Tirofiban for Stroke without Large or Medium-Sized Vessel Occlusion.

BACKGROUND: The effects of the glycoprotein IIb/IIIa receptor inhibitor tirofiban in patients with acute ischemic stroke but who have no evidence of complete occlusion of large or medium-sized vessels have not been extensively studied. METHODS: In a multicenter trial in China, we enrolled patients with ischemic stroke without occlusion of large or medium-sized vessels and with a National Institutes of Health Stroke Scale score of 5 or more and at least one moderately to severely weak limb. Eligible patients had any of four clinical presentations: ineligible for thrombolysis or thrombectomy and within 24 hours after the patient was last known to be well; progression of stroke symptoms 24 to 96 hours after onset; early neurologic deterioration after thrombolysis; or thrombolysis with no improvement at 4 to 24 hours. Patients were assigned to receive intravenous tirofiban (plus oral placebo) or oral aspirin (100 mg per day, plus intravenous placebo) for 2 days; all patients then received oral aspirin until day 90. The primary efficacy end point was an excellent outcome, defined as a score of 0 or 1 on the modified Rankin scale (range, 0 [no symptoms] to 6 [death]) at 90 days. Secondary end points included functional independence at 90 days and a quality-of-life score. The primary safety end points were death and symptomatic intracranial hemorrhage. RESULTS: A total of 606 patients were assigned to the tirofiban group and 571 to the aspirin group. Most patients had small infarctions that were presumed to be atherosclerotic. The percentage of patients with a score of 0 or 1 on the modified Rankin scale at 90 days was 29.1% with tirofiban and 22.2% with aspirin (adjusted risk ratio, 1.26; 95% confidence interval, 1.04 to 1.53, P = 0.02). Results for secondary end points were generally not consistent with the results of the primary analysis. Mortality was similar in the two groups. The incidence of symptomatic intracranial hemorrhage was 1.0% in the tirofiban group and 0% in the aspirin group. CONCLUSIONS: In this trial involving heterogeneous groups of patients with stroke of recent onset or progression of stroke symptoms and nonoccluded large and medium-sized cerebral vessels, intravenous tirofiban was associated with a greater likelihood of an excellent outcome than low-dose aspirin. Incidences of intracranial hemorrhages were low but slightly higher with tirofiban. (Funded by the National Natural Science Foundation of China; RESCUE BT2 Chinese Clinical Trial Registry number, ChiCTR2000029502.).

Deficiency of FRMD5 results in neurodevelopmental dysfunction and autistic-like behavior in mice.

The pathophysiology of autism spectrum disorders (ASDs) is causally linked to postsynaptic scaffolding proteins, as evidenced by numerous large-scale genomic studies [1, 2] and in vitro and in vivo neurobiological studies of mutations in animal models [3, 4]. However, due to the distinct phenotypic and genetic heterogeneity observed in ASD patients, individual mutation genes account for only a small proportion (<2%) of cases [1, 5]. Recently, a human genetic study revealed a correlation between de novo variants in FERM domain-containing-5 (FRMD5) and neurodevelopmental abnormalities [6]. In this study, we demonstrate that deficiency of the scaffolding protein FRMD5 leads to neurodevelopmental dysfunction and ASD-like behavior in mice. FRMD5 deficiency results in morphological abnormalities in neurons and synaptic dysfunction in mice. Frmd5-deficient mice display learning and memory dysfunction, impaired social function, and increased repetitive stereotyped behavior. Mechanistically, tandem mass tag (TMT)-labeled quantitative proteomics revealed that FRMD5 deletion affects the distribution of synaptic proteins involved in the pathological process of ASD. Collectively, our findings delineate the critical role of FRMD5 in neurodevelopment and ASD pathophysiology, suggesting potential therapeutic implications for the treatment of ASD.

A Therapeutic Vaccine in Combination with Cyclic GMP-AMP Cures More Differentiated Melanomas in Mice.

We have identified a combinational immunotherapy termed TheraVac vaccine (TheraVac) that can cure multiple large established mouse tumors, but it failed to cure melanoma in mice. TheraVac consists of an immunostimulating arm containing an agonist (HMGN1 [N1]) for TLR4 and an agonist (R848) for TLR7/8 that synergize to activate tumor-infiltrating dendritic cells (DCs) and promote Th1 immune responses. The second arm uses an immune checkpoint blockade, anti-PDL-1, to diminish tumor-associated immunosuppression. In this study, we investigated supplementation of TheraVac by a stimulator of IFN genes (STING) agonist, cyclic GMP-AMP (cGAMP), because together they synergize in activating DCs and produced more immunostimulating IL-12p70 and TNF-α cytokines. The synergistic activation and maturation of DCs is dependent on the activation of tank binding kinase-1 (TBK1). Treatment of three different melanin-producing mouse melanomas (B16F1, M3, and M4) with intratumoral delivery of cGAMP and TheraVac eradicated 60-80% of these melanomas. Immunoprofiling of M3 tumor treated with TheraVac plus cGAMP showed an increase in CD8+ CTLs and macrophages in the tumor. There was also a marked increase of CD4, CD8 effector and memory T cells and generation of functional tumor-specific CTLs in tumor-draining lymph nodes. The resultant tumor-free mice were selectively resistant to subsequent challenge with the same tumors, indicating long-term tumor-specific protective immunity. Overall, our findings have important implications for clinical trials with a combination of these immunotherapeutics to cure melanin-producing human melanomas, without the need for exogenous tumor Ags and no clear toxic effects in mice.

Genome-Wide Identification of Gene Loss Events Suggests Loss Relics as a Potential Source of Functional lncRNAs in Humans.

Gene loss is a prevalent source of genetic variation in genome evolution. Calling loss events effectively and efficiently is a critical step for systematically characterizing their functional and phylogenetic profiles genome wide. Here, we developed a novel pipeline integrating orthologous inference and genome alignment. Interestingly, we identified 33 gene loss events that give rise to evolutionarily novel long noncoding RNAs (lncRNAs) that show distinct expression features and could be associated with various functions related to growth, development, immunity, and reproduction, suggesting loss relics as a potential source of functional lncRNAs in humans. Our data also demonstrated that the rates of protein gene loss are variable among different lineages with distinct functional biases.

Quantitative model suggests both intrinsic and contextual features contribute to the transcript coding ability determination in cells.

Gene transcription and protein translation are two key steps of the 'central dogma.' It is still a major challenge to quantitatively deconvolute factors contributing to the coding ability of transcripts in mammals. Here, we propose ribosome calculator (RiboCalc) for quantitatively modeling the coding ability of RNAs in human genome. In addition to effectively predicting the experimentally confirmed coding abundance via sequence and transcription features with high accuracy, RiboCalc provides interpretable parameters with biological information. Large-scale analysis further revealed a number of transcripts with a variety of coding ability for distinct types of cells (i.e. context-dependent coding transcripts), suggesting that, contrary to conventional wisdom, a transcript's coding ability should be modeled as a continuous spectrum with a context-dependent nature.

Identification and Characterization of Pectate Lyase as a Novel Allergen in Artemisia sieversiana Pollen.

INTRODUCTION: Artemisia species are widely spread in north hemisphere. Artemisia sieversiana pollen is one of the common pollen allergens in the north of China. At present, seven allergens were identified and had been listed officially from A. sieversiana pollen, but the remaining allergens are still insufficiently studied, which need to be found. METHODS: Pectate lyase was purified from the extracts of A. sieversiana pollen by anion exchange, size exclusion, and HPLC-hydrophobic interaction chromatography. The gene of A. sieversiana pectate lyase (Art si pectate lyase) was cloned and expressed in Escherichia coli. The enzyme activity and circular dichroism (CD) spectrum of natural and recombinant proteins were analyzed. The allergenicity of Art si pectate lyase was characterized by enzyme-linked immunosorbent assay (ELISA), Western blot, inhibition ELISA, and basophil activation test. The allergen's physicochemical properties, three-dimensional structure, sequence profiles with homologous allergens and phylogenetic tree were analyzed by in silico methods. RESULTS: Natural Art si pectate lyase (nArt si pectate lyase) was purified from A. sieversiana pollen extracts by three chromatographic strategies. The cDNA sequence of Art si pectate lyase had a 1191-bp open reading frame encoding 396 amino acids. Both natural and recombinant pectate lyase (rArt si pectate lyase) exhibited similar CD spectrum, and nArt si pectate lyase had higher enzymatic activity. Moreover, the specific immunoglobulin E (IgE) binding rate against nArt si pectate lyase and rArt si pectate lyase was determined as 40% (6/15) in patients' serum with Artemisia species pollen allergy by ELISA. The nArt si pectate lyase and rArt si pectate lyase could inhibit 76.11% and 47.26% of IgE binding activities to the pollen extracts, respectively. Art si pectate lyase was also confirmed to activate patients' basophils. Its structure contains a predominant motif of classic parallel helical core, consisting of three parallel ß-sheets, and two highly conserved features (vWiDH, RxPxxR) which may contribute to pectate lyase activity. Moreover, Art si pectate lyase shared the highest sequence identity of 73.0% with Art v 6 among currently recognized pectate lyase allergen, both were clustered into the same branch in the phylogenetic tree. CONCLUSION: In this study, pectate lyase was identified and comprehensively characterized as a novel allergen in A. sieversiana pollen. The findings enriched the allergen information for this pollen and promoted the development of component-resolved diagnosis and molecular therapy of A. sieversiana pollen allergy.

Combinational delivery of TLR4 and TLR7/8 agonist enhanced the therapeutic efficacy of immune checkpoint inhibitors to colon tumor.

Immunotherapy is regarded as a potent cancer treatment, with DC vaccines playing a crucial role. Although clinical trials have demonstrated the safety and efficacy of DC vaccines, loading antigens in vitro is challenging, and their therapeutic effects remain unpredictable. Moreover, the diverse subtypes and maturity states of DCs in the body could induce both immune responses and immune tolerance, potentially affecting the vaccine's efficacy. Hence, the optimization of DC vaccines remains imperative. Our study discovered a new therapeutic strategy by using CT26 and MC38 mouse colon cancer models, as well as LLC mouse lung cancer models. The strategy involved the synergistic activation of DCs through intertumoral administration of TLR4 agonist high-mobility group nucleosome binding protein 1 (HMGN1) and TLR7/8 agonist (R848/resiquimod), combined with intraperitoneal administration of TNFR2 immunosuppressant antibody. The experimental results indicated that the combined use of HMGN1, R848, and α-TNFR2 had no effect on LLC cold tumors. However, it was effective in eradicating CT26 and MC38 colon cancer and inducing long-term immune memory. The combination of these three drugs altered the TME and promoted an increase in anti-tumor immune components. This may provide a promising new treatment strategy for colon cancer.

Association of antenatal corticosteroids with mortality and morbidities in very preterm infants born to women with hypertensive disorders of pregnancy: a multicenter prospective cohort study.

BACKGROUND: Hypertensive disorders of pregnancy (HDP) is the most common cause of indicated preterm delivery, but the impact of prenatal steroid exposure on the outcomes of preterm infants born to HDP mothers, who may be at risk for intrauterine hypoxia-ischemia, remains uncertain. The study objective is to evaluate the mortality and morbidities in HDP for very preterm infants (VPIs) exposed to different course of ANS. METHODS: This is a prospective cohort study comprising infants with < 32 weeks gestation born to women with HDP only from 1 Jan. 2019 to 31 Dec. 2021 within 40 participating neonatal intensive care units (NICUs) in Sino-northern network. ANS courses included completed, partial, repeated, and no ANS. Univariate and multivariable analyses were performed on administration of ANS and short-term outcomes before discharge. RESULTS: Among 1917 VPIs born to women with HDP only, 987(51.4%) received a complete course of ANS within 48 h to 7 days before birth, 560(29.2%) received partial ANS within 24 h before delivery, 100(5.2%) received repeat ANS and 270 (14.1%) did not receive any ANS. Compared to infants who received complete ANS, infants unexposed to ANS was associated with higher odds of death (AOR 1.85; 95%CI 1.10, 3.14), Severe Neurological Injury (SNI) or death (AOR 1.68; 95%CI 1.29,3.80) and NEC or death (AOR 1.78; 95%CI 1.55, 2.89), the repeated ANS group exhibits a significant negative correlation with the duration of oxygen therapy days (correlation coefficient - 18.3; 95%CI-39.2, -2.1). However, there were no significant differences observed between the full course and partial course groups in terms of outcomes. We can draw similar conclusions in the non-SGA group, while the differences are not significant in the SGA group. From KM curve, it showed that the repeated group had the highest survival rate, but the statistical analysis did not indicate a significant difference. CONCLUSIONS: Even partial courses of ANS administered within 24 h before delivery proved to be protective against death and other morbidities. The differences mentioned above are more pronounced in the non-SGA group. Repeat courses demonstrate a trend toward protection, but this still needs to be confirmed by larger samples.

Methylprednisolone as Adjunct to Endovascular Thrombectomy for Large-Vessel Occlusion Stroke: The MARVEL Randomized Clinical Trial.

Importance: It is uncertain whether intravenous methylprednisolone improves outcomes for patients with acute ischemic stroke due to large-vessel occlusion (LVO) undergoing endovascular thrombectomy. Objective: To assess the efficacy and adverse events of adjunctive intravenous low-dose methylprednisolone to endovascular thrombectomy for acute ischemic stroke secondary to LVO. Design, Setting, and Participants: This investigator-initiated, randomized, double-blind, placebo-controlled trial was implemented at 82 hospitals in China, enrolling 1680 patients with stroke and proximal intracranial LVO presenting within 24 hours of time last known to be well. Recruitment took place between February 9, 2022, and June 30, 2023, with a final follow-up on September 30, 2023. Interventions: Eligible patients were randomly assigned to intravenous methylprednisolone (n = 839) at 2 mg/kg/d or placebo (n = 841) for 3 days adjunctive to endovascular thrombectomy. Main Outcomes and Measures: The primary efficacy outcome was disability level at 90 days as measured by the overall distribution of the modified Rankin Scale scores (range, 0 [no symptoms] to 6 [death]). The primary safety outcomes included mortality at 90 days and the incidence of symptomatic intracranial hemorrhage within 48 hours. Results: Among 1680 patients randomized (median age, 69 years; 727 female [43.3%]), 1673 (99.6%) completed the trial. The median 90-day modified Rankin Scale score was 3 (IQR, 1-5) in the methylprednisolone group vs 3 (IQR, 1-6) in the placebo group (adjusted generalized odds ratio for a lower level of disability, 1.10 [95% CI, 0.96-1.25]; P = .17). In the methylprednisolone group, there was a lower mortality rate (23.2% vs 28.5%; adjusted risk ratio, 0.84 [95% CI, 0.71-0.98]; P = .03) and a lower rate of symptomatic intracranial hemorrhage (8.6% vs 11.7%; adjusted risk ratio, 0.74 [95% CI, 0.55-0.99]; P = .04) compared with placebo. Conclusions and Relevance: Among patients with acute ischemic stroke due to LVO undergoing endovascular thrombectomy, adjunctive methylprednisolone added to endovascular thrombectomy did not significantly improve the degree of overall disability. Trial Registration: ChiCTR.org.cn Identifier: ChiCTR2100051729.

[Research progress in pharmacological effects of Gypsum Fibrosum and material basis for its heat-clearing effect].

Gypsum Fibrosum, as a classic heat-clearing medicine, is widely used in the clinical practice of traditional Chinese medicine(TCM). However, debates exist about the material basis and mechanism of its efficacy. Therefore, this paper reviewed the recent research progress in the heat-clearing effect and mechanism of Gypsum Fibrosum and discussed the material basis for the heat-clearing effect of this medicine. Ca~(2+) may inhibit the upward movement of temperature set point by regulating the Na~+/Ca~(2+) level in the heat-regulating center. Moreover, trace elements may inhibit the rise of body temperature by regulating the immune system, promoting the absorption of Ca~(2+), and affecting the synthesis of prostaglandin E2(PGE2). This review aims to enrich the knowledge about the mechanism of Gypsum Fibrosum in clearing heat and provides a scientific basis for the clinical application and further development of Gypsum Fibrosum.

Anti-TNFR2 enhanced the antitumor activity of a new HMGN1/3M-052 stimulated dendritic cell vaccine in a mouse model of colon cancer.

Immunotherapy is the new approach for cancer treatment that can be achieved through several strategies, one of which is dendritic cells (DCs) vaccine therapy. However, traditional DC vaccination lacks accurate targeting, so DC vaccine preparation needs to be optimized. Immunosuppressive CD4+Foxp3+ regulatory T cells (Tregs) in the tumor microenvironment can promote tumor immune escape. Therefore, targeting Tregs has become a strategy for tumor immunotherapy. In this study, we found that HMGN1 (N1, a dendritic cell-activating TLR4 agonist) and 3M-052 (a newly synthesized TLR7/8 agonist) synergistically stimulate DCs maturation and increase the production of proinflammatory cytokines TNFα and IL-12. In a colon cancer mice model, vaccination with N1 and 3M-052 stimulated and tumor antigen-loaded DCs combined with anti-TNFR2 inhibited tumor growth in mice, and the antitumor effect was mainly achieved through stimulation of cytotoxic CD8 T cell activation and depletion of Tregs. Overall, the combinating of DC activation by N1 and 3M-052 with inhibition of Tregs by antagonizing TNFR2 as a therapeutic strategy may represent a more effective strategy for cancer treatment.

Development of a novel modified vaccine (TheraVacM) for curative treatment of mouse solid tumors.

Monotherapy with immune checkpoint blockade (ICB) antibodies (anti-CTLA4 and anti-PD1/PDL-1) is only effective for 20% to 30% of patients with certain cancers. Patients with cancers harboring few effector T cells (Teffs) are insensitive to ICB therapy. The lack of tumor-specific Teffs is predominantly caused by the paralysis of tumor-infiltrating dendritic cells (TiDCs) resulting from immunosuppression in the tumor microenvironment. We have identified a potent combination of high mobility group nucleosome binding domain 1 (HMGN1, N1) and fibroblast stimulating lipopeptide-1 (FSL-1) that can synergistically trigger maturation of both mouse and human DCs. Accordingly, we designed a combinational anti-cancer immunotherapy with two arms: an immune-activating arm consisting of N1 and FSL-1 to stimulate the generation of Teffs by triggering full maturation of TiDCs, and an ICB arm using anti-PDL-1 or anti-CTLA4 to prevent Teffs from being silenced in the tumor tissue. This combinational immunotherapeutic vaccination regimen dubbed modified TheraVac (TheraVacM) has proved particularly effective as it cured 100% of mice bearing established ectopic CT26 colon and RENCA kidney tumors. The resultant tumor-free mice were resistant to subsequent re-challenge with the same tumors, indicating the generation of long-term tumor specific protective immunity. Since the immune-activating arm also induces full maturation of human DCs, and anti-PDL-1 or anti-CTLA4 have been FDA-approved, this combinational immunotherapy has the potential to be an effective clinical therapy for patients with solid tumors.

Iron-Catalyzed Synthesis of Peroxylpyrrolo[2,1-a]isoquinolines through Oxidative Dearomatization.

A mild late-stage modification of pyrrolo[2,1-a]isoquinolines was established through iron-catalyzed oxidative dearomatization and peroxidation. Peroxylated pyrroloisoquinolines have been prepared readily with hydroperoxide in low to good yields (up to 72%) at room temperature. Interestingly, the treatment of fully aromatized pyrrolo[1,2-a]quinolines under the current reaction system resulted in the formation of ring-opening products.

Effects of site-directed mutagenesis of GLP-1 and glucagon receptors on signal transduction activated by dual and triple agonists.

The paradigm of one drug against multiple targets, known as unimolecular polypharmacology, offers the potential to improve efficacy while overcoming some adverse events associated with the treatment. This approach is best exemplified by targeting two or three class B1 G protein-coupled receptors, namely, glucagon-like peptide-1 receptor (GLP-1R), glucagon receptor (GCGR) and glucose-dependent insulinotropic polypeptide receptor for treatment of type 2 diabetes and obesity. Some of the dual and triple agonists have already shown initial successes in clinical trials, although the molecular mechanisms underlying their multiplexed pharmacology remain elusive. In this study we employed structure-based site-directed mutagenesis together with pharmacological assays to compare agonist efficacy across two key signaling pathways, cAMP accumulation and ERK1/2 phosphorylation (pERK1/2). Three dual agonists (peptide 15, MEDI0382 and SAR425899) and one triple agonist (peptide 20) were evaluated at GLP-1R and GCGR, relative to the native peptidic ligands (GLP-1 and glucagon). Our results reveal the existence of residue networks crucial for unimolecular agonist-mediated receptor activation and their distinct signaling patterns, which might be useful to the rational design of biased drug leads.

Formyl peptide receptor 2 as a potential therapeutic target for inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a global health burden whose existing treatment is largely dependent on anti-inflammatory agents. Despite showing some therapeutic actions, their clinical efficacy and adverse events are unacceptable. Resolution as an active and orchestrated phase of inflammation involves improper inflammatory response with three key triggers, specialized pro-resolving mediators (SPMs), neutrophils and phagocyte efferocytosis. The formyl peptide receptor 2 (FPR2/ALX) is a human G protein-coupled receptor capable of binding SPMs and participates in the resolution process. This receptor has been implicated in several inflammatory diseases and its association with mouse model of IBD was established in some resolution-related studies. Here, we give an overview of three reported FPR2/ALX agonists highlighting their respective roles in pro-resolving strategies.

Molecular recognition of two endogenous hormones by the human parathyroid hormone receptor-1.

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) are two endogenous hormones recognized by PTH receptor-1 (PTH1R), a member of class B G protein- coupled receptors (GPCRs). Both PTH and PTHrP analogs including teriparatide and abaloparatide are approved drugs for osteoporosis, but they exhibit distinct pharmacology. Here we report two cryo-EM structures of human PTH1R bound to PTH and PTHrP in the G protein-bound state at resolutions of 2.62 Å and 3.25 Å, respectively. Detailed analysis of these structures uncovers both common and unique features for the agonism of PTH and PTHrP. Molecular dynamics (MD) simulation together with site-directed mutagenesis studies reveal the molecular basis of endogenous hormones recognition specificity and selectivity to PTH1R. These results provide a rational template for the clinical use of PTH and PTHrP analogs as an anabolic therapy for osteoporosis and other disorders.

A high-throughput screening campaign against PFKFB3 identified potential inhibitors with novel scaffolds.

The growth of solid tumors depends on tumor vascularization and the endothelial cells (ECs) that line the lumen of blood vessels. ECs generate a large fraction of ATP through glycolysis, and elevation of their glycolytic activity is associated with angiogenic behavior in solid tumors. 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) positively regulates glycolysis via fructose-2/6-bisphosphate, the product of its kinase activity. Partial inhibition of glycolysis in tumor ECs by targeting PFKFB3 normalizes the otherwise abnormal tumor vessels, thereby reducing metastasis and improving the outcome of chemotherapy. Although a limited number of tool compounds exist, orally available PFKFB3 inhibitors are unavailable. In this study we conducted a high-throughput screening campaign against the kinase activity of PFKFB3, involving 250,240 chemical compounds. A total of 507 initial hits showing >50% inhibition at 20 µM were identified, 66 of them plus 1 analog from a similarity search consistently displayed low IC50 values (<10 µM). In vitro experiments yielded 22 nontoxic hits that suppressed the tube formation of primary human umbilical vein ECs at 10 µM. Of them, 15 exhibited binding affinity to PFKFB3 in surface plasmon resonance assays, including 3 (WNN0403-E003, WNN1352-H007 and WNN1542-F004) that passed the pan-assay interference compounds screening without warning flags. This study provides potential leads to the development of new PFKFB3 inhibitors.

Porcupine inhibitor CGX1321 alleviates heart failure with preserved ejection fraction in mice by blocking WNT signaling.

Heart failure with preserved ejection fraction (HFpEF) is highly prevalent, and lacks effective treatment. The aberration of WNT pathway underlies many pathological processes including cardiac fibrosis and hypertrophy, while porcupine is an acyltransferase essential for the secretion of WNT ligands. In this study we investigated the role of WNT signaling pathway in HFpEF as well as whether blocking WNT signaling by a novel porcupine inhibitor CGX1321 alleviated HFpEF. We established two experimental HFpEF mouse models, namely the UNX/DOCA model and high fat diet/L-NAME ("two-hit") model. The UNX/DOCA and "two-hit" mice were treated with CGX1321 (3 mg·kg-1·d-1) for 4 and 10 weeks, respectively. We showed that CGX1321 treatment significantly alleviated cardiac hypertrophy and fibrosis, thereby improving cardiac diastolic function and exercise performance in both models. Furthermore, both canonical and non-canonical WNT signaling pathways were activated, and most WNT proteins, especially WNT3a and WNT5a, were upregulated during the development of HEpEF in mice. CGX1321 treatment inhibited the secretion of WNT ligands and repressed both canonical and non-canonical WNT pathways, evidenced by the reduced phosphorylation of c-Jun and the nuclear translocation of ß-catenin and NFATc3. In an in vitro HFpEF model, MCM and ISO-treated cardiomyocytes, knockdown of porcupine by siRNA leads to a similar inhibitory effect on WNT pathways, cardiomyocyte hypertrophy and cardiac fibroblast activation as CGX1321 did, whereas supplementation of WNT3a and WNT5a reversed the anti-hypertrophy and anti-fibrosis effect of CGX1321. We conclude that WNT signaling activation plays an essential role in the pathogenesis of HFpEF, and porcupine inhibitor CGX1321 exerts a therapeutic effect on HFpEF in mice by attenuating cardiac hypertrophy, alleviating cardiac fibrosis and improving cardiac diastolic function.

Two new iridoid glycosides from Odontites vulgaris.

Two new iridoid glycosides, named 3'-O-benzoyl-dolichocymboside D (1) and dolichocymboside E (2), along with ten known glycosides (3-12), were isolated from the ethanol extract of the whole plants of Odontites vulgaris Moench. The structures of the isolated compounds were elucidated by 1D and 2D NMR and HR-ESI-MS spectra and by comparison with those reported in the literature. This is the first report on compounds 11 and 12 isolated from the family Scrophulariaceae, and compounds 8-10 were isolated from the genus Odontites.

Interleukin-8: An evolving chemokine.

Early in the 1980s several laboratories mistakenly reported that partially purified interleukin-1 (IL-1) was chemotactic for neutrophils. However, further investigations by us, revealed that our purified IL-1 did not have neutrophil chemotactic activity and this activity in the LPS-stimulated human monocyte conditioned media could clearly be separated from IL-1 activity on HPLC gel filtration. This motivated Teizo Yoshimura and Kouji Matsushima to purify the monocyte-derived neutrophil chemotactic factor (MDNCF), present in LPS conditioned media and molecularly clone the cDNA for MDNCF. They found that MDNCF protein (later renamed IL-8, and finally termed CXCL8) is first translated as a precursor form consisting of 99 amino acid residues and the signal peptide is then removed, leading to the secretion and processing of biologically active IL-8 of 72 amino acid form (residues 28-99). There are four cysteine residues forming two disulfide linkage and 14 basic amino acid residues which result in a very basic property for the binding of IL-8 to heparan sulfate-proteoglycan. The IL-8 gene consists of 4 exons and 3 introns. IL-8 is produced by various types of cells in inflammation. The 5'-flanking region of IL-8 gene contains several nuclear factor binding sites, and NF-κB in combination with AP-1 or C/EBP synergistically activates IL-8 gene in response to IL-1 and TNFα. Two receptors exist for IL-8, CXCR1 and CXCR2 in humans, which belong to γ subfamily of GTP binding protein (G-protein) coupled rhodopsin-like 7 transmembrane domain receptors. Rodents express CXCR2 and do not produce IL-8, but produce numerous homologues instead. Once IL-8 binds to the receptor, ß and γ subunits of G-protein are released from Gα (Gαi2 in neutrophils) and activate PI3Kγ, PLCß2/ß3, PLA2 and PLD. Gαi2 inhibits adenyl cyclase to decrease cAMP levels. Small GTPases Ras/Rac/Rho/cdc42/Rap1, PKC and AKT (PKB) exist down-stream of ß and γ subunits and regulate cell adhesion, actin polymerization, membrane protrusion, and eventually cell migration. PLCß activation generates IP3 and induces Ca++ mobilization, DAG generation to activate protein kinase C to lead granule exocytosis and respiratory burst. MDNCF was renamed interleukin 8 (IL-8) at the International Symposium on Novel Neutrophil Chemotactic Activating Polypeptides, London, UK in 1989. The discovery of IL-8 prompted us to also purify and molecularly clone the cDNA of MCAF/MCP-1 responsible for monocyte chemotaxis, and other groups to identify a large family of chemotactic cytokines capable of attracting other types of leukocytes. In 1992, most of the investigators contributing to the discovery of this new family of chemotactic cytokines gathered in Baden, Austria and agreed to name this family "chemokines" and subsequently established the CXCL/CCL and CXCR/CCR nomenclature. The discovery of chemokines resulted in solving the long-time enigma concerning the mechanism of cell type specific leukocyte infiltration into inflamed tissues and provided a molecular basis for immune and hematopoietic cell migration and interactions under physiological as well as pathological conditions. To our surprise based on its recently identified multifunctional activities, IL-8 has evolved from a neutrophil chemoattractant to a promising therapeutic target for a wide range of inflammatory and neoplastic diseases. IL-8 was initially characterized as a chemoattractant of neutrophils engaged in acute inflammation and then discovered to also be chemotactic for endothelial cells with a major role in angiogenesis. These two activities of IL-8 foster its stimulatory effect on tumor growth. This is abetted by recent additional discoveries showing that IL-8 has stimulatory effects on stem cells and can therefore directly promote the growth of receptor expressing cancer stem cells. IL-8 by interacting with bone marrow stem/progenitor cells has also the capacity to mobilize and release hematopoietic cells into the peripheral circulation. This includes the mobilization of neutrophilic myeloid-derived suppressor cells (N-MDSC) to infiltrate into tumors and thus further promotes the immune escape of tumors. Finally, the capacity of IL-8 to induce trans-differentiation of epithelial cancer cells into mesenchymal phenotype (EMT) increases the malignancy of tumors by promoting their metastatic spread and resistance to chemotherapeutics and cytotoxic immune cells. These observations have stimulated considerable current efforts to develop receptor antagonists for IL-8 and humanized anti-IL-8 antibody for the therapy of cancer, particularly in combination with immune checkpoint inhibitors, such as anti-PD-1/PD-L1 antibodies.