Clicking gallic acid into chitosan prolongs its antioxidant activity and produces intracellular Ca2+ responses in rat brain cells.

Gallic acid is a vegetable-derived and highly bioactive phenolic acid, but its antioxidant capacity is sensitive to environmental conditions. Chitosan is a biopolymer capable of exerting significant protection to various molecules, including phenolic compounds. A chitosan derivative that extends the antioxidant activity of gallic acid was synthesized by click chemistry and characterized by FT-IR, 1H NMR, and antioxidant capacity assays. Our results show that synthesized polymeric solutions and nanoparticles of N-(gallic acid) chitosan were both internalized by rat brain cells, processes that were modulated by extracellular Ca2+ and Na+. Their internalization was supported by dynamic light scattering and ζ-potential analyses, while Ca2+ imaging recordings performed in brain cells revealed the potential biological effect of N-(gallic acid) chitosan. We conclude that the synthesis of an N-(gallic acid) chitosan derivative through click chemistry is viable and may serve as strategy to prolong its antioxidant activity and to study its biological effects in vivo.

Functionalized Protein Binders in Developmental Biology.

Developmental biology has greatly profited from genetic and reverse genetic approaches to indirectly studying protein function. More recently, nanobodies and other protein binders derived from different synthetic scaffolds have been used to directly dissect protein function. Protein binders have been fused to functional domains, such as to lead to protein degradation, relocalization, visualization, or posttranslational modification of the target protein upon binding. The use of such functionalized protein binders has allowed the study of the proteome during development in an unprecedented manner. In the coming years, the advent of the computational design of protein binders, together with further advances in scaffold engineering and synthetic biology, will fuel the development of novel protein binder-based technologies. Studying the proteome with increased precision will contribute to a better understanding of the immense molecular complexities hidden each step along the way to generate form and function during development.

Seamless knockins in Drosophila via CRISPR-triggered single-strand annealing.

CRISPR-Cas greatly facilitated the integration of exogenous sequences into specific loci. However, knockin generation in multicellular animals remains challenging, partially due to the complexity of insertion screening. Here, we describe SEED/Harvest, a method to generate knockins in Drosophila, based on CRISPR-Cas and the single-strand annealing (SSA) repair pathway. In SEED (from "scarless editing by element deletion"), a switchable cassette is first integrated into the target locus. In a subsequent CRISPR-triggered repair event, resolved by SSA, the cassette is seamlessly removed. Germline excision of SEED cassettes allows for fast and robust knockin generation of both fluorescent proteins and short protein tags in tandem. Tissue-specific expression of Cas9 results in somatic cassette excision, conferring spatiotemporal control of protein labeling and the conditional rescue of mutants. Finally, to achieve conditional protein labeling and manipulation of short tag knockins, we developed a genetic toolbox by functionalizing the ALFA nanobody.

Agro-Industrial By-Products of Plant Origin: Therapeutic Uses as well as Antimicrobial and Antioxidant Activity.

The importance of bioactive compounds in agro-industrial by-products of plant origin lies in their direct impacts on human health. These compounds have been shown to possess antioxidant, anti-inflammatory, and antimicrobial properties, contributing to disease prevention and strengthening the immune system. In particular, the antimicrobial action of these compounds emerges as an important tool in food preservation, providing natural alternatives to synthetic preservatives and contributing to combating antimicrobial resistance. Using agro-industrial by-products of plant origin not only addresses the need to reduce waste and promote sustainability but also inaugurates a new era in the formulation of functional foods. From fruit peels to pulps and seeds, these by-products are emerging as essential ingredients in the creation of products that can promote health. Continued research in this area will unveil new applications and properties of these by-products and open doors to a food paradigm in which health and sustainability converge, paving the way to a healthier and more equitable future. The present review presents an overview of our knowledge of agro-industrial by-products and some of their more relevant health-promoting bioactivities.

Protocol for generating in-frame seamless knockins in Drosophila using the SEED/Harvest technology.

The generation of knockins is fundamental to dissect biological systems. SEED/Harvest, a technology based on CRISPR-Cas9, offers a powerful approach for seamless genome editing in Drosophila. Here, we present a protocol to tag any gene in the Drosophila genome using SEED/Harvest technology. We describe knockin design, plasmid preparation, injection, and insertion screening. We then detail procedures for germline harvesting. The technique combines straightforward cloning and robust screening of insertions, while still resulting in scarless gene editing. For complete details on the use and execution of this protocol, please refer to Aguilar et al.1.

Lyophilized Avocado Paste Improves Corn Chips' Nutritional Properties and Sensory Acceptability.

Avocado paste (AP) is an industrial byproduct and a potential source of bioactive compounds, so there is great interest in its valorization. The objective of the present study was to evaluate the effects of adding AP to corn chips regarding their nutritional profile and sensory acceptability. Three AP-supplemented corn chip samples were prepared (C-2%, C-6%, and C-10%), along with a control chip (C), whose total phenolics, flavonoids, antioxidant capacity, proximate composition, minerals, fatty acids, and sensory acceptability were evaluated. Regarding the content of phenolic compounds and flavonoids, significant increases were found between all samples (p < 0.05), particularly between C and C-10% (from 0.93 to 3.56 mg GAE/g dw and 1.17 to 6.61 mg QE/g dw, respectively). Their antioxidant capacity also increased significantly (p < 0.05) with all methods used (FRAP, DPPH, ORAC, and TEAC). Regarding the sensory analysis, no significant differences were found (p > 0.05) between C and C-2% in the parameters of smell, color, flavor, and overall acceptability; however, the texture of C-2% was better evaluated. The C-2% sample also had the highest acceptability; 82% of the participants mentioned that they would buy the C-2%, higher than the rest of the samples. These results suggest the feasibility of adding 2% AP as a strategy to improve the nutritional properties of corn chips without compromising their sensory acceptability; therefore, AP may be used as a food ingredient.

Avocado Paste Phenolics Mitigate a High-Fat Diet-Induced Plasma HDL Decrease in Male Wistar Rats, by Altering the mRNA Expression of Hepatic SCARB1.

Avocado paste (AP) is the main industrial byproduct of its processing, and retains various phenolic compounds (PCs). PCs are known to normalize the plasma lipid profile, but those from avocado byproducts have been minimally studied. We report the normalizing effects of an AP-derived phenolic extract (PE) on the plasma lipid profile of male Wistar rats. A standard (SD) and high-fat diet (HFD) were formulated, and the same diets were supplemented with 1 g/kg of diet of PE (SD + PE and HFD + PE). Rats were fed these diets during an 8-week period. The HFD induced signs of dyslipidemia, but PE treatment countered the decrease in HDL. Relative mRNA expression (real-time PCR) of the hepatic HDL receptor (SCARB1) increased in both groups (SD + PE and HFD + PE), while the LDR receptor (LDLR) increased in SD + PE group. The mRNA expression of apolipoproteins APOA1 and APOB was unaffected. We conclude that PCs from AP can counter a diet-induced decrease in plasma HDL by acting on the mRNA expression of its hepatic receptor.

Oregano Essential Oil versus Conventional Disinfectants against Salmonella Typhimurium and Escherichia coli O157:H7 Biofilms and Damage to Stainless-Steel Surfaces.

This study compared the effect of oregano essential oil versus sodium hypochlorite, hydrogen peroxide, and benzalkonium chloride against the viability of adhered Salmonella Typhimurium and Escherichia coli O157:H7 on 304 stainless steel. Oregano essential oil was effective in disrupting the biofilms of both bacteria at concentrations ranging from 0.15 to 0.52 mg mL-1. In addition, damage to stainless-steel surfaces following disinfection treatments was assessed by weight loss analysis and via visual inspection using light microscopy. Compared to the other treatments, oregano oil caused the least damage to stainless steel (~0.001% weight loss), whereas sodium hypochlorite caused the most severe damage (0.00817% weight loss) when applied at 0.5 mg mL-1. Moreover, oregano oil also had an apparent protective impact on the stainless steel as weight losses were less than for the control surfaces (distilled water only). On the other hand, sodium hypochlorite caused the most severe damage to stainless steel (0.00817% weight loss). In conclusion, oregano oil eliminated monoculture biofilms of two important foodborne pathogens on 304 stainless-steel surfaces, while at the same time minimizing damage to the surfaces compared with conventional disinfectant treatments.

N-(levodopa) chitosan derivative based on click chemistry shows biological functionality in brain cells.

OBJECTIVE: To develop N-(levodopa) chitosan derivatives through click chemistry to study their effect in brain cells.Significance: This study presents a proof-of-concept that macromolecules such as N-(Levodopa) chitosan derivatives traverse brain cell membranes and induce biomedical functionalities. METHODS: Through click chemistry, we developed N-(levodopa) chitosan derivatives. They were physically and chemically characterized by FT-IR, 1H-NMR, TGA and Dynamic Light Scattering analyses. Solution and nanoparticles of N-(levodopa) chitosan derivatives were tested in primary cell cultures from the postnatal rat olfactory bulb, substantia nigra and corpus callosum. Ca2+ imaging and UPLC experiments were used to investigate if the biomaterial modulated the brain cell physiology. RESULTS: N-(levodopa) chitosan derivatives induced intracellular Ca2+ responses in primary cell cultures of the rat brain. UPLC experiments indicated that levodopa attached to chitosan was converted into dopamine by brain cells. CONCLUSION: The present study shows that N-(levodopa) chitosan may be useful to develop new treatment strategies, which could serve as molecular reservoirs of biomedical drugs to treat degenerative disorders of the nervous system.

Heterodimerization-dependent secretion of bone morphogenetic proteins in Drosophila.

Combinatorial signaling is key to instruct context-dependent cell behaviors. During embryonic development, adult homeostasis, and disease, bone morphogenetic proteins (BMPs) act as dimers to instruct specific cellular responses. BMP ligands can form both homodimers or heterodimers; however, obtaining direct evidence of the endogenous localization and function of each form has proven challenging. Here, we make use of precise genome editing and direct protein manipulation via protein binders to dissect the existence and functional relevance of BMP homodimers and heterodimers in the Drosophila wing imaginal disc. This approach identified in situ the existence of Dpp (BMP2/4)/Gbb (BMP5/6/7/8) heterodimers. We found that Gbb is secreted in a Dpp-dependent manner in the wing imaginal disc. Dpp and Gbb form a gradient of heterodimers, whereas neither Dpp nor Gbb homodimers are evident under endogenous physiological conditions. We find that the formation of heterodimers is critical for obtaining optimal signaling and long-range BMP distribution.

Could Naringenin Participate as a Regulator of Obesity and Satiety?

Obesity is a serious health problem worldwide, since it is associated with multiple metabolic disorders and complications such as cardiovascular disease, type 2 diabetes, fatty liver disease and overall metabolic dysfunction. Dysregulation of the hunger-satiety pathway, which includes alterations of central and peripheral signaling, explains some forms of obesity by favoring hyperphagia and weight gain. The present work comprehensively summarizes the mechanisms by which naringenin (NAR), a predominant flavanone in citrus fruits, could modulate the main pathways associated with the development of obesity and some of its comorbidities, such as oxidative stress (OS), inflammation, insulin resistance (IR) and dyslipidemia, as well as the role of NAR in modulating the secretion of enterohormones of the satiety pathway and its possible antiobesogenic effect. The results of multiple in vitro and in vivo studies have shown that NAR has various potentially modulatory biological effects against obesity by countering IR, inflammation, OS, macrophage infiltration, dyslipidemia, hepatic steatosis, and adipose deposition. Likewise, NAR is capable of modulating peptides or peripheral hormones directly associated with the hunger-satiety pathway, such as ghrelin, cholecystokinin, insulin, adiponectin and leptin. The evidence supports the use of NAR as a promising alternative to prevent overweight and obesity.

Revisiting the high-fat diet/low streptozotocin prediabetic rat model: A bioanalytical adjustment.

Insulin resistance (IR) is the main feature of prediabetes (PD), which ultimately leads to diabetes. High-dose streptozotocin-treated rodents often show irreversible ß-cell mass loss and function, leaving the premorbid diabetic state (PD/IR) unnoticed. This study aimed to re-evaluate the synergistic/independent effect of a sub-chronic consumption (1-5 weeks) of a high-fat diet (60% gross energy from fat, 3.8 kcal.g-1) with [PD/IR-2 (week 2) to PD/IR-5 week five)] or without [HFD-5 (week five)] a single intraperitoneal dose (35 mg.kg-1) of streptozotocin in Wistar rats. Bioassay performance and clinical/histological features suggesting PD/IR or diabetes, were documented weekly and compared to standard chow-fed (3.5 kcal.g-1) rats (healthy controls, HC). PD/IR1-5 (fed with HFD for 1 to 5 weeks plus a single dose of streptozotocin) and HFD-5 (just fed with HFD for 5 weeks) groups reduced their food intake yet gained more body weight than HC. Groups exhibited hyperglycemia, dyslipidemia, and impaired glucose tolerance in decreasing order as follows: PD/IR-5, PD/IR-4, HFD-5, PD/IR-2-3, and HC. Histological disturbances in the pancreas, Soleus muscle, and liver were mostly observed in HFD-5 and PD/IR4-5 groups. HFD administration for 4 weeks white a single moderate dose of streptozotocin four days before sacrifice, leads to a convenient PD/IR rat model.

Optimization and In Vitro Digestion of a Guava (Psidium guajava), Mamey (Pouteria sapota) and Stevia (Stevia rebaudiana) Functional Beverage.

Guava and mamey are phenolic- and carotenoid-rich fruits with potential health benefits, but are minimally used as ingredients in functional beverages. The objectives of the present work are to optimize the content of guava and mamey pulps and a stevia solution in the formulation of a functional beverage with high content of bioactive compounds and sensory acceptability using a mixture design analysis, and to analyze its composition after in vitro digestion. The optimized formulation (17.77 and 19.23 g of guava and mamey pulps, respectively; 1% stevia solution) yielded a beverage with 418.21 mg gallic acid equivalents (GAE)/100 mL and 0.20 mg ß-carotene/100 mL, and an antioxidant capacity of 213.58, 78.90 and 234.03 mg Trolox equivalents (TE)/100 mL using three methodologies. The mathematical model developed was significant (p < 0.05), according to R2 values between 0.70 and 0.75. α- and ß-carotene were quantified during the oral phase of in vitro digestion. Gallic, p-coumaric, ferulic and chlorogenic acids were also identified. The beverage had a general acceptability of 6.72. We conclude that the mathematical model developed was a good predictor of the experimental data and that the optimized beverage contained high bioactive concentrations (phenolics and carotenoids) and was well-accepted by potential consumers.

Prevalence of Antibiotic-Resistant E. coli Strains in a Local Farm and Packing Facilities of Honeydew Melon in Hermosillo, Sonora, Mexico.

Pathogenic strains of Escherichia coli threaten public health due to their virulence factors and antibiotic resistance. Additionally, the virulence of this bacterium varies by region depending on environmental conditions, agricultural practices, and the use of antibiotics and disinfectants. However, there is limited research on the prevalence of antibiotic-resistant E. coli in agriculture. Therefore, this research aimed to determine the antibiotic resistance of E. coli isolated from the Honeydew melon production system in Hermosillo, Sonora, Mexico. Thirty-two E. coli strains were isolated from 445 samples obtained from irrigation water, harvested melons, the hands of packaging workers, boxes, and discarded melons. The resistance profile of the E. coli strains was carried out to 12 antibiotics used in antimicrobial therapeutics against this bacterium; a high level of resistance to ertapenem (100%) was detected, followed by meropenem (97%), and ampicillin (94%); 47% of the strains were classified as multidrug-resistant. It was possible to identify the prevalence of the extended-spectrum ß-lactamase (ESBLs) gene blaTEM (15.6%), as well as the non-ESBL genes qepA (3.1%) and aac(6')lb-cr (3.1%). The E. coli strains isolated from irrigation water were significantly associated with resistance to aztreonam, cefuroxime, amikacin, and sulfamethoxazole/trimethoprim. Irrigation water, packing workers' hands, and discarded melons showed a higher prevalence of antibiotic-resistant, ESBL, and non-ESBL genes of E. coli strains in a farm and packing facility of Honeydew melon in Hermosillo, Sonora.

Single-Cell Protein Production as a Strategy to Reincorporate Food Waste and Agro By-Products Back into the Processing Chain.

Food waste is a serious problem with negative environmental and economic consequences. Unused food (either as waste or by-products and referred to as food residues in the present work) is a source of carbohydrates, lipids, proteins, vitamins, minerals and bioactive compounds that could be used in an alternate or secondary life cycle to avoid discarding it. The present work reviews the potential use of food residues for the bioengineering of single-cell protein (SCP), addressing aspects of production, nutrition and safety, as well as the main challenges and perspectives. SCP is obtained from various microorganisms, including fungi, bacteria, yeasts and algae, in pure or mixed form. SCP generally contains a higher percentage of protein (30-80%) compared to soy (38.6%), fish (17.8%), meat (21.2%) and whole milk (3.28%). SCP is a source of essential amino acids, including methionine, threonine and lysine. The use of food residues as substrates for the production of SCP would reduce production costs (35-75%); however, optimization and industrial scaling are some of the main challenges to its sustainable production. The use food waste and agro by-products from the food industry could be a promising alternative to obtain protein according to a circular production scheme.

Lesser-Consumed Tropical Fruits and Their by-Products: Phytochemical Content and Their Antioxidant and Anti-Inflammatory Potential.

Açaí, lychee, mamey, passion fruit and jackfruit are some lesser-consumed tropical fruits due to their low commercial production. In 2018, approximately 6.8 million tons of these fruits were harvested, representing about 6.35% of the total world production of tropical fruits. The present work reviews the nutritional content, profile of bioactive compounds, antioxidant and anti-inflammatory capacity of these fruits and their by-products, and their ability to modulate oxidative stress due to the content of phenolic compounds, carotenoids and dietary fiber. Açaí pulp is an excellent source of anthocyanins (587 mg cyanidin-3-glucoside equivalents/100 g dry weight, dw), mamey pulp is rich in carotenoids (36.12 mg ß-carotene/100 g fresh weight, fw), passion fruit peel is rich in dietary fiber (61.16 g/100 dw). At the same time, jackfruit contains unique compounds such as moracin C, artocarpesin, norartocarpetin and oxyresveratrol. These molecules play an important role in the regulation of inflammation via activation of mitogen-activated protein kinases (including p38, ERK and JNK) and nuclear factor κB pathways. The properties of the bioactive compounds found in these fruits make them a good source for use as food ingredients for nutritional purposes or alternative therapies. Research is needed to confirm their health benefits that can increase their marketability, which can benefit the primary producers, processing industries (particularly smaller ones) and the final consumer, while an integral use of their by-products will allow their incorporation into the circular bioeconomy.

Engineered kinases as a tool for phosphorylation of selected targets in vivo.

Reversible protein phosphorylation by kinases controls a plethora of processes essential for the proper development and homeostasis of multicellular organisms. One main obstacle in studying the role of a defined kinase-substrate interaction is that kinases form complex signaling networks and most often phosphorylate multiple substrates involved in various cellular processes. In recent years, several new approaches have been developed to control the activity of a given kinase. However, most of them fail to regulate a single protein target, likely hiding the effect of a unique kinase-substrate interaction by pleiotropic effects. To overcome this limitation, we have created protein binder-based engineered kinases that permit a direct, robust, and tissue-specific phosphorylation of fluorescent fusion proteins in vivo. We show the detailed characterization of two engineered kinases based on Rho-associated protein kinase (ROCK) and Src. Expression of synthetic kinases in the developing fly embryo resulted in phosphorylation of their respective GFP-fusion targets, providing for the first time a means to direct the phosphorylation to a chosen and tagged target in vivo. We presume that after careful optimization, the novel approach we describe here can be adapted to other kinases and targets in various eukaryotic genetic systems to regulate specific downstream effectors.

Pomegranate (Punica granatum L.) Peel Extracts as Antimicrobial and Antioxidant Additives Used in Alfalfa Sprouts.

Aqueous and ethanolic pomegranate peel extracts (PPE) were studied as a source of phenolic compounds with antimicrobial, anti-quorum sensing, and antioxidant properties. The aqueous extract showed higher total phenolic and flavonoid content (153.43 mg GAE/g and 45.74, respectively) and antioxidant capacity (DPPH radical inhibition: 86.12%, ABTS radical scavenging capacity: 958.21 mg TE/dw) compared to the ethanolic extract. The main phenolic compounds identified by UPLC-DAD were chlorogenic and gallic acids. The aqueous PPE extract showed antimicrobial activity against Listeria monocytogenes, Salmonella Typhimurium, Candida tropicalis (MICs 19-30 mg/mL), and anti-quorum sensing activity expressed as inhibition of Chromobacterium violaceum violacein production (%). The aqueous PPE extracts at 25 mg/mL applied on alfalfa sprouts reduced psychrophilic bacteria (1.12 Log CFU/100 g) and total coliforms (1.23 Log CFU/100 g) and increased the antioxidant capacity of the treated sprouts (55.13 µmol TE/100 g (DPPH) and 126.56 µmol TE/100 g (ABTS)) compared to untreated alfalfa. This study emphasizes PPE's antioxidant and antimicrobial activities in alfalfa sprouts preservation.

Studying Protein Function Using Nanobodies and Other Protein Binders in Drosophila.

The direct manipulation of proteins by nanobodies and other protein binders has become an additional and valuable approach to investigate development and homeostasis in Drosophila. In contrast to other techniques, that indirectly interfere with proteins via their nucleic acids (CRISPR, RNAi, etc.), protein binders permit direct and acute protein manipulation. Since the first use of a nanobody in Drosophila a decade ago, many different applications exploiting protein binders have been introduced. Most of these applications use nanobodies against GFP to regulate GFP fusion proteins. In order to exert specific protein manipulations, protein binders are linked to domains that confer them precise biochemical functions. Here, we reflect on the use of tools based on protein binders in Drosophila. We describe their key features and provide an overview of the available reagents. Finally, we briefly explore the future avenues that protein binders might open up and thus further contribute to better understand development and homeostasis of multicellular organisms.