High Intracellular Concentrations Research Articles

Lysteria Monocytogenes Infection during Monochorionic Twin Pregnancy: Case Report and Review of the Literature

Listeriosis is a rare but severe foodborne disease caused by Listeria Monocytogenes (LM), a small facultative intracellular bacillus. When occurring in pregnant women, it can be vertically transmitted to the fetus and the newborn. Infected women usually display aspecific and mild symptoms, and rarely develop the severe forms of the disease (such as neurolisteriosis). On the contrary, fetal and neonatal listeriosis can lead to complications such as fetal loss, preterm birth, neonatal sepsis, and respiratory distress syndrome (RDS). Prompt diagnosis is one of the main challenges because of the aspecific presentation of the disease; therapy relies on antibiotics that reach high intracellular concentration and can penetrate and pass the placenta reaching the fetus. Herein we report an infrequent case of LM infection involving a woman with monochorionic diamniotic twin pregnancy, followed by a comprehensive review of the available literature on listeriosis in pregnancy.

Identification of key genes participating in copper-diethyldithiocarbamate-related cell death process and predicting the development of prostate cancer

Copper (Cu) is used as a cofactor in all organisms, and yet it can be toxic at high intracellular concentrations, causing cell death. Diethyldithiocarbamate (DDC) is a Cu ionophore that can transport Cu effectively into the cell. Copper-diethyldithiocarbamate (Cu-DDC) can treat prostate cancer (PCa) and may correlate with the cell death process. However, the specific Cu-DDC-related cell death genes in PCa are still unknown. Information about the Cu-DDC-related cell death genes was obtained from a previous study. Concurrently, the RNA expression profiles and clinical data were downloaded from public databases such as GEO, TCGA, and CPGEA. Using data from TCGA database, the logistic and lasso regression models were generated using R software. The influence of these genes in affecting PCa progression and prognosis was analyzed. Finally, the expression of these genes was verified in clinical samples. We found five Cu-DDC-related cell death genes associated with the occurrence of PCa from GSE35988, a gene dataset, namely, CDKN2A, PRC1, CDK1, SOX2, and ZNF365. CDKN2A, PRC1, and CDK1 are known to influence PCa patients’ disease-free survival (DFS) status and were overexpressed, whereas SOX2 and ZNF365 were under-expressed in PCa in the different databases. Some of these genes can affect PCa progression. Consistent with the database results, the mRNA and protein expression of CDKN2A, PRC1, and CDK1 was also higher in clinical samples. In conclusion, we identified five hub genes which are important for Cu-DDC-related cell death process that can predict the development of PCa.

A-345 Effect of Hemolysis on Routine Blood Gas Analytes

Abstract Background Hemolysis is a major pre-analytical concern for most laboratory analytes. Detection of hemolysis and mitigation efforts are especially important for analytes, such as potassium, with high intracellular concentrations. Routine serum and plasma chemistry tests are performed on analyzers with the capacity to detect and measure the degree of hemolysis. However, for whole blood chemistries and blood gas measurements, the instruments utilized lack this capacity. The aim of this study was to evaluate the effect of hemolysis on whole blood and blood gas analytes and to compare visual assessments of hemolysis to measured hemolysis indices (H-index). Methods Remnant whole blood samples were split into two portions and each portion was aspirated and dispensed through a syringe one or more times. For the mock portion, only the syringe was used, while the hemolyzed portion had a needle affixed to the end of the syringe. The needle provided shear stress on the red blood cells to induce hemolysis, while the mock procedure was used to assess the impact of aspirating/dispensing on the sample in the absence of hemolysis. Each portion was then analyzed on a Radiometer ABL800 series instrument, spun down, and the H-index of the plasma portion was measured on a Roche cobas 8000 instrument. Medical technologists recorded their visual assessment of the specimens, with two technologists agreeing to the categorization of the specimen as either slightly, moderately, or severely hemolyzed. Degree of hemolysis was categorized by the delta (hemolyzed - mock) of the measured H-index: slight hemolysis was defined as an H-index delta of <100, moderate as 100-500 and severe as >500. Results The effect of hemolysis, with H-indices ranging from 2 to 3861, on 13 routine blood gas analytes was studied for 85 whole blood specimens. Hemolysis had little effect on metabolites, as percent bias was within ±3% at all levels of hemolysis for glucose, creatinine, and lactate. Similarly, most cooximetry components were minimally affected, with total hemoglobin, oxyhemoglobin, carboxyhemoglobin, and oxygen saturation within ±5% bias at all levels of hemolysis. Methemoglobin had a larger overall negative bias, with slight, moderate, and severe hemolysis levels yielding percent biases of -6.6, -12.3, and -13.3%, respectively. As expected, potassium displayed a significant positive bias with increasing hemolysis, with a generally linear trend. At moderate levels of hemolysis, the average potassium bias was 24.0% and at severe levels, over 100%. Sodium and ionized calcium also displayed overall linear trends but with a negative bias. At slight, moderate, and severe levels of hemolysis, sodium had a -0.56, -1.10, and -3.96% bias, and ionized calcium had a -2.99, -5.65, and -15.5% bias respectively. Conclusions Hemolysis can falsely increase or decrease a range of blood gas analytes and lead to misinterpretation of results and adversely affect clinical decision-making. Therefore, equipping current blood gas analyzers with hemolysis detection capabilities is crucial to enable laboratories to mitigate this effect and ensure accurate results for patient care.

Depolarization of mouse DRG neurons by GABA does not translate into acute pain or hyperalgesia in healthy human volunteers.

The majority of somatosensory DRG neurons express GABAA receptors (GABAAR) and depolarise in response to its activation based on the high intracellular chloride concentration maintained by the Na-K-Cl cotransporter type 1 (NKCC1). The translation of this response to peripheral nerve terminals in people is so far unclear. We show here that GABA (EC50 = 16.67μM) acting via GABAAR produces an influx of extracellular calcium in approximately 20% (336/1720) of isolated mouse DRG neurons. In contrast, upon injection into forearm skin of healthy volunteers GABA (1mM, 100μl) did not induce any overt sensations nor a specific flare response and did not sensitize C-nociceptors to slow depolarizing electrical sinusoidal stimuli. Block of the inward chloride transporter NKCC1 by furosemide (1mg/100μl) did not reduce electrically evoked pain ratings nor did repetitive GABA stimulation in combination with an inhibited NKCC1 driven chloride replenishment by furosemide. Finally, we generated a sustained period of C-fiber firing by iontophoretically delivering codeine or histamine to induce tonic itch. Neither the intensity nor the duration of histamine or codeine itch was affected by prior injection of furosemide. We conclude that although GABA can evoke calcium transients in a proportion of isolated mouse DRG neurons, it does not induce or modify pain or itch ratings in healthy human skin even when chloride gradients are altered by inhibition of the sodium coupled NKCC1 transporter.

ATP, the 31P Spectral Modulus, and Metabolism.

Adenosine triphosphate (ATP) has a high intracellular millimolar concentration (ca. 2.4 mM) throughout the phylogenetic spectrum of eukaryotes, archaea, and prokaryotes. In addition, the function of ATP as a hydrotrope in the prevention of protein aggregation and maintenance of protein solubilization is essential to cellular, tissue, and organ homeostasis. The 31P spectral modulus (PSM) is a measure of the health status of cell, tissue, and organ systems, as well as of ATP, and it is based on in vivo 31P nuclear magnetic resonance (31P NMR) spectra. The PSM is calculated by dividing the area of the 31P NMR integral curve representing the high-energy phosphates by that of the low-energy phosphates. Unlike the difficulties encountered in measuring organophosphates such as ATP or any other phosphorylated metabolites in a conventional 31P NMR spectrum or in processed tissue samples, in vivo PSM measurements are possible with NMR surface-coil technology. The PSM does not rely on the resolution of individual metabolite signals but uses the total area derived from each of the NMR integral curves of the above-described spectral regions. Calculation is based on a simple ratio of the high- and low-energy phosphate bands, which are conveniently arranged in the high- and low-field portions of the 31P NMR spectrum. In practice, there is essentially no signal overlap between these two regions, with the dividing point being ca. -3 δ. ATP is the principal contributor to the maintenance of an elevated PSM that is typically observed in healthy systems. The purpose of this study is to demonstrate that (1) in general, the higher the metabolic activity, the higher the 31P spectral modulus, and (2) the modulus calculation does not require highly resolved 31P spectral signals and thus can even be used with reduced signal-to-noise spectra such as those detected as a result of in vivo analyses or those that may be obtained during a clinical MRI examination. With increasing metabolic stress or maturation of metabolic disease in cells, tissues, or organ systems, the PSM index declines; alternatively, with decreasing stress or resolution of disease states, the PSM increases. The PSM can serve to monitor normal homeostasis as a diagnostic tool and may be used to monitor disease processes with and without interventional treatment.

Anoctamin 1, a multi-modal player in pain and itch

Anoctamin 1 (ANO1/TMEM16A) encodes a Ca2+-activated Cl- channel. Among ANO1′s many physiological functions, it plays a significant role in mediating nociception and itch. ANO1 is activated by intracellular Ca2+ and depolarization. Additionally, ANO1 is activated by heat above 44 °C, suggesting heat as another activation stimulus. ANO1 is highly expressed in nociceptors, indicating a role in nociception. Conditional Ano1 ablation in dorsal root ganglion (DRG) neurons results in a reduction in acute thermal pain, as well as thermal and mechanical allodynia or hyperalgesia evoked by inflammation or nerve injury. Pharmacological interventions also lead to a reduction in nocifensive behaviors. ANO1 is functionally linked to the bradykinin receptor and TRPV1. Bradykinin stimulates ANO1 via IP3-mediated Ca2+ release from intracellular stores, whereas TRPV1 stimulates ANO1 via a combination of Ca2+ influx and release. Nerve injury causes upregulation of ANO1 expression in DRG neurons, which is blocked by ANO1 antagonists. Due to its role in nociception, strong and specific ANO1 antagonists have been developed. ANO1 is also expressed in pruritoceptors, mediating Mas-related G protein-coupled receptors (Mrgprs)-dependent itch. The activation of ANO1 leads to chloride efflux and depolarization due to high intracellular chloride concentrations, causing pain and itch. Thus, ANO1 could be a potential target for the development of new drugs treating pain and itch.

Dual approach: micellar delivery of chlorambucil prodrug with concurrent glutathione depletion and GST inhibition for enhanced anticancer activity

Although chlorambucil (CHL) is a long-established anticancer drug, the drug failure of CHL, mediated by the intracellular defense system consisting of glutathione (GSH) and GSH S-transferase pi (GST-pi), has significantly limited the application of CHL. To overcome this issue, we first designed a GSH-responsive small-molecule prodrug (EA-SS-CHL) by combining CHL and ethacrynic acid (EA). Subsequently, drug-loaded nanoparticles (ECPP) were formed by the self-assembly between EA-SS-CHL and amphiphilic PEG-PDLLA to improve the water solubility of the prodrug and its ability to target tumor sites. Upon exposure to high intracellular GSH concentration, EA-SS-CHL gradually degrades, leading to the release of EA and CHL. The presence of EA facilitates the depletion of GSH and inhibition of GST-pi, ultimately attenuating the detoxification of the intracellular defense system to CHL. Cytotoxicity studies and apoptosis assays demonstrate that ECPP exhibits higher therapeutic efficiency than CHL. Additionally, in vivo tumor suppression effects and biocompatibility provide further evidence for the superiority of ECPP. This work presents a promising strategy to enhance the efficacy of CHL in cancer therapy.

ASP210: a potent oligonucleotide-based inhibitor effective against TKI-resistant CML cells.

Clinical experience with tyrosine kinase inhibitors (TKIs) over the past two decades has shown that, despite the apparent therapeutic benefit, nearly 30% of patients with chronic myelogenous leukemia (CML) display primary resistance or intolerance to TKIs, and approximately 25% of those treated are forced to switch TKIs at least once during therapy due to acquired resistance. Safe and effective treatment modalities targeting leukemic clones that escape TKI therapy could hence be game changers in the professional management of these patients. Here, we aimed to investigate the efficacy of a novel therapeutic oligonucleotide of unconventional design, called ASP210, to reduce BCR-ABL1 mRNA levels in TKI-resistant CML cells, with the assumption of inducing their apoptosis. Imatinib- and dasatinib-resistant sublines of BCR-ABL1-positive MOLM-7 and CML-T1 cells were established and exposed to 0.25 and 2.5 µM ASP210 for 10 days. RT-qPCR showed a remarkable reduction of the target mRNA level by >99% after a single application. Cell viability was monitored daily by trypan blue staining. In response to the lack of driver oncoprotein BCR-ABL1, TKI-resistant CML cells underwent apoptosis regardless of the presence of the clinically relevant T315I mutation by day 5 after redosing with ASP210. The effect was selective for cancer cells, indicating a favorable safety profile for this therapeutic modality. Furthermore, the spontaneous uptake and high intracellular concentrations of ASP210 suggest its potential to be effective at relatively low doses. The present findings suggest that ASP210 is a promising therapeutic avenue for patients with CML who fail to respond to TKI therapy.NEW & NOTEWORTHY Effective treatment modalities targeting leukemic clones that escape tyrosine kinase inhibitor (TKI) therapy could be game changers in the professional management of patients displaying primary resistance, intolerance, or acquired resistance to TKIs. Although delivering authentic innovations today is more complex than ever, we developed a highly potent and safe oligonucleotide-based modality against BCR-ABL1 mRNA named ASP210 that effectively induces cell death in BCR-ABL1-positive TKI-resistant cells while sparing BCR-ABL1-negative healthy cells.

Kalium channelrhodopsins effectively inhibit neurons

The analysis of neural circuits has been revolutionized by optogenetic methods. Light-gated chloride-conducting anion channelrhodopsins (ACRs)—recently emerged as powerful neuron inhibitors. For cells or sub-neuronal compartments with high intracellular chloride concentrations, however, a chloride conductance can have instead an activating effect. The recently discovered light-gated, potassium-conducting, kalium channelrhodopsins (KCRs) might serve as an alternative in these situations, with potentially broad application. As yet, KCRs have not been shown to confer potent inhibitory effects in small genetically tractable animals. Here, we evaluated the utility of KCRs to suppress behavior and inhibit neural activity in Drosophila, Caenorhabditiselegans, and zebrafish. In direct comparisons with ACR1, a KCR1 variant with enhanced plasma-membrane trafficking displayed comparable potency, but with improved properties that include reduced toxicity and superior efficacy in putative high-chloride cells. This comparative analysis of behavioral inhibition between chloride- and potassium-selective silencing tools establishes KCRs as next-generation optogenetic inhibitors for in vivo circuit analysis in behaving animals.

Polymer brush construction with interface-mediated RAFT polymerization technique for glutathione determination

Reactive polymeric thin films designed on solid surfaces have widespread applications in many fields involving the immobilization of biomolecules. Efficient and specific designs for biomolecule immobilization on polymer-modified solid surfaces are central to biosensor technologies such as protein and gene chips. Research areas related to the functionalization of surfaces include polymer brushes whose functionality is increased by the Diels-Alder (DA) reaction, which have been made usable as rewritable platforms with their thermal transformation properties and have found a lot of use, especially in biomolecule determination studies. Glutathione (GSH), one of the biomolecules, is found in in very high intracellular concentrations. It is the most important antioxidant molecule in the environment and its determination is a good indicator for us to obtain information about the status of the antioxidant defense mechanism in various diseases. When studies in were examined in detail, no study was found on the synthesis of polymer brushes with thiol functional groups covalently bonded to the silicon surface using the interfacial-mediated RAFT polymerization technique and their use in GSH adsorption. Therefore, in this study, to fill this gap in the literature, polymer brushes with thiol (SH) functional groups to be used in GSH adsorption were synthesized. Poly(furfurylmethacrylate) [poly(FMA)] brushes, formed by polymer chains covalently bonded to the silicon surface, were synthesized by interface-mediated RAFT polymerization. Then, by maleimide conjugation using hexylamine, polymeric brushes with thiol functional groups covalently bonded to the silicon surface which can be used in GSH adsorption were obtained for determination purposes. It is thought that the prepared polymeric brushes will find a wide application area for the determination of many new molecules, especially the determination of glutathione and will contribute to the literature.

Hexokinase-linked glycolytic overload and unscheduled glycolysis in hyperglycemia-induced pathogenesis of insulin resistance, beta-cell glucotoxicity, and diabetic vascular complications.

Hyperglycemia is a risk factor for the development of insulin resistance, beta-cell glucotoxicity, and vascular complications of diabetes. We propose the hypothesis, hexokinase-linked glycolytic overload and unscheduled glycolysis, in explanation. Hexokinases (HKs) catalyze the first step of glucose metabolism. Increased flux of glucose metabolism through glycolysis gated by HKs, when occurring without concomitant increased activity of glycolytic enzymes-unscheduled glycolysis-produces increased levels of glycolytic intermediates with overspill into effector pathways of cell dysfunction and pathogenesis. HK1 is saturated with glucose in euglycemia and, where it is the major HK, provides for basal glycolytic flux without glycolytic overload. HK2 has similar saturation characteristics, except that, in persistent hyperglycemia, it is stabilized to proteolysis by high intracellular glucose concentration, increasing HK activity and initiating glycolytic overload and unscheduled glycolysis. This drives the development of vascular complications of diabetes. Similar HK2-linked unscheduled glycolysis in skeletal muscle and adipose tissue in impaired fasting glucose drives the development of peripheral insulin resistance. Glucokinase (GCK or HK4)-linked glycolytic overload and unscheduled glycolysis occurs in persistent hyperglycemia in hepatocytes and beta-cells, contributing to hepatic insulin resistance and beta-cell glucotoxicity, leading to the development of type 2 diabetes. Downstream effector pathways of HK-linked unscheduled glycolysis are mitochondrial dysfunction and increased reactive oxygen species (ROS) formation; activation of hexosamine, protein kinase c, and dicarbonyl stress pathways; and increased Mlx/Mondo A signaling. Mitochondrial dysfunction and increased ROS was proposed as the initiator of metabolic dysfunction in hyperglycemia, but it is rather one of the multiple downstream effector pathways. Correction of HK2 dysregulation is proposed as a novel therapeutic target. Pharmacotherapy addressing it corrected insulin resistance in overweight and obese subjects in clinical trial. Overall, the damaging effects of hyperglycemia are a consequence of HK-gated increased flux of glucose metabolism without increased glycolytic enzyme activities to accommodate it.

The Renaissance of Spiramycin in Clinical Practice

Spiramycin is a natural 16-membered macrolide antibiotic that has been used in clinical practice for about 70 years. Despite long-term use, spiramycin retains its position in clinical practice; the resistance of the main respiratory and urogenital pathogens is lower to spiramycin than to 14- and 15-membered macrolides. The interest in spiramycin is due to the fact that its physico-chemical and pharmacokinetic properties are similar to the last semi-synthetic macrolides, and it lacks the disadvantages of erythromycin. Special biological properties of spiramycin explain its consistently high clinical efficacy that has lasted for 70 years of clinical use in various infections. It is necessary to emphasize the uniqueness of spiramycin as an antibiotic with not only the direct effect on microbes, but also non-antibiotic properties. This allowed the experts at the time to declare the «paradox» of spiramycin — the presence of a higher real clinical efficacy than expected according to in vitro studies. Such properties of spiramycin include pro-antibiotic and post-antibiotic effects, immunomodulatory effect, as well as high intracellular and extracellular concentrations of the antibiotic. The review discusses antimicrobial, pharmacokinetic, and pharmacodynamic characteristics of spiramycin and its place in the treatment of various infections — respiratory, ENT, urogenital, gynecological, and dental.

Adenosine Triphosphate (ATP) and Protein Aggregation in Age-Related Vision-Threatening Ocular Diseases.

Protein aggregation is the etiopathogenesis of the three most profound vision-threatening eye diseases: age-related cataract, presbyopia, and age-related macular degeneration. This perspective organizes known information on ATP and protein aggregation with a fundamental unrecognized function of ATP. With recognition that maintenance of protein solubility is related to the high intracellular concentration of ATP in cells, tissues, and organs, we hypothesize that (1) ATP serves a critical molecular function for organismal homeostasis of proteins and (2) the hydrotropic feature of ATP prevents pathological protein aggregation while assisting in the maintenance of protein solubility and cellular, tissue, and organismal function. As such, the metabolite ATP plays an extraordinarily important role in the prevention of protein aggregation in the leading causes of vision loss or blindness worldwide.

Valine and nonessential amino acids affect bidirectional transport rates of leucine and isoleucine in bovine mammary epithelial cells

A more complete understanding of the mechanisms controlling AA transport in mammary glands of dairy cattle will help identify solutions to increase nitrogen feeding efficiency on farms. It was hypothesized that Ala, Gln, and Gly (NEAAG), which are actively transported into cells and exchanged for all branched-chain AA (BCAA), may stimulate transport of BCAA, and that Val may antagonize transport of the other BCAA due to transporter competition. Thus, we evaluated the effects of varying concentrations of NEAAG and Val on transport and metabolism of the BCAA Ala, Met, Phe, and Thr by bovine mammary epithelial cells. Primary cultures of bovine mammary epithelial cells were assigned to treatments of low (70% of mean in vivo plasma concentrations of lactating dairy cows) and high (200%) concentrations of Val and NEAAG (LVal and LNEAAG, HVal and HNEAAG, respectively) in a 2 × 2 factorial design. Cells were preloaded with treatment media containing [15N]-labeled AA for 24 h. The [15N]-labeled media were replaced with treatment media containing [13C]-labeled AA. Media and cells were harvested from plates at 0, 0.5, 1, 5, 15, 30, 60, and 240 min after application of the [13C]-labeled AA and assessed for [15N]- and [13C]-AA label concentrations. The data were used to derive transport, transamination, irreversible loss, and protein-synthesis fluxes. All Val fluxes, except synthesis of rapidly exchanging tissue protein, increased with the HVal treatment. Interestingly, the rapidly exchanging tissue protein, transamination, and irreversible-loss rate constants decreased with HVal, indicating that the significant flux increases were primarily driven by mass action with the cells resisting the flux increases by downregulating activity. However, the decreases could also reflect saturation of processes that would drive down the mass-action rate constants. This is supported by decreases in the same rate constants for Ile and Leu with HVal. This could be due to either competition for shared transamination and oxidation reactions or a reduction in enzymatic activity. Also, NEAAG did not affect Val fluxes, but influx and efflux rate constants increased for both Val and Leu with HNEAAG, indicating an activating substrate effect. Overall, AA transport rates generally responded concordantly with extracellular concentrations, indicating the transporters are not substrate-saturated within the in vivo range. However, BCAA transamination and oxidation enzymes may be approaching saturation within in vivo ranges. In addition, System L transport activity appeared to be stimulated by as much as 75% with high intracellular concentrations of Ala, Gln, and Gly. High concentrations of Val antagonized transport activity of Ile and Leu by 68% and 15%, respectively, indicating competitive inhibition, but this was only observable at HNEAAG concentrations. The exchange transporters of System L transport 8 of the essential AA that make up approximately 40% of milk protein, so better understanding this transporter is an important step for increased efficiency.

Zero-Mode Waveguide Nanowells for Single-Molecule Detection in Living Cells.

Single-molecule fluorescence imaging experiments generally require sub-nanomolar protein concentrations to isolate single protein molecules, which makes such experiments challenging in live cells due to high intracellular protein concentrations. Here, we show that single-molecule observations can be achieved in live cells through a drastic reduction in the observation volume using overmilled zero-mode waveguides (ZMWs- subwavelength-size holes in a metal film). Overmilling of the ZMW in a palladium film creates a nanowell of tunable size in the glass layer below the aperture, which cells can penetrate. We present a thorough theoretical and experimental characterization of the optical properties of these nanowells over a wide range of ZMW diameters and overmilling depths, showing an excellent signal confinement and a 5-fold fluorescence enhancement of fluorescent molecules inside nanowells. ZMW nanowells facilitate live-cell imaging as cells form stable protrusions into the nanowells. Importantly, the nanowells greatly reduce the cytoplasmic background fluorescence, enabling the detection of individual membrane-bound fluorophores in the presence of high cytoplasmic expression levels, which could not be achieved with TIRF microscopy. Zero-mode waveguide nanowells thus provide great potential to study individual proteins in living cells.

Identification of cuproptosis-related genes for predicting the development of prostate cancer.

Copper can be toxic at very high intracellular concentrations and can inhibit prostate cancer (PCa) progression. Recently, a study reported the mechanism of cuproptosis and the potentially associated genes. However, the function of these cuproptosis-related genes in PCa remains unknown. Based on the RNA sequence and clinical data from public databases, we analyzed the clinical value of cuproptosis-related genes in PCa. DLD, DLAT, PDHA1, and CDKN2A were expressed differently between normal and PCa tissues. The FDX1, LIAS, DLAT, GLS, and CDKN2A genes can affect PCa progression, while PDHA1 and CDKN2A influence the patients' disease-free survival (DFS) status. The expression of LIAS, LIPT1, DLAT, and PDHB did not alter upon the incidence of PCa in Chinese patients. A constructed regression model showed that FDX1, PDHA1, MTF1, and CDKN2A can be risk factors leading to PCa in both Western and Chinese patients with PCa. The lasso regression model reflected that these genes can affect the patients' DFS status. Additionally, the cuproptosis-related genes were associated with immune cell infiltration. We also verified the high expression of PDHA1 and CDKN2A, in clinical samples. In conclusion, we identified a novel cuproptosis-related gene signature for predicting the development of PCa.

Dual receptor specific nanoparticles targeting EGFR and PD-L1 for enhanced delivery of docetaxel in cancer therapy

Dual-receptor targeted (DRT) nanoparticles which contain two distinct targeting agents may exhibit higher cell selectivity, cellular uptake, and cytotoxicity toward cancer cells than single-ligand targeted nanoparticle systems without additional functionality. The purpose of this study is to prepare DRT poly(lactic-co-glycolic acid) (PLGA) nanoparticles for targeting the delivery of docetaxel (DTX) to the EGFR and PD-L1 receptor positive cancer cells such as human glioblastoma multiform (U87-MG) and human non-small cell lung cancer (A549) cell lines. Anti-EGFR and anti-PD-L1 antibody were decorated on DTX loaded PLGA nanoparticles to prepare DRT-DTX-PLGA via. single emulsion solvent evaporation method. Physicochemical characterizations of DRT-DTX-PLGA, such as particle size, zeta-potential, morphology, and in vitro DTX release were also evaluated. The average particle size of DRT-DTX-PLGA was 124.2 ± 1.1 nm with spherical and smooth morphology. In the cellular uptake study, the DRT-DTX-PLGA endocytosed by the U87-MG and A549 cells was single ligand targeting nanoparticle. From the in vitro cell cytotoxicity, and apoptosis studies, we reported that DRT-DTX-PLGA exhibited high cytotoxicity and enhanced the apoptotic cell compared to the single ligand-targeted nanoparticle. The dual receptor mediated endocytosis of DRT-DTX-PLGA showed a high binding affinity effect that leads to high intracellular DTX concentration and exhibited high cytotoxic properties. Thus, DRT nanoparticles have the potential to improve cancer therapy by providing selectivity over single-ligand-targeted nanoparticles.

How to Increase Cellular Glutathione.

Glutathione (GSH) has special antioxidant properties due to its high intracellular concentration, ubiquity, and high reactivity towards electrophiles of the sulfhydryl group of its cysteine moiety. In most diseases where oxidative stress is thought to play a pathogenic role, GSH concentration is significantly reduced, making cells more susceptible to oxidative damage. Therefore, there is a growing interest in determining the best method(s) to increase cellular glutathione for both disease prevention and treatment. This review summarizes the major strategies for successfully increasing cellular GSH stores. These include GSH itself, its derivatives, NRf-2 activators, cysteine prodrugs, foods, and special diets. The possible mechanisms by which these molecules can act as GSH boosters, their related pharmacokinetic issues, and their advantages and disadvantages are discussed.

Strategies to target the cancer driver MYC in tumor cells.

The MYC oncoprotein functions as a master regulator of cellular transcription and executes non-transcriptional tasks relevant to DNA replication and cell cycle regulation, thereby interacting with multiple proteins. MYC is required for fundamental cellular processes triggering proliferation, growth, differentiation, or apoptosis and also represents a major cancer driver being aberrantly activated in most human tumors. Due to its non-enzymatic biochemical functions and largely unstructured surface, MYC has remained difficult for specific inhibitor compounds to directly address, and consequently, alternative approaches leading to indirect MYC inhibition have evolved. Nowadays, multiple organic compounds, nucleic acids, or peptides specifically interfering with MYC activities are in preclinical or early-stage clinical studies, but none of them have been approved so far for the pharmacological treatment of cancer patients. In addition, specific and efficient delivery technologies to deliver MYC-inhibiting agents into MYC-dependent tumor cells are just beginning to emerge. In this review, an overview of direct and indirect MYC-inhibiting agents and their modes of MYC inhibition is given. Furthermore, we summarize current possibilities to deliver appropriate drugs into cancer cells containing derailed MYC using viral vectors or appropriate nanoparticles. Finding the right formulation to target MYC-dependent cancers and to achieve a high intracellular concentration of compounds blocking or attenuating oncogenic MYC activities could be as important as the development of novel MYC-inhibiting principles.

Role of the NMDAR-GluN2B subunits in the function of supramolecular NMDAR-BK channel complexes.

Large conductance calcium- and voltage-activated potassium channels (BK) are widely expressed across many tissues, contributing to many physiological functions. Membrane depolarization and relatively high (micromolar) intracellular concentrations of Ca2+ are required for their activation. Such concentrations are reached in the vicinity of Ca2+-permeant ion channels, to which BK proteins are closely located in various tissues, including the brain. N-methyl-D-aspartate receptors (NMDAR) are sodium (Na+)- and Ca2+-conducting glutamate-activated ion channels that are functionally coupled to BK channels in the olfactory bulb and the dentate gyrus, contributing to decreased neuronal intrinsic excitability. Recently, our group demonstrated that postsynaptic NMDAR-BK complexes at basal dendrites of somatosensory cortex layer 5 pyramidal neurons regulate synaptic transmission and long-term plasticity. However, the specific contribution of different NMDAR subunits (GluN1 and GluN2A/GluN2B) to the NMDAR-BK interaction remains elusive. Defects in this association may be of pathological relevance in the context of EIEE27 syndrome (epileptic encephalopathy, early infantile, 27), which has been related to mutations in the GluN2B-encoding gene (GRIN2B). In this work I aimed to understand the role of GluN2B subunits in the function of NMDAR-BK complexes and, more specifically, if GluN2B-related human mutations modify NMDAR-BK coupling. Function of NMDAR-BK complexes containing different EIEE27-related GluN2B mutations was examined using electrophysiology in heterologous expression systems. Proximity between channel proteins within complexes was assessed using in situ proximity ligation assay (PLA). Membrane expression efficiency was studied employing TIRF microscopy (Total Internal Reflection Fluorescence). Our results reveal some disease-related GluN2B mutations reduce the NMDAR-BK interaction, either by altered interactions with BK channels and/or by functional uncoupling between the channels within the NMDAR-BK complexes.