Choice of minimally invasive method of treatment of pancreatic pseudocysts: a single center, retrospective study

Aim of the study is to evaluate efficacy of different methods of minimally invasive treatment of pancreatic pseudocysts (PPC). Methods. A single center retrospective study of patients with pancreatic pseudocysts (n = 17): 90 males (76.9 %), 27 females (23.1 %) aged 25 to 72 years. The patients underwent external percutaneous drainage (group 1, n = 96) or internal drainage (group 2, n = 21). The diagnosis of pseudocysts included clinical, laboratory (biochemical and bacteriological) and special investigation methods: radiological, endoscopic, ultrasound examination of hepatobiliary zone, computer tomography. Results. Complications in the early postoperative period were observed in patients from both groups 1 and 2. They were related to inefficacy of cystodigestive anastomosis, which required percutaneous drainage in 2 cases (9.5 %), or to formation of pancreatic fistula. Lethal outcomes were not observed. Readmission to surgical department for removal of the drainage was required in 28 (23.9 %) patients from group 1. Internal drainage is considered more advantageous for PPC decompression compared to external one due to persistence of pancreatic fluid passage through gastrointestinal tract. External drainage is associated with frequent external pancreatic fistulae formation as well as prolonged hospital stay and treatment in an outpatient setting worsening the quality of life, but it is an intervention of choice in somatically severely ill patients, in fast growing cyst, imperfectly formed wall and threatening cyst rupture into abdominal cavity or abscess. These aspects prevent from refusal from external drainage for PPC treatment. Conclusion. When choosing the optimal time and type of surgical intervention in PPC, the surgeon should evaluate localization, sizes, maturation of PC wall and its relation to pancreatic duct, somatic state and patient’s individual features

СЕЛЕКТИВНОЕ ИНГИБИРОВАНИЕ KRAS-СИГНАЛЬНОГО КАСКАДА ПРИ КОМБИНИРОВАННОМ ВОЗДЕЙСТВИИ НИЗКИХ ДОЗ РАПАМИЦИНА И ПАКЛИТАКСЕЛА IN VIVO

Background. Therapy with compounds potentially capable to block KRAS oncogene signaling pathway is perspective direction in modern oncopharmacology. The aim of current study was to investigate effects of the combined treatment with rapamycin (RAP) and paclitaxel (PAC) in transgenic zebrafish (Danio rerio) with constant expression of mutant KRASV12 oncogene conjugated to green fluorescent protein (GFP) in epidermal cells. This strain has a modified phenotype due to epidermal hyperplasia and expression of GFP reporter at skin of embryos and adult fish.Materials and methods. Fish embryos 6 hpf were exposed to 0.1 % DMSO solution (control) and various doses of the drugs or combinations thereof. GFP expression in epidermal cells was morphometrically measured at 72 hpf.Results. Dose-related decrease in phenotypic changes up to complete epidermal normalization under RAP 50–400 nM treatment was observed. Treatment with nontoxic for embryos doses of PAC 50–250 nM increased fluorescence level in a dose-dependent manner, indicating an activation of KRAS signaling. Using of lower doses of RAP (10 and 25 nM) or PAC (10 nM) had no statistically significant effect on expression of transformed phenotype. Whereas combined treatment (RAP 10–25 nM and PAC 10–50 nM) dramatically decreased level of epidermal fluorescence and completely normalized phenotype of transgenic fish.Conclusions. Thus, mutual potentiating effect of RAP and PAC in low doses which leads to selective inhibition of the KRAS signaling pathway was revealed, indicating the prospect of further studies of these drugs combination for targeted cancer therapy.Введение. Применение соединений, потенциально способных блокировать функционирование сигнального каскада онкогена KRAS, является одним из перспективных направлений современной онкофармакологии.Цель исследования – изучить эффекты комбинированного действия рапамицина (RAP) и паклитаксела (РАС) на трансгенной линии зебрафиш (Danio rerio), характеризующейся постоянной экспрессией в клетках эпидермиса онкогена KRASV12, конъюгированного c зеленым флуоресцентным белком (green fluorescent protein, GFP). Эта линия имеет измененный фенотип, обусловленный гиперплазией кератиноцитов и флуоресценцией в них GFP-репортера.Материалы и методы. Эмбрионы рыб в возрасте 6 ч помещали в среду с добавлением 0,1 % раствора диметилсульфоксида (контроль) и различных доз исследуемых препаратов или их комбинаций. Время инкубации составило 72 ч, после чего проводили количественную оценку интенсивности флуоресценции GFP-репортера в клетках эпидермиса с помощью компьютерной морфометрии.Результаты. При воздействии RAP выраженность фенотипических изменений уменьшалась вплоть до полной нормализации фенотипа в дозе 50–400 нмоль. РАС в дозе 50–250 нмоль не оказывал токсического влияния на развитие эмбрионов, однако дозозависимо повышал уровень флуоресценции репортера, что свидетельствует об усилении экспрессии онкогена KRAS. Воздействие низких доз RAP (10–25 нмоль), а также PAC (10 нмоль) по отдельности не оказывало статистически значимого влияния на выраженность трансформированного фенотипа. В то же время использование различных комбинаций низких доз этих препаратов (RAP в дозе 10–25 нмоль в сочетании с PAC в дозе 10–50 нмоль) существенно снижало регистрируемый уровень флуоресценции, полностью нормализуя фенотип трансгенных рыб.Заключение. Выявлено взаимное потенцирующее действие низких доз RAP и PAC, приводящее к избирательному ингибированию сигнального каскада онкогена KRAS, что свидетельствует о перспективности дальнейших исследований комбинации этих препаратов для таргетной терапии опухолей

Robotic injection of zebrafish embryos for high-throughput screening in disease models

The increasing use of zebrafish larvae for biomedical research applications is resulting in versatile models for a variety of human diseases. These models exploit the optical transparency of zebrafish larvae and the availability of a large genetic tool box. Here we present detailed protocols for the robotic injection of zebrafish embryos at very high accuracy with a speed of up to 2000 embryos per hour. These protocols are benchmarked for several applications: (1) the injection of DNA for obtaining transgenic animals, (2) the injection of antisense morpholinos that can be used for gene knock-down, (3) the injection of microbes for studying infectious disease, and (4) the injection of human cancer cells as a model for tumor progression. We show examples of how the injected embryos can be screened at high-throughput level using fluorescence analysis. Our methods open up new avenues for the use of zebrafish larvae for large compound screens in the search for new medicines

Transfer learning for road-based location classification of non-residential property

This article reveals the method of the property classification by its location relative to highways using transfer learning approach. Solving this problem is crucial for non-residential real estate market value assessment automation in the course of market analysis, pre-trial appraisal and other aspects of making managerial decisions in the field of financing and lending. Instead of a standard approach based on models developed using machine learning libraries and programming, this work considers the use of Google's Teachable Machine service. This article examines the aspects of initial data preparation, the use of Teachable Machine for model training and the results obtained. The parameters and results of training classification models in different conditions are presented, the classification accuracy is analyzed. The results obtained generally indicate the validity of this approach and recommends it for solving similar problems. Copyright © 2021 for this paper by its authors. Use permitted under Creative Commons License Attribution 4.0 International (CC BY 4.0)

SELECTIVE INHIBITION OF KRAS SIGNALING BY COMBINATION OF LOW DOSE RAPAMYCIN AND PACLITAXEL IN VIVO

Background. Therapy with compounds potentially capable to block KRAS oncogene signaling pathway is perspective direction in modern oncopharmacology. The aim of current study was to investigate effects of the combined treatment with rapamycin (RAP) and paclitaxel (PAC) in transgenic zebrafish (Danio rerio) with constant expression of mutant KRASV12 oncogene conjugated to green fluorescent protein (GFP) in epidermal cells. This strain has a modified phenotype due to epidermal hyperplasia and expression of GFP reporter at skin of embryos and adult fish.Materials and methods. Fish embryos 6 hpf were exposed to 0.1 % DMSO solution (control) and various doses of the drugs or combinations thereof. GFP expression in epidermal cells was morphometrically measured at 72 hpf.Results. Dose-related decrease in phenotypic changes up to complete epidermal normalization under RAP 50–400 nM treatment was observed. Treatment with nontoxic for embryos doses of PAC 50–250 nM increased fluorescence level in a dose-dependent manner, indicating an activation of KRAS signaling. Using of lower doses of RAP (10 and 25 nM) or PAC (10 nM) had no statistically significant effect on expression of transformed phenotype. Whereas combined treatment (RAP 10–25 nM and PAC 10–50 nM) dramatically decreased level of epidermal fluorescence and completely normalized phenotype of transgenic fish.Conclusions. Thus, mutual potentiating effect of RAP and PAC in low doses which leads to selective inhibition of the KRAS signaling pathway was revealed, indicating the prospect of further studies of these drugs combination for targeted cancer therapy

SpCas9- and LbCas12a-Mediated DNA Editing Produce Different Gene Knockout Outcomes in Zebrafish Embryos

CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein) genome editing is a powerful technology widely used in current genetic research. In the most simple and straightforward way it can be applied for a gene knockout resulting from repair errors, induced by dsDNA cleavage by Cas nuclease. For decades, zebrafish (Danio rerio) has been known as a convenient model object of developmental biology. Both commonly used nucleases SpCas9 (Streptococcus pyogenes Cas9) and LbCas12a (Lachnospiraceae bacterium Cas12a) are extensively used in this model. Among them, LbCas12a is featured with higher specificity and efficiency of homology-directed editing in human cells and mouse. But the editing outcomes for these two nucleases in zebrafish are still not compared quantitatively. Therefore, to reveal possible advantages of one nuclease in comparison to the other in the context of gene knockout generation, we compare here the outcomes of repair of the DNA breaks introduced by these two commonly used nucleases in zebrafish embryos. To address this question, we microinjected the ribonucleoprotein complexes of the both nucleases with the corresponding guide RNAs in zebrafish zygotes and sequenced the target gene regions after three days of development. We found that LbCas12a editing resulted in longer deletions and more rare inserts, in comparison to those generated by SpCas9, while the editing efficiencies (percentage of mutated copies of the target gene to all gene copies in the embryo) of both nucleases were the same. On the other hand, overlapping of protospacers resulted in similarities in repair outcome, although they were cut by two different nucleases. Thus, our results indicate that the repair outcome depends both on the nuclease mode of action and on protospacer sequence

Characterization of CoCas9 nuclease from <i>Capnocytophaga ochracea</i>

Cas9 nucleases are widely used for genome editing and engineering. Cas9 enzymes encoded by CRISPR-Cas defence systems of various prokaryotic organisms possess different properties such as target site preferences, size, and DNA cleavage efficiency. Here, we biochemically characterized CoCas9 from Capnocytophaga ochracea, a bacterium that inhabits the oral cavity of humans and contributes to plaque formation on teeth. CoCas9 recognizes a novel 5’-NRRWC-3’ PAM and efficiently cleaves DNA in vitro. Functional characterization of CoCas9 opens ways for genetic engineering of C. ochracea using its endogenous CRISPR-Cas system. The novel PAM requirement makes CoCas9 potentially useful in genome editing applications.</p