imagine (Institute of molecular genetics and genetic engineering)
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Unveiling the Roles of Cysteine Proteinases F and W: From Structure to Pathological Implications and Therapeutic Targets
Get PDFCysteine cathepsins F and W are members of the papain-like cysteine protease family, which have distinct structural features and functional roles in various physiological and pathological processes. This review provides a comprehensive overview of the current understanding of the structure, biological functions, and pathological implications of cathepsins F and W. Beginning with an introduction to these proteases, we delve into their structural characteristics and elucidate their unique features that dictate their enzymatic activities and substrate specificity. We also explore the intricate involvement of cathepsins F and W in malignancies, highlighting their role as potential biomarkers and therapeutic targets in cancer progression. Furthermore, we discuss the emerging roles of these enzymes in immune response modulation and neurological disorders, shedding light on their implications in autoimmune and neurodegenerative diseases. Finally, we review the landscape of inhibitors targeting these proteases, highlighting their therapeutic potential and challenges in clinical translation. This review brings together the diverse facets of cysteine cathepsins F and W, providing insights into their roles in health and disease and guiding future investigations for therapeutic advances
LACTONASE MEDIATED QUORUM QUENCHING OF PSEUDOMONAS AERUGINOSA VIRULENCE
Get PDFSolving the problem of the antimicrobial resistance crisis is one
of the primary challenges currently confronting the healthcare
system. One of the most promising new strategies to combat
antimicrobial resistance is the antivirulence therapy, based on
silencing bacterial cell-to-cell communication (quorum quenching -
QQ). QQ enzymes lactonases represent a diverse group of enzymes
capable of inactivating signaling molecules of bacterial
communication – N-acyl homoserine lactones (AHLs), resulting in
alterations ofbacterial virulence. The numerous virulence factors and
resistance to most conventional antibiotics have led to Pseudomonas
aeruginosa being listed as one of the top-priority pathogens on the
ESCAPE pathogen list, highlighting the urgent need for the development of new therapies to combat this pathogen. P.
aeruginosauses cell-to-cell communication known as quorum sensing
(QS) that allows bacteria to monitor their own population density via
signal molecules and subsequently control bacterial pathogenesis.
Our hypothesis was that bacterial pathogens which share the same
ecological niche with P. aeruginosa during infection have developed a
system to disrupt its QS system, in order to survive in polymicrobial
communities alongside this successful pathogen. In our research we
identified QQ enzymes lactonases originating from two Gramnegative
bacterial pathogens Burkholderiacepacia and Stenotrophomonas
maltophilia. The genes encoding for the enzymes were cloned and
expressed in pQE30 expression vector. B. cepacia BCC4135 synthesizes
two lactonases YtnP and Y2-aiiA, that have the different cellular
localization, but also different substrate specificity, which could imply
the difference in their biological roles. S. maltophilia 6960 YtnP
lactonase has several advantageous biotechnological properties, such
as high thermostability, activity in a wide pH range, and no cytotoxic microscopy analysis showed a strong effect of analyzed
lactonases on preventing biofilm formationand initiating the
decomposition of the preformed biofilm of P. aeruginosa MMA83.
Functional assays showed that lactonases have the ability to
significantly reduce extracellular virulence factors production –
elastase, pyocyanin and rhamnolipid. Additionally, the results
obtained by real-time quantitative PCR showed that analyzed
recombinant enzymes significantly downregulated all three analyzed
P. aeruginosa QS networks at the transcriptional level. Finally, S.
maltophilia 6960 YtnP lactonase significantly prolonged survival of
Caenorhabditis elegans by reducing virulence of P. aeruginosa using fastkilling
liquid assay.
The described properties make B. cepacia and S. maltophilia lactonases
the promising therapeutic candidates for the development of nextgeneration
antivirulence agents.Book of abstracts and conference proceedings / 3rd International
Conference Antimicrobial Resistance - Current State and Perspectives, 16-18.
May 2024, Novi Sad, Serbia
Enzyme stabilization for biodegradation of organophosphate plastic additives and pesticides (orgOPhix)
Get PDFOrganophosphates (OPs) enjoy wide application in numerous industrial sectors, including plastic
production, agriculture, and the arms industry. These chemicals serve as plastic additives to improve
product properties, as pesticides to protect crops from pests and diseases, and as chemical weapons in
form of nerve agents. The widespread use of OPs has led to the near-ubiquitous accumulation of OPs in
soils and waters across the globe, with profound health and environmental repercussions. Acute exposure
to anthropogenic OPs causes toxicity in insects, plants, animals, and humans, while chronic exposure has
been linked to neurotoxic effects, developmental abnormalities, and increased risk of certain cancers. The
widespread pollution and high toxicity of OPs require development of efficient and eco-friendly
decontamination methods. Currently, only enzymatic degradation of OPs meets these criteria. However,
just a few enzymes involved in OPs degradation have been discovered. Among them are two novel
enzymes that we identified, which independently and efficiently degrade a wide range of OP plastic
additives, pesticides, and nerve agents1. Nonetheless, their soluble expression and storage stability was
highly problematic, limiting cost-effective production and broad OP decontamination potential.
Therefore, we plan to improve their soluble expression and environmental stability using protein
engineering tools such as ancestral sequence reconstruction and stability optimization algorithms.
Stabilized enzymes will have а wide range of potential applications, from water and soil remediation to
prophylactic protection against OP poisoning. Finally, we will develop bacteria carrying novel
phosphotriesterases as part of the OPs metabolic pathway. The bacteria will be used for OPs
bioremediation converting these toxic compounds into phosphate, a molecule essential for life.Principal Investigator: Dr Dragana Despotović, IMGGEDuration period: 2024-202
Suppressors of Blm-deficiency identify three novel proteins that facilitate DNA repair in Ustilago maydis
No full textTo identify new molecular components of the Brh2-governed homologous recombination (HR)-network in the highly radiation-resistant fungus Ustilago maydis, we undertook a genetic screen for suppressors of blm-KR hydroxyurea (HU)-sensitivity. Twenty DNA-damage sensitive mutants were obtained, three of which showing slow-growth phenotypes. Focusing on the “normally” growing candidates we identified five mutations, two in previously well-defined genes (Rec2 and Rad51) and the remaining three in completely uncharacterized genes (named Rec3, Bls9 and Zdr1). A common feature among these novel factors is their prominent role in DNA repair. Rec3 contains the P-loop NTPase domain which is most similar to that found in U. maydis Rec2 protein, and like Rec2, Rec3 plays critical roles in induced allelic recombination, is crucial for completion of meiosis, and with regard to DNA repair Δrec3 and Δrec2 are epistatic to one another. Importantly, overexpression of Brh2 in Δrec3 can effectively restore DNA-damage resistance, indicating a close functional connection between Brh2 and Rec3. The Bls9 does not seem to have any convincing domains that would give a clue as to its function. Nevertheless, we present evidence that, besides being involved in DNA-repair, Bls9 is also necessary for HR between chromosome homologs. Moreover, Δbls9 showed epistasis with Δbrh2 with respect to killing by DNA-damaging agents. Both, Rec3 and Bls9, play an important role in protecting the genome from mutations. Zdr1 is Cys2-His2 zinc finger (C2H2-ZF) protein, whose loss does not cause a detectable change in HR. Also, the functions of both Bls9 and Zdr1 genes are dispensable in meiosis and sporulation. However, Zdr1 appears to have overlapping activities with Blm and Mus81 in protecting the organism from methyl methanesulfonate- and diepoxybutane-induced DNA-damage. Finally, while deletion of Rec3 and Zdr1 can suppress HU-sensitivity of blm-KR, Δgen1, and Δmus81 mutants, interestingly loss of Bls9 does not rescue HU-sensitivity of Δgen1
Designing a functional nanoemulsion using lactose esters and modified monomers of bacterial polyhydroxyalkanoates to improve the anticancer activity of SN-38
Get PDFColon cancer is one of the most frequently diagnosed and deadliest cancers worldwide
hence the number of drug discoveries for the disease is still growing [1]. To improve the
solubility and in vivo stability of promising chemotherapeutics numerous delivery systems
are being designed and tested. Our study involved chemo-enzymatic methods to perform a
novel emulsion to provide sufficient solubility and delivery for one of the most promising
anticancer agents: SN-38 [2][3]. The oil phase consisted of selectively fluorinated monomers
from bacterial poly-(R)-3-hydroxynononanoate-co-heptanoate (PHN) which are fully
described in our previous studies (involving
1H NMR,
19F NMR, IR, UHPLC-MS/MS analysis).
A mixture of PHN-monomer-based lactose esters (also previously described) and DMSO
served as an emulsion stabiliser and water was the hydrophilic solvent. Dynamic Light
Scattering analysis indicated the average size of micelles was 500–1000 nm and their
stability was maintained for seven days. MTT assay showed that the novel emulsion
significantly improved the activity of the investigated chemotherapeutic by decreasing the
viability (IC50 and IC80
) of the selected colon cancer cells (and concentrations were: 0.125
and 0.25 mg ml
-1
). An experiment on zebrafish embryos (Danio rerio) showed relatively low
toxicity of the carrier alone (embryos were treated with >0.4 mg ml
-1
for 5 days). Experiments
on mice with colon cancer explants (HCT116) indicated a drop in tumour growth inhibition
factor (TGI) while treating them with SN-38 in the novel emulsion system compared to those
treated only with SN-38 in DMSO. The results allowed for patent claim nr P.447923.Book of abstracts: 6th Symposium on Biotransformations for Pharmaceutical and Cosmetic Industry June 17-21, 2024, Kraków, Polan
European Rare Diseases Research Alliance (ERDERA)
No full textThe European Rare Diseases Research Alliance (ERDERA) aims to improve the health and well-
being of the 30 million people living with a rare disease in Europe, by making Europe a world
leader in Rare Disease (RD) research and innovation, to support concrete health benefits to rare
disease patients, through better prevention, diagnosis and treatment. This Partnership will
deliver a RD ecosystem that builds on the successes of previous programmes by supporting
robust patient need-led research, developing new diagnostic methods and pathways,
spearheading the digital transformational change connecting the dots between care, patient
data and research, while ensuring strong alignment of strategies in RD research across
countries and regions. Structuring goal-oriented public-private collaborations targeted at
interventions all along the R&D value chain will ensure that the journey from knowledge to
patient impact is expedited, thereby optimising EU innovation potential in RD. To support its
ambition and missions ERDERA has been designed as a comprehensive and integrated
ecosystem of which structure can be compared to an institute encompassing three main parts:
(i) funding, (ii) internal (in house) Clinical Research Network that implements research activities
targeting clinical trial readiness of RDs and accelerating diagnosis and translation of research
discovery into improved patient care, and (iii) related supporting services (Data, Expertise,
Education and Training) as well as an acceleration hub that serve external and internal RD
community, all supported by all-embracing coordination and strategy and foundational
(inter)national alignment.Principal Investigator: Daria Julkowska, INSERM, FranceParticipants from IMGGE: dr Maja Stojiljković, dr Milena UgrinDuration period: 2024 - 203
Cell Types of Adult Mouse Brain: Definition and Experimental Access
No full textThe human brain is an incredibly complex organ, composed of over 150 billion cells that
work together to create consciousness and ultimately, define who we are. Abnormal
function or death of specialized cell types cause various brain diseases.
Understanding how brain structure produces its function is the key goal of neuroscience.
To define brain structure, we need to identify the types of building blocks (cell types)
and their relationships. Then we need to eliminate or inactivate them and observe the
consequences (e.g., inability to perform an action, like movement). Ethical barriers prevent
us from using this approach in humans.
Mus musculus, the house mouse, is a dominant model for studying mammalian brains.
Despite its small size, the mouse performs diverse behaviors common across mammals,
including sophisticated movements and learning.
The Allen Institute for Brain Science stands at the forefront of defining cell type identity
and function in the mammalian brain. Starting with single-cell transcriptomics followed by
measurements of other cellular properties including morphology and electrophysiology,
we created an extensive repository of brain single-cell data. We employed various
bioinformatics approaches to analyze these multidimensional and multimodal data to
define cell identity and cell types. We showed that the mouse brain contains at least 5000
cell types of which many exist in humans.
To assess cell type function, we used our single-cell measurements of chromatin
accessibility to define putative enhancer elements in the mouse and human genome.
When included in innocuous viruses, these enhancers can instruct expression of various
molecular tools in specific cell types to probe their function in the brain.
Starting with mice and with an eye towards humans, the Allen Institute is building genetic
tools for all cell types in mammalian brains. Coupled with advanced computational tools,
our ability to understand the roles of all brain cell types in health and disease and modify
their function toward cures is within reach.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024
20 years of work on exosomal DNA fragments
No full textIn the year 1948, French authors Mandel and Metais first described the presence of
cell-free DNA molecules (cfDNA) in the blood of mammals and also made a link of the
quantities of this DNA fraction to various diseases. Until the advent of high-throughput
DNA sequencing, this finding was mostly ignored, but since then, work on cfDNA has
increased.
The Sensen laboratory has performed several studies on the DNA molecules present
in plasma or serum, respectively, mostly using data obtained from paired-end highthroughput
Illumina sequencing experiments, followed by Bioinformatics analysis and
ultimately qPCR. This includes studies on Chronic Wasting disease in Canadian Wapitis,
work on Mad Cow Disease, radiation experiments with non-lethal doses in rats as well as
work on human disease conditions.
The work of more than 20 years in Canada, Austria and Hungary will be reviewed, which
has ultimately led to the development of a qPCR test, which can be used to detect the
onset of human sepsis up to two days before the first clinical signs. A special focus of this
talk will be on the close interconnection between the medical and laboratory work and
the Bioinformatics analyses, which are required for the development of a new molecular
diagnostic assay, which can be performed with standard equipment already available in
the clinics using non-invasive methods.
The presentation will end with an outlook on the work that is currently ongoing at the
Hungarian Center for Molecular Medicine in Szeged, which is focused on the stratification
of COVID-19 patients.Book of abstracts: 5th Belgrade Bioinformatics Conference, Serbia, Belgrade,17-20 june 2024
The Effects of Electrolytic Technology Toothbrush Application on the Clinical Parameters and Bacteria Associated with Periodontal Disease in Dogs
No full textThe aim of this study was to compare the effects of electrolytic and nonelectrolytic toothbrushing on dogs’ oral health and the presence of common bacteria associated with periodontal disease. Periodontal disease in dogs is a common problem worldwide. A toothbrushing procedure is recommended to prevent periodontal disease, with additional benefits if electrolytic toothbrushes are used in dog oral hygiene practices. A total of 26 dogs were enrolled in this eight-week study and were divided into two groups—treatment and control. Daily toothbrushing was performed on all dogs using the same dog toothbrush, with the power source disengaged in the control group. Oral examination was conducted on anesthetized dogs before and at 4 and 8 weeks after commencing the study, with sampling for bacterial analysis. This study was designed to be blind for owners, veterinarians, and laboratory staff. Improvements in the average gingival index (from 0.55 to 0.31) and calculus index (from 0.55 to 0.38) in the treatment group were recorded. In the control group, after an initial improvement in the plaque index (from 0.97 to 0.53), at week 8, it significantly rose to 1.21 (p < 0.05). Relative bacterial abundance revealed a reduction in all four tested bacteria in the treatment group, while in the control group, Campylobacter rectus levels rose by 3.67 log2 compared to before the study and at week 8. No adverse effects were recorded in either group
ISCHEMIA AFFECTS THE TERMINAL DIFFERENTIATION OF HUMAN PLURIPOTENT STEM CELLS AND NEURONAL PROGENITORS
No full textHypoxia/ischemia underpins a broad range of brain pathologies, including stroke and shortterm
cardiac arrest that lead to the induction of neural stem cell proliferation and the migration
of young neurons to injured areas. However, these processes are insufficient to fully restore
neuronal function. This study aimed to investigate the effect of hypoxia-ischemia on the
neurogenic potential of human pluripotent stem cells and the terminal differentiation of
neuronal progenitors.
NT2/D1 cell line was used as an in vitro model system of human neurogenesis. Ischemic stress
was achieved by exposure to glucose deprivation and/or cobalt chloride. To analyze the mRNA
expression level of target genes, quantitative RT-PCR was performed. Protein expression was
determined by Western blot and immunocytochemistry.
The analysis revealed that ischemia, induced in pluripotent cells or neural progenitors, led to a
significant decrease in SOX gene expression in these cells and a notably reduced number of
terminally differentiated neurons. In contrast, there was an increase in the expression level of
miR-21. These insights contribute to the understanding of SOX transcription factors and miR-
21, as well as their potential interplay in diseases related to ischemia. This positions them as
promising candidates for biomarkers and targets in the development of new diagnostic and
treatment strategies.VII Congress of the Serbian Genetic Society Zlatibor; October 2 to 5, 2024