REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering

Mutagenesis is an important tool to study gene regulation, model disease-causing mutations and for functional characterisation of proteins. Most of the current methods for mutagenesis involve multiple step procedures. One of the most accurate methods for genetically altering DNA is recombineering, which uses bacteria expressing viral recombination proteins. Recently, the use of in vitro seamless assembly systems using purified enzymes for multiple-fragment cloning as well as mutagenesis is gaining ground. Although these in vitro isothermal reactions are useful when cloning multiple fragments, for site-directed mutagenesis it is unnecessary. Moreover, the use of purified enzymes in vitro is not only expensive but also more inaccurate than the high-fidelity recombination inside bacteria. Here we present a single-step method, named REPLACR-mutagenesis (Recombineering of Ends of linearised PLAsmids after PCR), for creating mutations (deletions, substitutions and additions) in plasmids by in vivo recombineering. REPLACR-mutagenesis only involves transformation of PCR products in bacteria expressing Red/ET recombineering proteins. Modifications in a variety of plasmids up to bacterial artificial chromosomes (BACs; 144 kb deletion) have been achieved by this method. The presented method is more robust, involves fewer steps and is cost-efficient.</p

Serumska razina za neuron specifične enolaze kao biljeg ishemijsko-reperfuzijskog oštećenja u bolesnika podvrgnutih karotidnoj endarterektomiji

In patients with atherosclerotic stenosis of the extracranial segment of internal carotid artery, surgical intervention is an effective method to prevent cerebral ischemic stroke. However, this surgical procedure may cause vascular brain damage. The aim of the study was to investigate consequential brain ischemia-reperfusion injury by measuring the cerebral specific marker, neuronspecific enolase (NSE), in serum of patients having undergone internal carotid endarterectomy (CEA). The study involved 25 patients that underwent CEA due to internal carotid artery stenosis. Blood samples were obtained from each patient on three occasions: within 24 h prior to surgery, 12 h after surgery, and 48 h after surgery. Serum NSE levels were measured by a commercially available enzymelinked immunosorbent assay. The study showed that serum NSE level was statistically significantly increased 48 h after CEA as compared with the level 12 h after surgery and the level before surgery (p0.05). Data from our study showed CEA to affect serum NSE in patients with significant internal carotid artery stenosis. Thus, serum NSE may be used as a biochemical marker of brain ischemia-reperfusion injury following CEA.Kirurška intervencija je učinkovita metoda sprječavanja ishemijskog moždanog udara u bolesnika s aterosklerotskom stenozom ekstrakranijskog dijela unutarnje karotidne arterije. Međutim, ovaj kirurški zahvat može uzrokovati oštećenje moždanog krvožilja. Cilj ovoga istraživanja bio je ispitati posljedično ishemijsko-reperfuzijsko oštećenje mozga mjerenjem za neuron specifične enolaze (neuron-specific enolase, NSE) kao specifičnog moždanog biljega u serumu bolesnika podvrgnutih endarterektomiji unutarnje karotide (internal carotid endarterectomy, CEA). Istraživanje je obuhvatilo 25 bolesnika podvrgnutih CEA zbog stenoze unutarnje karotidne arterije. Uzorci krvi uzeti su u svakog bolesnika tri puta: unutar 24 h prije operacije, 12 h nakon operacije i 48 h nakon operacije. Serumske razine NSE mjerene su komercijalnim testom ELISA. Rezultati su pokazali da je serumska razina NSE bila statistički značajno povišena 48 h nakon CEA u usporedbi s razinom izmjerenom 12 h nakon operacije, kao i u usporedbi s razinom prije operacije (p0,05). Podaci dobiveni u ovom istraživanju pokazuju da CEA utječe na razinu NSE u serumu kod bolesnika sa značajnom stenozom unutarnje karotidne arterije. Stoga bi serumska razina NSE mogla poslužiti kao biokemijski biljeg ishemijsko-reperfuzijskog oštećenja mozga nakon CEA

Serum microtubule associated protein tau and myelin basic protein as the potential markers of brain ischaemia-reperfusion injury in patients undergoing carotid endarterectomy

Introduction. In the prevention of ischaemic stroke the recommended surgical procedure is carotid endarterectomy (CEA). However, surgical treatment of atherosclerotic stenosis may cause neurological complications. The aim of the study was to investigate consequential brain ischaemia-reperfusion injury by measuring the cerebral specific markers, the microtubule associated protein tau (MAPt) and myelin basic protein (MBP) in the serum of patients that underwent CEA. Material and methods. This study involved 25 participants who underwent CEA due to internal carotid artery stenosis. Blood samples were taken from each patient at three different intervals; within 24 hours prior to surgery, 12 hours after the surgery, and 48 hours after the surgery. Serum MAPt and MBP levels were measured by a commercially available enzyme-linked immunosorbent assay (ELISA). Results. The study showed that serum MAPt and MBP levels were statistically significantly decreased 12 hours after CEA compared to the level before the surgery (p &amp;lt; 0.05), but MAPt and MBP levels were normalized 48 hours after CEA. There was statistically significant correlation in serum MAPt levels with the velocity of blood flow in the internal carotid artery 12 and 48 hours after CEA (p &amp;lt; 0.05). Conclusions. Data from our study showed that CEA affects serum neuromarkers levels, such as MAPt and MBP, in patients with significant internal carotid artery stenosis. MAPt and MBP levels showed characteristic time curve in patients who underwent CEA and did not experience any neurological deficit in perioperative period. Possible alterations of this time curve may potentially be an index of a neurological event occurrence.  Introduction. In the prevention of ischaemic stroke the recommended surgical procedure is carotid endarterectomy (CEA). However, surgical treatment of atherosclerotic stenosis may cause neurological complications. The aim of the study was to investigate consequential brain ischaemia-reperfusion injury by measuring the cerebral specific markers, the microtubule associated protein tau (MAPt) and myelin basic protein (MBP) in the serum of patients that underwent CEA. Material and methods. This study involved 25 participants who underwent CEA due to internal carotid artery stenosis. Blood samples were taken from each patient at three different intervals; within 24 hours prior to surgery, 12 hours after the surgery, and 48 hours after the surgery. Serum MAPt and MBP levels were measured by a commercially available enzyme-linked immunosorbent assay (ELISA). Results. The study showed that serum MAPt and MBP levels were statistically significantly decreased 12 hours after CEA compared to the level before the surgery (p &amp;lt; 0.05), but MAPt and MBP levels were normalized 48 hours after CEA. There was statistically significant correlation in serum MAPt levels with the velocity of blood flow in the internal carotid artery 12 and 48 hours after CEA (p &amp;lt; 0.05). Conclusions. Data from our study showed that CEA affects serum neuromarkers levels, such as MAPt and MBP, in patients with significant internal carotid artery stenosis. MAPt and MBP levels showed characteristic time curve in patients who underwent CEA and did not experience any neurological deficit in perioperative period. Possible alterations of this time curve may potentially be an index of a neurological event occurrence.

Deacetylation of transcription factors in carcinogenesis

Reversible Nε-lysine acetylation/deacetylation is one of the most common post-translational modifications (PTM) of histones and non-histone proteins that is regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). This epigenetic process is highly involved in carcinogenesis, affecting histone and non-histone proteins’ properties and their biological functions. Some of the transcription factors, including tumor suppressors and oncoproteins, undergo this modification altering different cell signaling pathways. HDACs deacetylate their targets, which leads to either the upregulation or downregulation of proteins involved in the regulation of cell cycle and apoptosis, ultimately influencing tumor growth, invasion, and drug resistance. Therefore, epigenetic modifications are of great clinical importance and may constitute a new therapeutic target in cancer treatment. This review is aimed to present the significance of HDACs in carcinogenesis through their influence on functions of transcription factors, and therefore regulation of different signaling pathways, cancer progression, and metastasis.</p

Serum glial fibrillary acidic protein as a marker of brain damage in patients after carotid endarterectomy

Introduction. Surgical treatment of the extracranial section of internal carotid artery stenosis is an effective method of preventing cerebral ischaemic stroke. However, this surgical procedure may cause vascular brain damage. The aim of the study was to measure glial fibrillary acidic protein (GFAP) as a marker of brain damage in the serum of patients that underwent internal carotid endarterectomy (CEA). Material and methods. This study involved 25 participants who underwent CEA because of internal carotid artery stenosis. Blood samples were taken from each patient at three different times; within 24 hours prior to surgery, 12 hours after the surgery, and 48 hours after the surgery. Serum GFAP levels were measured by a commercially available enzyme-linked immunosorbent assay (ELISA). Results. The study showed that serum GFAP levels were not statistically different between all the three measurements (p &gt; 0.05). There was also no statistical significant difference in serum GFAP levels between symptomatic and asymptomatic patients (p &gt; 0.05). There was no statistically significant correlation in serum GFAP level 12 and 48 hours after the surgery with the clamping time (p &gt; 0.05). There was also no significant correlation in the serum GFAP levels with the velocity of blood flow in the internal carotid artery before CEA and after surgery (p &gt; 0.05). Conclusions. The study revealed that CEA does not change serum GFAP levels. Thus, GFAP cannot be a biochemical marker of brain damage after this surgery

Mapping of the three-dimensional lymphatic microvasculature in bladder tumours using light-sheet microscopy

Background: Cancers are heterogeneous and contain various types of irregular structures that can go undetected when examining them with standard two-dimensional microscopes. Studies of intricate networks of vasculature systems, e.g., the tumour lymphatic microvessels, benefit largely from three-dimensional imaging data analysis.Methods: The new DIPCO (Diagnosing Immunolabeled Paraffin-Embedded Cleared Organs) imaging platform uses three-dimensional light-sheet microscopy and whole-mount immunolabelling of cleared samples to study proteins and micro-anatomies deep inside of tumours.Results: Here, we uncovered the whole three-dimensional lymphatic microvasculature of formalin-fixed paraffin-embedded (FFPE) tumours from a cohort of 30 patients with bladder cancer. Our results revealed more heterogeneous spatial deviations in more advanced bladder tumours. We also showed that three-dimensional imaging could determine tumour stage and identify vascular or lymphatic system invasion with higher accuracy than standard two-dimensional histological diagnostic methods. There was no association between sample storage times and outcomes, demonstrating that the DIPCO pipeline could be successfully applied on old FFPE samples.Conclusions: Studying tumour samples with three-dimensional imaging could help us understand the pathological nature of cancers and provide essential information that might improve the accuracy of cancer staging.</div

Sensitization of MCF7 Cells with High Notch1 Activity by Cisplatin and Histone Deacetylase Inhibitors Applied Together

Histone deacetylase inhibitors (HDIs) are promising anti-cancer agents that inhibit proliferation of many types of cancer cells including breast carcinoma (BC) cells. In the present study, we investigated the influence of the Notch1 activity level on the pharmacological interaction between cisplatin (CDDP) and two HDIs, valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA, vorinostat), in luminal-like BC cells. The type of drug-drug interaction between CDDP and HDIs was determined by isobolographic analysis. MCF7 cells were genetically modified to express differential levels of Notch1 activity. The cytotoxic effect of SAHA or VPA was higher on cells with decreased Notch1 activity and lower for cells with increased Notch1 activity than native BC cells. The isobolographic analysis demonstrated that combinations of CDDP with SAHA or VPA at a fixed ratio of 1:1 exerted additive or additive with tendency toward synergism interactions. Therefore, treatment of CDDP with HDIs could be used to optimize a combined therapy based on CDDP against Notch1-altered luminal BC. In conclusion, the combined therapy of HDIs and CDDP may be a promising therapeutic tool in the treatment of luminal-type BC with altered Notch1 activity