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Donkey Anti-Rabbit IgG (H+L) Polyclonal Antibody, Alexa Fluor ?? 555 Conjugated


Radiologic and Genomic Evolution of Individual Metastases during HER2 Blockade in Colorectal Cancer.

  • Siravegna G
  • Cancer Cell
  • 2018 Jul 9

Literature context: body Thermo Fisher Cat#A-31572; RRID:AB_162543 Mouse monoclonal anti-Neu (9G6)


Targeting HER2 is effective in 24% of ERBB2 amplified metastatic colorectal cancer; however, secondary resistance occurs in most of the cases. We studied the evolution of individual metastases during treatment to discover spatially resolved determinants of resistance. Circulating tumor DNA (ctDNA) analysis identified alterations associated with resistance in the majority of refractory patients. ctDNA profiles and lesion-specific radiographic reports revealed organ- or metastasis-private evolutionary patterns. When radiologic assessments documented progressive disease in target lesions, response to HER2 blockade was retained in other metastases. Genomic and functional analyses on samples and cell models from eight metastases of a patient co-recruited to a postmortem study unveiled lesion-specific evolutionary trees and pharmacologic vulnerabilities. Lesion size and contribution of distinct metastases to plasma ctDNA were correlated.

Funding information:
  • NIEHS NIH HHS - ES06694(United States)

Cancer Lipid Metabolism Confers Antiangiogenic Drug Resistance.

  • Iwamoto H
  • Cell Metab.
  • 2018 Jul 3

Literature context: uor 555 Invitrogen Cat# A31572; RRID:AB_162543 Donkey anti-Rabbit IgG (H+L) Hi


Intrinsic and evasive antiangiogenic drug (AAD) resistance is frequently developed in cancer patients, and molecular mechanisms underlying AAD resistance remain largely unknown. Here we describe AAD-triggered, lipid-dependent metabolic reprogramming as an alternative mechanism of AAD resistance. Unexpectedly, tumor angiogenesis in adipose and non-adipose environments is equally sensitive to AAD treatment. AAD-treated tumors in adipose environment show accelerated growth rates in the presence of a minimal number of microvessels. Mechanistically, AAD-induced tumor hypoxia initiates the fatty acid oxidation metabolic reprogramming and increases uptake of free fatty acid (FFA) that stimulates cancer cell proliferation. Inhibition of carnitine palmitoyl transferase 1A (CPT1) significantly compromises the FFA-induced cell proliferation. Genetic and pharmacological loss of CPT1 function sensitizes AAD therapeutic efficacy and enhances its anti-tumor effects. Together, we propose an effective cancer therapy concept by combining drugs that target angiogenesis and lipid metabolism.

Funding information:
  • British Heart Foundation - G0802266(United Kingdom)

The Striatum Organizes 3D Behavior via Moment-to-Moment Action Selection.

  • Markowitz JE
  • Cell
  • 2018 Jun 28

Literature context: IgG Thermo-Fisher Cat# A-31572; RRID:AB_162543 Bacterial and Virus Strains


Many naturalistic behaviors are built from modular components that are expressed sequentially. Although striatal circuits have been implicated in action selection and implementation, the neural mechanisms that compose behavior in unrestrained animals are not well understood. Here, we record bulk and cellular neural activity in the direct and indirect pathways of dorsolateral striatum (DLS) as mice spontaneously express action sequences. These experiments reveal that DLS neurons systematically encode information about the identity and ordering of sub-second 3D behavioral motifs; this encoding is facilitated by fast-timescale decorrelations between the direct and indirect pathways. Furthermore, lesioning the DLS prevents appropriate sequence assembly during exploratory or odor-evoked behaviors. By characterizing naturalistic behavior at neural timescales, these experiments identify a code for elemental 3D pose dynamics built from complementary pathway dynamics, support a role for DLS in constructing meaningful behavioral sequences, and suggest models for how actions are sculpted over time.

Funding information:
  • NHLBI NIH HHS - HL24415(United States)
  • NICHD NIH HHS - P30 HD018655()
  • NIDCD NIH HHS - R01 DC011558()
  • NIDCD NIH HHS - R01 DC016222()
  • NINDS NIH HHS - U01 NS094190()
  • NINDS NIH HHS - U01 NS094191()

Evolution of Cortical Neurogenesis in Amniotes Controlled by Robo Signaling Levels.

  • Cárdenas A
  • Cell
  • 2018 Jun 20

Literature context: Invitrogen Cat# A-31572, RRID:AB_162543 Donkey Alexa488 anti-chicken Ig


Cerebral cortex size differs dramatically between reptiles, birds, and mammals, owing to developmental differences in neuron production. In mammals, signaling pathways regulating neurogenesis have been identified, but genetic differences behind their evolution across amniotes remain unknown. We show that direct neurogenesis from radial glia cells, with limited neuron production, dominates the avian, reptilian, and mammalian paleocortex, whereas in the evolutionarily recent mammalian neocortex, most neurogenesis is indirect via basal progenitors. Gain- and loss-of-function experiments in mouse, chick, and snake embryos and in human cerebral organoids demonstrate that high Slit/Robo and low Dll1 signaling, via Jag1 and Jag2, are necessary and sufficient to drive direct neurogenesis. Attenuating Robo signaling and enhancing Dll1 in snakes and birds recapitulates the formation of basal progenitors and promotes indirect neurogenesis. Our study identifies modulation in activity levels of conserved signaling pathways as a primary mechanism driving the expansion and increased complexity of the mammalian neocortex during amniote evolution.

Funding information:
  • Wellcome Trust - (United Kingdom)

Derivation and characterization of the NIH registry human stem cell line NYSCF100 line under defined feeder-free conditions.

  • Sevilla A
  • Stem Cell Res
  • 2018 Apr 10

Literature context: Scientific Alexa Fluor® 555 Cat#A-31572Secondary antibodyDonkey anti-Go


The human embryonic stem cell line NYSCFe001-A was derived from a day 6 blastocyst in feeder-free and antibiotic free conditions. The blastocyst was voluntarily donated for research as surplus after in vitro fertilization treatment following informed consent. The NYSCFe001-A line, registered as NYSCF100 on the NIH registry, presents normal karyotype, is mycoplasma free, expresses all the pluripotency markers and has the potential to differentiate into all three germ layers in vitro.

Funding information:
  • NCI NIH HHS - R01CA138998(United States)

Phase Separation of FUS Is Suppressed by Its Nuclear Import Receptor and Arginine Methylation.

  • Hofweber M
  • Cell
  • 2018 Apr 19

Literature context: a 555 Donkey anti-rabbit Thermo RRID:AB_162543 Alexa 647 Donkey anti-rabbit Th


Cytoplasmic FUS aggregates are a pathological hallmark in a subset of patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). A key step that is disrupted in these patients is nuclear import of FUS mediated by the import receptor Transportin/Karyopherin-β2. In ALS-FUS patients, this is caused by mutations in the nuclear localization signal (NLS) of FUS that weaken Transportin binding. In FTD-FUS patients, Transportin is aggregated, and post-translational arginine methylation, which regulates the FUS-Transportin interaction, is lost. Here, we show that Transportin and arginine methylation have a crucial function beyond nuclear import-namely to suppress RGG/RG-driven phase separation and stress granule association of FUS. ALS-associated FUS-NLS mutations weaken the chaperone activity of Transportin and loss of FUS arginine methylation, as seen in FTD-FUS, promote phase separation, and stress granule partitioning of FUS. Our findings reveal two regulatory mechanisms of liquid-phase homeostasis that are disrupted in FUS-associated neurodegeneration.

Funding information:
  • NIGMS NIH HHS - R01 GM48435(United States)

ATP and Odor Mixture Activate TRPM5-Expressing Microvillous Cells and Potentially Induce Acetylcholine Release to Enhance Supporting Cell Endocytosis in Mouse Main Olfactory Epithelium.

  • Fu Z
  • Front Cell Neurosci
  • 2018 Apr 5

Literature context: Fisher Scientific Cat# A-31572, RRID:AB_162543) for 1 h at room temperature. S


The main olfactory epithelium (MOE) functions to detect odor molecules, provide an epithelial surface barrier, and remove xenobiotics from inhaled air. Mechanisms coordinating the activities of different cell types within the MOE to maintain these functions are poorly understood. Previously, we showed that superficially located microvillous cells (MCs) in the MOE expressing transient receptor potential channel M5 (TRPM5) are cholinergic and chemoresponsive and that they play an important role in maintaining odor responses and olfactory-guided behavior under challenging chemical environment. Here we investigated TRPM5-MC activation and subsequent paracrine regulation. Ca2+ imaging showed that TRPM5-MCs dose-dependently increase their intracellular Ca2+ levels in response to ATP, an important signaling molecule for airway mucociliary movement, and to an odor mixture. Pharmacological examination showed that the ATP responses are primarily mediated by P2X purinergic receptors. Interestingly, using the endocytosis dye pHrodo Red dextran, we found that chemical-activated TRPM5-MCs significantly increase the number of pHrodo-labeled puncta compared to controls without stimulation and compared to cells that do not respond to ATP or to the odor mixture. These results indicate potential vesicle recycling after release of the signaling molecule acetylcholine (ACh). Interestingly, TRPM5 knockout (KO) results in a decrease in ATP-induced pHrodo internalization. We further investigated cholinergic regulation of neighboring supporting cells (SCs). We found that ACh strongly elevates intracellular Ca2+ and potentiates pHrodo endocytosis in SCs. The ACh effects are diminished in the presence of atropine or M3 muscarinic receptor antagonist and in SCs lacking M3 receptors. Collectively, these data suggest that TRPM5-MCs may regulate the MOE's multicellular network activity via cholinergic paracrine signaling for functional maintenance and adaptive plasticity.

Funding information:
  • NCI NIH HHS - CA094060(United States)
  • NIDCD NIH HHS - R01 DC012831()

Kir4.1-Dependent Astrocyte-Fast Motor Neuron Interactions Are Required for Peak Strength.

  • Kelley KW
  • Neuron
  • 2018 Apr 18

Literature context: vitrogen Cat# A-31572, RRID:AB_162543 Alexa donkey anti-rabbit 647 In


Diversified neurons are essential for sensorimotor function, but whether astrocytes become specialized to optimize circuit performance remains unclear. Large fast α-motor neurons (FαMNs) of spinal cord innervate fast-twitch muscles that generate peak strength. We report that ventral horn astrocytes express the inward-rectifying K+ channel Kir4.1 (a.k.a. Kcnj10) around MNs in a VGLUT1-dependent manner. Loss of astrocyte-encoded Kir4.1 selectively altered FαMN size and function and led to reduced peak strength. Overexpression of Kir4.1 in astrocytes was sufficient to increase MN size through activation of the PI3K/mTOR/pS6 pathway. Kir4.1 was downregulated cell autonomously in astrocytes derived from amyotrophic lateral sclerosis (ALS) patients with SOD1 mutation. However, astrocyte Kir4.1 was dispensable for FαMN survival even in the mutant SOD1 background. These findings show that astrocyte Kir4.1 is essential for maintenance of peak strength and suggest that Kir4.1 downregulation might uncouple symptoms of muscle weakness from MN cell death in diseases like ALS.

Funding information:
  • FIC NIH HHS - K01 TW000001(United States)

Anterior Cingulate Cortex Contributes to Alcohol Withdrawal- Induced and Socially Transferred Hyperalgesia.

  • Smith ML
  • eNeuro
  • 2018 Apr 25

Literature context: A-31572 also A31572 lot number RRID:AB_162543) and Alexa Fluor 488-labeled se


Pain is often described as a "biopsychosocial" process, yet social influences on pain and underlying neural mechanisms are only now receiving significant experimental attention. Expression of pain by one individual can be communicated to nearby individuals by auditory, visual, and olfactory cues. Conversely, the perception of another's pain can lead to physiological and behavioral changes in the observer, which can include induction of hyperalgesia in "bystanders" exposed to "primary" conspecifics in which hyperalgesia has been induced directly. The current studies were designed to investigate the neural mechanisms responsible for the social transfer of hyperalgesia in bystander mice housed and tested with primary mice in which hyperalgesia was induced using withdrawal (WD) from voluntary alcohol consumption. Male C57BL/6J mice undergoing WD from a two-bottle choice voluntary alcohol-drinking procedure served as the primary mice. Mice housed in the same room served as bystanders. Naïve, water-drinking controls were housed in a separate room. Immunohistochemical mapping identified significantly enhanced Fos immunoreactivity (Fos-ir) in the anterior cingulate cortex (ACC) and insula (INS) of bystander mice compared to naïve controls, and in the dorsal medial hypothalamus (DMH) of primary mice. Chemogenetic inactivation of the ACC but not primary somatosensory cortex reversed the expression of hyperalgesia in both primary and bystander mice. These studies point to an overlapping neural substrate for expression of socially transferred hyperalgesia and that expressed during alcohol WD.

Generation and characterization of two human iPSC lines from patients with methylmalonic acidemia cblB type.

  • Richard E
  • Stem Cell Res
  • 2018 Apr 17

Literature context: 00 Thermo Fischer Cat# A-31572, RRID:AB_162543 Alexa 555 Donkey anti-Mouse IgG


Two human induced pluripotent stem cell (iPSC) lines were generated from fibroblasts of two siblings with methylmalonic acidemia cblB type carrying mutations in the MMAB gene: c.287T➔C (p.Ile96Thr) and a splicing loss-of-function variant c.584G➔A affecting the last nucleotide of exon 7 in MMAB (p.Ser174Cysfs*23). Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability.

Funding information:
  • Telethon - GGP08051(Italy)

Generation of Duchenne muscular dystrophy patient-specific induced pluripotent stem cell line lacking exons 45-50 of the dystrophin gene (IITi001-A).

  • Eisen B
  • Stem Cell Res
  • 2018 Apr 14

Literature context: Life Technologies Cat# A31572, RRID:AB_162543


Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle degenerative disease caused by mutations in the dystrophin gene. We generated induced pluripotent stem cells (iPSCs) from a 13-year-old male patient carrying a deletion mutation of exons 45-50; iPSCs were subsequently differentiated into cardiomyocytes. iPSCs exhibit expression of the pluripotent markers (SOX2, NANOG, OCT4), differentiation capacity into the three germ layers, normal karyotype, genetic identity to the skin biopsy dermal fibroblasts and the patient-specific dystrophin mutation.

Funding information:
  • Howard Hughes Medical Institute - (United States)

Immunohistochemical Procedures for Characterizing the Retinal Expression Patterns of Cre Driver Mouse Lines.

  • Lu Q
  • Methods Mol. Biol.
  • 2018 Apr 26

Literature context: nkeyRabbitIgG(H + L)555Life TechA31572DonkeyRabbitIgG(H + L)488Life Te


The retina is a thin neural tissue sitting on the backside of the eye, composed of light-sensing cells, interneurons, and output ganglion neurons. The latter send electrical signals to higher visual centers in the brain. Transgenic mouse lines are becoming one of the most valuable mammalian animal models for the study of visual signal processing within the retina. Especially, the generation of Cre recombinase transgenic mouse lines provides a powerful tool for genetic manipulation. A key step for the utilization of transgenic lines is the characterization of their transgene expression patterns in the retina. Here we describe a standard protocol for characterizing the expression pattern of the Cre recombinase or fluorescent proteins in the retina with an immunohistochemical approach.

Insm1 Induces Neural Progenitor Delamination in Developing Neocortex via Downregulation of the Adherens Junction Belt-Specific Protein Plekha7.

  • Tavano S
  • Neuron
  • 2018 Mar 21

Literature context: Fisher Scientific Cat# A-31572, RRID:AB_162543 Alexa Fluor 647 AffiniPure Donk


Delamination of neural progenitor cells (NPCs) from the ventricular surface is a crucial prerequisite to form the subventricular zone, the germinal layer linked to the expansion of the mammalian neocortex in development and evolution. Here, we dissect the molecular mechanism by which the transcription factor Insm1 promotes the generation of basal progenitors (BPs). Insm1 protein is most highly expressed in newborn BPs in mouse and human developing neocortex. Forced Insm1 expression in embryonic mouse neocortex causes NPC delamination, converting apical to basal radial glia. Insm1 represses the expression of the apical adherens junction belt-specific protein Plekha7. CRISPR/Cas9-mediated disruption of Plekha7 expression suffices to cause NPC delamination. Plekha7 overexpression impedes the intrinsic and counteracts the Insm1-induced, NPC delamination. Our findings uncover a novel molecular mechanism underlying NPC delamination in which a BP-genic transcription factor specifically targets the integrity of the apical adherens junction belt, rather than adherens junction components as such.

Funding information:
  • Intramural NIH HHS - ZIA BC010763-05(United States)

Analysis of the distribution of spinal NOP receptors in a chronic pain model using NOP-eGFP knock-in mice.

  • Ozawa A
  • Br. J. Pharmacol.
  • 2018 Mar 28

Literature context: G Alexa Fluor 555, Cat# A31572, RRID:AB_162543; donkey anti-sheep IgG Alexa Fl


BACKGROUND AND PURPOSE: The nociceptin/orphanin FQ opioid peptide (NOP) receptor system plays a significant role in the regulation of pain. This system functions differently in the spinal cord and brain. The mechanism by which the NOP receptor agonists regulate pain transmission in these regions is not clearly understood. Here, we investigate the peripheral and spinal NOP receptor distribution and antinociceptive effects of intrathecal nociceptin/orphanin FQ (N/OFQ) in chronic neuropathic pain. EXPERIMENTAL APPROACH: We used immunohistochemistry to determine changes in NOP receptor distribution triggered by spinal nerve ligation (SNL) using NOP-eGFP knock-in mice. Antinociceptive effects of intrathecal N/OFQ on SNL-mediated allodynia and heat/cold hyperalgesia were assessed in wild-type mice. KEY RESULTS: NOP-eGFP immunoreactivity was decreased by SNL in the spinal laminae I and II outer, regions that mediate noxious heat stimuli. In contrast, immunoreactivity of NOP-eGFP was unchanged in the ventral border of lamina II inner, which is an important region for the development of allodynia. NOP-eGFP expression was also decreased in a large number of primary afferents in the L4 dorsal root ganglion (DRG) of SNL mice. However, SNL mice showed increased sensitivity, compared to sham animals to the effects of i.t administered N/OFQ with respect to mechanical as well as thermal stimuli. CONCLUSIONS AND IMPLICATIONS: Our findings suggest that the spinal NOP receptor system attenuates injury-induced hyperalgesia by direct inhibition of the projection neurons in the spinal cord that send nociceptive signals to the brain and not by inhibiting presynaptic terminals of DRG neurons in the superficial lamina.

Funding information:
  • NIDA NIH HHS - R01 DA023281()
  • Wellcome Trust - 089457/Z/09/Z(United Kingdom)

Generation of two induced pluripotent stem cell (iPSC) lines from p.F508del Cystic Fibrosis patients.

  • Fleischer A
  • Stem Cell Res
  • 2018 Mar 20

Literature context: IgG 1:500 Invitrogen; A-31572; RRID:AB_162543 Alexa Fluor 546 Donkey Anti-Goa


Cystic Fibrosis (CF) is a monogenic, lethal disease caused by mutations in the cystic fibrosis transmembrane conductance (CFTR) gene. Here we report the production of CF-iPS cell lines from two different p.F508del homozygous female patients (Table 1). Two different primary cell types, skin fibroblasts and keratinocytes, were transfected with retroviral cocktails containing four: c-MYC, KLF4, OCT4 and SOX2 (MKOS) or three: KLF4, OCT4 and SOX2 (KOS) reprogramming factors. Two fibroblast-derived MKOS lines are described in the main text. The lines carry the p.F508del mutation, have a normal karyotype, express pluripotency markers and are able to differentiate into the three germ layers.

Funding information:
  • NIDDK NIH HHS - R01-DK069983(United States)

Chromatin Modification and Global Transcriptional Silencing in the Oocyte Mediated by the mRNA Decay Activator ZFP36L2.

  • Dumdie JN
  • Dev. Cell
  • 2018 Feb 5

Literature context: hermoFisher Scientific A-31572; RRID:AB_162543 Alexa Fluor 488 conjugated donk


Global transcriptional silencing is a highly conserved mechanism central to the oocyte-to-embryo transition. We report the unexpected discovery that global transcriptional silencing in oocytes depends on an mRNA decay activator. Oocyte-specific loss of ZFP36L2 an RNA-binding protein that promotes AU-rich element-dependent mRNA decay prevents global transcriptional silencing and causes oocyte maturation and fertilization defects, as well as complete female infertility in the mouse. Single-cell RNA sequencing revealed that ZFP36L2 downregulates mRNAs encoding transcription and chromatin modification regulators, including a large group of mRNAs for histone demethylases targeting H3K4 and H3K9, which we show are bound and degraded by ZFP36L2. Oocytes lacking Zfp36l2 fail to accumulate histone methylation at H3K4 and H3K9, marks associated with the transcriptionally silent, developmentally competent oocyte state. Our results uncover a ZFP36L2-dependent mRNA decay mechanism that acts as a developmental switch during oocyte growth, triggering wide-spread shifts in chromatin modification and global transcription.

Funding information:
  • NICHD NIH HHS - K12 HD001259()
  • NIGMS NIH HHS - R35 GM118069()
  • NIMH NIH HHS - R33MH083521(United States)

A Metabolic Basis for Endothelial-to-Mesenchymal Transition.

  • Xiong J
  • Mol. Cell
  • 2018 Feb 15

Literature context: Fisher Scientific Cat#A-31572; RRID:AB_162543 Alexa Fluor 647 goat anti-Rabbi


Endothelial-to-mesenchymal transition (EndoMT) is a cellular process often initiated by the transforming growth factor β (TGF-β) family of ligands. Although required for normal heart valve development, deregulated EndoMT is linked to a wide range of pathological conditions. Here, we demonstrate that endothelial fatty acid oxidation (FAO) is a critical in vitro and in vivo regulator of EndoMT. We further show that this FAO-dependent metabolic regulation of EndoMT occurs through alterations in intracellular acetyl-CoA levels. Disruption of FAO via conditional deletion of endothelial carnitine palmitoyltransferase II (Cpt2E-KO) augments the magnitude of embryonic EndoMT, resulting in thickening of cardiac valves. Consistent with the known pathological effects of EndoMT, adult Cpt2E-KO mice demonstrate increased permeability in multiple vascular beds. Taken together, these results demonstrate that endothelial FAO is required to maintain endothelial cell fate and that therapeutic manipulation of endothelial metabolism could provide the basis for treating a growing number of EndoMT-linked pathological conditions.

Funding information:
  • Intramural NIH HHS - Z01 HL005012-11()
  • NHLBI NIH HHS - K08 HL121174()
  • NIA NIH HHS - P30 AG024827()
  • NIDDK NIH HHS - T32 DK007052()
  • NIGMS NIH HHS - GM084445(United States)
  • NINDS NIH HHS - R01 NS072241()

Graded Arrays of Spinal and Supraspinal V2a Interneuron Subtypes Underlie Forelimb and Hindlimb Motor Control.

  • Hayashi M
  • Neuron
  • 2018 Feb 21

Literature context: Life technologies Cat#A-31572; RRID:AB_162543 Donkey anti-rabbit secondary an


The spinal cord contains neural networks that enable regionally distinct motor outputs along the body axis. Nevertheless, it remains unclear how segment-specific motor computations are processed because the cardinal interneuron classes that control motor neurons appear uniform at each level of the spinal cord. V2a interneurons are essential to both forelimb and hindlimb movements, and here we identify two major types that emerge during development: type I neurons marked by high Chx10 form recurrent networks with neighboring spinal neurons and type II neurons that downregulate Chx10 and project to supraspinal structures. Types I and II V2a interneurons are arrayed in counter-gradients, and this network activates different patterns of motor output at cervical and lumbar levels. Single-cell RNA sequencing (RNA-seq) revealed type I and II V2a neurons are each comprised of multiple subtypes. Our findings uncover a molecular and anatomical organization of V2a interneurons reminiscent of the orderly way motor neurons are divided into columns and pools.

Funding information:
  • European Commission - Advanced Grant 294354(United States)
  • NIA NIH HHS - R01 AG036040(United States)

Generation of FHL2 homozygous knockout lines from human embryonic stem cells by CRISPR/Cas9-mediated ablation.

  • Chang CW
  • Stem Cell Res
  • 2018 Jan 2

Literature context: RRID:AB_162543 Secondary antibody Donkey Anti-


Cardiovascular disease is the leading cause of morbidity and mortality in the world. Mutations in the FHL2 (Four and a half LIM domains protein 2) gene are associated with cardiomyopathy in patients. Here, we generated two homozygous knockout lines using CRISPR/Cas9-mediated ablation in a human embryonic stem cell (hESC) WA09 line. These knockout lines exhibit a normal karyotype without expressing FHL2 protein, while maintaining pluripotency and differentiation properties. These isogenic mutation lines will be provided as a disease model for cardiomyopathy studies and drug screening.

Funding information:
  • NCRR NIH HHS - S10RR027926(United States)

Zebrafish Regulatory T Cells Mediate Organ-Specific Regenerative Programs.

  • Hui SP
  • Dev. Cell
  • 2017 Dec 18

Literature context: Fisher Scientific Cat# A-31572; RRID:AB_162543 Donkey anti-mouse Alexa Fluor 5


The attenuation of ancestral pro-regenerative pathways may explain why humans do not efficiently regenerate damaged organs. Vertebrate lineages that exhibit robust regeneration, including the teleost zebrafish, provide insights into the maintenance of adult regenerative capacity. Using established models of spinal cord, heart, and retina regeneration, we discovered that zebrafish Treg-like (zTreg) cells rapidly homed to damaged organs. Conditional ablation of zTreg cells blocked organ regeneration by impairing precursor cell proliferation. In addition to modulating inflammation, infiltrating zTreg cells stimulated regeneration through interleukin-10-independent secretion of organ-specific regenerative factors (Ntf3: spinal cord; Nrg1: heart; Igf1: retina). Recombinant regeneration factors rescued the regeneration defects associated with zTreg cell depletion, whereas Foxp3a-deficient zTreg cells infiltrated damaged organs but failed to express regenerative factors. Our data delineate organ-specific roles for Treg cells in maintaining pro-regenerative capacity that could potentially be harnessed for diverse regenerative therapies.

Funding information:
  • NIEHS NIH HHS - ES016005(United States)

Molecular Dissection of Neuroligin 2 and Slitrk3 Reveals an Essential Framework for GABAergic Synapse Development.

  • Li J
  • Neuron
  • 2017 Nov 15

Literature context: isher Scientific Cat#: A-31572, RRID:AB_162543 Alexa 405 goat anti-rabbit IgG


In the brain, many types of interneurons make functionally diverse inhibitory synapses onto principal neurons. Although numerous molecules have been identified to function in inhibitory synapse development, it remains unknown whether there is a unifying mechanism for development of diverse inhibitory synapses. Here we report a general molecular mechanism underlying hippocampal inhibitory synapse development. In developing neurons, the establishment of GABAergic transmission depends on Neuroligin 2 (NL2), a synaptic cell adhesion molecule (CAM). During maturation, inhibitory synapse development requires both NL2 and Slitrk3 (ST3), another CAM. Importantly, NL2 and ST3 interact with nanomolar affinity through their extracellular domains to synergistically promote synapse development. Selective perturbation of the NL2-ST3 interaction impairs inhibitory synapse development with consequent disruptions in hippocampal network activity and increased seizure susceptibility. Our findings reveal how unique postsynaptic CAMs work in concert to control synaptogenesis and establish a general framework for GABAergic synapse development.

Tonotopic alterations in inhibitory input to the medial nucleus of the trapezoid body in a mouse model of Fragile X syndrome.

  • McCullagh EA
  • J. Comp. Neurol.
  • 2017 Nov 1

Literature context: A-31572, RRID:AB_162543 Thermo-Fisher Donkey anti-Rabbi


Hyperexcitability and the imbalance of excitation/inhibition are one of the leading causes of abnormal sensory processing in Fragile X syndrome (FXS). The precise timing and distribution of excitation and inhibition is crucial for auditory processing at the level of the auditory brainstem, which is responsible for sound localization ability. Sound localization is one of the sensory abilities disrupted by loss of the Fragile X Mental Retardation 1 (Fmr1) gene. Using triple immunofluorescence staining we tested whether there were alterations in the number and size of presynaptic structures for the three primary neurotransmitters (glutamate, glycine, and GABA) in the auditory brainstem of Fmr1 knockout mice. We found decreases in either glycinergic or GABAergic inhibition to the medial nucleus of the trapezoid body (MNTB) specific to the tonotopic location within the nucleus. MNTB is one of the primary inhibitory nuclei in the auditory brainstem and participates in the sound localization process with fast and well-timed inhibition. Thus, a decrease in inhibitory afferents to MNTB neurons should lead to greater inhibitory output to the projections from this nucleus. In contrast, we did not see any other significant alterations in balance of excitation/inhibition in any of the other auditory brainstem nuclei measured, suggesting that the alterations observed in the MNTB are both nucleus and frequency specific. We furthermore show that glycinergic inhibition may be an important contributor to imbalances in excitation and inhibition in FXS and that the auditory brainstem is a useful circuit for testing these imbalances.

Regulation and effects of neurotrophic factors after neural stem cell transplantation in a transgenic mouse model of Alzheimer disease.

  • Li B
  • J. Neurosci. Res.
  • 2017 Nov 9

Literature context: AB_306031;AB_726362;AB_2242334;RRID:AB_162543;AB_2536180


According to much research, neurodegeneration and cognitive decline in Alzheimer disease (AD) are correlated with alternations of neurotrophic factors such as nerve growth factor, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor. The experimental illumination of neural stem cell (NSC) transplantation to eliminate AD symptoms is being explored frequently, and we have acknowledged that neurotrophic factors may play a pivotal role in cognitive improvement. However, the relation between the reversal of cognitive deficits after NSC transplantation and directed alternations of neurotrophic factors is not clearly expounded. Meanwhile, reduced inflammatory response, promoted vessel density, and vascular endothelial growth factor (VEGF) can be reflections of improvement in cerebrovascular function. Three weeks after NSC transplantation, spatial learning and memory function in NSC-injected (Tg-NSC) mice were significantly improved compared with vehicle-injected (Tg-Veh) mice. Meanwhile, results obtained by immunofluorescence and Western blot analyses demonstrated that the levels of neurotrophic factors, VEGF, and vessel density in the cortex of Tg-NSC mice were significantly enhanced compared with Tg-Veh mice, while the levels of proinflammatory cytokines interleukin (IL)-1β, tumor necrosis factor-α, and IL-6 were significantly decreased. Our results suggest that elevated concentrations of neurotrophic factors probably play a critical role in rescuing cognitive dysfunction in APP/PS1 transgenic mice after NSC transplantation, and neurotrophic factors may improve cerebrovascular function by means such as reducing inflammatory response and promoting angiogenesis.

MYC Controls Human Pluripotent Stem Cell Fate Decisions through Regulation of Metabolic Flux.

  • Cliff TS
  • Cell Stem Cell
  • 2017 Oct 5

Literature context: IgG Thermo Fisher Cat#:A31572; RRID:AB_162543 Alexa Fluor 647 donkey anti-goa


As human pluripotent stem cells (hPSCs) exit pluripotency, they are thought to switch from a glycolytic mode of energy generation to one more dependent on oxidative phosphorylation. Here we show that, although metabolic switching occurs during early mesoderm and endoderm differentiation, high glycolytic flux is maintained and, in fact, essential during early ectoderm specification. The elevated glycolysis observed in hPSCs requires elevated MYC/MYCN activity. Metabolic switching during endodermal and mesodermal differentiation coincides with a reduction in MYC/MYCN and can be reversed by ectopically restoring MYC activity. During early ectodermal differentiation, sustained MYCN activity maintains the transcription of "switch" genes that are rate-limiting for metabolic activity and lineage commitment. Our work, therefore, shows that metabolic switching is lineage-specific and not a required step for exit of pluripotency in hPSCs and identifies MYC and MYCN as developmental regulators that couple metabolism to pluripotency and cell fate determination.

Funding information:
  • NCRR NIH HHS - S10 RR027097()
  • NIGMS NIH HHS - P01 GM085354()

Suppression of Red Blood Cell Autofluorescence for Immunocytochemistry on Fixed Embryonic Mouse Tissue.

  • Whittington NC
  • Curr Protoc Neurosci
  • 2017 Oct 23

Literature context: er Scientific, cat. no. A31572, RRID:AB_162543)


Autofluorescence is a problem that interferes with immunofluorescent staining and complicates data analysis. Throughout the mouse embryo, red blood cells naturally fluoresce across multiple wavelengths, spanning the emission and excitation spectra of many commonly used fluorescent reporters, including antibodies, dyes, stains, probes, and transgenic proteins, making it difficult to distinguish assay fluorescence from endogenous fluorescence. Several tissue treatment methods have been developed to bypass this issue with varying degrees of success. Sudan Black B dye has been commonly used to quench autofluorescence, but can also introduce background fluorescence. Here we present a protocol for an alternative called TrueBlack Lipofuscin Autofluorescence Quencher. The protocol described in this unit demonstrates how TrueBlack efficiently quenches red blood cell autofluorescence across red and green wavelengths in fixed embryonic tissue without interfering with immunofluorescent signal intensity or introducing background staining. We also identify optimal incubation, concentration, and multiple usage conditions for routine immunofluorescence microscopy. © 2017 by John Wiley & Sons, Inc.

Funding information:
  • Intramural NIH HHS - ZIA NS002824-26()
  • NINDS NIH HHS - Z01 NS002824()

Generation of Mouse and Human Organoid-Forming Intestinal Progenitor Cells by Direct Lineage Reprogramming.

  • Miura S
  • Cell Stem Cell
  • 2017 Oct 5

Literature context: gG Molecular Probes Cat#A31572, RRID:AB_162543 Alexa 647-conjugated donkey ant


Intestinal organoids hold great promise as a valuable tool for studying and treating intestinal diseases. The currently available sources of human intestinal organoids, tissue fragments or pluripotent stem cells, involve invasive procedures or complex differentiation protocols, respectively. Here, we show that a set of four transcription factors, Hnf4α, Foxa3, Gata6, and Cdx2, can directly reprogram mouse fibroblasts to acquire the identity of fetal intestine-derived progenitor cells (FIPCs). These induced FIPCs (iFIPCs) form spherical organoids that develop into adult-type budding organoids containing cells with intestinal stem cell properties. The resulting stem cells produce all intestinal epithelial cell lineages and undergo self-renewing cell divisions. After transplantation, the induced spherical and budding organoids can reconstitute colonic and intestinal epithelia, respectively. The same combination of four defined transcription factors can also induce human iFIPCs. This alternative approach for producing intestinal organoids may well facilitate application for disease analysis and therapy development.

Assembly of the WHIP-TRIM14-PPP6C Mitochondrial Complex Promotes RIG-I-Mediated Antiviral Signaling.

  • Tan P
  • Mol. Cell
  • 2017 Oct 19

Literature context: y Ab ThermoFisher Cat#: A31572; RRID:AB_162543 IF: Goat anti-Mouse IgG, Alexa


Mitochondrial antiviral signaling platform protein (MAVS) acts as a central hub for RIG-I receptor proximal signal propagation. However, key components in the assembly of the MAVS mitochondrial platform that promote RIG-I mitochondrial localization and optimal activation are still largely undefined. Employing pooled RNAi and yeast two-hybrid screenings, we report that the mitochondrial adaptor protein tripartite motif (TRIM)14 provides a docking platform for the assembly of the mitochondrial signaling complex required for maximal activation of RIG-I-mediated signaling, consisting of WHIP and protein phosphatase PPP6C. Following viral infection, the ubiquitin-binding domain in WHIP bridges RIG-I with MAVS by binding to polyUb chains of RIG-I at lysine 164. The ATPase domain in WHIP contributes to stabilization of the RIG-I-dsRNA interaction. Moreover, phosphatase PPP6C is responsible for RIG-I dephosphorylation. Together, our findings define the WHIP-TRIM14-PPP6C mitochondrial signalosome required for RIG-I-mediated innate antiviral immunity.

Funding information:
  • Canadian Institutes of Health Research - (Canada)

A Modular Platform for Differentiation of Human PSCs into All Major Ectodermal Lineages.

  • Tchieu J
  • Cell Stem Cell
  • 2017 Sep 7

Literature context: ermo Fisher Scientific A-31572, RRID:AB_162543 AlexaFluor Donkey Anti-Rabbit 6


Directing the fate of human pluripotent stem cells (hPSCs) into different lineages requires variable starting conditions and components with undefined activities, introducing inconsistencies that confound reproducibility and assessment of specific perturbations. Here we introduce a simple, modular protocol for deriving the four main ectodermal lineages from hPSCs. By precisely varying FGF, BMP, WNT, and TGFβ pathway activity in a minimal, chemically defined medium, we show parallel, robust, and reproducible derivation of neuroectoderm, neural crest (NC), cranial placode (CP), and non-neural ectoderm in multiple hPSC lines, on different substrates independently of cell density. We highlight the utility of this system by interrogating the role of TFAP2 transcription factors in ectodermal differentiation, revealing the importance of TFAP2A in NC and CP specification, and performing a small-molecule screen that identified compounds that further enhance CP differentiation. This platform provides a simple stage for systematic derivation of the entire range of ectodermal cell types.

Funding information:
  • NINDS NIH HHS - R01 NS072381()

Inter-adipocyte Adhesion and Signaling by Collagen IV Intercellular Concentrations in Drosophila.

  • Dai J
  • Curr. Biol.
  • 2017 Sep 25

Literature context: Life Technologies Cat#A-31572; RRID:AB_162543 Rabbit anti-GFP polyclonal anti


Sheet-forming Collagen IV is the main component of basement membranes, which are planar polymers of extracellular matrix underlying epithelia and surrounding organs in all animals. Adipocytes in both insects and mammals are mesodermal in origin and often classified as mesenchymal. However, they form true tissues where cells remain compactly associated. Neither the mechanisms providing this tissue-level organization nor its functional significance are known. Here we show that discrete Collagen IV intercellular concentrations (CIVICs), distinct from basement membranes and thicker in section, mediate inter-adipocyte adhesion in Drosophila. Loss of these Collagen-IV-containing structures in the larval fat body caused intercellular gaps and disrupted continuity of the adipose tissue layer. We also found that Integrin and Syndecan matrix receptors attach adipocytes to CIVICs and direct their formation. Finally, we show that Integrin-mediated adhesion to CIVICs promotes normal adipocyte growth and prevents autophagy through Src-Pi3K-Akt signaling. Our results evidence a surprising non-basement membrane role of Collagen IV in non-epithelial tissue morphogenesis while demonstrating adhesion and signaling functions for these structures.

Human Pluripotent Stem Cell-Derived Atrial and Ventricular Cardiomyocytes Develop from Distinct Mesoderm Populations.

  • Lee JH
  • Cell Stem Cell
  • 2017 Aug 3

Literature context: ated ThermoFisher Cat.# A31572; RRID:AB_162543 Donkey anti-mouse IgG (H+L), Al


The ability to direct the differentiation of human pluripotent stem cells (hPSCs) to the different cardiomyocyte subtypes is a prerequisite for modeling specific forms of cardiovascular disease in vitro and for developing novel therapies to treat them. Here we have investigated the development of the human atrial and ventricular lineages from hPSCs, and we show that retinoic acid signaling at the mesoderm stage of development is required for atrial specification. Analyses of early developmental stages revealed that ventricular and atrial cardiomyocytes derive from different mesoderm populations that can be distinguished based on CD235a and RALDH2 expression, respectively. Molecular and electrophysiological characterization of the derivative cardiomyocytes revealed that optimal specification of ventricular and atrial cells is dependent on induction of the appropriate mesoderm. Together these findings provide new insights into the development of the human atrial and ventricular lineages that enable the generation of highly enriched, functional cardiomyocyte populations for therapeutic applications.

Funding information:
  • NIDA NIH HHS - DA021801(United States)
  • NIGMS NIH HHS - R01 GM090158(United States)

Neuronal Depolarization Drives Increased Dopamine Synaptic Vesicle Loading via VGLUT.

  • Aguilar JI
  • Neuron
  • 2017 Aug 30

Literature context: Fisher Scientific Cat# A-31572; RRID:AB_162543 Goat anti-mouse Alexa 647 Therm


The ability of presynaptic dopamine terminals to tune neurotransmitter release to meet the demands of neuronal activity is critical to neurotransmission. Although vesicle content has been assumed to be static, in vitro data increasingly suggest that cell activity modulates vesicle content. Here, we use a coordinated genetic, pharmacological, and imaging approach in Drosophila to study the presynaptic machinery responsible for these vesicular processes in vivo. We show that cell depolarization increases synaptic vesicle dopamine content prior to release via vesicular hyperacidification. This depolarization-induced hyperacidification is mediated by the vesicular glutamate transporter (VGLUT). Remarkably, both depolarization-induced dopamine vesicle hyperacidification and its dependence on VGLUT2 are seen in ventral midbrain dopamine neurons in the mouse. Together, these data suggest that in response to depolarization, dopamine vesicles utilize a cascade of vesicular transporters to dynamically increase the vesicular pH gradient, thereby increasing dopamine vesicle content.

Basement Membrane Manipulation in Drosophila Wing Discs Affects Dpp Retention but Not Growth Mechanoregulation.

  • Ma M
  • Dev. Cell
  • 2017 Jul 10

Literature context: A-31572; RRID:AB_162543 Chemicals,


Basement membranes (BMs) are extracellular matrix polymers basally underlying epithelia, where they regulate cell signaling and tissue mechanics. Constriction by the BM shapes Drosophila wing discs, a well-characterized model of tissue growth. Recently, the hypothesis that mechanical factors govern wing growth has received much attention, but it has not been definitively tested. In this study, we manipulated BM composition to cause dramatic changes in tissue tension. We found that increased tissue compression when perlecan was knocked down did not affect adult wing size. BM elimination, decreasing compression, reduced wing size but did not visibly affect Hippo signaling, widely postulated to mediate growth mechanoregulation. BM elimination, in contrast, attenuated signaling by bone morphogenetic protein/transforming growth factor β ligand Dpp, which was not efficiently retained within the tissue and escaped to the body cavity. Our results challenge mechanoregulation of wing growth, while uncovering a function of BMs in preserving a growth-promoting tissue environment.

Funding information:
  • NIDDK NIH HHS - R01DK076233-01(United States)

Recruited Monocytes and Type 2 Immunity Promote Lung Regeneration following Pneumonectomy.

  • Lechner AJ
  • Cell Stem Cell
  • 2017 Jul 6

Literature context: o A31572; RRID:AB_162543 Goat anti-


To investigate the role of immune cells in lung regeneration, we used a unilateral pneumonectomy model that promotes the formation of new alveoli in the remaining lobes. Immunofluorescence and single-cell RNA sequencing found CD115+ and CCR2+ monocytes and M2-like macrophages accumulating in the lung during the peak of type 2 alveolar epithelial stem cell (AEC2) proliferation. Genetic loss of function in mice and adoptive transfer studies revealed that bone marrow-derived macrophages (BMDMs) traffic to the lung through a CCL2-CCR2 chemokine axis and are required for optimal lung regeneration, along with Il4ra-expressing leukocytes. Our data suggest that these cells modulate AEC2 proliferation and differentiation. Finally, we provide evidence that group 2 innate lymphoid cells are a source of IL-13, which promotes lung regeneration. Together, our data highlight the potential for immunomodulatory therapies to stimulate alveologenesis in adults.

Funding information:
  • NHLBI NIH HHS - F30 HL131198()
  • NHLBI NIH HHS - R01 HL127002()
  • NHLBI NIH HHS - U01 HL134766()
  • NIGMS NIH HHS - T32 GM007618()

RPL10L Is Required for Male Meiotic Division by Compensating for RPL10 during Meiotic Sex Chromosome Inactivation in Mice.

  • Jiang L
  • Curr. Biol.
  • 2017 May 22

Literature context: A-31572; RRID:AB_162543 Donkey Ant


The mammalian sex chromosomes have undergone profound changes during their evolution from an ancestral pair of autosomes [1-4]. Specifically, the X chromosome has acquired a paradoxical sex-biased function by redistributing gene contents [5, 6] and has generated a disproportionately high number of retrogenes that are located on autosomes and exhibit male-biased expression patterns [6]. Several selection-based models have been proposed to explain this phenomenon, including a model of sexual antagonism driving X inactivation (SAXI) [6-8] and a compensatory mechanism based on meiotic sex chromosome inactivation (MSCI) [6, 8-11]. However, experimental evidence correlating the function of X-chromosome-derived autosomal retrogenes with evolutionary forces remains limited [12-17]. Here, we show that the deficiency of Rpl10l, a murine autosomal retrogene of Rpl10 with testis-specific expression, disturbs ribosome biogenesis in late-prophase spermatocytes and prohibits the transition from prophase into metaphase of the first meiotic division, resulting in male infertility. Rpl10l expression compensates for the lack of Rpl10, which exhibits a broad expression pattern but is subject to MSCI during spermatogenesis. Importantly, ectopic expression of RPL10L prevents the death of cultured RPL10-deficient somatic cells, and Rpl10l-promoter-driven transgenic expression of Rpl10 in spermatocytes restores spermatogenesis and fertility in Rpl10l-deficient mice. Our results demonstrate that Rpl10l plays an essential role during the meiotic stage of spermatogenesis by compensating for MSCI-mediated transcriptional silencing of Rpl10. These data provide direct evidence for the compensatory hypothesis and add novel insight into the evolution of X-chromosome-derived autosomal retrogenes and their role in male fertility.

Oriented clonal cell dynamics enables accurate growth and shaping of vertebrate cartilage.

  • Kaucka M
  • Elife
  • 2017 Apr 17

Literature context: , 1:1000, AB_162543, RRID:AB_1


Cartilaginous structures are at the core of embryo growth and shaping before the bone forms. Here we report a novel principle of vertebrate cartilage growth that is based on introducing transversally-oriented clones into pre-existing cartilage. This mechanism of growth uncouples the lateral expansion of curved cartilaginous sheets from the control of cartilage thickness, a process which might be the evolutionary mechanism underlying adaptations of facial shape. In rod-shaped cartilage structures (Meckel, ribs and skeletal elements in developing limbs), the transverse integration of clonal columns determines the well-defined diameter and resulting rod-like morphology. We were able to alter cartilage shape by experimentally manipulating clonal geometries. Using in silico modeling, we discovered that anisotropic proliferation might explain cartilage bending and groove formation at the macro-scale.

Funding information:
  • NIBIB NIH HHS - R01 EB014877()

Immunosuppression via Loss of IL2rγ Enhances Long-Term Functional Integration of hESC-Derived Photoreceptors in the Mouse Retina.

  • Zhu J
  • Cell Stem Cell
  • 2017 Mar 2

Literature context: s A31572; RRID:AB_162543 Alexa Fluo


Loss of photoreceptors is a common endpoint in degenerative retinal diseases. Human pluripotent stem cells provide a potential source for photoreceptor replacement, but, even in mouse models, the efficiency and efficacy of transplantation-based repair remains poor. In this study, we examined the degree to which immune rejection contributes to these disappointing outcomes using an immunodeficient IL2 receptor γ (IL2rγ)-null mouse model. Our results show that prevention of cell rejection in the normal and degenerating retinal environment significantly improves long-term survival and integration of hESC-derived donor retinal cells. Transplanted cells are able to differentiate into mature photoreceptors expressing various opsins and can functionally integrate into congenitally blind mice. Our work suggests that even though the retina is often considered immune-privileged, suppression of host immune-mediated cell rejection may well be a useful approach for improving long-term integration of transplanted cells with a view to successful clinical outcomes.

Morphology, innervation, and peripheral sensory cells of the siphon of aplysia californica.

  • Carrigan ID
  • J. Comp. Neurol.
  • 2015 Nov 1

Literature context: # A31572) RRID:AB_162543 1:200


The siphon of Aplysia californica has several functions, including involvement in respiration, excretion, and defensive inking. It also provides sensory input for defensive withdrawals that have been studied extensively to examine mechanisms that underlie learning. To better understand the neuronal bases of these functions, we used immunohistochemistry to catalogue peripheral cell types and innervation of the siphon in stage 12 juveniles (chosen to allow observation of tissues in whole-mounts). We found that the siphon nerve splits into three major branches, leading ultimately to a two-part FMRFamide-immunoreactive plexus and an apparently separate tyrosine hydroxylase-immunoreactive plexus. Putative sensory neurons included four distinct types of tubulin-immunoreactive bipolar cells (one likely also tyrosine hydroxylase immunoreactive) that bore ciliated dendrites penetrating the epithelium. A fifth bipolar neuron type (tubulin- and FMRFamide-immunoreactive) occurred deeper in the tissue, associated with part of the FMRFamide-immunoreactive plexus. Our observations emphasize the structural complexity of the peripheral nervous system of the siphon, and the importance of direct tests of the various components to better understand the functioning of the entire organ, including its role in defensive withdrawal responses.

Funding information:
  • NEI NIH HHS - T32 EY024234(United States)