Despite the recent breakthroughs in targeted and immunotherapy for melanoma, the overall survival rate remains low. In recent years, considerable attention has been paid to the gut microbiota and other modifiable patient factors (e.g., diet and body composition), though their role in influencing therapeutic responses has yet to be defined. Here, we characterized a cohort of 31 patients with unresectable IIIC-IV-stage cutaneous melanoma prior to initiation of targeted or first-line immunotherapy via the following methods: (i) fecal microbiome and metabolome via 16S rRNA amplicon sequencing and gas chromatography/mass spectrometry, respectively, and (ii) anthropometry, body composition, nutritional status, physical activity, biochemical parameters, and immunoprofiling. According to our data, patients subsequently classified as responders were obese (i.e., with high body mass index and high levels of total, visceral, subcutaneous, and intramuscular adipose tissue), non-sarcopenic, and enriched in certain fecal taxa (e.g., Phascolarctobacterium) and metabolites (e.g., anethole), which were potentially endowed with immunostimulatory and oncoprotective activities. On the other hand, non-response was associated with increased proportions of Streptococcus, Actinomyces, Veillonella, Dorea, Fusobacterium, higher neutrophil levels (and a higher neutrophil-to-lymphocyte ratio), and higher fecal levels of butyric acid and its esters, which also correlated with decreased survival. This exploratory study provides an integrated list of potential early prognostic biomarkers that could improve the clinical management of patients with advanced melanoma, in particular by guiding the design of adjuvant therapeutic strategies to improve treatment response and support long-term health improvement.

The microbiota of a cheese play a critical role in influencing its sensory and physicochemical properties. In this study, traditional Apulian Caciocavallo cheeses coming from 4 different dairies in the same area and produced following standardized procedures were examined, as well as the different bulk milks and natural whey starter (NWS) cultures used. Moreover, considering the cheese wheels as the blocks of Caciocavallo cheeses as whole, these were characterized at different layers (i.e., core, under-rind, and rind) of the block using a multi-omics approach. In addition to physical-chemical characterization, culturomics, quantitative PCR, metagenomics, and metabolomics analysis were carried out after salting and throughout the ripening time (2 mo) to investigate major shifts in the succession of the microbiota and flavor development. Culture-dependent and 16S rRNA metataxonomics results clearly clustered samples based on micro- biota biodiversity related to the production dairy plant as a result of the use of different NWS or the intrinsic conditions of each production site. At the beginning of the ripening, cheeses were dominated by Lactobacillus, and in 2 dairies (Art and SdC), Streptococcus genera were associated with the NWS. The analysis allowed us to show that although the diversity of identified genera did not change significantly between the rind, under- rind, and core fractions of the same samples, there was an evolution in the relative abundance and absolute quantification, modifying and differentiating profiles during ripening. The real-time PCR, also known as quantitative or qPCR, mainly differentiated the temporal adaptation of those species originating from bulk milks and those provided by NWS. The primary starters detected in NWS and cheeses contributed to the high relative concentration of 1-butanol, 2-butanol, 2-heptanol, 2-butanone, acetoin, delta-dodecalactone, hexanoic acid ethyl ester, octanoic acid ethyl ester, and volatile free fatty acids during ripening, whereas cheeses displaying low abundances of Streptococcus and Lactococcus (dairy Del) had a lower total concentration of acetoin compared with Art and SdC. However, the subdominant strains and nonstarter lactic acid bacteria present in cheeses are responsible for the production of secondary metabolites belonging to the chemical classes of ketones, alcohols, and organic acids, reaffirming the importance and relevance of autochthonous strains of each dairy plant although only considering a delimited production area.

Fructose intolerance (FI) is a widespread non-genetic condition in which the incomplete absorption of fructose leads to gastro-intestinal disorders. The crucial role of microbial dysbiosis on the onset of these intolerance symptoms together with their persistence under free fructose diets are driving the scientific community towards the use of probiotics as a novel therapeutic approach. In this study, we evaluated the prevalence of FI in a cohort composed of Romanian adults with Functional Grastrointestinal Disorders (FGIDs) and the effectiveness of treatment based on the probiotic formulation EQBIOTA^® (Lactiplantibacillus plantarum CECT 7484 and 7485 and Pediococcus acidilactici CECT 7483). We evaluated the impact of a 30-day treatment both on FI subjects and healthy volunteers. The gastrointestinal symptoms and fecal volatile metabolome were evaluated. A statistically significant improvement of symptoms (i.e., bloating, and abdominal pain) was reported in FI patient after treatment. On the other hand, at the baseline, the content of volatile metabolites was heterogeneously distributed between the two study arms, whereas the treatment led differences to decrease. From our analysis, how some metabolomics compounds were correlated with the improvement and worsening of clinical symptoms clearly emerged. Preliminary observations suggested how the improvement of gastrointestinal symptoms could be induced by the increase of anti-inflammatory and protective substrates. A deeper investigation in a larger patient cohort subjected to a prolonged treatment would allow a more comprehensive evaluation of the probiotic treatment effects.

Viroids are small, circular non-coding RNAs that autonomously replicate in plants, exploiting host cellular machinery for replication and spread. Recent studies reveal that viroid-like agents can infect filamentous fungi, suggesting cross-kingdom interactions. In this study, we report the discovery and the characterization of TsvlRNA1 in Trichoderma spirale . TsvlRNA1 is a 712-nt-long viroid-like RNA containing a hammerhead ribozyme in one polarity strand. Bioinformatic data, molecular validation, and reverse genetics experiments demonstrate that TsvlRNA1 is circular with an active ribozyme essential for replication. TsvlRNA1 replicates autonomously and transmits between Trichoderma species, eliciting 20–22 nt viroid-derived small RNAs consistent with RNA silencing targeting. The biocontrol capacity of Trichoderma against Rhizoctonia solani is variably modulated by TsvlRNA1, with effects ranging from positive to negative depending on host strain. In T. spirale , data suggest that TsvlRNA1 negatively affects antagonistic potential. TsvlRNA1 transmission via horizontal routes is prevalent, and the viroid-like RNA fails to infect plant hosts experimentally. These results highlight the as-yet underappreciated ecological and functional diversity of viroid-like agents in fungi, with implications for fungal biology, biocontrol, and genotype-phenotype relationships in eukaryotes. IMPORTANCE: Species of the fungal genus Trichoderma play a central role in sustainable agriculture by controlling fungal plant pathogens and supporting plant growth. For this reason, Trichoderma -based products represent a substantial share of the global market for microbial biofungicides. Viroids are the smallest known infectious agents, and their presence in filamentous fungi has only recently been discovered. Consequently, little is known about their biology, transmission, or interactions with fungal hosts. In this study, we describe TsvlRNA1, a viroid-like RNA associated with T. spirale , representing only the second viroid-like RNA to be biologically characterized in fungi. We show that TsvlRNA1 can influence the ability of Trichoderma to inhibit Rhizoctonia solani , a major plant pathogen, demonstrating its biological relevance. Unexpectedly, TsvlRNA1 can be transmitted between different Trichoderma species. This finding raises concerns about the possible transfer of genetic traits between fungi, including potentially those related to fungicide resistance, with important implications for agricultural biocontrol.

Viroids are small, circular non-coding RNAs that autonomously replicate in plants, exploiting host cellular machinery for replication and spread. Recent studies reveal that viroid-like agents can infect filamentous fungi, suggesting cross-kingdom interactions. In this study, we report the discovery and the characterization of TsvlRNA1 in Trichoderma spirale , a transmissible viroid-like RNA containing a hammerhead ribozyme in one polarity strand. Bioinformatic data, molecular validation, and reverse genetics experiments demonstrate that TsvlRNA1 is circular with an active ribozyme essential for replication. TsvlRNA1 replicates autonomously and transmits horizontally between Trichoderma species, eliciting 21–23 nt viroid-derived small RNAs consistent with RNA silencing targeting. The biocontrol capacity of Trichoderma against Rhizoctonia solani is variably modulated by TsvlRNA1, with effects ranging from positive to negative depending on host strain. In T. spirale , data suggests genotype-by-agent interactions influence antagonistic potential negatively. TsvlRNA1 transmission via horizontal routes is prevalent, and the viroid-like RNA fails to infect plant hosts experimentally. These results highlight so-far the underappreciated ecological and functional diversity of viroid-like agents in fungi, with implications for fungal biology, biocontrol, and genotype-phenotype relationships in eukaryotes. Importance: Species of the fungal genus Trichoderma play a central role in sustainable agriculture by controlling fungal plant pathogens and supporting plant growth. For this reason, Trichoderma -based products represent a substantial share of the global market for microbial biofungicides. Viroids are the smallest known infectious agents, and their presence in filamentous fungi has only recently been discovered. Consequently, little is known about their biology, transmission, or interactions with fungal hosts. In this study, we describe TsvlRNA1, a viroid-like RNA associated with T. spirale , representing only the second viroid-like RNA to be biologically characterized in fungi. We show that TsvlRNA1 can influence the ability of Trichoderma to inhibit Rhizoctonia solani , a major plant pathogen, demonstrating its biological relevance. Unexpectedly, TsvlRNA1 can be transmitted between different Trichoderma species. This finding raises concerns about the possible transfer of genetic traits between fungi, including those related to fungicide resistance, with important implications for agricultural biocontrol. Abstract Figure:

Ambiviruses are fungal-infecting circular RNA viruses that uniquely combine viroid-like and viral features, yet the function of their conserved ORF-B protein and their effects on hosts remain unknown, hindered by the lack of a reverse genetics system. Here, we constructed the first infectious cDNA clone of an ambivirus, Fusarium graminearum ambivirus 1 (FgAV1), using a head-to-tail dimer placed downstream of a fungal promoter. FgAV1 was horizontally transmitted via hyphal anastomosis to virus-free Fusarium graminearum strains. Notably, a reverse-oriented dimer construct was also infectious and transmissible. Targeted mutagenesis revealed that both ORF-A- and ORF-B-encoded proteins and the presence of embedded ribozymes are indispensable for ambivirus replication. Our results further demonstrate that FgAV1 infection triggers a fungal RNAi response, extending the antiviral role of host sRNAs to circular RNA viruses. Furthermore, FgAV1 infection suppressed fungal growth and significantly reduced the virulence of F. graminearum on wheat. These findings provide novel insights into ambivirus replication and their potential in fungal pathogen biocontrol.