Numerical analysis of shells. Volume 3 - Engineer's program manual for ''STARS-2'' - Shell Theory Automated for Rotational Structures-2, digital computer program

Manual of engineering programming information for Shell Theory Automated for Rotational Structures /STARS 2/ - Vol.

Lysines Acetylome and Methylome Profiling of H3 and H4 Histones in Trichostatin A-Treated Stem Cells

Trichostatin A ([R-(E,E)]-7-[4-(dimethylamino) phenyl]-N-hydroxy- 4,6-dimethyl- 7-oxo-2,4-heptadienamide, TSA) affects chromatin state through its potent histone deacetylase inhibitory activity. Interfering with the removal of acetyl groups from lysine residues in histones is one of many epigenetic regulatory processes that control gene expression. Histone deacetylase inhibition drives cells toward the differentiation stage, favoring the activation of specific genes. In this paper, we investigated the effects of TSA on H3 and H4 lysine acetylome and methylome profiling in mice embryonic stem cells (ES14), treated with trichostatin A (TSA) by using a new, untargeted approach, consisting of trypsin-limited proteolysis experiments coupled with MALDI-MS and LC-MS/MS analyses. The method was firstly set up on standard chicken core histones to probe the optimized conditions in terms of enzyme:substrate (E:S) ratio and time of proteolysis and, then, applied to investigate the global variations of the acetylation and methylation state of lysine residues of H3 and H4 histone in the embryonic stem cells (ES14) stimulated by TSA and addressed to differentiation. The proposed strategy was found in its simplicity to be extremely effective in achieving the identification and relative quantification of some of the most significant epigenetic modifications, such as acetylation and lysine methylation. Therefore, we believe that it can be used with equal success in wider studies concerning the characterization of all epigenetic modifications

Antimalarial Transmission-Blocking Interventions: Past, Present, and Future.

Malaria remains a major global health challenge. Appropriate use of current antimalarial tools has reduced the disease burden, but morbidity and mortality remain unacceptably high. It is widely accepted that, to achieve long-term control/eradication, it will be necessary to use interventions that inhibit the transmission of parasites to mosquitoes - these tools are termed transmission-blocking interventions (TBIs). This article aims to outline the rationale for the development of TBIs, with a focus on transmission-blocking drugs and (parasite-derived) transmission-blocking vaccines. We describe and summarise the current status of each of these intervention classes and attempt to identify future requirements in development, with a focus on the challenges of establishing each method within an integrated malarial control programme in the future

Detection of malaria sporozoites expelled during mosquito sugar feeding.

Malaria is a severe disease of global importance transmitted by mosquitoes of the genus Anopheles. The ability to rapidly detect the presence of infectious mosquitoes able to transmit malaria is of vital importance for surveillance, control and elimination efforts. Current methods principally rely on large-scale mosquito collections followed by labour-intensive salivary gland dissections or enzyme-linked immunosorbent (ELISA) methods to detect sporozoites. Using forced salivation, we demonstrate here that Anopheles mosquitoes infected with Plasmodium expel sporozoites during sugar feeding. Expelled sporozoites can be detected on two sugar-soaked substrates, cotton wool and Whatman FTA cards, and sporozoite DNA is detectable using real-time PCR. These results demonstrate a simple and rapid methodology for detecting the presence of infectious mosquitoes with sporozoites and highlight potential laboratory applications for investigating mosquito-malaria interactions. Our results indicate that FTA cards could be used as a simple, effective and economical tool in enhancing field surveillance activities for malaria

Subcompartmentalisation of Proteins in the Rhoptries Correlates with Ordered Events of Erythrocyte Invasion by the Blood Stage Malaria Parasite

Host cell infection by apicomplexan parasites plays an essential role in lifecycle progression for these obligate intracellular pathogens. For most species, including the etiological agents of malaria and toxoplasmosis, infection requires active host-cell invasion dependent on formation of a tight junction - the organising interface between parasite and host cell during entry. Formation of this structure is not, however, shared across all Apicomplexa or indeed all parasite lifecycle stages. Here, using an in silico integrative genomic search and endogenous gene-tagging strategy, we sought to characterise proteins that function specifically during junction-dependent invasion, a class of proteins we term invasins to distinguish them from adhesins that function in species specific host-cell recognition. High-definition imaging of tagged Plasmodium falciparum invasins localised proteins to multiple cellular compartments of the blood stage merozoite. This includes several that localise to distinct subcompartments within the rhoptries. While originating from the same organelle, however, each has very different dynamics during invasion. Apical Sushi Protein and Rhoptry Neck protein 2 release early, following the junction, whilst a novel rhoptry protein PFF0645c releases only after invasion is complete. This supports the idea that organisation of proteins within a secretory organelle determines the order and destination of protein secretion and provides a localisation-based classification strategy for predicting invasin function during apicomplexan parasite invasion. © 2012 Zuccala et al

Cell-Cell Communication between Malaria-Infected Red Blood Cells via Exosome-like Vesicles

Cell-cell communication is an important mechanism for information exchange promoting cell survival for the control of features such as population density and differentiation. We determined that Plasmodium falciparum-infected red blood cells directly communicate between parasites within a population using exosome-like vesicles that are capable of delivering genes. Importantly, communication via exosome-like vesicles promotes differentiation to sexual forms at a rate that suggests that signaling is involved. Furthermore, we have identified a P. falciparum protein, PfPTP2, that plays a key role in efficient communication. This study reveals a previously unidentified pathway of P. falciparum biology critical for survival in the host and transmission to mosquitoes. This identifies a pathway for the development of agents to block parasite transmission from the human host to the mosquito.Neta Regev-Rudzki, Danny W. Wilson, Teresa G. Carvalho, Xavier Sisquella, Bradley M. Coleman, Melanie Rug, Dejan Bursac, Fiona Angrisano, Michelle Gee, Andrew F. Hill, Jake Baum, Alan F. Cowma

Super-Resolution Dissection of Coordinated Events during Malaria Parasite Invasion of the Human Erythrocyte

Erythrocyte invasion by the merozoite is an obligatory stage in Plasmodium parasite infection and essential to malaria disease progression. Attempts to study this process have been hindered by the poor invasion synchrony of merozoites from the only in vitro culture-adapted human malaria parasite, Plasmodium falciparum. Using fluorescence, three-dimensional structured illumination, and immunoelectron microscopy of filtered merozoites, we analyze cellular and molecular events underlying each discrete step of invasion. Monitoring the dynamics of these events revealed that commitment to the process is mediated through merozoite attachment to the erythrocyte, triggering all subsequent invasion events, which then proceed without obvious checkpoints. Instead, coordination of the invasion process involves formation of the merozoite-erythrocyte tight junction, which acts as a nexus for rhoptry secretion, surface-protein shedding, and actomyosin motor activation. The ability to break down each molecular step allows us to propose a comprehensive model for the molecular basis of parasite invasion. © 2011 Elsevier Inc

Dysregulation of principal cell miRNAs facilitates epigenetic regulation of AQP2 and results in nephrogenic diabetes insipidus

Background MicroRNAs (miRNAs), formed by cleavage of pre-microRNA by the endoribonuclease Dicer, are critical modulators of cell function by post-transcriptionally regulating gene expression. Methods Selective ablation of Dicer in AQP2-expressing cells (DicerAQP2Cre1 mice) was used to investigate the role of miRNAs in the kidney collecting duct of mice. Results The mice had severe polyuria and nephrogenic diabetes insipidus, potentially due to greatly reduced AQP2 and AQP4 levels. Although epithelial sodium channel levels were decreased in cortex and increased in inner medulla, amiloride-sensitive sodium reabsorption was equivalent in DicerAQP2Cre1 mice and controls. Small-RNA sequencing and proteomic analysis revealed 31 and 178 significantly regulated miRNAs and proteins, respectively. Integrated bioinformatic analysis of the miRNAome and proteome suggested alterations in the epigenetic machinery and various transcription factors regulating AQP2 expression in DicerAQP2Cre1 mice. The expression profile and function of three miRNAs (miR-7688-5p, miR-8114, and miR-409-3p) whose predicted targets were involved in epigenetic control (Phf2, Kdm5c, and Kdm4a) or transcriptional regulation (GATA3, GATA2, and ELF3) of AQP2 were validated. Luciferase assays could not demonstrate direct interaction of AQP2 or the three potential transcription factors with miR-7688-5p, miR-8114, and miR-409-3p. However, transfection of respective miRNA mimics reduced AQP2 expression. Chromatin immunoprecipitation assays demonstrated decreased Phf2 and significantly increased Kdm5c interactions at the Aqp2 gene promoter in DicerAQP2Cre1 mice, resulting in decreased RNA Pol II association. Conclusions Novel evidence indicates miRNA-mediated epigenetic regulation of AQP2 expression

Targeted DNA methylation by homology-directed repair in mammalian cells. Transcription reshapes methylation on the repaired gene.

We report that homology-directed repair of a DNA double-strand break within a single copy Green Fluorescent Protein (GFP) gene in HeLa cells alters the methylation pattern at the site of recombination. DNA methyl transferase (DNMT)1, DNMT3a and two proteins that regulate methylation, Np95 and GADD45A, are recruited to the site of repair and are responsible for selective methylation of the promoter-distal segment of the repaired DNA. The initial methylation pattern of the locus is modified in a transcription-dependent fashion during the 15\u201320 days following repair, at which time no further changes in the methylation pattern occur. The variation in DNA modification generates stable clones with wide ranges of GFP expression. Collectively, our data indicate that somatic DNA methylation follows homologous repair and is subjected to remodeling by local transcription in a discrete time window during and after the damage. We propose that DNA methylation of repaired genes represents a DNA damage code and is source of variation of gene expression

POZ-, AT-hook-, and Zinc Finger-containing Protein (PATZ) Interacts with Human Oncogene B Cell Lymphoma 6 (BCL6) and Is Required for Its Negative Autoregulation.

The PATZ1 gene encoding a POZ/AT-hook/Kruppel zinc finger (PATZ) transcription factor, is considered a cancer-related gene because of its loss or misexpression in human neoplasias. As for other POZ/domain and Kruppel zinc finger (POK) family members, the transcriptional activity of PATZ is due to the POZ-mediated oligomer formation, suggesting that it might be not a typical transactivator but an architectural transcription factor, thus functioning either as activator or as repressor depending on the presence of proteins able to interact with it. Therefore, to better elucidate PATZ function, we searched for its molecular partners. By yeast two-hybrid screenings, we found a specific interaction between PATZ and BCL6, a human oncogene that plays a key role in germinal center (GC) derived neoplasias. We demonstrate that PATZ and BCL6 interact in germinal center-derived B lymphoma cells, through the POZ domain of PATZ. Moreover, we show that PATZ is able to bind the BCL6 regulatory region, where BCL6 itself acts as a negative regulator, and to contribute to negatively modulate its activity. Consistently, disruption of one or both Patz1 alleles in mice causes focal expansion of thymus B cells, in which BCL6 is up-regulated. This phenotype was almost completely rescued by crossing Patz1(+/-) with Bcl6(+/-) mice, indicating a key role for Bcl6 expression in its development. Finally, a significant number of Patz1 knock-out mice (both heterozygous and homozygous) also develop BCL6-expressing lymphomas. Therefore, the disruption of one or both Patz1 alleles may favor lymphomagenesis by activating the BCL6 pathway