness and screening. Forty-two papers were on the subject of signs and symptoms. Twelve of these were epidemiological assessments describing the signs and symptoms of different populations around the world. Nine papers described the signs and symptoms of patients diagnosed with pathological myopia. Seven papers were on school children and teenagers, two investigated adults, and the rest included the entire population. Forty-one papers described treatment modalities, including myopia control and refractive surgery. Studies on myopia control included papers on optical treatment, which were the most popular (n = 22), including papers on multifocal/progressive addition lenses (n = 10), orthokeratology (n = 8), and peripheral defocusing lenses (n = 9). One third of the papers were on pharmacological treatment (n = 14), mostly with atropine. Finally, two papers investigated behavioral modifications. Refractive surgery for myopic patients seeking to achieve spectacle independence (n = 18) was second in popularity.The topics assessed in the papers were on the following subjects: etiology (n = 28), Ophthalmology (n = 26), American Journal of Ophthalmology (n = 7), Archives of Ophthalmology (n = 6), Journal of Cataract and Refractive Surgery (n = 5), British Journal of Ophthalmology (n = 4) and Progress in Retinal and Eye Research (n = 3). All other journals published only a single paper. Eighty-nine of the 100 most cited papers were published in a journal with a Q1 impact factor and were written in English.The 100 most cited papers on myopia are listed in Supplementary n = 20), Australia (n = 17), and Singapore (n = 11). The first and last author's countries of affiliation were identical, except in 2 papers. The University of Sydney and the National University of Singapore are the origins of the greatest number of articles on the list (n = 8)  .P=0.028) and Singapore (P=0.028) achieved a significantly greater mean rank than the other groups.The mean rank of the countries was determined by the number of papers on the list as well as the number of citations of each paper. Only the US (n = 34). The mean number of citations was greatest for North America , followed by Australia , Asia , Europe , and Africa, with only one paper (189 ± 0). There was a significant difference in the mean number of citations between North America and Europe (P=0.004).When we compared the affiliation of the 100 most cited papers by continent, the majority of the papers were affiliated with Asia . p < 0.001). The earlier the paper was published, the more likely it was to receive a higher rate of citations. The distribution of the papers by year is presented in a 3rd degree function curve in n = 28). The categorization of papers by the type of article is presented in The 100 papers on myopia were published between 1999 and 2016. The number of citations dropped considerably after 2012, and a significant correlation was found between the year of publication and the mean number of citations per year (n = 10) received 228 citations [The number of eyes analysed in the papers varied, with a mean of 396 ± 1057 eyes . The paper with the least number of eyes (itations . This stitations . The numitations . This stP=0.034).Citations for multicenter studies were significantly higher than that for single-center studies (Previous studies have evaluated the most cited articles on ophthalmology in general  and on oThe topics of the papers were equally distributed between etiology, signs and symptoms, and treatment, with less attention paid to the prevention of myopia. However, papers that addressed prevention had a higher mean rank and were significantly more frequently cited than papers that did not. The ATOM studies in Singapore are an example attesting to this approach: the first and second studies sought an effective treatment , whereasIn this study, most papers on prevention discussed public health awareness compared to the screening of myopia. This might be due to the enhanced role that public health awareness plays in managing myopia progression by promoting increased outdoor activity for children, in addition to reducing screen time and near work, both of which are linked to myopia progression , 25. IncPapers on signs and symptoms had a higher mean rank and were significantly more frequently cited than papers that did not address this subject. Studies describing the signs and symptoms of myopia patients were mainly conducted on the entire population of myopia patients. This could be explained by the great interest in mapping the increase in the incidence of myopia in different countries and populations; thus, these studies include all age groups. Twenty percent of these papers described signs and symptoms of pathological myopia, emphasizing the importance given to studying the complications of myopia and the risks caused by the increasing numbers of high myopes, since this major health problem is projected to keep on expanding .Refractive surgery for myopic patients came second to papers on methods to reduce myopia progression. Four of the refractive surgery papers , 27–29 wOver 2/3 of the papers about treatment investigated the reduction of myopia progression. Optical treatments were the most popular and were equally divided between multifocal/progressive spectacle lenses, peripheral defocusing spectacle lenses, and orthokeratology although only the latter 2 modalities have superior efficacy in decreasing myopia progression. Papers on pharmacological treatment were mostly about diluted atropine drops and behavioral modifications, including health behavior programs to promote reduced screen time and increased outdoor activities for slowing myopia progression.Randomized clinical trials have shown that the rate of myopia progression is lower in children given atropine eye drops, compared with those treated with placebo . Other sThere were 2 behavioral treatment studies including outdoor interventions , 30 and Q1 impact factor and were written in English. Also, the seven journals with the highest number of publications were American journals. The optometry societies in Australia are very vigilant in their research and in promoting a means to control myopia progression, in particular through the Brian Holden research institute.Asia, with the highest prevalence of myopia in the world and with an incidence of myopia that has increased considerably in the past 50–60 years, was the source of the largest group of authors. Three countries published half of the papers and included the US, Australia, and Singapore. Singapore came second after the US in the mean rank mostly because of its leading studies on diluted atropine therapy. The US is the world leader in the quantity and quality of scientific papers as can be seen as well by the fact that most papers were published in the Intuitively, the earlier the paper was published, the more likely it was to have a higher citation index, allowing more exposure time and therefore more time to be cited. One study discovered that, over the years, about 40% of citations were cited for historical reasons, but in the remaining 60% of the cases, the old paper was still being actively used . InteresThe limitations of this study pertain to the methods of the study: Examining the number of citations of an article is not an objective measure of a paper's quality. In addition, sampling other databases may have produced a different list of the most cited articles on myopia. Future research should use other tools such as VosViewer to provide more information on changes in topics covered over time and add information on the relationships between countries, concepts, and authors. Moreover, examining the number of citations of an article is a limited and objective measure of the relevance of a study. Finally, recently published studies were not included in our analysis. These studies may have greatly influenced the field of myopia but have not generated a large number of citations yet as they have only recently been published. One possible solution could be to include the most publications by using the number of citations per year as the parameter for comparison in order to help the most recent publications gain importance. Yet, our list was formed by the 100 most cited publications, and the most recent publications, for example, from 2019 to 2022, did not gain a lot of citations and thus were not included even if divided by year.In summary, to our knowledge, this is the first report of the top-cited articles on myopia. There is a predominance of epidemiological assessments and multicenter studies assessing etiology, signs and symptoms, and prevention. Papers about myopia control were twice as common as those on refractive surgery to correct myopia. This emphasizes the great interest in mapping the increase in the incidence of myopia in different countries, public health awareness, and the reduction of myopia progression. The most prominent change in the most cited publications over time is the increase in papers on the treatment and prevention of myopia emphasizing the burden of this disease and the massive academic focus on trying to prevent and treat it. Half of the publications came from 3 countries: the US, a world leader in quantity and quality of scientific papers; Australia, which holds one of the largest and most vigilant optometry societies in the world; and Singapore, which is the origin of the leading studies on diluted atropine therapy, while Asia published a third. Additionally, the highest ranked and cited papers came from the US and Singapore. The increase in screen time during the COVID-19 epidemic, which exacerbated the myopia epidemic, may have resulted in publications from other parts of the world, but this remains to be seen.


Preeclampsia is a pregnancy complication Aim of this study was to investigate expression of Beclin1 and tumor necrosis factor (TNF)-α in normotensive and preeclamptic placentas of pregnant women patients.Twenty normotensive and 20 preeclamptic patients placentas were dissected for paraffin- wax processing. Placental samples were embedded in parafin blocks. Sections were stained with Hematoxylin-Eosin staining and TNF-α and Beclin1 immunostaining.In control group, root and floating villi were normal in histological perspectives, syncytial node number was low, vessels were normal with connective tissue. No hemorrhage was observed in the intervillous area. In preeclampsia group, decidual cell degeneration and fibrinoid accumulation increased. Vascular dilatation and congestion with mononuclear cell infiltration were observed. Beclin1 reaction was generally negative in control group. In preeclampsia group, Beclin1 reaction was increased in decidual cells, syncytial nodes and bridges and in chorionic villi and in some Hoffbauer cells. In control group, TNF-α expression was mainly negative but only in some decidual cells. In preeclampsia, TNF-α reaction was observed in degenerated decidua cells, in leukocytes and in villi.In preeclampsia placentas, degenerated decidua cells and inflammation increased. It was thought that Beclin1 and TNF-α signals could be used as a marker in affecting the fetal structure of blood flow in preeclamptic placentas. The abnormal placement of placenta in the uterine wall can cause pregnancy related mortalities and morbidities. Moreover, deterioration in blood circulation can histological cause some anomalies in placental structures such as fetal capillaries abnormalities, increased fibrin deposition and fibrotic tissue, degenerated villous structures.,4Maternal morbidity is a general term that defines any health problem related to pregnancy complications gestational diabetes mellitus and hypertension, preeclampsia, infections and hemorrhage. Regular clinical follow-up of pregnancy may help early diagnosis and treatment of those complications however there are stills open gap for exact diagnosis.th weeaks of gestations. PE is clinically dignosed as systolic hypertension (blood pressure (BP) > 140/90 mm Hg), diastolic hypertension (BP > 90 mm Hg) after 20 weeks of gestation with > 300 mg 24-hour urine protein output. PE is a multifactorial disease that adversely affects kidney and liver function, leading to end-organ injury. In case of severe PE, systolic/diastolic BP pressure ≥ 160/110 mm Hg with low thrombocyte count, increased hepatic aspartate transaminase and alanine transaminase enzymes are observed. Acute necrotizing arteriopathy (acute atherosis) is seen in the spiral arteries in the placentas of women with preeclampsia. These cases are characterized by fibrinoid necrosis in the wall of the vessels, accumulation of lipid-filled cells and mononuclear cell infiltration in the perivascular space. Infarct and retroplacental hematoma mass status in women with preeclampsia is estimated to be a direct result of acute atherosis and related vascular changes.Preeclampsia (PE) is defined as pregnancy hypertension that affects 3% to 8% of all pregnancies. PE is a leading cause of maternal morbidity and mortality which is characterized after 20 Apoptosis in the placental part increases in pregnancies complicated by preeclampsia or fetal growth retardation. Placental oxidative stress is an effective stimulant for apoptosis. As a result of many studies, the presence of cytokines and receptors of TNF in different human reproductive tissues (amnion and placenta) has been detected.Tumor necrosis factor (TNF)-α is effective on placental invasion and apoptosis.,12Beclin1 is a 450-amino acid protein that modulate the initiation of autophagy process during starvation. It is also a tumor suppressor gene and takes role in membrane trafficking. Beclin1 interacts with BCL2 to regulate cell survival and death. Expression of Beclin1 is dependent of many factors and involves in neurodegenerative disease and cancers and other disease.In this study, we examined the inflammation and autophagy processes between the placentas of preeclamptic and healthy pregnant women immunohistochemically.In our study, placentas belonging to 20 normotensive and 20 preeclampsia patients were included. Preeclampsia criteria were defined on the basis of American College of Obstetricians and Gynecologists. Placentas were obtained from Dicle University Medical Faculty Hospital Gynecology and Obstetrics Clinic. Ethical approval was obtained from Dicle University, Non-interventional Local Ethical Committee 2023/174). An informed consent form was obtained from all patients participating in the study. Permission was obtained from the local ethics committee for the study. Age (year), gravida, parity, mean systolic pressure (mm Hg), mean diastolic pressure (mm Hg), hemoglobin (g/dL) of pregnant women, platelet (103/µL), glucose, urea, creatinine, alanine aminotransferase enzyme (U/L), aspartate aminotransferase enzyme aspartate aminotransferase (U/L) and 24-hour urine output was recorded and shown in Table 74. An inAfter the placentas were fixed in 10% formaldehyde, they were passed through the rising alcohol series and included in the routine paraffin-wax tissue protocol. Hematoxylin-Eosin was performed by taking 5 µm sections from paraffin blocks.,15The obtained placental sections were washed with distilled water to perform immunohistochemistry. Then, for the antigen application process, the application was carried out by waiting at 700 W in a microwave oven for 10 minutes in citrate buffer solution with pH: 6.0. After this process, the obtained sections were allowed to cool for up to 25 minutes at room temperature and washed in distilled water for 2 × 4 minutes. As a result of these procedures, 3% hydrogen peroxide was used for 10 minutes in order to apply endogenous peroxidase blockade. The tissues we obtained were both washed with distilled water and washed with PBS. After performing the washing procedures, the sections obtained from the sections were incubated overnight at + 4°C with mouse monoclonal anti-TNF-α and anti-Beclin1 antibodies . The sections that were kept the next day were cleaned with PBS and applied with a secondary antibody solution  for 20 minutes. After PBS treatment, streptavidin peroxidase solution  was applied for 15 minutes. After the obtained sections were washed 3 times in PBS, DAB chromogen solution was applied for 8 minutes. Afterwards, the sections were washed with distilled water and counterstained with Harris hematoxylin for 2 minutes. The obtained sections were then allowed to be viewed under a light microscope with the imager A2 Zeiss.U test was used to make a pairwise comparison in the SPSS program. A P value <.05 was accepted as statistically significant at the threshold of evaluation and results obtained.In order to perform the statistical analysis of the obtained data, IBM SPSS Statistics version 25 software was used and evaluated. The obtained data were analyzed by applying normality tests. In order to make a better evaluation of the obtained data, the Mann–Whitney Figure Except for some syncytial cells in roots and floating villi, especially in areas where the maternal region is located, the Beclin1 reaction was generally evaluated as negative. Some syncytial areas and bridges were also evaluated as negative for Beclin1 reaction (Fig. In the section passing through the maternal region, clearly negative TNF-α expression was observed in decidual cells. It is negative in syncytial regions as well as in some cytotrophoblast cells. However, TNF-α expression was positive in some end nodes, especially towards the intervillous area. In general terms, TNF-α reaction was found to be negative Fig. E.TNF-α reaction was observed in parallel with significant degenerative changes in decidua cells. TNF-α reaction was positive in the vessel wall, especially in endothelial cells, in leukocyte structures in intervillous areas and in connective tissue cells inside the villi. It was observed that TNF-α reaction was positive with degenerative changes in the regions Fig. F.,17The most important difference between preeclampsia and normal pregnancy is that cytotrophoblasts invade spiral arteries both in decidual sections and myometrium and increase uteroplacental blood supply by decreasing vascular resistance in normal pregnancies, whereas in preeclampsia cytotrophoblasts invade spiral arteries only in decidual section and cannot penetrate the myometrium, there are differences in hypoperfusion and remodeling of vessels In preeclampsia, it has been reported that spiral artery invasion is incomplete in many regions of the placenta and endovascular cytotrophoblasts are very few. Akhlaq et al studied placentas of patients with preeclampsia. They found that histopathological alterations can vary depending on the severity of preeclampsia. They observed hypertrophic muscle and decidual cells, in the maternal-fetal region, hyperplasic placental villi, elevated number of syncytial nodes and leukocyte infiltration in connective tissue, and accumulation of chorionic villi in syncytial regions of chorionic villi. In our study, in control group, root and floating villi were normal in histological perspectives, syncytial node number was low, vessels were normal with connective tissue. No hemorrhage was observed in the intervillous area (Fig. Preeclampsia can induce many cellular events such as inflammatory pathway and oxidative stress mechanism. Some authors have found a reduced degree of trophoblast invasion in both the spiral arteries and the myometrium in severe preeclampsia.rea Fig. A. In prerea Fig. B. Unlike apoptotic tissue, necrotic tissues contribute to the pathophysiology of preeclampsia by forming an inflammatory response when taken up by endothelial cells. These conditions fail to explain the role of apoptosis in the development of placental pathology. In our study, Beclin1 reaction was generally negative in control group (Fig. Various studies have shown that apoptosis is increased in pregnancies complicated by some pathologies such as preeclampsia, fetal growth restriction and diabetes. in their study, stated that the failure of the increase in TNF-α protein expression in trophoblast cells in the second trimester of normal pregnancy alters or disrupts the remodeling process of the spiral arteries, prevents the placental angiogenic-antiangiogenic balance, and may induce syncytiotrophoblast stress. It has been suggested that migration activity, increase in syncytial node and endocrine functions after high TNF-α levels in risky pregnancies such as gestational diabetes, mild preeclampsia, and severe preeclampsia affect trophoblast biology. In our study, in control group, TNF-α expression was mainly negative but only in some decidual cells. (Fig. Placental trophoblastic cells and fetoplacental macrophages normally express TNF-α, which leads to endothelial cell activation and dysfunction. Basu et al,ls. Fig. E. In prels. Fig. F.In conclusion, in preeclampsia placentas, degenerative changes in decidua cells in the maternal region continued in placental villi and an increase in inflammation was observed in the intervillous area. It was observed that the apoptotic change due to inflammation increased and the angiogenic effect changed. It was thought that Beclin1 TNF-α signals could be used as a marker in affecting the fetal structure of blood flow in preeclamptic placentas.Conceptualization: Firat Aşir, Veysel Toprak, Senem Alkan Akalin, Engin Deveci.Data curation: Ece Öcal.Formal analysis: Ece Öcal, Veysel Toprak.Funding acquisition: Veysel Toprak, Engin Deveci.Investigation: Senem Alkan Akalin, Engin Deveci.Methodology: Ece Öcal.Project administration: Firat Aşir, Senem Alkan Akalin.Resources: Veysel Toprak, Engin Deveci.Software: Ece Öcal.Supervision: Firat Aşir.Validation: Ece Öcal, Engin Deveci.Visualization: Senem Alkan Akalin.Writing – original draft: Firat Aşir, Veysel Toprak, Senem Alkan Akalin.Writing – review & editing: Firat Aşir, Senem Alkan Akalin.


The assessment of functional effect of amino acid variants is a critical biological problem in proteomics for clinical medicine and protein engineering. Although natively occurring variants offer insights into deleterious variants, high-throughput deep mutational experiments enable comprehensive investigation of amino acid variants for a given protein. However, these mutational experiments are too expensive to dissect millions of variants on thousands of proteins. Thus, computational approaches have been proposed, but they heavily rely on hand-crafted evolutionary conservation, limiting their accuracy. Recent advancement in transformers provides a promising solution to precisely estimate the functional effects of protein variants on high-throughput experimental data. Here, we introduce a novel deep learning model, namely Rep2Mut-V2, which leverages learned representation from transformer models. Rep2Mut-V2 significantly enhances the prediction accuracy for 27 types of measurements of functional effects of protein variants. In the evaluation of 38 protein datasets with 118,933 single amino acid variants, Rep2Mut-V2 achieved an average Spearman’s correlation coefficient of 0.7. This surpasses the performance of six state-of-the-art methods, including the recently released methods ESM, DeepSequence and EVE. Even with limited training data, Rep2Mut-V2 outperforms ESM and DeepSequence, showing its potential to extend high-throughput experimental analysis for more protein variants to reduce experimental cost. In conclusion, Rep2Mut-V2 provides accurate predictions of the functional effects of single amino acid variants of protein coding sequences. This tool can significantly aid in the interpretation of variants in human disease studies.  Their biological activities could be affected by numerous variants. Although most of these variants have negligible effects on protein functions, a small fraction of native amino acid variants of human proteins are closely associated with human diseases influenza Hemagglutininand influenza A virus PA polymerase subunit[7], nonstructural protein 5A (NS5A) inhibitor for Hepatitis C virus[8], Hsp90[9], beta-lactamase TEM-1[13], a bacterial DNA methyltransferase M.HaeIII[14], BRCA1[15], hYAP65 WW domain[16], a Tn5 transposon-derived kinase APH(3′)II[17], yeast transcription factor Gal4[18], human tumor suppressor p53[18], PDZ domain[19], Saccharomyces cerevisiae poly(A)-binding protein Pab1[20], yeast ubiquitin geneand a tRNA gene[24], β-glucosidase enzyme Bgl3 from Streptomyces sp.[25], FAS/CD95[26], 20 ParD-ParE TA family members[27], murine ubiquitination factor E4Band HIV TatHigh-throughput experimental approaches, deep mutational scanningThus, computational approaches were proposed to overcome this limitation. These approaches can be classified into four groups. The first group, which includes PolyPhen-2Here, we propose a deep learning method, named Rep2Mut-V2, to use protein sequences as the sole input to accurately predict 27 types of measurements of mutational effects of protein variants. Rep2Mut-V2 is an improvement of our previous model Rep2Mut 22.1A total of 38 protein datasets, comprising 118,933 single amino acid variants , were us2.2., a wildtype sequence (WT), and a mutated sequence with a substitution of an amino acid at a position of interest  each with several pretrained models. For ESM-v1, we employed two models in this evaluation: esm1v_t33_650M_UR90S_1 DeepSequence: DeepSequence SIFT: Sorting Intolerant From Tolerant (SIFT) CPT: Cross-protein transfer (CPT) VariPred: VariPred EVE: evolutionary model of variant effect (EVE) 2.5X and Y be experimental and predicted estimates for a list of variants. SRCC is calculated using We use the Spearman's correlation coefficient (SRCC) to measure the performance of each tested method across the 38 datasets. For the implementation, we used the Python package scipy to calculate the SRCC between the predicted and experimental estimates. Specifically, we let 33.1Rep2Mut-V2 was first evaluated under three strategies: zero-shot transfer learning, few-shot learning and leave-one position-out cross-validation. The evaluation was conducted on the six fitness datasets  to 0.711 (on POLG_HCVJF), with an average SRCC improvement of 0.29. Furthermore, Rep2Mut-V2 demonstrated better performance than ESM-M2 on 36 datasets, with a median SRCC improvement of 0.209. The SRCC improvement ranged from 0.002 (on BG_STRSQ) to 0.50 (on RL401_2014). The datasets of PA_FLU and UBE4B were exceptions where ESM-M2 outperformed Rep2Mut-V2 by 0.022 and 0.015, respectively. But overall, Rep2Mut-V2’s predictions outshine ESM. For example, on the BG505 dataset in When compared to SIFT and EVE, Rep2Mut-V2 exhibited a consistent trend of performance improvement, outperforming both SIFT and EVE on all tested datasets. The SRCC improvement over SIFT ranged from 0.013 (PA_FLU) to 0.649 (GAL4), with an average improvement of 0.378. The SRCC improvement over EVE ranged from 0.002 (BRCA1_BRCT) to 0.348 (RASH), averaging 0.142.In the evaluation against VariPred, Rep2Mut-V2 excelled, surpassing VariPred on all 11 human datasets. The average improvement varied from 0.149 (TPK1_2017) to 0.643 (RASH), averaging 0.319. Similarly, Rep2Mut-v2 improved the prediction variants’ effects on 8 out of 11 human datasets when compared to CPT, with the SRCC improvement ranging from 0.009 (CALM1_Roth2017) to 0.4 (MK01).In summary, the estimation of Rep2Mut-V2 is substantially superior to DeepSequence, ESM, SIFT, CPT, VariPred and EVE. The averaged SRCC of Rep2Mut-V2 is 0.703, while DeepSequence, ESM-M1, ESM-M2, ESM-V2, SIFT, CPT, VariPred, and EVE had averaged SRCC score of 0.496, 0.412, 0.496, 0.461, 0.325, 0.484, 0.283 and 0.438 respectively. Rep2Mut-V2 significantly outperforms the state-of-the-art methods.3.3The above assessment of Rep2Mut-V2 used 90% of the dataset for training and 10% for testing. However, it is generally expensive and time consuming to generate more variant data through wet-lab experiments. Therefore, we tested Rep2Mut-V2’s performance with less variants for training. In detail, we used 30% of the variants from a dataset for fine-tuning Rep2Mut-V2 and the remaining 70% variants for testing.We compared this Rep2Mut-V2 model to DeepSequence and ESM. As illustrated in 4Rep2Mut-V2 was evaluated on 118,933 single amino acid variants from 38 protein datasets with 27 types of measurements of functional effects. The evaluation was conducted under various cross-validation strategies, and the performance of Rep2Mut-V2 was compared against six existing methods. The results consistently highlighted the superiority of Rep2Mut-V2 in accurately predicting the mutational effects upon protein variants. Even using limited variants for training, Rep2Mut-V2 maintained superior performance over existing methods. Notably, Rep2Mut-V2 relies solely on protein sequences and does not require protein 3D structures for precise prediction. Given the availability of millions of protein sequences compared to the limited number of proteins with experimental 3D structures, Rep2Mut-V2 proves to be a highly valuable tool, especially for those proteins which have experimentally determined functional effects only for a small fraction of variants.It is important to note that Rep2Mut-V2 uses representation vectors generated from an ESM framework as input, but its prediction performance is higher than the prediction performance of both ESM and DeepSequence. This success is partially attributed to the fact that ESM prediction relies solely on representation vectors of WT sequences, whereas our method uses representation vectors of both WT sequences and mutant sequences as inputs. Our design allows the models to learn the difference between WT and mutant vectors for accurate prediction. On the other hand, DeepSequence depends on evolutionary data generated from multiple sequencing alignments to infer mutational effects. Its performance is thus limited by the availability of similar sequences to refine the model.Our method has some limitations. First, our method was designed to predict the functional effects of single amino acid variants and is not currently equipped to handle high-order variants. We are presently extending our framework to address the prediction of functional effects of double/triple variants, although there are limited datasets with high-order variants. Second, we tested transfer learning with Rep2Mut-V2. However, transfer learning generally treats the contribution of each dataset equally, while the reliability of measuring functional effects of variants is different across those datasets. This uniform treatment may mislead transfer learning. To overcome this, a potential solution is to weigh the contribution of each dataset to shared layers in  based on5In this study, we proposed and tested Rep2Mut-V2 across 38 protein datasets with various effect measurements. Our approach was compared with six existing methods, and the evaluation demonstrates that our approach can achieve much better performance on most of the datasets. By relying solely on protein sequences, our approach achieved accurate prediction of functional effects even with a limited number of variants for training. These observations strongly suggest that Rep2Mut-V2 has the potential to study mutational effects across a broader spectrum of proteins, thereby benefiting human disease studies.The authors claim that there is no conflict of interest.


Theatre personnel can spread healthcare-associated infections through contaminated surgical scrubs. Decontamination of surgical scrubs through optimal methods is important to minimise transmission of microorganisms from theatre personnel’s clothing to different areas in the hospital and their homes.This study aimed to review the literature on the optimal home and hospital laundering methods for the decontamination of reusable surgical scrubs worn by theatre personnel.A systematic literature review of previous studies on laundering reusable surgical scrubs was performed. A review question was formulated using the patient, intervention, comparison and outcome (PICO) framework. A literature search was performed using ScienceDirect, Web of Science, ProQuest, EBSCOhost and Google Scholar.A direct link could be established between the cycle length and water temperature. The higher the water temperature, the shorter the washing cycle required. After a load has been washed in low or medium water temperatures, tumble drying and ironing should follow. Despite the water temperature, a disinfectant must be added to the load.Health professionals and hospital management should be aware of optimal laundering guidelines for hospital and home laundering as part of infection control. Water temperature, time, mechanical action, type of disinfectant and heat are factors influencing the successful removal of bacteria and other pathogens and represent the baseline of this article.Home-laundering of reusable surgical scrubs should follow strict guidelines. When these specific guidelines are applied, the effects of home-laundered scrubs will not negatively impact either the theatre or the home environment. The World Health Organization  defines Knowledge, education and training are some of the aspects that may influence healthcare professionals’ compliance or noncompliance with infection prevention and control procedures  proposed a set of guidelines compiled from the literature for best practices for laundering surgical scrubs AST . In the The aim of this study was to review the literature on the optimal home and hospital laundering methods for the decontamination of reusable surgical scrubs worn by theatre personnel. The objective was to critically synthesise and summarise the evidence available to determine the optimal home and hospital laundering methods for the decontamination of reusable surgical scrubs.A systematic literature review was chosen as a research method to identify and retrieve international evidence on the optimal home and hospital laundering methods for the decontamination of reusable surgical scrubs worn by theatre personnel  model is a widely used tool for structuring research questions for systematic literature reviews  online library: PubMed, ProQuest, the EBSCOhost platform  and the Web of Science (WoS) platform , Google Scholar and ScienceDirect, as indicated in Inclusion criteria: The inclusion criteria for this study were:Articles that referred to hospital or home laundering of surgical scrubsPrimary studies in EnglishResearch studies such as systematic reviewsBooksPublished journal articles on primary studiesPublished theses and dissertationsConference abstractsNo restriction was placed on the publication dates of the articles from the search to ensure that significant seminal studies were included.Exclusion criteria: The exclusion criteria for this study were:Studies in foreign languages other than EnglishConsumer/newspaper articlesArticles that did not relate to hospital or home laundering of surgical scrubsArticles for which only the abstracts were available and full articles could not be accessed or obtained with the assistance of the librarian.Reflection from the pilot study: When conducting a systematic literature review, the researchers should pilot the various steps of the process to increase efficiency and minimise error  independently screening the titles and abstracts of the retrieved studies for eligibility against the inclusion criteria. After reading the abstract, the studies that adhered to the inclusion criteria were selected. Following that, the entire articles of the abstracts that were selected as potentially relevant were screened. After the full-text screening, disagreements between the three reviewers were reconciled by the consensus of the fourth and fifth reviewers (J.H.L. and T.W.). To improve rigour, accurate record-keeping was ensured throughout the procedure for verification purposes. The data extracted were transferred into Microsoft Excel 2013 spreadsheets to record and summarise the data collection.The purpose of a quality assessment is to filter the data and exclude studies that were interpreted with biases. A quality assessment also strengthens the evidence that the researcher wants to use for the article  flow chart summarises the literature review results  Moher e. A totalThe temperature of the water, duration of the washing cycle, type of disinfectant, and whether heat has been applied after laundering, are all factors indicated as affecting an optimal laundering process. Although all 35 articles were included in The findings of this study could provide valuable information to healthcare workers and hospitals on optimal home and hospital laundering to improve optimal infection control practices, for example, how the healthcare worker can prevent contaminating their home environment and how the hospital can protect the personnel transporting the soiled scrubs to the laundry facility from bacteria and pathogens. Knowledge of successful decontamination of reusable surgical scrubs is important because bacteria and other pathogens present on the scrubs can spread to the patient, the person wearing the scrubs and to their home environment Dix .Occupational Health and Safety Act No. 85 of 1993 (OSHA)  for a minimum of 10 min Al-Benna .A combination of peroxyacetic acid (7.5%), hydrogen peroxide (20%) and acetic acid (7.5%) should be added to the wash load to facilitate the eradication of pathogenic bacteria that are difficult to eliminate, such as staphylococci, on the scrubs. The scrubs should be tumble-dried for a minimum of 10 min (Halliwell The scrubs should be covered and transported back to the hospital without coming into contact with other objects.Even though several articles did not recommend home laundering of reusable surgical scrubs, no scientific evidence could be found that this has an influence on the infection rate in healthcare facilities. The guidelines in this review should be followed to successfully remove pathogens from soiled scrubs to ensure good infection control practices. The hospital and home laundering environments of surgical scrubs are different, as well as the equipment that is used. Consequently, recommendations in the guidelines for surgical scrubs laundering at home and in hospitals are varying. These optimal laundering guidelines compiled from the literature include the correct water temperature, time, mechanical action, type of disinfectant and heat. These are all factors that determine whether pathogens are successfully removed from the soiled scrubs. The findings in this review can serve as a basic guideline on recommendations to reduce the transmission of pathogens when home and hospital laundering is performed.


Virus-like particles (VLPs) have gained a lot of interest within the past two decades. The use of VLP-based vaccines to protect against three infectious agents—hepatitis B virus, human papillomavirus, and hepatitis E virus—has been approved; they are very efficacious and offer long-lasting immune responses. Besides these, VLPs from other viral infectious agents  are under development. These VLPs, especially those from human and animal viruses, serve as stand-alone vaccines to protect against viruses from which the VLPs were derived. Additionally, VLPs, including those derived from plant and bacterial viruses, serve as platforms upon which to display foreign peptide antigens from other infectious agents or metabolic diseases such as cancer, i.e., they can be used to develop chimeric VLPs. The goal of chimeric VLPs is to enhance the immunogenicity of foreign peptides displayed on VLPs and not necessarily the platforms. This review provides a summary of VLP vaccines for human and veterinary use that have been approved and those that are under development. Furthermore, this review summarizes chimeric VLP vaccines that have been developed and tested in pre-clinical studies. Finally, the review concludes with a snapshot of the advantages of VLP-based vaccines such as hybrid/mosaic VLPs over conventional vaccine approaches such as live-attenuated and inactivated vaccines. Viruses infect a wide range of organisms, ranging from humans, plants, animals, birds, and insects to microorganisms . One unique feature of viruses is that their structural proteins, envelope proteins, or capsid proteins, along with other structural proteins, can, either independently or collectively, spontaneously self-assemble to form virus-like particles (VLPs) without the viral genome. Thus, any virus can be utilized to develop VLPs; VLPs can be developed by cloning the structural genes that code for the proteins of a virus of interest into an expression vector . The expVLPs have many features, unlike conventional vaccines, which make them very attractive platforms for vaccine design. They mimic the viruses from which the VLPs are derived in terms of size (20–200 nm) , geometrMoreover, VLPs are considered safe, since they do not contain the viral genome, and therefore, they cannot replicate. It is worth mentioning that, like any vaccine, VLPs can cause side effects such as pain and swelling at the injection site. Hence, it is no surprise that VLPs have gained considerable attention over the past two decades as an attractive platform for vaccine design. While VLPs derived from human and animal viruses are developed to protect humans and animals against viruses from which the VLPs are derived, VLPs from other organisms (including plants and prokaryotic microorganisms) are developed to display heterologous antigens from human and animal viruses, i.e., to develop chimeric VLPs , bottom.VLPs have been developed (while others remain under development) using viruses that infect humans . In factThree VLP-based vaccines—Gardasil-9, Gardasil-4, and Cervarix—have been approved around the world to protect against human papillomaviruses ,27,28,29One VLP-based vaccine (Hecolin) used to protect against HEV is approved in China; clinical trials to assess its efficacy in other countries—Bangladesh and the U.S.—are ongoing ,37. The 7 [In addition to the approved VLP-based vaccines above, several candidate VLP-based vaccines are under development. An excellent new book by Pumpens and Pusko provides a comprehensive summary of candidate VLP-based vaccines that have been developed from various viruses . VLPs th7 . Similar7 ,45.Helicobacter pylori (H. pylori) bacteria, hepatitis C virus (HCV), HPV-associated cancers, and four serotypes of dengue virus , physalis mottle virus (PhMV), potato virus Y (PVY), cucumber mosaic virus, and malva mosaic virus (MaMV) . VLPs fral trial ,111. PhMal trial ,113. In  antigen . Moreove antigen . On the other side of viruses that infect bacteria (bacteriophages or phages), VLPs have been developed from bacteriophage AP205, MS2, PP7, Qβ, P22, etc. . ImmunizOverall, VLPs from viruses that infect humans, animals, plants, and bacteria have been used to develop vaccines/candidate vaccines against infectious agents, as well as metabolic diseases. VLP vaccines are proteins, and thus, they cannot be amplified in the body  like other recombinant vaccines such as viral vector vaccines and mRNA vaccines. Nevertheless, VLP vaccines are better alternatives to the two recombinant vaccines. VLP vaccines do not need freezing conditions of −20 to −80 °C for transportation and storage, like mRNA vaccines. In addition to this, VLPs are immediately processed by APCs once they are injected into the body, unlike DNA and mRNA vaccines, whereby cells first need to transcribe (for DNA vaccines) and translate their mRNAs into proteins before they can be processed by APCs to be presented to the immune system.VLP vaccines are also better alternatives to conventional vaccines (live-attenuated vaccines and inactivated vaccines) for several reasons: First, VLP vaccines do not replicate, and thus, they can be used for everyone, including women who are pregnant or people with a compromised immune system. Additionally, coat proteins—including other structural proteins—from viruses that have segmented genomes  can also be used to develop hybrid/mosaic VLP-based vaccines without fear of genetic reassortment, like in live-attenuated vaccines. Viruses with segmented genomes have the ability to undergo genetic reassortment; unfortunately, a live-attenuated polyvalent vaccine derived from an AHSV led to the virulent reversion of AHSV type 1 and genomic reassortments with segments from AHSV types 1, 3, and 4, leading sporadic outbreaks of AHSV . Second,6 plaque forming units) of an ASFV vaccine offers complete cross-protection against an ASFV strain compared to partial cross-protection following immunization with half the dose [Although VLP vaccines are immunogenic at smaller doses, it is unclear whether smaller doses of VLPs can provide long-term protection against viruses with different genotypes such as HBV (which has at least nine genotypes ), HPV  prevalent in humans in different geographical regions around the world. As mentioned above, the Hecolin vaccine is derived from HEV genotype 1 and it cross-protects against genotypes 2 and 4. The vaccine is also expected to offer some degree of cross-protection to genotype 3 ,42,136. As mentioned earlier, VLPs  have also been used as display platforms to enhance the immunogenicity of peptides derived from other infectious agents  and non-infectious agents  ,82,139, Overall, VLPs are versatile and efficient platforms for vaccine development. They can be used to develop vaccines which act not only against the viruses from which they are derived, but also against other infectious and non-infectious agents. Furthermore, they are excellent platforms for the development of hybrid/mosaic vaccines against viruses with segmented genomes, especially those that are transmitted by insects . Their inability to replicate in insects reduces the risk of reassortment events that can occur with live-attenuated vaccines developed from viruses with segmented genomes. Additionally, a VLP-based vaccine for viruses such as AHSV may also help in differentiating vaccinated horses from infected horses, which may be challenging with a live-attenuated vaccine.


With a sample of 228 psychology papers that failed to replicate, we tested whether the trajectory of citation patterns changes following the publication of a failure to replicate. Across models, we found consistent evidence that failing to replicate predicted lower future citations and that the size of this reduction increased over time. In a 14-y postpublication period, we estimated that the publication of a failed replication was associated with an average citation decline of 14% for original papers. These findings suggest that the publication of failed replications may contribute to a self-correcting science by decreasing scholars’ reliance on unreplicable original findings. Scholars have long been concerned about the reliability and validity of scientific findings . In the To date, four investigations have addressed similar questions. Although these studies produced important advances, each was limited by small samples of failed replications, and together, they forward an ambiguous picture. Two papers investigating citations of studies replicated in the Open Science Collaboration’s Reproducibility Project: Psychology  found thThese papers used unique procedures and so are not directly comparable, but the general interpretations for the relationship between replication failures and citations appear mixed. We seek to provide a clearer picture by analyzing changes in citation patterns to original papers (OPs) after publication of a failed replication paper (FRP) and with more failed replications than earlier investigations. Additionally, we adopt a different approach. Given potentially important differences between 1) papers chosen and not chosen for replication and 2) papers that successfully replicate or fail to, it is challenging to estimate the counterfactual number of citations papers with unsuccessful replications would receive absent their failures. We handle this challenge by exclusively considering papers with failed replications and comparing their average citation trends over time before and after failures to replicate, effectively treating papers with failed replications as their own control group. We can thus estimate average expected citation trends over time for this specific group of papers before and after replication failures.We logged citation counts (after adding 1) to normalize residuals and restricted analyses to the first 14 y postpublication (83.3% of the data) due to a substantial drop-off in data beyond that. This resulted in a total sample size of 2,919 logged citation counts .2(1) = 26.16, P < 0.001, ΔR2 = 0.01, with a significant negative slope on FRP, b = −0.17, SE = 0.03, t = −5.12, P < 0.001. We then tested whether this effect increased over time by adding years since FRP . Doing so again improved model fit, Δχ2(1) = 119.17, P < 0.001, ΔR2 = 0.03, with a significant negative slope on both FRP, b = −0.15, SE = 0.03, t = −4.72, P < 0.001 and YS_FRP, b = −0.09, SE = 0.009, t = −10.76, P < 0.001.We fitted a series of hierarchical linear models (HLMs) including random intercepts for OPs. An initial model predicted logged citation counts from OPs’ number of years since publication and its first five polynomials (model 1). To account for differences in citation trends between relatively older and more recent OPs, we modeled effects of publication year (mean-centered), plus an interaction term between publication year and years since publication (model 2). We then estimated the effect of FRPs by adding an indicator coded 0 for years prior to FRP and 1 for subsequent years (model 3). This improved model fit, ΔχH of To interpret these effects, we compared each paper's predicted citations following replication failures with counterfactual predicted citations assuming that they had not failed to replicate. Reconverting predictions from logged to real units, average predicted citations were reduced from 24.09 to 20.82 following FRPs, a 14% reduction. Panel To test the robustness of these results, we explored various further model specifications using additional control variables, data, and modeling techniques .One important limitation of the present work is its observational design. OPs are not randomly assigned to be replicated, so we cannot rule out the possibility that confounding factors besides FRPs  may have produced the observed citation declines.Another limitation is that we binarily classified failed replications based on FRP authors’ and our own subjective assessments. Results of replication attempts, however, exist on a continuum, ranging from ambiguous to strong evidence for or against an original finding. Often, it is not obvious whether a replication effort did or should cast doubt on original findings . AdditioWe also did not assess whether OP citations were positive/affirmative vs. negative/critical. Given that we see an overall decline in citations, and some postfailure citations are negative/critical [estimates range from 3%  to 12% ], the trFuture work could also test whether failed replications are associated with steeper or shallower citation declines when published as part of a large-scale replication vs. a standalone replication. Large-scale replication efforts seem to attract attention, but they also tend to de-emphasize the specific papers that failed to replicate. Thus, large-scale projects may have a relatively larger or smaller impact on the perceived reliability of OPs.Although matching papers with and without failed replications presents some challenges, future work should seek to replicate our results using alternative approaches that compare citation patterns among papers that failed to replicate to two additional classes of papers : 1) papers that successfully replicated and 2) papers with no known replication attempts. Our results suggest that papers that fail to replicate likely receive fewer citations post-failure-to-replicate relative to these classes of papers.Recent replication efforts may indeed be contributing to a more self-correcting science.https://osf.io/8ustx/ and https://osf.io/8ustx/?view_only=a6e84a79e33f4a848d5ebb97d4936fc2. See SI Appendix for our expanded Open Science Statement describing analytic decisions as well as Procedure details sufficient for replication.This study was not preregistered. Data and analysis code are available here: In late 2021, we collected FRPs from major replication projects n = 154) (4 (5) 1111, and aAppendix 01 (PDF)Click here for additional data file.