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Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone which is essential in eukaryotes. It is required for the activation and stabilization of a wide variety of client proteins and many of them are involved in important cellular pathways. Since Hsp90 affects numerous physiological processes such as signal transduction, intracellular transport, and protein degradation, it became an interesting target for cancer therapy. Structurally, Hsp90 is a flexible dimeric protein composed of three different domains which adopt structurally distinct conformations. ATP binding triggers directionality in these conformational changes and leads to a more compact state. To achieve its function, Hsp90 works together with a large group of cofactors, termed co-chaperones. Co-chaperones form defined binary or ternary complexes with Hsp90, which facilitate the maturation of client proteins. In addition, posttranslational modifications of Hsp90, such as phosphorylation and acetylation, provide another level of regulation. They influence the conformational cycle, co-chaperone interaction, and inter-domain communications. In this review, we discuss the recent progress made in understanding the Hsp90 machinery.

Background and aimsIntralipid (ILP) has been demonstrated in animal models and humans to mitigate the cardio-depressant effects of local anesthetics and I/R injury, possibly via restoration of metabolic dysfunction, activation of cardio-protective signaling and augmentation of contractility.Methods8 adult female SD rats received a single intraperitoneal injection of LPS (20 mg/kg). Echocardiography was performed on the rats at baseline prior to injection of LPS, and then at 6h post-LPS, in order to assess LV ejection fraction. Rats were randomly divided to receive either 20% ILP (n=4) or phosphate buffered saline (PBS; n=4) as a 5 ml/kg bolus followed by a 0.5 ml/kg/min infusion over 10 min and echocardiography was conducted at 1, 5 and 10 min to reassess LVEF. At 10 min, LV tissue was collected and Western blots were performed to assess for GSK and STAT3 phosphorylation. Values are expressed as mean±SEM. P<0.05 is considered statistically significant.ResultsBaseline LVEF in PBS and ILP before LPS were 75.7±1.1% and 74.2±1.2%, respectively. 6 h after LPS, LVEF was decreased (LVEF= 54.3±4.8% in PBS, and 46.0±2.5% in ILP; both p<0.05 vs. baseline). Rats treated with ILP had improved EF at 5 min (LVEF=63±3.9% p<0.05 vs.6h post LPS) that peaked at 10 min (LVEF=70.5±2.3%, p<0.05 vs.6h post LPS). PBS group had no significant improvement in LVEF at 5 and 10 min (LVEF=58.4±6.4% and 58.9±7.8%, respectively; both p=ns vs. 6h post LPS). Western blots demonstrated increased phosphorylation of STAT3 (∼2-fold) in ILP treated rats (p<0.05), not GSK phosphorylation.ConclusionsAdministration of ILP significantly improves LVF likely mediated via STAT3 phosphorylation.

T cell exhaustion presents one of the major hurdles to cancer immunotherapy. Among exhausted CD8 + tumor-infiltrating lymphocytes, the terminally exhausted subset contributes directly to tumor cell killing owing to its cytotoxic effector function. However, this subset does not respond to immune checkpoint blockades and is difficult to be reinvigorated with restored proliferative capacity. Here, we show that a half-life-extended interleukin-10–Fc fusion protein directly and potently enhanced expansion and effector function of terminally exhausted CD8 + tumor-infiltrating lymphocytes by promoting oxidative phosphorylation, a process that was independent of the progenitor exhausted T cells. Interleukin-10–Fc was a safe and highly efficient metabolic intervention that synergized with adoptive T cell transfer immunotherapy, leading to eradication of established solid tumors and durable cures in the majority of treated mice. These findings show that metabolic reprogramming by upregulating mitochondrial pyruvate carrier-dependent oxidative phosphorylation can revitalize terminally exhausted T cells and enhance the response to cancer immunotherapy. Tang and colleagues show that a half-life-extended IL-10–Fc fusion protein acts directly on terminally exhausted PD1 + TIM-3 + CD8 + T cells to enhance their proliferation and effector function by reprogramming the cellular metabolism to oxidative phosphorylation in a mitochondrial pyruvate carrier–dependent manner. Treatment of tumor-bearing mice with IL-10–Fc and adoptive T cell therapy led to eradication of their established solid tumors and durable cures.

Background Chronic undernutrition, a condition currently affecting more than 17 millions of children under five years of age, has severe long-term consequences including stunting. Epidemiologic studies have emphasized that undernutrition cannot be ascribed to food insecurity alone and gut microbiota has been shown to play an active role in disease aetiology. In mammals, post-natal growth is controlled by the activity of the somatotropic axis. Undernutrition leads to a decrease of the Insulin like Growth Factor 1 (IGF-1) and a state of Growth Hormon (GH) resistance. Aims Previously we have shown the capacity of selected Lactobacillus plantarum strain to maintain growth in infant mono-colonized mice during chronic undernutrition. Here we show that L. plantarum retains its growth promoting capabilities also in a conventional mouse model. Methods C57BL6 male mice were weaned at 21 days and bred on a standard or an experimental (isocaloric, hypoprotidic and hypolipidic) diet until young adulthood. One group of mice on experimental diet received an LpWJL oral supplementation (2*10^8 CFU/day; 5 days per week) and the other group received placebo. Length and weight were measured weekly. Mice were sacrificed at day 56 to study the impact of the LpWJL oral supplementation on IGF-1 levels and organ growth. Somatotropic axis activity was tested at Day 28 by injecting the mice with GH and measured by the STAT5 phosphorylation level. Results At D56, mice fed with the experimental diet were smaller than the standard diet group (7.7 vs 8.9 cm; p< 0.01). Undernourished mice had a lower hepatic level of IGF-1 (113 ± 39 vs 174 ± 35 pg/mg tissues; p<0.01) and a lower plasmatic level of IGF-1 (150 ± 50 vs 388 ± 103 ng/mL; p< 0.01) compared with the standard diet group. Body length of mice fed experimental diet was longer in LpWJL –supplemented group compared to the placebo supplementation (8.02 ± 0.19 vs 7.73 ± 0.16 cm; p<0.0001). LpWJL-treated mice showed 23% increase in daily growth gain compared to placebo without the change in the mean daily food intake. In the LpWJL group, mice had a higher hepatic IGF-1 level (108 ± 12.5 vs 59.8 ± 18.5 pg/mg tissues; p< 0.0001) and a higher plasmatic IGF-1 level (209 ± 51 vs 148 ± 32 ng/mL; p< 0.001) compared to the placebo group. At day 28, mice exposed to LpWJL during starvation process showed an increase of the sensibility of the hepatic GH receptor to GH according to the STAT5 phosphorylation level. Conclusions Oral supplementation by LpWJL alleviates the GH resistance and improves juvenile growth of conventional infant mice upon undernutrition. Funding Agencies None

BackgroundInterleukin 13 (IL-13) and IL-4 are archetypal cytokines that drive type 2 immune responses, which are dysregulated in a large proportion of patients with asthma. The regulation of IL-13 and IL-4 signalling has focused on their downstream events (e.g. phosphorylation of STAT6 upon receptor binding) leading to transcription of type 2 mediators. Further regulators of this fundamental pathway remain poorly understood. Likewise, molecular markers of type 2 disease remain scarce.MethodsWe used RNA immunoprecipitation, siRNA and shRNA depletion in cell lines and primary bronchial epithelium, as well as RT-qPCR and protein analyses (western blotting, MSD). We also employed Frac-seq (subcellular fractionation and RNA-sequencing) to compare steady (transcription) vs polyribosome-bound (translation) mRNA expression in response to IL-13 in an IGF2BP3-dependent manner. We explored several datasets of ‘type 2 high’ disease cohorts.ResultsUsing RNA immunoprecipitation, we detected IGF2BP3 directly bound to the mRNAs encoding for IL4R and IL13Ra1. Depletion of IGF2BP3 led to an increase of IL-4Ra and IL-13Ra1 cell surface expression, without affecting IL4R or IL13RA1 mRNA levels, methylation or stability in human bronchial epithelial BEAS-2B cells. Concomitantly, IGF2BP3 depletion led to increased IL-13-dependent STAT6 phosphorylation and activity as determined by a STAT6-reporter and increased expression of IL-13-dependent eotaxins CCL26, CCL24 and CCL11 mRNAs, as well as the classically associated Th2 marker GATA3. IGF2BP3 regulation of eotaxin and GATA3 expression was recapitulated in primary airway cells. Frac-seq demonstrated that IL-13 triggers different transcriptional and post-transcriptional responses, with IGF2BP3 levels mainly modulating steady mRNA levels. We also found IGF2BP3 upregulated in multiple cohorts of atopic and type 2 high asthmatic patients.ConclusionsWe show for the first time that an RNA binding protein modulates IL-13/IL-4 signalling and offer a novel approach for suppressing IL-13-dependent signalling in bronchial epithelium. IGF2BP3 is increased in type 2 high disease, where it may be part of a feedback loop to halt type 2 inflammation and serve as a novel biomarker.

Background and AimsIPF is a progressive, incurable scarring disease of the lung. A common genetic variant near AKAP13 has been associated with IPF susceptibility and elevated gene and protein expression. How this common variant contributes to the pathogenesis of IPF is not known. AKAP13 is a multifunctional scaffold protein, with high expression in lung epithelial cells and integrates intracellular signalling through its interactions with RhoA and protein kinase A (PKA). A13 is a selective small-molecule inhibitor of AKAP13’s RhoGEF domain. This study investigates how an AKAP13 variant alters epithelial signalling and evaluates the therapeutic potential of targeting AKAP13 using A13.MethodsCRISPR-Cas9 editing was used to introduce the AKAP13 variant into immortalized human bronchial epithelial cells (iHBECs). Overexpression constructs modelled a truncated AKAP13 isoform lacking the PKA-binding domain. Real-time adhesion and proliferation were measured (xCELLigence). Cell transcriptional profiling is performed using ncounter. Intracellular cAMP was measured by FRET. RhoA activity was measured by G-LISA, and SMAD2 phosphorylation was measured by immunoblotting, both in the presence or absence of LPA stimulation. IPF patient-derived precision-cut lung slices (PCLS) were treated with A13 and analysed for profibrotic gene expression by real-time PCR analysis and SMAD2 nuclear translocation using immunofluorescence and cellpose single cell segmentation.ResultsiHBECs carrying the AKAP13 variant showed increased expression of truncated isoform of AKAP13. These cells exhibited a ~50% increase in cell adhesion and ~30% reduction in proliferation compared to cells not expressing the AKAP13 variant. These cells showed a profibrotic gene signature had increased RhoA activation and SMAD2 phosphorylation following LPA stimulation. Furthermore, cells harbouring the AKAP13 variant displayed a reduced capacity to generate intracellular cAMP. A13 treatment reversed the pro-adhesive phenotype and significantly reduced RhoA activation. In IPF-derived PCLS (n=4), A13 suppressed SERPINE1, CCN2, and MMP7 expression and reduced SMAD2 nuclear translocation.ConclusionsPresence of an AKAP13 variant disrupts epithelial homeostasis and promotes pro-fibrotic signalling. Inhibition of AKAP13’s RhoGEF domain with A13 restores epithelial function and attenuates fibrotic activation, supporting AKAP13 as a therapeutic target in IPF.

IntroductionIdiopathic Pulmonary Fibrosis (IPF) is a progressive fibrosing interstitial lung disease with limited treatment options and a grave prognosis. Treprostinil, a synthetic prostacyclin analogue used in pulmonary hypertension, is under investigation for its antifibrotic potential. The antifibrotic mechanisms of Treprostinil are yet to be fully defined. Treprostinil activates the IP receptor to generate cAMP but may also target intracellular PPARβδ receptors. PPARβδ activation can inhibit the RhoA pathway which is amplified in IPF. These studies aim to explore the antifibrotic mechanisms of Treprostinil.MethodsExpression of IP and PPARβδ receptors in primary human lung fibroblasts (pHLFs) was confirmed via RT-qPCR and Western blotting. RhoA activation was measured using G-LISAä and TGFβ activity via TMLC coculture assay and Western blotting for phospho-SMAD2. Profibrotic gene expression was measured by RT-qPCR. Fibroblast proliferation was assessed using BrdU incorporation by ELISA and time-lapse live cell imaging. In IPF patient-derived precision-cut lung slices (PCLS), nuclear phospho-SMAD2 accumulation was assessed via immunofluorescence and hydroxyproline levels quantified using high-performance liquid chromatography in PCLS conditioned media.ResultsBoth IP and PPARβδ receptors were present in pHLFs, with PPARβδ more abundant. Treprostinil inhibited LPA-induced RhoA activation and this effect was not reversed by the presence of an IP antagonist. Treprostinil did not inhibit LPA-induced TGFβ activation in the TMLC assay or SMAD2 phosphorylation. However, Treprostinil depletes total SMAD2/3 likely via cAMP and this correlates with a rise in phospho-YAP. Fibroblast proliferation was also inhibited in a concentration-dependent manner by Treprostinil and there were additive effects when combined with nintedanib, pirfenidone, or nerandomilast. Treprostinil downregulated TGFβ-response genes in pHLFs including FN and SERPINE1, as well as nuclear phospho-SMAD2 and hydroxyproline content levels in the PCLS model.ConclusionsTreprostinil inhibits RhoA activation and this may be via PPARβδ activation. Treprostinil did not affect SMAD2 phosphorylation but did deplete total SMAD2/3 in a cAMP dependent manner in pHLFs. Antifibrotic effects of Treprostinil were detected in IPF derived PCLS. Further studies are ongoing to establish the underlying mechanism as there were additive effects of Treprostinil with established antifibrotic agents which may have important clinical implications.

The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixture events for extinct taxa 1 . However, the irreversible post-mortem degradation 2 of ancient DNA has so far limited its recovery—outside permafrost areas—to specimens that are not older than approximately 0.5 million years (Myr) 3 . By contrast, tandem mass spectrometry has enabled the sequencing of approximately 1.5-Myr-old collagen type I 4 , and suggested the presence of protein residues in fossils of the Cretaceous period 5 —although with limited phylogenetic use 6 . In the absence of molecular evidence, the speciation of several extinct species of the Early and Middle Pleistocene epoch remains contentious. Here we address the phylogenetic relationships of the Eurasian Rhinocerotidae of the Pleistocene epoch 7 – 9 , using the proteome of dental enamel from a Stephanorhinus tooth that is approximately 1.77-Myr old, recovered from the archaeological site of Dmanisi (South Caucasus, Georgia) 10 . Molecular phylogenetic analyses place this Stephanorhinus as a sister group to the clade formed by the woolly rhinoceros ( Coelodonta antiquitatis ) and Merck’s rhinoceros ( Stephanorhinus kirchbergensis ). We show that Coelodonta evolved from an early Stephanorhinus lineage, and that this latter genus includes at least two distinct evolutionary lines. The genus Stephanorhinus is therefore currently paraphyletic, and its systematic revision is needed. We demonstrate that sequencing the proteome of Early Pleistocene dental enamel overcomes the limitations of phylogenetic inference based on ancient collagen or DNA. Our approach also provides additional information about the sex and taxonomic assignment of other specimens from Dmanisi. Our findings reveal that proteomic investigation of ancient dental enamel—which is the hardest tissue in vertebrates 11 , and is highly abundant in the fossil record—can push the reconstruction of molecular evolution further back into the Early Pleistocene epoch, beyond the currently known limits of ancient DNA preservation. Palaeoproteomic analysis of dental enamel from an Early Pleistocene Stephanorhinus resolves the phylogeny of Eurasian Rhinocerotidae, by enabling the reconstruction of molecular evolution beyond the limits of ancient DNA preservation.

IntroductionHeart failure (HF) is marked by impaired calcium (Ca2 +) handling, which compromises contractility and heightens arrhythmogenic risk. Ca2 + buffering within cardiac myocytes is essential for stabilizing intracellular Ca2 +. It is underexplored in HF, especially in large-animal models. This study examines how HF alters Ca2 + buffering power and investigates the potential of phosphodiesterase inhibition in restoring Ca2 + dynamics.MethodsHF was induced in adult sheep through tachypacing, and animals were divided into three groups: control, HF (n=10), and HF treated with tadalafil (20 mg daily, n=6). Ventricular cardiac myocytes were isolated, and intracellular Ca2 + was measured using Fluo-5F. Voltage clamp was achieved via a perforated patch clamp. Sarcoplasmic reticulum (SR) Ca2 + content and cellular buffering properties were measured by applying a 5 mM caffeine and 20 mM 2,3-butanedione monoxime solution. Protein levels were quantified using western blotting. All data were analysed with nested statistical models.ResultsHF significantly reduced Ca2 + transient amplitude compared to controls (325.2±34.6nmol/L vs. 169.0±16.8nmol/L, p=0.005), with tadalafil treatment showing no notable improvement (149.4±30.5nmol/L, p=0.96 vs. HF). Ca2 + influx via the L-type Ca2 + channel was also reduced in HF (peak current density 1.26±0.17pA/pF vs. 2.10±0.16pA/pF in controls, p=0.007), this remained impaired in the tadalafil group (1.32±0.22pA/pF, p=0.99 vs. HF). SR Ca2 + content showed no change between controls (77.9±9.6nmol/L) and HF (73.9±9.2nmol/L, p=0.99), but decreased significantly in tadalafil-treated HF (49.0±2.9nmol/L, p=0.018 vs. HF).Ca2 + buffering properties were markedly altered in HF, Bmax decreased from 150.1±19.9µM in controls to 105.2±10.7µM (p=0.02). Kd dropped significantly in HF to 0.61±0.1µM (vs. 1.69±0.20µM in controls, p=0.0002), with an overall increase in buffer power (59.2±6.4 in control and 93.5±7.9 in HF p=0.02). Tadalafil treatment resulted in an increase in Kd to 1.25±0.13 µM (p=0.015 vs. HF), Bmax remained similar to HF at 101.8±4.6 µM (p=0.99 vs. HF). Overall buffer power was restored to baseline levels (61.3±6.5 p=0.02 vs HF.) Western blot analysis showed that HF led to a reduction in TnI phosphorylation (p=0.01), which tadalafil partially restored. SERCA levels were equally reduced in both HF and tadalafil groups (p=0.007 and p=0.003 vs. control, respectively).Abstract BS33 Figure 1Buffer properties with tadalafil treatment. Ai: Ca2+ recordings during application of caffeine + BDM solution. Aii: NCX currents during the same application of caffeine. Aiii: Integral of NCX currents in Aii. Aiv: Representative buffer slopes derived from the caffeine method. Bi: Bmax. Bii: Kd, in control (N=12), HF (N=9) and Tadalafil (N=6) groups. Mean±SEM compared using a nested one-way ANOVA[Image Omitted. See PDF.]Abstract BS33 Figure 2Interplay between buffer power and free and total Ca2+ and tadalafil treatment. Representative trace of free calcium transients (Ai), of total calcium transients (Aii) and the buffer power of cell across the calcium transient (Aiii). Bi: Total calcium amplitude. Bii: Change of total/change of free calcium: ergo cellular buffer power across calcium transient. Biii: Diastolic buffer power. Ci: The relationship between buffer power and free calcium. Cii The relationship between total and free calcium. In control (N=12), HF (N=9) and tadalafil groups (N=6) Mean±SEM compared using nested one-way ANOVA[Image Omitted. See PDF.]ConclusionThis study demonstrates that HF results in an increase in Ca2 + buffering by decreasing Kd, likely due to reduced TnI phosphorylation. Tadalafil restores Ca2 + buffering to baseline by restoring Kd values. This represents a potential therapeutic target for both arrhythmia and contractility in HF. Despite the reduced SR content in tadalafil-treated HF, Ca2 + transient amplitude was preserved, due to the reduction of Ca2 + buffering power seen. These findings propose that targeting Ca2 + buffering with tadalafil may offer a novel strategy to modulate Ca2 + handling in HF.