imagine (Institute of molecular genetics and genetic engineering)
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A novel spi1 mutation in a patient with agammaglobulinemia
Get PDFAgammaglobulinemia is a primary immunodeficiency characterized by a low
number or absence of mature B lymphocytes and consequently by immunoglobulin deficiency.
In 2021, six patients with pathogenic variants in SPI1 gene associated with
agammaglobulinemia type 10 (PU.MA) were described for the first time. This gene encodes the
pioneer transcription factor PU.1, which plays an important role in the differentiation of B
lymphocytes, monocytes, and conventional dendritic cells. Here we present a female patient
with a novel mutation in SPI1 gene which has not been previously found in patients with PU.MA.
Case description: A 37-year-old female patient with frequent middle ear infections in early
childhood was diagnosed with agammaglobulinemia at the age of 15 when she started
immunoglobulin replacement therapy (IgRT). One year later, an allogeneic hematopoietic stem
cell transplant from a healthy sibling donor was performed. Unfortunately, chimerism analysis
found no DNA material from the donor in the patient's blood, suggesting graft rejection, so she
remained dependent on antibody replacement therapy. Years later, she was diagnosed with
protein-losing enteropathy, and despite escalating doses of IgRT, IgG levels remained low.
Subsequently, the patient developed persistent COVID -19 viremia and bacterial
meningoencephalitis. Clinical exome sequencing using the TruSight (Illumina) panel was
performed and in comparision with the human reference genome (hg19), has revealed a
heterozygous mutation in exon 4 of the SPI1 gene. This mutation is characterized by the
insertion of 2 nucleotides (c.441dup), a reading frame shift, and the insertion of a premature
stop codon. According to the American College of Medical Genetics and Genomics, this
mutation is described as a likely pathogenic-class 2 (PVS1_Very Strong).
Conclusion: From analysis of previous literature, we concluded that the mutant sequence in
exon 4 encodes the PEST region of the pioneer transcription factor PU.1, which is responsible
for interaction with other transcription factors. Immunophenotyping of peripheral blood cells did
not reveal CD19+ B cells, suggesting that a differentiation arrest may have developed between
the prepro-B and pro-B stages, where there is a high requirement for PU.1 activity. Nextgeneration
sequencing can be a very useful tool to uncover the causes of rare primary
immunodeficiencies, but further analysis is needed to explain the relationship between patient
genotype and clinical presentation
Circular Plastics: Upscaling the mixed plastic waste to biodegradable plastics
Get PDFIn a world where unsustainable anthropogenic activities are drastically affecting Earth’s
ecosystems and natural resources, threatening animal (including human) health and safety, the
sustainable management and use of resources, including preventing plastic pollution, have
become of paramount importance. Plastics, being at the core of many low cost and high
performing materials, are widely used despite being based on non-renewable petrochemicals.
Currently, in EU, only 7% of plastics are recycled out of 57.2 million tonnes produced, and
93% end up in incineration, landfills and oceans, releasing persistent, bio-accumulative, toxic
and hazardous chemicals.
Bio Innovation of a Circular Economy for Plastics 1 (BioICEP) an EU Horizon project focuses
on reducing the burden of plastic waste in the environment and also changing the linear value
chains of current plastic products. The project's overall objective is to demonstrate a seamless,
sustainable route to a circular economy for plastics by developing cost-effective and low-
energy demand processes for waste plastic biotransformation into high-value marketable
products such as biodegradable plastics. To address these challenges, the BioICEP project
focuses on developing physical, enzymatic and biocatalytic pre-treatments to convert the
mixed plastic waste to biodegradable plastics focusing on recycling and upcycling of the
products with a zero-waste and regenerative approach.Polymer Processing Society Asia-Australasia Regional
Conference, PPS – 2023
Hotel Uday Samudra, Kovalam, Kerala
29
th
November – 2
nd
December 202
The role of MIR-34 family members on the mucociliary process in the cellular respiratory model system
Get PDFIntroduction: Primary ciliary dyskinesia is a rare and heterogeneous disorder primarily affecting the respiratory organs, with impaired mucociliary clearance being a common characteristic. Recently, the importance of the miR34/449 family in ciliogenesisin animal models has been described. Thisstudy aimed
to establish a modelsystem to study respiratory diseases and assessfor the first time the role of the miR34 family on the mucociliary process in humans.
Methods: We cultured the primary Normal Human Bronchial Epithelial (NHBE) cells in the air-liquid interface system, enabling the differentiation of multiciliated cells(MCCs) and goblet cells(GCs). During the
differentiation process, transient overexpression of miR-34a/b/c members was conducted. The model
system and treatments were validated through confocal microscopy (β-tubulin, MUC5B, MUC5AC antibodies) and qRT-PCR of miRNAs,specifically ciliogenesis markers(NOTCH1, MCIDAS, GEMC1, CCNO, RFX3),
and differentiated cell markers (FOXJ1 and TFF3).
Results: Expression levels of ciliogenesis and differentiated cells markers and detection of cilia and
mucins at confocal microscopy confirmed the successful establishment of cellular modelsystem. During
the initial differentiation stage, an overexpression of miR34a/b/c changed the expression profile of ciliogenesis and differentiated cell markers.
Conclusion: The established model system provides a valuable platform for exploring innovative treatment approaches for lung diseases. These findings suggest that overexpression of miR34a/b/c has impact on mucociliary process by reducing the duration required for the process of ciliogenesis.
Furthermore, the expression levels of differentiated cell markerssuggest increased number of MCCs and
decreased number of GCs, indicating the role of miR34a/b/c in enhancing mucociliary clearance
Molecular basis of phenylketonuria in Serbia: an update
Get PDFIntroduction: Phenylketonuria (PKU) is the most frequent inborn disorder of amino acid metabolism
caused by variants in human phenylalanine hydroxylase gene (PAH).
Methods: In thisstudy (an update for the time period of 10 years, with patientsfrom our previousstudies included) a total of 109 PKU patients from Serbia were analyzed. They were classified into three phenotypic categories in accordance with pre-treatment plasma phenylalanine level: classic PKU, mild PKU
and mild hyperphenylalaninemia. For genetic analyses, we combined Sanger sequencing, MLPA and
next generation sequencing to identify disease-causing variantsin PAH gene, which were further classified using ACMG classification. Additionally, we used in silico and/or eukaryotic expression studiesto assess the effect of novel genetic variants identified in our patients.
Results: Disease-causing variants were identified in 217 of 218 alleles, reaching detection rate of 99.5%.
We detected a total of 32 different variants, of which 29 previously described and three novel ones:
p.Gln226Lys, p.Pro244His and p.Pro416Leu. In silico and/or eukaryotic expression studies confirmed pathogenic effect of all novel genetic variants. The most frequent variant was p.Leu48Ser (31.2%), followed
by p.Arg408Trp (13.8%), p.Ile306Val (9.2%). p.Glu390Gly (5%), p.Pro281Leu (4.6%), and p.Arg261Gln
(3.2%). All detected disease-causing variants were classified as pathogenic using ACMG classification.
Conclusion: Our study brings the updated spectrum of molecular genetic data, variant classification
and detailed phenotypic characteristicsfor PKU patientsfrom Serbia. Therefore, ourstudy contributesto
better understanding of molecular landscape of PKU in Europe and to general knowledge on genotype–
phenotype correlation in PKU
The role of the gut bacteria during host aging
Get PDFIntroduction: Microbial community established in the gut has been recognized as an important factor
which influence host aging. Bacteria from the gut co-evolved with the host resulting in mutually beneficial interactions essential for host’s wellbeing. This complex crosstalk reflects mainly through the interaction between bacterial macromolecules (e.g., exopolysaccharides) and the host receptors leading
to the activation of various cellular pathways. Here, we explore the potential of different lactobacilli,
commonly used as probiotics, to activate longevity signalling in Caenorhabditis elegans.
Methods: Evaluation of C. elegans lifespan and aging parameters (locomotion rate and pharyngeal
pumping) were performed by feeding N2 wild-type worms with different Lactobacillus species. Worms
fed with selected strains were subjected to RNAseq analysis, qPCR and Western blot to evaluate activation of autophagy, immunity, antioxidative response and mitochondrial function. Activation of autophagy was confirmed in DA2123 GFP-labelled LGG-1 transgenic strain and JIN1375 hlh-30 (tm1978)
mutant, while immunity activation was evaluated by using KU25 pmk-1 (km25) mutant and through
nematode killing assays.
Results: Selected strains of lactobacilli promoted health and lifespan of worms through activation of
TFEB/HLH-30 dependent autophagy and p38 MAPK/PMK-1 dependent immune response which provided resistance of worms exposed to pathogens. Moreover, RNAseq analysis identified core gene signature associate with exopolysaccharide-induced longevity highlighting involvement of fmo-2, gsto-1,
nlp-29, and clec-47 genes in increased lifespan of the worms.
Conclusion: Analyzed lactobacilli showed potential to promote healthy aging and could be further investigated in order to better understand application of lactobacilli as pro-longevity probiotics
Short-term effect of Brevibacillus laterosporus supplemented diet on worker honey bee microbiome
Get PDFIntroduction: Brevibacillus laterosporus is a promising microbiological agent that can be used to prevent and control destructive diseases affecting honey bee colonies. In the presentstudy, the short-term
effect of the B. laterosporus BGSP11 bee diet on microbiota and mycobiota was investigated.
Methods: The honey bee diet was supplemented with spores of B. laterosporus BGSP11 at a concentration of 1×108 CFU/mL in sucrose solution. Metabarcoding analysis of the bee microbial community profile was performed based on 16S RNA (bacteriobiota) and Internally Transcribes Spacer (ITS) region
(mycobiota) obtained using MiSeq Illumina sequencing. The QIIME2 v2021.4 pipeline was used to analyze the obtained amplicon data library.
Results: The results show that the BGSP11 bee diet slightly altered the bee microbiota and did not lead
to potentially harmful changes in the bacterial microbiota. Moreover, it can potentially induce positive
changes, mainly reflected in the reduction of opportunistic bacteria. On the other hand, the treatment
had a greater effect on mycobiota. However, the changesin the bee mycobiome caused by the treatment
cannot be considered a priori as beneficial or harmful,since the interaction between the bee and its mycobiome is not sufficiently studied. The observed positive changes in the bee mycobiome are mainly
reflected in the reduction of phytopathogenic fungi that may affect the organoleptic and techno-functional properties of honey.
Conclusion: This pilot study suggests that the introduction of BGSP11 in beekeeping practice as a biological agent could be considered due to no harmful effects observed on the microbiota of bees
POTENTIAL NEW GENES INVOLVED IN CYSTIC FIBROSIS PHENOTYPE
Get PDFBackground: Cystic fibrosis (CF) is autosomal
recessive disorder characterized by chronic
respiratory problems and poor growth. CF is caused
by defect in transmembrane conductance regulator
(CFTR) protein. CF is diagnosed by sweat chloride
analysis (>60 mmol/L) with the identification of
two CF-causing variants of CFTR gene. With a
longstanding history of CFTR gene analysis, our
laboratory identified several patients with elevated
sweat chloride and clinical manifestations of CF in
whom no CF-causing mutations were detected after
sequencing of whole coding region and testing for
large insertion/deletion of CFTR gene. In order to
elucidate genetic background of conditions that
mimic CF we performed whole exome sequencing
(WES) in two such patients.
Methods: Library preparation was done using
DNA nanoball technology. Produced fastq files
were mapped to hg38. VCF files were generated
using GATK and annotated with InterVar and AnnoVar
tools. Variants filtering for disease relevance was done using the following criteria: QC, GnomAD
Allele Frequency, Functional consequences
and phenotype-genotype relationship.
Results: CACNA1H and MUC5B genes were
found to be impaired in both patients. Similar number
of variants predicted to impair protein function
were detected (27 and 25) in each patient. Loss of
function variants were found in 7 and 11 genes,
respectively.
Conclusion: Further assessment of selected
variants will clarify their functional effect and relevance
for the patient’s clinical phenotype. WES
analysis will help identify genetic aspects of disease
and assist in optimal patient management in about
0.01% of patients with elevated sweat chloride and
high clinical suspicion of CF that do not carry any
CF-causing variants.ABSTRACT BOOK: “Genetic Diseases from Diagnostics to Prevention and Therapy” October 05-14th Balkan Congress of Human Genetics & 9th Rare Disease SEE Meeting 2023; Skopje, October 05-07, 202
Supplementary information for the article: Novović, K., Kuzmanović Nedeljković, S., Poledica, M., Nikolić, G., Grujić, B., Jovčić, B., Kojić, M., & Filipić, B. (2023). Virulence potential of multidrug-resistant Acinetobacter baumannii isolates from COVID-19 patients on mechanical ventilation: The first report from Serbia. Frontiers in Microbiology, 14. https://www.frontiersin.org/articles/10.3389/fmicb.2023.1094184
Get PDFWe would like to thank Dušan Melentić from General Hospital “Dr Laza K. Lazarević” Šabac for isolation of pure cultures of Acinetobacter baumannii and logistic support between Šabac hospital and University of Belgrade, Serbia.Related to published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1782]The Supplementary Material for this article can be found online at: [https://www.frontiersin.org/articles/10.3389/fmicb.2023.1094184/full#supplementary-material
2-Hydroxyethyl Methacrylate/Gelatin/Alginate Scaffolds Reinforced with Nano TiO2 as a Promising Curcumin Release Platform
Get PDFThe idea of this study was to create a new scaffolding system based on 2-hydroxyethyl methacrylate, gelatin, and alginate that contains titanium(IV) oxide nanoparticles as a platform for the controlled release of the bioactive agent curcumin. The innovative strategy to develop hybrid scaffolds was the modified porogenation method. The effect of the scaffold composition on the chemical, morphology, porosity, mechanical, hydrophilicity, swelling, degradation, biocompatibility, loading, and release features of hybrid scaffolds was evaluated. A porous structure with interconnected pores in the range of 52.33–65.76%, favorable swelling capacity, fully hydrophilic surfaces, degradability to 45% for 6 months, curcumin loading efficiency above 96%, and favorable controlled release profiles were obtained. By applying four kinetic models of release, valuable parameters were obtained for the curcumin/PHEMA/gelatin/alginate/TiO2 release platform. Cytotoxicity test results depend on the composition of the scaffolds and showed satisfactory cell growth with visible cell accumulation on the hybrid surfaces. The constructed hybrid scaffolds have suitable high-performance properties, suggesting potential for further in vivo and clinical studies
Trendovi u molekularnoj biologiji/Trends in Molecular Biology
No full textProšlo je 70 godina od otkrića sekundarne strukture molekula DNK. Od tog momenta molekularna biologija se razvija neverovatnom brzinom. „Trendovi u molekularnoj biologiji 3“ u svakom poglavlju pokazuju fascinantne domete koje dostiže molekularna biologija našeg vremena. I Nobelove nagrade se skoro svake godine dodeljuju za postignuća iz ove naučne discipline. A naši molekularni biolozi drže korak sa modernim trendovima. Jedan od autora ovog Zbornika govori : „Bez sumnje, ono što je otkriće elektrona bilo za 20. vek to su otkrića genomike za 21. vek.” Autori „Trendova u molekularnoj biologiji 3“ ni najmanje ne sumnjaju u to. Ove godine TMB3 prati i suplement, Knjiga apstrakata Drugog kongresa molekularnih biologa Srbije (CoMBoS2). Pod pokroviteljstvom Srpskog društva za molekularnu biologiju, Beograd je bio 2023. godine mesto susretanja molekularnih biologa Srbije, regiona i Evrope. Doprinos Kongresu, koji su obeležila inspirativna predavanja i inovativne naučne ideje, dali su svi molekularni biolozi Srbije. Formula uspešnosti i ovde je bila aktuelna: „Svi za jednog, jedan za svi!