Breast milk is the ideal food for infants and undoubtedly has immediate and long‑term benefits. Breast milk contains extracellular vesicles (EVs) i.e., exosomes secreted by maternal breast cells. Exosomes carry genetic material, such as long non‑coding RNAs (lncRNAs), which possibly participate in cell‑to‑cell communications, as they are known to regulate critical gene pathways. The aim of the present study was to screen human breastmilk exosomes for their lncRNA cargo and to examine exosomal lncRNA levels associated with milk obtained from mothers that gave birth at term or prematurely (<37 weeks of gestation). Samples were collected at 3 weeks postpartum from 20 healthy, breastfeeding mothers; 10 mothers had given birth at full‑term and 10 mothers preterm. Exosomal RNA was extracted from all samples and the expression of 88 distinct lncRNAs was determined using reverse transcription‑quantitative PCR. A total of 13 lncRNAs were detected in ≥85% of the samples, while 31 were detected in ≥50% of the samples. Differential expression analysis of the lncRNAs between the two groups revealed ≥2‑fold differences, with generally higher lncRNA concentrations found in the milk of the mothers that gave birth at term compared with those that gave birth preterm. Among these, the non‑coding RNA activated at DNA damage (NORAD) was prominently detected in both groups, and its expression was significantly downregulated in the breast milk exosomes of mothers who delivered preterm. On the whole, the present study demonstrates that breast milk lncRNAs may be important factors of normal early human development. Collectively, the presence of lncRNAs in human breast milk may explain the consistent inability of researchers to fully ‘humanize’ animal milk.

Human interferon alpha 2a (IFNα2a) is a secreted glycoprotein that exerts a wide spectrum of biological effects, such as triggering of antiviral, antitumor and immunosuppressive responses. IFNα2a is used as pharmaceutical polypeptide in chronic hepatitis C virus (HCV) infection, chronic myelogenous leukemia, advanced renal cell carcinoma, and metastatic malignant melanoma. So far, the pharmaceutical polypeptide of this cytokine is produced in prokaryotic expression systems (E. coli). Here we report the expression and purification of recombinant human IFNα2a in the methylotrophic yeast Pichia pastoris. The cDNA encoding for human IFNα2a, modified to bear the P. pastoris codon bias, was cloned into the pPinkα-HC vector in order to be expressed as a secreted protein upon induction. Proper expression and secretion of recombinant human IFNα2a (approximately 19 kDa) was confirmed by PCR-sequencing, SDS-PAGE and Western blot analysis following methanol-induced expression in a number of individual transformed strains. Purification of the recombinant protein was performed by affinity chromatography, achieving a robust yield of purified active form. The purified recombinant protein showed an impressive stability to thermal denaturation as observed by Differential Scanning Fluorimetry. The biological activity of the P. pastoris-produced IFNα2a was confirmed in A549 and HT29 cells by monitoring transcriptional up-regulation of a panel of known interferon-stimulated genes (ISGs). Our results document that the P. pastoris expression system is a suitable system for producing biologically functional IFNα2a in a secreted form.

Aspergillus mold is a ubiquitously found, airborne pathogen that can cause a variety of diseases from mild to life-threatening in severity. Limitations in diagnostic methods combined with anti-fungal resistance render Aspergillus a global emerging pathogen. In industry, Aspergilli produce toxins, such as aflatoxins, which can cause food spoilage and pose public health risk issues. Here, we report a multiplex qPCR method for the detection and identification of the five most common pathogenic Aspergillus species, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, and Aspergillus nidulans. Our approach exploits species-specific nucleotide polymorphisms within their ITS genomic regions. This novel assay combines multiplex single-color real time qPCR and melting curve analysis and provides a straight-forward, rapid, and cost-effective detection method that can identify five Aspergillus species simultaneously in a single reaction using only six unlabeled primers. Due to their unique fragment lengths, the resulting amplicons are directly linked to certain Aspergillus species like fingerprints, following either electrophoresis or melting curve analysis. Our method is characterized by high analytical sensitivity and specificity, so it may serve as a useful and inexpensive tool for Aspergillus diagnostic applications both in health care and the food industry.

Stress-induced promoter-associated and antisense lncRNAs (si-paancRNAs) originate from a reservoir of oxidative stress (OS)-specific promoters via RNAPII pausing-mediated divergent antisense transcription. Several studies have shown that the KDM7A divergent transcript gene (KDM7A-DT), which encodes a si-paancRNA, is overexpressed in some cancer types. However, the mechanisms of this overexpression and its corresponding roles in oncogenesis and cancer progression are poorly understood. We found that KDM7A-DT expression is correlated with highly aggressive cancer types and specific inherently determined subtypes (such as ductal invasive breast carcinoma (BRCA) basal subtype). Its regulation is determined by missense TP53 mutations in a subtype-specific context. KDM7A-DT transcribes several intermediate-sized ncRNAs and a full-length transcript, exhibiting distinct expression and localization patterns. Overexpression of KDM7A-DT upregulates TP53 protein expression and H2AX phosphorylation in nonmalignant fibroblasts, while in semi-transformed fibroblasts, OS superinduces KDM7A-DT expression in a TP53-dependent manner. KDM7A-DT knockdown and gene expression profiling in TP53-missense mutated luminal A BRCA variant, where it is abundantly expressed, indicate its significant role in cancer pathways. Endogenous over-expression of KDM7A-DT inhibits DNA damage response/repair (DDR/R) via the TP53BP1-mediated pathway, reducing apoptosis and promoting G2/M checkpoint arrest. Higher KDM7A-DT expression in BRCA is associated with KDM7A-DT locus gain/amplification, higher histologic grade, aneuploidy, hypoxia, immune modulation scores, and activation of the c-myc pathway. Higher KDM7A-DT expression is associated with relatively poor survival outcomes in patients with luminal A or Basal subtypes. In contrast, it is associated with favorable outcomes in patients with HER2+ER- or luminal B subtypes. KDM7A-DT levels are coregulated with critical transcripts and proteins aberrantly expressed in BRCA, including those involved in DNA repair via non-homologous end joining and epithelial-to-mesenchymal transition pathway. In summary, KDM7A-DT and its si-lncRNA exhibit several intrinsic biological and clinical characteristics that suggest important roles in invasive BRCA and its subtypes. KDM7A-DT-defined mRNA and protein subnetworks offer resources for identifying clinically relevant RNA-based signatures and prospective targets for therapeutic intervention.

Milk is a biological fluid with a dynamic composition of micronutrients and bioactive molecules that serves as a vital nutrient source for infants. Milk composition is affected by multiple factors, including genetics, geographical location, environmental conditions, lactation phase, and maternal nutrition, and plays a key role in dictating its microbiome. This study addresses a less-explored aspect, comparing the microbial communities in human breast milk with those in mature milk from species that are used for milk consumption. Since mature animal milk is used as a supplement for both the infant (formula) and the child/adolescent, our main aim was to identify shared microbial communities in colostrum and mature human milk. Using 16S rRNA metagenomic sequencing, we focused on characterizing the milk microbiota in the Northern Greek population by identifying shared microbial communities across samples and comparing the relative abundance of prevalent genera. We analyzed ten human milk samples (from five mothers), with five collected three days postpartum (colostrum) and five collected thirty to forty days postpartum (mature milk) from corresponding mothers. To perform an interspecies comparison of human milk microbiota, we analyzed five goat and five bovine milk samples from a local dairy industry, collected fifty to seventy days after birth. Alpha diversity analysis indicated moderate diversity and stability in bovine milk, high richness in goat milk, and constrained diversity in breast milk. Beta diversity analysis revealed significant distinctions among mammalian species, emphasizing both presence/absence and abundance-based clustering. Despite noticeable differences, shared microbial components underscore fundamental aspects across all mammalian species, highlighting the presence of a core microbiota predominantly comprising the Proteobacteria, Firmicutes, and Actinobacteriota phyla. At the genus level, Acinetobacter, Gemella, and Sphingobium exhibit significant higher abundance in human milk compared to bovine and goat milk, while Pseudomonas and Atopostipes are more prevalent in animal milk. Our comparative analysis revealed differences and commonalities in the microbial communities of various mammalian milks and unraveled the existence of a common fundamental milk core microbiome. We thus revealed both species-specific and conserved microbial communities in human, bovine, and goat milk. The existence of a common core microbiome with conserved differences between colostrum and mature human milk underscores fundamental similarities in the microbiota of milk across mammalian species, which could offer valuable implications for optimizing the nutritional quality and safety of dairy products as well as supplements for infant health.

The atlas of stress response activity (ASTRA) is an open-access, curated database cataloging transcriptomic responses to stress in human cell lines, organized by major stress categories to advance research on conserved and stress-specific pathways, RNA biomarkers, and disease mechanisms. It integrates and standardizes bulk RNA-seq datasets from the Gene Expression Omnibus (GEO), focusing on messenger RNA and long noncoding RNA expression under diverse stress conditions. Data are grouped into four categories—oxidative stress, hypoxia, heat shock, and DNA damage—with subcategories corresponding to H2O2, low oxygen, increased temperature, and UV radiation as molecular stressors, respectively. A harmonized computational pipeline ensures comparability across samples and studies. Each dataset contains stressed and matched control samples, annotated with detailed metadata, including cell line identity, tissue origin, cell-state classification (non-diseased, cancer, organoid), treatment parameters, and sequencing protocols. ASTRA allows users to query gene-level expression, perform differential expression analyses, and visualize transcriptional dynamics across datasets, conditions, and cell-type groups. By profiling both coding and noncoding transcriptomes, ASTRA facilitates the discovery of novel stress-responsive genes and RNA-mediated regulatory mechanisms. As a comprehensive resource, it supports functional genomics, systems biology, and translational research in the areas of cellular homeostasis, adaptation, and pathogenesis. ASTRA is freely accessible at https://astra-db.com/.