The effects of single doses of intravenous ciprofloxacin and rifampin, multiple doses of rifampin, on glyburide exposure and effect on blood glucose levels in 9 healthy volunteers were investigated. The single intravenous dose of rifampin significantly increased the AUCs of glyburide and metabolite. Blood glucose levels dropped significantly in comparison to when glyburide was dosed alone. Multiple doses of rifampin induced liver enzymes leading to a marked decrease in glyburide exposure and in blood glucose measurements. When intravenous rifampin was given after multiple doses of rifampin, the inhibition of hepatic uptake transporters masked the induction effect, however, relative changes in AUC for glyburide and its hydroxyl metabolite were the same as that seen under non-induced conditions. The studies reported here demonstrate how measurements of both the parent drug and its primary metabolite are useful in unmasking simultaneous drug-drug induction and inhibition effects and characterizing enzymatic versus transporter mechanisms.
glyburide; rifampin; induction; inhibition; transporter-enzyme interplay
Activating mutations of Fms-like tyrosine kinase 3 (FLT3) constitute a major driver in the pathogenesis of acute myeloid leukaemia (AML). Hence, pharmacological inhibitors of FLT3 are of therapeutic interest for AML.
The effects of inhibition of FLT3 activity by a novel potent FLT3 inhibitor, BPR1J-097, were investigated using in vitro and in vivo assays.
The 50% inhibitory concentration (IC50) of BPR1J-097 required to inhibit FLT3 kinase activity ranged from 1 to 10 n, and the 50% growth inhibition concentrations (GC50s) were 21±7 and 46±14 n for MOLM-13 and MV4-11 cells, respectively. BPR1J-097 inhibited FLT3/signal transducer and activator of transcription 5 phosphorylation and triggered apoptosis in FLT3-driven AML cells. BPR1J-097 also showed favourable pharmacokinetic property and pronounced dose-dependent tumour growth inhibition and regression in FLT3-driven AML murine xenograft models.
These results indicate that BPR1J-097 is a novel small molecule FLT-3 inhibitor with promising in vivo anti-tumour activities and suggest that BPR1J-097 may be further developed in preclinical and clinical studies as therapeutics in AML treatments.
acute myeloid leukaemia; FLT3; FLT3-ITD; MOLM-13; MV4-11; kinase inhibitor
Neurons, sensory cells and endocrine cells secrete neurotransmitters and hormones to communicate with other cells and to coordinate organ and system function. Validation that a substance is used as an extracellular signaling molecule by a given cell requires a direct demonstration of its secretion. In this protocol we describe the use of biosensor cells to detect neurotransmitter release from endocrine cells in real-time. Chinese hamster ovary cells expressing the muscarinic acetylcholine (ACh) receptor M3 were used as ACh biosensors to record ACh release from human pancreatic islets. We show how ACh biosensors loaded with the Ca2+ indicator Fura-2 and pressed against isolated human pancreatic islets allow the detection of ACh release. The biosensor approach is simple; the Ca2+ signal generated in the biosensor cell reflects the presence (release) of a neurotransmitter. The technique is versatile because biosensor cells expressing a variety of receptors can be used in many applications. The protocol takes ~3 h.
Hereford is a major beef breed in the USA, and a sub-population, known as Line 1 (L1), was established in 1934 using two paternal half-sib bulls and 50 unrelated females. L1 has since been maintained as a closed population and selected for growth to 1 year of age. Objectives were to characterize the molecular genetic architecture of L1 (n = 240) by comparing a cross-section of L1 with the general US. Hereford population (AHA, n = 311), estimating effects of imposed selection within L1 based on allele frequencies at 50 K SNP loci, and examining loci-specific effects of heterozygosity on the selection criterion. Animals were genotyped using the Illumina BovineSNP50 Beadchip, and SNP were mapped to UMD3.0 assembly of the bovine genome sequence. Average linkage disequilibrium (LD), measured by square of Pearson correlation, of adjacent SNP was 0.36 and 0.16 in L1 and AHA, respectively. Difference in LD between L1 and AHA decreased as SNP spacing increased. Persistence of phase between L1 and AHA decreased from 0.45 to 0.14 as SNP spacing increased from 50 to 5,000 kb. Extended haplotype homozygosity was greater in L1 than in AHA for 95.6% of the SNP. Knowledge of selection applied to L1 facilitated a novel approach to QTL discovery. Minor allele frequency was (FDR < 0.01) affected by cumulative selection differential at 191 out of 25,901 SNP. With the FDR relaxed to 0.05, 13 regions on BTA2, 5, 6, 9, 11, 14, 15, 18, 23, and 26 are co-located with previously identified QTL for growth. After adjustment of postweaning gain phenotypes for fixed effects and direct additive genetic effects, regression of residuals on genome-wide heterozygosity was −235.3 ± 91.6 kg. However, no SNP-specific loci where heterozygotes were significantly superior to the average of homozygotes were revealed (FDR ≥ 0.17). In conclusion, genome-wide SNP genotypes clarified effects of selection and inbreeding within L1 and differences in genomic architecture between the population segment L1 and the AHA population.
linkage disequilibrium; persistence of phase; extended haplotype homozygosity; QTL; genetic load
Recently a new definition of surrogate endpoint, the ‘principal surrogate’, was proposed based on causal associations between treatment effects on the biomarker and on the clinical endpoint. Despite its appealing interpretation, limited research has been conducted to evaluate principal surrogates, and existing methods focus on risk models that consider a single biomarker. How to compare principal surrogate value of biomarkers or general risk models that consider multiple biomarkers remains an open research question. We propose to characterize a marker or risk model’s principal surrogate value based on the distribution of risk difference between interventions. In addition, we propose a novel summary measure (the standardized total gain) that can be used to compare markers and to assess the incremental value of a new marker. We develop a semiparametric estimated-likelihood method to estimate the joint surrogate value of multiple biomarkers. This method accommodates two-phase sampling of biomarkers and ismore widely applicable than existing nonparametric methods by incorporating continuous baseline covariates to predict the biomarker(s), and is more robust than existing parametric methods by leaving the error distribution of markers unspecified. The methodology is illustrated using a simulated example set and a real data set in the context of HIV vaccine trials.
Estimated likelihood; Predictiveness curve; Principal stratification; Semiparametric; Surrogate marker; Total gain
Pinellia pedatisecta agglutinin (PPA) is a specific mannose-binding plant lectin accumulated in the tuber of P. pedatisecta. In the work presented, the cytotoxicity of PPA to cancer cells was investigated through exogenous expression. A PPA gene was transduced into normal and cancer cell lines through plasmid vectors, and the effect of PPA expression was examined. Results showed that PPA translocated into the nucleus, colocalized with DNA and induced cell death. A mannose-binding motif and a V103-W130 region directed the nuclear translocation of PPA. Coprecipitation, mass spectrometry and western blotting analysis further indentified that PPA was associated with the methylosome, which contains methylosome protein 50 and protein arginine methyltransferase 5 (PRMT5). Knockdown of PRMT5 significantly inhibited the PPA-induced cell death, suggesting that PPA used the methylosome as a target. Furthermore, Ad.surp-PPA, an adenovirus vector in which the PPA gene was controlled by a survivin promoter (surp), selectively inhibited the proliferation of cancer cell lines. Taken together, the expression of PPA gene elicited significant cytotoxicity to cancer cells through targeting the methylosome and might be developed into a novel agent in cancer gene therapy.
methylosome; Pinellia pedatisecta agglutinin; MEP50; PRMT5; nuclear translocation
Angiogenic factors have an essential role in normal and pathologic angiogenesis. However, the clinical implication of angiogenic factor expression in myelodysplastic syndromes (MDS) remains unclear.
In this study, we sought to investigate the prognostic impact of the expression of genes encoding angiopoietin-1 (Ang-1), Ang-2, the receptor Tie2, vascular endothelial growth factor-A (VEGF-A) and VEGF-C in the bone marrow (BM) in 208 patients with newly diagnosed primary MDS.
BM Ang-1 expression was significantly higher in MDS patients, especially those with higher-risk subtypes, than in normal controls. With a median follow-up time of 32.9 months, the disease transformed to acute leukaemia more frequently in the patients bearing higher Ang-1 expression than in those with lower expression (31.5% vs 18.6%, P=0.023). The MDS patients with higher Ang-1 expression had shorter overall survival than those with lower expression (median 20.8±4.5 months vs 63.3±17.8 months, P<0.001). Multivariate analyses showed that higher Ang-1 expression was an independent unfavourable prognostic factor for overall survival. There was no impact of the expression of other angiogenic factors on survival.
BM Ang-1 expression may serve as a new biomarker to predict clinical outcome in MDS patients.
angiopoietin; vascular endothelial growth factor; myelodysplastic syndromes; prognosis
Death-associated protein kinase (DAPK) is a key player in several modes of neuronal death/injury and has been implicated in the late-onset Alzheimer's disease (AD). DAPK promotes cell death partly through its effect on regulating actin cytoskeletons. In this study, we report that DAPK inhibits microtubule (MT) assembly by activating MARK/PAR-1 family kinases MARK1/2, which destabilize MT by phosphorylating tau and related MAP2/4. DAPK death domain, but not catalytic activity, is responsible for this activation by binding to MARK1/2 spacer region, thereby disrupting an intramolecular interaction that inhibits MARK1/2. Accordingly, DAPK−/− mice brain displays a reduction of tau phosphorylation and DAPK enhances the effect of MARK2 on regulating polarized neurite outgrowth. Using a well-characterized Drosophila model of tauopathy, we show that DAPK exerts an effect in part through MARK Drosophila ortholog PAR-1 to induce rough eye and loss of photoreceptor neurons. Furthermore, DAPK enhances tau toxicity through a PAR-1 phosphorylation-dependent mechanism. Together, our study reveals a novel mechanism of MARK activation, uncovers DAPK functions in modulating MT assembly and neuronal differentiation, and provides a molecular link of DAPK to tau phosphorylation, an event associated with AD pathology.
DAPK; MARK/PAR-1; tau phosphorylation; neurodegeneration; microtubules
Mesenchymal stem cells (MSCs) have been employed successfully to treat various immune disorders in animal models and clinical settings. Our previous studies have shown that MSCs can become highly immunosuppressive upon stimulation by inflammatory cytokines, an effect exerted through the concerted action of chemokines and nitric oxide (NO). Here, we show that MSCs can also enhance immune responses. This immune-promoting effect occurred when proinflammatory cytokines were inadequate to elicit sufficient NO production. When inducible nitric oxide synthase (iNOS) production was inhibited or genetically ablated, MSCs strongly enhance T-cell proliferation in vitro and the delayed-type hypersensitivity response in vivo. Furthermore, iNOS−/− MSCs significantly inhibited melanoma growth. It is likely that in the absence of NO, chemokines act to promote immune responses. Indeed, in CCR5−/−CXCR3−/− mice, the immune-promoting effect of iNOS−/− MSCs is greatly diminished. Thus, NO acts as a switch in MSC-mediated immunomodulation. More importantly, the dual effect on immune reactions was also observed in human MSCs, in which indoleamine 2,3-dioxygenase (IDO) acts as a switch. This study provides novel information about the pathophysiological roles of MSCs.
mesenchymal stem cells; chemokine; immunomodulation; tissue repair
Head and neck squamous cell carcinoma (HNSCC) cells exposed to cisplatin (CIS) displayed a dramatic ATM-dependent phosphorylation of ΔNp63α that leads to the transcriptional regulation of downstream mRNAs. Here, we report that phospho (p)-ΔNp63α transcriptionally deregulates miRNA expression after CIS treatment. Several p-ΔNp63α-dependent microRNA species (miRNAs) were deregulated in HNSCC cells upon CIS exposure, including miR-181a, miR-519a, and miR-374a (downregulated) and miR-630 (upregulated). Deregulation of miRNA expression led to subsequent modulation of mRNA expression of several targets (TP53-S46, HIPK2, ATM, CDKN1A and 1B, CASP3, PARP1 and 2, DDIT1 and 4, BCL2 and BCL2L2, TP73, YES1, and YAP1) that are involved in the apoptotic process. Our data support the notion that miRNAs are critical downstream targets of p-ΔNp63α and mediate key pathways implicated in the response of cancer cells to chemotherapeutic drugs.
p63; cisplatin; squamous cell carcinomas; DICER1; microRNA
We aimed to determine which combination of physical examination (pe), mammography (mam), and ultrasonography (us) would optimize breast cancer detection in China.
We conducted a trial of screening with pe, mam, and us among Chinese women 25 years of age and older. All initial screenings using the three modalities were completed within 30 days of each other, and subjects were followed approximately 1 year later. The performances of the three screening methods used alone, in parallel, or in series were compared. Data were analyzed using exact confidence intervals (cis) and the McNemar test.
Between March 2009 and July 2011, 3028 eligible women completed all study examinations. At a mean follow-up of 1.3 years, 33 breast cancers were identified in the study population. Mammography detected 28 cancers; us, 24 cancers; and pe, 22 cancers. During the follow-up period, 2 false-negative cases occurred clinically. The highest sensitivity for breast cancer screening (93.9%) was achieved by paralleling mam with us, but came at the cost of a higher recall rate (12.15%). Using us alone at the first stage, followed by mam when indicated, offered high specificity (99.4%) and the lowest recall rate (1.82%), which were not reached at the expense of sensitivity (84.8%). Used in series, us and mam achieved a sensitivity similar to that for the same modalities used in parallel (McNemar p > 0.05).
Taking limited health resources into consideration, the strategy of screening with us alone at the first stage, followed by mam when indicated, may optimize breast cancer detection in most regions of China.
Breast cancer; mass screening; mammography; ultrasonography; follow-up study; Chinese women
Efficient in vitro differentiation into specific cell types is more important than ever after the breakthrough in nuclear reprogramming of somatic cells and its potential for disease modeling and drug screening.
Key success factors for neuronal differentiation are the yield of desired neuronal marker expression, reproducibility, length, and cost. Three main neuronal differentiation approaches are stromal-induced neuronal differentiation, embryoid body (EB) differentiation, and direct neuronal differentiation. Here, we describe our neurodifferentiation protocol using small molecules that very efficiently promote neural induction in a 5-stage EB protocol from six induced pluripotent stem cells (iPSC) lines from patients with Parkinson's disease and controls. This protocol generates neural precursors using Dorsomorphin and SB431542 and further maturation into dopaminergic neurons by replacing sonic hedgehog with purmorphamine or smoothened agonist. The advantage of this approach is that all patient-specific iPSC lines tested in this study were successfully and consistently coaxed into the neural lineage.
Speckle-based methods have been developed to characterize tissue blood flow and perfusion. One such method, called modified laser speckle imaging (mLSI), enables computation of blood flow maps with relatively high spatial resolution. Although it is known that the sensitivity and noise in LSI measurements depend on image exposure time, a fundamental disadvantage of mLSI is that it does not take into account this parameter. In this work, we integrate the exposure time into the mLSI method and provide experimental support of our approach with measurements from an in vitro flow phantom.
The purpose of this study was to test the hypothesis that administration of epigallocatechin-3-gallate (EGCG), a polyphenol present in abundance in widely consumed tea, inhibits cell proliferation, invasion, and angiogenesis in breast cancer patients. EGCG in 400 mg capsules was orally administered three times daily to breast cancer patients undergoing treatment with radiotherapy. Parameters related to cell proliferation, invasion, and angiogenesis were analyzed while blood samples were collected at different time points to determine efficacy of the EGCG treatment. Compared to patients who received radiotherapy alone, those given radiotherapy plus EGCG for an extended time period (two to eight weeks) showed significantly lower serum levels of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and reduced activation of metalloproteinase-9 and metalloproteinase-2 (MMP9/MMP2). Addition of sera obtained from patients treated with combination of radiotherapy and EGCG feeding for 2–8 weeks to in vitro cultures of highly-metastatic human MDA-MB-231 breast cancer cells resulted in the following significant changes: (1) suppression of cell proliferation and invasion; (2) arrest of cell cycles at the G0/G1 phase; (3) reduction of activation of MMP9/MMP2, expressions of Bcl-2/Bax, c-Met receptor, NF-κB, and the phosphorylation of Akt. MDA-MB-231 cells exposed to 5–10 µM EGCG also showed significant augmentation of the apoptosis inducing effects of γ-radiation, concomitant with reduced NF-κB protein level and AKT phosphorylation. These results provide hitherto unreported evidence that EGCG potentiated efficacy of radiotherapy in breast cancer patients, and raise the possibility that this tea polyphenol has potential to be a therapeutic adjuvant against human metastatic breast cancer.
Adjuvant therapy; breast cancer patients; EGCG; γ-radiation; HGF; MMP9/MMP2; VEGF
The aim of this study was to explore the effects of the bisphosphonate zoledronate on calcification induced by inorganic phosphate (Pi) and/or bone morphogenetic protein 2 (BMP-2) and the underlying mechanisms. Primary vascular smooth muscle cells (VSMCs) from rats were treated with 3 mM Pi or 3 mM Pi/BMP-2, with and without addition of zoledronate; 1.4 mM Pi served as a control. Calcium deposits, expression of core binding factor α-1 (Cbfa-1), osteopontin (OPN), parathyroid pituitary-specific transcription factor (Pit)-1 and Pit-2, and Pi uptake of VSMCs was determined. The calcification of VSMCs induced by elevated Pi or Pi/BMP-2 was significantly inhibited by zoledronate. The expression of Cbfa-1, OPN and Pit-1 was increased significantly after treatment with an elevated level of Pi or Pi/BMP-2, and this expression was significantly suppressed by addition of zoledronate. Pi uptake of VSMCs increased following treatment with elevated Pi and significantly decreased by addition of zoledronate. These results indicated that zoledronate effectively inhibited calcification induced by Pi/BMP-2, and this may have been achieved by means of the downregulation of expression of calcification-related proteins and uptake of Pi.
vascular calcification; zoledronate; bone morphogenetic protein 2; osteopontin; core binding factor α-1; phosphate uptake
The live chimeric porcine circovirus type 2 (PCV2) vaccine with the capsid gene of the emerging subtype 2b cloned in the genomic backbone of the nonpathogenic PCV1 is attenuated in vivo and induces protective immunity against PCV2. To further determine the safety and efficacy of this experimental vaccine, we tested for evidence of pig-to-pig transmission by commingling nonvaccinated and vaccinated pigs, determined potential upregulation by simultaneous vaccination and infection with porcine parvovirus (PPV) and porcine reproductive and respiratory syndrome virus (PRRSV), and determined vaccine efficacy by challenging pigs 4 weeks after vaccination with PCV2b, PRRSV, and PPV. Forty-six 21-day-old, PCV2-naïve pigs were randomly assigned to one of six groups. Twenty-nine of 46 pigs were challenged with PCV2b, PRRSV, and PPV at day 28, 8/46 remained nonvaccinated and nonchallenged and served as negative controls, and 9/46 remained nonchallenged and served as vaccination controls. All animals were necropsied at day 49. PCV1-PCV2 viremia was detected in nonvaccinated contact pigs commingled with vaccinated pigs, indicating pig-to-pig transmission; however, PCV1-PCV2 DNA levels remained low in all vaccinated and contact pigs regardless of concurrent infection. Finally, vaccination 28 days before challenge resulted in significantly (P < 0.05) decreased amounts of PCV2 in tissues and sera and significantly (P < 0.05) reduced macroscopic and microscopic lesions. The results of this study indicate that the experimental live-attenuated chimeric PCV2 vaccine, although transmissible to contact pigs, remains attenuated in pigs concurrently infected with PRRSV and PPV and induces protective immunity against PCV2b when it is administered 28 days before PCV2 exposure.
The prevalence of family childhood adversities (FCAs) and their joint effects on the first onset of subsequent mental disorders throughout the life course are rarely examined, especially in Asian communities.
Face-to-face household interviews of 5201 people aged 18–70 years in Beijing and Shanghai were conducted by a multi-stage household probability sampling method. The first onsets of four broad groups of mental disorders and six categories of FCAs were assessed using The World Mental Health Composite International Diagnostic Interview (WMH-CIDI). Joint effects of FCAs were analyzed by the best fitting of several competitive multivariate models.
FCAs were highly prevalent and inter-correlated. Half of them were in a family-dysfunction cluster. The best-fitting model included each of six types of FCA (with family-dysfunction FCAs being the strongest predictors), number of family-dysfunction FCAs, and number of other FCAs. Family-dysfunction FCAs had a significant subadditive association with subsequent disorders. Little specificity was found for the effects of particular FCAs with particular disorders. Predictive effects of FCAs reached the highest in ages 13–24 compared to ages 4–12 and ⩾25. Estimates of population-attributable risk proportions indicated that all FCAs together explained 38.5% of all first-onset disorders.
Chinese children were exposed to a broad spectrum of inter-related FCAs, as found in Western countries. FCAs related to family dysfunction were especially associated with subsequent mental disorders. Biological and/or environmental factors that mediate these long-term effects should be studied in prospective research on broad groups of FCAs.
Childhood adversity (CA); China; family; first onset; mental disorders
The mechanical forces generated during the ventilation of patients with acute lung injury causes significant lung damage and inflammation. Low-volume ventilation protocols are commonly used to prevent stretch-related injury that occurs at high lung volumes. However, the cyclic closure and reopening of pulmonary airways at low lung volumes, i.e., derecruitment and recruitment, also causes significant lung damage and inflammation. In this review, we provide an overview of how biomedical engineering techniques are being used to elucidate the complex physiological and biomechanical mechanisms responsible for cellular injury during recruitment/derecruitment. We focus on the development of multiscale, multiphysics computational models of cell deformation and injury during airway reopening. These models, and the corresponding in vitro experiments, have been used to both elucidate the basic mechanisms responsible for recruitment/derecruitment injury and to develop alternative therapies that make the epithelium more resistant to injury. For example, models and experiments indicate that fluidization of the cytoskeleton is cytoprotective and that changes in cytoskeletal structure and cell mechanics can be used to mitigate the mechanotransduction of oscillatory pressure into inflammatory signaling. The continued application of biomedical engineering techniques to the problem of recruitment/derecruitment injury may therefore lead to novel and more effective therapies.
microbubble flows; cell mechanics; surface tension forces; ventilation-induced lung injury; power law rheology; pulmonary mechanobiology