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Protoplasts are plant cells from which the pectocellulosic cell wall has been removed, thus keeping the plasma membrane intact. For plant secondary metabolites research, this system is a powerful tool to study the metabolites' dynamics inside the cells, such as the subcellular localization of proteins, characterization of gene function, transcription factors involved in metabolite pathways, protein transport machinery, and to perform single-cell omics studies. Due to its lack of a cell wall, better images of the interior of the cell can be obtained compared to the whole tissue. This allows the identification of specific cell types involved in the accumulation of specialized metabolites, such as alkaloids, given their autofluorescence properties. Here is a simplified protocol to obtain protoplasts from leaves and in vitro cell cultures from Argemone mexicana, which produces the pharmacologically important alkaloids berberine and sanguinarine.

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Batoids (rays and skates) are cartilaginous fishes whose jaws are not articulated directly to the neurocranium. The only point of contact between them are the hyomandibular cartilages, resulting in a unique mandibular suspension called euhyostyly. Due to this decoupling of the jaws from the skull, muscles play an essential role in modulating mandibular movements during the feeding process, especially during mandibular protrusion. The main objectives of our study were: (1) to examine the mandibular musculature of eight batoid species from different orders in the Batoidea and (2) establish a standardized musclulature terminology for future comparative myological studies in batoids. For each muscle bundle, the general characteristics of each cranial muscle were described and their origin and insertions were identified. The number of muscle bundles differed intraspecifically. On the dorsal surface, we reported the first evidence of the presence of the precranial muscle (PCM) in U. halleri, as well as the ethmoideo-parethmoidalis muscle (ETM) in R. velezi, P. glaugostigma and Z. exasperata; in addition, the insertion of the spiracularis muscle (SP) extended to the ventral surface of the oropharyngeal tract in myliobatiforms. On the ventral surface of the head, both N. entemedor and M. californica exhibited additional muscles in the mandibular area. These muscles were renamed as part of the standardization of mandibular terminology: the depressor mandibularis minor (DMM) in N. entemedor and the adductor mandibulae profundus (AMP) in M. californica. The standardization of terminology is essential for futures studies of the mandibular apparatus in batoids, to facilitate the morphological description of muscles in species without anatomical accounts and for continuity in broader comparative analyses.

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Coffea arabica L. is a crucial crop globally, but its genetic homogeneity leads to its susceptibility to diseases and pests like the coffee berry borer (CBB). Chemical and cultural control methods are difficult due to the majority of the CBB life cycle taking place inside coffee beans. One potential solution is the use of the gene cyt1Aa from Bacillus thuringiensis as a biological insecticide. To validate candidate genes against CBB, a simple, rapid, and efficient transient expression system is necessary. This study uses cell suspensions as a platform for expressing the cyt1Aa gene in the coffee genome (C. arabica L. var. Catuaí) to control CBB. The Agrobacterium tumefaciens strain GV3101::pMP90 containing the bar and cyt1Aa genes are used to genetically transform embryogenic cell suspensions. PCR amplification of the cyt1Aa gene is observed 2, 5, and 7 weeks after infection. This chapter describes a protocol that can be used for the development of resistant varieties against biotic and abiotic stresses and CRISPR/Cas9-mediated genome editing.

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OBJECTIVE: A retrospective analysis was performed to explore the clinical effect of the Posterior Nasal Nerve (PNN) resection combined with hormone transnasal nebulization on Difficult-to-Treat Rhinosinusitis (DTRS). METHODS: A total of 120 DTRS patients were selected and divided into a control group (n = 60) and a study group (n = 60) according to different treatments. The control group patients were treated via PNN resection, followed by normal saline transnasal nebulization; the study group patients were given PNN resection and then treated with budesonide suspension transnasal nebulization. Subsequently, the comparison was performed between the two groups in terms of (1) Clinical baseline characteristics; (2) Sino-nasal Outcome Test (SNOT)-22 scores before treatment and after 3-months, 6-months and 12-months of treatment; (3) Lund-MacKay scores before treatment and after 10, 30, 90, and 180 days of treatment; (4) Incidence of adverse reactions during treatment. RESULTS: There was no significant difference in SNOT-22 or Lund-Kennedy scores between the two groups before treatment (p > 0.05). After treatment, the SNOT-22 and Lund-Kennedy scores of the control and the study groups were decreased, and compared with the control group, the SNOT-22 and Lund-Kennedy scores in the study group improved more significantly (p < 0.05). In addition, the study group and the control group presented with 1 and 4 cases of nasal adhesion, 2 and 3 cases of epistaxis, 1 and 4 cases of sinus orifice obstruction, 1 and 3 cases of lacrimal duct injuries, respectively. The incidence of adverse reactions in the study group was significantly lower than that in the control group (8.3% vs. 23.3%) (p < 0.05). CONCLUSION: PNN resection combined with hormone transnasal nebulization treatment can improve the symptoms and quality of life of DTRS patients, with good clinical efficacy but few adverse reactions. Therefore, such combination treatment deserves a promotion and application clinically. LEVEL OF EVIDENCE: Level 3.

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Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have shown increasing therapeutic potential in the last years. However, large production of EV is required for therapeutic purposes. Thereby, scaling up MSC cultivation in bioreactors is essential to allow culture parameters monitoring. In this study, we reported the establishment of a scalable bioprocess to produce MSC-EV in suspension cultures using spinner flasks and human collagen-coated microcarriers (3D culture system). We compared the EV production in this 3D culture system with the standard static culture using T-flasks (2D culture system). The EV produced in both systems were characterized and quantify by western blotting and nanoparticle tracking analysis. The presence of the typical protein markers CD9, CD63, and CD81 was confirmed by western blotting analyses for EV produced in both culture systems. The cell fold-increase was 5.7-fold for the 3D culture system and 4.6-fold for the 2D culture system, signifying a fold-change of 1.2 (calculated as the ratio of fold-increase 3D to fold-increase 2D). Furthermore, it should be noted that the total cell production in the spinner flask cultures was 4.8 times higher than that in T-flask cultures. The total cell production in the spinner flask cultures was 5.2-fold higher than that in T-flask cultures. While the EV specific production (particles/cell) in T-flask cultures (4.40 ± 1.21 × 108 particles/mL, p < 0.05) was higher compared to spinner flask cultures (2.10 ± 0.04 × 108 particles/mL, p < 0.05), the spinner flask culture system offers scalability, making it capable of producing enough MSC-EV at a large scale for clinical applications. Therefore, we concluded that 3D culture system evaluated here serves as an efficient transitional platform that enables the scaling up of MSC-EV production for therapeutic purposes by utilizing stirred tank bioreactors and maintaining xeno-free conditions.

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BACKGROUND: Achieving an aesthetically pleasant tip rotation is paramount in rhinoplasty, and these results should be long-lasting. However, even powerful structural techniques such as septal extension grafts can lose rotation over time, as most support depends on the attachment of the graft to the septal cartilage. METHODS: We describe a simple suture technique to strengthen tip support in rhinoplasty, by suspending it to the cartilaginous dorsum, providing additional support to the tip against forces, such as gravity, scar contraction and muscle tension. CONCLUSIONS: Combining traditional techniques with suspension sutures can improve intraoperative results and could enhance long-term tip stability.

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BACKGROUND/AIMS: Abexol is a mixture of primary aliphatic alcohols purified from beeswax (Apis mellifera), that produces anti-inflammatory, antioxidant and gastroprotective effects, as well as it is safe and well tolerated. To investigate and compare the efficacy and safety of Abexol (suspension versus tablets) in patients with gastrointestinal symptoms. METHODS: Monocentric study, open-label, randomized design, with two parallel groups receiving Abexol tablets (150 mg/d) or Abexol suspension (75 mg/d) for 8 weeks. Primary efficacy variable (significant improvement in the total score of Gastrointestinal Symptom Rating Scale [GSRS]). Significant reduction in the intensity of the gastrointestinal-symptoms and the reduction in the consumption of antacids are considered secondary efficacy variable. Short form-36 (SF-36) quality of life questiongenonaire was evaluated as collateral variable. Data were analyzed as per intention to treat. RESULTS: A significantly decrease in the overall score of the survey was observed with respect to the baseline level (p < 0.001) of 81.4% in the Abexol suspension group and 77.9% in the Abexol tablets group. At the end of the trial, most gastrointestinal- symptoms disappeared or reduced significantly. The frequency of consumption of neutralizing antacids was low. The significantly improvement in the perception of the state of health obtained in the Abexol is in correspondence with the improvement achieved in some of the components evaluate in the SF-36 questionnaire. Both treatments were safe and well tolerated. CONCLUSION: Abexol suspension showed efficacy and safety similar to Abexol tablets in patients with gastrointestinal symptoms, but using half the dose.

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The serotonin type 4 receptor (5-HT4R)shows promise as a target for treating major depressive disorder (MDD). Studies have demonstrated that 5-HT4R agonists have a faster antidepressant-like effect compared to conventional medications. Developing drugs that modulate this receptor could lead to faster and more effective MDD treatments. The compound N-(3-(phenylselanyl)prop-2-yn-1-yl)benzamide (SePB) induces an antidepressant-like effect in mice. The present study explored if the 5-HT4R mediates SePB's antidepressant effect. For this, male Swiss mice were treated with GR113808 (0.1 mg/kg, intraperitoneally - i.p.), a 5-HT4R antagonist, and SePB (10 mg/kg, intragastrically - i.g), and then subjected to the tail-suspension test (TST) and open-field test (OFT). In silico tests were conducted to analyze SePB's binding affinity to the 5-HT4R and identify participating amino acid residues. The administration of GR113808 blocked the antidepressant-like effect of SePB in the TST without changing locomotor activity in the OFT. Moreover, SePB exhibited a high binding affinity between the 5-HT4R (-7.9 kcal/mol) and the amino acid residues Leu298, Asp100, Thr97, Arg96, Glu80, Leu81, Cys184, Val185, and Phe186 seem to be important for this interaction. The involvement of the 5-HT4R in the antidepressant-like effect of SePB suggests potential for novel therapies in MDD.

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Purpose: Laparoscopic techniques to treat pelvic organ prolapse are gaining popularity around the globe due to their low recurrence rates and better functional results compared to perineal techniques. However, the optimum surgical procedures are not yet determined. In the current research, we suggest a novel surgical approach, laparoscopic vaginal suspension with suture rectopexy, to treat multiorgan pelvic prolapse. Methods: This prospective cohort trial was conducted from March 2018 to March 2022 and comprised 35 females with multiorgan pelvic organ prolapse with obstructed defecation symptoms. A residual rectal prolapse was still present despite the manual reduction of uterine prolapse. Patients' conditions before and after the operation were monitored regarding the obstructed defecation score, sexual function, need for laxatives, anorectal manometry pressures, anorectal sensations, and recurrence. The mean follow-up duration was one year. Results: Modified Longo score for obstructed defecation significantly decreased at six and twelve months after surgery. Additionally, a significant reduction was reported in the number of patients who needed laxatives at six and twelve months after surgery. Anorectal manometry pre- and post-surgery showed a significant elevation in the mean squeeze pressure and a decline in all rectal sensations. All parameters of the female sexual function scoring system increased postoperatively. No recurrence was reported during follow-up. Conclusion: For multiorgan pelvic prolapse, laparoscopic vaginal suspension combined with suture rectopexy has excellent functional outcomes, minimal morbidity, and low cost. (AU)

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OBJECTIVES: The present study investigated the anti-depressive-like (anti-immobility) effect of a lectin from Moringa oleifera seeds (WSMoL) in mice. METHODS: To evaluate an acute effect, the animals were treated with WSMoL (1, 2, and 4 mg/kg, i.p.) 30 min before the tail suspension test (TST). To investigate the involvement of monoaminergic and nitrergic signaling, the mice were pre-treated with selective antagonists. The role of the WSMoL carbohydrate-recognizing domain (CRD) was verified using previous blockage with casein (0.5 mg/mL). The subacute anti-immobility effect was also evaluated by administering WSMoL (1, 2, and 4 mg/kg, i.p.) once a day for 7 d. Finally, an open field test (OFT) was performed to identify possible interferences of WSMoL on animal locomotory behavior. RESULTS: WSMoL reduced the immobility time of mice in the TST at all doses, and combined treatment with fluoxetine (5 mg/kg, i.p.) and WSMoL (1 mg/kg) was also effective. The CRD appeared to be involved in the anti-immobility effect since the solution of WSMoL (4 mg/kg) pre-incubated with casein showed no activity. The lectin effect was prevented by the pre-treatment of mice with ketanserin, yohimbine, and SCH 23390, thereby demonstrating the involvement of monoaminergic pathways. In contrast, pre-treatment with L-NAME, aminoguanidine, and L-arginine did not interfere with lectin action. WSMoL exhibited a subacute effect in the TST, thereby reducing immobility time and increasing agitation time even on the seventh day. OFT data revealed that the anti-immobility effect was not caused by interference with locomotor behavior. CONCLUSION: WSMoL elicits an anti-depressant-like effect that is dependent on monoaminergic signaling.

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In a previous work, we proposed a vaccine chimeric antigen based on the fusion of the SARS-CoV-2 N protein to the extracellular domain of the human CD40 ligand (CD154). This vaccine antigen was named N-CD protein and its expression was carried out in HEK-293 stably transfected cells, grown in adherent conditions and serum-supplemented medium. The chimeric protein obtained in these conditions presented a consistent pattern of degradation. The immunization of mice and monkeys with this chimeric protein was able to induce a high N-specific IgG response with only two doses in pre-clinical experiments. In order to explore ways to diminish protein degradation, in the present work, the N and N-CD proteins were produced in suspension cultures and serum-free media following transient transfection of the HEK-293 clone 3F6, at different scales, including stirred-tank controlled bioreactors. The results showed negligible or no degradation of the target proteins. Further, clones stably expressing N-CD were obtained and adapted to suspension culture, obtaining similar results to those observed in the transient expression experiments in HEK-293-3F6. The evidence supports transient protein expression in suspension cultures and serum-free media as a powerful tool to produce in a short period of time high levels of complex proteins susceptible to degradation, such as the SARS-CoV-2 N protein.

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Major depressive disorder (MDD) is a debilitating illness that affects millions of people worldwide. Currently available antidepressants often take weeks to months to reach their full effect, which leads to an increased risk of suicidal behavior in patients with MMD. Intranasally, esketamine has emerged as an alternative to current antidepressants because of its rapid onset and long-lasting effects in patients with MDD. Animal models are useful for the initial pharmacological screening and for a better understanding of the mechanisms underlying the effects of new drugs with potential against MDD. There is a lack of data on alternative routes of drug administration, either oral or injectable, that can be used in preclinical studies. This study aimed to test whether ketamine has antidepressant-like effects in mice when administered via nebulization using a low-cost apparatus. When mice whose depressive-like behavior was induced by corticosterone were treated with nebulized ketamine at concentrations of 1.3, 2.6, and 5.2 mg/mL, immobility was reduced by 38.6 %, 62.0 %, and 61.1 %, respectively, in the forced swimming test (FST) and 43.6 %, 42.1 %, and 57.9 %, respectively, in the tail suspension test (TST). When depression-like behavior was induced by dexamethasone, nebulization with ketamine reduced immobility by 79.7 %, 49.2 %, and 44.4 % in the FST and 80.9 %, 71.4 %, and 80.4 %, respectively, in the TST. When depression-like behavior was induced by the association between dexamethasone and unpredictable chronic mild stress (UCMS) exposure, immobility was reduced by 26.1 %, 55.3 %, and 19.1 % in FST. Mice treated with nebulized ketamine did not show significant changes in the distance covered or in the time spent moving in the open field test. The efficacy of intraperitoneal and nebulized ketamine is equivalent, which shows that nebulization can be an alternative inexpensive route of drug administration for behavioral studies in rodents.

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Para el tratamiento de fracturas mandibulares existen protocolos cuyo propósito primordial es la función temprana. Existen diversos motivos por los cuales no se puede ejecutar ciertas técnicas en Venezuela, principalmente por problemas económicos para la adquisición de materiales de osteosíntesis especializados. Por esta razón, se ha recurrido a técnicas quirúrgicas antiguas. Se presenta un estudio con diseño longitudinal de tipo descriptivo, para el reporte de serie de 5 casos clínicos de fracturas de mandíbula, con los siguientes criterios de inclusión: presentar fractura mandibular en pacientes dentados parcialmente con imposibilidad al acceso al material de osteosíntesis del sistema de cargas soportadas. Tratados bajo procedimiento quirúrgico cerrado, la condición de edentulismo parcial confiere a la fractura de mandíbula inestabilidad, dificulta la reducción anatómica, pérdida de dimensión vertical y transversal, las que se recuperan a través de la elaboración de férulas tipo Gunning modificadas. Estas férulas se mantuvieron en posición con alambrados de suspensión ósea tipo circummandibulares y circumzigomáticos, como medios para establecer estabilidad en el tiempo. Se obtuvieron resultados satisfactorios, por lo que, aún hoy en día se pueden plantear como opciones de tratamiento.

For the treatment of mandibular fractures, there are protocols whose primary purpose is early function. However, several reasons prevent the execution of these techniques in Venezuela, mainly due to economic problems regarding the acquisition of specialized osteosynthesis materials. As a result, there is a necessity to resort to old surgical techniques. A study with a descriptive longitudinal design is presented, reporting 5 clinical cases of jaw fractures with the following inclusion criteria: presenting a mandibular fracture in partially dentate patients with impossibility of accessing the osteosynthesis material of the load-bearing system. The treatment conducted under closed surgical procedures. The condition of partial edentulism confers instability to the mandible fracture, hindering anatomical reduction and causing a loss of vertical and transverse dimension. These issues are addressed through the utilization of modified Gunning-type splints, which help in recovery. The splints were maintained in position with circum-mandibular and circum-zygomatic bone suspension wiring as a means to establish stability over time. Satisfactory results were obtained, indicating that these techniques can still be considered as treatment options today.

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Relapsing-remitting multiple sclerosis (RRMS) is an autoimmune neurological disease and is the most common subtype of MS. In addition, it is associated with the development of depression and anxiety. To date, depressive- and anxiety-like behaviours were only studied using models of progressive MS, which causes severe motor alterations. Thus, we sought to standardise the depressive and anxiety-like behaviours in an RRMS model induced by experimental autoimmune encephalomyelitis (RR-EAE) in mice. The RR-EAE model was induced in C57BL/6 female mice using myelin oligodendrocyte glycoprotein (MOG35-55) antigen and Quillaja saponin (Quil A) as an adjuvant. The immunisation of RR-EAE did not induce locomotor alteration but caused relapsing-remitting induction of clinical scores in mice until 35 post-immunization (p.i.). Also, increased levels of tumour necrosis factor alpha (TNF-α), astrocyte marker (GFAP), and microglial markers (IBA-1) were detected in the prefrontal cortex at 35 p.i. of RR-EAE. In the open field test, RR-EAE mice showed decreased time spent at the centre and sniffing behaviour (at days 21 and 34 p.i.). Also, on day 35 p.i. the RR-EAE group spent less time in the open arms and had decreased open-arm entries compared to control mice in the elevated plus maze (EPM) test, confirming the anxiety-like behaviour. At day 36° p.i. in the tail suspension test, mice showed depression-like behaviour with decreased latency time and increased immobility time. Thus, the RR-EAE model mimics the neuroinflammatory and behavioural features of the RRMS, including depression- and anxiety-like symptoms.

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The present study contains an experimental analysis of the vibratory response in a low-cylinder engine motorcycle at varying suspension preloads. Three different speed bumps of varying heights were used to subject the motorcycle to different vibrations. The analysis was carried out in three domains: time, frequency, and time-frequency. A triaxial accelerometer was used to measure the vibrations at the seat of the vehicle. The results indicated that the suspension system became more differentiated as the height of the bumps increased. However, for lower bumps, the action of the three spring preloads studied was quite similar. Quantitatively, only the higher bump showed a significant difference between the set preloads. The spectral distribution revealed that the frequency of interest was below 20 Hz for all the studied cases, which is in the same range of human body natural frequencies. The findings of this research can be utilized to enhance the design of low-cost motorcycles, thereby improving the safety and comfort of their drivers and passengers. This study constitutes a significant step towards developing an affordable system capable of gathering sufficient data to support the creation of evidence-based public health policies and propose new transport industry standards based on field measurements.

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The gastropod Pachyoliva columellaris (formerly Olivella columellaris) is particularly common on many sandy beaches in North Peru. In the studied region north of Paita, Piura province, the species reaches densities of over 80,000 individuals per beach meter. The snails perform tidal migrations throughout the lunar cycle although tidal amplitudes differ four-fold between neap and spring tide. Not all animals complete their tidal migrations, and significant numbers remain above the water line at low tide. Throughout the tidal cycle, the proportion of small individuals is increased near the water line whereas large animals dominate in lower beach zones. I conclude that the sandy beaches north of Paita are well-suited for further studies of P. columellaris and the mechanisms underlying tidal migrations in invertebrates.

El gasterópodo Pachyoliva columellaris (antes Olivella columellaris) es particularmente común en las playas arenosas del norte de Perú. En la región estudiada al norte de Paita, provincia de Piura, la especie alcanza densidades de más de 80.000 individuos por metro de playa. Los caracoles realizan migraciones mareales durante todo el ciclo lunar, aunque las amplitudes de marea difieren cuatro veces entre la marea muerta y la marea viva. No todos los animales completan sus migraciones mareales y un número significativo permanece por encima del nivel del agua en marea baja. Durante todo el ciclo de marea, la proporción de individuos pequeños aumenta cerca del nivel del agua, mientras que los animales grandes dominan en las zonas bajas de la playa. Concluyo que las playas arenosas al norte de Paita son apropiadas para estudios adicionales de P. columellaris y los mecanismos de las migraciones de marea en invertebrados.

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OBJECTIVE: To assess the willingness of patients with infertility to continue with their in vitro fertilization (IVF) treatment during the COVID-19 pandemic. METHODS: This cross-sectional survey was conducted in the reproductive, endocrine, and infertility medicine department (REIMD) at King Fahad Medical City (KFMC), Riyadh, Saudi Arabia. Patients that were planned to undergo IVF treatment at REIMD were contacted and asked about whether they would like to start IVF treatment during the COVID-19 pandemic from August 2020 to August 2021. Data was analyzed using the SPSS version 24. Statistics obtained as means and standard deviations from continuous variables correlated with the Chi-square test and results were considered significant at p≤0.05. RESULTS: Of the 400 participants, 245 (61.25%) were between the ages of 30-39 years. About 42.75% (n=171) of the patients had 6-10 years of infertility, and 18% (n=72) had at least one pregnancy but no living children. While 64.7% (n=259) of the participants responded on the first call, 83% (n=332) agreed to continue their treatment. Of those, 13% (n=43) preferred to book appointments as soon as possible; 29.8% (n=99) preferred booking within three months; while 57.2% (n=190) chose to book after three months. From our sample, 86.8% (n=59) were afraid to contract the virus and the choice to delay the IVF treatment correlated with the patient's age (p<0.001) and duration of infertility (p=0.007). CONCLUSIONS: The COVID-19 pandemic affected IVF treatment courses, and many patients were afraid to be infected during this pandemic.

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Prior to clinical use, extensive in vitro proliferation of human adipose-derived stem cells (ASCs) is required. Among the current options, spinner-type stirred flasks, which use microcarriers to increase the yield of adherent cells, are recommended. Here, we propose a methodology for ASCs proliferation through cell suspension culture using Cultispher-S® microcarriers (MC) under agitation in a spinner flask, with the aim of establishing a system that reconciles the efficiency of cell yield with high viability of the culture during two distinct phases: seeding and proliferation. The results showed that cell adhesion was potentiated under intermittent stirring at 70 rpm in the presence of 10% FBS for an initial cell concentration of 2.4 × 104 cells/mL in the initial 24 h of cultivation. In the proliferation phase, kinetic analysis showed that cell growth was higher under continuous agitation at 50 rpm with a culture medium renewal regime of 50% every 72 h, which was sufficient to maintain the culture at optimal levels of nutrients and metabolites for up to nine days of cultivation, representing an 11.1-fold increase and a maximum cell productivity of 422 cells/mL/h (1.0 × 105 viable cells/mL). ASCs maintained the immunophenotypic characteristics and mesodermal differentiation potential of both cell lines from different donors. The established protocol represents a more efficient and cost-effective method to obtain a high proliferation rate of ASCs in a microcarrier-based system, which is necessary for large-scale use in cell therapy, highlighting that the manipulation of critical parameters optimizes the ASCs production process.

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Ageratina pichichensis, is commonly used in traditional Mexican medicine. In vitro cultures were established from wild plant (WP) seeds, obtaining in vitro plant (IP), callus culture (CC), and cell suspension culture (CSC) with the objective to determine total phenol content (TPC) and flavonoids (TFC), as well as their antioxidant activity by DPPH, ABTS and TBARS assays, added to the compound's identification and quantification by HPLC, from methanol extracts obtained by sonication. CC showed significantly higher TPC and TFC than WP and IP, while CSC produced 2.0-2.7 times more TFC than WP, and IP produced only 14.16% TPC and 38.8% TFC compared with WP. There were identified compounds such as epicatechin (EPI), caffeic acid (CfA), and p-coumaric acid (pCA) in in vitro cultures that were not found in WP. The quantitative analysis shows gallic acid (GA) as the least abundant compound in samples, whereas CSC produced significantly more EPI and CfA than CC. Despite these results, in vitro cultures show lower antioxidant activity than WP, for DPPH and TBARS WP > CSC > CC > IP and ABTS WP > CSC = CC > IP. Overall, A. pichichensis WP and in vitro cultures produce phenolic compounds with antioxidant activity, especially CC and CSC, which are shown to be a biotechnological alternative for obtaining bioactive compounds.

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Pentacyclic triterpenes, including lupeol, α- amyrin, and ß-amyrin, present a large range of biological activities including anti-inflammatory, anti-cancer, and gastroprotective properties. The phytochemistry of dandelion (Taraxacum officinale) tissues has been widely described. Plant biotechnology offers an alternative for secondary metabolite production and several active plant ingredients are already synthesized through in vitro cultures. This study aimed to establish a suitable protocol for cell growth and to determine the accumulation of α-amyrin and lupeol in cell suspension cultures of T. officinale under different culture conditions. To this end, inoculum density (0.2% to 8% (w/v)), inoculum age (2- to 10-week-old), and carbon source concentration (1%, 2.3%, 3.2%, and 5.5% (w/v)) were investigated. Hypocotyl explants of T. officinale were used for callus induction. Age, size, and sucrose concentrations were statistically significant in cell growth (fresh and dry weight), cell quality (aggregation, differentiation, viability), and triterpenes yield. The best conditions for establishing a suspension culture were achieved by using a 6-week-old callus at 4% (w/v) and 1% (w/v) of sucrose concentration. Results indicate that 0.04 (±0.02) α-amyrin and 0.03 (±0.01) mg/g lupeol can be obtained in suspension culture under these starting conditions at the 8th week of culture. The results of the present study provide a backdrop for future studies in which an elicitor could be incorporated to increase the large-scale production of α-amyrin and lupeol from T. officinale.