bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanically Strong Globular‐Protein‐Based Fibers Obtained Using a Microfluidic Spinning Technique
Authors: He Haonan , Yang Chenjing , Wang Fan , Wei Zheng , Shen Jianlei , Chen Dong , Fan Chunhai , Zhang Hongjie , Liu Kai
Published: December 2019

Proteins used for the formation of light weight and mechanically strong biological fibers are typically composed of folded rigid and unfolded flexible units. In contrast to fibrous proteins, globular proteins are generally not regarded as a good candidate for fiber production due to their intrinsic structural defects. Thus, it is challenging to develop an efficient strategy for the construction of mechanically strong fibers using spherical proteins. Herein, we demonstrate the production of robust protein fibers from bovine serum albumin (BSA) using a microfluidic technique. Remarkably, the toughness of the fibers was up to 143 MJ m−3, and after post‐stretching treatment, their breaking strength increased to almost 300 MPa due to the induced long‐range ordered structure in the fibers. The performance is comparable to or even higher than that of many recombinant spider silks or regenerated silkworm fibers. Thus, this work opens a new way for making biological fibers with high performance.
Transformation of Receptor Tyrosine Kinases into Glutamate Receptors and Photoreceptors
Authors: Leippe Philipp , Broichhagen Johannes , Cailliau Katia , Mougel Alexandra , Morel Marion , Dissous Colette , Trauner Dirk , Vicogne Jérôme
Published: December 2019

Receptor tyrosine kinases (RTKs) are key regulators of cellular functions in metazoans. In vertebrates, RTKs are mostly activated by polypeptides but are not naturally sensitive to amino acids or light. Taking inspiration from Venus kinase receptors (VKRs), an atypical family of RTKs found in nature, we have transformed the human insulin (hIR) and hepatocyte growth factor receptor (hMET) into glutamate receptors by replacing their extracellular binding domains with the ligand‐binding domain of metabotropic glutamate receptor type 2 (mGluR2). We then imparted light sensitivity through covalent attachment of a synthetic glutamate‐based photoswitch via a self‐labelling SNAP tag. By employing a Xenopus laevis oocyte kinase activity assay, we demonstrate how these chimeric RTKs, termed light‐controlled human insulin receptor (LihIR) and light‐controlled human MET receptor (LihMET), can be used to exert optical control over the insulin or MET signaling pathways. Our results outline a potentially general strategy to convert RTKs into photoreceptors.
Waterborne polyurethane‐based dye with covalently bonded to Disperse blue 60
Authors: Hu Xianhai , Li Mingjun , Xian Yuxi , Liu Xiang , Liu Manli , Li Gen , Hu Pengwei , Cheng Congliang
Published: December 2019

A series of sulfonated waterborne polyurethane‐based dyes (DB60‐SWPUs) with covalently bonded to different proportions of Disperse blue 60 (DB60) were prepared by a modified acetone process. The expected structures of DB60‐SWPUs are characterized by Fourier transform infrared spectroscopy and1H‐NMR spectra. The obtained DB60‐SWPUs show high viscosity and intriguing storage stability. The fluorescent performances, thermal properties, and mechanical properties of DB60‐SWPUs were characterized. Compared with DB60, the UV–vis spectra of DB60‐WPUs have obvious hypochromic shift, and the fluorescence intensities are obviously enhanced. The migration rates of DB60‐SWPUs are much lower than that of DB60. DB60‐SWPUs have thermal migration resistance and excellent color fastness. The polyester fiber dyed by DB60‐SWPUs has not only high color yield, good fracture strength, excellent breaking strength retention, but also good permeability and wrinkle resistance. Moreover, DB60‐SWPUs are polymeric dyes with water as a solvent, which are especially suitable for environmental protection. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci.2020,137, 48862.
Ultrasound‐Assisted Enzyme‐Catalyzed Hydrolysis of Collagen to Produce Peptides With Biomedical Potential: Collagenase From Aspergillus terreus UCP1276
Authors: Costa‐Junior Romualdo B. , Brandão‐Costa Romero M. P. , Albuquerque Wendell W. C. , Batista Juanize M. S. , Pedrosa Raquel B. , Porto Ana L. F.
Published: December 2019

Ultrasound has been applied for varied purposes as it provides additional mechanical energy to a system, and is still profitable and straightforward, which are advantages for industrial applications. In this work, ultrasonic treatments were applied to purified collagenase fractions from a fermented extract by Aspergillus terreus UCP 1276 aiming to evaluate the potential effect on collagen hydrolysis. The physical agent was evaluated as an inductor of collagen degradation and consequently as a producer of peptides with anticoagulant activity. The sodium dodecyl sulphate‐polyacrylamide gel electrophoresis analyses were also carried out to compare the hydrolysis techniques. The ultrasound (40 kHz, 47.4 W/L) processing was conducted under the same conditions of pH and temperature at different times. The ultrasound‐assisted reaction was accelerated in relation to conventional processing. Collagenolytic activity was enhanced and tested in the presence of phenylmethanesulfonyl fluoride inhibitor. Underexposure, the activity was enhanced, reaching more than 72.0% of improvement in relation to the non‐exposed enzyme. A period of 30 min of incubation under ultrasound exposure was enough to efficiently produce peptides with biological activity, including anticoagulation and effect on prothrombin time at about 60%. The results indicate that low‐frequency ultrasound is an enzymatic inducer with likely commercial applicability accelerating the enzymatic reaction. Bioelectromagnetics. 2020;41:113–120. © 2019 Bioelectromagnetics Society.
A Phenazine based colorimetric and fluorescent chemosensor for sequential detection of Ag+ and I− in aqueous media
Authors: Dongare Pravin R. , Gore Anil H. , Kolekar Govind B. , Ajalkar Balu D.
Published: December 2019

A new colorimetric and fluorescent probe MNTPZ based on 1H‐imidazo[4,5‐b]phenazine derivative has been designed and synthesized for successive detection of Ag+ and I−. The probe MNTPZ shows selective colorimetric response by a change in color from yellow to orange and “turn‐off” fluorometric response upon binding with Ag+ in DMSO: Water (pH = 7, 1:1, v/v) over other cations. The binding mode of probe MNTPZ to Ag+ was studied by Job's plot, 1H NMR studies, FT‐IR spectroscopy and DFT calculations. Moreover, the situ generated probe MNTPZ+ Ag+ complex acted as an efficient fluorometric “turn‐on” probe for I− via Ag+ displacement approach. The detection limit of probe MNTPZ for Ag+ and the resultant complex probe MNTPZ+ Ag+ for I− were determined to be 1.36 μmol/L and 1.03 μmol/L respectively. Notably, the developed probe was successfully used for quantitative determination of I− in real samples with satisfactory results.
Short-term outcomes of a multicentre randomized clinical trial comparing D2 versus D3 lymph node dissection for colonic cancer (COLD trial)
Authors: Karachun A , Panaiotti L , Chernikovskiy I , Achkasov S , Gevorkyan Y , Savanovich N , Sharygin G , Markushin L , Sushkov O , Aleshin D , Shakhmatov D , Nazarov I , Muratov I , Maynovskaya O , Olkina A , Lankov T , Ovchinnikova T , Kharagezov D , Kaymakchi D , Milakin A , Petrov A
Published: December 2019

Background

It remains unclear whether extended lymphadenectomy provides oncological advantages in colorectal cancer. This multicentre RCT aimed to address this issue.

Methods

Patients with resectable primary colonic cancer were enrolled in four hospitals registered in the COLD trial, and randomized to D2 or D3 dissection in a 1 : 1 ratio. Data were analysed to assess the safety of D3 dissection.

Results

The study included the first 100 patients randomized in this ongoing trial. Ninety-nine patients were included in the intention-to-treat (ITT) analysis (43 D2, 56 D3). Ninety-two patients received the allocated treatment and were included in the per-protocol (PP) analysis: 39 of 43 in the D2 group and 53 of 56 in the D3 group. There were no deaths. The 30-day postoperative morbidity rate was 47 per cent in the D2 group and 48 per cent in the D3 group, with a risk ratio of 1·04 (95 per cent c.i. 0·68 to 1·58) (P = 0·867). There were two anastomotic leaks (5 per cent) in the D2 group and none in the D3 group. Postoperative recovery, complication and readmission rates did not differ between the groups in ITT and PP analyses. Mean lymph node yield was 26·6 and 27·8 in D2 and D3 procedures respectively. Good quality of complete mesocolic excision was more frequently noted in the D3 group (P = 0·048). Three patients in the D3 group (5 per cent) had metastases in D3 lymph nodes. D3 was never the only affected level of lymph nodes. N-positive status was more common in the D3 group (46 per cent versus 26 per cent in D2), with a risk ratio of 1·81 (95 per cent c.i. 1·01 to 3·24) (P = 0·044).

Conclusion

D3 lymph node dissection is feasible and may be associated with better N staging. Registration number: NCT03009227 (http://www.clinicaltrials.gov).
Protective Effects of Anthocyanins from Coreopsis tinctoria against Oxidative Stress Induced by Hydrogen Peroxide in MIN6 Cells
Authors: Liu Jianli , Tian Siqi , Fu Mingyang , He Yin , Yu Hui , Cao Xiangyu , Cao Yiyang , Xu Hanyuan
Published: December 2019

Anthocyanins (AC) from Coreopsis tinctoria possesses strong antioxidant properties, while the effects of AC on cells damage induced by reactive oxygen species (ROS) in diabetes mellitus diseases progression have not been reported. The present study was carried out to evaluate the protective property of AC against cellular oxidative stress with an experimental model, H2O2‐exposed MIN6 cells. AC could reverse the decrease of cell viability induced by H2O2 and efficiently suppressed cellular ROS production and cell apoptosis. In addition, Real‐time PCR and Western blot analyses indicated that AC could protect MIN6 cells against oxidative injury through increasing the translocation of Nrf2 into nuclear, decreasing the phosphorylation level of p38 and up‐regulating the protein expression of antioxidant enzyme (SOD1, SOD2 and CAT). Thus, this study provides evidence to support the beneficial effect of AC in inhibiting MIN6 cells from H2O2‐induced oxidative injury.
Exosomal miR‐663b targets Ets2‐repressor factor to promote proliferation and the epithelial–mesenchymal transition of bladder cancer cells
Authors: Yin Xinbao , Zheng Xueping , Liu Ming , Wang Dong , Sun Hui , Qiu Yue , Chen Jun , Shi Benkang
Published: December 2019

Exosomes circulating in biological fluids have the potential to be utilized as cancer biomarkers and are associated with cancer progression and metastasis. MicroRNA (miR)‐663b has been found to be elevated in plasma from patients with bladder cancer (BC). However, the functional role of exosomal miR‐663b in BC processes remains unknown. Here, we isolated exosomes from plasma and found that the miR‐663b level was elevated in exosomes from plasma of patients with BC compared with healthy controls. Exosomal miR‐663b from BC cells promoted cell proliferation and epithelial–mesenchymal transition. Moreover, exosomal miR‐663b targeted Ets2‐repressor factor and acted as a tumor promoter in BC cells. Taken together, our findings suggested that exosomal miR‐663b is a promising potential biomarker and target for clinical detection and therapy in BC.
Impact of dual antiplatelet therapy duration on 1‐year clinical outcomes in diabetic patients with acute coronary syndrome undergoing percutaneous coronary intervention: Insights from the real‐world OPT‐CAD study
Authors: Li Jing , Li Yi , Qiu Miaohan , Li Zhiguo , Yang Jing , Wang Xiaozeng , Bao Dan , Wang Xu , Han Yaling
Published: December 2019

Objectives

To investigate the optimal dual antiplatelet therapy (DAPT) duration for diabetic patients with acute coronary syndrome (ACS) after percutaneous coronary intervention (PCI).

Background

Diabetic patients are at high risk for ischemic and bleeding events when treated with antithrombotic agents after an ACS episode.

Methods

This post hoc study analyzed DAPT duration and clinical outcomes in diabetic patients with ACS who underwent PCI in the Optimal antiPlatelet Therapy for Chinese patients with Coronary Artery Disease (OPT‐CAD) registry. The primary outcome was patient‐oriented composite endpoints (PoCE) at 12 months; it was defined as a composite of all‐cause mortality, all myocardial infarction (MI), stroke, or any revascularization. The safety outcome was bleeding events.

Results

In total, 1,773 diabetic patients with ACS were enrolled and treated with PCI in the OPT‐CAD study. Premature DAPT discontinuation before 12 months was an independent PoCE predictor in diabetic patients (hazard ratio = 1.33, 95% confidence interval: 1.02–1.74, p = .006). It was associated with a significantly higher risk of PoCE (17.3 vs. 11.2%, p = .014) in diabetic patients with high risk factors (age ≥ 65 years or history of cardiovascular events including MI, stroke, or peripheral artery disease), but this association was not observed in those without high risk factors (10.4 vs. 9.1, p = .554). No excess bleeding risk was found in patients who received 12‐month DAPT compared with those who discontinued DAPT prematurely.

Conclusions

Premature DAPT discontinuation before 12 months was associated with increased risk of clinical events in diabetic patients with ACS after PCI, particularly in those with high‐risk profiles.
Electrochemical Water Oxidation Catalyzed by N4‐Coordinate Copper Complexes with Different Backbones: Insight into the Structure‐Activity Relationship of Copper Catalysts
Authors: Shen Junyu , Zhang Xiongfei , Cheng Minglun , Jiang Jian , Wang Mei
Published: December 2019

Copper complexes with a general formula of [(L)Cu(OH2)](BF4)2 (1, L1=N,N′‐dimethyl‐N,N′‐bis(pyridin‐2‐ylmethyl)‐1,2‐diaminoethane; 2, L2=N,N′‐bis(pyridin‐2‐ylmethyl)piperazine); 3, L3=N,N′‐bis(pyridin‐2‐ylmethyl)diazepane) were prepared as molecular catalysts for oxygen evolution reaction. These catalysts have the same first coordination environment but different backbones of diamine‐dipyridine N4‐ligands. Single crystal X‐ray diffraction studies on the molecular structures of 1–3 revealed that the backbone rigidity of the N4‐ligand has apparent influence on the Npy−Cu−N′py open angle of copper complexes. Comparative studies manifested that the subtle structural change caused by the backbone rigidity of N4‐ligands has an important influence on the catalytic performance and reaction pathway of copper catalysts for electrochemical water splitting.
Non‐Conventional Media as Strategy to Overcome the Solvent Dilemma in Chemoenzymatic Tandem Catalysis
Authors: Kourist Robert , González‐Sabín Javier
Published: December 2019

The amazing potential of multi‐catalytic cascade reactions to reduce the number of reaction steps and to solve synthetic problems brings the challenge of an increasing complexity that must be controlled. Particularly chemo‐enzymatic reactions are prone to conflicts regarding different optimal reaction conditions for chemical and biological catalysts. The preference of many chemical catalysts for hydrophobic (and often water‐free) solvent and of many enzymes for water poses a solvent dilemma. Recently, non‐conventional solvents have been very successful to provide suitable reaction media for catalysts that otherwise require very different solvents, and to alleviate fundamental problems such as the low solubility of many substrates in aqueous solvents. In the last few years, several examples underlined the considerable potential of ionic liquids and deep eutectic solvents for the engineering of cascade reactions. This mini‐review showcases the recent developments on the implementation of the so‐called non‐conventional media in such processes.
Intramolecular Metal‐Free N−N Bond Formation with Heteroaromatic Amines: Mild Access to Fused‐Triazapentalene Derivatives
Authors: Daniel Matthieu , Hiebel Marie‐Aude , Guillaumet Gérald , Pasquinet Eric , Suzenet Franck
Published: December 2019

Formation of N−N bonds may offer an original approach to various nitrogen‐containing heterocycles with numerous applications. For this purpose, we found that readily available heteroaromatic amines are appropriate substrates for providing an efficient and innovative approach for the formation of N−N bonds in the presence of iodine (III) reagent in very mild conditions. This method makes it possible to synthesize nitrogen rich triazapentalene derivatives exhibiting fluorescent properties, inaccessible with existing approaches.
Efficacy and safety of dronedarone in patients with a prior ablation for atrial fibrillation/flutter: Insights from the ATHENA study
Authors: Vamos Mate , Calkins Hugh , Kowey Peter R. , Torp‐Pedersen Christian T. , Corp dit Genti Valérie , Wieloch Mattias , Koren Andrew , Hohnloser Stefan H.
Published: December 2019

Background

The role of antiarrhythmic drugs for atrial fibrillation/atrial flutter (AF/AFL) after catheter ablation is not well established.

Hypothesis

We hypothesized that changing the myocardial substrate by ablation may alter the responsiveness to dronedarone.

Methods

We assessed the efficacy and safety of dronedarone in the treatment of paroxysmal/persistent atrial fibrillation/atrial flutter (AF/AFL) post‐ablation, based on a post hoc analysis of the ATHENA study. A total of 196 patients (dronedarone 90, placebo 106) had an ablation for AF/AFL before study entry. In these patients, the effect of treatment on the first hospitalization because of cardiovascular (CV) events/all‐cause death was assessed, as was AF/AFL recurrence in individuals with sinus rhythm at baseline. The safety of dronedarone vs placebo was also determined.

Results

In patients with prior ablation, dronedarone reduced the risk of AF/AFL recurrence (hazard ratio [HR]: 0.65 [95% confidence interval [CI]: 0.42, 1.00]; P < .05) as well as the median time to first AF/AFL recurrence (561 vs 180 days) compared with placebo. The HR for first CV hospitalization/all‐cause death with dronedarone vs placebo was 0.98 (95% CI: 0.62, 1.53; P = .91). Rates of treatment‐emergent adverse events were 83.1% vs 75.5% and rates of serious TEAEs were 27.0% vs 18.9% in the dronedarone and placebo groups, respectively. One death occurred with dronedarone (not treatment‐emergent) and five occurred with placebo.

Conclusion

In patients with prior ablation for AF/AFL, dronedarone reduced the risk of AF/AFL recurrence compared with placebo, but not the risk of first CV hospitalization/all‐cause death. Safety outcomes were consistent with those of the overall ATHENA study.
Antibodies to the RNA binding protein heterogeneous nuclear ribonucleoprotein A1 contribute to neuronal cell loss in an animal model of multiple sclerosis
Authors: Libner Cole D. , Salapa Hannah E. , Hutchinson Catherine , Lee Sangmin , Levin Michael C.
Published: December 2019

Neurodegeneration, including loss of neurons and axons, is a feature of progressive forms of multiple sclerosis (MS). The mechanisms underlying neurodegeneration are mostly unknown. Research implicates autoimmunity to nonmyelin self‐antigens as important contributors to disease pathogenesis. Data from our lab implicate autoimmunity to the RNA binding protein (RBP) heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) as a possible mechanism of neurodegeneration in MS. MS patients make antibodies to hnRNP A1, which have been shown to lead to neuronal dysfunction in vitro. Using an animal model of MS, experimental autoimmune encephalomyelitis (EAE), we show here that injection of anti‐hnRNP A1 antibodies, in contrast to control antibodies, resulted in worsened disease and increased neurodegeneration. We found a reduction of NeuN+ neuronal cell bodies in areas of the ventral gray matter of the spinal cord where anti‐hnRNP A1 antibodies localized. Neurons displayed increased levels of hnRNP A1 nucleocytoplasmic mislocalization and stress granule formation, both markers of neuronal injury. Anti‐hnRNP A1 antibodies were found to surround neuronal cell bodies and interact with CD68+ immune cells via Fc receptors. Additionally, anti‐hnRNP A1 antibodies were found within neuronal cell bodies including those of the ventral spinocerebellar tract (VSCT), a tract previously shown to undergo neurodegeneration in anti‐hnRNP A1 antibody injected EAE mice. Finally, both immune cells and neurons showed increased levels of inducible nitric oxide synthase, another indicator of cell damage. These findings suggest that autoimmunity to RBPs, such as hnRNP A1, play a role in neurodegeneration in EAE with important implications for the pathogenesis of MS.
Ablation of spinal cord estrogen receptor α‐expressing interneurons reduces chemically induced modalities of pain and itch
Authors: Tran May , Braz Joao Manuel , Hamel Katherine , Kuhn Julia , Todd Andrew J. , Basbaum Allan I.
Published: December 2019

Estrogens are presumed to underlie, at least in part, the greater pain sensitivity and chronic pain prevalence that women experience compared to men. Although previous studies revealed populations of estrogen receptor‐expressing neurons in primary afferents and in superficial dorsal horn neurons, there is little to no information as to the contribution of these neurons to the generation of acute and chronic pain. Here we molecularly characterized neurons in the mouse superficial spinal cord dorsal horn that express estrogen receptor α (ERα) and explored the behavioral consequences of their ablation. We found that spinal ERα‐positive neurons are largely excitatory interneurons and many coexpress substance P, a marker for a discrete subset of nociceptive, excitatory interneurons. After viral, caspase‐mediated ablation of spinal ERα‐expressing cells, we observed a significant decrease in the first phase of the formalin test, but in male mice only. ERα‐expressing neuron‐ablation also reduced pruritogen‐induced scratching in both male and female mice. There were no ablation‐related changes in mechanical or heat withdrawal thresholds or in capsaicin‐induced nocifensive behavior. In chronic pain models, we found no change in Complete Freund's adjuvant‐induced thermal or mechanical hypersensitivity, or in partial sciatic nerve injury‐induced mechanical allodynia. We conclude that ERα labels a subpopulation of excitatory interneurons that are specifically involved in chemically evoked persistent pain and pruritogen‐induced itch.
Spatial models of cell distribution in human lumbar dorsal root ganglia
Authors: Sperry Zachariah J. , Graham Robert D. , Peck‐Dimit Nicholas , Lempka Scott F. , Bruns Tim M.
Published: December 2019

Dorsal root ganglia (DRG), which contain the somata of primary sensory neurons, have increasingly been considered as novel targets for clinical neural interfaces, both for neuroprosthetic and pain applications. Effective use of either neural recording or stimulation technologies requires an appropriate spatial position relative to the target neural element, whether axon or cell body. However, the internal three‐dimensional spatial organization of human DRG neural fibers and somata has not been quantitatively described. In this study, we analyzed 202 cross‐sectional images across the length of 31 human L4 and L5 DRG from 10 donors. We used a custom semi‐automated graphical user interface to identify the locations of neural elements in the images and normalize the output to a consistent spatial reference for direct comparison by spinal level. By applying a recursive partitioning algorithm, we found that the highest density of cell bodies at both spinal levels could be found in the inner 85% of DRG length, the outer‐most 25–30% radially, and the dorsal‐most 69–76%. While axonal density was fairly homogeneous across the DRG length, there was a distinct low density region in the outer 7–11% radially. These findings are consistent with previous qualitative reports of neural distribution in DRG. The quantitative measurements we provide will enable improved targeting of future neural interface technologies and DRG‐focused pharmaceutical therapies, and provide a rigorous anatomical description of the bridge between the central and peripheral nervous systems.
RALB‐HC: A resource‐aware load balancer for heterogeneous cluster
Authors: Ahmed Usman , Aleem Muhammad , Noman Khalid Yasir , Arshad Islam Muhammad , Azhar Iqbal Muhammad
Published: December 2019

In the heterogeneous computing environment, programmers map the applications either on CPUs or GPUs. However, this default mapping process does not produce improved results, particularly on the heterogeneous clusters. If one resource of the cluster is more compute capable, then most of the scheduling schemes favor that powerful device. In this scenario, the scheduling schemes overload the powerful resources while making all other compute resources remain under utilized. This load imbalance problem results in higher energy consumption and increased execution time. In this research, a novel Resource‐Aware Load Balancer for the Heterogeneous Cluster (RALB‐HC) is proposed that distributes workload based on resources computing capabilities and applications computing needs. The RALB‐HC uses supervised machine learning approach to classify applications using the static code‐features. The RALB‐HC framework comprises of two phases: (1) job mapping based on the availability of the resources and (2) the resource‐aware load balancing to achieve the higher resource utilization ratio. The experimental results on a large set of real‐world and synthetic workloads show that the RALB‐HC reduces execution time by 31.61%, increased resource utilization ratio by 67.8% and improved throughout 147.35% as compared to baseline scheduling schemes.
Use of the CRISPR‐Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes
Authors: Bosch Justin A. , Knight Shannon , Kanca Oguz , Zirin Jonathan , Yang‐Zhou Donghui , Hu Yanhui , Rodiger Jonathan , Amador Gabriel , Bellen Hugo J. , Perrimon Norbert , Mohr Stephanie E.
Published: December 2019

The CRISPR‐Cas9 system makes it possible to cause double‐strand breaks in specific regions, inducing repair. In the presence of a donor construct, repair can involve insertion or ‘knock‐in’ of an exogenous cassette. One common application of knock‐in technology is to generate cell lines expressing fluorescently tagged endogenous proteins. The standard approach relies on production of a donor plasmid with ∼500 to 1000 bp of homology on either side of an insertion cassette that contains the fluorescent protein open reading frame (ORF). We present two alternative methods for knock‐in of fluorescent protein ORFs into Cas9‐expressing Drosophila S2R+ cultured cells, the single‐stranded DNA (ssDNA) Drop‐In method and the CRISPaint universal donor method. Both methods eliminate the need to clone a large plasmid donor for each target. We discuss the advantages and limitations of the standard, ssDNA Drop‐In, and CRISPaint methods for fluorescent protein tagging in Drosophila cultured cells. © 2019 by John Wiley & Sons, Inc.

Basic Protocol 1: Knock‐in into Cas9‐positive S2R+ cells using the ssDNA Drop‐In approach

Basic Protocol 2: Knock‐in into Cas9‐positive S2R+ cells by homology‐independent insertion of universal donor plasmids that provide mNeonGreen (CRISPaint method)

Support Protocol 1: sgRNA design and cloning

Support Protocol 2: ssDNA donor synthesis

Support Protocol 3: Transfection using Effectene

Support Protocol 4: Electroporation of S2R+‐MT::Cas9 Drosophila cells

Support Protocol 5: Single‐cell isolation of fluorescent cells using FACS
Automated Dynamic Clamp for Simulation of IK1 in Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes in Real Time Using Patchliner Dynamite8
Authors: Becker Nadine , Horváth András , De Boer Teun , Fabbri Alan , Grad Christian , Fertig Niels , George Michael , Obergrussberger Alison
Published: December 2019

Current in vitro assays typically poorly predict cardiac liability as they focus on single ion channels overexpressed in cell lines. Human induced pluripotent stem cell–derived cardiomyocytes (hiPSC‐CMs), on the other hand, provide a unique opportunity for drug testing on human cardiomyocytes using high‐throughput systems. However, these cells can differ from adult cardiomyocytes in their ion channel expression and, therefore, electrophysiologic properties. One of the main challenges of hiPSC‐CMs is the physiologic expression of ion channels such as the inward rectifiers (e.g., Kir2.1–2.3), which conduct the cardiac inward rectifier potassium current (IK1). IK1 is one of the primary contributors in maintaining a stable resting membrane potential in cardiac cells, which is essential for excitability. This is only expressed in low levels, or sometimes not at all, in hiPSC‐CMs as shown by patch clamp studies. Dynamic clamp is a method of electronically introducing ion currents (e.g., IK1) into cells to compensate for the lack of endogenous expression, thus offering the potential to record more stable action potentials in hiPSC‐CMs. In this article, we describe the method of using hiPSC‐CMs on an automated patch clamp device (Patchliner) coupled with the automated dynamic clamp add‐on (Dynamite8). We describe protocols for optimized cell handling and harvesting for use on the Patchliner and the steps required for automated execution of experiments and data analysis in dynamic clamp mode. © 2019 by John Wiley & Sons, Inc.

Basic Protocol: Recording action potential pharmacology from human induced pluripotent stem cell–derived cardiomyocytes in automated patch clamp combined with dynamic clamp to introduce simulated IK1 and compensate seal resistance

Support Protocol 1: Cardiomyocyte plating and culture

Support Protocol 2: Cell harvesting and dissociation

Alternate Protocol: Recording action potential pharmacology at physiologic temperatures
Magnetic Resonance Relaxometry for Determination of Protein Concentration and Aggregation
Authors: Taraban Marc B. , Briggs Katharine T. , Yu Yihua Bruce
Published: December 2019

The water‐proton signal, overwhelmingly considered a nuisance in nuclear magnetic resonance spectroscopy, is advantageously used as a tool to assess protein concentration and to detect protein aggregates in aqueous solutions. The protocols in this article describe use of the water‐proton transverse relaxation rate to determine concentration and aggregate content in protein solutions. Detailed recommendations and description of the parameter settings and data processing ensure successful implementation of this technique, even by a user with limited experience in magnetic resonance relaxometry. All measurements are done noninvasively, in a sealed container, without sampling or otherwise aliquoting the solution. The magnetic resonance relaxometry approach offered in this article could be advantageous for analysis of biologics formulations or when use of conventional analytical techniques is not possible. © 2019 by John Wiley & Sons, Inc.

Basic Protocol 1: Nuclear magnetic resonance (NMR) relaxometry to measure protein concentration

Basic Protocol 2: NMR relaxometry to measure protein aggregation
A potential association between IL‐3 and type I and III interferons in systemic lupus erythematosus
Authors: Oon Shereen , Monaghan Katherine , Ng Milica , Hoi Alberta , Morand Eric , Vairo Gino , Maraskovsky Eugene , Nash Andrew D , Wicks Ian P , Wilson Nicholas J
Published: December 2019

Objectives

Plasmacytoid dendritic cells (pDCs), through the production of type 1 interferons (IFNs) and other cytokines, are major contributors to systemic lupus erythematosus (SLE) pathogenesis. IL‐3 promotes pDC survival, but its role in SLE is not well characterised. This study investigated serum IL‐3 and IFN levels, and a whole blood ‘IL‐3 gene signature’, in human SLE.

Methods

Serum cytokine levels were measured by ELISA in n = 42 SLE patients, and n = 44 healthy donors. IL‐3‐regulated genes were determined by RNASeq of healthy donor whole blood cells (WBCs) stimulated in vitro with IL‐3 for 6 or 24 h. Whole blood cell RNASeq analysis was undertaken in a separate cohort of n = 31 SLE patients, and n = 28 healthy donors.

Results

Serum IL‐3 levels correlated with IFNα (r = 0.612, 95% CI 0.455–0.733, P < 0.001) and type III IFN (r = 0.585, 95% CI 0.406–0.720, P < 0.0001). IL‐3 stimulation of WBC in vitro altered 794 genes (−1 ≥ logFC ≥ 1, FDR < 0.05), of which 35 overlapped with genes differentially expressed between SLE and healthy donors. These 35 genes were expressed in 27/31 SLE donors, revealing the presence of an ‘IL‐3 gene signature’. There was strong correlation between the IL‐3 signature and an IFN signature, as determined by hierarchical clustering of the 500 most variable genes in SLE donors (r = 0.939, 95% CI 0.898–0.964, P < 0.0001).

Conclusion

A dual IL‐3/IFN gene signature is a feature of SLE. An association between IL‐3 and IFN raises the possibility that dual blockade of IL‐3 and IFN may be especially useful for SLE patients with this dual cytokine gene signature.
Bone marrow infiltration by flow cytometry at diffuse large B‐cell lymphoma NOS diagnosis implies worse prognosis without considering bone marrow histology
Authors: Martín‐Moro Fernando , Piris‐Villaespesa Miguel , Marquet‐Palomanes Juan , García‐Cosío Mónica , Villarrubia Jesús , Lario Ana , García Irene , Michael Berta , Roldán Ernesto , García‐Vela José Antonio , Lopez‐Jiménez Javier
Published: December 2019

Background

The significance of discrepant findings between histology (BMB) and flow cytometry (FC) in bone marrow (BM) examination at diffuse large B‐cell lymphoma (DLBCL) diagnosis is uncertain.

Methods

We performed a 5‐year retrospective single‐center study of patients diagnosed by DLBCL not otherwise specified (n = 82), divided into three groups according to BM infiltration at diagnosis: BMB−/FC− (75.6%), BMB+/FC+ (13.4%), and BMB−/FC+ (11%).

Results

Median infiltration by FC analysis of the BMB−/FC+ group was 0.8% and if we considered BM infiltration as positive in all cases, 4/9 would be upstaged. Median follow was 33 months. Event‐free survival (EFS) after 18 months was 82, 23, and 27% for BMB−/FC−, BMB−/FC+, and BMB+/FC+, respectively (p < .001). After 18 months of observation, OS was 87, 46, and 55% for BMB−/FC−, BMB−/FC+, and BMB+/FC+, respectively (p = .001). In multivariate analysis (BM infiltration vs. cell‐of‐origin according to Hans algorithm and standard IPI), BM infiltration was independently associated with EFS (HR: 1.94, 95% CI: 1.3–2.9) and overall survival (HR: 1.69, 95% CI: 1.1–2.7).

Conclusion

In summary, minimal BM infiltration, detected by FC but not by BMB, has same prognostic implications than overt BM infiltration and should be considered as extranodal involvement regardless the infiltration quantity.
Fine needle aspiration of malignant melanoma with myxoid features: A case report with molecular analysis
Authors: Felty Cameron C. , Jackson Christopher R. , Marotti Jonathan D. , Liu Xiaoying
Published: December 2019

Malignant melanoma with myxoid features (MMM) is a rare melanoma variant in which tumor cells are embedded within a basophilic myxoid stroma. The stromal matrix is composed of acidic mucopolysaccharides, which are thought to be produced by mesenchymal stromal cells in response to melanoma invasion. Interestingly, this myxoid matrix is more often seen in metastasis from a primary tumor that does not have a myxoid stroma. The diagnosis of MMM on fine needle aspiration (FNA) can be confused with other myxoid tumors. Herein, we present a case of MMM diagnosed by FNA of a peri‐auricular lymph node in an 89‐year‐old man with a history of resected malignant melanoma. We describe the clinical, cytohistological, and immunohistochemical findings, and present the unique molecular alterations that were identified. We also discuss the differential diagnosis and potential diagnostic pitfalls associated with MMM.
Tcf7l2 transcription factor is required for the maintenance, but not the initial specification, of the neurotransmitter identity in the caudal thalamus
Authors: Tran Hong‐Nhung , Park Wonbae , Seong Sojeong , Jeong Ji‐eun , Nguyen Quy‐Hoai , Yoon Jaeseung , Baek Kwanghee , Jeong Yongsu
Published: December 2019

Background

Dysfunction of GABAergic and glutamatergic neurons in the brain, which establish inhibitory and excitatory networks, respectively, may cause diverse neurological disorders. The mechanism underlying the determination of GABAergic vs. glutamatergic neurotransmitter phenotype in the caudal diencephalon remains largely unknown.

Results

In this study, we investigated the consequence of Tcf7l2 (transcription factor 7‐like 2) ablation on the neurotransmitter identity of GABAergic and glutamatergic neurons in the caudal diencephalon. We identified positive and negative activity in the control of glutamatergic and GABAergic neuronal gene expression by Tcf7l2. Loss of Tcf7l2 did not alter the initial acquisition of the neurotransmitter identity in thalamic neurons. However, glutamatergic thalamic neurons failed to maintain their excitatory neurotransmitter phenotype in the absence of Tcf7l2. Moreover, a subset of Tcf7l2‐deficient thalamic neurons underwent a glutamatergic to GABAergic neurotransmitter identity switch. Our data indicate that Tcf7l2 may promote glutamatergic neuronal differentiation and repress GABAergic neurotransmitter identity in the caudal thalamus.

Conclusions

This study provides evidence for a novel and crucial role of Tcf7l2 in the molecular mechanism by which the neurotransmitter identity of glutamatergic thalamic neurons is established. Our findings exemplify a clear case of neurotransmitter identity regulation that is partitioned into initiation and maintenance phases.
Generation of a hypoxia‐sensing mouse model
Authors: Lin Qun , Huang Yan , Giordano Frank J. , Yun Zhong
Published: December 2019

Oxygen (O2) homeostasis is essential to the metazoan life. O2‐sensing or hypoxia‐regulated molecular pathways are intimately involved in a wide range of critical cellular functions and cell survival from embryogenesis to adulthood. In this report, we have designed an innovative hypoxia sensor (O2CreER) based on the O2‐dependent degradation domain of the hypoxia‐inducible factor‐1α and Cre recombinase. We have further generated a hypoxia‐sensing mouse model, R26‐O2CreER, by targeted insertion of the O2CreER‐coding cassette in the ROSA26 locus. Using the ROSAmTmG mouse strain as a reporter, we have found that this novel hypoxia‐sensing mouse model can specifically identify hypoxic cells under the pathological condition of hind‐limb ischemia in adult mice. This model can also label embryonic cells including vibrissal follicle cells in E13.5–E15.5 embryos. This novel mouse model offers a valuable genetic tool for the study of hypoxia and O2 sensing in mammalian systems under both physiological and pathological conditions.
Integrated assessment of the spatial distribution and structural dynamics of deep benthic marine communities
Authors: Jansen Jan , Dunstan Piers K. , Hill Nicole A. , Koubbi Philippe , Melbourne‐Thomas Jessica , Causse Romain , Johnson Craig R.
Published: December 2019

Characterizing the spatial distribution and variation of species communities and validating these characteristics with data from the field are key elements for an ecosystem‐based approach to management. However, models of species distributions that yield community structure are usually not linked to models of community dynamics, constraining understanding and management of the ecosystem, particularly in data‐poor regions. Here we use a qualitative network model to predict changes in Antarctic benthic community structure between major marine habitats characterized largely by seafloor depth and slope, and use multivariate mixture models of species distributions to validate the community dynamics. We then assess how future increases in primary production associated with anticipated loss of sea‐ice may affect the ecosystem. Our study shows how both spatial and structural features of ecosystems in data‐poor regions can be analyzed and possible futures assessed, with direct relevance for ecosystem‐based management.
Species‐level image classification with convolutional neural network enables insect identification from habitus images
Authors: Hansen Oskar L. P. , Svenning Jens‐Christian , Olsen Kent , Dupont Steen , Garner Beulah H. , Iosifidis Alexandros , Price Benjamin W. , Høye Toke T.
Published: December 2019

Changes in insect biomass, abundance, and diversity are challenging to track at sufficient spatial, temporal, and taxonomic resolution. Camera traps can capture habitus images of ground‐dwelling insects. However, currently sampling involves manually detecting and identifying specimens. Here, we test whether a convolutional neural network (CNN) can classify habitus images of ground beetles to species level, and estimate how correct classification relates to body size, number of species inside genera, and species identity.

We created an image database of 65,841 museum specimens comprising 361 carabid beetle species from the British Isles and fine‐tuned the parameters of a pretrained CNN from a training dataset. By summing up class confidence values within genus, tribe, and subfamily and setting a confidence threshold, we trade‐off between classification accuracy, precision, and recall and taxonomic resolution.

The CNN classified 51.9% of 19,164 test images correctly to species level and 74.9% to genus level. Average classification recall on species level was 50.7%. Applying a threshold of 0.5 increased the average classification recall to 74.6% at the expense of taxonomic resolution. Higher top value from the output layer and larger sized species were more often classified correctly, as were images of species in genera with few species.

Fine‐tuning enabled us to classify images with a high mean recall for the whole test dataset to species or higher taxonomic levels, however, with high variability. This indicates that some species are more difficult to identify because of properties such as their body size or the number of related species.

Together, species‐level image classification of arthropods from museum collections and ecological monitoring can substantially increase the amount of occurrence data that can feasibly be collected. These tools thus provide new opportunities in understanding and predicting ecological responses to environmental change.
Scientist’s guide to developing explanatory statistical models using causal analysis principles
Authors: Grace James B. , Irvine Kathryn M.
Published: December 2019

Recent discussions of model selection and multimodel inference highlight a general challenge for researchers: how to convey the explanatory content of a hypothesized model or set of competing models clearly. The advice from statisticians for scientists employing multimodel inference is to develop a well‐thought‐out set of candidate models for comparison, though precise instructions for how to do that are typically not given. A coherent body of knowledge, which falls under the general term causal analysis, now exists for examining the explanatory scientific content of candidate models. Much of the literature on causal analysis has been recently developed, and we suspect may not be familiar to many ecologists. This body of knowledge comprises a set of graphical tools and axiomatic principles to support scientists in their endeavors to create “well‐formed hypotheses,” as statisticians are asking them to do. Causal analysis is complementary to methods such as structural equation modeling, which provides the means for evaluation of proposed hypotheses against data. In this paper, we summarize and illustrate a set of principles that can guide scientists in their quest to develop explanatory hypotheses for evaluation. The principles presented in this paper have the capacity to close the communication gap between statisticians, who urge scientists to develop well‐thought‐out coherent models, and scientists, who would like some practical advice for exactly how to do that.
Soil nitrogen cycling is determined by the competition between mycorrhiza and ammonia‐oxidizing prokaryotes
Authors: Tatsumi Chikae , Taniguchi Takeshi , Du Sheng , Yamanaka Norikazu , Tateno Ryunosuke
Published: December 2019

Mycorrhizal fungi have considerable effects on soil carbon (C) storage, as they control the decomposition of soil organic matter (SOM), by modifying the amount of soil nitrogen (N) available for free‐living microbes. Through their access to organic N, ectomycorrhizal (ECM) fungi compete with free‐living soil microbes; this competition is thought to slow down SOM decomposition. However, arbuscular mycorrhizal (AM) fungi cannot decompose SOM, and therefore must wait for N to first be processed by free‐living microbes. It is unclear what form of N the ECM fungi and free‐living microbes compete for, or which microbial groups compete for N with ECM fungi. To investigate this, we focused on the N transformation steps (i.e., the degradation of high‐molecular‐weight organic matter, mineralization, and nitrification) and the microbes driving each step. Simple comparisons between AM forests and ECM forests are not sufficient to assert that mycorrhizal types would determine the N transformation steps in soil, because soil physiochemistry, which strongly affects N transformation steps, differs between the forests. We used an aridity gradient with large differences in soil moisture, pH, and SOM quantity and quality, to distinguish the mycorrhizal and physicochemical effects on N transformation. Soil samples (0–10 cm depth) were collected from AM‐symbiotic black locust forests under three aridity levels, and from ECM‐symbiotic oak forests under two aridity levels. Soil physicochemical properties, extractable N dynamics and abundance, composition, and function of soil microbial communities were measured. In ECM forests, the ammonia‐oxidizing prokaryotic abundance was low, whereas that of ECM fungi was high, resulting in lower nitrate N content than in AM forests. Since ECM forests did not have lower saprotrophic fungal abundance and prokaryotic decompositional activity than the AM forests, the hypothesis that ECM fungi could reduce SOM decay and ammonification by free‐living microbes, might not hold in ECM forests. However, the limitation of ECM fungi on nitrate N production would result in a feedback that will accelerate plant dependence on these fungi, thereby raising soil C storage through an increase in the ECM biomass and plant C investment in soils.
The Potential of β‐Hydroxy‐γ‐vinyl‐γ‐lactone in the Synthesis of Natural Products and Beyond
Authors: Fernandes Rodney A.
Published: December 2019

In this mini‐review, we have abstracted the syntheses of β‐hydroxy‐γ‐vinyl‐γ‐lactone and the different tactics and strategies adopted using this important building block in the synthesis of various natural products. Apart from this, the lactone in having a strategic vinyl bond was contemplated as allyl donor in Pd‐catalyzed allylic arylation and also in a cascade Prins‐Ritter reaction. This building block was visualized to be a desymmetrized 3,4‐dihydroxy‐hexyl unit providing orthogonal functional groups for various useful transformations. The active α‐position is available for alkylation, the γ‐vinyl bond, the lactone carbonyl and the β‐hydroxy groups add value to this building block for various possible conversions to natural products and beyond.
Asymmetric Remote Addition Reactions of Heterocycle‐Based Dearomative Dienamine or Trienamine Species to 1‐Azadienes: Application to Construct Chiral Azocanes and Azecanes
Authors: Hu Di , Gao Yang , Song Xue , Du Wei , Chen Ying‐Chun
Published: December 2019

The dearomative dienamines between a few five‐membered heteroaryl aldehydes and chiral secondary amine catalysts underwent highly regio‐, chemo‐ and stereoselective Michael additions to 1‐azadienes derived from 2‐methylenebenzofuran‐3(2H)‐ones or 2‐methylenebenzo[b]thiophen‐3(2H)‐ones. The very remote ε‐regioselective Michael‐type additions could be similarly realized by employing the analogous dearomative trienamine species from β‐indolyl unsaturated aldehydes. The resulting multifunctional products enabled the efficient construction of enantioenriched chiral azocane or even azecane frameworks with high molecular complexity.