bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficient Uptake of 177Lu‐Porphyrin‐PEG Nanocomplexes by Tumor Mitochondria for Multimodal‐Imaging‐Guided Combination Therapy
Authors: Yu Bo , Wei Hao , He Qianjun , Ferreira Carolina A. , Kutyreff Christopher J. , Ni Dalong , Rosenkrans Zachary T. , Cheng Liang , Yu Faquan , Engle Jonathan W. , Lan Xiaoli , Cai Weibo
Published: November 2017

The benefits to intracellular drug delivery from nanomedicine have been limited by biological barriers and to some extent by targeting capability. We investigated a size‐controlled, dual tumor‐mitochondria‐targeted theranostic nanoplatform (Porphyrin‐PEG Nanocomplexes, PPNs). The maximum tumor accumulation (15.6 %ID g−1, 72 h p.i.) and ideal tumor‐to‐muscle ratio (16.6, 72 h p.i.) was achieved using an optimized PPN particle size of approximately 10 nm, as measured by using PET imaging tracing. The stable coordination of PPNs with 177Lu enables the integration of fluorescence imaging (FL) and photodynamic therapy (PDT) with positron emission tomography (PET) imaging and internal radiotherapy (RT). Furthermore, the efficient tumor and mitochondrial uptake of 177Lu‐PPNs greatly enhanced the efficacies of RT and/or PDT. This work developed a facile approach for the fabrication of tumor‐targeted multi‐modal nanotheranostic agents, which enables precision and radionuclide‐based combination tumor therapy.
Pd‐Catalyzed Cyanation of (Hetero)Aryl Halides by Using Biphosphine Ligands
Authors: Zhang Shaoke , Neumann Helfried , Beller Matthias
Published: November 2017

Tetraadamantylbiphosphine (TABP; L1), which showed superior activity in the palladium‐catalyzed cyanation of 4‐chloroanisole compared to standard phosphines, was synthesized as a new ligand. The generality of the new catalytic system was shown by the cyanation reaction of approximately 30 (hetero)aryl halides including hindered, electron‐rich, and electron‐poor aryl chlorides. These reactions constitute the first examples of using biphosphine ligands in Pd‐catalyzed coupling reactions.
Examining the importance of the level of computational theory used in solution‐phase structural determinations by NMR using residual dipolar couplings
Authors: Tabada Diana , Mahmoudi Faranak , Carroll William R.
Published: November 2017

The structures of many small molecules have been determined using residual dipolar couplings (RDCs) and the singular value decomposition method (SVD). Here, a key part of the SVD method is examined. The methods used to predict the 3‐dimensional structures of molecules used in the SVD methodology for comparison with RDCs were examined for their impact on the quality of agreement that those structures had with observed RDCs. It was observed that the method of generating a proposed structure had a great impact on the quality of agreement with observed spectral data and that more computationally intensive methods of proposing structures provided better agreement with the observed RDCs in the control molecule.
Analysis of Cellular DNA Content by Flow Cytometry
Authors: Darzynkiewicz Zbigniew , Huang Xuan , Zhao Hong
Published: November 2017

Cellular DNA content can be measured by flow cytometry with the aim of : (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing DNA ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and/or proteolytic treatment to permeabilize cells and make DNA accessible to fluorochrome. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells based on DNA‐content differences for their subsequent culturing, is described. Also presented are methods for staining cell nuclei isolated from paraffin‐embedded tissues. Available algorithms are listed for deconvolution of DNA‐content‐frequency histograms to estimate percentage of cells in major phases of the cell cycle and frequency of apoptotic cells with fractional DNA content. © 2017 by John Wiley & Sons, Inc.
High‐Dimensional Fluorescence Cytometry
Authors: Ashhurst Thomas Myles , Smith Adrian Lloyd , King Nicholas Jonathan Cole
Published: November 2017

The immune system consists of a complex network of cells, all expressing a wide range of surface and/or intracellular proteins. Using flow cytometry, these cells can be analyzed by labeling with fluorophore‐conjugated antibodies. The recent expansion of fluorescence flow cytometry technology, in conjunction with the ever‐expanding understanding of the complexity of the immune system, has led to the generation of larger high‐dimensional fluorescence flow cytometry panels. However, as panel size and complexity increases, so too does the difficulty involved in constructing high‐quality panels, in addition to the challenges of analyzing such high‐dimensional datasets. As such, this unit seeks to review the key principles involved in building high‐dimensional panels, as well as to guide users through the process of building and analyzing quality panels. Here, cytometer configuration, fluorophore brightness, spreading error, antigen density, choosing the best conjugates, titration, optimization, and data analysis will all be addressed. © 2017 by John Wiley & Sons, Inc.
Screening Fusion Tags for Improved Recombinant Protein Expression in E. coli with the Expresso® Solubility and Expression Screening System
Authors: Steinmetz Eric J. , Auldridge Michele E.
Published: November 2017

The simplicity, speed, and low cost of bacterial culture make E. coli the system of choice for most initial trials of recombinant protein expression. However, many heterologous proteins are either poorly expressed in bacteria, or are produced as incorrectly folded, insoluble aggregates that lack the activity of the native protein. In many cases, fusion to a partner protein can allow for improved expression and/or solubility of a difficult target protein. Although several different fusion partners have gained favor, none are universally effective, and identifying the one that best improves soluble expression of a given target protein is an empirical process. This unit presents a strategy for parallel screening of fusion partners for enhanced expression or solubility. The Expresso® Solubility and Expression Screening System includes a panel of seven distinct fusion partners and utilizes an extremely simple cloning strategy to enable rapid screening and identification of the most effective fusion partner. © 2017 by John Wiley & Sons, Inc.
Correlation between maternal anxiety, reactivity of fetal cerebral circulation to auditory stimulation, and birth outcome in normotensive and gestational hypertensive women
Authors: Vujović Marina , Sovilj Mirjana , Jeličić Ljiljana , Stokić Miodrag , Plećaš Darko , Plešinac Snežana , Nedeljković Nadežda
Published: November 2017

This study investigated the correlation between maternal anxiety and blood flow changes through the fetal middle cerebral artery (MCA) after defined acoustic stimulation in 43 normotensive (C) and 40 gestational hypertensive (GH) subjects. Neonatal outcomes (gestational age at birth, Apgar score, birth weight) in the C and GH groups were analyzed. State (STAI‐S) and trait (STAI‐T) anxiety was assessed using Spielberger's questionnaire. The MCA blood flow was assessed once between 28 and 41 weeks of gestation using color Doppler ultrasound before and after application of defined acoustic stimulus. Relative size of the Pulsatility index (Pi) change (RePi) was calculated. The general hypotheses were: (1) women in GH group would have higher anxiety; (2) higher anxiety correlates with higher RePi change and poorer neonatal outcome; (3) fetuses from the GH group would have poorer neonatal outcome. Subjects from the GH group had higher STAI‐T and RePi compared to the C group. A positive correlation between RePi and STAI‐S, STAI‐T, and systolic/diastolic blood pressure was found in both groups. There were more preterm deliveries in the GH group compared to the C group. A significant effect of STAI‐T on body weight was observed in the C and GH group. There was a predictive effect of STAI‐T and RePi on the C group, and STAI‐S, STAI‐T, diastolic blood pressure, and RePi on the GH group in terms of neonatal body weight. This study demonstrates an association between antenatal anxiety in GH women and increased fetal cerebral circulation in response to defined auditory stimulation.
Characterization of calbindin D28k expressing interneurons in the ventral horn of the mouse spinal cord
Authors: Floyd Taylor L. , Dai Yiyun , Ladle David R.
Published: November 2017

Background: Expression of the calcium binding protein, calbindin (CB), is well established as a hallmark of Renshaw cells, a class of interneurons found in spatially restricted areas in the ventral spinal cord that directly modulate motor neuron activity. CB expression, however, is not restricted only to Renshaw cells in the ventral horn, and within this population other interneuron subtypes may be identifiable on the basis of cell position and the potential for coexpression of other calcium binding proteins. Results: Here we have quantified the changing CB expression pattern in the ventral spinal cord across postnatal development in the mouse. Fewer neurons express CB as postnatal development progresses, and those neurons frequently coexpress other calcium binding proteins (calretinin and parvalbumin) in subpopulations with distinct spatial distributions. We also found a significant portion of CB‐expressing interneurons receive putative synaptic contacts from primary sensory afferents. Conclusions: These findings suggest CB labels a heterogeneous group of interneurons in the ventral horn, some of which may process sensory information. Based on cellular position, CB expression may be a shared feature of subsets of interneurons arising from multiple ventral progenitor domains. Developmental Dynamics 247:185–193, 2018. © 2017 Wiley Periodicals, Inc.
Evaluating porewater polycyclic aromatic hydrocarbon–related toxicity at a contaminated sediment site using a spiked field-sediment approach
Authors: Hartzell Sharon E. , Unger Michael A. , Vadas George G. , Yonkos Lance T.
Published: November 2017

Although the complexity of contaminant mixtures in sediments can confound the identification of causative agents of adverse biological response, understanding the contaminant(s) of primary concern at impacted sites is critical to sound environmental management and remediation. In the present study, a stock mixture of 18 polycyclic aromatic hydrocarbon (PAH) compounds was prepared to reflect the variety and relative proportions of PAHs measured in surface sediment samples collected from discrete areas of a historically contaminated industrial estuary. This site-specific PAH stock mixture was spiked into nontoxic in-system and out-of-system field-collected reference sediments in dilution series spanning the range of previously measured total PAH concentrations from the region. Spiked sediments were evaluated in 10-d Leptocheirus plumulosus tests to determine whether toxicity in laboratory-created PAH concentrations was similar to the toxicity found in field-collected samples with equivalent PAH concentrations. The results show that toxicity of contaminated sediments was not explained by PAH exposure, while indicating that toxicity in spiked in-system (fine grain, high total organic carbon [TOC]) and out-of-system (course grain, low TOC) sediments was better explained by porewater PAH concentrations, measured using an antibody-based biosensor that quantified 3- to 5-ring PAHs, than total sediment PAH concentrations. The study demonstrates the application of site-specific spiking experiments to evaluate sediment toxicity at sites with complex mixtures of multiple contaminant classes and the utility of the PAH biosensor for rapid sediment-independent porewater PAH analysis. Environ Toxicol Chem 2018;37:893–902. © 2017 SETAC
Linking in vitro estrogenicity to adverse effects in the inland silverside (Menidia beryllina)
Authors: Mehinto Alvine C. , Kroll Kevin J. , Jayasinghe B. Sumith , Lavelle Candice M. , VanDervort Darcy , Adeyemo Olanike K. , Bay Steven M. , Maruya Keith A. , Denslow Nancy D.
Published: November 2017

High-throughput cell assays that detect and integrate the response of multiple chemicals acting via a common mode of action have the potential to enhance current environmental monitoring practices. Establishing the linkage between in vitro and in vivo responses is key to demonstrating that in vitro cell assays can be predictive of ecologically relevant outcomes. The present study investigated the potency of 17β-estradiol (E2), estrone (E1), nonylphenol (NP), and treated wastewater effluent using the readily available GeneBLAzer® estrogen receptor transactivation assay and 2 life stages of the inland silverside (Menidia beryllina). In vitro estrogenic potencies were ranked as follows: E2 > E1 >> NP. All 3 model estrogens induced vitellogenin and choriogenin expression in a dose-dependent manner in larvae and juveniles. However, apical effects were only found for E2 and E1 exposures of juveniles, which resulted in female-skewed sex ratios. Wastewater effluent samples exhibiting low in vitro estrogenicity (below the 10% effective concentration [EC10]), did not cause significant changes in M. beryllina. Significant induction of estrogen-responsive genes was observed at concentrations 6 to 26 times higher than in vitro responses. Gonadal feminization occurred at concentrations at least 19 to 26 times higher than the in vitro responses. These findings indicated that in vitro cell assays were more sensitive than the fish assays, making it possible to develop in vitro effect thresholds protective of aquatic organisms. Environ Toxicol Chem 2018;37:884–892. © 2017 SETAC

Abstract
Sustainable coffee‐based CO2 adsorbents: toward a greener production via hydrothermal carbonization
Authors: Querejeta Nausika , Gil M. Victoria , Rubiera Fernando , Pevida Covadonga
Published: November 2017

Activated carbons for adsorption of CO2 under flue gas conditions were prepared by means of hydrothermal carbonization (HTC) and subsequent CO2 activation of spent coffee grounds. The HTC of the samples consisted of their heating at moderate temperature with a high water content in autoclave. A preliminary screening concluded that 1:2 biomass/water ratio (spent coffee grounds as received) and no chemicals added during HTC with further activation in CO2 at 800°C for 1 h are suitable conditions to produce the CO2 adsorbents. In addition, the response surface methodology (RSM) successfully evaluated the combined effect of HTC temperature and dwell time, to maximize the CO2 capture capacity within the experimental region. Both the lowest temperature and dwell time (120°C, 3 h) resulted in the maximum CO2 capture capacity (2.95 wt.%). Two activated carbons (ACs) were then produced: one via hydrothermal carbonization optimized by means of response surface methodology (RSM) followed by CO2 activation (HC‐Co) and the other one by single‐step CO2 activation as described in the Group's patent ES2526259 (AC‐Co). Analysis of the features and performances of the two ACs revealed superior chemical and textural characteristics on HC‐Co for CO2 adsorption under post‐combustion capture conditions; HTC process is the sole responsible of this enhancement. Moreover, the proposed methodology to produce CO2 adsorbents from spent coffee grounds represents a more energy‐efficient approach. © 2017 Society of Chemical Industry and John Wiley & Sons, Ltd.
Motion artifact in studies of functional connectivity: Characteristics and mitigation strategies
Authors: Satterthwaite Theodore D. , Ciric Rastko , Roalf David R. , Davatzikos Christos , Bassett Danielle S. , Wolf Daniel H.
Published: November 2017

Motion artifacts are now recognized as a major methodological challenge for studies of functional connectivity. As in‐scanner motion is frequently correlated with variables of interest such as age, clinical status, cognitive ability, and symptom severity, in‐scanner motion has the potential to introduce systematic bias. In this article, we describe how motion‐related artifacts influence measures of functional connectivity and discuss the relative strengths and weaknesses of commonly used denoising strategies. Furthermore, we illustrate how motion can bias inference, using a study of brain development as an example. Finally, we highlight directions of ongoing and future research, and provide recommendations for investigators in the field. Hum Brain Mapp, 40:2033–2051, 2019. © 2017 Wiley Periodicals, Inc.
Four in vivo g‐ratio‐weighted imaging methods: Comparability and repeatability at the group level
Authors: Ellerbrock Isabel , Mohammadi Siawoosh
Published: November 2017

A recent method, denoted in vivo g‐ratio‐weighted imaging, has related the microscopic g‐ratio, only accessible by ex vivo histology, to noninvasive MRI markers for the fiber volume fraction (FVF) and myelin volume fraction (MVF). Different MRI markers have been proposed for g‐ratio weighted imaging, leaving open the question which combination of imaging markers is optimal. To address this question, the repeatability and comparability of four g‐ratio methods based on different combinations of, respectively, two imaging markers for FVF (tract‐fiber density, TFD, and neurite orientation dispersion and density imaging, NODDI) and two imaging markers for MVF (magnetization transfer saturation rate, MT, and, from proton density maps, macromolecular tissue volume, MTV) were tested in a scan–rescan experiment in two groups. Moreover, it was tested how the repeatability and comparability were affected by two key processing steps, namely the masking of unreliable voxels (e.g., due to partial volume effects) at the group level and the calibration value used to link MRI markers to MVF (and FVF). Our data showed that repeatability and comparability depend largely on the marker for the FVF (NODDI outperformed TFD), and that they were improved by masking. Overall, the g‐ratio method based on NODDI and MT showed the highest repeatability (90%) and lowest variability between groups (3.5%). Finally, our results indicate that the calibration procedure is crucial, for example, calibration to a lower g‐ratio value (g = 0.6) than the commonly used one (g = 0.7) can change not only repeatability and comparability but also the reported dependency on the FVF imaging marker. Hum Brain Mapp 39:24–41, 2018. © 2017 Wiley Periodicals, Inc.
MicroRNA‐21 and Dicer are dispensable for hepatic stellate cell activation and the development of liver fibrosis
Authors: Caviglia Jorge Matias , Yan Jun , Jang Myoung‐Kuk , Gwak Geum‐Youn , Affo Silvia , Yu Lexing , Olinga Peter , Friedman Richard A. , Chen Xin , Schwabe Robert F.
Published: November 2017

Fibrosis and cancer represent two major complications of chronic liver disease. MicroRNAs have been implicated in the development of fibrosis and cancer, thus constituting potential therapeutic targets. Here, we investigated the role of microRNA‐21 (miR‐21), a microRNA that has been implicated in the development of fibrosis in multiple organs and has also been suggested to act as an “oncomir.” Accordingly, miR‐21 was the microRNA that showed the strongest up‐regulation in activated hepatic stellate cells (HSCs) in multiple models of fibrogenesis, with an 8‐fold to 24‐fold induction compared to quiescent HSCs. However, miR‐21 antisense inhibition did not suppress the activation of murine or human HSCs in culture or in liver slices. Moreover, genetic deletion of miR‐21 in two independently generated knockout mice or miR‐21 antisense inhibition did not alter HSC activation or liver fibrosis in models of toxic and biliary liver injury. Despite a strong up‐regulation of miR‐21 in injury‐associated hepatocellular carcinoma and in cholangiocarcinoma, miR‐21 deletion or antisense inhibition did not reduce the development of liver tumors. As inhibition of the most up‐regulated microRNA did not affect HSC activation, liver fibrosis, or fibrosis‐associated liver cancer, we additionally tested the role of microRNAs in HSCs by HSC‐specific Dicer deletion. Although Dicer deletion decreased microRNA expression in HSCs and altered the expression of select genes, it only exerted negligible effects on HSC activation and liver fibrosis. Conclusion: Genetic and pharmacologic manipulation of miR‐21 does not inhibit the development of liver fibrosis and liver cancer. Moreover, suppression of microRNA synthesis does not significantly affect HSC phenotype and activation. (Hepatology 2018;67:2414‐2429).
Does HPV status influence survival after vulvar cancer?
Authors: Rasmussen Christina Louise , Sand Freja Lærke , Hoffmann Frederiksen Marie , Kaae Andersen Klaus , Kjær Susanne K.
Published: November 2017

High‐risk human papillomavirus (HPV) infection is essential in the carcinogenesis of a substantial part of anogenital and oropharyngeal cancers and has additionally been shown to be a possible predictive marker for survival, especially in oropharyngeal cancer. Studies examining the influence of HPV status on survival after vulvar cancer have been conflicting and limited by small study populations. Therefore, the aim of this review and meta‐analysis was to examine whether HPV status influences survival after vulvar cancer, which, to our knowledge, has not been done before. We conducted a systematic search of PubMed, Cochrane Library and Embase to identify studies examining survival after histologically verified and HPV tested vulvar cancer. A total of 18 studies were eligible for inclusion. Study‐specific and pooled HRs of the 5‐year OS and DFS were calculated using a fixed effects model. The I2 statistic was used to describe heterogeneity. The studies included a total of 1,638 women with HPV tested vulvar cancers of which 541 and 1,097 were HPV‐positive and HPV‐negative, respectively. Fifteen studies included only squamous cell carcinomas. We found a pooled HR of 0.61 (95% CI: 0.48–0.77) and 0.75 (95% CI: 0.57–1.00) for 5‐year OS and DFS, respectively. Across study heterogeneity was moderate to high (OS: I2 = 51%; DFS: I2 = 73%). In conclusion, women with HPV‐positive vulvar cancers have a superior survival compared to women with HPV‐negative, which could be of great clinical interest and provides insight into the differences in the natural history of HPV‐positive and negative vulvar cancers.
Osteoblast-Derived Extracellular Vesicles Are Biological Tools for the Delivery of Active Molecules to Bone
Authors: Cappariello Alfredo , Loftus Alexander , Muraca Maurizio , Maurizi Antonio , Rucci Nadia , Teti Anna
Published: November 2017

Extracellular vesicles (EVs) are newly appreciated regulators of tissue homeostasis and a means of intercellular communication. Reports have investigated the role of EVs and their cargoes in cellular regulation and have tried to fine-tune their biotechnological use, but to date very little is known on their function in bone biology. To investigate the relevance of EV-mediated communication between bone cells, we isolated EVs from primary mouse osteoblasts and assessed membrane integrity, size, and structure by transmission electron microscopy (TEM) and fluorescence-activated cell sorting (FACS). EVs actively shuttled loaded fluorochromes to osteoblasts, monocytes, and endothelial cells. Moreover, osteoblast EVs contained mRNAs shared with donor cells. Osteoblasts are known to regulate osteoclastogenesis, osteoclast survival, and osteoclast function by the pro-osteoclastic cytokine, receptor activator of nuclear factor κ-B ligand (Rankl). Osteoblast EVs were enriched in Rankl, which increased after PTH treatment. These EVs were biologically active, supporting osteoclast survival. EVs isolated from rankl–/– osteoblasts lost this pro-osteoclastic function, indicating its Rankl-dependence. They integrated ex vivo into murine calvariae, and EV-shuttled fluorochromes were quickly taken up by the bone upon in vivo EV systemic administration. Rankl–/– mice lack the osteoclast lineage and are negative for its specific marker tartrate-resistant acid phosphatase (TRAcP). Treatment of rankl–/– mice with wild-type osteoblast EVs induced the appearance of TRAcP-positive cells in an EV density-dependent manner. Finally, osteoblast EVs internalized and shuttled anti-osteoclast drugs (zoledronate and dasatinib), inhibiting osteoclast activity in vitro and in vivo. We conclude that osteoblast EVs are involved in intercellular communication between bone cells, contribute to the Rankl pro-osteoclastic effect, and shuttle anti-osteoclast drugs, representing a potential means of targeted therapeutic delivery. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.
Retracted: Effect of microRNA‐128 on cisplatin resistance of glioma SHG‐44 cells by targeting JAG1
Authors: Yi Dong‐Ye , Su Qing , Zhang Fang‐Cheng , Fu Peng , Zhang Qing , Cen Yong‐Cun , Zhao Hong‐Yang , Xiang Wei
Published: November 2017

This current study intends to investigate the effect of microRNA‐128 (miR‐128) on cisplatin (DDP) resistance in glioma SHG‐44 cells. SHG‐44/DDP cells were transfected with miR‐128 antisense oligonucleotide (ASO) and assigned into blank, resistance, NC, anti‐miR‐128, miR‐128 mimic, si‐JAG1, and anti‐miR‐128 + si‐JAG1 groups. qRT‐PCR and Western blotting were employed for determining expression of miR‐128, JAG1, Bax and Bcl‐2. MTT assay, Giemsa staining, and flow cytometry were applied to detect DDP resistance, cellular morphology, and cell cycle, respectively. JAG1 is targeted and negatively regulated by miR‐128. In in vitro experiments, compared with the blank group, the rest groups exhibited declined miR‐28 and Bax expression, lowered cell inhibition rate and apoptosis rate, but elevated JAG1 and Bcl‐2 expression with cells arrested in the S phase. Compared with the resistance group, the anti‐miR‐128 group showed decreasedBax expression along with a lowered cell inhibition rate and apoptosis rate, but increased JAG1 and Bcl‐2 expression with reduced cells arrested in the S phase; while the miR‐128 mimic group showed an opposite trend; the si‐JAG1 group showed decreased Bcl‐2 expression and reduced cells in the S phase. In in vivo experiments, compared with the resistance group, the tumor growth rate, tumor volume, and weight as well as JAG1 expression accelerated in the anti‐miR‐128 group; whereas the miR‐128 mimic and si‐JAG1 groups exhibited an opposite trend. Our findings demonstrated that miR‐128 ASO transfection might down‐regulate the expression of miR‐128 in SHG‐44/DDP and up‐regulate the DDP resistance in SHG‐44/DDP cells, providing a potential treatment target for glioma.
Expanding the CRISPR/Cas9 toolkit for Pichia pastoris with efficient donor integration and alternative resistance markers
Authors: Weninger Astrid , Fischer Jasmin E. , Raschmanová Hana , Kniely Claudia , Vogl Thomas , Glieder Anton
Published: November 2017

Komagataella phaffii (syn. Pichia pastoris) is one of the most commonly used host systems for recombinant protein expression. Achieving targeted genetic modifications had been hindered by low frequencies of homologous recombination (HR). Recently, a CRISPR/Cas9 genome editing system has been implemented for P. pastoris enabling gene knockouts based on indels (insertion, deletions) via non‐homologous end joining (NHEJ) at near 100% efficiency. However, specifically integrating homologous donor cassettes via HR for replacement studies had proven difficult resulting at most in ∼20% correct integration using CRISPR/Cas9. Here, we demonstrate the CRISPR/Cas9 mediated integration of markerless donor cassettes at efficiencies approaching 100% using a ku70 deletion strain. The Ku70p is involved in NHEJ repair and lack of the protein appears to favor repair via HR near exclusively. While the absolute number of transformants in the Δku70 strain is reduced, virtually all surviving transformants showed correct integration. In the wildtype strain, markerless donor cassette integration was also improved up to 25‐fold by placing an autonomously replicating sequence (ARS) on the donor cassette. Alternative strategies for improving donor cassette integration using a Cas9 nickase variant or reducing off targeting associated toxicity using a high fidelity Cas9 variant were so far not successful in our hands in P. pastoris. Furthermore we provide Cas9/gRNA expression plasmids with a Geneticin resistance marker which proved to be versatile tools for marker recycling. The reported CRSIPR‐Cas9 tools can be applied for modifying existing production strains and also pave the way for markerless whole genome modification studies in P. pastoris.
Retracted: MicroRNA‐29b‐3p prevents Schistosoma japonicum‐induced liver fibrosis by targeting COL1A1 and COL3A1
Authors: Tao Ran , Fan Xiang‐Xue , Yu Hai‐Jing , Ai Guo , Zhang Hong‐Yue , Kong Hong‐Yan , Song Qi‐Qin , Huang Yu , Huang Jia‐Quan , Ning Qin
Published: November 2017

Schistosomiasis is one of the world's major public health problems in terms of morbidity and mortality, causing granulomatous inflammation and cumulative fibrosis. This study explored in vivo and vitro effects of miR‐29b‐3p in granulomatous liver fibrosis by targeting COL1A1 and COL3A1 in Schistosoma japonicum infection. Thirty male Balb/c mice were assigned to normal control and model (percutaneous infection of cercariae of S. japonicum) groups. NIH‐3T3 mouse embryonic fibroblasts were designated into blank, NC, miR‐29b‐3p mimic, TGF‐β1, TGF‐β1 + NC, and TGF‐β1 + miR‐29b‐3p mimic groups. HE and Masson staining were employed to observe the pathological changes and collagenous fibrosis. The expression of α‐SMA, COL1A1, COL3A1, TIMP‐1 was determined by immunohistochemistry. The RT‐qPCR, Western blotting and immunofluorescence staining were conducted to determine expression of miR‐29b‐3p, COL1A1, and COL3A1. CCK‐8 assay and flow cytometry were performed to evaluate viability and apoptosis. The relative expression of miR‐29b‐3p decreased in the model group. The model group showed marked fibrosis in liver tissues. The expression of α‐SMA, COL1A1, COL3A1, TIMP‐1 was higher in the model group than that in the normal control group. Dual luciferase reporter gene assay revealed that miR‐29b‐3p directly targeted COL1A1 and COL3A1. Compared with the blank, NC, TGF‐β1 and TGF‐β1 + NC groups, the miR‐29b‐3p mimic group exhibited up‐regulated expression of miR‐29b‐3p and MMP‐9 but down‐regulated expression of TIMP‐1, HSP47, α‐SMA, COL1A1, and COL3A1; while lower cell viability but higher apoptosis rate showed. It indicated that miR‐29b‐3p prevents S. japonicum‐induced liver fibrosis by inhibiting COL1A1 and COL3A1.
The mechanism and potential targets of class II HDACs in angiogenesis
Authors: Hou Fei , Li Dandan , Yu Honglian , Kong Qingsheng
Published: November 2017

Angiogenesis refers to the new blood vessels deriving from the existing blood vessels, and it is a complex regulatory process. Angiogenesis is associated with the normal development of the body and tumor growth and migration. The imbalance of histone deacetylase, as an epigenetic modification, could induce the production of diseases, such as cancer, metabolic diseases, etc., and it also plays an important role in angiogenesis. Many researches indicate that class II HDACs nuclear shuttle and its phosphorylation are necessary for the diseases and the protection of the collective itself. This paper will make a review for the relationship between II HDACs and angiogenesis under physiological and pathologic categories, looking forward to the disease treatment in the future.
Retracted: In vivo and in vitro effects of PTH1‐34 on osteogenic and adipogenic differentiation of human bone marrow‐derived mesenchymal stem cells through regulating microRNA‐155
Authors: Han Huan‐Sheng , Ju Fang , Geng Shuo
Published: November 2017

This study aimed to investigate the effect of parathyroid hormone (PTH1‐34) on osteogenic and adipogenic differentiation of hBMSCs by the regulation of miR‐155. A total of 36 adult volunteers were selected in this study. Effects of PTH1‐34 on the proliferation of hBMSCs and miR‐155 expression were investigated using a MTT assay. The hBMSCs were divided into blank, PTH1‐34, miR‐155 mimic, miR‐155 mimic negative control (NC), miR‐155 inhibitor, miR‐155 inhibitor NC, PTH1‐34 + miR‐155 mimic, PTH1‐34 + miR‐155 inhibitor, and NPS R‐568 groups. Postmenopausal osteoporosis (PMOP) mouse models were established by ovariectomy (OVX) and divided into ten groups. The RT‐qPCR and Western blotting assay were carried out to detect the expression of osteogenesis/adipogenic‐related genes and miR‐155, and osteogenesis/adipogenic‐related proteins. PTH1‐34 could promote hBMSCs proliferation and inhibit miR‐155 expression in a dose‐dependent manner. Compared with the blank control group, expressions of Runx2, and BSP was up‐regulated in the PTH1‐34 and miR‐155 inhibitor groups, while expressions of miR‐155, PPAR‐γ, lipoprotein lipase (LPL), fatty acid binding protein 4 (Fabp4), adiponectin, and CCAAT/enhancer binding protein α (C/EBPα) were down‐regulated. In the in vivo experiment, compared with the OVX group, the BMD and expressions of Runx2 and BSP was up‐regulated in the PTH1‐34, miR‐155 inhibitor, and PTH1‐34 + miR‐155 inhibitor groups, while expressions of PPAR‐γ, LPL, Fabp4, adiponectin, and C/EBPα were down‐regulated. The miR‐155 mimic and NPS R‐568 groups followed opposite trends. PTH1‐34 could promote the osteogenic differentiation and inhibits adipogenic differentiation of hBMSCs through down‐regulating miR‐155 in PMOP mice.
LncRNA CASC2 inhibited the viability and induced the apoptosis of hepatocellular carcinoma cells through regulating miR‐24‐3p
Authors: Fan Ji‐Chang , Zeng Fei , Le Yi‐Guan , Xin Lin
Published: November 2017

Cancer susceptibility candidate 2 (CASC2), a recently discovered long non‐coding RNA (lncRNA), was confirmed to play numerous roles in several human cancers. However, the involvement and concrete mechanism of CASC2 in hepatocellular carcinoma (HCC) still need to be further elucidated. The relative expressions of CASC2 and miR‐24‐3p in HCC tissue and cell lines were determined by quantitative real‐time PCR (qRT‐PCR). The effects of CASC2 and miR‐24‐3p on HCC cells were further assessed via cell viability and apoptosis. In vivo tumorigenesis assay was performed to verify the inhibition effect of CASC2 on the tumor growth and further clarify the important role of miR‐24‐3p in this mechanism. Compared with the paired normal tissues, the relative expression of CASC2 significantly reduced in the HCC tissues, while miR‐24‐3p as determined by qRT‐PCR obviously increased in the HCC tissues. This observation was also found in HCC cell lines. Meanwhile, the expression of CASC2 was negatively related to miR‐24‐3p expression in the HCC tissues (r = −0.804, P < 0.001). CASC2 could negatively regulate the expression of miR‐24‐3p in vitro. Moreover, CASC2 overexpression resulted in the growth inhibitory and apoptosis‐inducing effects on HCC cells, but the up‐regulation of miR‐24‐3p greatly eliminated the CASC2‐induced effects. The tumorigenesis of HCC cells was restrained significantly by CASC2 overexpression as shown by decreased tumor volume and growth rate. However, miR‐24‐3p up‐regulation rescued the inhibition of CASC2 on the tumor growth in tumor‐bearing mice. LncRNA CASC2 inhibited the viability and induced the apoptosis of HCC cells through regulating miR‐24‐3p.
Design of inhibitory peptide targeting Toxoplasma gondii RON4‐human β‐tubulin interactions by implementing structural bioinformatics methods
Authors: Vetrivel Umashankar , Nagarajan Hemavathy , Thirumudi Indhuja
Published: November 2017

Toxoplasma gondii an obligate intracellular parasite causes toxoplasmosis in homeothermic animals. Host invasion of this parasite is mediated by the formation of Moving Junction (MJ) complex which encompasses a network of microneme and Rhoptry Neck proteins (RONs) 2/4/5/8. Among these proteins, RON4 is the only cytosolic secretory protein that is considered as a crucial member, as it directly facilitates the motility of MJ complex by interacting with host tubulin. It is also prominently localized at the host‐pathogen interface during the invasion, thus projecting it as a potential drug target. The structure of RON4 is yet to be crystallized. Hence, in this study, fold recognition and Free Energy Landscape sampling was performed to predict the plausible 3D structure of RON4. Further, its interacting pattern with the reported crystal structure of human tubulin was analyzed using molecular docking. Subsequently, a β‐tubulin based inhibitory peptides were derived based on its interacting interface observed in RON4‐β‐tubulin docked complex. Following which, a stepwise validation of these peptides for various physico‐chemical properties and its homology with antimicrobial peptides were also screened. The peptide (RT_pep) surpassing all these validation filters was modeled and its stability was analysed by Molecular Dynamics simulation. To validate further, the stable conformation of the RT_pep was docked to RON4. Finally, essential molecular dynamics simulation was conducted to determine the stability and atomic motions of native RON4 and also to decipher its association with β‐tubulin and RT_pep. All these analyses cumulatively suggest the therapeutic potential of RT_pep in targeting toxoplasmosis.
HIF‐2α activated lncRNA NEAT1 promotes hepatocellular carcinoma cell invasion and metastasis by affecting the epithelial‐mesenchymal transition
Authors: Zheng Xiaowei , Zhang Yiwen , Liu Yujia , Fang Luo , Li Li , Sun Jiao , Pan Zongfu , Xin Wenxiu , Huang Ping
Published: November 2017

The aim of this study was to investigate the role of NEAT1 in hepatocellular carcinoma (HCC), and probe whether NEAT1 participate in the epithelial‐mesenchymal transition (EMT) and metastasis regulated by HIF‐2α. Expression of lncRNA NEAT1 was initially assessed in HCC tissues and in a series of HCC cell lines. The correlations between NEAT1 levels and HIF‐2α were analyzed through plasmid vector construction. The potential underlying mechanisms of NEAT1 in HCC were performed through in vitro and in vivo functional assays. Expression of NEAT1, HIF‐2α were significantly increased in HCC tissues and cell lines. Then, in vitro assays revealed that NEAT1 promotes EMT and metastasis by stimulating the inactivation of HIF‐2α in HCC. An in vivo animal model also demonstrated the cancer promotion mechanism of NEAT1. In this study, we found that the NEAT1 was high expression in HCC. NEAT1 promotes tumor cell EMT, migration and invasion capacities by stimulating the activation of HIF‐2α in HCC.
Epiregulin (EREG) is upregulated through an IL‐1β autocrine loop in Caco‐2 epithelial cells with reduced CFTR function
Authors: Massip‐Copiz Macarena , Clauzure Mariángeles , Valdivieso Ángel G. , Santa‐Coloma Tomás A.
Published: November 2017

CFTR is a cAMP‐regulated chloride channel, whose mutations produce cystic fibrosis. The impairment of CFTR activity increases the intracellular Cl− concentration, which in turn produces an increased interleukin‐1β (IL‐1β) secretion. The secreted IL‐1β then induces an autocrine positive feedback loop, further stimulating IL‐1β priming and secretion. Since IL‐1β can transactivate the epidermal growth factor receptor (EGFR), we study here the levels of expression for different EGFR ligands in Caco‐2/pRS26 cells (expressing shRNA against CFTR resulting in a reduced CFTR expression and activity). The epiregulin (EREG), amphiregulin (AREG), and heparin binding EGF like growth factor (HBEGF) mRNAs, were found overexpressed in Caco‐2/pRS26 cells. The EREG mRNA had the highest differential expression and was further characterized. In agreement with its mRNA levels, Western blots (WB) showed increased EREG levels in CFTR‐impaired cells. In addition, EREG mRNA and protein levels were stimulated by incubation with exogenous IL‐1β and inhibited by the Interleukin 1 receptor type I (IL1R1) antagonist IL1RN, suggesting that the overexpression of EREG is a consequence of the autocrine IL‐1β loop previously described for these cells. In addition, the JNK inhibitor SP600125, and the EGFR inhibitors AG1478 and PD168393, also had an inhibitory effect on EREG expression, suggesting that EGFR, activated in Caco‐2/pRS26 cells, is involved in the observed EREG upregulation. In conclusion, in Caco‐2 CFTR‐shRNA cells, the EGFR ligand EREG is overexpressed due to an active IL‐1β autocrine loop that indirectly activates EGFR, constituting new signaling effectors for the CFTR signaling pathway, downstream of CFTR, Cl−, and IL‐1β.
A high throughput method to study the physiology of E2:ERα signaling in breast cancer cells
Authors: Leone Stefano , Busonero Claudia , Acconcia Filippo
Published: November 2017

17β‐estradiol (E2) regulates diverse physiological effects including cell proliferation through the estrogen receptor α (ERα), which as a transcription factor drives gene transcription and as an extra‐nuclear localized receptor triggers the membrane‐dependent activation of diverse kinase cascades. E2 also modifies ERα intracellular levels via diverse intracellular mechanisms. In this way, the E2‐acivated ERα integrates signaling cascades with the modulation of receptor intracellular concentration and with the induction of DNA synthesis and ultimately drives cell proliferation. In turn, E2 signaling deregulation can cause many diseases including breast cancer (BC). Recently, we performed a Western blotting (WB)‐based screen to identify novel pathways affecting ERα intracellular levels and BC cell proliferation. However, because WB lacks high throughput potential, a high‐content method to detect all aspects of E2:ERα signaling (nuclear and extra‐nuclear receptor activity, ERα levels, E2‐induced DNA synthesis) is desirable. Here, we set up a rapid way to measure E2:ERα signaling in 96‐well plate format. To demonstrate its robustness, we also challenged 4OH‐tamoxifen resistant (Tam‐Res) BC cells with a library of anti‐cancer drugs and identified methotrexate (MTX) as a molecule inducing ERα degradation and preventing BC cell proliferation. Overall, our research provides a high‐content technique to study the physiology of E2:ERα signaling in cells and further suggests a possible anti‐ERα and anti‐proliferative use for MTX in Tam‐Res BCs.
Complete graph immersions and minimum degree
Authors: Dvořák Zdeněk , Yepremyan Liana
Published: November 2017

An immersion of a graph H in another graph G is a one‐to‐one mapping and a collection of edge‐disjoint paths in G, one for each edge of H, such that the path corresponding to the edge has endpoints and . The immersion is strong if the paths are internally disjoint from . We prove that every simple graph of minimum degree at least contains a strong immersion of the complete graph . This improves on previously known bound of minimum degree at least 200t obtained by DeVos et al. Our result supports a conjecture of Lescure and Meyniel (also independently proposed by Abu‐Khzam and Langston), which is the analogue of famous Hadwiger’s conjecture for immersions and says that every graph without a ‐immersion is ‐colorable.
Serologic and viral genome prevalence of HSV, EBV, and HCMV among healthy adults in Wuhan, China
Authors: Wen Le , Qiu Yong , Cheng Shuang , Jiang Xuan , Ma Yan‐Ping , Fang Wei , Wang Wei , Cui Jie , Ruan Qiang , Zhao Fei , Hu Fei , Luo Min‐Hua
Published: November 2017

The worldwide infection rate of herpesvirus is high, but the detailed prevalence in China, especially the central area, remains unclear. In the present study, the prevalence of herpes simplex virus (HSV), Epstein‐Barr virus (EBV), and human cytomegalovirus (HCMV) was investigated in 303 healthy adults in Wuhan, a representative city in Central China. Viral‐specific IgG and IgM titers were examined in the serum by chemiluminescent immunoassay, and the existence of viral genomic DNA in blood cells was determined by nested PCR. The overall IgG seroprevalences were 81.5%, 95.4%, and 93.7% for HSV, EBV, and HCMV, while the corresponding IgM seroprevalences were only 6.3%, 2.3%, and 0. The viral genomic DNA of HSV, EBV, and HCMV was identified in the blood samples of 5.9%, 14.2%, and 22.8% of the tested donors, respectively. Significantly, less HSV IgM‐positive samples were found in the population over 20 years old than below 20 group; female displayed higher chances for HSV IgG and genome positivity; and occupations such as waiters and medical staffs were shown to be with higher risk for HCMV genome positivity. This study provided useful reference data for the HSV, EBV, and HCMV prevalence in central China, and suggested the potential importance of detecting viral genome to complement serum test data.
Dynamics of viral load in Crimean Congo hemorrhagic fever
Authors: Hasanoglu Imran , Guner Rahmet , Carhan Ahmet , K. Tufan Zeliha , Y. Caglayik Dilek , Yilmaz Gul R. , Tasyaran Mehmet A.
Published: November 2017

Crimean Congo hemorrhagic fever (CCHF) is a viral zoonotic disease with high mortality rate. There are only a few studies on viral load in CCHF. In our study, we revealed the dynamics of viral load and its relationship with mortality in early phase of the disease. A total of 138 serum samples were collected from 23 patients. All patients had positive PCR for CCHF on admission. Serum samples were obtained daily from all patients for the first 6 days of hospitalization and stored at −80°C for viral load measurement. We found statistically significant difference between mean number of viremic serum samples of fatal and non‐fatal patients. Furthermore, non‐fatal cases' viral loads demonstrated statistically significant decreases over time; however, we could not observe a similar trend in viral loads of fatal cases. Limited number of studies on CCHF indicate that score of the contest between CCHF virus and immune system determines the survival in CCHF and viral load is found to be the most prognostic factor. In our study, we found that there is a notable decrease trend in viral loads of non‐fatal patients over time and this clearance of CCHF virus is significantly related with survival.
Using 3D printing techniques to create an anthropomorphic thorax phantom for medical imaging purposes
Authors: Hazelaar Colien , van Eijnatten Maureen , Dahele Max , Wolff Jan , Forouzanfar Tymour , Slotman Ben , Verbakel Wilko F.A.R.
Published: November 2017

Purpose

Imaging phantoms are widely used for testing and optimization of imaging devices without the need to expose humans to irradiation. However, commercially available phantoms are commonly manufactured in simple, generic forms and sizes and therefore do not resemble the clinical situation for many patients.

Methods

Using 3D printing techniques, we created a life‐size phantom based on a clinical CT scan of the thorax from a patient with lung cancer. It was assembled from bony structures printed in gypsum, lung structures consisting of airways, blood vessels >1 mm, and outer lung surface, three lung tumors printed in nylon, and soft tissues represented by silicone (poured into a 3D‐printed mold).

Results

Kilovoltage x‐ray and CT images of the phantom closely resemble those of the real patient in terms of size, shapes, and structures. Surface comparison using 3D models obtained from the phantom and the 3D models used for printing showed mean differences <1 mm for all structures. Tensile tests of the materials used for the phantom show that the phantom is able to endure radiation doses over 24,000 Gy.

Conclusions

It is feasible to create an anthropomorphic thorax phantom using 3D printing and molding techniques. The phantom closely resembles a real patient in terms of spatial accuracy and is currently being used to evaluate x‐ray‐based imaging quality and positional verification techniques for radiotherapy.