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New 2-pyrrolamidobenzothiazole-based inhibitors of mycobacterial DNA gyrase were discovered. Among these, compounds 49 and 51, show excellent antibacterial activity against Mycobacterium tuberculosis and Mycobacterium abscessus with a notable preference for mycobacteria. Both compounds can penetrate infected macrophages and reduce intracellular M. tuberculosis load. Compound 51 is a potent inhibitor of DNA gyrase (M. tuberculosis DNA gyrase IC 50  = 4.1 nM, Escherichia coli DNA gyrase IC 50 of <10 nM), selective for bacterial topoisomerases. It displays low MIC 90 values (M. tuberculosis: 0.63 μM; M. abscessus: 2.5 μM), showing specificity for mycobacteria, and no apparent toxicity. Compound 49 not only displays potent antimycobacterial activity (MIC 90 values of 2.5 μM for M. tuberculosis and 0.63 μM for M. abscessus) and selectivity for mycobacteria but also exhibits favorable solubility (kinetic solubility = 55 μM) and plasma protein binding (with a fraction unbound of 2.9 % for human and 4.7 % for mouse). These findings underscore the potential of fine-tuning molecular properties to develop DNA gyrase B inhibitors that specifically target the mycobacterial chemical space, mitigating the risk of resistance development in non-target pathogens and minimizing harm to the microbiome., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Masson SAS.)

The KCNH family of potassium channels serves relevant physiological functions in both excitable and non-excitable cells, reflected in the massive consequences of mutations or pharmacological manipulation of their function. This group of channels shares structural homology with other voltage-gated K + channels, but the mechanisms of gating in this family show significant differences with respect to the canonical electromechanical coupling in these molecules. In particular, the large intracellular domains of KCNH channels play a crucial role in gating that is still only partly understood. Using KCNH1 (K V 10.1) as a model, we have characterized the behavior of a series of modified channels that could not be explained by the current models. With electrophysiological and biochemical methods combined with mathematical modeling, we show that the uncovering of an open state can explain the behavior of the mutants. This open state, which is not detectable in wild-type channels, appears to lack the rapid flicker block of the conventional open state. Because it is accessed from deep closed states, it elucidates intermediate gating events well ahead of channel opening in the wild type. This allowed us to study gating steps prior to opening, which, for example, explain the mechanism of gating inhibition by Ca 2+ -Calmodulin and generate a model that describes the characteristic features of KCNH channels gating., Competing Interests: RA, AT, AN, LP No competing interests declared, (© 2023, Abdelaziz et al.)

Background and Objectives: Medical registries evolved from a basic epidemiological data set to further applications allowing deriving decision making. Revision rates after non-traumatic amputation are high and dramatically impact the following rehabilitation of the amputee. Risk scores for revision surgery after non-traumatic lower limb amputation are still missing. The main objective was to create an amputation registry allowing us to determine risk factors for revision surgery after non-traumatic lower-limb amputation and to develop a score for an early detection and decision-making tool for the therapeutic course of patients at risk for non-traumatic lower limb amputation and/or revision surgery. Materials and Methods : Retrospective data analysis was of patients with major amputations lower limbs in a four-year interval at a University Hospital of maximum care. Medical records of 164 patients analysed demographics, comorbidities, and amputation-related factors. Descriptive statistics analysed demographics, prevalence of amputation level and comorbidities of non-traumatic lower limb amputees with and without revision surgery. Correlation analysis identified parameters determining revision surgery. Results: In 4 years, 199 major amputations were performed; 88% were amputated for non-traumatic reasons. A total of 27% of the non-traumatic cohort needed revision surgery. Peripheral vascular disease (PVD) (72%), atherosclerosis (69%), diabetes (42%), arterial hypertension (38%), overweight (BMI > 25), initial gangrene (47%), sepsis (19%), age > 68.2 years and nicotine abuse (17%) were set as relevant within this study and given a non-traumatic amputation score. Correlation analysis revealed delayed wound healing (confidence interval: 64.1% (47.18%; 78.8%)), a hospital length of stay before amputation of longer than 32 days (confidence interval: 32.3 (23.2; 41.3)), and a BKA amputation level (confidence interval: 74.4% (58%; 87%)) as risk factors for revision surgery after non-traumatic amputation. A combined score including all parameters was drafted to identify non-traumatic amputees at risk for revision surgery. Conclusions : Our results describe novel scoring systems for risk assessment for non-traumatic amputations and for revision surgery at non-traumatic amputations. It may be used after further prospective evaluation as an early-warning system for amputated limbs at risk of revision.

Voltage-gated potassium channel K V 1.3 inhibitors have been shown to be effective in preventing T-cell proliferation and activation by affecting intracellular Ca 2+ homeostasis. Here, we present the structure-activity relationship, K V 1.3 inhibition, and immunosuppressive effects of new thiophene-based K V 1.3 inhibitors with nanomolar potency on K + current in T-lymphocytes and K V 1.3 inhibition on Ltk - cells. The new K V 1.3 inhibitor trans-18 inhibited K V 1.3 -mediated current in phytohemagglutinin (PHA)-activated T-lymphocytes with an IC 50 value of 26.1 nM and in mammalian Ltk - cells with an IC 50 value of 230 nM. The K V 1.3 inhibitor trans-18 also had nanomolar potency against K V 1.3 in Xenopus laevis oocytes (IC 50  = 136 nM). The novel thiophene-based K V 1.3 inhibitors impaired intracellular Ca 2+ signaling as well as T-cell activation, proliferation, and colony formation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The immune response against a tumor is characterized by the interplay among components of the immune system and neoplastic cells. Here, we bioprinted a model with two distinct regions containing gastric cancer patient-derived organoids (PDOs) and tumor-infiltrated lymphocytes (TILs). The initial cellular distribution allows for the longitudinal study of TIL migratory patterns concurrently with multiplexed cytokine analysis. The chemical properties of the bioink were designed to present physical barriers that immune T-cells must breech during infiltration and migration toward a tumor with the use of an alginate, gelatin, and basal membrane mix. TIL activity, degranulation, and regulation of proteolytic activity reveal insights into the time-dependent biochemical dynamics. Regulation of the sFas and sFas-ligand present on PDOs and TILs, respectively, and the perforin and granzyme longitudinal secretion confirms TIL activation when encountering PDO formations. TIL migratory profiles were used to create a deterministic reaction-advection diffusion model. The simulation provides insights that decouple passive from active cell migration mechanisms. The mechanisms used by TILs and other adoptive cell therapeutics as they infiltrate the tumor barrier are poorly understood. This study presents a pre-screening strategy for immune cells where motility and activation across ECM environments are crucial indicators of cellular fitness.

Competing Interests: L.A.P. holds the position of Editorial Board Member for Function and is blinded from reviewing or making decisions for the manuscript.

Constant matrix remodeling and cellular heterogeneity in cancer are key contributors to its development and can profoundly alter treatment efficacy. Developing in-vitro models containing relevant features that can recapitulate these aspects of the tumor microenvironment and that are well characterized can circumvent the limitations of conventional 2D cultures and animal models. Automated fabrication methods combined with biomimetic biomaterials have provided the opportunity to create platforms that can potentially incorporate a heterogeneous population of cells in a 3D environment that allows cell-cell and cell-ECM interactions with reproducibility. This study used 3D extrusion bioprinting and a composite bioink containing a reinforced decellularized extracellular matrix (ECM) hydrogel to fabricate a head and neck cancer in-vitro model. The constituents of this model included fibroblasts and active ECM proteins to represent the stroma, along with HNSCC cells to represent the tumor component. The topographical characterization of the bioink showed a fibrous network with nanometer-sized pores. After cell encapsulation and model fabrication, we observed spheroid development and growth over time with cancer cells in the core and fibroblasts in the periphery. Our model is compatible with matrix metalloproteinase (MMP) quantification techniques and showed significant differences in the presence of MMP-9 and MMP-10 compared to the control groups. This characterized model is proposed as a tool for further translational and drug discovery applications since it provides a biomimetic scenario that allows the study of the tumor microenvironment in-vitro using nondestructive longitudinal monitoring over time., (Creative Commons Attribution license.)

The mechanisms linking tumor microenvironment acidosis to disease progression are not understood. Here, we used mammary, pancreatic, and colon cancer cells to show that adaptation to growth at an extracellular pH (pH e ) mimicking acidic tumor niches is associated with upregulated net acid extrusion capacity and elevated intracellular pH at physiological pH e , but not at acidic pH e . Using metabolic profiling, shotgun lipidomics, imaging and biochemical analyses, we show that the acid adaptation-induced phenotype is characterized by a shift toward oxidative metabolism, increased lipid droplet-, triacylglycerol-, peroxisome content and mitochondrial hyperfusion. Peroxisome proliferator-activated receptor-α (PPARA, PPARα) expression and activity are upregulated, at least in part by increased fatty acid uptake. PPARα upregulates genes driving increased mitochondrial and peroxisomal mass and β-oxidation capacity, including mitochondrial lipid import proteins CPT1A, CPT2 and SLC25A20, electron transport chain components, peroxisomal proteins PEX11A and ACOX1, and thioredoxin-interacting protein (TXNIP), a negative regulator of glycolysis. This endows acid-adapted cancer cells with increased capacity for utilizing fatty acids for metabolic needs, while limiting glycolysis. As a consequence, the acid-adapted cells exhibit increased sensitivity to PPARα inhibition. We conclude that PPARα is a key upstream regulator of metabolic changes favoring cancer cell survival in acidic tumor niches., (© 2022 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Finely tuned regulation of transport protein localization is vital for epithelial function. The Na+-HCO3- co-transporter NBCn1 (also known as SLC4A7) is a key contributor to epithelial pH homeostasis, yet the regulation of its subcellular localization is not understood. Here, we show that a predicted N-terminal β-sheet and short C-terminal α-helical motif are essential for NBCn1 plasma membrane localization in epithelial cells. This localization was abolished by cell-cell contact disruption, and co-immunoprecipitation (co-IP) and proximity ligation (PLA) revealed NBCn1 interaction with E-cadherin and DLG1, linking it to adherens junctions and the Scribble complex. NBCn1 also interacted with RhoA and localized to lamellipodia and filopodia in migrating cells. Finally, analysis of native and GFP-tagged NBCn1 localization, subcellular fractionation, co-IP with Arl13B and CEP164, and PLA of NBCn1 and tubulin in mitotic spindles led to the surprising conclusion that NBCn1 additionally localizes to centrosomes and primary cilia in non-dividing, polarized epithelial cells, and to the spindle, centrosomes and midbodies during mitosis. We propose that NBCn1 traffics between lateral junctions, the leading edge and cell division machinery in Rab11 endosomes, adding new insight to the role of NBCn1 in cell cycle progression., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

A new series of dual low nanomolar benzothiazole inhibitors of bacterial DNA gyrase and topoisomerase IV were developed. The resulting compounds show excellent broad-spectrum antibacterial activities against Gram-positive Enterococcus faecalis , Enterococcus faecium and multidrug resistant (MDR) Staphylococcus aureus strains [best compound minimal inhibitory concentrations (MICs): range, <0.03125-0.25 μg/mL] and against the Gram-negatives Acinetobacter baumannii and Klebsiella pneumoniae (best compound MICs: range, 1-4 μg/mL). Lead compound 7a was identified with favorable solubility and plasma protein binding, good metabolic stability, selectivity for bacterial topoisomerases, and no toxicity issues. The crystal structure of 7a in complex with Pseudomonas aeruginosa GyrB24 revealed its binding mode at the ATP-binding site. Expanded profiling of 7a and 7h showed potent antibacterial activity against over 100 MDR and non-MDR strains of A. baumannii and several other Gram-positive and Gram-negative strains. Ultimately, in vivo efficacy of 7a in a mouse model of vancomycin-intermediate S. aureus thigh infection was also demonstrated.

Malignant tumor tissues exhibit inter- and intratumoral heterogeneities, aberrant development, dynamic stromal composition, diverse tissue phenotypes, and cell populations growing within localized mechanical stresses in hypoxic conditions. Experimental tumor models employing engineered systems that isolate and study these complex variables using in vitro techniques are under development as complementary methods to preclinical in vivo models. Here, advances in extrusion bioprinting as an enabling technology to recreate the three-dimensional tumor milieu and its complex heterogeneous characteristics are reviewed. Extrusion bioprinting allows for the deposition of multiple materials, or selected cell types and concentrations, into models based upon physiological features of the tumor. This affords the creation of complex samples with representative extracellular or stromal compositions that replicate the biology of patient tissue. Biomaterial engineering of printable materials that replicate specific features of the tumor microenvironment offer experimental reproducibility, throughput, and physiological relevance compared to animal models. In this review, we describe the potential of extrusion-based bioprinting to recreate the tumor microenvironment within in vitro models.

Tissue development, wound healing, pathogenesis, regeneration, and homeostasis rely upon coordinated and dynamic spatial and temporal remodeling of extracellular matrix (ECM) molecules. ECM reorganization and normal physiological tissue function, require the establishment and maintenance of biological, chemical, and mechanical feedback mechanisms directed by cell-matrix interactions. To replicate the physical and biological environment provided by the ECM in vivo , methods have been developed to decellularize and solubilize tissues which yield organ and tissue-specific bioactive hydrogels. While these biomaterials retain several important traits of the native ECM, the decellularizing process, and subsequent sterilization, and solubilization result in fragmented, cleaved, or partially denatured macromolecules. The final product has decreased viscosity, moduli, and yield strength, when compared to the source tissue, limiting the compatibility of isolated decellularized ECM (dECM) hydrogels with fabrication methods such as extrusion bioprinting. This review describes the physical and bioactive characteristics of dECM hydrogels and their role as biomaterials for biofabrication. In this work, critical variables when selecting the appropriate tissue source and extraction methods are identified. Common manual and automated fabrication techniques compatible with dECM hydrogels are described and compared. Fabrication and post-manufacturing challenges presented by the dECM hydrogels decreased mechanical and structural stability are discussed as well as circumvention strategies. We further highlight and provide examples of the use of dECM hydrogels in tissue engineering and their role in fabricating complex in vitro 3D microenvironments.

Vibrotactile sensation is an essential part of the sense of touch. In this study, the localized vibrotactile sensation of the arm-shoulder region was quantified in 10 able-bodied subjects. For this analysis, the six relevant dermatomes (C3-T2) and three segments-the lower arm, the upper arm, and the shoulder region were studied. For psychometric evaluation, tasks resulting in the quantification of sensation threshold, just noticeable difference, Weber fraction, and perception of dynamically changing vibrotactile stimuli were performed. We found that healthy subjects could reliably detect vibration in all tested regions at low amplitude (2-6% of the maximal amplitude of commonly used vibrotactors). The detection threshold was significantly lower in the lower arm than that in the shoulder, as well as ventral in comparison with the dorsal. There were no significant differences in Weber fraction (20%) detectable between the studied locations. A compensatory tracking task resulted in a significantly higher average rectified error in the shoulder than that in the upper arm, while delay and correlation coefficient showed no difference between the regions. Here, we presented a conclusive map of the vibrotactile sense of the healthy upper limb. These data give an overview of the sensory bandwidth that can be achieved with vibrotactile stimulation at the arm and may help in the design of vibrotactile feedback interfaces (displays) for the hand/arm/shoulder-region., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pardo, Markovic, Schilling, Wilke and Ernst.)

Expression of the voltage-gated potassium channel K V 10.1 (Eag1) has been detected in over 70% of human cancers, making the channel a promising new target for new anticancer drug discovery. A new structural class of K V 10.1 inhibitors was prepared by structural optimisation and exploration of the structure-activity relationship of the previously published hit compound ZVS-08 ( 1 ) and its optimised analogue 2 . The potency and selectivity of the new inhibitors between K V 10.1 and hERG were investigated using whole-cell patch-clamp experiments. We obtained two new optimised K V 10.1 inhibitors, 17a and 18b , with improved nanomolar IC 50 values of 568 nM and 214 nM, respectively. Compound 17a exhibited better ratio between IC 50 values for hEAG1 and hERG than previously published diarylamine inhibitors. Compounds 17a and 18b moderately inhibited the growth of the K V 10.1-expressing cell line MCF-7 in two independent assays. In addition, 17a and 18b also inhibited the growth of hERG-expressing Panc-1 cells with higher potency compared with MCF-7 cells. The main obstacle for newly developed diarylamine K V 10.1 inhibitors remains the selectivity toward the hERG channel, which needs to be addressed with targeted drug design strategies in the future.

Airway mucociliary regeneration and function are key players for airway defense and are impaired in chronic obstructive pulmonary disease (COPD). Using transcriptome analysis in COPD-derived bronchial biopsies, we observed a positive correlation between cilia-related genes and microRNA-449 ( miR449) . In vitro, miR449 was strongly increased during airway epithelial mucociliary differentiation. In vivo, miR449 was upregulated during recovery from chemical or infective insults. miR0449 -/- mice (both alleles are deleted) showed impaired ciliated epithelial regeneration after naphthalene and Haemophilus influenzae exposure, accompanied by more intense inflammation and emphysematous manifestations of COPD. The latter occurred spontaneously in aged miR449 -/- mice. We identified Aurora kinase A and its effector target HDAC6 as key mediators in miR449 -regulated ciliary homeostasis and epithelial regeneration. Aurora kinase A is downregulated upon miR449 overexpression in vitro and upregulated in miR449 -/- mouse lungs. Accordingly, imaging studies showed profoundly altered cilia length and morphology accompanied by reduced mucociliary clearance. Pharmacological inhibition of HDAC6 rescued cilia length and coverage in miR449 -/- cells, consistent with its tubulin-deacetylating function. Altogether, our study establishes a link between miR449 , ciliary dysfunction, and COPD pathogenesis.

Bioelectricity goes far beyond electrical signaling in the nervous system, but this was initially not obvious for me. This article describes the journey from studying the biophysics of ion channels in classical electrically excitable tissues to focusing on the pathogenic roles of the Kv10.1 potassium channel in cancers., Competing Interests: No competing financial interests exist., (© Luis A. Pardo 2023; Published by Mary Ann Liebert, Inc.)

The voltage-gated potassium channel K V 1.3 has been recognized as a tumor marker and represents a promising new target for the discovery of new anticancer drugs. We designed a novel structural class of K V 1.3 inhibitors through structural optimization of benzamide-based hit compounds and structure-activity relationship studies. The potency and selectivity of the new K V 1.3 inhibitors were investigated using whole-cell patch- and voltage-clamp experiments. 2D and 3D cell models were used to determine antiproliferative activity. Structural optimization resulted in the most potent and selective K V 1.3 inhibitor 44 in the series with an IC 50 value of 470 nM in oocytes and 950 nM in Ltk - cells. K V 1.3 inhibitor 4 induced significant apoptosis in Colo-357 spheroids, while 14 , 37 , 43 , and 44 significantly inhibited Panc-1 proliferation.

Dengue virus (DENV) disease has become one of the major challenges in public health. Currently, there is no antiviral treatment for this infection. Since human transmission occurs via mosquitoes of the Aedes genus, most efforts have been focused on the control of this vector. However, these control strategies have not been totally successful, as reflected in the increasing number of DENV infections per year, becoming an endemic disease in more than 100 countries worldwide. Consequently, the development of a safe antiviral agent is urgently needed. In this sense, rational design approaches have been applied in the development of antiviral compounds that inhibit one or more steps in the viral replication cycle. The entry of viruses into host cells is an early and specific stage of infection. Targeting either viral components or cellular protein targets are an affordable and effective strategy for therapeutic intervention of viral infections. This review provides an extensive overview of the small organic molecules, peptides, and inorganic moieties that have been tested so far as DENV entry direct-acting antiviral agents. The latest advances based on computer-aided drug design (CADD) strategies and traditional medicinal chemistry approaches in the design and evaluation of DENV virus entry inhibitors will be discussed. Furthermore, physicochemical drug properties, such as solubility, lipophilicity, stability, and current results of pre-clinical and clinical studies will also be discussed in detail., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Two decades of research have proven the relevance of ion channel expression for tumor progression in virtually every indication, and it has become clear that inhibition of specific ion channels will eventually become part of the oncology therapeutic arsenal. However, ion channels play relevant roles in all aspects of physiology, and specificity for the tumor tissue remains a challenge to avoid undesired effects. Eag1 (K V 10.1) is a voltage-gated potassium channel whose expression is very restricted in healthy tissues outside of the brain, while it is overexpressed in 70% of human tumors. Inhibition of Eag1 reduces tumor growth, but the search for potent inhibitors for tumor therapy suffers from the structural similarities with the cardiac HERG channel, a major off-target. Existing inhibitors show low specificity between the two channels, and screenings for Eag1 binders are prone to enrichment in compounds that also bind HERG. Rational drug design requires knowledge of the structure of the target and the understanding of structure-function relationships. Recent studies have shown subtle structural differences between Eag1 and HERG channels with profound functional impact. Thus, although both targets' structure is likely too similar to identify leads that exclusively bind to one of the channels, the structural information combined with the new knowledge of the functional relevance of particular residues or areas suggests the possibility of selective targeting of Eag1 in cancer therapies. Further development of selective Eag1 inhibitors can lead to first-in-class compounds for the treatment of different cancers., (© 2021 The Authors. Medicinal Research Reviews Published by Wiley Periodicals LLC.)

Levan is a high-valued polysaccharide of fructose produced by several microbial species. These polysaccharides have been described as effective therapeutic agents in some human disease conditions, such as cancer, heart diseases and diabetes. The objective of this study was to examine the effect of levan (β-(2 → 6)-fructan) produced through sucrose fermentation by B. subtilis var. natto on the proliferation rate, cytotoxicity, and apoptosis of human neuroblastoma SH-SY5Y cells. It was obtained 41.44 g/L of levan in 18 h by biotechnological fermentation and SH-SY5Y cells were exposed to 1000 μg/mL of levan. The treatment with 1000 μg/mL of levan induced apoptosis in SH-SY5Y cancer cells by the significant increase in Annexin V/7-AAD and caspase 3/7 activation, but did not decrease proliferation or triggered a cytotoxic effect. 1000 μg/mL levan treatment is a promising therapeutic strategy for SH-SY5Y neuroblastoma cells., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The K V 10.1 voltage-gated potassium channel is highly expressed in 70% of tumors, and thus represents a promising target for anticancer drug discovery. However, only a few ligands are known to inhibit K V 10.1, and almost all also inhibit the very similar cardiac hERG channel, which can lead to undesirable side-effects. In the absence of the structure of the K V 10.1-inhibitor complex, there remains the need for new strategies to identify selective K V 10.1 inhibitors and to understand the binding modes of the known K V 10.1 inhibitors. To investigate these binding modes in the central cavity of K V 10.1, a unique approach was used that allows derivation and analysis of ligand-protein interactions from molecular dynamics trajectories through pharmacophore modeling. The final molecular dynamics-derived structure-based pharmacophore model for the simulated K V 10.1-ligand complexes describes the necessary pharmacophore features for K V 10.1 inhibition and is highly similar to the previously reported ligand-based hERG pharmacophore model used to explain the nonselectivity of K V 10.1 pore blockers. Moreover, analysis of the molecular dynamics trajectories revealed disruption of the π-π network of aromatic residues F359, Y464, and F468 of K V 10.1, which has been reported to be important for binding of various ligands for both K V 10.1 and hERG channels. These data indicate that targeting the K V 10.1 channel pore is also likely to result in undesired hERG inhibition, and other potential binding sites should be explored to develop true K V 10.1-selective inhibitors as new anticancer agents.

The K V 1.3 voltage-gated potassium ion channel is involved in many physiological processes both at the plasma membrane and in the mitochondria, chiefly in the immune and nervous systems. Therapeutic targeting K V 1.3 with specific peptides and small molecule inhibitors shows great potential for treating cancers and autoimmune diseases, such as multiple sclerosis, type I diabetes mellitus, psoriasis, contact dermatitis, rheumatoid arthritis, and myasthenia gravis. However, no K V 1.3-targeted compounds have been approved for therapeutic use to date. This review focuses on the presentation of approaches for discovering new K V 1.3 peptide and small-molecule inhibitors, and strategies to improve the selectivity of active compounds toward K V 1.3. Selectivity of dalatazide (ShK-186), a synthetic derivate of the sea anemone toxin ShK, was achieved by chemical modification and has successfully reached clinical trials as a potential therapeutic for treating autoimmune diseases. Other peptides and small-molecule inhibitors are critically evaluated for their lead-like characteristics and potential for progression into clinical development. Some small-molecule inhibitors with well-defined structure-activity relationships have been optimized for selective delivery to mitochondria, and these offer therapeutic potential for the treatment of cancers. This overview of K V 1.3 inhibitors and methodologies is designed to provide a good starting point for drug discovery to identify novel effective K V 1.3 modulators against this target in the future., (© 2021 The Authors. Medicinal Research Reviews published by Wiley Periodicals LLC.)

Purpose: Exome- and whole-genome sequencing of muscle-invasive bladder cancer has revealed important insights into the molecular landscape; however, there are few studies of non-muscle-invasive bladder cancer with detailed risk factor information., Experimental Design: We examined the relationship between smoking and other bladder cancer risk factors and somatic mutations and mutational signatures in bladder tumors. Targeted sequencing of frequently mutated genes in bladder cancer was conducted in 322 formalin-fixed paraffin-embedded bladder tumors from a population-based case-control study. Logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (CI), evaluating mutations and risk factors. We used SignatureEstimation to extract four known single base substitution mutational signatures and Poisson regression to calculate risk ratios (RR) and 95% CIs, evaluating signatures and risk factors., Results: Non-silent KDM6A mutations were more common in females than males (OR = 1.83; 95% CI, 1.05-3.19). There was striking heterogeneity in the relationship between smoking status and established single base substitution signatures: current smoking status was associated with greater ERCC2- Signature mutations compared with former ( P = 0.024) and never smoking (RR = 1.40; 95% CI, 1.09-1.80; P = 0.008), former smoking was associated with greater APOBEC-Signature13 mutations ( P = 0.05), and never smoking was associated with greater APOBEC-Signature2 mutations (RR = 1.54; 95% CI, 1.17-2.01; P = 0.002). There was evidence that smoking duration (the component most strongly associated with bladder cancer risk) was associated with ERCC2- Signature mutations and APOBEC-Signature13 mutations among current ( P trend = 0.005) and former smokers ( P = 0.0004), respectively., Conclusions: These data quantify the contribution of bladder cancer risk factors to mutational burden and suggest different signature enrichments among never, former, and current smokers., (©2021 American Association for Cancer Research.)

Twin studies suggest a familial aggregation of bladder cancer, but elements of this increased familial risk of bladder cancer are not well understood. To characterize familial risk of bladder cancer, we examined the relationship between family history of bladder and other types of cancer among first-degree relatives and risk of bladder cancer in 1193 bladder cancer cases and 1418 controls in a large population-based case-control study. Multivariate logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the association between family history of bladder cancer (defined as at least one first-degree family member with bladder cancer or a cancer of any other site). We also evaluated cancer aggregation of specific sites in family members. Participants with a first-degree relative with bladder cancer had nearly double the risk of bladder cancer (OR = 1.8, 95% CI 1.2-2.9) as those without a family history of bladder cancer. Risk was increased for having a sibling with bladder cancer (OR = 2.6, 95% CI 1.3-5.3) compared to no siblings with cancer. Bladder cancer risk was elevated when participants reported a first-degree relative with a history of female genital cancer (OR = 1.5, 95% CI 1.1-2.1), melanoma (OR = 1.9, 95% CI 1.02-3.6), and tobacco-associated cancer (OR = 1.3, 95% CI 1.06-1.6). These findings add to evidence of a familial predisposition to bladder cancer. Clarification of the aggregation of bladder cancer in families and with other cancer sites will be of interest as many loci and common polymorphisms related to bladder cancer have yet to be identified in large genomic studies., (© 2021 UICC.)

Transport of ions and nutrients is a core mitochondrial function, without which there would be no mitochondrial metabolism and ATP production. Both ion homeostasis and mitochondrial phenotype undergo pervasive changes during cancer development, and both play key roles in driving the malignancy. However, the link between these events has been largely ignored. This review comprehensively summarizes and critically discusses the role of the reciprocal relationship between ion transport and mitochondria in crucial cellular functions, including metabolism, signaling, and cell fate decisions. We focus on Ca 2+ , H + , and K + , which play essential and highly interconnected roles in mitochondrial function and are profoundly dysregulated in cancer. We describe the transport and roles of these ions in normal mitochondria, summarize the changes occurring during cancer development, and discuss how they might impact tumorigenesis.

(1) Background: The voltage-gated potassium channel K V 10.1 (Eag1) is considered a near- universal tumour marker and represents a promising new target for the discovery of novel anticancer drugs. (2) Methods: We utilized the ligand-based drug discovery methodology using 3D pharmacophore modelling and medicinal chemistry approaches to prepare a novel structural class of K V 10.1 inhibitors. Whole-cell patch clamp experiments were used to investigate potency, selectivity, kinetics and mode of inhibition. Anticancer activity was determined using 2D and 3D cell-based models. (3) Results: The virtual screening hit compound ZVS-08 discovered by 3D pharmacophore modelling exhibited an IC 50 value of 3.70 µM against K V 10.1 and inhibited the channel in a voltage-dependent manner consistent with the action of a gating modifier. Structural optimization resulted in the most potent K V 10.1 inhibitor of the series with an IC 50 value of 740 nM, which was potent on the MCF-7 cell line expressing high K V 10.1 levels and low hERG levels, induced significant apoptosis in tumour spheroids of Colo-357 cells and was not mutagenic. (4) Conclusions: Computational ligand-based drug design methods can be successful in the discovery of new potent K V 10.1 inhibitors. The main problem in the field of K V 10.1 inhibitors remains selectivity against the hERG channel, which needs to be addressed in the future also with target-based drug design methods.

Heterozygous mutations in the ATP1A3 gene, coding for an alpha subunit isoform (α3) of Na + /K + -ATPase, are the primary genetic cause for rapid-onset dystonia-parkinsonism (RDP) and alternating hemiplegia of childhood (AHC). Recently, cerebellar ataxia, areflexia, pes cavus, optic atrophy and sensorineural hearing loss (CAPOS), early infantile epileptic encephalopathy (EIEE), childhood rapid onset ataxia (CROA) and relapsing encephalopathy with rapid onset ataxia (RECA) extend the clinical spectrum of ATP1A3 related disorders. AHC and RDP demonstrate distinct clinical features, with AHC symptoms being generally more severe compared to RDP. Currently, it is largely unknown what determines the disease severity, and whether severity is linked to the degree of functional impairment of the α3 subunit. Here we compared the effect of twelve different RDP and AHC specific mutations on the expression and function of the α3 Na + /K + -ATPase in transfected HEK cells and oocytes. All studied mutations led to functional impairment of the pump, as reflected by lower survival rate and reduced pump current. No difference in the extent of impairment, nor in the expression level, was found between the two phenotypes, suggesting that these measures of pump dysfunction do not exclusively determine the disease severity., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Kv10.1 (potassium voltage-gated channel subfamily H member 1, known as EAG1 or Ether-à-go-go 1), is a voltage-gated potassium channel, prevailingly expressed in the central nervous system. The aberrant expression of Kv10.1 is detected in over 70% of all human tumor tissues and correlates with poorer prognosis. In peripheral tissues, Kv10.1 is expressed almost exclusively during the G2/M phase of the cell cycle and regulates its progression-downregulation of Kv10.1 extends the duration of the G2/M phase both in cancer and healthy cells. Here, using biochemical and imaging techniques, such as live-cell measurements of microtubule growth and of cytosolic calcium, we elucidate the mechanisms of Kv10.1-mediated regulation at the G2/M phase. We show that Kv10.1 has a dual effect on mitotic microtubule dynamics. Through the functional interaction with ORAI1 (calcium release-activated calcium channel protein 1), it modulates cytosolic calcium oscillations, thereby changing microtubule behavior. The inhibition of either Kv10.1 or ORAI1 stabilizes the microtubules. In contrast, the knockdown of Kv10.1 increases the dynamicity of mitotic microtubules, resulting in a stronger spindle assembly checkpoint, greater mitotic spindle angle, and a decrease in lagging chromosomes. Understanding of Kv10.1-mediated modulation of the microtubule architecture will help to comprehend how cancer tissue benefits from the presence of Kv10.1, and thereby increase the efficacy and safety of Kv10.1-directed therapeutic strategies.

Antibody-based therapies hold promise for a safe and efficient treatment of cancer. The identification of target tumor cells through a specific antigen enriched on their surface and the subsequent delivery of the therapeutic agent only to those cells requires, besides the efficacy of the therapeutic agent itself, the identification of an antigen enriched on the surface of tumor cells, the generation of high affinity antibodies against that antigen. We have generated single-domain antibodies (nanobodies) against the voltage-gated potassium channel Kv10.1, which outside of the brain is detectable almost exclusively in tumor cells. The nanobody with highest affinity was fused to an improved form of the tumor necrosis factor-related apoptosis inducing ligand TRAIL, to target this cytokine to the surface of tumor cells. The resulting construct, VHH-D9-scTRAIL, shows rapid and strong apoptosis induction in different tumor models in cell culture. The construct combines two sources of specificity, the expression of the antigen restricted to tumor cells and the tumor selectivity of TRAIL. Such specificity combined with the high affinity obtained through nanobodies make the novel agent a promising concept for cancer therapy., (Copyright © 2020 Hartung, Krüwel, Shi, Pfizenmaier, Kontermann, Chames, Alves and Pardo.)

EAG ( ether-à-go-go or KCNH ) are a subfamily of the voltage-gated potassium (Kv) channels. Like for all potassium channels, opening of EAG channels drives the membrane potential toward its equilibrium value for potassium, thus setting the resting potential and repolarizing action potentials. As voltage-dependent channels, they switch between open and closed conformations (gating) when changes in membrane potential are sensed by a voltage sensing domain (VSD) which is functionally coupled to a pore domain (PD) containing the permeation pathway, the potassium selectivity filter, and the channel gate. All Kv channels are tetrameric, with four VSDs formed by the S1-S4 transmembrane segments of each subunit, surrounding a central PD with the four S5-S6 sections arranged in a square-shaped structure. Structural information, mutagenesis, and functional experiments, indicated that in "classical/ Shaker -type" Kv channels voltage-triggered VSD reorganizations are transmitted to PD gating via the α-helical S4-S5 sequence that links both modules. Importantly, these Shaker -type channels share a domain-swapped VSD/PD organization, with each VSD contacting the PD of the adjacent subunit. In this case, the S4-S5 linker, acting as a rigid mechanical lever (electromechanical lever coupling), would lead to channel gate opening at the cytoplasmic S6 helices bundle. However, new functional data with EAG channels split between the VSD and PD modules indicate that, in some Kv channels, alternative VSD/PD coupling mechanisms do exist. Noticeably, recent elucidation of the architecture of some EAG channels, and other relatives, showed that their VSDs are non-domain swapped. Despite similarities in primary sequence and predicted structural organization for all EAG channels, they show marked kinetic differences whose molecular basis is not completely understood. Thus, while a common general architecture may establish the gating system used by the EAG channels and the physicochemical coupling of voltage sensing to gating, subtle changes in that common structure, and/or allosteric influences of protein domains relatively distant from the central gating machinery, can crucially influence the gating process. We consider here the latest advances on these issues provided by the elucidation of eag1 and erg1 three-dimensional structures, and by both classical and more recent functional studies with different members of the EAG subfamily., (Copyright © 2020 Barros, de la Peña, Domínguez, Sierra and Pardo.)

Reprogramming of energy metabolism constitutes one of the hallmarks of cancer and is, therefore, an emerging therapeutic target. We describe here that the potassium channel Kv10.1, which is frequently overexpressed in primary and metastatic cancer, and has been proposed a therapeutic target, participates in metabolic adaptation of cancer cells through regulation of mitochondrial dynamics. We used biochemical and cell biological techniques, live cell imaging and high-resolution microscopy, among other approaches, to study the impact of Kv10.1 on the regulation of mitochondrial stability. Inhibition of Kv10.1 expression or function led to mitochondrial fragmentation, increase in reactive oxygen species and increased autophagy. Cells with endogenous overexpression of Kv10.1 were also more sensitive to mitochondrial metabolism inhibitors than cells with low expression, indicating that they are more dependent on mitochondrial function. Consistently, a combined therapy using functional monoclonal antibodies for Kv10.1 and mitochondrial metabolism inhibitors resulted in enhanced efficacy of the inhibitors. Our data reveal a new mechanism regulated by Kv10.1 in cancer and a novel strategy to overcome drug resistance in cancers with a high expression of Kv10.1.

Background: Prostate cancer (PCa) is one of the most commonly diagnosed cancers among men in developed countries; however, little is known about modifiable risk factors. Some studies have implicated organochlorine and organophosphate insecticides as risk factors (particularly the organodithioate class) and risk of clinically significant PCa subtypes. However, few studies have evaluated other pesticides. We used data from the Agricultural Health Study, a large prospective cohort of pesticide applicators in North Carolina and Iowa, to extend our previous work and evaluate 39 additional pesticides and aggressive PCa., Methods: We used Cox proportional hazards models, with age as the time scale, to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for the association between ever use of individual pesticides and 883 cases of aggressive PCa (distant stage, poorly differentiated grade, Gleason score ≥ 7, or fatal prostate cancer) diagnosed between 1993 and 2015. All models adjusted for birth year, state, family history of PCa, race, and smoking status. We conducted exposure-response analyses for pesticides with reported lifetime years of use., Results: There was an increased aggressive PCa risk among ever users of the organodithioate insecticide dimethoate (n = 54 exposed cases, HR = 1.37, 95% CI = 1.04, 1.80) compared to never users. We observed an inverse association between aggressive PCa and the herbicide triclopyr (n = 35 exposed cases, HR = 0.68, 95% CI = 0.48, 0.95), with the strongest inverse association for those reporting durations of use above the median (≥ 4 years; n = 13 exposed cases, HR=0.44, 95% CI=0.26, 0.77)., Conclusion: Few additional pesticides were associated with prostate cancer risk after evaluation of extended data from this large cohort of private pesticide applicators.

Background: Fuel oil-derived volatile organic compounds (VOCs) inhalation is associated with accidental marine spills. After the Prestige petroleum tanker sank off northern Spain in 2002 and the Deepwater Horizon oil rig catastrophe in 2009, subjects involved in environmental decontamination showed signs of ongoing or residual lung disease up to 5 y after the exposure., Objectives: We aimed at investigating mechanisms driving persistent respiratory disease by developing an animal model of inhalational exposure to fuel oil-derived VOCs., Methods: Female Wistar and Brown Norway (BN) rats and C57BL mice were exposed to VOCs produced from fuel oil mimicking the Prestige spill. Exposed animals inhaled the VOCs 2 h daily, 5 d per week, for 3 wk. Airway responsiveness to methacholine (MCh) was assessed, and bronchoalveolar lavage (BAL) and lung tissues were analyzed after the exposure and following a 2-wk washout., Results: Consistent with data from human studies, both strains of rats that inhaled fuel oil-derived VOCs developed airway hyperresponsiveness that persisted after the washout period, in the absence of detectable inflammation in any lung compartment. Histopathology and quantitative morphology revealed the development of peripherally distributed pulmonary emphysema, which persisted after the washout period, associated with increased alveolar septal cell apoptosis, microvascular endothelial damage of the lung parenchyma, and inhibited expression of vascular endothelial growth factor (VEGF)., Discussion: In this rat model, fuel oil VOCs inhalation elicited alveolar septal cell apoptosis, likely due to DNA damage. In turn, the development of a peculiar pulmonary emphysema pattern altered lung mechanics and caused persistent noninflammatory airway hyperresponsiveness. Such findings suggest to us that humans might also respond to VOCs through physiopathological pathways different from those chiefly involved in typical cigarette smoke-driven emphysema in chronic obstructive pulmonary disease (COPD). If so, this study could form the basis for a novel disease mechanism for lasting respiratory disease following inhalational exposure to catastrophic fuel oil spills. https://doi.org/10.1289/EHP4178.

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a genetically encoded marker for the positive ends of microtubules (EB3 labelled with tdTomato fluorescent protein) and high-speed, high-resolution, live-cell imaging using spinning disk confocal microscopy. Cell cycle synchronization and increased density of microtubules are achieved by inhibiting centrosomal separation in mitotic cells, and analysis of growth is performed using open-source U-Track software. The use of a bright and red-shifted fluorescent protein, in combination with the lower laser power and reduced exposure time required for spinning disk microscopy reduce phototoxicity and the probability of light-induced artifacts. This allows for imaging a larger number of cells in the same preparation while maintaining the cells in a growth medium under standard culture conditions. Because the analysis is performed in a supervised automatic fashion, the results are statistically robust and reproducible.

Voltage-gated potassium channels are transmembrane proteins that allow flow of potassium across the membrane to regulate ion homeostasis, cell proliferation, migration, cell volume, and specific processes such as muscular contraction. Aberrant function or expression of potassium channels can underlie pathologies ranging from heart arrhythmia to cancer; the expression of potassium channels is altered in many types of cancer and that alteration correlates with malignancy and poor prognosis. Targeting potassium channels therefore constitutes a promising approach for cancer therapy. In this review, we discuss strategies to target a particular family of potassium channels, the voltage-gated potassium channels (K V ) where a reasonable structural understanding is available. We also discuss the possible obstacles and advantages of such a strategy., Competing Interests: No competing financial interests exist., (Copyright 2019, Mary Ann Liebert, Inc., publishers.)

Introduction: New sampling techniques to analyse lung diseases, such as exhaled breath condensate (EBC), are a breakthrough in research field since they are less invasive and less traumatic for the patients compared to lung biopsies. Nevertheless, there is an increasing need to optimize not only the sampling protocols but the storage and processing of specimens to get accurate results., Methods: Exhaled breath condensate was sampled employing the ECoScreen device. Concentrated protein was obtained after ultracentrifugation, lyophilization and reversed-phase chromatography. MALDI-time of flight (TOF)/TOF mass spectrometry (MS) was applied to determine the protein profile in EBC. Commercially available ELISA kits were used to detect the selected biomarker in the EBC after MALDI-MS proteins identification., Results: The obtained EBC volume after two periods of 10 min doubled the amount obtained after 20 min. One hundred peptides were detected by MALDI-MS, and 18 proteins were identified after reversed-phase chromatography concentration. Dermcidin (P81605), S100A9 (P06702) and Cathepsin G (P08311) were selected to be analysed by ELISA. Dermcidin and S100A9 expression were statistically higher in lung cancer versus healthy volunteers. VEGF concentrations decreased, respectively, by 5.94 and 11.42-fold after 1 and 2 years of frozen EBC preservation in parallel with the declined number of proteins identified by MALDI-MS., Conclusion: Exhaled breath condensate analysis combined with MS technique may become a valuable method for lung cancer screening and Dermcidin and S100A9 may serve as biomarkers for lung cancer diagnosis or prognosis.

Organophosphates and carbamates have been among the most commonly used insecticides, with both agricultural and residential uses. Previous studies have suggested associations of non-Hodgkin lymphoma (NHL) with some of these chemicals; however, many studies have been limited in their ability to evaluate associations with lymphoma subtypes. We evaluated the use of eleven organophosphate and two carbamate insecticides in association with NHL in the North American Pooled Project, which includes data from case-control studies in the United States and Canada (1690 cases/5131 controls). We used unconditional logistic regression adjusting for potential confounders, including use of other pesticides, to estimate odds ratios (OR) and 95% confidence intervals (CI) for associations between these chemicals and NHL overall, and NHL subtypes, i.e., follicular (FL), diffuse large B-cell (DLBCL), small lymphocytic lymphoma (SLL) and others. Ever use of malathion was associated with increased risk of NHL overall (OR = 1.43; 95% CI: 1.14-1.81) compared with never users. Categories using tertiles of duration (<4 yrs., 4-12 yrs., and >12 yrs) also showed a significant exposure-response for increasing years of use of malathion and risk of NHL (OR <4vsUnex  = 1.33 (0.88, 2.03), OR 4-12vsUnex  = 1.42 (1.02, 1.96), OR >12vsUnex  = 1.55 (1.05, 2.28, p-trend < 0.01)). In addition, malathion use was statistically significantly associated with FL (OR = 1.58; 95% CI: 1.11-2.27) and DLBCL (OR = 1.61; 95% CI: 1.16-2.22) while there were no apparent associations with SLL or other subtypes, the p-value for heterogeneity across subtypes, however, was not significant. These results support previous studies suggesting an association between insecticide use and NHL overall, and provide new information on associations with NHL subtypes., (Published by Elsevier Ltd.)