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Control of Listeria monocytogenes and Staphylococcus aureus in meat and meat products by cell-free supernatant of Brevibacillus laterosporus BGSP7 and BGSP9
No full textThe presence of pathogens in food has increased awareness of food safety, but it also causes large economic losses. Fresh meat and meat products contain a sufficient quantity of proteins, lipids, water, and a favorable pH that stimulates the growth of various microorganisms, including pathogens.The aim of this study was to investigate the efficacy of Brevibacillus laterosporus BGSP7 (CFS-BGSP7) and BGSP9 (CFS-BGSP9) cell-free supernatants in the control of Listeria monocytogenes and Staphylococcus aureus in raw meat and meat products.Raw meat and meat products were sliced and then aseptically treated by immersion for 2 minutes into solutions containing: i) CFS-BGSP7; ii) CFS-BGSP9; iii) no treatment. The samples were then artificially contaminated with: Group I – L. monocytogenes (~4 log cfu g-1); Group II – S. aureus LMM322 (~4 log cfu g-1). Each sample was individually aseptically vacuum-packed and stored at 4°C for 8 weeks. The number of surviving bacteria in the samples were analyzed immediately after contamination with L. monocytogenes and S. aureus and at regular time-intervals: after 1, 3, 5 and 8 weeks of storage at 4°C.Meat samples treated with CFS-BGSP7 and CFS-SP9 showed a significant decrease in the cell counts of L. monocytogenes and S. aureus. When meat samples treated with CFS-BGSP7 and CFS-BGSP9 are compared, the results show a more intense reduction rate of both L. monocytogenes and S. aureus in all samples treated with CFS-BGSP7
The influence of environmental pollution on the allergenic potential of grass pollen
No full textGrass pollen is the most common cause of pollen allergies in Europe. However, growing evidence suggests that air pollution and climate change may contribute to the rising number of allergic cases and worsening symptoms. This narrative review article aims to summarize the impacts of increased health complications based on pollution research in recent years, obtained from ecological, molecular and clinical studies to provide a new perspective on the impact of pollutants on the environment and human health. Our detailed literature review includes studies on pollution and its effect on pollen allergens, which cause allergy symptoms, but only in the case of three grass species: Dactylis glomerata, Lolium perenne and Phleum pratense
Raw data for the article: Stanojević, S.; Vujić, V.; Ćuruvija, I.; Vasić, M.; Bogdanović, A.; Prijić I.; Blagojević, V. Diverse inflammatory responses in two rat strains as preclinical model for studying therapies targeting inflammatory processes in colitis. 2024.
No full textWe provide one excel (with eight sheets) and 1 jpg file. Data file named ’Raw data_Fig. 1,2,3,5,6,7,8,9.xls’ and ’Raw data _histology_Fig.4.jpg’ contain raw data on analyzed traits in the submitted manuscript. Data files named ’Raw data_Fig. 1,2,3,5,6,7,8,9.xls’ consists of 8 sheets. The first sheet named ’Fig. 1’ contains data showing the number of AO and DA rats (A) and the proportion of their survival (B) over 7 days following intrarectal instillation of 300 µl of saline, ethanol and TNBS (10 and 40 mg/kg b.w.). The second sheet named ’Fig. 2’ contains data showing length of colon segment of AO and DA rats affected by necrosis or hyperemia/edema 7 days following intrarectal instillation of 300 µl of saline, ethanol and TNBS (10 and 40 mg/kg b.w.). The third sheet named ’Fig. 3’ contains data showing consumption of food (A), water (B) and weight loss (C) of AO and DA rats following intrarectal instillation of 300 µl of saline, ethanol and TNBS (10 and 40 mg/kg b.w.). The fourth sheet named ’Fig. 5’ contains data showing the level of NO, urea (A), cytokines (B) and relative IL-17/IFN-γ balance of production, in colon tissue homogenates of AO and DA rats 7 days following intrarectal instillation of 300 µl of saline, ethanol and TNBS (10 and 40 mg/kg b.w.). The fifth sheet named ’Fig. 6’ contains data showing the expression of CD11b and CD4 (B), the proportion of HIS48hiCD43+ cells among CD11bintCD4low cells, and the proportion of CD163+MHCIIlo and CD163-MHCIIhi cells among resident CD11bhi CD4hi cells (C) in peritoneal washing of AO and DA rats 7 days following intrarectal instillation of 300 µl of saline, ethanol and TNBS (10 and 40 mg/kg b.w.). The sixth sheet name ’Fig. 7’ contains data showing the concentration of NO, Urea in unstimulated (A), and of TNF, IL-6 and IL-10 in LPS-stimulated (B) adherent peritoneal cells of AO and DA rats 7 days following intrarectal instillation of 300 µl of saline, ethanol and TNBS (10 and 40 mg/kg b.w.). The seventh sheet name ’Fig. 8’ contains data showing
the proportion of CD4+, CD4+CD25+, CD8+ and CD45RA+ cells in mesenteric lymph nodes (A), the concentration of IL-17 and IFN-γ (B) and the relative balance of IL-17/IFN-γ production (C) in Con A-stimulated mesenteric lymph node cells of AO and DA rats 7 days following intrarectal instillation of 300 µl of saline, ethanol and TNBS (10 and 40 mg/kg b.w.). The eight sheet named ’Fig. 9’ contains data showing the concentration of proteins in sera of AO and DA rats 7 days following intrarectal instillation of 300 µl of saline, ethanol and TNBS (10 and 40 mg/kg b.w.) (A), the levels of specific anti-E. coli antibodies of IgG1, IgG2a, IgG2b and IgA classes in sera of saline-injected AO and DA rats (B), and the levels of specific anti-E. coli antibodies of IgG1 (C), IgG2a (D), IgG2b (E) and IgA (F) classes in sera of AO and DA rats 7 days following intrarectal instillation of 300 µl of saline, ethanol and TNBS (10 and 40 mg/kg b.w.). Data file names ’Raw data _histology_Fig.4.jpg’ contains original photomicrographs od HE-stained slides with colon tissue obtained from AO and DA rats 7 days following intrarectal instillation of 300 µl of saline, ethanol and TNBS (10 and 40 mg/kg b.w.)
The Venom of Vipera ammodytes ammodytes: Proteomics, Neurotoxic Effect and Neutralization by Antivenom
No full textDeep proteomic analyses identified, in total, 159 master proteins (with 1% FDR and 2 unique peptides) from 26 protein families in the venom of Vipera ammodytes ammodytes (Vaa). Data are available via ProteomeXchange with the identifier PXD056495. The relative abundance of PLA2s is 11.60% of the crude venom, of which 4.35% are neurotoxic Ammodytoxins (Atxs). The neurotoxicity of the venom of Vaa and the neutralizing effect of the antivenom were tested on the neuromuscular preparation of the diaphragm (NPD) of rats. The activity of PLA2 in the venom of Vaa and its neutralization by the antivenom were determined under in vitro conditions. The Vaa venom leads to a progressive decrease in NPD contractions. We administered pre-incubated venom/antivenom mixtures at various ratios of 1:2, 1:10 and 1:20 (w/w) and observed the effects of these mixtures on NPD contractions. The results show that the mean effective time (ET50) for NPD contractions with the 1:20 mixture is highly significantly different (p < 0.001) from the ET50 for the venom and the ET50 for the 1:2 and 1:10 mixture ratios. We also found a highly significant (p < 0.001) reduction in Na+/K+-ATPase activity in the NPD under the influence of the venom. The reduction in the activity of this enzyme was reversible by the antivenom. Under in vitro conditions, we have achieved the complete neutralization of PLA2 by the antivenom. In conclusion, the antivenom abolished the venom-induced progressive decrease in NPD contractions in a concentration-dependent manner. Antivenom with approximately the same mass proportion almost completely restores Na+/K+-ATPase activity in the NPD and completely neutralizes the PLA2 activity of the venom in vitro
Analysis of AlphaFold2 Predicted Structures of Aggregation Factors in Lactic Acid Bacteria
No full textAutoaggregation is the ability of identical bacterial cells to adhere to each other, which is
important for colonization, kin and kind recognition, and bacterial survival.
There is a group of aggregation factors in lactic acid bacteria that have several distinctive
features: these are large proteins with a molecular mass greater than 150 kDa, containing an
N-terminal signal sequence and an LPXTG-like cell wall anchor domain, as well as a different
number of repetitive domains. These aggregation-promoting proteins are also called/known
as Snow-flake Forming Collagen Binding Aggregation Factors (SFCBAF) due to their unique
aggregation phenotype (PMIDs: 22182285, 29018422, 25955159, 30027759, 38014957).
Predictions for different members of this group (predicted by the InterPro program) indicate
a varying number and, in some cases, different compositions of repetitive domains.
Comparison of the predicted structures of known aggregation factors (AggL from Lactococcus
lactis; AggE from Enterococcus faecium; AggLb from Lacticaseibacillus paracasei; AggLr from
Lactococcus raffinolactis; and AggA from Tetragenococcus halophilus) using AlphaFold2
revealed structural similarities, which may explain the similar phenotype despite low identity
(e.g., AggLb is identical to AggA at 21.73% and AggL at 41.14%, while AggA is identical to AggL
at 32.43%). The structure itself resembles a shoe-like structure: with a heel and a sole in
the form of a loop, consisting of 6-7 adhesion domains superfamily (InterPro: IPR008966).
The most structurally dissimilar among them is AggLb, which is the largest of the described
aggregation factors of this type and has a greater number of repeats in the second half
of the protein compared to other members of this group. The calculation of electrostatic
potential shows that the protein surfaces predominantly have negative potential, which is
consistent with previously shown data for the AggLb protein, where the strain producing
AggLb demonstrated higher affinities for chloroform and a lower percentage of adhesion
to ethyl acetate, indicating that AggLb is able to provide strong electron-donor and weaker
electron-acceptor features to the bacteria.
In summary, this research enhances our comprehension of the structure of aggregation
factors in lactic acid bacteria
mRNA vaccine manufacturing – challenges in plasmid DNA cloning vector design
No full textIn the post-COVID-19 era, there has been a significant increase in the development of mRNA vaccines not only against various diseases besides SARS-CoV-2, but also to treat cancer and genetic disorders. These vaccines, revolutionizing vaccinology, offer rapid pandemic response, high efficacy, minimal side effects, and cost-effectiveness. Achieving these benefits hinges on seamlessly integrating mRNA production steps, from plasmid DNA cloning to lipid nanoparticle formulation. This overview aims to comprehend or circumvent pitfalls in plasmid DNA cloning, a critical initial step in mRNA vaccine production. The focus is on achieving accurate insert sequence and gene expression, and it highlights the critical role of plasmid DNA design in ensuring vaccine effectiveness. Our research project entitled “Role of macroautophagy in lipid nanoparticle mRNA delivery and adjuvanticity” recognized the significance of this aspect. During our research, we designed a plasmid DNA cloning vector to incorporate the GFP-SARS-CoV-2 Spike gene. The vector was carefully constructed with several key features, including a high-copy plasmid, pUC18/pUC19 vector backbone with a robust T7 promoter, origin of replication, multiple cloning sites, polyadenylation signal, and ampicillin resistance for bacterial selection. Despite careful design, challenges like poly-A tail deletion may arise, prompting the exploration of stable large-size and low-copy vectors, as well as linear and bacteriophage vectors. But, for largescale production and regulatory compliance, vector systems must be scalable and well-documented. Commercial vectors and automated synthesis facilitate gene construction, with artificial intelligence ensuring sequence accuracy. Precision is crucial for complex antigens, as seen in tuberculosis mRNA vaccine development. Addressing these challenges demands a combining of molecular biology techniques, computational tools, and collaboration with experts in microbiology, molecular biology, and vaccine development. The design’s scalability and documentation are vital for large-scale production and regulatory compliance, emphasizing the multifaceted approach required for successful mRNA vaccine development
Bacteriophages of multidrug-resistant nosocomial pathogens – Belgrade experience
No full textAntimicrobial resistance (AMR) arises whenbacteria and other microbes stop respondingto medications. AMR is now recognized as oneof serious global health threats, repeatedlyappearing in the World Health Organization’s(WHO) lists of urgent global health challenges,including the 2024 list. It is taking a fatal toll– nearly 5 million deaths globally per year areassociated with AMR, encompassing 1.27 milliondirectly attributed to AMR. The COVID-19pandemic paved the way for aggravation ofbacterial AMR – primarily due to enhancementin unspecific and unjustified prescription anduse of broad-spectrum antibiotics, resulting inwhat is now recognized as „silent pandemic ofAMR“. Bacteriophages (phages) are natural andspecific predators of bacteria - viruses that caninfect, replicate inside and lyse arguably anybacteria. Their therapeutic potential is beinghastily evaluated through different approaches:in silico, in vitro, ex vivo and in vivo – in laboratoryanimals as well as in human case and clinicalstudies. Although the results are promising,bacteria rapidly develop resistance againstphages, which why the isolation and researchof new phages is needed. Our work is concentratedon three bacterial species for which criticalpriority by WHO has been declared – carbapenem-resistant Acinetobacter baumannii,Pseudomonas aeruginosa and Klebsiella pneumoniae.Twenty distinct pathogenic strains ofA. baumannii, 6 K. pneumoniae and 6 P. aeruginosawere used as targets for bacteriophageisolation, and total of 14, 22 and 8 potentiallydistinct phages were collected, respectively. Allstrains were nosocomial isolates obtained fromvarious tissues, including from terminally ill patients.Six phages were characterized in detail.In particular, phage vB_AbaM_ISTD was appliedagainst A. baumannii in zebrafish embryomodel of systemic infection, and demonstratedpowerful therapeutic potential, eradicating theinfection. Interestingly, its DNA was characterizedwith highly modified thymidine (amassing1228 Da), making it the largest non-canonicaldeoxynucleoside reported so far.Book of abstract: From biotechnology to human and planetary health XIII congress of microbiologists of Serbia with international participation Mikromed regio 5, ums series 24: 4th – 6th april 2024, Mona Plaza hotel, Belgrade, Serbi
Dysregulation of transcripts SMAD4-209 and SMAD4-213 and their respective promoters in colon cancer cell lines
No full textackground: The pervasive role of alternative promoters in context-specific isoform expression and
the importance of promoter choice over its level of transcriptional activity have been recently implied
based on pan-cancer in silico studies. We aimed to explore this phenomenon at the cellular level on the
example of a major tumor suppressor SMAD4 in search of molecular mechanisms in colorectal cancer
that could be exploited for novel biomarkers or therapeutic approaches.
Methods: Multi-omics technologies, in silico tools and in vitro functional assays were applied to analyze
the transcripts expression and the alternative promoters’ function of the SMAD4 gene in colon cell lines
HCEC-1CT, HCT116, DLD-1, SW480 and SW620.
Results: High expression of the transcript SMAD4-213 emerged as a hallmark of colon cancer cells,
while in silico tools point to its possible additional role and potential for sponging miRNAs. Based on the
observed dysregulation of SMAD4-209 and SMAD4-213 in malignant vs. non-malignant colon cells, we
propose that their expression ratio might be a solid biomarker candidate for colorectal cancer detection.
Conclusions: A differential pattern of the respective promoters’ activity was observed that corresponds
to the expression of transcripts, confirming the role of alternative promoters in context-specific isoform
expression. The investigated SMAD4 promoters and transcripts harbor translational potential that should
be further investigated
The effect of Pb(ii), Cd(ii) and Pd(ii) ions on the conformational changes of ovalbumin
No full textProteinske strukture su prirodno dinamičke i fleksibilne. Ova univerzalna karakteristika je
apsolutno neophodna za biološku funkciju i relativnu stabilnost nativnih proteina. U bilo kom
trenutku, proteini zauzimaju odgovarajuću strukturu, odnosno konformaciju. Promene u ovoj
strukturi su krajnje odreĎene od strane faktora sredine u kojoj se dati proteini nalaze. Ove
promene mogu biti minorne i funkcionalno neophodne, dok neke promene mogu biti
drastične, krajnje izazivajući gubitak nativne strukture i prirodne funkcije polipeptida. Iako je
gubitak nativne konformacije proteina u većini bioloških slučajeva nepogodan, sami
konformacioni prelazi u termodinamički stabilniju formu predstavljaju potpuno prirodnu
pojavu i osnovu za mnoge biohemijske procese.
Glavni cilj ove disertacije je dalje rasvetljenje uticaja faktora koji utiču na konformacione
prelaze proteina, konkretno dejstva jona teških metala i organometalnih kompleksa primenom
ovalbumina kao model sistema. Radi ostvarenja ovog cilja, izvedena je strukturna analiza
ovalbumina primenom izvesnih metoda poput FTIR spektroskopije za praćenje promena
sekundarnih struktura, uključujući DLS i AFM metode za analizu dimenzija i morfologije
novonastalih struktura. Spektrofluorimetrijske probe su primenjene za analizu hidrofobnosti
ovalbumina, kao i za praćenje formiranja amiloidnih struktura.
Rezultati analize su pokazali da je stepen formiranja amiloidnih fibrila srazmeran sa
koncentracijom prisutnih jona teških metala, čije nespecifične interakcije su različito uticale
na krajnju morfologiju fibrila. Dobijeni rezultati strukturne analize su takoĎe pokazali da
ovalbumin predstavlja adekvatan model sistem za predviĎanje dejstva univerzalnih protein-
ligand interakcija sa potencijalno novim hemoterapeuticima na bazi metala.Protein structures are dynamic and flexible by nature. This universal characteristic is
absolutely necessary for the proper biological function and relative stability of native
proteins. Proteins, at any given moment, assume a certain structure, i.e. conformation.
Changes in this structure are ultimately determined by the environmental factors by which a
protein is affected. The changes induced by these factors may be minor, but functionally
mandatory, while others might be drastic and ultimately result in the loss of the native fold
and biological function of a polipeptide. Even though the loss of a protein’s native
conformation is generally viewed as a negative scenario, conformational changes towards
more thermodynamically stable forms represent entirely natural phenomena and the basis for
many biochemical processes.
The main goal of this dissertation is to further elucidate the effects of these factors on protein
conformational changes, specifically the effects of heavy metal ions and organometallic
complexes on ovalbumin as a model system. To this end, methods such as FTIR spectroscopy
were used for secondary structure analysis, including DLS and AFM techniques for
determining the protein's dimensions and morphology. Spectrofluorimetric dyes were also
used to monitor protein hydrophobicity and amyloid formation.
The obtained results show that amyloid formation is directly proportional to the concentration
of present heavy metal ions, the non-specific binding of which had differing effects on the
fibrils’ morphologies. Structural analyses results also showed that ovalbumin shows great
promise as an adequate model system for the prediction of universal protein-ligand
interactions with metal-based chemotherapeutic agents
Banana Lectin: A Novel Immunomodulatory Strategy for Mitigating Inflammatory Bowel Disease
No full textCompared to the general population, patients with inflammatory bowel disease (IBD) are less likely to be vaccinated, putting them at an increased risk of vaccine-preventable illnesses. This risk is further compounded by the immunosuppressive therapies commonly used in IBD management. Therefore, developing new treatments for IBD that maintain immune function is crucial, as successful management can lead to better vaccination outcomes and overall health for these patients. Here, we investigate the potential of recombinant banana lectin (rBanLec) as a supporting therapeutic measure to improve IBD control and possibly increase vaccination rates among IBD patients. By examining the therapeutic efficacy of rBanLec in a murine model of experimental colitis, we aim to lay the foundation for its application in improving vaccination outcomes. After inducing experimental colitis in C57BL/6 and BALB/c mice with 2,4,6-trinitrobenzene sulfonic acid, we treated animals orally with varying doses of rBanLec 0.1–10 µg/mL (0.01—1 µg/dose) during the course of the disease. We assessed the severity of colitis and rBanLec’s modulation of the immune response compared to control groups. rBanLec administration resulted in an inverse dose–response reduction in colitis severity (less pronounced weight loss, less shortening of the colon) and an improved recovery profile, highlighting its therapeutic potential. Notably, rBanLec-treated mice exhibited significant modulation of the immune response, favoring anti-inflammatory pathways (primarily reduction in a local [TNFα]/[IL-10]) crucial for effective vaccination. Our findings suggest that rBanLec could mitigate the adverse effects of immunosuppressive therapy on vaccine responsiveness in IBD patients. By improving the underlying immune response, rBanLec may increase the efficacy of vaccinations, offering a dual benefit of disease management and prevention of vaccine-preventable illnesses. Further studies are required to translate these findings into clinical practice