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Molecular diagnostics involving nucleic acids (DNA and RNA) are regarded as extremely functional tools. During the 2020 global health crisis, efforts intensified to optimize the production and delivery of molecular diagnostic kits for detecting SARS-CoV-2. During this period, RT-LAMP emerged as a significant focus. However, the thermolability of the reagents used in this technique necessitates special low-temperature infrastructure for transport, storage, and conservation. These requirements limit distribution capacity and necessitate cost-increasing adaptations. Consequently, this report details the development of a lyophilization protocol for reagents in a colorimetric RT-LAMP diagnostic kit to detect SARS-CoV-2, facilitating room-temperature transport and storage. We conducted tests to identify the ideal excipients that maintain the molecular integrity of the reagents and ensure their stability during room-temperature storage and transport. The optimal condition identified involved adding 5% PEG 8000 and 75 mM trehalose to the RT-LAMP reaction, which enabled stability at room temperature for up to 28 days and yielded an analytical and diagnostic sensitivity and specificity of 83.33% and 90%, respectively, for detecting SARS-CoV-2. This study presents the results of a lyophilized colorimetric RT-LAMP COVID-19 detection assay with diagnostic sensitivity and specificity comparable to RT-qPCR, particularly in samples with high viral load.

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The present work aimed at the development and characterization of aroeira leaf flour (Schinus terebinthifolius Raddi), obtained by lyophilization and drying in an air circulation oven. The technological, physical, physico-chemical, morphological, functional, and microbiological aspects were analyzed. Physico-chemical analysis identified the following properties with values provided respectively for fresh leaves (FOin) and flours (FES and FLIO): low water activity (0.984, 0.370, 0.387 g/100 g), moisture (64.52, 5.37, 7.97 g /100 g), ash (2.69, 6.51, and 6.89 g/100 g), pH (0.89, 4.45, 4.48 g/100 g), lipids (0.84, 1.67, 5.23 g/100 g), protein (3.29, 8.23, 14.12 g/100 g), carbohydrates (17.02, 53.12, 33.02 g/100 g), ascorbic acid (19.70, 34.20, 36.90 mg/100 g). Sources of fiber from plant leaves and flours (11.64, 25.1, 32.89 g/100 g) showed increased levels of luminosity. For NMR, the presence of aliphatic and aromatic compounds with olefinic hydrogens and a derivative of gallic acid were detected. The most abundant minerals detected were potassium and calcium. Micrographs identified the presence of irregular, non-uniform, and sponge-like particles. The main sugars detected were: fructose, glucose, and maltose. Malic, succinic, citric, lactic, and formic acids were found. Fifteen phenolic compounds were identified in the samples, highlighting: kaempferol, catechin, and caffeic acid. The values ​​found for phenolics were (447, 716.66, 493.31 mg EAG/100 g), flavonoids (267.60, 267.60, 286.26 EC/100 g). Antioxidant activity was higher using the ABTS method rather than FRAP for analysis of FOin, FES, and FLIO. Since the flours of the aroeira leaf have an abundant matrix of nutrients with bioactive properties and antioxidant activity, they have a potential for technological and functional use when added to food.

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Research on innovative mucosal adjuvants is essential to develop new vaccines for safe mucosal application. In this work, we propose the development of a Lactococcus lactis that expresses a variant of flagellin on its surface (FliC131*), to increase the adjuvanticity of the living cell and cell wall-derived particles (CWDP). We optimized the expression of FliC131*, and confirmed its identity and localization by Western blot and flow cytometry. We also generated CWDP containing FliC131* (CDWP-FliC131*) and evaluated their storage stability. Lastly, we measured the human TLR5 stimulating activity in vitro and assessed the adjuvanticity in vivo using ovalbumin (OVA) as a model antigen. As a result, we generated L. lactis/pCWA-FliC131*, that expresses and displays FliC131* on its surface, obtained the corresponding CWDP-FliC131*, and showed that both activated hTLR5 in vitro in a dose-dependent manner. Furthermore, CWDP-FliC131* retained this biological activity after being lyophilized and stored for a year. Finally, intranasal immunization of mice with OVA plus live L. lactis/pCWA-FliC131* or CWDP-FliC131* induced OVA-specific IgG and IgA in serum, intestinal lavages, and bronchoalveolar lavages. Our work demonstrates the potential of this recombinant L. lactis with an enhanced adjuvant effect, prompting its further evaluation for the design of novel mucosal vaccines.

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Lyophilization is a widely employed long-term preservation method in which the bacterial survival rate largely depends on the cryoprotectant used. Bacillus cereus strain PBC was selected for its ability to thrive in environments contaminated with arsenic, lead, and cadmium, tolerate 500 ppm of free cyanide, and the presence of genes such as ars, cad, ppa, dap, among others, associated with the bioremediation of toxic compounds and enterotoxins (nheA, nheB, nheC). Following lyophilization, the survival rates for Mannitol 2.5%, Mannitol 10%, and Glucose 1% were 98.02%, 97.12%, and 96.30%, respectively, with the rates being lower than 95% for other sugars. However, during storage, for the same sugars, the survival rates were 78.71%, 97.12%, and 99.97%, respectively. In the cake morphology, it was found that the lyophilized morphology showed no relationship with bacterial survival rate. The best cryoprotectant for the PBC strain was 1% glucose since it maintained constant and elevated bacterial growth rates during storage, ensuring that the unique characteristics of the bacterium were preserved over time. These findings hold significant implications for research as they report a new Bacillus cereus strain with the potential to be utilized in bioremediation processes.

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C. berlandieri ssp. berlandieri (C. berlandieri) is one of the most common members of the group of plants known as quelites, which are dark leafy greens widely consumed in Mexico. This study aimed to evaluate the impact of two drying procedures (oven drying and freeze-drying/lyophilization) on the polyphenolic composition, antioxidant capacity, and proximal chemical analysis of C. berlandieri leaves and inflorescences (raw or boiled). The results indicated that the raw freeze-dried samples had higher amounts (p < 0.05) of total phenolic compounds, total flavonoids, and antioxidant capacity, mainly in the inflorescence. The oven-dried samples showed an increased concentration of polyphenols after boiling, while the lyophilized samples showed a slightly decreased concentration. The drying process was observed to have little impact on the proximal chemical composition. Quantification by UPLC-DAD-ESI-QToF/MS identified up to 23 individual phenolic compounds, with freeze-dried samples showing higher amounts of individual compounds compared with oven-dried. Procyanidin B2 was found exclusively in the inflorescences. The inflorescences have a higher content of phenolic compounds and greater antioxidant capacity than the leaves. Regardless of the drying process, the leaves and inflorescences of C. berlandieri contain an interesting variety of phenolic compounds that may have beneficial effects on health.

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Curcumin and vitamin D3 are bioactive molecules of great importance for the food industry. However, their low stability in several processing conditions hampers their proper incorporation into powdered food formulations. This study proposes the enrichment of a common raw material (cornstarch) with curcumin and vitamin D3 by using high-shear wet agglomeration. The bioactives were initially encapsulated into liposome dispersions and then subjected to lyophilization. The resulting dried vesicles were later incorporated into cornstarch by wet agglomeration using maltodextrin as the binder solution. The phospholipid content and the amount of added liposomes were evaluated to characterize the enriched cornstarch samples. The lyophilized vesicles showed a high retention rate of 99 % for curcumin and vitamin D3, while the enriched cornstarch samples retained above 96 % (curcumin) and 98 % (vitamin D3) after 30 days of controlled storage. All in all, the presence of dried liposomes improved the flowability and delayed retrogradation phenomenon in agglomerated cornstarch. Therefore, this study introduced a novel and reliable method of incorporating hydrophobic and thermosensitive molecules into powdered food formulations by using readily available materials and a straightforward high-shear wet agglomeration process.

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Molecular biology is a widely used and widespread technique in research and as a laboratory diagnostic tool, aiming to investigate targets of interest from the obtainment, identification, and analysis of genetic material. In this context, methods, such as Polymerase Chain Reaction (PCR), Reverse Transcription Polymerase Chain Reaction (RT-PCR), real-time PCR, loopmediated isothermal amplification (LAMP), and loop-mediated isothermal amplification with reverse transcription (RT-LAMP), can be cited. Such methods use enzymes, buffers, and thermosensitive reagents, which require specific storage conditions. In an attempt to solve this problem, the lyophilization procedure (dehydration process by sublimation) can be applied, aiming to preserve and prolong the useful life of the reaction components in cases of temperature variation. In this review, we present a synthesis of the lyophilization process, describing the events of each step of the procedure and providing general information about the technique. Moreover, we selected lyophilization protocols found in the literature, paying attention to the conditions chosen by the authors for each step of the procedure, and structured the main data in tables, facilitating access to information for researchers who need material to produce new functional protocols.

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The maintenance of viable and stable Xanthomonascells is crucial for the xanthan reliable research and industrial production. The method, storage and recovery conditions should preserve bothviability and phenotypical and genotypical features. Here, the effectiveness classical methods on the long-term preservation of different Xanthomonas arboricola pathovar pruni strains was to determine.Strains were preserved by monthly sub-culturing in solid medium and lyophilization. After 12 years the viability of the strains, was assessed, as well as their productive capacity and the viscosity of the xanthan gum produced by these strains kept by lyophilization and sub-culturing. Among the lyophilized strains, only those stored at -18 °C were viable after 12 years. The productive capacity of the strains were poorly affected by lyophilization, the passage of the cultures into a solid nutrition medium being sufficient for them to return to their normal metabolism. The viscosity of the synthesized xanthan gum was method-dependent and higher for the lyophilized strains. The work and its findings arenew and original because a work on this topic has never been published before. The results obtained allow the breaking of paradigms regarding the preservation of Xanthomonas.(AU)

A manutenção de células de Xanthomonas viáveis e estáveis é crucial para se obter uma pesquisa confiável e para a produção de xantana industrial.O método, o armazenamento eascondições de recuperação devem preservar tanto a viabilidade quanto as características fenotípicas e genotípicas. O objetivo do estudo foi determinar a eficácia dos métodos clássicos na preservação a longo prazo de diferentes cepas de Xanthomonas arboricolapatovarpruni. As cepas foram preservadas por subcultivo mensal em meio sólido e liofilização. Após 12 anos,avaliou-se a viabilidade das linhagens, bem como a capacidade produtiva e a viscosidade da goma xantana produzida por essas linhagens mantidas por liofilização e subcultivo. Entre as cepas liofilizadas, somente foram viáveis, após 12 anos, as armazenadas a -18°C. A capacidade produtiva das cepas foi pouco afetada pela liofilização, sendo suficiente a passagem das culturas para um meio de cultivosólido para que elas voltassem ao seu metabolismo normal. A viscosidade da goma xantana sintetizada foi dependente do método e maior para as cepas liofilizadas. O estudo e suas descobertas sãonovos e originais porque um trabalho sobre este tópico nunca foi publicado antes. Os resultados obtidos permitem quebrar paradigmas quanto à preservação de Xanthomonas.(AU)

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Abstract The purpose of the present study was to develop stable lyophilized formulation of peginterferon alfa-2b which is acquiescent to the short lyophilization process. The present study evaluates the effect of buffering components and cryoprotectant(s) on depegylation of the peginterferon alfa-2b in combination with lyophilization process. Finally, a short lyophilization process was identified which can produce a stable pharmaceutical form of peginterferon alfa-2b without any depegylation during long-term storage. Formulations were analyzed mainly for depegylation by HP-size exclusion chromatography and in-vitro antiviral activity. Residual moisture content in the lyophilized product was also used as a key indicating parameter to check its role with respect to depegylation upon storage under various temperature conditions. It was observed that the peginterferon alfa-2b when formulated in presence of cryoprotectant like sucrose requires longer lyophilization process of about 5 days, irrespective of the buffering components used, to reduce the level of residual moisture content and thereby to produce the stable formulation without depegylation. A stable formulation in presence of high concentration of lactose as a cryoprotectant was developed which can withstand stresses exerted to protein-polymer conjugate during lyophilization phases without any significant depegylation. A short lyophilization process of about 48 hours can be utilized for peginterferon alfa-2b when formulated in presence of lactose as a cryoprotectant through which a stable lyophilized formulation can be produced as against longer process required when sucrose is used a cryoprotectant, which is essential from commercial point of view as lyophilization is a costly process.

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Probiotic bacteria are widely used to prepare pharmaceutical products and functional foods because they promote and sustain health. Nonetheless, probiotic viability is prone to decrease under gastrointestinal conditions. In this investigation, Lactiplantibacillus plantarum spp. CM-CNRG TB98 was entrapped in a gelatin−poly (vinyl alcohol) (Gel−PVA) hydrogel which was prepared by a "green" route using microbial transglutaminase (mTGase), which acts as a crosslinking agent. The hydrogel was fully characterized and its ability to entrap and protect L. plantarum from the lyophilization process and under simulated gastric and intestine conditions was explored. The Gel−PVA hydrogel showed a high probiotic loading efficiency (>90%) and survivability from the lyophilization process (91%) of the total bacteria entrapped. Under gastric conditions, no disintegration of the hydrogel was observed, keeping L. plantarum protected with a survival rate of >94%. While in the intestinal fluid the hydrogel is completely dissolved, helping to release probiotics. A Gel−PVA hydrogel is suitable for a probiotic oral administration system due to its physicochemical properties, lack of cytotoxicity, and the protection it offers L. plantarum under gastric conditions.

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La sandía vanessa, la luffa y la cassabanana son cucurbitáceas que poseen compuestos con potencial bioactivo, esto es, presencia de compuestos que ejercen efectos benéficos para la salud. En Colombia, estas frutas son desaprovechadas, debido a su escasa popularidad; dar a conocer la información de sus compuestos nutricionales incentiva su aprovechamiento y consumo. El objetivo del presente estudio fue realizar la caracterización fisicoquímica y evaluar el efecto de la liofilización y la extracción asistida por ultrasonido sobre el contenido de vitamina C, polifenoles totales y capacidad antioxidante de las partes de cada fruto. Los desechos generados entre cortezas y semillas son de 28,3 % (sandía), 68,76 % (luffa) y 25,39 % (cassabanana); estos, a su vez, presentaron contenidos de polifenoles totales y capacidad antioxidante mayores que en la pulpa. El tratamiento de liofilización mejoró la extracción % en capacidad antioxidante, vitamina C y polifenoles totales, comparado con las muestras frescas. Por otro lado, tanto la corteza como la pulpa de luffa son una buena fuente de compuestos con capacidad antioxidante, mientras que la sandía y la cassabanana alcanzaron una buena aceptación sensorial, lo cual, se atribuye al contenido de sólidos solubles y el alto contenido de agua, que las hace frutas dulces y refrescantes.

Vanessa watermelon, luffa and cassabanana are cucurbits that have compounds with bioactive potential, that is, compounds that have beneficial effects on health. In Colombia, these fruits are underutilized due to their low popularity; making known the information on their nutritional compounds encourages their use and consumption. The objective of this study was to perform the physicochemical characterization and evaluate the effect of freeze-drying and ultrasound-assisted extraction on the vitamin C content, total polyphenols and antioxidant capacity of the parts of each fruit. The wastes generated between rinds and seeds are 28.3 % (watermelon), 68.76 % (luffa) and 25.39 % (cassabanana), these in turn presented higher total polyphenol contents and antioxidant capacity than in the pulp. The freeze-drying treatment improved the extraction % in antioxidant capacity, vitamin C and total polyphenols compared to fresh samples. On the other hand, both rind and pulp of luffa are a good source of compounds with antioxidant capacity, while watermelon and cassabanana reached a good sensory acceptance, which is attributed to the soluble solids content and the high-water content, which makes them sweet and refreshing fruits.

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Salta province, northwestern Argentina, produces blueberries for export and discards fruits with a potential quantity of bioactive compounds. These bioactive compounds have health-promoting properties that prevent or delay the appearance of chronic diseases. This study aimed to formulate blueberry microcapsules using discarded fruit, to determine and evaluate the effect of spray-drying and lyophilization on the bioactive compounds and their physical properties. Fourteen capsule prototypes were obtained by applying a randomized full factorial design with two factors: type of drying and type of wall material. The former factor had two levels (spray-drying and lyophilization) and the latter had three levels, each with defined quantities to be used, namely maltodextrin (0%, 10%, 15%, and 30%), gum Arabic (0%, 10%, 15%, and 30%), and modified starch (0%, 10%, 15%, and 30%). Spray-drying, lyophilization, total polyphenols, anthocyanins, proanthocyanidins, antioxidant activity, and the physical properties of the microcapsules were analyzed using ANOVA, PCA, and cluster analysis. Results showed significant differences between the two processes (P < 0,05), with lyophilization being better at preserving bioactive compounds. The PCA test also showed a positive association between lyophilization and bioactive compounds, while spray-drying powders were related to negative characteristics, like moisture and water activity.

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BACKGROUND: Fish oil is an important source of healthy ω-3 fatty acids to be used in functional foods. However, its autoxidation susceptibility, aroma and solubility make it difficult to use. Its encapsulation could reduce these disadvantages. This manuscript focuses on the drying stage of the encapsulation process. Its objective was to study the encapsulation of fish oil with soy proteins by emulsification and lyophilization and compare microparticles characteristics with those processed identically but spray dried. RESULTS: Microparticles with different protein/oil ratios were prepared by emulsification and lyophilization. Soy proteins encapsulated fish oil in matrix-type microcapsules masking its typical odor and oily appearance. Microparticles dried by lyophilization showed a better solid recovery but lower encapsulation efficiency than those spray dried. Increasing protein/oil mass ratio of initial formulations seemed to favor initial lipid oxidation, but these differences were not appreciated when analyzing the oxidative stability over time (measured by Rancimat test). Porous structure and large surface area of lyophilized samples would favor oxygen easy penetration and exposition to free radicals, increasing lipid oxidation over time, while spray dried microparticles showed a good oxidative stability over time, like that of free oil. CONCLUSION: Drying processes were determinants in the morphology of microcapsules, the efficiency of encapsulation and protection exerted on the oil. Although emulsifying and drying processes caused certain initial oil oxidation, soy proteins managed to mask fish oil flavors and spray dried systems showed a good perspective of oxidative stability of fish oil over time, better than that of lyophilized microparticles. © 2021 Society of Chemical Industry.

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Abstract A cationic liposomal gene delivery system comprising DOTAP, DOPE, and cholesterol was prepared and optimized. The results showed that the liposome/DNA (LP/DNA) system had spherical morphology, with a particle size of around 150 nm and zeta potential of approximately 30 mV. Cytotoxicity experiments showed that cells treated with all of the liposome carriers- with the exception of LP1-had more than 80% viability even at a weight ratio of 30. The in vitro transfection efficiency was measured using a Promega™ Luciferase Assay System. Of the tested lipoplexes, LP2/DNA showed the highest cell transfection efficiency (at a weight ratio of 10)-which was similar to or slightly lower than that of Lipofectamine® 2000 in HeLa, A549, and SPC-A1 cell lines. After freeze-drying, the cell transfection efficiency decreased slightly (P>0.05). The cell uptake mechanism study showed that LP/DNA lipoplexes mainly entered cells via clathrin-mediated and caveolin-mediated endocytic pathways. The results confirmed that LP2 has potential for use as an effective gene carrier, and provides experimental evidence to support its further development as a safe and effective gene delivery system.

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Early abortion is one of the most common complications during pregnancy. However, the frequent handling of the genital region, more precisely the vagina, which causes discomfort to patients in this abortion process due to the frequency of drug insertion, as four pills are inserted every six hours, has led to the search for alternatives to alleviate the suffering caused by this practice in patients who are already in a shaken emotional state. Hence, this work aimed to develop composites of gelatin and misoprostol, using a conventional single-dose drug delivery system. These composites were prepared by freeze/lyophilization technique, by dissolving the gelatin in distilled water, with a concentration of 2.5% (w/v), and misoprostol was incorporated into the gelatin solution at the therapeutic concentration (800 mcg). They were subsequently molded, frozen and lyophilized. The samples of the composites were then crosslinked with sodium tripolyphosphate (TPP) 1% (v/v) with respect to the gelatin mass for 5 min. The characterization techniques used were: Optical Microscopy (OM), Fourier Transformed Infrared Spectroscopy (FTIR), Thermogravimetry (TG), Swelling, Biodegradation and Cytotoxicity. In OM it was observed that the addition of the drug improved the cylindrical appearance of the compounds, in comparison with the sample that was composed of only gelatin. There was a reduction in the degree of swelling with the addition of the drug and crosslinking. The cytotoxicity test indicated the biocompatibility of the material. Based on the results obtained in these tests, the composites have therapeutic potential for uterine emptying in pregnancy failures, especially in the first trimester.

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Beet has been used as an ingredient for functional foods due to its high antioxidant activity, thanks to the betalains it contains. The effects of the addition of beet extract (liquid and lyophilized) on the physicochemical characteristics, color, antioxidant activity (AA), total betalains (TB), total polyphenols (TP), and total protein concentration (TPC) were evaluated on stirred yogurt. The treatments (T1-yogurt natural, T2-yogurt added with beet juice, T3-added extract of beet encapsulated with maltodextrin, and T4-yogurt added with extract of beet encapsulated with inulin) exhibited results with significant differences (p < 0.05). The highest TB content was observed in T2 (209.49 ± 14.91), followed by T3 (18.65 ± 1.01) and later T4 (12.96 ± 0.55). The highest AA was observed on T2 after 14 days (ABTS˙ 0.819 mM TE/100 g and DPPH˙ 0.343 mM TE/100 g), and the lowest was found on T1 at day 14 (ABTS˙ 0.526 mM TE/100 g and DPPH˙ 0.094 mM TE/100 g). A high content of TP was observed (7.13 to 9.79 mg GAE/g). The TPC varied between 11.38 to 12.56 µg/mL. The addition of beet extract significantly increased AA in yogurt, betalains being the main compounds responsible for that bioactivity.

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OBJECTIVE: Lyophilization is potentially more practical and cost-effective alternative for sperm preservation. However, there are no studies that evaluate the ultrastructure of human spermatozoa after lyophilization. Therefore, the aim of our study was to evaluate the ultrasctructure of lyophilized spermatozoa using Transmission Electron Microscopy. METHODS: From a total of 21 donated seminal samples, 30 aliquots were originated and divided into two aliquots so that one could have been submitted to cryopreservation/thaw and the other for lyophilization/rehydration. The liquefied aliquots were homogenized at room temperature. Samples assigned for cryopreservation were placed in straws and samples assigned for lyophilization were placed in the appropriate vials. Cryopreservation samples were placed at -30oC for 30 minutes subsequently for 30 minutes at vapour phase and then plunged into liquid nitrogen. Lately, were warmed in water bath at 37oC for 10 minutes followed by 10 minutes centrifugation. The pellet was resuspended and analysed in a Makler chamber. The semen vials assigned for lyophilization were loaded into a pre-fixed freeze-drying chamber. Following lyophilization, vials were removed from the freeze-drying chamber and kept at 4oC until rehydration. TEM was performed after rehydration and thawing. Sperm samples were fixed, rinsed in buffer, post fixed and dehydration was carried out in escalating concentrations of alcohol solution, acetone and then, embedding in Epon resin. Ultrathin sections were stained and examined in a Transmission Electron Microscope. RESULTS: Analysis of sperm after freezing/thawing using Transmission Electron Microscopy showed lesions to the midpiece, with some mitochondria degeneration and random rupture of plasma membrane. In the head, we identified intact plasma membrane, nucleus and acrosome, as in the flagellum all main structures remained intact including the plasma membrane, the longitudinal columns of dense fibers and the semicircular fibers. Analysis by Transmission Electron Microscopy showed that spermatozoa heads had ruptured plasma membranes, absence of acrosomes, nuclei with heterogeneous and decompressed chromatin. Mitochondria were deteriorated in the midpiece. Longitudinal columns of dense fibers were absent in the flagellum. Axonemes, in cross-sections, were disrupted with disorganized structures. CONCLUSIONS: To our knowledge, our study demonstrated, for the first time, the structure of the human spermatozoa after lyophilization using Transmission Electron Microscopy. The use of a fixed lyophilization protocol with media containing cryoprotectants might explain the damage to the structures. More studies are necessary to improve the results of sperm lyophilization. In the future, the use of lyophilization of spermatozoa might reduce the costs of fertility preservation, since there will be no need for storage space and transportation is simpler.

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Platelet-rich plasma (PRP) showed positive results in the improvement of skin aging. Lyophilized PRP can be interesting in clinical practice due to the facility to obtain many samples in a single blood collection and can be used in multiple injections. To evaluate the effect of lyophilized PRP in the treatment of skin aging, through a Phase II pilot study. Nineteen women (54 years ± 7 years) with Glogau photoaging II and III types were select for this non-randomized, split-face controlled study. They received monthly intradermal injections of lyophilized PRP and saline solution (as control) into the facial skin, during a period of 2 months. The evaluation was performed by imaging method, histological techniques, and multiphoton microscopy. Although lyophilized PRP presented 10 times the platelet baseline value (P < .0001) and growth factors in adequate levels, only saline solution showed an increase of dermis thickness (p = .0009). Collagen pre and post-application remained the same for both types of treatments. The use of lyophilized PRP by mesotherapy showed no improvement on skin aging. TRIAL REGISTRATION APPROVAL: RBR-3n9wxw, UTN U1111-1226-6093-retrospectively registered.

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Biopharmaceutical products are of great importance in the treatment or prevention of many diseases and represent a growing share of the global pharmaceutical market. The usual technology for protein synthesis (cell-based expression) faces certain obstacles, especially with 'difficult-to-express' proteins. Cell-free protein synthesis (CFPS) can overcome the main bottlenecks of cell-based expression. This review aims to present recent advances in the production process of biologic products by CFPS. First, key aspects of CFPS systems are summarized. A description of several biologic products that have been successfully produced using the CFPS system is provided. Finally, the CFPS system's ability to scale up and scale down, its main limitations and its application for biologics production are discussed.

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Unlipidated outer membrane protein 19 (U-Omp19) is a novel mucosal adjuvant in preclinical development to be used in vaccine formulations. U-Omp19 holds two main properties, it is capable of inhibiting gastrointestinal and lysosomal peptidases, increasing the amount of co-administered antigen that reaches the immune inductive sites and its half-life inside cells, and it is able to stimulate antigen presenting cells in vivo. These activities enable U-Omp19 to enhance the adaptive immune response to co-administrated antigens. To characterize the stability of U-Omp19 we have performed an extensive analysis of its physicochemical and biological properties in a 3-year long-term stability study, and under potentially damaging freeze-thawing and lyophilization stress processes. Results revealed that U-Omp19 retains its full protease inhibitor activity, its monomeric state and its secondary structure even when stored in solution for 36 months or after multiple freeze-thawing cycles. Non-enzymatic hydrolysis resulted the major degradation pathway for storage in solution at 4 °C or room temperature which can be abrogated by lyophilization yet increasing protein tendency to form aggregates. This information will play a key role in the development of a stable formulation of U-Omp19, allowing an extended shelf-life during manufacturing, storage, and shipping of a future vaccine containing this pioneering adjuvant.

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