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Highly heterogenous cancers, such as triple-negative breast cancer (TNBC), remain challenging immunotherapeutic targets. Herein, we describe the synthesis and evaluation of immunotherapeutic liposomal spherical nucleic acids (SNAs) for TNBC therapy. The SNAs comprise immunostimulatory oligonucleotides (CpG-1826) as adjuvants and encapsulate lysates derived from TNBC cell lines as antigens. The resulting nanostructures (Lys-SNAs) enhance the codelivery of adjuvant and antigen to immune cells when compared to simple mixtures of lysates with linear oligonucleotides both in vitro and in vivo, and reduce tumor growth relative to simple mixtures of lysate and CpG-1826 (Lys-Mix) in both Py230 and Py8119 orthotopic syngeneic mouse models of TNBC. Furthermore, oxidizing TNBC cells prior to lysis and incorporation into SNAs (OxLys-SNAs) significantly increases the activation of dendritic cells relative to their nonoxidized counterparts. When administered peritumorally in vivo in the EMT6 mouse mammary carcinoma model, OxLys-SNAs significantly increase the population of cytotoxic CD8+ T cells and simultaneously decrease the population of myeloid derived suppressor cells (MDSCs) within the tumor micro-environment, when compared with Lys-SNAs and simple mixtures of oxidized lysates with CpG-1826. Importantly, animals administered OxLys-SNAs exhibit significant antitumor activity and prolonged survival relative to all other treatment groups, and resist tumor rechallenge. Together, these results show that the way lysates are processed and packaged has a profound impact on their immunogenicity and therapeutic efficacy. Moreover, this work points toward the potential of oxidized tumor cell lysate-loaded SNAs as a potent class of immunotherapeutics for cancers lacking common therapeutic targets.

Platelets are small anucleated blood cells primarily known for their vital hemostatic role. Allogeneic platelet concentrates (PCs) collected from healthy donors are an essential cellular product transfused by hospitals to control or prevent bleeding in patients affected by thrombocytopenia or platelet dysfunctions. Platelets fulfill additional essential functions in innate and adaptive immunity and inflammation, as well as in wound-healing and tissue-repair mechanisms. Platelets contain mitochondria, lysosomes, dense granules, and alpha-granules, which collectively are a remarkable reservoir of multiple trophic factors, enzymes, and signaling molecules. In addition, platelets are prone to release in the blood circulation a unique set of extracellular vesicles (p-EVs), which carry a rich biomolecular cargo influential in cell–cell communications. The exceptional functional roles played by platelets and p-EVs explain the recent interest in exploring the use of allogeneic PCs as source material to develop new biotherapies that could address needs in cell therapy, regenerative medicine, and targeted drug delivery. Pooled human platelet lysates (HPLs) can be produced from allogeneic PCs that have reached their expiration date and are no longer suitable for transfusion but remain valuable source materials for other applications. These HPLs can substitute for fetal bovine serum as a clinical grade xeno-free supplement of growth media used in the in vitro expansion of human cells for transplantation purposes. The use of expired allogeneic platelet concentrates has opened the way for small-pool or large-pool allogeneic HPLs and HPL-derived p-EVs as biotherapy for ocular surface disorders, wound care and, potentially, neurodegenerative diseases, osteoarthritis, and others. Additionally, allogeneic platelets are now seen as a readily available source of cells and EVs that can be exploited for targeted drug delivery vehicles. This article aims to offer an in-depth update on emerging translational applications of allogeneic platelet biotherapies while also highlighting their advantages and limitations as a clinical modality in regenerative medicine and cell therapies.

Background Foetal bovine serum (FBS), is the most commonly used culture medium additive for in vitro cultures, despite its undefined composition, its potential immunogenicity and possible prion/zoonotic transmission. For these reasons, significant efforts have been targeted at finding a substitute, such as serum free-media or human platelet-lysates (hPL). Our aim is to critically appraise the state-of-art for hPL in the published literature, comparing its impact with FBS. Materials and methods In June 2019 a systematic search of the entire Web of Science, Medline and PubMed database was performed with the following search terms: (mesenchymal stem cells) AND (fetal bovine serum OR fetal bovine calf) AND (human platelet lysate). Excluded from this search were review articles that were published before 2005, manuscripts in which mesenchymal stem cells (MSCs) were not from human sources, and when the FBS controls were missing. Results Based on our search algorithm, 56 papers were selected. A review of these papers indicated that hMSCs cultured with hPL showed a spindle-shaped elongated morphology, had higher proliferation indexes, similar cluster of differentiation (CD) markers and no significant variation in differentiation lineage (osteocyte, adipocyte, and chondrocyte) compared to those cultured with FBS. Main sources of primary hMSCs were either fat tissue or bone marrow; in a few studies cells isolated from alternative sources showed no relevant difference in their response. Conclusion Despite the difference in medium choice and a lack of standardization of hPL manufacturing, the majority of publications support that hPL was at least as effective as FBS in promoting adhesion, survival and proliferation of hMSCs. We conclude that hPL should be considered a viable alternative to FBS in hMSCs culture—especially with a view for their clinical use.

Asthma is a complex disease that often starts in childhood. Genomic and environmental factors as well as aberrant immune maturation early in life can contribute to the onset of disease, with great disparity over time and geographical regions. Epidemiological studies have scrutinised environmental exposures and attempted to translate these exposures into prevention strategies. Some approaches for patients with asthma have been successful (eg, smoking ban, the Finnish Asthma Programme), and primary prevention of wheeze in pre-school children (age 0–5 years) by the supplementation of vitamin D or fish oil, or both, to pregnant women seems promising. Several recent prevention initiatives are based on strong asthma-protective environmental microbial exposures associated with traditional rural lifestyles. Preclinical studies with various bacterial lysates, bacterial and dietary metabolites, or helminthic compounds have yielded promising results that await translation into clinical practice. Given the immense societal and individual burden of asthma, there is an urgent need to further develop novel strategies to eradicate the disease.

Ubiquitin-binding proteins play an important role in eukaryotes by translating differently linked polyubiquitin chains into proper cellular responses. Current knowledge about ubiquitin-binding proteins and ubiquitin linkage-selective interactions is mostly based on case-by-case studies. We have recently reported a method called ubiquitin interactor affinity enrichment-mass spectrometry (UbIA-MS), which enables comprehensive identification of ubiquitin interactors for all ubiquitin linkages from crude cell lysates. One major strength of UbIA-MS is the fact that ubiquitin interactors are enriched from crude cell lysates, in which proteins are present at endogenous levels, contain biologically relevant post-translational modifications (PTMs) and are assembled in native protein complexes. In addition, UbIA-MS uses chemically synthesized nonhydrolyzable diubiquitin, which mimics native diubiquitin and is inert to cleavage by endogenous deubiquitinases (DUBs). Here, we present a detailed protocol for UbIA-MS that proceeds in five stages: (i) chemical synthesis of ubiquitin precursors and click chemistry for the generation of biotinylated nonhydrolyzable diubiquitin baits, (ii) in vitro affinity purification of ubiquitin interactors, (iii) on-bead interactor digestion, (iv) liquid chromatography (LC)-MS/MS analysis and (v) data analysis to identify differentially enriched proteins. The computational analysis tools are freely available as an open-source R software package, including a graphical interface. Typically, UbIA-MS allows the identification of dozens to hundreds of ubiquitin interactors from any type of cell lysate, and can be used to study cell type or stimulus-dependent ubiquitin interactions. The nonhydrolyzable diubiquitin synthesis can be completed in 3 weeks, followed by ubiquitin interactor enrichment and identification, which can be completed within another 2 weeks.

BackgroundPostbiotics, non-viable microbial components with health benefits, may offer potential advantages in the management of IBS but the literature is limited. This very first systematic review and meta-analysis evaluates the efficacy and safety of postbiotics in the treatment of IBS.MethodsWe searched PubMed, Scopus, Embase, and Web of Science from inception to April 2025 for randomized controlled trials (RCTs) assessing the efficacy and safety of postbiotics in IBS diagnosed based on Rome III/IV criteria. Primary outcomes were IBS Symptom Severity Scale (IBS-SSS) scores and pain intensity reduction; secondary outcomes include quality of life (QoL) and treatment-emergent adverse events (TEAE). The risk of bias was assessed using Cochrane RoB 2.0. Random-effects meta-analysis was performed using RevMan 5.4.ResultsOf 1,053 records, 768 duplicates were removed, 281 articles were excluded, and eventually, four RCTs were analysed (IDDF2025-ABS-0349 figure 1. PRISMA Flow Diagram). All studies were parallel-group RCTs conducted in Europe (2020–2024). Postbiotic formulations varied: heat-killed bacteria (2 studies), bacterial lysates (1), and fermented preparations (1). Administration routes included oral capsules (3 studies) and enema (1) (IDDF2025-ABS-0349 table 1). The risk of bias was low overall (Cochrane RoB 2.0), except for one study with unclear allocation concealment. (IDDF2025-ABS-0349 figure 2. Risk of bias assessment)Abstract IDDF2025-ABS-0349 Table 1Characteristics of included studies POPULATIONINTERVENTIONCOMPARATOR Author/yearLocationYearDesignRome CriteriaIBS subtypeSample SizeAgePostbiotic usedDose&DurationSample SizeAgeMAAndersen 2020Germany2020RCTROME II all types22140–1 (12–8)Non-viable B bifidum HI-MMBb75 cells2 capsules once daily for 8 weeks22242–6 (13–8)A,B,C,DMack 2021Germany2021RCTROME IIall types19147.47 (15.66)Suspension of Nonviable bacterial lysate of E coli(DSM17252) and E faecalis (DSM16440)10 drops*3/day for week then m20*3/day for week then 30*3/day for 24 weeks (total 26weeks)19845.86(15.90)B,DBednarska 2022Seweden2022RCTROME IVIBS-D, mixed1837 (19–55)Referm oat gruel fermented with Lactobacillus plantarum 299vEnema, 250 ml twice daily, for 2 weeks1237(19–55)CSrivastavaIndia2024RCTROME IVIBS-D6834.88 (9.56)Heat-treated Bifidobacterium longum CECT 7347 (HT-ES1)2 capsules once daily for 12 weeksProbiotic: 65,placebo: 67Probiotic: 37.28 (11.04), placebo: 35.73 (11.42)A,B,C,DPostbiotics significantly improved IBS symptom severity (P < 0.001) with moderate heterogeneity (I2 = 69%; P = 0.07). For pain intensity, postbiotics also showed significant reduction (P = 0.02) with substantial heterogeneity (I2 = 87%; P = 0.0005). QoL improvement was significant after excluding one study using postbiotic enema preparation (P < 0.001) with no residual heterogeneity (I2 = 0%). Safety analysis found no significant difference in TEAE between postbiotics and placebo (RR 0.68, 95% CI 0.35–1.34; P = 0.26; I2 = 54%) (IDDF2025-ABS-0349 figure 3. Meta-analyses of outcomes).Abstract IDDF2025-ABS-0349 Figure 1PRISMA flow diagram[Figure omitted. See PDF]Abstract IDDF2025-ABS-0349 Figure 2Risk of bias assessment[Figure omitted. See PDF]Abstract IDDF2025-ABS-0349 Figure 3Meta-analyses of outcomes[Figure omitted. See PDF]ConclusionsPostbiotics are efficacious in alleviating IBS symptom severity and intensity, QoL is better and exhibits a favorable safety profile. Heterogeneity in study outcomes is due to variability in formulations and study protocols.

Chemoproteomics is a key technology to characterize the mode of action of drugs, as it directly identifies the protein targets of bioactive compounds and aids in the development of optimized small-molecule compounds. Current approaches cannot identify the protein targets of a compound and also detect the interaction surfaces between ligands and protein targets without prior labeling or modification. To address this limitation, we here develop LiP-Quant, a drug target deconvolution pipeline based on limited proteolysis coupled with mass spectrometry that works across species, including in human cells. We use machine learning to discern features indicative of drug binding and integrate them into a single score to identify protein targets of small molecules and approximate their binding sites. We demonstrate drug target identification across compound classes, including drugs targeting kinases, phosphatases and membrane proteins. LiP-Quant estimates the half maximal effective concentration of compound binding sites in whole cell lysates, correctly discriminating drug binding to homologous proteins and identifying the so far unknown targets of a fungicide research compound. Proteomics is often used to map protein-drug interactions but identifying a drug’s protein targets along with the binding interfaces has not been achieved yet. Here, the authors integrate limited proteolysis and machine learning for the proteome-wide mapping of drug protein targets and binding sites.

With the emphasis on intestinal health, probiotics have exploded into a vast market potential. However, new scientific evidence points out that the beneficial health benefits of probiotics are not necessarily directly related to viable bacteria. However, the metabolites or bacterial components of the live bacteria are the driving force behind health promotion. Therefore, scientists gradually noticed that the beneficial effects of probiotics are based on bacteria itself, metabolites, or cell lysates, and these factors are officially named “postbiotics” by the ISAPP. Postbiotic components are diverse and outperform live probiotics in terms of technology, safety, and cost due to their good absorption, metabolism, and organismal distribution. Postbiotics have been shown to have bioactivities such as antimicrobial, antioxidant, anti-inflammatory, anti-proliferative, and immunomodulation. Moreover, numerous studies have revealed the significant potential of postbiotics for disease treatment. This paper first presents the production and classification of postbiotics with examples from lactic acid bacteria (LAB), followed by the mechanisms of action with the most recent pre-clinical and clinical studies and the wide range of non-clinical and clinical applications of postbiotics. Furthermore, the current and future prospects of the postbiotic market with commercial available products are discussed. Finally, we comment on the knowledge gaps and future clinical applications with several examples.

A great deal of evidence has accumulated suggesting an important role of mucosal immunity not only in preventing COVID-19 but also in the pathogenesis of this infection. The aim of the study was to evaluate the levels of secretory immunoglobulin A (sIgA) in different compartments of the upper respiratory tract in COVID-19 patients in relation to the severity of the disease and treatment with a bacteria-based immunomodulating agent (Immunovac VP4). The titers of sIgA were determined by ELISA in nasal epithelial swabs, pharyngeal swabs, and salivary gland secretions at baseline and on days 14 and 30 of treatment. The levels of nasal, pharyngeal and salivary sIgA were significantly lower in more severe patients (subgroup A) than in less severe patients (subgroup B), p  < 0.01. In subgroup A, the patients who received Immunovac VP4 had higher pharyngeal sIgA levels in convalescent period than those who did not receive the therapy p  < 0.05. In subgroup B patients, an increase in immunoglobulin levels was observed from baseline to day 14 of treatment whether they received the add-on therapy or not, p  < 0.01. On day 30 of treatment, the sIgA levels in the standard treatment group, however, decreased, while the patients receiving the immunomodulating agent maintained high sIgA levels, p  < 0.05. Oxygen saturation significantly increased by day 14 in both groups, p  < 0.001. However, it was higher in the Immunovac VP4 group than in the standard treatment group, p  < 0.01. Thus, addition of a bacterial lysate-based immunomodulating agent to the treatment regimen for moderate-to-severe COVID-19 induces the production of pharyngeal and salivary sIgA. SIgA production is inversely correlated to CRP levels and percentage of lung involvement on CT scan and is directly correlated to SpO 2 levels.

The design and optimization of biosynthetic pathways for industrially relevant, non-model organisms is challenging due to transformation idiosyncrasies, reduced numbers of validated genetic parts and a lack of high-throughput workflows. Here we describe a platform for in vitro prototyping and rapid optimization of biosynthetic enzymes (iPROBE) to accelerate this process. In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE by screening 54 different cell-free pathways for 3-hydroxybutyrate production and optimizing a six-step butanol pathway across 205 permutations using data-driven design. Observing a strong correlation ( r  = 0.79) between cell-free and cellular performance, we then scaled up our highest-performing pathway, which improved in vivo 3-HB production in Clostridium by 20-fold to 14.63 ± 0.48 g l −1 . We expect iPROBE to accelerate design–build–test cycles for industrial biotechnology. The iPROBE platform accelerates the design and optimization of engineered biosynthetic pathways using a combination of cell-free protein synthesis, in vitro pathway assembly and a scoring system to identify high-performing combinations.