Colorectal tumours that are wild type for KRAS are often sensitive to EGFR blockade, but almost always develop resistance within several months of initiating therapy. The mechanisms underlying this ...acquired resistance to anti-EGFR antibodies are largely unknown. This situation is in marked contrast to that of small-molecule targeted agents, such as inhibitors of ABL, EGFR, BRAF and MEK, in which mutations in the genes encoding the protein targets render the tumours resistant to the effects of the drugs. The simplest hypothesis to account for the development of resistance to EGFR blockade is that rare cells with KRAS mutations pre-exist at low levels in tumours with ostensibly wild-type KRAS genes. Although this hypothesis would seem readily testable, there is no evidence in pre-clinical models to support it, nor is there data from patients. To test this hypothesis, we determined whether mutant KRAS DNA could be detected in the circulation of 28 patients receiving monotherapy with panitumumab, a therapeutic anti-EGFR antibody. We found that 9 out of 24 (38%) patients whose tumours were initially KRAS wild type developed detectable mutations in KRAS in their sera, three of which developed multiple different KRAS mutations. The appearance of these mutations was very consistent, generally occurring between 5 and 6 months following treatment. Mathematical modelling indicated that the mutations were present in expanded subclones before the initiation of panitumumab treatment. These results suggest that the emergence of KRAS mutations is a mediator of acquired resistance to EGFR blockade and that these mutations can be detected in a non-invasive manner. They explain why solid tumours develop resistance to targeted therapies in a highly reproducible fashion.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The RNA-binding protein TDP-43 regulates RNA metabolism at multiple levels, including transcription, RNA splicing, and mRNA stability. TDP-43 is a major component of the cytoplasmic inclusions ...characteristic of amyotrophic lateral sclerosis and some types of frontotemporal lobar degeneration. The importance of TDP-43 in disease is underscored by the fact that dominant missense mutations are sufficient to cause disease, although the role of TDP-43 in pathogenesis is unknown. Here we show that TDP-43 forms cytoplasmic mRNP granules that undergo bidirectional, microtubule-dependent transport in neurons in vitro and in vivo and facilitate delivery of target mRNA to distal neuronal compartments. TDP-43 mutations impair this mRNA transport function in vivo and in vitro, including in stem cell-derived motor neurons from ALS patients bearing any one of three different TDP-43 ALS-causing mutations. Thus, TDP-43 mutations that cause ALS lead to partial loss of a novel cytoplasmic function of TDP-43.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor nervous system. We show using multielectrode array and patch-clamp recordings that hyperexcitability detected by ...clinical neurophysiological studies of ALS patients is recapitulated in induced pluripotent stem cell-derived motor neurons from ALS patients harboring superoxide dismutase 1 (SOD1), C9orf72, and fused-in-sarcoma mutations. Motor neurons produced from a genetically corrected but otherwise isogenic SOD1(+/+) stem cell line do not display the hyperexcitability phenotype. SOD1(A4V/+) ALS patient-derived motor neurons have reduced delayed-rectifier potassium current amplitudes relative to control-derived motor neurons, a deficit that may underlie their hyperexcitability. The Kv7 channel activator retigabine both blocks the hyperexcitability and improves motor neuron survival in vitro when tested in SOD1 mutant ALS cases. Therefore, electrophysiological characterization of human stem cell-derived neurons can reveal disease-related mechanisms and identify therapeutic candidates.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Among the disciplines of medicine, the study of neurological disorders is particularly challenging. The fundamental inaccessibility of the human neural types affected by disease prevents their ...isolation for in vitro studies of degenerative mechanisms or for drug screening efforts. However, the ability to reprogram readily accessible tissue from patients into pluripotent stem (iPS) cells may now provide a general solution to this shortage of human neurons. Gradually improving methods for directing the differentiation of patient-specific stem cells has enabled the production of several neural cell types affected by disease. Furthermore, initial studies with stem cell lines derived from individuals with pediatric, monogenic disorders have validated the stem cell approach to disease modeling, allowing relevant neural phenotypes to be observed and studied. Whether iPS cell-derived neurons will always faithfully recapitulate the same degenerative processes observed in patients and serve as platforms for drug discovery relevant to common late-onset diseases remains to be determined.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The findings that amyotrophic lateral sclerosis (ALS) patients almost universally display pathological mislocalization of the RNA-binding protein TDP-43 and that mutations in its gene cause familial ...ALS have nominated altered RNA metabolism as a disease mechanism. However, the RNAs regulated by TDP-43 in motor neurons and their connection to neuropathy remain to be identified. Here we report transcripts whose abundances in human motor neurons are sensitive to TDP-43 depletion. Notably, expression of STMN2, which encodes a microtubule regulator, declined after TDP-43 knockdown and TDP-43 mislocalization as well as in patient-specific motor neurons and postmortem patient spinal cord. STMN2 loss upon reduced TDP-43 function was due to altered splicing, which is functionally important, as we show STMN2 is necessary for normal axonal outgrowth and regeneration. Notably, post-translational stabilization of STMN2 rescued neurite outgrowth and axon regeneration deficits induced by TDP-43 depletion. We propose that restoring STMN2 expression warrants examination as a therapeutic strategy for ALS.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
This study attempts a new identification of mechanisms of secondary ice production (SIP) based on the observation of small faceted ice crystals (hexagonal plates or columns) with typical sizes ...smaller than 100µm. Due to their young age, such small ice crystals can be used as tracers for identifying the conditions for SIP. Observations reported here were conducted in oceanic tropical mesoscale convective systems (MCSs) and midlatitude frontal clouds in the temperature range from 0 to −15◦C and heavily seeded by aged ice particles. It was found that in both MCSs and frontal clouds, SIP was observed right above the melting layer and extended to higher altitudes with colder temperatures. The roles of six possible mechanisms to generate the SIP particles are assessed using additional observations. In most observed SIP cases, small secondary ice particles spatially correlated with liquid-phase, vertical updrafts and aged rimed ice particles. However, in many cases, neither graupel nor liquid drops were observed in the SIP regions, and therefore, the conditions for an active Hallett–Mossop process were not met. In many cases, large concentrations of small pristine ice particles were observed right above the melting layer, starting at temperatures as warm as −0.5◦C. It is proposed that the initiation of SIP above the melting layer is stimulated by the recirculation of large liquid drops through the melting layer with convective turbulent updrafts. After re-entering a super cooled environment above the melting layer, they impact with aged ice, freeze, and shatter. The size of the splinters generated during SIP was estimated as 10µm or less. A principal conclusion of this work is that only the freezing drop-shattering mechanism could be clearly supported by the airborne in situ observations.
How to make spinal motor neurons Davis-Dusenbery, Brandi N; Williams, Luis A; Klim, Joseph R ...
Development (Cambridge),
02/2014, Volume:
141, Issue:
3
Journal Article
Peer reviewed
Open access
All muscle movements, including breathing, walking, and fine motor skills rely on the function of the spinal motor neuron to transmit signals from the brain to individual muscle groups. Loss of ...spinal motor neuron function underlies several neurological disorders for which treatment has been hampered by the inability to obtain sufficient quantities of primary motor neurons to perform mechanistic studies or drug screens. Progress towards overcoming this challenge has been achieved through the synthesis of developmental biology paradigms and advances in stem cell and reprogramming technology, which allow the production of motor neurons in vitro. In this Primer, we discuss how the logic of spinal motor neuron development has been applied to allow generation of motor neurons either from pluripotent stem cells by directed differentiation and transcriptional programming, or from somatic cells by direct lineage conversion. Finally, we discuss methods to evaluate the molecular and functional properties of motor neurons generated through each of these techniques.
Although many distinct mutations in a variety of genes are known to cause Amyotrophic Lateral Sclerosis (ALS), it remains poorly understood how they selectively impact motor neuron biology and ...whether they converge on common pathways to cause neuronal degeneration. Here, we have combined reprogramming and stem cell differentiation approaches with genome engineering and RNA sequencing to define the transcriptional and functional changes that are induced in human motor neurons by mutant SOD1. Mutant SOD1 protein induced a transcriptional signature indicative of increased oxidative stress, reduced mitochondrial function, altered subcellular transport, and activation of the ER stress and unfolded protein response pathways. Functional studies demonstrated that these pathways were perturbed in a manner dependent on the SOD1 mutation. Finally, interrogation of stem-cell-derived motor neurons produced from ALS patients harboring a repeat expansion in C9orf72 indicates that at least a subset of these changes are more broadly conserved in ALS.
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•iPSC-derived motor neurons harboring SOD1 mutations exhibit cell survival deficits•Genetic correction rescues ALS-related phenotypes•RNA-seq reveals expression changes and mitochondrial and ER stress disturbances•Motor neurons exhibit inherent ER stress linked to electrical activity
Motor neurons differentiated from human-ALS-patient-derived iPSCs were used to define transcriptional and functional changes arising from SOD1 mutations, which could be reversed by genome engineering of the SOD1 locus.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The contributions of diverse cell populations in the human lung to pulmonary fibrosis pathogenesis are poorly understood. Single-cell RNA sequencing can reveal changes within individual cell ...populations during pulmonary fibrosis that are important for disease pathogenesis.
To determine whether single-cell RNA sequencing can reveal disease-related heterogeneity within alveolar macrophages, epithelial cells, or other cell types in lung tissue from subjects with pulmonary fibrosis compared with control subjects.
We performed single-cell RNA sequencing on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and on one bronchoscopic cryobiospy sample from a patient with idiopathic pulmonary fibrosis. We validated these data using
RNA hybridization, immunohistochemistry, and bulk RNA-sequencing on flow-sorted cells from 22 additional subjects.
We identified a distinct, novel population of profibrotic alveolar macrophages exclusively in patients with fibrosis. Within epithelial cells, the expression of genes involved in Wnt secretion and response was restricted to nonoverlapping cells. We identified rare cell populations including airway stem cells and senescent cells emerging during pulmonary fibrosis. We developed a web-based tool to explore these data.
We generated a single-cell atlas of pulmonary fibrosis. Using this atlas, we demonstrated heterogeneity within alveolar macrophages and epithelial cells from subjects with pulmonary fibrosis. These results support the feasibility of discovery-based approaches using next-generation sequencing technologies to identify signaling pathways for targeting in the development of personalized therapies for patients with pulmonary fibrosis.
Oral contraceptive pills are used by approximately 250 million women worldwide, however a clear understanding of the concentrations of endogenous and exogenous hormones across a 28-day oral ...contraceptive pill pack is not well described. In our study of 16 female participants taking various monophasic oral contraceptive pills, we found significant fluctuations in endogenous and exogenous hormone levels throughout the pill cycle, challenging the previous assumption of hormonal stability in oral contraceptive users. The results from this study have wide ranging implications for research and treatment in women's health including: considerations in research design and interpretation for studies including women taking oral contraceptives, the potential for more precise and personalized methods of dosing to reduce unwanted side effects and adverse events, and the potential treatment of a variety of disorders ranging from musculoskeletal to neurological with exogenous hormones.
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