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Unveiling novel insights into Bacillus velezensis 16B pectin lyase for improved fruit juice processing
No full textMicrobial pectinolytic enzymes are important in various industries, particularly food processing. This study focuses on uncovering insights into a novel pectin lyase, BvPelB, from Bacillus velezensis 16B, with the aim of enhancing fruit juice processing. The study examines the structural and functional characteristics of pectinolytic enzyme, underscoring the critical nature of substrate specificity and enzymatic reaction mechanisms. BvPelB was successfully expressed and purified, exhibiting robust activity under alkaline conditions and thermal stability. Structural analysis revealed similarities with other pectin lyases, despite limited sequence identity. Biochemical characterization showed BvPelB's preference for highly methylated pectins and its endo-acting mode of cleavage. Treatment with BvPelB significantly increased juice yield and clarity without generating excessive methanol, making it a promising candidate for fruit juice processing. Overall, this study provides valuable insights into the enzymatic properties of BvPelB and its potential industrial applications in improving fruit juice processing efficiency and quality
Comparison of cytotoxicity methods for studying Vipera ammodytes venom and the anticytotoxic potency of antivenom
No full textIntroduction: Alternative in vitro tests that can be used instead of animal experiments are those that can most closely evaluate the biological activity of the drug of interest. For testing the potency of antivenom, these are the methods used to assess cytotoxicity. The aim of this study was to evaluate the most commonly used cytotoxicity methods for determining the protective potency of the antivenom Viekvin, which neutralizes Vipera ammodytes venom. Material and methods: The selected methods are based on different biological mechanisms: MTT assay, based on the activity of cell oxidoreductase enzymes; crystal violet staining, based on the degree of cell adhesion; trypan blue staining, based on cell membrane permeability, and propidium iodide staining, based on measurement of nucleic acids of dead cells. The pro-apoptotic effect of the venom was also determined with annexin V staining. Results: The IC50 value of V. ammodytes venom obtained by these methods was very similar, while the EC50 values differed significantly. Conclusions: We concluded that the choice of the method used to measure the anticytotoxic antivenom potency depends on the immunogenicity of the venom components that cause cell death; for each venom/antivenom pair, it is necessary to select the appropriate assay separately, and at present, none of the standard cytotoxic methods can be universally applied to determine antivenom potency
Long live the macrophages – memantine as cellular youth elixir?
No full textMemantine is an uncompetitive NMDA antagonist, blocking the ionic channels of NMDA receptors, influencing the Ca2+
metabolism. This drug is clinically used for treating Alzheimer’s disease, and there have been studies on animal models
covering a variety of disease models and tissues, including the heart, liver and brain. Results presented here are from a
peculiar observation made in the following study: female Dark Agouti rats of two ages (young – 3 months, and old – 24
months) were fed a memantine solution (60 mg/kg body weight/day for 7 consecutive days) through oral gavage from the
first day after experimental autoimmune encephalomyelitis was induced, and were sacrificed on day 8 (inductive phase
of disease). We confirmed the effects of systemic memantine application through histological analysis – livers of young
control group rats showed signs of steatosis, which was ameliorated in the treated group; rats from both old and young
control groups showed signs of liver mononuclear infiltration, which was absent in all rats from treated groups, regardless
of age. However, the curious unexpected result was of a technical nature – peritoneal macrophages isolated from the rats
showed substantially greater viability even 11 days after the experiment. Memantine had a stronger effect on peritoneal
cells of young rats – the peritoneal cell yield was two times lower in the memantine treated group, the percentage of
CD45RA+ peritoneal B-cells was substantially higher than in the control group, and 11 days post-experiment there were
3 times more live macrophages (CD11b+CD163+
). Macrophages are usually short living cells upon extraction from
tissues, and these results may point in a direction of an intrinsic mechanism for longevity of macrophages and potentially
other cells expressing the NMDAR. Since memantine is known to block extravasation of blood mononuclear cells into
peripheral tissues, it is possible that the dynamic of peritoneal cells replenishment has been disturbed. The implications
of our observations are unclear and further research will be need to establish the full scope of possibilities it unlocks,
however we feel confident that memantine could be the proverbial shamrock for macrophages, bringing them long and
happy lives
mRNA Vaccines Development: From Experimental Beginnings to the Nobel Prize
No full textRazvoj vakcina ima bogatu istoriju koja je obeležena revolucionarnim iskoracima i razvojem tehnologija koje su značajno uticale na javno zdravlje. Ovaj rad prikazuje istorijski razvoj vakcina, sa posebnim fokusom na nastanak i razvoj iRNK vakcina. Počevši od ranih inovacija u oblasti vakcina, rad pruža sveobuhvatan pregled kako je iRNK tehnologija unela revoluciju ovu oblast. Razmatra se jedinstveni aspekt imunološkog odgovora koji izazivaju iRNK vakcine, ističući njihovu sposobnost da proizvedu snažan i ciljan imunitet protiv patogena. Takođe, rad analizira proces proizvodnje iRNK vakcina, objašnjavajući korake od dizajna plazmida preko sinteze iRNK do enkapsulacije u lipidne nanopartikule. Diskusija uključuje analizu izazova tokom faza razvoja i kako su ti izazovi prevaziđeni. Praćenjem evolucije različitih tehnologija vakcina i naglašavanjem napretka koji su donele iRNK vakcine, ovaj rad naglašava njihov transformativni potencijal u prevenciji infektivnih i tretmanu malignih bolesti.The development of vaccines has a rich history marked by groundbreaking innovations and evolving technologies that have significantly improved public health. This paper explores the historical progression of vaccines, with a particular focus on the emergence and development of mRNA vaccines. Starting from early vaccine innovations, the study provides a comprehensive overview of how mRNA technology has revolutionized the field. It delves into the unique aspects of the immune response elicited by mRNA vaccines, highlighting their ability to produce robust and targeted immunity against pathogens. Additionally, the paper examines the production process of mRNA vaccines, from plasmid design and the synthesis of mRNA strands to their encapsulation in lipid nanoparticles. The discussion includes an analysis of the challenges faced during the development phases and how these have been addressed. By tracing the evolution of vaccine technology and emphasizing the advancements brought by mRNA vaccines, this paper underscores their transformative potential in preventing infectious diseases and treating various conditions. The findings suggest that mRNA vaccines represent a pivotal shift in vaccine development, offering new avenues for future research and application in both infectious diseases and personalized medicine
Pertussis vaccine: overview
No full textAbstract: Pertussis or whooping cough is a highly contagious, vaccine-preventable, respiratory disease caused by Bordetella pertussis, and transmitted through the respiratory tract (aerosol). According to the re- ports of the World Health Organization and Centers for Disease Control and Prevention, the incidence of pertussis shows periodical variations in certain regions of the world. As humans are the sole reservoir of this bacteria complete vaccination against pertussis and high vaccination coverage is of utmost importance for reducing the incidence and severity of the disease.
Two types of pertussis vaccine are available: whole- cell (wP) and acellular pertussis (aP) vaccine. wP contains whole nonviable bacteria, while aP usually contains two or more protein components. These pro- tein components include inactivated pertussis toxin, filamentous hemagglutinin, pertactin, and fimbriae. The acellular vaccine was developed in response to reports of adverse reactions upon administering the whole-cell vaccine in certain countries.
Both vaccines are usually formulated with diphtheria and tetanus toxoids, and more recently a trend of combin- ing more antigenic sources such as Haemophilus influen- zae type b, hepatitis B, and inactivated poliovirus vaccine has been accepted in many countries, including Serbia.
The wP vaccine stimulates a strong immune response more similar to infection, while the response to aP vac- cine differs in this respect. Due to the difference in the types of immune response predominating with different types of pertussis vaccines, there are differences in the duration of protection, and it has been reported that wP induces more durable protection. For countries that have adopted aP increased monitoring is advised as well as the inclusion of booster doses. The special focus is on the vaccination of pregnant women to protect the newborns.
Incited by the recent surge in pertussis cases in Serbia here we provide a comprehensive literature overview of pertussis vaccines
Improvement of fruit juice quality: novel endo-polygalacturonase II from Aspergillus tubingensis FAT 43 for enhanced liquefaction, clarification, and antioxidant potential
No full textThis study focuses on the isolation, purification, and characterisation of endo-polygalacturonase II from Aspergillus tubingensis FAT43, particularly emphasising its potential applications in the fruit juice industry. A comprehensive screening test revealed the temporal dynamics of endo-polygalacturonase production during a 96-hour fermentation process. The purification process, involving ammonium sulfate and ethanol precipitation followed by ion-exchange chromatography, resulted in a 3.3-fold purification of PG II with a yield of 16% and a specific activity of 6001.67 U mg−1. Molecular analysis confirmed the identity of PG II, its gene (pgaII), and a high degree of sequence identity with Aspergillus tubingensis in the SWISS-PROT database. The optimal pH for PG II activity was 3.5–4.5, with robust stability across a broad pH spectrum (3–7). The enzyme exhibited optimal temperature activity at 45 °C, with a retention of 90% activity at 50 °C. The calculated activation energy for PG II was 62.1 kJ mol−1, indicating good stability. Inactivation kinetics revealed a half-life of 13.7 h at 40 °C, 5.4 h at 50 °C, and 0.85 h at 60 °C, with an activation energy of denaturation of 32.8 kJ mol−1. Compared to literature-reported PGs, PG II from A. tubingensis FAT43 demonstrated superior thermal stability. Hydrolysis experiments on different pectins revealed the highest specificity for non-methylated substrates (polygalacturonic acid). In fruit juice processing, PG II significantly increased juice yield and clarity, with the highest impact observed in strawberry juice. Antioxidant activity assays indicated enhanced antioxidant potential in enzyme-treated juices, especially strawberry, quince, and apple juices. The study highlights PG II's potential as an industrially valuable enzyme for fruit juice processing, offering improved thermostability and versatility across various fruit types
Exploring E. coli-based expression of genetically inactivated tetanus toxin for vaccine development
No full textTetanus toxin, a highly potent neurotoxin produced by Clostridium tetani, is the primary agent responsible for causing tetanus. This serious, potentially fatal disease can be effectively prevented through vaccination. Thanks to successful vaccination campaigns, tetanus has become exceedingly rare in both developed and most developing countries. However, the widespread presence of C. tetani spores in the environment means that tetanus cannot be completely eradicated, underscoring the ongoing need for vaccination. Traditionally, tetanus vaccines are produced by cultivating C. tetani, extracting a crude form of the tetanus toxin, and then chemically inactivating it for use in immunization. This method has proven clinically effective and is in widespread use. A challenge with this approach, however, is that the vaccine contains hundreds of various C. tetani proteins, with the active component making up only a variable and small fraction of the overall vaccine mass. To improve the current tetanus vaccine, there is potential in the recombinant production of a genetically inactivated tetanus vaccine. Prior studies have demonstrated the feasibility of engineering the full-length tetanus toxin in E. coli, and our current work builds on this foundation. We have successfully cloned the complete tetanus toxin open reading frame into the pMAL expression vector. This step was followed by the creation of a genetically inactivated protein, achieved through standard site-directed mutagenesis which altered 8 critical amino acid residues. These mutations have been confirmed via sequencing, ensuring that the toxin is genetically inactivated and thus does not require chemical inactivation for vaccine production. Our present focus is on optimizing the expression of this protein in E. coli. Following this, we intend to conduct thorough assessments of the biochemical and immunological properties of the recombinant tetanus toxin. This research represents a promising avenue towards enhancing the efficacy and specificity of tetanus vaccines, potentially improving global health outcomes
Beneficial effects of a new probiotic formulation on adipocytokines, appetite-regulating hormones, and metabolic parameters in obese women
No full textObesity is often accompanied by low-grade chronic inflammation and metabolic syndrome. It has been established that microbiota influences many physiological processes, including the development of obesity, and dysbiosis has been observed in obese individuals. In this study, we aimed to evaluate the impact of a new probiotic formulation, containing two probiotic strains and the bioactive compound octacosanol, on body weight, metabolic parameters, and concentrations of certain adipocytokines and appetite-regulating hormones in obese women. This double blind placebo-controlled supplementary intervention study included twenty-five women in the intervention group and twenty-three in the placebo group, and it lasted 12 weeks. Daily oral supplementation included 7 × 1010 CFU of Lactiplantibacillus plantarum 299v (DSM9843), 5 × 109 CFU of Saccharomyces cerevisiae var. boulardii (DBVPG6763), and 40 mg of octacosanol or placebo. Body weight, metabolic parameters, adipocytokines, and appetite-regulating hormones were assessed before (T0) and after the intervention (T1). After the intervention, significantly lower median concentrations of CRP (p = 0.005) and IL-6 (p = 0.012) were measured in the intervention group than the baseline, while the median concentrations of ghrelin (p = 0.026) and HDL-cholesterol (p = 0.03) were significantly increased. The intervention group had lower CRP levels (p = 0.023) and higher ghrelin levels (p = 0.006) than the placebo group. Significant changes in BMI between groups were not observed. In summary, although the new probiotic formulation showed beneficial effects on IL-6, CRP, HDL, and ghrelin levels, its potential effects on regulating triglyceride, insulin, and glucose levels require further studies before the novel dietary intervention could be considered a useful adjuvant therapy and an effective strategy for the management of obesity and obesity-associated comorbidities
A Longitudinal Study of Escherichia coli Clinical Isolates from the Tracheal Aspirates of a Paediatric Patient—Strain Type Similar to Pandemic ST131
No full textEscherichia coli is a Gram-negative bacterium and part of the intestinal microbiota. However, it can cause various diarrhoeal illnesses, i.e., traveller’s diarrhoea, dysentery, and extraintestinal infections when the bacteria are translocated from the intestine to other organs, such as urinary tract infections, abdominal and pelvic infections, pneumonia, bacteraemia, and meningitis. It is also an important pathogen in intensive care units where cross-infection may cause intrahospital spread with serious consequences. Within this study, four E. coli isolates from the tracheal aspirates of a tracheotomised paediatric patient on chronic respiratory support were analysed and compared for antibiotic resistance and virulence potential. Genomes of all four isolates (5381a, 5381b, 5681, 5848) were sequenced using Oxford Nanopore Technology. According to PFGE analysis, the clones of isolates 5681 and 5848 were highly similar, and differ from 5381a and 5381b which were isolated first chronologically. All four E. coli isolates belonged to an unknown sequence type, related to the E. coli ST131, a pandemic clone that is evolving rapidly with increasing levels of antimicrobial resistance. All four E. coli isolates in this study exhibited a multidrug-resistant phenotype as, according to MIC data, they were resistant to ceftriaxone, ciprofloxacin, doxycycline, minocycline, and tetracycline. In addition, principal component analyses revealed that isolates 5681 and 5848, which were recovered later than 5381a and 5381b (two weeks and three weeks, respectively) possessed more complex antibiotic resistance genes and virulence profiles, which is concerning considering the short time period during which the strains were isolated.Supplementary information: [https://hdl.handle.net/21.15107/rcub_intor_987
Supplementary information for the article: Filipic, B.; Kojic, M.; Vasiljevic, Z.; Sovtic, A.; Dimkic, I.; Wood, E.; Esposito, A. A Longitudinal Study of Escherichia Coli Clinical Isolates from the Tracheal Aspirates of a Paediatric Patient—Strain Type Similar to Pandemic ST131. Microorganisms 2024, 12 (10), 1990. https://doi.org/10.3390/microorganisms12101990.
No full textSupplementary Figure S1: Pan-genome analyses of assembled genomes and number of core and shell genes. Supplementary Figure S2. Average nucleotide identity between genome sequences of E. coli isolates. Supplementary Figure S3. Alignment of the plasmid homologous to pMB2910_1 in the four strains, in order from top: 5381a, 5381b, 5681 and 5848. Tables: 5381b_VFDB_Jan_23-9085838786, 5381aVFDB_Jan_23-3173772185, 58848_VFDB_Jan_23-7074876386, 5681_VFDB_Jan_23-2749062099Supplementary material for: [https://intor.torlakinstitut.com/handle/123456789/987]Related to the published version: [https://doi.org/10.3390/microorganisms12101990