Primary Lines Research Articles

Relación entre talla y longitud del paso, en niños de Educación Primaria, a través de una variante del test Ten Metres Fast Walk Test

El presente trabajo analizó la relación existente entre la talla de los niños de las líneas de Educación primaria y la longitud en su zancada. Para lograrlo, se ha tenido en cuenta una muestra de ciento treinta y ocho estudiantes (n= 138) de primero a sexto de primaria por ambas líneas del colegio C.E.I.P. Los Morales durante el curso 2017/2018. Se ha utilizado la observación y la puesta en práctica y adaptación del test “Ten Metres walk test (10 MWT)”. El tratamiento estadístico que se ha aplicado ha sido descriptivo y de comparación entre las medias obtenidas. Los resultados, si se toma la media global de todos los cursos, indican que la zancada de los niños en edad escolar equivale al 36,43% de su altura. Siendo la media del primer ciclo 37,85%, del Segundo ciclo de 36,41 % y Tercer ciclo de 36,32%.

Surface-pressure pattern of separating flows over inclined slender bodies

Surface-pressure measurements and literature review of crossflows over inclined slender bodies at high Reynolds numbers (4.2 × 106 to 17 × 106, based on body length and freestream velocity) show that the constructed pressure maps have a distinct pattern, which relates to large-scale separation. The separation envelope is defined by a pair of primary and secondary surface-bifurcation lines as described by Lee [“Longitudinal development of flow-separation lines on slender bodies in translation,” J. Fluid Mech. 837, 627–639 (2018)]. The present study shows that the separation envelope falls in a “plateau” region in the circumferential distribution of the mean pressure. In the distribution of circumferential pressure gradient, there are two local maxima, where one is located on the windward side of the separation envelope and the other is located on the leeward side. Pattern tracking identifies that the locus of maximum circumferential pressure gradient resembles the locus of separation, but is located further upstream along the slender body.

Abstract 874: Adoptive transfer of cytotoxic lymphocytes generated by engineered human dendritic cells for AML treatment

Abstract Introduction: Acute myeloid leukemia (AML) is a devastating blood cancer. The clinical benefit of allogeneic stem cell transplantation (alloSCT) greatly relies on the graft-versus-leukemia (GVL) effect mediated by donor T cells, but it may also cause graft-versus-host disease (GVHD). One strategy to improve GVL without promoting GVHD is to adoptive transfer of cytotoxic T cells (CTLs) against leukemia-associated antigen (LAA). Generating potent CTLs is challenging. Stimulating naïve T cells with monocyte-derived dendritic cells loaded with LAA is commonly used, but it is severely limited due to low efficacy. We have developed a novel DC methodology and established immortalized human primary blood DC lines (ihv-DCs) by transducing human peripheral blood mononuclear cells (PBMCs) with a viral protein Tax. The constitutively activated ihv-DCs expand efficiently in vitro and can potently prime naïve T cells. They can be genetically modified to deliver given tumor antigens and stimulate antigen-specific CTLs in high efficiency. Here, we generate CTLs by stimulating donor PBMCs with ihv-DCs that carry HLA-A2 restrict human telomerase reverse transcriptase (hTERT), a known LAA. We investigate, in vitro and in vivo, the CTL killing efficacy. Methods: ihv-DCs that carry HLA-A2 restrict hTERT was engineered as previously described (Wu et al., PNAS 2018). HLA-A2 restrict hTERT-specific CTLs were generated by co-culturing ihv-DCs in the presence of IL-2 with donor PBMCs. In vitro cytotoxicity was performed by culturing CTLs with target cells that labeled with cell-track dye at different E:T ratio. Cell lysis was tested by flow-cytometry-based cell apoptosis analysis. In vivo cytotoxicity was examined by adoptive transfer CTLs into xenograft AML model. Flow cytometry was used to detect the leukemia burden. Results: Although variable, THP-1 cell line and blasts from all HLA-A2+ AML patients (11/11) in our study express significant intracellular hTERT. CTLs were successfully established by co-culturing ihv-DCs carrying hTERT with PBMCs from HLA-A2+ healthy donors. We observed an increasing proportion of effector memory T cells over time. We observed significant cytotoxic activities of purified CD8 T cells to U2OS that engineered to express hTERT, THP-1 cells, and primary leukemia blasts from AML patients in vitro. The cytotoxicity is predominantly hTERT specific as the killing to the primary U2OS cell line (negative for hTERT) is minimal. In addition, the cytotoxic effect was significantly diminished upon adding blockade antibody against HLA-A2, indicating the killing is through an HLA-A2 restrict manner. To study whether the CTLs can eradicate AML in vivo, we adoptively transferred CTLs into a xenograft AML model generated by engrafting primary AML into NSG mice (n=5), 5 × 106 cells were injected every 5 days for 4 times. Mice injected with PBS were set as control. On day 20, we found significant reduction of leukemia burden in blood and bone marrow from mice treated with CTLs. Importantly, no GVHD was observed. Persistent CTLs were detected in all organs (blood, bone marrow, liver, spleen, and lungs). Conclusion: Using a novel ihv-DC delivering LAA, we established CTLs that are highly efficient in killing primary leukemia blasts derived from AML patients both in vitro and in vivo. Our study provides a proof of principle for applying the novel DC methodology to AML treatment. Citation Format: Chenchen Zhao, Bei Jia, Yixing Jiang, Hiroko Shike, David F. Claxton, W Christopher Ehmann, Todd D. Schell, Hua Cheng, Bei Liu, Hong Zheng. Adoptive transfer of cytotoxic lymphocytes generated by engineered human dendritic cells for AML treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 874.

Emerging Strategies to Protect the Skin from Ultraviolet Rays Using Plant-Derived Materials.

Sunlight contains a significant amount of ultraviolet (UV) ray, which leads to various effects on homeostasis in the body. Defense strategies to protect from UV rays have been extensively studied, as sunburn, photoaging, and photocarcinogenesis are caused by excessive UV exposure. The primary lines of defense against UV damage are melanin and trans-urocanic acid, which are distributed in the stratum corneum. UV rays that pass beyond these lines of defense can lead to oxidative damage. However, cells detect changes due to UV rays as early as possible and initiate cell signaling processes to prevent the occurrence of damage and repair the already occurred damage. Cosmetic and dermatology experts recommend using a sunscreen product to prevent UV-induced damage. A variety of strategies using antioxidants and anti-inflammatory agents have also been developed to complement the skin’s defenses against UV rays. Researchers have examined the use of plant-derived materials to alleviate the occurrence of skin aging, diseases, and cancer caused by UV rays. Furthermore, studies are also underway to determine how to promote melanin production to protect from UV-induced skin damage. This review provides discussion of the damage that occurs in the skin due to UV light and describes potential defense strategies using plant-derived materials. This review aims to assist researchers in understanding the current research in this area and to potentially plan future studies.

Activation of C-reactive protein proinflammatory phenotype in the blood retinal barrier in vitro: implications for age-related macular degeneration.

The retinal pigment epithelium (RPE) is considered one of the main targets of age-related macular degeneration (AMD), the leading cause of irreversible vision loss among the ageing population worldwide. Persistent low grade inflammation and oxidative stress eventually lead to RPE dysfunction and disruption of the outer blood-retinal barrier (oBRB). Increased levels of circulating pentameric C-reactive protein (pCRP) are associated with higher risk of AMD. The monomeric form (mCRP) has been detected in drusen, the hallmark deposits associated with AMD, and we have found that mCRP induces oBRB disruption. However, it is unknown how mCRP is generated in the subretinal space. Using a Transwell model we found that both pCRP and mCRP can cross choroidal endothelial cells and reach the RPE in vitro and that mCRP, but not pCRP, is able to cross the RPE monolayer in ARPE-19 cells. Alternatively, mCRP can originate from the dissociation of pCRP in the surface of lipopolysaccharide-damaged RPE in both ARPE-19 and primary porcine RPE lines. In addition, we found that the proinflammatory phenotype of mCRP in the RPE depends on its topological localization. Together, our findings further support mCRP contribution to AMD progression enhancing oBRB disruption.

Abstract A42: EphB2 a potential therapeutic target for pediatric medulloblastoma

Abstract Aim: Medulloblastoma (MB) is the most frequent malignant brain tumor to occur in children and remains the leading cause of cancer-related mortality in childhood. The current standard of care for patients with MB includes surgical resection and radiation therapy followed by high-dose chemotherapy. Overall survival rates have reached 70%, but the outcome for young children, especially infants, is worse. Those who do survive suffer from long-term therapy-induced side effects. Thus, more effective and less toxic therapies are needed. Eph family receptors predominantly function during embryonic development and are typically either not expressed or expressed at very low levels in normal healthy tissues. However, it is now established that many Eph receptors are re-expressed and functional in human cancers, making them attractive, relatively tumor-specific targets. Thus, in MB anti-Eph therapy has the potential to target tumor cells and not normal brain, reducing off-target toxicity and potentially increasing response rates. We have generated compelling preliminary data showing that EphB2 is functional and overexpressed in pediatric MB. We now propose to extend these studies and validate antibody-based strategies to target this receptor. Methods: 1. EphB receptor expression was characterized using a number of approaches: RNA (RNASeq and QPCR), protein (flow cytometry and Western blot) and receptor localization by immunofluorescence in MB ATCC lines, primary lines, and patient tissue. 2. To better understand the function of the EphB2, gene expression was downregulated using shRNA in primary MB cell line models. Following EphB downregulation, we assessed in vitro growth and cell death induction and tumor-forming capacity using orthotopic MB intracranial xenografts. Moreover, following EphB activation, using soluble ligand we assessed in vitro growth via direct cell count and MTT assay, cell migration via scratch assay, and cell death induction via Annexin V staining. Following EphB inhibition, using peptide strategies we assessed in vitro growth, cell migration, and cell death induction. Results: Our study shows that EphB2 is both functional and highly expressed in MB. Furthermore, EphB2 appears to be expressed in a subtype-specific manner. We further confirmed our expression studies using the MB Advanced Genomics International Consortium (MAGIC) dataset. The EphB2 high-affinity ligands ephrinB1/B2-Fc were used to induce kinase activation. Following receptor activation, we observed reduced tumor cell proliferation and invasion in vitro. EphB2 receptor blocking studies, using the SNEW blocking peptide, effectively inhibited kinase phosphorylation and subsequently FAK phosphorylation. Most importantly, the small peptide rescues MB cell growth upon ephrin B ligand stimulation, indicating the specific mitogenic capacity of this receptor. Conclusion: In summary, this study furthers our understanding of EphB2 expression and function in MB and paves the way for future receptor-targeting studies in MB. Citation Format: Yuchen Li, Carolin Offenhauser, Rochelle D’Souza, Seckin Akgul, Bryan W. Day. EphB2 a potential therapeutic target for pediatric medulloblastoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A42.

The nature of the companion in the Wolf-Rayet system EZ Canis Majoris

Context. EZ Canis Majoris is a classical Wolf-Rayet star whose binary nature has been debated for decades. It was recently modeled as an eccentric binary with a periodic brightening at periastron of the emission originating in a shock heated zone near the companion. Aims. The focus of this paper is to further test the binary model and to constrain the nature of the unseen close companion by searching for emission arising in the shock-heated region. Methods. We analyze over 400 high resolution International Ultraviolet Explorer spectra obtained between 1983 and 1995 and XMM-Newton observations obtained in 2010. The light curve and radial velocity (RV) variations were fit with the eccentric binary model and the orbital elements were constrained. Results. We find RV variations in the primary emission lines with a semi-amplitude K1 ∼ 30 km s−1 in 1992 and 1995, and a second set of emissions with an anti-phase RV curve with K2 ∼ 150 km s−1. The simultaneous model fit to the RVs and the light curve yields the orbital elements for each epoch. Adopting a Wolf-Rayet mass M1 ∼ 20 M⊙ leads to M2 ∼ 3−5 M⊙, which implies that the companion could be a late B-type star. The eccentric (e = 0.1) binary model also explains the hard X-ray light curve obtained by XMM-Newton and the fit to these data indicates that the duration of maximum is shorter than the typical exposure times. Conclusions: The anti-phase RV variations of two emission components and the simultaneous fit to the RVs and the light curve are concrete evidence in favor of the binary nature of EZ Canis Majoris. The assumption that the emission from the shock-heated region closely traces the orbit of the companion is less certain, although it is feasible because the companion is significantly heated by the WR radiation field and impacted by the WR wind.

2111-P: Role of G-Protein Coupled Receptor 31 (GPR31) in ß-Cell Health and Disease

Islet β-cell inflammation, dysfunction, and death are characteristic of diabetes. 12/15-Lipoxygenase (12/15-LOX) produces 12-HETE and is found activated under conditions of insulin resistance and systemic inflammation. 12-HETE acts as an inflammatory mediator that promotes β-cell dysfunction and death through oxidative stress. Since 12-HETE has been shown to bind the G-protein coupled receptor 31 (GPR31), we hypothesized that this binding activates pathways leading to β-cell dysfunction. To test our hypothesis, we screened primary islets and β-cell lines for the presence of GPR31 and studied islets and glucose homeostasis in Gpr31b-/- mice. We observed that GPR31 is present in human and mouse islets, and the mouse β cell-derived cell line (βTC3). Consistent with a role in inflammation, GPR31 expression was increased upon stimulation with a cocktail of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β). We also created a germline knockout of the gene encoding GPR31 (Gpr31b) in mice. Similar to mice with deletion of the gene for 12/15-LOX (Alox15-/-), Gpr31b-/- mice were viable and healthy, with body weight and glucose tolerance indistinguishable from control littermates. To mimic conditions of insulin resistance in vitro, we treated isolated islets Gpr31-/- and control littermates with 10 µM S961, an insulin receptor inhibitor. Treated control islets showed an expected decrease in β-cell identity genes (Ins1/2, Pdx1), genes encoding antioxidant enzymes (Gpx1), and an increase in genes associated with ER stress (Chop, sXbp1). In contrast, Gpr31b-/- islets expression of these genes was resistant to S961. Compared to control littermates, Gpr31-/- mice were resistant to glucose intolerance induced by the β-cell inflammatory toxin streptozotocin. Together these data support the hypothesis that the major product of 12/15-LOX, 12-HETE, acts via GPR31 in promoting β-cell dysfunction in the setting of insulin resistance and inflammation. Disclosure M. Hernandez-Perez: None. I. Haider: None. R.M. Anderson: None. S.A. Tersey: None. R. Mirmira: Advisory Panel; Self; Hibercell, Sigilon Therapeutics, Veralox Therapeutics. Employee; Spouse/Partner; Eli Lilly and Company. Funding JDRF (3-PDF-2019-750-A-N); National Institutes of Health (T32DK064466, R01DK105588, (P30DK097512)

СХЕМА ПЕРЕРАБОТКИ ТЕХНИЧЕСКОЙ КОНОПЛИ В ОДНОТИПНУЮ ПЕНЬКУ

Industrial hemp as a source of fiber is a valuable industrial raw material. In modern conditions, the lines of its primary processing should produce the same type of fiber with different characteristics, depending on the requirements for the properties of the finished fiber. The purpose of the work is to substantiate the scheme for processing industrial hemp based on the characteristics of hemp. Five primary processing lines were compared, including domestic flax equipment (M-110L2 grinding machine, DLV-2 disintegrators, and TG-135L shaker machines with a lower comb field). As raw materials the industrial hemp from Penza region, harvested in the spring, was used. The mass of one handful is 100 g, the loading density is 0.5 kg/m, the twist is three times. Raw materials were loaded into each machine manually. After primary processing by instrumental methods, hemp grade and characteristics were determined according to GOST 9993-2014. The scheme of the line for primary processing of industrial hemp in the form of tangled mass of stems of various lengths, which allows the production of the same type of hemp with different values of technological characteristics, has been developed. For the production of the same type of hemp with an average mass length of fiber 142 ... 144 mm, a mass fraction of bonfire of 1.6 ... 2.2%, a weighted average linear density of 11 ... 12 tex, the line should include a pulping machine, a disintegrator, two shaking machines; with characteristics respectively 235 mm, 7 ... 8% and 12 ... 13 tex - a disintegrator and two shaking machines; 219 mm and 15 ... 16%, 18 ... 19 tex - pulley and two shaking machines. Using the studied set of equipment provides a reduction in capital costs, in comparison with the lines of other manufacturers, by 3 ... 4 times, energy costs - up to 40%

SAT-749 Defining The Role Of Androgens In Hernia Associated Skeletal Muscle Fibrosis

Introduction: Inguinal hernia is a highly prevalent condition occurring in 27% of adult men in their lifetime. The recurrence rate of hernia is 5-20%, resulting in a substantial cost burden in surgical repair procedures. Until recently, the mechanisms leading to the lower abdominal muscle (LAM) weakening characteristic of hernia were unknown. Our group developed the first mouse model of inguinal hernia through expression of the human aromatase enzyme in male mice (AromHum). Aromatase converts androgens to estrogens, and is expressed in the skeletal muscle in humans, but not mice. We found that locally formed estrogen from aromatase activity in LAM and decreased circulating testosterone levels are associated with muscle atrophy and fibrosis resulting in hernia. However, it is unclear how decreasing androgen levels might affect muscle fibrosis, and defining this potential mechanism could impact hernia treatment. We hypothesized that low androgen levels promote muscle fibroblast proliferation and fibrosis, and that androgen treatment would prevent hernia progression in AromHum mice. Methods: Arom Hum mice (3 weeks old) were treated with high-dose dihydrotestosterone (DHT) via injection for 7.5 weeks with hernia volume continuously recorded (n=5/group). Primary fibroblasts were isolated from LAM from WT and AromHum mice (n=5/genotype). Cells were treated for 24 hours with increasing doses (0.001, 0.01, 0.1, 1, 5, 10 and 100 nM) of R1881, a synthetic androgen, and compared to untreated cells by western blot. Results: Hernia volume was significantly decreased in AromHum mice treated with DHT compared to vehicle-treated mice, and volume remained consistently suppressed after DHT treatment (p < 0.005). In both primary fibroblast lines, R1881 treatment increased AR levels in a dose dependent manner, indicating that the treatment was effective. Preliminary data indicated that low doses of R1881 (0.001 and 0.01 nM) increased PCNA levels in LAM WT and LAM AromHum fibroblasts. Densitometry normalized to GAPDH showed 80% and 60% increases for 0.001 nM and 0.01 nM respectively in LAM WT fibroblasts, and 20% and 30% increases at these doses in LAM AromHum fibroblasts. Higher doses of R1881 decreased PCNA levels in LAM AromHum fibroblasts by 40% (10 nM) and 30% (100 nM), whereas a 25% decrease was detected in LAM WT fibroblasts at 100 nM. Conclusion: These data suggest that low androgen doses increase LAM fibroblast proliferation, which possibly contributes to hernia formation. Androgen treatment at higher doses can partially block the progression of hernia in vivo. However, it is unclear whether and how androgen deficiency in combination with excess estrogen affects fibroblast proliferation and hernia formation. Additional research is required to determine if androgen supplementation in sufficient doses is a potential therapeutic for inguinal hernia and other muscle weakness diseases.

Class Legislation, Fundamental Rights, and the Origins of Lochner and Liberty of Contract

While legal scholars and historians have criticized many judicial doctrines from the pre-New Deal period, critics have been especially scathing in their attacks on the “liberty of contract” doctrine enforced most famously in Lochner v. New York. Until recently, academics routinely asserted that the Lochner Court’s Justices simply made up the doctrine based on a combination of belief in laissez-faire economics and hostility to workers’ rights. Contemporary scholars, by contrast, have reconstructed the period’s due-process jurisprudence, finding in it a principled commitment to a conception of justice with philosophical and jurisprudential roots dating back to the Founding and beyond. There are two primary lines of this revisionist literature. One emphasizes traditional Anglo-American hostility to “class legislation” — legislation that arbitrarily favors or disfavors particular factions. The other emphasizes the influence of the natural rights tradition, tempered by precedent and historicism, on the Court’s due-process decisions. Part I of this Article reviews the debate that emerged in the 1990s and early 2000s between partisans of these interpretations. Part II of this Article discusses subsequent developments in the class legislation vs. fundamental rights debate through the present time, noting an increasing convergence between the two sides; both sides acknowledge that both class legislaton and fundamental rights played significant roles in the development of the Supreme Court’s due process jurisprudence, with the remaining debate primarily over which doctrine deserves more emphasis in histortical recountings. This Article concludes by noting that as this debate has progressed, certain areas of historical consensus have emerged. First, both sides agree that the Court did not attempt to enforce anything approaching a night watchman-type laissez-faire policy on government. Second, both sides agree that the Supreme Court’s fundamental-rights jurisprudence, often traced to the 1930s, in fact began to emerge in the pre–New Deal period. Finally, they agree that the Supreme Court Justices who adopted and applied the liberty of contract doctrine did not have the cartoonish reactionary motives attributed to them by Progressive and New Deal critics. Rather, the Justices, faced with constitutional challenges to novel assertions of government power, sincerely tried to protect liberty as they understood it, consistent with longstanding constitutional doctrines that reflected the notion that governmental authority had limits enforceable via the Due Process Clause.

Abstract A41: Nucleotide metabolism heterogeneity in mutant KRAS pancreatic cancer

Abstract Mutant KRAS orchestrates major metabolic adaptations critical for pancreatic ductal adenocarcinoma (PDAC) growth and survival, including changes in glucose, amino acid, lipid and energy metabolism. However, how nucleotide metabolism in PDAC is impacted by mutant KRAS has received much less attention, even though nucleotides play critical roles in major biologic processes including nucleic acid synthesis, phospholipid biosynthesis and protein glycosylation. Pyrimidine nucleotide metabolism is a highly complex and regulated system with multiple pathways contributing to its plasticity and robustness. Pyrimidine nucleotides can be produced via the de novo pathway (DNP) that utilizes glucose and amino acids, and the nucleoside scavenging pathway (NSP) that utilizes extracellular uridine and cytidine. We hypothesize that nucleotide products of RNA turnover can fuel a third route for pyrimidine biosynthesis, which we term the nucleotide recycling pathway (NRP). Among these three major pyrimidine nucleotide biosynthetic pathways, the DNP has been studied most extensively. The biologic and therapeutic significance of the NSP and NRP in PDAC, as well as their regulation and coordination with DNP in the context of mutant KRAS signaling, are poorly understood. We further hypothesize that since PDAC tumor microenvironment is characterized by insufficient supply of nutrients and oxygen, these tumors have limited DNP capacity and increased reliance on alternate pyrimidine biosynthetic pathways. Here, we describe a novel mass spectrometric (MS) method to measure the contributions of pyrimidine nucleotide convergent pathways to RNA and DNA synthesis in PDAC models. Applying this method to a panel of PDAC models, including patient-derived primary lines, revealed previously unappreciated pyrimidine biosynthetic heterogeneity, suggesting the existence of distinct pyrimidine nucleotide metabolic subtypes. Identifying and characterizing these subtypes in the context of oncogenic KRAS signaling may enable stratification of PDAC tumors and guide the development of novel therapeutic approaches. Citation Format: Thuc M. Le, Woosuk Kim, Joseph R. Capri, Anthony E. Cabebe, Wes Armstrong, Evan Abt, Soumya Poddar, Shili Xu, Dave Dawson, Timothy R. Donahue, Caius G. Radu. Nucleotide metabolism heterogeneity in mutant KRAS pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A41.

WNT signalling supported by MEK/ERK inhibition is essential to maintain pluripotency in bovine preimplantation embryo

Capturing stable embryonic stem cell (ESC) lines from domesticated animals still remains one of the challenges of non-rodent embryology. The stake is high, as stable ESCs derived from species such as cattle present high economic and scientific value. Understanding of the processes leading to the embryonic lineage segregation is crucial to provide species-orientated molecular environment capable of supporting self-renewal and pluripotency. Therefore, the aim of this study was to validate the action of the two core regulatory pathways (WNT and MEK/ERK) during bovine embryo development. In vitro produced bovine embryos were obtained in the presence of inhibitors (i), which enable activation of the WNT pathway (via GSK3i, CHIR99021) and suppression of MEK signalling by PD0325901 in the 2i system and PD184325 and SU5402 in the 3i system. We have followed the changes in the distribution of the key lineage specific markers both at the transcript and protein level. Our results showed that WNT signalling promotes the expression of key inner cell mass (ICM) specific markers in bovine embryos, regardless of the MEK/ERK inhibitor cocktail used. MEK/ERK downregulation is crucial to maintain OCT4 and NANOG expression within the ICM and to prevent their exclusion from the trophectoderm (TE). At the same time, the classical TE marker (CDX2) was downregulated at the mRNA and protein level. As a follow up for the observed pluripotency stimulating effect of the inhibitors, we have tested the potential of the 2i and the 3i culture conditions (supported by LIF) to derive primary bovine ESC lines. As a result, we propose a model in which all of the primary signalling pathways determining embryonic cell fate are active in bovine embryos, yet the requirement for pluripotency maintenance in cattle may differ from the described standards. WNT activation leads to the formation (and stabilisation of the ICM) and MEK/ERK signalling is maintained at low levels. Unlike in the mouse, GATA6 is expressed in both ICM and TE. MEK/ERK signalling affects HP formation in cattle, but this process is activated at the post-blastocyst stage. With regard to self-renewal, 2i is preferable, as 3i also blocks the FGF receptor, what may prevent PI3K signalling, important for pluripotency and self-renewal.

Cryopreservation of Viable Human Tissues: Renewable Resource for Viable Tissue, Cell Lines, and Organoid Development.

The availability of viable human tissues is critical to support translational research focused on personalized care. Most studies have relied on fresh frozen or formalin-fixed paraffin-embedded tissues for histopathology, genomics, and proteomics. Yet, basic, translational, and clinical research downstream assays such as tumor progression/invasion, patient-derived xenograft, organoids, immunoprofiling, and vaccine development still require viable tissue, which are time-sensitive and rare commodities. We describe the generation of two-dimensional (2D) and three-dimensional (3D) cultures to validate a viable freeze cryopreservation technique as a standard method of highest quality specimen preservation. After surgical resection, specimens were minced, placed in CryoStor™ media, and frozen using a slow freezing method (-1°C/min in -80°C) for 24 hours and then stored in liquid nitrogen. After 15-18 months, the tissues were thawed, dissociated into single-cell suspensions, and evaluated for cell viability. To generate primary 2D cultures, cells were plated onto Collagen-/Matrigel-coated plates. To develop 3D cultures (organoids), the cells were plated in reduced serum RPMI media on nonadherent plates or in Matrigel matrix. The epithelial nature of the cells was confirmed by using immunohistochemistry for cytokeratins. DNA and RNA isolation was performed using QIAGEN AllPrep kits. We developed primary lines (2D and 3D) of colon, thyroid, lung, renal, and liver cancers that were positive for cytokeratin staining. 3D lines were developed from the same cohort of tumor types in both suspended media and Matrigel matrix. Multiple freeze-thaw cycles did not significantly alter the viability and growth of 2D and 3D lines. DNA/RNA recovery was similar to its fresh frozen cohort. In this study, we validated 2D and 3D tissue cultures as methods to corroborate the feasibility of viable cryopreservation of tumor tissue. This proof-of-principle study, if more widely implemented, should improve accessibility of human viable tumor tissue/cells in a time-independent manner for many basic, preclinical, and translational assays.

Cross Interaction between M2 Muscarinic Receptor and Notch1/EGFR Pathway in Human Glioblastoma Cancer Stem Cells: Effects on Cell Cycle Progression and Survival.

Glioblastomas (GBM) are the most aggressive form of primary brain tumors in humans. A key feature of malignant gliomas is their cellular heterogeneity. In particular, the presence of an undifferentiated cell population of defined Glioblastoma Stem cells (GSCs) was reported. Increased expression of anti-apoptotic and chemo-resistance genes in GCSs subpopulation favors their high resistance to a broad spectrum of drugs. Our previous studies showed the ability of M2 muscarinic receptors to negatively modulate the cell growth in GBM cell lines and in the GSCs. The aim of this study was to better characterize the inhibitory effects of M2 receptors on cell proliferation and survival in GSCs and investigate the molecular mechanisms underlying the M2-mediated cell proliferation arrest and decreased survival. Moreover, we also evaluated the ability of M2 receptors to interfere with Notch1 and EGFR pathways, whose activation promotes GSCs proliferation. Our data demonstrate that M2 receptors activation impairs cell cycle progression and survival in the primary GSC lines analyzed (GB7 and GB8). Moreover, we also demonstrated the ability of M2 receptor to inhibit Notch1 and EGFR expression, highlighting a molecular interaction between M2 receptor and the Notch-1/EGFR pathways also in GSCs.

Information reservation of cyber-physical production of the Industry 4.0

An actual task has been studied to organize informative reserving of the production data and link channels in the Industry 4.0 smart factory. Informative reserving principles in a digital company are done by duplicating informative link channels into a single company communication environment. Connection channels duplicating creates the production division informative redundancy for the technological operations completion results data. The smart factory informative reserving increase company functionality reliability and provides functional and informative smart factory functionality. There are schemes of cyber and physical production components informative reserving and types of connection channels are defined which are used as primary and reserve lines of the information transmission. There are ways of informative reserving based on the technologies of Bluetooth, Ethernet and Internet of Things (IoT). Main way of cyber and physical production informative reserving is done in system level and is duplicating of the IoT wireless channel with a wired channel of the Ethernet connection and transmitting of duplicating messages. Auxiliary way of cyber and physical production informative reserving is done in local level of the production data exchange among cyber and physical systems with a Bluetooth channel.

A synopsis of prostate organoid methodologies, applications, and limitations.

Current in vitro modeling systems donot fully reflect the biologic and clinical diversity of prostate cancer (PCa). Organoids are 3D in vitro cell cultures that recapitulate disease heterogeneity, retain prostate gland architecture, and mirror parental tumor characteristics. To make better use of organoid models in the PCa research field, we provide a review of cutting-edge prostate organoid methodologies, applications, and limitations. We summarize methodologies for the establishment of benign prostate and PCa organoids and describe some of the model's practical applications and challenges. We highlight the patient-derived xenograft (PDX)-organoid interface model, which may allow for the generation of organoids from primary and rare PCa subtypes. Finally, we discuss potential future utilizations of PCa organoids in the realms of drug development and precision oncology. Organoids represent a quasi in vivo modeling system that can be easily amenable to genetic modification and functional studies. As such, organoids may serve as an intermediate preclinical model for studying PCa. Future directions may include the refinement of culturing conditions to increase drug response fidelity in PCa organoids. The PDX-organoid interface model may enable the future establishment of primary and rare subtype PCa organoid lines.

CD1d expression and invariant natural killer T-cell numbers are reduced in patients with upper gastrointestinal cancers and are further impaired by commonly used chemotherapies.

Esophageal and gastric cancers collectively cause over 1.1 million deaths annually and only 20-30% of patients respond favorably to current therapies. Cellular therapies using invariant natural killer T (iNKT) cells are showing promise for patients with other cancers; therefore, we investigated if these cells are altered in esophageal and gastric cancer patients. Flow cytometric analysis of peripheral blood from 139 patients revealed that iNKT cells are depleted from patients with esophageal and gastric adenocarcinoma and esophageal squamous cell carcinoma, both before and after treatment. Interrogation of the KMPlot database of transcriptomic data from 876 gastric cancer patients revealed that low CD1d expression is associated with poor prognosis. These observations suggest that therapies that boost CD1d expression and iNKT cell responses may benefit these patients. However, we found that chemotherapies used for esophageal and gastric cancers have adverse effects on iNKT cells in vitro. Cisplatin caused a significant reduction of CD1d expression by esophageal tumor cell lines. Cisplatin, 5-fluorouracil and carboplatin induced dose-dependent apoptosis in primary lines of iNKT cells and inhibited CD1d-dependent interferon-γ production and cytolytic degranulation by viable iNKT cells. Interestingly, cisplatin increased granzyme B and perforin production and decreased the production of the granzyme B inhibitor PI9, which protects cytotoxic cells from self-damage by granzyme B. Thus, cisplatin-induced apoptosis of iNKT cells may be mediated in part by altering granzyme B and PI9 expression. Our data suggest that iNKT cell-based immunotherapies may benefit patients with gastrointestinal cancers, but may be negatively affected by chemotherapies used for these cancers.

Stress Memory of Recurrent Environmental Challenges in Marine Invasive Species: Ciona robusta as a Case Study.

Fluctuating environmental changes impose tremendous stresses on sessile organisms in marine ecosystems, in turn, organisms develop complex response mechanisms to keep adaptive homeostasis for survival. Physiological plasticity is one of the primary lines of defense against environmental challenges, and such defense often relies on the antioxidant defense system (ADS). Hence, it is imperative to understand response mechanisms of ADS to fluctuating environments. Invasive species provide excellent models to study how species cope with environmental stresses, as invasive species encounter sudden, and often recurrent, extensive environmental challenges during the whole invasion process. Here, we studied the roles of ADS on rapid response to recurrent cold challenges in a highly invasive tunicate (Ciona robusta) by simulating cold stresses during its invasion process. We assessed antioxidative indicators, including malondialdehyde (MDA), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), as well as transcriptional changes of ADS-related genes to reveal the physiological plasticity under recurring cold stresses. Our results demonstrated that physiological homeostasis relied on the resilience of ADS, which further accordingly tuned antioxidant activity and gene expression to changing environments. The initial cold stress remodeled baselines of ADS to promote the development of stress memory, and subsequent stress memory largely decreased the physiological response to recurrent environmental challenges. All results here suggest that C. robusta could develop stress memory to maintain physiological homeostasis in changing or harsh environments. The results obtained in this study provide new insights into the mechanism of rapid physiological adaption during biological invasions.

From surface microrelief to big wrinkles in skin: A mechanical in-silico model

A major concern associated with skin, the largest organ of our body, is how to prevent it from wrinkling and aging. Understanding the mechanics of skin wrinkling can provide useful insight into skin aging prevention. However, despite decades of endeavors the underlying mechanism of skin wrinkling and aging remains poorly understood. This paper explores the effect of geometrical and mechanical properties of skin on its wrinkling via an integrated theoretical and computational analysis. The skin is modeled with a soft structure having different layers with various thicknesses and material properties. Innovatively, the pattern of skin microrelief is generated and mapped on the model to investigate its effect on the formation of primary lines, secondary lines, and big wrinkles of skin. Analytical interpretation provides preliminary insight into the critical compressive strain for the model skin to start wrinkling, while advanced computational models with surface microrelief offer clues for the skin’s post-wrinkling complex morphology. In particular, tissue geometry, material properties, and microrelief pattern are explored as the determinant parameters to control the location, size, and patterns of skin wrinkles. Our findings allude that the characteristics of compression-induced wrinkles are primarily determined by the geometrical and material property of skin layers rather than the genuine skin microrelief. However, microrelief plays a pivotal role in regulating and determining the locations of primary and secondary wrinkle lines. The edges of the microrelief units are favorable paths for evolving primary and secondary lines. Post-wrinkling analysis reveals that in addition to the periodic sinusoidal pattern, several secondary complex patterns such as non-symmetric periodic, period-doubling and self-contacting folds are observed in the compressed model of skin. Results of the study also show that wrinkle patterns highly depend on the thickness and material property of Stratum Corneum (SC), the outermost layer of skin.