A Simulation Optimization Approach to Epidemic Forecasting

Reliable forecasts of influenza can aid in the control of both seasonal and pandemic outbreaks. We introduce a simulation optimization (SIMOP) approach for forecasting the influenza epidemic curve. This study represents the final step of a project aimed at using a combination of simulation, classification, statistical and optimization techniques to forecast the epidemic curve and infer underlying model parameters during an influenza outbreak. The SIMOP procedure combines an individual-based model and the Nelder-Mead simplex optimization method. The method is used to forecast epidemics simulated over synthetic social networks representing Montgomery County in Virginia, Miami, Seattle and surrounding metropolitan regions. The results are presented for the first four weeks. Depending on the synthetic network, the peak time could be predicted within a 95% CI as early as seven weeks before the actual peak. The peak infected and total infected were also accurately forecasted for Montgomery County in Virginia within the forecasting period. Forecasting of the epidemic curve for both seasonal and pandemic influenza outbreaks is a complex problem, however this is a preliminary step and the results suggest that more can be achieved in this area.     

Type-1 (CB1) Cannabinoid Receptor Promotes Neuronal Differentiation and Maturation of Neural Stem Cells

Neural stem cells (NSCs) are self-renewing cells that can differentiate into multiple neural lineages and repopulate regions of the brain after injury. We have investigated the role of endocannabinoids (eCBs), endogenous cues that modulate neuronal functions including neurogenesis, and their receptors CB₁ and CB₂ in mouse NSCs. Real-time PCR and Western blot analyses indicated that CB₁ is present at higher levels than CB₂ in NSCs. The eCB anandamide (AEA) or the CB₁-specific agonist ACEA enhanced NSC differentiation into neurons, but not astrocytes and oligodendrocytes, whereas the CB₂-specific agonist JWH133 was ineffective. Conversely, the effect of AEA was inhibited by CB₁, but not CB₂, antagonist, corroborating the specificity of the response. CB₁ activation also enhanced maturation of neurons, as indicated by morphometric analysis of neurites. CB₁ stimulation caused long-term inhibition of the ERK1/2 pathway. Consistently, pharmacological inhibition of the ERK1/2 pathway recapitulated the effects exerted by CB₁ activation on neuronal differentiation and maturation. Lastly, gene array profiling showed that CB₁ activation augmented the expression of genes involved in neuronal differentiation while decreasing that of stemness genes. These results highlight the role of CB₁ in the regulation of NSC fate and suggest that its activation may represent a pro-neuronal differentiation signal.     

Gastrointestinal Cell Lines Form Polarized Epithelia with an Adherent Mucus Layer when Cultured in Semi-Wet Interfaces with Mechanical Stimulation

Mucin glycoproteins are secreted in large quantities by mucosal epithelia and cell surface mucins are a prominent feature of the glycocalyx of all mucosal epithelia. Currently, studies investigating the gastrointestinal mucosal barrier use either animal experiments or non-in vivo like cell cultures. Many pathogens cause different pathology in mice compared to humans and the in vitro cell cultures used are suboptimal because they are very different from an in vivo mucosal surface, are often not polarized, lack important components of the glycocalyx, and often lack the mucus layer. Although gastrointestinal cell lines exist that produce mucins or polarize, human cell line models that reproducibly create the combination of a polarized epithelial cell layer, functional tight junctions and an adherent mucus layer have been missing until now. We trialed a range of treatments to induce polarization, 3D-organization, tight junctions, mucin production, mucus secretion, and formation of an adherent mucus layer that can be carried out using standard equipment. These treatments were tested on cell lines of intestinal (Caco-2, LS513, HT29, T84, LS174T, HT29 MTX-P8 and HT29 MTX-E12) and gastric (MKN7, MKN45, AGS, NCI-N87 and its hTERT Clone5 and Clone6) origins using Ussing chamber methodology and (immuno)histology. Semi-wet interface culture in combination with mechanical stimulation and DAPT caused HT29 MTX-P8, HT29 MTX-E12 and LS513 cells to polarize, form functional tight junctions, a three-dimensional architecture resembling colonic crypts, and produce an adherent mucus layer. Caco-2 and T84 cells also polarized, formed functional tight junctions and produced a thin adherent mucus layer after this treatment, but with less consistency. In conclusion, culture methods affect cell lines differently, and testing a matrix of methods vs. cell lines may be important to develop better in vitro models. The methods developed herein create in vitro mucosal surfaces suitable for studies of host-pathogen interactions at the mucosal surface.     

Immediate and Heterogeneous Response of the LiaFSR Two-Component System of Bacillus subtilis to the Peptide Antibiotic Bacitracin

Background

Two-component signal transduction systems are one means of bacteria to respond to external stimuli. The LiaFSR two-component system of Bacillus subtilis consists of a regular two-component system LiaRS comprising the core Histidine Kinase (HK) LiaS and the Response Regulator (RR) LiaR and additionally the accessory protein LiaF, which acts as a negative regulator of LiaRS-dependent signal transduction. The complete LiaFSR system was shown to respond to various peptide antibiotics interfering with cell wall biosynthesis, including bacitracin.

Methodology and Principal Findings

Here we study the response of the LiaFSR system to various concentrations of the peptide antibiotic bacitracin. Using quantitative fluorescence microscopy, we performed a whole population study analyzed on the single cell level. We investigated switching from the non-induced ‘OFF’ state into the bacitracin-induced ‘ON’ state by monitoring gene expression of a fluorescent reporter from the RR-regulated liaI promoter. We found that switching into the ‘ON’ state occurred within less than 20 min in a well-defined switching window, independent of the bacitracin concentration. The switching rate and the basal expression rate decreased at low bacitracin concentrations, establishing clear heterogeneity 60 min after bacitracin induction. Finally, we performed time-lapse microscopy of single cells confirming the quantitative response as obtained in the whole population analysis for high bacitracin concentrations.

Conclusion

The LiaFSR system exhibits an immediate, heterogeneous and graded response to the inducer bacitracin in the exponential growth phase.     

Effects of Warm Ischemic Time on Gene Expression Profiling in Colorectal Cancer Tissues and Normal Mucosa

Background

Genome-wide gene expression analyses of tumors are a powerful tool to identify gene signatures associated with biologically and clinically relevant characteristics and for several tumor types are under clinical validation by prospective trials. However, handling and processing of clinical specimens may significantly affect the molecular data obtained from their analysis. We studied the effects of tissue handling time on gene expression in human normal and tumor colon tissues undergoing routine surgical procedures.

Methods

RNA extracted from specimens of 15 patients at four time points (for a total of 180 samples) after surgery was analyzed for gene expression on high-density oligonucleotide microarrays. A mixed-effects model was used to identify probes with different expression means across the four different time points. The p-values of the model were adjusted with the Bonferroni method.

Results

Thirty-two probe sets associated with tissue handling time in the tumor specimens, and thirty-one in the normal tissues, were identified. Most genes exhibited moderate changes in expression over the time points analyzed; however four of them were oncogenes, and two confirmed the effect of tissue handling by independent validation.

Conclusions

Our results suggest that a critical time point for tissue handling in colon seems to be 60 minutes at room temperature. Although the number of time-dependent genes we identified was low, the three genes that already showed changes at this time point in tumor samples were all oncogenes, hence recommending standardization of tissue-handling protocols and effort to reduce the time from specimen removal to snap freezing accounting for warm ischemia in this tumor type.     

Environmental Factors Associated with Disease Progression after the First Demyelinating Event: Results from the Multi-Center SET Study

Objectives

To investigate the associations of environmental MS risk factors with clinical and MRI measures of progression in high-risk clinically isolated syndromes (CIS) after the first demyelinating event.

Methods

We analyzed 211 CIS patients (age: 28.9±7.8 years) enrolled in the SET study, a multi-center study of high-risk CIS patients. Pre-treatment samples were analyzed for IgG antibodies against cytomegalovirus (anti-CMV), Epstein Barr virus (EBV) early nuclear antigen-1 (EBNA-1), viral capsid antigen (VCA), early antigen-diffuse (EA-D), 25 hydroxy-vitamin D3 and cotinine levels and HLA DRB1*1501 status. The inclusion criteria required evaluation within 4 months of the initial demyelinating event, 2 or more brain MRI lesions and the presence of two or more oligoclonal bands in cerebrospinal fluid. All patients were treated with interferon-beta. Clinical and MRI assessments were obtained at baseline, 6, 12, and 24 months.

Results

The time to first relapse decreased and the number of relapses increased with anti-CMV IgG positivity. Smoking was associated with increased number and volume of contrast-enhancing lesions (CEL) during the 2-year period. The cumulative number of CEL and T2 lesions during the 2-year period was greater for individuals in the highest quartile of anti-EBV VCA IgG antibodies. The percent loss of brain volume was increased for those in the highest quartile of with anti-EBV VCA IgG antibodies.

Conclusions

Relapses in CIS patients were associated with CMV positivity whereas anti-EBV VCA positivity was associated with progression on MRI measures, including accumulation of CEL and T2 lesions and development of brain atrophy.     

Increased Cardiac Myocyte PDE5 Levels in Human and Murine Pressure Overload Hypertrophy Contribute to Adverse LV Remodeling

Background

The intracellular second messenger cGMP protects the heart under pathological conditions. We examined expression of phosphodiesterase 5 (PDE5), an enzyme that hydrolyzes cGMP, in human and mouse hearts subjected to sustained left ventricular (LV) pressure overload. We also determined the role of cardiac myocyte-specific PDE5 expression in adverse LV remodeling in mice after transverse aortic constriction (TAC).

Methodology/Principal Findings

In patients with severe aortic stenosis (AS) undergoing valve replacement, we detected greater myocardial PDE5 expression than in control hearts. We observed robust expression in scattered cardiac myocytes of those AS patients with higher LV filling pressures and BNP serum levels. Following TAC, we detected similar, focal PDE5 expression in cardiac myocytes of C57BL/6NTac mice exhibiting the most pronounced LV remodeling. To examine the effect of cell-specific PDE5 expression, we subjected transgenic mice with cardiac myocyte-specific PDE5 overexpression (PDE5-TG) to TAC. LV hypertrophy and fibrosis were similar as in WT, but PDE5-TG had increased cardiac dimensions, and decreased dP/dt_max and dP/dt_min with prolonged tau (P<0.05 for all). Greater cardiac dysfunction in PDE5-TG was associated with reduced myocardial cGMP and SERCA2 levels, and higher passive force in cardiac myocytes in vitro.

Conclusions/Significance

Myocardial PDE5 expression is increased in the hearts of humans and mice with chronic pressure overload. Increased cardiac myocyte-specific PDE5 expression is a molecular hallmark in hypertrophic hearts with contractile failure, and represents an important therapeutic target.