Development of a scalable single process for producing SARS-CoV-2 RBD monomer and dimer vaccine antigens.

We have developed a single process for producing two key COVID-19 vaccine antigens: SARS-CoV-2 receptor binding domain (RBD) monomer and dimer. These antigens are featured in various COVID-19 vaccine formats, including SOBERANA 01 and the licensed SOBERANA 02, and SOBERANA Plus. Our approach involves expressing RBD (319-541)-His6 in Chinese hamster ovary (CHO)-K1 cells, generating and characterizing oligoclones, and selecting the best RBD-producing clones. Critical parameters such as copper supplementation in the culture medium and cell viability influenced the yield of RBD dimer. The purification of RBD involved standard immobilized metal ion affinity chromatography (IMAC), ion exchange chromatography, and size exclusion chromatography. Our findings suggest that copper can improve IMAC performance. Efficient RBD production was achieved using small-scale bioreactor cell culture (2 L). The two RBD forms - monomeric and dimeric RBD - were also produced on a large scale (500 L). This study represents the first large-scale application of perfusion culture for the production of RBD antigens. We conducted a thorough analysis of the purified RBD antigens, which encompassed primary structure, protein integrity, N-glycosylation, size, purity, secondary and tertiary structures, isoform composition, hydrophobicity, and long-term stability. Additionally, we investigated RBD-ACE2 interactions, in vitro ACE2 recognition of RBD, and the immunogenicity of RBD antigens in mice. We have determined that both the monomeric and dimeric RBD antigens possess the necessary quality attributes for vaccine production. By enabling the customizable production of both RBD forms, this unified manufacturing process provides the required flexibility to adapt rapidly to the ever-changing demands of emerging SARS-CoV-2 variants and different COVID-19 vaccine platforms.

Molecular reshaping of phage-displayed Interleukin-2 at beta chain receptor interface to obtain potent super-agonists with improved developability profiles.

Interleukin-2 (IL-2) engineered versions, with biased immunological functions, have emerged from yeast display and rational design. Here we reshaped the human IL-2 interface with the IL-2 receptor beta chain through the screening of phage-displayed libraries. Multiple beta super-binders were obtained, having increased receptor binding ability and improved developability profiles. Selected variants exhibit an accumulation of negatively charged residues at the interface, which provides a better electrostatic complementarity to the beta chain, and faster association kinetics. These findings point to mechanistic differences with the already reported superkines, characterized by a conformational switch due to the rearrangement of the hydrophobic core. The molecular bases of the favourable developability profile were tracked to a single residue: L92. Recombinant Fc-fusion proteins including our variants are superior to those based on H9 superkine in terms of expression levels in mammalian cells, aggregation resistance, stability, in vivo enhancement of immune effector responses, and anti-tumour effect.

FKBP25 regulates myoblast viability and migration and is differentially expressed in in vivo models of muscle adaptation.

FKBP25 (FKBP3 gene) is a dual-domain PPIase protein that consists of a C-terminal PPIase domain and an N-terminal basic tilted helix bundle (BTHB). The PPIase domain of FKBP25 has been shown to bind to microtubules, which has impacts upon microtubule polymerisation and cell cycle progression. Using quantitative proteomics, it was recently found that FKBP25 was expressed in the top 10% of the mouse skeletal muscle proteome. However, to date there have been few studies investigating the role of FKBP25 in non-transformed systems. As such, this study aimed to investigate potential roles for FKBP25 in myoblast viability, migration and differentiation and in adaptation of mature skeletal muscle. Doxycycline-inducible FKBP25 knockdown in C2C12 myoblasts revealed an increase in cell accumulation/viability and migration in vitro that was independent of alterations in tubulin dynamics; however, FKBP25 knockdown had no discernible impact on myoblast differentiation into myotubes. Finally, a series of in vivo models of muscle adaptation were assessed, where it was observed that FKBP25 protein expression was increased in hypertrophy and regeneration conditions (chronic mechanical overload and the mdx model of Duchenne muscular dystrophy) but decreased in an atrophy model (denervation). Overall, the findings of this study establish FKBP25 as a regulator of myoblast viability and migration, with possible implications for satellite cell proliferation and migration and muscle regeneration, and as a potential regulator of in vivo skeletal muscle adaptation.

Potent immunomodulatory and antitumor effect of anti-CD20-IL2no-alpha tri-functional immunocytokine for cancer therapy.

Introduction: The anti-CD20 antibody rituximab (RTX) has substantially improved outcomes of patients with B-cell lymphomas, although more efficient therapies are needed for refractory or relapsing lymphomas. An approach to increase the clinical effectiveness of anti-tumor therapy is the use of antibody-cytokine fusion proteins (immunocytokines (ICKs)) to deliver at the tumor site the antibody effector functions and cytokines that trigger anti-tumor activities. In particular, IL-2-based ICKs have shown significant results in preclinical studies but not in clinical trials due to the toxicity profile associated to high doses IL-2 and the undesired expansion of Tregs. Methods: To improve the efficacy of RTX therapy, we fused a murine (mIgG2a) or a human (hIgG1) version of RTX to a mutated IL-2 (no-alpha mutein), which has a disrupted affinity for the high affinity IL-2 receptor (IL-2R) to prevent the stimulation of Tregs and reduce the binding to endothelial cells expressing CD25, the α chain of high affinity IL-2R. Characterization of anti-CD20-IL2no-alpha ICKs was performed by SDS-PAGE, Western-blotting and SEC-HPLC and also by several functional in vitro techniques like T-cell proliferation assays, apoptosis, CDC and ADCC assays. The in vivo activity was assessed by using murine tumor cells expressing huCD20 in C57/Bl6 mice. Results: Both ICKs exhibited similar in vitro specific activity of their IL2no-alpha mutein moieties and kept CD20-binding capacity. Anti-CD20-IL2no-alpha (hIgG1) retained antibody effector functions as complement-dependent cytotoxicity and enhanced direct apoptosis, NK cell activation and antibody-dependent cellular cytotoxicity relative to RTX. In addition, both ICKs demonstrated a higher antitumor efficacy than parental molecules or their combination in an EL4-huCD20 tumor model in immunocompetent mice. Anti-CD20-IL2no-alpha (hIgG1) strongly expanded NK and CD8+ T cells but not Tregs in tumor-bearing mice. Discussion: These findings suggest that anti-CD20-IL2no-alpha could represent an alternative treatment for B cell lymphoma patients, mainly those refractory to RTX therapy.

Biology of primordial germ cells in vertebrates with emphasis in urodeles amphibians.

Primordial germ cells (PGCs) are highly specialized cells that play a relevant role in the maintenance and evolution of the species, since they create new combinations of genetic information between the organisms. Amphibians are a class of amniote vertebrates that are divided into three subclasses, the anurans (frogs and toads), the urodeles (salamanders and newts), and the gymnophiones (caecilians). The study of PGCs in amphibians has been addressed in more detail in anurans while little is known about the biology of this cell lineage in urodeles. Studies in some urodeles species have suggested that PGCs are of mesodermal origin, specifying in the lateral plate mesoderm at the late gastrula stage. With classical experiments it shown that, there is an induction of mesoderm, therefore most likely urodeles PGCs develop from unspecialized mesodermal tissue that responds to extracellular signals. However, some fundamental biological processes of PGCs such as the analysis of their specification, arrival, and colonization to the gonads, and their maintenance and differentiation into mature and fertile gametes remain to be elucidated. Therefore, knowledge about the biology of PGCs is of great importance to ensure the perpetuation of urodeles amphibians, as some species are in danger of becoming extinct.

The regulation of polyamine pathway proteins in models of skeletal muscle hypertrophy and atrophy: a potential role for mTORC1.

Polyamines have been shown to be absolutely required for protein synthesis and cell growth. The serine/threonine kinase, the mechanistic target of rapamycin complex 1 (mTORC1), also plays a fundamental role in the regulation of protein turnover and cell size, including in skeletal muscle, where mTORC1 is sufficient to increase protein synthesis and muscle fiber size, and is necessary for mechanical overload-induced muscle hypertrophy. Recent evidence suggests that mTORC1 may regulate the polyamine metabolic pathway, however, there is currently no evidence in skeletal muscle. This study examined changes in polyamine pathway proteins during muscle hypertrophy induced by mechanical overload (7 days), with and without the mTORC1 inhibitor, rapamycin, and during muscle atrophy induced by food deprivation (48 h) and denervation (7 days) in mice. Mechanical overload induced an increase in mTORC1 signaling, protein synthesis and muscle mass, and these were associated with rapamycin-sensitive increases in adenosylmethione decarboxylase 1 (Amd1), spermidine synthase (SpdSyn), and c-Myc. Food deprivation decreased mTORC1 signaling, protein synthesis, and muscle mass, accompanied by a decrease in spermidine/spermine acetyltransferase 1 (Sat1). Denervation, resulted increased mTORC1 signaling and protein synthesis, and decreased muscle mass, which was associated with an increase in SpdSyn, spermine synthase (SpmSyn), and c-Myc. Combined, these data show that polyamine pathway enzymes are differentially regulated in models of altered mechanical and metabolic stress, and that Amd1 and SpdSyn are, in part, regulated in a mTORC1-dependent manner. Furthermore, these data suggest that polyamines may play a role in the adaptive response to stressors in skeletal muscle.

Interaction between oocytes, cortical germ cells and granulosa cells of the mouse and bat, following the dissociation-re-aggregation of adult ovaries.

It is widely accepted that the oocyte plays a very active role in promoting the growth of the follicle by directing the differentiation of granulosa cells and secreting paracrine growth factors. In turn, granulosa cells regulate the development of the oocytes, establishing close bidirectional communication between germ and somatic cells. The presence of cortical cells with morphological characteristics, similar to primordial germ cells that express specific germline markers, stem cells and cell proliferation, known as adult cortical germ cells (ACGC) have been reported in phyllostomid bats. Using magnetic cell separation techniques, dissociation-cellular re-aggregation and organ culture, the behaviour of oocytes and ACGC was analyzed by interacting in vitro with mouse ovarian cells. Bat ACGC was mixed with disaggregated ovaries from a transgenic mouse that expressed green fluorescent protein. The in vitro reconstruction of the re-aggregates was evaluated. We examined the viability, integration, cellular interaction and ovarian morphogenesis by detecting the expression of Vasa, pH3, Cx43 and Laminin. Our results showed that the interaction between ovarian cells is carried out in the adult ovary of two species, without them losing their capacity to form follicular structures, even after having been enzymatically dissociated.

Dietary protein-bound or free amino acids differently affect intestinal morphology, gene expression of amino acid transporters, and serum amino acids of pigs exposed to heat stress.

Pigs exposed to heat stress (HS) increase body temperature in which can damage the intestinal epithelia and affect the absorption and availability of amino acids (AA). Protein digestion and metabolism further increase body temperature. An experiment was conducted with six pairs of pigs (of 47.3 ± 1.3 kg initial body weight) exposed to natural HS to assess the effect of substituting dietary protein-bound AA by free AA on morphology and gene expression of intestinal epithelial and serum concentration (SC) of free AA. Treatments were: high protein, 21.9% crude protein (CP) diet (HShp) and low protein, 13.5% CP diet supplemented with crystalline Lys, Thr, Met, Trp, His, Ile, Leu, Phe, and Val (HSaa). The HShp diet met or exceeded all AA requirements. The HSaa diet was formulated on the basis of ideal protein. Pigs were fed the same amount at 0700 and 1900 hours during the 21-d study. Blood samples were collected at 1700 hours (2.0 h before the evening meal), 2030 hours, and 2130 hours (1.5 and 2.5 h after the evening meal). At the end, all pigs were sacrificed to collect intestinal mucosa and a 5-cm section from each segment of the small intestine from each pig. Villi measures, expression of AA transporters (y+L and B0) in mucosa, and SC of AA were analyzed. Ambient temperature fluctuated daily from 24.5 to 42.6 °C. Weight gain and G.F were not affected by dietary treatment. Villi height tended to be larger (P ≤ 0.10) and the villi height:crypt depth ratio was higher in duodenum and jejunum of pigs fed the HSaa diet (P < 0.05). Gene expression of transporter y+L in jejunum tended to be lower (P < 0.10) and transporter B0 in the ileum was lower (P < 0.05) in HSaa pigs. Preprandial (1700 hours) SC of Arg, His, Ile, Leu, Thr, Trp, and Val was higher (P < 0.05), and Phe tended to be higher (P < 0.10) in HShp pigs. At 2030 hours (1.5 h postprandial), serum Lys, Met, and Thr were higher in the HSaa pigs (P < 0.05). At 2130 hours (2.5 h), Arg, His, Ile, Phe, and Trp were lower (P < 0.05); Met was higher (P < 0.05); and Lys tended to be higher (P < 0.10) in HSaa pigs. In conclusion, feeding HS pigs with low protein diets supplemented with free AA reduces the damage of the intestinal epithelia and seems to improve its absorption capacity, in comparison with HS pigs fed diets containing solely protein-bound AA. This information is useful to formulate diets that correct the reduced AA consumption associated with the decreased voluntary feed intake of pigs under HS.

La terapia familiar como vía de la resignificación en la enfermedad / Family therapy as a way to redefine disease

Se realizó un rastreo bibliográfico sobre la reacomodación de las dinámicas internas en familias que tienen un infante en cuidados paliativos, haciendo hincapié en la importancia de la terapia familiar para entender la enfermedad y resignificarla mediante la técnica narrativa de la metáfora. Teniendo en cuenta lo anterior, cuando llega una crisis significativa al núcleo familiar, como una enfermedad, esta cambia la dinámica familiar, puesto que obliga a movilizarse de manera inesperada para buscar posibles soluciones a la situación que aqueja. En muchos casos, la familia recurre a la terapia familiar como estrategia para fortalecer sus recursos, y la metáfora como herramienta narrativa, es uno de los recursos que se usa para ayudar a resignificar la enfermedad. Ahora bien, en cuanto al rastreo bibliográfico, fue la base del texto reflexivo construido, ya que en el rastreo se evidenció que en la terapia familiar no se encuentra un abordaje específico en los procesos de salud que requieren acompañamiento paliativo, por lo que se hace necesario visibilizar la utilidad de la terapia familiar en estos procesos. Como conclusión, se presenta el uso de la metáfora como posible vía de trabajo con las familias que están pasando por una situación de enfermedad, puesto que facilita la externalización del problema y logran así nuevas narrativas orientadas a la resignificación.

A literature review was carried out on the rearrangement of inner dynamics in families with an infant in palliative care, emphasizing the importance of family therapy to understand the disease and resignify it through the narrative technique of metaphor. Considering the above, when a significant crisis, such as a disease, reaches the family core, family dynamics change since it forces unexpected mobilization to find possible solutions to the situation. In many cases, the family uses family therapy as a strategy to strengthen their resources, and the metaphor, as a narrative tool, is one of the resources used to help resignify the disease. Bibliographic review was the basis of the reflexive text constructed, since the review showed that in family therapy there is no specific approach in health processes that require palliative accompaniment, reason why it is necessary to draw attention to the usefulness of family therapy in this type of processes. In conclusion, the use of metaphor is presented as a possible way of addressing families who are going through a situation of disease, since it facilitates the externalization of the problem and the achievement of new narratives aimed at redefinition of the disease.

Enhancing surface coil sensitive volume with hybridized electric dipoles at 17.2 T.

Preclinical MR applications at 17.2 T can require field of views on the order of a few square centimeters. This is a challenging task as the proton Larmor frequency reaches 730 MHz. Most of the protocols at such frequencies are performed with surface transceiver coils for which the sensitive volume and the signal to noise ratio (SNR) is given by their size. Here we propose an approach based on metamaterials in order to enhance the sensitive volume of a commercial surface coil for small animal imaging at 17.2 T. We designed a passive resonator composed of four hybridized electric dipoles placed onto the floor of the MRI bed. Combining numerical and experimental results on a phantom and in vivo, we demonstrate a 20% increase of the sensitive volume in depth and 25% along the rostro-caudal axis while maintaining more than 85% of the local SNR right beneath the surface coil plane. Moreover, our solution gives the ability to double the average SNR in the region between 1.2 and 2 cm away from the loop using a single layer of 1 mm thick metallic wires easy to design and manufacture.

Association of the Vascular Endothelial Growth Factor Gene Polymorphism +936 C/T with Diabetic Neuropathy in Patients with Type 2 Diabetes Mellitus.

BACKGROUND: Peripheral neuropathy is one of the most common late complications of diabetes. Vascular endothelial growth factor (VEGF) gene polymorphisms have been associated with the development of peripheral neuropathy in different populations of patients with type 2 diabetes mellitus (DM2). OBJECTIVE: To analyze the prevalence of the +936 C/T VEGF gene polymorphism among patients with DM2 with and without peripheral neuropathy. STUDY DESIGN AND METHODOLOGY: 218 unrelated DM2 patients, 90 with and 128 without peripheral neuropathy were genotyped for the +936 C/T VEGF gene polymorphism using PCR amplification followed by restriction length polymorphism analysis. RESULTS: The CC homozygous VEGF+936 C/T (rs3025039) was the predominant genotype in DM2 patients with peripheral neuropathy, whereas the predominant genotype in patients without neuropathy was the heterozygous C/T. No statistical association was found between genotype distribution and the presence of neuropathy (p = 0.063). The distribution of the genotypes according to the dominant (CC vs. CT + TT) and recessive (TT vs. CT + CC) models showed that the homozygous CC and TT genotypes, respectively, are not risk factors for neuropathy. The CT genotype conferred a protective effect as seen in the over-dominant model (CT vs. CC + TT) (OR = 0.52; 95% CI = 0.300-0.90; p = 0.019). CONCLUSION: We conclude that the VEGF+936 C/T (rs3025039) gene polymorphisms are related to peripheral neuropathy in Mexican DM2 patients, with the heterozygous genotype potentially conferring a protective effect.

Wireless coils based on resonant and nonresonant coupled-wire structure for small animal multinuclear imaging.

Earlier work on RF metasurfaces for preclinical MRI has targeted applications such as whole-body imaging and dual-frequency coils. In these studies, a nonresonant loop was used to induce currents into a metasurface that was operated as a passive inductively powered resonator. However, as we show in this study, the strategy of using a resonant metasurface reduces the impact of the loop on the global performance of the assembled coil. To mitigate this deficiency, we developed a new approach that relies on the combination of a commercial surface coil and a coupled-wire structure operated away from its resonance. This strategy enables the extension of the sensitive volume of the surface coil while maintaining its local high sensitivity without any hardware modification. A wireless coil based on a two parallel coupled-wire structure was designed and electromagnetic field simulations were carried out with different levels of matching and coupling between both components of the coil. For experimental characterization, a prototype was built and tested at two frequencies, 300 MHz for 1 H and 282.6 MHz for 19 F at 7 T. Phantom and in vivo MRI experiments were conducted in different configurations to study signal and noise figures of the structure. The results showed that the proposed strategy improves the overall sensitive volume while simultaneously maintaining a high signal-to-noise ratio (SNR). Metasurfaces based on coupled wires are therefore shown here as promising and versatile elements in the MRI RF chain, as they allow customized adjustment of the sensitive volume as a function of SNR yield. In addition, they can be easily adapted to different Larmor frequencies without loss of performance.

Logros y desafíos de la red universitaria REDMA: por la sostenibilidad ambiental de la educación superior en Cuba / Achievements and challenges of the REDMA university network: for the environmental sustainability of higher education in Cuba

Entre los temas más citados que hacen parte de las preocupaciones del mundo actual están la sostenibilidad ambiental y el desarrollo, no solo desde la reflexión de teóricos, humanistas, políticos y otros, sino también desde las agendas y agencias internacionales que los han posicionado como prioridad básica a ser atendida como salvaguarda de la vida en el planeta, en este sentido, la internacionalización de la economía y de los problemas socioculturales como la crisis ambiental y de valores, se tornan en asuntos medulares a considerar en la formación de profesionales. Desde la Educación Superior cubana, el logro de los fines mencionados está estrechamente relacionado con la capacidad de movilización colectiva y de proyección responsable de acciones que ofrece el trabajo en redes, de ahí que el objetivo de la presente disertación sea exponer los principales avances y resultados que ofrece hoy la Red de Medio Ambiente del MES (REDMA), en sus más de 20 años de conformada donde, desde la investigación y el intercambio científico, se integran procesos y políticas en la gestión del conocimiento y la innovación en favor del medioambiente, la prevención de riesgos y peligros, y la adaptación al cambio climático; las vías utilizadas para la compilación y selección de las mejores experiencias fueron el registro de información y la sistematización; los resultados expuestos evidencian sus contribuciones a la formación integral y multifacética de profesionales comprometidos con el desarrollo económico-social y sostenible del país.

Among the most cited issues that are part of the concerns of today's world are environmental sustainability and development, not only from the reflection of theorists, humanists, politicians and others, but also from international agendas and agencies which have positioned them as a basic priority to be addressed as a safeguard of life on the planet. In this sense, the internationalization of the economy and socio-cultural problems, such as the environmental and values crises, become core issues to be considered in the training of professionals. From the Cuban Higher Education, the achievement of the aforementioned purposes is closely related to the capacity of collective mobilization and responsible projection of actions offered by networking. Hence, the objective of this reflection is to present the main advances and results offered today by the Environment Network of the MES (REDMA), in its more than twenty years of formation where, from research and scientific exchange, processes and policies are integrated into knowledge management and innovation in favor of the environment, the prevention of risks and dangers, and the adaptation to climate change. The routes used for the compilation and selection of the best experiences were the registration of information and systematization. The exposed results show their contributions to the comprehensive and multifaceted training of professionals committed to the economic-social sustainable development of the country.

Novel HLA class I associations with HIV-1 control in a unique genetically admixed population.

Associations between HLA class I alleles and HIV progression in populations exhibiting Amerindian and Caucasian genetic admixture remain understudied. Using univariable and multivariable analyses we evaluated HLA associations with five HIV clinical parameters in 3,213 HIV clade B-infected, ART-naïve individuals from Mexico and Central America (MEX/CAM cohort). A Canadian cohort (HOMER, n = 1622) was used for comparison. As expected, HLA allele frequencies in MEX/CAM and HOMER differed markedly. In MEX/CAM, 13 HLA-A, 24 HLA-B, and 14 HLA-C alleles were significantly associated with at least one clinical parameter. These included previously described protective (e.g. B*27:05, B*57:01/02/03 and B*58:01) and risk (e.g. B*35:02) alleles, as well as novel ones (e.g. A*03:01, B*15:39 and B*39:02 identified as protective, and A*68:03/05, B*15:30, B*35:12/14, B*39:01/06, B*39:05~C*07:02, and B*40:01~C*03:04 identified as risk). Interestingly, both protective (e.g. B*39:02) and risk (e.g. B*39:01/05/06) subtypes were identified within the common and genetically diverse HLA-B*39 allele group, characteristic to Amerindian populations. While HLA-HIV associations identified in MEX and CAM separately were similar overall (Spearman's rho = 0.33, p = 0.03), region-specific associations were also noted. The identification of both canonical and novel HLA/HIV associations provides a first step towards improved understanding of HIV immune control among unique and understudied Mestizo populations.

Expression of the Sox9, Foxl2, Vasa, and TRPV4 genes in the ovaries and testes of the Morelet's crocodile, Crocodylus moreletii.

The Sox9 gene is important for determining sex in vertebrates, as well as for maintaining testis morphology and fertility during adult life. In the same way, Vasa is an important gene for the maintenance of the germinal lineage and has been highly conserved throughout evolution, as it is expressed in germ cells of both vertebrates and invertebrates. In the particular case of crocodiles, the expression of Sox9 during gonadal morphogenesis and in the testes of 3-month-old Alligator mississippiensis has been studied. However, it is interesting to carry out studies on other species of crocodiles in relation to their particular mechanism for sex determination influenced by temperature. In this work, we investigated the expression of the Sox9, Vasa, Foxl2, and TRPV4 genes in the ovaries and testes of 5-year-old juvenile crocodiles from Crocodylus moreletii. As expected, Sox9 expression was found in males, but surprisingly, it was also found in females. For the first time, the expression of Vasa was reported in spermatogonia, oogonia, and oocytes of 5-year-old crocodiles. Foxl2 is important for the development and maintenance of the ovary during adult life in vertebrates; moreover, Foxl2 protein and transcripts are both highly expressed in the ovaries compared to the testes. A possible upstream regulator of the Sox9 gene in reptiles has not yet been discovered; as such, the expression of the TRPV4 ion channel was evaluated. The TRPV4 ion channel was expressed in the cytoplasm of Sertoli and follicular cells and was therefore proposed as a possible regulator of SOX9.

Neo-oogenesis in mammals.

Recently, the existence of a mechanism for neo-oogenesis in the ovaries of adult mammals has generated much controversy within reproductive biology. This mechanism, which proposes that the ovary has cells capable of renewing the follicular reserve, has been described for various species of mammals. The first evidence was found in prosimians and humans. However, these findings were not considered relevant because the predominant dogma for reproductive biology at the time was that of Zuckerman. This dogma states that female mammals are born with finite numbers of oocytes that decline throughout postnatal life. Currently, the concept of neo-oogenesis has gained momentum due to the discovery of cells with mitotic activity in adult ovaries of various mammalian species (mice, humans, rhesus monkeys, domestic animals such as pigs, and wild animals such as bats). Despite these reports, the concept of neo-oogenesis has not been widely accepted by the scientific community, generating much criticism and speculation about its accuracy because it has been impossible to reproduce some evidence. This controversy has led to the creation of two positions: one in favour of neo-oogenesis and the other against it. Various animal models have been used in support of both camps, including both classic laboratory animals and domestic and wild animals. The aim of this review is to critically present the current literature on the subject and to evaluate the arguments pro and contra neo-oogenesis in mammals.

Gonadal morphogenesis and establishment of the germline in the phyllostomid bat Sturnira lilium.

In vertebrates such as the mouse and the human, primordial germ cells (PGCs) arise at the base of the allantois and are carried to the epithelium of the posterior intestine, to later migrate to the primordial gonads. In the case of bats, almost nothing is known about this process. To clarify the dynamics of PGCs during gonadal morphogenesis in the phyllostomid bat Sturnira lilium, the proteins for the Ddx4, Sox9 and Mis genes were detected in the gonads of embryos at different stages of development. We identified 15 stages (St) of embryonic development in Sturnira lilium. We found that the formation of the genital ridge and the establishment of the undifferentiated gonad take place between stages 11 and 14. The onset of morphological differentiation in the gonad is first detected in the male gonads at St17. The first PGCs in meiosis are detected in the ovary at St19, whereas in the testicles, the PGCs were in mitotic arrest. Structural changes leading to testicular and ovarian development in Sturnira lilium are observed to be similar to those described for the mouse; however, differences will be established concerning the time taken for these processes to occur.

Alternative Access to Functionalized 2,8-Ethanonoradamantane Derivatives.

7a-(Methoxycarbonyl)-N-methyl-1,3a,5,6,7,7a-hexahydro-4H-1,4,6-(epiethane[1,1,2]triyl)indene-4,9-dicarboximide has been prepared through a modification of a previous synthetic sequence, in which the benzyloxymethyl hydroxyl protecting group has been replaced by methoxymethyl, to avoid the apparent formation of a benzyl ester derivative as a side product. The overall yield of the new synthetic sequence is comparable to the previous one. Two advantages of the new procedure are: (a) no benzyl ester was formed and (b) a stereoisomeric mixture of syn- and anti-alcohols at the beginning of the synthetic sequence could be separated and the rest of the synthesis could be carried out with the main syn-stereoisomer instead of the corresponding stereoisomeric mixture as it was the case in the previous process. Additionally, several functional 2,8-ethanonoradamantane derivatives have been prepared.

In vitro functional assessment of natural HIV-1 group M Vpu sequences using a universal priming approach.

The HIV-1 accessory protein Vpu exhibits high inter- and intra- subtype genetic diversity that may influence Vpu function and possibly contribute to HIV-1 pathogenesis. However, scalable methods to evaluate genotype/phenotype relationships in natural Vpu sequences are limited, particularly those expressing the protein in CD4+ T-cells, the natural target of HIV-1 infection. A major impediment to assay scalability is the extensive genetic diversity within, and immediately upstream of, Vpu's initial 5' coding region, which has necessitated the design of oligonucleotide primers specific for each individual HIV-1 isolate (or subtype). To address this, we developed two universal forward primers, located in relatively conserved regions 38 and 90 bases upstream of Vpu, and a single universal reverse primer downstream of Vpu, which are predicted to cover the vast majority of global HIV-1 group M sequence diversity. We show that inclusion of up to 90 upstream bases of HIV-1 genomic sequence does not significantly influence in vitro Vpu expression or function when a Rev/Rev Response Element (RRE)-dependent expression system is used. We further assess the function of four diverse HIV-1 Vpu sequences, revealing reproducible and significant differences between them. Our approach represents a scalable option to measure the in vitro function of genetically diverse natural Vpu isolates in a CD4+ T-cell line.

Effect of fish oil on glutathione redox system in multiple sclerosis.

UNLABELLED: Multiple sclerosis (MS) is a chronic, inflammatory and autoimmune disease of the central nervous system. Dysregulation of glutathione homeostasis and alterations in glutathione-dependent enzyme activities are implicated in the induction and progression of MS. Evidence suggests that Omega-3 polyunsaturated fatty acids (PUFAs) have anti-inflammatory, antioxidant and neuroprotective effects. The aim of the present work was to evaluate the effect of fish oil on the activity of glutathione reductase (GR), content of reduced and oxidized glutathione, and GSH/GSSG ratio in MS. 50 patients with relapsing-remitting MS were enrolled. The experimental group received orally 4 g/day of fish oil for 12 months. Fish oil supplementation resulted in a significant increase in n-3 fatty acids and a decrease n-6 fatty acids. No differences in glutathione reductase activity, content of reduced and oxidized glutathione, and GSH/GSSG ratio were found. CONCLUSION: Glutathione reductase activity was not significantly different between the groups; however, fish oil supplementation resulted in smaller increase in GR compared with control group, suggesting a possible effect on antioxidant defence mechanisms.