The impact of enriched environments on cerebral oxidative balance in rodents: a systematic review of environmental variability effects.

Introduction: The present review aimed to systematically summarize the impacts of environmental enrichment (EE) on cerebral oxidative balance in rodents exposed to normal and unfavorable environmental conditions. Methods: In this systematic review, four databases were used: PubMed (830 articles), Scopus (126 articles), Embase (127 articles), and Science Direct (794 articles). Eligibility criteria were applied based on the Population, Intervention, Comparison, Outcomes, and Study (PICOS) strategy to reduce the risk of bias. The searches were carried out by two independent researchers; in case of disagreement, a third participant was requested. After the selection and inclusion of articles, data related to sample characteristics and the EE protocol (time of exposure to EE, number of animals, and size of the environment) were extracted, as well as data related to brain tissues and biomarkers of oxidative balance, including carbonyls, malondialdehyde, nitrotyrosine, oxygen-reactive species, and glutathione (reduced/oxidized). Results: A total of 1,877 articles were found in the four databases, of which 16 studies were included in this systematic review. The results showed that different EE protocols were able to produce a global increase in antioxidant capacity, both enzymatic and non-enzymatic, which are the main factors for the neuroprotective effects in the central nervous system (CNS) subjected to unfavorable conditions. Furthermore, it was possible to notice a slowdown in neural dysfunction associated with oxidative damage, especially in the prefrontal structure in mice. Discussion: In conclusion, EE protocols were determined to be valid tools for improving oxidative balance in the CNS. The global decrease in oxidative stress biomarkers indicates refinement in reactive oxygen species detoxification, triggering an improvement in the antioxidant network.

CLSI-based verification and de novo establishment of reference intervals for common biochemical assays in Croatian newborns.

Introduction: This study aimed to examine whether the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) reference intervals for 19 commonly used biochemical assays (potassium, sodium, chloride, calcium, magnesium, inorganic phosphorous, glucose, urea, creatinine, direct and total bilirubin, C-reactive protein (CRP), total protein, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP) and lactate dehydrogenase (LD)) could be applied to the newborn population of one Croatian clinical hospital. Materials and methods: Reference interval verification was performed according to the CLSI EP28-A3c guidelines. Samples of healthy newborns were selected using the direct a posteriori sampling method and analyzed on the Beckman Coulter AU680 biochemical analyzer. If verification wasn't satisfactory, further procedure included de novo determination of own reference intervals by analyzing 120 samples of healthy newborns. Results: After the first set of measurements, 14/19 tested reference intervals were adopted for use: calcium, inorganic phosphorous, glucose, urea, creatinine, total bilirubin, CRP, total protein, albumin, AST, ALT, GGT, ALP and LD. A second set of samples was tested for 5 analytes: potassium, sodium, chloride, magnesium and direct bilirubin. The verification results of the additional samples for sodium and chloride were satisfactory, while the results for potassium, magnesium and direct bilirubin remained unsatisfactory and new reference intervals were determined. Conclusions: The CALIPER reference intervals can be implemented into routine laboratory and clinical practice for the tested newborn population for most of the analyzed assays, while own reference intervals for potassium, magnesium and direct bilirubin have been determined.

Two cases with discrepancy in the quantitative cytological assessment of cerebrospinal fluid in neonatal samples using light microscopy in comparison with Sysmex XN-1000.

We present two cases from the neonatal department with cerebrospinal fluid examination. We revealed a striking discrepancy in polymorphonuclear (PMN) and mononuclear (MN) cell counts using conventional light microscopy in comparison with automated analyzer Sysmex XN-1000 (PMNs - 13 vs. 173x106/L, MNs - 200 vs. 67x106/L in case 1 and PMNs - 13 vs. 372x106/L, MNs - 411 vs. 179x106/L in case 2). We revealed the dominant presence of hemosiderophages in both cases in cytospin slide. Even though Sysmex XN-1000 offers fast examination with a low sample volume, there is possibility of misdiagnosis, with negative impact on the patient.

Craniofacial chondrogenesis in organoids from human stem cell-derived neural crest cells.

Knowledge of cell signaling pathways that drive human neural crest differentiation into craniofacial chondrocytes is incomplete, yet essential for using stem cells to regenerate craniomaxillofacial structures. To accelerate translational progress, we developed a differentiation protocol that generated self-organizing craniofacial cartilage organoids from human embryonic stem cell-derived neural crest stem cells. Histological staining of cartilage organoids revealed tissue architecture and staining typical of elastic cartilage. Protein and post-translational modification (PTM) mass spectrometry and snRNA-seq data showed that chondrocyte organoids expressed robust levels of cartilage extracellular matrix (ECM) components: many collagens, aggrecan, perlecan, proteoglycans, and elastic fibers. We identified two populations of chondroprogenitor cells, mesenchyme cells and nascent chondrocytes, and the growth factors involved in paracrine signaling between them. We show that ECM components secreted by chondrocytes not only create a structurally resilient matrix that defines cartilage, but also play a pivotal autocrine cell signaling role in determining chondrocyte fate.

Evolutionary analysis reveals the role of a non-catalytic domain of peptidyl arginine deiminase 2 in transcriptional regulation.

Peptidyl arginine deiminases (PADIs) catalyze protein citrullination, a post-translational conversion of arginine to citrulline. The most widely expressed member of this family, PADI2, regulates cellular processes that impact several diseases. We hypothesized that we could gain new insights into PADI2 function through a systematic evolutionary and structural analysis. Here, we identify 20 positively selected PADI2 residues, 16 of which are structurally exposed and maintain PADI2 interactions with cognate proteins. Many of these selected residues reside in non-catalytic regions of PADI2. We validate the importance of a prominent loop in the middle domain that encompasses PADI2 L162, a residue under positive selection. This site is essential for interaction with the transcription elongation factor (P-TEFb) and mediates the active transcription of the oncogenes c-MYC, and CCNB1, as well as impacting cellular proliferation. These insights could be key to understanding and addressing the role of the PADI2 c-MYC axis in cancer progression.

Protocol for HiBiT tagging endogenous proteins using CRISPR-Cas9 gene editing.

We present a method of in vitro/in vivo protein detection by pairing CRISPR-Cas9 genome editing with the NanoBiT system. We describe steps for cell culturing, in vitro CRISPR-Cas9 ribonucleoprotein delivery, cell monitoring, efficiency assessments, and edit analysis through HiBiT assays. We then detail procedures to determine edit specificity through genomic DNA analysis, small interfering RNA reverse transfection, and HiBiT blotting. This protocol is simple to execute and multifunctional, and it enables high-throughput screens on endogenous proteins to be conducted with ease.

The single D380 amino acid substitution increases pneumolysin cytotoxicity toward neuronal cells.

Bacterial meningitis, frequently caused by Streptococcus pneumoniae (pneumococcus), represents a substantial global health threat leading to long-term neurological disorders. This study focused on the cholesterol-binding toxin pneumolysin (PLY) released by pneumococci, specifically examining clinical isolates from patients with meningitis and comparing them to the PLY-reference S. pneumoniae strain D39. Clinical isolates exhibit enhanced PLY release, likely due to a significantly higher expression of the autolysin LytA. Notably, the same single amino acid (aa) D380 substitution in the PLY D4 domain present in all clinical isolates significantly enhances cholesterol binding, pore-forming activity, and cytotoxicity toward SH-SY5Y-derived neuronal cells. Scanning electron microscopy of human neuronal cells and patch clamp electrophysiological recordings on mouse brain slices confirm the enhanced neurotoxicity of the PLY variant carrying the single aa substitution. This study highlights how a single aa modification enormously alters PLY cytotoxic potential, emphasizing the importance of PLY as a major cause of the neurological sequelae associated with pneumococcal meningitis.

X-ray structure and enzymatic study of a bacterial NADPH oxidase highlight the activation mechanism of eukaryotic NOX.

NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2's requirement for activation.

Sequential trypsin and ProAlanase digestions unearth immunological protein biomarkers shrouded by skeletal collagen.

This study investigates the efficacy of proteomic analysis of human remains to identify active infections in the past through the detection of pathogens and the host response to infection. We advance leprosy as a case study due to the sequestering of sufferers in leprosaria and the suggestive skeletal lesions that can result from the disease. Here we present a sequential enzyme extraction protocol, using trypsin followed by ProAlanase, to reduce the abundance of collagen peptides and in so doing increase the detection of non-collagenous proteins. Through our study of five individuals from an 11th to 18th century leprosarium, as well as four from a contemporaneous non-leprosy associated cemetery in Barcelona, we show that samples from 2 out of 5 leprosarium individuals extracted with the sequential digestion methodology contain numerous host immune proteins associated with modern leprosy. In contrast, individuals from the non-leprosy associated cemetery and all samples extracted with a trypsin-only protocol did not. Through this study, we advance a palaeoproteomic methodology to gain insights into the health of archaeological individuals and take a step toward a proteomics-based method to study immune responses in past populations.

Protein degradation of Lsd1 is mediated by Bre1 yet opposed by Lsd1-interacting lncRNAs during fly follicle development.

Tissue development, homeostasis, and repair all require efficient progenitor expansion. Lysine-specific demethylase 1 (Lsd1) maintains plastic epigenetic states to promote progenitor proliferation while overexpressed Lsd1 protein causes oncogenic gene expression in cancer cells. However, the precise regulation of Lsd1 protein expression at the molecular level to drive progenitor differentiation remains unclear. Here, using Drosophila melanogaster oogenesis as our experimental system, we discovered molecular machineries that modify Lsd1 protein stability in vivo. Through genetic and biochemical analyses, an E3 ubiquitin ligase, Bre1, was identified as required for follicle progenitor differentiation, likely by mediating Lsd1 protein degradation. Interestingly, specific Lsd1-interacting long non-coding RNAs (LINRs) were found to antagonize Bre1-mediated Lsd1 protein degradation. The intricate interplay discovered among the Lsd1 complex, LINRs and Bre1 provides insight into how Lsd1 protein stability is fine-tuned to underlie progenitor differentiation in vivo.

Suppression of Pseudomonas aeruginosa type III secretion system by a novel calcium-responsive signaling pathway.

Expression of the type III secretion system (T3SS) in Pseudomonas aeruginosa is exquisitely controlled by diverse environmental or host-related signals such as calcium (Ca2+), however, the signal transduction pathways remain largely elusive. In this study, we reported that FleR, the response regulator of the two-component system FleS/FleR, inhibits T3SS gene expression and virulence of P. aeruginosa uncoupled from its cognate histidine kinase FleS. Interestingly, FleR was found to repress T3SS gene expression under Ca2+-rich conditions independently of its DNA-binding domain. FleR activates the elevation of intracellular c-di-GMP contents and FleQ serves as the c-di-GMP effector to repress T3SS gene expression through the Gac/Rsm pathway. Remarkably, we found that AmrZ, a member of the FleR regulon, inhibits T3SS gene expression by directly targeting the promoter of exsCEBA in an expression level-dependent manner. This study revealed an intricate regulatory network that connects P. aeruginosa T3SS gene expression to the Ca2+ signal.

Non-invasive PET imaging of liver fibrogenesis using a RESCA-conjugated Affibody molecule.

Non-invasive assessment of fibrogenic activity, rather than fibrotic scars, could significantly improve the management of fibrotic diseases and the development of anti-fibrotic drugs. This study explores the potential of an Affibody molecule (Z09591) labeled with the Al(18)F-restrained complexing agent (RESCA) method as a tracer for the non-invasive detection of fibrogenic cells. Z09591 was functionalized with the RESCA chelator for direct labeling with [18F]AlF. In vivo positron emission tomography/magnetic resonance imaging scans on U-87 tumor-bearing mice exhibited high selectivity of the resulting radiotracer, [18F]AlF-RESCA-Z09591, for platelet-derived growth factor receptor ß (PDGFRß), with minimal non-specific background uptake. Evaluation in a mouse model with carbon tetrachloride-induced fibrotic liver followed by a disease regression phase, revealed the radiotracer's high affinity and specificity for fibrogenic cells in fibrotic livers (standardized uptake value [SUV] 0.43 ± 0.05), with uptake decreasing during recovery (SUV 0.29 ± 0.03) (p < 0.0001). [18F]AlF-RESCA-Z09591 accurately detects PDGFRß, offering non-invasive assessment of fibrogenic cells and promising applications in precise liver fibrogenesis diagnosis, potentially contributing significantly to anti-fibrotic drug development.

The activation of LBH-CRYAB signaling promotes cardiac protection against I/R injury by inhibiting apoptosis and ferroptosis.

Myocardial ischemia-reperfusion (I/R) injury stands out among cardiovascular diseases, and current treatments are considered unsatisfactory. For cardiomyocytes (CMs) in ischemic tissues, the upregulation of Limb-bud and Heart (LBH) and αB-crystallin (CRYAB) and their subsequent downregulation in the context of cardiac fibrosis have been verified in our previous research. Here, we focused on the effects and mechanisms of activated LBH-CRYAB signaling on damaged CMs during I/R injury, and confirmed the occurrence of mitochondrial apoptosis and ferroptosis during I/R injury. The application of inhibitors, ectopic expression vectors, and knockout mouse models uniformly verified the role of LBH in alleviating both apoptosis and ferroptosis of CMs. p53 was identified as a mutual downstream effector for both LBH-CRYAB-modulated apoptosis and ferroptosis inhibition. In mouse models, LBH overexpression was confirmed to exert enhanced cardiac protection against I/R-induced apoptosis and ferroptosis, suggesting that LBH could serve as a promising target for the development of I/R therapy.

Overexpression of the potato VQ31 enhances salt tolerance in Arabidopsis.

Plant-specific VQ proteins have crucial functions in the regulation of plant growth and development, as well as in plant abiotic stress responses. Their roles have been well established in the model plant Arabidopsis thaliana; however, the functions of the potato VQ proteins have not been adequately investigated. The VQ protein core region contains a short FxxhVQxhTG amino acid motif sequence. In this study, the VQ31 protein from potato was cloned and functionally characterized. The complete open reading frame (ORF) size of StVQ31 is 672 bp, encoding 223 amino acids. Subcellular localization analysis revealed that StVQ31 is located in the nucleus. Transgenic Arabidopsis plants overexpressing StVQ31 exhibited enhanced salt tolerance compared to wild-type (WT) plants, as evidenced by increased root length, germination rate, and chlorophyll content under salinity stress. The increased tolerance of transgenic plants was associated with increased osmotic potential (proline and soluble sugars), decreased MDA accumulation, decreased total protein content, and improved membrane integrity. These results implied that StVQ31 overexpression enhanced the osmotic potential of the plants to maintain normal cell growth. Compared to the WT, the transgenic plants exhibited a notable increase in antioxidant enzyme activities, reducing cell membrane damage. Furthermore, the real-time fluorescence quantitative PCR analysis demonstrated that StVQ31 regulated the expression of genes associated with the response to salt stress, including ERD, LEA4-5, At2g38905, and AtNCED3. These findings suggest that StVQ31 significantly impacts osmotic and antioxidant cellular homeostasis, thereby enhancing salt tolerance.

Inhibitors to degraders: Changing paradigm in drug discovery.

The chemical understanding of biological processes provides not only a deeper insight but also a solution to abnormal biological functioning. Protein degradation, a natural biological process for debris removal in the cell, has been studied for years. The recent finding that natural degradation pathways can be utilized for therapeutic purposes is a paradigm shift in the drug discovery approach. Methods such as Proteolysis Targeting Chimera (PROTAC), lysosomal targeting chimera, hydrophobic tagging, AUtophagy TArgeting Chimera, AUTOphagy TArgeting Chimera and several other variants of these methods have made a considerable impact on the way of drug design. Few selected examples testify that a huge wave of change is on the way. The drug design based on the targeted protein degradation is a powerful tool in our arsenal. More molecules will be invented that will uncover the hidden secrets of biological functioning and provide enduring solutions to several unmet medical needs.

Approach to Abnormal Liver Biochemistries in the Primary Care Setting.

Liver biochemistries are commonly ordered in the primary care setting, and they may return abnormal even in an asymptomatic patient. Primary care physicians need to have a systematic way of interpreting any derangement in these tests so that further investigations, referrals, and management can be arranged appropriately. This review dwells into patterns of liver biochemistry derangement, common aetiologies to consider, history and examinations that are required, initial investigations to order, and when to refer urgently to the emergency department.

Exogenous melatonin delays leaves senescence and enhances saline and alkaline stress tolerance in grape seedlings.

Saline and alkaline stress is one of the major abiotic stresses facing agricultural production, which severely inhibits the growth and yield of plant. The application of plant growth regulators can effectively prevent crop yield reduction caused by saline and alkaline stress. Exogenous melatonin (MT) can act as a signaling molecule involved in the regulation of a variety of physiological processes in plants, has been found to play a key role in enhancing the improvement of plant tolerance to abiotic stresses. However, the effects of exogenous MT on saline and alkaline tolerance of table grape seedlings and its mechanism have not been clarified. The aim of this study was to investigate the role of exogenous MT on morphological and physiological growth of table grape seedlings (Vitis vinifera L.) under saline and alkaline stress. The results showed that saline and alkaline stress resulted in yellowing and wilting of grape leaves and a decrease in chlorophyll content, whereas the application of exogenous MT alleviated the degradation of chlorophyll in grape seedling leaves caused by saline and alkaline stress and promoted the accumulation of soluble sugars and proline content. In addition, exogenous MT increased the activity of antioxidant enzymes, which resulted in the scavenging of reactive oxygen species (ROS) generated by saline and alkaline stress. In conclusion, exogenous MT was involved in the tolerance of grape seedlings to saline and alkaline stress, and enhanced the saline and alkaline resistance of grape seedlings to promote the growth and development of the grape industry in saline and alkaline areas.

A multi-hierarchical approach reveals d-serine as a hidden substrate of sodium-coupled monocarboxylate transporters.

Transporter research primarily relies on the canonical substrates of well-established transporters. This approach has limitations when studying transporters for the low-abundant micromolecules, such as micronutrients, and may not reveal physiological functions of the transporters. While d-serine, a trace enantiomer of serine in the circulation, was discovered as an emerging biomarker of kidney function, its transport mechanisms in the periphery remain unknown. Here, using a multi-hierarchical approach from body fluids to molecules, combining multi-omics, cell-free synthetic biochemistry, and ex vivo transport analyses, we have identified two types of renal d-serine transport systems. We revealed that the small amino acid transporter ASCT2 serves as a d-serine transporter previously uncharacterized in the kidney and discovered d-serine as a non-canonical substrate of the sodium-coupled monocarboxylate transporters (SMCTs). These two systems are physiologically complementary, but ASCT2 dominates the role in the pathological condition. Our findings not only shed light on renal d-serine transport, but also clarify the importance of non-canonical substrate transport. This study provides a framework for investigating multiple transport systems of various trace micromolecules under physiological conditions and in multifactorial diseases.

Verifying the nonreporting hemolysis index for potassium, phosphate, magnesium, AST, LDH, iron, CA 19-9, and vitamin D, using Beckman Coulter AU5800 and DxI800 automated analyzers.

BACKGROUND: Hemolysis is a common reason for nonreporting results in biochemistry and is measured using the hemolysis index (HI), with nonreporting limits set for analytes by manufacturers. OBJECTIVE: To verify the nonreporting HI limit for potassium, phosphate, magnesium, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), iron, CA19-9, and vitamin D on the Beckman Coulter AU5800/DxI800 analyzers. METHOD: Hemolysate was created from EDTA-lined tubes of whole blood using an osmotic shock procedure. The hemolysate underwent serial dilutions with saline and was spiked in paired serum. The delta changes in HI and analyte concentration were measured, assessed using regression analysis, and compared against calculated reference change values. RESULTS: A linear relationship between increasing HI and increasing analyte concentration (R2 > 0.9) was observed for potassium (y = 0.8864x), phosphate (y = 0.1079x), magnesium (y = 0.0678x), AST (y = 29.035x), and LDH (y = 350x). Increasing HI values did not have a linear effect on iron (y = -0.2544x), CA19-9 (y = 2.7019x), or vitamin D (y = 8.036x) concentrations. CONCLUSION: The results from this experiment support increasing the HI nonreporting limit to 100 mg/dL for potassium; 200 mg/dL for magnesium; and 300 mg/dL for phosphate, CA19-9, and vitamin D. The iron assay is not affected by hemolysis as high as 500 mg/dL. The current HI nonreporting limit of 50 mg/dL is appropriate for LDH.

Postprandial effects following a semielemental critical-care diet lead to changes in uric acid, bile acids, and glucose in the central bearded dragon (Pogona vitticeps).

OBJECTIVE: To assess the prandial effects of a semielemental diet on plasma uric acid, bile acid, and glucose concentrations in the central bearded dragon (Pogona vitticeps). ANIMALS: 13 healthy adult male bearded dragons. METHODS: Following a 72-hour fasting period, blood was collected to measure preprandial uric acid, bile acid, and glucose concentrations. The animals were then gavage fed 1.2% body weight of an omnivore critical-care diet containing 20% protein, 9.5% fat, 2.5% fiber, and 2.39 kcal/mL. Blood was collected for repeat concentrations at 4 and 24 hours. RESULTS: Median (IQR) uric acid concentration (mg/dL) increased from 3.8 preprandial (2.8 to 4.3) to 4.7 4 hours postprandial (4 to 7; P = .0001). Median (IQR) bile acid concentration (mg/dL) increased from 1.8 preprandial (1 to 3.4) to 9.5 24 hours postprandial (5.6 to 10.4; P = .004). Median (IQR) glucose concentration (mg/dL) was 209 at time 0 (193 to 216), 287 at 4 hours (258 to 312), and 393 at 24 hours (361 to 464). Significant increases were seen between pre- and 4-hours-postprandial (P < .0001), pre- and 24-hours-postprandial (P < .0001), and 4-hours- and 24-hours-postprandial (P < .0001) glucose concentrations. CLINICAL RELEVANCE: Results suggest that postprandial status and diet composition should be considered during the interpretation of some biochemical analytes in the bearded dragon.