The Effect of "Proanthocyanidin" on Ischemia-Reperfusion Injury in Skeletal Muscles of Rats.

Background and Objectives: Lower limb skeletal muscle ischemia-reperfusion (IR) injury is associated with increased morbidity and mortality, and it is common in several clinical situations such as aortic aneurysms repairment, peripheral arterial surgery, vascular injury repairment, and shock. Although it is generally accepted that oxidative stress mediators have a significant role in IR injury, its precise mechanism is still unknown. Anecdotally, it is sustained not only by structural and functional changes in the organ it affects but also by damage to distant organs. The purpose of this report is to illustrate the effect of proanthocyanidin on IR injury. Materials and Methods: In our study, 18 male Wistar albino rats were used. The subjects were divided into three groups containing six mice each (control, C; ischemia-reperfusion, IR; ischemia-reperfusion and proanthocyanidin; IR-PRO). Intraperitoneal proanthocyanidin was given to the IR and proanthocyanidin groups 30 min before laparotomy, and 1 h ischemia led to these two groups. After one hour, reperfusion started. Muscle atrophy-hypertrophy, muscle degeneration-congestion, fragmentation-hyalinization, muscle oval-central nucleus ratio, leukocyte cell infiltration, catalase enzyme activity, and TBARS were all examined in lower-limb muscle samples after one hour of reperfusion. Results: When skeletal muscle samples were evaluated histopathologically, it was discovered that muscle atrophy-hypertrophy, muscle degeneration-congestion, fragmentation-hyalinization, and leukocyte cell infiltration with oval-central nucleus standardization were significantly higher in the IR group than in the C and IR-P groups. Oval-central nucleus standardization was significantly higher in the IR and IR-PRO groups than in the control group. TBARS levels were significantly higher in the IR group than in the control and IR-PRO groups, while catalase enzyme activity was found to be significantly lower in the IR group than in the control and IR-PRO groups. Conclusions: As a consequence of our research, we discovered that proanthocyanidins administered before IR have a protective impact on skeletal muscle in rats. Further research in this area is required.

The Role of Monocyte Distribution Width in the Early Prediction of Sepsis in Patients Undergoing Cardiovascular Surgery: A Cross-Sectional Study.

Background and Objectives: This is the first study to examine the role of monocyte distribution width (MDW) in predicting sepsis after cardiovascular surgery. Methods: This study included 43 consecutive patients who had undergone cardiovascular surgery between July 2021 and July 2022. All patients were examined at the following three time points (TPs): preoperative period (TP1), postoperative at 24 h (TP2), and discharge (TP3). SOFA score, leukocyte count, neutrophil-to-lymphocyte ratio (NLR), MDW, C-reactive protein (CRP), and procalcitonin (PCT) levels were tested at each TPs. The Sepsis-3 criteria were used to diagnose patients with sepsis. Results: The mean values of all variables (leukocyte count, NLR, MDW, CRP, and PCT levels) were significantly higher at TP2 and TP3 than at TP1 (p < 0.05). All these values were significantly higher at TP2 than at TP3 (p < 0.05). Patients with sepsis had significantly higher mean values for leukocyte count, NLR, MDW, CRP, and PCT levels than those without sepsis (p < 0.05). There was a significant correlation between MDW and inflammatory markers (CRP, PCT, and NLR) during the three time periods (p < 0.05). According to the ROC analysis, the optimal MDW cutoff value with the highest sensitivity and specificity for predicting sepsis in the postoperative period was 20.5. Conclusions: Our findings indicate that elevated MDW levels may be a valuable predictor of sepsis in patients following cardiovascular surgery.

The Effect of Cerium Oxide (CeO2) on Ischemia-Reperfusion Injury in Skeletal Muscle in Mice with Streptozocin-Induced Diabetes.

Objective: Lower extremity ischemia-reperfusion injury (IRI) may occur with trauma-related vascular injury and various vascular diseases, during the use of a tourniquet, in temporary clamping of the aorta in aortic surgery, or following acute or bilateral acute femoral artery occlusion. Mitochondrial dysfunction and increased basal oxidative stress in diabetes may cause an increase in the effects of increased reactive oxygen species (ROS) and mitochondrial dysfunction due to IRI. It is of great importance to examine therapeutic approaches that can minimize the effects of IRI, especially for patient groups under chronic oxidative stress such as DM. Cerium oxide (CeO2) nanoparticles mimic antioxidant enzymes and act as a catalyst that scavenges ROS. In this study, it was aimed to investigate whether CeO2 has protective effects on skeletal muscles in lower extremity IRI in mice with streptozocin-induced diabetes. Methods: A total of 38 Swiss albino mice were divided into six groups as follows: control group (group C, n = 6), diabetes group (group D, n = 8), diabetes-CeO2 (group DCO, n = 8), diabetes-ischemia/reperfusion (group DIR, n = 8), and diabetes-ischemia/reperfusion-CeO2 (group DIRCO, n = 8). The DCO and DIRCO groups were given doses of CeO2 of 0.5 mg/kg intraperitoneally 30 min before the IR procedure. A 120 min ischemia-120 min reperfusion period with 100% O2 was performed. At the end of the reperfusion period, muscle tissues were removed for histopathological and biochemical examinations. Results: Total antioxidant status (TAS) levels were found to be significantly lower in group DIR compared with group D (p = 0.047 and p = 0.022, respectively). In group DIRCO, total oxidant status (TOS) levels were found to be significantly higher than in group DIR (p < 0.001). The oxidative stress index (OSI) was found to be significantly lower in group DIR compared with group DCO (p < 0.001). Paraoxanase (PON) enzyme activity was found to be significantly increased in group DIR compared with group DCO (p < 0.001). The disorganization and degeneration score for muscle cells, inflammatory cell infiltration score, and total injury score in group DIRCO were found to be significantly lower than in group DIR (p = 0.002, p = 0.034, and p = 0.001, respectively). Conclusions: Our results confirm that CeO2, with its antioxidative properties, reduces skeletal muscle damage in lower extremity IRI in diabetic mice.

RNA aptamer capture of macromolecular complexes for mass spectrometry analysis.

Specific genomic functions are dictated by macromolecular complexes (MCs) containing multiple proteins. Affinity purification of these complexes, often using antibodies, followed by mass spectrometry (MS) has revolutionized our ability to identify the composition of MCs. However, conventional immunoprecipitations suffer from contaminating antibody/serum-derived peptides that limit the sensitivity of detection for low-abundant interacting partners using MS. Here, we present AptA-MS (aptamer affinity-mass spectrometry), a robust strategy primarily using a specific, high-affinity RNA aptamer against Green Fluorescent Protein (GFP) to identify interactors of a GFP-tagged protein of interest by high-resolution MS. Utilizing this approach, we have identified the known molecular chaperones that interact with human Heat Shock Factor 1 (HSF1), and observed an increased association with several proteins upon heat shock, including translation elongation factors and histones. HSF1 is known to be regulated by multiple post-translational modifications (PTMs), and we observe both known and new sites of modifications on HSF1. We show that AptA-MS provides a dramatic target enrichment and detection sensitivity in evolutionarily diverse organisms and allows identification of PTMs without the need for modification-specific enrichments. In combination with the expanding libraries of GFP-tagged cell lines, this strategy offers a general, inexpensive, and high-resolution alternative to conventional approaches for studying MCs.

Chromatin conformation remains stable upon extensive transcriptional changes driven by heat shock.

Heat shock (HS) initiates rapid, extensive, and evolutionarily conserved changes in transcription that are accompanied by chromatin decondensation and nucleosome loss at HS loci. Here we have employed in situ Hi-C to determine how heat stress affects long-range chromatin conformation in human and Drosophila cells. We found that compartments and topologically associating domains (TADs) remain unchanged by an acute HS. Knockdown of Heat Shock Factor 1 (HSF1), the master transcriptional regulator of the HS response, identified HSF1-dependent genes and revealed that up-regulation is often mediated by distal HSF1 bound enhancers. HSF1-dependent genes were usually found in the same TAD as the nearest HSF1 binding site. Although most interactions between HSF1 binding sites and target promoters were established in the nonheat shock (NHS) condition, a subset increased contact frequency following HS. Integrating information about HSF1 binding strength, RNA polymerase abundance at the HSF1 bound sites (putative enhancers), and contact frequency with a target promoter accurately predicted which up-regulated genes were direct targets of HSF1 during HS. Our results suggest that the chromatin conformation necessary for a robust HS response is preestablished in NHS cells of diverse metazoan species.

The effect of neutrophil-lymphocyte ratio on the postoperative course of coronary artery bypass graft surgery

Background/aim: Recovery after coronary artery bypass graft surgery (CABG) can be complicated, leading to postoperative morbidity. The roles of hematologic and surgery-related parameters are important. The main purpose of this study is to determine the role of preoperative and postcardiopulmonary bypass neutrophil/lymphocyte ratio (NLR) on postoperative recovery. Materials and methods: Sixty-two patients aged between 41 and 80 years, scheduled for elective CABG surgery with ASA I-II risk and without a history of preoperative blood transfusion, were included in the study. Three patients were excluded due to their need for additional surgical procedures other than CABG. The patients were divided into two groups that were formed depending on preoperative NLR cut-off values below (Group 1, n = 37) and above 4 (Group 2, n = 22). Postoperative data such as length of stay in the hospital and in the intensive care unit (ICU), chest tube drainage, and incidence of atrial fibrillation were recorded for all patients. Results: Preoperative NLR was significantly lower in Group 1 (P < 0.0001), and there was no significant difference between the groups in terms of postoperative NLR (P = 0.217) when the two groups were compared. The patients in Group 2 had a longer length of stay in the ICU (P = 0.035) and in the hospital (P = 0.034). There was a positive correlation between preoperative NLR and length of stay in the ICU (P = 0.017) and the hospital (P = 0.014). No statistically significant differences in postoperative drainage or incidence of postoperative atrial fibrillation were detected between the two groups. Conclusion: The results of our study demonstrate that the postoperative NLR may be useful to predict the length of hospital and ICU stays and help the management of follow-up and treatment processes in patients undergoing CABG surgery.

Comprehensive analysis of RNA-protein interactions by high-throughput sequencing-RNA affinity profiling.

RNA-protein interactions play critical roles in gene regulation, but methods to quantitatively analyze these interactions at a large scale are lacking. We have developed a high-throughput sequencing-RNA affinity profiling (HiTS-RAP) assay by adapting a high-throughput DNA sequencer to quantify the binding of fluorescently labeled protein to millions of RNAs anchored to sequenced cDNA templates. Using HiTS-RAP, we measured the affinity of mutagenized libraries of GFP-binding and NELF-E-binding aptamers to their respective targets and identified critical regions of interaction. Mutations additively affected the affinity of the NELF-E-binding aptamer, whose interaction depended mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depended primarily on secondary structure.

Defining NELF-E RNA binding in HIV-1 and promoter-proximal pause regions.

The four-subunit Negative Elongation Factor (NELF) is a major regulator of RNA Polymerase II (Pol II) pausing. The subunit NELF-E contains a conserved RNA Recognition Motif (RRM) and is proposed to facilitate Poll II pausing through its association with nascent transcribed RNA. However, conflicting ideas have emerged for the function of its RNA binding activity. Here, we use in vitro selection strategies and quantitative biochemistry to identify and characterize the consensus NELF-E binding element (NBE) that is required for sequence specific RNA recognition (NBE: CUGAGGA(U) for Drosophila). An NBE-like element is present within the loop region of the transactivation-response element (TAR) of HIV-1 RNA, a known regulatory target of human NELF-E. The NBE is required for high affinity binding, as opposed to the lower stem of TAR, as previously claimed. We also identify a non-conserved region within the RRM that contributes to the RNA recognition of Drosophila NELF-E. To understand the broader functional relevance of NBEs, we analyzed promoter-proximal regions genome-wide in Drosophila and show that the NBE is enriched +20 to +30 nucleotides downstream of the transcription start site. Consistent with the role of NELF in pausing, we observe a significant increase in NBEs among paused genes compared to non-paused genes. In addition to these observations, SELEX with nuclear run-on RNA enrich for NBE-like sequences. Together, these results describe the RNA binding behavior of NELF-E and supports a biological role for NELF-E in promoter-proximal pausing of both HIV-1 and cellular genes.

The effect of levosimendan on lung damage after myocardial ischemia reperfusion in rats in which experimental diabetes was induced.

BACKGROUND: It is known that diabetic complications and lipid peroxidation are closely associated. During ischemia and reperfusion (IR), injury may occur in distant organs, as well as in tissues next to the region exposed to the ischemia, and the lungs can be one of the most affected of these organs. Therefore, this study investigated the effects of levosimendan on lung tissue and the oxidant-antioxidant system in diabetic rats. MATERIALS AND METHODS: The study was conducted in 24 Wistar albino rats that were separated into four groups (C, control; DC, diabetic control; DIR, diabetic IR; and DIRL, diabetic IR levosimendan). Diabetes was induced in 18 rats using streptozotocin (55 mg/kg), and the animals were randomly separated into three groups after the effects of the diabetes became apparent. After a left thoracotomy, ischemia was performed on the myocardial muscle with the left main coronary artery (LAD) for 30 min in the DIR and DIRL groups. After ischemia, the LAD ligation was removed, and reperfusion was applied for 120 min. Single-dose intraperitoneal 12 µg/kg levosimendan was administered to group DIRL before the ischemia. Group DC was evaluated as the diabetic control group, and six rats were considered to be the control group (group C), in which thoracotomy was performed and then closed with no induction of myocardial ischemia. We measured the levels of malondialdehyde, as a lipid peroxidation end product, as well as catalase and glutathione S-transferase activities, as antioxidant enzymes in the lung tissue. Tissue samples were also examined histopathologically. RESULTS: Neutrophil infiltration or aggregation in lung tissue was significantly higher in the DIR group compared with the C, DC, and DIRL groups (P = 0.003, P = 0.026, and P = 0.026, respectively). Alveolar wall thickening in lung tissue was significantly higher in the DIR group compared with the C, DC, and DIRL groups (P = 0.002, P = 0.002, and P = 0.006, respectively). In addition, the lung tissue damage score was significantly higher in the DIR group compared with the C, DC, and DIRL groups (P = 0.001, P = 0.004, and P = 0.007, respectively). Finally, catalase and glutathione S-transferase activity levels were significantly higher in the DIR group compared with those observed in the C, DC, and DIRL groups. CONCLUSIONS: Although diabetes increases lipid peroxidation, it suppresses antioxidant activity. Our results showed that levosimendan had a protective effect against lung damage secondary to IR in the rats with induced diabetes. We recommend that experimental and clinical studies be conducted to examine the effects of levosimendan at different doses and different IR durations on various organs for clinical use.

Density-dependent cooperative non-specific binding in solid-phase SELEX affinity selection.

The non-specific binding of undesired ligands to a target is the primary factor limiting the enrichment of tight-binding ligands in affinity selection. Solution-phase non-specific affinity is determined by the free-energy of ligand binding to a single target. However, the solid-phase affinity might be higher if a ligand bound concurrently to multiple adjacent immobilized targets in a cooperative manner. Cooperativity could emerge in this case as a simple consequence of the relationship between the free energy of binding, localization entropy and the spatial distribution of the immobilized targets. We tested this hypothesis using a SELEX experimental design and found that non-specific RNA aptamer ligands can concurrently bind up to four bead-immobilized peptide targets, and that this can increase their effective binding affinity by two orders-of-magnitude. Binding curves were quantitatively explained by a new statistical mechanical model of density-dependent cooperative binding, which relates cooperative binding to both the target concentration and the target surface density on the immobilizing substrate. Target immobilization plays a key role in SELEX and other ligand enrichment methods, particularly in new multiplexed microfluidic purification devices, and these results have strong implications for optimizing their performance.

High-throughput binding characterization of RNA aptamer selections using a microplate-based multiplex microcolumn device.

We describe a versatile 96-well microplate-based device that utilizes affinity microcolumn chromatography to complement downstream plate-based processing in aptamer selections. This device is reconfigurable and is able to operate in serial and/or parallel mode with up to 96 microcolumns. We demonstrate the utility of this device by simultaneously performing characterizations of target binding using five RNA aptamers and a random library. This was accomplished through 96 total selection tests. Three sets of selections tested the effects of target concentration on aptamer binding compared to the random RNA library using aptamers to the proteins green fluorescent protein (GFP), human heat shock factor 1 (hHSF1), and negative elongation factor E (NELF-E). For all three targets, we found significant effects consistent with steric hindrance with optimum enrichments at predictable target concentrations. In a fourth selection set, we tested the partitioning efficiency and binding specificity of our three proteins' aptamers, as well as two suspected background binding sequences, to eight targets running serially. The targets included an empty microcolumn, three affinity resins, three specific proteins, and a non-specific protein control. The aptamers showed significant enrichments only on their intended targets. Specifically, the hHSF1 and NELF-E aptamers enriched over 200-fold on their protein targets, and the GFP aptamer enriched 750-fold. By utilizing our device's plate-based format with other complementary plate-based systems for all downstream biochemical processes and analysis, high-throughput selections, characterizations, and optimization were performed to significantly reduce the time and cost for completing large-scale aptamer selections.

RNA aptamers that functionally interact with green fluorescent protein and its derivatives.

Green Fluorescent Protein (GFP) and related fluorescent proteins (FPs) have been widely used to tag proteins, allowing their expression and subcellular localization to be examined in real time in living cells and animals. Similar fluorescent methods are highly desirable to detect and track RNA and other biological molecules in living cells. For this purpose, we have developed a group of RNA aptamers that bind GFP and related proteins, which we term Fluorescent Protein-Binding Aptamers (FPBA). These aptamers bind GFP, YFP and CFP with low nanomolar affinity and binding decreases GFP fluorescence, whereas slightly augmenting YFP and CFP brightness. Aptamer binding results in an increase in the pKa of EGFP, decreasing the 475 nm excited green fluorescence at a given pH. We report the secondary structure of FPBA and the ability to synthesize functional multivalent dendrimers. FPBA expressed in live cells decreased GFP fluorescence in a valency-dependent manner, indicating that the RNA aptamers function within cells. The development of aptamers that bind fluorescent proteins with high affinity and alter their function, markedly expands their use in the study of biological pathways.

Assessment of Aspirin and Clopidogrel Resistance in Patients Undergoing Cardiovascular Surgery: A Single-Center Cross-Sectional Study

Objective: We aimed to investigate antiplatelet drug resistance utilizing light transmission-lumiaggregometry (LT-LA) and the Platelet Function Analyzer-100 (PFA-100) in patients undergoing cardiovascular surgery. Materials and Methods: The study included 60 patients diagnosed with stable coronary artery disease and peripheral vascular diseases that required surgery. Participants were divided into three groups: patients receiving aspirin (ASA) (n=21), patients receiving clopidogrel (CLO) (n=19), and patients receiving dual therapy (ASA+CLO) (n=20). Aggregation and secretion tests by LT-LA and closure time by the PFA-100 were used to measure antiplatelet drug resistance. Results: Based on the adenosine diphosphate (ADP)-induced aggregation test, 43% of patients were resistant to ASA, 22% to CLO, and 15% to dual therapy. Diabetes, hypertension, and hyperlipidemia were the most commonly identified comorbid disorders. In patients with comorbid risk factors, the median value of platelet aggregation response to ADP was significantly higher in the ASA group than in the CLO and dual therapy groups (p=0.0001). In patients receiving ASA monotherapy, the maximum amplitude of aggregation response to platelet agonists was ≥70% in 43% of patients for ADP and 28% for collagen by LT-LA. Elevated ADP (≥0.29 nmol) and collagen (≥0.41 nmol)-induced adenosine triphosphate release were found by LT-LA in 66% of patients utilizing an ADP agonist and 80% of patients using a collagen agonist undergoing ASA therapy. Closure times obtained with the PFA-100 were normal in 28% of patients using collagen-ADP cartridges and 62% of patients using collagen-epinephrine (CEPI) cartridges who received ASA. Recurrent thrombosis and bleeding were observed in 12 (20%) patients with cardiovascular disease. Three of these individuals (25%) showed ASA resistance with normal responses to ADP-induced aggregation (≥70%) and secretion (≥0.29 nmol), as well as normal CEPI closure times. Conclusion: Our findings suggest that antiplatelet drug monitoring by LT-LA and PFA-100 may be useful for high-risk and complicated cardiovascular patients.

Ozone Administration Reduces Myocardial Ischemia Reperfusion Injury in Streptozotocin Induced Diabetes Mellitus Rat Model.

Objective: This study aimed to demonstrate whether ozone has cardioprotective effects on the myocardial ischemia-reperfusion injury (IRI) in rats with streptozotocin(STZ)-induced diabetes. Methods: A total of 38 male Wistar Albino rats were divided into five groups as follows: control group (group C,n=6), diabetic group (group D,n=6), diabetic ozone group (group DO,n=6), diabetic-ischemia/reperfusion (group DIR,n=6), diabetic-ischemia/reperfusion-ozone (group DIRO,n=6). Six rats died during this period and two died because of surgical complications. A myocardial ischemia-reperfusion model was created using a thoracotomy incision from 4th intercostal space. The LAD was ligated using an 8-0 prolene suture for 30min. Ozone was administered intraperitoneally(1mg/kg) 5min before reperfusion. The reperfusion time was 120 min. At the end of the reperfusion procedure, myocardial tissue histopathological examinations, and serum biochemical analyses were performed. Results: The percentage of TUNEL(+) cardiomyocytes/HPF was significantly higher in the DIR group than in the C, D, and DO groups. Conversely, TUNEL positivity was significantly lower in the DIRO group than in the DIR group. The IRI score was significantly higher in the DIR and DIRO groups than that in the C, D, and DO groups. In contrast, the IRI damage score in the DIRO group was significantly lower than that in the DIR group. Serum MDA levels were significantly higher in the DIR group than in the C, D, and DO groups. Similarly, MDA levels were significantly higher in the DIRO group than in the C and D groups. CAT activity was significantly higher in the DIR group than in the C and D groups. SOD activity was significantly higher in the DIR group than in the C and DO groups. Conclusion: Our study showed that ozone exerts cardioprotective effects in STZ-induced diabetic rats through its antioxidant role against oxidative stress. Both biochemical and histological analyses clearly revealed that ozone has beneficial effects against IRI in the diabetic rat myocardium.

Pericardiectomy after constrictive pericarditis associated with second dose of BNT162b2 vaccine: A case report.

Constrictive pericarditis is quite rare complication of messenger ribonucleic acid-based severe acute respiratory syndrome-Coronavirus 2 (SARS-CoV-2) vaccine. It is a severe clinical picture with clinical symptoms of right ventricular failure. Initial physical examination, laboratory work-up, and chest X-ray may yield non-specific findings. Echocardiography, computed tomography, and cardiac catheterization are other diagnostic tools. Surgery with pericardiectomy is the definitive treatment option. Herein, we report a case of pericardiectomy after constrictive pericarditis associated with the second dose of BNT162b2 vaccine.

Evaluation of the effect of enriched hydrogen saline solution on distant organ (lung) damage in skeletal muscle ischemia reperfusion in rats.

INTRODUCTION: Ischemia-reperfusion (IR) injury is a major concern that frequently occurs during vascular surgeries. Hydrogen-rich saline (HRS) solution exhibits antioxidant and anti-inflammatory properties. This study aimed to examine the effects of HRS applied before ischemia in the lungs of rats using a lower extremity IR model. MATERIAL AND METHODS: After approval was obtained from the ethics committee, 18 male Wistar albino rats weighing 250-280 g were randomly divided into three groups: control (C), IR and IR-HRS. In the IR and IR-HRS groups, an atraumatic microvascular clamp was used to clamp the infrarenal abdominal aorta, and skeletal muscle ischemia was induced. After 120 min, the clamp was removed, and reperfusion was achieved for 120 min. In the IR-HRS group, HRS was administered intraperitoneally 30 min before the procedure. Lung tissue samples were examined under a light microscope and stained with hematoxylin-eosin (H&E). Malondialdehyde (MDA) levels, total sulfhydryl (SH) levels, and histopathological parameters were evaluated in the tissue samples. RESULTS: MDA and total SH levels were significantly higher in the IR group than in the control group (p < 0.0001 and p = 0.001, respectively). MDA and total SH levels were significantly lower in the IR-HRS group than in the IR group (p < 0.0001 and p = 0.013, respectively). A histopathological examination revealed that neutrophil infiltration/aggregation, alveolar wall thickness, and total lung injury score were significantly higher in the IR group than in the control group (p < 0.0001, p = 0.001, and p < 0.0001, respectively). Similarly, alveolar wall thickness and total lung injury scores were significantly higher in the IR-HRS group than in the control group (p = 0.009 and p = 0.004, respectively). A statistically significant decrease was observed in neutrophil infiltration/aggregation and total lung injury scores in the IR-HRS group compared to those in the IR group (p = 0.023 and p = 0.022, respectively). CONCLUSION: HRS at a dose of 20 mg/kg, administered intraperitoneally 30 min before ischemia in rats, reduced lipid peroxidation and oxidative stress, while also reducing IR damage in lung histopathology. We believe that HRS administered to rats prior to IR exerts a lung-protective effect.

Endovascular recanalization of infra-popliteal TASC C and TASC D lesions in patients with critical limb-threatening ischemia: a single-center experience.

PURPOSE: The present study aims to (1) assess the technical success and limb salvage rates of endovascular therapy in patients with critical limb-threatening ischemia (CLTI) and infra-popliteal Trans-Atlantic Inter-Society Consensus (TASC) C/D lesions according to the updated 2015 TASC II classification and (2) to present our institutional experience. METHODS: A single-center retrospective study was conducted on patients with TASC C/D CLTI who underwent endovascular treatment between 2012 and 2017. The follow-up protocol consisted of Doppler ultrasound conduction every 3 months for the first year unless patients showed symptoms of CLTI. Patients with at least 1 year of follow-up data were included in the study, and if applicable their 3-year results were evaluated in terms of primary patency, absence of amputation, amputation-free survival, and overall survival. RESULTS: A total of 248 patients and 287 limbs (238 TASC D lesions and 49 TASC C lesions) were treated via infra-popliteal percutaneous transluminal angioplasty. The overall technical success was 87%, the primary patency rate was 41.5% in the first year, and the freedom from amputation rates were 80.8% in 1 year and 67.7% in 3 years. CONCLUSION: In patients with infra-popliteal arterial occlusive diseases, endovascular treatment methods demonstrate a high rate of technical success and favorable outcomes in limb preservation.

A lineage-specific nascent RNA assay unveils principles of gene regulation in tissue biology.

Gene regulatory mechanisms that modulate RNA Polymerase II activity are difficult to access in mammalian tissues composed of multiple cell lineages. Here, we develop a nascent RNA assay (PReCIS-seq) that measures lineage-specific transcriptionally-engaged Pol II on genes and DNA enhancer elements in intact mouse tissue. By employing keratinocytes as a prototype lineage, we unearth Pol II promoter-recruitment versus pause-release mechanisms operating in adult skin homeostasis. Moreover, we relate active enhancer proximity and transcription factor binding motifs on promoters to Pol II activity and promoter-proximal pausing level. Finally, we find Pol II firing rapidly into elongation on lineage identity genes and highly paused on cellular safeguarding genes in a context-dependent manner. Our work provides a basic platform to investigate mechanistic principles of gene regulation in individual lineages of complex mammalian tissues.

Multiplexed microcolumn-based process for efficient selection of RNA aptamers.

We describe a reusable microcolumn and process for the efficient discovery of nucleic acid aptamers for multiple target molecules. The design of our device requires only microliter volumes of affinity chromatography resin-a condition that maximizes the enrichment of target-binding sequences over non-target-binding (i.e., background) sequences. Furthermore, the modular design of the device accommodates a multiplex aptamer selection protocol. We optimized the selection process performance using microcolumns filled with green fluorescent protein (GFP)-immobilized resin and monitoring, over a wide range of experimental conditions, the enrichment of a known GFP-binding RNA aptamer (GFPapt) against a random RNA aptamer library. We validated the multiplex approach by monitoring the enrichment of GFPapt in de novo selection experiments with GFP and other protein preparations. After only three rounds of selection, the cumulative GFPapt enrichment on the GFP-loaded resin was greater than 10(8) with no enrichment for the other nonspecific targets. We used this optimized protocol to perform a multiplex selection to two human heat shock factor (hHSF) proteins, hHSF1 and hHSF2. High-throughput sequencing was used to identify aptamers for each protein that were preferentially enriched in just three selection rounds, which were confirmed and isolated after five rounds. Gel-shift and fluorescence polarization assays showed that each aptamer binds with high-affinity (KD < 20 nM) to the respective targets. The combination of our microcolumns with a multiplex approach and high-throughput sequencing enables the selection of aptamers to multiple targets in a high-throughput and efficient manner.

Protective effects of hydrogen rich saline solution in rats with experimental myocardial ischemia reperfusion injury.

Aim: The aim of our study is to show whether the administration of hydrogen-rich saline solution (HRSS) intraperitoneally before left main coronary artery (LAD) ischemia protects the myocardium against ischemia-reperfusion (IR) injury. Materials and methods: After ethics committee approval, 24 Wistar Albino rats were divided into 4 groups, 6 rats in each group. For experimental IR, myocardial ischemia was performed by LAD ligation. Left thoracotomy was performed without ischemia in the Control group (Group C). Left thoracotomy was performed without myocardial ischemia to the rats in the HRSS group, and HRSS was given intraperitoneally (ip) at a rate of 10 ml/kg throughout the procedure. In the MIR-HRSS group, a single dose of 10 ml/kg HRSS was administered 5 min before reperfusion. Histopathological and biochemical parameters were compared in myocardial tissue samples taken at the end of the reperfusion period. Results: When the groups were compared among themselves in terms of TOS and TAS levels, there was a significant difference between the groups (p = 0.006, p = 0.002). The severity of cardiomyocyte degeneration was significantly greater in MIR group than that in the control and HRSS groups (p = 0.002 and p = 0.001, respectively), as well as severity score of cardiomyocyte degeneration was higher in MIR-HRSS group compared with HRSS group (p = 0.035). Conclusion: Our study shows that HRSS is protective in IR injury, with the application of HRSS 5 min before reperfusion, interstitial edema severity, subendocardial haemorrhage are reduced, and oxidant status parameters are increased, while antioxidant status parameters are decreased. We believe that when it is supported by other studies, the protective effects of HRSS on IR damage will be shown in detail and its indications will be expanded.