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Additional file 2: Fig. S2 Subgroup analysis showed via SHAP plot stratified by dialysis. A: Impact of each feature on the in-hospital mortality in non-dialysis patients; B: Impact of each feature on the in-hospital mortality in dialysis patients.

Supplemental material, sj-docx-1-cat-10.1177_10760296231179439 for An Updated Pooled Analysis of Off-Label Under and Over-Dosed Direct Oral Anticoagulants in Patients with Atrial Fibrillation by Nan-Nan Shen, Eliana Ferroni, Claudio Barbiellini Amidei, Cristina Canova, Viviana Peron, Jia-Liang Wang, Hou-Wen Lin and Zhi-Chun Gu in Clinical and Applied Thrombosis/Hemostasis

Additional file 1: Fig. S1 Subgroup analysis showed via SHAP plot stratified by ACS. A: Impact of each feature on the in-hospital mortality in non-ACS patients; B: Impact of each feature on the in-hospital mortality in ACS patients.

Additional file 1: Figure S1 Feature selection for the relationship between various TyG indices and one-year mortality analyzed by the Boruta algorithm.

BackgroundThe study of genetic predisposition to pediatric systemic lupus erythematosus (pSLE) has brought new insights into the pathophysiology of SLE, as it is hypothesized that genetic predisposition is greater in children. Furthermore, identifying genetic variants and linking disrupted genes to abnormal immune pathways and clinical manifestations can be beneficial for both diagnosis and treatment. Here, we identified genetic alterations in a patient with childhood-onset SLE and analyzed the immunological mechanisms behind them to support future diagnosis, prognosis, and treatment.MethodsWhole exome sequencing (WES) was adopted for genetic analysis of a patient with childhood-onset SLE. Gene mutations were confirmed by Sanger sequencing. Clinical data of this patient were collected and summarized. Ingenuity Pathway Analysis was used to provide interacting genes of the perturbed genes. Online Enrichr tool and Cytoscape software were used to analysis the related pathways of these genes.ResultsWe present a case of a 2-year-old girl who was diagnosed with idiopathic thrombocytopenic purpura (ITP) and SLE. The patient was characterized by cutaneous bleeding spots on both lower extremities, thrombocytopenia, decreased serum complements levels, increased urinary red blood cells, and positive ANA and dsDNA. The patient was treated with methylprednisolone and mycophenolate, but clinical remission could not be achieved. The genomic analysis identified three novel mutations in this pSLE patient, a double-stranded missense mutation in ACP5 (c.1152G>T and c.420G>A) and a single-stranded mutation in SAMHD1 (c.1423G>A). Bioinformatic analysis showed that these two genes and their interacting genes are enriched in the regulation of multiple immune pathways associated with SLE, including cytokine signaling and immune cell activation or function. Analysis of the synergistic regulation of these two genes suggests that abnormalities in the type I interferon pathway caused by genetic variants may contribute to the pathogenesis of SLE.ConclusionThe combined complexity of polymorphisms in the coding regions of ACP5 and SAMHD1 influences the susceptibility to SLE. Alterations in these genes may lead to abnormalities in the type I interferon pathway. Our study extends the spectrum of mutations in the ACP5 and SAMHD1 genes. The identification of these mutations could aid in the diagnosis of SLE with genetic counseling and suggest potential precise treatments for specific pathways.

IntroductionMushroom-forming fungi comprise diverse species that develop complex multicellular structures. In cultivated species, both ecological adaptation and artificial selection have driven genome evolution. However, little is known about the connections among genotype, phenotype and adaptation in mushroom-forming fungi.ObjectivesThis study aimed to (1) uncover the population structure and demographic history of Lentinula edodes, (2) dissect the genetic basis of adaptive evolution in L. edodes, and (3) determine if genes related to fruiting body development are involved in adaptive evolution.MethodsWe analyzed genomes and fruiting body-related traits (FBRTs) in 133 L. edodes strains and conducted RNA-seq analysis of fruiting body development in the YS69 strain. Combined methods of genomic scan for divergence, genome-wide association studies (GWAS), and RNA-seq were used to dissect the genetic basis of adaptive evolution.ResultsWe detected three distinct subgroups of L. edodes via single nucleotide polymorphisms, which showed robust phenotypic and temperature response differentiation and correlation with geographical distribution. Demographic history inference suggests that the subgroups diverged 36,871 generations ago. Moreover, L. edodes cultivars in China may have originated from the vicinity of Northeast China. A total of 942 genes were found to be related to genetic divergence by genomic scan, and 719 genes were identified to be candidates underlying FBRTs by GWAS. Integrating results of genomic scan and GWAS, 80 genes were detected to be related to phenotypic differentiation. A total of 364 genes related to fruiting body development were involved in genetic divergence and phenotypic differentiation.ConclusionAdaptation to the local environment, especially temperature, triggered genetic divergence and phenotypic differentiation of L. edodes. A general model for genetic divergence and phenotypic differentiation during adaptive evolution in L. edodes, which involves in signal perception and transduction, transcriptional regulation, and fruiting body morphogenesis, was also integrated here.

Background Cancer patients are at a high risk of being infected with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), and are more likely to develop severe illness and have higher mortality once infected. In the COVID‐19 pandemic, it is urgent to understand the effects of antitumor therapy on the prognosis of patients with COVID‐19. Methods A systematic literature search was conducted in PubMed, Cochrane Library, Embase, MedRxiv, and Chinese National Knowledge Infrastructure (CNKI) until 21 June 2020. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were evaluated using a random effects model to analyze the effects of antitumor therapies on COVID‐19 patients. Results For cancer patients with COVID‐19, the death events related to antitumor treatment were higher than those with no antitumor treatment (OR = 1.55; 95% CI 1.07–2.25; p = 0.021). Compared with patients in the survival group, the non‐survival group showed no significant differences in patients who received antitumor therapy. Compared with patients in the non‐severe group, the severe group was more likely to receive antitumor therapy (OR = 1.50; 95% CI 1.02–2.19; p = 0.037) and there was a significant difference. The incidence of severe events was higher in the subgroup of chemotherapy (OR = 1.73; 95% CI 1.09–2.73). Conclusion The synthesized evidence suggests that cancer patients with COVID‐19 who received antitumor treatment shortly before symptom onset are more likely to experience severe symptoms and have high mortality. Receiving chemotherapy is an unfavorable factor for the prognosis of cancer patients with COVID‐19., Patients with cancer and COVID‐19 who received anti‐tumor treatment shortly before symptom onset are more likely to develop severe illness and have high a mortality. Receiving chemotherapy is an unfavorable factor for the prognosis of patients with COVID‐19.

Finely tuned mitogen-activated protein kinase (MAPK) signaling is important for cancer cell survival. Perturbations that push cells out of the MAPK fitness zone result in cell death. Previously, in a screen of the North China Pharmaceutical Group Corporation’s pure compound library of microbial origin, we identified elaiophylin as an autophagy inhibitor. Here, we demonstrated a new role for elaiophylin in inducing excessive endoplasmic reticulum (ER) stress, ER-derived cytoplasmic vacuolization, and consequent paraptosis by hyperactivating the MAPK pathway in multiple cancer cells. Genome-wide CRISPR/Cas9 knockout library screening identified SHP2, an upstream intermediary of the MAPK pathway, as a critical target in elaiophylin-induced paraptosis. The cellular thermal shift assay (CETSA) and surface plasmon resonance (SPR) assay further confirmed the direct binding between the SHP2 and elaiophylin. Inhibition of the SHP2/SOS1/MAPK pathway through SHP2 knockdown or pharmacological inhibitors distinctly attenuated elaiophylin-induced paraptosis and autophagy inhibition. Interestingly, elaiophylin markedly increased the already-elevated MAPK levels and preferentially killed drug-resistant cells with enhanced basal MAPK levels. Elaiophylin overcame drug resistance by triggering paraptosis in multiple tumor-bearing mouse models resistant to platinum, taxane, or PARPi, suggesting that elaiophylin might offer a reasonable therapeutic strategy for refractory ovarian cancer.

Additional file 1: Supplemental Figure 1. Urine G3BP levels in different groups of patients after excluding top 8 uG3BP level active LN patients. (a) uG3BP levels were significantly increased in active LN patients (n=78) compared to those in inactive LN (n=33, p=0.008), CKD patients (n=30, p=0.03) and healthy controls (n=27, p

Clear cell renal cell carcinoma (ccRCC) is one of the most common cancers worldwide. Despite intense efforts to elucidate its pathogenesis, the molecular mechanisms and genetic characteristics of this cancer remain unknown. In this study, three expression profile data sets (GSE15641, GSE16441 and GSE66270) were integrated to identify candidate genes that could elucidate functional pathways in ccRCC. Expression data from 63 ccRCC tumors and 54 normal samples were pooled and analyzed. The GSE profiles shared 379 differentially expressed genes (DEGs), including 249 upregulated genes, and 130 downregulated genes. A protein-protein interaction network (PPI) was constructed and analyzed using STRING and Cytoscape. Functional and signaling pathways of the shared DEGs with significant p values were identified. Kaplan-Meier plots of integrated expression scores were used to analyze survival outcomes. These suggested that FN1, ICAM1, CXCR4, TYROBP, EGF, CAV1, CCND1 and PECAM1/CD31 were independent prognostic factors in ccRCC. Finally, to investigate early events in renal cancer, we screened for the hub genes CCND1 and PECAM1/CD31. In summary, integrated bioinformatics analysis identified candidate DEGs and pathways in ccRCC that could improve our understanding of the causes and underlying molecular events of ccRCC. These candidate genes and pathways could be therapeutic targets for ccRCC.

BACKGROUND The serine peptidase inhibitor Kazal type 13 (SPINK13) gene has tumor suppressor activity, but its role in renal cell carcinoma (RCC) remains unknown. This study aimed to investigate mRNA expression of SPINK13 in clear cell renal cell carcinoma (CCRCC) in human tissue and to use bioinformatics data to investigate the role of SPINK13 expression as a clinicopathological and prognostic biomarker for patients with CCRCC. MATERIAL AND METHODS Patients with CCRCC (N=533) with available RNA sequence data from The Cancer Genome Atlas (TCGA)-CCRCC database were analyzed with patients who had a tissue diagnosis of CCRCC (N=305) at the Fudan University Shanghai Cancer Center (FUSCC). Differential transcriptional and proteome expression profiles were obtained from the ONCOMINE cancer microarray database, TCGA, and the Human Protein Atlas (HPA) database. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) measured SPINK13 mRNA expression in 305 samples of CCRCC tissue from the FUSCC. The effects of clinicopathological parameters on progression-free survival (PFS) and overall survival (OS) were analyzed using the Kaplan-Meier and log-rank test. RESULTS Transcriptional and proteome expression of SPINK13 were significantly increased CCRCC tissue samples. Increased SPINK13 mRNA expression was significantly associated with reduced PFS and OS in 838 patients with CCRCC patients from the two independent cohorts, the FUSCC and the TCGA-CCRCC cohorts (p

Background: Gastric cancer is the third most lethal cancer worldwide. Finding a novel marker is essential to targeted therapy and the diagnosis of gastric cancer. As newly discovered markers, circRNAs have aroused widespread attention on a global scale. Our research aims to understand the role of circRNAs in gastric cancer and to explore the underlying pathogenesis. Methods: Raw expression data of circRNAs were obtained from the GEO database. Integrated bioinformatics analysis was used to screen differentially expressed circRNAs (DECs) by RobustRankAggreg package. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to predict the functions of DECs. Then, the miRNAs and mRNAs at the downstream of DECs were predicted. Expression data of miRNAs and mRNAs were downloaded from The Cancer Genome Atlas (TCGA). The aberrantly expressed miRNAs and mRNAs were selected using the edgeR package. Results: Four datasets (GSE78092, GSE83521, GSE89143, and GSE93541) were downloaded from the GEO database. Among them, two DECs ( hsa_circ_0007991 and hsa_circ_0067934 ) were screened. The functional analyses of DECs confirmed that they were cancer-related circRNAs. Furthermore, hsa-mir-4705 (miRNA) and BMPR1B (mRNA) at the downstream of hsa_circ_0067934 were found differentially expressed in gastric cancer by expression data from TCGA database. Conclusions: Our study discovered the critical roles of hsa_circ_0007991 and hsa_circ_0067934 in the development of gastric cancer, and they could be novel markers for targeted therapy and assist the diagnosis of early-stage gastric cancer. Moreover, we discovered that the hsa_circ_0067934/hsa-mir-4705/BMPR1B axis might be involved in the carcinogenesis of gastric cancer.

Hepatitis B surface antigen (HBsAg) seroclearance is the most important milestone indicating favourable clinical outcomes in patients with chronic hepatitis B (CHB). However, it is difficult to achieve due to the impaired HBV-specific immunity, such as programmed cell death 1 (PD-1)-associated T cell exhaustion. We assessed soluble PD-1 (sPD-1) as a novel seromarker for predicting spontaneous HBsAg loss.Serial serum levels of sPD-1 were evaluated in 1046 untreated hepatitis B e antigen (HBeAg)-seronegative individuals who had achieved undetectable serum HBV DNA. Multiple regression analyses were applied to assess associations among baseline and subsequent sPD-1 levels, HBsAg decline during follow-up, and spontaneous HBsAg seroclearance.A total of 390 individuals achieved spontaneous HBsAg seroclearance during 6464.4 person-years of follow-up. Baseline sPD-1 levels were inversely associated with baseline HBsAg levels (qHBsAg) as well as a greater decline in qHBsAg during follow-up. Incidence rates of HBsAg seroclearance were 11.5, 61.7, 96.7 and 151.0 per 1000 person-years for sPD-1 levels of ≥4000, 536-3999, 125-535 and125 pg/mL, respectively (PsPD-1 level is a novel marker which independently predicts spontaneous HBsAg seroclearance of HBeAg-negative inactive CHB patients with undetectable HBV DNA. (word count: 234,250).

After the detailed analysis of the application of electronic reconnaissance satellite in peacetime and wartime, this paper puts forward the effectiveness index and calculation model (reconnaissance minimum power) of the interception ability of electronic reconnaissance satellite on ground-based radars, considering if radiation signals of ground-based radars can be effectively intercepted by electronic reconnaissance satellite. Furthermore, this paper sets the typical tactical scenario in which HEO, LEO electronic reconnaissance satellites implement surveillance on ground-based warning radars and guided radars, and then quantitatively calculates the reconnaissance minimum power of two kinds of electronic reconnaissance satellites on typical ground-based radars operating in different power gears. The analysis of examples show that the effectiveness index and calculation model is reasonable and effective, providing a better tool for ground-based radars’ quantitative analysis on the space-based electronic reconnaissance threats.

This thesis transforms the choice of marching route into risky two-level decision-making by analyzing feasible alternative routes, road conditions and their occurrence probability. Based on the decision tree method, the thesis depicts a two-level decision tree of marching route. After calculating the expected marching time along each alternative route, the thesis identifies two-level optimal marching route based on the minimal expected loss criterion. The result of example analysis proves that the method is effective, and describes the process of multi-level decision-making of marching route in a clearer way.

The use of Bayesian information criterion (BIC) in the model selection procedure is under the assumption that the observations are independent and identically distributed (i.i.d.). However, in practice, we do not always have i.i.d. samples. For example, clustered observations tend to be more similar within the same group, and longitudinal data is collected by measuring the same subject repeatedly. In these scenarios, the assumption in BIC is not satisfied. The concept of effective sample size is brought up and improved BIC is defined by replacing the sample size in the original BIC expression with the effective sample size, which will give us a better theoretical foundation in the circumstance that mixed-effects models involve. Numerical experiment results are also given by comparing the performance of our new BIC with other widely used BICs.

Iron oxide is ntensively studied as a potential anode material of lithium ion battery. Although it has a high theoretical capacity, the potential application is still limited due to its volumetric instability, relative high working voltage and low 1st cycle efficiency. In this thesis, strategies to alleviate these issues in iron oxide are addressed, such as morphology design, elemental modification and lithium addition. 1D rod-like morphology of iron oxide is firstly investigated as one of the optimized structure to overcome volumetric instability. It is seen that tuning the size of the nanorod led to optimal electrochemical performance, which is due to better structural retaining ability of the particles. It was seen that intermediate sized nanorod showed ~1000mAh g-1 capacity at the end of 50 cycles which is more than powder iron oxide. Elemental modification of iron oxide has also proven to be an effective strategy to lower the reaction voltage of hematite. Among several of tested material, Mg and Ti modified sample has shown the greatest potential also on enhancing the capacity at higher current density region. A capacity of 556 mAh g-1 at a delithiation current rate of 3C is achieved for Mg modified nanorod, which is five times higher than for pure hematite. Further synthesis of iron oxide microsphere, nanosphere and chunky-like particles showed has shown an unexpected high reversible capacity of ~800mAh g-1, which is not fully understood up to date. However, it can be inferred from these studies that in addition to particle morphology, there are other intrinsic factors that dominate the electrochemical behavior of iron oxide. The last section presents a lithium addition strategy that successfully forms a pre-lithiated form of iron oxide that has proven to increase the 1st cycle efficiency of iron oxide material from ~68% to ~80%. Doctor of Philosophy (IGS)

Necroptosis is a form of programmed, caspase-independent cell death that is involved in various pathologic disorders such as ischemia/reperfusion injury, acute kidney injury and inflammatory bowel diseases. Identification of necroptosis inhibitors has great therapeutic potential for the treatment of necroptosis-associated diseases. In this study, we identified that the Bcr-Abl inhibitor GNF-7 was a potent inhibitor of necroptosis. GNF-7 inhibited necroptosis in both human and mouse cells, while not protecting cells from apoptosis. Drug affinity responsive target stability assay (DARTS) demonstrated that it binded with RIPK1 and RIPK3. GNF-7 inhibited RIPK1 and RIPK3 kinase activities and thus disrupted RIPK1-RIPK3 necrosome complex formation. In vivo, GNF-7 ameliorated both cisplatin- and ischemia/reperfusion-induced AKI. Orally administration of GNF-7 attenuated renal cell necrosis and reduced pro-inflammatory responses in mouse models of AKI. Taken together, our study shows that GNF-7 is a novel necroptosis inhibitor and has great potential for the treatment of acute renal inflammatory disorders by targeting both RIPK1 and RIPK3 kinases.

Additional file 1 : Supplemental Table 1. Correlation analysis of ALCAM and patients’ characteristics.

Background/Aims: The biological effects of periodic mechanical stress on the mitogenesis of chondrocytes have been studied extensively over the past few years. However, the mechanisms underlying the ability of chondrocytes to sense and respond to mechanical stimuli remain to be determined. In the current study, we analyzed the mechanisms by which periodic mechanical stress is translated into biochemical signals and verified the key role of non-integrin mechanosensors including Caveolin-1 (Cav-1), and insulin-like growth factor-1 receptor (IGF-1R) in chondrocyte proliferation. Methods: Two steps were undertaken in the experiment. In the first step, the cells were maintained under static conditions or periodic mechanical stress for 0 h and 1 h prior to Western blot analysis. In the second step, the cells were pretreated with short hairpin RNA (shRNA) targeted to Cav-1 or IGF-1R or control scrambled shRNA. Moreover, they were pretreated with their selective inhibitors methyl β-cyclodextrin (MCD) or Linsitinib (OSI-906). They were maintained under static conditions or periodic mechanical stress for 1 h prior to Western blot analysis, and for 3 days, 8 h per day, prior to direct cell counting and CCK-8 assay, respectively. Results: Periodic mechanical stress significantly induced sustained phosphorylation of Cav-1 at Tyr14 and IGF-1R at Tyr1135/1136. Proliferation was inhibited by pretreatment with Cav-1 inhibitor MCD and by shRNA targeted to Cav-1 in chondrocytes in response to periodic mechanical stress. Meantime, MCD and shRNA targeted to Cav-1 also attenuated IGF-1R, and extracellular signal-regulated kinase (ERK)1/2 activation. In addition, inhibiting IGF-1R activity by Linsitinib and shRNA targeted to IGF-1R abrogated chondrocyte proliferation and phosphorylation level of ERK1/2 subjected to periodic mechanical stress, while the phosphorylation site of Cav-1 was not affected. Conclusion: These findings collectively suggested that periodic mechanical stress promoted chondrocyte proliferation through Cav-1-IGF-1R-ERK1/2.

Cisplatin (cis-dichloro-diammine platinum, CDDP) is a well-known chemotherapeutic drug against a broad spectrum of human malignancies. However, the clinical utility of this effective chemotherapy agent is dose limited by its toxic side effects such as nephrotoxicity and ototoxicity. Necroptosis is a form of programmed necrotic cell death that is mediated by serine/threonine kinases, RIPK1 and RIPK3, together with MLKL. In this study, we identified that the multitargeted kinase inhibitor KW-2449 inhibited cisplatin-induced necroptosis, while potentiated cisplatin-induced apoptosis in cancer cells. Mechanistic studies indicated that KW-2449 directly inhibited RIPK1 kinase activity to block necroptosis. Oral administration of KW-2449 attenuated renal cell necrosis and reduced pro-inflammatory responses in mouse models of cisplatin-induced nephrotoxicity. Taken together, our study shows that KW-2449 is a novel necroptosis inhibitor by targeting RIPK1 kinase activity and has great clinic potential for the treatment of cisplatin-induced nephrotoxicity.

In recent years, more and more studies have shown that the expression of miRNAs is closely related to the occurrence of tumors and plays an irreplaceable role in the development and metastasis of tumors. The research was focused on lung squamous cell carcinoma. The data information is downloaded from the TCGA database and analyzed for variance, which is then verified in the GEO database. Then differential expression of miRNAs was found and survival analysis was performed, through the cut – off standard(PAuthor SummaryMicroRNAs are single-stranded small molecular RNA that participate in the regulation of various biological functions through indirect regulation of gene expression, and have been reported to play an important role in the occurrence, development, invasion and metastasis of tumors. In recent years, the research on miRNAs has become increasingly hot, and the role of miRNAs in tumor has been proved more and more. The subjects of this study were squamous cell carcinoma in lung cancer with relatively few studies on miRNAs. Through high-throughput data analysis, miRNAs with differential expression between lung squamous cell carcinoma tissues and normal tissues were found. These differentially expressed miRNAs provide a new direction for the early diagnosis of patients. Then, survival analysis was conducted to find the miRNAs significantly correlated with the total survival time of patients, and multi-factor analysis was conducted to exclude the influence of other clinical factors, and independent risk factors (miRNAs) affecting the survival of patients were determined, so as to provide new targets for the treatment and survival prediction of patients.

Figure S4. Effect of IFN-α on sociability in NZB/NZW F1 mice, as assessed in the three-chamber social interaction test. (a) Total distance travelled in all of the three chambers. (b) Percentage of distance travelled in the two chambers. Percentage of time spent (c) and distance travelled (d) around the two cages. Number of animals per group =10. *P

Figure S2. The expression levels of IFN-α, IL-6 and CXCL10 in the brain. Number of animals per group =5. *P

Figure S1. Effect of IFN-α on lupus nephritis in NZB/NZW F1 mice. (a) Proteinuria, as quantified by 24-h urinary protein. (b) H&E- or PAS-stained kidney sections. Number of animals per group =5. *P

Figure S3. Effect of IFN-α on anxiety-like phenotypes in NZB/NZW F1 mice, as assessed by the elevated plus-maze test. (a) Total distance travelled in the elevated plus-maze. (b) Percentage of distance travelled in the open arm of the elevated plus-maze. (c) Total number of entries into the arms of the elevated plus-maze. (d) Percentage of entries into the open arms. (e) Total distance travelled in the open field arena. (f) Percentage of distance travelled in the margin area in the open field test. (g) Number of entries into the centre of the open field. Number of animals per group =10. *P

Additional file 3: Table S1. Clinicopathological characteristics baseline in relation to AQP9 expression status in TCGA cohort. Table S2. Univariate Cox logistic regression analysis of PFS in TCGA and FUSCC cohort (PFS: progression-free survival; TCGA: the Cancer Genome Atlas; FUSCC: Fudan university shanghai cancer center). Table S3. Univariate Cox logistic regression analysis of OS in TCGA and FUSCC cohort (OS: overall survival; TCGA: the Cancer Genome Atlas; FUSCC: Fudan university shanghai cancer center). Table S4. Correlation analysis between AQP9 and immune cell infiltrations in ccRCC samples using TIMER.

Figure S5. Effect of IFN-α on sociability in NZB/NZW F1 mice, as assessed by the novelty Y-maze task. (a) Total distance travelled. (b) Total number of arm entries. (c) Distance travelled in the arms per entry. Number of animals per group =10. *P

Hot topic of research today is to overcome the irreversible capacity loss (ICL) in high capacity anodes used in Li-ion battery. Several attempts have been exploited to overcome the ICL issue, but all of them involved time consuming and inevitable electrolyte decomposition. Here, we attempted to utilize the octahedral site of spinel cathode ( ex. LiMn 2 O 4 ) for the compensation of ICL when paired with conversion type anode, α-Fe 2 O 3 nanorods. The performance of Li 1.26 Mn 2 O 4 /α-Fe 2 O 3 nanorods cell is compared with similar configurations fabricated without any electrode treatment and α-Fe 2 O 3 treated one. Amongst, LiMn 2 O 4 treated assembly showed promising results as compared with anode treated configuration.

The comprehensive recycling of zinc and iron from smelting waste containing zinc ferrite (SWCZF) remains a challenge. A study is to be the first proposed that hazardous SWCZF is directionally converted with SO2 for simultaneous recycling of zinc and iron. In the first step, zinc and iron phases in SWCZF were transformed into zinc sulfate and ferric oxide via oriented transformation with SO2. The decomposition rate of zinc ferrite and the yield of zinc sulfate were 94% and 95%, respectively. In the second step, zinc and iron in SWCZF were directionally enriched to obtain zinc sulfate solution and iron concentrate via water leaching-magnetic separation. The leaching rate of zinc from water leaching was 93.2% and the iron recovery from magnetic separation was 83.8%. The zinc sulfate solution can be directly returned to the traditional hydrometallurgical process to produce electrolytic zinc or be used to produce zinc sulfate with no generation of iron residues. The iron concentrate can be used as a raw material for iron making. Meanwhile, a clean slag was produced and can be safe for storage or potentially used as construction materials. Therefore, the deep reclamation, reducing quantity and harmlessness of SWCZF were realized via this cleaner technical route.

Background/Aims: The mitogenic effects of periodic mechanical stress on chondrocytes have been studied extensively, but the mechanisms whereby chondrocytes sense and respond to mechanical stimuli remain to be determined. We explored the question and verified the key role of G protein coupled receptor kinase interacting protein 1 (GIT1) signaling in periodic mechanical stress-induced chondrocyte proliferation. Methods: Two steps were undertaken in the experiment. In the first step, the cells were maintained under non-pressure conditions or periodic mechanical stress for 1 h prior to Western blot analysis. In the second step, the cells were pretreated with short hairpin RNA (shRNA) targeted to GIT1 or Src or control scrambled shRNA, or transfected with GIT1 wild-type or GIT1 mutant Y321F, or focal adhesion kinase (FAK) wild-type or FAK mutants Y397F or Y576F/Y577, respectively. Moreover, the cells were pretreated with blocking antibody against integrin β1 or PP2. Then the cells were maintained under non-pressure conditions or periodic mechanical stress for 1 h prior to Western blot analysis, and for 3 days, 8 h per day, prior to direct cell counting and CCK-8 assay, respectively. Results: Periodic mechanical stress significantly induced sustained phosphorylation of GIT1 at Tyr321. Reduction of GIT1 with shRNA targeted to GIT1 and GIT1 mutant Y321F inhibited periodic mechanical stress-promoted chondrocyte proliferation, accompanied by attenuated extracellular signal-regulated kinase (ERK)1/2 and FAK phosphorylation at Tyr576/577. However, activation of Src and FAK-Tyr397 was not prevented upon GIT1 suppression. Furthermore, pretreatment with blocking antibody against integrin β1, Src-selective inhibitor, PP2, and shRNA targeted to Src blocked GIT1 activation under periodic mechanical stress. In addition, GIT1 phosphorylation at Tyr321 was not reduced upon pretreatment with FAK mutants Y397F or Y576F/Y577 under conditions of periodic mechanical stress. Conclusion: These findings collectively suggested that periodic mechanical stress promoted chondrocyte proliferation through at least two separate pathways, integrin β1-Src-GIT1-FAK(Tyr576/577)-ERK1/2, and the other parallel GIT1-independent integrin β1-FAK(Tyr397)-ERK1/2.

Four common species of Dalbergia genus (D. bariensis, D. oliveri, D. cochinchinensis, and D. retusa), which are traded as “Rosewood” or “Hongmu” commonly throughout East and Southeast Asia, were extracted with ethanol-benzene and the extractives were analyzed by means of three IR spectroscopic methods aiming at their chemotaxonomic differentiation. Conventional FTIR and 2nd derivative IR (SD-IR) showed defined spectral ranges, where the differences are pronounced. Two-dimensional correlation infrared (2D-IR) spectroscopy revealed significant auto-peaks suitable for reliable differentiation of the four Dalbergia species. The gas chromatography-mass spectrometry (GC-MS) evaluation of the extractives was also useful. It can be concluded that the analysis of the extractives is the best chemotaxonomic approach for identification of anatomically similar wood species.

The properties of wood can be improved through steam-heat treatment. There are many studies about mechanical properties of steam-heat treated wood, but very few studies are on the aspects of chemical modifications. In this study, FT-IR spectra combined with SD-IR spectra, correlation coefficients and 2D-IR spectra are employed to analyze the chemical modifications of teak (Tectona grandis L.F.) wood during steam-heat treatment under treatment temperatures from 120 °C to 220 °C at intervals of 20 °C. Acetic acid, which is produced during steam-heat treatment, acts as a catalyst of condensation and degradation reactions of wood components. The changes of wood components are more and more intense with increasing the treatment temperature. The sensitivity of wood samples to thermal perturbation rises initially with increasing treatment temperature before falling back. The steam-heat treated wood under 180 °C is the most sensitive.

T-bet+ CD11c+ B cells are critical for antichromatin immunoglobulin G production in the development of lupus. Figure S1. The fraction of T-bet and CD11c positive population was increased in CD138+ plasma cells. Figure S2. Correlation between the percentage of T-bet+ CD11c+ CD19+ B cells and the titers of anti-ANA antibodies (A), anti-dsDNA antibodies (B) from 22 SLE patients. Figure S3. IFNÎł is required for T-bet+ CD11c+ B cell differentiation and activation. 5 Ă 107 splenocytes from Bm12 mice or B6 mice cells were injected intraperitoneally into B6 mice (n = 5). (PDF 361 kb)

// Nan Shen 1, * , Yuan Ruan 2, *, # , Yajun Lu 3 , Xuefeng Jiang 4 , Huiqing Sun 4 , Gongming Gao 5 , Luming Nong 5 , Kewei Ren 4 1 Department of Clinical Pharmacy, The Affiliated Jiangyin Hospital of Southeast University Medical School, Jiangyin 214400, China 2 Department of Minimally Invasive Spine Center, Renji Orthopedics Hospital, Shantou 515065, China 3 Department of Orthopedics, Jiangyin No 3 People’s Hospital, Jiangyin 214433, China 4 Department of Orthopedics, The Affiliated Jiangyin Hospital of Southeast University Medical School, Jiangyin 214400, China 5 Department of Orthopedics, The Affiliated Changzhou No 2 Hospital of Nanjing Medical University, Changzhou 213003, China * These authors have contributed equally to this work # Co-first author Correspondence to: Luming Nong, email: luming_nong@sina.com Kewei Ren, email: rkwmedicine@tom.com Keywords: TNFAIP3, single nucleotide polymorphism, rheumatoid arthritis, meta-analysis Received: December 30, 2016 Accepted: January 25, 2017 Published: February 10, 2017 ABSTRACT Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic destructive inflammation in synovial joints. To date, many studies explored the associations between tumor necrosis factor alpha inducible protein 3 (TNFAIP3) gene rs6920220, rs2230926, and rs5029937 polymorphisms and the risk of rheumatoid arthritis (RA), but with contradictory results. We therefore conducted a comprehensive meta-analysis to address the associations. We searched in the databases of PubMed and Embase. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by the Stata 11.0 software. A total of 21 case-control studies for these three single nucleotide polymorphisms (SNPs) were included in this meta-analysis. Meta-analysis indicated that TNFAIP3 gene rs6920220, rs2230926, and rs5029937 polymorphisms were associated with the increased risk of RA. Stratification analysis of ethnicity found that rs6920220 and rs5029937 polymorphisms increased the risk of RA among Caucasians, while rs2230926 polymorphism increased the risk of RA among Asians. In summary, this meta-analysis confirms that TNFAIP3 gene polymorphisms may play important roles in the pathogenesis of RA.

To identify a key protein that binds monomeric G protein RhoA and activates the RhoA/Rho kinase/MYPT1 axis in vascular smooth muscle cells (VSMCs) upon angiotensin II (Ang II) stimulation.Primary cultured VSMCs from Sprague-Dawley rats were transfected with siRNAs against leukemia-associated RhoGEF (LARG), and then treated with Ang II, losartan, PD123319, or Val(5)-Ang II. The target mRNA and protein levels were determined using qPCR and Western blot analysis, respectively. Rat aortic rings were isolated, and the isometric contraction was measured with a force transducer and recorder.Stimulation with Ang II (0.1 μmol/L) for 0.5 h significantly increased the level of LARG mRNA in VSMCs. At 3, 6, and 9 h after the treatment with Ang II (0.1 μmol/L) plus AT(2) antagonist PD123319 (1 μmol/L) or with AT(1) agonist Val(5)-Ang II (1 μmol/L), the LARG protein, RhoA activity, and phosphorylation level of myosin phosphatase target subunit 1 (MYPT1) in VSMCs were significantly increased. Knockdown of LARG with siRNA reduced these effects caused by AT(1) receptor activation. In rat aortic rings pretreated with LARG siRNA, Ang II-induced contraction was diminished.Ang II upregulates LARG gene expression and activates the LARG/RhoA/MYPT1 axis via AT(1), thereby maintaining vascular tone.

Exposure of newborns to the maternal vaginal microbiota is interrupted with cesarean birthing. Babies delivered by cesarean section (C-section) acquire a microbiota that differs from that of vaginally delivered infants, and C-section delivery has been associated with increased risk for immune and metabolic disorders. Here we conducted a pilot study in which infants delivered by C-section were exposed to maternal vaginal fluids at birth. Similarly to vaginally delivered babies, the gut, oral and skin bacterial communities of these newborns during the first 30 d of life was enriched in vaginal bacteria--which were underrepresented in unexposed C-section-delivered infants--and the microbiome similarity to those of vaginally delivered infants was greater in oral and skin samples than in anal samples. Although the long-term health consequences of restoring the microbiota of C-section-delivered infants remain unclear, our results demonstrate that vaginal microbes can be partially restored at birth in C-section-delivered babies.

The mitogenic effects of periodic mechanical stress on nucleus pulpous cells have been studied extensively but the mechanisms whereby nucleus pulpous cells sense and respond to mechanical stimulation remain a matter of debate. We explored this question by performing cell culture experiments in our self-developed periodic stress field and perfusion culture system. Under periodic mechanical stress, rat nucleus pulpous cell proliferation was significantly increased (p

Methadone is a μ-opioid receptor agonist that is used for the treatment of chronic pain and opioid dependence. Clinically it is used in the racemic form; (R)-methadone has higher affinity at μ and δ opioid receptors [1] and greater analgesic activity [2]. Pharmacokinetic studies in humans have also found differences between the two isomers, with (R)-methadone having significantly longer elimination half-life, greater volume of distribution, and lower protein binding [3–5]. Methadone is subject to numerous pharmacokinetic drug interactions; these are thought to primarily occur at cytochrome P450 (CYP) sites of methadone metabolism [6–9]. The stereo-selectivity of the drug interaction may be of importance; interactions that increase or decrease (R)-methadone may lead to toxicity (i.e. respiratory depression) or withdrawal, respectively; interactions that increase (S)-methadone may increase the incidence of prolonged QT intervals [10]. Since the initial studies on CYP involvement in the in vitro metabolism of methadone [11,12], five other laboratories have investigated comparative involvement of different CYPs in methadone metabolism [13–19]. Most of these in vitro studies focused on the predominant metabolic pathway, N-demethylation followed by spontaneous cyclization to form 2-ethyl-1,5-dimethyl-3,3,-diphenylpyrrolidine (EDDP). The involvement of CYP3A4 was noted in all of these studies; and all that studied stereo-selectivity noted none for 3A4-mediated methadone N-demethylation [13,15,16,18,19]. Two laboratories also studied methadone depletion, with contrasting results; Wang and De Vane [15] found CYP3A4 depleted (R)-methadone at a much higher rate, Gerber et al. [16] found no difference in the depletion of (R)- and (S)-methadone by CYP3A4. As tools to study CYP2B6 became available, its important role in methadone N-demethylation was also noted [16,17], and studies on the stereo-selectivity of the reaction consistently showed higher rates of (S)-methadone metabolism by CYP2B6 [16,18,19]. Other CYPs have also been reported to be involved in methadone N-demethylation including: 2D6 [11,15,16], 2C8 [15], 2C9 [11–13,16] and 2C19 [11,13,16,18,19]. Among these CYPs, 2C19 (R > S) [16,18,19], 2C8 (R > S) and 2D6 (S > R) [15] displayed stereo-selectivity. These studies leave some questions about the involvement of CYPs besides 3A4 and 2B6 in methadone N-demethylation and the stereo-selectivity of methadone depletion. Only one study on CYP2B6 stereo-selective methadone N-demethylation provided detailed evidence beyond metabolism in recombinant expressed CYP [19]. The current investigation has both confirmed and expanded on these findings. We have tested the stereo-selective metabolism of methadone in a large panel of CYP Supersomes with scaling of Supersome activities, correlation analysis and use of a number of specific chemical inhibitors and monoclonal antibodies. The relative contribution of 3A4 and 2B6, as well as other CYPs, to in vitro methadone metabolism and stereo-selective metabolism was determined.

This study was conducted to examine the pharmacokinetic interactions between buprenorphine/naloxone (BUP/NLX) and lopinavir/ritonavir (LPV/r) in HIV-seronegative subjects chronically maintained on BUP/NLX.This study was an open labeled pharmacokinetic study in twelve HIV-seronegative subjects stabilized on at least 3 weeks of BUP/NLX therapy. Subjects sequentially underwent baseline and steady-state pharmacokinetic evaluation of once-daily LPV/r (800/200 mg).Compared to baseline values, BUP AUC0-24h (46.8 vs. 46.2 ng*hr/mL) and Cmax (6.54 vs. 5.88 ng/mL) did not differ significantly after achieving steady-state LPV/r. Similar analyses of norBUP, the primary metabolite of BUP, demonstrated no significant difference in norBUP AUC0-24 hours (73.7 vs. 52.7 ng x h/mL); however, Cmax (5.29 vs. 3.11 ng/mL) levels were statistically different (P0.05) after LPV/r administration. Naloxone concentrations were similarly unchanged for AUC0-24 hours (0.421 vs. 0.374 ng x hr/mL) and Cmax (0.186 vs. 0.186 ng/mL). Using standardized measures, no objective opioid withdrawal was observed. The AUC0-24 hours and Cmin of LPV in this study did not significantly differ from historical controls (159.6 vs. 171.3 microg x hr/mL) and (2.3 vs. 1.3 microg/mL).The addition of LPV/r to stabilized patients receiving BUP/NLX did not affect buprenorphine pharmacokinetics but did increase the clearance of norbuprenorphine. Pharmacodynamic responses indicate that the altered norbuprenorphine clearance did not lead to opioid withdrawal. Buprenorphine/naloxone and LPV/r can be safely coadministered without need for dosage modification.

Cocaine withdrawal symptoms are thought to play a role in relapse; studies characterizing the symptomatology have yielded mixed findings. This study sought to examine the pharmacodynamic/pharmacokinetic profile of repeated high dose exposure to oral cocaine and characterize acute and protracted withdrawal in cocaine abusers. This study employed a repeated-dosing, single-blind design in which subjects (n = 9), resided for 40 days on a closed ward. They were maintained for two 4-day cocaine exposure periods (Days 1-4 & Days 9-12, cocaine 175 mg, p.o.; 5 hourly doses; 875 mg/day) separated by a 4-day matched placebo exposure period (Days 5-8). After these 12 days, an additional period of 28 days of placebo maintenance followed (Days 13-40). Test sessions were conducted during each phase; measures of mood, drug effects, sleep, pharmacokinetics, and prolactin were collected throughout the study. The dosing regimen produced cocaine plasma concentrations (Cmax of 680 ng/mL) two to threefold higher than typically seen in acute dose studies. Prototypic psychostimulant effects, including subjective ratings of euphoric effects (liking, high, good effects) and significant cardiopressor effects, were sustained during the active dosing periods, corresponding to the rise and fall of plasma cocaine. Withdrawal-like symptoms (i.e., disruptions of sleep, increased ratings of anxiety, irritability, crashing) were observed within 24-hr after cessation of dosing. Cocaine reduced prolactin acutely, but no sustained alterations were observed for this measure or for other signs or symptoms during the 28-day abstinence period. These findings indicate that exposure to controlled high doses of cocaine produces modest symptoms consistent with cocaine withdrawal within hours of cessation of dosing but provide no evidence of symptoms persisting beyond 24 hours.

A liquid chromatography-electrospray ionization-tandem mass spectrometry method has been developed and validated to detect (R)- and (S)-methadone and (R)- and (S)-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in human plasma with cross-validation to urine and liver microsomes. Use of deuterated internal standards and liquid-liquid extraction coupled with chiral separation provided baseline separation with a lower limit of quantitation (LLOQ) of 2.5 ng/mL. The LLOQ was established from comparison of signal in blanks from six different sources per matrix with the same sources fortified at the LLOQ (none exceeded 19% of LLOQ) and precision and accuracy at the LLOQ determined in the same six sources per matrix. The assay was precise (% coefficients of variation within 13.8%) and accurate (% targets within 15%) in all three matrices. No interference was seen from addition of other psychoactive drugs. Stability was determined in plasma (24 h at room temperature, 321 days at -20 degrees C, 3 freeze-thaw cycles); processed plasma samples (5 days at -20 degrees C, 12 days on autosampler); urine (24 h at room temperature); and stock solutions (20 h at room temperature, 61 days at -20 degrees C). Applications of varying degree are presented for each matrix. Plasma from five subjects maintained on 100 mg oral methadone per day permitted comparison of the pharmacokinetics of the enantiomers. The t(1/2) of (R)-methadone was significantly longer than for (S)-methadone, and (S)-methadone was more tightly protein bound. The C(max), AUC, C(min), and % protein bound of (S)-EDDP were significantly greater than (R)-EDDP, while the t(1/2) of (R)-EDDP was significantly greater than (S)-EDDP. In spot urines, (R)- was higher than (S)-methadone, and (S)- was generally higher than (R)-EDDP. (R)- and (S)-EDDP production was detected after incubation of therapeutic concentrations of racemic methadone with human liver microsomes, and (S)-EDDP production was twofold greater than (R)-EDDP in three human placental microsomes incubated with racemic methadone.

Female rats display a more robust behavioral response to acute cocaine administration than do male rats. However, a clear understanding of the biological mechanisms underlying these differences remains elusive. The present study investigated whether sexual dimorphisms in cocaine-induced motor behavior might be based on monoaminergic levels and/or cocaine pharmacokinetics. An acute injection of cocaine (5, 15, 20 or 30 mg/kg) or saline was administered to male and female rats, and behavioral activity was monitored for 3 h. Following acute cocaine or saline administration motor behavior varied according to dose and sex; overall, female rats displayed greater rearing counts and stereotypic scores, greater total locomotor counts at 15, 20, and 30 mg/kg of cocaine, and greater ambulatory counts at 20 and 30 mg/kg of cocaine than did male rats. Neurochemical determinations in post-mortem tissue showed that both male and female rats had increases in total dopamine (DA) in the caudate putamen (CPu) 15 min following cocaine administration. Additionally, male rats had a decrease in dihydroxyphenylacetic acid (DOPAC)/DA turnover. Female rats showed significant reductions in total levels of DA, DOPAC, HVA, serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA), and DOPAC/DA turnover in the nucleus accumbens (NAc). Male rats displayed a reduction only in DOPAC/DA turnover and increases in 5-HT in the NAc following cocaine administration. Furthermore, sex differences in cocaine metabolism were observed where females had greater brain/blood levels of norcocaine and ecgonine methyl ester while male rats had higher blood levels of benzoylecgonine. These results suggest that sex differences in the behavioral responses to cocaine administration could be explained in part by intrinsic differences in both monoaminergic levels and metabolic processes.

In order to support studies on various medication protocols for the treatment of cocaine abuse, an accurate, precise, and sensitive (2.5 to 750 ng/mL) liquid chromatography-tandem mass spectrometry assay was developed to determine cocaine and benzoylecgonine in human plasma. Cocaine-d3 and benzoylecgonine-d3 were added as internal standards and samples were subjected to solid-phase extraction. Cocaine recovery was 94.4% and benzoylecgonine was 80.3% at 2.5 ng/mL. The selected reaction monitoring of parent ions at m/z 304 and 290 resulted in strong fragments at m/z 182 and 168 for cocaine and benzoylecgonine, respectively. The method was fully validated. The mean measured concentration at the 2.5 ng/mL, the lower limit of quantitation, was within 10.8% of the target and the precision determined at the low (5 ng/mL), medium (50 ng/mL), and high (650 ng/mL) quality controls ranged from 0.9 to 6.2 %CV. Cocaine and benzoylecgonine concentrations in plasma treated with 1% NaF showed changes of less than 10% when maintained at room temperature for up to 7 h and no significant changes when subjected to three freeze-thaw cycles. The concentrations of cocaine and benzoylecgonine remained stable in plasma samples stored at -20 degrees C for up to 11 months. Methanolic stock solutions of both analytes are stable, staying within 2% of the freshly prepared stock solutions, when stored at -20 degrees C for up to 235 days. Both extracted analytes reconstituted in methanolic solutions are stable for up to seven days whether stored at -20 degrees C or at room temperature on the autosampler. The method is rugged, rapid, and robust and has been applied to the batch analysis of more than 700 samples during pharmacokinetic profiling to assess potential interactions between intravenous (i.v.) cocaine challenge and treament medications. Results from three of these subjects receiving 40 mg (i.v.) cocaine demonstrate the utility of the method.

Oxidative modifications of low-density lipoproteins (LDL) may contribute to the pathogenesis of atherosclerosis. Although the oxidation products of the lipid components of LDL have been studied extensively, less is known about the oxidation products of the apoprotein, apolipoprotein B-100. To identify the specific oxidative modifications, we oxidized LDL in the presence of Cu(2+), treated with DNPH, precipitated and delipidated the protein, digested the protein with trypsin, and analyzed the peptides by high-performance liquid chromatography. We isolated nine peptides that exhibited measurable absorbance at 365 nm, which is characteristic of hydrazones derived from DNPH and is not observed in peptides derived from unoxidized LDL. Unexpectedly, we obtained the same peptides with absorbance at 365 nm in Cu(2+)-oxidized LDL not treated with DNPH. N-terminal sequence analyses and mass spectrometry indicated that the peptides isolated from the Cu(2+)-oxidized LDL all contained kynurenine residues in place of Trp residues found in the native apoprotein. The product profile we observed in Cu(2+)-oxidized LDL was remarkably different from the profiles observed in LDL oxidized by HOCl or myeloperoxidase in vitro, and the preferential oxidation of Trp to kynurenine in Cu(2+)-catalyzed oxidation of LDL contrasts with the products observed following oxidation of LDL with HOCl or myeloperoxidase. Our studies to date support the working hypothesis that the specific products of protein oxidation are sufficiently distinct to be developed as biomarkers of proposed mechanisms of oxidation of LDL and biological molecules in other toxicities and diseases.

Therapeutic doses of Ritalin, a racemic mixture of d- and l-threo-methyphenidate, result in low plasma concentrations of methylphenidate. In order to assess the safety and efficacy of methylphenidate, a sensitive analytical method is needed. A gas chromatography-negative ion chemical ionization mass spectrometry (GC-NCI-MS) assay capable of measuring both d- and l-enantiomers in human plasma was developed and validated to support clinical studies involving administration of d,l-methylphenidate. d,l-Methylphenidate-d3 is added to 1-mL plasma samples. The plasma samples are made basic, mixed with isopropanol and extracted with hexane. The hexane extracts are then back-extracted into 0.1 N HCl. The acidified aqueous extract is made basic, cooled to ice temperature, and the methylphenidate derivatized with heptafluorobutyryl-l-prolyl chloride. The two diastereomeric derivatives are then extracted into hexane. The hexane extract is evaporated to dryness, reconstituted in ethyl acetate, and analyzed by GC-NCI-MS. This method can accurately (+/- 5% target) and precisely (< 11.1% coefficient of variation) quantitate enantiomers of threo-methylphenidate in human plasma and in the whole blood at concentrations ranging from 0.75 to 100 ng/mL. Plasma samples are stable for up to five freeze-thaw cycles when the duration of each cycle did not exceed 0.5 h. The drug degraded gradually when plasma samples were left at room temperature; a 6% loss at 3 h progressed to 17% at 12 h and to 35% at 24 h. Therefore, it is important that extraction of plasma samples begins within 0.5 h after samples are removed from the freezer. Whole blood stability results show that concentrations of methylphenidate in whole blood, with or without NaF, stored for up to 6 h at room temperature did not deviate from the target concentration by more than 13%. The derivatized methylphenidate in extract is stable at 4 degrees C for up to 10 days.