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1.
J Pharm Sci ; 107(2): 529-542, 2018 02.
Article En | MEDLINE | ID: mdl-29074375

The Biophorum Development Group (BPDG) is an industry-wide consortium enabling networking and sharing of best practices for the development of biopharmaceuticals. To gain a better understanding of current industry approaches for establishing biopharmaceutical drug product (DP) robustness, the BPDG-Formulation Point Share group conducted an intercompany collaboration exercise, which included a bench-marking survey and extensive group discussions around the scope, design, and execution of robustness studies. The results of this industry collaboration revealed several key common themes: (1) overall DP robustness is defined by both the formulation and the manufacturing process robustness; (2) robustness integrates the principles of quality by design (QbD); (3) DP robustness is an important factor in setting critical quality attribute control strategies and commercial specifications; (4) most companies employ robustness studies, along with prior knowledge, risk assessments, and statistics, to develop the DP design space; (5) studies are tailored to commercial development needs and the practices of each company. Three case studies further illustrate how a robustness study design for a biopharmaceutical DP balances experimental complexity, statistical power, scientific understanding, and risk assessment to provide the desired product and process knowledge. The BPDG-Formulation Point Share discusses identified industry challenges with regard to biopharmaceutical DP robustness and presents some recommendations for best practices.


Drug Industry/methods , Pharmaceutical Preparations/chemistry , Biopharmaceutics/methods , Chemistry, Pharmaceutical/methods , Clinical Trials as Topic , Drug Design , Humans , Intersectoral Collaboration , Risk Assessment , Technology, Pharmaceutical/methods
2.
J Neurosci ; 36(40): 10337-10355, 2016 10 05.
Article En | MEDLINE | ID: mdl-27707970

Axon-Schwann cell interactions are crucial for the development, function, and repair of the peripheral nervous system, but mechanisms underlying communication between axons and nonmyelinating Schwann cells are unclear. Here, we show that ER81 is functionally required in a subset of mouse RET+ mechanosensory neurons for formation of Pacinian corpuscles, which are composed of a single myelinated axon and multiple layers of nonmyelinating Schwann cells, and Ret is required for the maintenance of Er81 expression. Interestingly, Er81 mutants have normal myelination but exhibit deficient interactions between axons and corpuscle-forming nonmyelinating Schwann cells. Finally, ablating Neuregulin-1 (Nrg1) in mechanosensory neurons results in no Pacinian corpuscles, and an Nrg1 isoform not required for communication with myelinating Schwann cells is specifically decreased in Er81-null somatosensory neurons. Collectively, our results suggest that a RET-ER81-NRG1 signaling pathway promotes axon communication with nonmyelinating Schwann cells, and that neurons use distinct mechanisms to interact with different types of Schwann cells. SIGNIFICANCE STATEMENT: Communication between neurons and Schwann cells is critical for development, normal function, and regeneration of the peripheral nervous system. Despite many studies about axonal communication with myelinating Schwann cells, mostly via a specific isoform of Neuregulin1, the molecular nature of axonal communication with nonmyelinating Schwann cells is poorly understood. Here, we described a RET-ER81-Neuregulin1 signaling pathway in neurons innervating Pacinian corpuscle somatosensory end organs, which is essential for communication between the innervating axon and the end organ nonmyelinating Schwann cells. We also showed that this signaling pathway uses isoforms of Neuregulin1 that are not involved in myelination, providing evidence that neurons use different isoforms of Neuregulin1 to interact with different types of Schwann cells.


DNA-Binding Proteins/physiology , Neuregulin-1/physiology , Pacinian Corpuscles/growth & development , Pacinian Corpuscles/physiology , Proto-Oncogene Proteins c-ret/physiology , Signal Transduction/physiology , Transcription Factors/physiology , Animals , Axons/physiology , DNA-Binding Proteins/genetics , Mechanotransduction, Cellular/genetics , Mechanotransduction, Cellular/physiology , Mice , Mice, Inbred C57BL , Mutation/genetics , Myelin Sheath/physiology , Neuregulin-1/genetics , Neurons/physiology , Peripheral Nerve Injuries/physiopathology , Proto-Oncogene Proteins c-ret/genetics , Schwann Cells/physiology , Transcription Factors/genetics
4.
Neuron ; 91(5): 1137-1153, 2016 Sep 07.
Article En | MEDLINE | ID: mdl-27545714

The gate control theory (GCT) of pain proposes that pain- and touch-sensing neurons antagonize each other through spinal cord dorsal horn (DH) gating neurons. However, the exact neural circuits underlying the GCT remain largely elusive. Here, we identified a new population of deep layer DH (dDH) inhibitory interneurons that express the receptor tyrosine kinase Ret neonatally. These early RET+ dDH neurons receive excitatory as well as polysynaptic inhibitory inputs from touch- and/or pain-sensing afferents. In addition, they negatively regulate DH pain and touch pathways through both pre- and postsynaptic inhibition. Finally, specific ablation of early RET+ dDH neurons increases basal and chronic pain, whereas their acute activation reduces basal pain perception and relieves inflammatory and neuropathic pain. Taken together, our findings uncover a novel spinal circuit that mediates crosstalk between touch and pain pathways and suggest that some early RET+ dDH neurons could function as pain "gating" neurons.


Interneurons/physiology , Pain/physiopathology , Proto-Oncogene Proteins c-ret/metabolism , Spinal Cord/physiology , Touch/physiology , Animals , Clozapine/analogs & derivatives , Clozapine/pharmacology , Female , Interneurons/drug effects , Interneurons/metabolism , Male , Mice , Mice, Transgenic , Neural Inhibition/physiology , Neurons/physiology , Pain Measurement , Spinal Cord/metabolism , Spinal Cord Dorsal Horn/metabolism
5.
MAbs ; 8(3): 513-23, 2016.
Article En | MEDLINE | ID: mdl-26752675

Antibody-drug conjugates (ADCs) are of great interest as targeted cancer therapeutics. Preparation of ADCs for early stage screening is constrained by purification and biochemical analysis techniques that necessitate burdensome quantities of antibody. Here we describe a method, developed for the maytansinoid class of ADCs, enabling parallel conjugation of antibodies in 96-well format. The method utilizes ∼ 100 µg of antibody per well and requires <5 µg of ADC for characterization. We demonstrate the capabilities of this system using model antibodies. We also provide multiple examples applying this method to early-stage screening of maytansinoid ADCs. The method can greatly increase the throughput with which candidate ADCs can be screened in cell-based assays, and may be more generally applicable to high-throughput preparation and screening of different types of protein conjugates.


Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Neoplasm/pharmacology , Immunoconjugates/pharmacology , Maytansine/pharmacology , Neoplasms/drug therapy , Antibodies, Monoclonal, Humanized/immunology , Antibodies, Neoplasm/immunology , Cell Line, Tumor , Drug Screening Assays, Antitumor/methods , Humans , Immunoconjugates/immunology , Neoplasms/immunology
6.
Elife ; 4: e06828, 2015 Apr 02.
Article En | MEDLINE | ID: mdl-25838128

RET can be activated in cis or trans by its co-receptors and ligands in vitro, but the physiological roles of trans signaling are unclear. Rapidly adapting (RA) mechanoreceptors in dorsal root ganglia (DRGs) express Ret and the co-receptor Gfrα2 and depend on Ret for survival and central projection growth. Here, we show that Ret and Gfrα2 null mice display comparable early central projection deficits, but Gfrα2 null RA mechanoreceptors recover later. Loss of Gfrα1, the co-receptor implicated in activating RET in trans, causes no significant central projection or cell survival deficit, but Gfrα1;Gfrα2 double nulls phenocopy Ret nulls. Finally, we demonstrate that GFRα1 produced by neighboring DRG neurons activates RET in RA mechanoreceptors. Taken together, our results suggest that trans and cis RET signaling could function in the same developmental process and that the availability of both forms of activation likely enhances but not diversifies outcomes of RET signaling.


Ganglia, Spinal/metabolism , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Mechanoreceptors/metabolism , Mechanotransduction, Cellular , Proto-Oncogene Proteins c-ret/genetics , Animals , Cell Survival , Embryo, Mammalian , Ganglia, Spinal/cytology , Ganglia, Spinal/growth & development , Gene Expression Regulation, Developmental , Glial Cell Line-Derived Neurotrophic Factor Receptors/deficiency , Mechanoreceptors/cytology , Mice , Mice, Knockout , Morphogenesis/genetics , Neurturin/deficiency , Neurturin/genetics , Proto-Oncogene Proteins c-ret/deficiency
7.
Front Biol (Beijing) ; 8(4)2013 Aug 01.
Article En | MEDLINE | ID: mdl-24376457

Touch sensation is critical for our social and environmental interactions. In mammals, most discriminative light touch sensation is mediated by the Aß low-threshold mechanoreceptors. Cell bodies of Aß low-threshold mechanoreceptors are located in the dorsal root ganglia and trigeminal ganglia, which extend a central projection innervating the spinal cord and brain stem and a peripheral projection innervating the specialized mechanosensory end organs. These specialized mechanosensory end organs include Meissner's corpuscles, Pacinian corpuscles, lanceolate endings, Merkel cells, and Ruffini corpuscles. The morphologies and physiological properties of these mechanosensory end organs and their innervating neurons have been investigated for over a century. In addition, recent advances in mouse genetics have enabled the identification of molecular mechanisms underlying the development of Aß low-threshold mechanoreceptors, which highlight the crucial roles of neurotrophic factor signaling and transcription factor activity in this process. Here, we will review the anatomy, physiological properties, and development of mammalian low-threshold Aß mechanoreceptors.

8.
Development ; 140(23): 4672-82, 2013 Dec.
Article En | MEDLINE | ID: mdl-24173802

Mammalian neuroepithelial stem cells divide using a polarized form of cytokinesis, which is not well understood. The cytokinetic furrow cleaves the cell by ingressing from basal to apical, forming the midbody at the apical membrane. The midbody mediates abscission by recruiting many factors, including the Kinesin-6 family member Kif20b. In developing embryos, Kif20b mRNA is most highly expressed in neural stem/progenitor cells. A loss-of-function mutant in Kif20b, magoo, was found in a forward genetic screen. magoo has a small cerebral cortex, with reduced production of progenitors and neurons, but preserved layering. In contrast to other microcephalic mouse mutants, mitosis and cleavage furrows of cortical stem cells appear normal in magoo. However, apical midbodies show changes in number, shape and positioning relative to the apical membrane. Interestingly, the disruption of abscission does not appear to result in binucleate cells, but in apoptosis. Thus, Kif20b is required for proper midbody organization and abscission in polarized cortical stem cells and has a crucial role in the regulation of cerebral cortex growth.


Cerebral Cortex/metabolism , Cytokinesis/physiology , Kinesins/metabolism , Neural Stem Cells/metabolism , Animals , Cell Polarity/genetics , Gene Expression , Kinesins/genetics , Mice , Mice, Inbred C57BL , Microtubules/metabolism , RNA, Messenger/biosynthesis
9.
Mol Pain ; 8: 52, 2012 Jul 09.
Article En | MEDLINE | ID: mdl-22776446

BACKGROUND: Itch is one of the major somatosensory modalities. Some recent findings have proposed that gastrin releasing peptide (Grp) is expressed in a subset of dorsal root ganglion (DRG) neurons and functions as a selective neurotransmitter for transferring itch information to spinal cord interneurons. However, expression data from public databases and earlier literatures indicate that Grp mRNA is only detected in dorsal spinal cord (dSC) whereas its family member neuromedin B (Nmb) is highly expressed in DRG neurons. These contradictory results argue that a thorough characterization of the expression of Grp and Nmb is warranted. FINDINGS: Grp mRNA is highly expressed in dSC but is barely detectable in DRGs of juvenile and adult mice. Anti-bombesin serum specifically recognizes Grp but not Nmb. Grp is present in a small number of small-diameter DRG neurons and in abundance in layers I and II of the spinal cord. The reduction of dSC Grp after dorsal root rhizotomy is significantly different from those of DRG derived markers but similar to that of a spinal cord neuronal marker. Double fluorescent in situ of Nmb and other molecular markers indicate that Nmb is highly and selectively expressed in nociceptive and itch-sensitive DRG neurons. CONCLUSION: The majority of dSC Grp is synthesized locally in dorsal spinal cord neurons. On the other hand, Nmb is highly expressed in pain- and itch-sensing DRG neurons. Our findings provide direct anatomic evidence that Grp could function locally in the dorsal spinal cord in addition to its roles in DRG neurons and that Nmb has potential roles in nociceptive and itch-sensitive neurons. These results will improve our understanding about roles of Grp and Nmb in mediating itch sensation.


Gastrin-Releasing Peptide/biosynthesis , Neurokinin B/analogs & derivatives , Pain/metabolism , Pain/pathology , Pruritus/pathology , Sensory Receptor Cells/metabolism , Spinal Cord/metabolism , Aging/genetics , Amino Acid Sequence , Animals , Antibody Specificity/immunology , Bombesin/chemistry , Bombesin/immunology , Bombesin/metabolism , Cold Temperature , Ganglia, Spinal/metabolism , Ganglia, Spinal/pathology , Gastrin-Releasing Peptide/genetics , Gene Expression Regulation, Developmental , Humans , Immune Sera/immunology , Mechanotransduction, Cellular , Mice , Molecular Sequence Data , Neurokinin B/genetics , Neurokinin B/metabolism , Nociceptors/metabolism , Nociceptors/pathology , Pain/complications , Pain Threshold , Physical Stimulation , Protein Transport , Pruritus/complications , Pruritus/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Bombesin/genetics , Receptors, Bombesin/metabolism , Rhizotomy , Sensory Receptor Cells/pathology , Spinal Cord/pathology
10.
J Vis Exp ; (53)2011 Jul 27.
Article En | MEDLINE | ID: mdl-21841759

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology (1-5). In the last few years, several genomewide libraries of yeast strains and plasmids have been generated (6-10). These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches (11-16). Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.


Plasmids/genetics , Saccharomyces cerevisiae/genetics , Transformation, Genetic , Robotics/methods
11.
Neural Dev ; 6: 3, 2011 Jan 07.
Article En | MEDLINE | ID: mdl-21214893

BACKGROUND: The dorsal thalamus acts as a gateway and modulator for information going to and from the cerebral cortex. This activity requires the formation of reciprocal topographic axon connections between thalamus and cortex. The axons grow along a complex multistep pathway, making sharp turns, crossing expression boundaries, and encountering intermediate targets. However, the cellular and molecular components mediating these steps remain poorly understood. RESULTS: To further elucidate the development of the thalamocortical system, we first created a thalamocortical axon reporter line to use as a genetic tool for sensitive analysis of mutant mouse phenotypes. The TCA-tau-lacZ reporter mouse shows specific, robust, and reproducible labeling of thalamocortical axons (TCAs), but not the overlapping corticothalamic axons, during development. Moreover, it readily reveals TCA pathfinding abnormalities in known cortical mutants such as reeler. Next, we performed an unbiased screen for genes involved in thalamocortical development using random mutagenesis with the TCA reporter. Six independent mutant lines show aberrant TCA phenotypes at different steps of the pathway. These include ventral misrouting, overfasciculation, stalling at the corticostriatal boundary, and invasion of ectopic cortical cell clusters. An outcross breeding strategy coupled with a genomic panel of single nucleotide polymorphisms facilitated genetic mapping with small numbers of mutant mice. We mapped a ventral misrouting mutant to the Emx2 gene, and discovered that some TCAs extend to the olfactory bulbs in this mutant. Mapping data suggest that other lines carry mutations in genes not previously known for roles in thalamocortical development. CONCLUSIONS: These data demonstrate the feasibility of a forward genetic approach to understanding mammalian brain morphogenesis and wiring. A robust axonal reporter enabled sensitive analysis of a specific axon tract inside the mouse brain, identifying mutant phenotypes at multiple steps of the pathway, and revealing a new aspect of the Emx2 mutant. The phenotypes highlight vulnerable choice points and latent tendencies of TCAs, and will lead to a refined understanding of the elements and interactions required to form the thalamocortical system.


Axons/physiology , Cerebral Cortex , Gene Expression Regulation, Developmental/genetics , Homeodomain Proteins/genetics , Mutation/genetics , Phenotype , Thalamus , Transcription Factors/genetics , Alkylating Agents/pharmacology , Animals , Body Patterning/drug effects , Body Patterning/genetics , Cell Adhesion Molecules, Neuronal/genetics , Cell Adhesion Molecules, Neuronal/metabolism , Cerebral Cortex/embryology , Cerebral Cortex/growth & development , Cerebral Cortex/metabolism , Embryo, Mammalian , Ethylnitrosourea/pharmacology , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Female , Gene Expression Regulation, Developmental/drug effects , Genetic Testing/methods , Lac Operon/genetics , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neural Pathways/abnormalities , Neural Pathways/embryology , Neural Pathways/growth & development , Reelin Protein , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Thalamus/embryology , Thalamus/growth & development , Thalamus/metabolism , beta-Galactosidase/metabolism
12.
Anal Biochem ; 340(2): 272-8, 2005 May 15.
Article En | MEDLINE | ID: mdl-15840500

A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for detection and quantification of free maytansinoid drug in disulfide-linked conjugates between monoclonal antibodies and the maytansinoid drug DM1 (MAb-DM1). Mobile phases and gradient conditions were optimized for separation of several DM1-related free drug species from MAb-DM1 conjugates. The selectivity, linearity, and reproducibility of the method are reported. Reduction of the disulfide-linked DM1 followed by RP-HPLC allowed estimation of purity of MAb-linked DM1 as well as recovery of L-DM1. The method was also used to estimate drug per MAb ratios, which were consistent with those determined by UV spectroscopy.


Antibodies, Monoclonal/analysis , Chromatography, High Pressure Liquid/methods , Immunoconjugates/analysis , Maytansine/analogs & derivatives , Maytansine/analysis , Reproducibility of Results
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