Cellular Production Research Articles

Notoginsenoside R1 Attenuates H/R Injury in H9c2 Cells by Maintaining Mitochondrial Homeostasis

Mitochondrial homeostasis is crucial for maintaining cellular energy production and preventing oxidative stress, which is essential for overall cellular function and longevity. Mitochondrial damage and dysfunction often occur concomitantly in myocardial ischemia–reperfusion injury (MIRI). Notoginsenoside R1 (NGR1), a unique saponin from the traditional Chinese medicine Panax notoginseng, has been shown to alleviate MIRI in previous studies, though its precise mechanism remains unclear. This study aimed to elucidate the mechanisms of NGR1 in maintaining mitochondrial homeostasis in hypoxia/reoxygenation (H/R) H9c2 cells. The results showed that NGR1 pretreatment effectively increased cell survival rates post-H/R, reduced lactate dehydrogenase (LDH) leakage, and mitigated cell damage. Further investigation into mitochondria revealed that NGR1 alleviated mitochondrial structural damage, improved mitochondrial membrane permeability transition pore (mPTP) persistence, and prevented mitochondrial membrane potential (Δψm) depolarization. Additionally, NGR1 pretreatment enhanced ATP levels, increased the activity of mitochondrial respiratory chain complexes I–V after H/R, and reduced excessive mitochondrial reactive oxygen species (mitoROS) production, thereby protecting mitochondrial function. Further analysis indicated that NGR1 upregulated the expression of mitochondrial biogenesis-related proteins (PGC-1α, Nrf1, Nrf2) and mitochondrial fusion proteins (Opa1, Mfn1, Mfn2), while downregulating mitochondrial fission proteins (Fis1, Drp1) and reducing mitochondrial autophagy (mitophagy) levels, as well as the expression of mitophagy-related proteins (Pink1, Parkin, BNIP3) post-H/R. Therefore, this study showed that NGR1 can maintain mitochondrial homeostasis by regulating mitophagy, mitochondrial fission–fusion dynamics, and mitochondrial biogenesis, thereby alleviating H9c2 cell H/R injury and protecting cardiomyocytes.

Molecular dynamics at immune synapse lipid rafts influence the cytolytic behavior of CAR T cells.

Chimeric antigen receptor T cells (CART) targeting CD19 through CD28.ζ signaling induce rapid lysis of leukemic blasts, contrasting with persistent tumor control exhibited by 4-1BB.ζ-CART. We reasoned that molecular dynamics at the CART immune synapse (CARIS) could explain differences in their tumor rejection kinetics. We observed that CD28.ζ-CART engaged in brief highly lethal CARIS and mastered serial killing, whereas 4-1BB.ζ-CART formed lengthy CARIS and relied on robust expansion and cooperative killing. We analyzed CARIS membrane lipid rafts (mLRs) and found that, upon tumor engagement, CD28.ζ-CAR molecules rapidly but transiently translocated into mLRs, mobilizing the microtubular organizing center and lytic granules to the CARIS. This enabled fast CART recovery and sensitivity to low target site density. In contrast, gradual accumulation of 4-1BB.ζ-CAR and LFA-1 molecules at mLRs built mechanically tonic CARIS mediating chronic Fas ligand-based killing. The differences in CD28.ζ- and 4-1BB.ζ-CARIS dynamics explain the distinct cytolytic behavior of CART and can guide engineering of more adaptive effective cellular products.

Second-Generation Cap Analogue Prodrugs for Targeting Aberrant Eukaryotic Translation Initiation Factor 4E Activity in Cancer.

Dysregulation of translation is a hallmark of cancer that enables rapid changes in cellular protein production to shape oncogenic phenotypes. Translation initiation is governed by the m7GpppX cap-binding protein eukaryotic translation initiation factor 4E (eIF4E), the rate-limiting factor of cap-dependent translation initiation. eIF4E is overexpressed in many cancers and drives the production of oncoproteins that promote tumor growth and survival. Accordingly, eIF4E has been established as an attractive albeit challenging therapeutic target. Building upon our previous work of developing cell-permeable cap analogue prodrugs that inhibit eIF4E binding to the m7GpppX cap, herein we disclose the design of second-generation cap analogues with alternative N-9-substituted linkers which exhibit anticancer activity in BRAFV600E mutant melanoma cell lines.

‘Breaking the biofilm barrier: vitamin C as a novel strategy against multidrug-resistant salmonella Typhimurium’

Abstract The emergence of multidrug-resistant (MDR), biofilm-forming strains of Salmonella Typhimurium poses a significant threat to food safety due to their persistence on food surfaces and equipment, complicating eradication efforts and increasing the risk of contamination. This study explored the antibiofilm activity of Vitamin C (VC) against MDR biofilm-forming Salmonella Typhimurium. Vitamin C inhibited S. Typhimurium biofilms in a dose-dependent manner, with a minimum inhibitory concentration of 125 mM. Treatment with Vitamin C significantly disrupted the bacterial biofilm structure, creating cannonball-like pores in the cell wall as observed through electron microscopy. The antibiofilm mechanism of Vitamin C was mediated by reactive oxygen species (ROS) generation, leading to sugar and protein leakage, reduced exopolysaccharide (EPS) production, and decreased quorum sensing (QS) activity. Transcriptional analysis revealed that Vitamin C downregulated expression of biofilm-related genes responsible for initial cellular attachment and EPS production (fimA, adrA) and upregulated genes associated with adhesion and multidrug efflux pumps (csgA, ipfE, and arcA). Importantly, the application of Vitamin C on coated Paneer significantly extended its shelf life. These findings highlight the potential of Vitamin C in combating Salmonella biofilms, enhancing food safety, and providing a framework for the development of antibiotic-independent therapies to address MDR strains of Salmonella.

Novel insights into insect mediated polystyrene biodegradation through bacterial genome analyses

Plastic pollution is a significant environmental challenge of contemporary age. Polystyrene (PS), among the most commonly used plastic polymers worldwide, is highly durable and difficult to degrade. Despite various disposal strategies, PS continues to impact biodiversity, human health, and ecosystems. Recently, the scientific community has focused on the potential role of microorganisms for plastic biodegradation, particularly those from the gut of plastivorous insects. In a previous study, three bacterial strains, each representing a distinct taxonomic group (Klebsiella, Pseudomonas, and Stenotrophomonas), were isolated from Alphitobius diaperinus larvae after rearing on a PS diet and enriched in a medium with PS as the sole carbon source. The Stenotrophomonas sp. strain, here identified as S. indicatrix, showed the greatest potential for PS degradation. The present study investigates the genetic profile of the newly isolated S. indicatrix strain DAI2m/c through genome sequencing, to identify enzyme-encoding genes involved in the intracellular metabolic pathways responsible for the biodegradation of the styrene monomer. Our findings indicate that the genome of S. indicatrix strain DAI2m/c encodes all enzymes required for one of the two recognized styrene degradation pathways, suggesting its ability to convert styrene into byproducts that are then utilized for cellular energy production.

Minimal residual disease as a target for liquid biopsy in patients with solid tumours.

Metastasis is the leading cause of cancer-related death in patients with solid tumours. Current imaging technologies are not sufficiently sensitive to detect minimal residual disease (MRD; also known as measurable or molecular residual disease) after initial surgery or chemotherapy, pointing to the need for more sensitive tests to detect remaining traces of cancer in the body. Liquid biopsy, or the analysis of tumour-derived or tumour-induced cells or cellular products in the blood or other body fluids, has opened a new diagnostic avenue to detect and monitor MRD. Liquid biopsy is already used in clinical decision making for patients with haematological malignancies. Here, we review current knowledge on the use of circulating tumour DNA (ctDNA) to detect and monitor MRD in patients with solid tumours. We also discuss how ctDNA-guided MRD detection and characterization could herald a new era of novel 'post-adjuvant therapies' with the potential to eliminate MRD and cure patients before terminal metastatic disease is evident on imaging.

Protective effects of phosphocreatine against Doxorubicin-Induced cardiotoxicity through mitochondrial function enhancement and apoptosis suppression via AMPK/PGC-1α signaling pathway.

Doxorubicin (DOX), a potent chemotherapy drug, is limited by its cardiotoxic effects, which can lead to heart damage. This study explores the cardioprotective potential of Phosphocreatine (PCr) in vitro and in vivo models, focusing on its impact on the AMPK and PGC-1α pathways, apoptosis reduction, and mitochondrial function preservation. Advanced methodologies, including high-resolution respirometry (HRR), were employed to assess mitochondrial bioenergetics, AMPK activity, and apoptotic rates in cardiomyocytes. Electrocardiography (ECG) and echocardiography (echo) were used to monitor cardiac function in vivo. Results showed that PCr significantly activated the AMPK and PGC-1α pathways, reduced apoptosis, and stabilized mitochondrial function in cardiomyocytes exposed to DOX. There was an upregulation of AMPK and PGC-1α target genes, stabilization of mitochondrial membranes, and improvements in cellular energy production and antioxidant defenses. PCr also markedly reduced apoptotic markers, enhancing cardiomyocyte viability. ECG and echocardiography revealed that PCr preserved cardiac function, indicated by improved heart rate variability, reduced QT interval prolongation, and enhanced ejection fraction. These findings highlight PCr's potential in mitigating DOX-induced cardiotoxicity by enhancing mitochondrial function and reducing apoptosis. The study underscores the promise of PCr as an agent to reduce chemotherapy-related cardiac injuries, paving the way for further research to improve patient outcomes in cancer treatment.

Treatment of Long COVID With Enoxaparin

Background and Purpose: Long COVID is a complex multisystemic disease state, which represents a huge economic and health burden worldwide. Hypercoagulation and the formation of fibrinaloid microclots have been proposed as an underlying pathology, which may underpin a wide variety of presenting symptoms, via reduced blood flow and cellular energy production. Postexertional malaise (PEM) is common and exercise therapy should not be recommended without addressing the underlying pathology. Case Presentation: Patient was female, 43, with Long COVID for 21 months characterized primarily by extreme fatigue, exercise intolerance, PEM, cognitive dysfunction, and postural orthostatic tachycardia syndrome. She was unable to work or leave the house unless for essential medical appointments. Before her initial severe acute respiratory syndrome coronavirus 2 infection, she had no prior health conditions or comorbidities, a body mass index of 20.8, and engaged in regular vigorous exercise. Intervention: This case report describes a patient treated with enoxaparin, a low molecular weight heparin, at 20 mg per day subcutaneously, with assessment at 8 weeks. Outcomes: The patient's function and quality of life improved markedly, as reported subjectively, and with objective measurements at 8 weeks. Levels of activity improved markedly with no adverse events. The severity of PEM decreased dramatically, and postural orthostatic tachycardia syndrome was better controlled. The FUNCAP score, used to measure functional capacity, increased from 3.3 (moderately affected) to 5.07 (mildly affected) after treatment. Discussion: It is recommended that this treatment should be further investigated to elucidate the role of enoxaparin in addressing underlying pathology in Long COVID, including hypercoagulation and microclots.

HIF1α Plays a Crucial Role in the Development of TFE3–Rearranged Renal Cell Carcinoma by Orchestrating a Metabolic Shift Toward Fatty Acid Synthesis

ABSTRACTTumor development often requires cellular adaptation to a unique, high metabolic state; however, the molecular mechanisms that drive such metabolic changes in TFE3–rearranged renal cell carcinoma (TFE3‐RCC) remain poorly understood. TFE3‐RCC, a rare subtype of RCC, is defined by the formation of chimeric proteins involving the transcription factor TFE3. In this study, we analyzed cell lines and genetically engineered mice, demonstrating that the expression of the chimeric protein PRCC‐TFE3 induced a hypoxia‐related signature by transcriptionally upregulating HIF1α and HIF2α. The upregulation of HIF1α by PRCC‐TFE3 led to increased cellular ATP production by enhancing glycolysis, which also supplied substrates for the TCA cycle while maintaining mitochondrial oxidative phosphorylation. We crossed TFE3‐RCC mouse models with Hif1α and/or Hif2α knockout mice and found that Hif1α, rather than Hif2α, is essential for tumor development in vivo. RNA‐seq and metabolomic analyses of the kidney tissues from these mice revealed that ketone body production is inversely correlated with tumor development, whereas de novo lipid synthesis is upregulated through the HIF1α/SREBP1‐dependent mechanism in TFE3‐RCC. Our data suggest that the coordinated metabolic shift via the PRCC‐TFE3/HIF1α/SREBP1 axis is a key mechanism by which PRCC‐TFE3 enhances cancer cell metabolism, promoting tumor development in TFE3‐RCC.

Clinical application of a low-volume extracorporeal photochemotherapy for the treatment of graft-versus-host disease

Introduction. Extracorporeal photopheresis (ECP) is an attractive method of treating graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation due to the lack of systemic immunosuppression. However, the clinical use of ECP is often limited by the need for leukapheresis. There are modifications of ECP without the use of leukapheresis, in particular low-volume ECP.Aim: to analyze the use of lv-ECP for the treatment of patients with acute and chronic GvHD.Patients and methods. The results of treatment of 9 cases of acute GVHD and 15 cases of chronic GVHD using the lv-ECP from April 2021 to March 2024 are presented. The cellular product was obtained by effusion of 15-30 ml of whole blood followed by photochemical treatment. The processed autologous cell product was administered to the patient.Results. The overall response in patients with acute GVHD was 44%, organ-specific response for skin lesions was 56 %, and for gastrointestinal lesions, 40%. In the treatment of chronic GvHD, the overall response was 73.3 %; the effectiveness of therapy for skin lesions was 71 %; for gastrointestinal lesions 67 %; for lung damage 67 %; for eye damage 100 %, for liver damage 100 %.Conclusion. lv-ECP has shown promising efficacy results in the treatment of acute and chronic GvHD. lv-ECP is an attractive alternative to standard ECP when leukapheresis is not possible.

Newly Initiated Statin Treatment Is Associated with Decreased Plasma Coenzyme Q10 Level After Acute ST-Elevation Myocardial Infarction.

Coenzyme Q10 (CoQ10) plays a crucial role in facilitating electron transport during oxidative phosphorylation, thus contributing to cellular energy production. Statin treatment causes a decrease in CoQ10 levels in muscle tissue as well as in serum, which may contribute to the musculoskeletal side effects. Therefore, we aimed to assess the effect of newly initiated statin treatment on serum CoQ10 levels after acute ST-elevation myocardial infarction (STEMI) and the correlation of CoQ10 levels with key biomarkers of subclinical or clinically overt myopathy. In this study, we enrolled 67 non-diabetic, statin-naïve early-onset STEMI patients with preserved renal function. Plasma CoQ10 level was determined by ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC/MS-MS), while the myopathy marker serum fatty acid-binding protein 3 (FABP3) level was measured with enzyme-linked immunosorbent assay (ELISA) at hospital admission and after 3 months of statin treatment. The treatment significantly decreased the plasma CoQ10 (by 43%) and FABP3 levels (by 79%) as well as total cholesterol, low-density lipoprotein cholesterol (LDL-C), apolipoprotein B100 (ApoB100), and oxidized LDL (oxLDL) levels. The change in CoQ10 level showed significant positive correlations with the changes in total cholesterol, LDL-C, ApoB100, and oxLDL levels, while it did not correlate with the change in FABP3 level. Our results prove the CoQ10-reducing effect of statin treatment and demonstrate its lipid-lowering efficacy but contradict the role of CoQ10 reduction in statin-induced myopathy.

EFFECT OF MELATONIN ON PLASMA PYRUVATE LEVELS IN ALLOXAN-INDUCED DIABETIC RATS UNDER DIFFERENT PHOTOPERIODS

Some studies suggest that melatonin exerts a stimulating effect by reducing lactate dehydrogenase activity and shifting the balance of redox enzymes towards the predominance of the Krebs cycle, thereby favoring aerobic processes that are more efficient for cellular energy production. Therefore, an in-depth investigation of this effect of melatonin is warranted. The aim of this experimental study was to examine the impact of melatonin on the levels of pyruvic acid in the plasma of rats under physiological conditions and in alloxan-induced diabetes, with a focus on hypo- and hyperfunction of the pineal gland. Methods. The study was conducted on 50 sexually mature male outbred white rats weighing 0.18–0.20 kg. Photoperiodic changes were simulated over a one-week period, with the following conditions: 1) natural light/dark cycle from March 19 to 25, 2024, averaging 12:12 hours; 2) artificial 12:12 light/dark cycle (light from 8:00 a.m. to 8:00 p.m., with illumination at 500 lux); 3) constant light (500 lux) for 24 hours; 4) constant darkness for 24 hours. Alloxan diabetes was induced by intraperitoneal injection of a 5% alloxan monohydrate solution at a dose of 170 mg/kg. Fasting glucose levels were measured using the One Touch Ultra Easy device (Johnson & Johnson, USA). The rats were divided into four groups based on lighting conditions: 1) intact rats; 2) a control group, receiving daily intraperitoneal melatonin injections (10 mg/kg) at 8 a.m. for one week (Sigma, USA); 3) rats with diabetes mellitus; 4) rats with diabetes mellitus, receiving melatonin injections (10 mg/kg) starting on the 5th day post-alloxan for one week. Animals were euthanized by decapitation under light ether anesthesia on the 12th day of the experiment. Pyruvate levels in blood plasma were measured using standard methods. Statistical analysis was performed using Statistica 10 (StatSoft Inc.). Results. It is known that the presence of hyperglycemia under the conditions of diabetes intensifies the processes of free radical oxidation. According to the data obtained by us, in diabetic rats, a high level of glucose is accompanied by an increase in pyruvate in blood serum. This happens due to dysregulation of aerobic processes in the conditions of a decrease in insulin. In this case, pyruvate turns into lactate, creating conditions for the development of lactic acidosis. Staying under conditions of dark deprivation (light 24 hours) for a week led to a 15% increase in serum glucose and pyruvate levels in blood serum compared to intact animals. A similar situation was observed in a group of diabetic animals. Under conditions of round-the-clock lighting in diabetic animals, the indicators of glucose and pyruvate content increased even in comparison with the indicators of diabetic animals under equinox conditions and were on average 22% higher. However, exposure to light deprivation (24 hours of darkness) for a week led to a 10% decrease in glucose content and a 13% increase in pyruvate content when compared to intact animals. This happens due to the activation of the processes of aerobic oxidation of glucose under the conditions of an increase in melatonin. In diabetic rats, fasting pyruvate and glucose indicators under conditions of 24-hour darkness were negated by smaller deviations when compared with a group of diabetic animals that were under conditions of the equinox. It has been established that a short daytime photoperiod (8 hours of light: 16 hours of darkness) is characterized by an increase in melatonin secretion. Under these conditions, the indicators of antioxidant protection and the immune system improved in experimental animals. In addition, pinealectomized rats are characterized by reduced activity of glutathione peroxidase in tissues due to the absence of melatonin. The introduction of melatonin led to a decrease in glucose content in diabetic rats (under conditions of darkness - to normalization). The content of pyruvate in the blood serum of diabetic animals regardless of lighting conditions under the influence of melatonin injections did not differ from the control indicators under equinox conditions. The ability of melatonin to improve glucose uptake through glucose transporters has been investigated. In addition, there are data on melatonin as an activator of pyruvate dehydrogenase. It has been realised that melatonin has possibility to inhibit the activity of pyruvate dehydrogenase kinase and activate the pyruvate dehydrogenase thereby allowing the transformation of pyruvate to acetyl-CoA and sending glucose for oxidation in the mitochondria. Conclusion. Therefore, under both normal and diabetic conditions, melatonin enhances the efficiency of aerobic glucose oxidation pathways.

Recombinant Follicle-Stimulating Hormone and Luteinizing Hormone Enhance Mitochondrial Function and Metabolism in Aging Female Reproductive Cells

Ovarian aging significantly impacts female fertility, with mitochondrial dysfunction emerging as a key factor. This study investigated the effects of recombinant follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on mitochondrial function and metabolism in aging female reproductive cells. Human granulosa cells (HGL5) were treated with FSH/LH or not. Mitochondrial function was assessed through various assays, including mitochondrial mass, membrane potential, ROS levels, and ATP production. Mitochondrial dynamics and morphology were analyzed using MitoTracker staining. Cellular respiration was measured using a Seahorse Bioenergetics Analyzer. Metabolic reprogramming was evaluated through gene expression analysis and metabolite profiling. In vivo effects were studied using aging mouse oocytes. FSH/LH treatment significantly improved mitochondrial function in aging granulosa cells, increasing mitochondrial mass and membrane potential while reducing ROS levels. Mitochondrial dynamics showed a shift towards fusion and elongation. Cellular respiration, ATP production, and spare respiratory capacity were enhanced. FSH/LH-induced favorable alterations in cellular metabolism, favoring oxidative phosphorylation. In aging mouse oocytes, FSH/LH treatment improved in vitro maturation and mitochondrial health. In conclusion, FSH/LH supplementation ameliorates age-related mitochondrial dysfunction and improves cellular metabolism in aging female reproductive cells.

Comparative Analysis of Human Brain RNA-seq Reveals the Combined Effects of Ferroptosis and Autophagy on Alzheimer's Disease in Multiple Brain Regions.

Ferroptosis and autophagy are closely associated with Alzheimer's disease (AD). Elevated ferric ion levels can induce oxidative stress and chronic inflammatory responses, resulting in brain tissue damage and further neurological cell damage. Autophagy in Alzheimer's has a dual role. On one hand, it protects neurons by removing β-amyloid and cellular damage products caused by oxidative stress and inflammation. On the other hand, abnormal autophagy is linked to neuronal apoptosis and neurodegeneration. However, the intricate interplay between ferroptosis and autophagy in AD remains insufficiently explored. This study focuses on the roles of ferroptosis and autophagy in AD and their interconnection through bioinformatics analysis, shedding light on the disease. Ferroptosis and autophagy significantly correlate with the development and course of AD. Using PPI network analysis and unsupervised consistency clustering analysis, we uncovered a complex network of interactions between ferroptosis and autophagy during disease progression, demonstrating a significant congruence in their modification patterns. Functional analyses further demonstrated that ferroptosis and autophagy together affect the immunological status and synaptic regulation in hippocampal regions in patients with AD, which significantly impacts the start and progression of the disease.

Complexities, Benefits, Risks, and Clinical Implications of Sodium Bicarbonate Administration in Critically Ill Patients: A State-of-the-Art Review.

Sodium bicarbonate has been used in the treatment of different pathologies, such as hyperkalemia, cardiac arrest, tricyclic antidepressant toxicity, aspirin toxicity, acute acidosis, lactic acidosis, diabetic ketoacidosis, rhabdomyolysis, and adrenergic receptors' resistance to catecholamine in patients with shock. An ongoing debate about bicarbonate's efficacy and potential harm has been raised for decades because of the lack of evidence supporting its potential efficacy. Despite the guidelines' restrictions, sodium bicarbonate has been overused in clinical practice. The overuse of sodium bicarbonate could be because of the desire to correct the arterial blood gas parameters rapidly instead of achieving homeostasis by treating the cause of the metabolic acidosis. Moreover, it is believed that sodium bicarbonate may reverse acidosis-induced myocardial depression, hemodynamic instability, ventricular arrhythmias, impaired cellular energy production, resistance to catecholamines, altered metabolism, enzyme suppression, immune dysfunction, and ineffective oxygen delivery. On the other hand, it is crucial to pay attention to the potential harm that could be caused by excessive sodium bicarbonate administration. Sodium bicarbonate may cause paradoxical respiratory acidosis, intracellular acidosis, hypokalemia, hypocalcemia, alkalosis, impaired oxygen delivery, cerebrospinal fluid acidosis, and neurologic dysfunction. In this review, we discuss the pathophysiology of sodium bicarbonate-induced adverse effects and potential benefits. We also review the most recent clinical trials, observational studies, and guidelines discussing the use of sodium bicarbonate in different pathologies.

The vital role of biological standardization in ensuring efficacy and safety of biological products - Historical perspectives.

Biological Standardization has been pivotal to the success of traditional biological products, such as vaccines, antitoxins, and immune globulins, by ensuring their quality and consistency across manufacturers worldwide. The principles of biological standardization have similarly supported the development and manufacture of safe and effective modern biological products, including hormone, therapeutic protein, and monoclonal antibody products, and continue to play a vital role in advancing new cutting-edge biological products, such as tissue, cellular, and gene-therapy products. Biological standardization started with the physical standards ensuring the reliability and suitability of methods used to test biological products and science of bioassays or biological methods and related biostatistics providing a framework for evaluating biological, functional activity or potency of these products. It expanded to include written standards defining the quality requirements for manufacturing and regulation of biological standards. Due to the shift in the biologics industry from public health to commercial-driven enterprises during the past 50 years, the biological standardization program has evolved to include the product-specific reference standards and harmonization of physical standards. The global success of conventional vaccines in controlling numerous deadly infectious diseases can largely be attributed to the availability of physical and written international standards developed through a strong biological standardization program. This article explores the evolution of biological standardization for more than a century, its scientific and regulatory principles, challenges from disruption in international standardization efforts, and future perspectives for the field.

Inactivation of the conserved protease LonA increases production of xylanase and amylase in Bacillus subtilis

BackgroundBacillus subtilis is widely used for industrial enzyme production due to its capacity to efficiently secrete proteins. However, secretion efficiency of enzymes varies widely, and optimizing secretion is crucial to make production commercially viable. Previously, we have shown that overexpression of the xylanase XynA lowers expression of Clp protein chaperones, and that inactivation of CtsR, which regulates and represses clp transcription, increases the production of XynA. In the current study, we examined whether the same is the case for overexpression of the α-amylase AmyM from Geobacillus stearothermophilus by B. subtilis, and why XynA shows a different timing of secretion compared to AmyM.ResultsTranscriptome analyses revealed that B. subtilis cells overexpressing AmyM exhibited a distinct profile compared to XynA overexpressing cells, however there were also similarities and in both cases expression of CtsR controlled genes was downregulated. In contrast to XynA, inactivation of CtsR did not improve AmyM production. Upregulation of other protein chaperones, including GroEL/ES and DnaJ/K, by inactivating their transcriptional repressor HrcA, had almost no effect on XynA yields and in fact considerably lowered that of AmyM. Despite using the same promoter, the production of XynA peaks well before AmyM reaches its optimal secretion rate. Transcriptome and ribosome profiling indicated that this is neither related to transcription nor to translation regulation. We show that the reduced secretion in the stationary phase is partially due to the activity of secreted proteases, but also due to the activity of the intracellular protease LonA. The absence of this protein resulted in a 140% and 20% increased production for XynA and AmyM, respectively.ConclusionThe combination of transcriptome and ribosome profiling offered important information to determine at which cellular level production bottlenecks occurred. This helped us to identify LonA protease as an important factor influencing enzyme production yields in B. subtilis.

Bioluminescent Probes for the Detection of Superoxide and Nitric Oxide.

The regulation of reactive oxygen species (ROS) such as superoxide (SO) and nitric oxide (NO) is crucial in biology, influencing metabolism and signaling pathways. Imbalances in these species lead to oxidative stress and various diseases. Traditional methods for measuring SO and NO face challenges in terms of sensitivity and specificity, particularly in complex biological matrices. This report introduces bioluminescent probes that leverage the intrinsic sensitivity of bioluminescence for direct and selective detection of SO and NO. These probes release analogs of d-luciferin upon reaction with their target ROS. Following addition of luciferase, luminescence is generated proportional to the amount of accumulated luciferin, allowing for quantitation of SO or NO. Both probes exhibit high specificity, confirmed through cell-free assays and cell-based studies in macrophages, demonstrating their utility in measuring cellular SO and NO production. These assays offer a robust, high-throughput platform for studying ROS, providing direct insights into oxidative stress-related mechanisms.

Subcellular Localization Guides eNOS Function.

Nitric oxide synthases (NOS) are enzymes responsible for the cellular production of nitric oxide (NO), a highly reactive signaling molecule involved in important physiological and pathological processes. Given its remarkable capacity to diffuse across membranes, NO cannot be stored inside cells and thus requires multiple controlling mechanisms to regulate its biological functions. In particular, the regulation of endothelial nitric oxide synthase (eNOS) activity has been shown to be crucial in vascular homeostasis, primarily affecting cardiovascular disease and other pathophysiological processes of importance for human health. Among other factors, the subcellular localization of eNOS plays an important role in regulating its enzymatic activity and the bioavailability of NO. The aim of this review is to summarize pioneering studies and more recent publications, unveiling some of the factors that influence the subcellular compartmentalization of eNOS and discussing their functional implications in health and disease.

The Effects of Metformin on Weight Loss, Cardiovascular Health, and Longevity.

Metformin, a biguanide derived from Galega officinalis, was first synthesized by Werner and Bell in 1922. Metformin was approved for the treatment of diabetes by the US Food and Drug Administration in 1994. It has since become the most widely used oral antidiabetic agent. The exact mechanisms by which metformin exerts its clinical effects remain the subject of ongoing research. Metformin interacts with multiple molecular pathways, and the downstream effects of which affect weight, cardiovascular health, and longevity. Metformin reduces hunger by mitigating insulin resistance in the hypothalamic pro-opiomelanocortin neurons. It enhances satiety by stimulating the enteral release of glucagon-like peptide 1. It also induces favorable changes to enteric microbiota, enhancing metabolism. These effects cumulatively contribute to metformin-induced weight loss. Metformin use has shown associations with improved cardiovascular outcomes including reduced all-cause mortality, lower rates of myocardial infarctions, and improved heart failure outcomes. Many of these actions are mediated through the direct activation of adenosine monophosphate-activated kinase (AMPK), which, in turn, enhances cellular energy production and endothelial nitric oxide synthase-mediated vascular relaxation. It antagonizes proinflammatory cytokines, reducing cardiac fibrosis and remodeling. The metformin-AMPK pathway may also explain the potential utility of metformin in mitigating aging. Acting through AMPK, it inhibits the mammalian target of rapamycin, leading to increased autophagy and cell growth. The metformin-AMPK-sirtuin pathway may also contribute to longevity. In this review, we will discuss the use of metformin in weight loss, cardiovascular health, and longevity, highlighting the historic background, molecular mechanisms, and current evidence.