Dual-Slope Path Loss Model for Integrating Vehicular Sensing Applications in Urban and Suburban Environments.

The development of intelligent transportation systems (ITS), vehicular ad hoc networks (VANETs), and autonomous driving (AD) has progressed rapidly in recent years, driven by artificial intelligence (AI), the internet of things (IoT), and their integration with dedicated short-range communications (DSRC) systems and fifth-generation (5G) networks. This has led to improved mobility conditions in different road propagation environments: urban, suburban, rural, and highway. The use of these communication technologies has enabled drivers and pedestrians to be more aware of the need to improve their behavior and decision making in adverse traffic conditions by sharing information from cameras, radars, and sensors widely deployed in vehicles and road infrastructure. However, wireless data transmission in VANETs is affected by the specific conditions of the propagation environment, weather, terrain, traffic density, and frequency bands used. In this paper, we characterize the path loss based on the extensive measurement campaign carrier out in vehicular environments at 700 MHz and 5.9 GHz under realistic road traffic conditions. From a linear dual-slope path loss propagation model, the results of the path loss exponents and the standard deviations of the shadowing are reported. This study focused on three different environments, i.e., urban with high traffic density (U-HD), urban with moderate/low traffic density (U-LD), and suburban (SU). The results presented here can be easily incorporated into VANET simulators to develop, evaluate, and validate new protocols and system architecture configurations under more realistic propagation conditions.

Path Loss Characterization in an Outdoor Corridor Environment for IoT-5G in a Smart Campus University at 850 MHz and 3.5 GHz Frequency Bands.

The usage scenarios defined in the ITU-M2150-1 recommendation for IMT-2020 systems, including enhanced Mobile Broadband (eMBB), Ultra-reliable Low-latency Communication (URLLC), and massive Machine Type Communication (mMTC), allow the possibility of accessing different services through the set of Radio Interface Technologies (RITs), Long-term Evolution (LTE), and New Radio (NR), which are components of RIT. The potential of the low and medium frequency bands allocated by the Federal Communications Commission (FCC) for the fifth generation of mobile communications (5G) is described. In addition, in the Internet of Things (IoT) applications that will be covered by the case of use of the mMTC are framed. In this sense, a propagation channel measurement campaign was carried out at 850 MHz and 5.9 GHz in a covered corridor environment, located in an open space within the facilities of the Pedagogical and Technological University of Colombia campus. The measurements were carried out in the time domain using a channel sounder based on a Universal Software Radio Peripheral (USRP) to obtain the received signal power levels over a range of separation distances between the transmitter and receiver from 2.00 m to 67.5 m. Then, a link budget was proposed to describe the path loss behavior as a function of these distances to obtain the parameters for the close-in free space reference distance (CI) and the floating intercept (FI) path loss prediction models. These parameters were estimated from the measurements made using the Minimum Mean Square Error (MMSE) approach. The estimated path loss exponent (PLE) values for both the CI and FI path loss models at 850 MHz and 3.5 GHz are in the range of 2.21 to 2.41, respectively. This shows that the multipath effect causes a lack of constructive interference to the received power signal for this type of outdoor corridor scenario. These results can be used in simulation tools to evaluate the path loss behavior and optimize the deployment of device and sensor network infrastructure to enable 5G-IoT connectivity in smart university campus scenarios.

COVID-19 in lung transplant recipients: A multicenter study.

This study describes the clinical presentation, treatment, and outcomes of SARS-CoV-2 infection in lung transplant recipients (LTRs). This is a multicenter, retrospective study of all adult LTRs with confirmed SARS-CoV-2 infection from March 4 until April 28, 2020 in six Spanish reference hospitals for lung transplantation. Clinical and radiological data, treatment characteristics, and outcomes were reviewed. Forty-four cases were identified in that period. The median time from transplantation was 4.2 (interquartile range: 1.11-7.3) years. Chest radiography showed acute parenchymal abnormalities in 32 (73%) cases. Hydroxychloroquine was prescribed in 41 (93%), lopinavir/ritonavir (LPV/r) in 14 (32%), and tocilizumab in 19 (43%) patients. There was a strong interaction between tacrolimus and LPV/r in all cases. Thirty-seven (84%) patients required some degree of respiratory support and/or oxygen therapy, and 13 (30%) were admitted to intermediate or intensive critical care units. Seventeen (39%) patients had died and 20 (45%) had been discharged at the time of the last follow-up. Deceased patients had a worse respiratory status and chest X-ray on admission and presented with higher D-dimer, interleukin-6, and lactate dehydrogenase levels. In this multicenter LTR cohort, SARS-CoV-2 presented with high mortality. Additionally, the severity of disease on presentation predicted subsequent mortality.

DNA methylation profile of different clones of human adipose stem cells does not allow to predict their differentiation potential.

Human adipose stem cells can differentiate into various mesodermic lineages, including adipogenic, osteogenic, chondrogenic, myogenic and endothelial pathways. In addition, these cells types possess immunomodulatory properties, potentially useful for autoimmune and autoinflammatory diseases. However, single-cell expanded clones have shown that the cells can present a variety of differentiation potential, which may be partly due to epigenetic differences among them. The objective of this study was to assess if DNA methylation plays a role in the differentiation potential observed between different cell clones obtained from the same donor. To this end, the methylation profile of five clonal cell lines of human adipose stem cells obtained by liposuction from two donors was analyzed. Previous reports demonstrated that cell lines 1.7 and 1.22 from Donor 1 and 3.5 from Donor 3 were adipogenic-osteogenic, but not cell lines 1.10 and 3.10. The genes analyzed were neuronal, endothelial, myogenic, osteogenic, adipogenic, extracellular matrix, cell cycle, cytoskeleton and metabolic enzymes. All clones analyzed in this study displayed a similar pattern of methylation in most of the gene families: 85.5% were hypomethylated genes and 14.5% hypermethylated. In conclusion, the methylation pattern of the 1113 genes studied in this report was not a consistent tool to identify the differentiation potential of human adipose stem cells.

Negative neuronal differentiation of human adipose-derived stem cell clones.

AIMS: Adipose mesenchymal stem cells are a heterogeneous population. Therefore, the question posed in this study is whether the heterogenic differentiation potential exhibited by the different clones toward mesodermic lineages can be extended to nonmesodermic lineages, such as the neuroectoderm. MATERIALS & METHODS: Different single cell clones of human adipose mesenchymal stem cells from the same donor were isolated. Neuronal plasticity of the clones was assessed according to the pattern DNA methylation, gene expression and intracellular calcium responses. RESULTS: Under neurogenic culture conditions, clones presented variable expression of neuronal-specific genes, but still expressed osteogenic markers. No calcium response was exhibited in response to KCl incubation. The DNA methylation profile presented a very similar pattern in neuroectoderm gene promoters. CONCLUSIONS: Data indicate that there are no significant differences between the undifferentiated and supposedly neuronal-differentiated mesenchymal cells.

(α'(H))-Dicalcium silicate bone cement doped with tricalcium phosphate: characterization, bioactivity and biocompatibility.

The influence of phosphorus doping on the properties of (α'(H))-dicalcium silicate (C(2)S) bone cement was analyzed, in addition to bioactivity and biocompatibility. All the cements were composed of a solid solution of TCP in C(2)S ([Formula: see text]-C(2)S(ss)) as the only phase present. The compressive strength ranged from 3.8-16.3 MPa. Final setting times ranged from 10 to 50 min and were lower for cements with lower L/P content. Calcium silicate hydrate was the principal phase formed during the hydration process of the cements. The cement exhibited a moderate degradation and could induce carbonated hydroxyapatite formation on its surface and into the pores. The cell attachment test showed that the (α'(H))-C(2)SiO(4) solid solution supported human adipose stem cells adhesion and spreading, and the cells established close contacts with the cement after 24 h of culture. The novel (α'(H))-C(2)S(ss) cements might be suitable for potential applications in the biomedical field, preferentially as materials for bone/dental repair.

Phenotypic and functional characterization of glucagon-positive cells derived from spontaneous differentiation of D3-mouse embryonic stem cells.

BACKGROUND: Glucagon expression is being considered as a definitive endoderm marker in protocols aiming to obtain insulin-secreting cells from embryonic stem cells. However, it should be considered that in vivo glucagon is expressed both in definitive endoderm- and neuroectoderm-derived cells. Therefore, the true nature and function of in vitro spontaneously differentiated glucagon-positive cells remains to be established. METHODS: D3 and R1 mouse embryonic stem cells as well as α-TC1-9 cells were cultured and glucagon expression was determined by real-time PCR and immunocytochemistry. Functional analyses regarding intracellular calcium oscillations were performed to further characterize glucagon(+) cells. RESULTS: Specifically, 5% of D3 and R1 cells expressed preproglucagon, with a small percentage of these (<1%) expressing glucagon-like peptide 1. The constitutive expression of protein convertase 5 supports the expression of both peptides. Glucagon(+) cells co-expressed neurofilament middle and some glucagon-like peptide-1(+) cells, glial fibrillary acidic protein, indicating a neuroectodermic origin. However, few glucagon-like peptide-1(+) cells did not show coexpression with glial fibrillary acidic protein, suggesting a non-neuroectodermic origin for these cells. Finally, glucagon(+) cells did not display Ca(2+) oscillations typical of pancreatic α-cells. DISCUSSION: These results indicate the possible nondefinitive endodermal origin of glucagon-positive cells spontaneously differentiated from D3 and R1 cell lines, as well as the presence of cells expressing glucagon-like peptide-1 from two different origins.

Phenotypic differences during the osteogenic differentiation of single cell-derived clones isolated from human lipoaspirates.

Osteogenic precursors can be obtained from mesenchymal stem cells, which can be isolated from different sources, including adipose tissue. Optimal osteogenic differentiation in in vitro conditions and the selection of the potential precursors that could be further used in bone regeneration still have two main questions left to solve, viz. the heterogeneity of the mesenchymal cell population and the presence of a basal transcription level of several characteristic genes of the osteogenic lineage, which makes rapid and effective comparisons during cell differentiation difficult. Single-cell clones were isolated and expanded from human lipoaspirate cells. Osteogenic differentiation was induced and studied in defined media, using four representative isolated cell clones showing differences in the basal expression of a set of characteristic osteogenic genes. The clones showing a low constitutive expression of these genes were able to display comparatively higher levels of mineralization. In addition, the cells from these clones displayed a characteristic pattern of bundle fibres of collagen during osteogenic induction and showed a higher potency to differentiate towards the adipogenic lineage. These results demonstrate that specific multipotent precursors can be isolated from human lipoaspirate cells with a higher differentiation potential, allowing the maturation of specific lineages in a shorter time. These results additionally demonstrate that, since the basal expressions of the several genes were used as osteogenic markers, a phenotypic biochemical analysis should always be utilized to study optimal osteogenesis conditions. Copyright © 2010 John Wiley & Sons, Ltd.

Differentiation of embryonic stem cells using pancreatic bud-conditioned medium gives rise to neuroectoderm-derived insulin-secreting cells.

Human embryonic stem cells can be differentiated into insulin-secreting cells by emulating in vitro the key processes that occur during embryonic development. However, the resulting cells are generally immature; thus, further research must be performed to identify the necessary factors to complete the differentiation. To this end, we cultured mouse embryonic stem cells with pancreatic bud-conditioned medium, based on a recent publication. Unlike in humans, mouse cells present two types of insulin, which can be used to identify different cell lineages. As a result, the cell product presented a neuroectodermal genetic expression pattern, with no expression of any definitive endodermal marker analyzed. Also, nonglucose-dependent insulin release was detected. Altogether, this previously published protocol results in neuroectoderm, and not definitive endoderm, derived insulin-positive cells. This further confirms the difficulty of obtaining true cell types of this germ layer. Finally, we identified a 16-kDa protein band that was present in pancreatic bud-conditioned medium. Sequencing this band revealed the presence of Reg proteins. The role of pancreatic bud-conditioned medium remains to be tested in definitive endoderm committed cells.

Specific effect of 5-fluorouracil on alpha-fetoprotein gene expression during the in vitro mouse embryonic stem cell differentiation.

Embryonic stem (ES) cells are considered an important alternative to develop in vitro screening methods for embryotoxicity. Mouse ES cells can be cultured as cell suspension aggregates termed "embryoid bodies" (EBs) in which cells start to differentiate. We have studied the expression of several genes in the presence of a wide range of concentrations of 5-fluorouracil (5-FU). This well-established embryotoxic compound completely inhibited cell viability at 200 nmol/L in monolayer cultures. At lower concentrations, 5-FU led to decrease in the expression of the alpha-fetoprotein gene, a marker of the visceral endoderm, in the EBs. However, the expression of several mesodermal gene markers was not significantly affected at these concentrations. These results suggest a high sensitivity of the visceral endoderm differentiation to 5-FU. Therefore, the quantification of the alpha-fetoprotein gene after exposure to potential embryotoxicants should be considered an additional end point in future embryotoxicity assays in vitro with ES cells.

Expression of Neuropathy Target Esterase in mouse embryonic stem cells during differentiation.

Neuropathy Target Esterase (NTE) was initially identified as the primary target esterase of some organophosphorus compounds that cause delayed neuropathy. Some studies in vivo suggest that this protein may also perform a function in embryonic development and therefore also in cell differentiation. The aim of this work was to characterize embryonic stem cells (ESC) as cellular model before to approach to the role of NTE in embryotoxicity processes through mechanistic studies. Mouse D3 ESC in monolayer expressed an NTE activity of 23 nmol phenol/min/mg of protein, while mouse R1 ESC showed a specific NTE activity 3 times higher than D3. An increased expression of gene Pnpla6 (that codifies for NTE) was seen during differentiation in both the D3 cells in monolayer and embryonic bodies (EBS). The maximums of the Pnpla6 expression were reached after 30 h and 5 days of differentiation in monolayer and EBS cultures, respectively. This peak of the Pnpla6 expression correlated with the peak of the NTE enzymatic activity in D3 monolayers. NTE activity and Pnpla6 expression returned to basal levels after 48 h (in monolayer cultures) and 10 days (in EBS) of differentiation, respectively. The changes in the Pnpla6 expression did not correlate with changes noted in the expression of two endoderm, two ectoderm and one neuroectoderm gene markers. In conclusion, this manuscript reports about NTE expression in ESC and its variation during first stages of differentiation. Nevertheless, the role of this activity and the meaning of the variations detected during differentiation must be further studied.

Insulin-producing cells from embryonic stem cells experimental considerations.

The main objective of cell bioengineering is to generate customized tissues that allow recovering the lost functions in the organism in the absence of immune rejection. Although the possibility of in vitro generation of entire organs is technically very complex, obtaining specific cell types for replacement therapies seems to be a more realistic goal at mean time. In this context, those pathologies affected by the dysfunction of a specific cell type, as it is the case of beta-cell in diabetes, would be in principle candidates to benefit from cell transplantation protocols. Embryonic stem cells offer interesting possibilities in this context because they fulfill two important criteria: (i) High proliferation rate by symmetric cell division, overcoming the problem of biomass scarcity and (ii) Plasticity of differentiating to all cell types present in the adult organism, including the germ line. Different approaches have been developed in vitro to obtain insulin-producing cells from embryonic stem cells. Nevertheless, a definitive protocol does not exist yet. However, the experience accumulated in this field by the different laboratories has provided considering key points that would help to design a preferred protocol in the future.

Isolation and characterization of residual undifferentiated mouse embryonic stem cells from embryoid body cultures by fluorescence tracking.

The differentiation of mouse embryonic stem (ES) cells can be induced in vitro after leukemia inhibitory factor (LIF) withdrawal and further enhanced by the formation of "embryoid body" (EB) aggregates. This strategy is being used in order to optimize differentiation protocols that would result in functional cells for experimental cell replacement therapies. However, this study presents the possibility for residual undifferentiated cells to survive after standard in vitro procedures. Mouse ES cells were stably transfected with the enhanced green fluorescent protein (EGFP), under the control of the Oct4 promoter, a transcription factor that is expressed in undifferentiated ES cells but down-regulated on differentiation. Residual fluorescent cells were isolated from EBs that were cultured in standard conditions in absence of LIF. These residual cells displayed recurrent gain of chromosomes 8 and 9. Residual fluorescent cells, further expanded in absence of LIF and cultured as EBs, still displayed a significant Oct4 expression in comparison with parental transfected ES cells. Consequently, these residual cells have an intrinsic resistance to differentiate. The behavior of these cells, observed in vitro, can be overcome in vivo, as they were able to induce teratomas in subcutaneously injected nude mice. Residual undifferentiated cells displayed slight levels of VASA and DAZL expression. These results demonstrate that mouse ES cells cultured in vitro, in standard conditions, can spontaneously acquire recurrent karyotypical changes that may promote an undifferentiated stage, being selected in standard culture conditions in vitro.

Generation of insulin-producing cells from stem cells.

Islet transplantation as a potential treatment for diabetes will always be limited mainly because of the difficulty in obtaining sufficiently large numbers of purified islets from cadaveric donors. One alternative to organ or tissue transplantation is the use of a renewable source of cells. Stem cells are clonogenic cells capable of both self-renewal and multilineage differentiation. Therefore, these cells have the potential to proliferate and differentiate into any type of cell and to be genetically modified in vitro, thus providing cells which can be isolated and used for transplantation. Moreover, these derived cells have proven to be useful in different animal models. In this regard, insulin-secreting cells derived from mouse embryonic stem cells normalize blood glucose when transplanted into streptozotocin-induced diabetic animals. Using a combination of several differentiation methods and a 'cell trapping' system, we have obtained insulin-secreting cells from undifferentiated embryonic stem cells. The construct used allows the expression of a neomycin selection system under the control of the regulatory regions of insulin gene and other beta cell genes, such as Nkx6.1. Transplanted animals correct hyperglycaemia within 1 week and restore body weight in four weeks. Graft removal rescued the diabetic condition. Glucose tolerance test (IPGTT) and blood glucose normalization after a challenge meal was similar in control and in transplanted animals. This approach opens new possibilities for tissue transplantation in the treatment of type 1 and 2 diabetes.

Ectodermal commitment of insulin-producing cells derived from mouse embryonic stem cells.

Embryonic stem cells possess the ability to differentiate in vitro into a variety of cell lineages, including insulin-producing cells. Pancreatic beta-cells derive from foregut endoderm during embryonic development. However, previous reports using transgenic mice strongly indicate that insulin-positive cells may be generated also through the neuroectoderm pathway. To analyze this point, a culture system was performed in which only ectoderm committed cells were present. Based on published work, we achieved this by maintaining transfected clonal R1 mouse embryonic stem cells in monolayer in the absence of LIF. Contrary to differentiation protocols via embryoid body formation, monolayer cultured cells displayed ectodermal fates according to the marker gene expression pattern. Under these particular conditions, neomycin was added in order to select insulin-expressing cells. The cell lineage obtained expressed Pdx1, Pax6, Isl1, AChE, MBP, TH, and GS genes, confirming ectodermal commitment, even though some of these factors are also expressed in endoderm. In addition these cells displayed excitatory properties similar to astrocytes. Co-expression of insulin II and nestin was observed in monolayer culture and in the presence of specific conditioned media. No expression of early endodermal markers was detected along monolayer cultures. Altogether, these observations suggest that cells with ectoderm fates could participate in vitro in the derivation of insulin-producing cells. These results have implications for insulin gene regulation and hormone secretion in order to generate insulin-producing cells for replacement protocols in the treatment of diabetes.

Insulin-secreting cells derived from stem cells: clinical perspectives, hypes and hopes.

Diabetes is a degenerative disease that results from the selective destruction of pancreatic beta-cells. These cells are responsible for insulin production and secretion in response to increases in circulating concentrations of nutrients, such as glucose, fatty acids and amino acids. This degenerative disease can be treated by the transplantation of differentiated islets obtained from cadaveric donors, according to a new surgical intervention developed as Edmonton protocol. Compared to the classical double transplant kidney-pancreas, this new protocol presents several advantages, concerning to the nature of the implant, immunosuppressive drug regime and the surgical procedure itself. However, the main problem to face in any islet transplantation program is the scarcity of donor pancreases and the low yield of islets isolated (very often around 50%) from each pancreas. Nevertheless, transplanted patients presented no adverse effects and no progression of diabetic complications. In the search of new cell sources for replacement trials, stem cells from embryonic and adult origins represent a key alternative. In order to become a realistic clinical issue transplantation of insulin-producing cells derived from stem cells, it needs to overcome multiple experimental obstacles. The first one is to develop a protocol that may allow obtaining a pure population of functional insulin-secreting cells as close as possible to the pancreatic beta-cell. The second problem should concern to the transplantation itself, considering issues related to immune rejection, tumour formation, site for implant, implant survival, and biosafety mechanisms. Although transplantation of bioengineered cells is still far in time, experience accumulated in islet transplantation protocols and in experiments with appropriate animal models will give more likely the clues to address this question in the future.

Mitochondrial dysfunction is involved in apoptosis induced by serum withdrawal and fatty acids in the beta-cell line INS-1.

The potential toxic effects of high extracellular concentrations of fatty acids were tested in beta(INS-1)-cells cultured in the absence of serum, a condition known to alter cell survival in various systems. This may in part mimic the situation in type 1 or 2 diabetes where beta-cells are already insulted by various stressful conditions, such as cytokines and oxidative stress. Serum removal caused, over a 36-h period, oxidative stress and an early impairment of mitochondrial function, as revealed by increased superoxide production and markedly reduced mitochondrial membrane potential, but a lack of cytochrome c and apoptosis-inducing factor release in the cytosol. The fatty acids palmitate and oleate considerably accelerated the apoptosis process in serum-starved cells, as revealed by fluorescence-activated cell sorting analysis, morphological changes, chromatin condensation, DNA laddering, poly(ADP-ribose) polymerase cleavage, cytochrome c and apoptosis-inducing factor release, and increased levels of Bax and cytosolic caspase-2. The fatty acids also increased nitric oxide production, apparently independently of inducible nitric oxide synthase induction. Under the same experimental conditions, elevated glucose alone had only a marginal effect on beta-cell apoptosis. Together the results indicate that elevated concentrations of fatty acids are particularly efficient in accelerating the rate of apoptosis of already stressed beta(INS-1)-cells displaying altered mitochondrial function, and that the mitochondrial arm of the apoptosis process is involved in beta-cell lipotoxicity.

Direct visualization by confocal fluorescent microscopy of the permeation of myristoylated peptides through the cell membrane.

To study the permeability through the cellular membrane of synthetic peptides containing an hydrophobic moiety, we used a 13-mer myristoylated peptide labeled with a N-terminal fluorescent probe. After 2 h of incubation, the subcellular distribution was analyzed in intact chromaffin cells by confocal fluorescent microscopy. Our results demonstrate that myristoylated peptides diffuse into intact cells, showing an heterogeneous distribution, but they do not reach the cellular nucleous, at least during the time range used.