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The sinoatrial node (SAN), the leading pacemaker region, generates electrical impulses that propagate throughout the heart. SAN dysfunction with bradyarrhythmia is well documented in heart failure (HF). However, the underlying mechanisms are not completely understood. Mitochondria are critical to cellular processes that determine the life or death of the cell. The release of Ca 2+ from the ryanodine receptors 2 (RyR2) on the sarcoplasmic reticulum (SR) at mitochondria–SR microdomains serves as the critical communication to match energy production to meet metabolic demands. Therefore, we tested the hypothesis that alterations in the mitochondria–SR connectomics contribute to SAN dysfunction in HF. We took advantage of a mouse model of chronic pressure overload–induced HF by transverse aortic constriction (TAC) and a SAN-specific CRISPR-Cas9–mediated knockdown of mitofusin-2 ( Mfn2 ), the mitochondria–SR tethering GTPase protein. TAC mice exhibited impaired cardiac function with HF, cardiac fibrosis, and profound SAN dysfunction. Ultrastructural imaging using electron microscope (EM) tomography revealed abnormal mitochondrial structure with increased mitochondria–SR distance. The expression of Mfn2 was significantly down-regulated and showed reduced colocalization with RyR2 in HF SAN cells. Indeed, SAN-specific Mfn2 knockdown led to alterations in the mitochondria–SR microdomains and SAN dysfunction. Finally, disruptions in the mitochondria–SR microdomains resulted in abnormal mitochondrial Ca 2+ handling, alterations in localized protein kinase A (PKA) activity, and impaired mitochondrial function in HF SAN cells. The current study provides insights into the role of mitochondria–SR microdomains in SAN automaticity and possible therapeutic targets for SAN dysfunction in HF patients.

Introduction: Sinoatrial node (SAN), the leading pacemaker region, generates electrical impulses that propagate throughout the heart. SAN dysfunction with bradyarrhythmia is well documented in heart failure (HF). However, the underlying mechanisms are not entirely understood. Mitochondria are critical to cellular processes that determine life or death of the cell. Therefore, proper communication with the mitochondria is essential for normal cellular function. We tested the hypothesis that alterations in the mitochondria-sarcoplasmic reticulum (SR) connectomics underlie SAN dysfunction in HF. Methods: We took advantage of a mouse model of pressure-overload induced HF by transverse aortic constriction (TAC) and a SAN-specific CRISPR/Cas9 mediated gene silencing of mitofusin-2 ( Mfn2 ). Results: TAC mice exhibited impaired cardiac function with HF, cardiac fibrosis, and profound SAN dysfunction. Ultrastructural imaging using electron microscope (EM) tomography revealed abnormal mitochondrial structure with increased SR-mitochondria distance. The expression of the SR-mitochondria tether GTPase protein Mfn-2 was significantly down-regulated and showed reduced colocalization with ryanodine receptor (RyR2) in HF SAN cells. Indeed, SAN-specific gene silencing of Mfn-2 led to alterations in the mitochondrial-SR microdomains[MFN1] and SAN dysfunction. Finally, disruption in the mitochondrial-SR microdomains resulted in abnormal mitochondrial Ca 2+ handling, alterations in localized PKA activity, and impaired mitochondrial function in HF SAN cells. Conclusion: The study provides insight on the role of mitochondrial-SR microdomains in SAN automaticity and into possible therapeutic targets for SAN dysfunction in HF patients.

BACKGROUND: Long QT syndrome (LQTS) is a hereditary disease that predisposes patients to life-threatening cardiac arrhythmias and sudden cardiac death. Our previous study of the human ether-à-go-go related gene (hERG)–encoded K(+) channel (K(v)11.1) supports an association between hERG and RING finger protein 207 (RNF207) variants in aggravating the onset and severity of LQTS, specifically T613M hERG (hERG(T613M)) and RNF207 frameshift (RNF207(G603fs)) mutations. However, the underlying mechanistic underpinning remains unknown. OBJECTIVE: The purpose of the present study was to test the role of RNF207 in the function of hERG-encoded K(+) channel subunits. METHODS: Whole-cell patch-clamp experiments were performed in human embryonic kidney (HEK 293) cells and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) together with immunofluorescent confocal and high resolution microscopy, auto-ubiquitinylation assays, and co-immunoprecipitation experiments to test the functional interactions between hERG and RNF207. RESULTS: Here, we demonstrated that RNF207 serves as an E3 ubiquitin ligase and targets misfolded hERG(T613M) proteins for degradation. RNF207(G603fs) exhibits decreased activity and hinders the normal degradation pathway; this increases the levels of hERG(T613M) subunits and their dominant-negative effect on the wild-type subunits, ultimately resulting in decreased current density. Similar findings are shown for hERG(A614V), a known dominant-negative mutant subunit. Finally, the presence of RNF207(G603fs) with hERG(T613M) results in significantly prolonged action potential durations and reduced hERG current in human-induced pluripotent stem cell–derived cardiomyocytes. CONCLUSION: Our study establishes RNF207 as an interacting protein serving as a ubiquitin ligase for hERG-encoded K(+) channel subunits. Normal function of RNF207 is critical for the quality control of hERG subunits and consequently cardiac repolarization. Moreover, our study provides evidence for protein quality control as a new paradigm in life-threatening cardiac arrhythmias in patients with LQTS.

Balanced proliferation and differentiation of neural progenitor cells (NPCs) are critical for brain development, but how the process is regulated and what components of the cell division machinery is involved are not well understood. Here we report that SEPT7, a cell division regulator originally identified in Saccharomyces cerevisiae, interacts with KIF20A in the intercellular bridge of dividing NPCs and plays an essential role in maintaining the proliferative state of NPCs during cortical development. Knockdown of SEPT7 in NPCs results in displacement of KIF20A from the midbody and early neuronal differentiation. NPC-specific inducible knockout of Sept7 causes early cell cycle exit, precocious neuronal differentiation, and ventriculomegaly in the cortex, but surprisingly does not lead to noticeable cytokinesis defect. Our data uncover an interaction of SEPT7 and KIF20A during NPC divisions and demonstrate a crucial role of SEPT7 in cell fate determination. In addition, this study presents a functional approach for identifying additional cell fate regulators of the mammalian brain.

Purpose Nonsteroidal anti-inflammatory drugs (NSAIDs) are among one of the most commonly prescribed medications for pain and inflammation. Diclofenac (DIC) is a commonly prescribed NSAID that is known to increase the risk of cardiovascular diseases. However, the mechanisms underlying its cardiotoxic effects remain largely unknown. In this study, we tested the hypothesis that chronic exposure to DIC increases oxidative stress, which ultimately impairs cardiovascular function. Methods and Results Mice were treated with DIC for 4 weeks and subsequently subjected to in vivo and in vitro functional assessments. Chronic DIC exposure resulted in not only systolic but also diastolic dysfunction. DIC treatment, however, did not alter blood pressure or electrocardiographic recordings. Importantly, treatment with DIC significantly increased inflammatory cytokines and chemokines as well as cardiac fibroblast activation and proliferation. There was increased reactive oxygen species (ROS) production in cardiomyocytes from DIC-treated mice, which may contribute to the more depolarized mitochondrial membrane potential and reduced energy production, leading to a significant decrease in sarcoplasmic reticulum (SR) Ca2+ load, Ca2+ transients, and sarcomere shortening. Using unbiased metabolomic analyses, we demonstrated significant alterations in oxylipin profiles towards inflammatory features in chronic DIC treatment. Conclusions Together, chronic treatment with DIC resulted in severe cardiotoxicity, which was mediated, in part, by an increase in mitochondrial oxidative stress.

Natural structures are wonderful examples of what can be done with a fairly limited selection of resources. Natural structures have this feature due to their complex architecture, gradient chemical compositions and microstructure. Inspired by nature, many attempts have been made to manufacture man-made materials with similar gradients (e.g. surface nanocrystallization by severe surface plastic deformation (S2PD)). Gradient microstructure of alloys manufactured by the S2PD techniques has been very well studied. However, the distribution of chemical compositions within the alloy after the S2PD treatment has rarely been explored. The current work found that by using S2PD combined with one-year long natural aging treatments, the microstructure and chemical composition of the Al-Zn-Mg-Cu alloy (7034 Al alloy) exhibited a new gradient distribution measured from the treated surface to the pristine interior. Zn (Cu) content on the surface layer increased from 10.2 (1.3) to 57.7 (5.9) wt.%, along with an Mg surface layer depletion after treatments. The thickness of the observed chemical gradient layer was surprisingly as thick as 45 μm. Also observed were the similar phenomena in other alloys after the same treatments. The findings provide potential improvements in bioinspired materials manufacturing, rare resource saving and surface functionalization.

Highlights • The usage of antihypertensive drugs is not associated with the risk and severity of COVID-19. • Hypertensive patients with COVID-19 benefit from prior usage of angiotensin-converting enzyme inhibitors/angiotensin receptor blockers., Background The association of antihypertensive drugs with the risk and severity of COVID-19 remains unknown. Methods and Results We systematically searched PubMed, MEDLINE, The Cochrane Library, Cochrane Central Register of Controlled Trials (CENTRAL), ClinicalTrials.gov, and medRxiv for publications before July 13, 2020. Cohort studies and case-control studies that contain information on the association of antihypertensive agents including angiotensin-converting enzyme inhibitors (ACEIs), angiotensin II receptor blockers (ARBs), calcium-channel blockers (CCBs), β-blockers, and diuretics with the risk and severity of COVID-19 were selected. The random-effects or fixed-effects models were used to pool the odds ratio (OR) with 95% confidence interval (CI) for the outcomes. The literature search yielded 53 studies that satisfied our inclusion criteria, which comprised 39 cohort studies and 14 case-control studies. These studies included a total of 2,100,587 participants. We observed no association between prior usage of antihypertensive medications including ACEIs/ARBs, CCBs, β-blockers, or diuretics and the risk and severity of COVID-19. Additionally, when only hypertensive patients were included, the severity and mortality were lower with prior usage of ACEIs/ARBs (overall OR of 0.81, 95% CI 0.66-0.99, p

Background Chloroquine (CQ) and its derivative hydroxychloroquine (HCQ) have recently emerged as potential antiviral and immunomodulatory options for the treatment of 2019 coronavirus disease (COVID-19). To examine the safety profiles of these medications, we systematically evaluated the adverse events (AEs) of these medications from published randomized controlled trials (RCTs). Methods We systematically searched MEDLINE, the Cochrane library, the Cochrane Central Register of Controlled Trials (CENTRAL), and the ClinicalTrials.gov for all the RCTs comparing CQ or HCQ with placebo or other active agents, published before June 20, 2020. The random-effects or fixed-effects models were used to pool the risk estimates relative ratio (RR) with 95% confidence interval (CI) for the outcomes. Results The literature search yielded 23 and 19 studies for CQ and HCQ, respectively, that satisfied our inclusion criteria. Of these studies, we performed meta-analysis on 6 studies for CQ and 18 studies for HCQ. We did not limit our analysis to published records involving viral treatment alone; data also included the usage of either CQ or HCQ for the treatment of other diseases. The trials for the CQ consisted of a total of 2,137 participants (n = 1,077 CQ, n = 1,060 placebo), while the trials for HCQ involved 2,675 participants (n = 1,345 HCQ and n = 1,330 control). The overall mild and total AEs were significantly higher in CQ-treated non-COVID-19 patients, HCQ-treated non-COVID-19 patients, and HCQ-treated COVID-19 patients. The AEs were further categorized into four groups and analyses revealed that neurologic, gastrointestinal (GI), dermatologic, and sensory AEs were higher in participants taking CQ compared to placebo, while GI, dermatologic, sensory, and cardiovascular AEs were higher in HCQ-treated COVID-19 patients compared to control patients. Moreover, subgroup analysis suggested higher AEs with respect to dosage and duration in HCQ group. Data were acquired from studies with perceived low risk of bias, so plausible bias is unlikely to seriously affect the main findings of the current study. Conclusions Taken together, we found that participants taking either CQ or HCQ exhibited more AEs than participants taking placebo or control. Precautionary measures should be taken when using these drugs to treat COVID-19. The meta-analysis was registered on OSF (https://osf.io/jm3d9). Registration The meta-analysis was registered on OSF (https://osf.io/jm3d9).

BackgroundRecently, chloroquine (CQ) and its derivative hydroxychloroquine (HCQ) have emerged as potential antiviral and immunomodulatory options for the treatment of 2019 coronavirus disease (COVID-19). To examine the safety profiles of these medications, we systematically evaluated the adverse events (AEs) of these medications from published randomized controlled trials (RCTs).MethodsWe systematically searched PubMed, MEDLINE, Cochrane, the Cochrane Central Register of Controlled Trials (CENTRAL), EMBASE, and the ClinicalTrials.gov for all the RCTs comparing CQ or HCQ with placebo or other active agents, published before March 31, 2020. The random-effects or fixed-effects models were used to pool the risk estimates relative ratio (RR) with 95% confidence interval (CI) for the outcomes.ResultsThe literature search yielded 23 and 17 studies for CQ and HCQ, respectively, that satisfied our inclusion criteria. Of these studies, we performed meta-analysis on the ones that were placebo-controlled, which included 6 studies for CQ and 14 studies for HCQ. We did not limit our analysis to published reports involving viral treatment alone; data also included the usage of either CQ or HCQ for the treatment of other diseases. The trials for the CQ consisted of a total of 2,137 participants (n=1,077 CQ, n=1,060 placebo), while the trials for HCQ involved 1,096 participants (n=558 HCQ and n=538 placebo). The overall mild or total AEs were statistically higher comparing CQ or HCQ to placebo. The AEs were further categorized into four groups and analyses revealed that neurologic, gastrointestinal, dermatologic, and ophthalmic AEs were higher in participants taking CQ compared to placebo. Although this was not evident in HCQ treated groups, further analyses suggested that there were more AEs attributed to other organ system that were not included in the categorized meta-analyses. Additionally, meta-regression analyses revealed that total AEs was affected by dosage for the CQ group.ConclusionsTaken together, we found that participants taking either CQ or HCQ have more AEs than participants taking placebo. Precautionary measures should be taken when using these drugs to treat COVID-19.

Surface contamination layer mainly containing Fe, Ti and O with a thickness of ~ 20 μm was induced on AA 7150 surface during ultrasonic shot peening (USSP) treatment. The contaminated layer and its exfoliation evolution in solution containing HCl was investigated using immersion test, electrochemical impedance spectra (EIS), scanning kelvin probe force microscopy (SKPFM), SEM with energy-dispersive X-ray spectroscopy, XRD and TEM. USSPed AA 7150 with contamination layer showed ~ 3 times higher corrosion rate than that of controlled sample. However, after the exfoliation of the contamination layer, the corrosion rate of USSPed alloy actually is lower than that of its untreated counterpart. Additionally, as revealed by SKPFM, surface contamination layer is cathodic relative to the substrate, indicating that surface contamination layer is detrimental for the substrate alloy when galvanic corrosion occurs. Nonetheless, it should be noted that even in the presence of contamination layer, the localized corrosion resistance of peened alloy was greatly improved, which might be due to surface nanocrystallization, disappearance of rolled 〈220〉 texture and residual compressive stress effects caused by USSP.

The normal function of ion channels depends critically on the precise subcellular localization and the number of channel proteins on the cell surface membrane. Small-conductance, Ca2+-activated K+ channels (SK, KCa2) are expressed in human atrial myocytes and responsible for shaping atrial action potentials. Understanding the mechanisms of SK channel trafficking may provide new insights into the regulation controlling the repolarization of atrial myocytes. We have previously demonstrated that the C and N termini of SK2 channels interact with actin-binding proteins, α-actinin2 and filamin A, respectively. However, the roles of the interacting proteins on SK2 channel trafficking remain incompletely understood. Using Total Internal Reflection Fluorescence (TIRF) microscopy, we studied the mechanisms of surface membrane localization of SK2 (KCa2.2) channels. When SK2 channels were co-expressed with filamin A or α-actinin2, the membrane fluorescence intensity of SK2 channels increased significantly. We next tested the effects of primaquine and dynasore on SK2 channels expression. Treatment with primaquine significantly reduced the membrane expression of SK2 channels. In contrast, treatment with dynasore failed to alter the surface membrane expression of SK2 channels. Further investigations using constitutively-active or dominant-negative forms of Rab GTPases provided additional insights into the distinct roles of the two cytoskeletal proteins on the recycling processes of SK2 channels from endosomes. α-actinin2 facilitated recycling of SK2 channels from both early and recycling endosomes while filamin A likely aids the recycling of SK2 channels from recycling endosomes. This article is protected by copyright. All rights reserved

Whereas A380 series alloys are some of the most widely used aluminum alloys, iron can severely degrade the ductility of an Al–Si die-casting alloy because its presence leads to the precipitation of different AlFeSi intermetallic phases resulting in development of stress concentrations. Thus, controlling the fraction and morphology of AlFeSi phase, especially the β-AlFeSi phase, is an important way to refine the ductility of A380 die-casting alloys. This article describes how Sr element was added to A380 alloys to study the effect of Sr on the morphology change of the AlFeSi phase. The results show that high cooling rates and Sr addition can both change the morphology of β-AlFeSi and transfer β-AlFeSi to α-AlFeSi. In addition, both high cooling rates and Sr addition can modify the eutectic Al–Si phase. The study shows that the optimum Sr addition and cooling rate for the A380 series alloy are 0.1 wt% and 10 °C/s, respectively.

In most aluminum–silicon-based die casting alloys, iron is considered one of the most harmful elements. The presence of iron in Al–Si-based alloys leads to the precipitation of a series of iron-containing intermetallic phases. Of these, the β-AlFeSi phase is the most detrimental to the toughness of Al–Si-based alloys. Reducing the formation of the β-AlFeSi phase has the potential of significantly improving the ductility of die casting alloys. The purpose of this work is to show how combined use of high-temperature and short-time heat treatment can be an effective way of controlling the morphology and fraction of β-AlFeSi phase in A380 alloy. Experimental results indicate that if heat treatment temperature goes up to 500 °C (932 °F), the higher temperature can reduce formation of β-AlFeSi with shorter heat treatment time. Evidence of this reduction is proved in optical and scanning electron microscopy images showing both the decomposition of β-AlFeSi and the transformation of β to α-AlFeSi.

Malignant pleural mesothelioma (MPM) is an aggressive malignancy arising from mesothelial lining of pleura. It is associated with a poor prognosis, partly due to the lack of a precise understanding of the molecular mechanisms associated with its malignant behavior. In the present study, we expanded on our previous studies on cell cycle control of MPM cells by targeting CD26 molecule with humanized anti-CD26 monoclonal antibody (HuCD26mAb), focusing particularly on ubiquitin-specific protease 22 (USP22). We showed that USP22 protein expression is detected in clinical specimens of MPM and that USP22 knockdown, as well as CD26 knockdown, significantly inhibits the growth and proliferation of MPM cells in vitro and in vivo. Moreover, depletion of both USP22 and CD26 suppresses MPM cell proliferation even more profoundly. Furthermore, expression levels of USP22 correlate with those of CD26. HuCD26mAb treatment induces a decrease in USP22 level through its interaction with the CD26 molecule, leading to increased levels of ubiquitinated histone H2A and p21. By demonstrating a CD26-related linkage with USP22 in MPM cell inhibition induced by HuCD26mAb, our present study hence characterizes USP22 as a novel target molecule while concurrently suggesting a new therapeutic strategy for MPM.

This research investigated the effects of high-intensity ultrasound on the combustion synthesis of TiC particles in Al–Ti–C system. The process involved that high-intensity ultrasound was applied on the surface of a compacted Al–Ti–C pellet directly through a Nb probe during the thermal explosion reaction. By comparing with the sample without ultrasonic treatment, it was found that the thermal explosion reaction for synthesizing TiC phase could take place thoroughly in the ultrasonically treated sample. During the process of synthesizing TiC phase, the dissolution of solid graphite particles into the Al–Ti melt, as well as the nucleation and growth of TiC particles could be promoted effectively due to the effects of ultrasound, leading to an enhancement of the formation of TiC particles. Ultrasound assisted combustion synthesis as a simple and effective approach was proposed for synthesizing materials in this research.

In situ TiB2 particles can be synthesized via salts–metal reaction by adding K2TiF6 and KBF4 mixed salts into the Al melt. In this research, a novel ultrasound assisted technique was proposed with the aim of synthesizing TiB2 particles in the Al melt at low temperature by means of introducing high-intensity ultrasound into the salts–metal reaction at 700 °C, which was at least 100 °C lower than those in the conventional methods. The effects of ultrasound on the synthesis of TiB2 particles were investigated by comparing two groups of experiments with and without ultrasound. The results showed that the yield of TiB2 particles in the ultrasonically treated sample could reach 90.4%, whereas the value of which was around 28% in the control sample without ultrasound after 10 min of reaction, indicating that ultrasound was able to promote significantly the formation of TiB2 particles at low temperature. Most of in situ formed TiB2 particles (95%) were smaller than 300 nm in size. Our research showed that significantly enhanced formation of small TiB2 particles in ultrasonic field can be attributed to faster dissolution of mixed salts powders into Al melt, accelerated mass transfer of solutes, as well as the promoted nucleation and growth of TiB2 particles due to the effects of ultrasonic cavitation.

As a promising reinforcement of aluminum alloy, in situ formed Al3Ti particles have attracted more attention in the fabrication of aluminum matrix composites. In our research, in situ Al3Ti/7075 alloy composites were fabricated by adding K2TiF6 salt powders into molten 7075 alloy at 750 °C via casting method. The formation of in situ Al3Ti particles and their effects on the microstructure and mechanical properties of 7075 alloy, including hardness, ultimate tensile strength (UTS), and yield strength (YS), were investigated. The results showed that in situ formed Al3Ti particles were rod-like in morphology, with the average length and width of 15 µm and 5 µm, respectively. Due to the nucleating effect of Al3Ti particles, α-Al crystals of 7075 alloy transferred from dendritic to equiaxed structure in morphology, the size of which decreased obviously as well. Compared with 7075 alloy, the hardness, UTS, and YS of in situ Al3Ti/7075 alloy were improved by 14.3%, 18.1%, and 25.8%, respectively.

The tumor suppressor protein p53 functions as a transcriptional factor that activates genes controlling cell cycle arrest and apoptosis. Here, we report that protein inhibitor of activated Stat1 (PIAS1) interacts with the tetramerization and C-terminal regulatory domains of p53 in yeast two-hybrid analyses. Endogenous PIAS1 is also associated with endogenous p53 in mammalian cells. Ectopic expression of PIAS1 activates p53-mediated expression in mouse embryonic fibroblast cells (p53(-/-)) as well as a variety of other cell lines. Furthermore, ectopic expression of PIAS1 induces p53-mediated expression of cyclin-dependent kinase inhibitor p21 and G(1) arrest of the cell cycle in H1299 cells. In addition, a PIAS1 mutant without the RING-finger domain required for sumoylation could still activate p53-mediated gene expression, indicating that activation of p53 by PIAS1 does not require the RING-finger domain. Taken together, our results suggest that PIAS1 is a novel activator of p53.

This paper presents examples which show how computer animation may be used in teaching electrical power engineering subjects. The topics covered include: transmission system transients; power transformer saturation; and electric machine operation.

Although the prognosis of acute lymphoblastic leukemia (ALL) has improved considerably in recent years, some of the cases still exhibit therapy-resistant. We have previously reported that CD9 was expressed heterogeneously in B-ALL cell lines and CD9(+) cells exhibited an asymmetric cell division with greater tumorigenic potential than CD9(-) cells. CD9(+) cells were also serially transplantable in immunodeficient mice, indicating that CD9(+) cell possess self-renewal capacity. In the current study, we performed more detailed analysis of CD9 function for the cancer stem cell (CSC) properties. In patient sample, CD9 was expressed in the most cases of B-ALL cells with significant correlation of CD34-expression. Gene expression analysis revealed that leukemogenic fusion proteins and Src family proteins were significantly regulated in the CD9(+) population. Moreover, CD9(+) cells exhibited drug-resistance, but proliferation of bulk cells was inhibited by anti-CD9 monoclonal antibody. Knockdown of CD9 remarkably reduced the leukemogenic potential. Furthermore, gene ablation of CD9 affected the expression and tyrosine-phosphorylation of Src family proteins and reduced the expression of histone-deubiquitinase USP22. Taken together, our results suggest that CD9 links to several signaling pathways and epigenetic modification for regulating the CSC properties of B-ALL.

Resveratrol (3,4',5-trihydroxy-trans-stilbene), a polyphenol naturally occurring in grapes and other plants, has cancer chemo-preventive effects and therapeutic potential. Although resveratrol modulates multiple pathways in tumor cells, how resveratrol or its affected pathways converge on chromatin to mediate its effects is not known. Using glioma cells as a model, we showed here that resveratrol inhibited cell proliferation and induced cellular hypertrophy by transforming spindle-shaped cells to enlarged, irregular and flatten-shaped ones. We further showed that resveratrol-induced hypertrophic cells expressed senescence-associated-β-galactosidase, suggesting that resveratrol-induced cellular senescence in glioma cells. Consistent with these observations, we demonstrated that resveratrol inhibited clonogenic efficiencies in vitro and tumor growth in a xenograft model. Furthermore, we found that acute treatment of resveratrol inhibited mono-ubiquitination of histone H2B at K120 (uH2B) in breast, prostate, pancreatic, lung, brain tumor cells as well as primary human cells. Chronic treatment with low doses of resveratrol also inhibited uH2B in the resveratrol-induced senescent glioma cells. Moreover, we showed that depletion of RNF20, a ubiquitin ligase of histone H2B, inhibited uH2B and induced cellular senescence in glioma cells in vitro, thereby recapitulated the effects of resveratrol. Taken together, our results suggest that uH2B is a novel direct or indirect chromatin target of resveratrol and RNF20 plays an important role in inhibiting cellular senescence programs that are intact in glioma cells.

Histone modifications play an important role in transcriptional regulation and are intimately involved in important biological and disease processes. Despite their functional significance, whether and how extracellular signals modulate histone modifications are not well defined. Using mono-ubiquitination of histone H2B as a model system, we have previously shown that mono-ubiquitination of histone H2B is induced by glucose through glycolysis in budding yeast Sacchromyces cerevisiae [1]. Because histones are well conserved proteins among eukaryotes and glycolysis is the most conserved metabolic pathway, we reasoned that the glucose–glycolysis–uH2B signal pathway originally discovered in yeast may be conserved in human cells. Using cultured human glioma cells as a model, we show in this report that extracellular media modulated global levels of mono-ubiquitination of histone H2B at K120 (uH2B). Nutrient deprivation removed the ubiquitin moiety of uH2B. Glucose-containing media induced uH2B in the cells while media lacking glucose had no effect on the induction of uH2B, suggesting that glucose was required for inducing uH2B in the cells. In contrast, non-metabolic glucose analogs were defective in inducing uH2B, suggesting that glucose metabolism was required for glucose-induced mono-ubiquitination in the cultured glioma cells. Moreover, shRNA knockdown of PKM2, an essential enzyme for glycolysis in malignant tumors, inhibited mono-ubiquitination of histone H2B in these cells. Taken together, our previous and current results demonstrate that the novel glucose–glycolysis–uH2B signal pathway is well conserved from yeast to mammalian cells, providing an evolutionarily-conserved regulatory mechanism of histone modifications.

Whole-genome sequencing projects have generated a wealth of gene sequences from a variety of organisms. A major challenge is to rapidly uncover gene regulatory circuits and their functional manifestations at the cellular level. Here we report the coupled fabrication of nanocraters ranging in size from 100 pL to 1.5 nL on permeable membranes for culturing cells. Using this approach, we developed bacterial and yeast cell microarrays that allowed phenotypic determinations of gene activities and drug targets on a large scale. Cell microarrays will therefore be a particularly useful tool for studying phenotypes of gene activities on a genome-wide scale.

One challenge facing biology is the elucidation of the function of the estimated 30 000 human genes and their polymorphic variants. Reagents that affect the activity of specific genes will be useful in the dissection of cellular regulatory networks. Here, as a test case, we used a two-bait two-hybrid system to identify peptide aptamers that distinguish allelic forms of H-Ras. Some of these anti-Ras aptamters inhibit the interaction of oncogenic Ras with c-Raf1 in vitro, and abolish EGF-stimulated activation of c-Raf1 in vivo. These experiments show that the inactivation of protein function by peptide aptamers represents a viable approach to the understanding and control of signaling pathways and oncogenic missense alleles.

HLA-specific killer cell inhibitory receptors (KIR) are thought to impede natural killer (NK) and T cell activation programs through recruitment of the SH2 domain-containing tyrosine phosphatases, SHP-1 and SHP-2, to their cytoplasmic tails (CYT). To identify other SH2 domain-containing proteins that bind KIR CYT, we used the recently described yeast two-bait interaction trap and a modified version of this system, both of which permit tyrosine phosphorylation of bait proteins. Using these systems, we show that KIR CYT, once phosphorylated by the src-family tyrosine kinase LCK, additionally bind the p85α regulatory subunit of phosphatidylinositol (PI) 3-kinase. Furthermore, we show that in an NK cell line, NK3.3, cross-linking of KIR results in recruitment of p85α to KIR and activation of PI 3-kinase lipid kinase activity. One consequence of KIR coupling to PI 3-kinase is downstream activation of the antiapoptotic protein kinase AKT. Therefore, in addition to providing negative signals, KIR may also contribute positive signals for NK and T cell growth and/or survival.

Cellular therapy using embryonic stem cells has always been an area of great interest due to the pluripotent characteristics of stem cells. In 2006, Takahashi and Yamanaka (Cell 126, 663–676) demonstrated that somatic cells can be reprogrammed into a stem cell-like state, termed induced pluripotent stem (iPS) cells, by ectopic expression of Oct4, Sox2, Klf4 and c Myc. A later report (Nakagawa et al. Nat. Biotechnol.26:101–106, 2008) showed that iPS cells can be produced in the absence of the c Myc oncogene. We have used this latter strategy to successfully reprogram somatic cells derived from C57BL/6 mouse tail fibroblast to iPS cells. Retrovirus infected fibroblasts exhibited stem cell-like morphology by 14 days post infection. These iPS cells were then infected with a retrovirus that expressed HOXB4. Recombinant leukemia inhibitor factor (LIF) supplement was removed from media at this time and the cells allowed to differentiate into embryoid bodies. These cells were screened for specific differentiation stem cell markers, such as Oct4, Nanog, Sall4 and SSEA-1. iPS cells were converted into embryonic bodies and then infected with retroviruses expressing HOXB4. Embryoid bodies stably expressing HOXB4 were induced to hematopoietic differentiation by treatment of thrombopoietin (TPO), stem cell factor (SCF), vascular endothelial growth factor (VEGF), interferon gamma (IFNg) and fms-like tyrosine kinase (FLT3 ligand). Evaluation of iPS-derived hematopoietic cells on smears show strikingly similarity in morphology to the W4 mouse embryonic stem (ES) cells differentiated into hematopoietic cells as a control. Flow cytometry analysis of iPS-derived hematopoietic cells after 1 week exposure to cytokines revealed 7% B220+ cells (B cells), 11% Ter119+ cells (erythroid), and 13% Gr-1+ cells (granulocytes) similar to W4 ES cells. The iPS-derived hematopoietic cells were transplanted into irradiated immunodeficient mice via lateral tail vein injection. Transplantation of these iPS-derived hematopoietic progenitors tagged with GFP into irradiated SCID mice revealed that the hematopoietic progenitors were able to home to the bone marrow after 1 week of transplantation. Importantly, after 1 month, GFP+ engrafted cells remained in the bone marrow suggesting a long-term engraftment. This long term engraftment of the iPS-derived hematopoietic cells to the bone marrow constitutes an important step toward potential therapy of numerous patient-specific blood based diseases.

Hemophilia A, a sex-linked bleeding disorder, is caused by mutations within the genomic sequence of the Factor VIII (FVIII) gene. Mutations of FVIII lead to depleted or reduced protein production, and inefficient clotting. Because hemophilia A is caused by a single gene deficiency, several attempts at gene therapy have been made, including several phase I clinical trials. However, these have all failed for various reasons--including immune rejection. However, with the recent discovery of induced pluripotent stem (iPS) cells the immune rejection barrier can be circumvented. iPS cells can be generated from somatic cells by introducing the ectopic expression of three transcription factors Oct4, Sox2, Klf4 (Nakagawa et al., Nat Biotechnol.26: 101–106, 2008) To date, iPS cells have been indistinguishable from ES cells, and thus provide tremendous therapeutic potential. In this study, we differentiated iPS cells to endothelial and endothelial progenitor cells using the EB (embryonic bodies) differentiation method. iPS derived endothelial or endothelial progenitor cells express both endothelial or endothelial progenitor cell specific markers, such as CD31, and Flk1, as well as FVIII. These iPS-derived cells were directly injecting into the liver of irradiated hemophilia A model mice. Seven days after transplantation, hemophilia A and control mice were challenged by a tail-clip bleeding assay. Non-transplanted hemophilia A mice died within few hours, while transplanted mice survived for many weeks and more. Plasma FVIII expression in transplanted hemophilia A mice is significantly higher than in non-transplanted hemophilia mice and this elevated expression confers a correction on the hemophilia A phenotype in mice following a tail-clip. Our studies suggest that transplantation of iPS derived endothelial progenitor cells can rescue the phenotype of hemophilia in mouse models and thus potentially suitable for the future development of cell therapy of monogenetic disorders.