Surface engineering of metal-organic framework nanoparticles-based miRNA carrier: Boosting RNA stability, intracellular delivery and synergistic therapy.

MicroRNAs (miRNAs) are small noncoding RNAs that are critical for the regulation of multiple physiological and pathological processes, thus holding great clinical potential. However, the therapeutic applications of miRNAs are severely limited by their biological instability and poor intracellular delivery. Herein, we describe a dual-layers surface engineering strategy to design an efficient miRNA delivery nanosystem based on metal-organic frameworks (MOFs) incorporating lipid coating. The resulting nanoparticle system was demonstrated to protect miRNA from ribonuclease degradation, enhance cellular uptake and facilitate lysosomal escape. These ensured effective miRNA mediated gene therapy, which synergized with MOF-specific photodynamic therapy and pre-encapsulated doxorubicin (Dox) chemotherapy to provide a multifunctional with therapeutic effectiveness against cencer cells The mechanisms of miRNA binding and Dox loading were revealed, demonstrating the potential of the present MOFs surface-engineered strategy to overcome their inherent pore-size restriction for macromolecular miRNA carrying, enableefficient co-delivery. In vitro studies revealed the potential of our multifunctional system for miRNA delivery and the demonstrated the therapeutic effectiveness against cancer cells, thereby providing a versatile all-in-one MOFs strategy for delivery of nucleic acids and diverse therapeutic molecules in synergistic therapy.

Measuring GPCR-Induced Intracellular Calcium Signaling Using a Quantitative High-Throughput Assay.

Alterations in intracellular calcium are integral to signal transduction pathways for many G-protein-coupled receptors, but this signaling is not well studied. This is mostly due to a lack of reliable, robust, high-throughput, quantitative methods to monitor intracellular calcium concentrations in live cells. Recently, we developed a reliable, robust, quantitative method to measure intracellular calcium levels in which HEK293 cell suspensions loaded with Fura-2/AM are placed in 96-well plates. Minimum and maximum intracellular calcium levels, which are required for converting fluorescence into calcium concentrations, are calibrated using EGTA to chelate calcium and ionomycin to load calcium into cells, respectively. Fluorescence is monitored with a PHERAstar FS plate reader. We provide a detailed method for this high-throughput assay that can be used to quantitate intracellular calcium in endogenous and exogenously (stable or transient) expressed GPCRs in HEK293 cells.

Measuring Calcium Signaling at the Primary Cilia.

Cellular signaling is nature's ingenious way for cells to perceive their surroundings and transmit external cues to internal compartments. Due to its critical role in cellular functions, the intricate machinery of molecular signaling has been intensively studied. A diverse arsenal of techniques exists to quantify the molecules involved in these processes. Among them, calcium stands out as a ubiquitous signaling molecule with roles in countless biological pathways. To elucidate its function as a second messenger, methods for measuring intracellular calcium have steadily evolved. This chapter introduces various methods for investigating calcium signaling cascades in cells as well as in cilia (thin hairlike projections) specifically, where calcium signaling is triggered by different cilial manipulation techniques.

Unveiling mechanisms of antimicrobial peptide: Actions beyond the membranes disruption.

Antimicrobial peptides (AMPs) are a critical component of the innate immune system, playing a key role in defending against a variety of pathogenic microorganisms. While many AMPs act primarily on the cell membrane of target pathogens, leading to lysis and subsequent cell death, less is known about their nonlytic membrane activity. This nonlytic activity allows AMPs to target and disrupt bacterial cells without causing lysis, leading to bacterial death through alternative mechanisms.Understanding these nonlytic properties of AMPs is crucial, as they present a promising alternative to traditional antibiotics, which can induce bacterial resistance and have adverse effects on human health and the environment. The mechanisms by which AMPs exhibit nonlytic membrane activity are still being explored. However, it is believed that AMPs penetrate the bacterial membrane and interact directly with internal cellular components such as DNA, RNA, and various enzymes essential for microbial survival and replication. This interaction disrupts metabolic homeostasis, ultimately resulting in bacterial death.The nonlytic activity of AMPs also results in minimal damage to host cells and tissues, making them attractive candidates for the development of new, more effective antibiotics. This review emphasizes the mechanisms by which AMPs nonlytically target cellular components, including DNA, proteins, RNA, and other biomolecules, and discusses their clinical significance. Understanding these mechanisms may pave the way for developing alternatives to conventional antibiotics, offering a solution to the growing issue of antibiotic resistance.

Embracing multiple infection models to tackle Q fever: A review of in vitro, in vivo, and lung ex vivo models.

Multiple animal and cell culture models are employed to study pathogenesis of Coxiella burnetii, the causative agent of acute and chronic human Q fever. C. burnetii is a lung pathogen that is aerosolized in contaminated products and inhaled by humans to cause acute disease that can disseminate to other organs and establish chronic infection. Cellular models of Q fever include a variety of tissue-derived cell lines from mice and humans such as lung alveolar ex vivo cells. These models have the advantage of being cost-effective and reproducible. Similarly, animal models including mice and guinea pigs are cost-effective, although only immunocompromised SCID mice display a severe disease phenotype in response to Nine Mile I and Nine Mile II isolates of C. burnetii while immunocompetent guinea pigs display human-like symptoms and robust immune responses. Non-human primates such as macaques and marmosets are the closest model of human disease but are costly and largely used for adaptive immune response studies. All animal models are used for vaccine development but many differences exist in the pathogen's ability to establish lung infection when considering infection routes, bacterial isolates, and host genetic background. Similarly, while cellular models are useful for characterization of host-pathogen mechanisms, future developments should include use of a lung infection platform to draw appropriate conclusions. Here, we summarize the current state of the C. burnetii lung pathogenesis field by discussing the contribution of different animal and cell culture models and include suggestions for continuing to move the field forward.

Evolution of microbial community and resistance genes in denitrification system under single and combined exposure to benzethonium chloride and methylparaben.

Benzethonium chloride (BZC) and methylparaben (MeP) are commonly added into cosmetics as preservatives, which are frequently detected in wastewater treatment plants. Different response patterns of denitrification system were proposed under single and combined exposure to BZC and MeP (0, 0.5, 5 mg/L) by evaluating system performance, functional genes, extracellular polymeric substance (EPS), cytotoxicity, microbial community structure and resistance genes (RGs). The inhibition effect of BZC on denitrification system was stronger than MeP, and the co-exposure of BZC and MeP showed synergistic effect, enhancing the inhibition effect of BZC single exposure. BZC and/or MeP could promote the diffusion of RGs in sludge, including intracellular RGs (si-RGs) and extracellular RGs (se-RGs). Moreover, the single exposure of BZC and co-exposure of BZC and MeP increased the dissemination risks of RGs in water (w-RGs). IntI1 and tnpA-04, mobile genetic elements (MGEs), correlated positively with diverse RGs from different fractions. Notably, the spread of RGs through horizontal gene transfer mediated by MGEs and the flow of si-RGs into extracellular and water were observed in this study.

A Hydroxyquinoline Polymer with Excellent Amyloidosis Inhibition and Protein Delivery Ability to Combat Amyloid-ß-Mediated Neurotoxicity.

The accumulation of abnormal protein deposits known as amyloid-ß (Aß) plaques contributes to the development and progression of Alzheimer's disease. Aggregated Aß exacerbates oxidative stress by stimulating the production of reactive oxygen species (ROS) in a detrimental feedback loop. 8-Hydroxyquinoline (8-HQ) is recognized for its ability to inhibit or reverse Aß aggregation and reduce neurotoxicity. Here, an 8-HQ-based polymer, DHQ, was developed to combat Aß-mediated neurotoxicity by delivering an antioxidant enzyme. DHQ efficiently delivers superoxide dismutase into targeted cells, thereby downregulating the intracellular ROS level. Additionally, the polymer effectively inhibits the fibrillization of three proteins involved in fibrosis, ß-lactoglobulin (BLG), insulin, and Aß1-40, at nanomolar concentrations. Cell culture models demonstrated that DHQ reduces ROS levels induced by Aß1-40 aggregation, rescuing cell viability and preventing apoptosis. Intracellular delivery of SOD further enhanced the ability to maintain the ROS homeostasis. This polymer offers a multifaceted approach to treating diseases associated with amyloidosis.

Emerging role of MAPK signaling in glycosphingolipid-associated tumorigenesis.

Glycosphingolipids (GSLs) are a type of amphipathic lipid molecules consisting of hydrophobic ceramide backbone bound to carbohydrate moiety clustered in the cell surface microdomains named 'lipid rafts' and are known to participate in cell-cell communication as well as intra-cellular signaling, thereby facilitating critical normal cellular processes and functions. Over the past several decades, various GSLs have been reported to be aberrantly expressed in different cancers, many of which have been associated with their prognosis. The wide implication of MAPK signaling in controlling tumor growth, progression, and metastasis through activation of an upstream signaling cascade, often originating in the cell membrane, justifies the rationale for its plausible influence on MAPK signaling. This review highlights the role of GSLs and their metabolites in regulating different signaling pathways towards modulation of tumor cell growth, migration, and adhesion by interacting with various receptors [epidermal growth factor receptor (EGFR), and platelet derived growth factor receptor (PDGFR), and other receptor tyrosine kinases (RTKs)] leading to activation of the MAPK pathway. Furthermore, GSLs can influence the activity and localization of downstream signaling components in the MAPK pathway by regulating the activation state of kinases, which in turn, regulate the activity of MAPKs. Additionally, this review further consolidates the GSL-mediated modulation of MAPK pathway components through the regulation of gene expression. Finally, recent findings on GSL-MAPK crosstalk will be explored in this article for the identification of potential anti-cancer therapeutic targets.

Eliminating Intracellular MRSA via Mannosylated Lipid-Coated Calcium Phosphate Nanoparticles.

Methicillin-resistant Staphylococcus aureus (MRSA) within cells proves exceptionally challenging to eradicate using conventional antimicrobials, resulting in recurring infections and heightened resistance. Herein, we reported an innovative mannosylated lipid-coated photodynamic/photothermal calcium phosphate nanoparticle (MAN-LCaP@ICG) for eradicating intracellular MRSA. The MAN-LCaP functioned as the vehicle for drug delivery, exhibiting preferential uptake by macrophages and facilitating the transport of ICG to intracellular pathogens. The MAN units integrated into MAN-LCaP@ICG could promote binding with MAN residuals on macrophage cells, as evidenced by cellular uptake assays using fluorescence microscopy and flow cytometry. Following its targeted accumulation, MAN-LCaP@ICG could enter into the cytoplasm and efficiently eradicate intracellular MRSA by a combination of the lysosome escape capability of CaP and the photodynamic and photothermal therapeutic effects of ICG. Furthermore, MAN-LCaP@ICG could kill MRSA more effectively than LCaP@ICG without MAN units or free ICG in a mouse peritoneal infection model. Therefore, MAN-LCaP@ICG provided a promising direction for human clinical application in combating intracellular infections.

Morphology-Mediated Tumor Deep Penetration for Enhanced Near Infrared II Photothermal and Chemotherapy of Colorectal Cancer.

The low permeability and heterogeneous distribution of drugs (including nanomedicines) have limited their deep penetration into solid tumors. Herein we report the design of gold nanoparticles with virus-like spikes (AuNVs) to mimic viral shapes and facilitate tumor penetration. Mechanistic studies revealed that AuNVs mainly entered cells through macropinocytosis, then transported to the Golgi/endoplasmic reticulum system via Rab11-regulated pathway, and finally exocytosed through recycling endosomes, leading to high cellular uptake, effective transcytosis, and deep tumor penetration compared to gold nanospheres (AuNPs) and gold nanostars (AuNSs). The high tumor accumulation and deep tumor penetration of mitoxantrone (MTO) facilitated by AuNVs endowed effective chemophotothermal therapy when exposed to a near-infrared II laser, significantly reducing tumor sizes in a mouse model of colorectal cancer. This study reveals a potent mechanism of viral-like structures in tissue penetration and highlights their potential as effective drug delivery carriers.

InSeq analysis of defined Legionella pneumophila libraries identifies a transporter-encoding gene cluster important for intracellular replication in mammalian hosts.

Legionella pneumophila is an intracellular bacterial pathogen that replicates inside human alveolar macrophages to cause a severe pneumonia known as Legionnaires' disease. L. pneumophila requires the Dot/Icm Type IV secretion system to deliver hundreds of bacterial proteins to the host cytosol that manipulate cellular processes to establish a protected compartment for bacterial replication known as the Legionella-containing vacuole. To better understand mechanisms apart from the Dot/Icm system that support survival and replication in this vacuole, we used transposon insertion sequencing in combination with defined mutant sublibraries to identify L. pneumophila fitness determinants in primary mouse macrophages and the mouse lung. This approach validated that many previously identified genes important for intracellular replication were critical for infection of a mammalian host. Further, the screens uncovered additional genes contributing to L. pneumophila replication in mammalian infection models. This included a cluster of seven genes in which insertion mutations resulted in L. pneumophila fitness defects in mammalian hosts. Generation of isogenic deletion mutants and genetic complementation studies verified the importance of genes within this locus for infection of mammalian cells. Genes in this cluster are predicted to encode nucleotide-modifying enzymes, a protein of unknown function, and an atypical ATP-binding cassette (ABC) transporter with significant homology to multidrug efflux pumps that has been named Lit, for Legionella infectivity transporter. Overall, these data provide a comprehensive overview of the bacterial processes that support L. pneumophila replication in a mammalian host and offer insight into the unique challenges posed by the intravacuolar environment.IMPORTANCEIntracellular bacteria employ diverse mechanisms to survive and replicate inside the inhospitable environment of host cells. Legionella pneumophila is an opportunistic human pathogen and a model system for studying intracellular host-pathogen interactions. Transposon sequencing is an invaluable tool for identifying bacterial genes contributing to infection, but current animal models for L. pneumophila are suboptimal for conventional screens using saturated mutant libraries. This study employed a series of defined transposon mutant libraries to identify determinants of L. pneumophila fitness in mammalian hosts, which include a newly identified bacterial transporter called Lit. Understanding the requirements for survival and replication inside host cells informs us about the environment bacteria encounter during infection and the mechanisms they employ to make this environment habitable. Such knowledge will be key to addressing future challenges in treating infections caused by intracellular bacteria.

Intracellular TAS2Rs act as a gatekeeper for the excretion of harmful substances via ABCB1 in keratinocytes.

Bitter taste receptors (TAS2Rs) are not only expressed in the oral cavity but also in skin. Extraoral TAS2Rs are thought to be involved in non-taste perception and tissue-specific functions. Keratinocytes that express TAS2Rs in the skin provide a first-line defense against external threats. However, the functional roles of these receptors in host defense remain unclear. Here, we demonstrated the sensory role of intracellularly located TAS2Rs against toxic substances in keratinocytes. Although many G protein-coupled receptors elicit signals from the surface, TAS2Rs were found to localize intracellularly, possibly to the ER, in human keratinocytes and HaCaT cells. TAS2R38, one of the TAS2R members, activated the Gα12/13/RhoA/ROCK/p38 MAP kinase/NF-κB pathway upon stimulation by phenylthiocarbamide (PTC), an agonist for this receptor, leading to the production of ABC transporters, such as ABCB1, in these cells. Notably, treatment with bitter compounds, such as PTC and saccharin, induced the upregulation of ABCB1 in HaCaT cells. Mechanistically, intracellular TAS2R38 and its downstream signaling Gα12/13/RhoA/ROCK/p38 MAP kinase/NF-κB pathway were identified to be responsible for the above effect. Pretreatment with PTC prevented the accumulation of rhodamine 123 because of its excretion via ABCB1. Furthermore, pretreatment with PTC or saccharin counteracted the effect of the toxic compound, diphenhydramine, and pretreated HaCaT cells were found to proliferate faster than untreated cells. This anti-toxic effect was suppressed by treatment with verapamil, an ABCB1 inhibitor, indicating that enhanced ABCB1 helps clear toxic substances. Altogether, harmless activators of TAS2Rs may be promising drugs that enhance the excretion of toxic substances from the human skin.

Polyoxazolines with Cholesterol Lipid Anchor for Fast Intracellular Delivery.

Due to the increasing challenges posed by the growing immunity to poly(ethylene glycol) (PEG), there is growing interest in innovative polymer-based materials as viable alternatives. In this study, the advantages of lipids and polymers are combined to allow efficient and rapid cytoplasmic drug delivery. Specifically, poly(2-methyl-2-oxazoline) is modified with a cholesteryl hemisuccinate group as a lipid anchor (CHEMSPOx). The CHEMSPOx is additionally functionalized with a coumarin group (CHEMSPOx-coumarin). Both polymers self-assembled in water into vesicles of ≈100 nm and are successfully loaded with a hydrophobic model drug. The loaded vesicles reveal high cellular internalization across variant cell lines within 1 h at 37 °C as well as 4 °C, albeit to a lesser extent. A kinetic study confirms the fast internalization within 5 min after the sample's addition. Therefore, different internalization pathways are involved, e.g., active uptake but also nonenergy dependent mechanisms. CHEMSPOx and CHEMSPOx-coumarin further demonstrate excellent cyto-, hemo-, and membrane compatibility, as well as a membrane-protecting effect, which underlines their good safety profile for potential biological intravenous application. Overall, CHEMSPOx, as a lipopolyoxazoline, holds great potential for versatile biological applications such as fast and direct intracellular delivery or cellular lysis protection.

Amyloid nomenclature 2024: update, novel proteins, and recommendations by the International Society of Amyloidosis (ISA) Nomenclature Committee.

The ISA Nomenclature Committee met at the XIX International Symposium of Amyloidosis in Rochester, MN, 27 May 2024. The in-person event was followed by many electronic discussions, resulting in the current updated recommendations. The general nomenclature principles are unchanged. The total number of human amyloid fibril proteins is now 42 of which 19 are associated with systemic deposition, while 4 occur with either localised or systemic deposits. Most systemic amyloidoses are caused by the presence of protein variants which promote misfolding. However, in the cases of AA and ATTR the deposits most commonly consist of wild-type proteins and/or their fragments. One peptide drug, previously reported to create local iatrogenic amyloid deposits at its injection site, has been shown to induce rare instances of systemic deposition. The number of described animal amyloid fibril proteins is now 16, 2 of which are unknown in humans. Recognition of the importance of intracellular protein aggregates, which may have amyloid or amyloid-like properties, in many neurodegenerative diseases is rapidly increasing and their significance is discussed.

Effect of Hydroxyapatite Nanoparticle Crystallinity and Colloidal Stability on Cytotoxicity.

Hydroxyapatite nanoparticles (nHA) have gained attention as potential intracellular drug delivery vehicles due to their high binding affinity for various biomolecules and pH-dependent solubility. Yet, the dependence of nHA cytocompatibility on their physicochemical properties remains unclear since numerous studies have revealed starkly contrasting results. These discrepancies may be attributed to differences in size, shape, crystallinity, and aggregation state of nHA, which complicates fundamental understanding of the factors driving nHA cytotoxicity. Here, we hypothesize that nHA cytotoxicity is primarily driven by intracellular calcium levels following the internalization of nHA nanoparticles. By investigating the cytotoxicity of spherical nHA with different crystallinity and dispersity, we find that both lower crystallinity and increased agglomeration of nHA raise cytotoxicity, with nanoparticle agglomeration being the more dominant factor. We show that the internalization of nHA enhances intracellular calcium levels and increases the production of reactive oxygen species (ROS). However, only subtle changes in intracellular calcium are observed, and their physiological relevance remains to be confirmed. In conclusion, we show that nHA agglomeration enhances ROS production and the associated cytotoxicity. These findings provide important guidelines for the future design of nHA-containing formulations for biomedical applications, implying that nHA crystallinity and especially agglomeration should be carefully controlled to optimize biocompatibility and therapeutic efficacy.

Electro-mediated cathodic oxygen drives respiration chain electron transfer of electroactive bacteria to enhance refractory organic biological oxidation.

In electro-mediated biological system (EMBS), biological anode and cathode components were incorporated into an anaerobic bioreactor, providing a small amount of oxygen to the cathode as an electron acceptor. Oxygen diffusion also impacts the anode's anaerobic ecological environment. This study unraveled how oxygen influences the metabolism and electron transport chain during the biological oxidation of refractory organics. Under the influence of electromotive force, the straight-chain model pollutant N,N-dimethylformamide (DMF) showed rapid degradation and better ammonification, with maximum rates reaching 0.53 h-1 and 26.6 %, respectively. Elevated electromotive force promoted the enrichment of functional electroactive bacteria on the anode and enhanced the availability of electron storage sites, thereby facilitating electron transfer at the anode-biofilm interface. Conversely, the anodic micro-aerobic environment disrupted the anaerobic microbial community structure, and the competitive interactions among fermentative bacteria and electroactive bacteria inhibited DMF degradation. Metagenomic analysis confirmed that cathodic oxygen up-regulated the pyruvate metabolism and the tricarboxylic acid (TCA) cycle to generate NADH and synthesize ATP. The electromotive force induced by cathodic oxygen accelerated the electron transfer in respiratory chains of electroactive bacteria, driving the oxidation of NADH and enhancing the degradation of organics. This study improves our understanding of the regulatory mechanisms governing metabolic pathways under the influence of cathodic oxygen. It offers potential for developing more efficient EMBS in industrial wastewater pretreatment, ensuring that oxygen is prevented from diffusing to the anode during micro-aeration at the cathode.

Can Physical Therapy Techniques Slow Down the Progression of Lipedema?

Background: Lipedema is a disease characterized by an increase in extracellular fluid. In these patients, the increase in the amount of extracellular fluid may accelerate the progression of the disease. The aim of this study is to examine the effect of complex decongestive therapy (CDT) on intracellular/extracellular fluid balance. Methods and Results: Twenty-two female patients diagnosed with lipedema by a specialist lymphologist were included in the study. Patients were subjected to complex decongestive therapy and pneumatic compression therapy 6 days a week for 1 month. Extracellular and intracellular fluid volumes were assessed using bioimpedance spectroscopy before and after the treatment. A decrease in intracellular (p = 0.010) and extracellular (p = 0.002) fluid volumes was observed after the treatment. Conclusion: There is no completely curative treatment method for lipedema. Current treatments aim to slow down the progression of the disease. CDT is considered effective in reducing intracellular and extracellular fluid volume in lipedema patients. Therefore, it is thought to be effective in slowing down the progression of the disease.

Towards a high-rate operation of contact stabilization process: A microscopic view of carbon capture properties.

Carbon capture performance is a key factor determining the chemical energy recovery potential of the high-rate contact stabilization (HiCS) process. However, the mechanisms of organic carbon capture are complex, involving surface adsorption, extracellular adsorption, and intracellular storage. A unique characteristic of the HiCS process is its low sludge residence time (SRT). Unfortunately, the influence of SRT on carbon capture has not been thoroughly studied, especially in terms of the underlying mechanisms. In this study, the microscopic changes in carbon capture performance during the transition from a conventional contact stabilized (CS) system to a high-rate mode of operation were demonstrated using intracellular carbon sources, extracellular polymeric substances (EPS), signaling molecules, and microbial community assays. The results showed that the extracellular carbon adsorption and intracellular carbon storage performance increased, and the microbial community structure changed significantly with converting the CS system to the high-rate operation mode. The enhancement of extracellular carbon adsorption performance mainly relied on the growth of EPS, which was accomplished by the strong growth of the relative abundance of the dominant bacterial group Cloacibacterium within the HiCS system, offsetting the negative effect produced by the decline of acyl-homoserine lactones. 98 mgCOD/gSS, 343 mgCOD/gSS, and 500 mgCOD/gSS of polyhydroxyalkanoates (PHAs) per sludge unit were obtained at SRT-24d, 8d, and 2d, respectively, suggesting that the HiCS system is more advantageous for rapid PHAs production.

Trifluoperazine effect on human sperm: The accumulation of reactive oxygen species and the decrease in the mitochondrial membrane potential.

A strong link between antipsychotic drug use and reduced human sperm quality has been reported. Trifluoperazine (TFP), a commonly used antipsychotic, is now being explored for anticancer applications. Although there are hints that TFP might affect the male reproductive system, its impact on human sperm quality remains uncertain. Using a human sperm and TFP in vitro coculture system, we examined the effect of TFP (12.5, 25, 50 and 100 µM) on human sperm function and physiological parameters. The results showed that 50 µM and 100 µM TFP induced the accumulation of reactive oxygen species (ROS) and a decrease in the mitochondrial membrane potential (MMP) of human sperm, leading to decreased sperm viability, while 25 µM TFP inhibited only the penetration ability, total sperm motility, and progressive motility. Although 12.5 µM and 25 µM TFP increased [Ca2+]i in human sperm, they did not affect capacitation or the acrosome reaction. These results may be explained by the observation that 12.5 µM and 25 µM TFP did not increase tyrosine phosphorylation in human sperm, although TFP increased [Ca2+]i in a time-course traces similar to that of progesterone. Our results indicated that TFP could cause male reproductive toxicity by inducing the accumulation of ROS and a decrease in the MMP in human sperm.

Stiffening of the Cytoplasm in Response to Intracellularly Applied Forces.

Cells constantly encounter mechanical forces that regulate various cellular functions, such as migration, division, and differentiation. Understanding how cells respond to forces at the intracellular level is essential for elucidating the mechanical adaptability of living cells. This study investigates how the cytoplasm alters its mechanical properties in response to forces applied inside a cell. The mechanical properties were measured through in situ characterization using magnetic tweezers to apply mechanical forces on magnetic beads internalized into cells. The findings reveal that the cytoplasm stiffens within seconds when force is applied to the cytoplasm. Macromolecular crowding and cytoskeletal structures, particularly F-actin, were found to significantly contribute to cytoplasm stiffening. The stiffening response was also observed across multiple length scales by using magnetic beads of varying diameters. These results highlight the rapid adaptation of the cytoplasm to mechanical forces applied to the inside of a cell.