bluuebunny/crossref_metadata_2025 huggingface dataset:
Two novel PIWI families: roles in inter-genomic conflicts in bacteria and Mediator-dependent modulation of transcription in eukaryotes
Authors: Burroughs A Maxwell , Iyer Lakshminarayan M , Aravind L
Published: June 2013

Background

The PIWI module, found in the PIWI/AGO superfamily of proteins, is a critical component of several cellular pathways including germline maintenance, chromatin organization, regulation of splicing, RNA interference, and virus suppression. It binds a guide strand which helps it target complementary nucleic strands.

Results

Here we report the discovery of two divergent, novel families of PIWI modules, the first such to be described since the initial discovery of the PIWI/AGO superfamily over a decade ago. Both families display conservation patterns consistent with the binding of oligonucleotide guide strands. The first family is bacterial in distribution and is typically encoded by a distinctive three-gene operon alongside genes for a restriction endonuclease fold enzyme and a helicase of the DinG family. The second family is found only in eukaryotes. It is the core conserved module of the Med13 protein, a subunit of the CDK8 subcomplex of the transcription regulatory Mediator complex.

Conclusions

Based on the presence of the DinG family helicase, which specifically acts on R-loops, we infer that the first family of PIWI modules is part of a novel RNA-dependent restriction system which could target invasive DNA from phages, plasmids or conjugative transposons. It is predicted to facilitate restriction of actively transcribed invading DNA by utilizing RNA guides. The PIWI family found in the eukaryotic Med13 proteins throws new light on the regulatory switch through which the CDK8 subcomplex modulates transcription at Mediator-bound promoters of highly transcribed genes. We propose that this involves recognition of small RNAs by the PIWI module in Med13 resulting in a conformational switch that propagates through the Mediator complex.

Reviewers

This article was reviewed by Sandor Pongor, Frank Eisenhaber and Balaji Santhanam.
Mitral commissurotomy through the left ventricle apical orifice with Heart Ware left ventricular assist device implantation
Authors: Mohite Prashant Nanasaheb , Zych Bartlomiej , Popov Aron Frederik , Patil Nikhil , Luthra Suvitesh , Hedger Mike , Simon Andre Ruediger , Amrani Mohammed
Published: June 2013

Diseased, replaced or repaired mitral valve can lead to restricted blood flow to left ventricle and inadequate flow in left ventricular assist device (LVAD). A middle age woman with ‘burnt out’ hypertrophic cardiomyopathy had mitral valve repair for mitral regurgitation. She needed LVAD to support severe decompensating heart failure. Repaired mitral valve posed a risk of restricted flow through the device. Mitral commissurotomy was performed on beating heart through the left ventricular apical hole created for insertion of inflow cannula of LVAD.
The influence of applying insurance medicine guidelines for depression on disability assessments
Authors: Schellart Antonius JM , Zwerver Feico , Anema Johannes R , Van derBeek Allard J
Published: June 2013

Background

In the current study we report on the effects of an implementation strategy in the form of a training programme on the assessed work limitations of a client with depression by insurance physicians (IPs) participating in a RCT. These assessed work limitations of a client were in the form of scores on the List of Functional Abilities (LFA).

Method

We conducted a randomised controlled trial (RCT) for IPs in which we compared the intervention of a specially developed training programme with the usual methods of implementation and training currently used. The outcome was the mean sum score and the inter-rater reliability (Intraclass Correlation Coefficient, ICC) of the LFA scores. These LFA scores were scored by the IPs participating in the RCT for the work limitations of the cases presented in different videos, two videos before the training and two after the training of the intervention group.

Results

At baseline, the intervention group (IG) consisted of 21 IPs and the control group (CG) of 19. For one participant of the IG and for one of the CG the LFAs of the two case reports after training were not available. Before training the sum scores for the first case report did not differ significantly between the groups, while the mean sum score was higher in the IG than in the CG for the second case report. For both case reports after training a higher score was found in the IG than in the CG. The inter-rater reliability measured for the two case reports before training was about the same in the IG and the CG: 0.64 and 0.65, respectively. For the two case reports after training, the ICC was higher in the IG than in the CG: 0.69 and 0.54, respectively. This difference was not significant however.

Conclusion

It would appear that the implementation of a specially designed training programme on guidelines for depression may lead to greater inter-rater reliability in the assessments by insurance physicians of the work limitations of clients with depression. It is, however, important to note that insurance physicians who receive training may find more work limitations than those who do not.

Trial registration

Netherlands’ Trial Register NTR1863
T and B lymphocytes in the brains of dogs with concomitant seropositivity to three pathogenic protozoans: Leishmania chagasi, Toxoplasma gondii and Neospora caninum
Authors: Sakamoto Keila Priscilla , de Melo Guilherme Dias , Machado Gisele Fabrino
Published: June 2013

Background

Visceral leishmaniasis is a disease with great variability regarding the clinical manifestations in humans and dogs. Chronically infected dogs may develop neurological disorders, however, there are few reports that characterize the lesions and make clear the pathogenesis of the canine cerebral leishmaniasis. Concomitant with Leishmania chagasi, dogs may be infected by opportunistic pathogens, such as Toxoplasma gondii and Neospora caninum, which may contribute to the occurrence of lesions in the central nervous system. Hence, we aimed to compare the T and B lymphocytes population in the brains of infected dogs with seropositivity to L. chagasi, T. gondii and N. caninum concurrently (n = 24), seropositivity only to L. chagasi (n = 31), and seropositivity to T. gondii and N. caninum (n = 16). Uninfected dogs were used as control (n = 10).

Results

Inflammatory lesions, characterised by mononuclear cell accumulation, composed mainly of CD3+ T lymphocytes predominated in several encephalic regions of the dogs from all the three infected groups, with no difference among them (P = 0.0004), whereas CD79α+ B lymphocytes were detected in very small intensity and presented no difference among groups (P = 0.5313). Furthermore, no association among diseases was detected at the serological enquire.

Conclusions

We demonstrate that the peripheral infection by L. chagasi per se can promote the influx of lymphocytes within the nervous milieu as occurs during Toxoplasma and Neospora infections, and the concomitant seropositivity against these pathogens does not exacerbate the inflammatory brain lesions. Therefore, these findings give additional support that the brain should be included in the list of organs affected by visceral leishmaniasis and that even asymptomatic infected dogs may develop brain lesions.
The antiretroviral agent saquinavir enhances hTERT expression and telomerase activity in human T leukaemia cells in vitro
Authors: Adamo Riccardo , Comandini Alessandro , Aquino Angelo , Bonmassar Laura , Guglielmi Loredana , Bonmassar Enzo , Franzese Ornella
Published: June 2013

Background

Saquinavir, a protease inhibitor utilized in HIV infection, shows antitumor activity in various experimental models. In previous studies performed in our laboratory the drug was found to induce a substantial increase of telomerase activity in normal peripheral blood mononuclear cells. Aim of the present investigation was to test whether saquinavir was able to increase telomerase activity and the expression of the catalytic subunit of telomerase, hTERT, in human malignant hematopoietic cells.

Methods

Human Jurkat CD4+T cell leukaemia cell line was used throughout the present study. The antiproliferative effect of saquinavir was tested by the MTT assay. Telomerase activity was determined according to the telomeric repeat amplification protocol. The expression of hTERT mRNA was semi-quantitative evaluated by RT-PCR amplification and quantitative Real Time PCR. The binding of the transcription factor c-Myc to its specific E-Box DNA binding-site ofhTERTpromoter was analyzed by Electophoretic Mobility Shift Assay (EMSA). The amount of c-Myc in cytoplasm and nucleus of leukemia cells was determined by Western Blot analysis, and c-Myc down-regulation was obtained by siRNA transfection.

Results

Saquinavir produced a substantial increase of telomerase activity in Jurkat cells in vitro without increasing but rather reducing target cell proliferation rate. Telomerase up-regulation appeared to be the result of enhanced expression of hTERT. Saquinavir-mediated up-regulation of hTERT gene was the result of the increased binding of proteins to the E-Box sequence of the promoter. Moreover, saquinavir amplified the expression of c-Myc especially in the nuclear cell fraction. The direct influence of saquinavir on this transcription factor was also demonstrated by the antagonistic effect of the drug on siRNA induced c-Myc suppression. Since c-Myc is the main responsible for hTERT transcription, these findings suggest that the main mechanism underlying saquinavir-induced telomerase activation is mediated by c-Myc up-regulation.

Conclusions

Saquinavir augments hTERT expression while inhibiting leukemic cell growth. Experimental evidences show that this effect is mediated by saquinavir-influenced increase of c-Myc levels. This could have relevance in terms of enhanced hTERT-dependent tumor cell immunogenicity and suggests new paharmacological approaches interfering with c-Myc dependent pathways.
A framework incorporating the impact of exposure scenarios and application conditions on risk assessment of chemicals applied to skin
Authors: Dancik Yuri , Troutman John A , Jaworska Joanna
Published: June 2013

Purpose

1. To develop a framework for exposure calculation via the dermal route to meet the needs of 21st century toxicity testing and refine current approaches; 2. To demonstrate the impact of exposure scenario and application conditions on the plasma concentration following dermal exposure.

Method

A workflow connecting a dynamic skin penetration model with a generic whole-body physiologically-based pharmacokinetic (PBPK) model was developed. The impact of modifying exposure scenarios and application conditions on the simulated steady-state plasma concentration and exposure conversion factor was investigated for 9 chemicals tested previously in dermal animal studies which did not consider kinetics in their experimental designs.

Results

By simulating the animal study scenarios and exposure conditions, we showed that 7 studies were conducted with finite dose exposures, 1 with both finite and infinite dose exposures (in these 8 studies, an increase in the animal dose resulted in an increase in the simulated steady-state plasma concentrations (Cp,ss)), while 1 study was conducted with infinite dose exposures only (an increase in the animal dose resulted in identicalCp,ss). Steady-state plasma concentrations were up to 30-fold higher following an infinite dose scenario vs. a finite dose scenario, and up to 40-fold higher with occlusion vs. without. Depending on the chemical, the presence of water as a vehicle increased or decreased the steady-state plasma concentration, the largest difference being a factor of 16.

Conclusions

The workflow linking Kasting’s model of skin penetration and whole-body PBPK enables estimation of plasma concentrations for various applied doses, exposure scenarios and application conditions. Consequently, it provides a quantitative, mechanistic tool to refine dermal exposure calculations methodology for further use in risk assessment.
Predictors of dietary supplement use among female health workers in Tehran
Authors: Baygi Fereshteh , Sotoudeh Gity , Qorbani Mostafa , Sadrzadeh-Yeganeh Haleh , Rahimi Abbass , Koohdani Fariba , Asayesh Hamid
Published: June 2013

Background

Despite the lack of evidence on the necessity of dietary supplements to meet nutrients requirements, the majority of people use them all over the world. This study aimed to investigate factors associated with supplement use in women who work in health centers in the city of Tehran.

Methods

Five hundred sixty three female health workers participated in a cross-sectional study carried out in 2010 in health centers of Tehran. Weight, height, and waist circumference were measured and body mass index was calculated. Data on demographic characteristics, lifestyle, and dietary supplement use were collected by interviewing. The analysis was conducted using univariate and multivariate logistic regression (MLR) in SPSS version 16.

Results

The prevalence of dietary supplement use was 53.8%. In univariate logistic regression model, age, education, husband’s education, duration of employment, and tendency for changing weight at the time of the study were statistically significant predictors (P < 0.05). After MLR analysis, education (OR: 1.11, 95% CI: 1.05-1.17) and duration of employment (OR: 1.04, 95% CI: 1.02-1.06) remained significant in the model. Women with higher education and longer duration of employment had more tendency to use nutrient supplements.

Conclusion

Our findings showed that education and duration of employment were the most important predictors for taking dietary supplements in this population.
Human induced pluripotent stem cell-derived neural stem cells survive, migrate, differentiate, and improve neurologic function in a rat model of middle cerebral artery occlusion
Authors: Yuan Ting , Liao Wei , Feng Nian-Hua , Lou Yuan-Lei , Niu Xin , Zhang Ai-Jun , Wang Yang , Deng Zhi-Feng
Published: June 2013

Introduction

Stroke is a major cause of permanent neurologic damage, with few effective treatments available to restore lost function. Induced pluripotent stem cells (iPSCs) have the potential to generate all cell types in vitro and can be generated from a stroke patient. Therefore, iPSCs are attractive donor sources of genetically identical “patient-specific” cells to hold promise in therapy for stroke. In the present study, we established a four-stage culture system by using serum-free medium and retinoic acid (RA) to differentiate iPSCs into neural stem cells (NSCs) effectively and stably. Our hypothesis was that iPSC-derived NSCs would survive, migrate, and differentiate in vivo, and improve neurologic function after transplantation into the brains of rats with ischemic stroke.

Methods

Human iPSCs (iPS-S-01) and human ESCs (HuES17) were used to differentiate into NSCs by using our four-stage culture system. iPSCs and differentiated NSCs were characterized by immunocytochemistry staining and reverse transcription-polymerase chain reaction (RT-PCR) analysis. After establishment of focal cerebral ischemia with occlusion of the middle cerebral artery (MCA) and cell transplantation, animals were killed at 1 week and 2 weeks to analyze survival, migration, and differentiation of implanted cells in brain tissue. Animal behavior was evaluated via rope grabbing, beam walking, and Morris water maze tests.

Results

iPSCs were efficiently induced into NSCs by using a newly established four-stage induction system in vitro. iPSCs expressed pluripotency-associated genes Oct4, Sox2, and Nanog before NSC differentiation. The iPSC-derived NSCs spontaneously differentiated into neurons and astrocytes, which highly express β-tubulin and glial fibrillary acidic protein (GFAP), respectively. On transplantation into the striatum, CM-DiI labeled iPSC-derived NSCs were found to migrate into the ischemia area at 1 week and 2 weeks, and animal-function recovery was significantly improved in comparison with control groups at 3 weeks.

Conclusions

The four-stage induction system is stable and effective to culture, differentiate, and induce iPSCs to NSCs by using serum-free medium combined with retinoic acid (RA). Implanted iPSC-derived NSCs were able to survive, migrate into the ischemic brain area to differentiate into mature neural cells, and seem to have potential to restore lost neurologic function from damage due to stroke in a rat model.
Kupffer cells potentiate liver sinusoidal endothelial cell injury in sepsis by ligating programmed cell death ligand-1
Authors: Hutchins Noelle A , Wang Fei , Wang Yvonne , Chung Chun-Shiang , Ayala Alfred
Published: June 2013

PD-1 and PD-L1 have been reported to provide peripheral tolerance by inhibiting TCR-mediated activation. We have reported that PD-L1−/− animals are protected from sepsis-induced mortality and immune suppression. Whereas studies indicate that LSECs normally express PD-L1, which is also thought to maintain local immune liver tolerance by ligating the receptor PD-1 on T lymphocytes, the role of PD-L1 in the septic liver remains unknown. Thus, we hypothesized initially that PD-L1 expression on LSECs protects them from sepsis-induced injury. We noted that the increased vascular permeability and pSTAT3 protein expression in whole liver from septic animals were attenuated in the absence of PD-L1. Isolated LSECs taken from septic animals, which exhibited increased cell death, declining cell numbers, reduced cellular proliferation, and VEGFR2 expression (an angiogenesis marker), also showed improved cell numbers, proliferation, and percent VEGFR2+ levels in the absence of PD-L1. We also observed that sepsis induced an increase of liver F4/80+PD-1+-expressing KCs and increased PD-L1 expression on LSECs. Interestingly, PD-L1 expression levels on LSECs decreased when PD-1+-expressing KCs were depleted with clodronate liposomes. Contrary to our original hypothesis, we document here that increased interactions between PD-1+ KCs and PD-L1+ LSECs appear to lead to the decline of normal endothelial function—essential to sustain vascular integrity and prevent ALF. Importantly, we uncover an underappreciated pathological aspect of PD-1:PD-L1 ligation during inflammation that is independent of its normal, immune-suppressive activity.
Prepubertal Mouse Testis Growth and Maturation and Androgen Production Are Acutely Sensitive to Di-n-butyl Phthalate
Authors: Moody Sarah , Goh Hoey , Bielanowicz Amanda , Rippon Paul , Loveland Kate L. , Itman Catherine
Published: June 2013

Phthalates are plasticizers with widespread industrial, domestic, and medical applications. Epidemiological data indicating increased incidence of testicular dysgenesis in boys exposed to phthalates in utero are reinforced by studies demonstrating that phthalates impair fetal rodent testis development. Because humans are exposed to phthalates continuously from gestation through adulthood, it is imperative to understand what threat phthalates pose at other life stages. To determine the impact during prepuberty, we assessed the consequences of oral administration of 1 to 500 mg di-n-butyl phthalate (DBP)/kg/d in corn oil to wild-type (C57BL/6J) male mice from 4 to 14 days of age. Dose-dependent effects on testis growth correlated with reduced Sertoli cell proliferation. Histological and immunohistochemical analyses identified delayed spermatogenesis and impaired Sertoli cell maturation after exposure to 10 to 500 mg DBP/kg/d. Interference with the hypothalamic-pituitary-gonadal axis was indicated in mice fed 500 mg DBP/kg/d, which had elevated circulating inhibin but no change in serum FSH. Increased immunohistochemical staining for inhibin-α was apparent at doses of 10 to 500 mg DBP/kg/d. Serum testosterone and testicular androgen activity were lower in the 500 mg DBP/kg/d group; however, reduced anogenital distance in all DBP-treated mice suggested impaired androgen action at earlier time points. Long-term effects were evident, with smaller anogenital distance and indications of disrupted spermatogenesis in adult mice exposed prepubertally to doses from 1 mg DBP/kg/d. These data demonstrate the acute sensitivity of the prepubertal mouse testis to DBP at doses 50- to 500-fold lower than those used in rat and identify the upregulation of inhibin as a potential mechanism of DBP action.
Diet-Induced Obesity Increases Tumor Growth and Promotes Anaplastic Change in Thyroid Cancer in a Mouse Model
Authors: Kim Won Gu , Park Jeong Won , Willingham Mark C. , Cheng Sheue-yann
Published: June 2013

Recent epidemiological studies provide strong evidence suggesting obesity is a risk factor in several cancers, including thyroid cancer. However, the molecular mechanisms by which obesity increases the risk of thyroid cancer are poorly understood. In this study, we evaluated the effect of diet-induced obesity on thyroid carcinogenesis in a mouse model that spontaneously develops thyroid cancer (ThrbPV/PVPten+/− mice). These mice harbor a mutated thyroid hormone receptor-β (denoted as PV) and haplodeficiency of the Pten gene. A high-fat diet (HFD) efficiently induced the obese phenotype in ThrbPV/PVPten+/− mice after 15 weeks. Thyroid tumor growth was markedly greater and survival was significantly lower in ThrbPV/PVPten+/− mice fed an HFD than in controls fed a low-fat diet (LFD). The HFD increased thyroid tumor cell proliferation by increasing the protein levels of cyclin D1 and phosphorylated retinoblastoma protein to propel cell cycle progression. Histopathological analysis showed that the frequency of anaplasia of thyroid cancer was significantly greater (2.6-fold) in the HFD group than the LFD group. The HFD treatment led to an increase in parametrial/epididymal fat pad and elevated serum leptin levels in ThrbPV/PVPten+/− mice. Further molecular analyses indicated that the HFD induced more aggressive pathological changes that were mediated by increased activation of the Janus kinase 2-signaling transducer and activator of transcription 3 (STAT3) signaling pathway and induction of STAT3 target gene expression. Our findings demonstrate that diet-induced obesity exacerbates thyroid cancer progression in ThrbPV/PVPten+/− mice and suggest that the STAT3 signaling pathway could be tested as a potential target for the treatment of thyroid cancer.
P2 receptor networks regulate signaling duration over a wide dynamic range of ATP concentrations
Authors: Grol Matthew W. , Pereverzev Alexey , Sims Stephen M. , Dixon S. Jeffrey
Published: June 2013

The primordial intercellular signaling molecule ATP acts through two families of cell-surface P2 receptors – the P2Y family of G protein-coupled receptors and the P2X family of ligand-gated cation channels. Multiple P2 receptors are expressed in a variety of cell types. However, the significance of these networks of receptors in any biological system remains unknown. Using osteoblasts as a model system, we found that a low concentration of ATP (10 µM, ATPlow) induced transient elevation of cytosolic Ca2+; whereas, a high concentration of ATP (1 mM, ATPhigh) elicited more sustained elevation. Moreover, graded increases in the Ca2+ signal were achieved over a remarkable million-fold range of ATP concentrations (1 nM to 1 mM). Next, we demonstrated that ATPlow caused transient nuclear localization of NFATc1; whereas, ATPhigh elicited more sustained localization. When stimulated with ATPhigh, osteoblasts from P2X7 loss-of-function mice showed only transient Ca2+-NFATc1 signaling; in contrast, sustained signaling was observed in wild-type cells. Additional experiments revealed a role for P2Y receptors in mediating transient signaling induced by low ATP concentrations. Thus, distinct P2 receptors with varying affinities for ATP account for this wide range of sensitivity to extracellular nucleotides. Finally, ATPhigh, but not ATPlow, was shown to elicit robust expression of the NFAT target gene Ptgs2 (encoding COX-2), consistent with a critical role for duration of Ca2+-NFAT signaling in regulating target gene expression. Taken together, ensembles of P2 receptors provide a mechanism by which cells sense ATP over a wide concentration range, and transduce this input into distinct cellular signals.
Enhancement of c-Myc degradation by Bloom (BLM) helicase leads to delayed tumor initiation
Authors: Chandra Suruchika , Priyadarshini Raina , Madhavan Vinoth , Tikoo Shweta , Hussain Mansoor , Mudgal Richa , Modi Priyanka , Srivastava Vivek , Sengupta Sagar
Published: June 2013

The spectrum of tumors due to overexpression of c-Myc and the loss of BLM are very similar. Hence it was hypothesized that the presence of BLM may negatively regulate c-Myc functions. Using multiple isogenic cellines it was observed that the decreased endogenous c-Myc levels in presence of BLM were reversed by treatment with proteasomal inhibitors, thereby indicating that BLM enhanced c-Myc turnover. While the N-terminal region of BLM interacted with c-Myc, the rest of the helicase contacted with c-Myc's E3 ligase, Fbw7. The two BLM domains acted as “clamp/adaptor”, enhancing the binding of c-Myc to Fbw7. BLM promoted Fbw7-dependent K48-linked c-Myc ubiquitylation and its subsequent degradation in a helicase independent manner. A subset of BLM-regulated genes that were also c-Myc targets were determined and validated at both RNA and protein levels. To obtain in vivo validation of the effect of BLM on c-Myc mediated tumor initiation, isogenic cells from colon cancer cells either expressing or not expressing BLM were manipulated to inducibly shut down the expression of c-Myc. Using these cell lines the metastatic potential and the rate of initiation of tumors in nude mice were determined. The presence of BLM decreased c-Myc mediated invasiveness and delayed tumor initiation in mouse xenograft model. Consequently in tumors expressing BLM but not c-Myc, decreased ratio of proliferation over apoptosis was observed coupled with suppressed expression of angiogenesis marker, CD31. Hence BLM acts as a “caretaker tumor suppressor” due to its regulation of c-Myc stability.
Reprogrammed pancreatic progenitor-like intermediate state of hepatic cells is more susceptible to pancreatic beta cell differentiation
Authors: Wang Qiwei , Wang Hai , Sun Yu , Li Shi-Wu , Donelan William , Chang Lung-Ji , Jin Shouguang , Terada Naohiro , Cheng Henrique , Reeves Westley , Yang Li-Jun
Published: June 2013

Induced pluripotent stem cells (iPSCs) hold great promise for cell therapy. However, their low efficiency of lineage-specific differentiation and tumorigenesis severely hinder clinical translation. We hypothesized that reprogramming of somatic cells into lineage-specific progenitor cells might allow for large-scale expansion, avoiding the tumorigenesis inherent with iPSCs and simultaneously facilitating lineage-specific differentiation. Here we aimed at reprogramming rat hepatic WB cells, using four Yamanaka factors, into pancreatic progenitor cells (PPCs) or intermediate (IM) cells that have characteristics of PPCs. IM clones were selected based on their specific morphology and alkaline phosphatase activity and stably passaged under defined culture conditions. IM cells did not have iPSC properties, can be stably expanded in large quantity, and expressed all 14 genes that are used to define the PPC developmental stage. Directed differentiation of IM and WB cells by Pdx1-Ngn3-MafA (PNM) into pancreatic beta-like cells revealed that the IM cells are more susceptible to directed beta cell differentiation due to open chromatin configuration, as demonstrated by expression of key pancreatic beta cell genes, secretion of insulin in response to glucose stimulation, and easy access to exogenous PNM proteins at the rat insulin 1 and Pdx1 promoters. This notion of IM cells superior to their parental cells is further supported by the epigenetic demonstration of accessibility of Pdx1 and insulin 1 promoters. In conclusion, we have developed a strategy to derive and expand IM cells from hepatic WB cells via conventional cell reprogramming. This proof-of-principal study may offer a novel, safe, and effective way to generate autologous pancreatic beta cells for cell therapy of diabetes.
Septin rings act as template for myosin higher-order structures and inhibit redundant polarity establishment
Authors: Schneider Clemens , Grois Julia , Renz Christian , Gronemeyer Thomas , Johnsson Nils
Published: June 2013

The mechanisms of the coordinated assembly and disassembly of the septin/myosin ring is central for the understanding of polar growth and cytokinesis in yeast and other organisms. The septin- and myosin-binding protein Bni5p provides a dual function during the formation and disassembly of septin/myosin rings. Early in the cell cycle Bni5p captures Myo1p at the incipient bud site and actively transforms it into higher-order structures. Additionally, Bni5p stabilizes the septin/myosin ring and is released from the septins shortly before the onset of cytokinesis. Once this Bni5p-dissociation from the septins is artificially prevented, ring disassembly is impaired and the untimely appearance of septin/myosin ring is induced. The prematurely formed septin/myosin rings delay the establishment of a new polarity axis and the progression into a new cell cycle. This observation suggests a negative feedback between septin/myosin ring formation and polarity establishment that might help to guarantee the singular assembly of this structure and the synchronization of its formation with the cell cycle.
Differential and competitive regulation of human melanocortin 1 receptor signaling by β-arrestin isoforms
Authors: Abrisqueta Marta , Herraiz Cecilia , Pérez Oliva Ana B , Sanchez-Laorden Berta L , Olivares Concepción , Jiménez-Cervantes Celia , García-Borrón José C
Published: June 2013

The melanocortin 1 receptor (MC1R) is a G protein-coupled receptor (GPCR) crucial for the regulation of melanocyte proliferation and differentiation. MC1R activation by melanocortin hormones triggers the cAMP pathway and stimulates the extracellular signal-regulated protein kinases ERK1 and ERK2 to promote synthesis of photoprotective eumelanin pigments among other effects. Signaling from most GPCRs is regulated by the β-arrestin (ARRB) family of cytosolic multifunctional adaptor proteins which mediate signal termination and endocytosis of GPCR-agonist complexes. The ubiquitously expressed non-visual β-arrestin1 (ARRB1) and β-arrestin2 (ARRB2) are highly homologous but not functionally equivalent. Their role in the regulation of MC1R is unknown. Using a combination of co-immunoprecipitation, gel filtration chromatography, confocal microscopy, siRNA-mediated knockdown and functional assays, we demonstrated agonist-independent competitive interactions of ARRB1 and ARRB2 with MC1R, which might also be independent of phosphorylation of MC1R C-terminal Ser/Thr residues. The effects of ARRBs were isoform-specific. ARRB2 inhibited MC1R agonist-dependent cAMP production but not ERK activation, stimulated internalization and showed prolonged co-localization with the receptor in endocytic vesicles. Conversely, ARRB1 had no effect on internalization or functional coupling, but competed with ARRB2 for binding MC1R, which might increase signaling by displacement of inhibitory ARRB2. These data suggest a novel mechanism of MC1R functional regulation based on the relative expression of ARRB isoforms, with possible activatory ARRB1-dependent effects arising from partial relief of inhibitory ARRB2-MC1R interactions. Thus, competitive displacement of inhibitory ARRBs by functionally neutral ARRB isoforms might exert a paradigm-shifting signal-promoting effect to fine-tune signaling downstream of certain GPCRs.
Nonhomologous end-joining promotes resistance to DNA damage in the absence of an ADP-ribosyltransferase that signals DNA single strand breaks
Authors: Couto C. Anne-Marie , Hsu Duen-Wei , Teo Regina , Rakhimova Alina , Lempidaki Styliani , Pears Catherine J. , Lakin Nicholas D.
Published: June 2013

ADP-ribosylation of proteins at DNA lesions by ADP-ribosyltransferases (ARTs) is an early response to DNA damage. The best defined role of ADP-ribosylation in the DNA damage response is in repair of single strand breaks (SSBs). Recently, we initiated a study of how ADP-ribosylation regulates DNA repair in Dictyostelium and found that whilst two ARTs (Adprt1b and Adprt2) are required for tolerance of cells to SSBs, a third ART (Adprt1a) promotes nonhomologous end-joining (NHEJ). Here we report that disruption of adprt2 results in accumulation of DNA damage throughout the cell cycle following exposure to agents that induce base damage and DNA SSBs. Although ADP-ribosylation is evident in adprt2− cells exposed to MMS, disruption of adprt1a and adprt2 in combination abrogates this response and further sensitises cells to this agent, indicating that in the absence of Adprt2, Adprt1a signals MMS-induced DNA lesions to promote resistance of cells to DNA damage. As a consequence of defective signalling of SSBs by Adprt2, Adprt1a is required to assemble NHEJ factors in chromatin and disruption of the NHEJ pathway in combination with adprt2 increases sensitivity of cells to MMS. Taken together, these data indicate overlapping functions between different ARTs in signalling DNA damage and illustrate a critical requirement for NHEJ in maintaining cell viability in the absence of an effective SSB response.
Plectin-containing, centrally-localized focal adhesions exert traction forces in primary lung epithelial cells
Authors: Eisenberg Jessica L. , Beaumont Kristin G. , Takawira Desire , Hopkinson Susan B. , Mrksich Milan , Scott Budinger G. R. , Jones Jonathan C. R.
Published: June 2013

Receptor clustering upon cell-substratum attachment induces assembly of cytoplasmic protein complexes termed focal adhesions (FAs), which connect, albeit indirectly, extracellular matrix to the cytoskeleton. A subset of cultured primary alveolar epithelial cells (AEC) display a unique pattern of vinculin/paxillin/talin-rich FAs in two concentric circles when cultured on glass and micro-patterned substrates: one ring of FAs located at the cell periphery (pFAs), another FA ring located centrally in the cell (cFAs). Unusually, cFAs associate with an aster-like actin array as well as keratin bundles. Moreover, cFAs show rapid paxillin turnover rates following fluorescence recovery after photobleaching and exert traction forces similar to those generated by FAs at the cell periphery. The plakin protein plectin localizes to cFAs and is primarily absent from pFAs, whereas tensin, a marker of mature/fibrillar adhesions, is found in both cFAs and pFAs. In primary AEC in which plectin expression is depleted, cFAs are largely absent with an attendant reorganization of both the keratin and actin cytoskeletons. We suggest that the mechanical environment in the lung gives rise to the assembly of unconventional FAs in AEC. These FAs not only show a distinctive arrangement, but also possess unique compositional and functional properties.
Reciprocal cross-regulation between RNF41 and USP8 controls cytokine receptor sorting and processing
Authors: De Ceuninck Leentje , Wauman Joris , Masschaele Delphine , Peelman Frank , Tavernier Jan
Published: June 2013

The mechanisms controlling the steady-state cytokine receptor cell surface levels, and consequently the cellular response to cytokines, remain poorly understood. The number of surface-exposed receptors is a dynamic balance of de novo synthesis, transport to the plasma membrane, internalization, recycling, degradation and ectodomain shedding. We previously reported that the E3 ubiquitin ligase Ring Finger Protein 41 (RNF41) inhibits basal lysosomal degradation and enhance ectodomain shedding of JAK2-associated cytokine receptors. Ubiquitin-specific protease 8 (USP8), an RNF41 interacting deubiquitinating enzyme (DUB) stabilizes RNF41 and is involved in trafficking of various transmembrane proteins. The present study identifies USP8 as a substrate of RNF41 and reveals that loss of USP8 explains the aforementioned RNF41 effects. RNF41 redistributes and ubiquitinates USP8, and reduces USP8 levels. In addition, USP8 knockdown functionally matches the effects of RNF41 ectopic expression on the model leptin and leukemia inhibitory factor (LIF) receptors. Moreover, RNF41 indirectly destabilizes the ESCRT-0 complex via USP8 suppression. Collectively, our findings demonstrate that RNF41 controls JAK2-associated cytokine receptor trafficking by acting as a key regulator of USP8 and ESCRT-0 stability. Balanced reciprocal cross-regulation between RNF41 and USP8 thus decides if receptors are sorted for lysosomal degradation or recycling, this way regulating basal cytokine receptor levels.
New Ionic Plastic Crystals of NR4BEt3Me (R = Me and Et) and NRxR′4−xBEt3Me (R = Et, R′ = Me and Pr, x = 1–3) in a New Class of Plastic Crystals
Authors: Hayasaki Tomoyuki , Hirakawa Satoru , Honda Hisashi
Published: June 2013

New ionic plastic crystals of NR4BEt3Me (R = Me and Et) and NRxR′4−xBEt3Me (R = Et, R′ = Me and Pr, x = 1–3) were formed in a new region of plastic crystal. In this area, globular cations and anions are assembled by weak interactions. On the basis of our results of 1H and 13C NMR spectra and electrical conductivity measurements, we could conclude that isotropic reorientation and self-diffusion of globular cations and anions were detected in the crystal. X-ray diffraction (XRD) lines observed in RxR′4−xBEt3Me (R = Et, R′ = Me, x = 0–4) revealed a CsCl-type cubic crystal structure. Additionally, DSC spectra revealed that this new class of plastic crystals displays low phase-transition temperatures in the plastic phase and have high melting points. The former tendency is frequently detected in molecular plastic crystals while the latter is often found in ionic plastic crystals.
Different Photochromic Rates of Azopyridine Derivatives in New Dual Photoisomeric Cobaloxime Complex Crystals
Authors: Yamagiwa Hiroki , Sekine Akiko
Published: June 2013

Two cobaloxime complexes, namely (3-cyanopropyl)(3-phenylazopyridine)cobaloxime and (3-cyanopropyl)(4-phenylazopyridine)cobaloxime, were synthesized as new dual photoisomeric complexes for controlling the reactivity of trans–cis photoisomerization of phenylazopyridine derivatives in the solid state. The phenylazopyridine moieties of the crystals were transformed from the trans to the cis-form on exposure to UV light. In contrast, the 3-cyanopropyl groups of the crystals were isomerized to 1-cyanopropyl groups on irradiation with visible light. Moreover, the 3–1 photoisomerization of the 3-cyanopropyl group occurred with retention of the single-crystal form. The rates of the trans–cis transformations of the phenylazopyridine moieties of the crystals before the 3–1 phototransformation were compared with those after the phototransformation in the solid state. For the two crystals, the rates of the trans–cis phototransformations significantly increased after 3–1 photoisomerization. The crystal structures before and after 3–1 photoisomerization were analyzed. The free space around the phenylazopyridine moieties expanded as a result of 3–1 photoisomerization. It is clear that the reaction rates of the trans–cis transformations of the phenylazopyridine moieties on exposure to UV light can be modified by introducing another photoreactive group that is photoisomerized by visible light.
A Reworkable Photothermal Dual-curing System
Authors: Adachi Masanori , Okamura Haruyuki , Shirai Masamitsu
Published: June 2013

A reworkable photothermal dual-curing system was devised using an epoxide and methacrylates with acid anhydride units connected with tertiary ester linkages. The mixture of these compounds was curable by photoirradiation at 365 nm and subsequent heating. The cured sample can be redissolved in methanol after irradiation at 254 nm followed by baking above 100 °C. The system is useful for the development of high-performance UV-curing materials.
Rational Design of a Fluorescence-amplified Aptasensor Based on Enzyme-assisted Target Recycling Strategy
Authors: Nakao Shingo , Oishi Motoi
Published: June 2013

A fluorescence-amplified sensing system composed of a fluorophore-labeled adenosine DNA-aptamer/quencher-labeled capture DNA duplex, assistant DNA, and exonuclease III (Exo III) was designed and prepared. The newly constructed enzyme-assisted aptasensor exhibited amplification of fluorescent signal in response to adenosine (target) through an Exo III-assisted target recycling process.
Deregulated Serum Concentrations of Circulating Cell–Free MicroRNAs miR-17, miR-34a, miR-155, and miR-373 in Human Breast Cancer Development and Progression
Authors: Eichelser Corinna , Flesch-Janys Dieter , Chang-Claude Jenny , Pantel Klaus , Schwarzenbach Heidi
Published: June 2013

BACKGROUND

MicroRNAs (miRs) are small, noncoding RNAs that target genes involved in tumor development and progression. In the current study, we investigated the use of circulating miR concentrations as biomarkers in the serum of breast cancer patients.

METHODS

We analyzed serum samples from 120 patients with primary breast cancer after surgery and before chemotherapy (M0, classified into 3 subgroups of 40 patients with progesterone/estrogen-positive, HER2-positive, and triple-negative cancer), 32 patients with overt metastasis (M1), and 40 healthy women. Using quantitative TaqMan MicroRNA PCR, we measured the relative concentrations of 6 circulating microRNAs (miR-10b, -17, -34a, -93, -155, and -373) known to be relevant for tumor development and progression. The data were correlated with clinicopathologic risk factors, with particular reference to HER2 and hormone receptor status of the primary tumor and the presence of metastases.

RESULTS

The relative serum concentrations of circulating miR-34a [P = 0.013, area under the curve (AUC) 0.636], miR-93 (P = 0.001, AUC 0.699), and miR-373 (P = 0.0001, AUC 0.879) were significantly different between M0 breast cancer patients and healthy women, whereas miR-17 (P = 0.002, AUC 0.679) and miR-155 (P = 0.0001, AUC 0.781) were differently expressed between M0 and M1 patients. Increased concentrations of miR-373 were associated with negative HER2 status of the primary tumor (P = 0.0001). Deregulated concentrations of miR-17 (P = 0.019) and miR-34a (P = 0.029) were detected in patients with progesterone/estrogen receptor–positive and –negative status, respectively.

CONCLUSIONS

Our findings indicate that serum concentrations of deregulated microRNAs may be linked to a particular biology of breast carcinomas favoring progression and metastatic spread.
Complete Numerical Solution of the Diffusion Equation of Random Genetic Drift
Authors: Zhao Lei , Yue Xingye , Waxman David
Published: June 2013

A numerical method is presented to solve the diffusion equation for the random genetic drift that occurs at a single unlinked locus with two alleles. The method was designed to conserve probability, and the resulting numerical solution represents a probability distribution whose total probability is unity. We describe solutions of the diffusion equation whose total probability is unity as complete. Thus the numerical method introduced in this work produces complete solutions, and such solutions have the property that whenever fixation and loss can occur, they are automatically included within the solution. This feature demonstrates that the diffusion approximation can describe not only internal allele frequencies, but also the boundary frequencies zero and one. The numerical approach presented here constitutes a single inclusive framework from which to perform calculations for random genetic drift. It has a straightforward implementation, allowing it to be applied to a wide variety of problems, including those with time-dependent parameters, such as changing population sizes. As tests and illustrations of the numerical method, it is used to determine: (i) the probability density and time-dependent probability of fixation for a neutral locus in a population of constant size; (ii) the probability of fixation in the presence of selection; and (iii) the probability of fixation in the presence of selection and demographic change, the latter in the form of a changing population size.
Probing the Boundaries of Orthology: The Unanticipated Rapid Evolution ofDrosophila centrosomin
Authors: Eisman Robert C , Kaufman Thomas C
Published: June 2013

The rapid evolution of essential developmental genes and their protein products is both intriguing and problematic. The rapid evolution of gene products with simple protein folds and a lack of well-characterized functional domains typically result in a low discovery rate of orthologous genes. Additionally, in the absence of orthologs it is difficult to study the processes and mechanisms underlying rapid evolution. In this study, we have investigated the rapid evolution of centrosomin (cnn), an essential gene encoding centrosomal protein isoforms required during syncytial development in Drosophila melanogaster. Until recently the rapid divergence of cnn made identification of orthologs difficult and questionable because Cnn violates many of the assumptions underlying models for protein evolution. To overcome these limitations, we have identified a group of insect orthologs and present conserved features likely to be required for the functions attributed to cnn in D. melanogaster. We also show that the rapid divergence of Cnn isoforms is apparently due to frequent coding sequence indels and an accelerated rate of intronic additions and eliminations. These changes appear to be buffered by multi-exon and multi-reading frame maximum potential ORFs, simple protein folds, and the splicing machinery. These buffering features also occur in other genes in Drosophila and may help prevent potentially deleterious mutations due to indels in genes with large coding exons and exon-dense regions separated by small introns. This work promises to be useful for future investigations of cnn and potentially other rapidly evolving genes and proteins.
Resistência "in vivo" do Plasmodium falciparum às 4-amino - quinoleínas e à associação sulfadoxina-pirimetamina: I - estudo de Porto Velho, Rondônia, 1983
Authors: Reyes Santiago , Passos Afonso D. C. , Osanai Carlos H.
Published: June 2013

Através da prova de 7 dias foi estudado o grau de resistência do Plasmodium falciparum à cloroquina, amodiaquina e sulfadoxina-pirimetamina em Porto Velho, Estado de Rondônia, Brasil. Não se observaram diferenças significativas nas médias de parasitas nos dias de seguimento e nas proporções de resistência entre os três medicamentos testados, fazendo com que os autores recomendem a manutenção das 4-aminoquinoleínas como drogas a serem usadas atualmente em infecções não graves por P. falciparum na área de Porto Velho.
Método de strout utilizando diferentes velocidades de centrifugação no diagnóstico da fase aguda da doença de Chagas
Authors: Emanuel Antonio , Castro Cleudson Nery de
Published: June 2013

Empregando-se o método de Strout, analisou-se a eficácia de três diferentes velocidades de centrifugação (19 G, 165 G e 640 G) na avaliação da parasitemia de 20 chagásicos em fase aguda. Realizaram-se 309 exames nos 20 pacientes, com média de 15 exames para cada caso. Os exames realizados a 19 G evidenciaram a menor eficácia enquanto os realizados a 165 G e 640 G mostraram-se igualmente eficazes.
Infestaçao humana pelo Dipylidium caninum
Authors: Lemos Carlos Henrique , Oliveira Clarice de Resende
Published: June 2013

Os autores descrevem um caso de infestação pelo Dipylidium caninum numa criança residente em Araguari, Minas Gerais, que apresentou lesões cutâneas, insônia, irritabilidade, vômitos eperda do apetite. Relatam a eliminação abundante deproglotes quando a paciente foi tratada com medicamentos caseiros e a remissão dos sintomas, após dois tratamentos com mebendazol. Contudo não puderam concluir sobre a eficiência das drogas, devido à medicação empírica e ã administração de 200 mg/dia de mebendazol, contrariando a dosagem recomendada para Cestodas, que é de 400 mg/dia. Relatam, ainda, que exames das fezes do cão de propriedade da família revelaram apenas a presença de ancilostomídeos.