Although triptolide (TP) has been widely used for the treatment of inflammatory, autoimmune diseases, and various kinds of tumors, the long experimental and clinical applications have exhibited severe reproductive system toxicity in TP-treated animals and patients. More importantly, the underlying molecular mechanism involved in TP-induced reproductive system toxicity still needs more research. β, IL-6, and TNF-α), oxidative stress indicators , apoptosis rate, protein distribution and expression , cell viability, relative protein levels , autophagosome were detected by ELISA, commercial biochemical detection kits, flow cytometry, immunohistochemistry, CCK-8, western blotting, and transmission electron microscope, respectively.  Adult female Sprague Dawley rats and human ovarian granulosa cell lines were treated with TP and then treated with XinJiaCongRongTuSiZiWan (XJCRTSZW). Histological analysis and follicle count were executed using H&E staining. Hormone  concentrations, inflammation indicators (IL-1in vivo. Moreover, XJCRTSZW application significantly increased the TP-induced diminishment of E2, AMH, and LNH-B concentrations, apoptosis rate, SOD and GSH-Px concentrations, and p62 protein level; however, it declined the TP-induced augmentation of MDA level, the levels of IL-1β, IL-6, and TNF-α, autophagosome, beclin-1, LC3-II/LC3-I, cleaved-caspase-3, p-AMPKα-1, and p-SIRT1 protein levels both in vivo and in vitro. Besides, XJCRTSZW treatment prominently enhanced the TP-induced decrease of cell viability in vitro.  XJCRTSZW administration notably improved the TP-treated pathological symptoms, including few mature follicles in the ovary and less granular cell layer, and disordered the arrangement of the follicle, lymphocytes and plasma cells infiltration, and necrosis, shedding, and follicular cystic dilatation of the granular layer follicle cells in the ovarian stroma. Furthermore, XJCRTSZW treatment observably enhanced the TP-induced reduction of primary follicles and secondary follicles numbers and decreased the TP-induced elevation of atretic follicle numbers and the expression of AMPK, SIRT1, and 8-OhdG in GCs in vivo and in vitro. Moreover, XJCRTSZW ameliorates TP-induced reproductive toxicity through AMPK/SIRT and Akt signaling axis mediated autophagy both in vivo and in vitro. XJCRTSZW can alleviate TP-induced reproductive toxicity via apoptosis, inflammation, and oxidative stress both  Tripterygium wilfordii Hook F [Tripterygium wilfordii for 8 weeks. Hikim et al. [Triptolide (TP), a diterpene trioxide, is a core component obtained from Chinese herb i Hook F , which hi Hook F –4. Thus,i Hook F , 6 have m et al.  reportedm et al. . TherefoThe molecular mechanisms involved in the toxicity of TP might encompass oxidative stress, inflammation, apoptosis, autophagy, and other responses  . AutophaCyperus rotundus, Fructus Leonuri, wolfberry, eupatorium, Chinese yam, and dogwood. In the prescription, cistanche, cuscuta, and raspberry are used as the emperor medicines to invigorate the kidney and essence. Epimedium, dogwood, and rehmannia are minister medicines to strengthen the power of the emperor medicines to nourish the kidney. Among them, epimedium warms the kidney and yang, and rehmannia and dogwood nourish the essence and blood of liver and kidney. The rest of the medicines are all adjuvants. Among them, angelica, Caulis Spatholobi, Fructus Leonuri, wolfberry, and eupatorium nourish blood and promote blood circulation. Morus nourishes the liver and kidneys and strengthens muscles and bones; Chinese yam nourishes qi and invigorates the spleen; and Cyperus rotundus is the key to regulating menstruation in gynecology, which can regulate the qi and activate the blood and make all the medicines tonic without stagnation. Moreover, our group has been demonstrating the effectiveness of XJCRTSZW on premature ovarian failure (POF) [XinJiaCongRongTuSiZiWan (XJCRTSZW) is a traditional Chinese medicine (TCM) compound for invigorating the kidney, nourishing blood, and promoting blood circulation, which is created by Professor Keming Wu based on many years of clinical experience. The prescription is composed of cistanche, cuscuta, raspberry, morus, rehmannia, angelica, epimedium, Caulis Spatholobi, re (POF)  and polyre (POF) . Furtherin vivo and in vitro. Moreover, XJCRTSZW ameliorates TP-induced reproductive toxicity through AMPK/SIRT and Akt signaling axis mediated autophagy both in vivo and in vitro. The results of this study will provide new insights and methods for the therapy of reproductive toxicity.In the present study, we reported that XJCRTSZW could alleviate TP-induced reproductive toxicity via apoptosis, inflammation, and oxidative stress both ad libitum at (25 ± 2)°C and 40%–60% relative humidity. All the procedures were carried out strictly based on the National Institute of Health Guide for the Care and Use of Laboratory Animals. Also, the study was ratified by the Board and Ethics Committee of Chengdu University of Traditional Chinese Medicine.Adult female Sprague Dawley rats  were purchased and acclimated to standard laboratory conditions for 7 days before experiments. Rats were provided with a 12 h/12 h light-dark cycle and fed with a standard diet and water 2).Human ovarian granulosa cell lines (cat. no. CP-H192) were purchased from Procell . Cells were maintained in complete medium for human ovarian granulosa cells  at 37°C with 5% carbon dioxide , including control, TP, TP + XJCRTSZW-high, TP + XJCRTSZW-middle, and TP + XJCRTSZW-low. Rats in TP, TP + XJCRTSZW-high, TP + XJCRTSZW-middle, and TP + XJCRTSZW-low group were intragastrically administered with 400 μg/kg·d TP  for continuous 40 days to induce the reproductive toxicity, while rats in the control group were intragastrically administered with equal amount of saline including 5% DMSO. Then, rats in TP + XJCRTSZW-high, TP + XJCRTSZW-middle, and TP + XJCRTSZW-low group were intragastrically administered 24.15 g/(kg.d), 12.075 g/(kg.d), and 6.0375 g/(kg.d) XJCRTSZW , including control, XJCRTSZW, and coenzyme Q10 groups. Rats in XJCRTSZW and coenzyme Q10 group were intragastrically administered with 24.15 g/(kg.d) XJCRTSZW and 15 mg/(kg.d) coenzyme Q10, respectively, while rats in the control group were intragastrically administered with 1 ml/100 g saline 2 times a day for 5 consecutive days. Then, blood was taken from the abdominal aorta after the rats were intraperitoneally anesthetized with sodium pentobarbital (40 mg/kg). Serum was isolated and inactivated the complement for in vitro experiments. Human ovarian granulosa cell lines were inoculated into six-well plates and divided into nine groups including control, DMSO, TP, TP + coenzyme Q10, TP + XJCRTSZW, TP + XJCRTSZW + CQ, TP + XJCRTSZW + NAC, TP + NAC, and TP + CQ groups. Human ovarian granulosa cells in the TP group were treated with 200 μL 100 nM TP for 12 h and then treated with 200 μL medicated serum obtained from the control group as above described. Cells in the TP + coenzyme Q10 group were treated with 200 μL 100 nM TP for 12 h and then treated with 200 μL medicated serum obtained from the coenzyme Q10 group as above described. Cells in the TP + XJCRTSZW group were treated with 200 μL 100 nM TP for 12 h and then treated with 200 μL medicated serum obtained from the XJCRTSZW group as above described. Cells in the TP + XJCRTSZW + CQ group were treated with 200 μL 100 nM TP for 12 h and then treated with 200 μL medicated serum obtained from the XJCRTSZW group and 100 μM chloroquine (CQ), respectively. Cells in the TP + XJCRTSZW + NAC group were treated with 200 μL 100 nM TP for 12 h and then treated with 200 μL medicated serum obtained from the XJCRTSZW group and 10 μM N-acetylcysteine (NAC), respectively. Cells in the TP + NAC group were treated with 200 μL 100 nM TP for 12 h and then treated with 10 μM NAC. Cells in the TP + CQ group were treated with 200 μL 100 nM TP for 12 h and then treated with 100 μM chloroquine. Cells in the control and DMSO groups were treated with the same amount of saline and dimethyl sulfoxide (DMSO), respectively, and then treated with 200 μL medicated serum obtained from the control group as above described. Subsequently, cells were maintained at 37°C with 5% carbon dioxide (CO2) for further 48 h.For 5/well and cultured for 24 h at 37°C in 5% CO2. Subsequently, the cell count kit-8  was used to detect the proliferation of cells according to the operating manual. The absorbance was recorded at 450 nm by a microplate reader .Human ovarian granulosa cells were inoculated in 96-well plates with a density of 1 × 10μm sections were stained with hematoxylin and eosin (H&E). Pictures were obtained under a microscope . Then, the number of primary follicles, secondary follicles, and atretic follicles was measured.The ovarian tissue was separated, fixed, embedded, and cut into sections. 5 β, IL-6, and TNF-α were also determined by rat IL-1β ELISA kit (ZC-36391), rat IL-6 ELISA kit (ZC-36404), and rat TNF-α ELISA kit (ZC-37624)  based on the manufacturer's instructions. The absorbance of wells was determined with a microplate reader (Thermo Fisher Scientific) at 450 nm wavelength to analyze the sample concentration.The levels of estradiol (E2), anti-Mullerian hormone (AMH), follicle stimulating hormone (FSH), luteinizing hormone (LH), inhibin B (INH B) were detected using the Estradiol ELISA Kit , rat anti-Mullerian hormone (AMH) ELISA KIT , rat follicle stimulating hormone (FSH) ELISA KIT , rat luteinizing hormone (LH) ELISA KIT , and rat Inhibin B ELISA KIT  according to the manufacturer's instructions. The serum or supernatant levels of IL-1The levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were detected using the lipid peroxidation MDA assay kit , total superoxide dismutase assay Kit with NBT , and total glutathione peroxidase assay kit with NADPH  according to the manufacturer's instructions. The absorbance of wells was determined with a microplate reader (Thermo Fisher Scientific) at 532 nm (MDA), 560 nm (SOD), and 340 nm (GSH-Px) wavelengths to analyze the sample concentration.Apoptosis of GCs was evaluated by a flow cytometric assay. In brief, GCs were collected and stained with Annexin V-APC and PI  at room temperature for 20 min in the dark. The fluorescence of the cells was measured by flow cytometry .μm) were obtained for the IHC experiment. Sections were stained with rabbit polyclonal SIRT1 antibody , rat monoclonal AMPK antibodies , and rabbit polyclonal 8-OHdG antibody  overnight at 4°C. Subsequently, the sections were incubated with goat anti-rabbit IgG (H + L)-biotin  at 4°C for 30 min. The results were analyzed with the digital trinocular camera microscope  and image analysis software Image-Pro Plus 6.0 .GCs separated from the ovary were immobilized with 4% paraformaldehyde for 6 h at room temperature. After being dehydrated, embedded, and cut, sections , rabbit anti-p-AMPKα , rabbit polyclonal to pan-AKT , rabbit polyclonal to AKT (phospho T308) , and rabbit anti-β-actin .Protein samples from GCs separated from ovary or human ovarian granulosa cells were extracted using a total protein extraction kit . Then, the protein concentration was detected by a protein assay kit (Beyotime). Next, protein samples were separated by 10% SDS-PAGE gel and electrically transferred to PVDF membranes . After being blocked with 3% bovine serum albumin (BSA) for 1 h at room temperature, the membranes were incubated with the primary antibodies at 4°C overnight. After washing with TBST for 3 × 5 min, the membranes were incubated with goat anti-Rabbit IgG (H + L)-HRP  for 1 h at room temperature. Protein bands were analyzed by an electrochemiluminescence (ECL) chemiluminescence kit , and the band intensity was quantified with Image-Pro Plus 6.0 software. The primary antibodies used were as follows: rabbit anti-beclin-1 , rabbit anti-LC3II/I , rabbit anti-p62 , rabbit monoclonal (E61) to procaspase-3 , rabbit anticleaved caspase-3 , rabbit monoclonal (EPR2849Y) to SIRT1 (phospho S47) , rabbit monoclonal (EPR18239) to SIRT1 , rabbit anti-AMPKp > 0.05 and p < 0.05, respectively.Data were presented as the means ± standard deviation. Differences among multiple groups were analyzed using one-way analysis of variance and Duncan's test using the SPSS 20.0 package . The differences were considered as statistically nonsignificant and significant when H&E staining analysis showed that there were few mature follicles in the ovary, less granular cell layer and disordered arrangement of the follicle, lymphocytes and plasma cells infiltration, and necrosis, shedding, and follicular cystic dilatation of the granular layer follicle cells in the ovarian stroma in TP-treated rats, which was observably ameliorated with XJCRTSZW treatment . Moreoveβ, IL-6, and TNF-α, while low-does XJCRTSZW treatment just decreased the TP-increased the serum levels of IL-1β, IL-6, and TNF-α with no statistical difference  or XJCRTSZW treatment. Moreover, the use of CQ (inhibitor of autophagy) and NAC (inhibitor of oxidative stress) further significantly elevated human ovarian granulosa cells viability on the basis of the XJCRTSZW treatment, while the effect of CQ or NAC treatment alone was observably worse than that of coenzyme Q10 or XJCRTSZW treatment alone . Similarβ, IL-6, and TNF-α (Figures The apoptosis rate of human ovarian granulosa cells was significantly enhanced in the TP group compared to that in the control group, while coenzyme Q10 or XJCRTSZW treatment notably declined the TP-induced increase of apoptosis rate, and XJCRTSZW treatment combined with CQ or NAC treatment further prominently decreased the TP-induced elevation of apoptosis rate . The sam Figures . In addi Figures . In brieα-1, p-SIRT1, and p-Akt protein levels, whereas it observably elevated the TP-induced decrease of p62 protein levels. Similarly, XJCRTSZW treatment combined with CQ or NAC treatment further notably reduced the protein level of beclin-1, LC3-II/LC3-I, and cleaved-caspase-3, whereas it increased the protein level of p62. No statistical change was discovered in the protein level of procaspase-3, AMPKα-1, SIRT1, and Akt (Figures α-1/SIRT1/Akt signaling axis.Human ovarian granulosa cells treated with TP showed cell necrosis, fragmented nuclei, chromatin aggregation, as well as the disordered cytoplasmic content, the blurred structure, pyknotic mitochondria, and a lot of autophagosomes. However, coenzyme Q10 or XJCRTSZW treatment notably ameliorated these symptoms, and XJCRTSZW treatment combined with CQ or NAC treatment further prominently improved these symptoms of cultured human ovarian granulosa cells . In addi Figures . Therefoin vivo and in vitro. Moreover, XJCRTSZW ameliorates TP-induced reproductive toxicity through the AMPK/SIRT and Akt signaling axis mediated autophagy both in vivo and in vitro.The main results of the present study are that XJCRTSZW can alleviate TP-induced reproductive toxicity via apoptosis, inflammation, and oxidative stress both in vivo, as well as the dysregulation of some hormones both in vivo and in vitro. E2 is the most abundant and active estrogen in women. It is secreted by follicular granulosa cells and regulated by LH and FSH, which can promote the development of various organs of the reproductive system, facilitate endometrial hyperplasia and shedding, and maintain female secondary sexual characteristics [Although TP has been demonstrated to have anti-inflammatory, antitumor, antiarteriosclerosis, and analgesic effects, plenty of studies also report its reproductive toxicity both in human beings and animals. In line with the previous studies reported by Liu et al. , our reseristics . AMH is eristics . The leveristics . Both LHeristics . Therefoeristics , 22. Moreristics  show thaeristics . Thus, Xeristics . The abneristics . Howevereristics . Taken tβ, IL-6, and TNF-α both in vivo and in vitro, which suggested that XJCRTSZW can relieve TP-induced reproductive toxicity via apoptosis and inflammation. In addition, XJCRTSZW treatment also observably inverted the TP-induced alterations of SOD, GSH-Px, and MDA both in vivo and in vitro. SOD is an antioxidant enzyme that specifically scavenges oxygen-free radicals in the body that play an important role in the body's oxidation and antioxidant balance and can disproportionate superoxide anion-free radicals to produce hydrogen peroxide to be further converted into water by GSH-PX in the body [in vitro. Since NAC has been demonstrated to have antioxidant properties in a growing number of studies [Mechanically, several responses are associated with TP-induced reproductive toxicity, including inflammation, oxidative stress, apoptosis, and autophagy . In the the body . MDA is the body . Therefo studies , 30, we  studies . Further studies , cuscuta studies , and ras studies , as well studies , Caulis  studies , and eup studies  have exhα-1 and SIRT1, but elevated the TP-induced decrease of phosphorylated level of Akt both in vivo and in vitro. Also, XJCRTSZW treatment antagonized the TP-induced changes of beclin-1, LC3-II/LC3-I, p62, and cleaved-caspase-3 both in vivo and in vitro. Emerging evidence indicates that both the AMPK/SIRT1 [in vitro. Moreover, previous study has also reported that the AMPK/SIRT signaling pathway is associated with oxidative stress [in vitro. In brief, XJCRTSZW relieves TP-induced reproductive toxicity via the AMPK/SIRT and Akt signaling axis mediated autophagy.More and more evidence has been shown that TP is able to regulate the autophagic pathway in various cell lines and tissues, such as murine leukemia WEHI-3 cells and WEHI-3 generated leukemia mice , cardiomPK/SIRT1  and Akt PK/SIRT1  signalinPK/SIRT1 , 46. Thee stress . Oxidatie stress . Althouge stress , its spein vivo and in vitro. Moreover, XJCRTSZW ameliorates TP-induced reproductive toxicity through AMPK/SIRT and Akt signaling axis mediated autophagy both in vivo and in vitro. However, our study also has some limitations. For instance, it has been reported that there is crosstalk between autophagy and apoptosis, as well as autophagy and oxidative stress regulated by TP [In conclusion, XJCRTSZW can alleviate TP-induced reproductive toxicity via apoptosis, inflammation, and oxidative stress both ed by TP . Therefoed by TP . Thus, t


Women with evidence of ischemia and no obstructive coronary artery disease (INOCA) have an increased risk of major adverse cardiac events, including heart failure with preserved ejection fraction (HFpEF). To investigate potential links between INOCA and HFpEF, we examined pathophysiological findings present in both INOCA and HFpEF.vs. rest. All CMRI measures were quantified using CVI42 software (Circle Cardiovascular Imaging Inc). Statistical analysis was performed using linear regression models, Fisher’s exact tests, ANOVA, or Kruskal-Wallis tests.We performed adenosine stress cardiac magnetic resonance imaging (CMRI) in 56 participants, including 35 women with suspected INOCA, 13 women with HFpEF, and 8 reference control women. Myocardial perfusion imaging was performed at rest and with vasodilator stress with intravenous adenosine. Myocardial perfusion reserve index was quantified as the ratio of the upslope of increase in myocardial contrast at stress P = 0.007), Body surface area (0.05) were higher in the HFpEF group. Left ventricular ejection fraction (P = 0.02) was lower among the INOCA and HFpEF groups than reference controls after age adjustment. In addition, there was a graded reduction in myocardial perfusion reserve index in HFpEF vs. INOCA vs. reference controls , which was attenuated with age-adjustment.Age (Reduced myocardial perfusion reserve appears to be a common pathophysiologic feature in INOCA and HFpEF patients. Up to 50% of women with INOCA have coronary microvascular dysfunction (CMD) with impaired myocardial perfusion reserve, most often detected by reduced coronary flow reserve through invasive coronary function testing (CFT) or non-invasive imaging such as cardiac magnetic resonance imaging (CMRI) or positron emission tomography,5. Previous studies demonstrated that women with CMD often have left ventricular (LV) diastolic dysfunction, adverse ventricular remodeling, elevated inflammatory markers, and myocardial scar–8. However, the mechanism(s) linking HFpEF with INOCA remains to be elucidated. Accordingly, we hypothesized that CMD might be contributing to adverse ventricular remodeling in INOCA and subsequently to HFpEF. We tested our hypothesis by evaluating LV remodeling and myocardial perfusion abnormalities in participants with CMD, HFpEF, and reference controls.Women with evidence of ischemia and no obstructive coronary artery disease (INOCA) are at increased risk of developing major adverse cardiovascular events, most commonly heart failure with preserved ejection fraction (HFpEF)NCT02582021. All women with INOCA underwent clinically indicated invasive CFT after demonstration of no obstructive coronary artery disease (CAD), defined as < 50% stenosis. CFT is considered the gold standard invasive test to diagnose CMD and published previously. In brief, CFT was performed by infusing vasoactive substances through a guiding catheter placed in the left main coronary artery. The Doppler guide wire was positioned in the proximal left anterior descending coronary artery, and test results are interpreted in Table 1.Sixty-four participants were recruited, including 36 women with suspected INOCA, 20 with HFpEF (14 women and 6 men), and 8 reference control women. All study participants selection criteria and intervention are illustrated in , including symptoms of heart failure , left ventricular ejection fraction > 45% prior to study entry, structural evidence of cardiovascular abnormalities: evidence of abnormal filling or relaxation, left atrial enlargement or left ventricular hypertrophy documented by echocardiogram and evidence of elevated filling pressure pressures: left ventricular end-diastolic pressure or pulmonary capillary wedge pressure at rest > 15 mmHg, elevated brain natriuretic peptide, or use of diuretics. In addition, participants who had the following were excluded: LVEF < 45%, Acute coronary syndrome , primary cardiomyopathies , constrictive pericarditis, high-output heart failure and right ventricular myopathies, patients with concurrent cardiogenic shock or requiring inotropic or intra-aortic balloon support or current acute decompensated HF requiring therapy, alternative reason for dyspnea such as: significant pulmonary disease or severe COPD, hemoglobin less than 10 g/dL or body mass index more than 40 kg/m2, systolic blood pressure (SBP) ≥ 180 mmHg at entry, or SBP > 150 mmHg and < 180 mmHg at entry unless the patient is receiving 3 or more antihypertensive drugs, prior or planned percutaneous coronary intervention or coronary artery bypass grafting, Non-cardiac illness with a life expectancy less than 4 years, inability to give informed consent, contraindication to CMRI, end stage renal disease, end stage liver disease. In addition, obstructive CAD was excluded by noninvasive coronary computed tomography angiography in this group due to the lack of clinically indicated CFT. Reference control women were recruited from the Cardiac Magnetic Resonance Imaging Normal Reference Control Group Testing study NCT00573339.All participants hospitalized for HFpEF were enrolled. All participants met modified criteria for diagnosis of HFpEF based on the European Society of Cardiology guidelines (2012)Women had no symptoms suggestive of cardiovascular ischemia and cardiovascular risk factors and had a non-ischemic Bruce protocol exercise treadmill stress test. The study was approved by the institutional review board, and all participants provided written informed consent.n = 59) (140 μg/kg for 3 min) or regadenoson (n = 5) stress-rest perfusion (0.4 mg). CMRI was per) and a standardized gadolinium bolus of 0.05 mmoL/kg injected at 4 mformed using standardized product sequences  during stress and rest. First-pass perfusion images were obtained in three LV short-axis slices  with the following parameters: Gradient echo - EPI hybrid sequence, TR per slice 134.8 ms, TE 0.94 ms, BW 1240 Hz/pixel, readout flip angle 43°, slice thickness 8 mm, image matrix 155 × 224 pixels, in-plane resolution 1.34 mm × 1.34 mm × 8 mm2, parallel imaging (GRAPPA) factor 2, imaging three slices every heartbeat. In the event of a peak stress heart rate of > 120 bpm, two slices were obtained during stress first-pass imaging with the exclusion of the distal LV slice position. Cardiac morphology and function were assessed using a stack of short-axis cine images spanning the entire LV, together with a series of long-axis images in the horizontal, vertical, and LV outflow tract views. Typical scan parameters included contiguous 8 mm/0 mm slices, 1.34 mm × 1.34 mm × 8 mm voxel size, 155 mm × 224 mm matrix, 25 cardiac phases, 11 segments, 10 heartbeats/slice. Visual assessment of splenic switch-off for evaluation of stress test adequacy was performed in participants who received adenosine.For stress, all participants received either adenosine (. The Seattle Angina Questionnaire (SAQ), measuring five functional dimensions, was used to characterize clinical symptoms (higher scores indicate less angina). Kansas City Cardiomyopathy Questionnaire (KCCQ) was used to characterize heart failure symptoms (higher scores indicate better HF symptoms). Left ventricular mass and end-systolic and end-diastolic volumes were indexed to the body surface area.All CMRI data were quantified and analyzed using CVI42 software (Circle Cardiovascular Imaging Inc.). Myocardial perfusion reserve index (MPRI) was calculated as previously published.We analyzed a total of 56 women participants and excluded men participants and participants with an EF of less than 50%, as shown in P-values were obtained using either Fisher’s exact tests, ANOVA, or Kruskal-Wallis tests using SAS software.Age-adjusted p-values were statistically obtained using linear regression models, and non-adjusted P = 0.003, 30.8%, 6.5%, P = 0.05) while family history of CAD was prevalent among the INOCA group compared to the HFpEF group . Both groups had similar use of calcium channel blockers, aspirin, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and statins. The β-blocker use was more prevalent in the HFpEF group than in the INOCA . As expected, the INOCA group had lower SAQ scores compared to the HFpEF group, indicating relatively severe angina symptoms. Interestingly, KCCQ symptoms were modestly reduced among the INOCA and the HFpEF groups, and there were no statistically significant differences between them across all the domains.Pertinent baseline characteristics are summarized in CMRI results are shown in The data supports the hypothesis that reduced myocardial perfusion is a common pathophysiologic feature in INOCA and HFpEF patients. These data show a reduction in LVEF and impaired myocardial perfusion reserve in patients with suspected INOCA and patients with HFpEF. The perfusion abnormality was worse among participants with HFpEF than INOCA and raised the question of whether INOCA and HFpEF share similar pathophysiological processes but worse in HFpEF than INOCA. Although myocardial perfusion was reduced in both INOCA and HFpEF, only the epicardial MPRI remained statistically significantly different following age adjustment. Similar to women with HFpEF, women with INOCA showed significant symptoms related to HF evidenced by the KCCQ assessment.–3; however, the contributing mechanism(s) to subsequent heart failure progression in INOCA has not been well characterized. Recent results from the PROMIS-HFpEF trial showed a high prevalence of CMD evident from reduced coronary flow reserve measured using adenosine stress transthoracic Doppler echocardiography among HFpEF patients. Furthermore, emerging data support the hypothesis that myocardial ischemia, secondary to CMD, is a key mechanism leading to pathological remodeling in HFpEF,13,14. Indeed, the impaired coronary microvascular function has been independently associated with features of HFpEF such as LV diastolic dysfunction and elevated highsensitivity cardiac troponin concentrations, leading to worse clinical outcomes and recurrent hospitalizations,13. Like CMD, other cardiovascular risk factors such as essential hypertension and DM can adversely impact cardiac remodeling. Previous studies demonstrated that CMD and HFpEF share similar risk factors, including age, obesity, hypertension, and DM,16–19. The current investigation builds upon these prior observations by directly comparing indices of LV morphology and myocardial perfusion in INOCA and HFpEF as compared to reference control participants.Women with suspected INOCA are at increased risk of developing major adverse cardiovascular events, most commonly HFpEFA possible explanation for our findings is that participants in the HFpEF group are older, more obese, have a higher prevalence of DM, relatively more hypertensive, and therefore at greater risk of chronic myocardial microvascular ischemia evidenced by reduced perfusion. Thus, it is plausible that chronic exposure to myocardial ischemia may be linked to adverse remodeling.Our study is limited by relatively small sample size and, therefore, limited in adjusting for confounding variables. Our study is also limited by the lack of a longitudinal design and the inability to decide the temporality of our findings. In addition, our focus on the INOCA and reference control groups of exclusive women limits the generalizability of these results to men. Finally, our recruitment of symptomatic INOCA participants limits generalizability to asymptomatic patients.These data, taken together, support the hypothesis that CMD and other cardiovascular risk factors may play a pivotal role in driving heart failure progression in INOCA. However, future studies are needed to confirm the exact mechanistic link through longitudinal investigation of women with INOCA.


Caulobacter crescentus, we demonstrate that DciA plays a prominent role in DNA replication fork maintenance. Cell cycle analyses using a synchronized Caulobacter cell population showed that cells devoid of DciA exhibit a severe delay in fork progression. Biochemical characterization revealed that the DnaB helicase in its default state forms a hexamer that inhibits self-loading onto single-stranded DNA. We found that upon binding to DciA, the DnaB hexamer undergoes conformational changes required for encircling single-stranded DNA, thereby establishing the replication fork. Further investigation of the functional structure of DciA revealed that the C-terminus of DciA includes conserved leucine residues responsible for DnaB binding and is essential for DciA in vivo functions. We propose that DciA stimulates loading of DnaB onto single strands through topological isomerization of the DnaB structure, thereby ensuring fork progression. Given that the DnaB-DciA modules are widespread among eubacterial species, our findings suggest that a common mechanism underlies chromosome replication.The replicative DNA helicase translocates on single-stranded DNA to drive replication forks during chromosome replication. In most bacteria the ubiquitous replicative helicase, DnaB, co-evolved with the accessory subunit DciA, but how they function remains incompletely understood. Here, using the model bacterium  The unwinding of double-stranded (ds) DNA is fundamental for the semiconservative replication of chromosomes in all cellular life . In the Escherichia coli DnaB (DnaBEC) and its loading partner, DnaC .,24.Esche complex ,21,25. Wge ssDNA ,21,25. Sterminus . Recent terminus ,21. A siterminus  27). Im. ImEschesubtilis , the vassubtilis . Thus, hVibrio cholerae DciA homolog , whereas the motile swarmer progeny blocks replication initiation and consequently experiences an extended nonproliferating period, termed the G1 phase  medium supplemented with appropriate antibiotics, as described. When necessary, cumate (1 μM), glucose (0.1%), or xylose (0.1%) was added to the culture medium, as indicated. For synchronization experiments, newborn G1 cells were harvested using the LUDOX density-gradient centrifugation method  antibody was used as primary antibody, which was detected by AP-conjugated anti-rabbit second antibodies (Bio-rad). The immune reactions were performed in Immunoreaction Enhancer Solution (Toyobo) according to the manufacturer's instructions.C. crescentus proteins were expressed in E. coli and purified as described in the Supplemental Material. E. coli proteins (DnaBEC and DnaC) were purified as described previously  was used instead of a forked DNA substrate, and competitor DNA was replaced by a mixture of fluorescently-labeled 21-mer oligonucleotides . DNA samples were extracted and analyzed using 9% polyacrylamide gel electrophoresis.32P]ATP according to the manufacturer's instructions, followed by annealing with the complementary strands.When radioactively-labelled substrates were used, the synthetic DNA strands  were incubated with T4 polynucleotide kinase (Toyobo) and [γ-The ATPase assay was performed essentially as described previously . Brieflyin vitro pulldown assays, Rosetta™ 2(DE3) cells harboring pETsfTq2dciA(WT)-d or its derivatives were grown exponentially in PY  medium (200 ml) supplemented with kanamycin and chloramphenicol at 30°C, and expression of sfTq2-tagged proteins was induced at 30°C for 1 h by addition of 1 mM isopropyl-β-d-thiogalactoside. Cells were harvested by centrifugation and resuspended in Lysis buffer to adjust the cell density to OD600 of 200. Following cell lysis using lysozyme (0.2 mg/ml) and brief sonication, cleared cell lysate was prepared using ultracentrifugation. The sfTq2-tagged proteins were further purified through ammonium sulfate (0.25 g/ml) precipitation. Precipiate was dissolved in lysis buffer and a portion containing 4 μg of the sfTq2-tagged proteins and purified DnaB-His (6 μg) were incubated with Ni-conjugated magnetic beads (Promega) for 20 min at 4°C in R1 buffer. After washing the resin twice using W1 buffer, materials retained on the resin were eluted in Elution buffer (10 μl). A portion (2.5 μl) of the eluted sample was analyzed using SDS 12% polyacrylamide gel electrophoresis and Coomassie brilliant blue staining. Buffers used are as follows: Lysis buffer , R1 buffer , W1 buffer , and Elution buffer (Lysis buffer with 500 mM imidazole).To perform dciA  is an essential gene in C. crescentus, we investigated the colony forming ability of a C. crescentus strain depleted of DciA. To express DciA conditionally, we engineered the PxylX-dciA strain in which the xylose-dependent promoter (PxylX) was inserted upstream of the dciA-coding region  or CCNA_00381 (DNA glycosylase), because colony formation and cell morphology of the DciA-depleted cells were fully restored by introduction of a low copy number plasmid expressing the dciA gene, but not a control vector   Figure –D. Theses Figure , arguingC. crescentus cells. We reasoned that if DciA is a component of replication fork proteins, its absence may cause delay in S-phase entry and/or progression. We therefore assessed cellular DNA content over time in a synchronous population of cells. Moreover, to inactivate dciA in a timely manner, we made use of an inducible protein degradation system by which a DAS + 4-tagged target protein undergoes rapid proteolysis in a manner that is dependent on the E. coli SspBec adaptor . In the presence of the inducer, cumate, the amounts of DciA were increased by 1.3–20 fold in a dose-dependent manner (0.25–2 μM cumate) and reached a plateau (25-fold) at 10 μM cumate . Similar to DciA-depleted cells, increasing levels of DciA resulted in production of elongated cells . DNA staining with SYTOX Green fluorescence dye revealed that those elongated cells displayed fewer fluorescent signals compared to the cells with the control vector, suggesting that DNA replication is compromised in cells overexpressing DciA.To further investigate the role of To better resolve defects in chromosome replication, we further analyzed cells blocked for cell division with the antibiotic cephalexin, reasoning that cephalexin-treatment allows for additional rounds of chromosome replication in predivisional cells and thus, such over-replicating cells are easily distinguished from cells compromised for replication. As expected, when DNA replication in a synchronous population was monitored under cephalexin treatment, nearly all cells with an empty vector initiated chromosome replication within 30 min and completed it by 90 min Figure . SubsequC. crescentus DciA is a stable monomer in solution. This observation is consistent with recent reports that a DciA homolog from V. cholerae adopts a monomeric state in solution .To obtain molecular insights into DciA during chromosome replication, we set out to analyze the interaction between DciA and DnaB using size exclusion chromatography on the following purified recombinant proteins: DciA with an N-terminal hexahistidine tag  and DnaB with a C-terminal hexahistidine tag  Figure . The olisolution . Converssolution ,20,44. HTo assess the interaction between DciA and DnaB, a mixture of His-DciA and DnaB-His was analyzed using a Superdex 200 column. In this analysis, the two proteins coeluted earlier than either protein alone, indicating that DciA directly binds DnaB Figure . The coein vitro ATP hydrolysis assay using pooled peak fractions of DnaB-His from the size exclusion chromatography. In the absence of DciA, ATP hydrolysis was readily observed at various concentrations of wild-type DnaB-His  or each of the three domains . Unlike wild-type sfTq2-DciA FL, these variants severely reduced the affinity for DnaB-His Figure . MoreoveC. crescentus. While homologs of these proteins are highly conserved among bacterial species, it has been difficult to distinguish between their functions in replication initiation and elongation, and the molecular mechanisms underlying the DnaB-DciA system remain unclear. Here, we provide good evidence that the C. crescentus DciA acts as a replication fork protein to render DnaB competent for topological loading onto replication forks. We found that C. crescentus DnaB forms a homohexamer that is incapable of self-loading onto ssDNA unless a free ssDNA end is present for its threading, consistent with the idea that the DnaB hexamer takes on a closed ring-shaped structure as the default state. Similar results were reported using a DnaB homolog from Vibrio cholerae that expresses cognate DciA homolog  from P. aeruginosa was previously implicated in the early stage of chromosome replication; however, it did not clearly differentiate between a role for DciA in replication initiation and elongation  takes on various oligomeric states and exists preferentially as a monomer in solution. Upon biding to DnaI, the regulatory subunit belonging to the AAA + family, BsDnaC monomers are assembled into a hexameric ring around DNA, thereby exhibiting the helicase activity (C. crescentus DnaB-DciA system in that C. crescentus DnaB forms a stable hexamer and that the pre-formed DnaB hexamer is activated by DciA.The finding that DciA stimulates the ATPase activity of DnaB contrasts with the previous reports that i DnaBEC . These oreaction . Subsequ complex . Our dat complex . We specactivity . Becauseactivity , it has B. subtilis, primosomal DnaB, DnaD, and DnaI proteins comprise a helicase loader complex at the origin, while Helicobacter pylori employs the unique Hp0897 protein to operate the cognate replicative helicase during replication initiation (B. subtilis or H. pylori helicase operators have been annotated in C. crescentus, it is possible that in the course of evolution, this organism acquired structural homologs of these proteins with little sequence similarity.It remains unclear how DciA contributes to initiation of chromosome replication. We do not fully rule out DciA playing a specific role at the origin, although a G1 cell cycle arrest, a hallmark of replication initiation defects, was not observed in cells devoid of DciA in our experimental conditions. Alternatively, it is tempting to speculate that an as-of-yet unknown protein factor may operate at the origin. In some Gram-positive bacteria including itiation . AlthougThe data supporting the findings of this study are available from the corresponding author upon reasonable request.gkac1146_Supplemental_FileClick here for additional data file.


Heart failure (HF) is a significant cause of morbidity and mortality worldwide. HF with preserved ejection fraction (HFpEF) is a complex syndrome, often participated by several cardiac and extracardiac conditions, including chronic kidney disease, pulmonary disease, anaemia and advanced age. Circulating biomarkers reflecting pathophysiological pathways involved in HFpEF development and progression may assist clinicians in early diagnosis and management of this condition. Natriuretic peptides (NPs) are cardioprotective hormones released by cardiomyocytes in response to pressure or volume overload and in response to activation of neuro-endocrine-immune system. The relevance of B-type NP (BNP) and N-terminal pro-B-type NP (NT-proBNP) for diagnosis and risk stratification has been extensively demonstrated, and these biomarkers are emerging tools for population screening and as guides to the start of treatment in subclinical HF. On the contrary, conflicting evidence exists on the value of NPs to guide HF therapy. Among the other biomarkers, high-sensitivity troponins and soluble suppression of tumorigenesis-2 are the most promising biomarkers for risk stratification, predicting outcome independently from NPs. In this review, some novel biomarkers are being tested in such clinical scenario, more tightly linked to specific pathophysiological processes of cardiac damage. Heart failure (HF) is a progressive condition in which the heart muscle is not able to pump enough blood to meet the needs of the body. The prevalence of HF is in 1–2% of adults in industrialized countries and is increasing with population ageing. HF then represents one of the major public health problems . HFpEF iThe risk of all-cause death is comparable in HFpEF and HF with mildly reduced EF  and lower than in HFrEF, while the risk of death or HF hospitalization is lower for HFpEF than HFrEF or HFmrEF ,11. WhilCirculating biomarkers are possible tools to identify patients at higher risk, those with activation of specific pathways of organ damage and, possibly, to provide support for therapy optimization. Both conventional and novel biomarkers may be helpful to predict future disease development, for diagnosis and risk stratification, as well as inclusion and exclusion criteria for clinical trials . A broadB-type natriuretic peptide (BNP) and the N-terminal fragment of proBNP (NT-proBNP) are produced from the cleavage of their 108-aminoacid precursor proBNP by proprotein convertases, such as corin and furin. The biologically active BNP is degraded by several peptidases, such as dipeptidyl peptidase IV and neutral endopeptidases (NEP or neprylisin) ,20,21. BBNP and NT-proBNP are crucial biomarkers for the diagnosis of HFrEF, with a less established role for risk stratification and management ,23. NoneComorbidities influence NP circulating levels in both HFpEF and HFrEF, which is of great clinical importance, given the higher prevalence of non-cardiac conditions in HFpEF. Several conditions are associated with higher NPs, including chronic obstructive pulmonary disease (COPD), atrial fibrillation (AF), kidney disease, diabetic ketosis, while NP levels may be significantly reduced in obese patients ,30,31. FNP elevation has been used in many trials as an inclusion criterion to improve the diagnostic specificity and for risk enrichment ,34. BNP In a sub-analysis of the I-PRESERVE trial including 2162 patients, Jhund et al. have investigated the association between changes in NT-proBNP over a 6-month follow-up and clinical outcomes . Changes in NT-proBNP were associated with the risk of clinical outcomes, and particularly with HF-related outcomes. A 1000 ng/L elevation in NT-proBNP over 6 months was associated with a 2-fold higher risk of cardiovascular death or HF hospitalization  .The prognostic role of NT-proBNP has been also evaluated in a recent analysis of the EMPEROR-Preserved trial, which enrolled and randomized 5988 patients with a LVEF > 40% and NYHA class II-IV to receive empaglifozin or a placebo . 5986 (9p < 0.001) and ≥50% NT-proBNP reduction between admission and discharge  were independent predictors of death and rehospitalization for HF. Moreover, the combination of E/e’ and NT-proBNP values at discharge significantly improved the prognostic ability compared to each variable analyzed independently  [n = 283) compared to those with HFrEF (n = 776) followed up for 6 months, multivariable adjusted Cox regression analysis reported that for any 2.7-factor increase in NT-proBNP levels, the HR for mortality was 2.14 for HFpEF (95% CI 1.48 to 3.09) and 1.96 for HFrEF (95% CI 1.60 to 2.40). Furthermore, prognostically relevant comorbidities were more often present in patients with HFpEF than patients with HFrEF, but only in low (≤3000 ng/L) and not in high (>3000 ng/L) NT-proBNP discharge categories [Even in patients with acute HF and preserved EF, NT-proBNP was a strong predictor of all-cause mortality ,39,40. I < 0.01) . AnotherGuideline-recommended HF therapies reduce NP levels . The useAs for other NPs, the measurement of circulating atrial NP (ANP) is complicated due to its short half-life (2–5 min) related to the rapid cleavage by neprilysin, insulin-degrading enzyme and natriuretic peptide receptor-C. ANP precursor (proANP), which is stoichiometrically equimolar to ANP, has a longer half-life and it is more easily measurable by searching its mid-regional portion (MR-proANP) ,49. MR-pCardiac troponins are released from intracellular space to the bloodstream following alterations in membrane properties. The release of myocardial troponins may not require myocardial cell death, but the extrusion of proteins from reversibly injured cardiomyocytes may occur during transient increases in cell permeability due to cell wounds ,54. In pp < 0.04), after adjustment for age, sex and other clinical covariates , diabetes, hypertension, coronary artery disease, AF, and HF therapies) in 50 patients with HFpEF, 51 with HFrEF and 50 controls without HF [p = 0.03) [Santhanakrishnan et al. have shown that hs-TnT was higher in HFrEF than in HFpEF  . Both hs = 0.03) . The mecIn the Multi-Ethnic Study of Atherosclerosis (MESA), hs-TnT and NT-proBNP could identify the subset of patients with LV hypertrophy at a higher risk for incident HF, both with and without impaired systolic function ,61. In ap = 0.01) or HFrEF . hs-TnT displayed no significant association with 30-day outcome in the HFpEF group , as opposed to HFmrEF  and HFrEF , suggesting a lesser prognostic value of hs-TnT in HFpEF [p = 0.003). The AUC for hs-TnT was higher than BNP (0.739 vs. 0.631), and the optimal hs-TnT cut-off for adverse events  was 26 ng/L [Several studies investigated the prognostic value of troponin assay in patients hospitalized for HF and having a preserved EF. In a cohort of 500 patients with LVEF ≥ 40%, TnT was directly correlated with serum creatinine and symptom severity, and independently predicted all-cause death and HF rehospitalization . In a rein HFpEF . As for p = 0.03; PARAGON-HF: 9%, p < 0.001) [p = 0.046) [The phase 2 Prospective comparison of ARNI (angiotensin receptor–neprilysin inhibitor) with ARB (angiotensin-receptor blockers) on Management Of heart failUre with preserved ejectioN fraction (PARAMOUNT) and the phase 3 Prospective Comparison of ARNI with ARB Global Outcomes in HF With Preserved Ejection Fraction (PARAGON-HF) trials randomized patients with HFpEF to sacubitril/valsartan or valsartan, showing that patients receiving sacubitril/valsartan had a greater reduction in hs-TnT compared to those assigned to valsartan  ,69. MoreData regarding the prognostic ability of troponins also derive from a recent analysis of the EMPEROR-Preserved trial. Of 5988 study participants with LVEF > 40%, 5825 (97.3%) had available hs-TnT measurements, with an overall median baseline of 17.8 ng/L (Q1 and Q3 at 11.6 and 26.9 ng/L respectively) and 3767 (65.7%) patients showing hs-TnT > 14 ng/L . FinallyDifferent circulating biomarker profiles have emerged in patients with LVEF ≤ 40% or LVEF ≥ 50%. Patients with HFpEF display significantly higher levels of soluble suppression of tumorigenesis 2 (sST2), hs-C-reactive protein (CRP) and cystatin-C, while HFrEF patients presented higher concentrations of NT-proBNP, hs-TnT and hemoglobin . MoreovesST2 is a member of the Toll-like/interleukin-1 receptor superfamily, which is expressed following myocardial stretch and cardiovascular injury, together with its ligand, IL-33. The IL-33/ST2 interaction exerts anti-apoptotic, anti-hypertrophic and anti-fibrotic actions ,73, whiln = 50) than in sex-matched controls aged ≥55 years without HF or coronary artery disease (n = 50) [n = 68, 64%) to a control group with hypertension without HF , finding that sST2 was higher in HFpEF than controls (p < 0.001), although patients with HFpEF were also older [n = 70) with controls without HF (n = 4864), found that the median sST2 level was higher in HFpEF  compared to controls  [p = 0.049), hypertension (p = 0.023), AF (p = 0.049), renal dysfunction (p < 0.001) and congestion (p < 0.001) [Santhanakrishnan et al. reported that sST2 was not significantly higher in patients with HFpEF ((n = 50) . Conversso older . Even Si7 ng/mL) . Overall< 0.001) .p = 0.02) after adjustment for clinical covariates  [sST2 has emerged as a powerful predictor of outcome in chronic HF, and has been evaluated specifically in the setting of HFpEF ,80. In aure, AF) .p = 0.004; 36.6 ng/mL vs. 17.9 ng/mL for non- cardiovascular death, p = 0.02) [n = 86; HFrEF n = 86, control without HF n = 21) including acute decompensated HF subjects. In both crude analyses  and adjusted model  of HFpEF, sST2 was significantly associated with death from any cause or hospitalization for HF [In a cohort of 191 patients with acute decompensated HFpEF, sST2 emerged as a significant predictor of non-cardiovascular mortality , during a median follow-up of 445 days. Indeed, sST2 levels were increased in patients with all-cause death and non-cardiovascular death compared to those without events  . Similarn for HF . Moreoven for HF .Galectin-3  is a soluble β-galactoside binding lectin produced by macrophages, which plays an important role in the development of cardiovascular damage and remodeling by promoting the processes of cardiovascular inflammation, fibroblast proliferation and fibrosis, which drive the progression from subclinical structural cardiac involvement toward the development of HFpEF.Gal-3 levels in plasma are increased in patients with HFpEF compared to controls . Gal-3 an = 107; HFrEF n = 485) followed up for 18 months, but a similar elevation in plasma Gal-3 levels represents a much higher risk for all-cause death and hospitalization due to HF in patients with HFpEF vs. HFrEF (p < 0.001) [Gal-3 also predicted long-term outcome in HFpEF patients ,91,92,93< 0.001) .p = 0.009) after adjustment for clinical factors and NT-proBNP [A study on 247 patients (70% with HFpEF) reported that Gal-3 was strongly correlated with an increased risk of CV death and HF hospitalization in HFpEF patients, and the HR was 2.33 per 1 standard deviation increase in Gal-3  cytokine superfamily. It is synthesized as an inactive precursor that undergoes proteolytic activation, releasing the N-terminal pro-peptide from the mature GDF-15 protein, which is then secreted as a dimer ,97. GDF-n = 142) showed higher GDF-15 levels than controls (n = 188): 1.66 ng/mL (IQR:1.26–2.34 ng/mL) vs. 0.9 ng/mL (IQR: 0.70–1.09 ng/mL) (p < 0.001), respectively [Circulating GDF-15 levels are higher in patients with HFpEF compared to controls ,101,102.ectively . The NT-ectively . Nonetheectively .p < 0.001) and NT-proBNP  [The increase in plasma GDF-15 seems to hold prognostic significance in HF . In a co< 0.001) .n = 51,196) revealed a significant association between high CRP levels and new-onset HFpEF  [p = 0.004), but not with HFrEF  [Systemic inflammation is a crucial determinant of HFpEF development , but onl = 0.04) . A case- = 0.03) . In a st = 0.03) .p < 0.001) and a higher pulmonary artery systolic pressure (p = 0.002) [p = 0.037). The predictive value of MIF for the occurrence of death or hospitalization at 180 days was inferior to NPs  [Macrophage migration inhibitory factor (MIF) has recently attracted some attention. MIF is a pleiotropic mediator of the innate immune system, which induces the release of several cytokines, including TNF-α which activates matrix metalloproteinases and triggers collagen degradation ,112. In HFpEF is a heterogeneous clinical syndrome, with different cardiovascular and non-cardiovascular determinants participating in its development and presentation. Many biomarkers have been tested as possible tools for the diagnosis and management of HFpEF in the past years, only rarely showing promising results. B-type NPs have an established diagnostic role, and both B-type NPs and hs-Tn may help predict future disease trajectories. The notion of HFpEF as a systemic disorder has led to the assessment of multiple biomarkers exploring systemic inflammation, fibrosis or multiple mechanisms (as in the case of sST2 and GDF-15). Non-cardiac-specific biomarkers have no diagnostic role but may refine risk prediction in patients with established HFpEF, or signal an increased risk of HFpEF in patients with predisposing conditions. Biomarkers could also be measured serially over time, to gain insight on disease evolution and the response to treatment. Furthermore, the combination of multiple biomarkers might allow to investigate different disease pathways.n = 47) compared with HFpEF (n = 43), whereas 54 proteins  were differentially expressed in patients with HFmrEF (n = 83) compared with HFpEF [The development of molecular biology techniques provides the opportunity to investigate and increase our understanding of HFpEF. Proteomics allows the identification and quantification of all proteins of a cell, tissue or an organism . The advth HFpEF .A proteomic approach enables us to achieve a deeper understanding of the pathophysiological pathways of diseases, as showed by the proteomic identification of quiescin Q6, a protein involved in the formation of disulphide bonds, which allows to discriminate acute HF from non-cardiac dyspnea . MoreoveSome Authors have also performed -omic profiling to find novel biomarkers associated with incident HF or HF prognosis . For exa


N′N′-diacetylchitobiose and directly target the α subunit of the epithelial sodium channel. Solnatide was shown to have anti-inflammatory actions in acute lung injury and glomerulonephritis models. In this study, we evaluated whether the lectin-like domain of TNF can mitigate the development of immune-mediated arthritis in mice. In an antigen-induced arthritis model, solnatide reduced cell influx and release of pro-inflammatory mediators into the joints, associated with reduction in edema and tissue damage, as compared to controls indicating that TNF has anti-inflammatory effects in an acute model of joint inflammation via its lectin-like domain.In view of the crucial role of tumor necrosis factor (TNF) in joint destruction, TNF inhibitors, including neutralizing anti-TNF antibodies and soluble TNF receptor constructs, are commonly used therapeutics for the treatment of arthropathies like rheumatoid arthritis (RA). However, not all patients achieve remission; moreover, there is a risk of increased susceptibility to infection with these agents. Spatially distinct from its receptor binding sites, TNF harbors a lectin-like domain, which exerts unique functions that can be mimicked by the 17 residue solnatide peptide. This domain binds to specific oligosaccharides such as  Tumor necrosis factor (TNF) is a major cytokine, existing as a transmembrane and soluble ligand, which is involved in various acute and chronic inflammatory diseases. Robust evidence has linked overexpression of TNF to arthritis severity in animal models. TNF inhibitors (TNFi), including neutralizing monoclonal anti-TNF antibodies and soluble TNF receptor constructs, have revolutionized the treatment of rheumatoid arthritis (RA) and spondyloarthritis (SpA) in the clinical setting. TNF is not only a main mediator of pro-inflammatory mediator release, cell migration, and pain development, but the cytokine also activates enzymes that are involved in joint destruction, by eroding cartilage while inducing inflammatory bone resorption . ParadoxIn sharp contrast to its deleterious role in numerous pathophysiological phenomena, including the above-described arthropathies, TNF also represents a major component of the first-line defense against invasion by intracellular microorganisms, in addition to its beneficial function in tumor surveillance .N,N′-diacetylchitobiose (NAc) and branched trimannoses, is spatially distinct from its receptor binding sites  and in ARDS patients –18 upon Chondrocytes, osteoblasts, and mesenchymal stem cells have been shown to express ENaC-a, to which solnatide binds –25. AlthAll reagents were purchased from Sigma Chem Co., São Paulo, Brazil, unless stated otherwise. Solnatide was kindly donated by Apeptico Forschung und Entwicklung, Vienna, Austria, and was dissolved at the time of use using Roswell Park Memorial Institute (RPMI) 1640 supplemented with L-glutamine.A total of 42 Balb-C mice of either sex (25–30 g) were provided by the central animal facilities of our institution. Animals were housed in cages (six per cage) in temperature-controlled rooms with a 12-h light/dark cycle with free access to water and food. At the start of any experiments, mice were 2.5 months of age. All animal procedures and experimental protocols were approved by our local ethics committee on animal experimentation, which follows the recommendations of the Brazilian Council on Animal Experimentation (CONCEA) (protocol number 113/07). All efforts were made to minimize animal suffering and the number of animals used.Groups of mice received either 500 μg subcutaneous (s.c.) methylated bovine serum albumin (mBSA) s.c. mixed with Freund’s complete adjuvant (immunized) or incomplete Freund’s adjuvant [false-immunized (FI)], followed by a booster injection 7 days later. Twenty-one days following this immunization process, mice received intra-articular (i.a.) 90 μg mBSA. Groups were then sacrificed 7 h (acute phase) or 3 days (chronic phase) following the i.a. mBSA challenge. All injections were done under xylazine (10 mg/kg)/ketamine (80 mg/kg) intra-peritoneal (i.p.) anesthesia, and sacrifice was done under terminal anesthesia.In order to investigate the effect of blocking the endogenous lectin-like domain of TNF, groups of mice received 100 μg of NAc in 10 μl saline i.a. or 10 μl saline, 30 min prior to mBSA i.a. In a strategy to evaluate a possible preventive local anti-inflammatory activity of solnatide, groups of naive mice subjected to mBSA arthritis received 10–20 μg of 10 μl i.a. solnatide solutions. As a local therapeutic strategy, another group of mice received 10 μg/10 μl solnatide or 10 μl saline given i.a. 2 days after i.a. challenge with mBSA followed by sacrifice, under terminal anesthesia, after 24 h. In a last set of experiments, as a systemic therapeutic strategy, another group of mice subjected to mBSA arthritis received intravenous (i.v.) solnatide (1–10 μg) immediately prior to i.a. challenge with mBSA.Nociceptive behavior was assessed using the electronic pressure meter nociception paw test by an observer blinded to group allocation . AnimalsAnimals were sacrificed under anesthesia, and the synovial cavity of the knee joints was washed with 0.05 ml saline containing 10 mmol/L EDTA. Joint washes were collected by aspiration, and total cell counts were performed using a Neubauer chamber. Differential cell counts were performed using the panoptic Instant Prov™ staining kit (New ProvBrasil™). After centrifuging (500 g/10 min), the supernatants were stored at −80°C until used for measuring the concentrations of interleukin (IL)-1β, IL-6, CCL-2, and CXCL1 using commercially available kits .Knee joint tissues were excised for the histological study. After fixation in 10% v/v formaldehyde solution and decalcification , the whole joint, comprising the distal femoral and proximal tibial extremities, was processed for paraffin-embedding and staining with hematoxylin-eosin (HE) and safranin-O. Analysis was expressed as one result/sample. Semi-quantitative histopathological evaluations were performed by an independent observer (VCCG) blinded to group allocation considering synovial proliferation and cell infiltration and glycosaminglycan content (safranin staining intensity), ranging from 0 to 3 . Results were expressed as the median value for each group of four animals.p < 0.05 was considered as significant.Results were presented as means ± SD for pain behavior and cell counts in joint washings or medians for histology of measurements made on at least three animals in each group. Differences between means and medians were compared using Student’s “t” or Mann–Whitney tests, respectively; Administration of solnatide (10 or 20µg) into the joints of naive mice promoted a mild, acute cell influx, with predominance (>85%) of polymorphonuclear cells, which was similar to that observed in FI animals  administration of AP301 led to a significant decrease in joint edema and of the acute cell influx into joint exudates, with a decrease in polymorphonuclear cell, lymphocyte, and mononuclear cell counts  2 days after intra-articular challenge with mBSA. These mice were sacrificed after 24 h, and the joints were excised for histopathological examination. Results in via the p38 MAP kinase pathway , which encompasses the amino acid sequence of the lectin-like domain of TNF, is a valuable tool to dissect TNF receptor-dependent from TNF receptor-independent effects. Similar to what happens in the above-described arthropathies, activation of TNF RI has been shown to be crucial to TNF effects in immune glomerulonephritis , where i pathway , 30–32.  pathway . Similar pathway .Our present data for the first time demonstrate that solnatide significantly reduces inflammatory cell infiltration and joint damage in an immune-mediated (mBSA) experimental arthritis model in mice. The effect of solnatide was observed using both prophylactic and therapeutic strategies and following local (intra-articular) and systemic (intravenous) administration. In keeping with data from other studies, solnatide actions were also associated with a reduction in pro-inflammatory cytokine (IL-1β and IL-6) and chemokine (CCL2 and CXCL-1) levels in joint exudates.via resident cells inside the joint. In addition to a rich blood supply, with fenestrated capillaries, the synovial tissue harbors type I, fibroblast-like, and type II, macrophage-like synoviocytes  , 21, 22.viocytes . Althougviocytes , 25, areviocytes , 36. Couviocytes , it is lviocytes  and the viocytes  led us tIn summary, these data show that solnatide, a synthetic peptide mimicking the lectin-binding domain of TNF, reduces inflammation in an immune-mediated arthritis model. The anti-inflammatory activity of solnatide is associated with the reduction in joint damage. Since local therapeutic applications of solnatide have already been carried out in humans and have shown to dampen lung inflammation, there is a strong rationale to use solnatide as local treatment in inflammatory arthritis to achieve resolution of arthritis.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The animal study was reviewed and approved by Brazilian Council on Animal Experimentation (CONCEA).FR, FA and GS contributed to the conception of the protocol. FR, RL, AP, IN, VG-C and VO performed animal studies, including histology reading (VG-C). FR, FA and VO performed cytokine measurements. FR, FA, RL, BF and GS wrote the manuscript. All authors contributed to the article and approved the submitted version.RL received funding from NIH/NHLBI grant R01 HL138410A; FAA received funding from Fundação de Amparo à Pesquisa de Minas Gerais .We thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ)—Brazil for providing partial support for Prof. Rocha. We think Apeptico™ for kindly providing solnatide. We acknowledge partial support of this work from CNPq (CNPQ grants 313860/2021-1 and 403767/2021-0).We received the peptide solnatide from Apeptico as a kind donation but all experiments were run in Brazil Fortaleza and Belo Horizonte and Germany Erlangen. Author BF is an employee of APEPTICO Forschung und Entwicklung. Author RL is an inventor of patents with solnatide.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Obstructive sleep apnea (OSA) and inflammation are closely related. This study aimed to evaluate the associations and causal effect between C-reactive protein (CRP) and tumour necrosis factor-alpha (TNF-α) levels and OSA.Pooled analysis was conducted to compare the expression differences of CRP and TNF-α between OSA patients with different severity and controls, and between continuous positive airway pressure (CPAP) and non-CPAP interventions for OSA patients. Using published GWAS summary statistics, we conducted a bidirectional two-sample Mendelian Randomization (MR) to estimate the causal relationships between CRP and TNF-α levels and OSA risk. Effect estimates were evaluated using inverse-variance weighted (IVW) as primary method, and several other MR methods as sensitivity analysis.p < .00001) and CRP , showed a significant increase in OSA patients compared with controls and this increasing trend was associated with OSA severity. Besides, compared to blank control (non-CPAP), CPAP treatment can reduce high TNF-α  and CRP  in OSA. Moreover, the primary MR analysis by IVW showed that OSA was the genetically predicted cause of elevated CRP  using six SNPs as the instrument variable, which were repeated by weighted median  and MR RAPS . Besides, the causal effect from elevated CRP on increased OSA risk was almost significant by IVW . However, there were no causal associations between TNF-α and OSA from both directions.Both TNF-α (WMD [95%CI] = 5.86 [4.80–6.93] pg/ml, Increased CRP and TNF-α were associated with OSA severity and sensible to CPAP treatment. Also, OSA had a suggestive causal effect on elevated CRP. Obstructive sleep apnea (OSA) is a widespread sleep disorder that affects about 10–25% of the global population . During Common biomarkers serving as indicators of systemic inflammation include CRP and TNF-αThe majority of previous studies on OSA have only shown an inflammatory profile in patients with OSA; it is unclear whether the altered inflammatory profile occurs before or after the onset of OSA, or both. Mendelian Randomization (MR) is a reliable genetic epidemiology method that uses genetic variants as instrumental variable (IV) to assure whether causality exists between OSA and inflammation . BecauseIn this study, we first explored the association between the levels of CRP, TNF-α and OSA severity, and whether this aberration can be reduced by CPAP treatment. Then, we applied a bidirectional MR analysis to estimate the causal relationships between these two cytokines and OSA risk.Supplementary Methods.To compare the expression differences of TNF-α and CRP between OSA patients with different severity and controls, and between CPAP treatment and non-CPAP intervention for OSA patients, studies were identified from published literature. Detailed descriptions of searching strategy, inclusive and exclusive criteria and data extraction were presented in 2 statistic to estimate heterogeneity. If I2 > 50%, the data were pooled by random effect model, otherwise by fixed effect model. We also performed a sensitivity analysis by removing article one by one to see its effect on the p value. Moreover, a funnel diagram was conducted to evaluate publication biasAll data were analysed in the Review manager 5.3 . The measurement units of CRP and TNF-α were standardised. For CRP, mg/dl was converted to mg/l, TNF-α was pg/ml. As for continuous outcomes, the weighted mean differences (WMD) and 95% confidence interval (CI) were used as measures of the effect between the two groups in this study. We calculate an IA two-sample MR model was used to evaluate the causal effect between OSA and protein levels of CRP and TNF-α respectively. The GWAS summary statistics for OSA were from a recent publication using FinnGen Study which contained 217,955 individuals with 16,761 OSA patients from European . In thisp < 5 × 10−8 [p < 5 × 10−6 [2=0.1 to include independently associated variants as IV. After harmonising with responsive outcome, each pair was obtained for subsequent analysis. Besides, F-statistic was used to assess the strength of the association between SNP and exposure, where score > 10 indicates that the instrument is sufficiently strong [There are three assumptions for instrumental variable (IV) selection in two-sample MR analysis: (1) the selected variants are associated with exposure; (2) the IV variants are not associated with the confounding factors of exposure and outcome; (3) the effect from IV variants to outcome was exclusively through exposure . In deta5 × 10−8 . Because5 × 10−6 . Then, cy strong .We used several two-sample MR approaches to evaluate the causal effect between OSA and CRP, also OSA and TNF-α. First, we select SNPs of OSA as exposure to evaluate the causal effect of OSA on CRP and TNF-α. Then, the SNPs of OSA were performed as the outcome to assess the causal effect of CRP and TNF-α on OSA.We used inverse-variance weighted (IVW) as the main outcome which assumes that all SNPs are valid instruments . In sensTo compare the association between TNF-α and CRP levels and OSA patients, a total of 104 studies were included in the pooled qualitative analysis , and theSupplemental Tables 1–4). We found both TNF-α  and CRP  showed a significant increase in OSA patients compared with controls . Besides, there were increasing trends with statistical significance from mild to moderate and then to severe for TNF-α and CRP . Sensitivity analysis suggested significantly higher trends of TNF-α and CRP in OSA. The funnel plot results indicated no apparent publication bias .Firstly, we compared the level of TNF-α between 1981 OSA patients and 1112 controls in 34 studies and the level of CRP between 4285 OSA patients and 3692 controls in 64 studies . First, we analysed there were no statistical differences at baselines between the two groups for both TNF-α  and CRP . After CPAP intervention, compared with non-CPAP group, TNF-α was decreased significantly , while CRP was decreased slightly  . Sensitivity analysis suggested that the trend of reduction in CRP and TNF-α remain unchanged. The funnel plot results indicated no apparent publication bias .Next, we compared the changes in TNF-α of four studies and CRP of eight studies in OSA participants between the CPAP group and the non-CPAP group . Similar results were observed using Weighted median  and MR RAPS  except for MR-Egger . There was no evidence of pleiotropy  0.0196, p = .599). However, there was high heterogeneity . After removal the identified outliers of SNPs, there was no heterogeneity  and the results were still significant with similar trends as before . MR RAPS instead of MR-Egger and Weighted median was positive   . As for  = .025) .Cumulative evidence supports that systematic inflammation may play a critical role in OSA. However, the relationship between the changes in inflammatory proteins and OSA is not completely clear. Through comparisons of protein differences and bidirectional causality analysis, the main findings of this study were that patients with OSA tended to have increased CRP and TNF-α levels, and elevated CRP and TNF-α were positively associated with OSA severity. Besides, effective CPAP intervention can reduce abnormal TNF-α in OSA, but this effect on CRP was mild. Moreover, OSA was found to be a suggestive causality for elevated CRP by MR analysis. And the reverse causal effect of CRP on OSA may not be excluded. But there was no causality between OSA and TNF-α.As expected, the levels of CRP and TNF-α were higher in OSA and positively associated with OSA severity. Both cellular experiments and histological biopsy suggested that OSA led to vascular inflammation with increasing CRP and TNF-α ,32. Cons5. Recent research reported that OSA caused significant alteration in serum extracellular microvesicles protein composition including CRP which may participate in OSA-related injury [In this study, we used MR to overcome the influence of unmeasured confounders from observational study, and we found the suggestive causality from OSA on CRP through several complementary MR methods. There are several pathophysiologic mechanisms implying such causal relationship. Recurrent closure and narrowing of the upper airway can result in inflammation of mucosa ,41. And d injury . Interesd injury , whereasd injury . These rd injury ,47.p = .053) suggested we cannot exclude the possibility that CRP may lead to OSA. Consistently, a current longitudinal study reported that CRP was prospectively associated with increased OSA risk [In our reverse direction about causality from CRP to OSA, the almost significant association . Second, the diagnosis of OSA GWAS was from medical electronic records, which may have selection bias and impaired MR power. Because only people with obvious clinical symptoms will go to the hospital, and those with subclinical symptoms may be mistakenly included in the control group. Third, the sample size of TNF-α GWAS was relatively small which may be a reason why we did not find a causal relationship between TNF-α and OSA.In conclusion, our results confirmed that both CRP and TNF-α were increased in OSA and these increasing trends were severity-dependent. Though gently, elevated CRP and TNF-α can be reduced by effective CPAP treatment. In addition, MR analysis revealed suggestive causal direction from OSA to elevated CRP but not TNF-α. Further researches are needed to verify these findings.Click here for additional data file.


Globally, heart failure with preserved ejection fraction (HFpEF) is quickly becoming the dominant form of heart failure (HF) in ageing populations. However, there are still multiple gaps and challenges in making a firm diagnosis of HFpEF in many low-to-middle income Asian countries. In response to this unmet need, the Malaysian HFpEF Working Group (MY-HPWG) gathered and reviewed evidence surrounding the use of different diagnostic modalities indicated for patients with HFpEF to identify diagnostic tools that could be conveniently accessed across different healthcare settings. As a result, five recommendation statements were proposed and an accompanying algorithm was developed, with the aim of improving the diagnostic rate of HFpEF. The MY-HPWG recommends using more easily accessible and non-invasive tools, such as natriuretic peptide (NP) biomarkers and basic echocardiogram (ECHO), to ensure timely HFpEF diagnosis in the primary and secondary care settings, and prompt referral to a tertiary care centre for more comprehensive assessments in uncertain cases. Heart failure (HF) is a debilitating syndrome that causes reduced functional capacity and quality of life, disability, recurrent hospitalisation and premature mortality; notably, patients with HF often need increasingly intensive care with the deterioration of their condition , 2. It hHeart failure with preserved ejection fraction (HFpEF), a subset of HF, is quickly becoming the dominant form of HF in ageing populations worldwide . AccordiAt present, there are still multiple gaps and challenges in the management of patients with HFpEF in many low-to-middle income Asian countries, including Malaysia , 9, 10. In response to this unmet need, the Malaysia HFpEF Working Group (MY-HPWG) was convened to collate and review evidence surrounding the use of different diagnostic modalities indicated for patients with HF, with the aim of identifying diagnostic tools that could be conveniently accessed across different healthcare settings. To improve the diagnostic rate of patients at risk of HFpEF who are managed by primary care providers (PCPs), the MY-HPWG proposed five recommendation statements to facilitate the early diagnosis of patients seen at primary and secondary healthcare facilities, and to encourage early referral of patients suspected of HFpEF to tertiary care. An accompanying algorithm was also developed to succinctly present the key message of the recommendations . The recThe MY-HPWG, formed to promote quality management of HFpEF in Malaysia, comprises nine cardiologists and one internal medicine specialist; all of whom have special interest in HF. Additionally, among the cardiologists, two of them have special interest in interventional cardiology, one in heart transplant, one in imaging and one in device therapy. These experts from public, private and university hospitals in Malaysia, were tasked with reviewing current literature on the management of HFpEF (i.e. focusing on the diagnosis of the condition) and synthesising a set of recommendation statements based on collated evidence to guide the diagnosis and referral of patients with HFpEF seen at different healthcare levels in the country.https://pubmed.ncbi.nlm.nih.gov/) and Google Scholar (https://scholar.google.com/) to appraise the value of different diagnostic modalities in diagnosing HFpEF. Relevant studies published between 2004 and 2021 were identified using these search terms: HF, HFpEF, diagnosis, natriuretic peptide (NP), echocardiogram (ECHO) and cardiac function. The search results were then screened for relevance based on the following criteria: i) evidence-based diagnosis of HF/HFpEF; ii) availability of systematic review, meta-analysis or randomised controlled trial and iii) studies of Asian or Malaysian populations.The MY-HPWG performed a literature review using PubMed  enrolled in the ASIAN-HF registry showed that 70% of patients with HFpEF had a least two comorbidities, most commonly hypertension, followed by anaemia, CKD, diabetes mellitus (DM), coronary artery disease (CAD), atrial fibrillation (AF) and obesity  and comorbid illnesses  19–21).–21.19–21 obesity .N = 581), approximately 32% of patients were found to have previously unknown HF; up to 88% of these patients had HFpEF, while the rest had HF with reduced ejection fraction (HFrEF) (In a Dutch study aimed at screening older patients aged > 60 years old at risk of HF due to type 2 DM ( (HFrEF) . TherefoPatients with HF often present first in primary care. A population-based study conducted in Canada revealed that more patients with HF were diagnosed in outpatient than in a hospital setting (45.7% versus 36.6%) over a period of 9 years , highligPatients suspected of having HF should undergo a standard set of assessments, comprising chest X-ray (CXR), electrocardiogram (ECG) and laboratory tests, to establish a provisional diagnosis of HF.The scope of initial diagnostic work-up should consider the availability of resources in different healthcare settings . Access Patients suspected of HF should proceed to additional investigations to confirm their diagnosis with NP measurement and ECHO examination.The use of blood test to measure plasma levels of NPs that is simple and easy to interpret has tremendous clinical significance in many clinical settings , 33, espOwing to the high negative predictive value (NPV) but lower positive predictive values in both acute and non-acute settings, the use of NPs is recommended to exclude HF diagnosis, but not to confirm the presence of HF . The 202Patients with normal plasma NP concentrations are unlikely to have HF; in contrast, higher plasma NP concentration increases the likelihood that dyspnoea is due to HF . NonetheSeveral studies have shown that NP levels are affected by several factors, such as age , obesityThe presence of AF impairs the diagnostic performance of NPs. It is well established that AF is associated with higher concentrations of NPs; these levels may exceed the threshold for ‘HF’ even in the absence of further clinical support for HF diagnosis . The ana2  is inversely correlated with NT-proBNP level ; therefo2 . Neverth2 .Of note, a cross-sectional analysis of 633 patients with HF revealed that the BNP level was lower for HFpEF than for HFrEF (93 pg/mL versus 266 pg/mL) . CompareThe introduction of point-of-care testing (POCT) devices allows for rapid and robust detection of plasma NP concentration . ConsequECHO is the most useful tool to establish the diagnosis of HF. An ECHO machine can be used to assess at least the left ventricular ejection fraction (LVEF) of patients suspected of HF unless all risk factors  are abseWhile LVEF measurement may be influenced by several factors  , 46, it N = 203,135 patients) showed that deviation of LVEF below 60%–65% was associated with poorer survival irrespective of age, sex or presence of other relevant comorbidities, such as HF; importantly, similar trends were observed for HF patients in both the inpatient and outpatient settings  reported that an LVEF of 60%–64.9% was associated with greater risk of cardiovascular-related mortality in women than men   . The Ame< 0.001) .In the ED, focused cardiac ultrasound (FOCUS) has become an indispensable tool to facilitate the diagnostic evaluation of patients suspected of HF. The ASE and American College of Emergency Physicians support the use of FOCUS to primarily assess pericardial effusion, right ventricular enlargement, global cardiac systolic function and volume status . A recenTherefore, FOCUS performed in the ED setting could serve as an objective, rapid and non-invasive tool in assessing patients suspected of HF, especially when cardiologists and ECHO are not readily available.Patients with uncertain HFpEF diagnosis should be referred to tertiary care for standard/comprehensive echocardiography and/or further investigations.As specified in the 2021 ESC guideline, the criteria for HFpEF diagnosis include the presence of typical signs and symptoms, a preserved EF (LVEF ≥ 50%), elevated levels of BNP (≥ 35 pg/mL) and/or NT-proBNP (≥ 125 pg/mL), as well as objective evidence of cardiac functional and structural alterations which are consistent with HF . HoweverAt the tertiary care level, a standard ECHO to assess changes in the left ventricular (LV)/left atrial (LA) size or volume and diastolic function is recommended for patients with uncertain HFpEF diagnosis . A diagnThe importance of determining a patient’s diastolic function is highlighted in a retrospective analysis of patients with HFpEF . The stuIn some cases, a standard ECHO may still give inconclusive results and more detailed echocardiographic measurements are required . A compr2 is one of the criteria to diagnose HFpEF —maximal volume of the LA, measured at end-systole from biplane or three-dimensional images, indexed to body surface area (BSA)—should be determined as it is indirectly correlated to LV filling pressure; a marked feature of HFpEF . It is ase HFpEF .2 for men and > 95 g/m2 for women indicate HFpEF, while the ESC categorised LV changes into four different groups (N = 745) had either LVH or LV concentric remodelling when LVMI and RWT were measured  and left ventricular mass index (LVMI) normalised to a patient’s BSA or height. According to the Malaysian CPG , an LVMIt groups  14, 52), 522 formeasured .Elevation of filling pressure is a hallmark feature of HFpEF and could lead to fluid retention and an expanded plasma volume , 63, 64.Additional diagnostic investigations such as a stress ECHO may be considered if a diagnosis is still not established , 66. TheP < 0.0001)  points to a diagnosis of HFpEF , 61, 63. 0.0001) .Information about filling pressure also helps to ascertain the severity of the disease and subsequently, the treatment response. Although cardiac catheterisation remains the gold standard to determine filling pressure owing to its high accuracy that supports early patient diagnosis, it is impractical for all patients presenting with dyspnoea and suspicion of HF to undergo such an invasive procedure , 61, 63.HFpEF is a heterogeneous condition with various underlying aetiologies and pathophysiological abnormalities—both cardiovascular and non-cardiovascular. Identifying the final aetiology of HFpEF should be part of the diagnostic workup as it allows for the establishment of a possible specific, secondary cause of HFpEF or alternative explanations , 56.Aetiological workup may include a standard exercise stress test that could detect myocardial ischaemia, an abnormal blood pressure response to exercise, chronotropic incompetence, or supraventricular and ventricular arrhythmias. The detection of these conditions could influence management strategies . Other mPatients with HFpEF and multiple comorbidities, non-responders to treatment, or those in need of specialised/multidisciplinary care should be referred to tertiary care for optimal management.An assessment by a specialist is often recommended to establish a HF diagnosis. In the real world, a large population-based cohort study found that patients with HFpEF were less likely to have a cardiologist as their primary physician and were also less likely to have had a cardiology consultation compared with patients with HFrEF even though both groups had similar rates of in-hospital complications .Most published guidelines, including those from American Heart Association, ESC and National Institute for Health and Care Excellence, state that the diagnosis of HF should be timely and accurate –13; adheThe involvement of a cardiologist to confirm a HFpEF diagnosis is usually necessary owing to the need for a standard or comprehensive ECHO , 76. As Patients with HFpEF often have multiple comorbidities which could affect a patient’s prognosis. Thus, the optimal management of comorbidities in HFpEF plays a key role in determining patients’ outcome. A systematic review of 29 randomised trials that investigated strategies to improve patient outcome found that specialised follow-up provided by multidisciplinary healthcare teams significantly reduced mortality rate  in patients with HF compared with regular telephone contact and follow-up by primary care practitioners alone  . TherefoThere is an increasing awareness that HFpEF is a heterogenous disease with various phenotypes and comorbidities, making it difficult to establish a diagnosis based on the measurement of LVEF alone. As such, individually tailored approaches to disease diagnosis should be actively considered. The MY-HPWG recommends using more easily accessible and non-invasive tools, such as NP biomarkers and basic ECHO, to ensure timely HFpEF diagnosis in the primary and secondary care settings. Additionally, patients with uncertain diagnosis should be promptly referred to a tertiary care centre to undergo more comprehensive assessments and receive appropriate care.


The designer nucleases, including Zinc Finger Nuclease (ZFN), Transcription Activator-Like Effector Nuclease (TALEN), and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas), have been widely used for mechanistic studies, animal model generation, and gene therapy development. Clinical trials using designer nucleases to treat genetic diseases or cancers are showing promising results. Despite rapid progress, potential off-targets and host immune responses are challenges to be addressed for in vivo uses, especially in clinical applications. Short-term expression of the designer nucleases is necessary to reduce both risks. Currently, delivery methods enabling transient expression of designer nucleases are being pursued. Among these, virus-like particles as delivery vehicles for short-term designer nuclease expression have received much attention. This review will summarize recent developments in using virus-like particles (VLPs) for safe delivery of gene editing effectors to complement our last review on the same topic. First, we introduce some background information on how VLPs can be used for safe and efficient CRISPR/Cas9 delivery. Then, we summarize recently developed virus-like particles as genome editing vehicles. Finally, we discuss applications and future directions. Designer endonucleases enable scientists to edit target genomes and achieve gene knockout and DNA addition with unprecedented precision . Early gvirus-like particles (VLPs) for delivering mRNA, protein, or ribonucleoprotein of designer endonucleases to improve genome editing safety in mammalian cells and animals. Since our last review on VLPs as delivery tools for genome editing . Ap. ApPseudnthetase  and mammnthetase  for RNA Aptamer BoxB and ABP λ peptide N22 were found in bacteriophage λ . Peptidemom gene. Binding Com protein to the com sequence stimulates the translation of mom gene [The RNA aptamer com and its interacting protein Com were found in the bacteriophage Mu  68]. Th. Th68]. mom gene . The commom gene  and DNA mom gene .Since our last review of various types of VLPs developed for genome editing delivery , many neBefore being developed as genome editing delivery tools, VLPs were used for protein delivery for the purposes of viral-particle tracking and cell elimination ,70,71. TRecently, VLPs were harnessed to deliver Cas9 RNPs and DNA templates for both targeted genetic disruption and stable gene addition . In thisSec gene in multiple cell types [Recently, there are also new applications of VLPs originally reported by Mangeot et al. . Vindry ll types . They obll types  with babll types . Thus, wPcsk9 in mouse liver and resulted in a 78% reduction in serum Pcsk9 protein levels [Base editors (BEs), which mediate targeted single-nucleotide conversions, can minimize the risks of generating double-strand breaks compared with artificial endonucleases. They were commonly delivered by plasmid DNA, AAV (needs to be split into two AAV vectors due to the large size of adenine base editors), and LV. Previously Lyu et al. developed VLPs to deliver an adenine base editor (ABE), using the interactions between aptamer and ABP . They fon levels .Previously, Lyu et al. and Lu et al. used the specific interactions between aptamers and ABPs to package and deliver Cas9 and ABE RNPs by VLPs ,78,80. RIn the past, VLPs for mRNA delivery were developed from viral capsids ,83,84,85Several studies have found that sgRNA packaged alone could not be functionally delivered by VLPs ,15,63,84Two different strategies were used to develop VLPs for co-delivering Cas9 mRNA and sgRNA . In the + stem and progenitor cells [Knopp et al. originally used VLPs developed from gammaretroviral murine leukemia virus to package Cas9 mRNA or co-package Cas9 mRNA and sgRNA . Recentlor cells ,15.The RNA-delivery VLP system originally reported by Prel et al.  was alsoIn the studies described above using aptamer/ABP interactions to co-deliver Cas9 mRNA and sgRNA ,15,16, dVegfa) to suppress aberrant development of blood vessels in a mouse model of wet age-related macular degeneration [Two groups used a different strategy to co-deliver Cas9 mRNA and sgRNA by VLPs ,7,13 Fi. Ling etneration . In anotneration ,7.Yadav et al. used a similar strategy to co-deliver Cas9 mRNA and sgRNA  but a diVLPs as delivery vehicles for genome editing are still in preclinical development. However, successful applications have been reported in clinically relevant primary cells and disease animal models. Below, we summarize these applications.Myd88 gene [+ hematopoietic stem and progenitor cells; they showed little effects on cell viability or differentiation [+ T cells [Currently, genome editing in human blood cells relies on electroporation of Cas9 RNPs into them, which may affect cell viability. VLPs can deliver Cas9 RNPs into human hematopoietic stem cells to disrupt the d88 gene , whose m T cells .B2M or TRAC) disruption [+ T cells compared to CD8+ T cells (53.20% versus 2.51% gene disruption efficiency).Currently, generating CAR-expressing T cells with gene disruption depends on transduction of retroviral vectors and electroporation of Cas9 RNPs. Hybrid VLPs containing Cas9 RNPs and CAR-expressing lentiviral genomic RNAs have been used for simultaneous CAR insertion and gene (for example sruption . PseudotTyr) gene, and insertions and deletions were observed in 16 of 40 blastocysts injected [loxp-targeting Cas9 RNPs into the perivitelline space of R26R-EYFP embryos, and successfully removed the “lox-stop-lox” cassette to enable EYFP expression in mice. Finally, they delivered VLPs with RNPs targeting the hydroxyphenylpyruvate dioxygenase (Hpd) gene into Fah-/- mice via retro-orbital injection and obtained gene disruption in 7% to 13% of alleles [VLPs have been used to deliver Cas9 RNPs into mouse zygotes to disrupt the tyrosinase  need sgRNAs to be provided separately. The number of effectors packaged in RNP VLPs is pre-determined and controllable, whereas the number of effectors expressed from RNA VLPs may reach a much higher level during a short period of time. This high level of effectors is not desirable when off-targets are concerned, but it could be desirable when CRISPR/Cas9 cleavage is inhibited by heterochromatin ,88,89 anRNPs are packaged into capsids via two different mechanisms: the fusion mechanism and the aptamer/ABP interaction mechanism. With the fusion mechanism, fusing a large protein to Gag may impair capsid assembly efficiency and result in effector degradation by proteinase. With the aptamer/ABP interaction mechanism, inserting a small ABP into the NC protein of Gag has little effect on capsid assembly, whereas adding aptamers in sgRNA may impair genome editing activity with some target sequences. In addition, the release of RNPs from the ABPs cannot be controlled. It is hard to simply claim that one method is superior to another. The users have to decide which method best meets their specific needs.Although VLPs have not been tested in clinical trials for genome editing, they have been successfully used in vitro in clinically relevant human cells and in vivo in disease mouse models. We hope that in the near future VLPs are tested as delivery vehicles for genome editing in clinical applications to benefit patients.One common issue in using VLP as a genome editing delivery vehicle is the dependence of VSV-G or other viral envelope proteins to facilitate cell entry and endosomal escape. VSV-G and other viral envelopes could be toxic at high concentrations . In addiFinally, we would like to take this opportunity to encourage authors to try their best to include “virus-like particles (VLPs)” as one of the keywords while reporting related findings. Currently over twenty types of VLPs have been developed as genome editing delivery vehicles see . Many au