In vitro anti-tumoral and immunomodulatory effects of acidic polysaccharides isolated from the fungus Gloeosoma mirabile.

Polysaccharides from wood-rooting fungi have attracted attention due to their broad pharmacological properties. Herein, we report the antitumor and immunomodulatory activities of acid polysaccharides isolated from fungi Gloeosoma mirabile. The polysaccharide extracts displayed significant antiproliferative activity against cancer cell lines (MCF-7, HCT-116, U-937) in a dose-dependent manner and induction of IL-6 in macrophage RAW 264.7. Furthermore, flow cytometry analysis showed that high polysaccharide concentrations induced apoptosis by 83% in HL-60 cells. Based on gas chromatography-mass spectrometry (GC-MS) and Fourier transform infra-red (FT-IR) spectroscopy studies, acidic polysaccharides from G. mirabile were mainly composed of arabinose, α-D-galactopyranose and methyl ß-D-galactopyranoside.

Antioxidant Responses and Phytochemical Accumulation in Raphanus Species Sprouts through Elicitors and Predictive Models under High Temperature Stress.

Crop production is being impacted by higher temperatures, which can decrease food yield and pose a threat to human nutrition. In the current study, edible and wild radish sprouts were exposed to elevated growth temperatures along with the exogenous application of various elicitors to activate defense mechanisms. Developmental traits, oxidative damage, glucosinolate and anthocyanin content, and antioxidant capacity were evaluated alongside the development of a predictive model. A combination of four elicitors (citric acid, methyl jasmonate-MeJa, chitosan, and K2SO4) and high temperatures were applied. The accumulation of bioactives was significantly enhanced through the application of two elicitors, K2SO4 and methyl jasmonate (MeJa). The combination of high temperature with MeJa prominently activated oxidative mechanisms. Consequently, an artificial neural network was developed to predict the behavior of MeJa and temperature, providing a valuable projection of plant growth responses. This study demonstrates that the use of elicitors and predictive analytics serves as an effective tool to investigate responses and enhance the nutritional value of Raphanus species sprouts under future conditions of increased temperature.

Biosynthesis and function of 7-deazaguanine derivatives in bacteria and phages.

SUMMARYDeazaguanine modifications play multifaceted roles in the molecular biology of DNA and tRNA, shaping diverse yet essential biological processes, including the nuanced fine-tuning of translation efficiency and the intricate modulation of codon-anticodon interactions. Beyond their roles in translation, deazaguanine modifications contribute to cellular stress resistance, self-nonself discrimination mechanisms, and host evasion defenses, directly modulating the adaptability of living organisms. Deazaguanine moieties extend beyond nucleic acid modifications, manifesting in the structural diversity of biologically active natural products. Their roles in fundamental cellular processes and their presence in biologically active natural products underscore their versatility and pivotal contributions to the intricate web of molecular interactions within living organisms. Here, we discuss the current understanding of the biosynthesis and multifaceted functions of deazaguanines, shedding light on their diverse and dynamic roles in the molecular landscape of life.

Elephant Black Garlic's Beneficial Properties for Hippocampal Neuronal Network, Chemical Characterization and Biological Evaluation.

Garlic has been used for decades as an important food and additionally for its beneficial properties in terms of nutrition and ancestral therapeutics. In this work, we compare the properties of fresh (WG) and aged (BG) extract obtained from elephant garlic, harvested on Chiloe Island, Chile. BG was prepared from WG with a 20-day aging process under controlled temperature and humidity conditions. We observed that in BG, compounds such as diallyl disulfide decrease, and compounds of interest such as 5-hydroxymethylfurfural (69%), diallyl sulfide (17%), 3H-1,2-Dithiole (22%) and 4-Methyl-1,2,3-trithiolane (16%) were shown to be increased. Using 2,2-diphenyl-1-picrylhydrazyl (DPPH, BG: 51 ± 5.7%, WG: 12 ± 2.6%) and 2,20-azino-bis-(3-ethylbenzothiazoline-6 sulfonate) diammonium salt (ABTS, BG: 69.4 ± 2.3%, WG: 21 ± 3.9%) assays, we observed that BG possesses significantly higher antioxidant activity than WG and increased cell viability in hippocampal slices (41 ± 9%). The effects of WG and BG were shown to improve the neuronal function through an increased in intracellular calcium transients (189 ± 4%). In parallel, BG induced an increase in synaptic vesicle protein 2 (SV-2; 75 ± 12%) and brain-derived neurotrophic factor (BDNF; 32 ± 12%) levels. Thus, our study provides the initial scientific bases to foster the use of BG from Chiloe Island as a functional food containing a mixture of bioactive compounds that may contribute to brain health and well-being.

Nanocarrier of α-Tocopheryl Succinate Based on a Copolymer Derivative of (4,7-dichloroquinolin-2-yl)methanol and Its Cytotoxicity against a Breast Cancer Cell Line.

In order to improve the water solubility and, therefore, bioavailability and therapeutic activity of anticancer hydrophobic drug α-tocopherol succinate (α-TOS), in this work, copolymers were synthesized via free radicals from QMES (1-[4,7-dichloroquinolin-2-ylmethyl]-4-methacryloyloxyethyl succinate) and VP (N-vinyl-2-pirrolidone) using different molar ratios, and were used to nanoencapsulate and deliver α-TOS into cancer cells MCF-7. QMES monomer was chosen because the QMES pendant group in the polymer tends to hydrolyze to form free 4,7-dichloro-2-quinolinemethanol (QOH), which also, like α-TOS, exhibit anti-proliferative effects on cancerous cells. From the QMES-VP 30:70 (QMES-30) and 40:60 (QMES-40) copolymers obtained, it was possible to prepare aqueous suspensions of empty nanoparticles (NPs) loaded with α-TOS by nanoprecipitation. The diameter and encapsulation efficiency (%EE) of the QMES-30 NPs loaded with α-TOS were 128.6 nm and 52%; while for the QMES-40 NPs loaded with α-TOS, they were 148.8 nm and 65%. The results of the AlamarBlue assay at 72 h of treatment show that empty QMES-30 NPs (without α-TOS) produced a marked cytotoxic effect on MCF-7 breast cancer cells, corresponding to an IC50 value of 0.043 mg mL-1, and importantly, they did not exhibit cytotoxicity against healthy HUVEC cells. Furthermore, NP-QMES-40 loaded with α-TOS were cytotoxic with an IC50 value of 0.076 mg mL-1, demonstrating a progressive release of α-TOS; however, the latter nanoparticles were also cytotoxic to healthy cells in the range of the assayed concentrations. These results contribute to the search for a new polymeric nanocarrier of QOH, α-TOS or other hydrophobic drugs for the treatment of cancer or others diseases treatable with these drugs.

Comparison of serum creatinine, point-of-care symmetric dimethylarginine and renal imaging with glomerular filtration rate measured by renal scintigraphy in healthy and early chronic kidney diseased cats.

The aim of this study was to evaluate routinely used tests to diagnose cats in early stages of chronic kidney disease (CKD) and to describe a model for evaluating these variables simultaneously. Apparently healthy cats were screened using serum creatinine (sCr), point-of-care symmetric dimethylarginine (POC SDMA), urinalysis, urine protein/creatinine ratio (UPC) and imaging evaluation. Those parameters were compared to glomerular filtration rate (GFR) assessed by renal scintigraphy. Forty-four cats were included and consisted of 14 (31.8%) healthy cats (absence of abnormalities in renal morphology and sCr less than 1.6 mg/dL), 20 (45.5%) cats classified as CKD I (presence of abnormalities in renal morphology and sCr less than 1.6 mg/dL) and ten (22.7%) as CKD II (sCr equal to or greater than 1.6 mg/dL, with or without abnormalities in renal morphology). A large number (40.9%) of apparently healthy cats presented reduction in GFR, which included half of CKD I patients. Point-of-care SDMA was not a good predictor for decreased GFR, nor was it correlated with the variables GFR and sCr. Glomerular filtration rate was significantly lower in CKD I and II groups in comparison with healthy cats, but there was no significant difference between the CKD I and II groups. Multivariate logistic regression model identified three variables that affected the odds of a cat having decreased GFR (< 2.5 mL/min/kg): sCr (OR = 18.3; p = 0.019; CI = 1.6-207.2), and the ultrasonographic findings 'reduced corticomedullary definition' (OR = 19.9; p = 0.022; CI = 1.6-254.0) and 'irregular contour' (OR = 65.6; p = 0.003; CI = 4.2-1038.2). Renal ultrasonography evaluation should always be considered for screening early CKD in apparently healthy cats.

Influence of Constant Rate Infusions of Fentanyl Alone or in Combination With Lidocaine and Ketamine on the Response to Surgery and Postoperative Pain in Isoflurane Anesthetized Dogs Undergoing Unilateral Mastectomy: A Randomized Clinical Trial.

The aim of this study was to compare the effects of constant rate infusions (CRI) of fentanyl alone or combined with lidocaine and ketamine (FLK), on physiological parameters, isoflurane requirements and the number of postoperative analgesic rescues in dogs undergoing unilateral mastectomy. Twenty-two dogs were premedicated with acepromazine 0.02 mg/kg and morphine 0.5 mg/kg and anesthetized with propofol and isoflurane. Dogs were randomly assigned to 1 of 2 groups: Fentanyl group (fentanyl 5 µg/kg loading dose [LD] and 9 µg/kg/h CRI; n = 11); FLK group (fentanyl [same doses]; lidocaine 2 mg/kg LD and 3 mg/kg/h CRI; ketamine 1.0 mg/kg LD and 0.6 mg/kg/h CRI; = 11). Intraoperative evaluations were performed before the start of surgery and administration of the treatments (T0); three minutes after the LD (T1); during incision and tissue divulsion (T2); during closure of the surgical wound (T3). Meloxicam (0.1 mg/kg) was administered at T3. Blood samples were collected for determination of plasma concentrations of fentanyl, lidocaine and ketamine. Pain scores and the number of postoperative analgesic rescues with morphine (0.5 mg/kg) were evaluated for 24 hours postoperatively using the short form of the Glasgow Composite Measure Pain Scale. Compared to T0, significant decreases in heart rate (from 84 ± 28 to 53 ± 16 bpm in the Fentanyl group and from 93 ± 16 to 63 ± 15 bpm in FLK) and mean arterial pressure (from 61 ± 5 to 49 ± 10 mmHg in Fentanyl and from 59 ± 3 to 38 ± 6 mmHg in FLK) were observed at T1. Arterial hypotension was transient, with normalization of values at T2 and T3. The expired fraction of isoflurane did not differ significantly between the groups. Plasma concentrations of fentanyl, lidocaine and ketamine remained within the therapeutic range. Postoperatively, the number of dogs requiring analgesic rescue was significantly lower in the FLK (0/11, 0%) than in the Fentanyl group (5/11, 45%). In dogs administered morphine and meloxicam as part of the anesthesia protocol, an intraoperative CRI of FLK abolished the requirement for postoperative analgesic rescue for 24 hours in dogs undergoing mastectomy.

Phytotoxic activity of Ulex europaeus, an invasive plant on Chilean ecosystems: separation and identification of potential allelochemicals.

Despite its worldwide relevance as an invasive plant, there are few studies on Ulex europaeus (gorse) and its allelopathic activity is almost unexplored. The allelochemical profile of gorse was analysed through methanolic extract of pods and roots, and its phytotoxic effects on Lactuca sativa germination. The methanolic extract of pods had no effect in germination, while extract of roots resulted in a U-shaped dose-response curve: reducing the germination at concentration 0.5 mg mL-1. GC-MS analysis detected compounds with proven antimicrobial and antioxidant activities in the pods and cytotoxic compounds in the roots, which could explain the bioassay results. The quinolizidine alkaloids (QAs) composition was evaluated to predict possible biological functions. It showed the presence of QAs in gorse that are absent in their native range, indicating broad defense strategies against bacteria, fungi, plants, and insects in the Chilean ecosystem. This could explain the superiority of gorse in the invaded areas.

Synthesis, characterisation, crystal structure and antimicrobial evaluation of novel 6-alkoxyergosta-4,6,8(14),22-tetraen-3-one derived from natural ergosta-5,7,22-trien-3ß-ol.

In this study, we report a facile transformation starting from 5α-hydroxyergosta-7,22-dien-3,6-dione (1) to afford two novel compounds: 6-methoxyergosta-4,6,8(14),22-tetraen-3-one (2) and 6-ethoxyergosta-4,6,8(14),22-tetraen-3-one (3) using alcoholic acid catalysis. Their structures were elucidated using NMR experiments, FT-IR, MS and X-ray analysis. These compounds were evaluated for antibacterial activity using the disk and broth diffusion test. In those tests, compound 3 was found to be the most significant antibacterial agent. In general, compounds 1-3 showed inhibition zone in the range of 7.00-12.3 mm for S. aureus and S. mutans, meanwhile for Gram-negative bacteria E. coli and Pseudomonas sp. was found to be in the range of 7.00-8.00 mm. For the most active, compound 3, MIC was significantly lower than that reported for ergosterol, in a value of 160 µg/mL. Overall, these compounds were more active than their natural precursor.

Bromination of eudesmin isolated from araucaria araucana induces epimerization and give bromine derivatives with loss of anti-Candida activity.

Furofuran lignanes show important biological activities for the treatment of infectious diseases, inflammatory and metabolic pathologies. They have been isolated from leaves and barks of many plants. In Chile the native conifer Araucaria araucana produces eudesmin, matairesinol, secoisolariciresinol and lariciresinol in stemwood, branchwood and knotwood. These compounds were previously isolated by laborious flash chromatography on silica gel. Here we report the easy isolation of eudesmin by soxhlet extraction from milled knots of Araucaria araucana with hexane, followed by cryo-crystallization at -20 °C. Upon bromination of the isolated eudesmin epimerization at one benzylic position occurs, giving epieudesmin and the corresponding mono and di-brominated derivatives. The structures were determined by 1D, 2D NMR and X-ray diffraction. The analysis of products against Candida yeast showed that eudesmin has a moderate activity against different strains of Candida from 62.5 to 500 µg/mL. This activity decreases for epieudesmin, while bromine derivatives are not active.

In vitro cytotoxic capacity against tumor cell lines and antioxidant activity of acidic polysaccharides isolated from the Andean Patagonian fungus Phylloporia boldo.

Fungal polysaccharides possess a broad biological activity, including cytotoxic and antioxidant activities. This work aimed to evaluate the cytotoxic and antioxidant activity of the acidic polysaccharides of Phylloporia boldo strain (named PBAP40). Cytotoxic activity of polysaccharide was evaluated determining the viability of three tumor cell lines by MTT assay. The effect of acidic polysaccharide on the cell cycle of HL-60 cell line was evaluated by flow cytometry, and the antioxidant activity was determined by DPPH and ABTS assays. PBAP40 showed cytotoxic effects in tumor cell lines. Results suggest that P. boldo acidic polysaccharides arrested tumor cells in the cell cycle Sub G1 phase. The acidic polysaccharides of PBAP40 strain were not cytotoxic for the non-tumor cell line. PBAP40 also showed excellent antioxidant activity. The FT-IR analysis of the acidic polysaccharides indicated the presence of glucans bearing α- and ß- type glycosidic bonds.

Neuroprotective Properties of Eudesmin on a Cellular Model of Amyloid-ß Peptide Toxicity.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive impairment and memory loss. One of the hallmarks in AD is amyloid-ß peptide (Aß) accumulation, where the soluble oligomers of Aß (AßOs) are the most toxic species, deteriorating the synaptic function, membrane integrity, and neuronal structures, which ultimately lead to apoptosis. Currently, there are no drugs to arrest AD progression, and current scientific efforts are focused on searching for novel leads to control this disease. Lignans are compounds extracted from conifers and have several medicinal properties. Eudesmin (Eu) is an extractable lignan from the wood of Araucaria araucana, a native tree from Chile. This metabolite has shown a range of biological properties, including the ability to control inflammation and antibacterial effects. OBJECTIVE: In this study, the neuroprotective abilities of Eu on synaptic failure induced by AßOs were analyzed. METHODS: Using neuronal models, PC12 cells, and in silico simulations we evaluated the neuroprotective effect of Eu (30 nM) against the toxicity induced by AßOs. RESULTS: In primary cultures from mouse hippocampus, Eu preserved the synaptic structure against AßOs toxicity, maintaining stable levels of the presynaptic protein SV2 at the same concentration. Eu also averted synapsis failure from the AßOs toxicity by sustaining the frequencies of cytosolic Ca2+ transients. Finally, we found that Eu (30 nM) interacts with the Aß aggregation process inducing a decrease in AßOs toxicity, suggesting an alternative mechanism to explain the neuroprotective activity of Eu. CONCLUSION: We believe that Eu represents a novel lead that reduces the Aß toxicity, opening new research venues for lignans as neuroprotective agents.

Cellular stress modulates severity of the inflammatory response in lungs via cell surface BiP.

Inflammation is a central pathogenic feature of the acute respiratory distress syndrome (ARDS) in COVID-19. Previous pathologies such as diabetes, autoimmune or cardiovascular diseases become risk factors for the severe hyperinflammatory syndrome. A common feature among these risk factors is the subclinical presence of cellular stress, a finding that has gained attention after the discovery that BiP (GRP78), a master regulator of stress, participates in the SARS-CoV-2 recognition. Here, we show that BiP serum levels are higher in COVID-19 patients who present certain risk factors. Moreover, early during the infection, BiP levels predict severe pneumonia, supporting the use of BiP as a prognosis biomarker. Using a mouse model of pulmonary inflammation, we observed increased levels of cell surface BiP (cs-BiP) in leukocytes during inflammation. This corresponds with a higher number of neutrophiles, which show naturally high levels of cs-BiP, whereas alveolar macrophages show a higher than usual exposure of BiP in their cell surface. The modulation of cellular stress with the use of a clinically approved drug, 4-PBA, resulted in the amelioration of the lung hyperinflammatory response, supporting the anti-stress therapy as a valid therapeutic strategy for patients developing ARDS. Finally, we identified stress-modulated proteins that shed light into the mechanism underlying the cellular stress-inflammation network in lungs.

Traditional Medicinal Plants as a Source of Inspiration for Osteosarcoma Therapy.

Osteosarcoma is one of the most common types of bone cancers among paediatric patients. Despite the advances made in surgery, chemo-, and radiotherapy, the mortality rate of metastatic osteosarcoma remains unchangeably high. The standard drug combination used to treat this bone cancer has remained the same for the last 20 years, and it produces many dangerous side effects. Through history, from ancient to modern times, nature has been a remarkable source of chemical diversity, used to alleviate human disease. The application of modern scientific technology to the study of natural products has identified many specific molecules with anti-cancer properties. This review describes the latest discovered anti-cancer compounds extracted from traditional medicinal plants, with a focus on osteosarcoma research, and on their cellular and molecular mechanisms of action. The presented compounds have proven to kill osteosarcoma cells by interfering with different pathways: apoptosis induction, stimulation of autophagy, generation of reactive oxygen species, etc. This wide variety of cellular targets confer natural products the potential to be used as chemotherapeutic drugs, and also the ability to act as sensitizers in drug combination treatments. The major hindrance for these molecules is low bioavailability. A problem that may be solved by chemical modification or nano-encapsulation.

Sulfated Polysaccharide Extracted from the Green Algae Codium bernabei: Physicochemical Characterization and Antioxidant, Anticoagulant and Antitumor Activity.

Codium bernabei is a green alga that grows on Chilean coasts. The composition of its structural polysaccharides is still unknown. Hence, the aim of this work is to isolate and characterize the hot water extracted polysaccharide fractions. For this purpose, the water extracts were further precipitated in alcohol (TPs) and acid media (APs), respectively. Both fractions were characterized using different physicochemical techniques such as GC-MS, GPC, FTIR, TGA, and SEM. It is confirmed that the extracted fractions are mainly made of sulfated galactan unit, with a degree of sulfation of 19.3% (TPs) and 17.4% (ATs) and a protein content of 3.5% in APs and 15.6% in TPs. Other neutral sugars such as xylose, glucose, galactose, fucose, mannose, and arabinose were found in a molar ratio (0.05:0.6:1.0:0.02:0.14:0.11) for TPs and (0.05:0.31:1.0:0.03:0.1:0.13) for ATs. The molecular weight of the polysaccharide samples was lower than 20 kDa. Both polysaccharides were thermally stable (Tonset > 190 °C) and showed antioxidant activity according to the ABTS•+ and DPPH tests, where TPs fractions had higher scavenging activity (35%) compared to the APs fractions. The PT and APTTS assays were used to measure the anticoagulant activity of the polysaccharide fractions. In general, the PT activity of the TPs and APs was not different from normal plasma values. The exception was the TPs treatment at 1000 µg mL−1 concentration. The APTTS test revealed that clotting time for both polysaccharides was prolonged regarding normal values at 1000 µg mL−1. Finally, the antitumor test in colorectal carcinoma (HTC-116) cell line, breast cancer (MCF-7) and human leukemia (HL-60) cell lines showed the cytotoxic effect of TPs and APs. Those results suggest the potential biotechnological application of sulfate galactan polysaccharides isolated from a Chilean marine resource.

Evaluation of Adipose Tissue Zinc-Alpha 2-Glycoprotein Gene Expression and Its Relationship with Metabolic Status and Bariatric Surgery Outcomes in Patients with Class III Obesity.

Zinc-α2 glycoprotein (ZAG) is an adipokine involved in adipocyte metabolism with potential implications in the pathogenesis of metabolic disorders. Our aim was to evaluate the relationship between visceral (VAT) and subcutaneous adipose tissue (SAT) ZAG expression and metabolic parameters in patients with class III obesity, along with the impact of basal ZAG expression on short- and medium-term outcomes related to bariatric surgery. 41 patients with class III obesity who underwent bariatric surgery were included in this study. ZAG gene expression was quantified in SAT and VAT. Patients were classified into two groups according to SAT and VAT ZAG percentile. Anthropometric and biochemical variables were obtained before and 15 days, 45 days, and 1 year after surgery. The lower basal SAT ZAG expression percentile was associated with higher weight and waist circumference, while the lower basal VAT ZAG expression percentile was associated with higher weight, waist circumference, insulin, insulin resistance, and the presence of metabolic syndrome. Basal SAT ZAG expression was inversely related to weight loss at 45 days after surgery, whereas no associations were found between basal VAT ZAG expression and weight loss after surgery. Additionally, a negative association was observed between basal SAT and VAT ZAG expression and the decrease of gamma-glutamyl transferase after bariatric surgery. Therefore, lower SAT and VAT ZAG expression levels were associated with an adverse metabolic profile. However, this fact did not seem to confer worse bariatric surgery-related outcomes. Further research is needed to assess the clinical significance of the role of ZAG expression levels in the dynamics of hepatic enzymes after bariatric surgery.

Secretory Profile of Adipose-Tissue-Derived Mesenchymal Stem Cells from Cats with Calicivirus-Positive Severe Chronic Gingivostomatitis.

The feline calicivirus (FCV) causes infections in cats all over the world and seems to be related to a broad variety of clinical presentations, such as feline chronic gingivostomatitis (FCGS), a severe oral pathology in cats. Although its etiopathogeny is largely unknown, FCV infection is likely to be a main predisposing factor for developing this pathology. During recent years, new strategies for treating FCGS have been proposed, based on the use of mesenchymal stem cells (MSC) and their regenerative and immunomodulatory properties. The main mechanism of action of MSC seems to be paracrine, due to the secretion of many biomolecules with different biological functions (secretome). Currently, several pathologies in humans have been shown to be related to functional alterations of the patient's MSCs. However, the possible roles that altered MSCs might have in different diseases, including virus-mediated diseases, remain unknown. We have recently demonstrated that the exosomes produced by the adipose-tissue-derived MSCs (fAd-MSCs) from cats suffering from FCV-positive severe and refractory FCGS showed altered protein contents. Based on these findings, the goal of this work was to analyze the proteomic profile of the secretome produced by feline adipose-tissue-derived MSCs (fAd-MSCs) from FCV-positive patients with FCGS, in order to identify differences between them and to increase our knowledge of the etiopathogenesis of this disease. We used high-resolution mass spectrometry and functional enrichment analysis with Gene Ontology to compare the secretomes produced by the fAd-MSCs of healthy and calicivirus-positive FCGS cats. We found that the fAd-MSCs from cats with FCGS had an increased expression of pro-inflammatory cytokines and an altered proteomic profile compared to the secretome produced by cells from healthy cats. These findings help us gain insight on the roles of MSCs and their possible relation to FCGS, and may be useful for selecting specific biomarkers and for identifying new therapeutic targets.

Nanoscale ligand density modulates gap junction intercellular communication of cell condensates during chondrogenesis.

Aim: To unveil the influence of cell-matrix adhesions in the establishment of gap junction intercellular communication (GJIC) during cell condensation in chondrogenesis. Materials & methods: Previously developed nanopatterns of the cell adhesive ligand arginine-glycine-aspartic acid were used as cell culture substrates to control cell adhesion at the nanoscale. In vitro chondrogenesis of mesenchymal stem cells was conducted on the nanopatterns. Cohesion and GJIC were evaluated in cell condensates. Results: Mechanical stability and GJIC are enhanced by a nanopattern configuration in which 90% of the surface area presents adhesion sites separated less than 70 nm, thus providing an onset for cell signaling. Conclusion: Cell-matrix adhesions regulate GJIC of mesenchymal cell condensates during in vitro chondrogenesis from a threshold configuration at the nanoscale.

Determination of the effective dosage of tiletamine-zolazepam-ketamine-xylazine, with or without methadone, in dogs.

OBJECTIVE: To determine the effective dosage of the combination tiletamine-zolazepam-ketamine-xylazine (TKX), with or without methadone, in dogs. STUDY DESIGN: Prospective, randomized, experimental study. ANIMALS: A total of 29 dogs. METHODS: Dogs were randomly administered TKX (group TKX, n = 13) or combined with 0.3 mg kg-1 of methadone (group TKXM, n = 16) intramuscularly. The TKX solution contained tiletamine (50 mg mL-1), zolazepam (50 mg mL-1), ketamine (80 mg mL-1) and xylazine (20 mg mL-1). The effective dosages for immobility in 50% and 95% of the population (ED50 and ED95) were estimated using the up-and-down method. Approximately 20 minutes after drug administration, a skin incision was performed and the response was judged as positive or negative if the dogs moved or did not move, respectively. The TKX volume for the subsequent dog in the same group was increased or decreased by 0.005 mL kg-1 if the response of the previous dog was positive or negative, respectively. Heart and respiratory rates, and sedation/anesthesia scores (range 0-21) were recorded before and 15 minutes after drug administration. RESULTS: Estimated ED50 and ED95 (95% confidence intervals) were: TKX, 0.025 (0.020-0.029) and 0.026 (0.010-0.042) mL kg-1; TKXM, 0.022 (0.018-0.025) and 0.033 (0.017-0.049) mL kg-1. Median (interquartile range) scores for sedation/anesthesia were 17 (16-18) and 17 (15-20), and times until lateral recumbency were 5 (4-6) and 6 (4-10) minutes in TKX and TKXM, respectively (p > 0.05). In both groups heart and respiratory rates decreased, but values remained acceptable for anesthetized dogs. CONCLUSIONS AND CLINICAL RELEVANCE: The results provide a guide for volumes of TKX and TKXM in dogs requiring restraint for minimally invasive procedures. Inclusion of methadone in the TKX combination did not influence ED50.

Suicide gene therapy by canine mesenchymal stem cell transduced with thymidine kinase in a u-87 glioblastoma murine model: Secretory profile and antitumor activity.

The role played by certain domestic species such as dogs as a translational model in comparative oncology shows great interest to develop new therapeutic strategies in brain tumors. Gliomas are a therapeutic challenge that represents the most common form of malignant primary brain tumors in humans and the second most common form in dogs. Gene-directed enzyme/prodrug therapy using adipose mesenchymal stem cells (Ad-MSCs) expressing the herpes simplex virus thymidine kinase (TK) has proven to be a promising alternative in glioblastoma therapy, through its capacity to migrate and home to the tumor and delivering local cytotoxicity avoiding other systemic administration. In this study, we demonstrate the possibility for canine Ad-MSCs (cAd-MSCs) to be genetically engineered efficiently with a lentiviral vector to express TK (TK-cAd-MSCs) and in combination with ganciclovir (GCV) prodrug demonstrated its potential antitumor efficacy in vitro and in vivo in a mice model with the human glioblastoma cell line U87. TK-cAd-MSCs maintained cell proliferation, karyotype stability, and MSCs phenotype. Genetic modification significantly affects its secretory profile, both the analyzed soluble factors and exosomes. TK-cAd-MSCs showed a high secretory profile of some active antitumor immune response cytokines and a threefold increase in the amount of secreted exosomes, with changes in their protein cargo. We also found that the prodrug protein is not released directly into the culture medium by TK-cAd-MSCs. We believe that our work provides new perspectives for glioblastoma gene therapy in dogs and a better understanding of this therapy in view of its possible implantation in humans.