Entamoeba histolytica causes amoebiasis which is a major health concern in developing countries. E. histolytica pathogenicity has been implicated to a large repertoire of small GTPases which switch ...between the inactive GDP bound state and the active GTP bound state with the help of guanine nucleotide exchange factors (GEFs) and GTPase activating protein (GAPs). Rho family of small GTPases are well known to modulate the actin cytoskeletal dynamics which plays a major role in E. histolytica pathogenicity. Here, we report an atypical amoebic RhoGEF, and its preferred substrate EhRho6, which, upon overexpression abrogated the pathogenic behavior of the amoeba such as adhesion to host cell, monolayer destruction, erythrophagocytosis, and formation of actin dots. A causative immunoblot analysis revealed actin degradation in the EhRho6 overexpressing trophozoites that could be inhibited by blocking the amoebic proteasomal pathway. A careful analysis of the results from a previously published transcriptomics study, in conjunction with our observations, led to the identification of a clade of Rho GTPases in this pathogenic amoeba which we hypothesize to have implications during the amoebic encystation.
The transmission of Entamoeba histolytica from one host to another depends on its ability to undergo encystation. In this study, we identified a novel EhRhoGEFduo and its substrate RhoGTPases which are involved in the actin degradation and we hypothesize its implications in amoebic encystation. This study is the first report of Rho family members associated with the degradation of actin in E. histolytica.
Pluripotent embryonic stem cells (ESCs) maintain self-renewal while ensuring a rapid response to differentiation cues. The identification of genes maintaining ESC identity is important to develop ...these cells for their potential therapeutic use. Here we report a genome-scale RNAi screen for a global survey of genes affecting ESC identity via alteration of Oct4 expression. Factors with the strongest effect on Oct4 expression included components of the Paf1 complex, a protein complex associated with RNA polymerase II. Using a combination of proteomics, expression profiling, and chromatin immunoprecipitation, we demonstrate that the Paf1C binds to promoters of key pluripotency genes, where it is required to maintain a transcriptionally active chromatin structure. The Paf1C is developmentally regulated and blocks ESC differentiation upon overexpression, and the knockdown in ESCs causes expression changes similar to Oct4 or Nanog depletions. We propose that the Paf1C plays an important role in maintaining ESC identity.
DNA repair is essential to maintain genome integrity, and genes with roles in DNA repair are frequently mutated in a variety of human diseases. Repair via homologous recombination typically restores ...the original DNA sequence without introducing mutations, and a number of genes that are required for homologous recombination DNA double-strand break repair (HR-DSBR) have been identified. However, a systematic analysis of this important DNA repair pathway in mammalian cells has not been reported. Here, we describe a genome-scale endoribonuclease-prepared short interfering RNA (esiRNA) screen for genes involved in DNA double strand break repair. We report 61 genes that influenced the frequency of HR-DSBR and characterize in detail one of the genes that decreased the frequency of HR-DSBR. We show that the gene KIAA0415 encodes a putative helicase that interacts with SPG11 and SPG15, two proteins mutated in hereditary spastic paraplegia (HSP). We identify mutations in HSP patients, discovering KIAA0415/SPG48 as a novel HSP-associated gene, and show that a KIAA0415/SPG48 mutant cell line is more sensitive to DNA damaging drugs. We present the first genome-scale survey of HR-DSBR in mammalian cells providing a dataset that should accelerate the discovery of novel genes with roles in DNA repair and associated medical conditions. The discovery that proteins forming a novel protein complex are required for efficient HR-DSBR and are mutated in patients suffering from HSP suggests a link between HSP and DNA repair.
Organoid cultivation in suspension culture requires agitation at low shear stress to allow for nutrient diffusion, which preserves tissue structure. Multiplex systems for organoid cultivation have ...been proposed, but whether they meet similar shear stress parameters as the regularly used spinner flask and its correlation with the successful generation of brain organoids has not been determined.
Here we used computational fluid dynamics (CFD) to simulate two multiplex culture conditions: steering plates on an orbital shaker and the use of a previously described bioreactor. The bioreactor had low speed and high shear stress regions that may affect cell aggregate growth, depending on volume, whereas the computed variables of the steering plates were closer to those of the spinning flask.
Our protocol improves the initial steps of the standard brain organoid formation, and the produced organoids displayed regionalized brain structures, including retinal pigmented cells. Overall, we conclude that suspension culture on orbital steering plates is a cost-effective practical alternative to previously described platforms for the cultivation of brain organoids for research and multiplex testing.
Zika virus (ZIKV) is a global public health emergency due to its association with microcephaly, Guillain-Barré syndrome, neuropathy, and myelitis in children and adults. A total of 87 countries have ...had evidence of autochthonous mosquito-borne transmission of ZIKV, distributed across four continents, and no antivirus therapy or vaccines are available. Therefore, several strategies have been developed to target the main mosquito vector,
, to reduce the burden of different arboviruses. Among such strategies, the use of the maternally-inherited endosymbiont
has been applied successfully to reduce virus susceptibility and decrease transmission. However, the mechanisms by which
orchestrate resistance to ZIKV infection remain to be elucidated. In this study, we apply isobaric labeling quantitative mass spectrometry (MS)-based proteomics to quantify proteins and identify pathways altered during ZIKV infection;
infection; co-infection with
ZIKV in the
heads and salivary glands. We show that
regulates proteins involved in reactive oxygen species production, regulates humoral immune response, and antioxidant production. The reduction of ZIKV polyprotein in the presence of
in mosquitoes was determined by MS and corroborates the idea that
helps to block ZIKV infections in
The present study offers a rich resource of data that may help to elucidate mechanisms by which
orchestrate resistance to ZIKV infection in
, and represents a step further on the development of new targeted methods to detect and quantify ZIKV and
directly in complex tissues.
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•Differentiation analysis of SH-SY5Y cells with iTRAQ strategy is proposed.•Differentiated SH-SY5Y cells are more appropriated as a neuronal model.•Upregulated proteins are mainly ...related to ECM-interaction and apoptosis.•Proteins to explore as differentiation markers: AGRN, EMILIM-1, AIFM, STMN1.
SH-SY5Y neuroblastoma cells are susceptible to differentiation using retinoic acid (RA) and brain-derived neurotrophic factor (BDNF), providing a model of neuronal differentiation. We compared SH-SY5Y cells proteome before and after RA/BDNF treatment using iTRAQ and phosphopeptide enrichment strategies. We identified 5587 proteins, 366 of them with differential abundance. Differentiated cells expressed proteins related to neuronal development, and, undifferentiated cells expressed proteins involved in cell proliferation. Interactive network covered focal adhesion, cytoskeleton dynamics and neurodegenerative diseases processes and regulation of mitogen-activated protein kinase-related signaling pathways; key proteins involved in those processes might be explored as markers for neuronal differentiation.
Efficient chromosome segregation during mitosis relies on the coordinated activity of molecular motors with proteins that regulate kinetochore attachments to dynamic spindle microtubules
1. CLASPs ...are conserved kinetochore- and microtubule-associated proteins encoded by two paralog genes,
clasp1 and
clasp2, and have been previously implicated in the regulation of kinetochore microtubule dynamics
2–4. However, it remains unknown how CLASPs work in concert with other proteins to form a functional kinetochore microtubule interface. Here we have identified mitotic interactors of human CLASP1 via a proteomic approach. Among these, the microtubule plus-end-directed motor CENP-E
5 was found to form a complex with CLASP1 that colocalizes to multiple structures of the mitotic apparatus in human cells. We found that CENP-E recruits both CLASP1 and CLASP2 to kinetochores independently of its motor activity or the presence of microtubules. Depletion of CLASPs or CENP-E by RNA interference in human cells causes a significant and comparable reduction of kinetochore microtubule poleward flux and turnover rates and rescues spindle bipolarity in Kif2a-depleted cells. We conclude that CENP-E integrates two critical functions that are important for accurate chromosome movement and spindle architecture: one relying directly on its motor activity, and the other involving the targeting of key microtubule regulators to kinetochores.
Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin America caused by the thermodimorphic fungi of the genus
Paracoccidioides
spp.
Paracoccidioides lutzii
(PL) is one of the 5 species that ...constitute the
Paracoccidioides
genus. PL expresses low amounts of glycoprotein (Gp) 43 (PLGp43) and PLGp43 displays few epitopes in common with the
P. brasiliensis
(PB) immunodominant antigen PBGp43, which is commonly used for serological diagnosis of PCM. This difference in structure between the glycoproteins markedly reduces the efficiency of serological diagnosis in patients infected with PL. We previously demonstrated that peptide 10 (P10) from the PBGp43 induces protective immune responses in
in vitro
and
in vivo
models of PB PCM. Since, P10 has proven to be a promising therapeutic to combat PB, we sought to identify peptides in PL that could similarly be applied for the treatment of PCM. PL yeast cell proteins were isolated from PL: dendritic cell co-cultures and subjected to immunoproteomics. This approach identified 18 PL peptides that demonstrated
in silico
predictions for immunogenicity. Eight of the most promising peptides were synthesized and applied to lymphocytes obtained from peptide-immunized or PL-infected mice as well as to
in vitro
cultures with peptides or dendritic cells pulsed the peptides. The peptides LBR5, LBR6 and LBR8 efficiently promoted CD4
+
and CD8
+
T cell proliferation and dendritic cells pulsed with LBR1, LBR3, LBR7 or LBR8 stimulated CD4
+
T cell proliferation. We observed increases of IFN-γ in the supernatants from primed T cells for the conditions with peptides without or with dendritic cells, although IL-2 levels only increased in response to LBR8. These novel immunogenic peptides derived from PL will be employed to develop new peptide vaccine approaches and the proteins from which they are derived can be used to develop new diagnostic assays for PL and possibly other
Paracoccidioides
spp. These findings identify and characterize new peptides with a promising therapeutic profile for future against this important neglected systemic mycosis.
Zika is a vector-borne disease caused by an arbovirus (ZIKV) and overwhelmingly transmitted by
Ae. aegypti
. This disease is linked to adverse fetal outcomes, mostly microcephaly in newborns, and ...other clinical aspects such as acute febrile illness and neurologic complications, for example, Guillain-Barré syndrome. One of the most promising strategies to mitigate arbovirus transmission involves releasing
Ae. aegypti
mosquitoes carrying the maternally inherited endosymbiont bacteria
Wolbachia pipientis
. The presence of
Wolbachia
is associated with a reduced susceptibility to arboviruses and a fitness cost in mosquito life-history traits such as fecundity and fertility. However, the mechanisms by which
Wolbachia
influences metabolic pathways leading to differences in egg production remains poorly known. To investigate the impact of coinfections on the reproductive tract of the mosquito, we applied an isobaric labeling-based quantitative proteomic strategy to investigate the influence of
Wolbachia w
Mel and ZIKV infection in
Ae. aegypti
ovaries. To the best of our knowledge, this is the most complete proteome of
Ae. aegypti
ovaries reported so far, with a total of 3913 proteins identified, were also able to quantify 1044
Wolbachia
proteins in complex sample tissue of
Ae. aegypti
ovary. Furthermore, from a total of 480 mosquito proteins modulated in our study, we discuss proteins and pathways altered in
Ae. aegypti
during ZIKV infections,
Wolbachia
infections, coinfection
Wolbachia
/ZIKV, and compared with no infection, focusing on immune and reproductive aspects of
Ae. aegypti
. The modified aspects mainly were related to the immune priming enhancement by
Wolbachia
presence and the modulation of the Juvenile Hormone pathway caused by both microorganism’s infection.
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