Heat Shock Proteins (HSPs) and Cardiovascular Complications of Obesity: Searching for Potential Biomarkers.

Heat shock proteins (HSPs), a family of proteins that support cellular proteostasis and perform a protective function under various stress conditions, such as high temperature, intoxication, inflammation, or tissue hypoxia, constitute a promising group of possible biochemical markers for obesity and cardiovascular diseases. HSP27 is involved in essential cellular processes occurring in conditions of obesity and its cardiometabolic complications; it has protective properties, and its secretion may indicate a cellular response to stress. HSP40 plays a controversial role in the pathogenesis of obesity. HSP60 is involved in various pathological processes of the cardiovascular, immune, excretory, and nervous systems and is associated with obesity and concomitant diseases. The hypersecretion of HSP60 is associated with poor prognosis; hence, this protein may become a target for further research on obesity and its cardiovascular complications. According to most studies, intracellular HSP70 is an obesity-promoting factor, whereas extracellular HSP70 exhibited inconsistent dynamics across different patient groups and diagnoses. HSPs are involved in the pathogenesis of cardiovascular pathology. However, in the context of cardiovascular and metabolic pathology, these proteins require further investigation.

Polycondensed Peptide-Based Polymers for Targeted Delivery of Anti-Angiogenic siRNA to Treat Endometriosis.

Endometriosis (EM) is a prevalent gynecological disease characterized by the abnormal growth of tissue similar to the endometrium outside of the uterus. This condition is accompanied by the development of new blood vessels in endometriotic lesions. While surgical intervention is effective in removing endometriotic lesions, some patients require multiple surgeries. Therefore, finding non-surgical treatments for EM is of great interest. One of the promising approaches is anti-angiogenic therapy using siRNA-therapeutics to target the expression of the VEGFA gene. Peptide-based polymers have shown promise as siRNA delivery systems due to their biocompatibility and ease of modification. We conducted a study to evaluate the effectiveness of the R6p-cRGD peptide carrier as a non-viral vehicle for delivering siRNA to endothelial cells in vitro and endometrial implants in vivo. We investigated the physicochemical properties of the siRNA-complexes, assessed cellular toxicity, and examined the efficiency of GFP and VEGFA genes silencing. Furthermore, we tested the anti-angiogenic effects of these complexes in cellular and animal models. The transfection with siRNA complexes led to a significant increase in VEGFA gene knockdown efficiency and a decrease in the migration of endothelial cells. For the animal model, we induced endometriosis in rats by transplanting endometrial tissue subcutaneously. We evaluated the efficiency of anti-angiogenic therapy for EM in vivo using anti-VEGF siRNA/R6p-RGD complexes. During this assessment, we measured the volume of the implants, analyzed VEGFA gene expression, and conducted CD34 immunohistochemical staining. The results showed a significant decrease in the growth of endometriotic implants and in VEGFA gene expression. Overall, our findings demonstrate the potential of the R6p-cRGD peptide carrier as a delivery system for anti-angiogenic therapy of EM.

Non-Alcoholic Fatty Liver Disease and Bone Tissue Metabolism: Current Findings and Future Perspectives.

Non-alcoholic fatty liver disease (NAFLD) is reaching epidemic proportions worldwide. Moreover, the prevalence of this liver disease is expected to increase rapidly in the near future, aligning with the rise in obesity and the aging of the population. The pathogenesis of NAFLD is considered to be complex and to include the interaction between genetic, metabolic, inflammatory, and environmental factors. It is now well documented that NAFLD is linked to the other conditions common to insulin resistance, such as abnormal lipid levels, metabolic syndrome, and type 2 diabetes mellitus. Additionally, it is considered that the insulin resistance may be one of the main mechanisms determining the disturbances in both bone tissue metabolism and skeletal muscles quality and functions in patients with NAFLD. To date, the association between NAFLD and osteoporosis has been described in several studies, though it worth noting that most of them included postmenopausal women or elderly patients and originated from Asia. However, taking into account the health and economic burdens of NAFLD, and the increasing prevalence of obesity in children and adolescents worldwide, further investigation of the relationship between osteopenia, osteoporosis and sarcopenia in NAFLD, including in young and middle-aged patients, is of great importance. In addition, this will help to justify active screening and surveillance of osteopenia and osteoporosis in patients with NAFLD. In this review, we will discuss various pathophysiological mechanisms and possible biologically active molecules that may interplay between NAFLD and bone tissue metabolism.

Serum-Resistant Ternary DNA Polyplexes for Suicide Gene Therapy of Uterine Leiomyoma.

Uterine leiomyoma (UL) is a prevalent benign tumor in women that frequently gives rise to a multitude of reproductive complications. The use of suicide gene therapy has been proposed as a highly promising method for treating UL. To achieve successful gene therapy, it is essential to develop carriers that can efficiently transport nucleic acids into targeted cells and tissues. The instability of polyplexes in blood and other biological fluids is a crucial factor to consider when using non-viral carriers. In this study, we present serum-resistant and cRGD-modified DNA complexes for targeted delivery genes to UL cells. Ternary polyplexes were formed by incorporating cystine-cross-linked polyglutamic acid modified with histidine residues. We employed two techniques in the production of cross-linked polyanionic coating: matrix polymerization and oxidative polycondensation. In this study, we investigated the physicochemical properties of ternary DNA complexes, including the size and zeta-potential of the nanoparticles. Additionally, we evaluated cellular uptake, toxicity levels, transfection efficiency and specificity in vitro. The study involved introducing the HSV-TK gene into primary UL cells as a form of suicide gene therapy modeling. We have effectively employed ternary peptide-based complexes for gene delivery into the UL organtypic model. By implementing in situ suicide gene therapy, the increase in apoptosis genes expression was detected, providing conclusive evidence of apoptosis occurring in the transfected UL tissues. The results of the study strongly suggest that the developed ternary polyplexes show potential as a valuable tool in the implementation of suicide gene therapy for UL.

Gut Microbiota Patterns in Patients with Non-Alcoholic Fatty Liver Disease: A Comprehensive Assessment Using Three Analysis Methods.

Non-alcoholic fatty liver disease (NAFLD) is considered the most common chronic liver disease worldwide, affecting nearly 25% of the global adult population. Increasing evidence suggests that functional and compositional changes in the gut microbiota may contribute to the development and promote the progression of NAFLD. 16S rRNA gene next-generation sequencing is widely used to determine specific features of the NAFLD microbiome, but a complex system such as the gut microbiota requires a comprehensive approach. We used three different approaches: MALDI-TOF-MS of bacterial cultures, qPCR, and 16S NGS sequencing, as well as a wide variety of statistical methods to assess the differences in gut microbiota composition between NAFLD patients without significant fibrosis and the control group. The listed methods showed enrichment in Collinsella sp. and Oscillospiraceae for the control samples and enrichment in Lachnospiraceae (and in particular Dorea sp.) and Veillonellaceae in NAFLD. The families, Bifidobacteriaceae, Lactobacillaceae, and Enterococcaceae (particularly Enterococcus faecium and Enterococcus faecalis), were also found to be important taxa for NAFLD microbiome evaluation. Considering individual method observations, an increase in Candida krusei and a decrease in Bacteroides uniformis for NAFLD patients were detected using MALDI-TOF-MS. An increase in Gracilibacteraceae, Chitinophagaceae, Pirellulaceae, Erysipelatoclostridiaceae, Muribaculaceae, and Comamonadaceae, and a decrease in Acidaminococcaceae in NAFLD were observed with 16S NGS, and enrichment in Fusobacterium nucleatum was shown using qPCR analysis. These findings confirm that NAFLD is associated with changes in gut microbiota composition. Further investigations are required to determine the cause-and-effect relationships and the impact of microbiota-derived compounds on the development and progression of NAFLD.

Polycondensed Peptide Carriers Modified with Cyclic RGD Ligand for Targeted Suicide Gene Delivery to Uterine Fibroid Cells.

Suicide gene therapy was suggested as a possible strategy for the treatment of uterine fibroids (UFs), which are the most common benign tumors inwomen of reproductive age. For successful suicide gene therapy, DNAtherapeutics should be specifically delivered to UF cells. Peptide carriers are promising non-viral gene delivery systems that can be easily modified with ligands and other biomolecules to overcome DNA transfer barriers. Here we designed polycondensed peptide carriers modified with a cyclic RGD moiety for targeted DNA delivery to UF cells. Molecular weights of the resultant polymers were determined, and inclusion of the ligand was confirmed by MALDI-TOF. The physicochemical properties of the polyplexes, as well as cellular DNA transport, toxicity, and transfection efficiency were studied, and the specificity of αvß3 integrin-expressing cell transfection was proved. The modification with the ligand resulted in a three-fold increase of transfection efficiency. Modeling of the suicide gene therapy by transferring the HSV-TK suicide gene to primary cells obtained from myomatous nodes of uterine leiomyoma patients was carried out. We observed up to a 2.3-fold decrease in proliferative activity after ganciclovir treatment of the transfected cells. Pro- and anti-apoptotic gene expression analysis confirmed our findings that the developed polyplexes stimulate UF cell death in a suicide-specific manner.

Peptide-Based Nanoparticles for αvß3 Integrin-Targeted DNA Delivery to Cancer and Uterine Leiomyoma Cells.

Uterine leiomyoma is the most common benign tumor of the reproductive system. Current therapeutic options do not simultaneously meet the requirements of long-term efficiency and fertility preservation. Suicide gene delivery can be proposed as a novel approach to uterine leiomyoma therapy. Non-viral vehicles are an attractive approach to DNA delivery for gene therapy of both malignant and benign tumors. Peptide-based vectors are among the most promising candidates for the development of artificial viruses, being able to efficiently cross barriers of DNA transport to cells. Here we described nanoparticles composed of cysteine-crosslinked polymer and histidine-arginine-rich peptide modified with iRGD moiety and characterized them as vehicles for plasmid DNA delivery to pancreatic cancer PANC-1 cells and the uterine leiomyoma cell model. Several variants of nanoparticles were formulated with different targeting ligand content. The physicochemical properties that were studied included DNA binding and protection, interaction with polyanions and reducing agents, size, structure and zeta-potential of the peptide-based nanoparticles. Cytotoxicity, cell uptake and gene transfection efficiency were assessed in PANC-1 cells with GFP and LacZ-encoding plasmids. The specificity of gene transfection via αvß3 integrin binding was proved in competitive transfection. The therapeutic potential was evaluated in a uterine leiomyoma cell model using the suicide gene therapy approach. The optimal formulation was found to be at the polyplex with the highest iRGD moiety content being able to transfect cells more efficiently than control PEI. Suicide gene therapy using the best formulation resulted in a significant decrease of uterine leiomyoma cells after ganciclovir treatment. It can be concluded that the application of iRGD-modified peptide-based nanoparticles has a high potential for cellular delivery of DNA therapeutics in favor of uterine leiomyoma gene therapy.

Low-frequency component of photoplethysmogram reflects the autonomic control of blood pressure.

The question of how much information the photoplethysmogram (PPG) signal contains on the autonomic regulation of blood pressure (BP) remains unsolved. This study aims to compare the low-frequency (LF) and high-frequency components of PPG and BP and assess their correlation with oscillations in interbeat (RR) intervals at similar frequencies. The PPG signal from the distal phalanx of the right index finger recorded using a reflective PPG sensor at green light, the BP signal from the left hand recorded using a Finometer, and RR intervals were analyzed. These signals were simultaneously recorded within 15 min in a supine resting condition in 17 healthy subjects (12 males and 5 females) aged 33 ± 9 years (mean ± SD). The study revealed the high coherence of LF components of PPG and BP with the LF component of RR intervals. The high-frequency components of these signals had low coherence. The analysis of the signal instantaneous phases revealed the presence of high-phase coherence between the LF components of PPG and BP. It is shown that the LF component of PPG is determined not only by local myogenic activity but also reflects the processes of autonomic control of BP.

Evaluating daily profile of central aortic pressure and reflected pulse wave parameters in climacteric women.

INTRODUCTION: Structural and functional changes of the vascular wall in women occur already at the very early stages of reproductive aging. An emergence of applanation tonometry made it possible to evaluate arterial stiffness and central hemodynamic parameters non-invasively, which considerably expanded the information that had been provided previously by invasive methods used for studying these parameters during cardiac catheterization. Whereas a few studies have assessed central aortic pressure (CAP) parameters and reflected pulse wave in women at different phases of their reproductive aging, none investigated the daily profile of CAP and reflected pulse wave parameters in women undergoing different stages of the menopause. BACKGROUND: assessment of the daily variability in CAP and daily profile of amplification and augmentation of pulse blood pressure (PBP) in women at different menopause phases. METHODS: The study involved 384 climacteric women. The first group included 168 women undergoing perimenopause, the second group comprised of 216 women in their early postmenopausal stage. A 24-h blood pressure (BP) monitoring in the brachial artery and aorta (BPLab® Vasotens® system, Petr Telegin LLC, Russia) was performed via the measurements of the following indicators: systolic blood pressure (SBP), pulse blood pressure (PBP), central aortic systolic pressure (CASP), central aortic pulse pressure (CAPP), aortic augmentation index (AIxao), and pulse pressure amplification (PPA). RESULTS: When investigating PPA values in the brachial artery and aorta, we detected smaller amplification and higher aortic augmentation index at night than in daytime, which reflected a disproportionately higher CAP level during night hours. This pattern was more pronounced in postmenopausal women. We calculated the logistic regression equation (adjusted R2 = 0.49, log-likelihood = - 50.3, chi-square (19) = 97.6, p < 0.001), in which dependent variable was represented by the menopausal status, whereas body mass index with all indicators of a 24-h BP monitoring represented independent variables. In this model, two indicators (body mass index and AIxao) were, independently of each other, associated significantly with the menopause phases. Differences among women at various climacteric phases in terms of remaining indicators of a 24-h BP monitoring, apparently, matched the differences in their body mass index values. CONCLUSION: Rising CAP, in combination with declining PPA and augmenting reflected pulse wave amplitude, may be associated with an increased risk of cardiovascular complications.

Automatic wavelet-based assessment of behavioral sleep using multichannel electrocorticography in rats.

PURPOSE: During the last decade, the reported prevalence of sleep-disordered breathing in adults has been rapidly increasing. Therefore, automatic methods of sleep assessment are of particular interest. In a framework of translational neuroscience, this study introduces a reliable automatic detection system of behavioral sleep in laboratory rats based on the signal recorded at the cortical surface without requiring electromyography. METHODS: Experimental data were obtained in 16 adult male WAG/Rij rats at the age of 9 months. Electrocorticographic signals (ECoG) were recorded in freely moving rats during the entire day (22.5 ± 2.2 h). Automatic wavelet-based assessment of behavioral sleep (BS) was proposed. The performance of this wavelet-based method was validated in a group of rats with genetic predisposition to absence epilepsy (n=16) based on visual analysis of their behavior in simultaneously recorded video. RESULTS: The accuracy of automatic sleep detection was 98% over a 24-h period. An automatic BS assessment method can be adjusted for detecting short arousals during sleep (microarousals) with various duration. CONCLUSIONS: These findings suggest that automatic wavelet-based assessment of behavioral sleep can be used for assessment of sleep quality. Current analysis indicates a temporal relationship between microarousals, sleep, and epileptic discharges in genetically prone subjects.

Modification of Joint Recurrence Quantification Analysis (JRQA) for assessing individual characteristics from short EEG time series.

This article proposes a modification of joint recurrence quantification analysis for identifying individual characteristics applied to human electroencephalography (EEG) using short time series. Statistical analysis of EEG characteristics facilitated the clarification of the spatial localization of identified individual characteristics. The method can be adapted for use as a stage of a rapid automatic configuration of brain-computer interface devices, which is especially relevant when working with children, due to limited opportunities for their long-term monitoring.

Wavelet skeletons in sleep EEG-monitoring as biomarkers of early diagnostics of mild cognitive impairment.

Many neuro-degenerative diseases are difficult to diagnose in their early stages. For example, early diagnosis of Mild Cognitive Impairment (MCI) requires a wide variety of tests to distinguish MCI symptoms and normal consequences of aging. In this article, we use the wavelet-skeleton approach to find some characteristic patterns in the electroencephalograms (EEGs) of healthy adult patients and patients with cognitive dysfunctions. We analyze the EEG activity recorded during natural sleep of 11 elderly patients aged between 60 and 75, six of whom have mild cognitive impairment, and apply a nonlinear analysis method based on continuous wavelet transformskeletons. Our studies show that a comprehensive analysis of EEG signals of the entire sleep state allows us to identify a significant decrease in the average duration of oscillatory patterns in the frequency band [12; 14] Hz in the presence of mild cognitive impairment. Thus, the changes in this frequency range can be interpreted as related to the activity in the motor cortex, as a candidate for developing the criteria for early objective MCI.

The intensity of oscillations of the photoplethysmographic waveform variability at frequencies 0.04-0.4 Hz is effective marker of hypertension and coronary artery disease in males.

Background: It is believed that the intensity of oscillations in the photoplethysmographic waveform variability reflects the activity of vascular regulatory mechanisms. However, the relationship of such fluctuations with the state of health is poorly understood.Purpose: The aim of our study was to assess the possibility of using spectral indices that reflect the intensity of oscillations of the photoplethysmographic waveform variability at frequencies 0.04-0.4 Hz as markers of hypertension and coronary artery disease. We did not study women to exclude the influence of menopause and sex hormones on the results.Materials and Methods: We compared synchronous 10-minute records of finger photoplethysmogram and respiration at rest in 30 healthy males (48.8 ± 4.5 years; data presented as Mean ± SD) versus 30 patients with hypertension (aged 49.0 ± 4.3 years) versus 30 patients with stable coronary artery disease (49.2 ± 4.8 years). Percentages of high-frequency and low-frequency ranges in the total power of photoplethysmographic waveform variability spectrum (HF% and LF%), and LF/HF ratio were assessed.Results: HF% are subject to by 2- to 5-fold increase in hypertensive patients (p < .001) and up to an 8-fold increase in patients with coronary artery disease (p < .001) when compared with healthy persons. On the contrary, LF% is reduced by 1.5-5 times in all patients when compared with healthy people (p < .001). We identified cut-off points for each photoplethysmographic index to distinguish patients with coronary artery disease or hypertension from healthy subjects. Multiple logistic regression models based on photoplethysmographic waveform variability indices had sufficient sensitivity and specificity for patients with hypertension or coronary artery disease.Conclusion: Frequency-domain indices of photoplethysmographic waveform variability (in particular, HF%, LF%, and LF/HF) are sufficiently sensitive and specific markers of hypertension and coronary artery disease in adult males.

DYNC1H1 gene methylation correlates with severity of spinal muscular atrophy.

Methylation profiles of CpG islands within the SLC23A2, CDK2AP1, and DYNC1H1 genes and their association with spinal muscular atrophy (SMA) severity were studied. High clinical heterogeneity of SMA suggests the existence of different factors modifying SMA phenotype with gene methylation as a plausible one. The genes picked up in our earlier genome-wide methylation studies of SMA patients demonstrated obvious differences in their methylation patterns, thus suggesting the likely involvement of their protein products in SMA development. Significantly decreased methylation of CpG islands within exon 37 of the DYNC1H1 gene was observed in patients with a severe SMA manifestation (type I) compared to mildly affected SMA patients (types III-IV). This finding provides new information on peculiarities of methylation in clinically different types of SMA patients and gives a clue for identification of new SMA modifiers.

Discrepancy between the European clinical guidelines and myocardial revascularization in patients with stable coronary artery disease in Russia.

OBJECTIVE: In European Society of Cardiology/European Association for Cardio-Thoracic Surgery (ESC/EACTS) guidelines, six indications have been proposed for making a decision on myocardial revascularization in patients with stable coronary artery disease (CAD). Our aim was to study a discrepancy between the actual clinical situation and ESC/EACTS indications on performing the revascularization in patients with CAD in Russia. DESIGN AND SETTING: We used retrospective clinical data on patients with stable CAD enrolled in the 2012-2015 Russian Registry of Hypertension, Coronary Artery Disease, and Chronic Heart Failure. PARTICIPANTS: A total of 1522 patients with CAD (aged 53.0 ± 8.5 years, 76.2% male) were used for analysis. INTERVENTIONS: All patients were divided into two groups: 591 patients with performed myocardial revascularization (named as R-CAD) and 931 patients refused from revascularization (named as NR-CAD). Factors associated with revascularization performance were identified by discriminant function analysis. MAIN OUTCOME MEASURES: ESC/EACTS indications for revascularization were assessed. RESULTS: A total of 1196 patients with CAD had any ESC/EACTS indication for revascularization, but only 40.2% of them had performed invasive coronary intervention. Myocardial revascularization was appropriate in 81.4% of R-CAD patients and 76.8% of NR-CAD patients. The main factor of revascularization performance was any stenosis >50% and grades III-IV of stable angina. With non-performed revascularization, the following factors were associated: limiting angina or angina equivalent, unresponsive to medical therapy, atherosclerotic peripheral arterial disease and increasing the New York Heart Association class of chronic heart failure. Most ESC/EACTS indications had little effect on decision-making on revascularization. CONCLUSION: There is a discrepancy between the actual clinical situation and ESC/EACTS guidelines on myocardial revascularization in patients with stable CAD in Russia.

Anti-angiogenic treatment of endometriosis via anti-VEGFA siRNA delivery by means of peptide-based carrier in a rat subcutaneous model.

Development of gene therapy for endometriosis requires inhibition of vascularization in endometrial lesions. We have previously developed CXCR4 receptor-targeted siRNA carrier L1 and observed efficient RNAi-mediated down-regulation of VEGFA gene expression in endothelial cells followed by decrease in VEGFA protein production and inhibition of cell migration. In this study we evaluated L1 carrier as non-viral vector for anti-VEGFA siRNA delivery into endometrial implants in rat subcutaneous endometriosis model created by subcutaneous auto-transplantation of uterus horn's fragments. Therapeutic anti-angiogenic efficiency of anti-VEGFA siRNA/L1 polyplexes was evaluated by lesion size measurement, histopathologic examination, immunohistochemical staining and real-time reverse transcriptase-PCR analysis. After in vivo administration of anti-VEGFA siRNA we observed a 55-60% inhibition of endometriotic lesions growth and approximately two-fold decrease in VEGFA gene expression in comparison with untreated implants. Results of immunohistochemical examination of endometriotic lesions confirmed anti-angiogenic effects of anti-VEGFA siRNA/L1 polyplexes. Ultimately, our results demonstrate the efficiency of anti-angiogenic treatment of EM by means of anti-VEGFA siRNA delivery with L1 peptide-based carrier.

Molecular Factors Involved in Spinal Muscular Atrophy Pathways as Possible Disease-modifying Candidates.

Spinal Muscular Atrophy (SMA) is a neuromuscular disorder caused by mutations in the SMN1 gene. Being a monogenic disease, it is characterized by high clinical heterogeneity. Variations in penetrance and severity of symptoms, as well as clinical discrepancies between affected family members can result from modifier genes influence on disease manifestation. SMN2 gene copy number is known to be the main phenotype modifier and there is growing evidence of additional factors contributing to SMA severity. Potential modifiers of spinal muscular atrophy can be found among the wide variety of different factors, such as multiple proteins interacting with SMN or promoting motor neuron survival, epigenetic modifications, transcriptional or splicing factors influencing SMN2 expression. Study of these factors enables to reveal mechanisms underlying SMA pathology and can have pronounced clinical application.

Arginine-rich cross-linking peptides with different SV40 nuclear localization signal content as vectors for intranuclear DNA delivery.

The major barriers for intracellular DNA transportation by cationic polymers are their toxicity, poor endosomal escape and inefficient nuclear uptake. Therefore, we designed novel modular peptide-based carriers modified with SV40 nuclear localization signal (NLS). Core peptide consists of arginine, histidine and cysteine residues for DNA condensation, endosomal escape promotion and interpeptide cross-linking, respectively. We investigated three polyplexes with different NLS content (10 mol%, 50 mol% and 90 mol% of SV40 NLS) as vectors for intranuclear DNA delivery. All carriers tested were able to condense DNA, to protect it from DNAase I and were not toxic to the cells. We observed that cell cycle arrest by hydroxyurea did not affect transfection efficacy of NLS-modified carriers which we confirmed using quantitative confocal microscopy analysis. Overall, peptide carrier modified with 90 mol% of SV40 NLS provided efficient transfection and nuclear uptake in non-dividing cells. Thus, incorporation of NLS into arginine-rich cross-linking peptides is an adequate approach to the development of efficient intranuclear gene delivery vehicles.

Development of a receptor-targeted gene delivery system using CXCR4 ligand-conjugated cross-linking peptides.

BACKGROUND: Success in gene therapy greatly depends on the efficiency of nucleic acid delivery. Important features of the carriers for gene delivery should include an enhanced transfection ability, targeting of specific receptors and low toxicity. In the present study, we characterized CXCR4-targeted cross-linking peptides modified with an N-terminal fragment of chemokine stromal cell-derived factor-1α as carriers for gene delivery. METHODS: We studied three variants of DNA/carrier complexes with different targeting ligand content. The physicochemical characteristics of the complexes, including their DNA-binding and protective ability, interaction with glycosaminoglycans and size, were determined. Transfection efficacy was studied in cell lines with different levels of CXCR4 expression (HeLa, A172, CHO, Е.А.hy926) and also in human mesenchymal stem cells (hMSCs). The influence of the ligand content on the efficacy of transfection was studied by means of chlorpromazine blockage of clathrin-mediated endocytosis, competition with CXCR4-antagonist AMD3100, and valproic acid treatment of hMSCs. RESULTS: CXCR4-targeted peptides were evaluated for their physicochemical properties and in vitro transfection capacities. Ligand-modified carriers were found to be 10- to 50-fold more effective than unmodified carriers in CXCR4-positive cells. By contrast, their transfection efficacy in CXCR4-negative cells was similar to unmodified carriers. Experiments with chlorpromazine demonstrated receptor-specific transfection in A172 cells. The transfection efficacy of CXCR4-targeted carriers in AMD3100-treated HeLa cells was reduced by two-fold compared to the untreated control. Valproic acid treatment resulted in a four- to 15-fold increase of transfection efficacy for ligand-modified carriers in hMSCs. CONCLUSIONS: CXCR4-targeted cross-linking peptides should be considered as useful tools for nonviral gene delivery into tumor and mesenchymal stem cells.

Assessment of Nuclear Gem Quantity for Evaluating the Efficacy of Antisense Oligonucleotides in Spinal Muscular Atrophy Cells.

Spinal muscular atrophy is a neuromuscular disorder caused by mutations in both copies of the survival motor neuron gene 1 (SMN1), which lead to reduction in the production of the SMN protein. Currently, there are several therapies that have been approved for SMA, with many more undergoing active research. While various biomarkers have been proposed for assessing the effectiveness of SMA treatment, a universally accepted one still has not been identified. This study aimed to describe a fast and reliable method using the number of gems in cell nuclei as a potential tool for assessment of splicing correction of oligonucleotide efficacy in SMA cells. To gain insight into whether the number of gems in cell nuclei varies based on their SMN genotype and whether the increase in gem number is associated with therapeutic response, we utilized fibroblast cell cultures obtained from a patient with SMA type II and from a healthy individual. We discovered a remarkable difference in the number of gems found in the nuclei of these cells, specifically when counting gems per 100 nuclei. The SMA fibroblasts treated with antisense oligonucleotide showed beneficial effects in correcting the abnormal splicing of SMN2 exon 7. It was observed that there was a significant increase in the number of gems in the treated cells compared to the intact SMA cells. The results obtained significantly correlate with an increase of full-length SMN transcript sharing. Based on our findings, we propose using the quantity of gems as a reliable biomarker for SMA drug development.