An Optimized Procedure for the Site-Directed Labeling of NGF and proNGF for Imaging Purposes

Neurotrophins are growth factors of fundamental importance for the development, survival and maintenance of different neuronal and non-neuronal populations. Over the years, the use of labeled neurotrophins has helped in the study of their biological functions, leading to a better understanding of the processes that regulate their transport, traffic, and signaling. However, the diverse and heterogeneous neurotrophin labeling strategies adopted so far have often led to poorly reproducible protocols and sometimes conflicting conclusions. Here we present a robust, reliable, and fast method to obtain homogeneous preparations of fluorescent proNGF and NGF with 1:1 labeling stoichiometry. This strategy is well suited for several applications, ranging from advanced imaging techniques such as single particle tracking, to analyses that require large amounts of neurotrophins such as in vivo monitoring of protein biodistribution. As a proof of the quality of the labeled NGF and proNGF preparations, we provide a quantitative analysis of their colocalization with proteins involved in the signaling endosome function and sorting. This new analysis allowed demonstrating that proNGF localizes at a sub-population of endosomes not completely overlapped to the one hosting NGF

Precursor and mature NGF live tracking: one versus many at a time in the axons

The classical view of nerve growth factor (NGF) action in the nervous system is linked to its retrograde axonal transport. However, almost nothing is known on the trafficking properties of its unprocessed precursor proNGF, characterized by different and generally opposite biological functions with respect to its mature counterpart. Here we developed a strategy to fluorolabel both purified precursor and mature neurotrophins (NTs) with a controlled stoichiometry and insertion site. Using a single particle tracking approach, we characterized the axonal transport of proNGF versus mature NGF in living dorsal root ganglion neurons grown in compartmentalized microfluidic devices. We demonstrate that proNGF is retrogradely transported as NGF, but with a lower flux and a different distribution of numbers of neurotrophins per vesicle. Moreover, exploiting a dual-color labelling technique, we analysed the transport of both NT forms when simultaneously administered to the axon tips

Precursor and mature NGF live tracking: one versus many at a time in the axons

The classical view of nerve growth factor (NGF) action in the nervous system is linked to its retrograde axonal transport. However, almost nothing is known on the trafficking properties of its unprocessed precursor proNGF, characterized by different and generally opposite biological functions with respect to its mature counterpart. Here we developed a strategy to fluorolabel both purified precursor and mature neurotrophins (NTs) with a controlled stoichiometry and insertion site. Using a single particle tracking approach, we characterized the axonal transport of proNGF versus mature NGF in living dorsal root ganglion neurons grown in compartmentalized microfluidic devices. We demonstrate that proNGF is retrogradely transported as NGF, but with a lower flux and a different distribution of numbers of neurotrophins per vesicle. Moreover, exploiting a dual-color labelling technique, we analysed the transport of both NT forms when simultaneously administered to the axon tips

Serum IgG antibodies from pregnant women reacting to mimotopes of simian virus 40 large T antigen, the viral oncoprotein

Simian virus 40 (SV40) large T antigen (LT) coding sequences were revealed in different human samples, whereas SV40 antibodies (Ab) were detected in human sera of cancer patients and healthy individuals, although with a lower prevalence. Previous studies carried out by the neutralization assay gave a SV40 seroprevalence, in the general population, up to 8%, although higher rates, 12%, were detected in kidney transplant children, in a group of HIV-positive patients, and in healthy females. In this study, serum samples from pregnant women, together with those from non-pregnant women, were analyzed to check the prevalence of IgG Ab reacting to SV40 LT antigens. Serum samples were collected from pregnant and non-pregnant women, with the same mean age. Women were in the range of 15-48 years old. Samples were assayed by an indirect ELISA employing specific SV40 LT mimotopes as antigens, whereas functional analysis was performed by neutralization of the viral infectivity in cell cultures. As a control, sera were analyzed for Ab against BK polyomavirus (BKPyV), which is a human polyomavirus homologous to SV40. Statistical analyses employed chi-square with Yates' correction, and Student's t tests. Indirect ELISAs indicated that pregnant women tested SV40 LT-positive with a prevalence of 17% (23/134), whereas non-pregnant women had a prevalence of 20% (36/180) (P > 0.05). Ab against BKPyV were detected with a prevalence of 80% in pregnant women and with a prevalence of 78% in non-pregnant women. These data indicate that SV40 infects at a low prevalence pregnant women. We may speculate that SV40, or a close human polyomavirus still undetected, could be transmitted from mother to fetus

Colorectal surgery in Italy during the Covid19 outbreak: a survey from the iCral study group

Background The COVID19 pandemic had a deep impact on healthcare facilities in Italy, with profound reorganization of surgical activities. The Italian ColoRectal Anastomotic Leakage (iCral) study group collecting 43 Italian surgical centers experienced in colorectal surgery from multiple regions performed a quick survey to make a snapshot of the current situation. Methods A 25-items questionnaire was sent to the 43 principal investigators of the iCral study group, with questions regard- ing qualitative and quantitative aspects of the surgical activity before and after the COVID19 outbreak. Results Two-thirds of the centers were involved in the treatment of COVID19 cases. Intensive care units (ICU) beds were partially or totally reallocated for the treatment of COVID19 cases in 72% of the hospitals. Elective colorectal surgery for malignancy was stopped or delayed in nearly 30% of the centers, with less than 20% of them still scheduling elective colo- rectal resections for frail and comorbid patients needing postoperative ICU care. A significant reduction of the number of colorectal resections during the time span from January to March 2020 was recorded, with significant delay in treatment in more than 50% of the centers. Discussion Our survey confirms that COVID19 outbreak is severely affecting the activity of colorectal surgery centers partici- pating to iCral study group. This could impact the activity of surgical centers for many months after the end of the emergency

In Vitro and In Vivo Human Herpesvirus 8 Infection of Placenta

Herpesvirus infection of placenta may be harmful in pregnancy leading to disorders in fetal growth, premature delivery, miscarriage, or major congenital abnormalities. Although a correlation between human herpesvirus 8 (HHV-8) infection and abortion or low birth weight in children has been suggested, and rare cases of in utero or perinatal HHV-8 transmission have been documented, no direct evidence of HHV-8 infection of placenta has yet been reported. The aim of this study was to evaluate the in vitro and in vivo susceptibility of placental cells to HHV-8 infection. Short-term infection assays were performed on placental chorionic villi isolated from term placentae. Qualitative and quantitative HHV-8 detection were performed by PCR and real-time PCR, and HHV-8 proteins were analyzed by immunohistochemistry. Term placenta samples from HHV-8-seropositive women were analyzed for the presence of HHV-8 DNA and antigens. In vitro infected histocultures showed increasing amounts of HHV-8 DNA in tissues and supernatants; cyto- and syncitiotrophoblasts, as well as endothelial cells, expressed latent and lytic viral antigens. Increased apoptotic phenomena were visualized by the terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end-labeling method in infected histocultures. Ex vivo, HHV-8 DNA and a latent viral antigen were detected in placenta samples from HHV-8-seropositive women. These findings demonstrate that HHV-8, like other human herpesviruses, may infect placental cells in vitro and in vivo, thus providing evidence that this phenomenon might influence vertical transmission and pregnancy outcome in HHV-8-infected women

Molecular dynamics in HIV-1 infection of the brain

The focus of the present thesis was to investigate the molecular dynamics in HIV-linfection of the brain. HIV-l infects the brain early during the infection and causesneurological syndromes and neuropathological modifications. To understand how thevirus replicates and evolves within the brain may be important to unravel pathogenesisof HIV-l associated neurological diseases. We analyzed HIV-lV3 sequences from thebiological material of infected patients classified in different clinical stagesof infection, we characterized the biological properties of the virus recovered fromthe brain and blood compartments and measured the viral load in these two compartments-moreover, we studied the tropism of HIV-l isolates from the blood and brain for microgliacells and astrocytes. We analysed published and unpublished HIV-l sequences derived from the brainsand CSF of neurologically healthy subjects to deterrnine the amino acid present atposition 305 of the V3 loop, which has been linked to development of AIDS dementia.Contrary to what previously reported by other authors, a proline was not presentat position 305 in any of the non demented subjects, while histidine, which was reportedto be a hallmark for sequences derived from demented patients, was present in 16of the 25 sequences. Furthermore, we studied whether the presence of neurologicalmanifestations was accompanied by appearance of viral genetic polymorphism. For thispurpose, we directy sequenced the V3 loop of the env gene obtained from brain tissuesof asymptomatics and patients with various neurological manifestations includingHIV-l encephalitis. The analysis of the V3 loop revealed the presence of very homogeneousviral populations in the brain tissues with different histopathological findings. To evaluate whether active viral replication in the brain compartment correlateswith the development of neurological symptoms, we selected eight brain tissues obtainedfrom infected patients deceaded at different stages of the disease and quantifiedthe HIV-l DNA copies by using a semiquantitative PCR analysis. We were able to detectHIV-l proviral DNA in 7 of the 8 samples independently of the stage of the disease:the highest levels of viral DNA however were found in the brain of subjects withhistopathological signs of HIV-l encephalitis. We quantified HIV-l RNA levels inpaired CSF and plasma samples of 30 infected patients with various neurological disordersor without . The method used in this study was a RT-PCR tecnique and the aim to deterrninewhether active replication of the virus in the brain is correlated to the neurologicalsymptoms. Plasma viral load did not differ significantly among patients with or withoutHIV-l encephalitis; conversely, higher levels of HIV-l RNA were detected in CSF samplescollected from patients with HIV-l encephalitis compared to subjects without CNSinvolvement or that presented with opportunistic infections of the CNS. These resultsshowed a striking correlation between productive viral infection of CNS and the developmentof HIV-l encephalitis. In order to understand whether a different evolution of HIV-l in the brain occurswith respect to the blood, we compared the biological characteristics of paired HIV-lisolates from CSF and PBMCs obtained from infected individuals.Using the MT-2 cellassay we detected that more than 50% of PBMC isolates were SI (syncytia inducing).The presence of SI variants in blood correlated to the advanced stage of the diseaseand to the low CD4 cell count of the patients studied. Conversely, the majority (77%)of primary HIV-I isolates from CSF were typed as NSI variants. In our study we observeddiscordant phenotype in 46% of paired CSF and PBMCs isolates- in particular NSI (nonsyncytia inducing) variants were cultured from CSF whereas the corrisponding virusin the blood was characterized as SI. Our data suggest that the virus populationsin the two compartments are different and may evolve apart from each other. Thatthis may be what happens during disease progression is confirmed by sequence analysisof HIV-l isolates from CSF and blood of patients treated with Zidovudine. To further invetigate the properties of HIV-I isolates obtained from blood andbrain compartment we infected cells derived from the brain. We found that a majorityof blood and CSF isolates (9/10) obtained from infected patients at different stageof the disease, infected and replicated in primary cultures of microglial cells derivedfrom the temporal lobe tissue resected surgically from patients with intractableepilepsy. Primary human astrocytes could also be infected in vitro by approximately1/3 of the isolates used in the study; in these cells, however, the infection waslatent and could be reactivated with cytokines. ISBN 91-85910-65-

The absence of culturable bacteria from at term placentae from HIV-negative and positive women argues against a placental unique microbiota

letter to the edito

Oncological Frontiers in the Treatment of Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is a rare malignancy characterized by very poor prognosis and lack of treatment options. Immunotherapy has rapidly emerged as an effective tool for MPM, particularly for tumors of non-epithelioid histology. At the same time, comprehensive genomic sequencing may open the way to new-generation targeted-drugs able to hit specific MPM molecular vulnerabilities. These innovations will possibly enrich, but also dramatically complicate, the elucidation of treatment algorithms. Multidisciplinary integration is urgently needed