An ancient polymorphic regulatory region within the BDNF gene associated with obesity modulates anxiety-like behaviour in mice and humans.

Obesity and anxiety are morbidities notable for their increased impact on society during the recent COVID-19 pandemic. Understanding the mechanisms governing susceptibility to these conditions will increase our quality of life and resilience to future pandemics. In the current study, we explored the function of a highly conserved regulatory region (BE5.1) within the BDNF gene that harbours a polymorphism strongly associated with obesity (rs10767664; p = 4.69 × 10-26). Analysis in primary cells suggested that the major T-allele of BE5.1 was an enhancer, whereas the obesity-associated A-allele was not. However, CRISPR/CAS9 deletion of BE5.1 from the mouse genome (BE5.1KO) produced no significant effect on the expression of BDNF transcripts in the hypothalamus, no change in weight gain after 28 days and only a marginally significant increase in food intake. Nevertheless, transcripts were significantly increased in the amygdala of female mice and elevated zero maze and marble-burying tests demonstrated a significant increase in anxiety-like behaviour that could be reversed by diazepam. Consistent with these observations, human GWAS cohort analysis demonstrated a significant association between rs10767664 and anxiousness in human populations. Intriguingly, interrogation of the human GTEx eQTL database demonstrated no effect on BDNF mRNA levels associated with rs10767664 but a highly significant effect on BDNF-antisense (BDNF-AS) gene expression and splicing. The subsequent observation that deletion of BE5.1 also significantly reduced BDNF-AS expression in mice suggests a novel mechanism in the regulation of BDNF expression common to mice and humans, which contributes to the modulation of mood and anxiety in both species.

Expression Profile of New Gene Markers and Signaling Pathways Involved in Immunological Processes in Human Cumulus-Oophorus Cells.

The function of the immune system extends from defense against external pathogens to the recognition and elimination of mutated or dying cells, aiding elimination of malignant potential and/or maintaining homeostasis. The many cell types of the immune system secrete a broad range of factors to enable cellular signaling that is vital to physiological processes. Additionally, in the ovary, follicular selection and maturation, as well as ovulation, are directly regulated by the nearby immune cells. Additionally, ovulation and rupture of the follicle have been observed to resemble a local inflammatory response. Cells of the cumulus-oocyte complex (COC) show evolving gene expression profiles throughout the oocytes' lifespan, including genes associated with immunological processes. Analysis of these genes allows the identification of useful molecular markers, as well as highlighting gene functions and interactions in these cells. Cumulus cells were obtained from hormonally stimulated patients undergoing an in vitro fertilization procedure and studied under long-term culture conditions. The microarray technique made it possible to compare the level of CCs' gene expression on the 1st, 7th, 15th and 30th day of cultivation. Additionally, RNA microarray analysis was performed to map gene expression in these cells, associated with immunological processes and associated cytokine signaling. Subsequently, the use of DAVID software allowed us to identify the "defense response to other organism", "defense response", "defense response to virus", "cytokine secretion", "cytokine production" and "cytokine-mediated signaling pathway" GO BP terms, as well as allowing further analysis of the most differentially expressed genes associated with these processes. Of the 122 genes involved, 121 were upregulated and only one was downregulated. The seven most upregulated genes related to the abovementioned terms were ANXA3, IFIT1, HLA-DPA1, MX1, KRT8, HLA-DRA and KRT18. Therefore, genes involved in immunological defense processes are upregulated in CC cultures and could serve as useful molecular markers of growth and development in the COC, as well as the proliferation of granulosa and cumulus cells.

Molecular Mechanisms Associated with ROS-Dependent Angiogenesis in Lower Extremity Artery Disease.

Currently, atherosclerosis, which affects the vascular bed of all vital organs and tissues, is considered as a leading cause of death. Most commonly, atherosclerosis involves coronary and peripheral arteries, which results in acute (e.g., myocardial infarction, lower extremities ischemia) or chronic (persistent ischemia leading to severe heart failure) consequences. All of them have a marked unfavorable impact on the quality of life and are associated with increased mortality and morbidity in human populations. Lower extremity artery disease (LEAD, also defined as peripheral artery disease, PAD) refers to atherosclerotic occlusive disease of the lower extremities, where partial or complete obstruction of peripheral arteries is observed. Decreased perfusion can result in ischemic pain, non-healing wounds, and ischemic ulcers, and significantly reduce the quality of life. However, the progressive atherosclerotic changes cause stimulation of tissue response processes, like vessel wall remodeling and neovascularization. These mechanisms of adapting the vascular network to pathological conditions seem to play a key role in reducing the impact of the changes limiting the flow of blood. Neovascularization as a response to ischemia induces sprouting and expansion of the endothelium to repair and grow the vessels of the circulatory system. Neovascularization consists of three different biological processes: vasculogenesis, angiogenesis, and arteriogenesis. Both molecular and environmental factors that may affect the process of development and growth of blood vessels were analyzed. Particular attention was paid to the changes taking place during LEAD. It is important to consider the molecular mechanisms underpinning vessel growth. These mechanisms will also be examined in the context of diseases commonly affecting blood vessel function, or those treatable in part by manipulation of angiogenesis. Furthermore, it may be possible to induce the process of blood vessel development and growth to treat peripheral vascular disease and wound healing. Reactive oxygen species (ROS) play an important role in regulation of essential cellular signaling pathways such as cell differentiation, proliferation, migration and apoptosis. With regard to the repair processes taking place during diseases such as LEAD, prospective therapeutic methods have been described that could significantly improve the treatment of vessel diseases in the future. Summarizing, regenerative medicine holds the potential to transform the therapeutic methods in heart and vessel diseases treatment.

A Clinical Trial of Photobiomodulation Effect on Orthodontic Microscrews Stability Using a 635 nm Red Laser Light.

Background: One of the procedures enhancing implants stability can be photobiomodulation. Objective: To assess the effect of a 635 nm wavelength on orthodontic microscrews stability, survival rate, and an individual patient's pain score. Materials and methods: The study was done with 15 subjects, 30 orthodontic microscrews with a length of 10 mm and diameter 1.4 mm. Implants inserted on the right and left side of the maxilla. The implants in the test group (G1) were irradiated with the laser at palatal and buccal part of peri-implant area (two points). The diode laser (SmartM PRO; Lasotronix, Warsaw, Poland) was used with the following settings: power: 100 mW, spot size: 0.5 cm2, mean power density (irradiance): 200 mW/cm2, in continuous wave (CW) mode, energy per point: 4 J, radiant exposure: 8 J/cm2, time per point: 40 sec, the total energy dose (radiant energy) per session: 8 J in contact mode, handpiece diameter: 8 mm. Laser sessions: day of surgery and 3, 6, 9, 12, 15, 30 days later. Cumulative radiant energy was 59 J. Periotest appliance was used to assess the microscrews stability at day of surgery and 3, 6, 9, 12, 15, 30, 60 days later. Each patient received a survey for individual pain assessment (NRS-11) promptly after the implantation. Results: The analysis after 60 days revealed significantly higher secondary stability for the laser-irradiated microscrews group in comparison with control implants (G2 group) (p = 0.0037). We observed significantly higher stability in the experimental group compared with the control after 30 days (p = 0.0218). Moreover, we noted significantly higher microimplants stability for the control group in comparison with the test group, 9 days after implantation (p = 0.0374). Laser application had no effect on pain reduction noted in the first day in comparison with the control sites (p = 0.6690). No microscrews were lost in the study. Conclusions: Application of a 635 nm laser on peri-implant soft tissue increased the microscrews stability after 30 and 60 days.

CRISPR/Cas9 in Cancer Immunotherapy: Animal Models and Human Clinical Trials.

Even though chemotherapy and immunotherapy emerged to limit continual and unregulated proliferation of cancer cells, currently available therapeutic agents are associated with high toxicity levels and low success rates. Additionally, ongoing multi-targeted therapies are limited only for few carcinogenesis pathways, due to continually emerging and evolving mutations of proto-oncogenes and tumor-suppressive genes. CRISPR/Cas9, as a specific gene-editing tool, is used to correct causative mutations with minimal toxicity, but is also employed as an adjuvant to immunotherapy to achieve a more robust immunological response. Some of the most critical limitations of the CRISPR/Cas9 technology include off-target mutations, resulting in nonspecific restrictions of DNA upstream of the Protospacer Adjacent Motifs (PAM), ethical agreements, and the lack of a scientific consensus aiming at risk evaluation. Currently, CRISPR/Cas9 is tested on animal models to enhance genome editing specificity and induce a stronger anti-tumor response. Moreover, ongoing clinical trials use the CRISPR/Cas9 system in immune cells to modify genomes in a target-specific manner. Recently, error-free in vitro systems have been engineered to overcome limitations of this gene-editing system. The aim of the article is to present the knowledge concerning the use of CRISPR Cas9 technique in targeting treatment-resistant cancers. Additionally, the use of CRISPR/Cas9 is aided as an emerging supplementation of immunotherapy, currently used in experimental oncology. Demonstrating further, applications and advances of the CRISPR/Cas9 technique are presented in animal models and human clinical trials. Concluding, an overview of the limitations of the gene-editing tool is proffered.

Muscle Cell Morphogenesis, Structure, Development and Differentiation Processes Are Significantly Regulated during Human Ovarian Granulosa Cells In Vitro Cultivation.

Granulosa cells (GCs) have many functions and are fundamental for both folliculogenesis and oogenesis, releasing hormones and communicating directly with the oocyte. Long-term in vitro cultures of GCs show significant stem-like characteristics. In the current study, RNA of human ovarian granulosa cells was collected at 1, 7, 15 and 30 days of long-term in vitro culture. Understanding the process of differentiation of GCs towards different cell lineages, as well as the molecular pathways underlying these mechanisms, is fundamental to revealing other possible stemness markers of this type of cell. Identifying new markers of GC plasticity may help to understand the aetiology and recurrence of a wide variety of diseases and health conditions and reveal possible clinical applications of the ovarian tissue cells, affecting not only the reproductive ability but also sex hormone production. Granulosa cells were the subject of this study, as they are readily available as remnant material leftover after in vitro fertilisation procedures and exhibit significant stem-like characteristics in culture. The change in gene expression was investigated through a range of molecular and bioinformatic analyses. Expression microarrays were used, allowing the identification of groups of genes typical of specific cellular pathways. This candidate gene study focused on ontological groups associated with muscle cell morphogenesis, structure, development and differentiation, namely, "muscle cell development", "muscle cell differentiation", "muscle contraction", "muscle organ development", "muscle organ morphogenesis", "muscle structure development", "muscle system process" and "muscle tissue development". The results showed that the 10 most upregulated genes were keratin 19, oxytocin receptor, connective tissue growth factor, nexilin, myosin light chain kinase, cysteine and glycine-rich protein 3, caveolin 1, actin, activating transcription factor 3 and tropomyosin, while the 10 most downregulated consisted of epiregulin, prostaglandin-endoperoxide synthase 2, transforming growth factor, interleukin, collagen, 5-hydroxytryptmine, interleukin 4, phosphodiesterase, wingless-type MMTV integration site family and SRY-box 9. Moreover, ultrastructural observations showing heterogeneity of granulosa cell population are presented in the study. At least two morphologically different subpopulations were identified: large, light coloured and small, darker cells. The expression of genes belonging to the mentioned ontological groups suggest the potential ability of GCs to differentiate and proliferate toward muscle lineage, showing possible application in muscle regeneration and the treatment of different diseases.

The Stemness of Human Ovarian Granulosa Cells and the Role of Resveratrol in the Differentiation of MSCs-A Review Based on Cellular and Molecular Knowledge.

Ovarian Granulosa Cells (GCs) are known to proliferate in the developing follicle and undergo several biochemical processes during folliculogenesis. They represent a multipotent cell population that has been differentiated to neuronal cells, chondrocytes, and osteoblasts in vitro. However, progression and maturation of GCs are accompanied by a reduction in their stemness. In the developing follicle, GCs communicate with the oocyte bidirectionally via gap junctions. Together with neighboring theca cells, they play a crucial role in steroidogenesis, particularly the production of estradiol, as well as progesterone following luteinization. Many signaling pathways are known to be important throughout the follicle development, leading either towards luteinization and release of the oocyte, or follicular atresia and apoptosis. These signaling pathways include cAMP, PI3K, SMAD, Hedgehog (HH), Hippo and Notch, which act together in a complex manner to control the maturation of GCs through regulation of key genes, from the primordial follicle to the luteal phase. Small molecules such as resveratrol, a phytoalexin found in grapes, peanuts and other dietary constituents, may be able to activate/inhibit these signaling pathways and thereby control physiological properties of GCs. This article reviews the current knowledge about granulosa stem cells, the signaling pathways driving their development and maturation, as well as biological activities of resveratrol and its properties as a pro-differentiation agent.

Human Cumulus Cells in Long-Term In Vitro Culture Reflect Differential Expression Profile of Genes Responsible for Planned Cell Death and Aging-A Study of New Molecular Markers.

In the ovarian follicle, maturation of the oocyte increases in the presence of somatic cells called cumulus cells (CCs). These cells form a direct barrier between the oocyte and external environment. Thanks to bidirectional communication, they have a direct impact on the oocyte, its quality and development potential. Understanding the genetic profile of CCs appears to be important in elucidating the physiology of oocytes. Long-term in vitro culture of CCs collected from patients undergoing controlled ovarian stimulation during in vitro fertilization procedure was conducted. Using microarray expression analysis, transcript levels were assessed on day 1, 7, 15, and 30 of culture. Apoptosis and aging of CCs strictly influence oocyte quality and subsequently the outcome of assisted reproductive technologies (ART). Thus, particular attention was paid to the analysis of genes involved in programmed cell death, aging, and apoptosis. Due to the detailed level of expression analysis of each of the 133 analyzed genes, three groups were selected: first with significantly decreased expression during the culture; second with the statistically lowest increase in expression; and third with the highest significant increase in expression. COL3A1, SFRP4, CTGF, HTR2B, VCAM1, TNFRSF11B genes, belonging to the third group, were identified as potential carriers of information on oocyte quality.

The Proliferation and Differentiation of Adipose-Derived Stem Cells in Neovascularization and Angiogenesis.

Neovascularization and angiogenesis are vital processes in the repair of damaged tissue, creating new blood vessel networks and increasing oxygen and nutrient supply for regeneration. The importance of Adipose-derived Mesenchymal Stem Cells (ASCs) contained in the adipose tissue surrounding blood vessel networks to these processes remains unknown and the exact mechanisms responsible for directing adipogenic cell fate remain to be discovered. As adipose tissue contains a heterogenous population of partially differentiated cells of adipocyte lineage; tissue repair, angiogenesis and neovascularization may be closely linked to the function of ASCs in a complex relationship. This review aims to investigate the link between ASCs and angiogenesis/neovascularization, with references to current studies. The molecular mechanisms of these processes, as well as ASC differentiation and proliferation are described in detail. ASCs may differentiate into endothelial cells during neovascularization; however, recent clinical trials have suggested that ASCs may also stimulate angiogenesis and neovascularization indirectly through the release of paracrine factors.

Epigenetic Research in Stem Cell Bioengineering-Anti-Cancer Therapy, Regenerative and Reconstructive Medicine in Human Clinical Trials.

The epigenome denotes all the information related to gene expression that is not contained in the DNA sequence but rather results from chemical changes to histones and DNA. Epigenetic modifications act in a cooperative way towards the regulation of gene expression, working at the transcriptional or post-transcriptional level, and play a key role in the determination of phenotypic variations in cells containing the same genotype. Epigenetic modifications are important considerations in relation to anti-cancer therapy and regenerative/reconstructive medicine. Moreover, a range of clinical trials have been performed, exploiting the potential of epigenetics in stem cell engineering towards application in disease treatments and diagnostics. Epigenetic studies will most likely be the basis of future cancer therapies, as epigenetic modifications play major roles in tumour formation, malignancy and metastasis. In fact, a large number of currently designed or tested clinical approaches, based on compounds regulating epigenetic pathways in various types of tumours, employ these mechanisms in stem cell bioengineering.

Human Wharton's Jelly-Cellular Specificity, Stemness Potency, Animal Models, and Current Application in Human Clinical Trials.

Stem cell therapies offer a great promise for regenerative and reconstructive medicine, due to their self-renewal and differentiation capacity. Although embryonic stem cells are pluripotent, their utilization involves embryo destruction and is ethically controversial. Therefore, adult tissues that have emerged as an alternative source of stem cells and perinatal tissues, such as the umbilical cord, appear to be particularly attractive. Wharton's jelly, a gelatinous connective tissue contained in the umbilical cord, is abundant in mesenchymal stem cells (MSCs) that express CD105, CD73, CD90, Oct-4, Sox-2, and Nanog among others, and have the ability to differentiate into osteogenic, adipogenic, chondrogenic, and other lineages. Moreover, Wharton's jelly-derived MSCs (WJ-MSCs) do not express MHC-II and exhibit immunomodulatory properties, which makes them a good alternative for allogeneic and xenogeneic transplantations in cellular therapies. Therefore, umbilical cord, especially Wharton's jelly, is a promising source of mesenchymal stem cells.

Bone Regeneration, Reconstruction and Use of Osteogenic Cells; from Basic Knowledge, Animal Models to Clinical Trials.

The deterioration of the human skeleton's capacity for self-renewal occurs naturally with age. Osteoporosis affects millions worldwide, with current treatments including pharmaceutical agents that target bone formation and/or resorption. Nevertheless, these clinical approaches often result in long-term side effects, with better alternatives being constantly researched. Mesenchymal stem cells (MSCs) derived from bone marrow and adipose tissue are known to hold therapeutic value for the treatment of a variety of bone diseases. The following review summarizes the latest studies and clinical trials related to the use of MSCs, both individually and combined with other methods, in the treatment of a variety of conditions related to skeletal health. For example, some of the most recent works noted the advantage of bone grafts based on biomimetic scaffolds combined with MSC and growth factor delivery, with a greatly increased regeneration rate and minimized side effects for patients. This review also highlights the continuing research into the mechanisms underlying bone homeostasis, including the key transcription factors and signalling pathways responsible for regulating the differentiation of osteoblast lineage. Paracrine factors and specific miRNAs are also believed to play a part in MSC differentiation. Furthering the understanding of the specific mechanisms of cellular signalling in skeletal remodelling is key to incorporating new and effective treatment methods for bone disease.

Analysis of visible-light-active Sn(II)-TiO2 photocatalysts.

The influence of Sn(II) species on TiO2 is investigated. The absorption spectra of these materials are red-shifted by 115 nm to the visible region of the solar spectrum compared with P25 TiO2. This prominent red-shift is attributed to the interaction of Sn(II) 5s orbitals with the TiO2 decreasing the band gap of TiO2 by raising its valence band. The tin oxidation state and the materials electronic structure are evaluated using Mössbauer spectroscopy and valence band X-ray photoelectron spectroscopy respectively. These materials are active for sacrificial photo-generation of hydrogen in visible light.