Marcin Jąkalski

Cold ischemia time (CIT), the interval between tissue excision and preservation, is a critical preanalytical variable that profoundly impacts gene expression profiles. Variability in CIT can lead to inconsistent transcriptomic results, making study interpretation challenging and undermining reproducibility in biomedical research. Our study aimed to evaluate the impact of CIT on the expression of cancer-related genes, particularly these involved in hypoxia, apoptosis, and epithelial-to-mesenchymal transition (EMT). We performed RNA sequencing on 54 normal colon mucosa samples from nine patients undergoing colorectal cancer surgeries, freezing samples at predefined intervals ranging from 0 to 60 minutes. A total of 44 differentially expressed genes (DEGs) (p < 0.05) were identified when comparing samples frozen immediately (T0) with those frozen after 60 minutes (T5). These DEGs were further analyzed through functional and pathway enrichment analyses and weighted gene co-expression network analysis (WGCNA). The enrichment analysis revealed significant alterations in pathways associated with apoptosis, hypoxia, EMT, and cancer progression, including p53 and HIF-1 signaling. WGCNA highlighted two co-expressed gene modules: ME2, which showed downregulation of apoptosis-related genes, and ME4, linked to apoptosis and cellular metabolism. Our findings highlight CIT as a critical preanalytical variable, showing that prolonged ischemia can induce transcriptomic changes that may mimic malignancy, and potentially confound research outcomes. To minimize such effects, we recommend keeping CIT under 30 minutes.

Cold ischemia time (CIT), the interval between tissue excision and preservation, is a critical preanalytical variable that profoundly impacts gene expression profiles. Variability in CIT can lead to inconsistent transcriptomic results, making study interpretation challenging and undermining reproducibility in biomedical research. Our study aimed to evaluate the impact of CIT on the expression of cancer-related genes, particularly these involved in hypoxia, apoptosis, and epithelial-to-mesenchymal transition (EMT). We performed RNA sequencing on 54 normal colon mucosa samples from nine patients undergoing colorectal cancer surgeries, freezing samples at predefined intervals ranging from 0 to 60 minutes. A total of 44 differentially expressed genes (DEGs) (p < 0.05) were identified when comparing samples frozen immediately (T0) with those frozen after 60 minutes (T5). These DEGs were further analyzed through functional and pathway enrichment analyses and weighted gene coexpression network analysis (WGCNA). The enrichment analysis revealed significant alterations in pathways associated with apoptosis, hypoxia, EMT, and cancer progression, including p53 and HIF-1 signaling. WGCNA highlighted two co-expressed gene modules: ME2, which showed downregulation of apoptosis-related genes, and ME4, linked to apoptosis and cellular metabolism. Our findings highlight CIT as a critical preanalytical variable, showing that prolonged ischemia can induce transcriptomic changes that may mimic malignancy, and potentially confound research outcomes. To minimize such effects, we recommend keeping CIT under 45–60 minutes.

Alzheimer’s disease (AD) is a common and increasing societal problem due to the extending human lifespan. In males, loss of chromosome Y (LOY) in leukocytes is strongly associated with AD. We studied here DNA methylation and RNA expression in sorted monocytes and granulocytes with and without LOY from male AD patients. Through multi-omic analysis, we identified new candidate genes along with those previously associated with AD. Global analyses of DNA methylation in samples with LOY vs. normal state showed that hypomethylation dominated both in granulocytes and monocytes. Our findings highlight LOY-related differences in DNA methylation that occur in gene regulatory regions. Specifically, we observed alterations in key genes involved in leukocyte differentiation: FLI1, involved in early hematopoiesis; RUNX1, essential for blood cell development; RARA, regulating gene expression in response to retinoic acid; CANX, crucial for protein folding; CEBPB, a transcription factor important for immune responses; and MYADM, implicated in cell adhesion and migration. Moreover, protein–protein interaction analysis in granulocytes identified that products of two of these genes, CANX and CEBPB, are key hub proteins. This research underscores the potential of multi-omic approach in pure hematopoietic cell populations to uncover the molecular underpinnings of AD. Finally, our results link previous analysis showing impact of LOY on leukocyte differentiation, LOY-associated transcriptional dysregulation and GWAS studies of LOY.

Histologically normal mammary tissue from breast cancer patients can harbor significant genetic alterations. We analyzed the spectrum of DNA variants in 408 matched histologically normal tissue and tumors from 77 poor-prognosis patients, 49 patients recruited without prognosis bias, and mammary gland samples from 15 non-cancerous individuals. Whole exome sequencing revealed a higher prevalence of pathogenic post-zygotic variants in cancer-associated genes: AKT1, PIK3CA, PTEN, TBX3, and TP53, affecting poor-prognosis patients (29%), compared to 12.5% in those without prognosis criteria (p=0.0008578). PIK3CA variants were recurrent across patients, while TP53 variants were restricted to those with adverse prognoses. Duplex sequencing detected low-frequency pathogenic PIK3CA and TP53 variants in distant normal tissues of poor-prognosis patients. Disease recurrence significantly reduced survival rates, with poor prognosis patients experiencing higher mortality within 24 months (p=0.0088), further worsened by the presence of pathogenic post-zygotic variants. These findings highlight the importance of genetic monitoring even in microscopically normal mammary tissue.

Alzheimer’s disease (AD) is a common and increasing societal problem due to the extending human lifespan. In males, loss of chromosome Y (LOY) in leukocytes is strongly associated with AD. We studied here DNA methylation and RNA expression in sorted monocytes and granulocytes with and without LOY from male AD patients. Through multi-omic analysis, we identified new candidate genes and confirmed the involvement of numerous genes previously associated with AD. Our findings highlight LOY-related differences in DNA methylation that occur in gene regulatory regions and are predominantly accompanied by down-regulation of affected genes. Specifically, we observed alterations in key genes involved in leukocyte differentiation: FLI1 , involved in early hematopoiesis; RUNX1 , essential for blood cell development; RARA , regulating gene expression in response to retinoic acid; CANX , crucial for protein folding; CEBPB , a transcription factor important for immune responses; and MYADM , implicated in cell adhesion and migration. Moreover, protein-protein interaction analysis in granulocytes identified that products of two of these genes, CANX and CEBPB , are key hub proteins. Thus, LOY appears to dysregulate genes involved in leukocyte differentiation and induce higher-level epigenetic changes. This research underscores the potential of multi-omic approaches in pure cell populations to uncover the molecular underpinnings of AD and reinforces the significance of LOY as a pathogenic factor in this disease. Overall, results support the hypothesis that age-related immune cell dysfunction contributes to AD development. Finally, our results link previous analysis showing impact of LOY on leukocyte differentiation, LOY-associated transcriptional dysregulation and GWAS studies of LOY.

Bladder urothelial carcinoma (BLCA) is the 10th most common cancer with a low survival rate and strong male bias. We studied the field cancerization in BLCA using multi-sample- and multi-tissue-per-patient protocol for sensitive detection of autosomal post-zygotic chromosomal alterations and loss of chromosome Y (LOY). We analysed 277 samples of histologically normal urothelium, 145 tumors and 63 blood samples from 52 males and 15 females, using the in-house adapted Mosaic Chromosomal Alterations (MoChA) pipeline. This approach allows identification of the early aberrations in urothelium from BLCA patients. Overall, 45% of patients exhibited at least one alteration in at least one normal urothelium sample. Recurrence analysis resulted in 16 hotspots composed of either gains and copy number neutral loss of heterozygosity (CN-LOH) or deletions and CN-LOH, encompassing well-known and new BLCA cancer driver genes. Conservative assessment of LOY showed 29%, 27% and 18% of LOY-cells in tumors, blood and normal urothelium, respectively. We provide a proof of principle that our approach can characterize the earliest alterations preconditioning normal urothelium to BLCA development. Frequent LOY in blood and urothelium-derived tissues suggest its involvement in BLCA.

Despite advances in early detection and treatment strategies, breast cancer recurrence and mortality remain a significant health issue. Recent insights suggest the prognostic potential of microscopically healthy mammary gland, in the vicinity of the breast lesion. Nonetheless, a comprehensive understanding of the gene expression profiles in these tissues and their relationship to patient outcomes is still missing. Furthermore, the increasing trend towards breast-conserving surgery may inadvertently lead to the retention of existing cancer-predisposing mutations within the normal mammary gland. This study assessed the transcriptomic profiles of 242 samples from 83 breast cancer patients with unfavorable outcomes, including paired uninvolved mammary gland samples collected at varying distances from primary lesions. As a reference, control samples from 53 mammoplasty individuals without cancer history were studied. A custom panel of 634 genes linked to breast cancer progression and metastasis was employed for expression profiling, followed by whole-transcriptome verification experiments and statistical analyses to discern molecular signatures and their clinical relevance. A distinct gene expression signature was identified in uninvolved mammary gland samples, featuring key cellular components encoding keratins, CDH1, CDH3, EPCAM cell adhesion proteins, matrix metallopeptidases, oncogenes, tumor suppressors, along with crucial genes (FOXA1, RAB25, NRG1, SPDEF, TRIM29 , and GABRP ) having dual roles in cancer. Enrichment analyses revealed disruptions in epithelial integrity, cell adhesion, and estrogen signaling. This signature, named KAOS for Keratin-Adhesion-Oncogenes-Suppressors, was significantly associated with reduced tumor size but increased mortality rates. Integrating molecular assessment of non-malignant mammary tissue into disease management could enhance survival prediction and facilitate personalized patient care.

Bacterial gene regulatory networks orchestrate responses to environmental challenges. Horizontal gene transfer can bring in genes with regulatory potential, such as new transcription factors (TFs), and this can disrupt existing networks. Serious regulatory perturbations may even result in cell death. Here, we show the impact on Escherichia coli of importing a promiscuous TF that has adventitious transcriptional effects within the cryptic Rac prophage. A cascade of regulatory network perturbations occurred on a global level. The TF, a C regulatory protein, normally controls a Type II restriction-modification system, but in E. coli K-12 interferes with expression of the RacR repressor gene, resulting in de-repression of the normally-silent Rac ydaT gene. YdaT is a prophage-encoded TF with pleiotropic effects on E. coli physiology. In turn, YdaT alters expression of a variety of bacterial regulons normally controlled by the RcsA TF, resulting in deficient lipopolysaccharide biosynthesis and cell division. At the same time, insufficient RacR repressor results in Rac DNA excision, halting Rac gene expression due to loss of the replication-defective Rac prophage. Overall, Rac induction appears to counteract the lethal toxicity of YdaT. We show here that E. coli rewires its regulatory network, so as to minimize the adverse regulatory effects of the imported C TF. This complex set of interactions may reflect the ability of bacteria to protect themselves by having robust mechanisms to maintain their regulatory networks, and/or suggest that regulatory C proteins from mobile operons are under selection to manipulate their host's regulatory networks for their own benefit.

Despite surging interest in space travel in recent decades, the impacts of prolonged, elevated exposure to galactic cosmic radiation (GCR) on human health remain poorly understood. This form of ionizing radiation causes significant changes to biological systems including damage to DNA structure by altering epigenetic phenotype with emphasis on DNA methylation. Building on previous work by Kennedy et al. (2018), we evaluated spatial DNA methylation patterns triggered by high-LET (⁵⁶Fe, ²⁸Si) and low-LET (X rays) and the influence of chromosome positioning and epigenetic architecture in distinct radial layers of cell nucleus. Next, we validated our results using gene expression data of mice and JAXA astronauts. We showed that primarily ⁵⁶Fe induces a persistent DNA methylation increase whereas ²⁸Si and X rays induce a decrease DNA methylation which is not persistent with time. Moreover, we highlighted the role of heterochromatin-associated histone modifications in absorbing GCR and protecting euchromatin-associated DNA fragments localized in inner parts of nucleus. In summary, our study provides novel insights towards epigenetic nuclear architecture and its role in limiting external radiation damage.

Alzheimer’s disease (AD) is a common and increasing societal problem due to the extending human lifespan. In males, loss of Y (LOY) in leukocytes is more prevalent in AD patients. We studied DNA methylation, gene expression and other epigenetic changes in monocytes and granulocytes with and without LOY from male AD patients and controls. New candidate genes were identified and numerous genes already implicated in AD pathogenesis were confirmed. We show that multi-omics of leukocytes can define AD candidate genes and we strengthen the role of LOY in disease development. The LOY-associated differences in DNA methylation levels were predominantly observed in regulatory regions, and supported by expression analysis showing down-regulation of immune genes. The single-cell transcriptomics highlighted that the AD patient-specific immune activation dominates over LOY-specific activation. Our findings agree with the hypothesis that age-related dysfunction of immune cells contribute to AD and that LOY is reflected by higher-level epigenetic changes with an AD-specific pattern.