bluuebunny/crossref_metadata_2025_split huggingface dataset:
A simple, cost-effective method for generating murine colonic 3D enteroids and 2D monolayers for studies of primary epithelial cell function
Authors: Fernando Elizabeth H. , Dicay Michael , Stahl Martin , Gordon Marilyn H. , Vegso Andrew , Baggio Cristiane , Alston Laurie , Lopes Fernando , Baker Kristi , Hirota Simon , McKay Derek M. , Vallance Bruce , MacNaughton Wallace K. 
Published: July 2017

Cancer cell lines have been the mainstay of intestinal epithelial experimentation for decades, due primarily to their immortality and ease of culture. However, because of the inherent biological abnormalities of cancer cell lines, many cellular biologists are currently transitioning away from these models and toward more representative primary cells. This has been particularly challenging, but recent advances in the generation of intestinal organoids have brought the routine use of primary cells within reach of most epithelial biologists. Nevertheless, even with the proliferation of publications that use primary intestinal epithelial cells, there is still a considerable amount of trial and error required for laboratories to establish a consistent and reliable method to culture three-dimensional (3D) intestinal organoids and primary epithelial monolayers. We aim to minimize the time other laboratories spend troubleshooting the technique and present a standard method for culturing primary epithelial cells. Therefore, we have described our optimized, high-yield, cost-effective protocol to grow 3D murine colonoids for more than 20 passages and our detailed methods to culture these cells as confluent monolayers for at least 14 days, enabling a wide variety of potential future experiments. By supporting and expanding on the current literature of primary epithelial culture optimization and detailed use in experiments, we hope to help enable the widespread adoption of these innovative methods and allow consistency of results obtained across laboratories and institutions. NEW & NOTEWORTHY Primary intestinal epithelial monolayers are notoriously difficult to maintain culture, even with the recent advances in the field. We describe, in detail, the protocols required to maintain three-dimensional cultures of murine colonoids and passage these primary epithelial cells to confluent monolayers in a standardized, high-yield and cost-effective manner.
Automated full-range pressure-volume curves in mice and rats
Authors: Robichaud Annette , Fereydoonzad Liah , Limjunyawong Nathachit , Rabold Richard , Allard Benoit , Benedetti Andrea , Martin James G. , Mitzner Wayne 
Published: July 2017

Pressure-volume (PV) curves constructed over the entire lung volume range can reliably detect functional changes in mouse models of lung diseases. In the present study, we constructed full-range PV curves in healthy and elastase-treated mice using either a classic manually operated technique or an automated approach using a computer-controlled piston ventilator [flexiVent FX; Scientific Respiratory Equipment (SCIREQ), Montreal, Quebec, Canada]. On the day of the experiment, subjects were anesthetized, tracheotomized, and mechanically ventilated. Following an initial respiratory mechanics scan and degassing of the lungs with 100% O2, full-range PV curves were constructed using either the classic or the automated technique. In control mice, superimposable curves were obtained, and statistical equivalence was attained between the two methodologies. In the elastase-treated ones, where significant changes in respiratory mechanics and lung volumes were expected, very small differences were observed between the two techniques, and the criteria for statistical equivalence were met in two out of four parameters assessed. The automated technique was adapted to rats and used to estimate the functional residual capacity (FRC) by volume subtraction. This novel approach generated FRC estimates consistent with the literature, with added accuracy relative to the existing method in diseased subjects. In conclusion, the automated technique generated full-range PV curves that were equivalent or very close to those obtained with the classic method under physiological or severe pathological conditions. The automation facilitated some technical aspects of the procedure, eased its use across species, and helped derive a more accurate estimate of FRC in preclinical models of respiratory disease.NEW & NOTEWORTHY Partial and full-range pressure-volume (PV) curves are frequently used to characterize lung disease models. Whereas automated techniques exist to construct partial PV curves, a manually operated approach is classically employed to build the full-range ones. In this study, the full-range PV curve technique was automated using a computer-controlled piston ventilator. The automation simplified the technique, facilitated its extension to other species, and inspired a novel way of estimating the functional residual capacity in laboratory rodents.
Infiltrating T Cells Increase IDO1 Expression in Glioblastoma and Contribute to Decreased Patient Survival
Authors: Zhai Lijie , Ladomersky Erik , Lauing Kristen L. , Wu Meijing , Genet Matthew , Gritsina Galina , Győrffy Balázs , Brastianos Priscilla K. , Binder David C. , Sosman Jeffrey A. , Giles Francis J. , James Charles D. , Horbinski Craig , Stupp Roger , Wainwright Derek A. 
Published: July 2017

AbstractPurpose: Indoleamine 2,3 dioxygenase 1 (IDO1) mediates potent immunosuppression in multiple preclinical models of cancer. However, the basis for elevated IDO1 expression in human cancer, including the most common primary malignant brain tumor in adults, glioblastoma (GBM), is poorly understood. The major objective of this study is to address this gap in our understanding of how IDO1 expression contributes to the biology of GBM, and whether its level of expression is a determinant of GBM patient outcome.Experimental Design: Patient-resected GBM, The Cancer Genome Atlas, human T-cell:GBM cocultures, as well as nu/nu, NOD-scid, and humanized (NSG-SGM3-BLT) mice-engrafted human GBM form the basis of our investigation.Results: In situ hybridization for IDO1 revealed transcript expression throughout patient-resected GBM, whereas immunohistochemical IDO1 positivity was highly variable. Multivariate statistical analysis revealed that higher levels of IDO1 transcript predict a poor patient prognosis (P = 0.0076). GBM IDO1 mRNA levels positively correlated with increased gene expression for markers of cytolytic and regulatory T cells, in addition to decreased patient survival. Humanized mice intracranially engrafted human GBM revealed an IFNγ-associated T-cell–mediated increase of intratumoral IDO1.Conclusions: Our data demonstrate that high intratumoral IDO1 mRNA levels correlate with a poor GBM patient prognosis. It also confirms the positive correlation between increased GBM IDO1 levels and human-infiltrating T cells. Collectively, this study suggests that future efforts aimed at increasing T-cell–mediated effects against GBM should consider combinatorial approaches that coinhibit potential T-cell–mediated IDO1 enhancement during therapy. Clin Cancer Res; 23(21); 6650–60. ©2017 AACR.
Mechanisms of Resistance to NTRK Inhibitors and Therapeutic Strategies in NTRK1-Rearranged Cancers
Authors: Fuse Miho J. , Okada Koutaroh , Oh-hara Tomoko , Ogura Hayato , Fujita Naoya , Katayama Ryohei 
Published: July 2017

Abstract
Neurotrophic receptor tyrosine kinase 1 (NTRK1) gene rearrangement leads to constitutive activation of NTRK1, which induces high-transforming ability. NTRK-rearranged cancers have been identified in several cancer types, such as glioblastoma, non–small cell lung cancer, and colorectal cancer. Although there are currently no clinically approved inhibitors that target NTRK1, several tyrosine kinase inhibitors (TKI), such as entrectinib and LOXO-101, are in clinical trials. The purpose of this study was to identify potential mechanisms of resistance to NTRK inhibitors and find potential therapeutic strategies to overcome the resistance. We examined the sensitivity of TPM3-NTRK1-transformed Ba/F3 cells and TPM3-NTRK1-harboring KM12 cells to multiple NTRK inhibitors. Acquired NTRK inhibitor-resistant mutations were screened by N-ethyl-N-nitrosourea mutagenesis with Ba/F3-TPM3-NTRK1 cells or by the establishment of NTRK-TKI-resistant cells from KM12 cells continuously treated with NTRK-TKIs. We identified multiple novel NTRK-TKI resistance mutations in the NTRK1 kinase domain, including G595R, and insulin growth factor receptor type 1 (IGF1R) bypass pathway-mediated resistance. After identifying the resistance mechanisms, we performed drug screening with small-molecule inhibitors to overcome the resistance. As a result, we found that ponatinib and nintedanib effectively inhibited the survival of TPM3-NTRK1-G667C but not G595R mutants, both of which showed resistance to entrectinib or larotrectinib (LOXO-101). Furthermore, cabozantinib with an IGF1R inhibitor such as OSI-906 could overcome bypass pathway-mediated resistance. We developed a comprehensive model of acquired resistance to NTRK inhibitors in cancer with NTRK1 rearrangement and identified cabozantinib as a therapeutic strategy to overcome the resistance. Mol Cancer Ther; 16(10); 2130–43. ©2017 AACR.
TTK Inhibitors as a Targeted Therapy forCTNNB1(β-catenin) Mutant Cancers
Authors: Zaman Guido J.R. , de Roos Jeroen A.D.M. , Libouban Marion A.A. , Prinsen Martine B.W. , de Man Jos , Buijsman Rogier C. , Uitdehaag Joost C.M. 
Published: July 2017

AbstractThe spindle assembly checkpoint kinase TTK (Mps1) is a key regulator of chromosome segregation and is the subject of novel targeted therapy approaches by small-molecule inhibitors. Although the first TTK inhibitors have entered phase I dose escalating studies in combination with taxane chemotherapy, a patient stratification strategy is still missing. With the aim to identify a genomic biomarker to predict the response of tumor cells to TTK inhibitor therapy, we profiled a set of preclinical and clinical TTK inhibitors from different chemical series on a panel of 66 genetically characterized cell lines derived from different tumors (Oncolines). Cell lines harboring activating mutations in the CTNNB1 gene, encoding the Wnt pathway signaling regulator β-catenin, were on average up to five times more sensitive to TTK inhibitors than cell lines wild-type for CTNNB1. The association of CTNNB1-mutant status and increased cancer cell line sensitivity to TTK inhibition was confirmed with isogenic cell line pairs harboring either mutant or wild-type CTNNB1. Treatment of a xenograft model of a CTNNB1-mutant cell line with the TTK inhibitor NTRC 0066-0 resulted in complete inhibition of tumor growth. Mutations in CTNNB1 occur at relatively high frequency in endometrial cancer and hepatocellular carcinoma, which are known to express high TTK levels. We propose mutant CTNNB1 as a prognostic drug response biomarker, enabling the selection of patients most likely to respond to TTK inhibitor therapy in proof-of-concept clinical trials. Mol Cancer Ther; 16(11); 2609–17. ©2017 AACR.
mTOR (Mechanistic Target of Rapamycin) Inhibition Decreases Mechanosignaling, Collagen Accumulation, and Stiffening of the Thoracic Aorta in Elastin-Deficient Mice
Authors: Jiao Yang , Li Guangxin , Li Qingle , Ali Rahmat , Qin Lingfeng , Li Wei , Qyang Yibing , Greif Daniel M. , Geirsson Arnar , Humphrey Jay D. , Tellides George 
Published: July 2017

Objective—

Elastin deficiency because of heterozygous loss of an
ELN
allele in Williams syndrome causes obstructive aortopathy characterized by medial thickening and fibrosis and consequent aortic stiffening. Previous work in
Eln
-null mice with a severe arterial phenotype showed that inhibition of mTOR (mechanistic target of rapamycin), a key regulator of cell growth, lessened the aortic obstruction but did not prevent early postnatal death. We investigated the effects of mTOR inhibition in
Eln
-null mice partially rescued by human
ELN
that manifest a less severe arterial phenotype and survive long term.

Approach and Results—
Thoracic aortas of neonatal and juvenile mice with graded elastin deficiency exhibited increased signaling through both mTOR complex 1 and 2. Despite lower predicted wall stress, there was increased phosphorylation of focal adhesion kinase, suggestive of greater integrin activation, and increased transforming growth factor-β–signaling mediators, associated with increased collagen expression. Pharmacological blockade of mTOR by rapalogs did not improve luminal stenosis but reduced mechanosignaling (in delayed fashion after mTOR complex 1 inhibition), medial collagen accumulation, and stiffening of the aorta. Rapalog administration also retarded somatic growth, however, and precipitated neonatal deaths. Complementary, less-toxic strategies to inhibit mTOR via altered growth factor and nutrient responses were not effective.

Conclusions—
In addition to previously demonstrated therapeutic benefits of rapalogs decreasing smooth muscle cell proliferation in the absence of elastin, we find that rapalogs also prevent aortic fibrosis and stiffening attributable to partial elastin deficiency. Our findings suggest that mTOR-sensitive perturbation of smooth muscle cell mechanosensing contributes to elastin aortopathy.
In Vivo Base Editing of PCSK9 (Proprotein Convertase Subtilisin/Kexin Type 9) as a Therapeutic Alternative to Genome Editing
Authors: Chadwick Alexandra C. , Wang Xiao , Musunuru Kiran 
Published: July 2017

Objective—

High-efficiency genome editing to disrupt therapeutic target genes, such as
PCSK9
(proprotein convertase subtilisin/kexin type 9), has been demonstrated in preclinical animal models, but there are safety concerns because of the unpredictable nature of cellular repair of double-strand breaks, as well as off-target mutagenesis. Moreover, precise knock-in of specific nucleotide changes—whether to introduce or to correct gene mutations—has proven to be inefficient in nonproliferating cells in vivo. Base editors comprising CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats [CRISPR]-CRISPR-associated 9) fused to a cytosine deaminase domain can effect the alteration of cytosine bases to thymine bases in genomic DNA in a sequence-specific fashion, without the need for double-strand DNA breaks. The efficacy of base editing has not been established in vivo. The goal of this study was to assess whether in vivo base editing could be used to modify the mouse
Pcsk9
gene in a sequence-specific fashion in the liver in adult mice.

Approach and Results—

We screened base editors for activity in cultured cells, including human-induced pluripotent stem cells. We then delivered a base editor into the livers of adult mice to assess whether it could introduce site-specific nonsense mutations into the
Pcsk9
gene. In adult mice, this resulted in substantially reduced plasma PCSK9 protein levels (>50%), as well as reduced plasma cholesterol levels (≈30%). There was no evidence of off-target mutagenesis, either cytosine-to-thymine edits or indels.

Conclusions—
These results demonstrate the ability to precisely introduce therapeutically relevant nucleotide variants into the genome in somatic tissues in adult mammals, as well as highlighting a potentially safer alternative to therapeutic genome editing.
ADPase CD39 Fused to Glycoprotein VI‐Fc Boosts Local Antithrombotic Effects at Vascular Lesions
Authors: Degen Heidrun , Borst Oliver , Ziegler Melanie , Mojica Munoz Ann‐Katrin , Jamasbi Janina , Walker Britta , Göbel Silvia , Fassbender Julia , Adler Kristin , Brandl Richard , Münch Götz , Lorenz Reinhard , Siess Wolfgang , Gawaz Meinrad , Ungerer Martin 
Published: July 2017

Background

GPVI (Glycoprotein VI) is the essential platelet collagen receptor in atherothrombosis. Dimeric
GPVI
‐Fc (Revacept) binds to
GPVI
binding sites on plaque collagen. As expected, it did not increase bleeding in clinical studies.
GPVI
‐Fc is a potent inhibitor of atherosclerotic plaque‐induced platelet aggregation at high shear flow, but its inhibition at low shear flow is limited. We sought to increase the platelet inhibitory potential by fusing
GPVI
‐Fc to the ectonucleotidase
CD
39 (fusion protein GPVI‐CD39), which inhibits local ADP accumulation at vascular plaques, and thus to create a lesion‐directed dual antiplatelet therapy that is expected to lack systemic bleeding risks.

Methods and Results

GPVI
‐
CD
39 effectively stimulated local
ADP
degradation and, compared with
GPVI
‐Fc alone, led to significantly increased inhibition of
ADP
‐, collagen‐, and human plaque–induced platelet aggregation in Multiplate aggregometry and plaque‐induced platelet thrombus formation under arterial flow conditions.
GPVI
‐
CD
39 did not increase bleeding time in an in vitro assay simulating primary hemostasis. In a mouse model of ferric chloride–induced arterial thrombosis,
GPVI
‐
CD
39 effectively delayed vascular thrombosis but did not increase tail bleeding time in vivo.

Conclusions

GPVI‐CD39 is a novel approach to increase local antithrombotic activity at sites of atherosclerotic plaque rupture or injury. It enhances
GPVI
‐Fc–mediated platelet inhibition and presents a potentially effective and safe molecule for the treatment of acute atherothrombotic events, with a favorable risk–benefit ratio.
The vascular basement membrane in the healthy and pathological brain
Authors: Thomsen Maj S , Routhe Lisa J , Moos Torben 
Published: July 2017

The vascular basement membrane contributes to the integrity of the blood-brain barrier (BBB), which is formed by brain capillary endothelial cells (BCECs). The BCECs receive support from pericytes embedded in the vascular basement membrane and from astrocyte endfeet. The vascular basement membrane forms a three-dimensional protein network predominantly composed of laminin, collagen IV, nidogen, and heparan sulfate proteoglycans that mutually support interactions between BCECs, pericytes, and astrocytes. Major changes in the molecular composition of the vascular basement membrane are observed in acute and chronic neuropathological settings. In the present review, we cover the significance of the vascular basement membrane in the healthy and pathological brain. In stroke, loss of BBB integrity is accompanied by upregulation of proteolytic enzymes and degradation of vascular basement membrane proteins. There is yet no causal relationship between expression or activity of matrix proteases and the degradation of vascular matrix proteins in vivo. In Alzheimer’s disease, changes in the vascular basement membrane include accumulation of Aβ, composite changes, and thickening. The physical properties of the vascular basement membrane carry the potential of obstructing drug delivery to the brain, e.g. thickening of the basement membrane can affect drug delivery to the brain, especially the delivery of nanoparticles.
Brain atrophy following hemiplegic migraine attacks
Authors: Pelzer Nadine , Hoogeveen Evelien S , Ferrari Michel D , Poll-The Bwee Tien , Kruit Mark C , Terwindt Gisela M 
Published: July 2017

Background Patients with hemiplegic migraine (HM) may sometimes develop progressive neurological deterioration of which the pathophysiology is unknown. Patient We report a 16-year clinical and neuroradiological follow-up of a patient carrying a de novo p.Ser218Leu CACNA1A HM mutation who had nine severe HM attacks associated with seizures and decreased consciousness between the ages of 3 and 12 years. Results Repeated ictal and postictal neuroimaging revealed cytotoxic oedema during severe HM attacks in the symptomatic hemisphere, which later showed atrophic changes. In addition, progressive cerebellar atrophy was observed. Brain atrophy halted after cessation of severe attacks, possibly due to prophylactic treatment with flunarizine and sodium valproate. Conclusion Severe HM attacks may result in brain atrophy and prophylactic treatment of these attacks might be needed in an early stage of disease to prevent permanent brain damage.
Cortical Button Versus Cross-pin Femoral Fixation for Hamstring Anterior Cruciate Ligament Reconstruction: A Meta-analysis of Randomized Controlled Trials
Authors: Jiang Hai , Ma Guangzhi , Li Qi , Hu Yanqing , Li Jian , Tang Xin 
Published: July 2017

Background: Incidences of graft rupture are associated with postoperative knee laxity after anterior cruciate ligament (ACL) reconstruction. Reports of postoperative knee laxity after ACL reconstruction using different femoral fixation techniques in several studies are controversial. Purpose: To compare, via meta-analysis of randomized controlled trials (RCTs), the clinical outcomes and postoperative knee laxity of autogenous hamstring ACL reconstruction using cortical button versus cross-pin femoral fixation. Study Design: Meta-analysis. Methods: This study followed the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. The online PubMed, EMBASE, and Cochrane Central Register of Controlled Trials databases were searched from inception to April 1, 2017. The study included only level 1 or 2 RCTs that compared cortical button and cross-pin femoral fixation for ACL reconstruction with hamstring autografts and that reported clinical outcomes or postoperative knee laxity. The Cochrane Collaboration’s risk of bias tool was used to assess the risk of bias for all included studies. For the meta-analysis, the investigators extracted data on clinical outcomes measured by postoperative International Knee Documentation Committee (IKDC) score or Lysholm score and postoperative knee laxity defined as >5 mm side-to-side difference by the arthrometric measurement, Lachman test ≥2+, and pivot-shift test ≥2+. The risk ratio (RR) and its corresponding 95% confidence interval (CI) were computed for dichotomous data. Heterogeneity was assessed by I2 tests. Results: A total of 6 RCTs with 445 patients were included. Statistical analysis of pooled data showed no significant difference between the cortical button and cross-pin groups on postoperative IKDC score (RR, 0.94; 95% CI, 0.88-1.02; P = .13; I2 = 4%) and Lysholm score (RR, 0.97; 95% CI, 0.91-1.04; P = .45; I2 = 0%). Postoperative knee laxity was reported in 5 studies, and no significant difference was found between the 2 groups (RR, 1.49; 95% CI, 0.83-2.68; P = .18; I2 = 37%). Conclusion: Cortical button femoral fixation for autogenous hamstring ACL reconstruction had no significant difference in terms of clinical outcomes and postoperative knee laxity compared with cross-pin femoral fixation.