21 research outputs found
The first myriapod genome sequence reveals conservative arthropod gene content and genome organisation in the centipede Strigamia maritima.
Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific life history.This work was supported by the following grants: NHGRIU54HG003273 to R.A.G; EU Marie Curie ITN #215781 “Evonet” to M.A.; a Wellcome Trust Value in People (VIP) award to C.B. and Wellcome Trust graduate studentship WT089615MA to J.E.G; Marine
rhythms of Life” of the University of Vienna, an FWF (http://www.fwf.ac.at/) START award (#AY0041321) and HFSP (http://www.hfsp.org/) research grant (#RGY0082/2010) to KT-‐R; MFPL Vienna International PostDoctoral Program for Molecular Life Sciences (funded by Austrian Ministry of Science and Research and City of Vienna, Cultural Department -‐Science and Research to T.K; Direct Grant (4053034) of the Chinese University of Hong Kong to J.H.L.H.; NHGRI HG004164 to G.M.; Danish Research Agency (FNU), Carlsberg Foundation, and Lundbeck Foundation to C.J.P.G.; U.S. National Institutes of Health R01AI55624 to J.H.W.; Royal Society University Research fellowship to F.M.J.; P.D.E. was supported by the BBSRC via the Babraham Institute;This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.pbio.100200
Investigating the Effects of Indirect Coculture of Human Mesenchymal Stem Cells on the Migration of Breast Cancer Cells: A Systematic Review and Meta-Analysis
Purpose: Breast cancer is the most diagnosed cancer and the leading cause of cancer death in women globally, and mesenchymal stem cells have been widely implicated in tumour progression. This systematic review and meta-analysis seeks to identify and summarise existing literature on the effects of human mesenchymal stem cells (hMSCs) on the migration of breast cancer cells (BCCs) in vitro, to determine the direction of this relationship according to existing research and to identify the directions for future research. Methods: A systematic literature search was conducting using a collection of databases, using the following search terms: in vitro AND mesenchymal stem cells AND breast cancer. Only studies that investigated the effects of human, unmodified MSCs on the migration of human, unmodified BCCs in vitro were included. Standardised mean differences (SMDs) were calculated to determine pooled effect sizes. Results: This meta-analysis demonstrates that hMSCs (different sources combined) increase the migration of both MDA-MB-231 and MCF-7 cell lines in vitro (SMD = 1.84, P = .03 and SMD = 2.69, P < .00001, respectively). Importantly, the individual effects of hMSCs from different sources were also analysed and demonstrated that MSCs derived from human adipose tissue increase BCC migration (SMD = 1.34, P = .0002) and those derived from umbilical cord increased both MDA-MB-231 and MCF-7 migration (SMD = 3.93, P < .00001 and SMD = 3.01, P < .00001, respectively). Conclusions: To our knowledge, this is the first systematic review and meta-analysis investigating and summarising the effects of hMSCs from different sources on the migration of BCCs, in vitro
Cryopreservation of human bone marrow derived mesenchymal stem cells at high concentration is feasible
Introduction: For stem cell therapies to be adopted in mainstream health care, robust, reliable, and cost-effective storage and transport processes must be developed. Cryopreservation remains the best current platform for this purpose, and freezing cells at high concentration may have many benefits, including savings on cost and storage space, facilitating transport logistics, and reducing cryoprotectant volume. Cells, such as mesenchymal stem cells (MSCs), are typically frozen at 1 million cells per milliliter (mL), but the aim of this study is to examine the post-thaw attributes of human bone marrow derived MSCs (hBM-MSCs) frozen at 1, 5, and 10 million cells per mL.
Methods: Thawed cells were assessed for their morphology, phenotypic marker expression, viability, apoptosis level, metabolic activity, proliferation, and osteogenic and adipogenic differentiation.
Results: In this study, for the first time, it is shown that all assessed cells expressed the typical MSC markers (CD90, CD105, and CD73) and lacked the expression of CD14, CD20, CD34, CD45, and HLA-DR. In addition, all cells showed elongated fibroblastic morphology. Post-thaw viability was retained with no difference among the three concentrations. Moreover, no significant statistical difference was observed in the post-thaw apoptosis level, metabolic activity, proliferation, and osteogenic potential, indicating that these cells are amenable to cryopreservation at higher concentrations.
Conclusion: The results of this study are of paramount importance to the development of manufacturing processes around a useful freezing concentration when cells are targeted to be stored for at least 6 months.</p
The effect of chronic high insulin exposure upon metabolic and myogenic markers in C2C12 skeletal muscle cells and myotubes
This is the peer reviewed version of the following article: TURNER, M.C. ...et al., 2018. The effect of chronic high insulin exposure upon metabolic and myogenic markers in C2C12 skeletal muscle cells and myotubes.. Journal of Cellular Biochemistry, 119 (7), pp.5686-5695, which has been published in final form at https://doi.org/10.1002/jcb.26748. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.Skeletal muscle is an insulin sensitive tissue and accounts for approximately 80% of
post-prandial glucose disposal. This study describes the effects of insulin, delivered
for 72 hours, to skeletal muscle myoblasts during differentiation or to skeletal muscle
myotubes. After chronic treatment, cultures were acutely stimulated with insulin and
analysed for total and phosphorylated Akt (Ser473), mRNA expression of metabolic
and myogenic markers and insulin-stimulated glucose uptake. Skeletal muscle cells
differentiated in the presence of insulin chronically, reduced acute insulin stimulated
phosphorylation of Akt Ser473. In addition, there was a reduction in mRNA expression
of Hexokinase II (HKII), GLUT4 and PGC-1α. Insulin-stimulated glucose uptake was
attenuated when cells were differentiated in the presence of insulin. In contrast,
myotubes exposed to chronic insulin showed no alterations in phosphorylation of Akt
Ser473. Both HKII and GLUT4 mRNA expression were reduced by chronic exposure
to insulin; while PGC-1α was not different between culture conditions and was
increased by acute insulin stimulation. These data suggest that there are differential
responses in insulin signalling, transcription and glucose uptake of skeletal muscle
cells when cultured in either the presence of insulin during differentiation or in myotube cultures
583. The effect of steam, hypochlorite and caustic soda used for treating direct-to-can milking equipment, on the bacteriological quality and flora of milk
Pharmacological characterization of type 1α metabotropic glutamate receptor-stimulated [(35)S]-GTPγS binding
1. The activation of G proteins by type 1α metabotropic glutamate receptors (mGluRs) in membranes from recombinant baby hamster kidney cells expressing the cloned rat mGluR1α receptor has been studied by use of a [(35)S]-guanosine 5′-[γ-thio]triphosphate ([(35)S]-GTPγS) binding assay. 2. L-Glutamate increased the rate of [(35)S]-GTPγS binding in a concentration-dependent manner (−logEC(50) (M) 5.25±0.07), with an optimal (62.4±1.6%) increase over basal binding being observed following 60 min incubation at 30°C with 70 pM [(35)S]-GTPγS, 1 μM GDP, 10 mM MgCl(2), 100 mM NaCl and 100 μg membrane protein ml(−1). The L-glutamate (100 μM)-stimulated increase in [(35)S]-GTPγS binding was totally prevented in the presence of the group I mGluR antagonist (S)-4-carboxy-3-hydroxyphenylglycine (300 μM). 3. Quantitative analysis of the affinity and number of G proteins activated by a maximally effective concentration of L-glutamate revealed an equilibrium dissociation constant (K(D)) for [(35)S]-GTPγS binding of 0.76±0.20 nM and a maximal number of GTPγS-liganded G proteins (B(max)) of 361±30 fmol mg(−1) protein. 4. Metabotropic glutamate receptor agonists, quisqualate (−logEC(50) (M) 6.74±0.06), 1S,3R-ACPD (4.64±0.08) and (S)-3,5-dihydroxyphenylglycine (5.16±0.23) also increased [(35)S]-GTPγS binding in a concentration-dependent manner, with the latter two agents behaving as partial agonists. 5. (+)-α-Methylcarboxyphenylglycine (300 μM) caused a parallel rightward shift of the L-glutamate concentration-effect curve for [(35)S]-GTPγS binding, allowing an antagonist equilibrium dissociation constant (K(D)) of 34.0±7.8 μM to be calculated for this mGluR antagonist. 6. Pretreatment of BHK-mGluR1α cells with a concentration of pertussis toxin (PTX) shown to be maximally effective (100 ng ml(−1), 24 h) before membrane preparation resulted in a marked decrease in agonist-stimulated [(35)S]-GTPγS binding (by 66.0±0.9%), and an altered concentration-effect relationship for agonist-stimulated [(35)S]-GTPγS binding by the residual PTX-insensitive G-protein population. 7. The modulation of [(35)S]-GTPγS binding by agonists and antagonists in membranes from recombinant cells provides an excellent system in which to study mGluR interactions with PTX-sensitive and -insensitive G proteins
Altered M1 Muscarinic Acetylcholine Receptor (CHRM1)-Gαq/11 Coupling in a Schizophrenia Endophenotype
Alterations in muscarinic acetylcholine receptor (CHRM) populations have been implicated in the pathology of schizophrenia. Here we have assessed whether the receptor function of the M1 subtype (CHRM1) is altered in a sub-population of patients with schizophrenia, defined by marked (60–80%) reductions in cortical [3H]-pirenzepine (PZP) binding, and termed ‘muscarinic receptor-deficit schizophrenia’ (MRDS). Using a [35S]-GTPγS-Gαq/11 immunocapture method we have assessed whether CHRM1 signalling in human cortex (Brodmann area 9 (BA9)) is altered in post mortem tissue from a MRDS group compared with a subgroup of patients with schizophrenia displaying normal PZP binding, and controls with no known history of psychiatric or neurological disorders. The CHRM agonist (oxotremorine-M) and a CHRM1-selective agonist (AC-42) increased Gαq/11-[35S]-GTPγS binding, with AC-42 producing responses that were ~50% of those maximally evoked by the full agonist, oxotremorine-M, in control and subgroups of patients with schizophrenia. However, the potency of oxotremorine-M to stimulate Gαq/11-[35S]-GTPγS binding was significantly decreased in the MRDS group (pEC50 (M)=5.69±0.16) compared with the control group (6.17±0.10) and the non-MRDS group (6.05±0.07). The levels of Gαq/11 protein present in BA9 did not vary with diagnosis. Maximal oxotremorine-M-stimulated Gαq/11-[35S]-GTPγS binding in BA9 membranes was significantly increased in the MRDS group compared with the control group. Similar, though non-statistically significant, trends were observed for AC-42. These data provide evidence that both orthosterically and allosterically acting CHRM agonists can stimulate a receptor-driven functional response ([35S]-GTPγS binding to Gαq/11) in membranes prepared from post mortem human dorsolateral prefrontal cortex of patients with schizophrenia and controls . Furthermore, in a subgroup of patients with schizophrenia displaying markedly decreased PZP binding (MRDS) we have shown that although agonist potency may decrease, the efficacy of CHRM1-Gαq/11 coupling increases, suggesting an adaptative change in receptor-G protein coupling efficiency in this endophenotype of patients with schizophrenia
The first myriapod genome sequence reveals conservative arthropod gene content and genome organisation in the centipede <i>Strigamia maritima</i>
Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific life history