Search Results (50)

Controlled delivery using nanoparticle-based systems has attracted considerable attention; however, achieving cell-type specificity remains a major challenge. To address this issue, we focused on the intrinsic cell tropism of viruses. The hepatocyte tropism of hepatitis B virus (HBV) is mediated by interactions between its large envelope protein (L protein) and host factors, including the sodium taurocholate cotransporting polypeptide (NTCP). In this study, we explored viral-like secretory vesicles (VLSVs) displaying HBV spike proteins as a virus-inspired vesicle platform for hepatocyte targeting. We previously established a method for producing VLSVs from HBV L- and S-expressing HEK293T cells. In the present study, we developed an improved protocol using exosome-depleted fetal calf serum and optimized ultracentrifugation, resulting in VLSVs with comparable particle numbers and sizes but approximately tenfold higher protein content per particle. VLSVs were concentrated using a two-layer sucrose cushion, labeled with DiI, and purified by sucrose density gradient ultracentrifugation. We evaluated DiI uptake in hepatocyte-derived cells (HepG2 and Huh7), non-hepatic cells (MDA-MB231, H1299, HeLa, and Vero), and NTCP-overexpressing HepG2 cells. VLSVs showed preferential uptake in the following order: NTCP-overexpressing HepG2 > HepG2 > Huh7 > non-hepatic cells. Furthermore, removal of the N-terminal Flag tag from the L protein enhanced hepatocyte-associated uptake, suggesting the importance of preserving the native structure of the preS1 domain. While vesicle characterization and mechanistic validation remain to be further investigated, these findings provide a proof-of-concept for a virus-inspired vesicle platform exhibiting preferential uptake in hepatocyte-derived cells. Full article

This study assessed trace mineral levels (Co, Cr, Cu, Fe, Mn, Mo, Se, and Zn) in paired serum samples from multiparous Holstein Friesian cows and their calves after colostrum intake, to explore potential relationships between maternal and neonatal mineral status. The acid-digested samples were analyzed by inductively coupled plasma mass spectrometry. Serum levels of Co, Cu, Fe, and Se were significantly higher in cows than in calves (p < 0.001), while Zn levels were higher in calves. The levels of Cr, Mn, and Mo were similar in both groups. Overall, mineral deficiencies were more prevalent in cows, with Se being the most deficient element, followed by Zn, Cu, and Co. Calves were more deficient in Co and Mn than their mothers but were not generally deficient in Se. Serum levels of Cr, Cu, Mn, Mo, and Se were positively correlated in cows and their calves, suggesting that maternal mineral status influences neonatal mineral levels. Overall, these results provide insights into trace mineral dynamics in cow–calf pairs. Further studies are needed to clarify the relative contribution of placental and colostral mineral transfer. Full article

Background: Cultured human skin explants provide preclinical models to investigate drug delivery and the efficacy of topical treatments for wound healing. However, different culture conditions may affect cell viability, proliferation, and even wound healing. Since animal-derived supplements can influence the investigation of human physiological responses, this study evaluated the effects of non-animal supplements on the ex vivo wound healing process to improve the use of this model for preclinical drug efficacy tests. Methods: In in vitro scratch assays using HaCaT cells and fibroblasts, for media supplemented with normal human serum (NHS), oxygen carriers (OCs) had a positive impact on cell migration, supporting the further evaluation in ex vivo skin culture models. Human skin explants with standardized superficial wounds were cultured in four supplemented media: (i) Dulbecco’s Modified Eagle Medium High Glucose (DMEM) with fetal calf serum (FCS), (ii) DMEM with NHS and OC, (iii) CnT-Prime^TM with NHS and OC, and (iv) EpiLife™ with NHS and an OC. Results: During the 12-day culture, we observed re-epithelialization in all groups with the exception of EpiLife + NHS + OC (with no Ca⁺⁺ supplement). For these samples, starting from day 6, we noticed a loosening of the dermal–epidermal junction and disruption of the upper epidermal layer. Furthermore, an immunohistochemical analysis of extracellular matrix components and remodeling factors, including type I and III collagen, transforming growth factor-β2, and matrix metalloproteinase-9, provided insights into tissue repair dynamics. Conclusions: NHS plus OC is comparable to FCS supplementation and represents a more physiological and ethical alternative. Full article

Natural killer (NK) cells hold promise in cancer treatment due to their ability to spontaneously lyse cancer cells. For clinical use, high quantities of pure, functional NK cells are necessary. Combining adherence-based isolation with specialized media showed the unreliability of the isolation method, but demonstrated the superiority of the NK MACS^® medium, particularly in suboptimal conditions. Neither human pooled serum, fetal calf serum (FCS), human platelet lysate, nor chemically defined serum replacement could substitute human AB serum. Interleukin (IL-)2, IL-15, IL-21, and combined CD2/NKp46 stimulation were assessed. IL-21 and CD2/NKp46 stimulation increased cytotoxicity, but reduced NK cell proliferation. IL-15 stimulation alone achieved the highest proliferation, but the more affordable IL-2 performed similarly. The RosetteSep™ human NK cell enrichment kit was effective for isolation, but the presence of peripheral blood mononuclear cells (PBMCs) in the culture enhanced NK cell proliferation, despite similar expression levels of CD16, NKp46, NKG2D, and ICAM-1. In line with this, purified NK cells cultured in NK MACS^® medium with human AB serum and IL-2 demonstrated high cytotoxicity against primary glioblastoma stem cells. Full article

Chagas disease is caused by the single-flagellated protozoan Trypanosoma cruzi, which affects several million people worldwide. Understanding the signal transduction pathways involved in this parasite’s growth, adaptation, and differentiation is crucial. Understanding the basic mechanisms of signal transduction in T. cruzi could help to develop new drugs to treat the disease caused by these protozoa. In the present work, we have demonstrated that Fetal Calf Serum (FCS) can quickly increase the levels of both phosphorylated and unphosphorylated forms of T. cruzi DNA polymerase beta (TcPolβ) in tissue-cultured trypomastigotes. The in vitro phosphorylation sites on TcPolβ by protein kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 have been identified by Mass Spectrometry (MS) analysis and with antibodies against phosphor Ser-Thr-Tyr. MS analysis indicated that these protein kinases can phosphorylate Ser and Thr residues on several sites on TcPolβ. Unexpectedly, it was found that TcCK1 and TcPKC1 can phosphorylate a different Tyr residue on TcPolβ. By using a specific anti-phosphor Tyr monoclonal antibody, it was determined that TcCK1 can be in vitro autophosphorylated on Tyr residues. In vitro and in vivo studies showed that phorbol 12-myristate 13-acetate (PMA) can activate the PKC to stimulate the TcPolβ phosphorylation and enzymatic activity in T. cruzi epimastigotes. Full article

As the beef industry moves towards efficient animal production to improve sustainability in agriculture, new production and management approaches are emerging. Among the many facets of the beef industry, cow–calf operations have the most opportunity for efficiency improvement, including improvements in fertility. This project accounts for measures and methods of (1) twinning reproductive technologies and (2) twin calf perinatal care and pre-weaning rearing. The overall objective was to produce twin calves using two reproductive technologies—embryo transfer and artificial insemination. The subobjectives were to determine accuracy of twin pregnancies embryo/fetal losses using ultrasonography, evaluate parturition and dystocia, and determine the effects of different twin-raising methods on neonatal behavior and growth. A fixed-time artificial insemination (FTAI) protocol was applied to 77 multiparous Angus-cross cows from a commercial beef herd in northcentral South Dakota during the summer of 2019. Cows were assigned to two different treatments groups: only artificially inseminated (AI) or received an embryo transfer following artificial insemination (ET + AI). They were estrous-synchronized, artificially inseminated (AI) with black Angus semen at day 0, and received and embryo transfer (ET) at day 7. Ultrasound examination detected 56% pregnancy risk for both groups, with sensitivity, specificity, and accuracy of 75%, 100%, and 90.5%, respectively, for bilateral twin detection. Calves were born during spring 2020. Twin calves (n = 34) and singleton calves (n = 11) were assigned to one of three raising methods: (1) twin born and twin raised (TT; n = 16), (2) twin born and single raised (TS; n = 18), and (3) single born and single raised (S; n = 11). Neonatal nursing behavior and birth weights were recorded, and adjusted day 200 and day 280 were calculated measures of vitality and growth. Blood samples were collected at age 24 h for colostrum intake measures (total serum protein, IgG1, and IgM). Twin calves were born 20% (p < 0.05) lighter in body weight than singletons; however, weights did not differ at day 280 between TT and S calves. TS calves had the shortest average latency to stand, but immunoglobulin concentrations did not differ among treatments. At weaning, cows that had birthed and raised twins produced more kilograms of live weight per pregnancy than cows birthing and raising singletons. Using ET + AI proved to increase twinning rate, and growth was maintained when raising both twins with their dam. Full article

The neuroblastoma cell lines SH-SY5Y and Neuro2A are commonly utilized models in neurobiological research. DMEM supplemented with different nutrients and 5–10% Fetal Calf Serum (FCS) is typically used for culturing these cell lines. During special treatments, a reduced FCS content is often deployed to reduce cellular proliferation or the content of bioactive compounds. The impact of the reduction of FCS in culture media on the metabolic profile of SH-SY5Y and Neuro2A cells is currently unknown. Using an Amplex Red Assay, this study showed that the consumption of L-glutamine decreased after FCS reduction. Glucose and pyruvate consumption increased in both cell lines after the reduction of FCS. Thus, lactate production also increased with reduced FCS concentration. The reduction of FCS in the cell culture medium resulted in a reduced aerobic ATP production for SH-SY5Y cells and a complete shut down of aerobic ATP production for Neuro2A cells, measured using the Seahorse XF Real-Time ATP Rate Assay. Utilizing the Seahorse XF Glutamine Oxidation Stress Test, Neuro2A cells showed an increased utilization of L-glutamine oxidation after reduction of FCS. These results indicate that changes in FCS concentration in culture media have an impact on the different energy production strategies of SH-SY5Y and Neuro2A cells which must be considered when planning special treatments. Full article

The investigation of adipose tissue-derived mesenchymal stem cells (ASCs) has received considerable interest in regenerative medicine. A nontoxic adipogenic induction protocol valid for cells of different mammalian species has not been described. This study aims to establish an adipogenic differentiation protocol suitable for horses, sheep, dogs, murines, and human cells. An optimized rosiglitazone protocol, consisting of 5% fetal calf serum in Dulbecco’s Modified Eagle’s Medium, 10 μg/mL insulin, 0.55 μg/mL transferrin, 6.8 ng sodium selenite, 1 μM dexamethasone, and 1–5 μM of rosiglitazone, is compared to the 3-isobutyl-1-methylxantine (IBMX) protocol, where rosiglitazone was replaced with 0.5 mM IBMX and 0.2 mM indomethacin. Cell viability, cytotoxicity, a morphometric analysis of the lipid, and the expression of adipogenic markers for 14 days were assessed. The data revealed that using 5 µM of rosiglitazone promotes the adipogenic differentiation capacity in horse, sheep, and dog cells compared to IBMX induction. Meanwhile, marked reductions in the cell viability and cell number with the IBMX protocol were detected, and rosiglitazone increased the cell number and lipid droplet size, prevented apoptosis, and upregulated FABP-4 and Leptin expression in the cells of most of the species. Our data revealed that the rosiglitazone protocol improves the adipogenesis of ASCs, together with having less toxicity, and should be considered for cell reproducibility and clinical applications targeting obesity. Full article

Chronic kidney disease (CKD) patients undergoing dialysis are at high risk of bone fractures. CKD-induced mineral and bone disorder is extended to periodontal disease due to changes in the ionic composition of saliva in CKD patients, dysregulating mineralization, hindering regeneration and thereby promoting the progression of dental complications. Despite the importance of cementum for overall oral health, the mechanisms that regulate its development and regeneration are not well comprehended, and a lack of sufficient in vitro experimental models has hindered research progress. In this study, the impact of experimental conditions on the calcification of cementoblasts was systematically investigated, aimed at establishing a standardized and validated model for the calcification of cementoblasts. The effects of phosphate, calcium, ascorbic acid, β-glycerolphosphate, dexamethasone, and fetal calf serum on the calcification process of cementoblasts were analyzed over a wide range of concentrations and time points by investigating calcium content, cell viability, gene expression and kinase activity. Cementoblasts calcified in a concentration- and time-dependent manner with higher concentrations of supplements cause a higher degree of calcification but decreased cell viability. Phosphate and calcium have a significantly stronger effect on cementoblast calcification processes compared to osteogenic supplements: ascorbic acid, β-glycerolphosphate, and dexamethasone induce calcification over a wide range of osteogenic signalling pathways, with osteopontin being a central target of gene regulation. Conversely, treatment with ascorbic acid, β-glycerolphosphate, and dexamethasone leads to activating only selected pathways, especially promoting bone sialoprotein expression. The developed and validated cementoblast calcification protocol, incubating up to 60% confluent cementoblasts with 1.9 mmol L⁻¹ of phosphate supplementation for a reasonable, multi-pathway calcification induction and 10 mmol L⁻¹ β-glycerolphosphate, 75 µmol L⁻¹ ascorbic acid and 10 nmol L⁻¹ dexamethasone for a reasonable osteogenic differentiation-based calcification induction, provides standard in vitro experimental models for better understanding cementoblast function and regeneration. Full article

The augmentation of adipocytes in the adipose tissues brings disordered pathophysiological conditions, including type 2 diabetes, hyperlipidemia, hypertension, cardiovascular disease, and cancer. The phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) prevents oxidative stress as radical scavenging in cells. However, the role of the disorder as a pharmacologic factor has been poorly understood. This study elucidates the regulatory effects of DHMBA on adipogenesis in mouse 3T3-L1 adipocytes in vitro. The 3T3-L1 preadipocytes were cultured in DMEM containing 10% calf fetal serum in the presence of DHMBA. Culturing with DHMBA repressed the growth of 3T3-L1 preadipocytes cultured in a medium without differentiation factors. Interestingly, when 3T3-L1 preadipocytes were cultured in a medium including differentiation factors containing insulin, DHMBA did not affect the number of cells with the differentiation process of adipogenesis. Culturing with DHMBA (1, 10, or 100 μM) inhibited lipid accumulation in adipocytes and repressed adipogenesis in 3T3-L1 cells. The potent inhibitory effects of DHMBA on adipogenesis were seen at the later stage of culture. Adipogenesis was inhibited by the presence of wortmannin, PD98059, or Bay 11-7082, which are inhibitors of pathways related to insulin signaling pathway. Notably, the suppressive effects of DHMBA on adipogenesis were expressed by the presence of these inhibitors. DHMBA treatment declined the levels of PPARy and C/EBPα related to preadipocyte differentiation and PI3 kinase 100α, Akt, MAPK, phosphor-MAPK, and mTOR implicated in the insulin signaling pathway, leading to adipogenesis promotion. Thus, DHMBA may inhibit adipogenesis via regulating diverse signaling pathways, providing a new strategy for the therapy of obesity. Full article

We evaluated the suitability of 2% human platelet lysate medium (2%HPL) as a replacement for 2% fetal bovine serum medium (2%FBS) for the xeno-free organ culture of human donor corneas. A total of 32 corneas from 16 human donors were cultured in 2%FBS for 3 days (TP1), then evaluated using phase contrast microscopy (endothelial cell density (ECD) and cell morphology). Following an additional 25-day culture period (TP2) in either 2%FBS or 2%HPL, the pairs were again compared using microscopy; then stroma and Descemet membrane/endothelium (DmE) were processed for next generation sequencing (NGS). At TP2 the ECD was higher in the 2%HPL group (2179 ± 288 cells/mm²) compared to 2%FBS (2113 ± 331 cells/mm²; p = 0.03), and endothelial cell loss was lower (ECL HPL = −0.7% vs. FBS = −3.8%; p = 0.01). There were no significant differences in cell morphology between TP1 and 2, or between 2%HPL and 2%FBS. NGS showed the differential expression of 1644 genes in endothelial cells and 217 genes in stromal cells. It was found that 2%HPL led to the upregulation of cytoprotective, anti-inflammatory and anti-fibrotic genes (HMOX1, SERPINE1, ANGPTL4, LEFTY2, GADD45B, PLIN2, PTX3, GFRA1/2), and the downregulation of pro-inflammatory/apoptotic genes (e.g., CXCL14, SIK1B, PLK5, PPP2R3B, FABP5, MAL, GATA3). 2%HPL is a suitable xeno-free substitution for 2%FBS in human cornea organ culture, inducing less ECL and producing potentially beneficial alterations in gene expression. Full article

To combine the excellent transfection properties of lipids with the high stability of polymeric nanoparticles, we designed a hybrid system with a polymeric core surrounded by a shell of different lipids. The aim is to use this technology for skin vaccination purposes where the transfection of dendritic cells is crucial. Based on a carrier made of PLGA and the positively charged lipid DOTMA, we prepared a panel of nanocarriers with increasing amounts of the zwitterionic phospholipid DOPE in the lipid layer to improve their cell tolerability. We selected a nomenclature accordingly with numbers in brackets to represent the used mol% of DOPE and DOTMA in the lipid layer, respectively. We loaded mRNA onto the surface and assessed the mRNA binding efficacy and the degree of protection against RNases. We investigated the influence of the lipid composition on the toxicity, uptake and transfection in the dendritic cell line DC 2.4 challenging the formulations with different medium supplements like fetal calf serum (FCS) and salts. After selecting the most promising candidate, we performed an immune stimulation assay with primary mouse derived dendritic cells. The experiments showed that all tested lipid–polymer nanoparticles (LPNs) have comparable hydrodynamic parameters with sizes between 200 and 250 nm and are able to bind mRNA electrostatically due to their positive zetapotential (20–40 mV for most formulations). The more of DOPE we add, the more free mRNA we find and the better the cellular uptake reaching approx. 100% for LPN(60/40)–LPN(90/10). This applies for all tested formulations leading to LPN(70/30) with the best performance, in terms of 67% of live cells with protein expression. In that case, the supplements of the medium did not influence the transfection efficacy (56% vs. 67% (suppl. medium) for live cells and 63% vs. 71% in total population). We finally confirmed this finding using mouse derived primary immune cells. We can conclude that a certain amount of DOTMA in the lipid coating of the polymer core is essential for complexation of the mRNA, but the zwitterionic phospholipid DOPE is also important for the particles’ performance in supplemented media. Full article

Conventional liposomes often lack stability, limiting their applicability and usage apart from intravenous routes. Nevertheless, their advantages in drug encapsulation and physicochemical properties might be helpful in oral and pulmonary drug delivery. This study investigated the feasibility and stability of liposomes containing tetraether lipids (TEL) from Thermoplasma acidophilum. Liposomes composed of different molar ratios of TEL:Phospholipon 100H (Ph) were produced and exposed to various temperature and pH conditions. The effects on size, polydispersity index, and zeta potential were examined by dynamic and electrophoretic light scattering. Autoclaving, which was considered an additional process step after fabrication, could minimize contamination and prolong shelf life, and the stability after autoclaving was tested. Moreover, 5(6)-carboxyfluorescein leakage was measured after incubation in the presence of fetal calf serum (FCS) and lung surfactant (Alveofact). The incorporation of TEL into the liposomes significantly impacted the stability against low pH, higher temperatures, and even sterilization by autoclaving. The stability of liposomes containing TEL was confirmed by atomic force microscopy as images revealed similar sizes and morphology before and after incubation with FCS. It could be concluded that increasing the molar ratio in the TEL:Ph liposome formulations improved the structural stability against high temperature, low pH, sterilization via autoclaving, and the presence of FCS and lung surfactant. Full article

The intrinsic healing following tendon injury is ideal, in which tendon progenitor cells proliferate and migrate to the injury site to directly bridge or regenerate tendon tissue. However, the mechanism determining why and how those cells are attracted to the injury site for tendon healing is not understood. Since the tenocytes near the injury site go through apoptosis or necrosis following injury, we hypothesized that secretions from injured tenocytes might have biological effects on cell proliferation and migration to enhance tendon healing. Tenocyte apoptosis was induced by 24 h cell starvation. Apoptotic body-rich media (T-ABRM) and apoptotic body-depleted media (T-ABDM) were collected from culture media after centrifuging. Tenocytes and bone marrow-derived stem cells (BMDSCs) were isolated and cultured with the following four media: (1) T-ABRM, (2) T-ABDM, (3) GDF-5, or (4) basal medium with 2% fetal calf serum (FCS). The cell activities and functions were evaluated. Both T-ABRM and T-ABDM treatments significantly stimulated the cell proliferation, migration, and extracellular matrix synthesis for both tenocytes and BMDSCs compared to the control groups (GDF-5 and basal medium). However, cell proliferation, migration, and extracellular matrix production of T-ABRM-treated cells were significantly higher than the T-ABDM, which indicates the apoptotic bodies are critical for cell activities. Our study revealed the possible mechanism of the intrinsic healing of the tendon in which apoptotic bodies, in the process of apoptosis, following tendon injury promote tenocyte and stromal cell proliferation, migration, and production. Future studies should analyze the components of the apoptotic bodies that play this role, and, thus, the targeting of therapeutics can be developed. Full article

In vitro maturation (IVM) of mammalian oocytes, which influences subsequent in vitro development of embryos, is affected by the macromolecule content in culture media for the success of oocyte maturation competence, in which the cytoplasmic and nuclear reprogramming events occur. The insulin-like growth factor family (IGFs) promotes the maturation of bovine oocytes and the expansion of cumulus cells and also inhibits apoptosis. This study was, therefore, designed to examine the effects of macromolecules (bovine serum albumin, BSA; fetal calf serum, FCS; and polyvinyl alcohol, PVA) on in vitro nuclear maturation, total cellular protein, glutathione peroxidase (GPx) enzyme activity, and the gene expression level of IGF1, IGF2, and their receptor in bovine oocytes. Oocytes obtained from bovine ovaries were cultured in bicarbonate-buffered medium 199 supplemented with 4 mg/mL BSA, 10% FCS, 1 mg/mL PVA, and without macromolecule supplement (control) during 22 h in the air with a humidified atmosphere and 5% CO₂ at 38.5 °C temperature. Supplementation of BSA and FCS increased (χ² = 9.84; p < 0.05) the percentages of oocytes that reached metaphase II compared to the control and PVA. The amount of protein per ml of cell extracts of oocytes matured in FCS supplemented culture media was higher (p < 0.05) than the oocytes in the PVA and control. The levels of GPx enzyme activity in cell extracts isolated from oocytes in each experimental group did not change over time, but the GPx enzyme activity in oocytes matured in PVA-supplemented culture media was lower (p < 0.05) than in oocytes in the other experimental groups. Transcript for the IGF1 gene was not detected in all experimental groups, but the supplementation of BSA and FCS significantly elevated the transcript level of the IGF2 gene. In addition, the maturation of oocytes with BSA-supplemented media increased the transcript level of the IGF1R gene, whereas the transcript level of the IGF2R gene was similar among macromolecule supplementation groups. The current study concluded that BSA and FCS could improve in vitro bovine oocyte development due to supporting nuclear maturation and increasing the total cellular protein content, GPx enzyme, and transcript activity. Full article