Supplementary Figure 5. The relative fungi abundance of melanoma and healthy samples at different taxonomical levels: (A) Phylum (B) Class (C) Order (D) Family.

Supplementary Table 1. Melanoma patients and healthy controls data.

Supplementary Figure Legends and Tables 2-9

Supplementary Figure 11. Relative abundance of selected species in responders and non responders (R vs. NR).

Supplementary Figure 6. Gut microbiota richness.

Supplementary Figure 10. Relative abundance of selected species in patients that exhibited benefits during anti-PD-1 treatment and patients who did not show benefits (CB vs. NB).

Supplementary Figure 7. Relative abundance of selected species during anti-PD-1 treatment.

Supplementary Figure 1. Baseline characteristics of the study populations with respect to (A) age groups, (B) BMI groups, and (C) sex.

Supplementary Figure 2. (A) Shannon diversity of patients with normal (n=102) and elevated level of serum LDH (n=16). Kruskal–Wallis test was performed to compare groups, p<0.05. Horizontal line shows median. The lower and upper hinges correspond to the first and third quartiles. The upper and the lower whiskers extend from the hinge to the largest and smallest value, respectively, no further than 1.5 * IQR from the hinge (where IQR is the inter-quartile range, or distance between the first and third quartiles). Dots represent potential outliers. (B) Relative abundance of Neurospora crassa and Sugiyamaella lignohabitans in patients with normal serum LDH level (n=102) and patients with elevated level of serum LDH (LDH>1.5x, what corresponds to >375 U/L, n=16). Welch Two Sample t-test was performed to compare groups, padj<0.05 (FDR). Horizontal line shows median. Red dot shows mean. Dots represent individual samples.

Supplementary Figure 4. Phylogenetic trees of mycobiota in (A) healthy and (B) melanoma subjects.

A growing number of mutations are being identified in the noncoding genome, including microRNA (miRNA) genes; however, little is known about the consequences of these mutations and how harmful they are to the functioning of miRNA genes. To evaluate the effects of miRNA gene mutations, we took advantage of a large collection of somatic mutations identified in miRNA genes in >10,000 The Cancer Genome Atlas cancer samples and compared them with the corresponding miRNA sequencing data. Using different analytical approaches and rigorous statistical criteria, we revealed that a substantial fraction of mutations is deleterious for the proper functioning of miRNA genes affecting the level of mature miRNAs, isomiR profiles (precision of DROSHA/DICER1 cleavage), and/or 5p/3p miRNA strand balance. We also showed that most mutations, especially those identified as deleterious, destabilize the structure of miRNA precursors. The analysis showed that many miRNA gene mutations can damage miRNA genes and, if located in disease-related miRNA genes, may be pathogenic variants.

Supplementary Figure 8. Relative abundance of statistically significant gut fungi species in overweight and normal BMI patients (A) before anti-PD-1 treatment, and (B) in the third month of anti-PD-1 treatment.

Supplementary Figure 9. Relative abundance of Yarrowia lipolytica in overweight patients during anti-PD-1 treatment.

Supplementary Figure 3. Relative abundance of Candida glabrata in normal (18.5

The gut microbiome has emerged as an important modulator of cancer progression and therapy response, yet the role of its fungal component, the gut mycobiome, remains poorly understood. In this review, we summarize current knowledge on the relationship between intestinal fungi and cancer, with a particular focus on the potential influence of the gut mycobiome on the efficacy of immune checkpoint inhibitors (ICIs). Drawing from both human studies and preclinical models, we discuss evidence linking fungal dysbiosis to altered immune responses and therapy outcomes in cancer patients. Specific fungal taxa, such as Candida and Malassezia, have been associated with changes in the tumor microenvironment and modulation of host immunity. We also review the proposed mechanisms through which fungi may impact antitumor immunity, including interactions with immune receptors and production of bioactive metabolites. Although research in this field is still in its early stages, emerging data suggest that the gut mycobiome may serve as a biomarker for immunotherapy response and a potential target for therapeutic intervention. A better understanding of host-fungus interactions in the gut could contribute to the development of personalized strategies to improve the effectiveness and safety of cancer immunotherapy.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular disorders associated with expansions of microsatellites, respectively, in DMPK and CNBP. Their pathogenesis is linked to the global aberrant alternative splicing (AAS) of many genes and marks mostly muscular and neuronal tissues, while blood is the least affected. Recent data in DM1 skeletal muscles indicated that abnormalities in RNA metabolism also include global upregulation of circular RNAs (circRNAs). CircRNAs are a heterogeneous group considered splicing errors and by-products of canonical splicing. To elucidate whether circRNA dysregulation is an inherent feature of the myotonic environment, we perform their analysis in the frontal cortex and whole blood of DM1 and DM2 patients. We find a global elevation of circRNAs in both tissues, and its magnitude is neither correlated with the differences in their parental gene expression nor is associated with AAS published earlier. Aberrantly spliced cassette exons of linear transcripts affected in DM1 and DM2 are not among the circularized exons, which unique genomic features prerequisite back-splicing. However, the blueprint of the AAS of linear RNAs is found in a variety of circRNA isoforms. The heterogeneity of circRNAs also originates from the utilization of exonic and intronic cryptic donors/acceptors in back splice junctions, and intron-containing circRNAs are more characteristic of the blood. Overall, this study reveals circRNA dysregulation in various tissues from DM1 and DM2; however, their levels do not correlate with the AAS in linear RNAs, suggesting a potential independent regulatory mechanism underlying circRNA upregulation in myotonic dystrophy.

Inflammatory bowel disease (IBD) refers to chronic, recurrent inflammatory intestinal disorders, primarily including Crohn’s disease (CD) and Ulcerative colitis (UC). Numerous studies have elucidated the importance of the gut microbiome in IBD. Recently, numerous studies have focused on the gut virome, an intriguing and enigmatic aspect of the gut microbiome. Alterations in the composition of phages, eukaryotic viruses, and human endogenous retroviruses that occur in IBD suggest potential involvement of the gut virome in IBD. Nevertheless, the mechanisms by which it maintains intestinal homeostasis and interacts with diseases are only beginning to be understood. Here, we thoroughly reviewed the composition of the gut virome in both healthy individuals and IBD patients, emphasizing the key viruses implicated in the onset and progression of IBD. Furthermore, the complex connections between the gut virome and the intestinal barrier, immunity, and gut microbiome were dissected to advance the interpretation of IBD pathogenesis. The updated discussion of the evidence regarding the gut virome will advance our knowledge in gut virome and chronic gastrointestinal diseases. Targeting the gut virome is a promising avenue for IBD treatment in future.

Interest in the genetic variation of noncoding genomic elements, including microRNAs (miRNAs), is growing, and several mutations in miRNA genes implicated in human diseases, including cancer, have already been detected. However, the lack of dedicated analytical tools severely hampers progress in this area. In this study, we developed the first whole-miRNome sequencing (WMS) platform, which enables the targeted sequencing of all human miRNA genes (n ∼2000) and 28 miRNA biogenesis genes. By sequencing various types of DNA samples, including ∼300 tumor/normal pairs, from lung, colorectal, ovarian, renal, and basal cell carcinomas, we identified ∼2000 mutations, including 879 in miRNA genes. These mutations were located in all parts of the genes, including seed or cleavage sites essential for the functioning of miRNA genes. The high reliability of the mutations was confirmed through various approaches, including different sequencing methods. The analysis identified several miRNA genes with functional enrichment of cancer mutations, including MIR3928, which was specifically mutated in basal cell carcinoma, suggesting its potential role in this cancer. WMS also allowed the identification of multiple copy number alterations, which often encompassed miRNA genes. WMS provides highly effective, low-cost sequencing of all miRNA genes in different types of samples, including highly degraded ones.

The gut virome, an essential component of the intestinal microbiome, constitutes ∼0.1% of the total microbial biomass but contains a far greater number of particles than bacteria, with phages making up 90%-95% of this virome. This review systematically examines the developmental patterns of the gut virome, focusing on factors influencing its composition, including diet, environment, host genetics, and immunity. Additionally, it explores the gut virome's associations with various diseases, its interactions with gut bacteria and the immune system, and its emerging clinical applications.