Natural pesticide, Azadirachta indica A. Juss. (Neem), disrupted reproductive parameters of male rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Neem (Azadirachta indica A. Juss.) is native to India and belongs to the Meliaceae family. It has been used for centuries in Eastern medicine, and more recently, as a natural pesticide. Although the use of bioinsecticides is supported by organic food production, further research is needed on each formulation, particularly on their effects on organisms. AIM OF THE STUDY: This study evaluated the reproductive toxicity of an aqueous extract of neem leaves in male Wistar rats of reproductive age after eight days of exposure. MATERIALS AND METHODS: For this study, 20 rats were divided into four groups of five animals each and treated with different concentrations: Group 1 received 10,000 ppm, Group 2 received 7500 ppm, Group 3 received 5000 ppm, and Group 4 served as the control, received distilled water. The animals were observed for eight days for any clinical signs of toxicity. Semen quality, including sperm motility and viability, was analyzed. RESULTS: No significant differences in physiological changes were observed between treatments. However, sperm motility and viability were affected in a concentration-dependent manner, with the group that received the highest dose exhibiting inviable spermatozoa. CONCLUSION: The data suggested that the administered doses had low toxicity. Nevertheless, concerns regarding reproductive toxicity remain because motility and sperm viability are negatively affected at the highest dose. Although bioinsecticides are widely used in organic food production, further research on each formulation, particularly regarding their long-term effects on organisms, is required.

Gene editing in small and large animals for translational medicine: a review.

The CRISPR/Cas9 system is a simpler and more versatile method compared to other engineered nucleases such as Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs), and since its discovery, the efficiency of CRISPR-based genome editing has increased to the point that multiple and different types of edits can be made simultaneously. These advances in gene editing have revolutionized biotechnology by enabling precise genome editing with greater simplicity and efficacy than ever before. This tool has been successfully applied to a wide range of animal species, including cattle, pigs, dogs, and other small animals. Engineered nucleases cut the genome at specific target positions, triggering the cell's mechanisms to repair the damage and introduce a mutation to a specific genomic site. This review discusses novel genome-based CRISPR/Cas9 editing tools, methods developed to improve efficiency and specificity, the use of gene-editing on animal models and translational medicine, and the main challenges and limitations of CRISPR-based gene-editing approaches.

3D culture of mesenchymal stem cells from the yolk sac to generate intestinal organoid.

Organoids are in vitro models that originated from the three-dimensional culture of stem cells with the ability to reproduce part of the in vivo structural and functional specificities of body organs. Intestinal organoids have great relevance in cell therapy, as they provide more accurate information about tissue composition and architecture in relation to two-dimensional culture, in addition to serving as a study model for host interaction and drug testing. The yolk sac (YS) is a promising source of mesenchymal stem cells (MSCs), which are multipotent stem cells with self-renewal ability and potential to differentiated into mesenchymal lineages. Besides this, the YS is responsible for the formation of intestinal epithelium during embryonic development. Thus, the aim of this study was to verify if the three-dimensional in vitro culture of stem cells derived from the canine YS is capable of developing intestinal organoids. MSCs from the canine YS and gut cells were isolated and characterized, then three-dimensionally cultured in Matrigel. In both cells lineages, spherical organoids were observed and after 10 days the gut cells formed crypt-like buds and villus-like structures. Despite having the same induction of differentiation process and having the expression of intestinal markers, the MSC from the YS morphology was not in the form of crypt budding. The hypothesis is that these cells could generate structures equivalent to the intestinal organoids of the colon that other studies showed formed only spherical structures. The culture of MSC from the YS, as well as the establishment of protocols for 3D cultivation of this tissue, is relevant, as it will serve as a tool in various applications in basic and scientific biology.

Isolation and characterization of neural stem cells from fetal canine spinal cord.

Neurogenesis in adult mammals occurs mainly in the subventricular and subgranular areas of the brain, but there are also reports of its occurrence in the spinal cord. In a study on rats, neural stem cells and neuroprogenitor cells could be obtained through primary spinal cord culture, but there are no studies on these cells in canine species, to date. Dogs represent an appropriate animal model for studies on neurogenesis and neurological disorders. In addition, they are animals of great affective value, and the therapeutic use of neural stem cells can represent a breakthrough in regenerative veterinary medicine. Therefore, this study aimed to determine a protocol for the isolation, culture, and characterization of neural and neuroprogenitor stem cells derived from the spinal cord of canine fetuses. The cells were isolated from spinal cord fragments and cultured in serum-free culture medium supplemented with EGF and FGF-2 growth factors. These cells were observed daily by optical microscopy to analyze their morphological characteristics. From the third day in vitro, it was possible to observe translucent cell groupings, similar to the neurospheres, which approximately ranged from 50 µm to 200 µm at seven days in vitro. Throughout the culture period, the neurospheres developed ribbons in their periphery that migrated and communicated with other neurospheres. RT-PCR revealed that the cells expressed the characteristic genes SOX2, NESTIN, and GFAP. In addition to gene expression, the cells were phenotypically marked in the immunofluorescence assay for the proteins Nestin, GFAP, and ß-tubulin III, characterizing them as neurospheres. Our results suggest that the spinal cord may be a source of neural stem cells and neural progenitor cells in canine fetuses. These cells may be an interesting option for neurogenesis and neuroregenerative therapy studies.

A recessive variant in TFAM causes mtDNA depletion associated with primary ovarian insufficiency, seizures, intellectual disability and hearing loss.

Mitochondrial disorders are collectively common, genetically heterogeneous disorders in both pediatric and adult populations. They are caused by molecular defects in oxidative phosphorylation, failure of essential bioenergetic supply to mitochondria, and apoptosis. Here, we present three affected individuals from a consanguineous family of Pakistani origin with variable seizures and intellectual disability. Both females display primary ovarian insufficiency (POI), while the male shows abnormal sex hormone levels. We performed whole exome sequencing and identified a recessive missense variant c.694C > T, p.Arg232Cys in TFAM that segregates with disease. TFAM (mitochondrial transcription factor A) is a component of the mitochondrial replisome machinery that maintains mtDNA transcription and replication. In primary dermal fibroblasts, we show depletion of mtDNA and significantly altered mitochondrial function and morphology. Moreover, we observed reduced nucleoid numbers with significant changes in nucleoid size or shape in fibroblasts from an affected individual compared to controls. We also investigated the effect of tfam impairment in zebrafish; homozygous tfam mutants carrying an in-frame c.141_149 deletion recapitulate the mtDNA depletion and ovarian dysgenesis phenotypes observed in affected humans. Together, our genetic and functional data confirm that TFAM plays a pivotal role in gonad development and expands the repertoire of mitochondrial disease phenotypes.

Characterization of hematopoietic stem cells from the canine yolk sac.

The characterization of hematopoietic stem cells (HSC) from the canine yolk sac (cYS) can contribute to future gene therapies because it is possible to obtain information about the beginning of the development of the circulatory system through the characterization. The cYS is a likely source of HSC, which is a source of blood cell development in mammals. Studies in this field have been conducted for decades; however, interest in cellular therapy is currently at its peak with greater visibility, and these cells are a promising therapeutic tool for the treatment of diseases related to animals and humans. The aim of this study was to isolate and characterize HSC from the cYS embryos at 30 to 45 days of gestational age. Our results showed that the cYS was macroscopically located in the ventral region with a central portion and extremities. The cells in culture presented a circular morphology and cell clusters. The average cell viability was 22.55% dead cells out of 6.5 × 104 total cells. The cells were also able to form colonies on methylcellulose. Flow cytometry analysis revealed the expression of CD34, CD117, and CD45. Our results suggest that the cYS can be used as a source of hematopoietic cells, and this study is very important to understand the mechanism and development of the hematopoietic system in dogs.

Human-modified landscapes narrow the isotopic niche of neotropical birds.

Deforestation and habitat loss resulting from land use changes are some of the utmost anthropogenic impacts that threaten tropical birds in human-modified landscapes (HMLs). The degree of these impacts on birds' diet, habitat use, and ecological niche can be measured by isotopic analysis. We investigated whether the isotopic niche width, food resources, and habitat use of bird trophic guilds differed between HMLs and natural landscapes (NLs) using stable carbon (δ13C) and nitrogen isotopes (δ15N). We analyzed feathers of 851 bird individuals from 28 landscapes in the Brazilian Atlantic Forest. We classified landscapes into two groups according to the percentage of forest cover (HMLs ≤ 30%; NLs ≥ 47%), and compared the isotopic niche width and mean values of δ13C and δ15N for each guild between landscape types. The niches of frugivores, insectivores, nectarivores, and omnivores were narrower in HMLs, whereas granivores showed the opposite pattern. In HMLs, nectarivores showed a reduction of 44% in niche width, while granivores presented an expansion of 26%. Individuals in HMLs consumed more resources from agricultural areas (C4 plants), but almost all guilds showed a preference for forest resources (C3 plants) in both landscape types, except granivores. Degraded and fragmented landscapes typically present a lower availability of habitat and food resources for many species, which was reflected by the reduction in niche width of birds in HMLs. Therefore, to protect the diversity of guilds in HMLs, landscape management strategies that offer birds more diverse habitats must be implemented in tropical regions.

HEK293T Cells with TFAM Disruption by CRISPR-Cas9 as a Model for Mitochondrial Regulation.

The mitochondrial transcription factor A (TFAM) is considered a key factor in mitochondrial DNA (mtDNA) copy number. Given that the regulation of active copies of mtDNA is still not fully understood, we investigated the effects of CRISPR-Cas9 gene editing of TFAM in human embryonic kidney (HEK) 293T cells on mtDNA copy number. The aim of this study was to generate a new in vitro model by CRISPR-Cas9 system by editing the TFAM locus in HEK293T cells. Among the resulting single-cell clones, seven had high mutation rates (67-96%) and showed a decrease in mtDNA copy number compared to control. Cell staining with Mitotracker Red showed a reduction in fluorescence in the edited cells compared to the non-edited cells. Our findings suggest that the mtDNA copy number is directly related to TFAM control and its disruption results in interference with mitochondrial stability and maintenance.

Somatic feather follicle cell culture of the gallus domesticus species for creating a wild bird genetic resource bank.

The creation of a genetic resource bank of avian species aims to prevent the decline and fragmentation of wild bird populations, which in turn lead to the loss of genetic diversity and, in more serious cases, the extinction of the most threatened species. In order for the collected genetic material to be stored in a bank and useful when necessary, it is essential to improve the technique ensuring its effectiveness. Thus, our study used feather follicle cells from the domestic gallus species to standardize the technique of cell culture and subsequent cryopreservation. This study aimed to establish a protocol, in vitro, of isolation and primary culture of somatic cells derived from the feather follicle, with the purpose of establishing a cell lineage, and evaluate its viability for the biobank formation. Developing feathers of gallus domesticus were collected at 12, 21 and 34 days of age. The feathers were morphologically analyzed and then we selected the region of the calamus due to the presence of pulp for cell culture and cryopreservation. The results showed that it is possible to find cells with distinct morphology; cells in elliptical shape with central nucleus also in elliptical shape, cells with shape and round nucleus, cells compatible with the fibers of the barbules, cell agglomerates and cells adhered to the bottom of the plate with fibroblastatoid shape. After 24 hours of culture there was the presence of primary culture with 80% of confluence and after cryopreservation the average viability after freezing was 68.8%, with cellular morphologies being maintained. Therefore, we proved the isolation of somatic cells from the follicle of bird's feathers, suggesting that this is a source of great value, viable and effective for obtaining biological material for the elaboration of a biobank.

Characterization of post-edited cells modified in the TFAM gene by CRISPR/Cas9 technology in the bovine model.

Gene editing in large animal models for future applications in translational medicine and food production must be deeply investigated for an increase of knowledge. The mitochondrial transcription factor A (TFAM) is a member of the HMGB subfamily that binds to mtDNA promoters. This gene maintains mtDNA, and it is essential for the initiation of mtDNA transcription. Lately, we generated a new cell line through the disruption of the TFAM gene in bovine fibroblast cells by CRISPR/Cas 9 technology. We showed that the CRISPR/Cas9 design was efficient through the generation of heterozygous mutant clones. In this context, once this gene regulates the mtDNA replication specificity, the study aimed to determine if the post-edited cells are capable of in vitro maintenance and assess if they present changes in mtDNA copies and mitochondrial membrane potential after successive passages in culture. The post-edited cells were expanded in culture, and we performed a growth curve, doubling time, cell viability, mitochondrial DNA copy number, and mitochondrial membrane potential assays. The editing process did not make cell culture unfeasible, even though cell growth rate and viability were decreased compared to control since we observed the cells grow well when cultured in a medium supplemented with uridine and pyruvate. They also exhibited a classical fibroblastoid appearance. The RT-qPCR to determine the mtDNA copy number showed a decrease in the edited clones compared to the non-edited ones (control) in different cell passages. Cell staining with Mitotracker Green and red suggests a reduction in red fluorescence in the edited cells compared to the non-edited cells. Thus, through characterization, we demonstrated that the TFAM gene is critical to mitochondrial maintenance due to its interference in the stability of the mitochondrial DNA copy number in different cell passages and membrane potential confirming the decrease in mitochondrial activity in cells edited in heterozygosis.

Edition of Prostaglandin E2 Receptors EP2 and EP4 by CRISPR/Cas9 Technology in Equine Adipose Mesenchymal Stem Cells.

In mesenchymal stem cells (MSCs), it has been reported that prostaglandin E2 (PGE2) stimulation of EP2 and EP4 receptors triggers processes such as migration, self-renewal, survival, and proliferation, and their activation is involved in homing. The aim of this work was to establish a genetically modified adipose (aMSC) model in which receptor genes EP2 and EP4 were edited separately using the CRISPR/Cas9 system. After edition, the genes were evaluated as to if the expression of MSC surface markers was affected, as well as the migration capacity in vitro of the generated cells. Adipose MSCs were obtained from Chilean breed horses and cultured in DMEM High Glucose with 10% fetal bovine serum (FBS). sgRNA were cloned into a linearized LentiCRISPRv2GFP vector and transfected into HEK293FT cells for producing viral particles that were used to transduce aMSCs. GFP-expressing cells were separated by sorting to obtain individual clones. Genomic DNA was amplified, and the site-directed mutation frequency was assessed by T7E1, followed by Sanger sequencing. We selected 11 clones of EP2 and 10 clones of EP4, and by Sanger sequencing we confirmed 1 clone knock-out to aMSC/EP2 and one heterozygous mutant clone of aMSC/EP4. Both edited cells had decreased expression of EP2 and EP4 receptors when compared to the wild type, and the edition of EP2 and EP4 did not affect the expression of MSC surface markers, showing the same pattern in filling the scratch. We can conclude that the edition of these receptors in aMSCs does not affect their surface marker phenotype and migration ability when compared to wild-type cells.

Morphological and Molecular Analysis of In Vitro Tubular Structures from Bovine Yolk Sac-Derived MSCs.

The yolk sac is an extraembryonic membrane, of saccular form, connected to the ventral region of the embryo. It is the main source of nutrition for the embryo during the period when the placenta is not fully formed. The aim of this study was to generate tubular structures using mesenchymal stem cells from the bovine yolk sac (bYS-MSCs) and determine if these structures can be a model for in vitro vasculogenesis. The evaluation of this tissue by histochemistry revealed a strong marking of collagen fibers and PAS technique negativity. In transmission electron microscopy, cytoplasmic organelles with large nuclei were observed. The vessel formation assay on a Matrigel substrate showed that the mesenchymal cells of the yolk sac without growth factors (VEGF) are capable of forming branches, sprouting cells, and tubular structures similar to capillary blood. These tubular structures were xenotransplanted subcutaneously into the mesentery of BALB/c/nude mice; after 45 days, vascularized tissue and extensions of blood vessels around the tubular structures could be observed. Real-time PCR (qPCR) demonstrated an expression of the VEGF gene in different gestational age groups. No difference in distribution or expression was detected among groups. Our results suggest that the spontaneous formation of tubules from the yolk sac can be an experimental model to elucidate initial organogenesis and the possible formation of blood capillaries from in vitro mesenchymal cells and possible route of organoid production.

Edition of TFAM gene by CRISPR/Cas9 technology in bovine model.

The mitochondrial transcription factor A (TFAM) is a mitochondrial DNA (mtDNA) binding protein essential for the initiation of transcription and genome maintenance. Recently it was demonstrated that the primary role of TFAM is to maintain the integrity of mtDNA and that it is a key regulator of mtDNA copy number. It was also shown that TFAM plays a central role in the mtDNA stress-mediated inflammatory response. In our study, we proposed to evaluate the possibility of editing the TFAM gene by CRISPR/Cas9 technology in bovine fibroblasts, as TFAM regulates the replication specificity of mtDNA. We further attempted to maintain these cells in culture post edition in a medium supplemented with uridine and pyruvate to mimic Rho zero cells that are capable of surviving without mtDNA, because it is known that the TFAM gene is lethal in knockout mice and chicken. Moreover, we evaluated the effects of TFAM modification on mtDNA copy number. The CRISPR gRNA was designed to target exon 1 of the bovine TFAM gene and subsequently cloned. Fibroblasts were transfected with Cas9 and control plasmids. After 24 h of transfection, cells were analyzed by flow cytometry to evaluate the efficiency of transfection. The site directed-mutation frequency was assessed by T7 endonuclease assay, and cell clones were analyzed for mtDNA copy number by Sanger DNA sequencing. We achieved transfection efficiency of 51.3%. We selected 23 successfully transformed clones for further analysis, and seven of these exhibited directed mutations at the CRISPR/Cas9 targeted site. Moreover, we also found a decrease in mtDNA copy number in the gene edited clones compared to that in the controls. These TFAM gene mutant cells were viable in culture when supplemented with uridine and pyruvate. We conclude that this CRISPR/Cas9 design was efficient, resulting in seven heterozygous mutant clones and opening up the possibility to use these mutant cell lines as a model system to elucidate the role of TFAM in the maintenance of mtDNA integrity.