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It is generally accepted that dry-aged buffalo (B. bubalis) meat becomes dark faster than bovine (B. taurus) meat, discouraging consumer purchase. We have investigated whether this faster darkening process might depend on structural and/or kinetic differences between buffalo and bovine myoglobins (Mbs). To this end, we have purified to homogeneity buffalo Mb from Longissimus dorsi muscle and obtained both its Mr (17,034.5) and the complete amino acid sequence, which, compared with the bovine one, showed three amino acid substitutions: Dbo141Ebu, Abo19Tbu and Abo117Dbu. As revealed by the 3D structure, they were located on the surface of the protein, far from the heme binding pocket, and did not cause appreciable structural changes. Autoxidation rates of purified buffalo and bovine myoglobins at 37 °C, pH 7.2, were almost identical (0.052±0.001 h−1 and 0.054±0.002 h−1, respectively), as were their oxygen-binding Kd values (3.7±0.1 μM and 3.5± 0.1 μM, respectively). These data indicate that the structure of the heme pockets in the two proteins is similar, with similar functional properties. Moreover, the percent of MetMb values in the purified buffalo and bovine samples, after the same time from slaughtering, were almost identical (57% and 47%, respectively). The results presented here suggest that the faster darkening of buffalo meat depends on factors other than the oxidation rate of its Mb, as, for example, the Mb content (0.393±0.005 g/100 g) and consequently its MetMb content, which is almost twice as high as bovine meat (0.209±0.003 g/100 g), and likely other factors.

Abstract Background The pattern recognition receptor long pentraxin-3 (PTX3) plays conflicting roles in cancer by acting as an oncosuppressor or as a pro-tumor mediator depending on tumor context. Triple negative breast cancer (TNBC) represents the most aggressive histotype of breast cancer, characterized by the lack of efficacious therapeutic targets/approaches and poor prognosis. Thus, the characterization of new molecular pathways and/or alternative druggable targets is of great interest in TNBC. Methods The expression of PTX3 in BC tumor samples and in BC cell lines has been analyzed using the Gene Expression-Based Outcome for Breast Cancer Online (GOBO), qPCR, Western blot and ELISA assay. The contribution of tumor and stromal cells to PTX3 production in TNBC was assessed by analyzing single cell RNA sequencing data and RNAscope performed on TNBC tumor samples. In order to investigate the effects of PTX3 in TNBC, different cell lines were engineered to knock-down (MDA-MB-231 and BT549 cells) or overexpress (MDA-MB-468 and E0771 cells) PTX3. Finally, using these engineered cells, in vitro (including gene expression profiling and gene set enrichment analyses) and in vivo (orthotopic tumor models in immune-compromised and immune competent mice) analyses were performed to assess the role and the molecular mechanism(s) exerted by PTX3 in TNBC. Results In silico and experimental data indicate that PTX3 is mainly produced by tumor cells in TNBC and that its expression levels correlate with tumor stage. Accordingly, gene expression and in vitro results demonstrate that PTX3 overexpression confers a high aggressive/proliferative phenotype and fosters stem-like features in TNBC cells. Also, PTX3 expression induces a more tumorigenic potential when TNBC cells are grafted orthotopically in vivo. Conversely, PTX3 downregulation results in a less aggressive behavior of TNBC cells. Mechanistically, our data reveal that PTX3 drives the activation of the pro-tumorigenic Toll-like receptor 4 (TLR4) signaling pathway in TNBC, demonstrating for the first time that the PTX3/TLR4 autocrine stimulation loop contributes to TNBC aggressiveness and that TLR4 inhibition significantly impacts the growth of PTX3-producing TNBC cells. Conclusion Altogether, these data shed light on the role of tumor-produced PTX3 in TNBC and uncover the importance of the PTX3/TLR4 axis for therapeutic and prognostic exploitation in TNBC. Graphical abstract

Ribosome-inactivating proteins (RIPs) are a group of proteins with rRNA N-glycosylase activity that irreversibly inhibit protein synthesis and consequently cause cell death. Recently, an RIP called ledodin has been found in shiitake; it is cytotoxic, strongly inhibits protein synthesis, and shows rRNA N-glycosylase activity. In this work, we isolated and characterized a 50 kDa cytotoxic protein from shiitake that we named edodin. Edodin inhibits protein synthesis in a mammalian cell-free system, but not in insect-, yeast-, and bacteria-derived systems. It exhibits rRNA N-glycosylase and DNA-nicking activities, which relate it to plant RIPs. It was also shown to be toxic to HeLa and COLO 320 cells. Its structure is not related to other RIPs found in plants, bacteria, or fungi, but, instead, it presents the characteristic structure of the fold type I of pyridoxal phosphate-dependent enzymes. Homologous sequences have been found in other fungi of the class Agaricomycetes; thus, edodin could be a new type of toxin present in many fungi, some of them edible, which makes them of great interest in health, both for their involvement in food safety and for their potential biomedical and biotechnological applications.

Nuzzo, Russo, Errico, D’Amico et al. investigate neuroinflammation in forty-eight pediatric spinal muscular atrophy patients before and after Nusinersen treatment. They find signatures of neuroinflammation that are specifically associated with severe disease and show that Nusinersen therapy has neuro-immunomodulatory effects.

Abstract Background Imprinting Control Regions (ICRs) are CpG-rich sequences acquiring differential methylation in the female and male germline and maintaining it in a parental origin-specific manner in somatic cells. Despite their expected high mutation rate due to spontaneous deamination of methylated cytosines, ICRs show conservation of CpG-richness and CpG-containing transcription factor binding sites in mammalian species. However, little is known about the mechanisms contributing to the maintenance of a high density of methyl CpGs at these loci. Results To gain functional insights into the mechanisms for maintaining CpG methylation, we sought to identify the proteins binding the methylated allele of the ICRs by determining the interactors of ZFP57 that recognizes a methylated hexanucleotide motif of these DNA regions in mouse ESCs. By using a tagged approach coupled to LC–MS/MS analysis, we identified several proteins, including factors involved in mRNA processing/splicing, chromosome organization, transcription and DNA repair processes. The presence of the post-replicative mismatch-repair (MMR) complex components MSH2 and MSH6 among the identified ZFP57 interactors prompted us to investigate their DNA binding profile by chromatin immunoprecipitation and sequencing. We demonstrated that MSH2 was enriched at gene promoters overlapping unmethylated CpG islands and at repeats. We also found that both MSH2 and MSH6 interacted with the methylated allele of the ICRs, where their binding to DNA was mediated by the ZFP57/KAP1 complex. Conclusions Our findings show that the MMR complex is concentrated on gene promoters and repeats in mouse ESCs, suggesting that maintaining the integrity of these regions is a primary function of highly proliferating cells. Furthermore, the demonstration that MSH2/MSH6 are recruited to the methylated allele of the ICRs through interaction with ZFP57/KAP1 suggests a role of the MMR complex in the maintenance of the integrity of these regulatory regions and evolution of genomic imprinting in mammalian species.

Herein, we developed an innovative and easily accessible solid-phase synthetic protocol for Peptide Nucleic Acid (PNA) oligomers by systematically investigating the ultrasonication effects in all steps of the PNA synthesis (US-PNAS). When compared with standard protocols, the application of the so-obtained US-PNAS approach succeeded in improving the crude product purities and the isolated yields of different PNA, including small or medium-sized oligomers (5-mer and 9-mer), complex purine-rich sequences (like a 5-mer Guanine homoligomer and the telomeric sequence TEL-13) and longer oligomers (such as the 18-mer anti-IVS2-654 PNA and the 23-mer anti-mRNA 155 PNA). Noteworthy, our ultrasound-assisted strategy is compatible with the commercially available PNA monomers and well-established coupling reagents and only requires the use of an ultrasonic bath, which is a simple equipment generally available in most synthetic laboratories.

Abstract Progressive myoclonus epilepsy (PME) of Unverricht–Lundborg type (EPM1) is an autosomal recessive neurodegenerative disorder with the highest incidence of PME worldwide. Mutations in the gene encoding cystatin B (CSTB) are the primary genetic cause of EPM1. Here, we investigate the role of CSTB during neurogenesis in vivo in the developing mouse brain and in vitro in human cerebral organoids (hCOs) derived from EPM1 patients. We find that CSTB (but not one of its pathological variants) is secreted into the mouse cerebral spinal fluid and the conditioned media from hCOs. In embryonic mouse brain, we find that functional CSTB influences progenitors’ proliferation and modulates neuronal distribution by attracting interneurons to the site of secretion via cell‐non‐autonomous mechanisms. Similarly, in patient‐derived hCOs, low levels of functional CSTB result in an alteration of progenitor's proliferation, premature differentiation, and changes in interneurons migration. Secretion and extracellular matrix organization are the biological processes particularly affected as suggested by a proteomic analysis in patients’ hCOs. Overall, our study sheds new light on the cellular mechanisms underlying the development of EPM1.

Abstract Background We have previously shown that HCC patients and healthy subjects are equally responsive to a RNAdjuvant®, a novel TLR-7/8/RIG-I agonist based on noncoding RNA developed by CureVac, by an ex vivo evaluation. However, the immunological effect of adjuvants on immune cells from cancer patients undergoing chemotherapy remains to be demonstrated. Different adjuvants currently used in cancer vaccine clinical trials were evaluated in the present study on immune cells from cancer patients before and after chemotherapy in an ex vivo setting. Methods PBMCs were obtained from 4 healthy volunteers and 23 patients affected by either colon (OMA) or lung cancer (OT). The effect of CpG, Poly I:C, Imiquimod and RNA-based adjuvant (RNAdjuvant®) was assessed using a multiparametric approach to analyze network dynamics of early immune responses. Evaluation of CD80, CD86 and HLA-DR expression as well as the downstream effect on CD4+ T cell phenotyping was performed by flow cytometry; cytokine and chemokine production was evaluated by Bio-Plex ProTM. Results Treatment with RNAdjuvant® induced the strongest response in cancer patients in terms of activation of innate and adoptive immunity. Indeed, CD80, CD86 and HLA-DR expression was found upregulated in circulating dendritic cells, which promoted a CD4+ T cell differentiation towards an effector phenotype. RNAdjuvant® was the only one to induce most of the cytokines/chemokines tested with a pronounced Th1 cytokine pattern. According to the different parameters evaluated in the study, no clear cut difference in immune response to adjuvants was observed between healthy subjects and cancer patients. Moreover, in the latter group, the chemotherapy treatment did not consistently correlate to a significant altered response in the different parameters. Conclusions The present study is the first analysis of immunological effects induced by adjuvants in cancer patients who undergo chemotherapy, who are enrolled in the currently ongoing cancer vaccine clinical trials. The results show that the RNAdjuvant® is a potent and Th1 driving adjuvant, compared to those tested in the present study. Most importantly, it is demonstrated that chemotherapy does not significantly impair the immune system, implying that cancer patients are likely to respond to a cancer vaccine even after a chemotherapy treatment.

Gamma-ray emission provides constraints on the nonthermal radiation processes at play in astrophysical particle accelerators. This allows both the nature of accelerated particles and the maximum energy that they can reach to be determined. Notably, it remains an open question to what extent supernova remnants contribute to the sea of Galactic cosmic rays. In the Galactic plane, at around 312° of Galactic longitude, Fermi-LAT observations show an extended source (4FGL J1409.1−6121e) around five powerful pulsars. This source is described by one large disk of 0.°7 radius with a high significance of 45 σ in the 4FGL-DR3 catalog. Using 14 yr of Fermi-LAT observations, we revisited this region with a detailed spectro-morphological analysis in order to disentangle its underlying structure. Three sources have been distinguished, including the supernova remnant G312.4−0.4 whose gamma-ray emission correlates well with the shell observed at radio energies. The hard spectrum detected by the LAT, extending up to 100 GeV without any sign of a cutoff, is well reproduced by a purely hadronic model.

Cetuximab is a monoclonal antibody blocking the epidermal growth factor receptor (EGFR) in metastatic colorectal cancer (mCRC). However, cetuximab treatment has no clinical benefits in patients affected by mCRC with KRAS mutation or in the presence of constitutive activation of signalling pathways acting downstream of the EGFR. The aim of this study was to improve cetuximab’s therapeutic action by conjugating cetuximab with the type 1 ribosome inactivating protein (RIP) quinoin isolated from quinoa seeds. A chemical conjugation strategy based on the use of heterobifunctional reagent succinimidyl 3-(2-pyridyldithio)propionate (SPDP) was applied to obtain the antibody-type 1 RIP chimeric immunoconjugate. The immunotoxin was then purified by chromatographic technique, and its enzymatic action was evaluated compared to quinoin alone. Functional assays were performed to test the cytotoxic action of the quinoin cetuximab immunoconjugate against the cetuximab-resistant GEO-CR cells. The novel quinoin cetuximab immunoconjugate showed a significant dose-dependent cytotoxicity towards GEO-CR cells, achieving IC50 values of 27.7 nM (~5.0 μg/mL) at 72 h compared to cetuximab (IC50 = 176.7 nM) or quinoin (IC50 = 149.3 nM) alone assayed in equimolar amounts. These results support the therapeutic potential of quinoin cetuximab immunoconjugate for the EGFR targeted therapy, providing a promising candidate for further development towards clinical use in the treatment of cetuximab-resistant metastatic colorectal cancer.

BackgroundRecent evidence suggests that a lower extent of the retronasal aroma release correspond to a higher amount of ad libitum food intake. This has been regarded as one of the bases of behavioral choices towards food consumption in obese people. In this pilot study we investigated the hypothesis that saliva from obese individuals could be responsible for an alteration of the retro-nasal aroma release. We tested this hypothesis in vitro, by comparing the release of volatiles from a liquid food matrix (wine) after its interaction with saliva from 28 obese (O) and 28 normal-weight (N) individuals.Methods and findingsAmplicon sequencing of the 16S rRNA V4 region indicated that Firmicutes and Actinobacteria were more abundant in O, while Proteobacteria and Fusobacteria dominated in N. Streptococcaceae were significantly more abundant in the O subjects and constituted 34% and 19% on average of the saliva microbiota of O and N subjects, respectively. The Total Antioxidant Capacity was higher in O vs N saliva samples. A model mouth system was used to test whether the in-mouth wine aroma release differs after the interaction with O or N saliva. In O samples, a 18% to 60% significant decrease in the mean concentration of wine volatiles was detected as a result of interaction with saliva, compared with N. This suppression was linked to biochemical differences in O and N saliva composition, which include protein content.ConclusionMicrobiological and biochemical differences were found in O vs N saliva samples. An impaired retronasal aroma release from white wine was detected in vitro and linked to compositional differences between saliva from obese and normal-weight subjects. Additional in vivo investigations on diverse food matrices could contribute to understanding whether a lower olfactory stimulation due to saliva composition can be a co-factor in the development/maintenance of obesity.

In order to prepare self-standing and flexible slow neutron reflectors made of graphite fluoride (GF) with high contents of (C2F)n structural phase, graphite foils of different thicknesses were used as starting materials for gas (F2)/solid fluorination. The maximal interlayer distance of GF was obtained with this phase thanks to the stacking sequence FCCF/FCCF; this is mandatory for the efficient reflection of slow neutrons. 71 and 77% of the (C2F)n phase were achieved for graphite foils with thicknesses of 1.0 and 0.1 mm, respectively. The interlayer distances were 8.6 Å as expected. The fluorination conditions (static mode, a long duration of 24 h, annealing in pure F2 gas for 24 h, and temperatures in the 390–460 °C range) were adapted to large pieces of graphite foils (7 × 7 cm2) in order to both avoid exfoliation and achieve a homogeneous dispersion of fluorine atoms. This process was also efficient for thinner (0.01 mm thick) graphitized graphene oxide foil. 56% of the (C2F)n phase and an interlayer of 8.6 Å were achieved for this foil when fluorination was performed at 430 °C. Whatever the nature and the thickness of the foil, their flexibilities are maintained. [ABSTRACT FROM AUTHOR]

Aberrant NF-κB activity drives oncogenesis and cell survival in multiple myeloma (MM) and many other cancers. However, despite an aggressive effort by the pharmaceutical industry over the past 30 years, no specific IκBα kinase (IKK)β/NF-κB inhibitor has been clinically approved, due to the multiple dose-limiting toxicities of conventional NF-κB-targeting drugs. To overcome this barrier to therapeutic NF-κB inhibition, we developed the first-in-class growth arrest and DNA-damage-inducible (GADD45)β/mitogen-activated protein kinase kinase (MKK)7 inhibitor, DTP3, which targets an essential, cancer-selective cell-survival module downstream of the NF-κB pathway. As a result, DTP3 specifically kills MM cells, ex vivo and in vivo, ablating MM xenografts in mice, with no apparent adverse effects, nor evident toxicity to healthy cells. Here, we report the results from the preclinical regulatory pharmacodynamic (PD), safety pharmacology, pharmacokinetic (PK), and toxicology programmes of DTP3, leading to the approval for clinical trials in oncology. These results demonstrate that DTP3 combines on-target-selective pharmacology, therapeutic anticancer efficacy, favourable drug-like properties, long plasma half-life and good bioavailability, with no target-organs of toxicity and no adverse effects preclusive of its clinical development in oncology, upon daily repeat-dose administration in both rodent and non-rodent species. Our study underscores the clinical potential of DTP3 as a conceptually novel candidate therapeutic selectively blocking NF-κB survival signalling in MM and potentially other NF-κB-driven cancers. Keywords: NF-κB, GADD45β, Multiple myeloma, Cancer, Pharmacology

Ribosome-inactivating proteins (RIPs) are a group of proteins with rRNA N-glycosylase activity that catalyze the removal of a specific adenine located in the sarcin–ricin loop of the large ribosomal RNA, which leads to the irreversible inhibition of protein synthesis and, consequently, cell death. The case of elderberry (Sambucus nigra L.) is unique, since more than 20 RIPs and related lectins have been isolated and characterized from the flowers, seeds, fruits, and bark of this plant. However, these kinds of proteins have never been isolated from elderberry leaves. In this work, we have purified RIPs and lectins from the leaves of this shrub, studying their main physicochemical characteristics, sequences, and biological properties. In elderberry leaves, we found one type 2 RIP and two related lectins that are specific for galactose, four type 2 RIPs that fail to agglutinate erythrocytes, and one type 1 RIP. Several of these proteins are homologous to others found elsewhere in the plant. The diversity of RIPs and lectins in the different elderberry tissues, and the different biological activities of these proteins, which have a high degree of homology with each other, constitute an excellent source of proteins that are of great interest in diagnostics, experimental therapy, and agriculture.

The involvement of sirtuins (SIRTs) in modulating metabolic and stress response pathways is attracting growing scientific interest. Some SIRT family members are located in mitochondria, dynamic organelles that perform several crucial functions essential for eukaryotic life. Mitochondrial dysfunction has emerged as having a key role in a number of human diseases, including cancer. Here, we investigated mitochondrial damage resulting from treatment with a recently characterized pan-SIRT inhibitor, MC2494. MC2494 was able to block mitochondrial biogenesis and function in terms of ATP synthesis and energy metabolism, suggesting that it might orchestrate cell response to metabolic stress and thereby interfere with cancer promotion and progression. Targeting mitochondrial function could thus be considered a potential anticancer strategy for use in clinical therapy.