Anatomical variations of the equine popliteal tendon

The function of the popliteal muscle and tendon in horses remains undescribed. In humans, it is considered a stabilizer of the posterior-lateral region of the knee; its function is closely related to that of the lateral collateral ligament (LCL) and meniscus. The popliteal tendon (PopT) constitutes the main proximal attachment of the popliteus muscle to the femur, and in humans, insertional variations have been described. Knowledge of anatomical variations is needed for the correct interpretation of diagnostic images and arthroscopic findings. To elucidate further the anatomy of the equine PopT, both hind limbs of 30 horses were dissected. Similar to humans, the equine PopT has 3 variants (types I, II, and III) depending on the number of components forming the tendon. Additionally, the area of insertion varies; the location can be either cranial, underneath, or caudal to the proximal insertion of the LCL. Furthermore, the PopT has a constant attachment to the lateral meniscus. The results of the present study are useful for clinicians working with equine orthopedics, as the tendon and insertional variants could affect the interpretation of diagnostic images and arthroscopic examinations

Análise bionformática do fosfoproteoma neuronal

Mestrado em Biomedicina MolecularA fosforilação anormal de proteínas é uma das características chave da Doença de Alzheimer (DA) que pode estar envolvida tanto na patogénese como na progressão da doença. A fosforilação reversível de proteínas representa um importante mecanismo regulador que envolve a atividade de fosfoproteínas fosfatases (FPF) e proteínas cinases (PC). Um desequilíbrio intracelular entre a actividade de FPF e PC pode alterar a atividade, localização subcelular e interacções de proteínas, contribuindo para a desregulação da função e sinalização neuronal e, consequentemente para a neurodegeneração. Assim, o estudo do fosfoproteoma neuronal da DA tornase relevante tanto do ponto de vista fisiológico como patológico. Culturas primárias corticais foram expostas ao ácido ocadáico (AO, um inibidor de PPP) ou ao péptido β amilóide (Aβ) para mimetizar as condições da DA. Os lisados celulares foram aplicados numa coluna de afinidade para fosfoproteínas. As frações enriquecidas em fosfoproteínas foram analisadas por espetrometria de massa tendo sido desenvolvido um script em linguagem python (http://sourceforge.net/projects/protdb/) para análise das proteínas identificadas. Os resultados provenientes das condições Controlo vs AO indicam que o tratamento com este inibidor de FPF leva a um aumento do número de fosfoproteínas (174 vs 242 proteínas totais e 32 vs 100 proteínas exclusivas). Os resultados do tratamento com Aβ indicam uma alteração qualitativa do fosfoproteoma neuronal (174 vs 166 proteínas totais) com um número considerável de proteínas exclusivas (42 vs 34 proteínas exclusivas). Subsequentemente, para a obtenção de informação detalhada e caracterização das proteínas identificadas em cada condição, foi realizada uma análise exploratória das fosfoproteínas organizando-as por classe proteica, processos biológicos, localização subcelular e funções moleculares. Os tratamentos com AO e Aβ levam a alterações em proteínas envolvidas em processos celulares que se encontram comprometidos na DA, tais como a actividade das PC e FPF, degradação proteica, stress oxidativo, folding proteico, dinâmica do citoesqueleto, síntese proteica e apoptose. A caracterização do fosfoproteoma neuronal da DA pode revelar ou elucidar os mecanismos moleculares subjacentes à transdução de sinais anormal associada com a patogénese da doença. A análise das fosfoproteínas exclusivas poderá, também, contribuir para a identificação de potenciais novos biomarcadores ou alvos terapêuticos para a DA.Abnormal protein phosphorylation is a characteristic hallmark of Alzheimer’s disease (AD) and may be implicated both in pathogenesis or disease progression. Reversible protein phosphorylation represents a key regulatory mechanism involving the activity of protein phosphatases (PPP) and protein kinases (PK). Imbalanced PPP and PK activity can alter protein action, subcellular localization and protein interactions, thus contributing to abnormal neuronal function and signaling and consequently to neurodegeneration. Hence, the study of the AD neuronal phosphoproteome is of physiological and pathological relevance. Primary cortical cultures were exposed to okadaic acid (OA, a PPP inhibitor) or amyloid-β peptide (Aβ), in order to mimic AD conditions. Cell lysates were applied to a phosphoprotein affinity column and phosphoprotein enriched fractions analyzed by mass spectrometry. A protein database management framework (http://sourceforge.net/projects/protdb/) was set up allowing for the development of a script to analyze the identified proteins. Data from Control vs OA conditions indicates that OA treatment leads to an increase in phosphoproteins (174 vs 242 proteins and 32 vs 100 exclusive proteins). Data indicates that Aβ treatment leads to a shift in neuronal phosphoproteome pool (174 vs 166 proteins) with noteworthy alterations in the exclusive neurophosphoproteome (42 vs 34 exclusive proteins). Subsequently, analysis of the protein classes, biological processes, subcellular localization and molecular functions allowed for detailed information regarding the proteins obtained in the different groups. Upon treatments an alteration in the proteins involved in critical processes impaired in AD such as PK and PPP activities, protein degradation, oxidative stress, protein folding, cytoskeleton network dynamics, protein synthesis and apoptosis was observed. The characterization of AD neuronal phosphoproteome may reveal or elucidate the molecular mechanisms underlying abnormal signal transduction associated with AD pathogenesis. Further, by analyzing the pool of exclusive proteins, this work may also contribute to identify potential novel biomarker candidates or AD targets for therapeutic intervention

A Plasmodium falciparum C-mannosyltransferase is dispensable for parasite asexual blood stage development

C-mannosylation was recently identified in the thrombospondin-related anonymous protein (TRAP) from Plasmodium falciparum salivary gland sporozoites. A candidate P. falciparum C-mannosyltransferase (Pf DPY-19) was demonstrated to modify thrombospondin type 1 repeat (TSR) domains in vitro, exhibiting a different acceptor specificity than their mammalian counterparts. According to the described minimal acceptor of Pf DPY19, several TSR domain-containing proteins of P. falciparum could be C-mannosylated in vivo. However, the relevance of this protein modification for the parasite viability remains unknown. In the present study, we used CRISPR/Cas9 technology to generate a Pf DPY19 null mutant, demonstrating that this glycosyltransferase is not essential for the asexual blood development of the parasite. Pf DPY19 gene disruption was not associated with a growth phenotype, not even under endoplasmic reticulum-stressing conditions that could impair protein folding. The data presented in this work strongly suggest that Pf DPY19 is unlikely to play a critical role in the asexual blood stages of the parasite, at least under in vitro conditions

The Apicomplexa-specific glucosamine-6-phosphate N-acetyltransferase gene family encodes a key enzyme for glycoconjugate synthesis with potential as therapeutic target

Apicomplexa form a phylum of obligate parasitic protozoa of great clinical and veterinary importance. These parasites synthesize glycoconjugates for their survival and infectivity, but the enzymatic steps required to generate the glycosylation precursors are not completely characterized. In particular, glucosamine-phosphate N-acetyltransferase (GNA1) activity, needed to produce the essential UDP-N-acetylglucosamine (UDP-GlcNAc) donor, has not been identified in any Apicomplexa. We scanned the genomes of Plasmodium falciparum and representatives from six additional main lineages of the phylum for proteins containing the Gcn5-related N-acetyltransferase (GNAT) domain. One family of GNAT-domain containing proteins, composed by a P. falciparum sequence and its six apicomplexan orthologs, rescued the growth of a yeast temperature-sensitive GNA1 mutant. Heterologous expression and in vitro assays confirmed the GNA1 enzymatic activity in all lineages. Sequence, phylogenetic and synteny analyses suggest an independent origin of the Apicomplexa-specific GNA1 family, parallel to the evolution of a different GNA1 family in other eukaryotes. The inability to disrupt an otherwise modifiable gene target suggests that the enzyme is essential for P. falciparum growth. The relevance of UDP-GlcNAc for parasite viability, together with the independent evolution and unique sequence features of Apicomplexa GNA1, highlights the potential of this enzyme as a selective therapeutic target against apicomplexans

Plasmodium falciparum Apicomplexan-Specific Glucosamine-6-Phosphate <i>N</i>-Acetyltransferase Is Key for Amino Sugar Metabolism and Asexual Blood Stage Development.

--- - i: - N - N - O - N - Plasmodium falciparum - Cryptosporidium parvum - P. falciparum - N - N - P. falciparum - C. parvum b: - IMPORTANCE content: - UDP- - "-acetylglucosamine (UDP-GlcNAc), the main product of the hexosamine biosynthetic pathway, is an important metabolite in protozoan parasites since its sugar moiety is incorporated into glycosylphosphatidylinositol (GPI) glycolipids and " - "- and " - "-linked glycans. Apicomplexan parasites have a hexosamine pathway comparable to other eukaryotic organisms, with the exception of the glucosamine-phosphate " - "-acetyltransferase (GNA1) enzymatic step that has an independent evolutionary origin and significant differences from nonapicomplexan GNA1s. By using conditional genetic engineering, we demonstrate the requirement of GNA1 for the generation of a pool of UDP-GlcNAc and for the development of intraerythrocytic asexual " - " parasites. Furthermore, we present the 1.95\xE2\x80\x89\xC3\x85 resolution structure of the GNA1 ortholog from " - ", an apicomplexan parasite which is a leading cause of diarrhea in developing countries, as a surrogate for " - " GNA1. The in-depth analysis of the crystal shows the presence of specific residues relevant for GNA1 enzymatic activity that are further investigated by the creation of site-specific mutants. The experiments reveal distinct features in apicomplexan GNA1 enzymes that could be exploitable for the generation of selective inhibitors against these parasites, by targeting the hexosamine pathway. This work underscores the potential of apicomplexan GNA1 as a drug target against malaria." - " Apicomplexan parasites cause a major burden on global health and economy. The absence of treatments, the emergence of resistances against available therapies, and the parasite's ability to manipulate host cells and evade immune systems highlight the urgent need to characterize new drug targets to treat infections caused by these parasites. We demonstrate that glucosamine-6-phosphate " - -acetyltransferase (GNA1), required for the biosynthesis of UDP- - "-acetylglucosamine (UDP-GlcNAc), is essential for " - " asexual blood stage development and that the disruption of the gene encoding this enzyme quickly causes the death of the parasite within a life cycle. The high-resolution crystal structure of the GNA1 ortholog from the apicomplexan parasite " - ", used here as a surrogate, highlights significant differences from human GNA1. These divergences can be exploited for the design of specific inhibitors against the malaria parasite.

Content and Feedback Analysis of YouTube Videos: Football Clubs and Fans as Brand Communities

The use of Web 2.0 tools has been transforming the interaction between companies and their clients, especially for those that are selling emotional products. Consumers are generating and sharing contents concerning their favourite products on the web. Even if this process has been widely acknowledged, only a few studies have been specifically devoted to the analysis of both the contents and the feedback the consumers receive from other users. This article analyzes the online presence of sport brands through contents that are generated by sport clubs (official contents) and their fans (User Generated Content, UGC) on YouTube. After a description and classification of video contents, it examines the factors that influence the performance of the videos in terms of passive (videos views) and active behaviour (any kinds of interaction with videos) among the viewers. In order to carry out this analysis, 125 YouTube channels were considered thereby accounting for a total of 375 videos. Results show that official contents are those preferred by the users/consumers and that if the video displays a passive/purely informative content, the chance of getting an active behaviour from the users tends to decrease. These findings may help companies manage their online presence, creating awareness about contents and information that should be spread and shared on the web

Reduced Placental Transfer of Antibodies Against a Wide Range of Microbial and Vaccine Antigens in HIV-Infected Women in Mozambique.

Transplacental transfer of antibodies is essential for conferring protection in newborns against infectious diseases. We assessed the impact of different factors, including gestational age and maternal infections such as HIV and malaria, on the efficiency of cord blood levels and placental transfer of IgG subclasses. We measured total IgG and IgG subclasses by quantitative suspension array technology against 14 pathogens and vaccine antigens, including targets of maternal immunization, in 341 delivering HIV-uninfected and HIV-infected mother-infant pairs from southern Mozambique. We analyzed the association of maternal HIV infection, Plasmodium falciparum exposure, maternal variables and pregnancy outcomes on cord antibody levels and transplacental transfer. Our results show that maternal antibody levels were the main determinant of cord antibody levels. Univariable and multivariable analysis showed that HIV reduced the placental transfer and cord levels of IgG and IgG1 principally, but also IgG2 to half of the antigens tested. P. falciparum exposure and prematurity were negatively associated with cord antibody levels and placental transfer, but this was antigen-subclass dependent. Our findings suggest that lower maternally transferred antibodies may underlie increased susceptibility to infections of HIV-exposed infants. This could affect efficacy of maternal vaccination, especially in sub-Saharan Africa, where there is a high prevalence of HIV, malaria and unfavorable environmental factors

Fuzzy segmentation of postmodern tourists.

In postmodern tourism, the experiences of each tourist could not be summarized only through a unique perspective but multiple and disjointed perspectives are necessary. The aim of this paper is to create a nexus between postmodern tourist and fuzzy clustering, and to propose a suitable clustering procedure to segment postmodern tourists. From a methodological perspective, the main contribution of this paper is related to the use of the fuzzy theory from the beginning to the end of the clustering process. Furthermore, the suggested procedure is capable of analysing the uncertainty and vagueness that characterise the experiences and perceptions of postmodern consumers. From a managerial perspective, fuzzy clustering methods offer to practitioners a more realistic multidimensional description of the market not forcing consumers to belong to one cluster. Moreover, the results are easy and comprehensible to read since they are similar to those obtained with more traditional clustering techniques

Amyotrophic Lateral Sclerosis Multiprotein Biomarkers in Peripheral Blood Mononuclear Cells

Amyotrophic lateral sclerosis (ALS) is a fatal progressive motor neuron disease, for which there are still no diagnostic/prognostic test and therapy. Specific molecular biomarkers are urgently needed to facilitate clinical studies and speed up the development of effective treatments.We used a two-dimensional difference in gel electrophoresis approach to identify in easily accessible clinical samples, peripheral blood mononuclear cells (PBMC), a panel of protein biomarkers that are closely associated with ALS. Validations and a longitudinal study were performed by immunoassays on a selected number of proteins. The same proteins were also measured in PBMC and spinal cord of a G93A SOD1 transgenic rat model. We identified combinations of protein biomarkers that can distinguish, with high discriminatory power, ALS patients from healthy controls (98%), and from patients with neurological disorders that may resemble ALS (91%), between two levels of disease severity (90%), and a number of translational biomarkers, that link responses between human and animal model. We demonstrated that TDP-43, cyclophilin A and ERp57 associate with disease progression in a longitudinal study. Moreover, the protein profile changes detected in peripheral blood mononuclear cells of ALS patients are suggestive of possible intracellular pathogenic mechanisms such as endoplasmic reticulum stress, nitrative stress, disturbances in redox regulation and RNA processing.Our results indicate that PBMC multiprotein biomarkers could contribute to determine amyotrophic lateral sclerosis diagnosis, differential diagnosis, disease severity and progression, and may help to elucidate pathogenic mechanisms