Stroke is a significant public health issue, and research has consistently focused on studying the mechanisms of injury and identifying new targets. As a CDK5 activator, p39 plays a crucial role in various diseases. In this article, we will explore the role and mechanism of p39 in cerebral ischemic injury. We measured the level of p39 using western blot and QPCR at various time points following cerebral ischemia-reperfusion (I/R) injury. The results indicated a significant reduction in the level of p39. TTC staining and behavioral results indicate that the knockout of p39 (p39KO) provides neuroprotection in the short-term. Interestingly, the behavioral dysfunction in p39KO mice was exacerbated after the repair phase of I/R. Further study revealed that this deterioration may be due to demyelination induced by elevated p35 levels. In summary, our study offers profound insights into the significance of p39 in both the acute and repair stages of ischemic injury recovery and a theoretical foundation for future therapeutic drug exploration.

Post-stroke depression (PSD) is a common and serious complication that is affecting one thirds of stroke patients which leaves them with a poor quality of life, high mortality rate, high recurrent rate, and slow recovery. Recent studies showed that serum interleukin-18 (IL-18) level is a biomarker for patients with PSD. However, the role of IL-18 in the pathology of PSD is still unclear. In this study, we demonstrated that the IL-18 level in the ischemic brain significantly increased in mice with depression-like behaviors that were caused by the combined use of chronic spatial restraint stress and middle cerebral artery occlusion. Interestingly, IL-18 expression was mainly found in neurons at early phase and in microglia at a later phase. Injection of the exogenous IL-18 into the amygdala, but not the hippocampus or the striatum caused severe depression-like behaviors. On the contrary, the blockage of endogenous IL-18 by IL-18 binding protein, a specific antagonist of IL-18, repressed depressive phenotypes in SIR mice. IL-18 KO mice exhibited the resistance to spatial restraint stress and cerebral ischemia injury. Finally, we found that IL-18 mediated depressive behaviors by the interaction of IL-18 receptor and NKCC1, a sodium-potassium chloride co-transporter that is related to GABAergic inhibition. Administration of NKCC1 antagonist bumetanide exerted a therapeutic effect on the in IL-18-induced depressive mice. In conclusion, we demonstrated that increased IL-18 in the brain causes depression-like behaviors by promoting the IL-18 receptor/NKCC1 signaling pathway. Targeting IL-18 and its downstream pathway is a promising strategy for the prevention and treatment of PSD.

Diabetes is a complex metabolic syndrome that is characterized by prolonged high blood glucose levels and frequently associated with life-threatening complications1,2. Epidemiological studies have suggested that diabetes is also linked to an increased risk of cancer3–5. High glucose levels may be a prevailing factor that contributes to the link between diabetes and cancer, but little is known about the molecular basis of this link and how the high glucose state may drive genetic and/or epigenetic alterations that result in a cancer phenotype. Here we show that hyperglycaemic conditions have an adverse effect on the DNA 5-hydroxymethylome. We identify the tumour suppressor TET2 as a substrate of the AMP-activated kinase (AMPK), which phosphorylates TET2 at serine 99, thereby stabilizing the tumour suppressor. Increased glucose levels impede AMPK-mediated phosphorylation at serine 99, which results in the destabilization of TET2 followed by dysregulation of both 5-hydroxymethylcytosine (5hmC) and the tumour suppressive function of TET2 in vitro and in vivo. Treatment with the anti-diabetic drug metformin protects AMPK-mediated phosphorylation of serine 99, thereby increasing TET2 stability and 5hmC levels. These findings define a novel ‘phospho-switch’ that regulates TET2 stability and a regulatory pathway that links glucose and AMPK to TET2 and 5hmC, which connects diabetes to cancer. Our data also unravel an epigenetic pathway by which metformin mediates tumour suppression. Thus, this study presents a new model for how a pernicious environment can directly reprogram the epigenome towards an oncogenic state, offering a potential strategy for cancer prevention and treatment. Modulation of DNA 5-hydroxymethylcytosine by glucose reveals an AMPK–TET2–5hmC axis that links diabetes to cancer.

The surface characteristic of the heat transfer channel of micro heat exchangers has a significant effect on the flow boiling performance of fluid.Solid surface energy is often used to describe the surface characteristics of the heat transfer channel, which can provide a theoretical basis for the research of the flow boiling performance of the fluid in micro-channels.In the investigation, the contact angles of deionized water, ethylene glycol and formamide with the left, right and bottom surfaces of the heat transfer channel were measured respectively, and the solid surface energy of the heat transfer channel was calculated on the basis of Young`s theory.Then, the forming mechanism of the contact angle difference between the inside and outside surfaces of the micro-channels was analyzed through the Wenzel model of droplet.The results show that the solid surface energy of the left, right and bottom surfaces of the heat transfer channel are respectively 5.2, 5.6 and 8.2MJ/m2, and the surface roughness difference causes the inside surface features to be different.

5-hydroxymethylcytosine (5hmC) is considered as a novel DNA modification and plays an important role in cancer, stem cells, and developmental diseases. In this study, we demonstrated the existence of RNA 5hmC modification in mouse brain RNA by using a dot blot analysis method. Our data indicated that 5hmC modification in RNA samples was less than that in DNA samples. Further, we optimized the conditions for 5hmC detection in RNA samples such as DNase treatment, denature reagents, denature time, sample air-dry time, and the cross-linking time between RNA and membrane. Our results demonstrated that DNase treatment and denature reagents were two important factors that affected the 5hmC detection in RNA samples. By using the optimal conditions for RNA 5hmC detection, we found that the brainstem, the hippocampus, and the cerebellum had high levels of 5hmC modification and 5mC modification in RNA. Finally, we found that RNA 5hmC modification decreased in MPTP-induced Parkinson's disease model in mice. These suggest that 5hmC modification in RNA might play an important regulative role on protein or microRNA expression in these brain tissues. Because DNA 5hmC modification plays an important role in neural differentiation and development as well as neurological diseases, the significance of 5hmC modification in RNA in different neurological diseases needs further investigation. In summary, our study demonstrated for the first time the abundance of 5hmC modification in brain RNA by using a dot blot analysis method and proved that dot blot analysis is a useful method for 5hmC detection in RNA samples.

The identification of the regulatory targets of transcription factors is central to our understanding of how transcription factors fulfill their many key roles in development and homeostasis. DNA-binding sites have been uncovered for many transcription factors through a number of experimental approaches, but it has proven difficult to use this binding site information to reliably predict transcription factor target genes in genomic sequence space. Using the nematode Caenorhabditis elegans and other related nematode species as a starting point, we describe here a bioinformatic pipeline that identifies potential transcription factor target genes from genomic sequences. Among the key features of this pipeline is the use of sequence conservation of transcription-factor-binding sites in related species. Rather than using aligned genomic DNA sequences from the genomes of multiple species as a starting point, TargetOrtho scans related genome sequences independently for matches to user-provided transcription-factor-binding motifs, assigns motif matches to adjacent genes, and then determines whether orthologous genes in different species also contain motif matches. We validate TargetOrtho by identifying previously characterized targets of three different types of transcription factors in C. elegans, and we use TargetOrtho to identify novel target genes of the Collier/Olf/EBF transcription factor UNC-3 in C. elegans ventral nerve cord motor neurons. We have also implemented the use of TargetOrtho in Drosophila melanogaster using conservation among five species in the D. melanogaster species subgroup for target gene discovery.

Introduction Hypoglycemia triggers the secretion of counter-regulatory hormones such as cortisol, growth hormone(GH), and glucagon, all of which are protective mechanisms to restore euglycemia. Congenital hyperinsulinism (CHI) is a group of genetic diseases with abnormal insulin secretion. The clinical phenotype of CHI is characterized by severe hypoglycemia. It has been suggested that CHI patients have abnormal glucagon secretion during hypoglycemia, but the data is limited. Methods In this study, twogroups of patients with CHI (n=79) and ketosis hypoglycemia (KH, n=25) were reviewed retrospectively. All patients were admitted to the Department of Endocrinology at the Beijing Children's Hospital. Blood was collected during hypoglycemia, serum glucagon was measured by radioimmunoassay, and insulin was measured by electrochemiluminescence immunoassay. Results CHI patients had more severe hypoglycemia than patients with KH. The mean glucose levels in CHI patients were 1.81±0.54 mmol/L, and the levels were 2.13±0.50 mmol/L(p<0.050.01) in KH patients. This severe hypoglycemia in CHI was the result of abnormal insulin secretion. Insulin levels of CHI patients were 9.20(5.71,16.95)(median (Interquartile)) IU/ml, whereas KH patients had very low insulin levels( 0.35 range0.20,0.10)IU/ml. However, glucagon secretion in response to hypoglycemia in CHI patients was not low: the median glucagon value in CHI patients was 222(range 165,355)pg/ml compared to153(range 116,215)pg/ml in KH patients (p< 0.05). We also compared the hormones secretion when blood glucose levels of the two groups had no significant difference, the results showed that Insulin levels of CHI patients were(13.05±12.30) IU/ml, but KH patients still had low insulin levels(0.58±0.47) IU/ml.Whereas glucagon levels of CHI patients were 216(174,262)pg/ml, which was higher than that of KH patients (142 range 114,197,p< 0.05).For CHI patients ,cortisol secretion was lower than that in KH patients, but growth hormone levels had the reverse result. Besides, we choosed three different glucose levels in order to discover the law of glucagon secretion, but the result suggested that glucagon had no obvious secretion peak in CHI patients.

Objective: Single nucleotide polymorphisms (SNPs) in apolipoprotein A5 (APOA5) gene are associated with triglyceride (TG) levels. However, the minor allele frequencies and linkage disequilibriums (LDs) of the SNPs in addition to their effects on TG levels vary greatly between Caucasians and East Asians. The distributions of the SNPs/haplotypes and their associations with TG levels in Uyghur population, an admixture population of Caucasians and East Asians, have not been reported to date. Here, we performed a cross-sectional study to address these.Methods: Genotyping of four SNPs in APOA5 (rs662799, rs3135506, rs2075291, and rs2266788) was performed in 1174 unrelated Uyghur subjects. SNP/haplotype and TG association analyses were conducted.Results: The frequencies of the SNPs in Uyghurs were in between those in Caucasians and East Asians. The LD between rs662799 and rs2266788 in Uyghurs was stronger than that in East Asians but weaker than that in Caucasians, and the four SNPs resulted in four haplotypes (TGGT, CGGC, TCGT, and CGTT arranged in the order of rs662799, rs3135506, rs2075291, and rs2266788) representing 99.2% of the population. All the four SNPs were significantly associated with TG levels. Compared with non-carriers, carriers of rs662799-C, rs3135506-C, rs2075291-T, and rs2266788-C alleles had 16.0%, 15.1%, 17.1%, and 12.4% higher TG levels, respectively. When haplotype TGGT was defined as the reference, the haplotypes CGGC, TCGT, and CGTT resulted in 16.1%, 19.0%, and 19.8% higher TG levels, respectively. The proportions of variance in TG explained by APOA5 locus were 2.5%, 0.3%, 0.4%, and 1.9% for single SNP rs662799, rs3135506, rs2075291, and rs2266788, respectively, and 3.0% for the haplotypes constructed by them.Conclusions: The association profiles between the SNPs and haplotypes at APOA5 locus and TG levels in this admixture population differed from those in Caucasians and East Asians. The functions of these SNPs and haplotypes need to be elucidated comprehensively.

BACKGROUND Patients with advanced hematologic malignancies remain at risk for relapse following reduced-intensity conditioning (RIC) allogeneic hematopoietic stem cell transplantation (allo-HSCT). We conducted a prospective clinical trial to test whether vaccination with whole leukemia cells early after transplantation facilitates the expansion of leukemia-reactive T cells and thereby enhances antitumor immunity. METHODS We enrolled 22 patients with advanced chronic lymphocytic leukemia (CLL), 18 of whom received up to 6 vaccines initiated between days 30 and 45 after transplantation. Each vaccine consisted of irradiated autologous tumor cells admixed with GM-CSF-secreting bystander cells. Serial patient PBMC samples following transplantation were collected, and the impact of vaccination on T cell activity was evaluated. RESULTS At a median follow-up of 2.9 (range, 1-4) years, the estimated 2-year progression-free and overall survival rates of vaccinated subjects were 82% (95% CI, 54%-94%) and 88% (95% CI, 59%-97%), respectively. Although vaccination only had a modest impact on recovering T cell numbers, CD8+ T cells from vaccinated patients consistently reacted against autologous tumor, but not alloantigen-bearing recipient cells with increased secretion of the effector cytokine IFN-γ, unlike T cells from nonvaccinated CLL patients undergoing allo-HSCT. Further analysis confirmed that 17% (range, 13%-33%) of CD8+ T cell clones isolated from 4 vaccinated patients by limiting dilution of bulk tumor-reactive T cells solely reacted against CLL-associated antigens. CONCLUSION Our studies suggest that autologous tumor cell vaccination is an effective strategy to advance long-term leukemia control following allo-HSCT. TRIAL REGISTRATION Clinicaltrials.gov NCT00442130. FUNDING NCI (5R21CA115043-2), NHLBI (5R01HL103532-03), and Leukemia and Lymphoma Society Translational Research Program.

Abstract 1892 This icon denotes a clinically relevant abstract

Whole tumor cell-based vaccines administered in the early post-transplant (HSCT) setting are a promising approach to enhance graft-versus-leukemia responses. Our previous studies described the detection of increased CD8+ T cells with specificity for autologous chronic lymphocytic leukemia (CLL) cells in association with attainment of molecular remission within 6 months post-HSCT that developed following early-post transplant tumor vaccination (initiating at day 30-45). The series of vaccines were comprised of irradiated autologous tumor admixed with irradiated GM-CSF secreting bystander cells. It is unknown how this early post-transplant intervention shapes the reconstituting donor T cell repertoire. One approach to characterize this is through examining the kinetics of recovery of T cell receptor (TCR) beta-chain diversity using recently developed methodologies that apply deep sequencing techniques for the analysis of the highly variable complementarity-determining region 3 (CDR3) of the TCR. We therefore isolated naïve and memory T cells from the CD8 and CD4 compartments from 12 patients (6 with vaccine, 6 without) at time points informative for thymic-independent (early; 4 month post-transplant) and thymic-dependent (late; 1 year post-transplant) T cell immune recovery. The isolated cell subsets directly underwent PCR amplification of the CDR3 region to generate a template library that was then sequenced using the Illumina GA2 system. We obtained a median of 24,191 productive reads per sample (range 0-8,900,000). To evaluate the TCR diversity, we calculated the normalized probability distribution ('entropy') of each T cell compartment. Overall, we observed that the 6 vaccinated subjects demonstrated a higher normalized entropy at day 120 (p = 0.008) and more unique CDR3 sequences (one-tailed t-test with unequal variance: p = 0.03) compared to non-vaccinated subjects. In particular, CD8+ memory and naïve cells exhibited greater TCR diversity at days 120 and 365 in vaccinated than in non-vaccinated individuals. These results suggest that early post-transplant vaccination exerts an active role in shaping T cell reconstitution. Ongoing analyses focus on the integration of TCR repertoire data with the assessment of minimal residue disease, immunophenotyping and TCR excision circle (TREC) analysis. The results gained within this study provide us with critical information as we formulate the next generation of post-transplant cancer vaccines.

6564 Background: CLL patients (pts) with del(17p13) [p53 gene] have a poor outcome and do not respond to most conventional therapies. Flavopiridol induces apoptosis in CLL cells in a p53-independent manner, but phase I/II studies using a 24–72-hr CIVI schedule showed no activity. Increased drug binding to human plasma proteins prompted pharmacokinetic modeling indicating an optimal dosing schedule of 30-min IV bolus (IVB) followed by 4-hr CIVI. Methods: We report preliminary results of an ongoing phase I study using this schedule. Results: Nine pts (median age 60, range 44–73) with fludarabine-refractory CLL have been enrolled (median prior therapies 7, range 2–13). Five pts had del(17p13); 6 pts were stage III/IV. Pts received 50% of the dose by 30-min IVB and 50% by 4-hr CIVI weekly for 4 doses on a 6-week cycle. Six pts in cohort 1 received 60 mg/m2/dose; 3 pts in cohort 2 received 80 mg/m2/dose. Grade 3–4 toxicity was seen (neutropenia 6, thrombocytopenia 1, dyspnea 1, edema 1, diarrhea 1), but most toxicity was grade 1–2 (fatigue 7, nausea 6, anorexia 6). Tumor lysis was seen in 3 pts in cohort 2. Two pts had tumor lysis to the first dose. One pt completed cycle 1 without further tumor lysis. The other pt died of overwhelming tumor lysis, with predominant hyperkalemia not responding to forced alkaline hydration and IV insulin, and cardiac arrhythmia. A third pt had tumor lysis to dose 1 of cycle 2 and responded to medical management. This pt also developed hypotension that resolved with steroids, suggesting a cytokine release syndrome. Pts completed a median of 1 cycle (range 0–2). Treatment was stopped due to disease progression (3), infection (1), tumor lysis (2), and study interruption to institute guidelines to prevent further tumor lysis (3). Two pts in cohort 1 experienced an NCI 96 partial response lasting 5+ months; 1 of these pts has del(17p13). Conclusions: Single agent flavopiridol given weekly by 30-min IVB followed by 4-hr CIVI is active in refractory CLL, resulting in clinical responses and tumor lysis. Further study of this pharmacokinetically modeled schedule is warranted. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Aventis Pharmaceuticals

Flavopiridol is a broad cyclin dependent kinase inhibitor that induces p53/IL4 independent apoptosis in CLL cells. Despite potent pre-clinical activity, phase I/II studies of both a 24 and 72-hour continuous IV (CI) schedule demonstrated no activity in CLL or other cancers. Discordant binding of flavopiridol to human plasma proteins as compared to fetal calf serum prompted us to perform pharmacokinetic modeling from the Aventis-sponsored CI studies. This suggested optimal dosing would be a 30-minute IV bolus followed by 4-hour infusion. We report a mature phase I dose escalation study of flavopiridol with this schedule. We enrolled 23 pts (median age 61, range 44–84, 8 female) previously treated for CLL (median prior therapies 3, range 2–13) . At study entry, 21 pts had no response to their last therapy, 9 had intermediate risk disease, and 14 were stage III/IV. Pts received 50% of the flavopiridol dose IV over 30 minutes, the remaining 50% followed over 4 hours. This was repeated weekly 4 times on a 6-week cycle. Six pts in cohort 1 received 60 mg/m2/dose with 1 dose limiting toxicity (DLT, neutropenic fever) and 3 pts in cohort 2 received 80 mg/m2/dose. The maximally tolerated dose was exceeded in cohort 2. Acute tumor lysis syndrome (TLS) following the first flavopiridol dose was the DLT. One pt developed TLS that was controlled with aggressive medical management. The 2nd pt with TLS died with hyperkalemia before dialysis could be initiated, and on autopsy had extensive apoptosis/necrosis of diffuse intra-abdominal lymphadenopathy. No additional pts were treated at this dose, but a 3rd pt previously without TLS at the 80 mg/m2/dose developed TLS on day 1,cycle 2 at the 60 mg/m2 dose. An inpatient management plan to prevent further life-threatening TLS was initiated. We enrolled 14 additional pts. Several pts developed transient tumor lysis upon initial treatment, with increased serum potassium, phosphate, and LDH, but only 1 pt required temporary dialysis. Other manageable toxicities observed included neutropenia, anemia, thrombocytopenia, fatigue, nausea, diarrhea, and anorexia. Twenty-two patients have been followed long enough for NCI 96 response assessment. Nine pts achieved a PR (41%); 7 pts remain in remission (3–11+ months), and 2 pts relapsed at 7 and 12 months, respectively. Of the 9 responding pts, 8 were fludarabine refractory or intolerant, 8 had bulky LN (> 5cm), and 8 had del(11q) [n=6] or del(17p) [n=3] abnormalities. Additionally, opportunistic infections have not been noted to date. Eight of 9 responding pts with enlarged LN had a 50% reduction with the 1st treatment, compared to 2 of 10 who did not ultimately achieve a PR. The AUC of flavopiridol did not increase proportionally with dose, but pharmacologic data support our hypothesis that the clinical activity and toxicity of flavopiridol may be directly related to the Cmax, AUC, and Css. In summary, single agent flavopiridol given with this novel, pharmacologically modeled schedule has significant clinical activity in pts with fludarabine-refractory, genetically high-risk CLL. Further study of flavopiridol in CLL and other B-cell diseases using this pharmacokinetically modeled schedule is warranted.

This paper is focused on modeling and analysis on the determination of the optimized L-ratio, or L/sub e//L/sub c/, for the length of evaporator section to the length of condenser section of cylindrical heat pipes, for a heat sink in the horizontal position. The independent variables (for an equipped heat pipe on which all fixtures are firmed) in the function of the total thermal resistance are L/sub e/ and L/sub c/. Given the fact that this function has a local minimum between two fixed end points, the minimum extremal of the thermal resistance function can be obtained by taking the derivative of this functions and setting it to zero. Since the total length of a heat pipe, L, is constant, therefore L/sub c/ may be replaced by L-L/sub e/, then the differential equation can be solved. In this paper, a formula to analyze the optimal L/sub e//L/sub c/ ratio is established. Table 2 to table 4 show analyzed results for diameters from 4 mm to 6 mm micro heat pipes in assemblies which are currently quite popular in applications.