Native Liquid Chromatography and Mass Spectrometry to Structurally and Functionally Characterize Endo-Xylanase Proteoforms

Xylanases are of great value in various industries, including paper, food, and biorefinery. Due to their biotechnological production, these enzymes can contain a variety of post-translational modifications, which may have a profound effect on protein function. Understanding the structure–function relationship can guide the development of products with optimal performance. We have developed a workflow for the structural and functional characterization of an endo-1,4-β-xylanase (ENDO-I) produced by Aspergillus niger with and without applying thermal stress. This workflow relies on orthogonal native separation techniques to resolve proteoforms. Mass spectrometry and activity assays of separated proteoforms permitted the establishment of structure–function relationships. The separation conditions were focus on balancing efficient separation and protein functionality. We employed size exclusion chromatography (SEC) to separate ENDO-I from other co-expressed proteins. Charge variants were investigated with ion exchange chromatography (IEX) and revealed the presence of low abundant glycated variants in the temperature-stressed material. To obtain better insights into the effect on glycation on function, we enriched for these species using boronate affinity chromatography (BAC). The activity measurements showed lower activity of glycated species compared to the non-modified enzyme. Altogether, this workflow allowed in-depth structural and functional characterization of ENDO-I proteoforms.     

Evaluation of Oxidative Stress Biomarkers,Pro-Inflammatory Cytokines,and Histological Changes in Experimental Hypertension,Dyslipidemia, and Type 1 Diabetes Mellitus

The present study aims to compare the oxidative stress biomarkers, pro-inflammatory cytokines, and histological changes induced by three cardiovascular risk factors, namely, hypertension, dyslipidemia, and type 1 diabetes mellitus. Hypertension was induced with 40 mg/kg body weight (b.w.) of N omega-nitro-L-arginine-methyl (L-NAME) administered orally. Dyslipidemia was induced by the administration of a diet with a high cholesterol (2%) content. Diabetes mellitus was induced by intraperitoneal administration of a single dose of streptozocin (65 mg/kg). Malondialdehyde (MDA) and total oxidative status (TOS) are increased by all three cardiovascular risk factors (up to 207%). The indirect assessment of NO synthesis (NOx) is observed to be reduced after L-NAME administration (43%), and dyslipidemia induction (16%), while type 1 diabetes mellitus is associated with the highest levels of NOx (increased 112%). Hypertension, dyslipidemia, and type 1 diabetes reduced the total antioxidative capacity (TAC) and total thiol (SH) levels (up to 57%). The values of evaluated pro-inflammatory cytokines, tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), assessed from the ascending aorta were elevated by all three cardiovascular risk factors, with the highest levels induced by type 1 diabetes mellitus (up to 259%). The histopathological examination of the ascending and descending aorta revealed reversible pro-atherogenic changes consisting of the accumulation of lipid droplets in the subendothelial connective tissue on rats with hypertension and dyslipidemia. Irreversible pro-atherogenic changes consisting of a reduction of the specific elasticity of the arteries were observed in rats with type 1 diabetes mellitus. Type 1 diabetes mellitus demonstrates an alteration of the oxidative stress parameters, the elevation of tissue levels of the pro-inflammatory cytokines and causing irreversible pro-atherogenic changes on the aortic wall.     

Gal3 Plays a Deleterious Role in a Mouse Model of Endotoxemia

Lipopolysaccharide (LPS)-induced endotoxemia induces an acute systemic inflammatory response that mimics some important features of sepsis, the disease with the highest mortality rate worldwide. In this work, we have analyzed a murine model of endotoxemia based on a single intraperitoneal injection of 5 mg/kg of LPS. We took advantage of galectin-3 (Gal3) knockout mice and found that the absence of Gal3 decreased the mortality rate oflethal endotoxemia in the first 80 h after the administration of LPS, along with a reduction in the tissular damage in several organs measured by electron microscopy. Using flow cytometry, we demonstrated that, in control conditions, peripheral immune cells, especially monocytes, exhibited high levels of Gal3, which were early depleted in response to LPS injection, thus suggesting Gal3 release under endotoxemia conditions. However, serum levels of Gal3 early decreased in response to LPS challenge (1 h), an indication that Gal3 may be extravasated to peripheral organs. Indeed, analysis of Gal3 in peripheral organs revealed a robust up-regulation of Gal3 36 h after LPS injection. Taken together, these results demonstrate the important role that Gal3 could play in the development of systemic inflammation, a well-established feature of sepsis, thus opening new and promising therapeutic options for these harmful conditions.     

The Role of the Lymphatic System in the Pathogenesis and Treatment of Inflammatory Bowel Disease

Although the number of therapeutic options for the treatment of inflammatory bowel disease (IBD) has increased in recent years, patients suffer from decreased quality of life due to non-response or loss of response to the currently available treatments. An increased understanding of the disease’s etiology could provide novel insights for treatment strategies in IBD. Lymphatic system components are generally linked to immune responses and presumably related to inflammatory diseases pathophysiology. This review aims to summarize findings on immune-mediated mechanisms in lymphoid tissues linked with IBD pathogenesis and (potential) novel treatments. Enhanced innate and adaptive immune responses were observed in mesenteric lymph nodes (MLNs) and other lymphoid structures, such as Peyer’s patches, in patients with IBD and in animal models. Furthermore, the phenomenon of lymphatic obstruction in the form of granulomas in MLNs and lymphatic vessels correlates with disease activity. There is also evidence that abnormalities in the lymphatic stromal components and lymph node microbiome are common in IBD and could be exploited therapeutically. Finally, novel agents targeting lymphocyte trafficking have been added to the treatment armamentarium in the field of IBD. Overall, gut-associated lymphoid tissue plays a key role in IBD immunopathogenesis, which could offer novel therapeutic targets.     

Preclinical Assessment of the Combination of PSMA-Targeting Radionuclide Therapy with PARP Inhibitors for Prostate Cancer Treatment

Prostate specific membrane antigen targeted radionuclide therapy (PSMA-TRT) is a promising novel treatment for prostate cancer (PCa) patients. However, PSMA-TRT cannot be used for curative intent yet, thus additional research on how to improve the therapeutic efficacy is warranted. A potential way of achieving this, is combining TRT with poly ADP-ribosylation inhibitors (PARPi), which has shown promising results for TRT of neuroendocrine tumor cells. Currently, several clinical trials have been initiated for this combination for PCa, however so far, no evidence of synergism is available for PCa. Therefore, we evaluated the combination of PSMA-TRT with three classes of PARPi in preclinical PCa models. In vitro viability and survival assays were performed using PSMA-expressing PCa cell lines PC3-PIP and LNCaP to assess the effect of increasing concentrations of PARPi veliparib, olaparib or talazoparib in combination with PSMA-TRT compared to single PARPi treatment. Next, DNA damage analyses were performed by quantifying the number of DNA breaks by immunofluorescent stainings. Lastly, the potential of the combination treatments was studied in vivo in mice bearing PC3-PIP xenografts. Our results show that combining PSMA-TRT with PARPi did not synergistically affect the in vitro clonogenic survival or cell viability. DNA-damage analysis revealed only a significant increase in DNA breaks when combining PSMA-TRT with veliparib and not in the other combination treatments. Moreover, PSMA-TRT with PARPi treatment did not improve tumor control compared to PSMA-TRT monotherapy. Overall, the data presented do not support the assumption that combining PSMA-TRT with PARPi leads to a synergistic antitumor effect in PCa. These results underline that extensive preclinical research using various PCa models is imperative to validate the applicability of the combination strategy for PCa, as it is for other cancer types.     

ICOSL Stimulation by ICOS-Fc Accelerates Cutaneous Wound Healing In Vivo

Background: ICOS and its ligand ICOSL are immune receptors whose interaction triggers bidirectional signals that modulate the immune response and tissue repair. Aim: The aim of this study was to assess the in vivo effects of ICOSL triggering by ICOS-Fc, a recombinant soluble form of ICOS, on skin wound healing. Methods: The effect of human ICOS-Fc on wound healing was assessed, in vitro, and, in vivo, by skin wound healing assay using ICOS^−/− and ICOSL^−/− knockout (KO) mice and NOD-SCID-IL2R null (NSG) mice. Results: We show that, in wild type mice, treatment with ICOS-Fc improves wound healing, promotes angiogenesis, preceded by upregulation of IL-6 and VEGF expression; increases the number of fibroblasts and T cells, whereas it reduces that of neutrophils; and increases the number of M2 vs. M1 macrophages. Fittingly, ICOS-Fc enhanced M2 macrophage migration, while it hampered that of M1 macrophages. ICOS^−/− and ICOSL^−/− KO, and NSG mice showed delayed wound healing, and treatment with ICOS-Fc improved wound closure in ICOS^−/− and NSG mice. Conclusion: These data show that the ICOS/ICOSL network cooperates in tissue repair, and that triggering of ICOSL by ICOS-Fc improves cutaneous wound healing by increasing angiogenesis and recruitment of reparative macrophages.     

Ferroptosis Modulation: Potential Therapeutic Target for Glioblastoma Treatment

Glioblastoma multiforme is a lethal disease and represents the most common and severe type of glioma. Drug resistance and the evasion of cell death are the main characteristics of its malignancy, leading to a high percentage of disease recurrence and the patients’ low survival rate. Exploiting the modulation of cell death mechanisms could be an important strategy to prevent tumor development and reverse the high mortality and morbidity rates in glioblastoma patients. Ferroptosis is a recently described type of cell death, which is characterized by iron accumulation, high levels of polyunsaturated fatty acid (PUFA)-containing phospholipids, and deficiency in lipid peroxidation repair. Several studies have demonstrated that ferroptosis has a potential role in cancer treatment and could be a promising approach for glioblastoma patients. Thus, here, we present an overview of the mechanisms of the iron-dependent cell death and summarize the current findings of ferroptosis modulation on glioblastoma including its non-canonical pathway. Moreover, we focused on new ferroptosis-inducing compounds for glioma treatment, and we highlight the key ferroptosis-related genes to glioma prognosis, which could be further explored. Thereby, understanding how to trigger ferroptosis in glioblastoma may provide promising pharmacological targets and indicate new therapeutic approaches to increase the survival of glioblastoma patients.     

Establishing In-House Cutoffs of CSF Alzheimer’s Disease Biomarkers for the AT(N) Stratification of the Alzheimer Center Barcelona Cohort

Background: Clinical diagnosis of Alzheimer’s disease (AD) increasingly incorporates CSF biomarkers. However, due to the intrinsic variability of the immunodetection techniques used to measure these biomarkers, establishing in-house cutoffs defining the positivity/negativity of CSF biomarkers is recommended. However, the cutoffs currently published are usually reported by using cross-sectional datasets, not providing evidence about its intrinsic prognostic value when applied to real-world memory clinic cases. Methods: We quantified CSF Aβ1-42, Aβ1-40, t-Tau, and p181Tau with standard INNOTEST^® ELISA and Lumipulse G^® chemiluminescence enzyme immunoassay (CLEIA) performed on the automated Lumipulse G600II. Determination of cutoffs included patients clinically diagnosed with probable Alzheimer’s disease (AD, n = 37) and subjective cognitive decline subjects (SCD, n = 45), cognitively stable for 3 years and with no evidence of brain amyloidosis in 18F-Florbetaben-labeled positron emission tomography (FBB-PET). To compare both methods, a subset of samples for Aβ1-42 (n = 519), t-Tau (n = 399), p181Tau (n = 77), and Aβ1-40 (n = 44) was analyzed. Kappa agreement of single biomarkers and Aβ1-42/Aβ1-40 was evaluated in an independent group of mild cognitive impairment (MCI) and dementia patients (n = 68). Next, established cutoffs were applied to a large real-world cohort of MCI subjects with follow-up data available (n = 647). Results: Cutoff values of Aβ1-42 and t-Tau were higher for CLEIA than for ELISA and similar for p181Tau. Spearman coefficients ranged between 0.81 for Aβ1-40 and 0.96 for p181TAU. Passing–Bablok analysis showed a systematic and proportional difference for all biomarkers but only systematic for Aβ1-40. Bland–Altman analysis showed an average difference between methods in favor of CLEIA. Kappa agreement for single biomarkers was good but lower for the Aβ1-42/Aβ1-40 ratio. Using the calculated cutoffs, we were able to stratify MCI subjects into four AT(N) categories. Kaplan–Meier analyses of AT(N) categories demonstrated gradual and differential dementia conversion rates (p = 9.815⁻²⁷). Multivariate Cox proportional hazard models corroborated these findings, demonstrating that the proposed AT(N) classifier has prognostic value. AT(N) categories are only modestly influenced by other known factors associated with disease progression. Conclusions: We established CLEIA and ELISA internal cutoffs to discriminate AD patients from amyloid-negative SCD individuals. The results obtained by both methods are not interchangeable but show good agreement. CLEIA is a good and faster alternative to manual ELISA for providing AT(N) classification of our patients. AT(N) categories have an impact on disease progression. AT(N) classifiers increase the certainty of the MCI prognosis, which can be instrumental in managing real-world MCI subjects.     

Genetic LGALS1 Variants Are Associated with Heterogeneity in Galectin-1 Serum Levels in Patients with Early Arthritis

Galectin 1 (Gal1) exerts immunomodulatory effects leading to therapeutic effects in autoimmune animal models. Patients with rheumatoid arthritis have been reported to show higher Gal1 serum levels than the healthy population. Our study aimed to find genetic variants on the Gal1 gene (LGALS1) modulating its expression and/or clinical features in patients with early arthritis (EA). LGALS1 was sequenced in 53 EA patients to characterize all genetic variants. Then, we genotyped rs9622682, rs929039, and rs4820293, which covered the main genetic variation in LGALS1, in 532 EA patients. Gal1 and IL-6 serum levels were measured by ELISA and Gal1 also by western blot (WB) in lymphocytes from patients with specific genotypes. Once disease activity improved with treatment, patients with at least one copy of the minor allele in rs9622682 and rs929039 or those with GG genotype in rs4820293 showed significantly higher Gal1 serum levels (p < 0.05). These genotypic combinations were also associated with higher Gal1 expression in lymphocytes by WB and lower IL-6 serum levels in EA patients. In summary, our study suggests that genetic variants studied in LGALS1 can explain heterogeneity in Gal1 serum levels showing that patients with higher Gal1 levels have lower serum IL-6 levels.     

Photorhabdus luminescens TccC3 Toxin Targets the Dynamic Population of F-Actin and Impairs Cell Cortex Integrity

Due to its essential role in cellular processes, actin is a common target for bacterial toxins. One such toxin, TccC3, is an effector domain of the ABC-toxin produced by entomopathogenic bacteria of Photorhabdus spp. Unlike other actin-targeting toxins, TccC3 uniquely ADP-ribosylates actin at Thr-148, resulting in the formation of actin aggregates and inhibition of phagocytosis. It has been shown that the fully modified F-actin is resistant to depolymerization by cofilin and gelsolin, but their effects on partially modified actin were not explored. We found that only F-actin unprotected by tropomyosin is the physiological TccC3 substrate. Yet, ADP-ribosylated G-actin can be produced upon cofilin-accelerated F-actin depolymerization, which was only mildly inhibited in partially modified actin. The affinity of TccC3-ADP-ribosylated G-actin for profilin and thymosin-β4 was weakened moderately but sufficiently to potentiate spontaneous polymerization in their presence. Interestingly, the Arp2/3-mediated nucleation was also potentiated by T148-ADP-ribosylation. Notably, even partially modified actin showed reduced bundling by plastins and α-actinin. In agreement with the role of these and other tandem calponin-homology domain actin organizers in the assembly of the cortical actin network, TccC3 induced intense membrane blebbing in cultured cells. Overall, our data suggest that TccC3 imposes a complex action on the cytoskeleton by affecting F-actin nucleation, recycling, and interaction with actin-binding proteins involved in the integration of actin filaments with each other and cellular elements.