The small molecule SI113 hinders epithelial-to-mesenchymal transition and subverts cytoskeletal organization in human cancer cells

The small molecule SI113 is an inhibitor of the kinase activity of SGK1, a key biological regulator acting on the PI3K/mTOR signal transduction pathway. Several studies demonstrate that this compound is able to strongly restrain cancer growth in vitro and in vivo, alone or in associative antineoplastic treatments, being able to elicit an autophagic response, either cytotoxic or cytoprotective. To elucidate more exhaustively the molecular mechanisms targeted by SI113, we performed activity-based protein profiling (ABPP) proteomic analysis using a kinase enrichment procedure. This technique allowed the identification via mass spectrometry of novel targets of this compound, most of them involved in functions concerning cell motility and cytoskeletal architecture. Using a glioblastoma multiforme, hepatocarcinoma and colorectal carcinoma cell line, we recognized an inhibitory effect of SI113 on cell migration, invading, and epithelial-to-mesenchymal transition. In addition, these cancer cells, when exposed to this compound, showed a remarkable subversion of the cytoskeletal architecture characterized by F-actin destabilization, phospho-FAK delocalization, and tubulin depolimerization. These results were definitely concordant in attributing to SI113 a key role in hindering cancer cell malignancy and, due to its negligible in vivo toxicity, can sustain performing a Phase I clinical trial to employ this drug in associative cancer therapy.     

SIRT6 stabilization and cytoplasmic localization in macrophages regulates acute and chronic inflammation in mice

Acute and chronic inflammations are key homeostatic events in health and disease. Sirtuins (SIRTs), a family of NAD-dependent protein deacylases, play a pivotal role in the regulation of these inflammatory responses. Indeed, SIRTs have anti-inflammatory effects through a myriad of signaling cascades, including histone deacetylation and gene silencing, p65/RelA deacetylation and inactivation, and nucleotide‑binding oligomerization domain, leucine rich repeat, and pyrin domain‑containing protein 3 inflammasome inhibition. Nevertheless, recent findings show that SIRTs, specifically SIRT6, are also necessary for mounting an active inflammatory response in macrophages. SIRT6 has been shown to positively regulate tumor necrosis factor alpha (TNFα) secretion by demyristoylating pro-TNFα in the cytoplasm. However, how SIRT6, a nuclear chromatin-binding protein, fulfills this function in the cytoplasm is currently unknown. Herein, we show by Western blot and immunofluorescence that in macrophages and fibroblasts there is a subpopulation of SIRT6 that is highly unstable and quickly degraded via the proteasome. Upon lipopolysaccharide stimulation in Raw 264.7, bone marrow, and peritoneal macrophages, this population of SIRT6 is rapidly stabilized and localizes in the cytoplasm, specifically in the vicinity of the endoplasmic reticulum, promoting TNFα secretion. Furthermore, we also found that acute SIRT6 inhibition dampens TNFα secretion both in vitro and in vivo, decreasing lipopolysaccharide-induced septic shock. Finally, we tested SIRT6 relevance in systemic inflammation using an obesity-induced chronic inflammatory in vivo model, where TNFα plays a key role, and we show that short-term genetic deletion of SIRT6 in macrophages of obese mice ameliorated systemic inflammation and hyperglycemia, suggesting that SIRT6 plays an active role in inflammation-mediated glucose intolerance during obesity.     

Double-antigen sandwich ELISA based on chimeric antigens for detection of antibodies to Trypanosoma cruzi in human sera

BackgroundEnzyme-linked immunosorbent assays (ELISA) are generally the chosen test for Chagas disease (CD) diagnosis; however, its performance depends on the antigen preparation adsorbed to the solid phase, which may lead to false-positive results and cross-reactions. The use of chimeric recombinant antigens can overcome this limitation. Four chimeric antigens from Trypanosoma cruzi (IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4) were developed and evaluated in phase I, II and III studies using indirect ELISA as diagnostic platform. However, peroxidase-labeled secondary anti-human IgG antibody, which is employed in indirect ELISAs, limits its use for the detection of species-specific and class-specific antibodies. To overcome this limitation, peroxidase-labeled antigens can be utilized, diagnosing both acute or chronic infection, in a species and immunoglobulin class-independent manner, through the use of a double-antigen sandwich ELISA (DAgS-ELISA). We aimed to evaluate and validate the diagnostic performance of the chimeric antigens IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4 in the DAgS-ELISA platform.Methodology/Principal findingsDAgS-ELISA was optimized by checkerboard titration. In phase I study, 207 positive and 205 negative samples were evaluated. Cross-reactivity to other infections was also assessed using 68 samples. The selected conditions for the tests utilized 25 ng of antigen per well and the conjugate diluted at 1:2,000 for all molecules. In the phase I study, the areas under the curve of IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4 were 98.7%, 99.5%, 98.6% and 98.8%, respectively. Among the positive samples, IBMP-8.1 antigen classified 53 (25.6%) as false negative, IBMP-8.2, 27 (13%), IBMP-8.3, 24 (11.6%) and IBMP-8.4, 43 (20.8%), giving sensitivities of 74.4%, 87%, 88.4% and 79.2%, respectively. The only antigen that did not reach 100% specificity was IBMP-8.3, with 96.6%. IBMP-8.3 was also the only molecule to show cross-reactivity with HTLV.Conclusions/SignificanceDAgS-ELISA is a promising tool for immunodiagnosis, and despite the high AUC values, the performance of this assay was different from the values obtained by our group when using these antigens in the indirect ELISA, for this reason, improvements are being considered to increase the sensitivity of the DAgS-ELISA.     

Stereotactic radiotherapy for brain oligometastases

Brain metastases, the most common metastases in adults, will develop in up to 40% of cancer patients, accounting for more than one-half of all intracranial tumors. They are most associated with breast and lung cancer, melanoma and, less frequently, colorectal and kidney carcinoma.Magnetic resonance imaging (MRI) is the gold standard for diagnosis. For the treatment plan, computed tomography (CT ) images are co-registered and fused with a gadolinium-enhanced T1-weighted MRI where tumor volume and organs at risk are contoured. Alternatively, plain and contrast-enhanced CT scans are co-registered. Single-fraction stereotactic radiotherapy (SRT ) is used to treat patients with good performance status and up to 4 lesions with a diameter of 30 mm or less that are distant from crucial brain function areas. Fractionated SRT (2–5 fractions) is used for larger lesions, in eloquent areas or in proximity to crucial or surgically inaccessible areas and to reduce treatment-related neurotoxicity. The single-fraction SRT dose, which depends on tumor diameter, impacts local control. Fractionated SRT may encompass different schedules. No randomized trial data compared the safety and efficacy of single and multiple fractions. Both single-fraction and fractionated SRT provide satisfactory local control rates, tolerance, a low risk of transient acute adverse events and of radiation necrosis the incidence of which correlated with the irradiated brain volume.     

Hyperinflammation after anti-SARS-CoV-2 mRNA/DNA vaccines successfully treated with anakinra: Case series and literature review

The current SARS-CoV-2 pandemic diffused worldwide has encouraged the rapid development of vaccines to counter the spread of the virus. At present in Italy, 75.01% of the population completed the vaccination course (AIFA.gov.it) and very few adverse events have been recorded by now. Side-effects related to a theoretical over-reaction of the immune system in response to vaccines administration have been described, and the possibility that an autoimmune or a hyperinflammatory condition may occur was recently observed. Herein, we report four cases of hyperinflammatory syndrome with features indicative of Adult-onset Still’s disease (AOSD) and macrophage activation syndrome (MAS), occurred after anti-SARS-CoV-2 vaccine injection and seen at our Unit between March and May 2021. Since interleukin (IL)-1 is one of the pivotal cytokines involved in AOSD pathogenesis, the inhibition of IL-1 is crucial in ameliorating the clinical symptoms of those patients. Moreover, it has been highlighted the central role of IL-1 as a hallmark of the hyperinflammatory status elicited by SARS-CoV-2 infection. In this case series, we successfully employed the IL-1 receptor antagonist anakinra to curb the cytokine release likely unleashed by the vaccine stimulation in potentially predisposed subjects. We also made a literature search to detect other patients with hyperinflammation temporally related to vaccines injection who benefited from IL-1 inhibition, while other AOSD/MAS-like described syndromes improved with other immunomodulatory strategies.     

The double-stranded RNA-binding protein,Staufen1, is an IRES-transacting factor regulating HIV-1 cap-independent translation initiation

Translation initiation of the viral genomic mRNA (vRNA) of human immunodeficiency virus-type 1 (HIV-1) can be mediated by a cap- or an internal ribosome entry site (IRES)-dependent mechanism. A previous report shows that Staufen1, a cellular double-stranded (ds) RNA-binding protein (RBP), binds to the 5’untranslated region (5′UTR) of the HIV-1 vRNA and promotes its cap-dependent translation. In this study, we now evaluate the role of Staufen1 as an HIV-1 IRES-transacting factor (ITAF). We first confirm that Staufen1 associates with both the HIV-1 vRNA and the Gag protein during HIV-1 replication. We found that in HIV-1-expressing cells, siRNA-mediated depletion of Staufen1 reduces HIV-1 vRNA translation. Using dual-luciferase bicistronic mRNAs, we show that the siRNA-mediated depletion and cDNA-mediated overexpression of Staufen1 acutely regulates HIV-1 IRES activity. Furthermore, we show that Staufen1-vRNA interaction is required for the enhancement of HIV-1 IRES activity. Interestingly, we find that only Staufen1 harboring an intact dsRNA-binding domain 3 (dsRBD3) rescues HIV-1 IRES activity in Staufen1 CRISPR-Cas9 gene edited cells. Finally, we show that the expression of Staufen1-dsRBD3 alone enhances HIV-1 IRES activity. This study provides evidence of a novel role for Staufen1 as an ITAF promoting HIV-1 vRNA IRES activity.     

Neuronal postdevelopmentally acting SAX-7S/L1CAM can function as cleaved fragments to maintain neuronal architecture in Caenorhabditis elegans

Whereas remarkable advances have uncovered mechanisms that drive nervous system assembly, the processes responsible for the lifelong maintenance of nervous system architecture remain poorly understood. Subsequent to its establishment during embryogenesis, neuronal architecture is maintained throughout life in the face of the animal’s growth, maturation processes, the addition of new neurons, body movements, and aging. The Caenorhabditis elegans protein SAX-7, homologous to the vertebrate L1 protein family of neural adhesion molecules, is required for maintaining the organization of neuronal ganglia and fascicles after their successful initial embryonic development. To dissect the function of sax-7 in neuronal maintenance, we generated a null allele and sax-7S-isoform-specific alleles. We find that the null sax-7(qv30) is, in some contexts, more severe than previously described mutant alleles and that the loss of sax-7S largely phenocopies the null, consistent with sax-7S being the key isoform in neuronal maintenance. Using a sfGFP::SAX-7S knock-in, we observe sax-7S to be predominantly expressed across the nervous system, from embryogenesis to adulthood. Yet, its role in maintaining neuronal organization is ensured by postdevelopmentally acting SAX-7S, as larval transgenic sax-7S(+) expression alone is sufficient to profoundly rescue the null mutants’ neuronal maintenance defects. Moreover, the majority of the protein SAX-7 appears to be cleaved, and we show that these cleaved SAX-7S fragments together, not individually, can fully support neuronal maintenance. These findings contribute to our understanding of the role of the conserved protein SAX-7/L1CAM in long-term neuronal maintenance and may help decipher processes that go awry in some neurodegenerative conditions.