Symplasmic and transmembrane zinc transport is modulated by cadmium in the Cd/Zn hyperaccumulator Sedum alfredii.

This study investigated the influence of Cd (25 µM) on Zn accumulation in a hyperaccumulating (HE) and a non-hyperaccumulating (NHE) ecotype of Sedum alfredii Hance at short-term supply of replete (Zn5, 5 µM) and excess (Zn400, 400 µM) Zn. Cd inhibited Zn accumulation in both ecotypes, especially under Zn400, in organs with active metal sequestration, i.e. roots of NHE and shoots of HE. Direct biochemical Cd/Zn competition at the metal-protein interaction and changes in transporter gene expression contributed to the observed accumulation patterns in the roots. Specifically, in HE, Cd stimulated SaZIP4 and SaPCR2 under Zn5, but downregulated SaIRT1 and SaZIP4 under Zn400. However, Cd downregulated related transporter genes, except for SaNRAMP1, in NHE, irrespective of Zn. Cadmium stimulated casparian strip (CSs) development in NHE, as part of the defense response, while it had a subtle effect on the (CS) in HE. Moreover, Cd delayed the initiation of the suberin lamellae (SL) in HE, but stimulated SL deposition in NHE under both Zn5 or Zn400. Changes in suberization were mainly ascribed to suberin-biosynthesis-related genes and hormonal signaling. Altogether, Cd regulated Zn accumulation mainly via symplasmic and transmembrane transport in HE, while Cd inhibited both symplasmic and apoplasmic Zn transport in NHE.

Modeling of brain efflux: Constraints of brain surfaces.

Fluid efflux from the brain plays an important role in solute waste clearance. Current experimental approaches provide little spatial information, and data collection is limited due to short duration or low frequency of sampling. One approach shows tracer efflux to be independent of molecular size, indicating bulk flow, yet also decelerating like simple membrane diffusion. In an apparent contradiction to this report, other studies point to tracer efflux acceleration. We here develop a one-dimensional advection-diffusion model to gain insight into brain efflux principles. The model is characterized by nine physiological constants and three efflux parameters for which we quantify prior uncertainty. Using Bayes' rule and the two efflux studies, we validate the model and calculate data-informed parameter distributions. The apparent contradictions in the efflux studies are resolved by brain surface boundaries being bottlenecks for efflux. To critically test the model, a custom MRI efflux assay measuring solute dispersion in tissue and release to cerebrospinal fluid was employed. The model passed the test with tissue bulk flow velocities in the range 60 to 190 [Formula: see text]m/h. Dimensional analysis identified three principal determinants of efflux, highlighting brain surfaces as a restricting factor for metabolite solute clearance.

A structure-functionality insight into the bioactivity of microbial polysaccharides toward biomedical applications: A review.

Microbial polysaccharides (MPs) are biopolymers secreted by microorganisms such as bacteria and fungi during their metabolic processes. Compared to polysaccharides derived from plants and animals, MPs have advantages such as wide sources, high production efficiency, and less susceptibility to natural environmental influences. The most attractive feature of MPs lies in their diverse biological activities, such as antioxidative, anti-tumor, antibacterial, and immunomodulatory activities, which have demonstrated immense potential for applications in functional foods, cosmetics, and biomedicine. These bioactivities are precisely regulated by their sophisticated molecular structure. However, the mechanisms underlying this precise regulation are not yet fully understood and continue to evolve. This article presents a comprehensive review of the most representative species of MPs, including their fermentation and purification processes and their biomedical applications in recent years. In particular, this work presents an in-depth analysis into the structure-activity relationships of MPs across multiple molecular levels. Additionally, this review discusses the challenges and prospects of investigating the structure-activity relationships, providing valuable insights into the broad and high-value utilization of MPs.

Bacterial Pathogenesis: Assessment of Intracellular Positioning of Pathogen-Containing Vacuoles During Infection.

Intracellular bacterial pathogens implement a diverse array of strategies to target host cells and establish infection. For vacuolar pathogens, the process of pathogen-containing vacuole movement within host cells, termed intracellular trafficking, is central to both pathogen survival and infection progression. Typically a process mediated by secreted virulence factors that manipulate the host cytoskeletal machinery, internalized pathogen-containing vacuoles traffic to the site of replication to establish a unique replicative niche, and if applicable, traffic back toward the host cell periphery for cell-to-cell spread. As such, the intracellular positioning of pathogen-containing vacuoles represents a fundamental measure of infection progression. Here, we describe a fluorescence microscopy-based method to quantitatively assess bacterial intracellular positioning, using Salmonella enterica serovar Typhimurium infection of epithelial cells as a model. This experimental approach can be modified to study infection in diverse host cell types, and with a broad array of pathogens. The system can also be adapted to examine the kinetics of infection, identify secreted virulence factors that mediate host trafficking, investigate host factors that are targeted by the pathogen for trafficking, and assess functional domains within a virulence factor responsible for mediating the phenotype. Collectively, these tools can provide fundamental insight into the pathogenesis of a diverse array of intracellular bacterial pathogens, and new host factors that are hijacked to mediate infection. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culture and preparation of host cells Alternate Protocol: Culture and preparation of host cells to assess host factor contribution to bacterial positioning Basic Protocol 2: Infection of epithelial cells with S. Typhimurium Basic Protocol 3: Fluorescence staining for analysis of bacterial positioning Basic Protocol 4: Fluorescence microscopy analysis of bacterial positioning.

A hard sphere model for single-file water transport across biological membranes.

We use Gürsey's statistical mechanics of a one-dimensional fluid to find a formula for the P f / P d ratio in the transport of hard spheres across a membrane through a narrow channel that can accommodate molecular movement only in single file. P f is the membrane permeability for osmotic flow and P d the permeability for exchange across the membrane in the absence of osmotic flow. The deviation of the ratio from unity indicates the degree of cooperative transport relative to ordinary diffusion of independent molecules. In contrast to an early idea that P f / P d must be equal to the number of molecules in the channel, regardless of the physical nature of the interactions among the molecules, we find a functional dependence on the fractional occupancy of the length of the channel by the hard spheres. We also attempt a random walk calculation for P d individually, which gives a result for P f as well when combined with the ratio.

Conformational free-energy landscapes of a Na+/Ca2+ exchanger explain its alternating-access mechanism and functional specificity.

Secondary-active transporters catalyze the movement of myriad substances across all cellular membranes, typically against opposing concentration gradients, and without consuming any ATP. To do so, these proteins employ an intriguing structural mechanism evolved to be activated only upon recognition or release of the transported species. We examine this self-regulated mechanism using a homolog of the cardiac Na+/Ca2+ exchanger as a model system. Using advanced computer simulations, we map out the complete functional cycle of this transporter, including unknown conformations that we validate against existing experimental data. Calculated free-energy landscapes reveal why this transporter functions as an antiporter rather than a symporter, why it specifically exchanges Na+ and Ca2+, and why the stoichiometry of this exchange is exactly 3:1. We also rationalize why the protein does not exchange H+ for either Ca2+ or Na+, despite being able to bind H+ and its high similarity with H+/Ca2+ exchangers. Interestingly, the nature of this transporter is not explained by its primary structural states, known as inward- and outward-open conformations; instead, the defining factor is the feasibility of conformational intermediates between those states, wherein access pathways leading to the substrate binding sites become simultaneously occluded from both sides of the membrane. This analysis offers a physically coherent, broadly transferable route to understand the emergence of function from structure among secondary-active membrane transporters.

Monocarboxylate Transporters 1 and 2 Are Responsible for L-Lactate Uptake in Differentiated Human Neuroblastoma SH-SY5Y Cells.

L-Lactate transport via monocarboxylate transporters (MCTs) in the central nervous system, represented by the astrocyte-neuron lactate shuttle (ANLS), is crucial for the maintenance of brain functions, including memory formation. Previously, we have reported that MCT1 contributes to L-lactate transport in normal human astrocytes. Therefore, in this study, we aimed to identify transporters that contribute to L-lactate transport in human neurons. SH-SY5Y cells, which are used as a model for human neurons, were differentiated using all-trans-retinoic acid. L-Lactate uptake was measured using radiolabeled L-lactate, and the expression of MCT proteins was confirmed Western blotting. L-Lactate transport was pH-dependent and saturated at high concentrations. Kinetic analysis suggested that L-lactate uptake was biphasic. Furthermore, MCT1, 2 selective inhibitors inhibited L-lactate transport. In addition, the expression of MCT1 and 2 proteins, but not MCT4, was confirmed. In this study, we demonstrated that MCT1 and 2 are major contributors to L-lactate transport in differentiated human neuroblastoma SH-SY5Y cells from the viewpoint of kinetic analysis. These results lead to a better understanding of ANLS in humans, and further exploration of the factors that can promote MCT1 and 2 functions is required.

Microbial hitchhikers on microplastics: The exchange of aquatic microbes across distinct aquatic habitats.

Microplastics (MPs) have the potential to modify aquatic microbial communities and distribute microorganisms, including pathogens. This poses a potential risk to aquatic life and human health. Despite this, the fate of 'hitchhiking' microbes on MPs that traverse different aquatic habitats remains largely unknown. To address this, we conducted a 50-day microcosm experiment, manipulating estuarine conditions to study the exchange of bacteria and microeukaryotes between river, sea and plastisphere using a long-read metabarcoding approach. Our findings revealed a significant increase in bacteria on the plastisphere, including Pseudomonas, Sphingomonas, Hyphomonas, Brevundimonas, Aquabacterium and Thalassolituus, all of which are known for their pollutant degradation capabilities, specifically polycyclic aromatic hydrocarbons. We also observed a strong association of plastic-degrading fungi (i.e., Cladosporium and Plectosphaerella) and early-diverging fungi (Cryptomycota, also known as Rozellomycota) with the plastisphere. Sea MPs were primarily colonised by fungi (70%), with a small proportion of river-transported microbes (1%-4%). The mere presence of MPs in seawater increased the relative abundance of planktonic fungi from 2% to 25%, suggesting significant exchanges between planktonic and plastisphere communities. Using microbial source tracking, we discovered that MPs only dispersed 3.5% and 5.5% of river bacterial and microeukaryotic communities into the sea, respectively. Hence, although MPs select and facilitate the dispersal of ecologically significant microorganisms, drastic compositional changes across distinct aquatic habitats are unlikely.

Kinesin KIF3A regulates meiotic progression and spindle assembly in oocyte meiosis.

Kinesin family member 3A (KIF3A) is a microtubule-oriented motor protein that belongs to the kinesin-2 family for regulating intracellular transport and microtubule movement. In this study, we characterized the critical roles of KIF3A during mouse oocyte meiosis. We found that KIF3A associated with microtubules during meiosis and depletion of KIF3A resulted in oocyte maturation defects. LC-MS data indicated that KIF3A associated with cell cycle regulation, cytoskeleton, mitochondrial function and intracellular transport-related molecules. Depletion of KIF3A activated the spindle assembly checkpoint, leading to metaphase I arrest of the first meiosis. In addition, KIF3A depletion caused aberrant spindle pole organization based on its association with KIFC1 to regulate expression and polar localization of NuMA and γ-tubulin; and KIF3A knockdown also reduced microtubule stability due to the altered microtubule deacetylation by histone deacetylase 6 (HDAC6). Exogenous Kif3a mRNA supplementation rescued the maturation defects caused by KIF3A depletion. Moreover, KIF3A was also essential for the distribution and function of mitochondria, Golgi apparatus and endoplasmic reticulum in oocytes. Conditional knockout of epithelial splicing regulatory protein 1 (ESRP1) disrupted the expression and localization of KIF3A in oocytes. Overall, our results suggest that KIF3A regulates cell cycle progression, spindle assembly and organelle distribution during mouse oocyte meiosis.

Drug Distribution After Intravitreal Injection: A Mathematical Model.

Purpose: Intravitreal injection of drugs is commonly used for treatment of chorioretinal ocular pathologies, such as age-related macular degeneration. Injection causes a transient increase in the intraocular volume and, consequently, of the intraocular pressure (IOP). The aim of this work is to investigate how intravitreal flow patterns generated during the post-injection eye deflation influence the transport and distribution of the injected drug. Methods: We present mathematical and computational models of fluid motion and mass transport in the vitreous chamber during the transient phase after injection, including the previously unexplored effects of globe deflation as ocular volume decreases. Results: During eye globe deflation, significant fluid velocities are generated within the vitreous chamber, which can possibly contribute to drug transport. Pressure variations within the eye globe are small compared to IOP. Conclusions: Even if significant fluid velocities are generated in the vitreous chamber after drug injection, these are found to have negligible overall effect on drug distribution.

Miro GTPases at the Crossroads of Cytoskeletal Dynamics and Mitochondrial Trafficking.

Miro GTPases are key components in the machinery responsible for transporting mitochondria and peroxisomes along microtubules, and also play important roles in regulating calcium homeostasis and organizing contact sites between mitochondria and the endoplasmic reticulum. Moreover, Miro GTPases have been shown to interact with proteins that actively regulate cytoskeletal organization and dynamics, suggesting that these GTPases participate in organizing cytoskeletal functions and organelle transport. Derailed mitochondrial transport is associated with neuropathological conditions such as Parkinson's and Alzheimer's diseases. This review explores our recent understanding of the diverse roles of Miro GTPases under cytoskeletal control, both under normal conditions and during the course of human diseases such as neuropathological disorders.

Quantum biochemical analysis of the TtgR regulator and effectors.

The recent expansion of multidrug-resistant (MDR) pathogens poses significant challenges in treating healthcare-associated infections. Although antibacterial resistance occurs by numerous mechanisms, active efflux of the drugs is a critical concern. A single species of efflux pump can produce a simultaneous resistance to several drugs. One of the best-studied efflux pumps is the TtgABC: a tripartite resistance-nodulation-division (RND) efflux pump implicated in the intrinsic antibiotic resistance in Pseudomonas putida DOT-T1E. The expression of the TtgABC gene is down-regulated by the HTH-type transcriptional repressor TtgR. In this context, by employing quantum chemistry methods based on the Density Functional Theory (DFT) within the Molecular Fragmentation with Conjugate Caps (MFCC) approach, we investigate the coupling profiles of the transcriptional regulator TtgR in complex with quercetin (QUE), a natural polyphenolic flavonoid, tetracycline (TAC), and chloramphenicol (CLM), two broad-spectrum antimicrobial agents. Our quantum biochemical computational results show the: [i] convergence radius, [ii] total binding energy, [iii] relevance (energetically) of the ligands regions, and [iv] most relevant amino acids residues of the TtgR-QUE/TAC/CLM complexes, pointing out distinctions and similarities among them. These findings improve the understanding of the binding mechanism of effectors and facilitate the development of new chemicals targeting TtgR, helping in the battle against the rise of resistance to antimicrobial drugs. These advances are crucial in the ongoing fight against rising antimicrobial drug resistance, providing hope for a future where healthcare-associated infections can be more beneficially treated.

Radiopharmaceutical transport in solid tumors via a 3-dimensional image-based spatiotemporal model.

Lutetium-177 prostate-specific membrane antigen (177Lu-PSMA)-targeted radiopharmaceutical therapy is a clinically approved treatment for patients with metastatic castration-resistant prostate cancer (mCRPC). Even though common practice reluctantly follows "one size fits all" approach, medical community believes there is significant room for deeper understanding and personalization of radiopharmaceutical therapies. To pursue this aim, we present a 3-dimensional spatiotemporal radiopharmaceutical delivery model based on clinical imaging data to simulate pharmacokinetic of 177Lu-PSMA within the prostate tumors. The model includes interstitial flow, radiopharmaceutical transport in tissues, receptor cycles, association/dissociation with ligands, synthesis of PSMA receptors, receptor recycling, internalization of radiopharmaceuticals, and degradation of receptors and drugs. The model was studied for a range of values for injection amount (100-1000 nmol), receptor density (10-500 nmol•l-1), and recycling rate of receptors (10-4 to 10-1 min-1). Furthermore, injection type, different convection-diffusion-reaction mechanisms, characteristic time scales, and length scales are discussed. The study found that increasing receptor density, ligand amount, and labeled ligands improved radiopharmaceutical uptake in the tumor. A high receptor recycling rate (0.1 min-1) increased radiopharmaceutical concentration by promoting repeated binding to tumor cell receptors. Continuous infusion results in higher radiopharmaceutical concentrations within tumors compared to bolus administration. These insights are crucial for advancing targeted therapy for prostate cancer by understanding the mechanism of radiopharmaceutical distribution in tumors. Furthermore, measures of characteristic length and advection time scale were computed. The presented spatiotemporal tumor transport model can analyze different physiological parameters affecting 177Lu-PSMA delivery.

Evaluating Bacterial Nanocellulose Interfaces for Recording Surface Biopotentials from Plants.

The study of plant electrophysiology offers promising techniques to track plant health and stress in vivo for both agricultural and environmental monitoring applications. Use of superficial electrodes on the plant body to record surface potentials may provide new phenotyping insights. Bacterial nanocellulose (BNC) is a flexible, optically translucent, and water-vapor-permeable material with low manufacturing costs, making it an ideal substrate for non-invasive and non-destructive plant electrodes. This work presents BNC electrodes with screen-printed carbon (graphite) ink-based conductive traces and pads. It investigates the potential of these electrodes for plant surface electrophysiology measurements in comparison to commercially available standard wet gel and needle electrodes. The electrochemically active surface area and impedance of the BNC electrodes varied based on the annealing temperature and time over the ranges of 50 °C to 90 °C and 5 to 60 min, respectively. The water vapor transfer rate and optical transmittance of the BNC substrate were measured to estimate the level of occlusion caused by these surface electrodes on the plant tissue. The total reduction in chlorophyll content under the electrodes was measured after the electrodes were placed on maize leaves for up to 300 h, showing that the BNC caused only a 16% reduction. Maize leaf transpiration was reduced by only 20% under the BNC electrodes after 72 h compared to a 60% reduction under wet gel electrodes in 48 h. On three different model plants, BNC-carbon ink surface electrodes and standard invasive needle electrodes were shown to have a comparable signal quality, with a correlation coefficient of >0.9, when measuring surface biopotentials induced by acute environmental stressors. These are strong indications of the superior performance of the BNC substrate with screen-printed graphite ink as an electrode material for plant surface biopotential recordings.

Aquaporins in the Cornea.

The cornea is an avascular, transparent tissue that allows light to enter the visual system. Accurate vision requires proper maintenance of the cornea's integrity and structure. Due to its exposure to the external environment, the cornea is prone to injury and must undergo proper wound healing to restore vision. Aquaporins (AQPs) are a family of water channels important for passive water transport and, in some family members, the transport of other small molecules; AQPs are expressed in all layers of the cornea. Although their functions as water channels are well established, the direct function of AQPs in the cornea is still being determined and is the focus of this review. AQPs, primarily AQP1, AQP3, and AQP5, have been found to play an important role in maintaining water homeostasis, the corneal structure in relation to proper hydration, and stress responses, as well as wound healing in all layers of the cornea. Due to their many functions in the cornea, the identification of drug targets that modulate the expression of AQPs in the cornea could be beneficial to promote corneal wound healing and restore proper function of this tissue crucial for vision.

Root-Applied Cerium Oxide Nanoparticles and Their Specific Effects on Plants: A Review.

With the pronounced increase in nanotechnology, it is likely that biological systems will be exposed to excess nanoparticles (NPs). Cerium oxide nanoparticles (CeO2 NPs) are among the most abundantly produced nanomaterials in the world. Their widespread use raises fundamental questions related to the accumulation in the environment and further interactions with living organisms, especially plants. NPs present in either soil or soilless environments are absorbed by the plant root systems and further transported to the aboveground parts. After entering the cytoplasm, NPs interact with chloroplast, nucleus, and other structures responsible for metabolic processes at the cellular level. In recent years, several studies have shown the impact of nanoceria on plant growth and metabolic processes. Research performed on different plants has shown a dual role for CeO2 NPs. The observed effects can be positive or negative and strongly depend on the plant species, characterization, and concentrations of NPs. This review describes the impact of root-applied CeO2 NPs on plant growth, photosynthesis, metal homeostasis, and parameters of induced oxidative stress.

Inhibition of Autophagy Alters Intracellular Transport of APP Resulting in Increased APP Processing.

Alzheimer's disease (AD) pathology is characterized by amyloid beta (Aß) plaques and dysfunctional autophagy. Aß is generated by sequential proteolytic cleavage of amyloid precursor protein (APP), and the site of intracellular APP processing is highly debated, which may include autophagosomes. Here, we investigated the involvement of autophagy, including the role of ATG9 in APP intracellular trafficking and processing by applying the RUSH system, which allows studying the transport of fluorescently labeled mCherry-APP-EGFP in a systematic way, starting from the endoplasmic reticulum. HeLa cells, expressing the RUSH mCherry-APP-EGFP system, were investigated by live cell imaging, immunofluorescence, and Western blot. We found that mCherry-APP-EGFP passed through the Golgi faster in ATG9 knockout cells. Furthermore, ATG9 deletion shifted mCherry-APP-EGFP from early endosomes and lysosomes toward the plasma membrane concomitant with reduced endocytosis. Importantly, this alteration in mCherry-APP-EGFP transport resulted in increased secreted mCherry-soluble APP and C-terminal fragment-EGFP. These effects were also phenocopied by pharmacological inhibition of ULK1, indicating that autophagy is regulating the intracellular trafficking and processing of APP. These findings contribute to the understanding of the role of autophagy in APP metabolism and could potentially have implications for new therapeutic approaches for AD.

Gaps in the wall of a perivascular space act as valves to produce a directed flow of cerebrospinal fluid: a hoop-stress model.

The flow of cerebrospinal fluid (CSF) along perivascular spaces (PVSs) is an important part of the brain's system for clearing metabolic waste. Astrocyte endfeet bound the PVSs of penetrating arteries, separating them from brain extracellular space. Gaps between astrocyte endfeet might provide a low-resistance pathway for fluid transport across the wall. Recent studies suggest that the astrocyte endfeet function as valves that rectify the CSF flow, producing the net flow observed in pial PVSs by changing the size of the gaps in response to pressure changes. In this study, we quantify this rectification based on three features of the PVSs: the quasi-circular geometry, the deformable endfoot wall, and the pressure oscillation inside. We provide an analytical model, based on the thin-shell hoop-stress approximation, and predict a pumping efficiency of about 0.4, which would contribute significantly to the observed flow. When we add the flow resistance of the extracellular space (ECS) to the model, we find an increased net flow during sleep, due to the known increase in ECS porosity (decreased flow resistance) compared to that in the awake state. We corroborate our analytical model with three-dimensional fluid-solid interaction simulations.

Spatial and Temporal Localization of Connexins in Cells Using Confocal Microscopy.

The 21-member connexin family found in humans is the building block of both single-membrane spanning channels (hemichannels) and double-membrane spanning intercellular channels. These large-pore channels are dynamic and typically have a short life span of only a few hours. Imaging connexins from the time of synthesis in the endoplasmic reticulum through to their degradation can be challenging given their distinct assembly states and transient residences in many subcellular compartments. Here, we describe how connexins can be effectively imaged on a confocal microscope in living cells when tagged with fluorescent proteins and when immunolabeled with high affinity anti-connexin antibodies in fixed cells. Temporal and spatial localization of multiple connexins and disease-linked connexin mutants at the subcellular level extensively informs on the mechanisms governing connexin regulation in health and disease.

A Dye Uptake Assay to Measure Large-Pore Channel Activity in Trypanosoma cruzi.

Large-pore channels allow the exchange of ions and molecules between the intra- and extracellular compartments. These channels are structures formed by several protein families with little or no evolutionary linkages that include connexins (Cxs), pannexins (Panxs), innexins (Inxs), CALHM1, and LRRC8 proteins. Recently, we have described the unnexins (Unxs) proteins expressed in Trypanosoma cruzi (T. cruzi) that also is like to form large-pore channels at the plasma membrane. In this chapter, we describe a dye uptake method for evaluating the unnexin-formed channel function in T. cruzi, as well as the methods for evaluating their participation in the transformation of trypomastigotes into amastigotes. These methods can facilitate understanding the role of large-pore channels in the parasite's biology.