Influence of the Mechanical Environment on the Engineering of Mineralised Tissues Using Human Dental Pulp Stem Cells and Silk Fibroin Scaffolds

Teeth constitute a promising source of stem cells that can be used for tissue engineering and regenerative medicine purposes. Bone loss in the craniofacial complex due to pathological conditions and severe injuries could be treated with new materials combined with human dental pulp stem cells (hDPSCs) that have the same embryonic origin as craniofacial bones. Optimising combinations of scaffolds, cells, growth factors and culture conditions still remains a great challenge. In the present study, we evaluate the mineralisation potential of hDPSCs seeded on porous silk fibroin scaffolds in a mechanically dynamic environment provided by spinner flask bioreactors. Cell-seeded scaffolds were cultured in either standard or osteogenic media in both static and dynamic conditions for 47 days. Histological analysis and micro-computed tomography of the samples showed low levels of mineralisation when samples were cultured in static conditions (0.16±0.1 BV/TV%), while their culture in a dynamic environment with osteogenic medium and weekly µCT scans (4.9±1.6 BV/TV%) significantly increased the formation of homogeneously mineralised structures, which was also confirmed by the elevated calcium levels (4.5±1.0 vs. 8.8±1.7 mg/mL). Molecular analysis of the samples showed that the expression of tooth correlated genes such as Dentin Sialophosphoprotein and Nestin were downregulated by a factor of 6.7 and 7.4, respectively, in hDPSCs when cultured in presence of osteogenic medium. This finding indicates that hDPSCs are able to adopt a non-dental identity by changing the culture conditions only. Also an increased expression of Osteocalcin (1.4x) and Collagen type I (1.7x) was found after culture under mechanically dynamic conditions in control medium. In conclusion, the combination of hDPSCs and silk scaffolds cultured under mechanical loading in spinner flask bioreactors could offer a novel and promising approach for bone tissue engineering where appropriate and rapid bone regeneration in mechanically loaded tissues is required.     

Light Respiratory Processes and Gross Photosynthesis in Two Scleractinian Corals

The light dependency of respiratory activity of two scleractinian corals was examined using O₂ microsensors and CO₂ exchange measurements. Light respiration increased strongly but asymptotically with elevated irradiance in both species. Light respiration in Pocillopora damicornis was higher than in Pavona decussata under low irradiance, indicating species-specific differences in light-dependent metabolic processes. Overall, the coral P. decussata exhibited higher CO₂ uptake rates than P. damicornis over the experimental irradiance range. P. decussata also harboured twice as many algal symbionts and higher total protein biomass compared to P. damicornis, possibly resulting in self-shading of the symbionts and/or changes in host tissue specific light distribution. Differences in light respiration and CO₂ availability could be due to host-specific characteristics that modulate the symbiont microenvironment, its photosynthesis, and hence the overall performance of the coral holobiont.     

Neurostimulation of the Cholinergic Anti-Inflammatory Pathway Ameliorates Disease in Rat Collagen-Induced Arthritis

Introduction

The inflammatory reflex is a physiological mechanism through which the nervous system maintains immunologic homeostasis by modulating innate and adaptive immunity. We postulated that the reflex might be harnessed therapeutically to reduce pathological levels of inflammation in rheumatoid arthritis by activating its prototypical efferent arm, termed the cholinergic anti-inflammatory pathway. To explore this, we determined whether electrical neurostimulation of the cholinergic anti-inflammatory pathway reduced disease severity in the collagen-induced arthritis model.

Methods

Rats implanted with vagus nerve cuff electrodes had collagen-induced arthritis induced and were followed for 15 days. Animals underwent active or sham electrical stimulation once daily from day 9 through the conclusion of the study. Joint swelling, histology, and levels of cytokines and bone metabolism mediators were assessed.

Results

Compared with sham treatment, active neurostimulation of the cholinergic anti-inflammatory pathway resulted in a 52% reduction in ankle diameter (p = 0.02), a 57% reduction in ankle diameter (area under curve; p = 0.02) and 46% reduction overall histological arthritis score (p = 0.01) with significant improvements in inflammation, pannus formation, cartilage destruction, and bone erosion (p = 0.02), accompanied by numerical reductions in systemic cytokine levels, not reaching statistical significance. Bone erosion improvement was associated with a decrease in serum levels of receptor activator of NF-κB ligand (RANKL) from 132±13 to 6±2 pg/mL (mean±SEM, p = 0.01).

Conclusions

The severity of collagen-induced arthritis is reduced by neurostimulation of the cholinergic anti-inflammatory pathway delivered using an implanted electrical vagus nerve stimulation cuff electrode, and supports the rationale for testing this approach in human inflammatory disorders.     

Cognitive Function in Ambulatory Patients with Systolic Heart Failure: Insights from the Warfarin versus Aspirin in Reduced Cardiac Ejection Fraction (WARCEF) Trial

We sought to determine whether cognitive function in stable outpatients with heart failure (HF) is affected by HF severity. A retrospective, cross-sectional analysis was performed using data from 2, 043 outpatients with systolic HF and without prior stroke enrolled in the Warfarin versus Aspirin in Reduced Cardiac Ejection Fraction (WARCEF) Trial. Multivariable regression analysis was used to assess the relationship between cognitive function measured using the Mini-Mental Status Exam (MMSE) and markers of HF severity (left ventricular ejection fraction [LVEF], New York Heart Association [NYHA] functional class, and 6-minute walk distance). The mean (SD) for the MMSE was 28.6 (2.0), with 64 (3.1%) of the 2,043 patients meeting the cut-off of MMSE <24 that indicates need for further evaluation of cognitive impairment. After adjustment for demographic and clinical covariates, 6-minute walk distance (β-coefficient 0.002, p<0.0001), but not LVEF or NYHA functional class, was independently associated with the MMSE as a continuous measure. Age, education, smoking status, body mass index, and hemoglobin level were also independently associated with the MMSE. In conclusion, six-minute walk distance, but not LVEF or NYHA functional class, was an important predictor of cognitive function in ambulatory patients with systolic heart failure.     

Synthetic Lethality in the Tobacco Plastid Ribosome and Its Rescue at Elevated Growth Temperatures

Consistent with their origin from cyanobacteria, plastids (chloroplasts) perform protein biosynthesis on bacterial-type 70S ribosomes. The plastid genomes of seed plants contain a conserved set of ribosomal protein genes. Three of these have proven to be nonessential for translation and, thus, for cellular viability: rps15, rpl33, and rpl36. To help define the minimum ribosome, here, we examined whether more than one of these nonessential plastid ribosomal proteins can be removed from the 70S ribosome. To that end, we constructed all possible double knockouts for the S15, L33, and L36 ribosomal proteins by stable transformation of the tobacco (Nicotiana tabacum) plastid genome. We find that, although S15 and L33 function in different ribosomal particles (30S and 50S, respectively), their combined deletion from the plastid genome results in synthetic lethality under autotrophic conditions. Interestingly, the lethality can be overcome by growth under elevated temperatures due to an improved efficiency of plastid ribosome biogenesis. Our results reveal functional interactions between protein and RNA components of the 70S ribosome and uncover the interdependence of the biogenesis of the two ribosomal subunits. In addition, our findings suggest that defining a minimal set of plastid genes may prove more complex than generally believed.     

Phenylcoumaran Benzylic Ether Reductase Prevents Accumulation of Compounds Formed under Oxidative Conditions in Poplar Xylem

Phenylcoumaran benzylic ether reductase (PCBER) is one of the most abundant proteins in poplar (Populus spp) xylem, but its biological role has remained obscure. In this work, metabolite profiling of transgenic poplar trees downregulated in PCBER revealed both the in vivo substrate and product of PCBER. Based on mass spectrometry and NMR data, the substrate was identified as a hexosylated 8–5-coupling product between sinapyl alcohol and guaiacylglycerol, and the product was identified as its benzyl-reduced form. This activity was confirmed in vitro using a purified recombinant PCBER expressed in Escherichia coli. Assays performed on 20 synthetic substrate analogs revealed the enzyme specificity. In addition, the xylem of PCBER-downregulated trees accumulated over 2000-fold higher levels of cysteine adducts of monolignol dimers. These compounds could be generated in vitro by simple oxidative coupling assays involving monolignols and cysteine. Altogether, our data suggest that the function of PCBER is to reduce phenylpropanoid dimers in planta to form antioxidants that protect the plant against oxidative damage. In addition to describing the catalytic activity of one of the most abundant enzymes in wood, we provide experimental evidence for the antioxidant role of a phenylpropanoid coupling product in planta.     

Chronic obstructive pulmonary disease (COPD): Evaluation from clinical,immunological and bacterial pathogenesis perspectives

Chronic obstructive pulmonary disease (COPD), a disease manifested by significantly impaired airflow, afflicts ～14.2 million cases in the United States alone with an estimated 63 million people world-wide. Although there are a number of causes, the predominant cause is excessive tobacco smoke. In fact, in China, there have been estimates of 315,000,000 people that smoke. Other less frequent causes are associated with indirect cigarette smoke, air pollutants, biomass fuels, and genetic mutations. COPD is often associated with heart disease, lung cancer, osteoporosis and conditions can worsen in patients with sudden falls. COPD also affects both innate and adaptive immune processes. Cigarette smoke increases the expression of matrix metalloproteases and proinflammatory chemokines and increases lung titers of natural killer cells and neutrophils. Yet, neutrophil reactive oxygen species (ROS) mediated by the phagocytic respiratory burst and phagocytosis is impaired by nicotine. In contrast to innate immunity in COPD, dendritic cells represent leukocytes recruited to the lung that link the innate immune responses to adaptive immune responses by activating naïve T cells through antigen presentation. The autoimmune process that is also a significant part of inflammation associated with COPD. Moreover, coupled with restricted FEV1 values, are the prevalence of patients with single or multiple infections by bacteria, viruses and fungi. Finally, we focus on one of the more problematic infectious agents, the Gram-negative opportunistic pathogenic bacterium, Pseudomonas aeruginosa. Specifically, we delve into the development of highly problematic biofilm infections that are highly refractory to conventional antibiotic therapies in COPD. We offer a non-conventional, biocidal treatment that may be effective for COPD airway infections as well as with combinations of current antibiotic regimens for more effective treatment outcomes and relief for patients with COPD.     

Characterization of Blockade Antibody Responses in GII.2.1976 Snow Mountain Virus-Infected Subjects

Snow Mountain virus (GII.2.1976) is the prototype strain of GII.2 noroviruses (NoVs), which cause an estimated 8% of norovirus outbreaks, yet little is known about the immunobiology of these viruses. To define the human immune response induced by SMV infection and the antigenic relationship between different GII.2 strains that have circulated between 1976 and 2010, we developed a panel of four GII.2 variant virus-like particles (VLPs) and compared their antigenicities by enzyme immunoassay (EIA) and surrogate antibody neutralization (blockade) assays. Volunteers infected with GII.2.1976 developed a mean 167-fold increase in blockade response against the homotypic VLP by day 8 postchallenge. Blockade extended cross-genotype activity in some individuals but not cross-genogroup activity. Polyclonal sera from GII.2.1976-infected volunteers blocked GII.2.1976 significantly better than they blocked GII.2.2002, GII.2.2008, and GII.2.2010, suggesting that blockade epitopes within the GII.2 strains have evolved in the past decade. To potentially map these epitope changes, we developed mouse monoclonal antibodies (MAbs) against GII.2.1976 VLPs and compared their reactivities to a panel of norovirus VLPs. One MAb had broad cross-genogroup EIA reactivity to a nonblockade, linear, conserved epitope. Six MAbs recognized conformational epitopes exclusive to the GII.2 strains. Two MAbs recognized GII.2 blockade epitopes, and both blocked the entire panel of GII.2 variants. These data indicate that the GII.2 strains, unlike the predominant GII.4 strains, have undergone only a limited amount of evolution in blockade epitopes between 1976 and 2010 and indicate that the GII.2-protective component of a multivalent norovirus vaccine may not require frequent reformulation.