Role of the Fungus Pneumocystis in IL1ß Pathway Activation and Airways Collagen Deposition in Elastase-Induced COPD Animals.

Inflammation and mucus production are prevalent characteristics of chronic respiratory conditions, such as asthma and chronic chronic obstructive pulmonary disease (COPD). Biological co-factors, including bacteria, viruses, and fungi, may exacerbate these diseases by activating various pathways associated with airway diseases. An example is the fungus Pneumocystis, which is linked to severe COPD in human patients. Recent evidence has demonstrated that Pneumocystis significantly enhanced inflammation and mucus hypersecretion in a rat model of elastase-induced COPD. The present study specifically aims to investigate two additional aspects associated with the pathology induced by Pneumocystis infection: inflammation and collagen deposition around airways. To this end, the focus was to investigate the role of the IL-1ß pro-inflammatory pathway during Pneumocystis infection in COPD rats. Several airway pathology-related features, such as inflammation, mucus hypersecretion, and fibrosis, were evaluated using histological and molecular techniques. COPD animals infected with Pneumocystis exhibited elevated inflammation levels, including a synergistic increase in IL-1ß and Cox-2. Furthermore, protein levels of the IL-1ß-dependent transcription factor cAMP response element-binding (CREB) showed a synergistic elevation of their phosphorylated version in the lungs of COPD animals infected with Pneumocystis, while mucus levels were notably higher in the airways of COPD-infected animals. Interestingly, a CREB responsive element (CRE) was identified in the Muc5b promoter. The presence of CREB in the Muc5b promoter was synergistically increased in COPD animals infected with Pneumocystis compared to other experimental groups. Finally, an increment of deposited collagen was identified surrounding the airways of COPD animals infected with Pneumocystis compared with the other experimental animal groups and correlated with the increase of Tgfß1 mRNA levels. These findings emphasize the role of Pneumocystis as a potential biological co-factor in chronic respiratory diseases like COPD or asthma, warranting new perspectives in the treatment of chronic respiratory diseases.

First detection of the potato cyst nematode, Globodera rostochiensis, infecting potato in the central region of Colombia.

Potato cyst nematodes (PCNs; Globodera spp.) cause significant losses in worldwide cultivated potato (Solanum tuberosum) crops. In Colombia, PCN was first reported in 1970 (Baeza 1972), although this report lacked a comprehensive species description and diagnosis. After that, G. pallida has been the only PCN species reported affecting potatoes in the main producing regions of Colombia (Evans et al. 1975; Nieto et al. 1983; Vallejo et al. 2021). However, in the survey conducted by Vallejo et al. (2021), a single sample from Chocontá, Cundinamarca in the central region of the country (N 5,22396046668291, W -73,6571338400244) showed molecular characters similar to G. rostochiensis. As correct identification is essential for effective pest management, the location was re-sampled in September 2022. From the soil samples collected, PCN cysts and second-stage juveniles (J2s) were retrieved from soil using Fenwick and centrifugation methods, respectively. Morphometric characters of cysts (n = 53) were consistent with G. rostochiensis, with a length without neck (L) ranging from 451 to 614 µm (X̅ = 546.9 ± 20.3 µm), width (W) from 424 to 658 µm (X̅ = 546.9 ± 25.5 µm) and L/W ratio was 1.00 ± 0.02. Distance from anus to vulva varied from 41 to 109 µm (X̅ =75.67 ± 13.8 µm), Granek's ratio from 2.3 to 5.5 µm (X̅ = 3.89 ± 0.7 µm), and the number of cuticular ridges between the vulva and the anus were 14 to 20 (X̅ = 16.19 ± 1.7). The second-stage juvenile (n = 90) length ranged from 394 to 547 µm (X̅ = 495.62 ± 31.0 µm), the stylet length varied from 18 to 24 µm (X̅ = 21.21 ± 0.9 µm) with rounded knobs. The length of the hyaline tail ranged from 20 - 31 µm (X̅ = 24.09 ± 1.92) and the true tail from 31- 56 µm (X̅ = 48.30 ± 5.71 µm). Molecular analyses confirmed morphological identification. DNA was extracted from cysts and J2s. PCR was performed for the 28S rDNA D2-D3 segment using primers D2A and D3B (Subbotin et al. 2006), and for the mitochondrial COI gene region using primers JB3 and JB5 (Derycke et al. 2005). BLAST analyses of target 28S rDNA D2-D3 sequences (OP293373-OP293380) showed 100% identity of the 658 bp to other sequences on Genbank, including isolates from Turkey, United Kingdom, and Iran (MK311329.1, MG994942.1, KU297659.1, and KU297658.1). Similarly, the target COI region sequences (OP297993-OP298001) were 100% identical to the 407 bp of G. rostochiensis POT01 isolate from Germany, and 99.75% identical to voucher NRM67 from Indonesia, and isolate CD2200 from USA (MF773722.1, MT240262.1, and MN095979.1). Phylogenetic analysis of both gene regions strongly supported G. rostochiensis, with the Colombian sequences clustering with MH399815.1, and KU297654.1 isolates for the COI and 28S regions, respectively (Fig. 1S). In addition, a pathogenicity test was conducted in the greenhouse. For this, ten cysts were inoculated to potato plants of Criolla variety grown in 5 pots of 15 cm diameter with sterile soil and sand (1:1). Noninoculated plants served as controls (three replicates each). After three months, 54 ± 23 cysts/100 g of soil were isolated from inoculated plants (Fig. 2S), resulting in a reproduction factor (R=Pf/Pi) of 4.54 ± 0.86, while no yellow females or cysts were observed on the control plants. To our knowledge, this is the first report of G. rostochiensis in Colombia. This is an important pest that causes serious yield losses of potatoes and is a quarantine nematode in many countries (EPPO 2017). Further studies are necessary to prevent the spread of this PCN species in the main producing potato regions of Colombia.

Pneumocystis Exacerbates Inflammation and Mucus Hypersecretion in a Murine, Elastase-Induced-COPD Model.

Inflammation and mucus hypersecretion are frequent pathology features of chronic respiratory diseases such as asthma and COPD. Selected bacteria, viruses and fungi may synergize as co-factors in aggravating disease by activating pathways that are able to induce airway pathology. Pneumocystis infection induces inflammation and mucus hypersecretion in immune competent and compromised humans and animals. This fungus is a frequent colonizer in patients with COPD. Therefore, it becomes essential to identify whether it has a role in aggravating COPD severity. This work used an elastase-induced COPD model to evaluate the role of Pneumocystis in the exacerbation of pathology, including COPD-like lung lesions, inflammation and mucus hypersecretion. Animals infected with Pneumocystis developed increased histology features of COPD, inflammatory cuffs around airways and lung vasculature plus mucus hypersecretion. Pneumocystis induced a synergic increment in levels of inflammation markers (Cxcl2, IL6, IL8 and IL10) and mucins (Muc5ac/Muc5b). Levels of STAT6-dependent transcription factors Gata3, FoxA3 and Spdef were also synergically increased in Pneumocystis infected animals and elastase-induced COPD, while the levels of the mucous cell-hyperplasia transcription factor FoxA2 were decreased compared to the other groups. Results document that Pneumocystis is a co-factor for disease severity in this elastase-induced-COPD model and highlight the relevance of STAT6 pathway in Pneumocystis pathogenesis.

Flow Diverter Treatment for Non-Ruptured Carotid Aneurysms: Efficacy and Safety.

PURPOSE: Internal carotid artery (ICA) aneurysm treatment with a flow diverter (FD) has shown an adequate efficacy and safety profile, presenting high complete occlusion or near occlusion rates with low complications during follow-up. The purpose of this study was to evaluate the efficacy and safety of FD treatment in non-ruptured internal carotid aneurysms. MATERIALS AND METHODS: This is a retrospective, single-center, observational study evaluating patients diagnosed with unruptured ICA aneurysms treated with an FD between January 1, 2014, and January 1, 2020. We analyzed an anonymized database. The primary effectiveness endpoint was complete occlusion (O'Kelly-Marotta D, OKM-D) of the target aneurysm through 1-year follow-up. The safety endpoint was the evaluation of modified Rankin Scale (mRS) 90 days after treatment, considering a favorable outcome an mRS 0-2. RESULTS: A total of 106 patients were treated with an FD, 91.5% were women; the mean follow- up was 427.2±144.8 days. Technical success was achieved in 105 cases (99.1%). All patients included had 1-year follow-up digital subtraction angiography control; 78 patients (73.6%) completed the primary efficacy endpoint by achieving total occlusion (OKM-D). Giant aneurysms had a higher risk of not achieving complete occlusion (risk ratio, 3.07; 95% confidence interval, 1.70 - 5.54]). The safety endpoint of mRS 0-2 at 90 days was accomplished in 103 patients (97.2%). CONCLUSION: Treatment of unruptured ICA aneurysms with an FD showed high 1-year total occlusion results, with very low morbidity and mortality complications.

Trypanosoma cruzi DNA Polymerase ß Is Phosphorylated In Vivo and In Vitro by Protein Kinase C (PKC) and Casein Kinase 2 (CK2).

DNA polymerase ß plays a fundamental role in the life cycle of Trypanosoma cruzi since it participates in the kinetoplast DNA repair and replication. This enzyme can be found in two forms in cell extracts of T. cruzi epimastigotes form. The H form is a phosphorylated form of DNA polymerase ß, while the L form is not phosphorylated. The protein kinases which are able to in vivo phosphorylate DNA polymerase ß have not been identified yet. In this work, we purified the H form of this DNA polymerase and identified the phosphorylation sites. DNA polymerase ß is in vivo phosphorylated at several amino acid residues including Tyr35, Thr123, Thr137 and Ser286. Thr123 is phosphorylated by casein kinase 2 and Thr137 and Ser286 are phosphorylated by protein kinase C-like enzymes. Protein kinase C encoding genes were identified in T. cruzi, and those genes were cloned, expressed in bacteria and the recombinant protein was purified. It was found that T. cruzi possesses three different protein kinase C-like enzymes named TcPKC1, TcPKC2, and TcPKC3. Both TcPKC1 and TcPKC2 were able to in vitro phosphorylate recombinant DNA polymerase ß, and in addition, TcPKC1 gets auto phosphorylated. Those proteins contain several regulatory domains at the N-terminus, which are predicted to bind phosphoinositols, and TcPKC1 contains a lipocalin domain at the C-terminus that might be able to bind free fatty acids. Tyr35 is phosphorylated by an unidentified protein kinase and considering that the T. cruzi genome does not contain Tyr kinase encoding genes, it is probable that Tyr35 could be phosphorylated by a dual protein kinase. Wee1 is a eukaryotic dual protein kinase involved in cell cycle regulation. We identified a Wee1 homolog in T. cruzi and the recombinant kinase was assayed using DNA polymerase ß as a substrate. T. cruzi Wee1 was able to in vitro phosphorylate recombinant DNA polymerase ß, although we were not able to demonstrate specific phosphorylation on Tyr35. Those results indicate that there exists a cell signaling pathway involving PKC-like kinases in T. cruzi.

The Catalytic Subunit of Schizosaccharomyces pombe CK2 (Cka1) Negatively Regulates RNA Polymerase II Transcription through Phosphorylation of Positive Cofactor 4 (PC4).

Positive cofactor 4 (PC4) is a transcriptional coactivator that plays important roles in transcription and DNA replication. In mammals, PC4 is phosphorylated by CK2, and this event downregulates its RNA polymerase II (RNAPII) coactivator function. This work describes the effect of fission yeast PC4 phosphorylation on RNAPII transcription in a cell extract, which closely resembles the cellular context. We found that fission yeast PC4 is strongly phosphorylated by the catalytic subunit of CK2 (Cka1), while the regulatory subunit (Ckb1) downregulates the PC4 phosphorylation. The addition of Cka1 to an in vitro transcription assay can diminish the basal transcription from the Ad-MLP promoter; however, the addition of recombinant fission yeast PC4 or Ckb1 can stimulate the basal transcription in a cell extract. Fission yeast PC4 is phosphorylated in a domain which has consensus phosphorylation sites for CK2, and two serine residues were identified as critical for CK2 phosphorylation. Mutation of one of the serine residues in PC4 does not completely abolish the phosphorylation; however, when the two serine residues are mutated, CK2 is no longer able to phosphorylate PC4. The mutant which is not phosphorylated is able to stimulate transcription even though it is previously phosphorylated by Cka1, while the wild type and the point mutant are inactivated by Cka1 phosphorylation, and they cannot stimulate transcription by RNAPII in cell extracts. Those results demonstrate that CK2 can regulate the coactivator function of fission yeast PC4 and suggests that this event could be important in vivo as well.

RNA Polymerase II Transcription in Pneumocystis: TFIIB from Pneumocystis carinii Can Replace the Transcriptional Functions of Fission Yeast Schizosaccharomyces pombe TFIIB In Vivo and In Vitro.

The Pneumocystis genus is an opportunistic fungal pathogen that infects patients with AIDS and immunocompromised individuals. The study of this fungus has been hampered due to the inability to grow it in a (defined media/pure) culture. However, the use of modern molecular techniques and genomic analysis has helped researchers to understand its complex cell biology. The transcriptional process in the Pneumocystis genus has not been studied yet, although it is assumed that it has conventional transcriptional machinery. In this work, we have characterized the function of the RNA polymerase II (RNAPII) general transcription factor TFIIB from Pneumocystis carinii using the phylogenetically related biological model Schizosaccharomyces pombe. The results of this work show that Pneumocystis carinii TFIIB is able to replace the essential function of S. pombe TFIIB both in in vivo and in vitro assays. The S. pombe strain harboring the P carinii TFIIB grew slower than the parental wild-type S. pombe strain in complete media and in minimal media. The S. pombe cells carrying out the P. carinii TFIIB are larger than the wild-type cells, indicating that the TFIIB gene replacement confers a phenotype, most likely due to defects in transcription. P. carinii TFIIB forms very weak complexes with S. pombe TATA-binding protein on a TATA box promoter but it is able to form stable complexes in vitro when S. pombe TFIIF/RNAPII are added. P. carinii TFIIB can also replace the transcriptional function of S. pombe TFIIB in an in vitro assay. The transcription start sites (TSS) of the endogenous adh gene do not change when P. carinii TFIIB replaces S. pombe TFIIB, and neither does the TSS of the nmt1 gene, although this last gene is poorly transcribed in vivo in the presence of P. carinii TFIIB. Since transcription by RNA polymerase II in Pneumocystis is poorly understood, the results described in this study are promising and indicate that TFIIB from P. carinii can replace the transcriptional functions of S. pombe TFIIB, although the cells expressing the P. carini TFIIB show an altered phenotype. However, performing studies using a heterologous approach, like this one, could be relevant to understanding the basic molecular processes of Pneumocystis such as transcription and replication.

Initiator-Directed Transcription: Fission Yeast Nmtl Initiator Directs Preinitiation Complex Formation and Transcriptional Initiation.

The initiator element is a core promoter element encompassing the transcription start site, which is found in yeast, Drosophila, and human promoters. This element is observed in TATA-less promoters. Several studies have defined transcription factor requirements and additional cofactors that are needed for transcription initiation of initiator-containing promoters. However, those studies have been performed with additional core promoters in addition to the initiator. In this work, we have defined the pathway of preinitiation complex formation on the fission yeast nmt1 gene promoter, which contains a functional initiator with striking similarity to the initiator of the human dihydrofolate reductase (hDHFR) gene and to the factor requirement for transcription initiation of the nmt1 gene promoter. The results show that the nmt1 gene promoter possesses an initiator encompassing the transcription start site, and several conserved base positions are required for initiator function. A preinitiation complex formation on the nmt1 initiator can be started by TBP/TFIIA or TBP/TFIIB, but not TBP alone, and afterwards follows the same pathway as preinitiation complex formation on TATA-containing promoters. Transcription initiation is dependent on the general transcription factors TBP, TFIIB, TFIIE, TFIIF, TFIIH, RNA polymerase II, Mediator, and a cofactor identified as transcription cofactor for initiator function (TCIF), which is a high-molecular-weight protein complex of around 500 kDa. However, the TAF subunits of TFIID were not required for the nmt1 initiator transcription, as far as we tested. We also demonstrate that other initiators of the nmt1/hDHFR family can be transcribed in fission yeast whole-cell extracts.

The Use of Antioxidants as Potential Co-Adjuvants to Treat Chronic Chagas Disease.

Chagas disease is a neglected tropical disease caused by the flagellated protozoa Trypanosome cruzi. This illness affects to almost 8-12 million people worldwide, however, is endemic to Latin American countries. It is mainly vectorially transmitted by insects of the Triatominae family, although other transmission routes also exist. T. cruzi-infected cardiomyocytes at the chronic stage of the disease display severe mitochondrial dysfunction and high ROS production, leading to chronic myocardial inflammation and heart failure. Under cellular stress, cells usually can launch mitochondrial biogenesis in order to restore energy loss. Key players to begin mitochondrial biogenesis are the PGC-1 (PPARγ coactivator 1) family of transcriptional coactivators, which are activated in response to several stimuli, either by deacetylation or dephosphorylation, and in turn can serve as coactivators for the NRF (nuclear respiratory factor) family of transcription factors. The NRF family of transcriptional activators, namely NRF1 and NRF2, can activate gene expression of oxidative phosphorylation (OXPHOS) components, mitochondrial transcriptional factor (Tfam) and nuclear encoded mitochondrial proteins, leading to mitochondrial biogenesis. On the other hand, NRF2 can activate gene expression of antioxidant enzymes in response to antioxidants, oxidants, electrophile compounds, pharmaceutical and dietary compounds in a mechanism dependent on KEAP1 (Kelch-like ECH-associated protein 1). Since a definitive cure to treat Chagas disease has not been found yet; the use of antioxidants a co-adjuvant therapy has been proposed in an effort to improve mitochondrial functions, biogenesis, and the antioxidant defenses response. Those antioxidants could activate different pathways to begin mitochondrial biogenesis and/or cytoprotective antioxidant defenses. In this review we discuss the main mechanisms of mitochondrial biogenesis and the NRF2-KEAP1 activation pathway. We also reviewed the antioxidants used as co-adjuvant therapy to treat experimental Chagas disease and their action mechanisms and finish with the discussion of antioxidant therapy used in Chagas disease patients.

T. cruzi DNA polymerase beta (Tcpolß) is phosphorylated in vitro by CK1, CK2 and TcAUK1 leading to the potentiation of its DNA synthesis activity.

The unicellular protozoan Trypanosoma cruzi is the causing agent of Chagas disease which affects several millions of people around the world. The components of the cell signaling pathways in this parasite have not been well studied yet, although its genome can encode several components able to transduce the signals, such as protein kinases and phosphatases. In a previous work we have found that DNA polymerase ß (Tcpolß) can be phosphorylated in vivo and this modification activates the synthesis activity of the enzyme. Tcpolß is kinetoplast-located and is a key enzyme in the DNA base excision repair (BER) system. The polypeptide possesses several consensus phosphorylation sites for several protein kinases, however, a direct phosphorylation of those sites by specific kinases has not been reported yet. Tcpolß has consensus phosphorylation sites for casein kinase 1 (CK1), casein kinase 2 (CK2) and aurora kinase (AUK). Genes encoding orthologues of those kinases exist in T. cruzi and we were able to identify the genes and to express them to investigate whether or no Tcpolß could be a substrate for in vitro phosphorylation by those kinases. Both CK1 and TcAUK1 have auto-phosphorylation activities and they are able to phosphorylate Tcpolß. CK2 cannot perform auto-phosphorylation of its subunits, however, it was able to phosphorylate Tcpolß. Pharmacological inhibitors used to inhibit the homologous mammalian kinases can also inhibit the activity of T. cruzi kinases, although, at higher concentrations. The phosphorylation events carried out by those kinases can potentiate the DNA polymerase activity of Tcpolß and it is discussed the role of the phosphorylation on the DNA polymerase and lyase activities of Tcpolß. Taken altogether, indicates that CK1, CK2 and TcAUK1 can play an in vivo role regulating the function of Tcpolß.

Occurrence and molecular characterization of cyst nematode species (Globodera spp.) associated with potato crops in Colombia.

Potato cyst nematodes (PCN) from the genus Globodera spp. cause major losses in the potato (Solanum tuberosum) industry worldwide. Despite their importance, at present little is known about the status of this plant pathogen in cultivated potatoes in Colombia. In this study, a total of 589 samples collected from 75 geographic localities in nine potato producing regions of Colombia (Cundinamarca, Boyacá, Antioquia, Nariño, Santander, Norte de Santander, Tolima, Caldas and Cauca) were assayed for the presence of potato cyst nematodes. Fifty-seven percent of samples tested positive for PCN. Based on phylogenetic analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2) of the rRNA gene and D2-D3 expansion segments of the 28S rRNA gene, all populations but one were identified as Globodera pallida. Sequences of G. pallida from Colombia formed a monophyletic group closely related to Peruvian populations, with the lowest average number of nucleotide substitutions per site (Dxy = 0.002) and net nucleotide substitutions per site (Da = 0.001), when compared to G. pallida populations from Europe, South and North America. A single sample formed a well-supported subclade along with G. rostochiensis and G. tabacum from Japan, USA and Argentina. To our knowledge this is the first comprehensive survey of Globodera populations from Colombia that includes genetic data. Our findings on species diversity and phylogenetic relationships of Globodera populations from Colombia may help elucidate the status and distribution of Globodera species, and lead to the development of accurate management strategies for the potato cyst nematodes.

The Oxidative Stress and Chronic Inflammatory Process in Chagas Disease: Role of Exosomes and Contributing Genetic Factors.

Chagas disease is a neglected tropical disease caused by the flagellated protozoa Trypanosoma cruzi that affects several million people mainly in Latin American countries. Chagas disease has two phases, which are acute and chronic, both separated by an indeterminate time period in which the infected individual is relatively asymptomatic. The acute phase extends for 40-60 days with atypical and mild symptoms; however, about 30% of the infected patients will develop a symptomatic chronic phase, which is characterized by either cardiac, digestive, neurological, or endocrine problems. Cardiomyopathy is the most important and severe result of Chagas disease, which leads to left ventricular systolic dysfunction, heart failure, and sudden cardiac death. Most deaths are due to heart failure (70%) and sudden death (30%) resulting from cardiomyopathy. During the chronic phase, T. cruzi-infected macrophages respond with the production of proinflammatory cytokines and production of superoxide and nitric oxide by the NADPH oxidase 2 (NOX2) and inducible nitric oxide synthase (iNOS) enzymes, respectively. During the chronic phase, myocardial changes are produced as a result of chronic inflammation, oxidative stress, fibrosis, and cell death. The cellular inflammatory response is mainly the result of activation of the NF-κB-dependent pathway, which activates gene expression of inflammatory cytokines, leading to progressive tissue damage. The persisting production of reactive oxygen species (ROS) is the result of mitochondrial dysfunction in the cardiomyocytes. In this review, we will discuss inflammation and oxidative damage which is produced in the heart during the chronic phase of Chagas disease and recent evidence on the role of macrophages and the production of proinflammatory cytokines during the acute phase and the origin of macrophages/monocytes during the chronic phase of Chagas disease. We will also discuss the contributing factors and mechanisms leading to the chronic inflammation of the cardiac tissue during the chronic phase of the disease as well as the innate and adaptive host immune response. The contribution of genetic factors to the progression of the chronic inflammatory cardiomyopathy of chronic Chagas disease is also discussed. The secreted extracellular vesicles (exosomes) produced for both T. cruzi and infected host cells can play key roles in the host immune response, and those roles are described. Lastly, we describe potential treatments to attenuate the chronic inflammation of the cardiac tissue, designed to improve heart function in chagasic patients.

Dual and Opposite Roles of Reactive Oxygen Species (ROS) in Chagas Disease: Beneficial on the Pathogen and Harmful on the Host.

Chagas disease is a neglected tropical disease, which affects an estimate of 6-7 million people worldwide. Chagas disease is caused by Trypanosoma cruzi, which is a eukaryotic flagellate unicellular organism. At the primary infection sites, these parasites are phagocytized by macrophages, which produce reactive oxygen species (ROS) in response to the infection with T. cruzi. The ROS produce damage to the host tissues; however, macrophage-produced ROS is also used as a signal for T. cruzi proliferation. At the later stages of infection, mitochondrial ROS is produced by the infected cardiomyocytes that contribute to the oxidative damage, which persists at the chronic stage of the disease. The oxidative damage leads to a functional impairment of the heart. In this review article, we will discuss the mechanisms by which T. cruzi is able to deal with the oxidative stress and how this helps the parasite growth at the acute phase of infection and how the oxidative stress affects the cardiomyopathy at the chronic stage of the Chagas disease. We will describe the mechanisms used by the parasite to deal with ROS and reactive nitrogen species (RNS) through the trypanothione and the mechanisms used to repair the damaged DNA. Also, a description of the events produced by ROS at the acute and chronic stages of the disease is presented. Lastly, we discuss the benefits of ROS for T. cruzi growth and proliferation and the possible mechanisms involved in this phenomenon. Hypothesis is put forward to explain the molecular mechanisms by which ROS triggers parasite growth and proliferation and how ROS is able to produce a long persisting damage on cardiomyocytes even in the absence of the parasite.

Primary infection by Pneumocystis induces Notch-independent Clara cell mucin production in rat distal airways.

Clara cells are the main airway secretory cells able to regenerate epithelium in the distal airways through transdifferentiating into goblet cells, a process under negative regulation of the Notch pathway. Pneumocystis is a highly prevalent fungus in humans occurring between 2 and 5 months of age, a period when airways are still developing and respiratory morbidity typically increases. Pneumocystis induces mucus hyperproduction in immunocompetent host airways and whether it can stimulate Clara cells is unknown. Markers of Clara cell secretion and Notch1 activation were investigated in lungs of immunocompetent rats at 40, 60, and 80 days of age during Pneumocystis primary infection with and without Valproic acid (VPA), a Notch inducer. The proportion of rats expressing mucin increased in Pneumocystis-infected rats respect to controls at 60 and 80 days of age. Frequency of distal airways Clara cells was maintained while mRNA levels for the mucin-encoding genes Muc5B and Muc5ac in lung homogenates increased 1.9 and 3.9 times at 60 days of infection (P. = 0.1609 and P. = 0.0001, respectively) and protein levels of the Clara cell marker CC10 decreased in the Pneumocystis-infected rats at 60 and 80 days of age (P. = 0.0118 & P. = 0.0388). CC10 and Muc5b co-localized in distal airway epithelium of Pneumocystis-infected rats at day 60. Co-localization of Muc5b and Ki67 as marker of mitosis in distal airways was not observed suggesting that Muc5b production by Clara cells was independent of mitosis. Notch levels remained similar and no transnucleation of activated Notch associated to Pneumocystis infection was detected. Unexpectedly, mucus was greatly increased at day 80 in Pneumocystis-infected rats receiving VPA suggesting that a Notch-independent mechanism was triggered. Overall, data suggests a Clara to goblet cell transdifferentiation mechanism induced by Pneumocystis and independent of Notch.

Increase in secreted airway mucins and partial Muc5b STAT6/FoxA2 regulation during Pneumocystis primary infection.

Airway mucus responses to subclinical infections may explain variations in progression of chronic lung diseases and differences in clinical expression of respiratory infections across individuals. Pneumocystis associates to more severe Chronic Obstructive Pulmonary Disease (COPD), asthma, respiratory distress of premature newborns, and is a consistent subclinical infection between 2 and 5 months of age when hospitalizations for respiratory cause and infant mortality are higher. This atypical fungus associates to increased mucin 5AC (MUC5AC), a central effector of Th2-type allergic inflammation, in infant lungs. However, mucus progression, expression of MUC5B essential for airway defense, and potential for pharmacologic modulation of mucus during Pneumocystis infection remain unknown. We measured MUC5B and Pneumocystis in infant lungs, and progression of mucin levels and effect of inhibition of the STAT6/FoxA2 mucus pathway using Kaempferol, a JAK/STAT6 inhibitor, in immunocompetent rats during Pneumocystis primary infection. Pneumocystis associated to increased MUC5B in infant lungs. Muc5b increased earlier and more abundantly than Muc5ac during experimental primary infection suggesting an acute defensive response against Pneumocystis as described against bacteria, while increased Muc5ac levels supports an ongoing allergic, Th2 lymphocyte-type response during primary Pneumocystis infection. Kaempferol partly reversed Muc5b stimulation suggesting limited potential for pharmacological modulation via the STAT6-FoxA2 pathway.

Comments on "Affective instability in those with and without mental disorders: A case control study" by Marwaha et al.

This letter has the purpose to comment the article by Marwaha et al. regarding affective instability and mental disorders. We wish to highlight the importance to report the proper measures of association in case-control studies, and the impact of adjusting the results when finding associations with possible confounders in the bivariate analysis.

Endogenous overexpression of an active phosphorylated form of DNA polymerase ß under oxidative stress in Trypanosoma cruzi.

Trypanosoma cruzi is exposed during its life to exogenous and endogenous oxidative stress, leading to damage of several macromolecules such as DNA. There are many DNA repair pathways in the nucleus and mitochondria (kinetoplast), where specific protein complexes detect and eliminate damage to DNA. One group of these proteins is the DNA polymerases. In particular, Tc DNA polymerase ß participates in kinetoplast DNA replication and repair. However, the mechanisms which control its expression under oxidative stress are still unknown. Here we describe the effect of oxidative stress on the expression and function of Tc DNA polymerase ß To this end parasite cells (epimastigotes and trypomastigotes) were exposed to peroxide during short periods of time. Tc DNA polymerase ß which was associated physically with kinetoplast DNA, showed increased protein levels in response to peroxide damage in both parasite forms analyzed. Two forms of DNA polymerase ß were identified and overexpressed after peroxide treatment. One of them was phosphorylated and active in DNA synthesis after renaturation on polyacrylamide electrophoresis gel. This phosphorylated form showed 3-4-fold increase in both parasite forms. Our findings indicate that these increments in protein levels are not under transcriptional control because the level of Tc DNA polymerase ß mRNA is maintained or slightly decreased during the exposure to oxidative stress. We propose a mechanism where a DNA repair pathway activates a cascade leading to the increment of expression and phosphorylation of Tc DNA polymerase ß in response to oxidative damage, which is discussed in the context of what is known in other trypanosomes which lack transcriptional control.

Progression of Type 2 Helper T Cell-Type Inflammation and Airway Remodeling in a Rodent Model of Naturally Acquired Subclinical Primary Pneumocystis Infection.

Subclinical primary Pneumocystis infection is the most common pulmonary infection in early infancy, making it important to determine whether it damages the lung. Pneumocystis peaks at 2 to 5 months of age, when respiratory morbidity coincidently increases. We have documented that Pneumocystis increases mucus production in infant lungs, and animal models reveal lung lesions that warrant characterization. Herein, immunocompetent rats infected at birth with Pneumocystis by cohabitation, to resemble community-acquired infection, underwent lung assessments at 45, 60, and 75 days of age. Lungs fixed by vascular perfusion to prevent collapse during necropsy were used for morphometry evaluations of mucus production, airway epithelial thickening, perivascular and peribronchiolar inflammation, and structural airway remodeling. Changes in these histologic features indicate lung disease. Selected immune markers were assessed in parallel using fresh-frozen lung tissue from sibling rats of the same cages. Sequential activation of NF-κB and an increased Gata3/T-bet mRNA level ratio, consistent with a type 2 helper T-cell-type inflammatory response, and subacute fibrosis were recognized. Therefore, documenting subclinical Pneumocystis infection induces lung disease in the immunocompetent host. Taken together with the peak age of primary Pneumocystis infection, results warrant investigating the clinical impact of this often subclinical infection on the severity of respiratory diseases in early infancy. This model can also be used to assess the effects of airway insults, including coinfections by recognized respiratory pathogens.

RNA polymerase II components and Rrn7 form a preinitiation complex on the HomolD box to promote ribosomal protein gene expression in Schizosaccharomyces pombe.

In Schizosaccharomyces pombe, ribosomal protein gene (RPG) promoters contain a TATA box analog, the HomolD box, which is bound by the Rrn7 protein. Despite the importance of ribosome biogenesis for cell survival, the mechanisms underlying RPG transcription remain unknown. In this study, we found that components of the RNA polymerase II (RNAPII) system, consisting of the initiation or general transcription factors (GTFs) TFIIA, IIB, IIE, TATA-binding protein (TBP) and the RNAPII holoenzyme, interacted directly with Rrn7 in vitro, and were able to form a preinitiation complex (PIC) on the HomolD box. PIC complex formation follows an ordered pathway on these promoters. The GTFs and RNAPII can also be cross-linked to HomolD-containing promoters in vivo. In an in vitro reconstituted transcription system, RNAPII components and Rrn7 were necessary for HomolD-directed transcription. The Mediator complex was required for basal transcription from those promoters in whole cell extract (WCE). The Med17 subunit of Mediator also can be cross-linked to the promoter region of HomolD-containing promoters in vivo, suggesting the presence of the Mediator complex on HomolD box-containing promoters. Together, these data show that components of the RNAPII machinery and Rrn7 participate in the PIC assembly on the HomolD box, thereby directing RPG transcription.

The Ski Protein is Involved in the Transformation Pathway of Aurora Kinase A.

Oncogenic kinase Aurora A (AURKA) has been found to be overexpresed in several tumors including colorectal, breast, and hematological cancers. Overexpression of AURKA induces centrosome amplification and aneuploidy and it is related with cancer progression and poor prognosis. Here we show that AURKA phosphorylates in vitro the transcripcional co-repressor Ski on aminoacids Ser326 and Ser383. Phosphorylations on these aminoacids decreased Ski protein half-life. Reduced levels of Ski resulted in centrosomes amplification and multipolar spindles formation, same as AURKA overexpressing cells. Importantly, overexpression of Ski wild type, but not S326D and S383D mutants inhibited centrosome amplification and cellular transformation induced by AURKA. Altogether, these results suggest that the Ski protein is a target in the transformation pathway mediated by the AURKA oncogene.