The accuracy of any analytical method is highly dependent on the selection of an appropriate calibration model. Here, we present CCWeights, an R package for automated assessment and selection of weighting factors for accurate quantification using linear calibration curve. Additionally, CCWeights includes a web application that allows users to analyze their data using an interactive graphical user interface, without any programming requirements. The workflow and features of CCWeights are illustrated by the analyses of two datasets acquired by liquid chromatography-mass spectrometry (LC-MS). The resulting quantification table can be directly utilized for further model assessment and subsequent data analysis.https://cran.r-project.org/web/packages/CCWeights), with source code available on GitHub (https://github.com/YonghuiDong/CCWeights) under a GPL-3 license. The web application can be run locally from R console using a simple command “runGui”. Alternatively, the web application can be freely accessed for direct online use at https://bcdd.shinyapps.io/CCWeights/.CCWeights is publicly available on CRAN repository (Bioinformatics Advances online. However(x-axis) .r2) should not be used as the criteria in selecting weighting factor because the large variances present at high concentrations dominate the correlation coefficient calculation. In addition, it is suggested that a weighting factor should only be used for heteroscedastic data (A simple and effective way to account for heteroscedasticity and improve the accuracy over the selected concentration range is to use weighted linear regression (WLR) with an appropriate weighting factor . UnfortuAlthough many commercial software, such as TraceFinder™  and Targetlynx™ , offer different weighting factors for users to choose for their gas chromatography or liquid chromatography mass spectrometry (GC- or LC-MS)-based targeted analyses, they do not provide functions to evaluate and select the appropriate weighting factor. To this end, we have developed CCWeights for automated selection of proper weighting factor for each individual compound, and analyte quantification using linear calibration curve. In particular, apart from the build-in weighting factors, CCWeights allows users to define their own weighting factors for model evaluation and analyte quantification. Furthermore, it provides a web interface which does not require any coding expertise.CCWeights is developed using R statistical language  and is rF-test  is smaller than corresponding F-table value (Ftab) at confidence of 99% . Users can customize the confidence levels according to their studies  is suggested. Otherwise, five commonly used weighting factors, that is 1/x0, 1/x, 1/x2, 1/y and 1/y2, together with user-defined weighting factors (if present) are tested. By applying regression with different weighting factors on a set of calibration curve standard data, the best weighting factor could be identified by choosing the one generating the smallest sum of the absolute relative errors (SRE%)  . Althougs (SRE%) .x0, 1/x, 1/x2, 1/y, 1/y2, and samples were quantified with each calibration model accordingly. The resulting linear regression models and quantification results were used to compare to those obtained from CCWeights, and CCWeights results are consistent with the ones obtained by TraceFinder™ (The features of CCWeights are illustrated by two examples: (i) targeted analysis without using any internal standards and (ii) targeted analysis with isotopically labeled counterparts as internal standards (Supplementary Files S1 and S2). Detailed sample preparation and data processing methods are provided in eFinder™ .CCWeights is an efficient and easy-to-use R package and web application allowing automated optimized weighting factor selection for accurate quantification using linear calibration curve. It provides a user-friendly output which can be used for further model assessment and subsequent data analysis. It is important to note that although the workflow and features of CCWeights are illustrated using two LC-MS datasets, it can be used for any analytical data in practice.This study was supported by the Blavatnik Center for Drug Discovery, Tel Aviv University, funded by the Blavatnik Family Foundation.Conflict of Interest: none declared.vbab029_Supplementary_DataClick here for additional data file.


The proinflammatory cytokine tumor necrosis factor (TNF) has been described as one of the main mediators of intestinal inflammatory diseases, affecting the composition of tight junction (TJ) proteins and leading to a disruption of the epithelial barrier. An intact intestinal barrier is mandatory, because the follicle-associated epithelium of Peyer’s patches represents the first defense line of the intestinal immune system and ensures a controlled uptake of antigens from the gut lumen. In the current study, we have analyzed the detailed effects of TNF on the follicle-associated epithelium of porcine Peyer’s patches by applying the Ussing chamber technique. Epithelial tissue specimens of Peyer’s patches and the surrounding villus epithelium were mounted into conventional Ussing chambers and incubated with TNF for 10 h. The transepithelial resistance, representing epithelial barrier function of the tissue, was recorded. A reduction of transepithelial resistance was detected after 8 h in Peyer’s patch tissue specimens, whereas the villus epithelium was not significantly affected by TNF. Subsequent molecular analysis of TJ protein expression revealed a marked decrease of claudin-1 and -4, and an increase of claudin-2. In neighboring villus epithelium, no significant changes in the expression of TJ proteins could be shown. A strong increase of TNF receptor-2 (TNFR-2) could also be detected in Peyer’s patches, in agreement with the major role of this receptor in Peyer’s patches. Our findings were in accordance with changes detected by confocal laser scanning immunofluorescence microscopy. The regulation of TNF effects via myosin light chain kinase (MLCK) was analyzed in blocking experiments. Our detailed analysis is the first to show that TNF affects the barrier function of the follicle-associated epithelium of porcine Peyer’s patches but has no effects on the villus epithelium. These findings reveal not only the basic differences of epithelial barrier function between the two structures, but also the significance of Peyer’s patches as a primary mucosal immune defense. The intestine is exposed to a multitude of exogenous substances, and therefore an efficient epithelial barrier is mandatory for limiting paracellular permeability. As a major part of the gut-associated lymphatic tissue (GALT), Peyer’s patches (PP) are located in the distal small intestine and hence represent a first line of immunological defense . It consin vivo and in vitro (Tumor necrosis factor (TNF) is released by macrophages and can subsequently mediate the production of other cytokines, which is the reason that it is described as a proinflammatory cytokine . It playin vitro . HoweverIn our current study, we hypothesized the functional and molecular difference of PP compared to VE under inflammatory conditions. Based on our previous findings , we have+ (149.4), Cl− (128.8), K+ (5), Ca2+ (1.2), Mg2+ (1.2), HCO3− (25), H2PO4− (0.6), HPO42- (2.4), d-Glucose (10)  and Enrofloxacin . The buffer was gassed with 95% O2 and 5% CO2, and the pH was adjusted to 7.4 before transport.Tissue specimens from the small intestine with and without PP from 28 adult pigs were taken immediately after slaughter at a slaughterhouse . The tunica serosa and muscularis externa were stripped off the mucosa, and the remaining tissue was rinsed with 0.9% NaCl to remove chyme and mucus. The tissue samples were transported to the laboratory in ice-cold buffer containing (in mmol/L): Na2 and 5% CO2 at a pH of 7.4. The area of the tissue exposed to the buffer was 0.96 cm2. After an equilibration period of 60 min, TNF was added at three different concentrations  to the serosal side of the tissue, and TER was recorded permanently for 10 h. A buffer exchange was carried out after 3, 6, and 8 h to ensure the viability of the tissue  was added to the mucosal side, and samples were taken directly after addition of the tracer and at the end of the flux periods from the same side. Samples of 600 μl were taken from the basolateral side every 40 min, giving three flux periods over 2 h. After removal of the samples, the absent volume was refilled with 600 μl fresh buffer containing the corresponding TNF concentration. Subsequently, Aquasafe 300 plus liquid scintillation cocktail  was added, and each sample was analyzed using a TriCarb 4910 TR liquid scintillation counter . Calculation of the specific activity and paracellular flux of the tracer was carried out as described previously . After 8 h of incubation with the cytokine, 2 μCi -D-Mannitol having been seen after incubation of the cells with 1000 U/ml TNF for 48 h (To strengthen our findings further, additional experiments were carried out using the non-transformed cell-line IPEC-J2. This porcine jejunal cell-line has recently been used for analysis of TNF effects on the intestinal barrier function. Significant effects on transepithelial resistance and paracellular flux of [for 48 h . In the ex vivo, as the tissue samples are more comparable with the intestine in vivo. Moreover, the PP samples allow us to investigate the direct effect of TNF on FAE. By employing the Ussing chamber technique, we performed a detailed analysis of the effects of TNF on TER for up to 10 h. Subsequently, tissues were stained with hematoxylin-eosin, and the size of PPs and the length of the surrounding villi were measured and compared between the control and TNF-treated groups. Apoptosis could be excluded as a reason for the observed changes, as the tissue samples still responded to vitality tests carried out using theophylline. Because no significant alterations in tissue quality after incubation with TNF were observed, and no evidence for apoptosis could be detected, other mechanisms for the drop in TER needed to be explored, with special attention being given to barrier perturbation, as described in detail above. It cannot be ruled out, that a longer incubation with TNF might also affect the functional barrier properties of VE additional to changes in PP FAE, as intestinal inflammation like in PWD go also in accordance with a disturbed morphology of VE in porcine (Our aim has been to analyze PP and neighboring VE  porcine .In our current study, we have been able to confirm the functional and molecular differences of PP and VE under intestinal inflammatory conditions. The importance of the porcine FAE as a first step in intestinal immunological defense has been established, as only PP tissue specimens reacted to the addition of the proinflammatory cytokine TNF with changes in barrier function, TJ protein composition, and the expression of TNFR-2, whereas VE did not respond. The outcome of our current study might allow further approaches focusing on the identification and analysis of counter-regulatory, beneficial, and therefore possibly therapeutic and preventative compounds for administration under inflammatory intestinal conditions.


Heart failure with preserved ejection fraction (HFpEF) is a complex, heterogeneous disease characterized by autonomic imbalance, cardiac remodeling, and diastolic dysfunction. One feature that has recently been linked to the pathology is the presence of macrovascular and microvascular dysfunction. Indeed, vascular dysfunction directly affects the functionality of cardiomyocytes, leading to decreased dilatation capacity and increased cell rigidity, which are the outcomes of the progressive decline in myocardial function. The presence of an inflammatory condition in HFpEF produced by an increase in proinflammatory molecules and activation of immune cells  has been proposed to play a pivotal role in vascular remodeling and endothelial cell death, which may ultimately lead to increased arterial elastance, decreased myocardium perfusion, and decreased oxygen supply to the tissue. Despite this, the precise mechanism linking low-grade inflammation to vascular alterations in the setting of HFpEF is not completely known. However, the enhanced sympathetic vasomotor tone in HFpEF, which may result from inflammatory activation of the sympathetic nervous system, could contribute to orchestrate vascular dysfunction in the setting of HFpEF due to the exquisite sympathetic innervation of both the macro and microvasculature. Accordingly, the present brief review aims to discuss the main mechanisms that may be involved in the macro- and microvascular function impairment in HFpEF and the potential role of the sympathetic nervous system in vascular dysfunction. Heart failure (HF) is a pathological condition affecting mainly the elderly population. A subcategory of this disease is HF with preserved ejection fraction (HFpEF), whose incidence has increased notably in recent years, particularly in the last two decades, from 48 to 57% compared with systolic HF (or reduced ejection fraction HF). Furthermore, HFpEF accounts for the death of 1 in 8 people over 65 years . Then, uPatients with HFpEF display several comorbidities associated with cardiac and vascular disturbances, including but not limited to diabetes mellitus, obesity, pulmonary hypertension, coronary artery disease, chronic renal failure, and systemic inflammation , all of Endothelium-dependent coronary microvascular dysfunction is present in approximately 30% of patients with HFpEF . In addirarefaction, is observed in patients with HFpEF , which acting at SMC induces cell relaxation resulting in blood vessel vasodilation (Both arms of the autonomic nervous system (ANS)  innervate blood vessel walls and regulate wall tension –31. SMCstriction , 31. In dilation , 31. BesThe role of vascular NO is essential for vasodilation, inhibition of platelet aggregation, and protection of the integrity of the endothelial layer given its anti-inflammatory, proangiogenic, anti-apoptotic, and anti-fibrotic properties, reducing vascular inflammation and atherosclerosis , 33. At 2-adrenergic receptors stimulate NO synthesis by the activation of endothelial nitric oxide synthase isoform (eNOS)  . Interesm (eNOS) , 39. Furm (eNOS) , 40. Indm (eNOS) , 41. Notm (eNOS) , 26. Them (eNOS) –44. Oxidm (eNOS) . Beyond m (eNOS) . Also, apairment . Thus, aculature , 48. Intculature , 50 but culature , suggestRisk factors in HF, such as diabetes mellitus, aging, and hypertension, among others, trigger systemic low-grade inflammation, characterized by chronic elevations in circulating immune cells, proinflammatory cytokines, and increased expression of endothelial adhesion molecules, such as vascular and intercellular cell adhesion molecules-1 (ICAM-1 and VCAM-1), and the corresponding ligands of circulating leukocytes, increasing myocardial infiltration of CD45+ and CD3+ T-lymphocytes . The lat3R1 activation and in consequence IP3 signal to increased Ca2+ release and vascular tone in VSMCs during HF (3R1 for elevations in cytosolic [Ca2+] (3R1) are upregulated in HF and increased receptor phosphorylation in HF, it has been suggested that IP3R1 may play an important role in Ca2+ regulation in VSMC (2+) mishandling in the vasculature and subsequent acceleration of cardiac remodeling and progression of HFpEF , and transient receptor potential cation channels (TRPC)  . For RyRnagement . In addiunctions . In addiunctions . Finallyn the SR . In addin the SR . The lathandling . TherefoLittle is known about the role of macro- and microvascular alterations during the onset, development, and progression of HFpEF. However, it is highly likely that vascular rarefaction takes place during the onset, maintenance and/or progression of HFpEF resulting in increases in microvascular resistance, reductions in tissue perfusion, and activation of vasomotor sympathetic fibers that ultimately create a feed-forward mechanism that promotes the further deterioration of vascular function by shifting the balance between vasoconstriction and vasodilation. On the contrary, proinflammatory and pro-oxidative molecules have been associated with the etiology of the disease. At the microvascular level, the decrease in the bioavailability of NO, alterations in the sGC-cGMP-PKG pathway, accumulation of ROS, and chronic low-grade inflammation are the main actions involved in the alteration of vascular function both at the systemic circulation and in the coronary territory, promoting a functional decrease in cardiomyocytes, evidenced by delayed myocardial relaxation, increased myocardial stiffness, cardiac hypertrophy, and interstitial fibrosis. The latter may have fundamental implications for the progressive decline in cardiac function during HFpEF.To date, there are only preventive and palliative actions to deal with HFpEF, such as exercise and a healthy lifestyle, which do not imply a remission of the disease. In this article, several molecular candidates rise as potential therapeutic targets to improve both vascular and cardiac functions in HFpEF, including but not limited to NO metabolic pathway, IP3R signaling, adrenergic pathways, and reduction of oxidative stress and vascular inflammation.AS-A wrote the first draft. KP, CT, RI, and RDR contributed to manuscript formulation and revision. All authors have read and approved the final manuscript.


Distinct molecular signatures were observed in different HFpEF forms. Clusters of biomarkers differentially abundant between HFpEF forms were predominantly associated with microvascular inflammation. New candidate protein markers were also identified, including leucine-rich alpha-2-glycoprotein 1 (LRG1), serum amyloid A1 (SAA1) and inter-alpha-trypsin inhibitor heavy chain 3 (ITIH3). Our study is the first to apply systematic, quantitative proteomic screening of plasma samples from patients with different subtypes of HFpEF and identify candidate biomarkers for improved management of acute and chronic HFpEF and HCM.Heart failure with preserved ejection fraction (HFpEF) accounts for around 50% of all heart failure cases. It is a heterogeneous condition with poorly understood pathogenesis. Here, we aimed to identify unique pathogenic mechanisms in acute and chronic HFpEF and hypertrophic cardiomyopathy (HCM). We performed unbiased, comprehensive proteomic analyses of plasma samples from gender- and BMI-matched patients with acute HFpEF ( Approximately 50% of patients with heart failure (HF) present with a preserved left ventricular ejection fraction (LVEF), a condition referred to as heart failure with preserved ejection fraction (HFpEF) . HF withHypertrophic cardiomyopathy (HCM) is often a key precursor of HF. The progression of HCM into HFpEF was described in a large population-based observational study , in whicCurrent ‘omics studies investigating the molecular mechanisms of HF are mainly focused on HFrEF , while oGiven the ease of access to plasma samples, the aim of this study was to identify perturbed proteins that could be translated into specific biomarkers for different forms of HFpEF including chronic and acute cases. We also identified associated pathways providing insight into the pathogenesis of different forms of HFpEF including HCF, acute and stable HFpEF patients.g for 10 min to obtain plasma. All participants provided written informed consent prior to inclusion. Ethical approvals were obtained from all hospitals and institutions involved in this study. The study was conducted in accordance with the Declaration of Helsinki.A total of 31 gender- and BMI-matched patients diagnosed with HFpEF in accordance with the latest guidelines for HF  were conn = 8), chronic HFpEF (n = 9) and HCM (n = 14) (The patients were stratified into acute HFpEF ((n = 14) . Briefly10p score that was determined by the false discovery rate (FDR) < 1%, in which decoy database search matches were <1% of the total matches.Plasma sample preparation was adjusted from the previously described methods using STop-And-Go-Extraction tips (STAGE Tips) ,11, wher2-transformed prior to entering pipelines in R. The packages or functions utilised in this study included (i) limma package  > 0.7), where the colour and the length of the edge are proportional to Pearson r. Overall, our data in terms of PPIs were consistent with those reported by broad evidence in the STRING database  were the key proteins across all groups. ECM proteins are slightly more influential in acute HFpEF vs. HCM and chronic HFpEF vs. HCM a,b. HoweUsing plasma samples from gender- and BMI-matched patients with different forms of HFpEF including HCM (cardiac-specific cause of HFpEF), chronic HFpEF (heterogeneous group) and acute HFpEF , we have identified a core set of plasma biomarkers and signalling pathways.2+ homeostasis in cardiomyocytes is a hallmark intramyocardial pathogenic mechanism of HF. Nevertheless, therapies targeting this aberrant intramyocardial biological process  are beneficial for HFrEF, while ineffective for HFpEF [2+. This finding suggests potential novel pathogenesis extrinsic to the heart in HFpEF.Aberrant Caor HFpEF , suggestor HFpEF . Therefo2+ induced by systemic microvascular impairment in HFpEF. Platelet activation is mediated by secreted molecules from alpha and dense granules. Within this pathway, we showed elevated abundance of a series of ECM proteins  and decreased abundance of APOA1, F5 and PLG. The link between inflammation and endothelial dysfunction in cardiovascular disease is well-established; vWF is key in this process where it is secreted into the circulation from endothelial cells in response to systemic inflammation. Furthermore, vWF possesses intrinsic proinflammatory properties capable of attracting leukocytes to the inflammatory site within the endothelial barrier [The effects on the myocardium as a result of systemic inflammation in HFpEF likely originates from systemic microvascular endothelial dysfunction. Our study demonstrated the importance of the platelet degranulation pathway in response to elevated intraplatelet Ca barrier , therefo barrier . ElevateWe have also shown a reduction in abundance in a number of key proteins including APOA1, the main component of high-density lipoprotein (HDL), in both acute and chronic HFpEF compared to HCM. External to the pathway of platelet activation, other apolipoprotein components of HDL  were also reduced in abundance in acute or chronic HFpEF compared to HCM. Interestingly, serpin family A member 1 (SERPINA1) or alpha-1-antitrypsin was decreased in chronic HFpEF vs. HCM, and increased in acute HFpEF vs. HCM or acute vs. chronic HFpEF, suggesting that SERPINA1 levels may be substantially low in chronic HFpEF. Given that the SERPINA1-HDL complex has a protective effect against inflammation , a decreAll the perturbed proteins in the platelet degranulation pathway together highlight potential underlying atherosclerosis in acute and chronic HFpEF. Microcirculatory dysfunction is closely associated with atherosclerosis ,28 and b2+ elevation is important in the interplay between systemic pathogenesis and local  abnormality in HFpEF. The progression of HF is related to increasing angiotensin-II formation in the heart as a result of enhanced LV end-systolic wall stress [2+ is well-characterised as a result of elevated angiotensin-II [2+ and LV diastolic dysfunction in HFpEF for the first time.Intraplatelet Cal stress , a key densin-II ,35. TherAnother key pathway is the regulation of IGF transport and uptake through IGFBP, centred by IGFBP complex acid labile subunit (IGFALS). The levels of IGFALS were significantly decreased in acute HFpEF vs. HCM. Given the pro-angiogenic function of the IGF pathway , our resIn terms of pathways perturbed in both acute and chronic HFpEF, we have identified amyloid fibre formation regulated by SAA1, which has also been detected in other multiomics studies using HF samples ,19,20. SThe vast majority of multiomics studies in the field of HF are mostly focused on HFrEF rather than HFpEF. A study by Raphael et al.  followedAnother HFpEF proteomic study utilised human myocardial tissues , specifiAlthough this study provides new information for personalised monitoring of HFpEF patients, there are a few limitations. We chose not to employ any depletion methods to remove highly abundant plasma proteins to prevent co-depleting any differentially abundant proteins, an approach that is becoming the standard in plasma/serum analysis . Follow-Our study is the first to apply comprehensive and quantitative proteomic screening of plasma samples from patients with different types of HFpEF, including HCM and acute and chronic HFpEF, representative of disease heterogeneity and hospitalisation. New potential protein markers were also identified for different HFpEF forms, including LRG1, SAA1 and ITIH3. We report perturbations in a series of systemic pathways including platelet activation and regulation of IGF transport and uptake. Many of these pathways are associated with microvascular inflammation, a well-established pathogenic process of HFpEF. We identified novel protein perturbations across HCM, acute and chronic HFpEF, including LRG1 and SAA1, which suggests the important roles of these proteins in acute exacerbation and hospitalisation of HFpEF. The network analyses showed the importance of ECM proteins in the pathogenesis of both heterogeneous forms of HFpEF (acute and chronic HFpEF) compared to the homogenous group with HCM. On the other hand, immune proteins appear to drive the acute decompensated HFpEF. In addition to biomarker potential, some of these proteins could have a therapeutic target potential, which should be explored in the future.


Phase-separated membraneless organelles or biomolecular condensates play diverse functions in cells, however recapturing their characteristics using small organic molecules has been a challenge. In the present study, cell-lysate-based screening of 843 self-assembling small molecules led to the discovery of a simple organic molecule, named huezole, that forms liquid droplets to selectively sequester tubulin. Remarkably, this small molecule enters cultured human cells and prevents cell mitosis by forming tubulin-concentrating condensates in cells. The present study demonstrates the feasibility of producing a synthetic condensate out of non-peptidic small molecules for exogenous control of cellular processes. The modular structure of huezole provides a framework for designing a class of organelle-emulating small molecules. R-huezole, phase separates to selectively sequester tubulin proteins to control the cell cycle. Its modular structure provides a framework for designing bioactive molecules to mimic membraneless organelles in cells.A non-peptidic small molecule,  Cells organize their contents into organelles, which have classically been considered as membrane-separated architectures. However, a growing number of studies have revealed newly defined types of organelles that form 3–5 One prominent role of membraneless organelles is the regulation of spindle formation during cell division at centrosomes and spindle formation sites.6 Centrosome is formed and expanded through phase separation at least in C. elegans,7 condensing tubulin to catalyze the generation of microtubules for assembly of the mitotic spindle.8 Spindle-defective protein 5 (SPD-5), one of the key centrosome proteins, forms phase-separated, spherical liquid condensates in vitro, which possess centrosomal activity for nucleating microtubules by recruiting tubulin and other centrosome proteins.8 A number of other spindle and centrosome-associated proteins, including BuGZ9 and TPX2,10 undergo liquid–liquid phase separation to recruit and concentrate tubulin for accelerating microtubule nucleation. Concentration of tubulin through liquid–liquid phase separation is a critical mechanism for regulating spindle formation during mitosis.The phase-separated membraneless structures are associated with diverse cellular functions, including enzymatic reactions, degradation pathways, cellular homeostasis, and transcriptional control.11–14 Such artificial organelle-like architectures would serve as models to further advance our understanding of the determinants of phase separation in cells and also open new opportunities for controlling biological processes. However, the artificial organelles that have been reported typically exploited biological components such as nucleic acids, proteins, and peptide building blocks.15–18 To date, only very limited success has been achieved using small molecules in formulating organelle-like structures that are capable of controlling biological processes.19 Creating artificial membraneless organelles with small organic molecules would offer simple models and alternatives for recreating or controlling cellular events and also enable unique opportunities for future drug discovery by interfering with disease processes. In the present study, we have taken a screening approach to search for small organic molecules that form artificial organelle-like condensates.The widespread involvement of membraneless organelles in cellular functions has inspired the design of synthetic, artificial molecules that capture the characteristics of membraneless organelles.20 Each of the compounds in our library form particles detectable by one of two environment-sensitive fluorescent probes, namely Nile Red and 8-anilinonaphthalene-1-sulfonic acid (ANS), in PBS buffer, and display a variety of particle morphologies such as micelle, fibers, crystals, and colloid aggregates.20 Each of the library compounds (20 μM) was incubated with human cell lysates. After this incubation period, the lysates were subjected to centrifugation  and an achiral analogue lacking the methyl group (4) (Huezole (1), as stored in the library, is a racemic mixture. To examine the importance of its stereochemistry, we synthesized its roup (4) . Dynamicroup (4) . Their aroup (4) , suggestroup (4) , and S3†R-huezole (3) for further in-depth analysis. The sub-micron size of the R-huezole particles allowed us to monitor their properties under a confocal microscope. The huezole assemblies were visualized by Nile Red (100 nM), a typical environment-sensitive fluorescent probe for molecular condensates.20,21R-huezole demixed in an aqueous solution and formed liquid-like spherical droplets, which can fuse with one another to generate larger spherical units in vitro  experiments, in which the center of a Nile Red-doped droplet of R-huezole was photo-bleached with high-intensity laser light in a total internal reflection fluorescence microscope, with the fluorescence recovery of the photobleached area was followed thereafter. The half-maximal recovery time (t1/2) of the fluorescence was 9.8 ± 0.06 s  but remained comparable to those reported for phase-separated fluidic liquid droplets of proteins.2,8,22 Collectively, these results indicate that R-huezole gradually forms phase-separated liquid-like droplets in an aqueous solution.We focused on in vitro . In PBS in vitro . Such a ± 0.06 s , demonstin vitro, HiLyte Fluor™ 488-labeled tubulin was incubated with R-huezole in PBS buffer. Confocal imaging indicated that tubulin was sequestered within the R-huezole droplets  containing Taxol, a microtubule stabilizer. In fact, under such a polymerization-enabling condition, we were able to observe the microtubule fiber formation of HiLyte Fluor™ 488-labeled tubulin under a confocal microscope  inhibited tubulin polymerization more potently than molecule 5 did , suggesting that R-huezole is capable of ectopically squelching tubulin but not fully disabling its polymerization. In fact, extended incubation of the R-huezole-tubulin condensates under a highly polymerization-inducing condition nucleated microtubules to form a centrosome-like microtubule aster similar to those previously observed in vitro with spindle-forming protein condensates8,25 .30 These probes exploit the cell cycle-dependent proteolysis of Cdt1 and geminin by E3 ligases, enabling the visualization of G1 and G2/M phases in red and green, respectively. Treatment with R-huezole induced a dose-dependent increase in the geminin/Cdt1 fluorescence ratio, suggesting an increase in the cell population in G2/M  of the interaction was estimated to be 3.5 μM, comparable to the IC50 value of 4.4 μM  had little impacts on the particle diameters of 1 and 3 as measured by DLS  in which the piperazine amide tail of huezole was replaced by TAMRA, a red-fluorescent small-molecule tag. The probe (0.1 μM) was incubated with increasing concentrations of tubulin, and the fluorescence polarization was measured. Tubulin increased fluorescence of the probe but not that of free TAMRA, indicating that the benzylphenyl triazole moiety interacts with tubulin Fig. S15. The disM Fig. S8 for the Fig. S16A.Given that molecule 5 has weak affinity to tubulin, it could influence the tubulin binding of 1 and 3. However, co-precipitation assays with increasing concentrations (up to 4 times excess) of 5 exhibited limited effects on the tubulin co-precipitation with 1 and 3 Fig. S16B. ConfocaR-huezole (3) on cell viability is unlikely to stem solely from an impact on microtubule polymerization. Intracellular formation of tubulin-condensing assemblies could also influence cell viability through microtubule-independent mechanisms.Consistent with the role of the benzylphenyl triazole moiety in tubulin binding, both molecules 3 and 5 displayed inhibition of tubulin polymerization, although molecule 3 did so more potently than molecule 5 Fig. S7. Given tThe present study demonstrates the feasibility of producing a synthetic condensate out of a non-peptidic small molecule for exogenous control of cellular processes. The modular structure of huezole presents an excellent framework for designing a class of bioactive self-assembling small molecules that possess two key features, a covalent conjugation of a protein ligand and a self-assembling unit. Such self-assembling small molecules, with their cell permeability, stability, and chemical tractability, may open new possibilities for organelle-emulating molecules that complement the more established peptide-or protein-based artificial organelles.The authors declare that all data supporting the findings of this study are available within the article and ESI.†MU conceptualised and supervised the project. GA performed the research. NN and ADM performed the synthesis. HY performed the FRAP experiment. DMP performed the MD simulation. FI and GA performed the Airyscan LSM-880 confocal imaging. GA, NN, HTV, AP, KPA, HY, DMP, FI, SS and MU contributed to the analysis and discussions during the work. GA and MU prepared the manuscript with edits from all co-authors. TO and MU obtained funding for this work. All authors have approved the final version of the manuscript.There are no conflicts to declare.SC-013-D1SC07151C-s001


Heart failure with preserved ejection fraction (HFpEF) accounts for almost half of all heart failure (HF) cases worldwide. Unfortunately, its incidence is expected to continue to rise, and effective therapy to improve clinical outcomes is lacking. Numerous efforts currently directed towards the pathophysiology of human HFpEF are uncovering signal transduction pathways and novel therapeutic targets. The nitric oxide-cyclic guanosine phosphate-protein kinase G (NO-cGMP-PKG) axis has been described as an important regulator of cardiac function. Suppression of the NO-cGMP-PKG signalling pathway is involved in the progression of HFpEF. Therefore, the NO-cGMP-PKG signalling pathway is a potential therapeutic target for HFpEF. In this review, we aim to explore the mechanism of NO-cGMP-PKG in the progression of HFpEF and to summarize potential therapeutic drugs that target this signalling pathway. In the past decade, heart failure with preserved ejection fraction (HFpEF) with left ventricular ejection fraction (LVEF) ≥50% has become more prevalent, accounting for approximately 50% of all cases of heart failure (HF) . The groThere is a fair amount of evidence demonstrating that the nitric oxide (NO) signalling pathway is disturbed in HFpEF patients, suggesting that it is, at least partly, the cause of cardiac dysfunction. Here, we compiled studies on the potential role of the nitric oxide-cyclic guanosine phosphate-protein kinase G (NO-cGMP-PKG) pathway in the occurrence and development of HFpEF to partially explain the pathogenesis of HFpEF. Additionally, unlike heart failure with reduced ejection fraction (HFrEF), which has effective and definitive interventions, there are limited therapeutic agents that can effectively improve clinical outcomes in patients with HFpEF -12. Due NO can be synthesized in vivo and is a crucial physiological transmitter that functions as an intercellular and intracellular messenger. As the main effector molecule that modulates vascular tone and maintains vascular integrity, NO is mainly produced and released by vascular endothelial cells . Under tcGMP, a universal intracellular second messenger, is widely distributed in various tissues. Guanylate cyclase (GC) catalyses the transition of guanosine triphosphate (GTP) to cGMP, and phosphodiesterases (PDEs) catalyse the degradation of cGMP , 25. cGMPKG, also known as cGMP-dependent protein kinase, is a serine/threonine protein kinase that is widely expressed in eukaryotic cells and is considered to be the most important downstream target of cGMP . By reguNO exerts its physiological functions through the cGMP-independent pathway and the cGMP-dependent pathway that is mediated by activation of sGC . Catalyset al. [A retrospective clinical study performed transcatheter left ventricular myocardial biopsy in patients with HFpEF to observe PKG activity and cGMP concentration. The cGMP content and PKG bioactivity were found to be significantly reduced in myocardial homogenates from the HFpEF patients . Thereafet al.  observedet al. . In anotet al. . The supet al. .The downregulation of the NO-cGMP-PKG pathway may be attributed to the reduction in myocardial NO bioavailability caused by the activation of coronary microvascular endothelial inflammation and nitrosation/oxidative stress . Common Excessive amounts of proinflammatory cytokines further give rise to endothelial production of ROS via NADPH oxidase activation , which mThe suppression of NO-cGMP-PKG signalling has been observed in clinical patient samples and a variety of experimental animal HFpEF models, which encourages investigators to pursue more targeted therapy strategies involving rescue of the NO-cGMP-PKG signalling pathway. Regarding HFpEF treatment strategies, over the last few years, many research institutions and personnel have made numerous efforts to identify drugs that act on this signalling pathway, and continuous efforts, which we will discuss below, are ongoing.Coronary microvascular endothelial inflammation and oxidative stress trigger endothelial dysfunction, which results in reduced NO signalling and affects neighbouring cardiomyocytes and cardiac fibroblasts via the sGC-cGMP-PKG signalling pathway -58. Decret al. [2+ channel current. However, mice with cardiomyocyte-restricted PGKI deletion were susceptible to dilated cardiomyopathy with adverse cardiac remodelling when subjected to angiotensin II injection or transverse aortic constriction [The impact of NO-cGMP-PKG signalling on cardiac hypertrophy and fibrosis has been identified in various preclinical experiments and clinical trials. NO exerts a direct antifibrotic and antihypertrophic effect on cardiovascular homeostasis by antagonizing endothelin-1, angiotensin II, and aldosterone. Low bioavailability of NO allows these molecules to exert their profibrotic effects and favours the proliferation of fibroblasts and myofibroblasts , 63. In et al. -68 firsttriction . In micetriction , 70.Insufficient NO-cGMP-PKG signalling between endothelial cells and cardiomyocytes affects myocardial relaxation and myocardial stiffness in addition to the regulation of cardiac hypertrophy and fibrosis.Low endomyocardial NO bioavailability attributed to reduce NO concentrations or increased superoxide production can impair LV distensibility and contribute to diastolic dysfunction . The impThe sarcomeric protein titin, which functions as a molecular spring, affects resting tension and passive stiffness in cardiomyocytes. Titin compliance is attributed to posttranscriptional changes and posttranslational modifications, including isoform expression and phosphorylation . AlternaSignificant progress has been made in pharmacological interventions against HFrEF in recent years. However, confirmation of the criteria for the diagnosis and management of HFpEF has been challenging because of the heterogeneity of the clinical manifestations and the complexity and diversity of the underlying pathophysiological mechanisms. Current treatment options for HFpEF patients mainly involve comprehensive strategies, such as amelioration of the patients’ symptoms, management of existing cardiovascular diseases and comorbidities, and early intervention of cardiovascular disease risk factors. Experimental data on the benefits of pharmacological therapy in HFpEF have indicated that targeting the NO-cGMP-PKG axis may serve as a promising therapeutic strategy in interventions for HFpEF. Preclinical evidence indicates that perturbation of this signal transduction cascade is a specific pathological mechanism that underlies HFpEF and is partly responsible for myocardial fibrosis and cardiomyocyte stiffness, eventually driving diastolic dysfunction.3 adrenergic receptor (β3AR)-selective agonists or eNOS enhancers, organic nitrates and inorganic nitrites/nitrates, has been attempted , angiotensin receptor neprilysin inhibitors (ARNIs), sodium-dependent glucose transporters 2 inhibitors (SGLT2is) and NO-inducing drugs, including βttempted   to patients with HFpEF. In the Neat-HFpEF trial with 110 HFpEF patients, the effect of isosorbide mononitrate on the activity tolerance of HFpEF patients was evaluated. Treatment with isosorbide mononitrate (120 mg/d) for 6 weeks did not improve quality of life (QOL) or submaximal exercise capacity and even led to a decrease in activity level . A possiOn the other hand, inorganic nitrate, which is considered an important NO reservoir in vivo, is an important alternative agent for rescuing NO signalling in HFpEF and seems to improve ventricular performance under stress, especially during exercise , 85.A haemodynamic trial randomly divided 28 HFpEF patients into an acute sodium nitrite infusion group and a matching placebo infusion group, showing that sodium nitrate could lower pulmonary capillary wedge pressure (PCWP) during exercise, which was related to the improvement of cardiac output reserve with exercise . Inorgan2), or QOL [In contrast to the above studies of favourable impacts of organic nitrates on HFpEF, a multicentre crossover trial showed that inorganic nitrate, administered via aerosol inhalation for 4 weeks, failed to improve the New York Heart Association (NYHA) functional classification, N-terminal pro-B-type natriuretic peptide (NT-proBNP) level, diastolic function (E/E'), peak oxygen consumption  targeting this pathway are underway to resolve the controversy.sGC stimulators and activators can activate sGC to treat cardiovascular diseases independently of NO, and sGC is expected to be a promising therapeutic target for HFpEF. Treatment with sGC activators or stimulators could correct the low myocardial NO bioavailability in HFpEF by restoring downstream signals.2, KCCQ-PLS, or 6-MWD [The DILATE-1 study, which enrolled 36 patients with LVEF>50% and a resting pulmonary artery wedge pressure (PAWP)>15 mmHg, aimed to assess the acute haemodynamic effects of riociguat in patients with diastolic heart failure. Treatment with a single oral dose of riociguat 2 mg resulted in a significant increase in stroke volume and a decrease in systolic blood pressure, indicating improvement of diastolic function, but there was no pronounced difference in PAWP . SOCRATEor 6-MWD . The reaor 6-MWD .Considering that sGC stimulators and activators show an encouraging effect on QOL for patients suffering from HFpEF , 97, lonRegulation of the intracellular concentration of cGMP mainly depends on the balance between synthesis by GC and hydrolysis by PDEs. By inhibiting the phosphodiesterase-mediated degradation of cGMP, PDE5is increase the level of intracellular cGMP, thus helping to attenuate the NO-sGC signalling deficiency.et al. [In 2011, Guazzi M et al.  first pret al. . Prospecet al. .2, or improve QOL in patients with HFpEF [2 and 6-MWD did not significantly differ between the placebo group and the sildenafil group, meaning that therapy with sildenafil for 24 weeks failed to obviously affect exercise ability or change clinical status [et al. [However, another clinical trial that included 52 HFpEF patients with PH and LVEF≥45% showed that compared with placebo, treatment with 60 mg sildenafil for 12 weeks failed to significantly reduce PAP or PAWP, increase cardiac output or peak VOth HFpEF , 105. Col status . In the  [et al.  analysed [et al. .However, whether PDE5i can benefit certain subgroups of HFpEF patients with PH remains an open question. Given that there is no consensus on the current findings, further study is warranted to provide more in-depth information regarding the impact of PDE5is on clinical outcomes and prognosis in patients diagnosed with HFpEF.The angiotensin receptor neprilysin inhibitor (ARNI) combines two different drugs—a neprilysin inhibitor and an angiotensin receptor blocker—where the neprilysin inhibitor can restore cGMP-PKG signalling in the cardiomyocytes of HFpEF patients by activating natriuretic peptide instead of the NO signalling pathway. We will discuss this class of medication therapy in HFpEF here because of its potential synergy in activating the cGMP-PKG pathway.The PARAMOUNT trial enrolled 307 participants with an ejection fraction ≥45% and elevated NT-proBNP levels greater than 400 pg/mL and compared the primary endpoint of NT-proBNP levels between the sacubitril-valsartan group and the valsartan group. This randomized, parallel-group, double-blind, multicentre trial provided evidence that patients taking sacubitril-valsartan had lower NT-proBNP levels and better cardiac performance than those taking valsartan . This stBriefly, many new studies  are expected to re-evaluate the efficacy and safety of ARNI therapy in patients suffering from HFpEF. For instance, the multicentre, randomized, double-blind, controlled study PARAGLIDE-HF (NCT03988634), which is designed to assess the safety and tolerability of sacubitril/valsartan and its effect on changes in NT-proBNP levels in HFpEF patients, is currently recruiting.Sodium-dependent glucose transporter 2 inhibitors (SGLT2is) were initially used as hypoglycaemic drugs for diabetic subjects with HF, and they were found to reduce the risk of cardiovascular events -116. FurIn the SOLOIST-WHF study, sotagliflozin was found to reduce the risk of the composite endpoint of cardiovascular death, hospitalization for HF, and emergency department visits in diabetic patients with LVEF>50% . The EMPSGLT2is have been confirmed to improve the prognosis of HFpEF, but the specific mechanisms have yet to be identified, and their pharmacological effects are currently limited to preclinical research. Numerous experimental studies have shown that SGLT2is can inhibit myocardial inflammation and oxidative stress and improve endothelial function, thereby correcting the pathological suppression of the NO-cGMP-PKG pathway and its downstream targets. The activation effect of SGLT2is on the NO-sGC-cGMP-PKG pathway is supported by further experimental evidence from studies involving a nondiabetic pig model of HFpEF established by injection of deoxycorticosterone acetate (DOCA) and Ang II combined with Western diet feeding for 18 weeks and a mouse HFpEF model of spontaneous type 2 diabetes (db/db mouse) , 123. ThBased on the publication of the above clinical research results, in February 2022, empagliflozin was approved by the Food and Drug Administration (FDA) for the new indication of reducing the risk of cardiovascular death and hospitalization in patients with HF regardless of ejection fraction. The 2022 AHA/ACC/HFSA Guideline for the Management of Heart Failure pointed out that SGLT2is may contribute to reducing the rates of HF hospitalization and cardiovascular mortality for patients with HFpEF and that SGLT2is firstly received the recommendation for the treatment of HFpEF , 126. Th3AR mitigated myocardial hypertrophy and fibrosis in mice subjected to isoproterenol or angiotensin II stimulation in a NOS-mediated manner [3AR with the selective agonist mirabegron as a therapeutic alternative in HFpEF, and two related clinical studies are currently ongoing .Utilizing a mouse model of hypertrophy induced by neurohormonal or haemodynamic stresses, investigators showed that activation of βd manner . This stIn an experimental rat model, treatment with the eNOS enhancer AVE3085 led to increased production of NO and a significant improvement in diastolic dysfunction . The resAt present, the underlying pathophysiological mechanism and aetiology of HFpEF are not fully understood, making drug development challenging. There are still some major obstacles in adopting the interventional approaches used in preclinical animal models in clinical practice, as some of these models do not precisely reproduce the diversity and heterogeneity of human HFpEF. Knowledge of the NO-cGMP-PKG axis from abundant animal HFpEF models and human HFpEF heart tissue samples not only improves our understanding of the aetiology and pathophysiology of HFpEF but also drives extensive efforts towards identifying novel therapeutic strategies targeting the NO-cGMP-PKG signalling pathway.The evidence of the benefits of pharmacological treatment in specific populations from subgroup analysis of the PARAGON-HF trial and (CHARM)-Preserved study  could enCurrently, therapy with pharmacological treatment to regulate the NO-cGMP-PKG axis is being further studied in a multitude of clinical trials , and theDespite the inspiring scientific evidence supporting activation of the NO-cGMP-PKG pathway in the treatment of HFpEF, most of the large randomized clinical trials that assessed the effect of agents targeting this pathway have yielded negative results. Therefore, drugs (except SGLT2i) that target NO-cGMP-PKG signalling are not recommended in current guidelines. One of the primary concerns is that these strategies may contribute to hypotension, which is a potential barrier to the application of NO donors and sGC stimulants/activators. Due to side effects such as hypotension caused by the systemic vascular action of NO, the development of heart-targeted nanoparticles coated with NO inducers to avoid adverse effects on peripheral vessels is highly anticipated. Additionally, inhibition of the NO-cGMP-PKG pathway is only part of the pathological mechanism of HFpEF. According to recently reported studies, concomitant metabolic and hypertensive stress triggers nitrosative stress, which is more likely to be one of the principal drivers in HFpEF in addition to low NO bioavailability and is a possible reason why most NO-inducing pharmacological agents have failed to date. Given the heterogeneity of HFpEF and the diversity of comorbidities, the need for the development of more drugs with different therapeutic targets is urgent. Consequently, combination with drugs that interfere with other signalling pathways may help to improve the prognosis and prolong the survival time of HFpEF patients.+/H+ exchanger 1 (NHE1), thereby reducing the intracellular Na+ concentration of cardiomyocytes [SGLT2is are currently the only drugs that have clearly been proven to improve cardiovascular outcomes in HFpEF and are recommended by clinical guidelines. Limited experimental studies have shown that SGLT2is could possibly exert their effects by activating NO-cGMP-PKG signalling. However, the direct therapeutic target of SGLT2is in improving cardiac function in HFpEF patients has not been fully elucidated. Given that their benefits on cardiovascular events are independent of their hypoglycaemic effects and that the SGLT2 protein is largely absent in cardiac cells , it is smyocytes -132. Nev


Heart failure with preserved ejection fraction (HFpEF) is a highly prevalent syndrome with multifaceted pathophysiology. All approaches to neurohormonal modulation were shown not to improve survival in HFpEF, despite their well-established efficacy in heart failure with reduced ejection fraction (HFrEF). This might be attributed to suboptimal study design, inadequate diagnostic criteria, or statistical power, but is also likely to reflect a lack of consideration for its clinical heterogeneity. The attention then shifted to the phenotypic heterogeneity of HFpEF, with the ultimate goal of developing therapies tailored to individual patient phenotypes. Recently, the sodium-glucose co-transporter-2 inhibitor (SGLT2i) empagliflozin has been found to reduce the combined risk of cardiovascular death or hospitalization for HF in patients with HFpEF, a result driven by a reduction in HF hospitalizations. This paper recapitulates the journey from the failure of trials on neurohormonal antagonists to the attempts of personalized approaches and the new perspectives of SGLT2i therapy for HFpEF. Heart failure with preserved ejection fraction (HFpEF) is a syndrome with complex and multifaceted pathophysiology, which includes a crucial role of comorbidities. The apparent impossibility to find a “one-size-fits-all” treatment for HFpEF prompted a dissection of the phenotypic spectrum of this condition, with the ultimate goal of tailoring treatment on individual phenotypes. Recently, a standardized therapy was found to confer a prognostic benefit in HFpEF. Indeed, the sodium-glucose co-transporter-2 inhibitor (SGLT2i) empagliflozin reduced the combined risk of cardiovascular death or hospitalization for HF in patients with HFpEF, a result driven by a reduction in HF hospitalizations . This paThe 2021 European Society of Cardiology (ESC) guidelines retained the classification of HF into 3 categories: HF with reduced EF , HF with mildly reduced EF , and HFpEF (EF ≥ 50%) ), with a fair number of events (n = 463), suggesting a real lack of benefit. No effects emerged on all-cause death or all-cause hospitalization. Empagliflozin slowed down the yearly eGFR decline but did not improve a more clinically significant renal endpoint  (HR 0.94 [0.73–1.24]), contrary to a 49% reduction in the empagliflozin outcome trial in patients with chronic HF and a reduced EF (EMPEROR-Reduced) trial   compared to placebo). Extracardiac effects are indeed of utmost clinical benefit in HFpEF patients, where comorbidities are highly prevalent.p = 0.001), 6-min walking distance by 20.1 m  and reduced body weight by 0.72 kg  at 12 weeks [Although some survival benefits have been reported with spironolactone and sacubitril/valsartan in patients with HFpEF, the magnitude of these effects has been modest, and the benefits have been apparent only in some patient subgroups. The EMPEROR-Preserved trial is a groundbreaking study showing for the first time a prognostic benefit in patients with HFpEF. Nonetheless, these positive results derived entirely from a reduction in the less clinically relevant endpoint, i.e., HF hospitalization. It will be interesting to see whether dapagliflozin reduces cardiovascular mortality in the dapagliflozin evaluation to improve the lives of patients with HFpEF (DELIVER) trial such that the results of the DELIVER and EMPEROR-Preserved trials would mirror the pattern of results of the dapagliflozin and prevention of adverse outcomes in HF (DAPA-HF) and EMPEROR-Reduced trials in patients with HFrEF. In the PRESERVED-HF trial, 324 HFpEF patients  were randomized to dapagliflozin or placebo. Dapagliflozin improved Kansas City Cardiomyopathy Questionnaire Clinical Summary Score (KCCQ-CS) by 5.8 points  2.3–9.2, 12 weeks . A meta-12 weeks . Further12 weeks –70. Phen12 weeks . More emIn summary, empagliflozin has emerged as a standardized therapy able to improve patient outcomes in HFpEF, although the survival benefit was driven by a reduction in HF hospitalization. The notion of tailoring the remaining therapy  according to the individual phenotype may deserve further consideration, especially if dapagliflozin is found to be no more effective than empagliflozin. Specific trials, possibly with a pragmatic design, should investigate treatment tailoring as an additional strategy to SGLT2 inhibition.6MWD, 6-min walking distance; ACEi, angiotensin-converting enzyme inhibitors; Aldo-DHF, aldosterone receptor blockade in diastolic heart failure; ARBs, angiotensin receptor blockers; ARNI, angiotensin receptor neprilysin inhibitor; CHARM-Preserved, effects of candesartan in patients with chronic heart failure and preserved left-ventricular ejection fraction; CIBIS-ELD, Cardiac Insufficiency Bisoprolol Study in Elderly; CV, cardiovascular; E/e′, transmitral early diastolic velocity (E) over tissue Doppler early diastolic myocardial velocity (E′); HF, heart failure; HFpEF, heart failure with preserved ejection fraction; HR, hazard ratio; I-Preserve, Irbesartan in Heart Failure with Preserved Ejection Fraction Study; LA, left atrium; LV, left ventricular; LVEF, left ventricular ejection fraction; MRA, mineralocorticoid receptor antagonists; NYHA, New York Heart Association; NS, nonsignificant; NT-proBNP, N-terminal pro-B-type natriuretic peptide; PARAGON-HF, angiotensin receptor neprilysin inhibition in heart failure with preserved ejection fraction; PARAMOUNT, Prospective comparison of ARNI with ARB on Management Of heart failUre with preserved ejectioN fraction; PEP-CHF, perindopril in elderly people with chronic heart failure; SENIORS, Study of Effects of Nebivolol Intervention on Outcomes and Rehospitalization in Seniors; TOPCAT, Treatment of Preserved Cardiac Function Heart Failure With an Aldosterone Antagonist.The table summarizes all clinical trials on neurohormonal drugs  to treat HFpEF, providing the main characteristics of patient populations, the endpoints and HR values with their 95% CI values. See text for further details.


Current data indicate that in the community, approximately 50% of patients with heart failure (HF) have preserved left ventricular (LV) ejection fraction (LVEF)—the so-called HFpEF. Treatment of HFpEF has been considered an unmet need for decades. We believe that the main underlying reasons have been (a) the ever-changing LVEF cut-offs used for HF classification; (b) controversies regarding the definition of the LVEF normal range; (c) the fact that HFpEF does not represent a phenotype, but a category of diseases with entirely different characteristics , hypertrophic cardiomyopathy (HCM) etc.); (d) the lack of recognition that hypertensive HFpEF is the most common and important HFpEF phenotype; (e) the assumption that neurohormonal overactivity is absent in HF patients with a LVEF > 45–50% which has been proven to be wrong. Current HFpEF trials, in which the vast majority of the participants suffered from hypertension (HTN), whereas VHD and HCM were absent, demonstrated that neurohormonal and sodium-glucose cotransporter 2 (SGLT2) inhibitors are effective in HF patients over a wide LVEF range. Thus, restricting these lifesaving treatments to HF patients with reduced LVEF is not justified anymore and it should be additionally considered for HFpEF patients suffering from HTN. The clinical syndrome of heart failure (HF) remains a worrying healthcare issue, as it affects more than 26 million people worldwide, despite the current drug and device therapies ,2. In thLVEF has been used for decades for HF classification and treatment guidance. The 2013 American College of Cardiology Foundation (ACCF)/American Heart Association (AHA) guidelines defined HFrEF by a LVEF ≤ 40%, borderline HFpEF by a LVEF 41–49%, and HFpEF by a LVEF ≥ 50% . FurtherA prerequisite for using the terms for mildly reduced, preserved, or normal LVEF is a definition of the normal LVEF range. According to the 2015 recommendations of the American Society of Echocardiography and the European Association of Cardiovascular Imaging, the normal reference range for LVEF is 52–72% for males and 54–74% for females . The latGladding et al., investigated the relationship between echocardiographically obtained LVEF and survival and observed that during follow-up the unadjusted hazard ratios (HR) for mortality demonstrated a U-shaped relationship for LVEF with a nadir of risk at an LVEF of 60–65%, with the results being similar after adjustments for conditions accompanied by an elevated LVEF  and when restricted to patients suffering from HF  15]..15].The findings of another study were similar, in which an increased risk for cardiovascular-related mortality persisted to a LVEF level of 60.0–64.9% in females, whereas in males the equivalent LVEF level was lower (55.0–59.9%)  16]..16].Differences related to sex were also reported in the COronary CT Angiography EvaluatioN For Clinical Outcomes: An InteRnational Multicenter (CONFIRM) registry in whom LVEF was measured by cardiac computed tomography. Females with high LVEF died more often from any cause as compared to females with normal LVEF, while an opposite trend was observed in males . Thus, tSeveral trials testing the effectiveness of renin-angiotensin-aldosterone system (RAAS) inhibition in HFpEF have reported positive results.n = 1322), HFrEF , and HFpEF  .,46.45,46Obesity has been associated with increased risk of HF development, irrespective of the LVEF. Patterns of LV remodeling in obese patients include concentric LV remodeling, concentric LV hypertrophy, and eccentric LV hypertrophy. The underlying mechanisms include hemodynamic and neurohormonal factors, the effect of coexisting morbidities and inflammation . The “obTreatment of HfpEF has been considered an unmet need for years. There are five main reasons in our opinion. (a) The ever-changing LVEF cut offs used for HF classification; (b) controversies regarding the definition of the LVEF normal range; (c) the fact that HFpEF does not represent a phenotype, but a category of diseases with entirely different characteristics ; (d) the lack of recognition that hypertensive HFpEF is the most common and important HFpEF phenotype; (e) the assumption that neurohormonal overactivity is absent in HF patients with LVEF > 45–50%, which has been proved wrong. The findings of recent studies indicate that the four major classes of medications used in HF—β-blockers, mineralocorticoid receptor antagonists, sacubitril-valsartan /ACEi/angiotensin receptor blockers (ARB), and SGLT2 inhibitors—are effective not only in HFrEF, but in HF including a wider range of LVEFs and especially in hypertensive HFpEF  [50]. EnTreatment of HFpEF, especially in hypertensive HFpEF which is the most common, should not be considered an unmet need anymore. Based on the contemporary evidence, the role of LVEF in the HF classification is doubtful. In this regard, a HF classification based on causes or risk factors would be the most reasonable approach. Despite the greater effectiveness of neurohormonal inhibitors in the lower end (vs. the higher end) of HF spectrum, restricting the four major classes of medications used for HF treatment in patients with a LVEF < 40% is not justified anymore.


The fibroblast is one of the ideal target cell candidates for cell-based gene therapy approaches to promote tissue repair. Gene delivery to fibroblasts by viral transfection has been confirmed to have high transfection efficiency. However, in addition to immunogenic effects of viruses, the random integration of viral genes may damage the genome, affect the cell phenotype or even cause cancerous mutations in the transfected cells. Due to these potential biohazards and unknown long-term risks, the clinical use of viral transfection has been very limited. In contrast, initial non-viral transfection methods have been simple and safe to implement, with low immunogenicity, insertional mutagenesis, and risk of carcinogenesis, but their transfection efficiency has been relatively low. Nucleofection, a more recent non-viral transfection method, now combines the advantages of high transfection efficiency and direct nucleic acid delivery to the nucleus with a high safety.Here, we reviewed recent articles on fibroblast nucleofection, summarized different research points, improved methods and application scopes, and opened up ideas for promoting the further improvement and development of fibroblast nucleofection to meet the needs of a variety of disease research and clinical applications. Fibroblasts are the most common cells in connective tissue, and are differentiated from mesenchymal cells. The morphology and function of fibroblasts often undergo dynamic changes during the processes of disease development and tissue repair . FibroblGene therapy utilizing fibroblasts has been proven feasible by in vitro and in vivo experiments. For example, primary fibroblasts modified by gene nucleofection were used to treat a rat hindlimb ischemia model. They promoted the formation of collateral vessels in the lower limbs of rats and the reconstruction of circulation in the ischemic area . Lwin etMany researchers have explored the transfection efficiency of nucleofection in fibroblasts and examined the efficacy of the transgenic fibroblasts used for further cell therapy. In this article, we review the published work on fibroblasts nucleofection in recent years, and summarize the different angles of researches, advancements of methods, and evolution of applications.Due to different research purposes, different species and tissues have been selected to be used for isolating and culturing fibroblasts for experiments. Some studies have pointed out that the number of passages, confluence state, growth rate, and division phase of fibroblasts before they are used for nucleofection can affect nucleofection efficiency. Although opinions differ on the optimal number of passages for fibroblasts, most studies agree that cells should be kept in a low-passage exponential growth phase prior to transfection. Reaching high confluency should be avoided, since cells in a high confluence state are more resistant to reprogramming. In addition, the composition of the fibroblast culture medium was also indicated to correlate with the final nucleofection efficiency by affecting the proliferation rate of fibroblasts. So far, an overview over experimental design and statistical data on specific effects of these factors on fibroblasts nucleofection efficiency is missing.Fibroblasts have been successfully extracted from diverse species and tissues for research by enzymatic and explant culture methods. The enzymatic method mostly refers to the use of collagenase, dispase, hyaluronidase or trypsin to digest the chopped tissue specimens. The typical explant method consists of cutting the sample into tissue fragments of about 0.5 mm in length, planting them in a petri dish with culture medium, and letting the cells grow out of the sample. For example, we have successfully isolated fibroblasts from the dermis of the skin obtained from the back of Lewis inbred rats by the enzymatic method in previous studies. In these experiments, the first 3 passages of the cultured cells were used for subsequent experiments , 13. Som2 humidified incubators. Some of these studies added basic fibroblast growth factor (bFGF) to the culture medium, but effects on subsequent transfection were not observed. Only Kuebler et al. and Teklemariam et al. chose Iscove's Modified Dubecco's Medium (IMDM) to culture fibroblasts [Notwithstanding some studies emphasize the necessity to use cells within three to five passages in the nucleofection process , some stroblasts , 24, IMDroblasts , 36, 41 roblasts .There are a few publications reporting that the subsequent nucleofection process is significantly affected by different sources of fibroblasts. Some studies mentioned that primary fibroblasts and NIH3T3 cells are difficult to transfect –44, but Cell contamination often severely affects transfection outcomes . BacteriNucleofection is an electroporation-based transfection modality with a variety of cell-specific transfection buffers and different programs that control voltage, frequency, and pulse duration. Skrzyszowska et al. used Sacl enzyme to cut the constructed plasmid DNA into linear conformation. They added fibroblast nucleofection buffer for nucleofection of pig fibroblasts, wherein the U-20 procedure was used for pig fetal fibroblasts, U-23 procedure was used for porcine adult dermal fibroblasts. In their work, they only give numbers for different protocols , while treating the exact procedure as a trade secret. The transferred target sequence carried an enhanced green fluorescent protein (eGFP) gene. This allowed to detect transfection efficiency of the gene by observing the fluorescence intensity of eGFP . Ko et aPrevious studies have compared and found that the cuvettes provided by the manufacturer of the Nucleofector device and cuvettes from Eppendorf had no significant difference in the nucleofection process. Under certain circumstances, the transfection solution provided by the manufacturer could even be improved . It was Skrzyszowska et al. compared cell viability and proliferative activity after nucleofection and lipofection, both of which were higher in the nucleofection group. However, in PCR screening results, only in 1 of the 5 analyzed groups of nucleofection, the gene was integrated into the nuclear genome, while the transgenes of 3 of the 5 analyzed groups of lipofection were successfully integrated . TherefoThe implementation conditions and transfection results of nucleofection in related studies are summarized in Table In order to more intuitively present the relationship between the sources of fibroblasts, the nucleofection procedures used in each study with the nucleofection efficiency and cell viability in Table The composition of the nucleofection standard solution is unreported, and may have a certain negative impact on cell survival and proliferation. Most studies stated that the cell viability after nucleofection is higher than other chemical or physical transfection methods, but it is always lower than the viability of untransfected cells. Thus, some researchers recommend that 24 h after nucleofection, the standard solution of nucleofection should be replaced with DMEM, IMDM or other inducible medium according to different experimental purposes , 50. ConGene editing of fibroblasts by nuclear transfection for clinical application is a possible direction of this technology. Although it has not yet been applied to the clinic, related studies on nucleofection of fibroblasts have been carried out in various ways. For example, in research about tissue repair and reconstruction, the genes encoding VEGF165 and bFGF were transferred into fibroblasts by nucleofection, and the cells were injected into the gracilis and adductor muscles of ischemia model rat lower limbs. This significantly improved the circulation reconstruction in this model . BiodegrThe applications of nucleofection in related studies are summarized in Table Making use of the characteristics of fibroblasts, nucleofection treatment of fibroblasts has been proven to be of potential in a lot of aspects. In addition to promoting the repair and reconstruction of soft tissue or bone tissue damage, it can also be used to support the survival of adjacent cells through its exocrine function, used as a nuclear donor to produce transgenic animals, induce it into other differentiated cells or iPSCs, etc.. The diverse application modes make fibroblast nucleofection of extraordinary significance in disease research and development of novel treatments. The high efficiency of nucleofection has been well confirmed, whereas the specific and detailed way in which nucleic acids "directly" enter the nucleus is still unknown. To clarify this process in the future may allow to further improve it. Although the details of the nucleofection procedure and standard solution formula are unknown, some methods have been developed to improve the nucleofection process. This can elevate the transfection efficiency while reducing the experimental cost and ensuring cell viability, which undoubtedly paved the way for the development of fibroblast nuclear transfection-related research. However, it should be noted that nucleofection like other gene transfection methods may cause IFN and DDR, affect the level of oxidative stress in cells, and interfere with subsequent experiments. Similarly, it is necessary to study the specific mechanism of cell damage during the gene transfection process, as it can help to find a better way to minimize the negative impact on experimental cells. This can further promote the improvement and development of fibroblast nucleofection to meet the needs of various researches and clinical applications.


It then assessed the impact of this inflammatory cytokine on an invitro model of human blood–brain barrier (BBB) and revealed dramatic impairments in both barrier integrity and function, the main cause of early death after an ischemic stroke. Co-treatment of BBB models in similar experiments with outgrowth endothelial cell-derived conditioned media (OEC-CM) negated the deleterious effects of TNF-α on BBB. Effective suppression of anti-angiogenic factor endostatin, stress fiber formation, oxidative stress, and apoptosis along with concomitant improvements in extracellular matrix adhesive and tubulogenic properties of brain microvascular endothelial cells and OECs played an important role in OEC-CM-mediated benefits. Significant increases in pro-angiogenic endothelin-1 and monocyte chemoattractant protein-1 in OEC-CM compared to the secretomes of OEC and HBMEC, detected by proteome profiling assay, accentuate the beneficial effects of OEC-CM. In conclusion, this reverse translational study identifies TNF-α as an important mediator of post-ischemic cerebral barrier damage and proposes OEC-CM as a potential vasculoprotective therapeutic strategy by demonstrating its ability to regulate a wide range of mechanisms associated with BBB function. Clinical trial registration NCT02980354.Improved understanding of the key mechanisms underlying cerebral ischemic injury is essential for the discovery of efficacious novel therapeutics for stroke. Through detailed analysis of plasma samples obtained from a large number of healthy volunteers (The online version contains supplementary material available at 10.1007/s12015-022-10439-4. Ischemic stroke (IS), stemming from an interference with blood supply to the brain, continues to be one of the leading causes of mortality and morbidity worldwide , 2. Throexvivo expansion and elevated risk of emboli formation and immune reaction associated with cell-based therapies have thus far hampered the widespread use of these particular cells in clinical practice -1.3-benzene disulfonate (WST-1) kit . For this, ~ 5 × 104 cells were then seeded to each well and subjected to respective experimental conditions. The specific characteristics of the network of tubules, defined as the sum of number or length of segments, isolated elements and branches detected in the analyzed area, were then assessed using ImageJ software  [Firstly, growth factor-reduced Matrigel  was added to 96-well plates and allowed to polymerize at 37 °C. 1 × 10nd, USA) .Equal number of cells, grown in 6-well plates and exposed to experimental conditions, were seeded on fibronectin (Sigma) or collagen (Sigma) pre-coated 96-well plates. Following 1 h incubation, the plates were gently washed with PBS and examined under light microscope. The adherent cells were counted in at least four random fields per well.Calcein AM  and Apo-ONE homogeneous caspase-3/7 assay kit  were used to measure cell viability and caspase 3/7 activity, respectively as described before .The organization of actin cytoskeleton was studied by staining of F-actin filaments with rhodamine phallodin as before . Cells gCells, subjected to different experimental conditions, were fixed in 4% paraformaldehyde/PBS for 20 min and stained for 5 min at room temperature with Hoechst 33,258 . The coverslips were mounted on glass slides and analyzed by fluorescence microscopy .t-test, Mann–Whitney, chi-square, or one-way analysis of variance followed by Tukey’s post hoc analysis, where appropriate. P < 0.05 was considered as significant.The statistical analyses were performed either with GraphPad Prism 8.0 statistical software package  or SPSS package 15.0 for Windows . Statistical tests were performed by A total of 90 HVs and 81 patients were recruited for the study. Table Similarly, the level of chemokine SDF-1, known to mediate trafficking and homing of EPCs to the site of injury, was also higher in patients which peaked on day 7, gradually decreased from then on and normalized on day 90 Fig. . In contOxidative stress, emerging from an imbalance between endogenous pro-oxidants and anti-oxidants, is thought to profoundly affect the pathogenesis and outcome of IS . AnalyseEffective neovascularization of infarct area, mediated by release of pro-angiogenic factors in the injured tissue, is crucial for recovery following an IS . GradualSince most IS patients have at least one vascular risk factor , this stDetection of higher TNF-α levels in HVs with DM, hypertension and hyperlipidemia on the other hand, proved these risk factors as the generators of TNF-α in the absence of an overt ischemic injury  and monocyte chemoattractant protein-1 (MCP-1) and a decrease in anti-angiogenic factor endostatin in OEC-CM Fig. , I. ET-1Compared to OECs, a specific increase in interleukin-8 IL-8) and decreases in urokinase plasminogen activator (uPA), serpin-E1, angiopoietin-2 (Ang2), and pentraxin-3 levels were also found in OEC-CM. Like endothelins, IL-8 also directly enhances EC proliferation and survival . Serpin-L-8 and dGiven the conspicuous differences in HV and IS patient plasma endostatin levels and the role of this anti-angiogenic factor in EPC functionality and post-stroke angiogenesis , 48, thiDespite introduction of various guidelines, such as ARRIVE and STEPS , 50 to fUnlike TNF-α and SDF-1, no significant variation was observed in the levels of VEGF and PDGF-BB between IS patients and HVs until day 90 post-stroke. Despite presence of reports showing dramatic increases in serum VEGF levels in IS patients , 53, theinvitro model of human BBB, the present study has shown that treatment with hypoxia-primed OEC-CM, a promising cell-based but cell-free strategy, mitigates the disruptive effects of TNF-α on vascular integrity and function, as proven by increases in TEER readings and decreases in paracellular flux of NaF. However, the capacity of EPCs to replace the dead or damaged cerebral ECs to maintain vascular homeostasis cannot be overlooked [Due their ability to re-endothelialize damaged endothelium and promote angiogenesis, EPCs and their functional subtype OECs have recently been at the forefront of clinical investigation , 57. Suberlooked , 13. Ind2.− production are involved in TNF-α-induced BBB damage [2.− production in HBMECs and OECs subjected to TNF-α with OEC-CM prove the anti-oxidant effect of this therapy. Suppression of ROS, apoptosis, and inflammatory mediator generation in ECs subjected to H2O2 by OEC-CM reinforces the modulatory role of this therapeutic regimen on pro-oxidative mechanisms [Enhanced NADPH oxidase activity and OB damage , 29. ObsB damage , 60. Inhchanisms .invivo following stroke and myocardial ischemia and restoring BBB integrity invitro [Apoptosis of cerebrovascular cells induced by inflammatory mediators may also contribute to cerebral barrier damage . Analyseinvitro –65.In addition to reparative processes, regenerative processes, notably angiogenesis involving EC proliferation, tubulogenesis, branching and anastomosis also contribute to neurological recovery after IS . ProgresThere are some limitations to this study. Since 88.2% of patients had mRS score of ≤ 3 on day 90 after stroke, the correlation between TNF-α levels and disease outcome could not be ascertained. As lacunar and cortical strokes made up almost half of the overall sample size, the neurological improvements was unlikely to derive from a particular stroke subtype. Considering that treatment with an anti-TNF-< antibody negate the ischemic injury-evoked BBB damage , analyseInvitro studies performed with a clinically-relevant concentration of TNF-< has proven that TNF-< compromises BBB integrity through a series of mechanisms, including EC apoptosis, oxidative stress, cytoskeletal reorganization and cell–matrix adhesion. Scrutiny of OEC-CM as an emerging cell-free therapeutic strategy has proven it to be very effective in negating the deleterious effects of TNF-< on BBB. Specific molecule(s) that contribute the most to the BBB-restorative effect of OEC-CM deserve further investigation in future studies. Similarly, it will be important to determine the therapeutic impact of OEC-CM in future invivo studies.In depth analysis of plasma samples collected for The Dunhill Medical Trust EPC study has shown a constant increase in TNF-α levels in patients with IS compared to HVs. Supplementary Figure captions (DOCX 16 KB)Supplementary Fig. 1 (JPG 167 KB)Supplementary Fig. 2 (JPG 46 KB)Supplementary Fig. 3 (JPG 785 KB)Supplementary Fig. 4 (JPG 96 KB)Supplementary Fig. 5 (JPG 230 KB)Supplementary Fig. 6 (JPG 3687 KB)Supplementary Fig. 7 (JPG 23 KB)Supplementary Fig. 8 (JPG 502 KB)Supplementary Fig. 9 (JPG 21 KB)Below is the link to the electronic supplementary material.


IntroductionRheumatoid arthritis and its treatment have different effects on weight gain. This study examined the effect of tumor necrosis factor (TNF) inhibitors on weight gain in patients with seropositive rheumatoid arthritis compared with non-TNF therapy and disease activity.MethodsA retrospective cohort study was conducted in Prince Mohammad bin Abdulaziz Hospital for all patients aged ≥ 18-year-old diagnosed with seropositive rheumatoid arthritis and started on TNF inhibitors or non-TNF biologics in outpatient clinics since 2015. Patients were excluded if they were pregnant, had an uncontrolled underlying metabolic disease, were post-bariatric, or had a history of malabsorption. We also excluded individuals on treatment for congestive heart failure or end-stage renal disease.ResultsA total of 116 patients with rheumatoid arthritis were reviewed between 2015 and 2019. Only 69 patients met the inclusion criteria (51 and 18) in the TNF-a  and non-TNF-a inhibitor groups, respectively. The weight change from pre-treatment to post-treatment showed an average increase of 1.2 kg (95% CI: -0.68-3.17) in the TNF-a inhibitor group while in the non- TNF-a inhibitor group, the average increase in weight was 2.67 kg (95% CI: -0.44-5.78). Over the period of two years, there was no statistical difference in weight gain in both groups or in relation to disease activity.ConclusionThe results of this study did not show a significant increase in weight gain in seropositive patients with rheumatoid arthritis who were treated with TNF or non-TNF inhibitors. Tumor necrosis factor-alpha (TNF-a) is expressed in each cell in our body. It plays a significant role in the pathophysiology and disease activity of various inflammatory diseases such as but not limited to rheumatoid arthritis (RA) and psoriatic arthritis, along with inflammatory bowel disease and ankylosing spondylitis. That explains its different activities and role in regulating the human body interaction. TNF-a inhibitors are used in patients with RA who failed to reach disease control by non-biological therapies .Overweight and obese were estimated to be around 35% of the world population. It was linked that this increasing prevalence was associated with a higher risk of negative impact on quality of life .In patients with rheumatoid arthritis, a higher body mass index (BMI) has been linked to worse outcomes in both disease activity and function .The effect of anti-TNF on weight is not well-established and is yet to be determined in correlation to disease activity. Different studies have shown the impact of TNF inhibitors on weight gain with variable and heterogeneous data; however, till now, we have no clear answer for the effect of these medications on seropositive patients with rheumatoid arthritis. We have also considered that seronegative rheumatoid arthritis can have some diagnostic confusion -4.This is the first study in the Middle East and North Africa where the effect of TNF inhibitors on weight gain in a specific group of patients (only seropositive rheumatoid arthritis) has been studied, and this result was compared and correlated with non-TNF therapy and disease activity.We conducted a retrospective cohort study using data from Prince Mohammad bin Abdulaziz Hospital Integrated Clinical Information System by collecting data of adult rheumatology patients diagnosed with seropositive rheumatoid arthritis and following in outpatient clinics since 2015. The study was conducted in accordance with the ethical principles of the Helsinki Declaration and Prince Mohammad bin Abdulaziz Hospital, Ministry of Health, policies and guidelines for clinical research. The patient's identity was protected and known only by the investigators. We identified all adult patients aged ≥ 18-year-old with a confirmed diagnosis of seropositive (positive serum cyclic citrullinated peptide or rheumatoid factor or both) rheumatoid arthritis who will be treated with TNF-a inhibitors and will be compared to those who receive non-TNF-a inhibitor therapy  as a control group at Prince Mohammad bin Abdulaziz Hospital Rheumatology Clinics.Patients' weight, along with their other demographics, the time of treatment initiation, disease activity assessment by Disease Activity Score (DAS-28), and type of treatment, were collected from the system every three to six months for 24 months from TNF-and non-TNF initiation.Patients were excluded if they were pregnant, had an uncontrolled underlying metabolic disease, were post-bariatric, or had a history of malabsorption. We also excluded individuals on treatment with congestive heart failure or end-stage renal disease (ESRD).The primary outcome was the change in body weight in patients initiated on TNF-a inhibitors compared with non-TNF-a inhibitors treated patients over the study period. Secondary outcomes were to assess the difference between TNF-a and non-TNF-a inhibitor groups regarding body weight change and to assess the relationship between the initiation of TNF-a inhibitors, weight change, and disease activity. Statistical analysishttp://www.rstudio.com/).Descriptive statistical parameters were reported as mean ± standard deviation (SD) for continuous variables and n (%) for categorical variables. To assess the normal distribution of data, The Shapiro-Wilk normality test was used. The study groups at baseline were compared using T-tests for continuous variables and chi-square (χ2) analysis for categorical variables. The T-test was used to compare pre and post-treatment weight changes in both groups. P < 0.05 was considered significant. All statistical analyses were performed using RStudio software . RStudio: Integrated Development for R. RStudio, PBC, Boston, MA. A total of 116 patients with RA were reviewed between 2015 and 2019. Only 69 patients met the inclusion criteria (51 and 18) in the TNF-a inhibitor and non-TNF-a inhibitor groups, respectively. Baseline characteristics were identical between the two groups  patients in the TNF-a inhibitor group were not on any active medications for other comorbidities compared to only seven (38.9%) from the non-TNF-a inhibitor group.On the other hand, comorbidities commonly reported in both groups were hypertension, diabetes, thyroid disease, and chronic kidney disease in 26.1%, 21.7%, 5.8%, and 4.3% of patients, respectively. There was no effect of baseline prednisolone, anti-hypertension, diabetes medications, and thyroid hormone on weight gain.We reported 19 (37.3%) patients on adalimumab, 30 (58.8%) patients on etanercept, and two (3.9%) patients on infliximab. In the non-TNF-a inhibitor group, data showed three (20%) patients on abatacept, four (20%) patients on rituximab, five (25%) patients on tocilizumab, and six (35%) patients on tofacitinib treatment.The body weight documented at baseline and then at follow-up at different time intervals over 24 months was collected  in the TNF-a inhibitor group while in the non-TNF-a inhibitor group, the average increase in weight was 2.67 kg (95% CI: -0.44-5.78). The conducted paired-sample t-test showed no significant difference in the mean weight pre and post-treatment in the TNF-a inhibitor group (P=0.199) and non-anti TNF groups (P=0.084).Over the period of two years, there was no statistical difference in weight gain between the two groups or in the same group. However, numerically, there was more weight gain in the non-TNF group compared to the TNF group.There was no statistically significant difference between TNF and non-TNF regarding disease activity. There was no significant statistical relationship between disease activity and weight gain, though there was a numerically proportional relationship between weight gain and improving disease activity Tables -3.Rheumatoid arthritis can affect around 1% of the population in the world. Chronic synovial inflammation can lead to joint destruction and function loss .The body weight in patients with rheumatoid arthritis independently impacts the disease activity, response to treatment, and quality of life ,6. Our pTNF-a inhibitors are one of the biological therapies that we used in RA patients to control the disease if non-biological therapy did not work . They weIn a recent analysis in 2020 by Patsalos O et al., 26 studies had been collected to look for the effect of anti-TNF on weight and BMI. There was a small increase in weight and BMI with diverse data  with infliximab, etanercept, and adalimumab, respectively. However, they included different disease groups  that can affect body weight by different mechanisms [A meta-analysis by Wu et al. on psoriatic patients showed a significant increase in body weight on anti-TNF initiation in comparison with interleukin (IL) 12/23 and IL-17 .Serelis et al. studied the effects of anti-TNF-α treatment in 19 women with rheumatoid arthritis, showing no significant impact on body composition; however, there was an increase in adiponectin levels that can affect the systemic inflammation . ConversThe mechanism by which TNF-a inhibitors affect weight is not well-understood, but TNF may inhibit lipoprotein lipase, leading to weight loss in RA patients .In RA patients who gain weight with TNF inhibitor (infliximab and etanercept) treatment, leptin and adiponectin levels have elevated ,14. In RIn contrast to previous research with heterogeneous patient populations, we eliminated seronegative rheumatoid arthritis to eliminate this heterogeneity. The effect of anti-TNF medication versus non-TNF therapy  on weight gain in patients with rheumatoid arthritis are compared. In our study, the data follow-up was for two years.In this retrospective study, there was a non-significant statistically increase in body weight over time. However, numerically, there was an increase in weight gain over therapy with non-TNF medications and to a lesser extent with the TNF group. That increase in body weight may also reflect an improvement in disease activity although it was not statistically significant.Till now, the exact mechanism of weight gain during rheumatoid arthritis treatment has not been well-studied. By reviewing the previous studies and with our study findings, we postulated the following hypothesis: In patients who gain weight, inhibition of inflammatory cytokines may enhance adipose tissue proliferation, which increases both adiponectin and leptin production. With time, this can cause a lack of sensitivity to both, leading to leptin and adiponectin resistance. Because the individual keeps eating, the fat cells produce more leptin to signal the feeling of satiety, leading to increased levels of both hormones. Another hypothesis is that neutralizing antibodies against both leptin and adiponectin can develop during biological therapy by looking at some studies showing an increase in adiponectin levels without an increase in body weight .The results of this study did not show a significant increase in the weight gain in seropositive rheumatoid arthritis patients who were treated with TNF or non-TNF inhibitors. However, numerically, there was a trend of weight gain in both groups, which was more in the non-TNF groups. This increase was going with improvement in disease activity. The effect of rheumatoid arthritis and treatment on weight gain is variable and needs to be monitored during treatment.


I congratulate Pellis and colleagues, and Dunbar and Held on their excellent papers describing a variety of mechanistic models of SARS‐Cov‐2 transmission, and more generally on their work to support policy formulation during the COVID‐19 pandemic. Both papers address the difficulties of predicting and then evaluating the impact if non‐pharmaceutical interventions (NPIs) against the transmission of severe respiratory pathogens. These are likely to remain key ongoing challenges for the analytical science of pandemic preparedness, with high demand from policy makers for accurate estimates of the epidemiological benefits of NPIs. Here, I would like to make one related methodological point.β. The parameter β is used to calculate the risk of infection per susceptible and is modified by other parameters to reflect differences in infectiousness, susceptibility and mixing (Keeling & Rohani, β as a smooth function of time in an alternative model, as is common practice for similar parameters in other analytical frameworks (Wood, There may be benefits in making the null hypotheses in mechanistic modelling studies of NPIs more explicit and more general. For example, models usually contain an underlying basic rate of transmissibility per unit time per infected individual, often denoted ks Wood, , so that