Inhibition of proteasome deubiquitinating activity as a new cancer therapy

Ubiquitin-tagged substrates are degraded by the 26S proteasome, which is a multisubunit complex comprising a proteolytic 20S core particle capped by 19S regulatory particles. The approval of bortezomib for the treatment of multiple myeloma validated the 20S core particle as an anticancer drug target. Here we describe the small molecule b-AP15 as a previously unidentified class of proteasome inhibitor that abrogates the deubiquitinating activity of the 19S regulatory particle. b-AP15 inhibited the activity of two 19S regulatory-particle-associated deubiquitinases, ubiquitin C-terminal hydrolase 5 (UCHL5) and ubiquitin-specific peptidase 14 (USP14), resulting in accumulation of polyubiquitin. b-AP15 induced tumor cell apoptosis that was insensitive to TP53 status and overexpression of the apoptosis inhibitor BCL2. We show that treatment with b-AP15 inhibited tumor progression in four different in vivo solid tumor models and inhibited organ infiltration in an acute myeloid leukemia model. Our results show that the deubiquitinating activity of the 19S regulatory particle is a new anticancer drug target.     

Association between rs4673 (C/T) and rs13306294 (A/G) haplotypes of NAD(P)H oxidase p22phox gene and severity of stenosis in coronary arteries

Phagocytic NADH/NADPH oxidase is an important enzyme producing reactive oxygen species within subendothelial space of vessels. Findings have shown that p22phox subunit is an essential element related to the enzyme activity. Since some p22phox polymorphisms are thought to have functional roles in the enzyme thus, we studied the association between rs4673 (C242T) and rs13306294 (A/G) haplotypes and the severity of stenosis in coronary arteries. One hundred eighty-two subjects undergoing coronary angiography were recruited on the base of study design. Patients (n=114) had at least a stenosed coronary artery (>50% stenosis) and subdivided into three subgroups; SVD (n=28), 2VD (n=31) and 3VD (n=55) while controls (n=68) had the normal coronary arteries (<5% stenosis). The direct haplotyping technique of SNPs was performed using ARMS-RFLP-PCR method. Furthermore, alphabet-based tools predicted the changes of secondary structure at the rs4673 position. All haplotypes being proposed theoretically were found in the study population. The distribution of two-allele haplotypes had no significant difference between patients and controls (P=0.1). Although the rs4673 allele frequency was not significant between the groups (P>0.5), chi square test and multinomial regression analysis showed an observed high risk for rs13306294 A allele among patients. The bioinformatics tools predicted that the p22phox secondary structure is not changed due to the substitution of Tyr→His at the rs4673 position. We concluded that the polymorphisms have no allele linkage on the chromosome. In addition, the rs13306294 A allele is a potential factor of stenosis of coronary arteries that increases susceptibility for the extent of disease.     

Balancing cohesin eviction and retention prevents aberrant chromosomal interactions,Polycomb-mediated repression,and X-inactivation

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Real-Time Monitoring of Electrically Stimulated Norepinephrine Release in Rat Thalamus: I. Resolution of Transmitter and Metabolite Signal Components

Abstract: Electrical stimulation of an ascending path of the locus ceruleus-norepinephrine system was used to elicit release of norepinephrine at noradrenergic terminal fields of the rat thalamus. Overflow into the extracellular fluid space was measured by fast in vivo chronoamperometry. At pretreated carbon fibers, the electrochemical signal consists of a sharp peak of ∼20–30-s duration followed by a slower, plateau-like decay to baseline. The peak, characterized by a variety of pharmacological manipulations and dialysis perfusion, is primarily due to norepinephrine. The plateau was shown to correspond to metabolite efflux of 3,4-dihydroxyphenylacetic acid. By varying the degree of electrochemical pretreatment, the response time and sensitivity of the fibers can be tuned to follow the entire signal or to select the separate components for detailed evaluation. This approach can be used to provide new information on the spatial and temporal characteristics of stimulated neurotransmitter release.     

Regulation of H(2)O(2)-induced necrosis by PKC and AMP-activated kinase signaling in primary cultured hepatocytes

Recent studies have suggested that, in certain cases, necrosis, like apoptosis, may be programmed, involving the activation and inhibition of many signaling pathways. In this study, we examined whether necrosis induced by H(2)O(2) is regulated by signaling pathways in primary hepatocytes. A detailed time course revealed that H(2)O(2) treated to hepatocytes is consumed within minutes, but hepatocytes undergo necrosis several hours later. Thus, H(2)O(2) treatment induces a "lag phase" where signaling changes occur, including PKC activation, Akt (PKB) downregulation, activation of JNK, and downregulation of AMP-activated kinase (AMPK). Investigation of various inhibitors demonstrated that PKC inhibitors were effective in reducing necrosis caused by H(2)O(2) (~80%). PKC inhibitor treatment decreased PKC activity but, surprisingly, also upregulated Akt and AMPK, suggesting that various PKC isoforms negatively regulate Akt and AMPK. Akt did not appear to play a significant role in H(2)O(2)-induced necrosis, since PKC inhibitor treatment protected hepatocytes from H(2)O(2) even when Akt was inhibited. On the other hand, compound C, a selective AMPK inhibitor, abrogated the protective effect of PKC inhibitors against necrosis induced by H(2)O(2). Furthermore, AMPK activators protected against H(2)O(2)-induced necrosis, suggesting that much of the protective effect of PKC inhibition was mediated through the upregulation of AMPK. Work with PKC inhibitors suggested that atypical PKC downregulates AMPK in response to H(2)O(2). Knockdown of PKC-alpha using antisense oligonucleotides also slightly protected (~22%) against H(2)O(2). Taken together, our data demonstrate that the modulation of signaling pathways involving PKC and AMPK can alter H(2)O(2)-induced necrosis, suggesting that a signaling "program" is important in mediating H(2)O(2)-induced necrosis in primary hepatocytes.     

Biomedical applications of carbon nanomaterials: Drug and gene delivery potentials

One of the major components in the development of nanomedicines is the choice of the right biomaterial, which notably determines the subsequent biological responses. The popularity of carbon nanomaterials (CNMs) has been on the rise due to their numerous applications in the fields of drug delivery, bioimaging, tissue engineering, and biosensing. Owing to their considerably high surface area, multifunctional surface chemistry, and excellent optical activity, novel functionalized CNMs possess efficient drug-loading capacity, biocompatibility, and lack of immunogenicity. Over the past few decades, several advances have been made on the functionalization of CNMs to minimize their health concerns and enhance their biosafety. Recent evidence has also implied that CNMs can be functionalized with bioactive peptides, proteins, nucleic acids, and drugs to achieve composites with remarkably low toxicity and high pharmaceutical efficiency. This review focuses on the three main classes of CNMs, including fullerenes, graphenes, and carbon nanotubes, and their recent biomedical applications.     

Computational fluid dynamics modeling,a novel,and effective approach for developing scalable cell therapy manufacturing processes

Induced pluripotent stem cells (iPSCs) hold great potential to generate novel, curative cell therapy products. However, current methods to generate these novel therapies lack scalability, are labor-intensive, require a large footprint, and are not suited to meet clinical and commercial demands. Therefore, it is necessary to develop scalable manufacturing processes to accommodate the generation of high-quality iPSC derivatives under controlled conditions. The current scale-up methods used in cell therapy processes are based on empirical, geometry-dependent methods that do not accurately represent the hydrodynamics of 3D bioreactors. These methods require multiple iterations of scale-up studies, resulting in increased development cost and time. Here we show a novel approach using computational fluid dynamics modeling to effectively scale-up cell therapy manufacturing processes in 3D bioreactors. Using a GMP-compatible iPSC line, we translated and scaled-up a small-scale cardiomyocyte differentiation process to a 3-L computer-controlled bioreactor in an efficient manner, showing comparability in both systems.     

Breast cancer-linked lncRNA u-Eleanor is upregulated in breast of healthy women with lack or short duration of breastfeeding

Recently, it has been revealed that estrogen-related reproductive factors are linked with some early gene expression lesions associated with malignancy in clinically healthy breasts. Accordingly, the aim of the current study was to evaluate the association of expression levels of estrogen-related long noncoding RNAs (lncRNAs) upstream Eleanor (u-Eleanor) and HOX antisense intergenic RNA (HOTAIR) with the different patterns of reproductive factors in breast tissue of healthy women. The subjects of this study were 98 cancer-free women who had undergone cosmetic mammoplasty. The expression levels of u-Eleanor and HOTAIR were measured using quantitative real-time polymerase chain reaction. The results of the current study showed that the women without a history of breastfeeding had a high-level expression of u-Eleanor compared with the women with a breastfeeding duration greater than 6 to 24 months (P = 0.03) as well as the women with a breastfeeding duration of more than 24 months (P = 0.005). Furthermore, a higher expression of u-Eleanor was found in the women with a short breastfeeding duration for 1 to 6 months than that in the women with a breastfeeding duration of greater than 24 months (P = 0.02). In the same way, the results of correlation test (r = −0.258; P = 0.036) and multivariate regression model (β = −0.321; P = 0.023) are indicative of a significant relationship of elevated expression of u-Eleanor with decreasing breastfeeding duration in the women. These findings could be important to identify the molecular mechanisms behind the relationship between a lack or short duration of the breastfeeding and the risk of breast cancer, which has previously been reported by epidemiological studies.