Multi-layered electrospinning and electrospraying approach: Effect of polymeric supplements on chondrocyte suspension

Articular cartilage was expected to be one of the first tissues to be successfully engineered, but replicating the complex fibril architecture and the cellular distribution of the native cartilage has proven difficult. While electrospinning has been widely used to reproduce the depth-dependent fibre architecture in 3D scaffolds, the chondrocyte-controlled distribution remains an unsolved problem. To incorporate cells homogeneously through the depth of scaffolds, a combination of polymer electrospinning and cell seeding is necessary. A multi-layer approach alternating between polymer electrospinning with chondrocyte electrospraying can be a solution. Still, the success of this process is related to the survival rate of the electrosprayed chondrocytes embedded within the electrospun mesh. In this regard, the present study investigated the impact of the multi-layered process and the supplementation of the electrospray chondrocyte suspension with different concentrations of Gelatin and Alginate on the viability of electrosprayed chondrocytes embedded within a Polycaprolactone/Gelatin electrospun mesh and on the mechanical properties of the resulting meshes. The addition of Gelatin in the chondrocyte suspension did not increase significantly ( p > 0.05) the percentage of viable electrosprayed chondrocytes (25%), while 3 wt% Alginate addition led to a significant ( p < 0.05) increase in chondrocyte viability (50%) relative to the case without polymer supplement (15%). Furthermore, the addition of both polymer supplements increased the mechanical properties of the multi-layer construct. These findings imply that this multi-layered approach can be applied to cartilage TE allowing for automated chondrocyte integration during scaffolds creation.

Cellular Distribution of C-C Motif Chemokine Ligand 2 Like Immunoreactivities in Frontal Cortex and Corpus Callosum of Normal and Lipopolysaccharide Treated Animal

Background: C-C motif chemokine ligand 2 (CCL2) is reported to be involved in the pathogenesis of various neurological and/or psychiatric diseases. Tissue or cellular expression of CCL2, in normal or pathological condition, may play an essential role in recruiting of monocytes or macrophages into the targeted organs, and be involved in a certain pathogenic mechanism. However, only a few studies focused on tissue and cellular distribution of the CCL2 peptide in the brain’s grey and white matters (GM, WM), and the changes of the GM and WM cellular CCL2 level in septic or endotoxic encephalopathy was not explored. Hence, the CCL2 cellular distribution in the front brain cortex and the corpus callosum (CC) WM was investigated in the present work by using immunofluorescent staining. Results: 1) Normally, CCL2 like immunoreactivity (CCL2-ir) in the CC is significantly higher than the cortex, especially when the measurement includes ependymal layer attached to the CC. 2) Structures surrounding the vasculatures contribute major CCL2-ir positive profiles in both GM and WM, but significantly more in the CC WM, in which they are bilaterally distributed and predominantly located in the lateral CC between the cingulate cortex and the lateral ventricles. 3) Following systemic lipopolysaccharide (LPS), the number of neuron-like CCL2-ir positive cells are increased significantly in the cortex, but not in the CC. 4) More CCL2-ir positive elements are accumulated inside microvasculature like structures in the CC WM, compared to those found in the cortex following systemic LPS. 5) Few macrophage/microglia marker-Iba-1 labeled structures exhibit CCL2-ir in normal cortex and CC, but the co-localization is significantly increased following systemic LPS. 6) Following saline or LPS injection, CCL2-ir and GFAP or Iba-1 double labeled structures are observed within the ependymal layer between the lateral ventricles and the CC. No accumulation of neutrophils was detected.Conclusion: there exist differences in the cellular distribution of the CCL2 peptide in the front brain cortex GM and the subcortical WM - the CC, in both the physiological condition and experimental endotoxemia. Which might cause different pathological change in the GM and WM.

A phenotypic rescue approach identifies lineage regionalization defects in a mouse model of DiGeorge syndrome

ABSTRACTTBX1 is a key regulator of pharyngeal apparatus (PhAp) development. Vitamin B12 treatment partially rescues aortic arch patterning defects of Tbx1+/- embryos. Here we show that it also improves cardiac outflow tract septation and branchiomeric muscle anomalies of Tbx1 hypomorphic mutants. At molecular level, the in vivo vB12 treatment let us to identify genes that were dysregulated by Tbx1 haploinsufficiency and rescued by treatment. We found that SLUG, encoded by the rescued gene Snai2, identified a population of mesodermal cells that was partially overlapping with but distinct from ISL1+ and TBX1+ populations. In addition, SLUG+ cells were mislocalized and had a greater tendency to aggregate in Tbx1+/- and Tbx1-/- embryos and vB12 treatment restore cellular distribution. Adjacent neural crest-derived mesenchymal cells, which do not express TBX1, were also affected, showing enhanced segregation from cardiopharyngeal mesodermal cells. We propose that TBX1 regulates cell distribution in core mesoderm and the arrangement of multiple lineages within the PhAp.

Endoplasmic reticulum stress-induced release and binding of calreticulin from human ovarian cancer cells

Background Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone, but can appear surface bound on cancers cells, including ovarian cancers (OC). We investigated at what stage of cell viability, CRT appeared associated with surface of human OC cells. CRT on pre-apoptotic tumour cells is thought to initiate their eradication via a process termed immunogenic cell death (ICD). Methods We treated OC cells with the chemotherapeutic—doxorubicin (DX) known to induce translocation of CRT to some tumour cell surfaces, with and without the ER stressor—thapsigargin (TG)—and/or an ER stress inhibitor—TUDCA. We monitored translocation/release of CRT in pre-apoptotic cells by flow cytometry, immunoblotting and ELISA. We investigated the difference in binding of FITC-CRT to pre-apoptotic, apoptotic and necrotic cells and the ability of extracellular CRT to generate immature dendritic cells from THP-1 monocytes. Results Dx-treatment increased endogenously released CRT and extracellular FITC_CRT binding to human pre-apoptotic OC cells. DX and TG also promoted cell death in OC cells which also increased CRT release. These cellular responses were significantly inhibited by TUDCA, suggesting that ER stress is partially responsible for the changes in CRT cellular distribution. Extracellular CRT induces maturation of THP-1 towards a imDC phenotype, an important component of ICD. Conclusion Collectively, these cellular responses suggest that ER stress is partially responsible for the changes in CRT cellular distribution. ER-stress regulates in part the release and binding of CRT to human OC cells where it may play a role in ICD.

The gammaherpesvirus 68 viral cyclin facilitates expression of LANA

Gammaherpesviruses establish life-long infections within their host and have been shown to be the causative agents of devastating malignancies. Chronic infection within the host is mediated through cycles of transcriptionally quiescent stages of latency with periods of reactivation into detectable lytic and productive infection. The mechanisms that regulate reactivation from latency remain poorly understood. Previously, we defined a critical role for the viral cyclin in promoting reactivation from latency. Disruption of the viral cyclin had no impact on the frequency of cells containing viral genome during latency, yet it remains unclear whether the viral cyclin influences latently infected cells in a qualitative manner. To define the impact of the viral cyclin on properties of latent infection, we utilized a viral cyclin deficient variant expressing a LANA-beta-lactamase fusion protein (LANA::βla), to enumerate both the cellular distribution and frequency of LANA gene expression. Disruption of the viral cyclin did not affect the cellular distribution of latently infected cells, but did result in a significant decrease in the frequency of cells that expressed LANA::βla across multiple tissues and in both immunocompetent and immunodeficient hosts. Strikingly, whereas the cyclin-deficient virus had a reactivation defect in bulk culture, sort purified cyclin-deficient LANA::βla expressing cells were fully capable of reactivation. These data emphasize that the γHV68 latent reservoir is comprised of at least two distinct stages of infection characterized by differential LANA expression, and that a primary function of the viral cyclin is to promote LANA expression during latency, a state associated with ex vivo reactivation competence.

Transport Pathways That Contribute to the Cellular Distribution of Phosphatidylserine

Phosphatidylserine (PS) is a negatively charged phospholipid that displays a highly uneven distribution within cellular membranes, essential for establishment of cell polarity and other processes. In this review, we discuss how combined action of PS biosynthesis enzymes in the endoplasmic reticulum (ER), lipid transfer proteins (LTPs) acting within membrane contact sites (MCS) between the ER and other compartments, and lipid flippases and scramblases that mediate PS flip-flop between membrane leaflets controls the cellular distribution of PS. Enrichment of PS in specific compartments, in particular in the cytosolic leaflet of the plasma membrane (PM), requires input of energy, which can be supplied in the form of ATP or by phosphoinositides. Conversely, coupling between PS synthesis or degradation, PS flip-flop and PS transfer may enable PS transfer by passive flow. Such scenario is best documented by recent work on the formation of autophagosomes. The existence of lateral PS nanodomains, which is well-documented in the case of the PM and postulated for other compartments, can change the steepness or direction of PS gradients between compartments. Improvements in cellular imaging of lipids and membranes, lipidomic analysis of complex cellular samples, reconstitution of cellular lipid transport reactions and high-resolution structural data have greatly increased our understanding of cellular PS homeostasis. Our review also highlights how budding yeast has been instrumental for our understanding of the organization and transport of PS in cells.

Sumoylation regulates the assembly and activity of the SMN complex

SMN is a ubiquitously expressed protein and is essential for life. SMN deficiency causes the neurodegenerative disease spinal muscular atrophy (SMA), the leading genetic cause of infant mortality. SMN interacts with itself and other proteins to form a complex that functions in the assembly of ribonucleoproteins. SMN is modified by SUMO (Small Ubiquitin-like Modifier), but whether sumoylation is required for the functions of SMN that are relevant to SMA pathogenesis is not known. Here, we show that inactivation of a SUMO-interacting motif (SIM) alters SMN sub-cellular distribution, the integrity of its complex, and its function in small nuclear ribonucleoproteins biogenesis. Expression of a SIM-inactivated mutant of SMN in a mouse model of SMA slightly extends survival rate with limited and transient correction of motor deficits. Remarkably, although SIM-inactivated SMN attenuates motor neuron loss and improves neuromuscular junction synapses, it fails to prevent the loss of sensory-motor synapses. These findings suggest that sumoylation is important for proper assembly and function of the SMN complex and that loss of this post-translational modification impairs the ability of SMN to correct selective deficits in the sensory-motor circuit of SMA mice.