[123I]CLINDE SPECT as a neuroinflammation imaging approach in a rat model of stroke.

Poststroke neuroinflammation exacerbates disease progression. [11C]PK11195-positron emission tomography (PET) imaging has been used to visualize neuroinflammation; however, its short half-life of 20 min limits its clinical use. [123I]CLINDE has a longer half-life (13h); therefore, [123I]CLINDE-single-photon emission computed tomography (SPECT) imaging is potentially more practical than [11C]PK11195-PET imaging in clinical settings. The objectives of this study were to 1) validate neuroinflammation imaging using [123I]CLINDE and 2) investigate the mechanisms underlying stroke in association with neuroinflammation using multimodal techniques, including magnetic resonance imaging (MRI), gas-PET, and histological analysis, in a rat model of ischemic stroke, that is, permanent middle cerebral artery occlusion (pMCAo). At 6 days post-pMCAo, [123I]CLINDE-SPECT considerably corresponded to the immunohistochemical images stained with the CD68 antibody (a marker for microglia/microphages), comparable to the level observed in [11C]PK11195-PET images. In addition, the [123I]CLINDE-SPECT images corresponded well with autoradiography images. Rats with severe infarcts, as defined by MRI, exhibited marked neuroinflammation in the peri-infarct area and less neuroinflammation in the ischemic core, accompanied by a substantial reduction in the cerebral metabolic rate of oxygen (CMRO2) in 15O-gas-PET. Rats with moderate-to-mild infarcts exhibited neuroinflammation in the ischemic core, where CMRO2 levels were mildly reduced. This study demonstrates that [123I]CLINDE-SPECT imaging is suitable for neuroinflammation imaging and that the distribution of neuroinflammation varies depending on the severity of infarction.

Unraveling the cryptic functions of mitogen-activated protein kinases Cpk2 and Mpk2 in Cryptococcus neoformans.

Mitogen-activated protein kinase (MAPK) pathways are fundamental to the regulation of biological processes in eukaryotic organisms. The basidiomycete Cryptococcus neoformans, known for causing fungal meningitis worldwide, possesses five MAPKs. Among these, Cpk1, Hog1, and Mpk1 have established roles in sexual reproduction, stress responses, and cell wall integrity. However, the roles of Cpk2 and Mpk2 are less understood. Our study elucidates the functional interplay between the Cpk1/Cpk2 and Mpk1/Mpk2 MAPK pathways in C. neoformans. We discovered that CPK2 overexpression compensates for cpk1Δ mating deficiencies via the Mat2 transcription factor, revealing functional redundancy between Cpk1 and Cpk2. We also found that Mpk2 is phosphorylated in response to cell wall stress, a process regulated by the MAPK kinase (MAP2K) Mkk2 and MAP2K kinases (MAP3Ks) Ssk2 and Ste11. Overexpression of MPK2 partially restores cell wall integrity in mpk1Δ by influencing key cell wall components, such as chitin and the polysaccharide capsule. Contrarily, MPK2 overexpression cannot restore thermotolerance and cell membrane integrity in mpk1Δ. These results suggest that Mpk1 and Mpk2 have redundant and opposing roles in the cellular response to cell wall and membrane stresses. Most notably, the dual deletion of MPK1 and MPK2 restores wild-type mating efficiency in cpk1Δ mutants via upregulation of the mating-regulating transcription factors MAT2 and ZNF2, suggesting that the Mpk1 and Mpk2 cooperate to negatively regulate the pheromone-responsive Cpk1 MAPK pathway. Our research collectively underscores a sophisticated regulatory network of cryptococcal MAPK signaling pathways that intricately govern sexual reproduction and cell wall integrity, thereby controlling fungal development and pathogenicity.IMPORTANCEIn the realm of fungal biology, our study on Cryptococcus neoformans offers pivotal insights into the roles of specific proteins called mitogen-activated protein kinases (MAPKs). Here, we discovered the cryptic functions of Cpk2 and Mpk2, two MAPKs previously overshadowed by their dominant counterparts Cpk1 and Mpk1, respectively. Our findings reveal that these "underdog" proteins are not just backup players; they play crucial roles in vital processes like mating and cell wall maintenance in C. neoformans. Their ability to step in and compensate when their dominant counterparts are absent showcases the adaptability of C. neoformans. This newfound understanding not only enriches our knowledge of fungal MAPK mechanisms but also underscores the intricate balance and interplay of proteins in ensuring the organism's survival and adaptability.

A Case of Palmoplantar Keratoderma in the Constellation of Connective Tissue Diseases.

Overlap syndrome is a clinical challenge and brings together a wide range of treatment options for the treating physician. Addressing each and every complaint of the patient is crucial. A 50-year-old female patient presented with skin thickening, blackening, and hyperkeratosis; dysphagia; joint pain; features of myopathy; Raynaud's phenomenon; and dry mouth. Inflammatory markers were raised along with a positive antinuclear antibody (ANA) with Golgi apparatus pattern, anti-Sjögren's-syndrome-related antigen A (anti-SSA)/Ro60 3+, anti-SSA/Ro52 3+, and anti-PM/Scl 2+ antibodies that suggested overlap syndrome. Although the patient had no respiratory complaints, a unique interstitial lung disease (ILD) pattern was noted during the evaluation. Skin manifestations were puzzling, but the histopathology analyses of skin biopsies taken from two different sites revealed distinguishing features of cutaneous lupus and dermatomyositis. Treatment with hydroxychloroquine, pilocarpine, nifedipine, methotrexate, and topical tacrolimus produced a dramatic improvement in the clinical features. This case highlights subtle and florid features of different autoimmune diseases. The hyperkeratotic skin changes were the most striking feature, but the whole evaluation process unveiled many rare presentations of known autoimmune conditions that can open doors to new areas of our understanding toward connective tissue diseases (CTDs). Our case report demonstrates significant heterogeneity in the ANA patterns, ILD patterns, clinical manifestations, and treatment approaches.

CPK10 protein kinase regulates Arabidopsis tolerance to boron deficiency through phosphorylation and activation of BOR1 transporter.

Boron (B) is crucial for plant growth and development. B deficiency can impair numerous physiological and metabolic processes, particularly in root development and pollen germination, seriously impeding crop growth and yield. However, the molecular mechanism underlying boron signal perception and signal transduction is rather limited. In this study, we discovered that CPK10, a calcium-dependent protein kinase in the CPK family, has the strongest interaction with the boron transporter BOR1. Mutations in CPK10 led to growth and root development defects under B-deficiency conditions, while constitutively active CPK10 enhanced plant tolerance to B deficiency. Furthermore, we found that CPK10 interacted with and phosphorylated BOR1 at the Ser689 residue. Through various biochemical analyses and complementation of B transport in yeast and plants, we revealed that Ser689 of BOR1 is important for its transport activity. In summary, these findings highlight the significance of the CPK10-BOR1 signaling pathway in maintaining B homeostasis in plants and provide targets for the genetic improvement of crop tolerance to B-deficiency stress.

Metformin blocks BIK1-mediated CPK28 phosphorylation and enhances plant immunity.

INTRODUCTION: Metformin (MET), derived from Galega officinalis, stands as the primary first-line medication for the treatment of type 2 diabetes (T2D). Despite its well-documented benefits in mammalian cellular processes, its functions and underlying mechanisms in plants remain unclear. OBJECTIVES: This study aimed to elucidate MET's role in inducing plant immunity and investigate the associated mechanisms. METHODS: To investigate the impact of MET on enhancing plant immune responses, we conducted assays measuring defense gene expression, reactive oxygen species (ROS) accumulation, mitogen-activated protein kinase (MAPK) phosphorylation, and pathogen infection. Additionally, surface plasmon resonance (SPR) and microscale thermophoresis (MST) techniques were employed to identify MET targets. Protein-protein interactions were analyzed using a luciferase complementation assay and a co-immunoprecipitation assay. RESULTS: Our findings revealed that MET boosts plant disease resistance by activating MAPKs, upregulating the expression of downstream defense genes, and fortifying the ROS burst. CALCIUM-DEPENDENT PROTEIN KINASE 28 (CPK28) was identified as a target of MET. It inhibited the interaction between BOTRYTIS-INDUCED KINASE 1 (BIK1) and CPK28, blocking CPK28 threonine 76 (T76) transphosphorylation by BIK1, and alleviating the negative regulation of immune responses by CPK28. Moreover, MET enhanced disease resistance in tomato, pepper, and soybean plants. CONCLUSION: Collectively, our data suggest that MET enhances plant immunity by blocking BIK1-mediated CPK28 phosphorylation.

Factors Associated with Acute Kidney Injury Occurrence and Prognosis in Rhabdomyolysis at the Emergency Department.

Background and Objectives: This study aimed to analyze patients with rhabdomyolysis who presented to emergency departments and identify their distribution of related disease and prognostic factors. Materials and Methods: A retrospective cohort study was conducted on patients with rhabdomyolysis who presented to emergency departments over a 10-year period. Patient data, including patients' demographic variables (sex and age), mode of arrival, final diagnosis, statin use, rhabdomyolysis trigger factors, and levels of serum creatine phosphokinase (CPK), myoglobin, creatinine, sodium, potassium, phosphate, calcium, and lactate, were analyzed. Univariate and multivariate logistic regression analyses were conducted to identify the predictive factors of acute kidney injury (AKI). Results: Among the patients, 268 (65.6%) were found to have trigger factors without underlying diseases. Furthermore, 115 (28.2%) patients developed AKI. This comprehensive study sheds light on the diverse factors influencing the occurrence of AKI in rhabdomyolysis and provides insights into AKI predictive markers. Furthermore, we analyzed the cases by dividing them into six groups: occurrence of AKI, occurrence of infection, and simple or complex rhabdomyolysis. CPK time course was found to be important in clinical prognosis, such as AKI occurrence, dialysis or not, and mortality. Conclusions: Age, statin use, elevated creatinine and lactate levels, and initial serum CPK level emerged as significant predictors of AKI. CPK time course was also found to be an important factor in predicting the clinical outcomes of patients with rhabdomyolysis.

Symptomatic creatine phosphokinase elevation in a Crohn's disease patient caused by upadacitinib.

We present a Crohn's disease patient receiving high dose upadacitinib treatment with elevated CPK levels and myopathy, and provide the reader with practical tips on stopping and restarting upadacitinib, emphasizing the need for adequate monitoring.

Diversified molecular adaptations of inorganic nitrogen assimilation and signaling machineries in plants.

Plants evolved sophisticated machineries to monitor levels of external nitrogen supply, respond to nitrogen demand from different tissues and integrate this information for coordinating its assimilation. Although roles of inorganic nitrogen in orchestrating developments have been studied in model plants and crops, systematic understanding of the origin and evolution of its assimilation and signaling machineries remains largely unknown. We expanded taxon samplings of algae and early-diverging land plants, covering all main lineages of Archaeplastida, and reconstructed the evolutionary history of core components involved in inorganic nitrogen assimilation and signaling. Most components associated with inorganic nitrogen assimilation were derived from the ancestral Archaeplastida. Improvements of assimilation machineries by gene duplications and horizontal gene transfers were evident during plant terrestrialization. Clusterization of genes encoding nitrate assimilation proteins might be an adaptive strategy for algae to cope with changeable nitrate availability in different habitats. Green plants evolved complex nitrate signaling machinery that was stepwise improved by domains shuffling and regulation co-option. Our study highlights innovations in inorganic nitrogen assimilation and signaling machineries, ranging from molecular modifications of proteins to genomic rearrangements, which shaped developmental and metabolic adaptations of plants to changeable nutrient availability in environments.

CPK12 and Ca2+-mediated hypoxia signaling.

Hypoxia triggers reactive oxygen species (ROS)-induced elevation in cytoplasmic calcium (Ca2+) in the plant cells. Calcium-dependent protein kinase 12 (CPK12) acts as a sensor to recognize the Ca2+ signature and is activated by autophosphorylation. Then, the CPK12 moves into the nucleus with the help of phosphatidic acid (PA) and phosphorylates ERF-VII family proteins that activate hypoxia signaling and response. The study provides a novel mechanism of hypoxia signaling in plants. Moreover, the mechanism of hypoxia-specific Ca2+ signature generation remains elusive.

Polymyositis: A Case Report.

Inflammatory myopathies are a group of diseases whose common pathway is immune-mediated muscle damage, one of which is polymyositis. The definition of polymyositis is controversial, with proponents advocating a definition based on immunohistochemical and histopathological findings in muscle biopsies, while other proponents advocate a definition based on clinical manifestations and histopathological findings. Polymyositis is a quite rare disease that is clinically characterized by progressive proximal muscle weakness with a symmetric distribution. Within the diagnostic approach, laboratory studies show elevation of sarcoplasmic enzymes; nerve conduction tests are performed, which may aid in distinguishing myopathic causes of weakness from neuropathic disorders; and muscle biopsy is considered the gold standard to diagnose inflammatory myopathy and to distinguish the subclasses. We report the case of a 61-year-old male patient who presented generalized symmetrical weakness, predominantly in the upper extremities, and dysphagia, whose laboratory studies, autoantibodies, and muscle biopsy were confirmatory of this entity.

Dysphagia: A Case Report of an Atypical Presentation of Statin-Induced Necrotizing Myositis.

Statin medications act by inhibiting the enzyme hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGCR), thus decreasing hepatic cholesterol synthesis. They are considered the mainstay treatment of hypercholesterolemia due to their tremendous efficacy and mortality benefit. Although generally well tolerated, statins may adversely affect skeletal muscle resulting in side effects ranging from mild myalgia to life-threatening necrotizing myositis. Statin-induced necrotizing autoimmune myositis is a rare yet devastating adverse effect that may occur shortly after initiation of therapy or after several years of use. Unfortunately, medication discontinuation has shown no impact on prevention or alleviation of symptoms. Though there is currently no definitive guidance for the treatment of this condition, corticosteroids are generally considered to be first line, via high-dose oral prednisone or intravenous methylprednisolone. In this case report, we discuss the case of a 72-year-old male with an unusual presentation of statin-induced necrotizing autoimmune myositis: dysphagia, weakness, and weight loss. His diagnosis was confirmed by muscle biopsy indicating necrotizing myositis and his serum was found to be strongly positive for anti-HMG-CoA reductase antibodies. This patient had a very brief history of statin use, but his primary care provider discontinued the medication a couple of months prior to symptom onset due to elevated liver function tests. He was treated with aggressive intravenous fluid hydration and intravenous corticosteroids during an extended inpatient hospital stay. He was discharged to a rehabilitation facility. This report demonstrates the importance of creating a wide differential for patients who present with fatigue, generalized weakness, and dysphagia. It is essential to always consider statin-induced necrotizing myositis if a patient has a history of statin use, even if the statin has been discontinued. Necrotizing myositis demands timely diagnosis and management to improve mortality.

Role of Biomarkers in the Management of Immune-Checkpoint Inhibitor-Related Myocarditis.

PURPOSE OF REVIEW: Immune checkpoint inhibitor (ICI)-related myocarditis poses a major clinical challenge given its non-specific presentation, rapid progression, and high mortality rate. Here, we review the role of blood-based biomarkers in the clinical management of patients with ICI-related myocarditis. RECENT FINDINGS: Myocardial injury, its unique pattern, and the co-occurrence with myositis are defining features of ICI-related myocarditis. Non-cardiac biomarkers, specifically creatinine phosphokinase, precedes the symptomatic presentation and is highly sensitive for diagnosing ICI-related myocarditis, making them useful screening biomarkers. Combined elevations in cardiac troponins and non-cardiac biomarkers improve the confidence of an ICI myocarditis diagnosis. High troponin and creatinine phosphokinase levels are strongly associated with severe outcomes. We propose biomarker-based algorithms for the monitoring and diagnosis of ICI-related myocarditis. Biomarkers, such as cardiac troponins and creatine phosphokinase, can be used in combination in the monitoring, diagnosis, and prognostication of patients with ICI-related myocarditis.

Arabidopsis calcium-dependent protein kinase CPK6 regulates drought tolerance under high nitrogen by the phosphorylation of NRT1.1.

Nitrogen (N) is an essential macronutrient for plant growth and development, and its availability is regulated to some extent by drought stress. Calcium-dependent protein kinases (CPKs) are a unique family of Ca2+ sensors with diverse functions in N uptake and drought-tolerance signaling pathways; however, how CPKs are involved in the crosstalk between drought stress and N transportation remains largely unknown. Here, we identify the drought-tolerance function of Arabidopsis CPK6 under high N conditions. CPK6 expression was induced by ABA and drought treatments. The mutant cpk6 was insensitive to ABA treatment and low N, but was sensitive to drought only under high N conditions. CPK6 interacted with the NRT1.1 (CHL1) protein and phosphorylated the Thr447 residue, which then repressed the NO3- transporting activity of Arabidopsis under high N and drought stress. Taken together, our results show that CPK6 regulates Arabidopsis drought tolerance through changing the phosphorylation state of NRT1.1, and improve our knowledge of N uptake in plants during drought stress.

Cystin is required for maintaining fibrocystin (FPC) levels and safeguarding proteome integrity in mouse renal epithelial cells: A mechanistic connection between the kidney defects in cpk mice and human ARPKD.

Autosomal recessive polycystic kidney disease (ARPKD) is caused primarily by mutations in PKHD1, encoding fibrocystin (FPC), but Pkhd1 mutant mice failed to reproduce the human phenotype. In contrast, the renal lesion in congenital polycystic kidney (cpk) mice, with a mutation in Cys1 and cystin protein loss, closely phenocopies ARPKD. Although the nonhomologous mutation diminished the translational relevance of the cpk model, recent identification of patients with CYS1 mutations and ARPKD prompted the investigations described herein. We examined cystin and FPC expression in mouse models (cpk, rescued-cpk (r-cpk), Pkhd1 mutants) and mouse cortical collecting duct (CCD) cell lines (wild type (wt), cpk). We found that cystin deficiency caused FPC loss in both cpk kidneys and CCD cells. FPC levels increased in r-cpk kidneys and siRNA of Cys1 in wt cells reduced FPC. However, FPC deficiency in Pkhd1 mutants did not affect cystin levels. Cystin deficiency and associated FPC loss impacted the architecture of the primary cilium, but not ciliogenesis. No reduction in Pkhd1 mRNA levels in cpk kidneys and CCD cells suggested posttranslational FPC loss. Studies of cellular protein degradation systems suggested selective autophagy as a mechanism. In support of the previously described function of FPC in E3 ubiquitin ligase complexes, we demonstrated reduced polyubiquitination and elevated levels of functional epithelial sodium channel in cpk cells. Therefore, our studies expand the function of cystin in mice to include inhibition of Myc expression via interaction with necdin and maintenance of FPC as functional component of the NEDD4 E3 ligase complexes. Loss of FPC from E3 ligases may alter the cellular proteome, contributing to cystogenesis through multiple, yet to be defined, mechanisms.

[18F]FDG and [11C]PK11195 PET imaging in the evaluation of brown adipose tissue - effects of cold and pharmacological stimuli and their association with crotamine intake in a male mouse model.

This study aimed to evaluate the role of positron emission tomography (PET) with [11C]PK11195 and [18F]FDG in the characterization of brown adipose tissue (BAT). METHODS: Male C57BL/6 mice were studied with the glucose analogue [18F]FDG (n = 21) and the TSPO mitochondrial tracer [11C]PK11195 (n = 28), without stimulus and after cold (6-9 °C) or beta-agonist (CL316243) stimuli. PET studies were performed at baseline and after 21 days of daily treatment with crotamine, which is a peptide described to induce adipocyte tissue browning and to increase BAT metabolism. Tracer uptake (SUVmax) was measured in the interscapular BAT and translocator protein 18 kDa (TSPO) expression was evaluated by immunohistochemistry. RESULTS: The cold stimulus increased [18F]FDG uptake compared to no-stimulus (5.21 ± 1.05 vs. 2.03 ± 0.21, p < 0.0001) and to beta-agonist stimulus (2.65 ± 0.39, p = 0.0003). After 21 days of treatment with crotamine, there was no significant difference in the [18F]FDG uptake compared to the baseline in the no-stimulus group and in the cold-stimulus group, with a significant increase in uptake after CL stimulus (baseline: 2.65 ± 0.39; 21 days crotamine: 4.77 ± 0.81, p = 0.0003). Evaluation of [11C]PK11195 at baseline shows that CL stimulus increases the BAT uptake compared to no-stimulus (4.47 ± 0.66 vs. 3.36 ± 0.68, p = 0.014). After 21 days of treatment with crotamine, there was no significant difference in the [11C]PK11195 uptake compared to the baseline in the no-stimulus group (2.94 ± 0.58, p = 0.7864) and also after CL stimulus (3.55 ± 0.79, p = 0.085). TSPO expression correlated with [11C]PK11195 uptake (r = 0.83, p = 0.018) but not with [18F]FDG uptake (r = 0.40, p = 0.516). CONCLUSIONS: [11C]PK11195 allowed the identification of BAT under thermoneutral conditions or after beta3-adrenergic stimulation in a direct correlation with TSPO expression. The beta-adrenergic stimulus, despite presenting a lower intensity of glycolytic activation compared to cold at baseline, allowed the observation of an increase in BAT uptake of [18F]FDG after 21 days of crotamine administration. Although some limitations were observed for the metabolic changes induced by crotamine, this study reinforced the potential of using [11C]PK11195 and/or [18F]FDG-PET to monitor the activation of BAT.

Textural properties of microglial activation in Alzheimer's disease as measured by (R)-[11C]PK11195 PET.

Alzheimer's disease is the most common form of dementia worldwide, accounting for 60-70% of diagnosed cases. According to the current understanding of molecular pathogenesis, the main hallmarks of this disease are the abnormal accumulation of amyloid plaques and neurofibrillary tangles. Therefore, biomarkers reflecting these underlying biological mechanisms are recognized as valid tools for an early diagnosis of Alzheimer's disease. Inflammatory mechanisms, such as microglial activation, are known to be involved in Alzheimer's disease onset and progression. This activated state of the microglia is associated with increased expression of the translocator protein 18 kDa. On that account, PET tracers capable of measuring this signature, such as (R)-[11C]PK11195, might be instrumental in assessing the state and evolution of Alzheimer's disease. This study aims to investigate the potential of Gray Level Co-occurrence Matrix-based textural parameters as an alternative to conventional quantification using kinetic models in (R)-[11C]PK11195 PET images. To achieve this goal, kinetic and textural parameters were computed on (R)-[11C]PK11195 PET images of 19 patients with an early diagnosis of Alzheimer's disease and 21 healthy controls and submitted separately to classification using a linear support vector machine. The classifier built using the textural parameters showed no inferior performance compared to the classical kinetic approach, yielding a slightly larger classification accuracy (accuracy of 0.7000, sensitivity of 0.6957, specificity of 0.7059 and balanced accuracy of 0.6967). In conclusion, our results support the notion that textural parameters may be an alternative to conventional quantification using kinetic models in (R)-[11C]PK11195 PET images. The proposed quantification method makes it possible to use simpler scanning procedures, which increase patient comfort and convenience. We further speculate that textural parameters may also provide an alternative to kinetic analysis in (R)-[11C]PK11195 PET neuroimaging studies involving other neurodegenerative disorders. Finally, we recognize that the potential role of this tracer is not in diagnosis but rather in the assessment and progression of the diffuse and dynamic distribution of inflammatory cell density in this disorder as a promising therapeutic target.

Calcium-dependent activation of CPK12 facilitates its cytoplasm-to-nucleus translocation to potentiate plant hypoxia sensing by phosphorylating ERF-VII transcription factors.

Calcium-dependent protein kinases (CDPKs/CPKs) are key regulators of plant stress signaling that translate calcium signals into cellular responses by phosphorylating diverse substrate proteins. However, the molecular mechanism by which plant cells relay calcium signals in response to hypoxia remains elusive. Here, we show that one member of the CDPK family in Arabidopsis thaliana, CPK12, is rapidly activated during hypoxia through calcium-dependent phosphorylation of its Ser-186 residue. Phosphorylated CPK12 shuttles from the cytoplasm to the nucleus, where it interacts with and phosphorylates the group VII ethylene-responsive transcription factors (ERF-VII) that are core regulators of plant hypoxia sensing, to enhance their stabilities. Consistently, CPK12 knockdown lines show attenuated tolerance of hypoxia, whereas transgenic plants overexpressing CPK12 display improved hypoxia tolerance. Nonethelss, loss of function of five ERF-VII proteins in an erf-vii pentuple mutant could partially suppress the enhanced hypoxia-tolerance phenotype of CPK12-overexpressing lines. Moreover, we also discovered that phosphatidic acid and 14-3-3κ protein serve as positive and negative modulators of the CPK12 cytoplasm-to-nucleus translocation, respectively. Taken together, these findings uncover a CPK12-ERF-VII regulatory module that is key to transducing calcium signals from the cytoplasm into the nucleus to potentiate hypoxia sensing in plants.

The influence of serial 50 µL microsampling on rats administered azathioprine, the immunosuppressive drug.

According to the ICH S3A Q&A, microsampling is applicable to pharmaceutical drugs and toxicological analysis. Few studies have reported the effect of microsampling on the toxicity of immunotoxicological drugs. The aim of this multicenter study was to evaluate the toxicological effects of serial microsampling on rats treated with azathioprine as a model drug with immunotoxic effects. Fifty microliters of blood were collected from the jugular vein of Sprague-Dawley rats at six time points from day 1 to 2 and 7 time points from day 27 to 28. The study was performed at three organizations independently. The microsampling effect on clinical signs, body weights, food consumption, hematological parameters, biochemical parameters, urinary parameters, organ weights, and tissue pathology was evaluated. Azathioprine-induced changes were observed in certain hematological and biochemical parameters and thymus weight and pathology. Microsampling produced minimal or no effects on almost all parameters; however, at 2 organizations, azathioprine-induced changes were apparently masked for two leukocytic, one coagulation, and two biochemical parameters. In conclusion, azathioprine toxicity could be assessed appropriately as overall profiles even with blood microsampling. However, microsampling may influence azathioprine-induced changes in certain parameters, especially leukocytic parameters, and its usage should be carefully considered.