Primary Localization and Tumor Thickness as Prognostic Factors of Survival in Patients with Mucosal Melanoma

Background

Data on survival with mucosal melanoma and on prognostic factors of are scarce. It is still unclear if the disease course allows for mucosal melanoma to be treated as primary cutaneous melanoma or if differences in overall survival patterns require adapted therapeutic approaches. Furthermore, this investigation is the first to present 10-year survival rates for mucosal melanomas of different anatomical localizations.

Methodology

116 cases from Sep 10 1984 until Feb 15 2011 retrieved from the Comprehensive Cancer Center and of the Central Register of the German Dermatologic Society databases in Tübingen were included in our analysis. We recorded anatomical location and tumor thickness, and estimated overall survival at 2, 5 and 10 years and the mean overall survival time. Survival times were analyzed with the Kaplan-Meier method. The log-rank test was used to compare survival times by localizations and by T-stages.

Principal Findings

We found a median overall survival time of 80.9 months, with an overall 2-year survival of 71.7%, 5-year survival of 55.8% and 10-year survival of 38.3%. The 10-year survival rates for patients with T₁, T₂, T₃ or T₄ stage tumors were 100.0%, 77.9%, 66.3% and 10.6% respectively. 10-year survival of patients with melanomas of the vulva was 64.5% in comparison to 22.3% of patients with non-vulva mucosal melanomas.

Conclusion

Survival times differed significantly between patients with melanomas of the vulva compared to the rest (p = 0.0006). It also depends on T-stage at the time of diagnosis (p<0.0001).     

Strategies to target SARS-CoV-2 entry and infection using dual mechanisms of inhibition by acidification inhibitors

Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH₄Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.     

miR-BAG: Bagging Based Identification of MicroRNA Precursors

Non-coding elements such as miRNAs play key regulatory roles in living systems. These ultra-short, ∼21 bp long, RNA molecules are derived from their hairpin precursors and usually participate in negative gene regulation by binding the target mRNAs. Discovering miRNA candidate regions across the genome has been a challenging problem. Most of the existing tools work reliably only for limited datasets. Here, we have presented a novel reliable approach, miR-BAG, developed to identify miRNA candidate regions in genomes by scanning sequences as well as by using next generation sequencing (NGS) data. miR-BAG utilizes a bootstrap aggregation based machine learning approach, successfully creating an ensemble of complementary learners to attain high accuracy while balancing sensitivity and specificity. miR-BAG was developed for wide range of species and tested extensively for performance over a wide range of experimentally validated data. Consideration of position-specific variation of triplet structural profiles and mature miRNA anchored structural profiles had a positive impact on performance. miR-BAG’s performance was found consistent and the accuracy level was observed to be >90% for most of the species considered in the present study. In a detailed comparative analysis, miR-BAG performed better than six existing tools. Using miR-BAG NGS module, we identified a total of 22 novel miRNA candidate regions in cow genome in addition to a total of 42 cow specific miRNA regions. In practice, discovery of miRNA regions in a genome demands high-throughput data analysis, requiring large amount of processing. Considering this, miR-BAG has been developed in multi-threaded parallel architecture as a web server as well as a user friendly GUI standalone version.     

Effect of Commiphora mukul extract on cardiac dysfunction and ventricular function in isoproterenol-induced myocardial infarction

In present study, hydroalcoholic extract of C. mukul significantly improved the cardiac function and prevented myocardial ischemic impairment manifested in the form of increased heart rate, decreased arterial pressure, increased left ventricular end diastolic pressure, and altered myocardial contractility indices. C. mukul treatment additionally also produced a significant increase in lactate dehydrogenase levels and prevented decline of protein content in heart. C. mukul preserved the structural integrity of myocardium. Reduced leakage of myocyte enzyme lactate dehydrogenase and maintenance of structural integrity of myocardium along with favorable modulation of cardiac function and improved cardiac performance indicate the salvage of myocardium with C. mukul treatment. Guggulsterones which are considered to be responsible for most of the therapeutic properties of C. mukul may underlie the observed cardioprotective effect of C. mukul against cardiac dysfunction in isoproterenol-induced ischemic rats.     

Nicotinamide inhibits Plasmodium falciparum Sir2 activity in vitro and parasite growth

Plasmodium falciparum sirtuin, PfSir2, contains histone deacetylase (HDAC) activity that may be central to the regulation of virulence gene expression in the parasites. Although a few reports have been published recently regarding in vitro and in vivo function of PfSir2, expression of the endogenous protein (c. 30 kDa) has not been shown yet. Here we report the presence of PfSir2 in the parasite at the protein level by specific antibodies. HDAC activity of PfSir2 can be inhibited by nicotinamide, a product of sirtuin reaction. Surprisingly, we find that nicotinamide also delays parasite growth significantly in culture. These findings further our knowledge on PfSir2 and raise the possibility of using an inexpensive agent like nicotinamide as an antimalarial in combination with other antiparasitic drugs.     

Plasmodium falciparum origin recognition complex subunit 5: functional characterization and role in DNA replication foci formation

The mechanism of DNA replication initiation and progression is poorly understood in the parasites, including human malaria parasite Plasmodium falciparum . Using bioinformatics tools and yeast complementation assay, we identified a putative homologue of Saccharomyces cerevisiae o rigin r ecognition c omplex subunit 5 in P. falciparum (PfORC5). PfORC5 forms distinct nuclear foci colocalized with the replication foci marker proliferating cell nuclear antigen (PfPCNA) and co-immunoprecipitates with PCNA during early-to-mid trophozoite stage replicating parasites. Interestingly, these proteins separate from each other at the non-replicating late schizont stage, citing the evidence of the presence of both PCNA and ORC components in replication foci during eukaryotic DNA replication. PfORC1, another ORC subunit, colocalizes with PfPCNA and PfORC5 at the beginning of DNA replication, but gets degraded at the late schizont stage, ensuring the regulation of DNA replication in the parasites. Further, we have identified putative PCNA-interacting protein box in PfORC1 that may explain in part the colocalization of PfORC and PfPCNA. Additionally, use of specific DNA replication inhibitor hydroxyurea affects ORC5/PCNA foci formation and parasitic growth. These results strongly favour replication factory model in the parasites and confer great potential to understand the co-ordination between ORC and PCNA during eukaryotic DNA replication in general.     

Global diversity and balancing selection of 23 leading Plasmodium falciparum candidate vaccine antigens

Investigation of the diversity of malaria parasite antigens can help prioritize and validate them as vaccine candidates and identify the most common variants for inclusion in vaccine formulations. Studies of vaccine candidates of the most virulent human malaria parasite, Plasmodium falciparum, have focused on a handful of well-known antigens, while several others have never been studied. Here we examine the global diversity and population structure of leading vaccine candidate antigens of P. falciparum using the MalariaGEN Pf3K (version 5.1) resource, comprising more than 2600 genomes from 15 malaria endemic countries. A stringent variant calling pipeline was used to extract high quality antigen gene ‘haplotypes’ from the global dataset and a new R-package named VaxPack was used to streamline population genetic analyses. In addition, a newly developed algorithm that enables spatial averaging of selection pressure on 3D protein structures was applied to the dataset. We analysed the genes encoding 23 leading and novel candidate malaria vaccine antigens including csp, trap, eba175, ama1, rh5, and CelTOS. Our analysis shows that current malaria vaccine formulations are based on rare haplotypes and thus may have limited efficacy against natural parasite populations. High levels of diversity with evidence of balancing selection was detected for most of the erythrocytic and pre-erythrocytic antigens. Measures of natural selection were then mapped to 3D protein structures to predict targets of functional antibodies. For some antigens, geographical variation in the intensity and distribution of these signals on the 3D structure suggests adaptation to different human host or mosquito vector populations. This study provides an essential framework for the diversity of P. falciparum antigens to be considered in the design of the next generation of malaria vaccines.     

Immunological unresponsiveness in leprosy and its relevance to immunoregulation in man

The varied forms of leprosy form a clinical and immunological spectrum which offers extraordinary possibilities for insight into immunoregulatory mechanisms in man. At one pole, tuberculoid leprosy, patients develop high levels of cell-mediated immunity which ultimately results in killing of bacilli in the tissues, albeit often with damage to nerves. At the lepromatous pole, patients exhibit selective immunological unresponsiveness to antigens ofMycobacterium leprae. Even though all currently known protein species ofMycobacterium leprae and BCG are cross-reactive, lepromatous patients unreactive toMycobacterium leprae antigens frequently respond strongly to tuberculin.In vitro experiments suggest the existence of lepromin-induced suppressor activity, mediated by both monocytes andT cells. TheT suppressor cells have the T₈ phenotype of which 50% express the activation markers,Ia and FcR. The one unique species of antigen of the leprosy bacillus is a phenolic glycolipid, and it appears that theT _s cells largely recognize the terminal trisaccharide of this unique antigen. Depletion ofT _s cells restoresin vitro reactivity of lymphocytes to lepromin in a portion of lepromatous patients, and addition of IL-2 containing supernatants partially restores responsiveness toMycobacterium leprae antigens. Vaccination of lepromatous patients with a mixture ofMycobacterium leprae and live BCG restores cell-mediated immunity in the majority of lepromatous patients, and concomitantly reduces thein vitro suppressor activity and number of activated T₈ cells. These experiments suggest the existence of stage-of-disease related suppressor cells in leprosy which appear to block the responsiveness ofT _H capable of responding to either specific or cross-reactive mycobacterial antigens. The mode of action of theseT _s appears to be the inhibition of production of IL-2 and other lymphokines. Successful immunotherapeutic vaccination appears to overcome this block in the majority of patients.     

The major native proteins of the leprosy bacillus

This study addresses a major obstacle to vaccine development for leprosy, the isolation and characterization of the native protein antigens of the leprosy bacillus. Mycobacterium leprae harvested from armadillos was subjected to a simple fractionation protocol to arrive at the three major subcellular fractions, cell walls, cytoplasmic membrane, and soluble cytoplasm. The application of extensive detergent phase separations to membrane fractions allowed removal of lipoarabinomannan and the mannosyl phosphatidylinositols, and the recognition and purification of two major membrane proteins (MMP) of molecular mass 35 kDa (MMP-I) and 22 kDa (MMP-II); recovery of these proteins was about 0.5 mg each per g of M. leprae. MMP-I is N-blocked and is perhaps a lipoprotein. End group analysis on MMP-II indicates a new protein. Three major cytoplasmic proteins (MCP) of molecular mass 14 kDa (MCP-I), 17 kDa (MCP-II), and 28 kDa (MCP-III) were also recognized. MCP-I, the most abundant protein in M. leprae, represents 1% of the bacterial mass. End group analysis of the first 30 residues and immunoblotting studies demonstrate sizeable structural homology to a protein from Mycobacterium tuberculosis but immunological distinctiveness. MCP-I, which also occurs in highly immunogenic peptidoglycan-bound form, is a primary candidate for future vaccine development. The cell walls of M. leprae are also characterized by one major extractable protein, also of molecular mass 17 kDa. Thus the major antigens of the leprosy bacillus, protein and carbohydrate alike, are now nearer to complete definition.