A Bioprinted Heart-on-a-Chip with Human Pluripotent Stem Cell-Derived Cardiomyocytes for Drug Evaluation

In this work, we show that valve-based bioprinting induces no measurable detrimental effects on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The aim of the current study was three-fold: first, to assess the response of hiPSC-CMs to several hydrogel formulations by measuring electrophysiological function; second, to customise a new microvalve-based cell printing mechanism in order to deliver hiPSC-CMs suspensions, and third, to compare the traditional manual pipetting cell-culture method and cardiomyocytes dispensed with the bioprinter. To achieve the first and third objectives, iCell2 (Cellular Dynamics International) hiPSC-CMs were used. The effects of well-known drugs were tested on iCell2 cultured by manual pipetting and bioprinting. Despite the results showing that hydrogels and their cross-linkers significantly reduced the electrophysiological performance of the cells compared with those cultured on fibronectin, the bio-ink droplets containing a liquid suspension of live cardiomyocytes proved to be an alternative to standard manual handling and could reduce the number of cells required for drug testing, with no significant differences in drug-sensitivity between both approaches. These results provide a basis for the development of a novel bioprinter with nanolitre resolution to decrease the required number of cells and to automate the cell plating process.

Formation of Benign Tumours by Stem Cell Misregulation

Our tissues usually have just the right number of cells to optimally fulfil their function. Not enough cells within a tissue can lead to dysfunction, while too many cells result in a tumour. Yet, how this homeostatic balance is maintained remains poorly defined. Most differentiated cells within tissues have a finite lifespan and need to be replaced at a corresponding pace to maintain tissue homeostasis. These new differentiated cells are generated by proliferation of the stem/progenitor cells that serve the tissue. Work in simple invertebrates clearly suggests stem cells respond to at least two types of signals: niche signaling and growth factors. Niche signals promote the undifferentiated state by preventing differentiation, and thus allow for stem cell self-renewal. Growth factor sources comprise a systemic input reflecting the animal’s nutritional status, and a localized, homeostatic feedback from the tissue that the stem cells serve. That homeostatic signal couples stem cell proliferation rates to the tissue’s need for new differentiated cells. Evidence from simple organisms suggests two types of benign tumours can arise from deregulation of either niche or homeostatic signaling. Namely, constitutive niche signaling promotes the formation of undifferentiated “stem cell” tumours, while defective homeostatic signaling leads to the formation of differentiated tumours. We propose that these principles may be conserved and underlie benign tumour formation in humans, while benign tumours can evolve into cancer.

Adipose-derived stem cells in the treatment of knee osteoarthritis: from extraction methods to the preparation of the transplant

Introduction: Recent studies have investigated the use of adipose tissue as source of mesenchymal stem cells in the treatment of knee osteoarthritis in humans. However, there are still several protocols being performed. Objective: Analyze the protocols published in the literature in the last ten years and to investigate how they are being carried out and if they are following the criteria adopted by the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT). Methodology: Articles from the PubMed, ScienceDirect and Lilacs database published in January / 2010 until the present time, which were evaluated in order to investigate the use of adipose-derived stem cells in the treatment of knee osteoarthritis. Results: Thirty four articles were evaluated in its entiraty. The abdominal area was the most choosen to do the liposuction, however the quantities of adipose tissue removed and the number of cells transplanted was variable. It is hightlited the enzimatic digestion of adipose tissue with collagenase as extraction method. Only 14 articles complied all the 3 criteria required to prove the real presence of mesenchymal stem cells in the samples that was transplanted. However, all the articles showed improvement of function and pain. Final considerations: Thus, even the results found are promising, the evidence is still limited in humans and the variability of the methodology makes it difficult to standardize the technique, also its implementation as a reference in the treatment of knee osteoarthritis.

The SARS-CoV-2 variant, Omicron, shows rapid replication in human primary nasal epithelial cultures and efficiently uses the endosomal route of entry.

At the end of 2021 a new SARS-CoV-2 variant, Omicron, emerged and quickly spread across the world. It has been demonstrated that Omicrons high number of Spike mutations lead to partial immune evasion from even polyclonal antibody responses, allowing frequent re-infection and vaccine breakthroughs. However, it seems unlikely these antigenic differences alone explain its rapid growth; here we show Omicron replicates rapidly in human primary airway cultures, more so even than the previously dominant variant of concern, Delta. Omicron Spike continues to use human ACE2 as its primary receptor, to which it binds more strongly than other variants. Omicron Spike mediates enhanced entry into cells expressing several different animal ACE2s, including various domestic avian species, horseshoe bats and mice suggesting it has an increased propensity for reverse zoonosis and is more likely than previous variants to establish an animal reservoir of SARS-CoV-2. Unlike other SARS-CoV-2 variants, however, Omicron Spike has a diminished ability to induce syncytia formation. Furthermore, Omicron is capable of efficiently entering cells in a TMPRSS2-independent manner, via the endosomal route. We posit this enables Omicron to infect a greater number of cells in the respiratory epithelium, allowing it to be more infectious at lower exposure doses, and resulting in enhanced intrinsic transmissibility.

Indication-based protocols with different solutions for mechanical stromal-cell transfer

Background Regenerative medicine is the fastest developing branch of plastic surgery in recent times. Adipose tissue is one of the largest and most important sources in the body for stromal cells. Although mechanical isolation methods are both very popular and have many advantages, they still have no accepted protocols. Objective We developed new protocols called indication-based protocols (IPs) for standardization and new techniques called mechanical stromal-cell transfer (MEST) by using ultra-sharp blades and dilution of adipose tissue with different solutions (saline, Ringer and 5% Dextrose) Methods & material: In order to obtain the desired physical structure (liquid, gel, solid) and the desired volume, four different types of IPs have been defined. Adipose tissue was prediluted with different solutions using 10 or 20 cc injectors in IPs 1 and 2, while condensed adipose tissue was used directly in IPs 3 and 4. Results In MEST, stromal cells were obtained from 100 mL of condensed fat using different IPs with 92% mean viability and cell counts of 26.80–91.90 × 106. Stromal cells can be obtained in the desired form and number of cells by using four different IPs. Conclusion Isolation of stromal cells by cutting fat with sharp blades will prevent the death of fat tissue and stromal cells and will allow high viability and cell count with our new technique. Predilution with different solutions: Diluting the condensed adipose tissue with the desired solutions (saline, Ringer or 5% Dextrose) before the adinizing process will provide even more stromal cells. Lay Summary Obtaining regenerative stromal cells from adipose tissue can be done by two methods: Enzymatic and mechanical. Mechanical methods have many advantages. Although mechanical stromal cell extraction from adipose tissue is very popular and many techniques have been described, there are still no accepted protocols, definition for the end product, and no consensus on the status of the stromal cells. In this study, stromal cells were obtained mechanically by using ultra-sharp blade systems, without exposing adipose tissue to blunt trauma. Thus, a higher number of cells and higher viability could be obtained. An “Indication based” protocol has been defined for the first time in order to obtain the desired number and status (solid, semi-solid, liquid) end product. Diluting the condensed adipose tissue with the desired solutions (saline, Ringer or 5% Dextrose) before the adinizing process will provide even more stromal cells. This will provide an opportunity for clinicians to obtain and apply a stromal cell solution for different indications in different anatomical regions.

Untraceable Authentication Protocol for IEEE802.11s Standard

In the current paper, a new handover authentication protocol for IEEE802.11s Wireless mesh networks is presented. The new protocol divides the network into a number of cells, each cell contains a number of access points and based on the concept of ticket authentication, the mesh user takes a new ticket when enters the region of a new cell which decreases the handover latency. Moreover, in the current paper, a new idea for ticket generation is proposed, called Chain Ticket Derivation Function (CTDF), which uses the concept of a chain. Using CTDF in our proposed protocol raises the level of privacy for the users. The security analysis presented in the paper showed more strengths in our proposed scheme. Two formal verification tools, AVISPA and BAN logic are used to test the proposed protocol.

Biotherapic of Trypanosoma cruzi 17d increases apoptosis in experimentally infected mice

Introduction: the mechanism of action of ultradiluted medicines has not yet been established[1,3]. Many basic research studies have focused on isopathic models using in vitro or in vivo designs [4,5]. Recent studies indicate that an ultradiluted (isopathic) antigen can transfer signals to the immune system and modulate its response when an organism is challenged against this same antigen [6]. Some studies on experimental infection of mice by T. cruzi identified apoptotic cells and showed that the increase of their number is associated with an increase also in the number of parasites in the blood of the infected animals, while blockage of apoptosis can be the target of therapeutic intervention [7,8]. Aim: to evaluate the development of apoptosis in mice treated with biotherapic of Trypanosoma cruzi in dilution 17d through in situ detection of fragmented DNA. Method: in a blind randomized controlled trial, 36 male Swiss mice age 4 or 8 weeks were distributed in groups control - treated with 7% hydroalcoholic solution(CI-4=9 animals or CI-8=9 animals); and treated with biotherapic 17d (BIOT-4=9 animals or BIOT-8=9 animals). Infection was performed with 1,400 trypomastigotes T. cruzi-strain Y via intraperitoneal. Biotherapic 17d was prepared through the addition of 0.9ml of concentrated T. cruzi (10E+7 trypomastigotes/ml) to 9.1 ml of distilled water. The following dilutions were prepared in 86% hydroalcoholic solution until dilution 16d. Dilution 17d was prepared with 7% hydroalcoholic solution. It was performed microbiological control and biological risk in vivo. Treatment: 0.2 ml in 3 consecutive days, oral route, from the moment infection was verified. Animals were sacrificed on the 3rd day of treatment in a chamber saturated with ether. The liver and spleen were removed and fixated in 4% paraformaldehyde for 24 hours and then included in paraffin. Apoptosis was evaluated through DNA fragmentation – TUNEL technique (TdT dUTP-biotin Nick End Labeling (ApopTag® Peroxidade-Chemicon). For statistical analysis software Statistica 8.0 was used. This study was approved by the Ethics Committee for Animal Experimentation of UEM. Results and Discussion: in the samples of liver of animals age 4 and 8 weeks either treated or not with biotherapic 17d it was found cells parasitized by amastigotes of T. cruzi with apoptotic bodies, or phagocytic cells with phagocytic vacuole with apoptotic marked material inside them. The number of cells in apoptosis in animals age 4 weeks was not significantly (p=0.03) larger in treated group BIOT-C4 than in control group CI-4 (Figure 1). In animals age 8 weeks, the number of cells in apoptosis was significantly (p

Calculate central limit theorem for the number of empty cells after allocation of particles

This work belongs to the field of limit theorems for separable statistics. In particular, this paper considers the number of empty cells after placing particles in a finite number of cells, where each particle is placed in a polynomial scheme. The statistics under consideration belong to the class of separable statistics, which were previously considered in (Mirakhmedov: 1985), where necessary statements for the layout of particles in a countable number of cells were proved. The same scheme was considered in (Asimov: 1982), in which the conditions for the asymptotic normality of random variables were established. In this paper, the asymptotic normality of the statistics in question is proved and an estimate of the remainder term in the central limit theorem is obtained. In summary, the demand for separable statistics systems is growing day by day to address large-scale databases or to facilitate user access to data management. Because such systems are not only used for data entry and storage, they also describe their structure: file collection supports logical consistency; provides data processing language; restores data after various interruptions; database management systems allow multiple users.

Method for predicting the course of peritonsillar abscess in patients with exacerbation of chronic tonsillitis

Objectives clinical and laboratory examination of patients with acute tonsillitis for early diagnosis and prognosis of peritonsillar abscess. Material and methods. The study included 101 patient with lacunar tonsillitis complicated by peritonsillar abscess and 64 donors (control group). Immunological studies were performed according to WHO recommendations, on the basis of the immunological department of the EMB Research Institute and the immunological laboratory of the SamSMU. Results. Immunological examination of patients with abscess showed an increase in: neutrophil phagocytic activity, CD4+/CD8+, the number of cells expressing HLA-DR+ markers, complement activity, IgA, IgM, IgG plasma concentration, fibronectin level, pro-inflammatory cytokines IL-8, IL-1, IL-1 and a decrease in: the level of TNF-, myeloperoxidase activity, number of cells containing CD4+, CD8+, CD16+, CD20+, CD25+ markers. High correlation was registered between total lymphocytes and CD3+ and CD4+ cells (p 0.01); between CD3+ and CD4+ markers (p 0.01); as well as high correlation of IL-1 levels with IL-8 and IL-1 (p 0.01). Cluster analysis revealed different types of immune homeostasis. The first type (cluster) had high values of leukocytes (total), lymphocytes (total), cells with CD3+, CD4+, CD8+, CD16+, CD20+, CD95+ and HLA-DR+ markers; the second type (cluster) was characterized by significantly lower levels of these immune status indicators. 41 patient had the first type of immune response, with an explicit clinical picture and rapid formation of an abscess. The second type of immune response was registered in 60 patients having a torpid course of the disease with delayed development of abscess. Further, to assess the type of immune reactions, it is necessary to substitute the values of indicators into the model and calculate the integral coefficient of the body's reaction (ICTROI and ICTROII).

A Bimodal Pattern and Age-Related Growth of Intra-Annual Wood Cell Development of Chinese Fir in Subtropical China

Age plays an important role in regulating the intra-annual changes in wood cell development. Investigating the effect of age on intra-annual wood cell development would help to understand cambial phenology and xylem formation dynamics of trees and predict the growth of trees accurately. Five intermediate trees in each stand (total of 5 stands) in five age groupings of Chinese fir (Cunninghamia lanceolata Hook.) plantations in subtropical China were monitored on micro-cores collected weekly or biweekly from January to December in 2019. We modeled the dynamics of wood cell development with a mixed effects model, analyzed the age effect on intra-annual wood cell development, and explored the contribution of rate and duration of wood cell development on intra-annual wood cell development. We found a bimodal pattern of wood cell development in all age classes, and no matter the date of peak or the maximal number of cells the bimodal patterns were similar in all age classes. In addition, compared with the older trees, the younger trees had the longest duration of wood cell development because of the later end of wood cell development and a larger number of wood cells. The younger trees had the faster growth rate than the older trees, but the date of the maximal growth rate in older trees was earlier than younger trees, which led to the production of more wood cells in the younger trees. Moreover, we found that the number of cells in wood cell formation was mostly affected by the rate (92%) rather than the duration (8%) of wood cell formation.