Optimizing Dosage of the Maurten Bicarbonate System for Peak Performance
in Hyrox-Specific FTP Testing | Viktorie Johánková
Optimizing Dosage of the Maurten Bicarb System
Authors: Viktorie Johánková (1), Daniel Follprecht (1), Jakub Vavřička (1), Aleš Kroužecký (1), Pavel
Brož (1,2)
Supervisor: Pavel Brož (1,2)
(1) Department of Sports Medicine and Active Health Sciences, Faculty of Medicine in Pilsen,
Charles University (2) Department of Clinical Biochemistry and Haematology, Faculty of Medicine
in Pilsen, Charles University and University Hospital, Pilsen
State-of-the-Art: Sodium bicarbonate (NaHCO3) is a well-known ergogenic aid, but its effectiveness
is often limited by gastrointestinal side effects. Traditional soda is long-established but
reportedly fails to reach an effective ergogenic threshold in approximately half of all cases. The
Maurten Bicarb System is a newer hydrogel-based delivery system designed to minimize these
side effects. However, data on its absorption kinetics compared to traditional soda is still limited,
necessitating pilot testing to establish baseline dosing protocols for future performance
research.
Objective: The primary aim of this pilot study was to track individual venous blood bicarbonate
(HCO3−) changes at 1 and 2 hours post-ingestion to determine how many subjects reach the
ergogenic threshold (increase of ≥5 mmol/L) using both delivery methods. This pilot serves as a
foundation for a future study.
Material and Methods: Four male subjects (aged 26–53) participated in this crossover pilot.
In two separate sessions, they received a dose of 0.25 g/kg of NaHCO3 via either the Maurten
Bicarb System or traditional soda. Acid-base balance parameters were measured from venous
blood samples at baseline (T0), 1 hour (T1), and 2 hours (T2) post-ingestion.
Results & Discussion: In the pilot study, blood bicarbonate (HCO3−) levels were tracked across
eight observations (4 subjects, each testing both systems). The results demonstrated significant
inter-individual variability: under the Maurten Bicarb System, two subjects reached the ergogenic
threshold of +5 mmol/L by the 2-hour mark, while the other two peaked at +4.2 mmol/L.
Similarly, when using traditional sodium bicarbonate, two subjects successfully crossed the +5
mmol/L threshold, while the remaining two showed lower increases. These findings indicate
that a standard weight-based dose of 0.25 g/kg results in an effective ergogenic blood profile in
only 50% of cases within the initial two-hour window, regardless of the delivery method.
Conclusion: Both the Maurten Bicarb System and traditional soda showed a 50% success rate(2
out of 4 subjects) in reaching the ergogenic threshold within the first two hours. These results
highlight the high inter-individual variability in bicarb kinetics and underscore the necessity of
our planned research.
Funding: The work was supported by the grant SVV 260774 and Cooperatio Program, research
area IMMU.
Single-cell Transcriptomics Reveals a Pro-coagulant B-cell Phenotype and ERO1A-driven Metabolic Remodelling in Metastatic Lung Adenocarcinoma | Zia Ullah
B-cell Coagulation and ERO1A in LUAD
Authors: Zia Ullah (1,2), Pavel Ostasov (1,2), Monika Holubová (1,3)
Supervisor: Pavel Ostasov (1,2)
(1) Biomedical Center, Faculty of Medicine in Pilsen, Charles University (2) Department of Histology
and Embryology, Faculty of Medicine in Pilsen, Charles University (3) Department of Haematology
and Oncology, Faculty of Medicine in Pilsen, Charles University and University Hospital,
Pilsen
State-of-the-Art: Lung adenocarcinoma (LUAD) metastasis is a complex process driven by the
interplay between tumour cells and the immune microenvironment. While cancer-associated
hypercoagulability is a known facilitator of systemic spread, the specific role of the adaptive
immune system—particularly B cells—in modulating haemostatic pathways remains poorly
characterized. Most studies focus on platelet-driven coagulation, leaving a gap in our understanding
of how immune cells actively contribute to pro-thrombotic states within the tumour
role. Furthermore, metabolic adaptations, such as those regulated by ERO1A, are essential for
tumour cell survival during dissemination. Identifying these cell-specific signatures is essential
for developing targeted therapies to intercept the metastatic cascade.
Objective: The aim is to characterize the transcriptomic landscape of B cells and epithelial cells
in primary versus metastatic LUAD, with a focus on the expression of coagulation-related markers
(S100A1, PF4) and metabolic drivers (ERO1A, ADGRL2).
Material and Methods: We analysed single-cell RNA sequencing (scRNA-seq) datasets from
primary and metastatic LUAD. Data integration and batch effect removal were performed using
the Harmony package in R. To ensure statistical robustness and account for biological variation
between samples, we employed a pseudo bulk differential expression analysis. Raw counts
were aggregated by sample and cell type, and differentially expressed genes (DEGs) were identified
using a Quasi-Likelihood F-test via the edgeR package. Functional enrichment was conducted
using MSigDB Hallmark 2020 and Oncogenic Signatures to identify pathway-level shifts
associated with metastasis.
Results & Discussion: Pseudobulk analysis shows the Coagulation hallmark is enriched in B
cells from metastatic sites versus primary tumours. This is driven by S100A1 and PF4 upregulation.
PF4 expression in B cells suggests a specialized immune-haemostatic role, potentially
facilitating tumour-cell-platelet aggregates that shield metastatic cells. In the epithelial compartment,
ERO1A is upregulated DEG ({logFC} > 1.5), correlating with Hypoxia and Glycolysis
enrichment. This indicates metabolic reprogramming for metastatic adaptation. Conversely,
ADGRL2 expression decreased during progression. Together, these results provide evidence for
a coordinated shift where B cells adopt a pro-coagulant phenotype alongside epithelial metabolic
remodelling potentially promoting LUAD dissemination.
Conclusion: We identified a novel pro-coagulant B-cell signature (S100A1+/PF4+) and confirmed
ERO1A and ADGRL2 as key markers of metastatic remodelling in LUAD which had not previously
been discussed. These findings highlight the immune-coagulation interface as a potential therapeutic
target.
Funding: The study was supported by Charles University Research Project No SVV 260 651 and by the Ministry of Health of the Czech Republic in cooperation with the Czech Health Research
Council under project No. NW24-03-00079.
Diazepam treatment and stress reactivity in Lurcher mouse, a model of cerebellar motor and cognitive affective syndrome | Nilpawan Roy Choudhury
Treatment of stress reactivity in Lurcher mice
Authors: Nilpawan Roy Choudhury (1)
Supervisor: Jan Cendelín (1,2)
(1) Department of Pathological Physiology, Faculty of Medicine in Pilsen, Charles University
(2) Biomedical Center, Faculty of Medicine in Pilsen, Charles University
State-of-the-Art: The cerebellum is involved in non-motor functions, beyond motor coordination.
Lurcher mice are one of the best models for studying cerebellar ataxia and behavioral and
cognitive impairments resulting from selective olivocerebellar degeneration. Altered behavior
in these mice is hypothesized to be caused in part by stress-induced behavioral disinhibition.
Such behavioral traits potentially impair the performance not only in behavioral but also in cognitive
and motor tests and abnormal stress response might lead to a different stress-related
gene expression profile in Lurcher mice. We have hypothesized that pharmacological suppression
of anxiety may improve performance on cognitive and motor tests. However, low diazepam
doses (0.5 & 1.0 mg/kg) were not effective in Lurcher mice.
Objective: This study aimed to investigate whether a higher dose of Diazepam could influence
stress-related gene expression profile, reduce signs of behavioral disinhibition, and thereby improve
pathological behavioral phenotypes in Lurcher mice.
Material and Methods: Lurcher and Wild-type B6CBA mice aged 4-5 months were used. Mice
were treated with saline (control) or diazepam at a dose of 2 mg/kg, 30 mins prior to the tests.
The tests were arranged in a three-week protocol that included the open-field test, elevated plus
maze test, pre-pulse inhibition and startle response, grip strength measurement, Morris water
maze test with a hidden and visible goal task, and rotarod test. To evaluate gene expression,
brains were collected from Lurcher and Wild-type mice 60 minutes after exposure to a stressor,
represented by 5 minutes of the forced swimming test, or from undisturbed naïve controls. For
this experiment, the mice were also injected with diazepam (2.0 mg/kg) or saline.
Results & Discussion: Saline-treated Lurcher mice exhibited impaired behavior compared to
Wild-type mice in the open field, grip strength, Morris water maze with a visual platform, and
rotarod tests, and didn’t show any significant differences compared to Wild-type mice in the
elevated plus maze and pre-pulse inhibition test. The effect of diazepam was observed in a scattered
manner across all tests. Gene expression analysis showed that stress exposure down-regulated
Avpr1 expression in the hippocampus of Wild-type mice, but not in Lurcher mice. While
diazepam up-regulated Crhr1 and Avpr1 expression in the hippocampus of Wild-type mice, it
down-regulated these genes in Lurcher mice. Diazepam upregulated Crhr1 expression in the
amygdala of Wild-type mice and downregulated Gabra1 in the cortex of Lurcher mice.
Conclusion: The effect of diazepam was observed in a scattered manner with different tests.
The higher dose of Diazepam treatment also didn’t improve overall behavior in Lurcher mice.
Stressor response and diazepam treatment were predominantly observed in the hippocampus.
Funding: This work was supported by the GAUK project No. 49724, Cooperatio (NEUR and MED/
DIAG research areas), and SVV262774.
Preoperative inflammatory profiling and mitochondrial DNA copy number predict
postoperative complications in colorectal cancer | Natálie Danešová
Inflammatory and mitochondrial markers in CRC
Authors: Natálie Danešová (1,2), Ladislav Sojka (3), Veronika Makajevová (3), Jaromír Šimša (3), Soňa
Vodenková (2), Veronika Vymetálková (1,2,4), Klára Vokáčová (2,4)
Supervisor: Soňa Vodenková (2)
(1) Biomedical Center, Faculty of Medicine in Pilsen, Charles University (2) Department of Molecular
Biology of Cancer, Institute of Experimental Medicine of the Czech Academy of Sciences,
Prague, Czech Republic (3) Department of Surgery, First Faculty of Medicine, Charles University
and Thomayer Hospital, Prague, Czech Republic (4) Institute of Biology and Medical Genetics,
First Faculty of Medicine, Charles University, Prague, Czech Republic
State-of-the-Art: Postoperative complications (POCs) after colorectal cancer (CRC) surgery
remain a major clinical problem, as they may negatively affect recovery and overall patient outcomes.
However, reliable preoperative biomarkers for identifying patients at higher risk of POCs
are still lacking. Surgical trauma triggers a systemic inflammatory response and may also disrupt
mitochondrial homeostasis. Therefore, inflammatory cytokines such as IL-6, IL-10, IL-1β, TGF- β1,
and TNF-α, together with mitochondrial DNA copy number (mtDNA-CN), represent promising
candidate biomarkers of systemic inflammation and mitochondrial stress. Early identification
of patients at increased risk could improve perioperative risk stratification, postoperative monitoring,
and individualized care.
Objective: The aim of this study was to evaluate perioperative changes in selected cytokines
and mtDNA-CN in patients undergoing CRC surgery and to assess their potential, individually
and in combination, for predicting POCs.
Material and Methods: This prospective study enrolled 75 patients with newly diagnosed CRC
undergoing surgical resection. Based on predefined inclusion and exclusion criteria, relative
mtDNA-CN was measured in whole blood collected preoperatively and ten days after surgery
in a subset of 51 patients using multiplex qPCR. In a clinically homogeneous subcohort of 20 patients,
plasma levels of IL-6, IL-10, TNF-α, IL-1β, and TGF-β1 were determined by ELISA. Statistical
analyses included paired comparisons between time points and receiver operating characteristic
(ROC) analyses to evaluate the predictive performance of individual biomarkers and their
combinations for POCs. To account for multiple testing, p-values were adjusted using the false
discovery rate (FDR) method and are reported as q-values.
Results & Discussion: Relative mtDNA-CN decreased significantly between the preoperative
and postoperative time points (p = 0.004). Preoperatively, obese patients had higher mtDNA-CN
and were also at greater risk of developing POCs (p = 0.006 and p = 0.009, respectively). After
surgery, plasma levels of IL-6, IL-10, and TNF-α increased significantly, indicating a sustained
postoperative inflammatory response (q = 0.022, q = 0.034, and q = 0.003, respectively). ROC
analyses further showed that combined biomarker models integrating cytokines and mtDNA-
CN had good discriminatory ability for identifying patients who developed POCs (AUC up to
0.91). Together, these findings support a link between mitochondrial alterations, postoperative
inflammation, and the risk of POCs in CRC.
Conclusion: Combined assessment of inflammatory cytokines and mtDNA-CN may improve
the identification of CRC patients at higher risk of POCs. However, further validation in larger
cohorts is needed.
Funding: Supported by the Ministry of Health of the Czech Republic in cooperation with the Czech Health Research Council (project No. NW24-03-00062), by the Charles University, project
GA UK No. 540225, and by the Cooperatio Program, research area „Oncology and Haematology”.
Chromatographic separation of bioactive molecules from edible mushroom species and their evaluation for the anti-inflammatory potential using nitric oxide production in LPS-stimulated macrophages | Eliška Fousková
Identification of mushroom bioactive compounds
Authors: Eliška Fousková (1,2)
Supervisor: Shashank Pandey (1)
(1) Department of Pharmacology and Toxicology Faculty of Medicine in Pilsen, Charles University
(2) Laboratoire BioCIS, CY Cergy Paris Université, Paris, France
State-of-the-Art: In recent decades, bioactive molecules from natural sources have attracted
increasing attention due to their health-promoting properties as alternatives to synthetic drugs.
Various biological activities, including anti-inflammatory have been reported. Mushrooms represent
a rich source of biologically active compounds such as peptides and polysaccharides
with immunomodulatory potential. To isolate these molecules, chromatographic techniques
are commonly used to separate complex mixtures into further fractions that can be screened
for the pharmacological activity. To complement necessary information for further biomedical
research, tested molecules are commonly analyzed by nuclear magnetic resonance (NMR) spectroscopy
to characterize functional groups within the molecules.
Objective: The aim of this study was to optimize chromatographic separation of complex mixtures
from edible mushroom species and evaluate fractions for their ability to modulate nitric
oxide production in LPS-stimulated RAW 264.7 cells. The most promising fractions were further
analyzed by NMR spectroscopy.
Material and Methods: Extracts from three edible mushroom species were prepared using
Milli-Q water. Ultracentrifugation was used to separate molecules by their molecular weight.
Two chromatographic methods, solid-phase extraction (SPE) and high-performance liquid chromatography
(HPLC), were then applied independently to fractionate the extracts. Murine RAW
264.7 macrophages were cultured as an in vitro inflammation model. Cells were stimulated with
lipopolysaccharide (LPS) to induce nitric oxide (NO) production. Fractions from both methods
were applied to LPS-stimulated cells, and nitrite levels in the supernatant were measured using
the Griess reaction. This NO assay enabled comparison of fractionation efficiency and bioassay
compatibility. Selected fractions were further analyzed by NMR spectroscopy.
Results & Discussion: RAW 264.7 macrophages were treated with fractions obtained from
both SPE and HPLC methods. Results were compared with positive and negative controls. HPLC
showed to be a non-invasive method compatible with the NO assay. Fractions showed different
NO inhibition levels. Some fractions reduced nitric oxide production in activated macrophages
up to 32 %, suggesting the presence of compounds that modulate inflammatory processes.
Therefore, HPLC fractions were further subjected to NMR spectroscopy to identify the functional
groups responsible for the observed activity.
Conclusion: Immunomodulatory differences among fractions suggest certain molecules reduce
NO levels. Findings support mushroom-derived bioactive molecules as anti-inflammatory
agents. Future work will focus on screening additional pharmacological effects of promising
fractions.
Methylation Analysis of Sialdenoma Papilliferum of the Salivary Glands, and Syringocystadenoma Papilliferum and Tubular adenoma of the Skin: Are They Similar Entities? | Kristýna Behenská
SP, SCAP, TA: are they similar entities?
Authors: Kristýna Behenská (1,2), Petr Martínek (2), Marián Grendár (2), Martin Hyrcza (3), Lester
D.R.Thompson (4), Tomáš Vaněček (2), Carlota Rossi (5), Luigi Corcione (6), Petr Slavík (1,2), Ljubov
Kastnerová (1,2), Michal Michal (1,2), Alena Skálová (1,2), Denisa Kacerovská (1,2)
Supervisor: Martina Bradová 1,2
(1) Department of Pathology, Faculty of Medicine in Pilsen, Charles University, Czech Republic
(2) Bioptical Laboratory, Ltd., Pilsen, Czech Republic (3) Department of Pathology and Laboratory
Medicine, University of Calgary, Canada (4) Head and Neck Pathology Consultations, Woodland
Hills, CA, USA (5) Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Milan, Italy
(6) University of Parma, Parma, Italy
State-of-the-Art: Sialadenoma papilliferum (SP) of the salivary gland, syringocystadenoma
papilliferum (SCAP), and tubular adenoma (TA) of the skin are distinguished as separate entities.
The pathogenesis of SP and SCAP, however, partially overlaps: the BRAF p.V600E mutation
is consistently reported in SP and in a subset of SCAP. TAs carry mutations in the MAPK signalling
pathway, affecting BRAF, KRAS, and HRAS genes. Despite their different locations, the morphology
of these three lesions is comparable. They are characterised by exo-/endophytic squamous
proliferation and inverted papillary/tubular/ductal proliferations, with a predominance of tubular
growth in TA.
Objective: To investigate potential underlying biological similarities among sialadenoma papilliferum,
syringocystadenoma papilliferum, and tubular adenoma of the skin by comparing their
DNA methylation profiles and BRAF expression, using selected adnexal and follicular tumors as
controls.
Material and Methods: We reviewed the authors’ archives and selected three groups of tumors
for study: cohort 1, comprising 14 cases of SP (including 4 recurrences); cohort 2, comprising
17 cases of SCAP; and cohort 3, comprising 15 cases of TA. All groups underwent classification
analysis based on methylation profiles and were investigated for BRAF immunohistochemistry
(IHC) using the VE1 antibody clone (ready-to-use, Ventana). The control cohort consisted of cylindromas
(n=23), basal cell carcinomas of the skin (n=32) and trichoblastomas (n=22).
Results & Discussion: The methylation analysis revealed partially overlapping clustering
among the predefined labels SCAP and TA categories, while SP was separated. Control cohorts
were distinctly separated from each other and from the test groups. All tumor types showed
BRAF IHC expression mostly in moderate to strong intensity.
Methylation profiles of SCAP and TA were not unambiguously separated indicating similarity of
underlying biology, while the SP seem to be more distinguishable. The three tested entities are
characterized by similar morphology with variable phenotype of subepithelial portion of the
tumor with predominance of papillary architecture in SCAP, tubular architecture in TA and both
in SP, however this was not recognizable in methylation analysis.
Conclusion: SCAP and TA showed overlapping methylation profiles, suggesting shared underlying
biology, whereas SP was more distinct. Despite characteristic papillary, tubular, or mixed architecture
on morphology, these structural differences were not reflected in methylation-based
classification.
Funding: This study was in part supported by study grant SVV 260773 from the Ministry of Education, Czech Republic, the Cooperatio Program, research area SURG, and the project National
Institute for Cancer Research – NICR (Programme EXCELES, ID Project No. LX22NPO5102)
Funded by the European Union – Next Generation EU
From Hamartoma to Carcinoma: Molecular Genetic Landscape of Seromucinous and
Respiratory Epithelial Adenomatoid Hamartomas as Precursor Lesions | Petr Slavík
Adenocarcinomas ex REAH/SH
Authors: Petr Slavík (1,2), Abbas Agaimy (3), Valerie Costes Martineau (4), Martin Hyrcza (5), Boris
Rychlý (6), Jan Laco (7), Niels J. Rupp (8), Tomáš Vaněček (2), Petr Martínek (2), Petr Grossmann (2),
Emilija Todorovic (9), Esther I. Hauben (10), Gisele de Rezende (11), Ilmo V. Leivo (12), Fredrik C. Andersson
(13),Kristýna Behenská (1,2), Alena Skálová (1,2), Michal Michal (1,2)
Supervisor: Martina Bradová (1, 2)
(1) Department of Pathology, Faculty of Medicine in Pilsen, Charles University, Czech Republic
(2) Bioptical Laboratory, Ltd., Pilsen, Czech Republic (3) Institute of Pathology, Universitätsklinikum
Erlangen, Germany (4) Pathology Department, CHU Montpellier, France (5) Charbonneau
Cancer Institute, University of Calgary, Canada (6) Department of Pathology, Unilabs Slovensko,
Bratislava, Slovakia (7) The Fingerland Department of Pathology, Charles University, Hradec
Králové, Czech Republic (8) Department of Pathology and Molecular Pathology, University
Hospital Zurich, Switzerland (9) Department of Pathology and Laboratory Medicine, University
of Calgary, Canada (10) Department of Pathology, University Hospitals Leuven, Belgium
(11) Niguarda Cancer Center, Milan, Italy (12) University of Turku and Turku University Hospital,
Finland (13) Skåne University Hospital, Lund, Sweden
State-of-the-Art: Since the first identification of seromucinous hamartomas (SH) and respiratory
epithelial adenomatoid hamartomas (REAH), there have been speculations about whether
these entities truly represent hamartomas or if they are true preneoplastic lesions. The subsequent
identification of an entity named atypical sinonasal glands arising in seromucinous
hamartoma (ASGSH) led to multiple studies to assess their morphological, immunohistochemical
and molecular genetic profile.
Objective: The aim of the study was to determine the correlation of ASGSH to several types of
malignant sinonasal tumors and to further warrant the importance of the detection of ASGSH
component in these tumors.
Material and Methods: Out of the authors’ archives 69 cases of varying lesions were selected
and subsequently subjected to histomorphological, immunohistochemical and molecular genetic
analysis (Next Generation Sequencing RNA and DNA panel). The histomorphological evaluation
focused mainly on the presence of SH, REAH and the true precancerous lesion ASGSH. The
antibodies tested in the immunohistochemical analysis were among others SOX10, S100 protein,
p63, p40, lysozyme and CK7. The correlation of all research methods was done subsequently.
Results & Discussion: A subset of tumors was found to show an apparent association with SH,
REAH or ASGSH, suggesting a possible histogenetic relationship and raising the hypothesis that
ASGSH may represent a precursor lesion in some cases. These findings indicate that certain
tumors may arise in this setting although such a pathway is unlikely to be universal. In instances
where ASGSH is not identified within the tumor mass, it may have been overgrown by the neoplasm
or may persist only focally within the lesion. Careful histologic evaluation is therefore
important to detect any preneoplastic component, even when present only in a limited area.
Molecular findings indicate a heterogeneous genetic background that at present shows no consistent
association with specific histologic or immunohistochemical features.
Conclusion: ASGSH may be a precursor to some sinonasal malignancies. Detection of ASGSH at
tumor periphery indicates potential developmental relationship which highlights the diagnostic value of morphologic evaluation in head and neck tumors. When ASGSH can’t be found, immunohistochemistry
may prove useful.
Funding: This study was supported by study grant SVV 260652 from the Ministry of Education.
Spatial profiling and prognostic value of CD66+ neutrophils in colorectal cancer patients with synchronous and metachronous liver metastases | Wenjing Ye
Spatial & prognostic analysis of CD66+ neutrophils
Authors: Wenjing Ye (1), Sergii Pavlov (1), Filip Ambrozkiewicz (1), Vaclav Liska (1,2), Kari Hemminki
(1,3), Andriy Trailin (1)
Supervisor: Kari Heimminki (1,3)
(1) Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Czech Republic
(2) Department of Surgery, Faculty of Medicine in Pilsen, Charles University and University Hospital,
Pilsen (3) Department of Cancer Epidemiology, German Cancer Research Center, Heidelberg,
Germany.
State-of-the-Art: Colorectal cancer (CRC) is among the most common and lethal cancers globally.
The development of distant metastases—either synchronous or metachronous—significantly
worsens prognosis, with each type displaying distinct biological and clinicopathological
features. Neutrophils have been shown to play dual roles in CRC progression, acting as either
pro- or anti-tumor effectors.
Objective: This study investigated the spatial distribution and prognostic significance of neutrophils
within primary tumor (pCRC) and paired liver metastases (LM).
Material and Methods: Formalin-fixed paraffin-embedded (FFPE) tissue sections from pCRC
and paired synchronous (N=55) or metachronous (N=44) LM were analyzed. CD66+ neutrophils
were detected via immunoperoxidase staining. Whole-slide images were analyzed using QuPath
software to quantify cell densities across four spatial regions: tumor center (TC), inner margin
(IM), outer margin (OM) and peritumor (PT) region of pCRC and LM. Cell densities were stratified
by the 25th percentile into low and high groups. Prognostic associations with disease-free survival
(DFS) after resection of LM in both groups and time to occurrence (TTO) of metachronous
LM were assessed using Cox regression.
Results & Discussion: In LM, higher cell densities of CD66+ neutrophils were observed in IM
and OM compared to TC and PT in both synchronous and metachronous groups. In pCRC, no
regional differences were found in the synchronous group, whereas in the metachronous group,
higher densities were found in TC, IM and OM compared to PT. Compared with pCRC, higher
CD66+ cell densities were observed in the OM and PT of LM in both groups. Additionally, a higher
cell density in TC of metachronous LM than that of synchronous LM was observed (Fig A).
A high IM/OM ratio in pCRC was associated with shorter TTO (Fig B). High neutrophil densities in
OM and PT of synchronous LM and OM of metachronous pCRC predicted shorter DFS (Fig C,D,E),
whereas high densities in TC of metachronous LM correlated with longer DFS (Fig F).
Conclusion: Neutrophils exhibited protumor prognostic associations in metachronous pCRC
and synchronous LM, and anti-tumor associations in metachronous LM. These findings suggest
that CD66 may serve as a potential biomarker for predicting metastatic progression and clinical
outcomes in CRC.
Funding: AZV NW24-03-00521
CRISPR/Cas9-mediated editing of the pathogenic CAG repeat in the ATXN1 gene
for spinocerebellar ataxia type 1. | Parvathi Satheesh
CRISPR Editing of ATXN1 CAG Repeat in SCA1
Authors: Parvathi Satheesh (1), Jan Tůma (1)
Supervisor: Jan Tůma (1)
(1) Department of Pathological Physiology, Faculty of Medicine in Pilsen, Charles University
State-of-the-Art: Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative
disorder characterized by cerebellar atrophy, progressive motor impairment, dysarthria,
and cognitive decline. The disease is caused by an expanded CAG trinucleotide repeat in the
ATXN1 gene, which produces a polyglutamine-expanded ataxin-1 protein that accumulates in
neuronal cells and leads to cellular stress and neurodegeneration. Despite the well-defined
genetic cause, there is currently no disease-modifying therapy for SCA1. CRISPR/Cas9 genome
editing has emerged as a promising strategy for targeting pathogenic repeat expansions and
correcting underlying genetic mutations. Precise targeting of the expanded CAG tract in ATXN1
could therefore represent a potential therapeutic approach for SCA1.
Objective: To develop and validate a CRISPR/Cas9 genome-editing strategy targeting the expanded
CAG repeat in the ATXN1 gene and evaluate its ability to generate precise DNA breaks
and enable removal of the pathogenic repeat in cellular models of SCA1.
Material and Methods: In vitro cellular models were established from SCA1^154Q/2Q transgenic
mice and wild-type controls, such as primary fibroblasts, embryonic neural stem cells (eNSCs),
and eNSC-derived neurons. Candidate single-guide RNAs (sgRNAs) targeting sequences
flanking the expanded CAG tract within the ATXN1 gene were designed and evaluated in silico
for specificity. Four sgRNAs were selected for in vitro validation. CRISPR/Cas9 components were
delivered into SCA1 fibroblasts and eNSCs as ribonucleoprotein (RNP) complexes and plasmids.
The CRISPR machineries were delivered into the cells by chemical transfection and electroporation
protocols, followed by genomic DNA isolation and analysis by cleavage assay to assess
editing activity and detect CRISPR-induced double-strand breaks at the target locus.
Results & Discussion: In vitro validation in SCA1 fibroblasts showed that two sgRNAs could
induce some double-strand breaks (DSBs) at the target locus, as confirmed by cleavage assay.
These results provide initial proof-of-concept that the expanded CAG tract can be targeted using
CRISPR/Cas9 tools. The two most effective sgRNAs are now being evaluated in combination to
excise the expanded repeat segment in SCA1 mouse eNSCs and differentiated neuronal cells to
assess editing efficiency and potential rescue of disease-associated cellular phenotypes. We are
also optimising the electroporation protocol for eNSC cells for better transformation efficiency.
The image attached shows eNSCs with primary cilia (green basal body, yellow actin filament)
and mitotic cells with actin filaments separating pink chromatids.
Conclusion: The site-specific DSB induction by CRISPR potentially enables excision of the CAG
repeat. This is a promising step toward developing therapeutic strategies for SCA1. These studies
will also help evaluate the feasibility and safety of repeat-targeting CRISPR strategies for
polyglutamine disorders.
Funding: This work was supported by the GAUK project No. 70124, AZV grant No. NW26-08-
00119, Cooperatio (NEUR and MED/DIAG research areas), and SVV262774.