Mathematical Models for Cell Migration with Real-Time Cell Cycle Dynamics

The fluorescent ubiquitination-based cell cycle indicator, also known as FUCCI, allows the visualization of the G1 and S/G2/M cell cycle phases of individual cells. FUCCI consists of two fluorescent probes, so that cells in the G1 phase fluoresce red and cells in the S/G2/M phase fluoresce green. FUCCI reveals real-time information about cell cycle dynamics of individual cells, and can be used to explore how the cell cycle relates to the location of individual cells, local cell density, and different cellular microenvironments. In particular, FUCCI is used in experimental studies examining cell migration, such as malignant invasion and wound healing. Here we present, to our knowledge, new mathematical models that can describe cell migration and cell cycle dynamics as indicated by FUCCI. The fundamental model describes the two cell cycle phases, G1 and S/G2/M, which FUCCI directly labels. The extended model includes a third phase, early S, which FUCCI indirectly labels. We present experimental data from scratch assays using FUCCI-transduced melanoma cells, and show that the predictions of spatial and temporal patterns of cell density in the experiments can be described by the fundamental model. We obtain numerical solutions of both the fundamental and extended models, which can take the form of traveling waves. These solutions are mathematically interesting because they are a combination of moving wavefronts and moving pulses. We derive and confirm a simple analytical expression for the minimum wave speed, as well as exploring how the wave speed depends on the spatial decay rate of the initial condition.     

Logistic Proliferation of Cells in Scratch Assays is Delayed

Scratch assays are used to study how a population of cells re-colonises a vacant region on a two-dimensional substrate after a cell monolayer is scratched. These experiments are used in many applications including drug design for the treatment of cancer and chronic wounds. To provide insights into the mechanisms that drive scratch assays, solutions of continuum reaction–diffusion models have been calibrated to data from scratch assays. These models typically include a logistic source term to describe carrying capacity-limited proliferation; however, the choice of using a logistic source term is often made without examining whether it is valid. Here we study the proliferation of PC-3 prostate cancer cells in a scratch assay. All experimental results for the scratch assay are compared with equivalent results from a proliferation assay where the cell monolayer is not scratched. Visual inspection of the time evolution of the cell density away from the location of the scratch reveals a series of sigmoid curves that could be naively calibrated to the solution of the logistic growth model. However, careful analysis of the per capita growth rate as a function of density reveals several key differences between the proliferation of cells in scratch and proliferation assays. Our findings suggest that the logistic growth model is valid for the entire duration of the proliferation assay. On the other hand, guided by data, we suggest that there are two phases of proliferation in a scratch assay; at short time, we have a disturbance phase where proliferation is not logistic, and this is followed by a growth phase where proliferation appears to be logistic. These two phases are observed across a large number of experiments performed at different initial cell densities. Overall our study shows that simply calibrating the solution of a continuum model to a scratch assay might produce misleading parameter estimates, and this issue can be resolved by making a distinction between the disturbance and growth phases. Repeating our procedure for other scratch assays will provide insight into the roles of the disturbance and growth phases for different cell lines and scratch assays performed on different substrates.     

A randomised, blinded, placebo-controlled trial in dementia patients continuing or stopping neuroleptics (the DART-AD trial)

Background

There have been increasing concerns regarding the safety and efficacy of neuroleptics in people with dementia, but there are very few long-term trials to inform clinical practice. The aim of this study was to determine the impact of long-term treatment with neuroleptic agents upon global cognitive decline and neuropsychiatric symptoms in patients with Alzheimer disease.

Methods and Findings

Design: Randomised, blinded, placebo-controlled parallel two-group treatment discontinuation trial.Setting: Oxfordshire, Newcastle and Gateshead, London and Edinburgh, United Kingdom.Participants: Patients currently prescribed the neuroleptics thioridazine, chlorpromazine, haloperidol trifluoperazine or risperidone for behavioural or psychiatric disturbance in dementia for at least 3 mo.Interventions: Continue neuroleptic treatment for 12 mo or switch to an identical placebo.Outcome measures: Primary outcome was total Severe Impairment Battery (SIB) score. Neuropsychiatric symptoms were evaluated with the Neuropsychiatric Inventory (NPI).Results: 165 patients were randomised (83 to continue treatment and 82 to placebo, i.e., discontinue treatment), of whom 128 (78%) commenced treatment (64 continue/64 placebo). Of those, 26 were lost to follow-up (13 per arm), resulting in 51 patients per arm analysed for the primary outcome. There was no significant difference between the continue treatment and placebo groups in the estimated mean change in SIB scores between baseline and 6 mo; estimated mean difference in deterioration (favouring placebo) −0.4 (95% confidence interval [CI] −6.4 to 5.5), adjusted for baseline value (p = 0.9). For neuropsychiatric symptoms, there was no significant difference between the continue treatment and placebo groups (n = 56 and 53, respectively) in the estimated mean change in NPI scores between baseline and 6 mo; estimated mean difference in deterioration (favouring continue treatment) −2.4 (95% CI −8.2 to 3.5), adjusted for baseline value (p = 0.4). Both results became more pronounced at 12 mo. There was some evidence to suggest that those patients with initial NPI ≥ 15 benefited on neuropsychiatric symptoms from continuing treatment.

Conclusions

For most patients with AD, withdrawal of neuroleptics had no overall detrimental effect on functional and cognitive status. Neuroleptics may have some value in the maintenance treatment of more severe neuropsychiatric symptoms, but this benefit must be weighed against the side effects of therapy.Trial registration: Cochrane Central Registry of Controlled Trials/National Research Register (#ISRCTN33368770).     

Glycogen synthase (GYS1) mutation causes a novel skeletal muscle glycogenosis

Polysaccharide storage myopathy (PSSM) is a novel glycogenosis in horses characterized by abnormal glycogen accumulation in skeletal muscle and muscle damage with exertion. It is unlike glycogen storage diseases resulting from known defects in glycogenolysis, glycolysis, and glycogen synthesis that have been described in humans and domestic animals. A genome-wide association identified GYS1, encoding skeletal muscle glycogen synthase (GS), as a candidate gene for PSSM. DNA sequence analysis revealed a mutation resulting in an arginine-to-histidine substitution in a highly conserved region of GS. Functional analysis demonstrated an elevated GS activity in PSSM horses, and haplotype analysis and allele age estimation demonstrated that this mutation is identical by descent among horse breeds. This is the first report of a gain-of-function mutation in GYS1 resulting in a glycogenosis.     

A GYS1 gene mutation is highly associated with polysaccharide storage myopathy in Cob Normand draught horses

Glycogen storage diseases or glycogenoses are inherited diseases caused by abnormalities of enzymes that regulate the synthesis or degradation of glycogen. Deleterious mutations in many genes of the glyco(geno)lytic or the glycogenesis pathways can potentially cause a glycogenosis, and currently mutations in fourteen different genes are known to cause animal or human glycogenoses, resulting in myopathies and/or hepatic disorders. The genetic bases of two forms of glycogenosis are currently known in horses. A fatal neonatal polysystemic type IV glycogenosis, inherited recessively in affected Quarter Horse foals, is due to a mutation in the glycogen branching enzyme gene ( GBE1 ). A second type of glycogenosis, termed polysaccharide storage myopathy (PSSM), is observed in adult Quarter Horses and other breeds. A severe form of PSSM also occurs in draught horses. A mutation in the skeletal muscle glycogen synthase gene ( GYS1 ) was recently reported to be highly associated with PSSM in Quarter Horses and Belgian draught horses. This GYS1 point mutation appears to cause a gain-of-function of the enzyme and to result in the accumulation of a glycogen-like, less-branched polysaccharide in skeletal muscle. It is inherited as a dominant trait. The aim of this work was to test for possible associations between genetic polymorphisms in four candidate genes of the glycogen pathway or the GYS1 mutation in Cob Normand draught horses diagnosed with PSSM by muscle biopsy.     

Use of mesenchymal stem cells or autologous conditioned serum to modulate the inflammatory response to spermatozoa in mares

Current treatments for Persistent mating-induced endometritis such as uterine lavage and oxytocin therapy focus on aiding the uterus in removal of inflammatory products, but these treatments do not modulate the inciting inflammatory response. Biological treatments, such as autologous conditioned serum (ACS) and mesenchymal stem cells (MSCs), have been used in human and veterinary medicine for immunomodulation for over 10 years. The objectives of this project were to evaluate the ability of ACS or MSCs to modulate the inflammatory response to spermatozoa after breeding. Two experiments were performed with six normal mares in each study to evaluate the effects of intrauterine administration of ACS, dexamethasone, or a placebo (experiment 1), or allogeneic MSCs or a placebo (experiment 2) on the inflammatory response to spermatozoa using clinical and biochemical endpoints. Treatment with ACS and MSCs significantly (P < 0.05) reduced the number of neutrophils in the uterine lumen 6 hours after the sperm challenge. An increase (P < 0.05) in the anti-inflammatory cytokine IL-1Ra was observed after treatment with MSCs before exposure to spermatozoa. There was no difference in IL-1Ra concentration in mares treated with ACS, dexamethasone, or a placebo. Mesenchymal stem cells and ACS were able to modulate the immune response to spermatozoa in normal mares. The effect may be due to an increase in IL-1Ra in MSCs-treated mares, but other bioactive molecules may be responsible for the decrease in neutrophils in ACS-treated mares. Autologous conditioned serum and bone-derived culture expanded MSCs were able to modulate the uterine inflammatory response to spermatozoa in normal mares. Treatment with allogeneic stem cells may be beneficial if a similar modulation in inflammatory cytokines occurs in mares affected by persistent mating–induced endometritis.     

Dietary fatty acid composition influences tissue lipid profiles and regulation of body temperature in Japanese quail

Many avian species reduce their body temperature (T _b) to conserve energy during periods of inactivity, and we recently characterized how ambient temperature (T _a) and nutritional stress interact with one another to influence physiologically controlled hypothermic responses in Japanese quail (Coturnix japonica). In the present study, we examined how the fatty acid (FA) composition of the diet influences the FA composition of phospholipids in major organs and how these affect controlled hypothermic responses and metabolic rates in fasted birds. For 5 weeks prior to fasting, quail were fed a standard diet and gavaged each morning with 0.7 ml of water (control), or a vegetable oil comprising saturated fatty acids (SFA; coconut oil), or unsaturated fatty acids (UFA; canola oil). Birds were then fasted for 4 days at a T _a of 15°C. We found that, while fasting, both photophase and scotophase T _b decreased significantly more in the SFA treatment group than in the control group; apparently the former down-regulated their T _b set point. This deeper hypothermic response was correlated with changes in the phospholipid composition of the skeletal muscle and liver, which contained significantly more oleic acid (18:1) and less arachidonic acid (20:4), respectively. Our data imply that these two FAs may be associated with thermoregulation.     

Expression of insulin‐like growth factor‐1 receptor in metastatic uveal melanoma and implications for potential autocrine and paracrine tumor cell growth

We investigated the importance of the insulin‐like growth factor‐1 receptor (IGF‐1R) in hepatic metastases of uveal melanoma. The expression pattern of IGF‐1R in archival tissue samples of hepatic metastasis from 24 patients was analyzed by immunohistochemistry. All the samples of hepatic metastases stained positive for IGF‐1R. To investigate the biological role of IGF‐1R on the growth of metastatic uveal melanoma, a long‐term cell line obtained from a hepatic metastasis (TJU‐UM001) was evaluated. TJU‐UM001 expressed cell surface IGF‐1R (>90%) and proliferated in response to exogenous and endogenous insulin‐like growth factor‐1 (IGF‐1). Correlatively, anti‐IGF‐1R antibody completely blocked IGF‐1‐induced growth of TJU‐UM001 cells. IGF‐1 preferentially induced phosphorylation of Akt (S473) in quiescent TJU‐UM001 cells, and this was blocked by anti‐IGF‐1R antibody. This study suggests that autocrine and paracrine mechanisms underlie IGF‐1‐induced growth of metastatic uveal melanoma and underscore the potential benefit of IGF‐1 or IGF‐1R antagonism in treatment for metastatic uveal melanoma.     

Enveloped virus inactivation using neutral arginine solutions and applications in therapeutic protein purification processes

For the manufacturing of recombinant protein therapeutics produced from mammalian cell culture, demonstrating the capacity of the purification process to effectively clear infectious viruses is a regulatory requirement. At least two process steps, using different mechanisms of virus removal and/or inactivation, should be validated in support of the regulatory approval process. For example, exposure of the product stream to low pH, detergents or solvent/detergent combinations is commonly incorporated in protein purification processes for the inactivation of lipid‐enveloped viruses. However, some proteins have limited stability at low pH or in the presence of the detergents, and alternative techniques for achieving the inactivation of enveloped viruses would be beneficial. We present here an alternative and novel approach for the rapid inactivation of enveloped viruses using pH‐neutral buffer solutions containing arginine. The implementation of this approach in a monoclonal antibody or Fc‐fusion protein purification process is described and illustrated with several different therapeutic proteins. The use of the neutral pH arginine solution was able to effectively inactivate two enveloped model viruses, with no measurable effect on the product quality of the investigated proteins. Thus, the use of pH‐neutral arginine containing buffer solutions provides an alternative means of virus inactivation where other forms of virus inactivation, such as low pH and/or solvent/detergent treatments are not possible or undesirable due to protein stability limitations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:108–112, 2014