EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells.

Introduction: Beta cell dysfunction by loss of beta cell identity, dedifferentiation, and the presence of polyhormonal cells are main characteristics of diabetes. The straightforward strategy for curing diabetes implies reestablishment of pancreatic beta cell function by beta cell replacement therapy. Aristaless-related homeobox (Arx) gene encodes protein which plays an important role in the development of pancreatic alpha cells and is a main target for changing alpha cell identity. Results: In this study we used CRISPR/dCas9-based epigenetic tools for targeted hypermethylation of Arx gene promoter and its subsequent suppression in mouse pancreatic αTC1-6 cell line. Bisulfite sequencing and methylation profiling revealed that the dCas9-Dnmt3a3L-KRAB single chain fusion constructs (EpiCRISPR) was the most efficient. Epigenetic silencing of Arx expression was accompanied by an increase in transcription of the insulin gene (Ins2) mRNA on 5th and 7th post-transfection day, quantified by both RT-qPCR and RNA-seq. Insulin production and secretion was determined by immunocytochemistry and ELISA assay, respectively. Eventually, we were able to induce switch of approximately 1% of transiently transfected cells which were able to produce 35% more insulin than Mock transfected alpha cells. Conclusion: In conclusion, we successfully triggered a direct, transient switch of pancreatic alpha to insulin-producing cells opening a future research on promising therapeutic avenue for diabetes management.

Oxidative stress-mediated beta cell death and dysfunction as a target for diabetes management.

The biggest drawback of a current diabetes therapy is the treatment of the consequences not the cause of the disease. Regardless of the diabetes type, preservation and recovery of functional pancreatic beta cells stands as the biggest challenge in the treatment of diabetes. Free radicals and oxidative stress are among the major mediators of autoimmune destruction of beta cells in type 1 diabetes (T1D) or beta cell malfunction and death provoked by glucotoxicity and insulin resistance in type 2 diabetes (T2D). Additionally, oxidative stress reduces functionality of beta cells in T2D by stimulating their de-/trans-differentiation through the loss of transcription factors critical for beta cell development, maturity and regeneration. This review summarizes up to date clarified redox-related mechanisms involved in regulating beta cell identity and death, underlining similarities and differences between T1D and T2D. The protective effects of natural antioxidants on the oxidative stress-induced beta cell failure were also discussed. Considering that oxidative stress affects epigenetic regulatory mechanisms involved in the regulation of pancreatic beta cell survival and insulin secretion, this review highlighted huge potential of epigenetic therapy. Special attention was paid on application of the state-of-the-art CRISPR/Cas9 technology, based on targeted epigenome editing with the purpose of changing the differentiation state of different cell types, making them insulin-producing with ability to attenuate diabetes. Clarification of the above-mentioned mechanisms could provide better insight into diabetes etiology and pathogenesis, which would allow development of novel, potentially more efficient therapeutic strategies for the prevention or reversion of beta cell loss.

TET-mediated DNA hydroxymethylation is negatively influenced by the PARP-dependent PARylation.

BACKGROUND: Poly(ADP-ribosyl)ation (PARylation), a posttranslational modification introduced by PARP-1 and PARP-2, has first been implicated in DNA demethylation due to its role in base excision repair. Recent evidence indicates a direct influence of PARP-dependent PARylation on TET enzymes which catalyse hydroxymethylation of DNA-the first step in DNA demethylation. However, the exact nature of influence that PARylation exerts on TET activity is still ambiguous. In our recent study, we have observed a negative influence of PARP-1 on local TET-mediated DNA demethylation of a single gene and in this study, we further explore PARP-TET interplay. RESULTS: Expanding on our previous work, we show that both TET1 and TET2 can be in vitro PARylated by PARP-1 and PARP-2 enzymes and that TET1 PARylation negatively affects the TET1 catalytic activity in vitro. Furthermore, we show that PARylation inhibits TET-mediated DNA demethylation at the global genome level in cellulo. CONCLUSIONS: According to our findings, PARP inhibition can positively influence TET activity and therefore affect global levels of DNA methylation and hydroxymethylation. This gives a strong rationale for future examination of PARP inhibitors' potential use in the therapy of cancers characterised by loss of 5-hydroxymethylcytosine.

The effects of the aerobic endurance running program on the morphological characteristics of adolescent girls with different nutritional status / Efectos del programa de carrera de resistencia aeróbica en las características morfológicas de las adolescentes con diferentes estados nutricionales

SUMMARY: This study aimed to explore the effects of a single moderate-intensity aerobic endurance running program on the morphological characteristics of normal-weighted and overweighted female adolescents and whether effects differ between individuals of different nutritional statuses. A total of 47 adolescent girls participated in this randomized controlled trial. Before and after 12 weeks of intervention, measurements were obtained for body height, body mass, BMI, triceps, subscapular, abdominal, thigh, and calf skinfolds, and chest, abdominal, thigh, and calf circumference. The total duration of the program was 12 weeks, with a frequency of 2 hours/week (24 sessions). The individual session lasted 60 minutes, and the intensity ranged from 60 % HRmax to 80 %. The findings from the present study suggest that the 12-week aerobic endurance running program induced changes in morphological characteristics of female adolescents. The significant reductions in body mass and BMI were small for normal-weight adolescents (-2,64 % and -3,48 %) and moderate for overweight adolescents (-4,14 % and -4,36 %) following the experimental program. The magnitude of reductions in the skinfold thickness was small for both groups (triceps, subscapular, abdominal, and calf) and moderate for thigh skinfold and a sum of skinfolds in the overweight group. The reductions in the magnitude of all circular measures were small for both groups. It ranged from - 1.59 % to -2.59 % for normal-weight and from -2.54 % to -3.92 % for overweight adolescents, respectively. Additionally, the effects of the applied program seem more favorable to overweight than normal-weight adolescents. This study's current findings indicate that the 12-week aerobic endurance running program is effective due to improvements in morphological characteristics, body mass, and BMI of female adolescents. Additionally, this study's findings show that applied aerobic endurance running program proved more effective for overweight adolescents.

RESUMEN: Este estudio tuvo como objetivo explorar los efectos de un programa único de carrera de resistencia aeróbica de intensidad moderada sobre las características morfológicas de mujeres adolescentes con peso normal y con sobrepeso y si los efectos difieren entre individuos con diferentes estados nutricionales. Un total de 47 niñas adolescentes participaron en este ensayo controlado aleatorio. Antes y después de 12 semanas de intervención, se obtuvieron mediciones de altura corporal, masa corporal, IMC, tríceps, pliegues cutáneos subescapular, abdominal, muslo y pantorrilla, y circunferencia torácica, abdominal, muslo y pantorrilla. La duración total del programa fue de12 semanas, con una frecuencia de 2 horas/semana (24 sesiones). La sesión individual tuvo una duración de 60 minutos y la intensidad osciló entre el 60 % y el 80 % de la FCmáx. Los hallazgos del presente estudio sugieren que el programa de carrera de resistencia aeróbica de 12 semanas indujo cambios en las características morfológicas de las adolescentes. Las reducciones significativas en la masa corporal y el IMC fueron menores para los adolescentes con peso normal (-2,64 % y -3,48 %) y moderadas para los adolescentes con sobrepeso (-4,14 % y -4,36 %) después del programa experimental. La magnitud de las reducciones en el grosor del pliegue cutáneo fue pequeña para ambos grupos (tríceps, subescapular, abdominal y pantorrilla) y moderada para el pliegue cutáneo del muslo y la suma de los pliegues cutáneos en el grupo con sobrepeso. Las reducciones en la magnitud de todas las medidas circulares fueron pequeñas para ambos grupos. Osciló entre -1,59 % y -2,59 % para normopeso y entre -2,54 % y -3,92 % para adolescentes con sobrepeso, respectivamente. Además, los efectos del programa aplicado parecen más favorables para los adolescentes con sobrepeso que para aquellos de peso normal. Los hallazgos actuales de este estudio indican que el programa de carreras de resistencia aeróbica de 12 semanas es eficaz, debido a las mejoras en las características morfológicas, la masa corporal y el IMC de las adolescentes. Además, los hallazgos de este estudio muestran que el programa de carrera de resistencia aeróbica aplicada demostró ser más efectivo para los adolescentes con sobrepeso.

Effects of Automation on Sustainability of Immunohistochemistry Laboratory.

The COVID-19 pandemic that hit the world recently caused numerous changes affecting the health system in every department. Reduced staff numbers, mostly due to illness, led to an increase in automation at every stage of laboratory work. The immunohistochemistry (IHC) laboratory conducts a high volume of slide staining every day. Therefore, we analyzed time and total costs required to obtain IHC slides in both the manual and automated way, comparing their efficiency by processing the same sample volume (48 microscope slides-the maximum capacity that an automated immunostainer-DAKO, Autostainer Link 48, Part No AS48030-can process over a single cycle). The total IHC procedure time to run 48 slides manually by one technician was 460 min, while the automated process finished a cycle within 390 min (15.22% less time). The final cost of a single manual IHC slide was 12.26 EUR and 7.69 EUR for slides labeled in the automated immunostainer, which reduced final costs by 37.27%. Thus, automation of the IHC procedure reduces the time and costs of the IHC process, contributing significantly to the sustainability of the healthcare system during the COVID-19 pandemic, overcoming insufficient human resources.

α-Lipoic Acid Increases Collagen Synthesis and Deposition in Nondiabetic and Diabetic Rat Kidneys.

α-Lipoic acid (ALA) is widely used as a nutritional supplement and therapeutic agent in diabetes management. Well-established antioxidant and hypoglycemic effects of ALA were considered to be particularly important in combating diabetic complications including renal injury. The present study evaluated the potential of ALA to affect profibrotic events in kidney that could alter its structure and functioning. ALA was administered intraperitoneally (10 mg/kg) to nondiabetic and streptozotocin-induced diabetic male Wistar rats for 4 and 8 weeks. The effects of ALA were assessed starting from structural/morphological alterations through changes that characterize profibrotic processes, to regulation of collagen gene expression in kidney. Here, we demonstrated that ALA improved systemic glucose and urea level, reduced formation of renal advanced glycation end products (AGEs), and maintained renal structural integrity in diabetic rats. However, profibrotic events provoked in diabetes were not alleviated by ALA since collagen synthesis/deposition and expression of transforming growth factor-ß1 (TGF-ß1) and α-smooth muscle actin (α-SMA) remained elevated in ALA-treated diabetic rats, especially after 8 weeks of diabetes onset. Moreover, 8 weeks treatment of nondiabetic rats with ALA led to the development of profibrotic features reflected in increased collagen synthesis/deposition. Besides the TGF-ß1 downstream signaling, the additional mechanism underlying the upregulation of collagen IV in nondiabetic rats treated with ALA involves decreased DNA methylation of its promoter that could arise from increased Tet1 expression. These findings emphasize the therapeutic caution in the use of ALA, especially in patients with renal diabetic complication.

DNA methylation of miR-200 clusters promotes epithelial to mesenchymal transition in human conjunctival epithelial cells.

Epithelial to mesenchymal transition (EMT) contributes to fibrosis associated pathologies including scarring of different ocular tissues. Recently targeting EMT is seen as an appropriate therapeutic approach for different fibrosis related eye diseases such as macular degeneration or glaucoma surgery related fibrosis. Nevertheless, for ocular surface diseases, target genes specific for particular cell type or condition are still undefined. This study aimed to expose the complex regulatory mechanisms that trigger EMT in human conjunctival epithelial (HCjE) cells. EMT was induced by prolonged treatment with two TGF-ß isoforms, TGF-ß1 and TGF-ß2, and their combination. TGF-ß1 showed the strongest potential for initiating EMT in HCjE cells, reflected on morphological changes, cell migration and the levels of mRNA expression of different epithelial (CDH1, OCLN, DSP) and mesenchymal (CDH2, FN1, VIM, SNAI1, ZEB2, TWIST1) marker genes. Co-treatment with the DNA demethylating agent 5-Azacytidine (5-AzaC) was capable of stopping the transition of HCjE cells towards a mesenchymal phenotype, based on morphological features, reduced cell mobility and mRNA and protein expression levels of epithelial and mesenchymal marker genes. An EMT qRT-PCR-based array revealed that EMT induced considerable alterations in gene expression, with downregulation of the majority of epithelial marker genes and upregulation of genes specific for the mesenchymal state. The major effect of 5-AzaC treatment was observed as a suppression of mesenchymal marker genes, suggesting the involvement of upstream negative regulator(s) whose promoter demethylation and subsequent expression will in turn promote EMT switch off. The expression level of miRNAs potentially important for EMT induction was determined using qRT-PCR-based array which pointed at members of miR-200 family as main regulators of EMT process in HCjE cells. 5-AzaC treatment induced increased expression of miR-200a, -200b, -200c and miR-141 towards the control level, indicating important role of DNA methylation in their regulation. The DNA methylation status of both miR-200 family clusters, analyzed with high-resolution melting (HRM) and bisulfite sequencing (Bis-Seq), revealed that TGF-ß1-induced EMT was accompanied by increase in promoter CpG methylation of both miR-200 loci, which was reverted after 5-AzaC treatment. In conclusion, our results indicate that DNA demethylation of promoters of miR-200 loci is critically important for stopping and reverting the EMT in human conjunctival epithelial cells, suggesting the potential for the development of novel epigenetic-based therapeutic strategies for treating conjunctival conditions associated with EMT.

[Abnormal ultrasonographic findings of the kidneys obtained with a portable echosonographic device in the patients with infectious diseases--a five-year study].

BACKGROUND/AIM: Ultrasonographic kidney changes might be a part of the clinical picture, or consequence of the various infections diseases. The aim of this study was to establish ultrasonographic findings obtained by portable devices, the frequency of abnormal findings of the kidneys in the non-selected group of the patients with infections diseases. METHODS: Over a five-year period (January 1, 2000-December 31, 2004), the kidneys were examined by ultrasonography in 2,718 patients, 1,452 males and 1,266 females, mean age 47.52 +/- 17 years, (16-92 years). The examination included the measurement of the size of the kidneys, evaluation of the condition of parenchyma and pyelo-calix, detection of simple cysts, calculi and tumor. The conventional portable ultrasonographic devices ALOKA SSD-500 and SSD-1000 (B-mode) with a convex 3.5 and 5 MHz sonde were used. RESULTS: The size of kidneys was normal in 95% of the patients, while they were enlarged in 1.3% and reduced in 1.5% of the patients. A normal ultrasonographic recording was noted in 68.9% of the patients, double pelvis in 0.1%, while hydronephrosis was revealed in 0.9% of the patients. A reduced renal parenchyma was observed in 16.1% of the patients. Nephrolithiasis was found in 10.9% of the patients, and simple cysts of kidneys in 8.9% of the patients. The finding of polycystic kidneys was seen in 0.5% of the patients. An ultrasonographic recording of angiomyolipoma was noted in 0.4% of the patients, and the finding of other tumors in 0.1% of the patients. Adrenal tumors were found in 0.1% of the patients. CONCLUSION: Portable ultrasonographic units may be highly useful for the standard morphological diagnostics of renal changes during infections, as well as in clinical-epidemiological studies and screening of hereditary and the acquired diseases of this organ.

[Elevated aminotransferse levels--diagnostic approach]. / Povisen nivo transaminaza--dijagnosticki pristup.

INTRODUCTION: Aminotransferase levels are a sensitive indicator of liver cell injury and is frequently used to identify patients with liver diseases such as hepatitis. Both aminotransferases are normally present in serum at low levels, usually less than 30 U per liter. Although these enzymes are present in tissues throughout the body, they are most often elevated in patients with liver diseases and may reflect liver injury. Raised aminotransferase levels of unknown origin are common problem in clinical practice. Serum alanine aminotransferase (ALT) activity, the variable most commonly measured to assess hepatic disease, fails to identify many patients with hepatic injury. On the other hand, elevated ALT level doesn't always confirm liver disease. The aim of this study was to show the most common reasons for elevated aminotransferase levels. MATERIAL AND METHODS: The study included 27 patients with elevated ALT. All of them were followed-up for six months before liver biopsy was performed. All patients underwent routine laboratory tests and ultrasound of the abdomen. RESULTS: We found that four patients had a nonalcoholic steatohepatitis (NASH), four patients had chronic hepatitis of unknown etiology, two of them had autoimmune hepatitis, four had mild lobular hepatitis (three of them unresolved acute hepatitis, one of them had nodular regenerative hyperplasia), six patients had normal results, and three had no specific changes. CONCLUSION: Elevated ALT level doesn't always mean that there is a liver disease. If aminotransferase levels are persistently more than twice the normal value, a biopsy is recommended. Although results of biopsy are unlikely to lead to a diagnosis or to changes in management, they often provide reassurance to the patient and the physician to exclude serious pathology.