Hemophilia, a congenital X linked disease, has the serious burden of bleeding, requiring life-long replacement with coagulation factors (CF). In the present day, there is a continuously improving treatment for this condition. Objective: Our observational, cross-sectional study aims at finding out whether a prophylactic replacement with CF is affordable from the point of view of its cost-effectiveness in our country. Material and methods: A cohort of 122 persons with hemophilia were included in this patient-reported outcome survey, and they answered a questionnaire consisting of 56 items, focused on 4 domains—socio-demographic, medical, quality of health/life and cost/cost-effectiveness. Results and discussion: The markers for quality of health/life were correlated with the direct and indirect costs of care, comparing subgroup 1 of patients with prophylactic vs. subgroup 2 with on-demand replacement. Based on the incremental quality adjusted life years and the incremental costs, we calc... [more]

The present study focuses on the inhibitory effect of volatile metabolites released by Bacillus velezensis CT32 on Verticillium dahliae and Fusarium oxysporum, the causal agents of strawberry vascular wilt. The CT32 strain was isolated from maize straw compost tea and identified as B. velezensis based on 16S rRNA gene sequence analysis. Bioassays conducted in sealed plates revealed that the volatile organic compounds (VOCs) produced by the strain CT32 possessed broad-spectrum antifungal activity against eight phytopathogenic fungi. The volatile profile of strain CT32 was obtained by headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS). A total of 30 volatile compounds were identified, six of which have not previously been detected in bacteria or fungi: (Z)-5-undecene, decyl formate, 2,4-dimethyl-6-tert-butylphenol, dodecanenitrile, 2-methylpentadecane and 2,2’,5,5’-tetramethyl-1,1’-biphenyl. Pure compounds were tested in vitro for th... [more]

Radiofrequency (RF)-based monopolar (MM) and bipolar mode (BM) applicators are used to thermally create coagulation zones (CZs) in biological tissues with the aim of destroying surface tumors and minimizing blood losses in surgical resection. Both modes have disadvantages as regards safely and in obtaining a sufficiently deep coagulation zone (CZ). In this study, we compared both modes versus a switching monopolar mode (SMM) in which the role of the active electrode changes intermittently between the two electrodes of the applicator. In terms of clinical impact, the three modes can easily be selected by the surgeon according to the surgical maneuver. We used computational and experimental models to study the feasibility of working in MM, BM, and SMM and to compare their CZ characteristics. We focused exclusively on BM and SMM, since MM only creates small coagulation zones in the area between the electrodes. The results showed that SMM produces the deepest CZ between both electrodes (33... [more]

Bioreactors have proven useful for a vast amount of applications. Besides classical large-scale bioreactors and fermenters for prokaryotic and eukaryotic organisms, micro-bioreactors, as specialized bioreactor systems, have become an invaluable tool for mammalian 3D cell cultures. In this systematic review we analyze the literature in the field of eukaryotic 3D cell culture in micro-bioreactors within the last 20 years. For this, we define complexity levels with regard to the cellular 3D microenvironment concerning cell−matrix-contact, cell−cell-contact and the number of different cell types present at the same time. Moreover, we examine the data with regard to the micro-bioreactor design including mode of cell stimulation/nutrient supply and materials used for the micro-bioreactors, the corresponding 3D cell culture techniques and the related cellular microenvironment, the cell types and in vitro models used. As a data source we used the National Library of Medicine and analyzed the s... [more]

Elementary Flux Modes (EFMs) provide a rigorous basis to systematically characterize the steady state, cellular phenotypes, as well as metabolic network robustness and fragility. However, the number of EFMs typically grows exponentially with the size of the metabolic network, leading to excessive computational demands, and unfortunately, a large fraction of these EFMs are not biologically feasible due to system constraints. This combinatorial explosion often prevents the complete analysis of genome-scale metabolic models. Traditionally, EFMs are computed by the double description method, an efficient algorithm based on matrix calculation; however, only a few constraints can be integrated into this computation. They must be monotonic with regard to the set inclusion of the supports; otherwise, they must be treated in post-processing and thus do not save computational time. We present aspefm, a hybrid computational tool based on Answer Set Programming (ASP) and Linear Programming (LP) th... [more]

Continuous manufacturing of biologics (biopharmaceuticals) has been an area of active research and development for many reasons, ranging from the demand for operational streamlining to the requirement of achieving obvious economic benefits. At the same time, biopharma strives to develop systems and concepts that can operate at similar scales for clinical and commercial production—using flexible infrastructures, such as single-use flow paths and small surge vessels. These developments should simplify technology transfer, reduce footprint and capital investment, and will allow to react readily to changing market pressures while maintaining quality attributes. Despite a number of clearly identified benefits compared to traditional batch processes, continuous bioprocessing is still not widely adopted for commercial manufacturing. This paper details how industry-specific technological, organizational, economic, and regulatory barriers that exist in biopharmaceutical manufacturing are hinder... [more]

Microalgae cultivation in liquid foams is a promising concept which requires the use of a surfactant as a foam stabilizing agent. The biodegradable character of a surfactant is a key aspect regarding its applicability in a liquid foam-bed photobioreactor (LF-PBR), since it might influence microalgal growth and the stability of the foam-based cultivation. In this work, the effects of the biodegradable surfactants bovine serum albumin (BSA), Saponin and Tween 20 on the whole microbial community of microalgal cultures (i.e., microalgal and bacterial populations) were studied. The three surfactants enhanced bacterial and microalgal growth in non-axenic microalgal cultures, but they differed in their efficiency to sustain bacterial growth. In this sense, Saponin was proven to enhance the growth of S. obliquus-associated bacteria in microalgae-free cultures, and to sustain it even when other nutrients were lacking, suggesting that Saponin can be used as an energy and nutrients source by thes... [more]

Blood is not always available in forensic autopsies, therefore, the search for alternative sampling materials is needed. This study aimed at examining if ethanol can be detected in costal cartilage and to investigate if different forms of costal cartilage can give accurate information about ethanol concentration in the blood or urine of human cadavers (n = 50). Ethanol concentration in samples of unground costal cartilage (UCC), ground costal cartilage (GCC), femoral venous blood, and urine was analyzed using a gas chromatography-flame ionization detector (GC-FID). Due to Polish law, we used two different cut-off points: the blood alcohol concentration >0.2 mg/mL defined as the ‘after use’ condition, and the blood alcohol concentration >0.5 mg/mL defined as the ‘state of insobriety’. Based on the constructed receiver operating characteristics (ROC) curves, the optimal cut-off point for ethanol content as the ‘after use’ condition was 0.273 mg/g for the UCC method and 0.069 mg/g for the... [more]

The development of an advanced and efficient drug delivery system with significant improvement in its efficacy and enhanced therapeutic value is one of the critical challenges in modern medicinal biology. The integration of nanomaterial science with molecular and cellular biology has helped in the advancement and development of novel drug delivery nanocarrier systems with precision and decreased side effects. The design and synthesis of nanocarriers using graphene oxide (GO) have been rapidly growing over the past few years. Due to its remarkable physicochemical properties, GO has been extensively used in efforts to construct nanocarriers with high specificity, selectivity, and biocompatibility, and low cytotoxicity. The focus of this review is to summarize and address recent uses of GO-based nanocarriers and the improvements as efficient drug delivery systems. We briefly describe the concepts and challenges associated with nanocarrier systems followed by providing critical examples of... [more]

Liposomes are bilayer membrane vesicles that can serve as vehicles for drug delivery. They are a good alternative to free drug administration that provides cell-targeted delivery into tumors, limiting the systemic toxicity of chemotherapeutic agents. Previous results from our group showed that an astrocytoma cell line exhibits selective uptake of sulfatide-rich (SCB) liposomes, mediated by the low-density lipoprotein receptor (LDL-R). The goal of this study was to assess the uptake of liposomes in a neuroblastoma cell line. For this purpose, we used two types of liposomes, one representing a regular cell membrane (DOPC) and another rich in myelin components (SCB). An astrocytoma cell line was used as a control. Characterization of liposome uptake and distribution was conducted by flow cytometry and fluorescence microscopy. Similar levels of LDL-R expression were found in both cell lines. The uptake of SCB liposomes was higher than that of DOPC liposomes. No alterations in cell viabilit... [more]

Protein phosphorylation is the most widespread type of post-translational modification and is properly controlled by protein kinases and phosphatases. Regarding the phosphorylation of serine (Ser) and threonine (Thr) residues, relatively few protein Ser/Thr phosphatases control the specific dephosphorylation of numerous substrates, in contrast with Ser/Thr kinases. Recently, protein Ser/Thr phosphatases were reported to have rigid substrate recognition and exert various biological functions. Therefore, identification of targeted proteins by individual protein Ser/Thr phosphatases is crucial to clarify their own biological functions. However, to date, information on the development of methods for identification of the substrates of protein Ser/Thr phosphatases remains scarce. In turn, substrate-trapping mutants are powerful tools to search the individual substrates of protein tyrosine (Tyr) phosphatases. This review focuses on the development of novel methods for the identification of S... [more]

A large portion of annual deaths worldwide are due to infections caused by disease-causing pathogens. These pathogens contain virulence genes, which encode mechanisms that facilitate infection and microbial survival in hosts. More recently, antimicrobial resistance (AMR) genes, also found in these pathogens, have become an increasingly large issue. While the National Center for Biotechnology Information (NCBI) Pathogen Detection Isolates Browser (NPDIB) database has been compiling genes involved in microbial virulence and antimicrobial resistance through isolate samples, few studies have identified the genes primarily responsible for virulence and compared them to those responsible for AMR. This study performed the first multivariate statistical analysis of the multidimensional NPDIB data to identify the major virulence genes from historical pathogen isolates for Australia, China, South Africa, UK, and US—the largely populated countries from five of the six major continents. The import... [more]

With the increasing spread of multidrug-resistant bacterial pathogens, it is of great importance to develop alternatives to conventional antibiotics. Here, we report the generation of a chimeric phage lysin, MLTphg, which was assembled by joining the lysins derived from Meiothermus bacteriophage MMP7 and Thermus bacteriophage TSP4 with a flexible linker via chimeolysin engineering. As a potential antimicrobial agent, MLTphg can be obtained by overproduction in Escherichia coli BL21(DE3) cells and the following Ni-affinity chromatography. Finally, we recovered about 40 ± 1.9 mg of MLTphg from 1 L of the host E. coli BL21(DE3) culture. The purified MLTphg showed peak activity against Staphylococcus aureus ATCC6538 between 35 and 40 °C, and maintained approximately 44.5 ± 2.1% activity at room temperature (25 °C). Moreover, as a produced chimera, it exhibited considerably improved bactericidal activity against Staphylococcus aureus (2.9 ± 0.1 log10 reduction was observed upon 40 nM MLTphg... [more]

The study investigated in situ biosynthesis of albumin capped 5-fluorouracil (5-FU) loaded gold nanoparticles (NPs) using bacterial extract for enhanced efficacy against MCF-7 and in silico prediction using a GastroPlus® software. The optimized formulations were characterized for morphology, size, zeta potential, drug loading (%DL) and entrapment (), compatibility, in vitro drug release, in vitro hemolysis, cellular toxicity and apoptosis studies. The results exhibited highly dispersed albumin capped mono-metallic stable NPs. Spherical size, negative zeta potential and polydispersity index were in range of 38.25−249.62 nm, 18.18−29.87 mV and 0.11−0.283, respectively. F11, F7 and F3 showed a progressive increase in %DL and with increased concentration of the cellular lysate (100% > 50% > 10%). The drug release was relatively extended over 48 h as compared to drug solution (96.64% release within 5 h). The hemolysis result ensured hemocompatibility (<14%) at the explored concentration.... [more]

Adipocyte-specific transcription factors and antioxidants are considered the best target of obesity. Aruncus dioicus var. kamtschaticus (A. dioicus, Samnamul) is easily available owing to edible and inexpensive. However, the anti-adipogenic effects of the underlying mechanism of A. dioicus extract (ADE) have not yet been reported. In the present study, we evaluate anti-adipogenic pathway in 3T3-L1 adipocytes, antioxidant activities and quantified phenolics using high-performance liquid chromatography of ADE. The results revealed ADE had reduced adipocyte differentiation (0.72-fold vs. MDI (media of differentiation) control), triglyceride (TG; 0.50-fold vs. MDI control, p < 0.001), and total cholesterol contents (0.77-fold vs. MDI control) by regulating adipocyte-specific transcription factors (C/EBPα, PPARγ, and SREBP1) and their downstream mRNA (AdipoQ, Ap2, SREBP1-c, and FAS) levels. Furthermore, ADE has higher total phenol and flavonoid contents and scavenging assay in the DPPH a... [more]

We use a system biology approach to translate the interaction of Bacillus Calmette-Gurin (BCG) + interleukin 2 (IL-2) for the treatment of bladder cancer into a mathematical model. The main goal of this research is to predict the outcome of BCG + IL-2 treatment combinations. We examined whether the delay effect caused by the proliferation of tumor antigen-specific effector cells after the immune system destroys BCG-infected urothelium cells after BCG and IL-2 immunotherapy influences success in bladder cancer treatment. To do this, we introduce a system of differential equations where the variables are the main participants in the immune response after BCG installations to fight cancer: the number of tumor cells, BCG cells, immune cells, and cytokines involved in the tumor-immune response. The relevant parameters describing the dynamics of the system are taken from a variety of biological, clinical literature and estimated using the mathematical models. We examine the local stability a... [more]

Exosomes are defined as a type of extracellular vesicle released when multivesicular bodies of the endocytic pathway fuse with the plasma membrane. They are characterized by their role in extracellular communication, partly due to their composition, and present the ability to recognize and interact with cells from the immune system, enabling an immune response. Their targeting capability and nanosized dimensions make them great candidates for cancer therapy. As chemotherapy is associated with cytotoxicity and multiple drug resistance, the use of exosomes targeting capabilities, able to deliver anticancer drugs specifically to cancer cells, is a great approach to overcome these disadvantages. The objective is to assess treatment efficiency in reducing tumor cells, as well as overall safety and response by cancer carriers. So far, results show exosomes as a promising therapeutic strategy in the fight against cancer. This review summarizes the characteristics and composition of exosomes,... [more]

Many venomous species, especially snakes, contain a variety of secreted phospholipases A2 that contribute to venom toxicity and prey digestion. We characterized a novel highly toxic phospholipase A2 of group II, WaPLA2-II, from the snake venom of Saudi Walterinnesia aegyptia (W. aegyptia). The enzyme was purified using a reverse phase C18 column. It is a monomeric protein with a molecular weight of approximately 14 kDa and an NH2-terminal amino acid sequence exhibiting similarity to the PLA2 group II enzymes. WaPLA2-II, which contains 2.5% (w/w) glycosylation, reached a maximal specific activity of 1250 U/mg at pH 9.5 and 55 °C in the presence of Ca2+ and bile salts. WaPLA2-II was also highly stable over a large pH and temperature range. A strong correlation between antimicrobial and indirect hemolytic activities of WaPLA2 was observed. Additionally, WaPLA2-II was found to be significantly cytotoxic only on cancerous cells. However, chemical modification with para-Bromophenacyl bromide... [more]

The modern view of the mechanism of intercellular movement of viruses is based largely on data from the study of the tobacco mosaic virus (TMV) 30-kDa movement protein (MP). The discovered properties and abilities of TMV MP, namely, (a) in vitro binding of single-stranded RNA in a non-sequence-specific manner, (b) participation in the intracellular trafficking of genomic RNA to the plasmodesmata (Pd), and (c) localization in Pd and enhancement of Pd permeability, have been used as a reference in the search and analysis of candidate proteins from other plant viruses. Nevertheless, although almost four decades have passed since the introduction of the term “movement protein” into scientific circulation, the mechanism underlying its function remains unclear. It is unclear why, despite the absence of homology, different MPs are able to functionally replace each other in trans-complementation tests. Here, we consider the complexity and contradictions of the approaches for assessment of the... [more]

Over the past decades, anaerobic biotechnology is commonly used for treating high-strength wastewaters from different industries. This biotechnology depends on interactions and co-operation between microorganisms in the anaerobic environment where many pollutants’ transformation to energy-rich biogas occurs. Properties of wastewater vary across industries and significantly affect microbiome composition in the anaerobic reactor. Methanogenic archaea play a crucial role during anaerobic wastewater treatment. The most abundant acetoclastic methanogens in the anaerobic reactors for industrial wastewater treatment are Methanosarcina sp. and Methanotrix sp. Hydrogenotrophic representatives of methanogens presented in the anaerobic reactors are characterized by a wide species diversity. Methanoculleus sp., Methanobacterium sp. and Methanospirillum sp. prevailed in this group. This work summarizes the relation of industrial wastewater composition and methanogen microbial communities present in... [more]

The spike protein of the pandemic human corona virus is essential for its entry into human cells. In fact, most neutralizing antibodies against Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) are directed against the Virus-surface exposed spike protein, making it the antigen of choice for use in vaccines and diagnostic tests. In the current pandemic context, global demand for spike proteins has rapidly increased and could exceed hundreds of grams to kilograms annually. Coronavirus spikes are large heavily glycosylated homo-trimeric complexes, with inherent instability. The poor manufacturability now threatens the availability of these proteins for vaccines and diagnostic tests. Here, we outline scalable, Good Manufacturing Practice (GMP) compliant, and chemically defined processes for the production of two cell-secreted stabilized forms of the trimeric spike proteins (Wuhan and D614G variant). The processes are chemically defined and based on clonal suspension-CHO cell po... [more]

Liposomes are well-investigated drug or gene delivery vehicles for chemotherapy, used by taking advantage of their biocompatibility and biodegradability. A central question on the construction of intracellular liposomal delivery systems is to entrap the liposomes of interest in the highly acidic and proteolytic endosomal environment. In the other words, it is essential that the liposomes release a therapeutic drug into the cytosol before they are degraded in the endosome. As a strategy to enhance the endosome escape, the self-lytic liposomes with acidic pH-selective membrane active polypeptide are considered highly effective. Here, an acidic pH-selective membrane-lytic polypeptide (LPE) and its retro isomer (rLPE) were designed, and then their membrane-lytic activities against EggPC liposomes were determined. It was noticed that the rLPE polypeptide showed an increase in activity compared with the LPE polypeptide. Furthermore, the rLPE polypeptide was conjugated to liposomes via a flex... [more]

Tuna blood (TB) was subjected to enzymatic hydrolysis. The effects of the relationship of hydrolysis time (30−180 min) and enzyme concentration (0.5−3.0% w/w protein) on the degree of hydrolysis (DH), yield, antioxidant and angiotensin-I-converting enzyme (ACE) inhibitory activities were determined. The response surface methodology (RSM) showed that TB hydrolysis’s optimum conditions were hydrolysis for 180 min and Alcalase, Neutrase or Flavourzyme at 2.81%, 2.89% or 2.87% w/w protein, respectively. The hydrolysates with good DH (40−46%), yield (3.5−4.6%), the IC50 of DPPH (0.8−1.6 mg/mL) and ABTS (1.0−1.4 mg/mL) radical scavenging activity, ferric reducing antioxidant power (FRAP) value (0.28−0.65 mmol FeSO4/g) and IC50 of ACE inhibitory activity (0.15−0.28 mg/mL) were obtained with those conditions. The TB hydrolysate using Neutrase (TBHN) was selected for characterization in terms of amino acid composition, peptide fractions and sensory properties. The essential, hydrophobic and hyd... [more]

Dried blood spot (DBS) technology has become a promising utility for the transportation and storage of biological fluids aimed for the subsequent clinical analysis. The basis of the DBS method is the adsorption of the components of a biological sample onto the surface of a membrane carrier, followed by drying. After drying, the molecular components of the biosample (nucleic acids, proteins, and metabolites) can be analyzed using modern omics, immunological, or genomic methods. In this work, we investigated the safety of proteins on a membrane carrier by tryptic components over time and at different temperatures (+4, 0, 25 °C) and storage (0, 7, 14, and 35 days). It was shown that the choice of a protocol for preliminary sample preparation for subsequent analytical molecular measurements affects the quality of the experimental results. The protocol for preliminary preparation of a biosample directly in a membrane carrier is preferable compared to the protocol with an additional stage of... [more]

In 2016, infectious microbes were one of the leading causes of death, especially in developing countries. Puriton® is a mineral mixture consisting of biotite, kaolinite, montmorillonite, serpentine, clinochlore, and vermiculite, and evaluated antimicrobial activity in vitro and safety in vivo. Nine pathogens and opportunistic bacteria, namely Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, Alcaligenes faecalis, Staphylococcus aureus, Enterococcus faecalis, Micrococcus luteus, Mycobacterium smegmatis, and Bacillus subtilis, and the two viruses Zika and Influenza A/Duck/MN/1525/81 were used. A 26-week oral repeated safety study of Puriton® was conducted. Puriton® suppressed the bacterial proliferation, with a minimum proliferative rate of 91.1% in B. subtilis ATCC6633. The virucidal efficacy of Puriton® against Zika virus after 4 h and 18 h of contact time was significant in all groups treated with Puriton®. Twenty-six-week repeated oral administration of Puriton® was c... [more]