Pharmacological Interference Research Articles

DRUG INTERFERENCE IN LABORATOTY TESTS

Drug Laboratory Tests Interference (DLTI) is currently considered one of the important problems of medicine. The ability of drugs to affect metabolism, displace endogenous and exogenous substances from bonds, interfere with chemical reactions during laboratory research can lead to false-positive or false-negative results, and as a result of incorrectly established diagnosis, incorrect therapy and deterioration of the patient’s condition.
 To highlight the problem of DLTI, summarize the information obtained from scientific sources for workers in the medical field.
 Medicines can affect the results of laboratory tests through technological or pharmacological (physiological) interference. Predicting DLTI is substantially difficult, since the sources of information on pharmacological interference consist mainly of instructions for medical use of drugs, for analytical - spontaneous reports. Very often, the information is fragmentary, insufficiently documented, and contradictory. In order to avoid diagnostic errors as much as possible, medical workers need to strictly follow the recommendations regarding the pre-analytical stage, emphasize to the laboratory workers what medicines the patient is taking. In order to facilitate the work of medical professionals in various countries, electronic databases with checked information about DLTI are being developed. However, in order to refer to the above databases, a medical professional must have a suspicion of a possible drug interaction with laboratory tests. Decision support programs are recommended to be developed and implemented to automate DLTI reporting.

Nicotine Decreases Nerve Regeneration and Pain Behaviors via PTEN and Downstream Inflammation-Related Pathway in Two Rat Nerve Injury Models.

Neuropathic pain is stubborn and associated with the peripheral nerve regeneration process. Nicotine has been found to reduce pain, but whether it is involved in the regulation of nerve regeneration and the underlying mechanism are unknown. In this study, we examined the mechanical allodynia thermal hyperalgesia together with the peripheral nerve regeneration after nicotine exposure in two rat neuropathic pain models. In the spinal nerve ligation model, in which anatomic nerve regeneration can be easily observed, nicotine reduced anatomic measures of regeneration as well as expression of regeneration marker growth-associated protein 43 (GAP43). In the tibial nerve crush model, nicotine treatment significantly suppressed GAP43 expression and functional reinnervation as measured by myelinated action potential and electromyography of gastrocnemius. In both models, nicotine treatment reduced macrophage density in the sensory ganglia and peripheral nerve. These effects of nicotine were reversed by the selective α7 nicotinic acetylcholine receptor (nAChR) blocker methyllycaconitine. In addition, nicotine significantly elevated expression of PTEN (the phosphatase and tensin homolog deleted on chromosome 10), a key player in both regeneration and pain. Pharmacological interference of PTEN could regulate GAP43 expression, pain-related behaviors, and macrophage infiltration in a nicotine-treated nerve crush model. Our results reveal that nicotine and its α7-nAChR regulate both peripheral nerve regeneration process and pain though PTEN and the downstream inflammation-related pathway.

Blueberry anthocyanins extract inhibits advanced glycation end-products (AGEs) production and AGEs-stimulated inflammation in RAW264.7 cells.

Pharmacological interference is considered to be a successful approach to inhibit advanced glycation end-products (AGEs) production and to block AGEs-induced diseases. Some synthetic medicines are effective for inhibiting the glycation reaction, but they cannot be widely applied in clinical as a result of their side effects and security concerns. The present study uses blueberry anthocyanins extract (BAE) to attenuate AGEs formation and AGEs-induced inflammatory response in vitro. In a bovine serum albumin-glucose model, BAE showed similar inhibitory activity on AGEs compared to the synthetic anti-glycation agent (aminoguanidine). The results showed that BAE exhibit strong anti-glycative action by scavenging glycosylated intermediates (Schiff base, fructosamine and α-dicarbonyl compounds), attenuating the molecular aggregation and amyloid-like fibrils formation, and preventing conformational modification. Additionally, BAE was found to dose-dependently inhibit the AGEs-induced secretions of nitric oxide and pro-inflammatory cytokines (interleukin-6, monocyte chemoattractant protein-1 and tumor necrosis factor-α) in RAW264.7 cells. The anti-inflammation activity of BAE was mediated by down-regulating the expressions of critical inflammatory markers, inducible nitric oxide synthase and cyclooxygenase-2, through nuclear factor-kappa B signaling pathways inhibition. BAE could serve as a natural inhibitor for controlling AGEs and AGEs-induced chronic inflammation. © 2023 Society of Chemical Industry.

Divergence of trafficking and polarization mechanisms for PIN auxin transporters during land plant evolution

The phytohormone auxin, and its directional transport through tissues, plays a fundamental role in the development of higher plants. This polar auxin transport predominantly relies on PIN-FORMED (PIN) auxin exporters. Hence, PIN polarization is crucial for development, but its evolution during the rise of morphological complexity in land plants remains unclear. Here, we performed a cross-species investigation by observing the trafficking and localization of endogenous and exogenous PINs in two bryophytes, Physcomitrium patens and Marchantia polymorpha, and in the flowering plant Arabidopsis thaliana. We confirmed that the GFP fusion did not compromise the auxin export function of all examined PINs by using a radioactive auxin export assay and by observing the phenotypic changes in transgenic bryophytes. Endogenous PINs polarize to filamentous apices, while exogenous Arabidopsis PINs distribute symmetrically on the membrane in both bryophytes. In the Arabidopsis root epidermis, bryophytic PINs have no defined polarity. Pharmacological interference revealed a strong cytoskeletal dependence of bryophytic but not Arabidopsis PIN polarization. The divergence of PIN polarization and trafficking is also observed within the bryophyte clade and between tissues of individual species. These results collectively reveal the divergence of PIN trafficking and polarity mechanisms throughout land plant evolution and the co-evolution of PIN sequence-based and cell-based polarity mechanisms.

Immunotherapies in MuSK-positive Myasthenia Gravis; an IgG4 antibody-mediated disease.

Muscle-specific kinase (MuSK) Myasthenia Gravis (MG) represents a prototypical antibody-mediated disease characterized by predominantly focal muscle weakness (neck, facial, and bulbar muscles) and fatigability. The pathogenic antibodies mostly belong to the immunoglobulin subclass (Ig)G4, a feature which attributes them their specific properties and pathogenic profile. On the other hand, acetylcholine receptor (AChR) MG, the most prevalent form of MG, is characterized by immunoglobulin (Ig)G1 and IgG3 antibodies to the AChR. IgG4 class autoantibodies are impotent to fix complement and only weakly bind Fc-receptors expressed on immune cells and exert their pathogenicity via interfering with the interaction between their targets and binding partners (e.g. between MuSK and LRP4). Cardinal differences between AChR and MuSK-MG are the thymus involvement (not prominent in MuSK-MG), the distinct HLA alleles, and core immunopathological patterns of pathology in neuromuscular junction, structure, and function. In MuSK-MG, classical treatment options are usually less effective (e.g. IVIG) with the need for prolonged and high doses of steroids difficult to be tapered to control symptoms. Exceptional clinical response to plasmapheresis and rituximab has been particularly observed in these patients. Reduction of antibody titers follows the clinical efficacy of anti-CD20 therapies, a feature implying the role of short-lived plasma cells (SLPB) in autoantibody production. Novel therapeutic monoclonal against B cells at different stages of their maturation (like plasmablasts), or against molecules involved in B cell activation, represent promising therapeutic targets. A revolution in autoantibody-mediated diseases is pharmacological interference with the neonatal Fc receptor, leading to a rapid reduction of circulating IgGs (including autoantibodies), an approach already suitable for AChR-MG and promising for MuSK-MG. New precision medicine approaches involve Chimeric autoantibody receptor T (CAAR-T) cells that are engineered to target antigen-specific B cells in MuSK-MG and represent a milestone in the development of targeted immunotherapies. This review aims to provide a detailed update on the pathomechanisms involved in MuSK-MG (cellular and humoral aberrations), fostering the understanding of the latest indications regarding the efficacy of different treatment strategies.

The enzymatic and neurochemical outcomes of a mutation in Mexican cavefish MAO reveal teleost-specific aspects of brain monoamine homeostasis.

Monoamine oxidases (MAO; MAO-A and MAO-B in mammals) are enzymes catalyzing the degradation of biogenic amines, including monoamine neurotransmitters. In humans, coding mutations in MAOs are extremely rare and deleterious. Here, we assessed the structural and biochemical consequences of a point mutation (P106L) in the single mao gene of the blind cavefish, Astyanax mexicanus. This mutation decreased mao enzymatic activity by ∼3-fold and affected the enzyme kinetics parameters, in line with potential structure-function alterations. HPLC measurements in brains of four A. mexicanus genetic lines (mutant and non-mutant cavefish, and mutant and non-mutant surface fish) showed major disturbances in serotonin, dopamine, noradrenaline and metabolite levels in mutants and demonstrated that the P106L mao mutation is responsible for monoaminergic disequilibrium in the P106L mao mutant cavefish brain. The outcomes of the mutation were different in the posterior brain (containing the raphe nucleus) and the anterior brain (containing fish-specific hypothalamic serotonergic clusters), revealing contrasting properties in neurotransmitter homeostasis in these different neuronal groups. We also discovered that the effects of the mutation were partially compensated by a decrease in activity of TPH, the serotonin biosynthesis rate-limiting enzyme. Finally, the neurochemical outcomes of the mao P106L mutation differed in many respects from a treatment with deprenyl, an irreversible MAO inhibitor, showing that genetic and pharmacological interference with MAO function are not the same. Our results shed light on our understanding of cavefish evolution, on the specificities of fish monoaminergic systems, and on MAO-dependent homeostasis of brain neurochemistry in general.

#3632 CHARACTERIZATION AND REGULATION OF THE PATHOGENIC MUCIN 1 FRAMESHIFT PROTEIN IN PATIENT DERIVED CELLS AFFECTED BY ADTKD-MUC1

Abstract Background and Aims Autosomal Dominant Tubulointerstitial Kidney Diseases (ADTKD) are caused by mutations in one of at least five genes (UMOD, MUC1, HNF1B, REN and SEC61A1) and are characterized by progressive decline in kidney function, reaching end-stage renal disease (ESRD) usually in mid adulthood. ADTKD-MUC1 is one of the most frequent subforms, exclusively caused by a characteristic frameshift mutation, leading to the de novo protein MUC1-fs. MUC1-fs is believed to play the distinct pathogenic role in terms of a toxic proteinopathy, accumulating in the early secretory pathway. The substance BRD4780 is reported to re-route MUC1-fs towards lysosomal degradation (Dvela-Levitt et al., Cell 2019), with interventional trials being in preparation. Therefore, we aimed to gain more insights into MUC1-fs temporal and spacial regulative characteristics, comparing pharmacological intervention vs. RNA interference in patient derived tubular cells. Methods Cell culture (HKC-8 cells and patient derived human urinary Primary Tubular Cells, huPTC); siRNA knockdown; Transient transfection; Immunofluorescence; immunogold electron microscopy (EM); Immunoblotting (IB); Lentiviral SV40/Large-T antigen immortalization of huPTC; Generation of a novel polyclonal MUC1-fs antibody with an independent antigen downstream of the VNTR. Results To analyze MUC1-fs protein in more detail, we generated iTCs (immortalized tubular cells) from huPTC of patients with ADTKD-MUC1. Clonal selection of cells was performed to gain immortalized clones with MUC1-fs expression. MUC1-fs mainly localizes to the secretory pathway. Co-localization was only partially observed with TMED9, which localizes to COP vesicles, being involved in protein trafficking within the early secretory pathway. TMED9 negative components of this pathway also showed MUC1-fs staining, such as the Golgi apparatus and Early Endosomes. Immunogold EM of huPTC reveals MUC1-fs expression within the ER and (secretory) vesicles. Ultrastructural analyses of biopsies by EM from ADTKD-MUC1 patients did not show specific protein accumulation, as previously described in ADTKD-UMOD. RNA interference for MUC1 and pharmacological application of BRD4780 was performed in huPTC and iTCs, with detailed description of MUC1-fs regulation kinetics. Both approaches led to strongly reduced MUC1-fs protein levels. MUC1-fs protein revealed a significantly shorter half life than wildtype mucin 1, possibly due to its different cellular distribution. Conclusion Our data confirm and extend previously published information on intracellular MUC1-fs localization and regulation (Dvela-Levitt et al., Cell 2019). The protein kinetics of MUC1-fs appear quite dynamic in terms of synthesis and decay, which should argue for it being a suitable pharmacological target. Both, BRD4780 and RNA interference potently downregulate MUC1-fs. Interestingly, in primary and immortalized patient derived cells MUC1-fs localization is not confined to the early secretory pathway. Should the effect of BRD4780 be restricted to TMED9 positive COP vesicles, this may implicate reduced effectivenes. Therefore, RNA interference may be more efficient, since it reduces MUC1-fs at an earlier stage and is not dependent on intracellular processing.

Abstract 3688: Exploring metabolic vulnerabilities of metastatic prostate cancer to bone

Abstract Currently, limited knowledge is available on subtype-specific metabolic features and their implications for treatment. Here, we investigated the metabolic determinants of the two major subtypes of castration-resistant prostate cancer (androgen receptor-expressing prostate cancer, ARPC; and aggressive variant prostate cancer, AVPC). Transcriptomic analyses revealed enrichment of gene sets involved in oxidative phosphorylation in ARPC tumor samples compared to AVPC. Unbiased screening of metabolic signaling pathways in PDX models by proteomic analyses further supported an enrichment of oxidative phosphorylation in ARPC compared to AVPC, and a skewing toward glycolysis by AVPC. Metabolic in vitro analyses showed that C4-2B cells depended on aerobic respiration, while PC3 cells relied more heavily on glycolysis, as further confirmed by pharmacological interference using IACS-010759, a clinical grade inhibitor of oxidative phosphorylation tested in patients. In vivo studies confirmed a significant reduction by IACS-010759 in C4-2B tumors implanted subcutaneously or in bone, and no effect in subcutaneous PC3 tumors. Unexpectedly, PC3 tumors in bone responded to IACS-010759, indicating a microenvironment-induced metabolic reprogramming. These results suggest that castration-resistant ARPC and AVPC exhibit different metabolic dependencies with aerobic respiration being a major driver of ARPC, while AVPC depends on glycolysis, but can undergo metabolic reprogramming in bone. Such metabolic vulnerabilities may be exploited to either introduce further lines of treatments or in combination with the existing therapies. These findings also underscore the important role that the tumor microenvironment can play in reprogramming prostate cancer metabolism and thus, influence the efficacy of metabolically targeted therapies. Citation Format: Federica Mossa, Daniele Robesti, Christopher Vellano, Mark Titus, Joe Marszalek, Daniel Frigo, Christopher Logothetis, Taranjit Gujral, Eleonora Dondossola. Exploring metabolic vulnerabilities of metastatic prostate cancer to bone. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3688.

Autocrine activation of MAPK signaling mediates intrinsic tolerance to androgen deprivation in LY6D prostate cancer cells

The emergence of castration-resistant prostate cancer remains an area of unmet clinical need. We recently identified a subpopulation of normal prostate progenitor cells, characterized by an intrinsic resistance to androgen deprivation and expression of LY6D. We here demonstrate that conditional deletion of PTEN in the murine prostate epithelium causes an expansion of transformed LY6D+ progenitor cells without impairing stem cell properties. Transcriptomic analyses of LY6D+ luminal cells identified an autocrine positive feedback loop, based on the secretion of amphiregulin (AREG)-mediated activation of mitogen-activated protein kinase (MAPK) signaling, increasing cellular fitness and organoid formation. Pharmacological interference with this pathway overcomes the castration-resistant properties of LY6D+ cells with a suppression of organoid formation and loss of LY6D+ cells invivo. Notably, LY6D+ tumor cells are enriched in high-grade and androgen-resistant prostate cancer, providing clinical evidence for their contribution to advanced disease. Our data indicate that early interference with MAPK inhibitors can prevent progression of castration-resistant prostate cancer.

Pharmacological Inhibition of Glycoprotein VI- and Integrin α2β1-Induced Thrombus Formation Modulated by the Collagen Type.

In secondary cardiovascular disease prevention, treatments blocking platelet-derived secondary mediators pose a risk of bleeding. Pharmacological interference of the interaction of platelets with exposed vascular collagens is an attractive alternative, with clinical trials ongoing. Antagonists of the collagen receptors, glycoprotein VI (GPVI), and integrin α2β1, include recombinant GPVI-Fc dimer construct Revacept, 9O12 mAb based on the GPVI-blocking reagent Glenzocimab, Syk tyrosine-kinase inhibitor PRT-060318, and anti-α2β1 mAb 6F1. No direct comparison has been made of the antithrombic potential of these drugs. Using a multiparameter whole-blood microfluidic assay, we compared the effects of Revacept, 9O12-Fab, PRT-060318, or 6F1 mAb intervention with vascular collagens and collagen-related substrates with varying dependencies on GPVI and α2β1. To inform on Revacept binding to collagen, we used fluorescent-labelled anti-GPVI nanobody-28. In this first comparison of four inhibitors of platelet-collagen interactions with antithrombotic potential, we find that at arterial shear rate: (1) the thrombus-inhibiting effect of Revacept was restricted to highly GPVI-activating surfaces; (2) 9O12-Fab consistently but partly inhibited thrombus size on all surfaces; (3) effects of GPVI-directed interventions were surpassed by Syk inhibition; and (4) α2β1-directed intervention with 6F1 mAb was strongest for collagens where Revacept and 9O12-Fab were limitedly effective. Our data hence reveal a distinct pharmacological profile for GPVI-binding competition (Revacept), GPVI receptor blockage (9O12-Fab), GPVI signaling (PRT-060318), and α2β1 blockage (6F1 mAb) in flow-dependent thrombus formation, depending on the platelet-activating potential of the collagen substrate. This work thus points to additive antithrombotic action mechanisms of the investigated drugs.

The role of vasoactive peptides in skin homeostasis-focus on adiponectin and the kallikrein-kinin system.

Vasoactive peptides often serve a multitude of functions aside from their direct effects on vasodynamics. This article will review the existing literature on two vasoactive peptides and their involvement in skin homeostasis: adiponectin and-as the main representative of the kallikrein-kinin system-bradykinin. Adiponectin is the most abundantly expressed adipokine in the human organism, where it is mainly localized in fat depots including subcutaneous adipose tissue, from where adiponectin can exert paracrine effects. The involvement of adiponectin in skin homeostasis is supported by a number of studies reporting the effects of adiponectin in isolated human keratinocytes, sebocytes, fibroblasts, melanocytes, and immune cells. Regarding skin pathology, the potential involvement of adiponectin in psoriasis, atopic dermatitis, scleroderma, keloid, and melanogenesis is discussed in this article. The kallikrein-kinin system is composed of a variety of enzymes and peptides, most of which have been identified to be expressed in the skin. This also includes the expression of bradykinin receptors on most skin cells. Bradykinin is one of the very few hormones that is targeted by treatment in routine clinical use in dermatology-in this case for the treatment of hereditary angioedema. The potential involvement of bradykinin in wound healing, psoriasis, and melanoma is further discussed in this article. This review concludes with a call for additional preclinical and clinical studies to further explore the therapeutic potential of adiponectin supplementation (for psoriasis, atopic dermatitis, wound healing, scleroderma, and keloid) or pharmacological interference with the kallikrein-kinin system (for wound healing, psoriasis, and melanoma).

Pollen tube invasive growth is promoted by callose.

Callose, a β-1,3-glucan, lines the pollen tube cell wall except for the apical growing region, and it constitutes the main polysaccharide in pollen tube plugs. These regularly deposited plugs separate the active portion of the pollen tube cytoplasm from the degenerating cell segments. They have been hypothesized to reduce the total amount of cell volume requiring turgor regulation, thus aiding the invasive growth mechanism. To test this, we characterized the growth pattern of Arabidopsis callose synthase mutants with altered callose deposition patterns. Mutant pollen tubes without callose wall lining or plugs had a wider diameter but grew slower compared to their respective wildtype. To probe the pollen tube's ability to perform durotropism in the absence of callose, we performed mechanical assays such as growth in stiffened media and assessed turgor through incipient plasmolysis. We found that mutants lacking plugs had lower invading capacity and higher turgor pressure when faced with a mechanically challenging substrate. To explain this unexpected elevation in turgor pressure in the callose synthase mutants we suspected that it is enabled by feedback-driven increased levels of de-esterified pectin and/or cellulose in the tube cell wall. Through immunolabeling we tested this hypothesis and found that the content and spatial distribution of these cell wall polysaccharides was altered in callose-deficient mutant pollen tubes. Combined, the results reveal how callose contributes to the pollen tube's invasive capacity and thus plays an important role in fertilization. In order to understand, how the pollen tube deposits callose, we examined the involvement of the actin cytoskeleton in the spatial targeting of callose synthases to the cell surface. The spatial proximity of actin with locations of callose deposition and the dramatic effect of pharmacological interference with actin polymerization suggest a potential role for the cytoskeleton in the spatial control of the characteristic wall assembly process in pollen tubes.

Recent approaches in the drug research and development of novel antimalarial drugs with new targets.

Malaria is a serious worldwide medical issue that results in substantial annual death and morbidity. The availability of treatment alternatives is limited, and the rise of resistant parasite types has posed a significant challenge to malaria treatment. To prevent a public health disaster, novel antimalarial agents with single-dosage therapies, extensive curative capability, and new mechanisms are urgently needed. There are several approaches to developing antimalarial drugs, ranging from alterations of current drugs to the creation of new compounds with specific targeting abilities. The availability of multiple genomic techniques, as well as recent advancements in parasite biology, provides a varied collection of possible targets for the development of novel treatments. A number of promising pharmacological interference targets have been uncovered in modern times. As a result, our review concentrates on the most current scientific and technical progress in the innovation of new antimalarial medications. The protein kinases, choline transport inhibitors, dihydroorotate dehydrogenase inhibitors, isoprenoid biosynthesis inhibitors, and enzymes involved in the metabolism of lipids and replication of deoxyribonucleic acid, are among the most fascinating antimalarial target proteins presently being investigated. The new cellular targets and drugs which can inhibit malaria and their development techniques are summarised in this study.

The lysophosphatidic acid-regulated signal transduction network in ovarian cancer cells and its role in actomyosin dynamics, cell migration and entosis.

Lysophosphatidic acid (LPA) species accumulate in the ascites of ovarian high-grade serous cancer (HGSC) and are associated with short relapse-free survival. LPA is known to support metastatic spread of cancer cells by activating a multitude of signaling pathways via G-protein-coupled receptors of the LPAR family. Systematic unbiased analyses of the LPA-regulated signal transduction network in ovarian cancer cells have, however, not been reported to date. Methods: LPA-induced signaling pathways were identified by phosphoproteomics of both patient-derived and OVCAR8 cells, RNA sequencing, measurements of intracellular Ca2+ and cAMP as well as cell imaging. The function of LPARs and downstream signaling components in migration and entosis were analyzed by selective pharmacological inhibitors and RNA interference. Results: Phosphoproteomic analyses identified > 1100 LPA-regulated sites in > 800 proteins and revealed interconnected LPAR1, ROCK/RAC, PKC/D and ERK pathways to play a prominent role within a comprehensive signaling network. These pathways regulate essential processes, including transcriptional responses, actomyosin dynamics, cell migration and entosis. A critical component of this signaling network is MYPT1, a stimulatory subunit of protein phosphatase 1 (PP1), which in turn is a negative regulator of myosin light chain 2 (MLC2). LPA induces phosphorylation of MYPT1 through ROCK (T853) and PKC/ERK (S507), which is majorly driven by LPAR1. Inhibition of MYPT1, PKC or ERK impedes both LPA-induced cell migration and entosis, while interference with ROCK activity and MLC2 phosphorylation selectively blocks entosis, suggesting that MYPT1 figures in both ROCK/MLC2-dependent and -independent pathways. We finally show a novel pathway governed by LPAR2 and the RAC-GEF DOCK7 to be indispensable for the induction of entosis. Conclusion: We have identified a comprehensive LPA-induced signal transduction network controlling LPA-triggered cytoskeletal changes, cell migration and entosis in HGSC cells. Due to its pivotal role in this network, MYPT1 may represent a promising target for interfering with specific functions of PP1 essential for HGSC progression.

Upregulation of KLF14 expression attenuates kidney fibrosis by inducing PPARα-mediated fatty acid oxidation

Tubulointerstitial fibrosis (TIF) is essential during the development of end-stage kidney disease (ESKD) and is associated with the impairment of fatty acid oxidation (FAO). Kruppel-like factor 14 (KLF14) is an important gene in lipid metabolism, but its role in TIF remains unknown. TGF-β-stimulated HK-2 cells and mouse unilateral ureteral obstruction (UUO) were used as renal fibrosis models. The role of KLF14 in the process of renal fibrosis was verified by gene knockout mice, genetic or pharmacological interference in animal model and cell model respectively. In the current study, we found that KLF14 expression increased after activation of the TGF-β signaling pathway during TIF. In KLF14−/− mice, more severe fibrosis was observed after unilateral ureteral obstruction (UUO) was induced. In human HK2 cells, knockdown of KLF14 led to more severe fibrosis induced by TGF-β1, while overexpression of KLF14 partially attenuated this process. Specifically, KLF14 deficiency decreased mitochondrial FAO activity, resulting in lipid accumulation. Thus, the energy supply to the cells was insufficient, finally resulting in TIF. We further proved that KLF14 could target peroxisome proliferator activated receptor alpha (PPARα) as a transcriptional activator. This study identified the upregulation of KLF14 expression in response to kidney stress during the process of fibrosis. Upon TIF, the activated TGF-β signaling pathway can enhance KLF14 expression, while the upregulation of KLF14 expression can decrease the degree of TIF by improving FAO activity in tubular epithelial cells and recovering the energy supply mediated by PPARα.

Autonomous rhythmic activity in glioma networks drives brain tumour growth

Diffuse gliomas, particularly glioblastomas, are incurable brain tumours1. They are characterized by networks of interconnected brain tumour cells that communicate via Ca2+ transients2-6. However, the networks' architecture and communication strategy and how these influence tumour biology remain unknown. Here we describe how glioblastoma cell networks include a small, plastic population of highly active glioblastoma cells that display rhythmic Ca2+ oscillations and are particularly connected to others. Their autonomous periodic Ca2+ transients preceded Ca2+ transients of other network-connected cells, activating the frequency-dependent MAPK and NF-κB pathways. Mathematical network analysis revealed that glioblastoma network topology follows scale-free and small-world properties, with periodic tumour cells frequently located in network hubs. This network design enabled resistance against random damage but was vulnerable to losing its key hubs. Targeting of autonomous rhythmic activity by selective physical ablation of periodic tumour cells or by genetic or pharmacological interference with the potassium channel KCa3.1 (also known as IK1, SK4 or KCNN4) strongly compromised global network communication. This led to a marked reduction of tumour cell viability within the entire network, reduced tumour growth in mice and extended animal survival. The dependency of glioblastoma networks on periodic Ca2+ activity generates a vulnerability7 that can be exploited for the development of novel therapies, such as with KCa3.1-inhibiting drugs.

RUNX1 isoform disequilibrium promotes the development of trisomy 21–associated myeloid leukemia

AbstractGain of chromosome 21 (Hsa21) is among the most frequent aneuploidies in leukemia. However, it remains unclear how partial or complete amplifications of Hsa21 promote leukemogenesis and why children with Down syndrome (DS) (ie, trisomy 21) are particularly at risk of leukemia development. Here, we propose that RUNX1 isoform disequilibrium with RUNX1A bias is key to DS-associated myeloid leukemia (ML-DS). Starting with Hsa21-focused CRISPR–CRISPR-associated protein 9 screens, we uncovered a strong and specific RUNX1 dependency in ML-DS cells. Expression of the RUNX1A isoform is elevated in patients with ML-DS, and mechanistic studies using murine ML-DS models and patient-derived xenografts revealed that excess RUNX1A synergizes with the pathognomonic Gata1s mutation during leukemogenesis by displacing RUNX1C from its endogenous binding sites and inducing oncogenic programs in complex with the MYC cofactor MAX. These effects were reversed by restoring the RUNX1A:RUNX1C equilibrium in patient-derived xenografts in vitro and in vivo. Moreover, pharmacological interference with MYC:MAX dimerization using MYCi361 exerted strong antileukemic effects. Thus, our study highlights the importance of alternative splicing in leukemogenesis, even on a background of aneuploidy, and paves the way for the development of specific and targeted therapies for ML-DS, as well as for other leukemias with Hsa21 aneuploidy or RUNX1 isoform disequilibrium.

Sulphide donors affect the expression of mucin and sulphide detoxification genes in the mucosal organs of Atlantic salmon (Salmo salar).

Hydrogen sulphide (H2S) is a gas that affects mucosal functions in mammals. However, its detrimental effects are less understood in fish despite being known to cause mass mortality. Here we used explant models to demonstrate the transcriptional responses of Atlantic salmon (Salmo salar) mucosa to the sulphide donor sodium hydrosulphide (NaHS). The study focused on two groups of genes: those encoding for sulphide detoxification and those for mucins. Moreover, we performed pharmacological studies by exposing the organ explants to mucus-interfering compounds and consequently exposed them to a sulphide donor. Exposure to NaHS significantly affected the expression of sulphide:quinone oxidoreductase (sqor1, sqor2) and mucin-encoding genes (muc5ac, muc5b). The general profile indicated that NaHS upregulated the expression of sulphide detoxification genes while a significant downregulation was observed with mucins. These expression profiles were seen in both organ explant models. Pharmacological stimulation and inhibition of mucus production used acetylcholine (ACh) and niflumic acid (NFA), respectively. This led to a significant regulation of the two groups of marker genes in the gills and olfactory rosette explants. Treatment of the mucosal organ explants with the mucus-interfering compounds showed that low dose NFA triggered more substantial changes while a dose-dependent response could not be established with ACh. Pharmacological interference demonstrated that mucins played a crucial role in mucosal protection against H2S toxicity. These results offer insights into how a sulphide donor interfered with mucosal responses of Atlantic salmon and are expected to contribute to our understanding of the least explored H2S-fish interactions-particularly at the mucosa.

Interference with Lipoprotein Maturation Sensitizes Methicillin-Resistant Staphylococcus aureus to Human Group IIA-Secreted Phospholipase A2 and Daptomycin

Methicillin-resistant Staphylococcus aureus (MRSA) has been classified as a high priority pathogen by the World Health Organization underlining the high demand for new therapeutics to treat infections. Human group IIA-secreted phospholipase A2 (hGIIA) is among the most potent bactericidal proteins against Gram-positive bacteria, including S. aureus. To determine hGIIA-resistance mechanisms of MRSA, we screened the Nebraska Transposon Mutant Library using a sublethal concentration of recombinant hGIIA. We identified and confirmed the role of lspA, encoding the lipoprotein signal peptidase LspA, as a new hGIIA resistance gene in both in vitro assays and an infection model in hGIIA-transgenic mice. Increased susceptibility of the lspA mutant was associated with enhanced activity of hGIIA on the cell membrane. Moreover, lspA deletion increased susceptibility to daptomycin, a last-resort antibiotic to treat MRSA infections. MRSA wild type could be sensitized to hGIIA and daptomycin killing through exposure to LspA-specific inhibitors globomycin and myxovirescin A1. Analysis of >26,000 S. aureus genomes showed that LspA is highly sequence-conserved, suggesting universal application of LspA inhibition. The role of LspA in hGIIA resistance was not restricted to MRSA since Streptococcus mutans and Enterococcus faecalis were also more hGIIA-susceptible after lspA deletion or LspA inhibition, respectively. Overall, our data suggest that pharmacological interference with LspA may disarm Gram-positive pathogens, including MRSA, to enhance clearance by innate host defense molecules and clinically applied antibiotics.

What evidence is there for implicating the brain orexin system in neuropsychiatric symptoms in dementia?

Neuropsychiatric symptoms (NPS) affect people with dementia (PwD) almost universally across all stages of the disease, and regardless of its exact etiology. NPS lead to disability and reduced quality of life of PwD and their caregivers. NPS include hyperactivity (agitation and irritability), affective problems (anxiety and depression), psychosis (delusions and hallucinations), apathy, and sleep disturbances. Preclinical studies have shown that the orexin neuropeptide system modulates arousal and a wide range of behaviors via a network of axons projecting from the hypothalamus throughout almost the entire brain to multiple, even distant, regions. Orexin neurons integrate different types of incoming information (e.g., metabolic, circadian, sensory, emotional) and convert them into the required behavioral output coupled to the necessary arousal status. Here we present an overview of the behavioral domains influenced by the orexin system that may be relevant for the expression of some critical NPS in PwD. We also hypothesize on the potential effects of pharmacological interference with the orexin system in the context of NPS in PwD.