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Implementation of analytical quality by design approach is a helpful tool in development of a robust analytical method which is applicable throughout the lifecycle of the product. This model is perfect strategy for analytical method development, in order to reduce outof-trend (OOT) and out-of-specification (OOS) results and to achieve high degree of robustness, providing cost-effective analytical method. The aim of this study was to implement a software which uses experimental design plans as an efficient and fast tool for development of method for quantitative determination of content of ACE inhibitor in tablets. Using statistical models in method development and optimization, the effect of each factor on responses of interest were calculated and the data were used to find the optimal chromatographic conditions. Best chromatographic separation within run time of 13 minutes was achieved on column Zorbax SB C18, 150 x 4.6 mm, (5 µm), using a mixture of 15 mM phosphate buffer pH 1.8 and acetonitrile as a mobile phase, with gradient elution and column temperature maintained at 60 °С. The validated method is specific, linear, accurate, precise and robust for the determination of angiotensin - converting enzyme inhibitor in tablets.

Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine1. As MIAs are difficult to chemically synthesize, the world's supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale2,3. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.

The synthetic biology toolkit for baker's yeast, Saccharomyces cerevisiae, includes extensive genome engineering toolkits and parts repositories. However, with the increasing complexity of engineering tasks and versatile applications of this model eukaryote, there is a continued interest to expand and diversify the rational engineering capabilities in this chassis by FAIR (findable, accessible, interoperable, and reproducible) compliance. In this study, we designed and characterised 41 synthetic guide RNA sequences to expand the CRISPR-based genome engineering capabilities for easy and efficient replacement of genomically encoded elements. Moreover, we characterize in high temporal resolution 20 native promoters and 18 terminators using fluorescein and LUDOX CL-X as references for GFP expression and OD600 measurements, respectively. Additionally, all data and reported analysis is provided in a publicly accessible jupyter notebook providing a tool for researchers with low-coding skills to further explore the generated data as well as a template for researchers to write their own scripts. We expect the data, parts, and databases associated with this study to support a FAIR-compliant resource for further advancing the engineering of yeasts.

Background/Aim. Carotid stenosis is a risk factor for cognitive impairment. The aim of the study was to evaluate the degree of cognitive impairment in patients with asymp-tomatic and symptomatic carotid stenosis and correlate it with the presence, location, and extent of cerebral ischemic lesions. Methods. A prospective analysis of 180 patients aged 50–70 years, divided into three groups (asymptomatic carotid stenosis, symptomatic carotid stenosis, and controls) was made. We assessed demographic characteristics, vascu-lar risk factors, ultrasound examination of the carotid arter-ies, computerized tomography (CT), magnetic resonance imaging (MRI) of the brain, and neuropsychological testing. Results. The brain CT findings on admission showed is-chemic lesions in the left hemisphere in 13.3% of patients in the asymptomatic group and in 41% of those in the symp-tomatic group. In the right hemisphere, lesions were regis-tered in 10% of the asymptomatic patients and in 46.7% of the symptomatic patients. The difference between groups was statistically significant. The lesion volumes measured on CT and MRI scans were significantly different (p < 0.001) between groups with asymptomatic and symptomatic carot-id stenosis. The degree of cognitive impairment, measured by the Addenbrooke's Cognitive Examination Revised (ACE-R), was significantly different between the groups (p < 0.05), with the most severe deficit in the symptomatic group. Conclusion. Our study has shown that cognitive impairment was more severe in patients with symptomatic carotid stenosis, compared to the patients with asymptomat-ic carotid stenosis.

Background Current stroke guidelines recommend advanced imaging (computed tomography [CT] perfusion or magnetic resonance imaging) prior to endovascular therapy (EVT) in patients with late presentation of large vessel occlusion. Adherence to guidelines may be constrained by resources or timely access to imaging. We sought to understand the factors which influence late window imaging selection for EVT candidates with large vessel occlusion. Methods We conducted an international survey from January to May 2022. The questions aimed to identify advanced imaging and treatment decisions based on access to imaging, time delays, and simulated patient scenarios. Results There were 3000 invited participants and 1506 respondents, the majority (89.6%) from comprehensive stroke centers in high‐income countries. Neurointerventionalists comprised 31.8% and noninterventionalists 68.2% of respondents. Overall, 70.7% reported routine use of advanced imaging for late EVT selection, and 63.6% reported its usage in every case. There was greater availability of advanced imaging in comprehensive stroke centers versus primary stroke centers (67.0% versus 33.7%; P

BackgroundTranscriptional reprogramming is a fundamental process of living cells in order to adapt to environmental and endogenous cues. In order to allow flexible and timely control over gene expression without the interference of native gene expression machinery, a large number of studies have focused on developing synthetic biology tools for orthogonal control of transcription. Most recently, the nuclease-deficient Cas9 (dCas9) has emerged as a flexible tool for controlling activation and repression of target genes, by the simple RNA-guided positioning of dCas9 in the vicinity of the target gene transcription start site.ResultsIn this study we compared two different systems of dCas9-mediated transcriptional reprogramming, and applied them to genes controlling two biosynthetic pathways for biobased production of isoprenoids and triacylglycerols (TAGs) in baker's yeast Saccharomyces cerevisiae. By testing 101 guide-RNA (gRNA) structures on a total of 14 different yeast promoters, we identified the best-performing combinations based on reporter assays. Though a larger number of gRNA-promoter combinations do not perturb gene expression, some gRNAs support expression perturbations up to ~threefold. The best-performing gRNAs were used for single and multiplex reprogramming strategies for redirecting flux related to isoprenoid production and optimization of TAG profiles. From these studies, we identified both constitutive and inducible multiplex reprogramming strategies enabling significant changes in isoprenoid production and increases in TAG.ConclusionTaken together, we show similar performance for a constitutive and an inducible dCas9 approach, and identify multiplex gRNA designs that can significantly perturb isoprenoid production and TAG profiles in yeast without editing the genomic context of the target genes. We also identify a large number of gRNA positions in 14 native yeast target pomoters that do not affect expression, suggesting the need for further optimization of gRNA design tools and dCas9 engineering.

This paper analyses how untapping women potential can influence the economic growth. Recent papers and discussions on this subject have shown that economic policies that empower women economically can stimulate outcome, productivity, diversification and ultimately economic growth of the economy. Therefore introducing "gender" or so called "pink" lens when designing economic policies can significantly improve economic performance of a country. The paper than shows how tackling different dimensions on gender inequality can influence economic development of Republic of North Macedonia. Designing policies aimed at closing gender gap in the country can significantly boost output and economic growth. [ABSTRACT FROM AUTHOR]

High growth enterprises are attracting the attention to both economic theoreticians and practitioners. Namely, among all enterprises, there is a group of enterprises which show high growth and contribute in large extent to the economic development of the countries. In fact high growth enterprises are considered as drivers of the innovation, twin transition and structural transformation of the countries. This is why, researchers and especially policy makers are interested to find out more behind the factors or determinants which triggers the growth of these enterprises. The reasoning behind this paper is to analyse what high growth enterprises represent, what are their characteristics, the different definitions used, than to analyse literature which focuses on the causality between high growth of companies and certain aspects of economic development, EU policies aimed at high growth potential (HGEs) enterprises, in order to be able to make a comparative analysis using available statistical data and draw conclusions and policy recommendations for fostering HGEs in Republic of North Macedonia. Analysis showed that only 2% of all companies in the country are contributing to 20% of employment. Still, most of them are found in traditional service sector followed by manufacturing, unlike the HGEs in EU where they are mostly found in knowledge intensive service sector. The main conclusion is that instead of policy mix to support local champions, government should be more focused on developing policy framework to back enterprises which have the potential to grow fast. To do that, government needs to create framework conditions which will enable companies to grow, but also to create tailored made instruments and policies which will back companies with high growth potential. The firms to be targeted are not those that would grow in any case, but those for which policy intervention would make a difference. Given the fact that HGEs are considered as drivers of innovation and technological advancement, EU is putting them high on their policy agenda. Therefore, aligning policy objectives of North Macedonia with the ones of EU in the area of SMEs with special focus on start-ups and scale-ups with potential to grow should be imperative for policy creators. [ABSTRACT FROM AUTHOR]

To determine the possible role of patent foramen ovale (PFO) as indicated by a right to left shunt and cryptogenic stroke in patients aged ≥ 60 years and to compare the prevalence of patent foramen ovale with stroke of known cause. We prospectively examined the prevalence of PFO using contrast enhanced colour Transcranial Doppler ultrasonography (bubble-cTCD) in 86 consecutive patients aged ≥60 years with transient ischaemic attack (TIA) or stroke and 86 consecutive patients aged <60 years with TIA/stroke as a control group. Patients with cryptogenic stroke were compared to stroke patients with known cause. A significantly higher prevalence of PFO in patients with cryptogenic compared to patients with stroke of known cause was registered in the patients aged < 60 years (56.6% vs.18.8%) and in patients aged ≥ 60 years (37.14% vs.11.76%), respectively. Multivariate analysis adjusted for age, hypertension and coronary disease showed that the existence of R-to-L shunt (RLS) was independently associated with cryptogenic stroke in both, in the younger group (odds ratio 4.012; 95% CI1.323 to 12.171, p =0.0143.70) and in the older group (odds ratio 3.197; 95% CI 1.140 to 10.877, p=0.037). Our findings suggest that PFO is strongly associated with cryptogenic stroke in patients aged ≥ 60 years. Bubble – cTCD was feasible and suitable as a first-line method for the detection of PFO in older patients. [ABSTRACT FROM AUTHOR]

Whole-cell biocatalysts have proven a tractable path toward sustainable production of bulk and fine chemicals. Yet the screening of libraries of cellular designs to identify best-performing biocatalysts is most often a low-throughput endeavor. For this reason, the development of biosensors enabling real-time monitoring of production has attracted attention. Here we applied systematic engineering of multiple parameters to search for a general biosensor design in the budding yeast Saccharomyces cerevisiae based on small-molecule binding transcriptional activators from the prokaryote superfamily of LysR-type transcriptional regulators (LTTRs). We identified a design supporting LTTR-dependent activation of reporter gene expression in the presence of cognate small-molecule inducers. As proof of principle, we applied the biosensors for in vivo screening of cells producing naringenin or cis,cis-muconic acid at different levels, and found that reporter gene output correlated with production. The transplantation of prokaryotic transcriptional activators into the eukaryotic chassis illustrates the potential of a hitherto untapped biosensor resource useful for biotechnological applications.

Advances in synthetic biology and our understanding of the rules of promoter architecture have led to the development of diverse synthetic constitutive and inducible promoters in eukaryotes and prokaryotes. However, the design of promoters inducible by specific endogenous or environmental conditions is still rarely undertaken. In this study, we engineered and characterized a set of strong, synthetic promoters for budding yeast Saccharomyces cerevisiae that are inducible under acidic conditions (pH ≤ 3). Using available expression and transcription factor binding data, literature on transcriptional regulation, and known rules of promoter architecture we improved the low-pH performance of the YGP1 promoter by modifying transcription factor binding sites in its upstream activation sequence. The engineering strategy outlined for the YGP1 promoter was subsequently applied to create a response to low pH in the unrelated CCW14 promoter. We applied our best promoter variants to low-pH fermentations, enabling ten-fold increased production of lactic acid compared to titres obtained with the commonly used, native TEF1 promoter. Our findings outline and validate a general strategy to iteratively design and engineer synthetic yeast promoters inducible to environmental conditions or stresses of interest.

BackgroundInterracial disparities in nephrolithiasis prevalence have been reported, but the interplay between genetics and the environment for urinary stone disease risk factors is poorly understood. To examine how environment may alter genetic predisposition for stone formation, we established the International Chinese Consortium on Nephrolithiasis (ICCON) as a multi-institutional collaboration to examine patterns of nephrolithiasis presentation between Chinese patients living in different countries.MethodsChinese patients undergoing percutaneous nephrolithotomy (PCNL) at six participating institutions in China and North America over 4 years were reviewed retrospectively. Patient demographics and clinical data were compared between Chinese patients living in China and North America.ResultsA total of 806 patients were included, encompassing 721 Chinese patients living in China and 85 living in North America. Nephrolithiasis patients living in China were more likely to be male (67% vs. 56%, P=0.02), present at a younger age (48.6±15.0 vs. 55.0±13.0 years, P

The validity of four gudgeon species known for the ichthyofauna of North Macedonia has been a matter of different taxonomic disputes (except for Gobio ohridanus Karaman, 1924). Recently, a restoration of the species status was proposed for Gobio balcanicus Dimovski et Grupče, 1977 and Romanogobio banarescui (Dimovski et Grupče, 1974). So far, Romanogobio stankoi (Karaman, 1974), has not been a part of any recent investigation and it is still considered as a junior synonym of Romanogobio elimeius (Kattoulas, Stephanidis et Economidis, 1973) or R. banarescui due to the lack of distinctive morphological characters. The osteological characters in the structure of the vertebral column, have significance to the systematics of the gudgeons, especially in elevation of Romanogobio at the level of genus. To contribute to resolving the taxonomic status in these gudgeon species, the goal of this paper is to identify distinctive characters by presenting and analyzing the structure of their vertebral column. Samples were inspected from the fish collections in the Institute of Biology (G. ohridanus), the Institute of Animal Science (G. balcanicus), and the Macedonian Museum of Natural History (both Romanogobio species). Fish samples were X-rayed as well as cleared and double-stained. Obtained data on the number of vertebrae were presented through the vertebral formula following Naseka (1996). Multiple Correspondence Analysis (MCA) was used to check the interrelation between taxa and the number of vertebrae in all units of the vertebral column. G. balcanicus is distinguished from G. ohridanus mainly in the predorsal and preanal subregion (10 vs. 11 vertebrae and 1 vs. 0 vertebrae, respectively), while R. stankoi distinguishes from R. banarescui by the number of vertebrae in the preanal and postanal subregion (2 vs. 4 vertebrae and 19 vs. 17 vertebrae, respectively). The structure of vertebral column offers distinctive morphological characters for the four gudgeon species from Ohird Lake and the Vardar River basin.

For more than half a century, organizational culture has represented a topic of increasing importance to both researchers and business practitioners. Despite its status as being an area of intensive research, the topic of organizational culture is over-investigated in certain sectors, while remaining unexplored and less understood in others. The purpose of this research is to address this gap and provide initial insight into the organizational culture specifics of the wood industry in Macedonia. The research utilized a quantitative approach with a questionnaire as the main research instrument to assess the link between organizational culture, job satisfaction and socio-demographic characteristics of employees. The data was gathered from one hundred participants from five companies. Descriptive statistics as well as chi-square and correlation analyses were used to explore gathered research data. The research findings provide valuable insight into the organizational culture of this industry that will benefit academics, business leaders and policy makers in their strategic decision making.

Objective: Acetylsalicylic acid (ASA) is the most widely used antiplatelet agent in treating ischemic strokes. ASA resistance varies between the populations, from 5% to 60%. We aimed to determine the prevalence of ASA resistance in our population, its association with demographic characteristics, risk factors, and the occurrence of recurrent ischemic stroke (IS). (IST). Methods: One hundred consecutive patients with primary or recurrent IS were prospectively included. Strokes were diagnosed with computed tomography (CT) or magnetic resonance imaging (MRI). In all patients a detailed history of cardiovascular risk factors and fasting blood analyzes (blood count, glycemia, HbA1c, degradation products, lipid profile) were taken, and the ASA resistance was examined with the Innovance PFA 200 system. The ASA resistance was examined after a minimum of 1 month of regular use of ASA 100 mg per day. Results: The prevalence of ASA resistance was 32%. ASA-resistant patients were statistically significantly older (69.9±7.5 vs, 61±1 y., p<0.05), and of male gender (75 vs, 45.6%, p<0.05). Previous cardiovascular diseases were statistically significantly more common in the ASA-resistant patients compared to the ASA-sensitive patients (46.8 vs 26.5%, p<0.05). The presence of recurrent stroke in the ASA-resistant versus ASA-sensitive patients was statistically significant (75% vs. 22.05%, p<0.0001). There was no statistically significant difference in the investigated laboratory parameters and the risk factors between the ASA-resistant and the ASA-sensitive patients. Conclusion. The prevalence of ASA resistance in our population is high. According to our results, ASA resistance testing should be done in the older male individuals with a previous burden of cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Chronic pain conditions impose a huge economic burden on affected individuals and society and is very significant. With this research paper we raise awareness of the effectiveness, safety and benefits of acupuncture for the treatment of chronic diseases. Acupuncture has been proven to be an exceptionally effective treatment option, especially for the following health issues: chronic pain, allergies, asthma, stress, anxiety, depression, migraines insomnia, hypertension, menopause and etc. Acupuncture has a great ability to create a feeling of relief from discomfort and pain. The treatment also targets the root of the illness and restores normal body functions. Acupuncture, as a treatment for chronic diseases incurable according to the WHO, works very effectively and positively, although the treatments are longer, the benefits for the patients are enormous on the one hand due to the improvement of the health condition and on the other hand due to the greater economic advantage and profitability. For these reasons, more and more people in the world decide to treat chronic diseases with acupuncture. A very important step is to share the knowledge and experience for TCM and enable the population to gain more knowledge about the treatment advantages and gradually accept it as a valuable health care treatment. [ABSTRACT FROM AUTHOR]