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Objective: Evaluate the real-world accuracy of Myxovirus resistance protein A (MxA) detected by the rapid, point-of-care FebriDx test during the second-wave pandemic in Italy in patients with acute respiratory infection (ARI) and a clinical suspicion of COVID-19. Design and methods: Prospective, observational, diagnostic accuracy study whereby hospitalized patients with ARI were consecutively enrolled in a single tertiary care center in Italy from August 1, 2020 to January 31, 2021. Results: COVID-19 was diagnosed in 136/200 (68.0%) patients and Non-COVID-19 was diagnosed in 64/200 (32.0%) patients. COVID-19 patients were younger and had a lower Charlson comorbidity index compared to Non-COVID-19 patients (p < 0.001). Concordance between FebriDx, MxA and rt-PCR for SARS-CoV-2 (gold standard) was good (k 0.93, 95% CI 0.87–0.99). Overall sensitivity and specificity were 97.8% [95% CI 93.7–99.5] and 95.3% [95% CI 86.9%–99.0%], respectively. FebriDx demonstrated a negative predictive value of 95.3% (95% CI 86.9–99.0) for an observed disease prevalence of 68%. Conclusions: FebriDx MxA showed high diagnostic accuracy to identify COVID-19 and could be considered as a real-time triage tool to streamline the management of suspected COVID-19 patients. FebriDx also detected bacterial etiology in Non-COVID-19 patients suggesting good performance to distinguish bacterial from viral respiratory infection.

Colorectal cancer (CRC) is a leading cause of cancer death worldwide. The risk of developing CRC is influenced by both environmental and genetic factors. Recently, chronic inflammation and gut microbiota modifications have been associated with increased CRC risk. Escherichia coli belongs to the commensal intestinal flora and can become highly pathogenic following the acquisition of genes coding for virulence factors, such as the cytotoxic necrotizing factor type 1 (CNF1). Numerous reports highlight that, besides exerting direct effects on epithelial cells, CNF1 can also act on immune cells, modulating their responses and possibly contributing to disease development. In the present review, we summarized the key studies addressing the immunomodulatory functions of CNF1 and discussed the contribution that CNF1 can bring about to CRC through the creation of a pro-inflammatory microenvironment.

Gastrointestinal (GI) manifestations are frequent in systemic sclerosis (SSc) with an impact on quality of life and morbidity. Bowel vasculopathy is a key pathogenetic factor responsible for GI involvement.To compare abdominal ultrasound (US) and Color Doppler Ultrasonography (CDU) features of splanchnic vessels of SSc patients with healthy controls.The charts of SSc patients who underwent an abdominal US and CDU study were retrospectively analyzed. For Superior Mesenteric Artery (SMA) and Inferior Mesenteric Artery (IMA) caliber, Peak Systolic Velocity (PSV), Reverse Velocity (RV), End-Diastolic Velocity (EDV), Mean Velocity (mV), Blood-flow, Resistive Index (RI) and Pulsatility Index (PI) were recorded.28 SSc patients and 28 controls were enrolled. In SSc, caliber of SMA was significantly smaller than in controls (5.75 ± 0.62 mm vs. 6.45 ± 0.60 mm, p 0.0001 - p adj =0.0002). The flow study of SMA and IMA showed a significant reduction of RV (SMA: 7.25 ± 6.37 cm/s vs. 18.52 ± 6.16 cm/s, p 0.0001 - p adj0.0001; IMA: 2.69 ± 6.10 cm/s vs. 17.06 ± 5.75 cm/s, p 0.0001 - p adj0.0001) and PI (SMA: 3.33 ± 0.75 vs. 4.53 ± 1.03, p 0.0001 - p adj =0.0002; IMA: 3.54 ± 0.95 vs. 6.08 ± 1.53, p 0.0001 - p adj0.0001) in SSc patients than controls.involvement of splanchnic vessels in SSc may be non-invasively investigated with abdominal US and CDU. Morphological and functional changes of Doppler parameters observed in SMA and IMA clearly demonstrate that these vessels are affected by SSc vasculopathy.

Simple Summary Colorectal cancer is a leading cause of death worldwide. Despite the development of novel surgical and therapeutic strategies, 50% of patients relapse after treatment. Therapy failure, due to low efficacy, adverse effects and drug resistance, is thus a major concern. The idea of combining standard therapy with non-toxic bioactive natural compounds is a recent topic in cancer research and aims to increase the efficacy of current antitumor therapies while reducing drug toxicity and adverse effects. In recent years, several studies have explored the capacity of polyphenols, dietary bioactive compounds enriched in fruit and vegetables, to act as adjuvants to improve colorectal cancer therapy. In the present review, we discuss these studies, highlighting the mechanisms underlying the adjuvant effect, and bring out the potential of this novel therapeutic approach as well as the critical issues related to clinical application. Abstract Colorectal cancer (CRC) is a major cancer type and a leading cause of death worldwide. Despite advances in therapeutic management, the current medical treatments are not sufficient to control metastatic disease. Treatment-related adverse effects and drug resistance strongly contribute to therapy failure and tumor recurrence. Combination therapy, involving cytotoxic treatments and non-toxic natural compounds, is arousing great interest as a promising more effective and safer alternative. Polyphenols, a heterogeneous group of bioactive dietary compounds mainly found in fruit and vegetables, have received great attention for their capacity to modulate various molecular pathways active in cancer cells and to affect host anticancer response. This review provides a summary of the most recent (i.e., since 2016) preclinical and clinical studies using polyphenols as adjuvants for CRC therapies. These studies highlight the beneficial effects of dietary polyphenols in combination with cytotoxic drugs or irradiation on both therapy outcome and drug resistance. Despite substantial preclinical evidence, data from a few pilot clinical trials are available to date with promising but still inconclusive results. Larger randomized controlled studies and polyphenol formulations with improved bioavailability are needed to translate the research progress into clinical applications and definitively prove the added value of these molecules in CRC management.

Colorectal cancer (CRC) is a leading cause of cancer death worldwide, and its incidence is correlated with infections, chronic inflammation, diet, and genetic factors. An emerging aspect is that microbial dysbiosis and chronic infections triggered by certain bacteria can be risk factors for tumor progression. Recent data suggest that certain bacterial toxins implicated in DNA attack or in proliferation, replication, and death can be risk factors for insurgence and progression of CRC. In this study, we recruited more than 300 biopsy specimens from people undergoing colonoscopy, and we analyzed to determine whether a correlation exists between the presence of bacterial genes coding for toxins possibly involved in CRC onset and progression and the different stages of CRC. We also analyzed to determine whether CRC-predisposing genetic factors could contribute to bacterial toxins response. Our results showed that CIF toxin is associated with polyps or adenomas, whereas pks+ seems to be a predisposing factor for CRC. Toxins from Escherichia coli as a whole have a higher incidence rate in adenocarcinoma patients compared to controls, whereas Bacteroides fragilis toxin does not seem to be associated with pre-cancerous nor with cancerous lesions. These results have been obtained irrespectively of the presence of CRC-risk loci.

Cytotoxic necrotizing factor 1 (CNF1) is a bacterial virulence factor, the target of which is represented by Rho GTPases, small proteins involved in a huge number of crucial cellular processes. CNF1, due to its ability to modulate the activity of Rho GTPases, represents a widely used tool to unravel the role played by these regulatory proteins in different biological processes. In this review, we summarized the data available in the scientific literature concerning the observed in vitro effects induced by CNF1. An article search was performed on electronic bibliographic resources. Screenings were performed of titles, abstracts, and full-texts according to PRISMA guidelines, whereas eligibility criteria were defined for in vitro studies. We identified a total of 299 records by electronic article search and included 76 original peer-reviewed scientific articles reporting morphological or biochemical modifications induced in vitro by soluble CNF1, either recombinant or from pathogenic Escherichia coli extracts highly purified with chromatographic methods. Most of the described CNF1-induced effects on cultured cells are ascribable to the modulating activity of the toxin on Rho GTPases and the consequent effects on actin cytoskeleton organization. All in all, the present review could be a prospectus about the CNF1-induced effects on cultured cells reported so far.

Aim Several studies suggest that the N-terminal fragment of pro-brain natriuretic peptide levels are quite different in wild-type transthyretin (TTR)-related amyloidosis (ATTRwt) and mutated TTR-related amyloidosis (ATTRm) compared with immunoglobulin light-chain cardiac amyloidosis. Our aim was to test this hypothesis in a cohort of patients with different types of cardiac amyloidosis. Patients and methods Seventy patients with ATTRwt, ATTRm, and light-chain cardiac amyloidosis matched for left ventricular (LV) mass index were studied by standard echocardiography, tissue Doppler imaging, and plasmatic cardiac biomarkers. Results Despite similar LV mass and renal function, patients with ATTR cardiac amyloidosis showed lower levels of N-terminal fragment of pro-brain natriuretic peptide than do light-chain amyloidosis ones, especially when expressed as a function of LV mass index. Conclusion Amyloidogenic light-chain-derived fibrils induce more severe myocardial dysfunction in light-chain amyloidosis than in ATTR, despite similar myocardial infiltration. Thus, the degree of cardiac dysfunction may be related not only to the amount of amyloid deposited, but also to qualitative differences among fibrils.

Some toxigenic bacteria produce protein toxins with carcinogenic signatures, which either directly damage DNA or stimulate signalling pathways related to cancer. So far, however, only a few of them have been proved to favour the induction or progression of cancer. In this work, we report that the Rho-activating Escherichia coli protein toxin, cytotoxic necrotising factor 1 (CNF1), induces epithelial to mesenchymal transition (EMT) in intestinal epithelial cells. EMT is a crucial step in malignant tumour conversion and invasiveness. In the case of CNF1, it occurs by up-regulation of the transcription factors ZEB1 and Snail1, delocalisation of E-cadherin and β-catenin, activation of the serine/threonine kinase mTOR, accelerated wound healing, and invasion. However, our results highlight that nontransformed epithelial cells entail the presence of inflammatory factors, in addition to CNF1, to acquire a mesenchymal-like behaviour. All this suggests that the surrounding microenvironment, as well as the cell type, dramatically influences the CNF1 ability to promote carcinogenic traits.

In hypertensive retinopathy, the retinal damage due to high blood pressure is accompanied by increased expression of Glial Fibrillary Acidic Protein (GFAP), which indicates a role of neuroinflammatory processes in such a retinopathy. Proteins belonging to the Rho GTPase family, particularly Rac1, are involved in the activation of Muller glia and in the progression of photoreceptor degeneration, and may thus represent a novel candidate for therapeutic intervention following central nervous system inflammation. In this paper, we have observed that topical administration as eye drops of Cytotoxic Necrotizing Factor 1 (CNF1), a Rho GTPase modulator, surprisingly improves electrophysiological and behavioral visual performances in aged spontaneously hypertensive rats. Furthermore, such functional improvement is accompanied by a reduction of Rac1 activity and retinal GFAP expression. Our results suggest that Rac1 inhibition through CNF1 topical administration may represent a new strategy to target retinal gliosis.

Natural compounds are emerging as agents for the treatment of malignant diseases. We previously showed that extracts from in vitro cell suspension cultures of strawberry reduced murine melanoma cell proliferation, as shown for fruit extracts. In this work, chromatographic, mass spectrometric, and spectrophotometric analyses were carried out to identify the bioactive compound exerting the detected cytotoxic activity. Moreover, aiming to confirm the anti-proliferative activity of the extracts against both paediatric and adult human tumors, cytotoxic experiments were performed on neuroblastoma, colon, and cervix carcinoma cell lines. Extracts from in vitro cell suspension cultures of strawberry induced a statistically significant reduction of cell growth in all the tumor cell lines tested. Interestingly, human fibroblasts from healthy donors were not subjected to this cytotoxic effect, highlighting the importance of further preclinical investigations. The accurate mass measurement, fragmentation patterns, and characteristic mass spectra and mass losses, together with the differences in chromatographic retention times and absorbance spectra, led us to hypothesize that the compound acting as an anti-proliferative agent could be a novel acetal dihydrofurofuran derivative (C8H10O3, molecular mass 154.0630 amu)

Background Glioblastomas are the most aggressive type of brain tumor. A successful treatment should aim at halting tumor growth and protecting neuronal cells to prevent functional deficits and cognitive deterioration. Here, we exploited a Rho GTPase-activating bacterial protein toxin, cytotoxic necrotizing factor 1 (CNF1), to interfere with glioma cell growth in vitro and vivo. We also investigated whether this toxin spares neuron structure and function in peritumoral areas. Methods We performed a microarray transcriptomic and in-depth proteomic analysis to characterize the molecular changes triggered by CNF1 in glioma cells. We also examined tumor cell senescence and growth in vehicle- and CNF1-treated glioma-bearing mice. Electrophysiological and morphological techniques were used to investigate neuronal alterations in peritumoral cortical areas. Results Administration of CNF1 triggered molecular and morphological hallmarks of senescence in mouse and human glioma cells in vitro. CNF1 treatment in vivo induced glioma cell senescence and potently reduced tumor volumes. In peritumoral areas of glioma-bearing mice, neurons showed a shrunken dendritic arbor and severe functional alterations such as increased spontaneous activity and reduced visual responsiveness. CNF1 treatment enhanced dendritic length and improved several physiological properties of pyramidal neurons, demonstrating functional preservation of the cortical network. Conclusions Our findings demonstrate that CNF1 reduces glioma volume while at the same time maintaining the physiological and structural properties of peritumoral neurons. These data indicate a promising strategy for the development of more effective antiglioma therapies.

Transthyterin amyloidosis is a life-threatening disorder caused by the deposition of hepatocyte-derived transthyretin (TTR) amyloid in various tissues and organs. The most common worldwide pathogenic variant with almost exclusive cardiac involvement is Val142Ile with an allele frequency of 3.5% in U.S. African-American population, but supposed extremely rare, with only sporadic cases in Caucasian patients. Unexpectedly, in our amyloidosis referral centre, we identified five patients (15.1% of all TTRm diagnosed patients, three families, two singleton) with Val142Ile variant belonging to unrelated families of Caucasian origin. Molecular study was performed in a total of 10 individuals of which three were Italian families (three affected individuals and five unaffected individuals) and two were singleton (one Italian patient and one patient from Argentine with Spanish ancestry). Sequence analysis of TTR gene revealed the presence of the heterozygous Val142Ile in the five affected patients and in five asymptomatic individuals. All probands underwent, at diagnosis, a complete clinical, echocardiographic and biohumoral evaluation. To the best of our knowledge, we describe the larger report of Caucasian patients with Val142Ile cardiomyopathy. All patients at diagnosis showed symptoms of heart failure with increased thickness of left ventricular walls and systo-diastolic left ventricular dysfunction. They also showed increased plasma values of NT-proBNP and troponin I. Our data confirm that Caucasian patients with the Val142Ile pathogenic variant have phenotypic manifestations similar to that of African-American one. Moreover, our data clearly show that Val142Ile pathogenic variant is not only an African-American mutation but could be also an underestimated Caucasian variant.

Eukaryotic cells secrete extracellular vesicles (EVs), either constitutively or in a regulated manner, which represent an important mode of intercellular communication. EVs serve as vehicles for transfer between cells of membrane and cytosolic proteins, lipids and RNA. Furthermore, certain bacterial protein toxins, or possibly their derived messages, can be transferred cell to cell via EVs. We have herein demonstrated that eukaryotic EVs represent an additional route of cell-to-cell propagation for the Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1). Our results prove that EVs from CNF1 pre-infected epithelial cells can induce cytoskeleton changes, Rac1 and NF-κB activation comparable to that triggered by CNF1. The observation that the toxin is detectable inside EVs derived from CNF1-intoxicated cells strongly supports the hypothesis that extracellular vesicles can offer to the toxin a novel route to travel from cell to cell. Since anthrax and tetanus toxins have also been reported to engage in the same process, we can hypothesize that EVs represent a common mechanism exploited by bacterial toxins to enhance their pathogenicity.

Accumulating evidence indicates that the human intestinal microbiota can contribute to the etiology of colorectal cancer. Triggering factors, including inflammation and bacterial infections, may favor the shift of the gut microbiota from a mutualistic to a pro-carcinogenic configuration. In this context, certain bacterial pathogens can exert a pro-tumoral activity by producing enzymatically-active protein toxins that either directly induce host cell DNA damage or interfere with essential host cell signaling pathways involved in cell proliferation, apoptosis, and inflammation. This review is focused on those toxins that, by mimicking carcinogens and cancer promoters, could represent a paradigm for bacterially induced carcinogenesis.

The Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1), which acts on the Rho GTPases that are key regulators of the actin cytoskeleton, is emerging as a potential therapeutic tool against certain neurological diseases characterized by cellular energy homeostasis impairment. In this brief communication, we show explorative results on the toxin’s effect on fibroblasts derived from a patient affected by myoclonic epilepsy with ragged-red fibers (MERRF) that carries a mutation in the m.8344A>G gene of mitochondrial DNA. We found that, in the patient’s cells, besides rescuing the wild-type-like mitochondrial morphology, CNF1 administration is able to trigger a significant increase in cellular content of ATP and of the mitochondrial outer membrane marker Tom20. These results were accompanied by a profound F-actin reorganization in MERRF fibroblasts, which is a typical CNF1-induced effect on cell cytoskeleton. These results point at a possible role of the actin organization in preventing or limiting the cell damage due to mitochondrial impairment and at CNF1 treatment as a possible novel strategy against mitochondrial diseases still without cure.

In this study, we report how the cholera toxin (CT) A subunit (CTA), the enzyme moiety responsible for signaling alteration in host cells, enters the exosomal pathway, secretes extracellularly, transmits itself to a cell population. The first evidence for long-term transmission of CT’s toxic effect via extracellular vesicles was obtained in Chinese hamster ovary (CHO) cells. To follow the CT intracellular route towards exosome secretion, we used a novel strategy for generating metabolically-labeled fluorescent exosomes that can be counted by flow cytometry assay (FACS) and characterized. Our results clearly show the association of CT with exosomes, together with the heat shock protein 90 (HSP90) and Protein Disulfide Isomerase (PDI) molecules, proteins required for translocation of CTA across the ER membrane into the cytoplasm. Confocal microscopy showed direct internalization of CT containing fluorescent exo into CHO cells coupled with morphological changes in the recipient cells that are characteristic of CT action. Moreover, Me665 cells treated with CT-containing exosomes showed an increase in Adenosine 3’,5’-Cyclic Monophosphate (cAMP) level, reaching levels comparable to those seen in cells exposed directly to CT. Our results prompt the idea that CT can exploit an exosome-mediated cell communication pathway to extend its pathophysiological action beyond an initial host cell, into a multitude of cells. This finding could have implications for cholera disease pathogenesis and epidemiology.

Cytotoxic necrotizing factor 1 (CNF1) is a bacterial protein toxin primarily expressed by pathogenic Escherichia coli strains, causing extraintestinal infections. The toxin is believed to enhance the invasiveness of E. coli by modulating the activity of Rho GTPases in host cells, but it has interestingly also been shown to promote inflammation, stimulate host immunity and function as a potent immunoadjuvant. The mechanisms underlying the immunostimulatory properties of CNF1 are, however, poorly characterized, and little is known about the direct effects of the toxin on immune cells. Here, we show that CNF1 induces expression of maturation markers on human immature monocyte-derived dendritic cells (moDCs) without compromising cell viability. Consistent with the phenotypic maturation, CNF1 further triggered secretion of proinflammatory cytokines and increased the capacity of moDCs to stimulate proliferation of allogenic naïve CD4+ T cells. A catalytically inactive form of the toxin did not induce moDC maturation, indicating that the enzymatic activity of CNF1 triggers immature moDCs to undergo phenotypic and functional maturation. As the maturation of dendritic cells plays a central role in initiating inflammation and activating the adaptive immune response, the present findings shed new light on the immunostimulatory properties of CNF1 and may explain why the toxin functions as an immunoadjuvant.

Pancreatic cancer (PC) has a very low average survival, but its prognosis is further reduced in the case of metastatic spread. Medical therapy in these cases is the only applicable methodology in the international guidelines. During anticancer treatments, common side effects are nausea, vomiting, arthralgia, neuropathy, and alopecia as well as a myelosuppressive effect. The toxicity of various drugs not only affects the quality of life of the patient, but often its severity requires a reduction in if not the termination of drug administration. Scientific studies have shown that a combined use of chemotherapy and certain natural substances, in the form of standardized extracts, can lead to an enhancement of the action of the chemotherapy. Here, we describe 2 cases of metastatic PC. The first case concerns the integrated treatment of a patient with cancer of the pancreas tail with metastatic involvement ab initio of peripancreatic lymph nodes and liver parenchyma, with numerous secondary lesions greater than 9.5 cm. The second case concerns the integrated treatment of a patient with cancer of the pancreatic body with metastatic involvement of the liver parenchyma, with a small secondary lesion. In both cases, an integrated cancer treatment approach, combining chemotherapy with natural remedies, extracts, and hyperthermia, induced a notable remission of primary and metastatic lesions.

Natural products may represent a rich source of new drugs. The enthusiasm toward this topic has recently been fueled by the 2015 Nobel Prize in Physiology or Medicine, awarded for the discovery of avermectin and artemisinin, natural products from Bacteria and Plantae, respectively, which have targeted one of the major global health issues, the parasitic diseases. Specifically, bacteria either living in the environment or colonizing our body may produce compounds of unexpected biomedical value with the potentiality to be employed as therapeutic drugs. In this review, the fascinating history of CNF1, a protein toxin produced by pathogenic strains of Escherichia coli, is divulged. Even if produced by bacteria responsible for a variety of diseases, CNF1 can behave as a promising benefactor to mankind. By modulating the Rho GTPases, this bacterial product plays a key role in organizing the actin cytoskeleton, enhancing synaptic plasticity and brain energy level, rescuing cognitive deficits, reducing glioma growth in experimental animals. These abilities strongly suggest the need to proceed with the studies on this odd drug in order to pave the way toward clinical trials.

Introduction Limited data are available on the natural history of high on treatment platelet reactivity (HPR) by arachidonic acid and ADP - markers of unfavorable prognosis in acute coronary syndrome patients -. Material and methods In a cohort of acute coronary syndrome male patients (n = 101), we evaluated the time-course of HPR by ADP (platelet aggregation by 10 μM ADP ≥ 70%) and arachidonic acid (platelet aggregation by 1 mmol arachidonic acid ≥ 20%) measuring platelet function in the acute phase (T0), at 6 months (T1) and 1 year (T2). Results We identified persistent (HPR at T0,T1 and T2), acute non persistent (HPR only at T0), and late (HPR only at T1 or T2). Patients with persistent HPR by ADP were more frequently with higher values of BMI. Patients carrying CYP2C19*2 variant were more prevalent in the group of persistent HPR (33%). Significant higher values of immature platelet fraction and high immature platelet fraction at 6 and 12 months and mean platelet volume were present in patients with late HPR. Immature platelet fraction was the only variable significantly associated with late HPR by ADP at multivariate analysis (OR = 1.6 (1.08-2.3), p = 0.016). Patients with persistent HPR by arachidonic acid were more frequently diabetics. Immature platelet fraction at 6 months and high immature platelet fraction at 6 and 12 months were the parameters associated with late HPR by AA (OR = 1.4 (1.0-1.9), p = 0.036; OR = 1.5 (1.08-2.4), p = 0.05; OR = 4.9 (1.3-18.8), p = 0.018, respectively). Conclusions About 25% of 101 patients has persistent HPR; they are more frequently diabetics, overweight or carriers of CYP2C19*2. The occurrence of an inflammatory state, indicated by the increase of immature platelet fraction, is associated with the occurrence of late HPR.

Background Few studies have analyzed the clinical and echocardiographic differences between light-chain (AL) and transthyretin (TTR) amyloidosis. Hypothesis The aim of the present research was to compare, in a real-world setting, the clinical and echocardiographic profiles of these kinds of amyloidosis, at the time of diagnosis, using new-generation echocardiography. Methods Seventy-nine patients with AL and 48 patients with TTR amyloidosis were studied. Results According to the criterion of mean left ventricular (LV) thickness >12 mm, 45 AL (C-AL) and all TTR patients had cardiac amyloidotic involvement, whereas 34 AL patients did not. TTR patients had increased right ventricular (RV) and LV chambers with increased RV and LV wall thickness and reduced LV ejection fraction and fractional shortening. Furthermore, TTR patients showed lower N-terminal pro Brain Natriuretic Peptide concentrations and New York Heart Association functional class when compared with C-AL. Conclusions Our data show that at time of first diagnosis, TTR patients have a more advanced amyloidotic involvement of the heart, despite less severe symptoms and biohumoral signs of heart failure. We can hypothesize that we observed different diseases at different stages. In fact, AL amyloidosis is a multiorgan disease with quick progression rate, that becomes rapidly symptomatic, whereas TTR amyloidosis might have a slow progression rate and might remain poorly symptomatic for a greater amount of time.

The Cytotoxic Necrotizing Factor 1 (CNF1) is a bacterial toxin secreted by certain Escherichia coli strains causing severe pathologies, making it a protein of pivotal interest in toxicology. In parallel, the CNF1 capability to influence important neuronal processes, like neuronal arborization, astrocytic support, and efficient ATP production, has been efficiently used in the treatment of neurological diseases, making it a promising candidate for therapy. Nonetheless, there are still some unsolved issues about the CNF1 mechanism of action and structuration probably caused by the difficulty to achieve sufficient amounts of the full-length protein for further studies. Here, we propose an efficient strategy for the production and purification of this toxin as a his-tagged recombinant protein from E. coli extracts (CNF1-H8). CNF1-H8 was expressed at the low temperature of 15°C to diminish its characteristic degradation. Then, its purification was achieved using an immobilized metal affinity chromatography (IMAC) and a size exclusion chromatography so as to collect up to 8 mg of protein per liter of culture in a highly pure form. Routine dynamic light scattering (DLS) experiments showed that the recombinant protein preparations were homogeneous and preserved this state for a long time. Furthermore, CNF1-H8 functionality was confirmed by testing its activity on purified RhoA and on HEp-2 cultured cells. Finally, a first structural characterization of the full-length toxin in terms of secondary structure and thermal stability was performed by circular dichroism (CD). These studies demonstrate that our system can be used to produce high quantities of pure recombinant protein for a detailed structural analysis. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:150-159, 2018.

In AL amyloidosis, the importance of right ventricle (RV) involvement has recently been underlined and its role in predicting prognosis has been emphasized. Little is known about the relationship between RV involvement, N-terminal pro-brain natriuretic peptide (NT-proBNP) and troponin levels. Aim of our study was to clarify the relationship between NT-proBNP and troponin and RV involvement and analyze their independent value as predictors of RV dysfunction.We examined 76 consecutive patients with biopsy-proven AL amyloidosis. Each patient received complete clinical evaluation, troponin I, NT-proBNP assay and comprehensive echocardiographic evaluation. Considering a tricuspidal annulus plane systolic excursion (TAPSE) value16 mm, 23 patients (30%) presented RV systolic dysfunction, whereas 53 (70%) did not. Patient with reduced TAPSE had thicker left ventricle (LV) walls and RV free walls, reduced LV fractional shortening, impaired LV diastolic function and worse LV and RV myocardial performance index. For RV dysfunction the best predictive value for NT-proBNP was identified as 2977 ng/l with sensitivity and specificity of 87% and 84%, respectively; best cut-off for troponin I was identified as 0.085 ng/l, with sensitivity and specificity of 85% and 90% respectively. At multivariable logistic regression analysis, both NT-proBNP and troponin I emerged as independent predictors of RV dysfunction presence but troponin appears to have a higher predictive power.Our study demonstrated that cut-off values of 2977 ng/ml for NT-proBNP and 0.085 ng/l for troponin were able to identify a subgroup of AL patients with RV dysfunction. Troponin I is more accurate and seems to be the best biohumoral marker of RV dysfunction.

The Cytotoxic Necrotizing Factor 1 (CNF1), a protein toxin from pathogenic E. coli, modulates the Rho GTPases, thus, directing the organization of the actin cytoskeleton. In the nervous system, the Rho GTPases play a key role in several processes, controlling the morphogenesis of dendritic spines and synaptic plasticity in brain tissues. This review is focused on the peculiar property of CNF1 to enhance brain plasticity in in vivo animal models of central nervous system (CNS) diseases, and on its possible application in therapy.