Computer simulations of in vitro morphogenesis

One of the most important challenges of contemporary biology is understanding how cells assemble into tissues. The complexity of morphogenesis calls for computational tools able to identify the dominant mechanisms involved in shaping tissues. This narrative review presents individual-based computational models that proved useful in simulating phenomena of interest in tissue engineering (TE), a research field that aims to create tissue replacements in the laboratory. First, we briefly describe morphogenetic mechanisms. Then, we present several computational models of cellular and subcellular resolution, along with applications that illustrate their potential to address problems of TE. Finally, we analyze experiments that may be used to validate computational models of tissue constructs made of cohesive cells. Our analysis shows that the models available in the literature are not exploited to their full potential. We argue that, upon validation, a computational model can be used to optimize cell culture conditions and to design new experiments.     

Fluidization, resolidification, and reorientation of the endothelial cell in response to slow tidal stretches

Mechanical stretch plays an important role in regulating shape and orientation of the vascular endothelial cell. This morphological response to stretch is basic to angiogenesis, neovascularization, and vascular homeostasis, but mechanism remains unclear. To elucidate mechanisms, we used cell mapping rheometry to measure traction forces in primary human umbilical vein endothelial cells subjected to periodic uniaxial stretches. Onset of periodic stretch of 10% strain amplitude caused a fluidization response typified by attenuation of traction forces almost to zero. As periodic stretch continued, the prompt fluidization response was followed by a slow resolidification response typified by recovery of the traction forces, but now aligned along the axis perpendicular to the imposed stretch. Reorientation of the cell body lagged reorientation of the traction forces, however. Together, these observations demonstrate that cellular reorientation in response to periodic stretch is preceded by traction attenuation by means of cytoskeletal fluidization and subsequent traction recovery transverse to the stretch direction by means of cytoskeletal resolidification.     

Elevated expression of cav-1 in a subset of SSc fibroblasts contributes to constitutive Alk1/Smad1 activation

Previous studies have shown that the transforming growth factor (TGF)β/Alk1/Smad1 signaling pathway is constitutively activated in a subset of systemic sclerosis (SSc) fibroblasts and this pathway is a critical regulator of CCN2 gene expression. Caveolin-1 (cav-1), an integral membrane protein and the main component of caveolae, has also been implicated in SSc pathogenesis. This study was undertaken to evaluate the role of caveolin-1 in Smad1 signaling and CCN2 expression in healthy and SSc dermal fibroblasts. We show that a significant subset of SSc dermal fibroblasts has up-regulated cav-1 expression in vitro, and that cav-1 up-regulation correlates with constitutive Smad1 phosphorylation. In addition, basal levels of phospho-Smad1 were down-regulated after inhibition of cav-1 in SSc dermal fibroblasts. Caveolin-1 formed a protein complex with Alk1 in dermal fibroblasts, and this association was enhanced by TGFβ. By using siRNA against cav-1 and adenoviral cav-1 overexpression we demonstrate that activation of Smad1 in response to TGFβ requires cav-1 and that cav-1 is sufficient for Smad-1 phosphorylation. We also show that cav-1 is a positive regulator of CCN2 gene expression, and that it is required for the basal and TGFβ-induced CCN2 levels. In conclusion, this study has revealed an important role of cav-1 in mediating TGFβ/Smad1 signaling and CCN2 gene expression in healthy and SSc dermal fibroblasts.     

Tracheal telocytes

Recently, a novel type of stromal cell - the telocytes (TC) - was identified in mouse trachea. These cells are known to possess the ultrastructural characteristics, which support their role in intercellular signaling. We found TC in all stromal compartments of the tracheal wall. TC with long prolongations (telopodes, Tp) were lining longitudinally the collagen bundles, and were serially arranged (end-to-end connections of Tp were found). Noteworthy, Tp frequently establish stromal synapses with mast cells (MC). Primary cilia were also identified in TC. In conclusion, tracheal TC could be involved in the tracheal regulation (e.g. secretion, contractility). The tandem TC-MC deserves further investigations.     

Angiotensin II induces skin fibrosis: a novel mouse model of dermal fibrosis

Introduction

Systemic sclerosis (SSc) is an autoimmune inflammatory disorder of unknown etiology characterized by fibrosis of the skin and internal organs. Ang II (angiotensin II), a vasoconstrictive peptide, is a well-known inducer of kidney, heart, and liver fibrosis. The goal of this study was to investigate the profibrotic potential of Ang II in the mouse skin.

Methods

Ang II was administered by subcutaneous osmotic mini pumps to C57BL/6 male mice. Collagen-content measurements were performed with Gomori Trichrome staining and hydroxyproline assay. The mRNA expression level of collagens, TGF-β1, TGF-β2, TGF-β3, CTGF, αSMA, CD3, Emr1, CD45/B220, MCP1, and FSP1 were quantified with real-time polymerase chain reaction (PCR). Immunostaining was performed for markers of inflammation and fibrosis, including, phospho-Smad2, αSMA, CD3, Mac3, CD45/B220, and CD163B. Fibrocytes were identified by double staining with CD45/FSP1 and CD45/PH4. Endothelial cells undergoing endothelial-to-mesenchymal transition (EndoMT) were identified by double staining with VE-cadherin/FSP1.

Results

Ang II-infused mice develop prominent dermal fibrosis in the area proximal to the pump, as shown by increased collagen and CTGF mRNA levels, increased hydroxyproline content, and more tightly packed collagen fibers. In addition, elevated mRNA levels of TGF-β2 and TGF-β3 along with increased expression of pSmad2 were observed in the skin of Ang II-treated mice. Dermal fibrosis was accompanied by an increased number of infiltrating fibrocytes, and an increased number of αSMA-positive cells, as well as CD163B⁺ macrophages in the upper dermis. This correlated with significantly increased mRNA levels of αSMA, Emr1, and MCP1. Infiltration of CD3-, CD45/B220-, and Mac3-positive cells was observed mainly in the hypodermis. Furthermore, an increased number of double-positive VE-cadherin/FSP1 cells were detected in the hypodermis only.

Conclusions

This work demonstrates that Ang II induces both inflammation and fibrosis in the skin via MCP1 upregulation and accumulation of activated fibroblasts. Additionally, our data suggest that populations of these fibroblasts originate from circulating blood cells. Ang II infusion via osmotic minipumps could serve as a useful mouse model of skin fibrosis to gain new insights into pathogenic mechanisms and to test new antifibrotic therapies.     

Where’s the Noise? Key Features of Spontaneous Activity and Neural Variability Arise through Learning in a Deterministic Network

Even in the absence of sensory stimulation the brain is spontaneously active. This background “noise” seems to be the dominant cause of the notoriously high trial-to-trial variability of neural recordings. Recent experimental observations have extended our knowledge of trial-to-trial variability and spontaneous activity in several directions: 1. Trial-to-trial variability systematically decreases following the onset of a sensory stimulus or the start of a motor act. 2. Spontaneous activity states in sensory cortex outline the region of evoked sensory responses. 3. Across development, spontaneous activity aligns itself with typical evoked activity patterns. 4. The spontaneous brain activity prior to the presentation of an ambiguous stimulus predicts how the stimulus will be interpreted. At present it is unclear how these observations relate to each other and how they arise in cortical circuits. Here we demonstrate that all of these phenomena can be accounted for by a deterministic self-organizing recurrent neural network model (SORN), which learns a predictive model of its sensory environment. The SORN comprises recurrently coupled populations of excitatory and inhibitory threshold units and learns via a combination of spike-timing dependent plasticity (STDP) and homeostatic plasticity mechanisms. Similar to balanced network architectures, units in the network show irregular activity and variable responses to inputs. Additionally, however, the SORN exhibits sequence learning abilities matching recent findings from visual cortex and the network’s spontaneous activity reproduces the experimental findings mentioned above. Intriguingly, the network’s behaviour is reminiscent of sampling-based probabilistic inference, suggesting that correlates of sampling-based inference can develop from the interaction of STDP and homeostasis in deterministic networks. We conclude that key observations on spontaneous brain activity and the variability of neural responses can be accounted for by a simple deterministic recurrent neural network which learns a predictive model of its sensory environment via a combination of generic neural plasticity mechanisms.     

Identification of the asymptomatic Plasmodium falciparum and Plasmodium vivax gametocyte reservoir under different transmission intensities

BackgroundUnderstanding epidemiological variables affecting gametocyte carriage and density is essential to design interventions that most effectively reduce malaria human-to-mosquito transmission.Methodology/Principal findingsPlasmodium falciparum and P. vivax parasites and gametocytes were quantified by qPCR and RT-qPCR assays using the same methodologies in 5 cross-sectional surveys involving 16,493 individuals in Brazil, Thailand, Papua New Guinea, and Solomon Islands. The proportion of infections with detectable gametocytes per survey ranged from 44–94% for P. falciparum and from 23–72% for P. vivax. Blood-stage parasite density was the most important predictor of the probability to detect gametocytes. In moderate transmission settings (prevalence by qPCR>5%), parasite density decreased with age and the majority of gametocyte carriers were children. In low transmission settings (prevalence<5%), >65% of gametocyte carriers were adults. Per survey, 37–100% of all individuals positive for gametocytes by RT-qPCR were positive by light microscopy for asexual stages or gametocytes (overall: P. falciparum 178/348, P. vivax 235/398).Conclusions/SignificanceInterventions to reduce human-to-mosquito malaria transmission in moderate-high endemicity settings will have the greatest impact when children are targeted. In contrast, all age groups need to be included in control activities in low endemicity settings to achieve elimination. Detection of infections by light microscopy is a valuable tool to identify asymptomatic blood stage infections that likely contribute most to ongoing transmission at the time of sampling.     

Spatial Distribution and Molecular Diversity of Archaeal Communities in the Extreme Hypersaline Meromictic Brâncoveanu Lake (Transylvanian Basin,Romania)

Dating from the Middle Miocene, the massive halite deposits lying beneath the Transylvanian Basin (Central Romania) have been valuable mineral resources quarried for millennia. Among the numerous hypersaline pit lakes that resulted from this mining, Brâncoveanu Lake is unique by its extreme salinity. Assessment of physicochemical variables, water chemistry and trophic status indicated that Brâncoveanu Lake is a permanently stratified, pH-neutral, NaCl-rich and eutrophied system. We investigated the abundance, molecular diversity and vertical distribution of archaeal community by culture-independent approaches. Additionally, the most relevant environmental parameters shaping the archaeal community composition were evaluated by statistical methods. Archaea appeared to largely outnumber Bacteria; altogether the great prevalence of Halobacteriaceae-related sequences could imply a major contribution of this group to the biogeochemical carbon turnover. The fairly distinct composition of archaeal communities reflects the lake's physicochemical stratification. Among the limnological factors, salinity and oxygen showed a significant impact on determining the composition and structure of archaeal assemblages. Furthermore, Brâncoveanu Lake might harbor novel microorganisms such as members of the recently described phylum Nanohaloarchaea. Overall, this study reported the occurrence of halophilic Archaea in a little explored hydrogeochemical system and provided a better insight into geomicrobiology of meromictic hypersaline pit lakes.     

Unveiling Ga(III) phthalocyanine—a different photosensitizer in neuroblastoma cellular model

Phthalocyanines (Pc) and their metallated derivatives are strongly considered for photodynamic therapy (PDT) possessing unique properties as possible new photosensitizers (PS). We have used toxicological assessments, real‐time monitoring of cellular impedance, and imagistic measurements for assessing the in vitro dark toxicity and PDT efficacy of Ga(III)‐Pc in SHSy5Y neuroblastoma cells. We have established the non‐toxic concentration range of Ga(III)‐Pc, a compound which shows a high intracellular accumulation, with perinuclear distribution in confocal microscopy. By choosing Ga(III)Pc non‐toxic dose, we performed in vitro experimental PDT hampering cellular proliferation. Our proposed Ga(III)‐Pc could complete a future PS panel for neuroblastoma alternate therapy.