The characteristics and prognostic significance of histone H1 expression in breast cancer.

Histone H1 (H.H1) is involved in chromatin organisation and gene regulation and is overexpressed in many malignant tumours, including breast cancer (BC). This study proposed and evaluated the prognostic role of H.H1 expression in BC. H.H1 mRNA expression was evaluated in publicly available BC dataset bc-GenExMiner database (n=4421). H.H1 protein expression was assessed immunohistochemically in a well-characterised early-stage BC cohort (n=1311), and associations with clinicopathological data and survival outcomes were evaluated. At the mRNA level, there was a significant association between high H.H1 mRNA and basal-like BC subtype and with poor outcome. The association with shorter survival was observed in the whole cohort and in the basal-like class. H.H1 protein expression was detected in both tumour cells and surrounding stroma. Total expression was detected in 72% of the cases, including 28% in tumour cell nuclei and 44% in the stroma. There was strong association between high tumour H.H1 expression and triple-negative BC (TNBC) subtype (p=0.007) and with shorter survival (p=0.019), independent of other variables including tumour size, histologic tumour grade, and lymph node status. H.H1 expression is associated with poor prognosis in BC. Given poor prognostic role of H.H1 in TNBC, it may represent a potential therapeutic target for patients with this aggressive disease.

Immunohistochemical Investigation into Protein Expression Patterns of FOXO4, IRF8 and LEF1 in Canine Osteosarcoma.

Osteosarcoma (OSA) is the most common type of primary bone malignancy in people and dogs. Our previous molecular comparisons of canine OSA against healthy bone resulted in the identification of differentially expressed protein-expressing genes (forkhead box protein O4 (FOXO4), interferon regulatory factor 8 (IRF8), and lymphoid enhancer binding factor 1 (LEF1)). Immunohistochemistry (IHC) and H-scoring provided semi-quantitative assessment of nuclear and cytoplasmic staining alongside qualitative data to contextualise staining (n = 26 patients). FOXO4 was expressed predominantly in the cytoplasm with significantly lower nuclear H-scores. IRF8 H-scores ranged from 0 to 3 throughout the cohort in the nucleus and cytoplasm. LEF1 was expressed in all patients with significantly lower cytoplasmic staining compared to nuclear. No sex or anatomical location differences were observed. While reduced levels of FOXO4 might indicate malignancy, the weak or absent protein expression limits its primary use as diagnostic tumour marker. IRF8 and LEF1 have more potential for prognostic and diagnostic uses and facilitate further understanding of their roles within their respective molecular pathways, including Wnt/beta-catenin/LEF1 signalling and differential regulation of tumour suppressor genes. Deeper understanding of the mechanisms involved in OSA are essential contributions towards the development of novel diagnostic, prognostic, and treatment options in human and veterinary medicine contexts.

Modulation of the pre-metastatic bone niche: molecular changes mediated by bone-homing prostate cancer extracellular vesicles.

Prostate cancer (PCa) is a leading male malignancy worldwide, often progressing to bone metastasis, with limited curative options. Extracellular vesicles (EVs) have emerged as key players in cancer communication and metastasis, promoting the formation of supportive microenvironments in distant sites. Our previous studies have highlighted the role of PCa EVs in modulating osteoblasts and facilitating tumor progression. However, the early pre-metastatic changes induced by PCa EVs within the bone microenvironment remain poorly understood. To investigate the early effects of repeated exposure to PCa EVs in vivo, mimicking EVs being shed from the primary tumor, PCa EVs isolated from cell line PC3MLuc2a were fluorescently labelled and repeatedly administered via tail vein injection to adult CD1 NuNu male mice for a period of 4 weeks. In vivo imagining, histological analysis and gene expression profiling were performed to assess the impact of PCa EVs on the bone microenvironment. We demonstrate for the first time that PCa EVs home to both bone and lymph nodes following repeated exposures. Furthermore, the accumulation of EVs within the bone leads to distinct molecular changes indicative of disrupted bone homeostasis (e.g., changes to signaling pathways such as Paxillin p = 0.0163, Estrogen Receptor p = 0.0271, RHOA p = 0.0287, Ribonucleotide reductase p = 0.0307 and ERK/MAPK p = 0.0299). Changes in key regulators of these pathways were confirmed in vitro on human osteoblasts. In addition, our data compares the known gene signature of osteocytes and demonstrates a high proportion of overlap (52.2%), suggesting a potential role for this cell type in response to PCa EV exposure. No changes in bone histology or immunohistochemistry were detected, indicating that PCa EV mediated changes were induced at the molecular level. This study provides novel insights into the alterations induced by PCa EVs on the bone microenvironment. The observed molecular changes indicate changes in key pathways and suggest a role for osteocytes in these EV mediated early changes to bone. Further research to understand these early events may aid in the development of targeted interventions to disrupt the metastatic cascade in PCa.

Quantitative expression of oestrogen receptor in breast cancer: Clinical and molecular significance.

BACKGROUND: Oestrogen receptor (ER) positive breast cancer (BC) patients are eligible for endocrine therapy (ET), regardless of ER immunohistochemical expression level. There is a wide spectrum of ER expression and the response to ET is not uniform. This study aimed to assess the clinical and molecular consequences of ER heterogeneity with respect to ET-response. METHODS: ER expression, categorised by percentage and staining intensity in a large BC cohort (n = 7559) was correlated with clinicopathological parameters and patient ET response. The Cancer Genome Atlas Data BC cohort (n = 1047) was stratified by ER expression and transcriptomic analysis completed to better understand the molecular basis of ER heterogeneity. RESULTS: The quantitative proportional increase in ER expression was positively associated with favourable prognostic parameters. Tumours with 1-9% ER expression were characteristically similar to ER-negative (<1%) tumours. Maximum ET-response was observed in tumours with 100% ER expression, with responses significantly different to tumours exhibiting ER at < 100% and significantly decreased survival rates were observed in tumours with 50% and 10% of ER expression. The Histochemical-score (H-score), which considers both staining intensity and percentage, added significant prognostic value over ER percentage alone with significant outcome differences observed at H-scores of 30, 100 and 200. There was a positive correlation between ER expression and ESR1 mRNA expression and expression of ER-regulated genes. Pathway analysis identified differential expression in key cancer-related pathways in different ER-positive groups. CONCLUSION: ET-response is statistically proportionally related to ER expression with significant differences observed at 10%, 50% and 100%. The H-score adds prognostic and predictive information.

Characterisation of luminal and triple-negative breast cancer with HER2 Low protein expression.

BACKGROUND: Breast cancer (BC) expressing low levels of human epidermal growth factor receptor 2 (HER2 Low) is an emerging category that needs further refining. This study aims to provide a comprehensive clinico-pathological and molecular profile of HER2 Low BC including response to therapy and patient outcome in the adjuvant and neoadjuvant settings. METHODS: Two different independent and well-characterised BC cohorts were included. Nottingham cohort (A) (n = 5744) and The Cancer Genome Atlas (TCGA) BC cohort (B) (n = 854). The clinical, molecular, biological and immunological profile of HER2 Low BC was investigated. Transcriptomic and pathway enrichment analyses were performed on the TCGA BC cohort and validated through next-generation sequencing in a subset of Nottingham cases. RESULTS: Ninety percent of HER2 Low tumours were hormone receptor (HR) positive (HR+), enriched with luminal intrinsic molecular subtype, lacking significant expression of HER2 oncogenic signalling genes and of favourable clinical behaviour compared to HER2 negative (HER2-) BC. In HR+ BC, no significant prognostic differences were detected between HER2 Low and HER2- tumours. However, in HR- BC, HER2 Low tumours were less aggressive with longer patient survival. Transcriptomic data showed that the majority of HR- /HER2 Low tumours were of luminal androgen receptor (LAR) intrinsic subtype, enriched with T-helper lymphocytes, activated dendritic cells and tumour associated neutrophils, while most HR-/HER2- tumours were basal-like, enriched with tumour associated macrophages. CONCLUSION: HER2 Low BC is mainly driven by HR signalling in HR+ tumours. HR-/HER2 Low tumours tend to be enriched with LAR genes with a unique immune profile.

Characterizing Two New Henneguya Species in the Respiratory Organs of African Sharptooth Catfish.

Henneguya species are myxozoans, a suborder of Cnidaria, which can affect the gills and extrarespiratory organs of the African sharptooth catfish, Clarias gariepinus. This research describes natural infection-induced histological alterations caused by the Henneguya species present. The Henneguya species were also identified molecularly using DNA sequenced from infected tissue cysts, and phylogenetically analyzed. Clinical investigations revealed cyst-like nodules on the fish gill filaments and extrarespiratory organs. Within a milky fluid inside the cysts were several Henneguya-like spores. Henneguya sp. infested 27.5% of the fish, with the highest prevalence in the gills compared to the extrarespiratory organs. The Henneguya species parasitized the gill and the dendritic tissues, resulting in histopathological characteristics. The plasmodia's developmental stages resulted in destructive damage which manifested as marked necrosis, which was replaced by a focal aggregation of inflammatory cells. Amplification of the 18S ribosomal DNA from the fish parasites was followed by sequencing, which confirmed their identities as new species Henneguya qenabranchiae n. sp. and Henneguya qenasuprabranchiae n. sp. with 99.53 and 99.64% identities, respectively, to Henneguya sp. 1 HS-2015. The two C. gariepinus myxozoans shared some characteristics based on morphologic and phylogenetic analysis as previously published, where it was proposed that they were a sister lineage to Henneguya species in Egypt, and it is now proposed that they are new species.

Encapsulated papillary carcinoma of the breast: does it have a native basement membrane?

BACKGROUND: Encapsulated papillary carcinoma (EPC) is surrounded by a thick fibrous capsule-like structure, which is interpreted as a thickened basement membrane (BM). This study aimed to describe the geometric characteristics of the EPC capsule and to refine whether it is an expansion of the BM or a stromal reactive process. MATERIAL AND METHODS: In all, 100 cases were divided into four groups: EPC, ductal carcinoma in situ (DCIS), normal breast tissue and invasive tumours, with an additional encapsulated papillary thyroid carcinoma (EPTC) control group. Representative slides from each case were stained with picrosirius red (PSR) stain and examined using polarised microscopy. Images were analysed using ImageJ, CT-FIRE, and Curve align image analysis programmes. RESULTS: Compared to the normal and DCIS BM, the EPC group showed a significant increase of collagen fibre width, straightness, and density, and a decrease of fibre length. The EPC capsule showed less alignment of fibres with a more perpendicular arrangement, and it was enriched with disorganised collagen type I (stromal collagen) fibres. Compared to other groups, the EPC capsule showed significant variation in the thickness, evenness, distribution of collagen fibres, and significant intracapsular heterogeneity. Compared to BM-like material in the invasive group, the EPC capsule showed a higher density of collagen fibres with longer, straighter, and more aligned fibres, but there was no difference in the distribution of both collagen types I and III. Conversely, compared to EPTC, there were no differences between both EPC and EPTC capsules except that the fibres in the EPC capsule were straighter. Although differences between normal ducts and lobules and DCIS BM collagen fibre density, straightness, orientation, and alignment were detected, both were significantly different from EPC capsule. CONCLUSION: This study provided evidence that the EPC capsule is a reactive process rather than a thickened native BM characteristic of normal and in situ lesions, which provides further evidence that EPC is an indolent invasive carcinoma based on capsule characteristics.

The KDM5B and KDM1A lysine demethylases cooperate in regulating androgen receptor expression and signalling in prostate cancer.

Histone H3 lysine 4 (H3K4) methylation is key epigenetic mark associated with active transcription and is a substrate for the KDM1A/LSD1 and KDM5B/JARID1B lysine demethylases. Increased expression of KDM1A and KDM5B is implicated in many cancer types, including prostate cancer (PCa). Both KDM1A and KDM5B interact with AR and promote androgen regulated gene expression. For this reason, there is great interested in the development of new therapies targeting KDM1A and KDM5B, particularly in the context of castrate resistant PCa (CRPC), where conventional androgen deprivation therapies and androgen receptor signalling inhibitors are no longer effective. As there is no curative therapy for CRPC, new approaches are urgently required to suppress androgen signalling that prevent, delay or reverse progression to the castrate resistant state. While the contribution of KDM1A to PCa is well established, the exact contribution of KDM5B to PCa is less well understood. However, there is evidence that KDM5B is implicated in numerous pro-oncogenic mechanisms in many different types of cancer, including the hypoxic response, immune evasion and PI3/AKT signalling. Here we elucidate the individual and cooperative functions of KDM1A and KDM5B in PCa. We show that KDM5B mRNA and protein expression is elevated in localised and advanced PCa. We show that the KDM5 inhibitor, CPI-455, impairs androgen regulated transcription and alternative splicing. Consistent with the established role of KDM1A and KDM5B as AR coregulators, we found that individual pharmacologic inhibition of KDM1A and KDM5 by namoline and CPI-455 respectively, impairs androgen regulated transcription. Notably, combined inhibition of KDM1A and KDM5 downregulates AR expression in CRPC cells. Furthermore, combined KDM1A and KDM5 inhibition impairs PCa cell proliferation and invasion more than individual inhibition of KDM1A and KDM5B. Collectively our study has identified individual and cooperative mechanisms involving KDM1A and KDM5 in androgen signalling in PCa. Our findings support the further development of KDM1A and KDM5B inhibitors to treat advanced PCa. Further work is now required to confirm the therapeutic feasibility of combined inhibition of KDM1A and KDM5B as a novel therapeutic strategy for targeting AR positive CRPC.

The importance of m6A topology in chicken embryo mRNA: a precise mapping of m6A at the conserved chicken ß-actin zipcode.

N6-methyladenosine (m6A) in mRNA regulates almost every stage in the mRNA life cycle, and the development of methodologies for the high-throughput detection of methylated sites in mRNA using m6A-specific methylated RNA immunoprecipitation with next-generation sequencing (MeRIPSeq) or m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) have revolutionized the m6A research field. Both of these methods are based on immunoprecipitation of fragmented mRNA. However, it is well documented that antibodies often have nonspecific activities, thus verification of identified m6A sites using an antibody-independent method would be highly desirable. We mapped and quantified the m6A site in the chicken ß-actin zipcode based on the data from chicken embryo MeRIPSeq results and our RNA-Epimodification Detection and Base-Recognition (RedBaron) antibody-independent assay. We also demonstrated that methylation of this site in the ß-actin zipcode enhances ZBP1 binding in vitro, while methylation of a nearby adenosine abolishes binding. This suggests that m6A may play a role in regulating localized translation of ß-actin mRNA, and the ability of m6A to enhance or inhibit a reader protein's RNA binding highlights the importance of m6A detection at nucleotide resolution.

Defining invasion in breast cancer: the role of basement membrane.

Basement membrane (BM) is an amorphous, sheet-like structure separating the epithelium from the stroma. BM is characterised by a complex structure comprising collagenous and non-collagenous proteoglycans and glycoproteins. In the breast, the thickness, density and composition of the BM around the ductal lobular system vary during differing development stages. In pathological conditions, the BM provides a physical barrier that separates proliferating intraductal epithelial cells from the surrounding stroma, and its absence or breach in malignant lesions is a hallmark of invasion and metastases. Currently, diagnostic services often use special stains and immunohistochemistry (IHC) to identify the BM in order to distinguish in situ from invasive lesions. However, distinguishing BM on stained sections, and differentiating the native BM from the reactive capsule or BM-like material surrounding some invasive malignant breast tumours is challenging. Although diagnostic use of the BM is being replaced by myoepithelial cell IHC markers, BM is considered by many to be a useful marker to distinguish in situ from invasive lesions in ambiguous cases. In this review, the structure, function and biological and clinical significance of the BM are discussed in relation to the various breast lesions with emphasis on how to distinguish the native BM from alternative pathological tissue mimicking its histology.

Canine osteosarcoma in comparative oncology: Molecular mechanisms through to treatment discovery.

Cancer is a leading cause of non-communicable morbidity and mortality throughout the world, similarly, in dogs, the most frequent cause of mortality is tumors. Some types of cancer, including osteosarcoma (OSA), occur at much higher rates in dogs than people. Dogs therefore not only require treatment themselves but can also act as an effective parallel patient population for the human disease equivalent. It should be noted that although there are many similarities between canine and human OSA, there are also key differences and it is important to research and highlight these features. Despite progress using chorioallantoic membrane models, 2D and 3D in vitro models, and rodent OSA models, many more insights into the molecular and cellular mechanisms, drug development, and treatment are being discovered in a variety of canine OSA patient populations.

Exploring anti-androgen therapies in hormone dependent prostate cancer and new therapeutic routes for castration resistant prostate cancer.

Androgen deprivation therapies (ADTs) are important treatments which inhibit androgen-induced prostate cancer (PCa) progression by either preventing androgen biosynthesis (e.g. abiraterone) or by antagonizing androgen receptor (AR) function (e.g. bicalutamide, enzalutamide, darolutamide). A major limitation of current ADTs is they often remain effective for limited durations after which patients commonly progress to a lethal and incurable form of PCa, called castration-resistant prostate cancer (CRPC) where the AR continues to orchestrate pro-oncogenic signalling. Indeed, the increasing numbers of ADT-related treatment-emergent neuroendocrine-like prostate cancers (NePC), which lack AR and are thus insensitive to ADT, represents a major therapeutic challenge. There is therefore an urgent need to better understand the mechanisms of AR action in hormone dependent disease and the progression to CRPC, to enable the development of new approaches to prevent, reverse or delay ADT-resistance. Interestingly the AR regulates distinct transcriptional networks in hormone dependent and CRPC, and this appears to be related to the aberrant function of key AR-epigenetic coregulator enzymes including the lysine demethylase 1 (LSD1/KDM1A). In this review we summarize the current best status of anti-androgen clinical trials, the potential for novel combination therapies and we explore recent advances in the development of novel epigenetic targeted therapies that may be relevant to prevent or reverse disease progression in patients with advanced CRPC.

The METTL3 RNA Methyltransferase Regulates Transcriptional Networks in Prostate Cancer.

Prostate cancer (PCa) is a leading cause of cancer-related deaths and is driven by aberrant androgen receptor (AR) signalling. For this reason, androgen deprivation therapies (ADTs) that suppress androgen-induced PCa progression either by preventing androgen biosynthesis or via AR signalling inhibition (ARSi) are common treatments. The N6-methyladenosine (m6A) RNA modification is involved in regulating mRNA expression, translation, and alternative splicing, and through these mechanisms has been implicated in cancer development and progression. RNA-m6A is dynamically regulated by the METTL3 RNA methyltransferase complex and the FTO and ALKBH5 demethylases. While there is evidence supporting a role for aberrant METTL3 in many cancer types, including localised PCa, the wider contribution of METTL3, and by inference m6A, in androgen signalling in PCa remains poorly understood. Therefore, the aim of this study was to investigate the expression of METTL3 in PCa patients and study the clinical and functional relevance of METTL3 in PCa. It was found that METTL3 is aberrantly expressed in PCa patient samples and that siRNA-mediated METTL3 knockdown or METTL3-pharmacological inhibition significantly alters the basal and androgen-regulated transcriptome in PCa, which supports targeting m6A as a novel approach to modulate androgen signalling in PCa.

Comparative pathology of dog and human prostate cancer.

Though relatively rare in dogs, prostate cancer (PCa) is the most common non-cutaneous cancer in men. Human and canine prostate glands share many functional, anatomical and physiological features. Due to these similarities, canine PCa has been proposed as a model for PCa in men. PCa is typically androgen-dependent at diagnosis in men and for this reason, androgen deprivation therapies (ADT) are important treatments for advanced PCa in men. In contrast, there is some evidence that PCa is diagnosed more commonly in castrate dogs, at which point, limited therapeutic options are available. In men, a major limitation of current ADT is that progression to a lethal and incurable form of PCa, termed castrate-resistant prostate cancer (CRPC), is common. There is, therefore, an urgent need for a better understanding of the mechanism of PCa initiation and progression to CRPC to enable the development of novel therapeutic approaches. This review focuses on the functional, physiological, endocrine and histopathological similarities and differences in the prostate gland of these species. In particular, we focus on common physiological roles for androgen signalling in the prostate of men and dogs, we review the short- and longer-term effects of castration on PCa incidence and progression in the dog and relate how this knowledge may be relevant to understanding the mechanisms of CRPC in men.

Clinical and molecular significance of the RNA m6A methyltransferase complex in prostate cancer.

N6-methyladenosine (m6A) is the most abundant internal mRNA modification and is dynamically regulated through distinct protein complexes that methylate, demethylate, and/or interpret the m6A modification. These proteins, and the m6A modification, are involved in the regulation of gene expression, RNA stability, splicing and translation. Given its role in these crucial processes, m6A has been implicated in many diseases, including in cancer development and progression. Prostate cancer (PCa) is the most commonly diagnosed non-cutaneous cancer in men and recent studies support a role for m6A in PCa. Despite this, the literature currently lacks an integrated analysis of the expression of key components of the m6A RNA methyltransferase complex, both in PCa patients and in well-established cell line models. For this reason, this study used immunohistochemistry and functional studies to investigate the mechanistic and clinical significance of the METTL3, METTL14, WTAP and CBLL1 components of the m6A methyltransferase complex in PCa specimens and cell lines. Expression of METTL3 and CBLL1, but not METTL14 and WTAP, was associated with poorer PCa patient outcomes. Expression of METTL3, METTL14, WTAP and CBLL1 was higher in PCa cells compared with non-malignant prostate cells, with the highest expression seen in castrate-sensitive, androgen-responsive PCa cells. Moreover, in PCa cell lines, expression of METTL3 and WTAP was found to be androgen-regulated. To investigate the mechanistic role(s) of the m6A methyltransferase complex in PCa cells, short hairpin RNA (shRNA)-mediated knockdown coupled with next generation sequencing was used to determine the transcriptome-wide roles of METTL3, the catalytic subunit of the m6A methyltransferase complex. Functional depletion of METTL3 resulted in upregulation of the androgen receptor (AR), together with 134 AR-regulated genes. METTL3 knockdown also resulted in altered splicing, and enrichment of cell cycle, DNA repair and metabolic pathways. Collectively, this study identified the functional and clinical significance of four essential m6A complex components in PCa patient specimens and cell lines for the first time. Further studies are now warranted to determine the potential therapeutic relevance of METTL3 inhibitors in development to treat leukaemia to benefit patients with PCa.

Recent Advances in Experimental Dendritic Cell Vaccines for Cancer.

The development of immunotherapeutic methods for the treatment of oncological diseases have made it possible to improve the effectiveness of standard therapies. There was no breakthrough after first using of personalized therapeutic vaccines based on dendritic cells in clinical practice. A deeper study of the biology of dendritic cells, as well as the use of new approaches and agents for antigenic work, have made it possible to expand the field of application of dendritic cell (DC) vaccines and improve the indicators of cancer patients. In addition, the low toxicity of DC vaccines in clinical trials makes it possible to use promising predictions of their applicability in wider clinical practice. This review examines new approaches and recent advances of the DC vaccine in clinical trials.

Immunohistochemical Characterisation of GLUT1, MMP3 and NRF2 in Osteosarcoma.

Osteosarcoma (OSA) is an aggressive bone malignancy. Unlike many other malignancies, OSA outcomes have not improved in recent decades. One challenge to the development of better diagnostic and therapeutic methods for OSA has been the lack of well characterized experimental model systems. Spontaneous OSA in dogs provides a good model for the disease seen in people and also remains an important veterinary clinical challenge. We recently used RNA sequencing and qRT-PCR to provide a detailed molecular characterization of OSA relative to non-malignant bone in dogs. We identified differential mRNA expression of the solute carrier family 2 member 1 (SLC2A1/GLUT1), matrix metallopeptidase 3 (MMP3) and nuclear factor erythroid 2-related factor 2 (NFE2L2/NRF2) genes in canine OSA tissue in comparison to paired non-tumor tissue. Our present work characterizes protein expression of GLUT1, MMP3 and NRF2 using immunohistochemistry. As these proteins affect key processes such as Wnt activation, heme biosynthesis, glucose transport, understanding their expression and the enriched pathways and gene ontologies enables us to further understand the potential molecular pathways and mechanisms involved in OSA. This study further supports spontaneous OSA in dogs as a model system to inform the development of new methods to diagnose and treat OSA in both dogs and people.

Proangiogenic Effect of 2A-Peptide Based Multicistronic Recombinant Constructs Encoding VEGF and FGF2 Growth Factors.

Coronary artery disease remains one of the primary healthcare problems due to the high cost of treatment, increased number of patients, poor clinical outcomes, and lack of effective therapy. Though pharmacological and surgical treatments positively affect symptoms and arrest the disease progression, they generally exhibit a limited effect on the disease outcome. The development of alternative therapeutic approaches towards ischemic disease treatment, especially of decompensated forms, is therefore relevant. Therapeutic angiogenesis, stimulated by various cytokines, chemokines, and growth factors, provides the possibility of restoring functional blood flow in ischemic tissues, thereby ensuring the regeneration of the damaged area. In the current study, based on the clinically approved plasmid vector pVax1, multigenic constructs were developed encoding vascular endothelial growth factor (VEGF), fibroblast growth factors (FGF2), and the DsRed fluorescent protein, integrated via picornaviruses' furin-2A peptide sequences. In vitro experiments demonstrated that genetically modified cells with engineered plasmid constructs expressed the target proteins. Overexpression of VEGF and FGF2 resulted in increased levels of the recombinant proteins. Concomitantly, these did not lead to a significant shift in the general secretory profile of modified HEK293T cells. Simultaneously, the secretome of genetically modified cells showed significant stimulating effects on the formation of capillary-like structures by HUVEC (endothelial cells) in vitro. Our results revealed that when the multicistronic multigene vectors encoding 2A peptide sequences are created, transient transgene co-expression is ensured. The results obtained indicated the mutual synergistic effects of the growth factors VEGF and FGF2 on the proliferation of endothelial cells in vitro. Thus, recombinant multicistronic multigenic constructs might serve as a promising approach for establishing safe and effective systems to treat ischemic diseases.

The treatment of articular cartilage injuries with mesenchymal stem cells in different animal species.

One of the major problems observed in veterinary practice is articular cartilage injuries in animals. In terms of agriculture, it leads to their culling from the herd, even if they are highly productive animals. With companion animals, owners usually have to decide between euthanasia or long-term sometimes lifelong treatment of the injury by a veterinarian. The use of mesenchymal stem cells (MSCs) for the treatment of cartilage injury in veterinary medicine is based on the good results observed in preclinical studies, where large animals have been used as experimental models to study the regenerative activity of MSCs. According to the literature, MSCs in veterinary medicine have been used to treat cartilage injury of dogs and horses, whereas sheep and goats are generally models for reproducing the disease in preclinical experimental studies.

Application of gene therapy in the treatment of superficial digital flexor tendon injury in horses.

Background: Tendon injuries are one of the most common causes of orthopedic disorders in horses. Such injuries involve a long course of treatment and recovery. The most promising method of treating these injuries is the use of recombinant proteins and gene therapy. Aim: In this work, we evaluated the therapeutic efficacy of plasmid DNA (pDNA) containing two species-specific coding sequences, i.e. vascular endothelial growth factor 164 (VEGF164) and fibroblast growth factor 2 (FGF2), in the treatment of severe damage to the tendon of the superficial digital flexor. Methods: A pDNA construct was used to restore the damaged superficial digital flexor tendon in the horse. Results: This study showed that the administration of pDNA encoding VEGF164 and FGF2 genes at the injury area increased the regenerative activities of the damaged tendon. Conclusion: This study shows the therapeutic properties of genetic constructs (pDNA) and contributes to the advancements in the use of these therapies.