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Parkinson's disease (PD) is a chronic neurological disorder that is identified by a characteristic combination of symptoms such as bradykinesia, resting tremor, rigidity, and postural instability. It is the second most common neurodegenerative disease after Alzheimer's disease and is characterized by the progressive loss of dopamine-producing neurons in the brain. Currently, available treatments for PD are symptomatic and do not prevent the disease pathology. There is growing interest in developing disease-modifying therapy that can reduce disease progression and improve patients' quality of life. One of the promising therapeutic approaches under evaluation is gene therapy utilizing a viral vector, adeno-associated virus (AAV), to deliver transgene of interest into the central nervous system (CNS). Preclinical studies in small animals and nonhuman primates model of PD have shown promising results utilizing the gene therapy that express glial cell line-derived neurotrophic factor (GDNF), cerebral dopamine neurotrophic factor (CDNF), aromatic L-amino acid decarboxylase (AADC), and glutamic acid decarboxylase (GAD). This study provides a comprehensive review of the current state of the above-mentioned gene therapies in various phases of clinical trials for PD treatment. We have highlighted the rationale for the gene-therapy approach and the findings from the preclinical and nonhuman primates studies, evaluating the therapeutic effect, dose safety, and tolerability. The challenges associated with gene therapy for heterogeneous neurodegenerative diseases, such as PD, have also been described. In conclusion, the review identifies the ongoing promising gene therapy approaches in clinical trials and provides hope for patients with PD., (© 2024. Fondazione Società Italiana di Neurologia.)

The author identified the genes and proteins of human enzymes involved in the biosynthesis of catecholamines (dopamine, norepinephrine, epinephrine) and tetrahydrobiopterin (BH4): tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), dopamine β-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), and GTP cyclohydrolase I (GCH1). In Parkinson's disease (PD), the activities and levels of mRNA and protein of all catecholamine-synthesizing enzymes are decreased, especially in dopamine neurons in the substantia nigra. Hereditary GCH1 deficiency results in reductions in the levels of BH4 and the activities of TH, causing decreases in dopamine levels. Severe deficiencies in GCH1 or TH cause severe decreases in dopamine levels leading to severe neurological symptoms, whereas mild decreases in TH activity in mild GCH1 deficiency or in mild TH deficiency result in only modest reductions in dopamine levels and symptoms of DOPA-responsive dystonia (DRD, Segawa disease) or juvenile Parkinsonism. DRD is a treatable disease and small doses of L-DOPA can halt progression. The death of dopamine neurons in PD in the substantia nigra may be related to (i) inflammatory effect of extra neuronal neuromelanin, (ii) inflammatory cytokines which are produced by activated microglia, (iii) decreased levels of BDNF, and/or (iv) increased levels of apoptosis-related factors. This review also discusses progress in gene therapies for the treatment of PD, and of GCH1, TH and AADC deficiencies, by transfection of TH, AADC, and GCH1 via adeno-associated virus (AAV) vectors., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Natural redox-regulated channel proteins often utilize disulfide bonds as redox sensors for adaptive regulation of channel conformations in response to diverse physiological environments. In this study, we developed novel synthetic ion channels capable of reversibly switching their ion-transport capabilities by incorporating multiple disulfide bonds into artificial systems. X-ray structural analysis and electrophysiological experiments demonstrated that these disulfide-bridged molecules possess well-defined tubular cavities and can be efficiently inserted into lipid bilayers to form artificial ion channels. More importantly, the disulfide bonds in these molecules serve as redox-tunable switches to regulate the formation and disruption of ion-permeation pathways, thereby achieving a transition in the transmembrane transport process between the ON and OFF states., (© 2024 Wiley-VCH GmbH.)

The problem of drug resistance in epilepsy means that in many cases, a surgical treatment may be advised. But this is only possible if there is an epileptic focus, and resective brain surgery may have adverse side effects. One of the promising alternatives is gene therapy, which allows the targeted expression of therapeutic genes in different brain regions, and even in specific cell types. In this review, we provide detailed explanations of some key terms related to genetic engineering, and describe various regulatory elements that have already been used in the development of different approaches to treating epilepsy using viral vectors. We compare a few universal promoters for their strength and duration of transgene expression, and in our description of cell-specific promoters, we focus on elements driving expression in glutamatergic neurons, GABAergic neurons and astrocytes. We also explore enhancers and some other cis-regulatory elements currently used in viral vectors for gene therapy, and consider future perspectives of state-of-the-art technologies for designing new, stronger and more specific regulatory elements. Gene therapy has multiple advantages and should become more common in the future, but there is still a lot to study and invent in this field. [ABSTRACT FROM AUTHOR]

Understanding the mechanisms that underlie the adaptive response of ectotherms to rising temperatures is key to mitigate the effects of climate change. We assessed the molecular and physiological processes that differentiate between rainbow trout (Oncorhynchus mykiss) with high and low tolerance to acute thermal stress. To achieve our goal, we used a critical thermal maximum trial in two strains of rainbow trout to elicit loss of equilibrium responses to identify high and low tolerance fish. We then compared the hepatic transcriptome profiles of high and low tolerance fish relative to untreated controls common to both strains to uncover patterns of differential gene expression and to gain a broad perspective on the interacting gene pathways and functional processes involved. We observed some of the classic responses to increased temperature (e.g., induction of heat shock proteins) but these responses were not the defining factors that differentiated high and low tolerance fish. Instead, high tolerance fish appeared to suppress growth-related functions, enhance certain autophagy components, better regulate neurodegenerative processes, and enhance stress-related protein synthesis, specifically spliceosomal complex activities, mRNA regulation, and protein processing through post-translational processes, relative to low tolerance fish. In contrast, low tolerance fish had higher transcript diversity and demonstrated elevated developmental, cytoskeletal, and morphogenic, as well as lipid and carbohydrate metabolic processes, relative to high tolerance fish. Our results suggest that high tolerance fish engaged in processes that supported the prevention of further damage by enhancing repair pathways, whereas low tolerance fish were more focused on replacing damaged cells and their structures. [ABSTRACT FROM AUTHOR]

Contemporary evolution was observed in a feral rainbow trout (Oncorhynchus mykiss) population of a thermal stream (Valcheta) in Northern Patagonia, in terms of juvenile thermal tolerance and preferred temperature. Other authors showed that high-temperature treatment applied to male rainbow trout juveniles increased the thermal tolerance in the next generation. This implies a high mutation rate and/or a modified epigenetic inheritance. Comparisons were made among a) a rainbow trout strain adapted in terms of upper thermal tolerance and higher preferred temperature (Valcheta stream), b) a wild temperate stream population (Guillelmo stream), and c) two temperate farmed strains. We examined: Oxidative damage (lipid peroxidation) and activities of antioxidant enzymes; Catalase (CAT), Glutathione S-Transferases (GST), and Superoxide Dismutase (SOD), in liver, testicle, and spermatozoa. Semen fatty acid composition, sperm morphology, sperm motility, and fertilization performance in samples before and after the application of cryopreservation procedures were also evaluated. The observed responses, mainly related to the sperm membrane, reinforces the idea that ROS can affect the germinal line of male rainbow trout juveniles subjected to high water temperature. Our results suggest that the acquired thermal tolerance traits may be part of a wide spectrum of novel characteristics produced as a consequence of an enhanced mutation rate and/or a different DNA methylation pattern, induced by higher temperatures and mediated by ROS. [ABSTRACT FROM AUTHOR]

Monogenic disorders are a group of human diseases caused by mutations in single genes. While some disease-altering treatments offer relief and slow the progression of certain conditions, the majority of monogenic disorders still lack effective therapies. In recent years, gene therapy has appeared as a promising approach for addressing genetic disorders. However, despite advancements in gene manipulation tools and delivery systems, several challenges remain unresolved, including inefficient delivery, lack of sustained expression, immunogenicity, toxicity, capacity limitations, genomic integration risks, and limited tissue specificity. This review provides an overview of the plasmid-based gene therapy techniques and delivery methods currently employed for monogenic diseases, highlighting the challenges they face and exploring potential strategies to overcome these barriers. [ABSTRACT FROM AUTHOR]

If the billions of oligodendrocytes (OLs) populating the central nervous system (CNS) of patients could express their feelings, they would undoubtedly tell gene therapists about their frustration with the other neural cell populations, neurons, microglia, or astrocytes, which have been the favorite targets of gene transfer experiments. This review questions why OLs have been left out of most gene therapy attempts. The first explanation is that the pathogenic role of OLs is still discussed in most CNS diseases. Another reason is that the so-called ubiquitous CAG, CBA, CBh, or CMV promoters—widely used in gene therapy studies—are unable or poorly able to activate the transcription of episomal transgene copies brought by adeno-associated virus (AAV) vectors in OLs. Accordingly, transgene expression in OLs has either not been found or not been evaluated in most gene therapy studies in rodents or non-human primates. The aims of the current review are to give OLs their rightful place among the neural cells that future gene therapy could target and to encourage researchers to test the effect of OL transduction in various CNS diseases. [ABSTRACT FROM AUTHOR]

Reduced muscle mass has been associated with the progression and prognosis of amyotrophic lateral sclerosis (ALS). However, it remains unclear whether decreased muscle mass is a risk factor for ALS or a consequence of motor neuron degeneration. Recently, serum creatinine-to-cystatin C ratio (CCR) have emerged as promising biomarkers for assessing muscle mass. We aimed to explore the association between CCR and the incidence of ALS using data from the UK Biobank. Between 2006 and 2010, 446,945 participants were included in the baseline. CCR was calculated as the ratio of serum creatinine to cystatin C. Cox regression models were used to analyze the relationship between CCR and ALS incidence. Furthermore, subgroup analyses were conducted to investigate potential covariates in these relationships. After adjusting for all covariates, the multivariate Cox regression analysis revealed a significant association between decreased CCR and an increased risk of ALS (hazard ratio (HR) = 0.990, 95% confidence interval (CI): 0.982–0.999, P = 0.026). Participants were stratified into groups based on CCR tertiles. Compared with participants in the highest tertiles of CCR, those in the lowest (HR = 1.388, 95% CI: 1.032–1.866, P = 0.030) and medium tertiles (HR = 1.348, 95% CI: 1.045–1.739, P = 0.021) had an increased risk of ALS incidence. Subgroup analysis showed that the relationship between CCR and ALS incidence was particularly significant among participants aged < 65 years (CCR tertile 1: HR = 1.916, 95% CI: 1.366–2.688, P < 0.001; CCR tertile 2: HR = 1.699, 95% CI: 1.267–2.278, P < 0.001). The present results demonstrate that lower CCR is significantly associated with a higher risk of ALS. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder. It is characterized by the progressive loss of dopaminergic (DAnergic) neurons in the substantia nigra and decreased dopamine (DA) levels, which lead to both motor and non-motor symptoms. Conventional PD treatments aim to alleviate symptoms, but do not delay disease progression. PD gene therapy offers a promising approach to improving current treatments, with the potential to alleviate significant PD symptoms and cause fewer adverse effects than conventional therapies. DA replacement approaches and DA enzyme expression do not slow disease progression. However, DA replacement gene therapies, such as adeno-associated virus (AAV)–glutamic acid decarboxylase (GAD) and L-amino acid decarboxylase (AADC) gene therapies, which increase DA transmitter levels, have been demonstrated to be safe and efficient in early-phase clinical trials. Disease-modifying strategies, which aim to slow disease progression, appear to be potent. These include therapies targeting downstream pathways, neurotrophic factors, and midbrain DAnergic neuronal factors, all of which have shown potential in preclinical and clinical trials. These approaches focus on maintaining the integrity of DAnergic neurons, not just targeting the DA transmitter level itself. In particular, critical midbrain developmental and maintenance factors, such as Nurr1 and Foxa2, can interact synergistically with neighboring glia, in a paracrine mode of action, to protect DAnergic neurons against various toxic factors. Similar outcomes could be achieved by targeting both DAnergic neurons and glial cells with other candidate gene therapies, but in-depth research is needed. Neurotrophic factors, such as neurturin, the glial-cell-line-derived neurotrophic factor (GDNF), the brain-derived neurotrophic factor (BDNF), and the vascular endothelial growth factor (VEGF), are also being investigated for their potential to support DAnergic neuron survival. Additionally, gene therapies targeting key downstream pathways, such as the autophagy–lysosome pathway, mitochondrial function, and endoplasmic reticulum (ER) stress, offer promising avenues. Gene editing and delivery techniques continue to evolve, presenting new opportunities to develop effective gene therapies for PD. [ABSTRACT FROM AUTHOR]

The basal ganglia are involved in motor control and action selection, and their impairment manifests in movement disorders such as Parkinson's disease (PD) and dystonia, among others. The complex neuronal circuitry of the basal ganglia is located deep inside the brain and presents significant treatment challenges. Conventional treatment strategies, such as invasive surgeries and medications, may have limited effectiveness and may result in considerable side effects. Non-invasive ultrasound (US) treatment approaches are becoming increasingly recognized for their therapeutic potential for reversibly permeabilizing the blood-brain barrier (BBB), targeting therapeutic delivery deep into the brain, and neuromodulation. Studies conducted on animals and early clinical trials using ultrasound as a therapeutic modality have demonstrated promising outcomes for controlling symptom severity while preserving neural tissue. These results could improve the quality of life for patients living with basal ganglia impairments. This review article explores the therapeutic frontiers of ultrasound technology, describing the brain mechanisms that are triggered and engaged by ultrasound. We demonstrate that this cutting-edge method could transform the way neurological disorders associated with the basal ganglia are managed, opening the door to less invasive and more effective treatments. [ABSTRACT FROM AUTHOR]

Recombinant adeno-associated viruses (rAAVs) have emerged as an attractive tool for gene delivery, and demonstrated tremendous promise in gene therapy and gene editing—therapeutic modalities with potential "one-and-done" treatment benefits compared to conventional drugs. Given their tropisms for the central nervous system (CNS) across various species including humans, rAAVs have been extensively investigated in both pre-clinical and clinical studies targeting neurodegenerative disease. However, major challenges remain in the application of rAAVs for CNS gene therapy, such as suboptimal vector design, low CNS transduction efficiency and specificity, and therapy-induced immunotoxicity. Therefore, continuing efforts are being made to optimize the rAAV vectors from their "core" genetic payloads to their "coat" or capsid structure. In this review, we describe current approaches for rAAV vector design tailored for transgene expression in the CNS, summarize the development of CNS-targeting AAV serotypes, and highlight recent advancements in AAV capsid engineering, aimed at generating a new generation of rAAVs with improved CNS tropism. Additionally, we discuss various administration routes for delivering rAAVs to the CNS and provide an overview of AAV-mediated gene therapies currently under investigation in clinical trials for the treatment of neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Our review paper evaluates the impact of plant-based products, primarily derived from plants from Serbia, on P-glycoprotein (P-gp) activity and their potential in modulating drug resistance in cancer therapy. We focus on the role and regulation of P-gp in cellular physiology and its significance in addressing multidrug resistance in cancer therapy. Additionally, we discuss the modulation of P-gp activity by 55 natural product drugs, including derivatives for some of them, based on our team's research findings since 2011. Specifically, we prospect into sesquiterpenoids from the genera Artemisia, Curcuma, Ferula, Inula, Petasites, and Celastrus; diterpenoids from the genera Salvia and Euphorbia; chalcones from the genera Piper, Glycyrrhiza, Cullen, Artemisia, and Humulus; riccardins from the genera Lunularia, Monoclea, Dumortiera, Plagiochila, and Primula; and diarylheptanoids from the genera Alnus and Curcuma. Through comprehensive analysis, we aim to highlight the potential of natural products mainly identified in plants from Serbia in influencing P-gp activity and overcoming drug resistance in cancer therapy, while also providing insights into future perspectives in this field. [ABSTRACT FROM AUTHOR]

Inspired by phase-separated biopolymers with enzymatic activity, we developed an amphiphilic catalyst consisting of alternating hydrophilic oligo(ethylene glycol) and hydrophobic aromatic units bearing a Hoveyda–Grubbs catalyst center (MAHGII). MAHGII served as both a droplet-forming scaffold and a catalyst for ring-closing metathesis reactions, providing a new biomimetic system that promotes organic reactions in an aqueous environment. [ABSTRACT FROM AUTHOR]

Background: Amyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron (MN) degeneration, leading to neuromuscular junction (NMJ) dismantling and severe muscle atrophy. The nuclear receptor interaction protein (NRIP) functions as a multifunctional protein. It directly interacts with calmodulin or α-actinin 2, serving as a calcium sensor for muscle contraction and maintaining sarcomere integrity. Additionally, NRIP binds with the acetylcholine receptor (AChR) for NMJ stabilization. Loss of NRIP in muscles results in progressive motor neuron degeneration with abnormal NMJ architecture, resembling ALS phenotypes. Therefore, we hypothesize that NRIP could be a therapeutic factor for ALS. Methods: We used SOD1 G93A mice, expressing human SOD1 with the ALS-linked G93A mutation, as an ALS model. An adeno-associated virus vector encoding the human NRIP gene (AAV-NRIP) was generated and injected into the muscles of SOD1 G93A mice at 60 days of age, before disease onset. Pathological and behavioral changes were measured to evaluate the therapeutic effects of AAV-NRIP on the disease progression of SOD1 G93A mice. Results: SOD1 G93A mice exhibited lower NRIP expression than wild-type mice in both the spinal cord and skeletal muscle tissues. Forced NRIP expression through AAV-NRIP intramuscular injection was observed in skeletal muscles and retrogradely transduced into the spinal cord. AAV-NRIP gene therapy enhanced movement distance and rearing frequencies in SOD1 G93A mice. Moreover, AAV-NRIP increased myofiber size and slow myosin expression, ameliorated NMJ degeneration and axon terminal denervation at NMJ, and increased the number of α-motor neurons (α-MNs) and compound muscle action potential (CMAP) in SOD1 G93A mice. Conclusions: AAV-NRIP gene therapy ameliorates muscle atrophy, motor neuron degeneration, and axon terminal denervation at NMJ, leading to increased NMJ transmission and improved motor functions in SOD1 G93A mice. Collectively, AAV-NRIP could be a potential therapeutic drug for ALS. [ABSTRACT FROM AUTHOR]

The Central Nervous System (CNS) is vulnerable to a range of diseases, including neurodegenerative and oncological conditions, which present significant treatment challenges. The blood–brain barrier (BBB) restricts molecule penetration, complicating the achievement of therapeutic concentrations in the CNS following systemic administration. Gene therapy using recombinant adeno-associated virus (rAAV) vectors emerges as a promising strategy for treating CNS diseases, demonstrated by the registration of six gene therapy products in the past six years and 87 ongoing clinical trials. This review explores the implementation of rAAV vectors in CNS disease treatment, emphasizing AAV biology and vector engineering. Various administration methods—such as intravenous, intrathecal, and intraparenchymal routes—and experimental approaches like intranasal and intramuscular administration are evaluated, discussing their advantages and limitations in different CNS contexts. Additionally, the review underscores the importance of optimizing therapeutic efficacy through the pharmacokinetics (PK) and pharmacodynamics (PD) of rAAV vectors. A comprehensive analysis of clinical trials reveals successes and challenges, including barriers to commercialization. This review provides insights into therapeutic strategies using rAAV vectors in neurological diseases and identifies areas requiring further research, particularly in optimizing rAAV PK/PD. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is a gradually worsening neurodegenerative disorder affecting the nervous system, marked by a slow progression and varied symptoms. It is the second most common neurodegenerative disease, affecting over six million people in the world. Its multifactorial etiology includes environmental, genomic, and epigenetic factors. Clinical symptoms consist of non-motor and motor symptoms, with motor symptoms being the classic presentation. Therapeutic approaches encompass pharmacological, non-pharmacological, and surgical interventions. Traditional pharmacological treatment consists of administering drugs (MAOIs, DA, and levodopa), while emerging evidence explores the potential of antidiabetic agents for neuroprotection and gene therapy for attenuating parkinsonian symptoms. Non-pharmacological treatments, such as exercise, a calcium-rich diet, and adequate vitamin D supplementation, aim to slow disease progression and prevent complications. For those patients who have medically induced side effects and/or refractory symptoms, surgery is a therapeutic option. Deep brain stimulation is the primary surgical option, associated with motor symptom improvement. Levodopa/carbidopa intestinal gel infusion through percutaneous endoscopic gastrojejunostomy and a portable infusion pump succeeded in reducing "off" time, where non-motor and motor symptoms occur, and increasing "on" time. This article aims to address the general aspects of PD and to provide a comparative comprehensive review of the conventional and the latest therapeutic advancements and emerging treatments for PD. Nevertheless, further studies are required to optimize treatment and provide suitable alternatives. [ABSTRACT FROM AUTHOR]

To advance the exploration of mechanisms underlying natural multi-gated ion channels, a novel butterfly-shaped biomimetic K + channel GnC7 (n = 3, 4) is developed with dual mechanical and pH responsiveness, exhibiting unprecedented K + /Na + selectivity (G3C7: 34.4; G4C7: 41.3). These channels constructed from poly(propylene imine) dendrimer and benzo-21-crown-7-ethers achieve high K + transport activity (EC 50 : 0.72 µm for G3C7; 0.9 µm for G4C7) due to their arc-like mechanical rotation. The dynamic mode relies on butterfly-shaped topology derived from the highly symmetrical core and multiple intramolecular hydrogen bonds. GnC7 can sense mechanical stimulus applied to liposomes/cells and then adapt the K + transport rate accordingly. Furthermore, reversible ON/OFF switching of K + transport is realized through the pH-controllable host-guest complexation. G4C7-induced ultrafast cellular K + efflux (70% within only 9 min) efficiently triggers mitochondrial-dependent apoptosis of cancer cells by provoking endoplasmic reticulum stress accompanied by drastic Ca 2+ sparks. This work embodies a multi-dimensional regulation of channel functions; it will provide insights into the dynamic behaviors of biological analogs and promote the innovative design of artificial ion channels and therapeutic agents., (© 2025 Wiley‐VCH GmbH.)

Reactive oxygen species (ROS)-responsive ion channels regulate the ion flow across the membranes in response to alterations in the cellular redox state, playing a crucial role in cellular adaptation to oxidative stress. Despite their significance, replicating ROS-responsive functionality in artificial ion channels remains elusive. In this study, we introduce a novel class of artificial H + /Cl - ion channels activatable by elevated ROS levels in cancer cells. ROS-induced decaging of the phenylboronate group triggers the rapid release of the channel-forming units, leading to self-assembly of the H-bonded cascades facilitating the synergistic transport of H + and Cl - ions, with H + /Cl - ion transport selectivity of 7.7. Upon activation, ROS-C-Cl exhibits significant apoptotic activity against human breast cancer cells, achieving an IC 50 of 2.8 μM, comparable to that of paclitaxel. Exploiting the intrinsic oxidative microenvironment of cancer cells, along with the enhanced oxidative stress arising from H + /Cl - co-transport, ROS-C-Cl demonstrates exceptional selectivity in targeting cancer cells with a selectivity index of 10.2 over normal breast cells, outperforming that of paclitaxel by 19.4 folds., (© 2024 Wiley-VCH GmbH.)

Spatial control over supramolecular self-assembly prevails in living system, yet remains difficult to replicate in synthetic scenarios. Here, on the basis of a hydrazone formation-mediated supramolecular hydrogelation system, access to patterning of supramolecular hydrogels is demonstrated via a light-triggered catalysis strategy. A photoacid generator that can produce protons in aqueous solutions upon irradiation is employed. The generated protons lead to a drop in pH of around three units (initial pH 7.0), effectively accelerating the formation and self-assembly of the hydrazone gelators. Because of the light-triggered catalysis, the hydrogelation samples in the presence of photoacid generator show lower critical gelation concentration, higher stiffness, and denser networks. Importantly, by performing selective irradiation using differently shaped masks, various spatially resolved supramolecular hydrogels following the shapes of the masks are fabricated. The concept of using light-triggered catalysis to realize spatial control over supramolecular self-assembly provides an alternative approach toward bottom-up fabrication of structured soft materials for various applications such as tissue engineering, single cell manipulation, and biosensing., (© 2025 Wiley‐VCH GmbH.)

Stimuli-responsive synthetic ionophores allow for spatial and temporal control over ion transport, with promise for applications in targeted therapy. Relay transporters have emerged as a new class of ion transporters - these are anchored carriers that sit in both leaflets of the bilayer and mediate transport across the membrane by passing ions between them. The relays are themselves membrane impermeable, and so must be incorporated into the membrane during vesicle preparation. Here we show that relay transporters can be delivered to both sides of the membrane of vesicles using a synthetic flippase. By incorporating an aryl azopyrazole photo-switch into the movable arm of the relay transporters the ion transport activity can be very efficiently and reversibly switched between off and on states. This control is achieved by extension and contraction of their movable arms via photo-isomerization of the central aryl azopyrazole moiety, hence modulating the ability of the relays to pass ions across the membrane., (© 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Compartmentalized models with coupled catalytic networks are considered as "protocells" in the context of research related to the origin of life. To model the kinetics of a simple cellular uptake-metabolism process, we use a compartmentalized protocell system that combines liposome-encapsulated intravesicular reporter pairs with co-encapsulated enzymes to monitor the membrane transport of a substrate (analyte uptake) and its subsequent enzymatic reaction inside the vesicles (metabolism to the product). The intravesicular chemosensing ensembles consist of the macrocycles cucurbit[7]uril or p-sulfonatocalix[4]arene and matching fluorescent dyes to set up suitable reporter pairs. When these macrocycle/dye reporter pairs are co-encapsulated with enzymes (trypsin, protein kinase A, or butyrylcholinesterase), it is possible to monitor first the transport of different substrates (polylysine, protamine, H-LRRWSLG-OH, or butyrylcholine) through added pores (outer membrane proteins F and C), with synthetic carriers (amphiphilic calixarenes), or by direct permeation (only for butyrylcholine). The subsequent enzymatic conversions of the substrates after they have entered the corresponding protocells can be monitored as consecutive reactions. The new type of in vitro assays can be applied to different enzymes and analytes, affording a comprehensive chemosensing system of high chemical complexity., (© 2025 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Natural channel proteins allow the selective permeation of ions, water or other nutritious entities across bilayer membranes, facilitating various essential physiological functions in living systems. Inspired by nature, chemists endeavor to simulate the structural features and transport behaviors of channel proteins through biomimetic strategies. In this review, we start from introducing the inherent traits of channel proteins such as their crystal structures, functions and mechanisms. Subsequently, different kind of synthetic ion channels including their design principles, dynamic regulations and therapeutic applications were carefully reviewed. Finally, the potential challenges and opportunities in this research field were also carefully discussed. It is anticipated that this review could provide some inspiring ideas and future directions towards the construction of novel bionic ion channels with higher-level structures, properties, functions and practical applications., (© 2024 Wiley-VCH GmbH.)

Background: Due to the broad application of next-generation sequencing, the molecular diagnosis of genetic disorders in pediatric neurology is no longer an unachievable goal. However, treatments for neurological genetic disorders in children remain primarily symptomatic. On the other hand, with the continuous evolution of therapeutic viral vectors, gene therapy is becoming a clinical reality. From this perspective, we wrote this review to illustrate the current state regarding viral-mediated gene therapy in childhood neurological disorders. Data sources: We searched databases, including PubMed and Google Scholar, using the keywords "adenovirus vector," "lentivirus vector," and "AAV" for gene therapy, and "immunoreaction induced by gene therapy vectors," "administration routes of gene therapy vectors," and "gene therapy" with "NCL," "SMA," "DMD," "congenital myopathy," "MPS" "leukodystrophy," or "pediatric metabolic disorders". We also screened the database of ClinicalTrials.gov using the keywords "gene therapy for children" and then filtered the results with the ones aimed at neurological disorders. The time range of the search procedure was from the inception of the databases to the present. Results: We presented the characteristics of commonly used viral vectors for gene therapy for pediatric neurological disorders and summarized their merits and drawbacks, the administration routes of each vector, the research progress, and the clinical application status of viral-mediated gene therapy on pediatric neurological disorders. Conclusions: Viral-mediated gene therapy is on the brink of broad clinical application. Viral-mediated gene therapy will dramatically change the treatment pattern of childhood neurological disorders, and many children with incurable diseases will meet the dawn of a cure. Nevertheless, the vectors must be optimized for better safety and efficacy. [ABSTRACT FROM AUTHOR]

Patients with neuromuscular diseases are particularly vulnerable in the perioperative period to the development of pulmonary and cardiac complications, or medication side effects. These risks could include hypoventilation, aspiration pneumonia, exacerbation of underlying cardiomyopathy, arrhythmias, adrenal insufficiency, prolonged neuromuscular blockade, issues related to thermoregulation, rhabdomyolysis, malignant hyperthermia, or prolonged mechanical ventilation. Interventions at each of the perioperative stages can be implemented to mitigate these risks. A careful pre-operative evaluation may help identify risk factors so that appropriate interventions are initiated, including cardiology consultation, pulmonary function tests, initiation of noninvasive ventilation, or implementation of preventive measures. Important intraoperative issues include positioning, airway and anesthetic management, and adequate ventilation. The postoperative period may require correction of electrolyte abnormalities, control of secretions with medications, manual or mechanical cough assistance, avoiding the risk of reintubation, judicious pain control, and appropriate medication management. The aim of this review is to increase awareness of the particular surgical challenges in this vulnerable population, and guide the clinician on the various evaluations and interventions that may result in a favorable surgical outcome. [ABSTRACT FROM AUTHOR]

Natural redox‐regulated channel proteins often utilize disulfide bonds as redox sensors for adaptive regulation of channel conformations in response to diverse physiological environments. In this study, we developed novel synthetic ion channels capable of reversibly switching their ion‐transport capabilities by incorporating multiple disulfide bonds into artificial systems. X‐ray structural analysis and electrophysiological experiments demonstrated that these disulfide‐bridged molecules possess well‐defined tubular cavities and can be efficiently inserted into lipid bilayers to form artificial ion channels. More importantly, the disulfide bonds in these molecules serve as redox‐tunable switches to regulate the formation and disruption of ion‐permeation pathways, thereby achieving a transition in the transmembrane transport process between the ON and OFF states. [ABSTRACT FROM AUTHOR]

Aromatic l-amino acid decarboxylase deficiency (AADC-DY) is caused by one or more mutations in the DDC gene, resulting in the deficit in catecholamines and serotonin neurotransmitters. The disease has limited therapeutic options with relatively poor clinical outcomes. Accumulated evidence suggests the involvement of neurodegenerative mechanisms in the etiology of AADC-DY. In the absence of neurotransmitters' neuroprotective effects, the accumulation and the chronic presence of several neurotoxic metabolites including 4-dihydroxy-L-phenylalanine, 3-methyldopa, and homocysteine, in the brain of subjects with AADC-DY, promote oxidative stress and reduce the cellular antioxidant and methylation capacities, leading to glial activation and mitochondrial dysfunction, culminating to neuronal injury and death. These pathophysiological processes have the potential to hinder the clinical efficacy of treatments aimed at increasing neurotransmitters' synthesis and or function. This review describes in detail the mechanisms involved in AADC-DY neurodegenerative etiology, highlighting the close similarities with those involved in other neurodegenerative diseases. We then offer novel strategies for the treatment of the disease with the objective to either reduce the level of the metabolites or counteract their prooxidant and neurotoxic effects. These treatment modalities used singly or in combination, early in the course of the disease, will minimize neuronal injury, preserving the functional integrity of neurons, hence improving the clinical outcomes of both conventional and unconventional interventions in AADC-DY. These modalities may not be limited to AADC-DY but also to other metabolic disorders where a specific mutation leads to the accumulation of prooxidant and neurotoxic metabolites. [ABSTRACT FROM AUTHOR]

In light of the unsuccessful traditional therapies for Parkinson's disease (PD) overmany years, there is an unmet need for the development of novel therapies to alleviate the symptoms of PD retardation or halt the progression of the disease itself. This systematic review aims to critically update some of the most promising novel treatments including gene therapy, cell-based therapies, targeted drug delivery, and neuroprotective agents, focusing on their challenges, limitations and future directions in PD research. Gene therapy in PD is encouraging, with AAV-based approaches targeting neurotrophic factors, dopamine production, and neuronal circuits in animal and clinical trials. A promising approach to targeted drug delivery for PD involves the use of nanotechnology to create drug delivery vehicles that can traverse the blood–brain barrier and deliver medications specifically to the regions of the brain affected by PD. Neuroprotective agents are compounds that have the ability to protect neurons from degeneration and death, and they hold great promise for the evolution of disease-modifying treatments for PD. Magnetic field therapy is a promising non-invasive method that promotes neural plasticity in PD. The establishment of standardized protocols for animal and human studies, safety, ethical considerations, and cost-effectiveness are the major challenges for the future research of novel PD therapies. The development of novel therapies for PD represents a promising path toward to effective personalized disease-modifying treatments for PD. [ABSTRACT FROM AUTHOR]

Early gene therapy studies held great promise for the cure of heritable diseases, but the occurrence of various genotoxic events led to a pause in clinical trials and a more guarded approach to progress. Recent advances in genetic engineering technologies have reignited interest, leading to the approval of the first gene therapy product targeting genetic mutations in 2017. Gene therapy (GT) can be delivered either in vivo or ex vivo. An ex vivo approach to gene therapy is advantageous, as it allows for the characterization of the gene-modified cells and the selection of desired properties before patient administration. Autologous cells can also be used during this process which eliminates the possibility of immune rejection. This review highlights the various stages of ex vivo gene therapy, current research developments that have increased the efficiency and safety of this process, and a comprehensive summary of Human Immunodeficiency Virus (HIV) gene therapy studies, the majority of which have employed the ex vivo approach. [ABSTRACT FROM AUTHOR]

Recombinant adeno-associated virus (rAAV) has been developed as a safe and effective gene delivery vehicle to treat rare genetic diseases. This study aimed to establish a novel biomanufacturing process to achieve high production and purification of various AAV serotypes (AAV2, 5, DJ, DJ8). First, a robust suspensive production process was developed and optimized using Gibco Viral Production Cell 2.0 in 30–60 mL shaker flask cultures by evaluating host cells, cell density at the time of transfection and plasmid amount, adapted to 60–100 mL spinner flask production, and scaled up to 1.2–2.0-L stirred-tank bioreactor production at 37 °C, pH 7.0, 210 rpm and DO 40%. The optimal process generated AAV titer of 7.52–8.14 × 1010 vg/mL. Second, a new AAV purification using liquid chromatography was developed and optimized to reach recovery rate of 85–95% of all four serotypes. Post-purification desalting and concentration procedures were also investigated. Then the generated AAVs were evaluated in vitro using Western blotting, transmission electron microscope, confocal microscope and bioluminescence detection. Finally, the in vivo infection and functional gene expression of AAV were confirmed in tumor xenografted mouse model. In conclusion, this study reported a robust, scalable, and universal biomanufacturing platform of AAV production, clarification and purification. [ABSTRACT FROM AUTHOR]

Neurodegenerative disorders are characterized by mitochondrial dysfunction and subsequently oxidative stress, inflammation, and apoptosis that contribute to neuronal cytotoxicity and degeneration. Huntington's (HD), Alzheimer's (AD), and Parkinson's (PD) diseases are three of the major neurodegenerative diseases. To date, researchers have found various natural phytochemicals that could potentially be used to treat neurodegenerative diseases. Particularly, the application of natural phenolic compounds has gained significant traction in recent years, driven by their various biological activities and therapeutic efficacy in human health. Polyphenols, by modulating different cellular functions, play an important role in neuroprotection and can neutralize the effects of oxidative stress, inflammation, and apoptosis in animal models. This review focuses on the current state of knowledge on phenolic compounds, including phenolic acids, flavonoids, stilbenes, and coumarins, as well as their beneficial effects on human health. We further provide an overview of the therapeutic potential and mechanisms of action of natural dietary phenolics in curing neurodegenerative diseases in animal models. [ABSTRACT FROM AUTHOR]

The sarcopenia index (SI), calculated as [(serum creatinine/serum cystatin C) × 100], maybe a simpler alternative for measuring muscle mass than computed tomography (CT) and bioelectrical impedance analysis (BIA). We enrolled 112 patients with head and neck cancers (HNC). The correlation of the SI with muscle surface area measured by CT (CTMSA, n = 82) and muscle mass by BIA (BIA-MM, n = 41) was tested. Cutoff values were set for SI, CTMSA, and BIA-MM. Overall survival (OS) was compared between the high- and low-SI/CTMSA/BIA-MM groups. The SI was correlated with CTMSA (r = 0.43) and BIA-MM (r = 0.52). The optimal cutoff values of SI, CTMSA, and BIA-MM were 76.1 (area under the curve [AUC] = 0.67), 129.2 (AUC = 0.59), and 46.1 (AUC = 0.62), respectively. OS was significantly lower in the low-SI group (78% at 1 year and 69% at 2 years) than in the high-SI group (94% at 1 year and 86% at 2 years; p = 0.006). There was no significant difference in OS between the low-and high-CTMSA and -BIA-MM groups. The SI, which only requires a blood sample, is a useful marker of muscle mass that correlates with short-term prognosis in patients with HNC. [ABSTRACT FROM AUTHOR]

Background: Cystatin C (CysC) levels in amyotrophic lateral sclerosis (ALS) have been found changes, however, the associations between serum CysC levels and the progression and survival of ALS remain largely unknown. Methods: A total of 1,086 ALS patients and 1,026 sex-age matched healthy controls (HCs) were enrolled in this study. Serum CysC, other renal function, and metabolic parameters were measured. Correlation analysis and binary logistic regression were used to explore the factors related to serum CysC. Kaplan-Meier curve and Cox regression model were used for survival analysis. Results: CysC levels were significantly higher in ALS patients compared to HCs (0.94 vs. 0.85 mg/L, p < 0.001). Compared with ALS patients with lower CysC levels, those with higher CysC levels had an older age of onset, significantly lower ALSFRS-R scores (40.1 vs. 41.3, p < 0.001), a faster disease progression rate (0.75 vs. 0.67, p = 0.011), and lower frontal lobe function scores (15.8 vs. 16.1, p = 0.020). In the correlation analysis, CysC levels were significantly negatively correlated with ALSFRS-R scores (r = -0.16, p < 0.001). Additionally, ALS patients with higher CysC levels had significantly shorter survival time (40.0 vs. 51.8, p < 0.001) compared to patients with lower CysC levels. Higher CysC levels were associated with a higher risk of death in Cox analysis (HR: 1.204, 95% CI: 1.012-1.433). However, when treatment was included in the model, the result was no longer significant. Conclusion: CysC levels in ALS patients were higher compared to HCs. Higher CysC levels were associated with greater disease severity, faster progression rate and shorter survival, needing early intervention. [ABSTRACT FROM AUTHOR]

Potassium channels represent the most prevalent class of ion channels, exerting regulatory control over numerous vital biological processes, including muscle contraction, neurotransmitter release, cell proliferation, and apoptosis. The seamless integration of astonishing functions into a sophisticated structure, as seen in these protein channels, inspires the chemical community to develop artificial versions, gearing toward simplifying their structure while replicating their key functions. In particular, over the past ten years or so, a number of elegant artificial potassium transporters have emerged, demonstrating high selectivity, high transport efficiency or unprecedented transport mechanisms. In this review, we will provide a detailed exposition of these artificial potassium transporters that are derived from a single molecular backbone or self-assembled from multiple components, with their respective structural designs, channel functions, transport mechanisms and biomedical applications thoroughly reviewed. [ABSTRACT FROM AUTHOR]

Inborn errors of neurotransmitter (NT) metabolism are a group of rare, heterogenous diseases with predominant neurological features, such as movement disorders, autonomic dysfunction, and developmental delay. Clinical overlap with other disorders has led to delayed diagnosis and treatment, and some conditions are refractory to oral pharmacotherapies. Gene therapies have been developed and translated to clinics for paediatric inborn errors of metabolism, with 38 interventional clinical trials ongoing to date. Furthermore, efforts in restoring dopamine synthesis and neurotransmission through viral gene therapy have been developed for Parkinson's disease. Along with the recent European Medicines Agency (EMA) and Medicines and Healthcare Products Regulatory Agency (MHRA) approval of an AAV2 gene supplementation therapy for AADC deficiency, promising efficacy and safety profiles can be achieved in this group of diseases. In this review, we present preclinical and clinical advances to address NT‐related diseases, and summarise potential challenges that require careful considerations for NT gene therapy studies. [ABSTRACT FROM AUTHOR]

Aquaculture offers solutions to meet the growing global demand for fish, and reports from the UN‐FAO indicate that aquaculture production in Latin America (LA) has grown at rates above the world average in recent years. One of the major constraints in the diversification of LA aquaculture is the control of reproduction in several popular native fish species for which difficulties in captive propagation have not yet been sufficiently overcome. This article reviews the use of hormone treatments to promote reproduction in females of these native fish species. LA has played a key role in the history of development of hormone administration, including the first hormonally induced spawning. That contribution is included in a historical overview of the discovery of the major hormones used in fish culture. The review provides a summary of difficulties to propagate females of various native fishes and the effects of administering hormones to enhance reproduction. Induced spawning of certain freshwater species was mainly achieved with pituitary extracts or human chorionic gonadotropin (hCG), although gonadotropin‐releasing hormone analogues (GnRHa) treatments are being researched, and successful studies suggest that low doses may be more effective. Research on new and emerging aquaculture species has applied both gonadotropins (Gths) and GnRHa‐based treatments, and GnRHa treatments have shown potential for marine species. However, native marine species new to aquaculture have also been conditioned to spawn spontaneously without hormones. Finally, we proposed future lines of research to examine reproductive strategies and GnRHa‐based hormone treatments to improve reproductive control for economically important fish species of LA. [ABSTRACT FROM AUTHOR]

Transmembrane ion transport modality has received a widespread attention due to its apoptotic activation toward anticancer cell activities. In this study, G-quadruplex-based potassium-specific transmembrane channels have been developed to facilitate the intracellular K + efflux, which perturbs the cellular ion homeostasis thereby inducing cancer cell apoptosis. Cholesterol-tag, a lipophilic anchor moiety, serves as a rudiment for the G-quadruplex immobilization onto the membrane, while G-quadruplex channel structure as a transport module permits ion binding and migration along the channels. A c-Myc sequence tagged with two-cholesterol is designed as a representative lipophilic G-quadruplex, which forms intramolecular parallel G-quadruplex with three stacks of G-quartets (Ch 2 -Para3). Fluorescence transport assay demonstrates Ch 2 -Para3 a high transport activity (EC 50 = 10.9 × 10 -6 m) and an ion selectivity (K + /Na + selectivity ratio of 84). Ch 2 -Para3 mediated K + efflux in cancer cells is revealed to purge cancer cells through K + efflux-mediated cell apoptosis, which is confirmed by monitoring the changes in membrane potential of mitochondria, leakage of cytochrome c, reactive oxygen species yield, as well as activation of a family of caspases. The lipophilic G-quadruplex exhibits obvious antitumor activity in vivo without systemic toxicity. This study provides a functional scheme aimed at generating DNA-based selective artificial membrane channels for the purpose of regulating cellular processes and inducing cell apoptosis, which shows a great promising for anticancer therapy in the future., (© 2024 Wiley‐VCH GmbH.)

It is established that neurogenesis of dentate gyrus is increased after ischemic insult, although the regulatory mechanisms have not yet been elucidated. In this study, we focused on Ezh2 which suppresses gene expression through catalyzing trimethylation of lysine 27 of histone 3. Male gerbils were injected with adeno-associated virus (AAV) carrying shRNA targeting to Ezh2 into right dentate gyrus 2 weeks prior to forebrain ischemia. One week after ischemia, animals were injected with thymidine analogue to label proliferating cells. Three weeks after ischemia, animals were killed for histological analysis. AAV-mediated knockdown of Ezh2 significantly decreased the ischemia-induced increment of proliferating cells, and the proliferated cells after ischemia showed significantly longer migration from subgranular zone (SGZ), compared to the control group. Furthermore, the number of neural stem cells in SGZ significantly decreased after ischemia with Ezh2 knockdown group. Of note, Ezh2 knockdown did not affect the number of proliferating cells or the migration from SGZ in the non-ischemic condition. Our data showed that, specifically after ischemia, Ezh2 knockdown shifted the balance between self-renewal and differentiation toward differentiation in adult dentate gyrus., (© 2024. The Author(s).)

It has long been an aspirational goal to create artificial channel structures that replicate the feat achieved by ion channel proteins. Biological ion channels occasionally demonstrate multiple conductance states (known as subconductance), remaining a challenging property to achieve in artificial channel molecules. We report a funnel-shaped single-molecule channel constructed by an electron-deficient macrocycle and two electron-deficient aromatic imide arms. Planar lipid bilayer measurements reveal distinct current recordings, including a closed state, two conducting states, and spontaneous transitions between the three states, resembling the events seen in biological ion channels. The transitions result from conformational changes induced by chloride transport in the channel molecule. Both opening states show a non-linear and rectifying I-V relationship, indicating voltage-dependent transport due to the asymmetrical channel structure. This work could enhance our understanding of ion permeation and channel opening mechanism., (© 2024 Wiley-VCH GmbH.)

Relaying conformational change over several nanometers is central to the function of allosterically regulated proteins. Replicating this mechanism artificially would provide important communication tools, but requires nanometer‐sized molecules that reversibly switch between defined shapes in response to signaling molecules. In this work, 1.8 nm long rigid rod oligo(phenylene‐ethynylene)s are scaffolds for switchable multi‐squaramide hydrogen‐bond relays. Each relay can adopt either a parallel or an antiparallel orientation relative to the scaffold; the preferred orientation is dictated by a director group at one end. An amine director responded to proton signals, with acid‐base cycles producing multiple reversible changes in relay orientation that were reported by a terminal NH, which is 1.8 nm distant. Moreover, a chemical fuel acted as a dissipative signal. As the fuel was consumed, the relay reverted to its original orientation, illustrating how information from out‐of‐equilibrium molecular signals can be communicated to a distant site. [ABSTRACT FROM AUTHOR]

The discovery of the neurotrophins and their potent survival and trophic effects led to great enthusiasm about their therapeutic potential to rescue dying neurons in neurodegenerative diseases. The further discovery that brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and glial cell linederived neurotrophic factor (GDNF) had potent survival-promoting activity on motor neurons led to the proposal for their use in motor neuron diseases such as amyotrophic lateral sclerosis (ALS). In this review we synthesize the literature pertaining to the role of NGF, BDNF, CNTF and GDNF on the development and physiology of spinal motor neurons, as well as the preclinical studies that evaluated their potential for the treatment of ALS. Results from the clinical trials of these molecules will also be described and, with the aid of decades of hindsight, we will discuss what can reasonably be concluded and how this information can inform future clinical development of neurotrophic factors for ALS. [ABSTRACT FROM AUTHOR]

Background: The Serum creatinine/cystatin C ratio (Cr/CysC ratio) is an emerging alternative index for muscle mass loss, a risk factor for cardiovascular diseases (CVDs). However, the association between the Cr/CysC ratio and CVD morbidity and mortality remains unknown. Methods: A total of 11,150 participants of the National Health and Nutrition Examination Survey (NHANES) were included in this study. Univariable and multivariable logistic regression models were employed to assess the association between the Cr/CysC ratio and self-reported CVD morbidity. Cox proportional hazard models were used to estimate the hazard ratio (HR) and 95% confidence interval (CI) of the Cr/CysC ratio for CVD mortality. Results: At baseline, 1181 (7.90%) participants had self-reported CVDs. Lower Cr/CysC ratios were found in participants with CVDs (1.18 ± 0.30 vs. 1.05 ± 0.23, p < 0.001). In the multivariable logistic regression model, the Cr/CysC ratio was inversely linked to CVD morbidity (odds ratio: 0.65, 95% CI: 0.52-0.81, p < 0.001, per standard deviation [SD] increase). 997 (8.94%) CVD deaths were documented during a median follow-up of 16.9 years. A higher Cr/CysC ratio was associated with a decreasing risk of CVD mortality (adjusted HR: 0.54, 95% CI: 0.46-0.65, p < 0.001, per SD increase). Conclusions: In NHANES participants, the Cr/CysC ratio had an inverse correlation with CVD morbidity and mortality. [ABSTRACT FROM AUTHOR]

Single molecules that co‐transport cations as well as anions across lipid membranes are few despite their high biological utility. The elegant yet simple lipidomimmetic peptide design herein enables efficient HCl transport without the use of any external additives for proton transport. The carboxylic acids in the dipeptide scaffold provide a handle to append two long hydrophobic tails and also provide a polar hydrophilic carboxylate group. The peptide central unit also provides NH sites for anion binding. Protonation of the carboxylate group coupled with the weak halide binding of the terminal NH group results in HCl transport with transport rates of H+>Cl−. The lipid‐like structure also facilitates seamless membrane integration and flipping of the molecule. The biocompatibility, design simplicity, and potential pH regulation of these molecules open up several avenues for their therapeutic use. [ABSTRACT FROM AUTHOR]

Simple Summary: P-glycoprotein (P-gp, ABCB1) is an ATP-binding cassette (ABC) transporter that contributes to the development of multidrug resistance (MDR) in cancer cells. P-gp pumps various amphipathic agents including anticancer drugs out of cells. The elucidation of the P-gp mechanism of drug transport is critical to develop strategies to overcome MDR. We used interdisciplinary approaches including cell biological, biochemical, mutational analysis, and molecular dynamics simulations to understand the conformational changes that occur in the homologous transmembrane helices (TMHs) 4 and 10 from the inward-open to the ATP-bound inward-closed states of P-gp during the transport cycle. We found that after substituting seven residues in either TMH4 or TMH10 with alanine, there was no significant effect on the transport function. However, the TMH4,10-14A mutant with 14 substitutions lost the ability to transport most of the substrates tested, revealing that conformational changes in both TMHs 4 and 10 are critical for P-gp's transport function. P-glycoprotein (P-gp, ABCB1) transports structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs, thus contributing to multidrug-resistant cancer. Cryo-EM structures of human P-gp revealed that TMHs 4 and 10 contribute to the formation of the drug-binding cavity and undergo conformational changes during drug transport. To assess the role of the conformational changes in TMH4 and TMH10 during drug transport, we generated two mutants (TMH4-7A and TMH10-7A), each containing seven alanine substitutions. Analysis of the drug efflux function of these mutants using 15 fluorescent substrates revealed that most of the substrates were transported, indicating that even seven mutations in an individual helix have no significant effect on transport function. We then designed the TMH4,10-14A mutant combining seven mutations in both TMHs 4 and 10. Interestingly, when the TMH4,10-14A mutant was tested with 15 substrates, there was no efflux observed for fourteen. The basal ATPase activity of the TMH4,10-14A mutant, similar to that of the WT protein, was inhibited by zosuquidar but was not stimulated by verapamil or rhodamine 6G. Molecular dynamics simulations indicated that the mutations cause TMHs 4 and 10 to pack tighter to their proximal helices, reducing their independent mobility. In aggregate, our findings demonstrate the critical role of the residues of homologous TMHs 4 and 10 for substrate transport, consistent with conformational changes observed in the structure of P-gp. [ABSTRACT FROM AUTHOR]