Biochimie最新文献

Chronic lead exposure poses severe threats to human health, which demands a rapid detection strategy beyond conventional instrumentation-dependent approaches. While CRISPR/Cas12a systems offer promising alternatives through trans-cleavage activity, conventional Pb²⁺ biosensors relying on DNAzyme-generated intact activators suffer from high background signals due to interference from uncleaved substrates. To address this limitation, we developed a steric-hindrance-controlled activation strategy by employing a chimeric DNAzyme substrate (Sub) that prevents Cas12a binding until Pb²⁺-dependent cleavage occurs. This DNAzyme-mediated splitting releases two fragments (A1/A2) that rearrange into split activators, triggering the CRISPR/Cas12a trans-cleavage of a quenched reporter (6-FAM/BHQ1). Under the optimal condition, the sensor achieved a linear detection range of 2.5-25 μM (R² = 0.998) with 2.18 μM LOD and high selectivity against interferents. Validation in tap water matrices demonstrated 98.6%-102.6% recovery (RSD 3.0%-7.5%), which showed robustness in real samples. This split-activator design paradigm eliminates background from uncleaved substrates without additional pretreatment steps to provide a versatile template for converting metal ions into CRISPR-detectable signals.

B₁₂ deficiency during pregnancy has been associated with shunted growth, neural tube defects, anemia, and neuro-cognitive disorders. The optimal prevention strategy remains to be found. Here we explore the effect of high doses of oral B₁₂ in early pregnancy followed by physiological doses of B₁₂ or placebo. The women (B₁₂ < 146 pmol/l) were recruited before week 17 and divided into three groups, all primed for 14 days with oral B₁₂ = 5 mg/day. Then onwards, the participants daily received 400 ml of milk (HO-B₁₂ = 1.6 μg; n = 31), B₁₂-pills (CN-B₁₂ = 1.6 μg; n = 32), or placebo (n = 13) until 4 months postpartum. Blood was drawn at baseline, ½ - 6 months after baseline; on the day of delivery (including cord blood); and at 4-month postpartum (child and mother). B₁₂ status was assessed by measuring plasma B₁₂, holotranscobalamin (holoTC), and homocysteine (Hcy). Birth parameters were recorded. Comparable changes were achieved following high dose administration of both B₁₂-forms. Plasma total B₁₂ and holoTC increased 2-3 and 6-10 times, respectively; while total Hcy decreased ≈2-fold. No clear difference was observed during and after interventions with milk, pills and placebo (including newborns). At the end of the study ⅞ of mothers and ⅔ of babies had adequate B₁₂ status despite its steady downward drift. Our study demonstrates the benefit of an early-stage supplementation to pregnant women with high oral B₁₂. A continuous low dose supplementation adds only minor advantages. The results may impact future strategies of ensuring a sufficient B₁₂ supply during pregnancy.

Mycobacterium tuberculosis (Mtb) can survive for a long time in vivo and evade host immune attacks, primarily through transcriptional regulation mediated by transcription regulators. JTY_2262 (homologous with Rv2250c) belongs to the Tetracycline Repressor (TetR) family of regulators, and its function is currently unclear. In this study, we combined JTY_2262 overexpressed transcriptomics and electrophoretic mobility shift assays (EMSA) to identify novel targets regulated by JTY_2262. The cofactors containing Cys, V_C, V_B1, V_B3, V_B6, Pb²⁺, Cu²⁺ and Li ⁺ inhibit the binding between JTY_2262 and the JTY_2045 (homologous with rv2031c) promoter. Overall, this study demonstrates that JTY_2262 is a versatile regulator. Under different environmental conditions, JTY_2262 senses concentration variations of specific cofactors. By modulating its DNA-binding affinity accordingly, JTY_2262 regulates targeted gene expression, enabling bacteria to adapt their metabolic machinery and physiological state to better adapt the changing environment.

Skeletal muscle is fundamental to human health, serving as the primary effector of movement and a central regulator of systemic metabolism. Age-related declines in muscle mass and mitochondrial function contribute to frailty, metabolic dysfunction, and loss of independence in older adults. While these changes are often attributed to reduced physical activity, chronic inflammation, and impaired regenerative capacity, emerging evidence implicates environmental and metabolic sensing pathways in muscle degeneration. The aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor best known for mediating responses to environmental pollutants such as dioxins, has recently been recognized as a key regulator of endogenous metabolic and redox processes. AHR activation occurs not only through xenobiotic exposure but also via endogenous ligands derived from tryptophan metabolism-including kynurenine and indole derivatives-whose levels rise in aging, chronic kidney disease (CKD), and other pollutant exposures. Sustained AHR activation in skeletal muscle has been shown to impair mitochondrial oxidative phosphorylation, promote proteolysis, and disrupt neuromuscular junction integrity, linking AHR signaling to muscle pathology. Experimental studies in rodent models demonstrate that pharmacologic or genetic inhibition of AHR can preserve muscle mass, mitochondrial function, and regenerative capacity. This review summarizes the molecular biology of the AHR, its emerging roles in skeletal muscle physiology and pathology, and the growing experimental toolkit for interrogating its function. Understanding how AHR signaling integrates environmental, metabolic, and aging cues may reveal new therapeutic opportunities to preserve skeletal muscle health and physical function across the lifespan.

HIV-associated mortality has been reduced by antiretroviral therapies (ART), but prolonged ART usage by people living with HIV (PLWH) is associated with frailty and poor healthspan. Mechanisms driving this phenomenon are not fully known, but clinical and preclinical studies suggest that HIV and ART may drive aberrant activation of the aryl hydrocarbon receptor (AhR) by kynurenine (KYN), an endogenous metabolite of tryptophan. Therefore, we investigated whether the combination of an HIV-like phenotype (Tg26 mice) and treatment with ART (emtricitabine; FTC) in female mice alters skeletal muscle homeostasis in an AhR-dependent manner to promote premature muscle aging phenotypes. Short-term FTC treatment increased serum KYN:tryptophan ratio and activated AhR signaling in skeletal muscle of Tg26 mice, although the study duration was not sufficient to induce significant FTC-related functional decline. FTC, alone or in combination with other ART (tenofovir alafenamide and tenofovir disproxil fumarate), activated AhR and induced senescence of female myoblasts in a manner comparable to KYN. Sequencing-based studies revealed targets and pathways related to the impacts of an HIV phenotype and ART in female skeletal muscle, including Gnas (encoding Gsα protein, critical for muscle glucose metabolism), inflammatory pathways, and lipid metabolism. Our studies suggest that the combined presence of HIV viral proteins and exposure to ART induced activation of AhR-mediated signaling in female muscle, as well as widespread changes across the skeletal muscle transcriptome and methylation landscape that may contribute to development of muscle dysfunction. This suggests AhR may represent a novel target for addressing persistent disparities in healthspan for PLWH.

Impaired or insufficient uptake of vitamin B₁₂ causes neurological disorders, anemia, and various diffuse symptoms, all historically called pernicious anemia (PA). Maintenance of adequate B₁₂ body store (B) is important, but the recommended doses and duration of peroral B₁₂-supplementation are usually half-empirical. The current work suggests physiologically realistic algebraic equations to express the changes in B via the daily B₁₂-uptake. The data from literature were used to estimate the relevant parameters of the equations. The modeled values of B showed that the daily ingestion of B₁₂ = 3 × 2.0 μg maintains the steady state value of B = 1316/1858/2683 μg in healthy individuals with low/mean/high types of B₁₂-uptake. The "classical" PA-patients have a highly decreased intrinsic factor (falling from 30 μg of B₁₂-equivalents to below 0.2 μg), giving B = 47/81/123 μg for the low/mean/high B₁₂-uptakes and the aforementioned doses. Alignment of B with the combined index of B₁₂-status (cB12) in a heterogeneous cohort indicated that B > 1250 μg provides the safe B₁₂-status for >84 % of the subjects. The risk of PA is associated with B < 400 μg. Repletion of a low body store from B = 400 μg to ≈1250 μg would require a prolonged supplementation of healthy individuals with B₁₂-pills, if they receive 1 × 5 μg/day or 1 × 50 μg/day. These schemes are expected to raise B after the time intervals of (not possible)/2200/780 days or 2330/860/480 days, respectively. Only a pill with high B₁₂ = 5000 μg/day would raise B to 1250 μg in 24/16/10 days.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is thought to play important roles in aging, oxidative stress, and cellular senescence. We have previously shown that the AhR agonist kynurenine (Kyn), a tryptophan metabolite that increases with age, can induce muscle atrophy in young mice. AhR overexpression can also lead to muscle atrophy and neuromuscular junction degradation. Here we utilized existing GEO data sets from skeletal muscles of aged mice to examine the impact of two longevity-related interventions, calorie restriction (CR) or treatment with the drug rapamycin (RM), on the expression of genes in the Kyn-AhR pathway. Data were examined in four skeletal muscles: soleus, gastrocnemius, tibialis anterior and triceps brachii. Results show that AhR expression increased with age in the triceps but was decreased with CR in the soleus and gastrocnemius. RM treatment did not significantly alter AhR expression in any of the four muscles of aged mice. Three enzymes that convert kynurenine to kynurenic acid in skeletal muscle, Kyat1, Kyat3 and Got2/Kyat4, are known to increase with endurance exercise and all three increased significantly with CR in aged skeletal muscle. In contrast, RM treatment did not increase Kyat1 expression in aged muscle and RM significantly decreased Kyat3 expression levels in muscles from aged mice. Together these data point to kynurenine aminotransferases as mediating some of the positive effects of CR on skeletal muscle with aging, and support prior research suggesting that CR and RM modulate different patterns of muscle-specific gene expression.

Tryptophan metabolism is a critical regulator of physiological and pathological processes, primarily through the kynurenine (KYN), serotonin and indole pathways. Dysregulation of indoleamine 2,3-dioxygenase 1 (IDO1) activity, serotonin and indole gut-microbial metabolism has been linked to a broad range of age-related chronic conditions, including cancer, cardiovascular disease, sarcopenia, and neurodegenerative disorders. Exercise emerges as a potent modulator of these pathways, redirecting tryptophan utilization to limit the accumulation of KYN metabolites while maintaining balanced indole and serotonin production. By regulating IDO1 activity and KYN flux, exercise alleviates inflammation, restores metabolic homeostasis, improved muscle integrity, neuroprotection, and overall systemic health. Mounting evidence supports the notion that lifestyle-based interventions targeting IDO1 and its downstream metabolites, particularly by physical activity, may offer a promising avenue for extending health span and mitigating the burden of chronic disease. This review synthesizes current advances in understanding the regulation of tryptophan metabolism (KYN, Serotonin and Indole) and highlights the unique capacity of exercise to remodel these pathways, underscoring their therapeutic potential in the context of healthy aging.

During transcription initiation, the holoenzyme of bacterial RNA polymerase (RNAP) specifically recognizes promoters using a dedicated σ factor. During transcription elongation, the core enzyme of RNAP interacts with nucleic acids mainly nonspecifically, by stably locking the DNA template and RNA transcript inside the main cleft. Here, we present a synthetic DNA aptamer that is specifically recognized by both core and holoenzyme RNAPs from extremophilic bacteria of the Deinococcus-Thermus lineage. The aptamer binds RNAP with subnanomolar affinities, forming extremely stable complexes even at high ionic strength conditions, blocks RNAP interactions with the DNA template and inhibits RNAP activity during transcription elongation. We propose that the aptamer binds at a conserved site within the downstream DNA-binding cleft of RNAP and traps it in an inactive conformation. The aptamer can potentially be used for structural studies to reveal RNAP conformational states, affinity binding of RNAP and associated factors, and screening of transcriptional inhibitors.