Bleaching melanin in formalin-fixed and paraffin-embedded melanoma specimens using visible light: a pilot study

In fluorescence microscopy, light radiation can be used to bleach fluorescent molecules in formalin-fixed and paraffin-embedded (FFPE) samples, in order to increase the ratio between signal of interest and background autofluorescence. We tested if the same principle can be exploited in bright field microscopy to bleach pigmented melanoma FFPE sections together with cell morphology maintenance. After dewaxing and rehydration, serial FFPE sections of a feline diffuse iris melanoma, a canine dermal melanoma, a gray horse dermal melanoma and a swine cutaneous melanoma were irradiated with visible light for I, 2, 3, 4 and 5 days, prior to Hematoxylin & Eosin staining. Complete bleaching was obtained after 1-day treatment in feline and swine melanomas, while 2 and 3 days were required in canine and equine neoplasms, respectively. In all treated samples, cell morphology was maintained. Photo-induced bleaching combined with immunohistochemistry was tested after a 3-day photo-treatment using five different markers. According to the literature, in all samples neoplastic cells stained positive for vimentin, S100 and PNL2, while negative for FVIII and pancytokeratin. in conclusion, visible light can be effectively exploited to bleach pigmented melanoma FFPE sections prior to perform routine histochemical and immunohistochemical stains

Systematic kinetic analysis of mitotic dis- and reassembly of the nuclear pore in living cells

During mitosis in higher eukaryotes, nuclear pore complexes (NPCs) disassemble in prophase and are rebuilt in anaphase and telophase. NPC formation is hypothesized to occur by the interaction of mitotically stable subcomplexes that form defined structural intermediates. To determine the sequence of events that lead to breakdown and reformation of functional NPCs during mitosis, we present here our quantitative assay based on confocal time-lapse microscopy of single dividing cells. We use this assay to systematically investigate the kinetics of dis- and reassembly for eight nucleoporin subcomplexes relative to nuclear transport in NRK cells, linking the assembly state of the NPC with its function. Our data establish that NPC assembly is an ordered stepwise process that leads to import function already in a partially assembled state. We furthermore find that nucleoporin dissociation does not occur in the reverse order from binding during assembly, which may indicate a distinct mechanism

Broad-Spectrum Inhibition of HIV-1 by a Monoclonal Antibody Directed against a gp120-Induced Epitope of CD4

To penetrate susceptible cells, HIV-1 sequentially interacts with two highly conserved cellular receptors, CD4 and a chemokine receptor like CCR5 or CXCR4. Monoclonal antibodies (MAbs) directed against such receptors are currently under clinical investigation as potential preventive or therapeutic agents. We immunized Balb/c mice with molecular complexes of the native, trimeric HIV-1 envelope (Env) bound to a soluble form of the human CD4 receptor. Sera from immunized mice were found to contain gp120-CD4 complex-enhanced antibodies and showed broad-spectrum HIV-1-inhibitory activity. A proportion of MAbs derived from these mice preferentially recognized complex-enhanced epitopes. In particular, a CD4-specific MAb designated DB81 (IgG1Κ) was found to preferentially bind to a complex-enhanced epitope on the D2 domain of human CD4. MAb DB81 also recognized chimpanzee CD4, but not baboon or macaque CD4, which exhibit sequence divergence in the D2 domain. Functionally, MAb DB81 displayed broad HIV-1-inhibitory activity, but it did not exert suppressive effects on T-cell activation in vitro. The variable regions of the heavy and light chains of MAb DB81 were sequenced. Due to its broad-spectrum anti-HIV-1 activity and lack of immunosuppressive effects, a humanized derivative of MAb DB81 could provide a useful complement to current preventive or therapeutic strategies against HIV-1

TRIS I: Absolute Measurements of the Sky Brightness Temperature at 0.6, 0.82 and 2.5 GHz

At frequencies close to 1 GHz the sky diffuse radiation is a superposition of radiation of Galactic origin, the 3 K Relic or Cosmic Microwave Background Radiation, and the signal produced by unresolved extragalactic sources. Because of their different origin and space distribution the relative importance of the three components varies with frequency and depends on the direction of observation. With the aim of disentangling the components we built TRIS, a system of three radiometers, and studied the temperature of the sky at $\nu =0.6$, $\nu = 0.82$ and $\nu = 2.5$ GHz using geometrically scaled antennas with identical beams (HPBW = $18^{\circ} \times 23^{\circ}$). Observations included drift scans along a circle at constant declination $\delta=+42^{\circ}$ which provided the dependence of the sky signal on the Right Ascension, and absolute measurement of the sky temperature at selected points along the same scan circle. TRIS was installed at Campo Imperatore (lat. = $42^{\circ}~26'$ N, long.= $13^{\circ}~33'$, elevation = 2000 m a.s.l.) in Central Italy, close to the Gran Sasso Laboratory.Comment: Accepted for publication in The Astrophysical Journa

The Influence of Catalysis on Mad2 Activation Dynamics

Mad2 is a key component of the spindle assembly checkpoint, a safety device ensuring faithful sister chromatid separation in mitosis. The target of Mad2 is Cdc20, an activator of the anaphase-promoting complex/cyclosome (APC/C). Mad2 binding to Cdc20 is a complex reaction that entails the conformational conversion of Mad2 from an open (O-Mad2) to a closed (C-Mad2) conformer. Previously, it has been hypothesized that the conversion of O-Mad2 is accelerated by its conformational dimerization with C-Mad2. This hypothesis, known as the Mad2-template hypothesis, is based on the unproven assumption that the natural conversion of O-Mad2 required to bind Cdc20 is slow. Here, we provide evidence for this fundamental assumption and demonstrate that conformational dimerization of Mad2 accelerates the rate of Mad2 binding to Cdc20. On the basis of our measurements, we developed a set of rate equations that deliver excellent predictions of experimental binding curves under a variety of different conditions. Our results strongly suggest that the interaction of Mad2 with Cdc20 is rate limiting for activation of the spindle checkpoint. Conformational dimerization of Mad2 is essential to accelerate Cdc20 binding, but it does not modify the equilibrium of the Mad2:Cdc20 interaction, i.e., it is purely catalytic. These results surpass previously formulated objections to the Mad2-template model and predict that the release of Mad2 from Cdc20 is an energy-driven process

Stability and expression levels of HLA-C on the cell membrane modulate HIV-1 infectivity

HLA-C expression is associated with a differential ability to control HIV-1 infection. Higher HLA-C levels may lead to a better control of HIV-1 infection through both a higher efficiency of antigen presentation to cytotoxic T lymphocytes (CTLs), as well as the triggering of activating Killer Immunoglobulin like receptors (KIR) on NK-cells, whereas lower levels may provide a poor HIV-1 control and a rapid progression toward AIDS.We characterized the relative amount of HLA-C heterotrimers (heavy chain/\u3b22m/peptide) and HLA-C free heavy chains on PBMC from healthy blood donors harboring both alleles with stable or unstable binding to \u3b22m/peptide. We analyzed the stability of HLA-C heterotrimers of different allotypes and the infectivity of HIV-1 virions produced by PBMC with various allotypes.We observed significant differences in HLA-C heterotrimers stability and in expression levels. We found that R5 HIV-1 virions produced by PBMC harboring unstable HLA-C alleles were more infectious than those produced by PBMC carrying the stable variants.We propose that HIV-1 infectivity might depend both on the amounts of HLA-C molecules and on their stability as trimeric complex. According to this model, individuals with low expressed HLA-C alleles and unstable binding to \u3b22m/peptide might have a worse control of HIV-1 infection and an intrinsically higher capacity to support viral replication.IMPORTANCE Following HIV-1 infection, some people advance rapidly toward AIDS while others have a slow disease progression. HLA-C, a molecule involved in immunity, is a key determinant of HIV-1 control.Here we reveal how HLA-C variants contribute to modulate viral infectivity. HLA-C is present on the cell surface in two different conformations: the immunologically active conformation is part of a complex that includes \u3b22-microglobulin/peptide; the other conformation is not bound to \u3b22-microglobulin/peptide and can associate with HIV-1, increasing its infectivity. Individuals with HLA-C variants with a predominance of immunologically active conformations would display a stronger immunity against HIV-1, a reduced viral infectivity and an effective control of HIV-1 infection, while subjects with HLA-C variants that easily dissociate from \u3b22-microglobulin/peptide would have a reduced immunological response to HIV-1 and produce more infectious virions.This study provides new information that could be useful to design novel vaccine strategies and therapeutic approaches against HIV-1

Effect of RNS60 in amyotrophic lateral sclerosis: a phase II multicentre, randomized, double-blind, placebo-controlled trial

Background and purpose Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with limited treatment options. RNS60 is an immunomodulatory and neuroprotective investigational product that has shown efficacy in animal models of ALS and other neurodegenerative diseases. Its administration has been safe and well tolerated in ALS subjects in previous early phase trials. Methods This was a phase II, multicentre, randomized, double-blind, placebo-controlled, parallel-group trial. Participants diagnosed with definite, probable or probable laboratory-supported ALS were assigned to receive RNS60 or placebo administered for 24 weeks intravenously (375 ml) once a week and via nebulization (4 ml/day) on non-infusion days, followed by an additional 24 weeks off-treatment. The primary objective was to measure the effects of RNS60 treatment on selected biomarkers of inflammation and neurodegeneration in peripheral blood. Secondary objectives were to measure the effect of RNS60 on functional impairment (ALS Functional Rating Scale-Revised), a measure of self-sufficiency, respiratory function (forced vital capacity, FVC), quality of life (ALS Assessment Questionnaire-40, ALSAQ-40) and survival. Tolerability and safety were assessed. Results Seventy-four participants were assigned to RNS60 and 73 to placebo. Assessed biomarkers did not differ between arms. The mean rate of decline in FVC and the eating and drinking domain of ALSAQ-40 was slower in the RNS60 arm (FVC, difference 0.41 per week, standard error 0.16, p = 0.0101; ALSAQ-40, difference -0.19 per week, standard error 0.10, p = 0.0319). Adverse events were similar in the two arms. In a post hoc analysis, neurofilament light chain increased over time in bulbar onset placebo participants whilst remaining stable in those treated with RNS60. Conclusions The positive effects of RNS60 on selected measures of respiratory and bulbar function warrant further investigation

Colorectal Cancer Stage at Diagnosis Before vs During the COVID-19 Pandemic in Italy

IMPORTANCE Delays in screening programs and the reluctance of patients to seek medical attention because of the outbreak of SARS-CoV-2 could be associated with the risk of more advanced colorectal cancers at diagnosis. OBJECTIVE To evaluate whether the SARS-CoV-2 pandemic was associated with more advanced oncologic stage and change in clinical presentation for patients with colorectal cancer. DESIGN, SETTING, AND PARTICIPANTS This retrospective, multicenter cohort study included all 17 938 adult patients who underwent surgery for colorectal cancer from March 1, 2020, to December 31, 2021 (pandemic period), and from January 1, 2018, to February 29, 2020 (prepandemic period), in 81 participating centers in Italy, including tertiary centers and community hospitals. Follow-up was 30 days from surgery. EXPOSURES Any type of surgical procedure for colorectal cancer, including explorative surgery, palliative procedures, and atypical or segmental resections. MAIN OUTCOMES AND MEASURES The primary outcome was advanced stage of colorectal cancer at diagnosis. Secondary outcomes were distant metastasis, T4 stage, aggressive biology (defined as cancer with at least 1 of the following characteristics: signet ring cells, mucinous tumor, budding, lymphovascular invasion, perineural invasion, and lymphangitis), stenotic lesion, emergency surgery, and palliative surgery. The independent association between the pandemic period and the outcomes was assessed using multivariate random-effects logistic regression, with hospital as the cluster variable. RESULTS A total of 17 938 patients (10 007 men [55.8%]; mean [SD] age, 70.6 [12.2] years) underwent surgery for colorectal cancer: 7796 (43.5%) during the pandemic period and 10 142 (56.5%) during the prepandemic period. Logistic regression indicated that the pandemic period was significantly associated with an increased rate of advanced-stage colorectal cancer (odds ratio [OR], 1.07; 95%CI, 1.01-1.13; P = .03), aggressive biology (OR, 1.32; 95%CI, 1.15-1.53; P < .001), and stenotic lesions (OR, 1.15; 95%CI, 1.01-1.31; P = .03). CONCLUSIONS AND RELEVANCE This cohort study suggests a significant association between the SARS-CoV-2 pandemic and the risk of a more advanced oncologic stage at diagnosis among patients undergoing surgery for colorectal cancer and might indicate a potential reduction of survival for these patients