IKKα is a serine/threonine kinase that acts as a tumour suppressor in non-small cell lung cancer (NSCLC) in an NF-κΒ-independent manner, however, little is known about its downstream signalling pathways. To interrogate the IKKα signalling network in NSCLC, we investigated the impact of IKKα silencing on the miRNA expression profile of NSCLC cells. Nanostring miRNome analysis identified miR-9-5p, a known oncomir, as the top upregulated miRNA upon IKKα depletion. We show that overexpression of miR-9-5p in human lung cancer cells increases cell migration and invasion and promotes epithelial-to-mesenchymal cell transition (EMT) through loss of E-cadherin and activation of the Akt1/β-catenin pathway. In vivo tumour xenograft models show that overexpression of miR-9-5p promotes tumour growth and widespread transcriptomic reprogramming, altering the expression of genes related to EMT, apoptosis and NF-κB signalling. Overall, we show that miR-9-5p acts as an oncogenic effector upon IKKα loss, promoting tumour progression through EMT and Akt-1/GSK-3β/β-catenin pathway activation, leading to increased invasiveness and tumour growth.

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/.

European hake, common sole and European eel represent economically important species in the Mediterranean basin and are commonly sold all over Europe. Reviewing of literature revealed that these species are highly prone to substitution, such as Merluccius merluccius by other hake species, Solea solea by other low-cost flatfish, and Anguilla anguilla by other eel species from China. Here we present the development of three methods for the rapid and accurate detection of possible substitutes using COI and cytb mtDNA genes. Conventional PCR through agarose screening and real-time PCR protocols are deployed, showing that all methods are able to provide accurate results and differentiate the target species. However, the results are strongly associated with DNA quantity, with High-Resolution-Melting (HRM) analysis showing the higher efficiency compared to the low-melting curve real-time PCR and conventional PCR. In our dataset we detected mislabeling of frozen M. hubbsi filet by M. capensis. Moreover, S. aegyptiaca specimens were mislabeled as S. solea in fish markets. However, the latter may be due to the extremely difficult morphological identification of the two sole species. The findings demonstrate the necessity of regulating processed fish-containing seafood and the feasibility of the suggested technology for tracing them. The findings are discussed in terms of public health hazards and conservation consequences, in addition to the financial losses brought on by mislabeling and seafood species substitution.

The Thracian Sea, a semi-enclosed coastal basin in the northeastern Aegean Sea, represents a dynamic marine environment influenced by freshwater inputs, stratification, and seasonal variability. Here, we investigated the spatiotemporal dynamics of microbial and ichthyofaunal communities using environmental DNA (eDNA) and high-throughput sequencing across various stations in the vicinity of the Thracian Sea, in consecutive months (through spring and summer). Seawater samples were collected from the surface and thermocline layers, and environmental parameters were recorded to examine their influence on biodiversity patterns. Microbial communities exhibited strong seasonal and depth-related structuring. Alpha diversity was highest in spring and declined during summer, while beta diversity analyses revealed clear clustering by month and depth. Dominant taxa included Alphaproteobacteria (SAR11), Cyanobacteria (Synechococcus, Prochlorococcus), with distinct core microbiomes. Fish communities, identified via CytB metabarcoding, displayed marked temporal turnover but limited spatial segregation. While alpha diversity metrics did not differ significantly, beta diversity analyses showed seasonal shifts with dominant taxa such as Raja spp., Engraulis spp., and Diplodus sargus. Multivariate and co-structure analyses (Mantel, Procrustes) revealed moderate but significant concordance between microbial and fish communities and support the existence of similar biodiversity responses to environmental parameters across temporal and spatial variability. Co-occurrence networks further present depth-specific associations, with surface communities being more cooperative and phototrophic, while thermocline networks showed modularity and potential ecological specialization. This study highlights the value of integrated eDNA-based monitoring in revealing seasonal biodiversity dynamics and ecological interactions in coastal marine ecosystems, supporting future spatial planning and conservation strategies in the Thracian Sea.

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.

Identification of a plant’s pollen components can be used to establish its geographical provenance, while also providing insights into the diet and foraging preferences of the honeybee (Apis mellifera L.). The diversity and amount of pollen represent crucial factors for pollinators. Here, we identified plant species visited by honeybees by analyzing the pollen pellets collected from honeybees in Kastoria, Greece. The results indicate that pollen from different periods was identified by means of floral composition. An interesting observation is that all identified plants belonged to different genera. Among the identified plants, native ones, such as the Macedonian pine, Pinus peuce, present a distinct foraging profile for local honeybees.

Immunogenetic evidence implicates the B cell receptor immunoglobulin (BcR IG) in the natural history of splenic marginal zone lymphoma (SMZL). Indeed, SMZL carries distinct features of somatic hypermutation (SHM) amongst cases utilizing particular IGHV genes in their BcR IG. Moreover, the BcR IG gene repertoire in SMZL is restricted, whereby ~30% of cases utilize the IGHV1-2*04 gene and allele, notable for carrying a tryptophan (W) residue at position VH FR3-75 instead of the arginine (R) residue that is encoded by all remaining IGHV1-2 gene alleles and almost all other human IGHV genes and alleles. IGHV1-2*04 cases display heterogeneous CDR3 features and light chains, as well as pronounced intraclonal diversification within the heavy chain gene rearrangements. Overall, this constellation of features argues for heavy chain dominance in this major immunogenetic subgroup of SMZL. Against that, scant information exists regarding the functional and structural properties of the clonotypic BcR IG in SMZL, while the relevance of the IGHV1-2*04 overuse remains an enigma. In order to address this knowledge gap, we performed immunological, biochemical and crystallographic studies of the clonotypic BcR IG from SMZL cases that we expressed as recombinant monoclonal antibodies (rmAbs). ELISA against molecules that are common antigenic targets of naturally-occurring autoantibodies and disease-occurring/related pathological autoantibodies, namely DNA, actin, myosin, β-amyloid, TNP, thyroglobulin, IgG F(ab)'2 fragments, and trinitrophenyl (TNP), revealed that SMZL rmAbs (n=42, of which 14 utilized the IGHV1-2*04) were poly/autoreactive, regardless of the expressed IGHV gene and their SHM status. Of note, comparison with CLL rmAbs (n=35), known for their poly/autoreactivity, revealed significantly (p<0.05) stronger binding of the SMZL rmAbs to all tested autoantigens on autoantigen protein microarrays. Autoreactivity was corroborated by flow cytometry showing that 38/42 SMZL rmAbs bound viable MEC1, HEK293 and HS-5 cells; and, by immunohistochemistry using the SMZL rmAbs as primary Abs, which documented binding to human tissues (appendix, tonsil, lymph nodes and kidney), albeit with distinct profiles depending on the expressed IGHV gene. Next, in order to assess the functional relevance of the W residue in IGHV1-2*04 BcR IG, we used PCR-based site-directed mutagenesis for modifying W75 to R (IGHV1-2*04 W75R, n=10), documenting significant (p<0.05) differences in antigen reactivity profile between the authentic IGHV1-2*04 rmAbs vs the respective IGHV1-2*04 W75R rmAbs. Further, in order to test the hypothesis of heavy chain dominance, we coupled the IGHV1-2*04 heavy chains with random light chains and compared their antigen reactivity profile versus the authentic rmAbs, observing no differences. Finally, we crystallized two different IGHV1-2*04 SMZL BcR Fabs and determined their structures to high resolution. In all crystals obtained, regardless of pH and precipitating agent, pairs of Fab molecules were found to interact homotypically through a symmetrical stacking cation-π interaction mediated by the allele-specific W75 residue and side chain of R95 in the IGHV1-2*04 heavy chain, burying an extensive surface of ~650 Å2. In silico analysis with FOLDX highlighted the high energetic contribution of these residues in promoting the formation of the homodimers in the crystals. In conclusion, we document pronounced poly/autoreactivity in SMZL suggesting that SMZL likely derives from a progenitor B-cell population with highly restricted BcR IG structures and autoreactive potential. We also provide experimental support to the hypothesis of heavy chain dominance in the IGHV1-2*04 subgroup which raises the intriguing possibility that the IGHV1-2*04 heavy chain may represent a target for immunotherapeutic interventions in a sizeable fraction of SMZL.

The adoptive transfer of T cell receptor-engineered (TCR-engineered) T cells (ACT) targeting the HLA-A2-restricted cancer-testis epitope NY-ESO-1157-165 (A2/NY) has yielded favorable clinical responses against several cancers. Two approaches to improve ACT are TCR affinity optimization and T cell coengineering to express immunomodulatory molecules that can exploit endogenous immunity. By computational design we previously developed a panel of binding-enhanced A2/NY-TCRs including A97L, which augmented the in vitro function of gene-modified T cells as compared with WT. Here, we demonstrated higher persistence and improved tumor control by A97L-T cells. In order to harness macrophages in tumors, we further coengineered A97L-T cells to secrete a high-affinity signal regulatory protein α (SiRPα) decoy (CV1) that blocks CD47. While CV1-Fc-coengineered A97L-T cells mediated significantly better control of tumor outgrowth and survival in Winn assays, in subcutaneous xenograft models the T cells, coated by CV1-Fc, were depleted. Importantly, there was no phagocytosis of CV1 monomer-coengineered T cells by human macrophages. Moreover, avelumab and cetuximab enhanced macrophage-mediated phagocytosis of tumor cells in vitro in the presence of CV1 and improved tumor control upon coadministration with A97L-T cells. Taken together, our study indicates important clinical promise for harnessing macrophages by combining CV1-coengineered TCR-T cells with targeted antibodies to direct phagocytosis against tumor cells.

PDF file - 1935K, Figure S3. shRNA based silencing of FRa in A1847 ovarian cancer cells. The parental human ovarian cancer cell line A1847 which expresses surface mesothelin and FRa protein was transduced with lentiviral particles encoding for an shRNA specific for silencing FRa gene expression (A1847 sh4 M+/F-). To determine surface antigen expression, cells were stained with an anti-mesothelin biobody reagent (P4 Bb) and anti-FRa antibody or proper isotype antibody controls. FRa expression was unaltered after engineering cells with control shRNA (A1847 csh M+/F+).

PDF file - 583K, Figure S6. Trans-, but not cis-, signaling CART cells exhibit more limited in vitro activity against cells bearing single antigen. IFN-γ secretion by trans M-z/F-28 CART cells was significantly reduced in response to A1847M+/F- cells compared with parental A1847M+/F+. M-z, M-z/F-28, or M-28z transduced T cells (1 x 105 T cells) were cultured alone (none) or stimulated overnight with an equal number of C30, A1847M+/F+ or A1847M+/F- cells. Cell-free supernatant was harvested after ~20 hours of incubation and the IFN- γ levels were measure with ELISA. Mean IFN- γ concentration plus-minus SEM (pg/ml) from triplicate cultures is shown. *P < 0.05 comparing the IFN- γ secretion produced by M-z/F-28 CART cells against A1847M+/F+ versus A1847M+/F- cells.

Background: CLL subset #4 is the largest stereotyped subset in IGHV-mutated CLL (M-CLL). The clonotypic B cell receptor immunoglobulin (BcR IG) in subset #4, encoded by the IGHV4-34/IGKV3-20 gene pair, displays long heavy complementarity determining region 3 (VH CDR3), enriched in positively charged residues; ubiquitous expression of gamma heavy chain isotype; distinctive imprint of somatic hypermutation (SHM), characterized by the frequent introduction of acidic residues and pronounced intraclonal diversification. These features are reminiscent of edited autoantibodies, implicating ongoing (auto)antigen interactions in the natural history of CLL subset #4. Aims: Here, we sought to explore the immune trajectory and clonal dynamics of CLL subset #4, particularly focused on the role of SHM. Methods: We studied longitudinal samples from 6 subset #4 and 6 non-subset #4 IGHV4-34-expressing M-CLL cases. Clonotypic IG heavy (HC) and light chain (LC) gene rearrangements were profiled by next generation sequencing (NGS). The clonotype accounting for the majority of NGS reads in a given sample was defined as dominant; related clonotypes expressing the same IGHV/IGKV gene and VH CDR3 amino acid (aa) sequence yet differing in the aa sequence of the VH and/or VK domains were defined as subclonotypes. The dominant clonotypes of all cases as well as 32 subclonotypes of subset #4 cases were expressed as recombinant monoclonal antibodies (rmAbs) and screened for antigen reactivity using ELISA and flow cytometry. Results: NGS revealed two distinct patterns of subclonal architecture in the IGH repertoire of subset #4. The first was characterized by the presence of a single dominant clonotype (4 cases) whereas the second by the co-existence of ≥2 significantly expanded clonotypes with comparable frequencies (2 cases). Turning to the LC, 5/6 cases carried a single dominant clonotype, while 1 case carried ≥1 expanded clonotypes. In contrast, a single dominant clonotype was detected in all non-subset #4 cases for both HC and LC. Longitudinal analysis revealed significantly (p<0.05) more pronounced subclonal drift (changing relative frequencies) of the expanded clonotypes in subset #4 vs non-subset #4 cases, particularly for the HC. Surprisingly, in all subset #4 cases we identified clonotypic IgG transcripts (same IGH gene and VH CDR3 length, high VH CDR3 aa sequence identity) with 100% germline IGHV identity (‘truly unmutated’), present at low frequency (median: 0.003%, range: 0.0003-0.03%). Prompted by this, we produced rmAbs for both the dominant, somatically hypermutated BcR IG clonotype of all 6 subset #4 cases, as well as 32 truly unmutated subclonotypes (from all studied subset #4 cases) and compared their antigen reactivity profiles. We report consistent, significantly (p<0.05) stronger reactivity of the truly unmutated, subclonotypic BcR IG compared to the mutated BcR IG expressed by the dominant CLL #4 clone. With few exceptions, all subclonotypic truly unmutated BcR IG displayed more intense recognition against dsDNA and lipopolysaccharides, antigenic elements from CMV, Influenza A and Mycoplasma pneumoniae as well as viable HEK293 and Jurkat T cells. Summary/Conclusion: Our findings support that SHM in CLL subset #4 is functionally driven by persistent selection by (auto)antigens, ultimately leading to a significant redemption from autoreactivity. The identification of truly unmutated clonotypic IgG gene transcripts likely reflects a complex trajectory of clonal evolution, offering hints about the precise timing of SHM in relation to class switch recombination in the natural history of CLL subset #4. Keywords: Antigen presentation, B cell chronic lymphocytic leukemia, B cell lymphoma, Antibody

Supplementary Figure and Table Legends - PDF file100K, Legend for Supplementary Figures S1-S5 and Supplementary Tables S1-S2

Supplementary Figure S4 - PDF file 1076K, ELISA validation of isolated anti-B7-H4 scFvs

Supplementary Materials and Methods - PDF file 113K, Additional experimental procedures including Western Blot, Flow Cytometry, and Xenograft models

Supplementary Table S1 - PDF file 1334K, B7-H4 expression on tumor cells derived from human ovarian cancer ascites and solid tumors

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.

Supplementary Figure S1 - PDF file 1189K, Cloning, expression and purification of recombinant B7-H4

Supplementary Figure S2 - PDF file 1219K, Analysis of B7-H4 expression in human ovarian cancer cells

PDF file - 514K, Figure S4. A1847 (M+/F+) and A1847 (M+/F-) cells exhibit similar in vitro growth rate. 1 x 106 A1847 (M+/F+) and A1847 (M+/F-) cells were seeded on day 0 and their in vitro growth kinetics were monitored by determining viable cells using the trypan blue exclusion method.