Periodontitis prevalence and associated factors: a comparison of two examination protocols

La variabilidad en la definición epidemiológica de la periodontitis y los protocolos de evaluación afectan la medición de la prevalencia y su asociación con ciertos factores. Si bien, el patrón oro para el examen periodontal es el registro de boca completa, que evalúa la pérdida de inserción (CAL, por sus siglas en inglés) y profundidad de sondaje (PS, por sus siglas en inglés), los recursos no siempre están disponibles para los sistemas de vigilancia epidemiológica. Objetivo: En este estudio se compararon diferentes protocolos y definiciones de periodontitis evaluando la prevalencia y la asociación de factores relacionados en pacientes adultos que solicitaron atención en la Facultad de Odontología de la UdelaR

FKBP52 is involved in the regulation of SOCE channels in the human platelets and MEG 01 cells

AbstractImmunophilins are FK506-binding proteins that have been involved in the regulation of calcium homeostasis, either by modulating Ca2+ channels located in the plasma membrane or in the rough endoplasmic reticulum (RE). We have investigated whether immunophilins would participate in the regulation of stored-operated Ca2+ entry (SOCE) in human platelets and MEG 01. Both cell types were loaded with fura-2 for determining cytosolic calcium concentration changes ([Ca2+]c), or stimulated and fixed to evaluate the protein interaction profile by performing immunoprecipitation and western blotting. We have found that incubation of platelets with FK506 increases Ca2+ mobilization. Thapsigargin (TG)-evoked, Thr-evoked SOCE and TG-evoked Mn2+ entry resulted in significant reduction by treatment of platelets with immunophilin antagonists. We confirmed by immunoprecipitation that immunophilins interact with transient receptor potential channel 1 (TRPC1) and Orai1 in human platelets. FK506 and rapamycin reduced the association between TRPC1 and Orai1 with FK506 binding protein (52) (FKBP52) in human platelets, and between TRPC1 and the type II IP3R, which association is known to be crucial for the maintenance of SOCE in human platelets. FKBP52 role in SOCE activation was confirmed by silencing FKBP52 using SiRNA FKBP52 in MEG 01 as demonstrated by single cell configuration imaging technique. TRPC1 silencing and depletion of cell of TRPC1 and FKBP52 simultaneously, impair activation of SOCE evoked by TG in MEG 01. Finally, in MEG 01 incubated with FK506 we observed a reduction in TRPC1/FKBP52 coupling, and similarly, FKBP52 silencing reduced the association between IP3R type II and TRPC1 during SOCE. All together, these results demonstrate that immunophilins participate in the regulation of SOCE in human platelets

Small-Scale Fluidized Bed Bioreactor for Long-Term Dynamic Culture of 3D Cell Constructs and in vitro Testing

With the increasing interest in three-dimensional (3D) cell constructs that better represent native tissues, comes the need to also invest in devices, i.e., bioreactors, that provide a controlled dynamic environment similar to the perfusion mechanism observed in vivo. Here a laboratory-scale fluidized bed bioreactor (sFBB) was designed for hydrogel (i.e., alginate) encapsulated cells to generate a dynamic culture system that produced a homogenous milieu and host substantial biomass for long-term evolution of tissue-like structures and “per cell” performance analysis. The bioreactor design, conceptualized through scale-down empirical similarity rules, was initially validated through computational fluid dynamics analysis for the distributor capacity of homogenously dispersing the flow with an average fluid velocity of 4.596 × 10–4 m/s. Experimental tests then demonstrated a consistent fluidization of hydrogel spheres, while maintaining shape and integrity (606.9 ± 99.3 μm diameter and 0.96 shape factor). It also induced mass transfer in and out of the hydrogel at a faster rate than static conditions. Finally, the sFBB sustained culture of alginate encapsulated hepatoblastoma cells for 12 days promoting proliferation into highly viable (>97%) cell spheroids at a high final density of 27.3 ± 0.78 million cells/mL beads. This was reproducible across multiple units set up in parallel and operating simultaneously. The sFBB prototype constitutes a simple and robust tool to generate 3D cell constructs, expandable into a multi-unit setup for simultaneous observations and for future development and biological evaluation of in vitro tissue models and their responses to different agents, increasing the complexity and speed of R&D processes

Two-pore channel 2 (TPC2) modulates store-operated Ca2+ entry

AbstractTwo-pore channels (TPCs) are NAADP-sensitive receptor channels that conduct Ca2+ efflux from the intracellular stores. Discharge of the internal Ca2+ pools results in the activation of store-operated Ca2+ entry (SOCE); however, the role of TPCs in the modulation of SOCE remains unexplored. Mammalian cells express three TPCs: TPC1, TPC2 and TPC3, a pseudogene in humans. Here we report that MEG01 and HEK293 cells endogenously express TPC1 and TPC2. Silencing TPC2 expression results in attenuation of the rate and extent of thapsigargin (TG)-evoked SOCE both in MEG01 and HEK293 cells, without having any effect on the ability of cells to accumulate Ca2+ into the TG-sensitive stores. Similarly, silencing of native TPC2 expression reduced thrombin-induced Ca2+ entry in MEG01 cells. In contrast, silencing of TPC1 expression was without effect either on TG or thrombin-stimulated Ca2+ entry both in MEG01 and HEK293 cells. Biotinylation analysis revealed that TPC1 and TPC2 are expressed in internal membranes. Finally, co-immunoprecipitation experiments indicated that endogenously expressed TPC2, but not TPC1, associates with STIM1 and Orai1, but not with TRPC1, in MEG01 cells with depleted intracellular Ca2+ stores, but not in resting cells. These results provide strong evidence for the modulation of SOCE by TPC2 involving de novo association between TPC2 and STIM1, as well as Orai1, in human cells

Applications and optimization of cryopreservation technologies to cellular therapeutics

Delivery of cell therapies often requires the ability to hold products in readiness whilst logistical, regulatory and potency considerations are dealt with and recorded. This requires reversibly stopping biological time, a process which is often achieved by cryopreservation. However, cryopreservation itself poses many biological and biophysical challenges to living cells that need to be understood in order to apply the low temperature technologies to their best advantage. This review sets out the history of applied cryopreservation, our current understanding of the various processes involved in storage at cryogenic temperatures, and challenges for robust and reliable uses of cryopreservation within the cell therapy arena

Non-invasive brain stimulation for dystonia: therapeutic implications

Dystonia is characterized by excessive muscle contractions giving rise to abnormal posture and involuntary twisting movements. Although dystonia syndromes are a heterogeneous group of disorders, certain pathophysiological mechanisms have been consistently identified across different forms. These pathophysiological mechanisms have subsequently been exploited for the development of non‐invasive brain stimulation (NIBS) techniques able to modulate neural activity in one or more nodes of the putative network that is altered in dystonia, and the therapeutic role of NIBS has hence been suggested. Here all studies that applied such techniques as a therapeutic intervention in any forms of dystonia, including the few works performed in children, are reviewed and emerging concepts and pitfalls of NIBS are discussed

Hsp83 loss suppresses proteasomal activity resulting in an upregulation of caspase-dependent compensatory autophagy

The 2 main degradative pathways that contribute to proteostasis are the ubiquitin-proteasome system and autophagy but how they are molecularly coordinated is not well understood. Here, we demonstrate an essential role for an effector caspase in the activation of compensatory autophagy when proteasomal activity is compromised. Functional loss of Hsp83, the Drosophila ortholog of human HSP90 (heat shock protein 90), resulted in reduced proteasomal activity and elevated levels of the effector caspase Dcp-1. Surprisingly, genetic analyses showed that the caspase was not required for cell death in this context, but instead was essential for the ensuing compensatory autophagy, female fertility, and organism viability. The zymogen pro-Dcp-1 was found to interact with Hsp83 and undergo proteasomal regulation in an Hsp83-dependent manner. Our work not only reveals unappreciated roles for Hsp83 in proteasomal activity and regulation of Dcp-1, but identifies an effector caspase as a key regulatory factor for sustaining adaptation to cell stress in vivo