Absence of Metabolic Cross-correction in Tay-Sachs Cells: IMPLICATIONS FOR GENE THERAPY

We have investigated the ability of a receptor-mediated gene transfer strategy (cross-correction) to restore ganglioside metabolism in fibroblasts from Tay-Sachs (TS) patients in vitro. TS disease is a GM2 gangliosidosis attributed to the deficiency of the lysosomal enzyme beta-hexosaminidase A (HexA) (beta-N-acetylhexosaminidase, EC ). The hypothesis is that transduced cells overexpressing and secreting large amounts of the enzyme would lead to a measurable activity in defective cells via a secretion-recapture mechanism. We transduced NIH3T3 murine fibroblasts with the LalphaHexTN retroviral vector carrying the cDNA encoding for the human Hex alpha-subunit. The Hex activity in the medium from transduced cells was approximately 10-fold higher (up to 75 milliunits) than observed in non-transduced cells. TS cells were cultured for 72 h in the presence of the cell medium derived from the transduced NIH3T3 cells, and they were analyzed for the presence and catalytic activity of the enzyme. Although TS cells were able to efficiently uptake a large amount of the soluble enzyme, the enzyme failed to reach the lysosomes in a sufficient quantity to hydrolyze the GM2 ganglioside to GM3 ganglioside. Thus, our results showed that delivery of the therapeutic HexA was not sufficient to correct the phenotype of TS cells

Enhancement of lysosomal glycohydrolase activity in human primary B lymphocytes during spontaneous apoptosis.

It has been shown that lysosomes are involved in B cell apoptosis but lysosomal glycohydrolases have never been investigated during this event. In this study we determined the enzymatic activities of some lysosomal glycohydrolases in human tonsil B lymphocytes (TBL) undergoing in vitro spontaneous apoptosis. Fluorimetric methods were used to evaluate the activities of β-hexosaminidases, α-mannosidase, β-mannosidase, β-galactosidase, β-glucuronidase and α-fucosidase. Results show that in TBL during spontaneous apoptosis, there is a significant increase in the activity of β-hexosaminidases, α-mannosidase, β-mannosidase and β-galactosidase. Also β-glucuronidase and α-fucosidase activities increase but not in a significant manner. Further studies on β-hexosaminidases revealed that also mRNA expression of the α- and β-subunits, which constitute these enzymes, increases during spontaneous TBL apoptosis. When TBL are protected from apoptosis by the thiol molecule N-acetyl-L-cysteine (NAC), there is no longer any increase in glycohydrolase activities and mRNA expression of β-hexosaminidase α- and β-subunits. This study demonstrates for the first time that the activities and expression of some lysosomal glycohydrolases are enhanced in TBL during spontaneous apoptosis and that these increases are prevented when TBL apoptosis is inhibited

Piezo1 – serine/threonine-protein phosphatase 2A – Cofilin1 biochemical mechanotransduction axis controls F-actin dynamics and cell migration

This study sheds light on a ground-breaking biochemical mechanotransduction pathway and reveals how Piezo1 channels orchestrate cell migration. We observed an increased cell migration rate in HEK293T (HEK) cells treated with Yoda1, a Piezo1 agonist, or in HEK cells overexpressing Piezo1 (HEK+P). Conversely, a significant reduction in cell motility was observed in HEK cells treated with GsMTx4 (a channel inhibitor) or upon silencing Piezo1 (HEK-P). Our findings establish a direct correlation between alterations in cell motility, Piezo1 expression, abnormal F-actin microfilament dynamics, and the regulation of Cofilin1, a protein involved in severing F-actin microfilaments. Here, the conversion of inactive pCofilin1 to active Cofilin1, mediated by the serine/threonine-protein phosphatase 2A catalytic subunit C (PP2AC), resulted in increased severing of F-actin microfilaments and enhanced cell migration in HEK+P cells compared to HEK controls. However, this effect was negligible in HEK-P and HEK cells transfected with hsa-miR-133b, which post-transcriptionally inhibited PP2AC mRNA expression. In summary, our study suggests that Piezo1 regulates cell migration through a biochemical mechanotransduction pathway involving PP2AC-mediated Cofilin1 dephosphorylation, leading to changes in F-actin microfilament dynamics

Identification and characterization of mature β-hexosaminidases associated with human placenta lysosomal membrane

International audienceβ-Hexosaminidase is a soluble glycohydrolase involved in glycoconjugate degradation into lysosomes, nevertheless its localization has also been described in cytosol and plasma membrane. Recently we demonstrated the presence of Hex associated to human fibroblast plasma membrane as mature form and functionally active towards GM2 ganglioside. In this study Hex was analysed in lysosomal membrane-enriched fraction, obtained by purification from highly purified human placenta lysosomes. Results demonstrate the presence of mature Hex associated to lysosomal membrane and displaying, as the plasma membrane (PM) associated form, an acidic optimum pH. When subjected to carbonate extraction, the enzyme behave as a peripheral membrane protein, while Triton X-114 phase separation confirmed its partial hydrophilic nature, characteristics that are in common with the PM-associated Hex. Moreover 2D electrophoresis indicated a slight difference in pI of β-subunits in the membrane and the soluble forms of the lysosomal Hex. These data reveal a new aspect of the Hex biology and suggest that a fully processed membrane-associated form of Hex is translocated from the lysosomal to the plasma membrane by an as yet unknown mechanism. We present a testable hypothesis that at the cell surface Hex changes the composition of glycoconjugates that are known to be involved in intercellular communication and signaling

Biologically driven cut-off definition of lymphocyte ratios in metastatic breast cancer and association with exosomal subpopulations and prognosis

High neutrophil to lymphocyte ratio (NLR) and monocyte to lymphocyte ratio (MLR) are respectively associated with systemic inflammation and immune suppression and have been associated with a poor outcome. Plasmatic exosomes are extracellular vesicles involved in the intercellular communication system that can exert an immunosuppressive function. Aim of this study was to investigate the interplay between the immune system and circulating exosomes in metastatic breast cancer (MBC). A threshold capable to classify patients according to MLR, NLR and PLR, was computed through a receiving operator curve analysis after propensity score matching with a series of female blood donors. Exosomes were isolated from plasma by ExoQuick solution and characterized by flow-cytometry. NLR, MLR, PLR and exosomal subpopulations potentially involved in the pre-metastatic niche were significantly different in MBC patients with respect to controls. MLR was significantly associated with number of sites at the onset of metastatic disease, while high levels of MLR and NLR were found to be associated with poor prognosis. Furthermore, exosomal subpopulations varied according to NLR, MLR, PLR and both were associated with different breast cancer subtypes and sites of distant involvement. This study highlights the nuanced role of immunity in MBC spread, progression and outcome. Moreover, they suggest potential interaction mechanisms between immunity, MBC and the metastatic niche

Monitoring of indoor bioaerosol for the detection of SARS-CoV-2 in different hospital settings

BackgroundSpore Trap is an environmental detection technology, already used in the field of allergology to monitor the presence and composition of potentially inspirable airborne micronic bioparticulate. This device is potentially suitable for environmental monitoring of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in hospital, as well as in other high-risk closed environments. The aim of the present study is to investigate the accuracy of the Spore Trap system in detecting SARS-CoV-2 in indoor bioaerosol of hospital rooms.MethodsThe Spore Trap was placed in hospital rooms hosting patients with documented SARS-CoV-2 infection (n = 36) or, as a negative control, in rooms where patients with documented negativity to a Real-Time Polymerase Chain Reaction molecular test for SARS-CoV-2 were admitted (n = 10). The monitoring of the bioaerosol was carried on for 24 h. Collected samples were analyzed by real-time polymerase chain reaction.ResultsThe estimated sensitivity of the Spore Trap device for detecting SARS-CoV-2 in an indoor environment is 69.4% (95% C.I. 54.3-84.4%), with a specificity of 100%.ConclusionThe Spore Trap technology is effective in detecting airborne SARS-CoV-2 virus with excellent specificity and high sensitivity, when compared to previous reports. The SARS-CoV-2 pandemic scenario has suggested that indoor air quality control will be a priority in future public health management and will certainly need to include an environmental bio-investigation protocol

A comparison of lysosomal enzymes expression levels in peripheral blood of mild- and severe-Alzheimer's disease and MCI patients: implications for regenerative medicine approaches

The association of lysosomal dysfunction and neurodegeneration has been documented in several neurodegenerative diseases, including Alzheimer's Disease (AD). Herein, we investigate the association of lysosomal enzymes with AD at different stages of progression of the disease (mild and severe) or with mild cognitive impairment (MCI). We conducted a screening of two classes of lysosomal enzymes: glycohydrolases (\u3b2-Hexosaminidase, \u3b2-Galctosidase, \u3b2-Galactosylcerebrosidase, \u3b2-Glucuronidase) and proteases (Cathepsins S, D, B, L) in peripheral blood samples (blood plasma and PBMCs) from mild AD, severe AD, MCI and healthy control subjects. We confirmed the lysosomal dysfunction in severe AD patients and added new findings enhancing the association of abnormal levels of specific lysosomal enzymes with the mild AD or severe AD, and highlighting the difference of AD from MCI. Herein, we showed for the first time the specific alteration of \u3b2-Galctosidase (Gal), \u3b2-Galactosylcerebrosidase (GALC) in MCI patients. It is notable that in above peripheral biological samples the lysosomes are more sensitive to AD cellular metabolic alteration when compared to levels of A\u3b2-peptide or Tau proteins, similar in both AD groups analyzed. Collectively, our findings support the role of lysosomal enzymes as potential peripheral molecules that vary with the progression of AD, and make them useful for monitoring regenerative medicine approaches for AD

Roles of the Amino Terminal Region and Repeat Region of the Plasmodium berghei Circumsporozoite Protein in Parasite Infectivity

The circumsporozoite protein (CSP) plays a key role in malaria sporozoite infection of both mosquito salivary glands and the vertebrate host. The conserved Regions I and II have been well studied but little is known about the immunogenic central repeat region and the N-terminal region of the protein. Rodent malaria Plasmodium berghei parasites, in which the endogenous CS gene has been replaced with the avian Plasmodium gallinaceum CS (PgCS) sequence, develop normally in the A. stephensi mosquito midgut but the sporozoites are not infectious. We therefore generated P. berghei transgenic parasites carrying the PgCS gene, in which the repeat region was replaced with the homologous region of P. berghei CS (PbCS). A further line, in which both the N-terminal region and repeat region were replaced with the homologous regions of PbCS, was also generated. Introduction of the PbCS repeat region alone, into the PgCS gene, did not rescue sporozoite species-specific infectivity. However, the introduction of both the PbCS repeat region and the N-terminal region into the PgCS gene completely rescued infectivity, in both the mosquito vector and the mammalian host. Immunofluorescence experiments and western blot analysis revealed correct localization and proteolytic processing of CSP in the chimeric parasites. The results demonstrate, in vivo, that the repeat region of P. berghei CSP, alone, is unable to mediate sporozoite infectivity in either the mosquito or the mammalian host, but suggest an important role for the N-terminal region in sporozoite host cell invasion

Biopolymer Nanoparticles for Nose-to-Brain Drug Delivery: A New Promising Approach for the Treatment of Neurological Diseases

Diseases affecting the central nervous system (CNS) are among the most disabling and the most difficult to cure due to the presence of the blood–brain barrier (BBB) which represents an impediment from a therapeutic and diagnostic point of view as it limits the entry of most drugs. The use of biocompatible polymer nanoparticles (NPs) as vehicles for targeted drug delivery to the brain arouses increasing interest. However, the route of administration of these vectors remains critical as the drug must be delivered without being degraded to achieve a therapeutic effect. An innovative approach for the administration of drugs to the brain using polymeric carriers is represented by the nose-to-brain (NtB) route which involves the administration of the therapeutic molecule through the neuro-olfactory epithelium of the nasal mucosa. Nasal administration is a non-invasive approach that allows the rapid transport of the drug directly to the brain and minimizes its systemic exposure. To date, many studies involve the use of polymer NPs for the NtB transport of drugs to the brain for the treatment of a whole series of disabling neurological diseases for which, as of today, there is no cure. In this review, various types of biodegradable polymer NPs for drug delivery to the brain through the NtB route are discussed and particular attention is devoted to the treatment of neurological diseases such as Glioblastoma and neurodegenerative diseases