Anabel Ortiz-Caltempa

The genus Plukenetia includes lianas or vines with oleaginous seeds rich in omega-3 and omega-6 fatty acids, proteins, and vitamin E, and the presence of flavonoids, steroids, and terpenoids has also been reported in leaves. Several species of Plukenetia have traditionally been cultivated in their native distribution areas, and their propagation is usually by seed. The aim of this work was to establish callus and cell suspension cultures of P. carabiaseae, an endemic species of Mexico, for the evaluation of the in vitro antioxidant and anti-inflammatory potential of its extracts. Three light conditions were evaluated for the establishment of P. carabiaseae callus lines from leaf explants. Friable calluses obtained under constant light were used to initiate a cell suspension cultures in Gamborg basal (B5) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (CIN), as growth regulators. After 35 days of cultivation, different polarity extracts from biomass were obtained, showing that the acetone extract had the highest antioxidant activity and a high total phenolic content (30.57 mg of gallic acid equivalent (GAE)/g dry weight). The anti-inflammatory activity of the methanolic extract, evaluated in murine macrophages induced with bacterial lipopolysaccharides, was dose-dependent, without cytotoxic effects. This is the first report of the establishment of P. carabiasiae cell suspension culture and demonstrates its potential as a biotechnological source of antioxidant and anti-inflammatory metabolites.

Parkinsonʼs disease is characterized by an abnormal accumulation of alpha synuclein (α-syn) in different regions of the central nervous system. At present, only palliative pharmacological treatments are available for Parkinson´s disease. Immunotherapy is considered an alternative to treat Parkinsonʼs disease, and plants are a convenient alternative platform for biopharmaceutical production. When compared to other systems, plants are particularly attractive because they offer cost-effectiveness, large-scale production, and enhanced safety. Therefore, this study aimed to establish a carrot cell suspension culture for the production of cLTB-Syn, a vaccine candidate against Parkinsonʼs disease. The convenience of MS medium optimization was demonstrated. Transgenic callus cultures were maintained and adapted on solid MSU9 medium without phytohormones, followed by growth kinetics in suspension cultures. The maximum biomass yield was 15.8 ± 0.35 g/L DW at 14 days of culture, with a growth rate of µ = 0.1034/d and td = 6.7 days. The cLTB-Syn protein production reached a maximum value of 2.62 ± 0.03 µg/g DW, representing a 1.6-fold increase over the initial culture time. Finally, the presence of the transgene was confirmed by PCR, and the integrity of cLTB-Syn protein was determined by dot blot assays. This study presents evidence of a promising system for a toxin-free biopharmaceutical production, which has the potential to be scaled up for large manufacturing, at a low cost.

Background: The callus cultures from the fronds of the lycophyte Phlegmariurus taxifolius produce the huperzine A (HupA) alkaloid, which is used in Alzheimer’s disease treatment. This study aimed to establish the growth kinetics and HupA production by the newly HupS21 cell line grown in 250 mL flasks and in a 2 L airlift bioreactor. Methods: Batch-type kinetics were carried out for 60 days in 250 mL flasks and for 20 days in a 2 L airlift bioreactor. Measurements of dry weight (DW), specific growth rate (μ), doubling time (dt), pH, carbohydrate consumption, and HupA quantification were performed. The acetylcholinesterase (AChE) inhibitory assay of the HupS21 alkaloidal extract was determined. Results: The 250 mL flasks kinetic reached a maximum cell growth of 8.17 g/L DW, with a μ of 0.045 day⁻¹ and a dt of 15.40 days. The maximum HupA production was of 2.03 μg/g DW at day 45. In the 2 L airlift reactor, a maximum growth of 16.70 g/L DW, a μ of 0.062 day⁻¹, a dt of 11.20 days, and HupA production of 2.48 μg/g DW at day 15 were obtained. The alkaloidal extract from the HupS21 cell line at 100 μg/mL showed an AChE inhibitory activity of 85.6 ± 1.27%. Conclusions: The airlift reactor outperformed the flask cultures in maximum cell growth, specific growth rate, doubling time, and HupA production. To our knowledge, this research is the first report on the establishment of suspension cell cultures of P. taxifolius in shaken flasks and in an airlift bioreactor, providing a foundation for scaling up HupA production for pharmaceutical use.

Bursera fagaroides, popularly used in México, possesses bioactive lignans. These compounds are low in the bark, and its extraction endangers the life of the trees. The aim of the present investigation was to search for alternative sources of cytotoxic compounds in B. fagaroides prepared as leaves and in vitro callus cultures. The friable callus of B. fagaroides was established using a combination of plant growth regulators: 4 mgL⁻¹ of 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mgL⁻¹ Naphthaleneacetic Acid (NAA) and 1 mgL⁻¹ Zeatin. The maximum cell growth was at day 28 with a specific growth rate of μ = 0.059 days⁻¹ and duplication time td = 11.8 days. HPLC quantification of the dichloromethane callus biomass extract showed that Scopoletin, with a concentration of 10.7 µg g⁻¹ dry weight, was the main compound inducible as a phytoalexin by the addition of high concentrations of 2,4-D, as well as by the absence of nutrients in the culture medium. In this same extract, the compounds γ-sitosterol and stigmasterol were also identified by GC-MS analysis. Open column chromatography was used to separate and identify yatein, acetyl podophyllotoxin and 7′,8′-dehydropodophyllotoxin in the leaves of the wild plant. Cytotoxic activity on four cancer cell lines was tested, with PC-3 prostate carcinoma (IC₅₀ of 12.6 ± 4.6 µgmL⁻¹) being the most sensitive to the wild-type plant extract and HeLa cervical carcinoma (IC₅₀ of 72 ± 5 µgmL⁻¹) being the most sensitive to the callus culture extract.

The carrot-made LTB-Syn antigen (cLTB-Syn) is a vaccine candidate against synucleinopathies based on carrot cells expressing the target antigen LTB and syn epitopes. Therefore, the development of an efficient production process is required with media culture optimization to increase the production yields as the main goal. In this study, the effect of two nitrogen sources (urea and glutamate) on callus cultures producing cLTB-Syn was studied, observing that the addition of 17 mM urea to MS medium favored the biomass yield. To optimize the MS media composition, the influence of seven medium components on biomass and cLTB-Syn production was first evaluated by a Plackett–Burman design (PBD). Then, three factors were further analyzed using a central composite design (CCD) and response surface methodology (RSM). The results showed a 1.2-fold improvement in biomass, and a 4.5-fold improvement in cLTB-Syn production was achieved at the shake-flask scale. At the bioreactor scale, there was a 1.5-fold increase in biomass and a 2.8-fold increase in cLTB-Syn yield compared with the standard MS medium. Moreover, the cLTB-Syn vaccine induced humoral responses in BALB/c mice subjected to either oral or subcutaneous immunization. Therefore, cLTB-Syn is a promising vaccine candidate that will aid in developing immunotherapeutic strategies to combat PD and other neurodegenerative diseases without the need for cold storage, making it a financially viable option for massive immunization.

Due to the increasing populations of anthelmintic-resistant gastrointestinal nematodes and as a consequence of the adverse effects of synthetic drugs, this study focuses on the search for secondary metabolites with nematocidal activity from the edible mushroom Pleurotus djamor using The proton nuclear magnetic resonance (¹H-NMR) metabolomics. The highest activity was shown by the ethyl acetate fractions of mycelium (EC₅₀ 290.8 µg/mL) and basidiomes (EC₅₀ 282.7 µg/mL). Principal component analysis (PCA) and hierarchical data analysis (HCA) of the ¹H-NMR metabolic profiles data showed that the ethanolic extracts, the ethyl acetate, butanol, and water fractions from mycelium have different metabolic profiles than those from basidiomes, while low polarity (hexane) fractions from both stages of fungal development show similar profiles. Orthogonal partial least squares discriminant analysis (OPLS-DA) allowed the identification of signals in the ¹H-NMR metabolic profile associated with nematocidal activity. The signals yielded via OPLS-DA and bidimensional NMR analysis allowed the identification of uracil as a component in the ethyl acetate fraction from basidiomes, with an EC₅₀ of 237.7 µg/mL. The results obtained showed that chemometric analyses of the ¹H-NMR metabolic profiles represent a viable strategy for the identification of bioactive compounds from samples with complex chemical profiles.

Bursera fagaroides popularly used in México possess bioactive lignans. These compounds are low in the bark, and its extraction endangers the life of the trees. The aim of the present investigation was to search for alternative sources of cytotoxic compounds from B. fagaroides as leaves and in vitro callus cultures. The friable callus of B. fagaroides were established using a combination of phytoregulators: 4 mgL-1 of 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mgL-1 Naphthaleneacetic Acid (NAA) and 1 mgL-1 Zeatin. The maximum cell growth was at day 28 with a specific growth rate μ= 0.059 days-1 and duplication time td=11.8 days. Scopoletin with a concentration of 10.7 µg/g dry weight was the main compound inducible as a phytoalexin by the addition of high concentrations of 2,4-D, as well as by the absence of nutrients in the culture medium. The com-pounds γ-sitosterol, and stigmasterol were also present. Open column chromatography was used to separate and identify: yatein, acetyl podophyllotoxin and 7'8'-dehydropodophyllotoxin in the leaves of the wild plant. Cytotoxic activity on four cancer cell lines was tested, being PC-3 prostate carcinoma (IC50 of 12.6±4.6) the most sensitive to the wild-type plant extract and HeLa cervical carcinoma (IC50 of 72±5) to the callus culture extract.

The development of virus-free, oral vaccines against poliovirus capable of inducing mucosal protective immunity is needed to safely combat this pathogen. In the present study, a carrot cell line expressing the poliovirus VP2 antigen was established at the level of callus and cell suspensions, exploring the effects of culture media (MS and B5), supplementation with urea, phytoregulators (2,4-D : KIN), and light conditions (continuous light, photoperiod, and total darkness). The best callus growth was obtained on B5 medium supplemented with 2 mg/L of 2,4-D + 2 mg/L kinetin and 0.0136 g/L of urea and in continuous light conditions. Suspension cultures of the SMC-1 line in 250 mL Erlenmeyer flasks had a maximum growth of 16.07 ± 0.03 g/L DW on day 12 with a growth rate of µ=0.3/d and a doubling time of 2.3 days. In a 2 L airlift bioreactor, the biomass yield achieved was 25.6 ± 0.05 g/L DW at day 10 with a growth rate of µ= 0.58/d and doubling time of 1.38 d. Cell growth was 1.5 times higher in bioreactors than in shake flasks, highlighting that both systems resulted in the accumulation of VP2 throughout the time in culture. The maximum VP2 yield in flasks was 387.8 µg/g DW at day 21, while in the reactor it was 550.2 µg/g DW at day 18. In conclusion, bioreactor-based production of the VP2 protein by the SMC-1 suspension cell line offers a higher productivity when compared to flask cultures, offering a key perspective to produce low-cost vaccines against poliomyelitis.

Carrot (Daucus carota) cells have been used to effectively manufacture recombinant biopharmaceuticals such as cytokines, vaccines, and antibodies. We generated the carrot cell line Z4, genetically modified to produce the LTB-Syn antigen, which is a fusion protein proposed for immunotherapy against synucleinopathies. In this work, the Z4 cell suspension line was cultivated to produce the LTB-Syn protein in a 250 mL shake flask and 2 L airlift bioreactor cultures grown for 45 and 30 days, respectively. Maximum biomass was obtained on day 15 in both the airlift bioreactor (35.00 ± 0.04 g/L DW) and shake flasks (17.00 ± 0.04 g/L DW). In the bioreactor, the highest LTB-Syn protein yield (1.52 ± 0.03 µg/g FW) was obtained on day 15; while the same occurred on day 18 for shake flasks (0.92 ± 0.02 µg/g FW). LTB-Syn protein levels were analyzed by GM1-ELISA and western blot. PCR analysis confirmed the presence of the transgene in the Z4 line. The obtained data demonstrate that the carrot Z4 cell suspension line grown in airlift bioreactors shows promise for a scale-up cultivation producing an oral LTB-Syn antigen.