Regulation of mitotic exit in higher eukaryotes:Drosophila mob genes

Tese de mestrado, Biologia (Biologia Evolutiva e do Desenvolvimento), 2009, Universidade de Lisboa, Faculdade de CiênciasO correcto processo de desenvolvimento depende da proliferação celular, apoptose e morfogénese. Esta dependência é, não só relativa a cada um destes processos isoladamente, mas essencialmente ao correcto balanceamento espacial e temporal entre eles. A desregulação destes processos ou da sinalização entre os mesmos pode levar ao desenvolvimento de doenças, como o cancro. Neste trabalho, pretendeu-se estudar aspectos relacionados com a regulação dos processos de divisão celular, usando como organismo modelo a mosca Drosophila melanogaster. Em concreto, pretendeu-se investigar o papel da saída de mitose durante o desenvolvimento. Nas leveduras S.cerevisae e S. Pombe os genes mob1 são essenciais à saída de mitose e citocinese, respectivamente. Estes dois processos estão altamente regulados por cascatas de sinalização: 'Mitotic Exit Network' (MEN) em S.cerevisae e 'Septation Initiation Network' (SIN) em S. Pombe. Desta forma, através do uso de mutantes para os genes mob1 e mob2 em Drosophila, investigou-se o seu papel na possível regulação da saída de mitose e a forma como este processo seria regulado. A caracterização fenotípica sugere que estes genes têm papéis diferentes dos seus ortólogos em levedura, i.e., não aparentam ter qualquer papel na regulação da saída de mitose ou citocinese. Em concreto, o gene dmob1 é um gene essencial, cujos mutantes morrem durante a embriogénese. Parece estar envolvido em processos de polaridade celular tanto durante o desenvolvimento embrionário, como no adulto. Durante as fases iniciais da embriogénese, sincício, as mitoses dos mutantes mob1 têm os centrossomas fora dos pólos do fuso mitótico, que se encontram mal focados. Curiosamente, estes defeitos na formação do fuso são ultrapassados com o avançar no desenvolvimento. Em estádios mais avançados do desenvolvimento verificou-se que nos mutantes mob1 processos morfogenéticos, como 'germband retration' falham. A análise de estruturas larvares permitiu perceber que este gene tem também um papel importante na regulação da proliferação celular. Finalmente, o estudo de mosaicos permitiu averiguar o papel deste gene na fase adulta. Verificou-se que é essencial para o estabelecimento da polaridade de células epiteliais, tal como já tinha sido verificado no embrião. Relativamente ao gene mob2, é também um gene essencial e mutantes morrem no terceiro estádio larvar. Tem um papel importante durante a mitose já que os mutantes para mob2 apresentam defeitos na segregação dos cromossomas e na formação do fuso mitótico. Estes defeitos estão associados a um crescimento reduzido do cérebro larvar. Este facto justifica-se pelo facto de ao falharem a divisão mitótica, as células embarcarem numa cascata apoptótica. Este trabalho mostra que os genes mob1 e mob2 são essenciais ao correcto desenvolvimento de Drosophila, e cria hipóteses acerca das suas funções e possíveis parceiros moleculares.Normal development requires the orchestration of cell proliferation, apoptosis and morphogenesis. These processes ultimately depend and influence each other. Errors in these processes are believed to constitute main causes for genomic instability and development of cancer. During the course of this work, the roles of two Drosophila genes of the conserved mob gene family were characterized. In the yeasts S. cerevisae and S. pombe, mob genes have been shown to play important roles in both mitosis exit, cytokinesis and cell polarity. The characterization of both dmob1 and dmob2 showed that they play distinct roles from their yeast orthologs. In fact, mob1 seems to have an important role in cell proliferation, cell polarity and tissue morphogenesis. Moreover, mob2 showed to be important for faithful cell division. This work shows that both these genes are crucial for Drosophila development and elaborates on their potential roles and partners that can be tested in future projects

The Toll-dorsal pathway is required for resistance to viral oral infection in Drosophila

Pathogen entry route can have a strong impact on the result of microbial infections in different hosts, including insects. Drosophila melanogaster has been a successful model system to study the immune response to systemic viral infection. Here we investigate the role of the Toll pathway in resistance to oral viral infection in D. melanogaster. We show that several Toll pathway components, including Spätzle, Toll, Pelle and the NF-kB-like transcription factor Dorsal, are required to resist oral infection with Drosophila C virus. Furthermore, in the fat body Dorsal is translocated from the cytoplasm to the nucleus and a Toll pathway target gene reporter is upregulated in response to Drosophila C Virus infection. This pathway also mediates resistance to several other RNA viruses (Cricket paralysis virus, Flock House virus, and Nora virus). Compared with control, viral titres are highly increased in Toll pathway mutants. The role of the Toll pathway in resistance to viruses in D. melanogaster is restricted to oral infection since we do not observe a phenotype associated with systemic infection. We also show that Wolbachia and other Drosophila-associated microbiota do not interact with the Toll pathway-mediated resistance to oral infection. We therefore identify the Toll pathway as a new general inducible pathway that mediates strong resistance to viruses with a route-specific role. These results contribute to a better understanding of viral oral infection resistance in insects, which is particularly relevant in the context of transmission of arboviruses by insect vectors.Biotechnology and Biological Sciences Research Council (UK) grant BB/E005470/1, Fundação para a Ciência e Tecnologia fellowships: SFRH/BPD/65985/2009, SFRH/BD/51881/2012, SFRH/51885/2012

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

DCV tissue tropism upon oral infection.

<p>(A) DCV is present in midgut lumen at 0 dpi. Adult male guts were immunostained with antibody against DCV (red), epithelial enterocytes were marked with GFP expression (green) driven by <i>Myo1A-Gal4</i> and DNA marked with TOTO3 (blue). (B) Fat body is infected with DCV 2 dpi. Midgut (C) and hindgut (D) muscle cells are infected with DCV at 5 dpi. Muscle cells of the ovarian (E) and testis (F) peritoneal sheath are also infected with DCV 5 dpi. (B–F) DCV was immunostained with an antibody (green), actin marked with phalloidin (red) and DNA marked with TOTO3 (blue). Haemocytes (G) are infected with DCV 5 dpi. Haemocytes were marked with GFP expression (green) driven <i>by hml(delta)-Gal4</i>, DCV was immunostained with an antibody (red), and DNA marked DAPI (blue). All tissues were dissected from adult flies. DCV infections (10¹¹ TCID₅₀/ml) were performed in 3–6 days old flies.</p

DCV oral and systemic infections have similar tissue tropism.

<p>(A and B) DCV tissue tropism 2 days after oral (A) and systemic (B) infection. (C and D) DCV tissue tropism 5 days after oral (C) and systemic (D) infection. DCV was at 10¹¹ TCID₅₀/ml for oral infection, 10⁵ TCID₅₀/ml for systemic infection. Tissues of twenty adult males per condition were dissected and immunostained with an antibody against DCV, actin marked with phalloidin and DNA marked TOTO3. Oesophagus, crop, proventriculus, midgut, Malpighian tubules, hindgut, testes, fat body, trachea and thorax skeletal muscle tissues of every individual was analysed for DCV presence and the intensity of the infection by confocal microscopy. “Not infected” - DCV not detected in any part of the tissue observed. “Weakly infected” - DCV was detected in less than one third of the tissue. “Moderately infected” - DCV was detected in one to two thirds of the tissue. “Strongly infected” - DCV was detected in more than two thirds of the tissue. DCV infections were performed in 3–6 days old flies.</p

Toll pathway activation by DCV infection.

<p>(A–C) Dorsal localization in fat body cells 5 days after DCV oral infection (10¹¹ TCID₅₀/ml) of <i>w¹¹¹⁸ iso</i> flies. All three fat body regions shown were dissected from the same fly and are representative of 14 DCV-positive flies (out of 20 total flies analysed). (A) DCV infected fat body region with nuclear import of Dorsal (white arrows). Nuclear import of Dorsal was observed in 4 out of the 14 DCV positive flies (B) DCV infected fat body region without nuclear import of Dorsal. (C) Fat body region not DCV infected without nuclear import of Dorsal. (D) Dorsal localization in fat body cells 2 days after DCV systemic infection (10⁷ TCID₅₀/ml) of <i>w¹¹¹⁸ iso</i> flies, showing nuclear import of Dorsal (white arrows). Nuclear import of Dorsal was observed in 5 of 10 DCV positive flies. (E) Dorsal localization in fat body cells of <i>pll^−/−</i> flies after DCV oral infection (Dorsal nuclear import was seen in 0 out of 16 DCV positive flies) (A–E) Adult male fat body was immunostained with an antibody against Dorsal (green), an antibody against DCV (red), and DNA was marked with DAPI (blue). (F–G) <i>Drs</i>-GFP expression in fat body 5 days after DCV oral infection. Both fat body regions shown were dissected from the same fly. (H) <i>Drs</i>-GFP expression in fat body after 5 days mock oral infection. (I) <i>Drs</i>-GFP in fat body after 2 days <i>Micrococcus luteus</i> oral infection. (F–I) Adult male fat body regions were immunostained with antibody against DCV (red), antibody against GFP (green), and DNA marked with TOTO3 (blue). DCV infections were performed in 3–6 days old flies.</p

Presence of DCV in haemocytes of infected flies.

<p>3–6 days-old <i>w¹¹¹⁸ iso</i> and <i>pll^−/−</i> males were orally or systemically infected with DCV and analyzed 2 or 5 dpi for the presence of virus in haemocytes. 10 males were analyzed for each condition.</p><p>Presence of DCV in haemocytes of infected flies.</p

Toll Pathway mutant flies are less resistant to other RNA viruses oral infection.

<p>(A) Survival of <i>pll^−/−</i> and <i>w¹¹¹⁸ iso</i> flies after CrPV oral infection (1.76×10¹⁰ TCID₅₀/ml) or buffer. <i>pll^−/−</i> flies were significantly more sensitive to CrPV than <i>w¹¹¹⁸ iso</i> (Cox proportional hazard mixed effect model, <i>p</i><0.001). (B) CrPV RNA levels in <i>pll^−/−</i> and <i>w¹¹¹⁸ iso</i> flies upon oral infection (1.76×10¹⁰ TCID₅₀/ml). CrPV loads are significantly different between <i>pll^−/−</i> and <i>w¹¹¹⁸ iso</i> line (Wilcoxon test, <i>p</i><0.005). (C) <i>pll^−/−</i> and <i>w¹¹¹⁸ iso</i> flies were systemically infected with CrPV at three different concentrations (10⁶, 10⁷, 10⁸ TCID₅₀/ml). <i>pll^−/−</i> mutant flies were not more susceptible to CrPV systemic infection than <i>w¹¹¹⁸ iso</i> control flies (Cox Proportional Hazards Model, <i>p</i> = 0.966, <i>p</i> = 1.000 and <i>p</i> = 0.974, respectively). (D) Survival of <i>pll^−/−</i> and <i>w¹¹¹⁸ iso</i> flies upon Nora oral infection or buffer. <i>pll^−/−</i> flies were not more sensitive than <i>w¹¹¹⁸ iso</i> (Cox proportional hazard mixed effect model, <i>p</i> = 0.887). (E) Nora RNA levels upon oral infection. Nora loads are significantly different between <i>pll^−/−</i> and <i>w¹¹¹⁸ iso</i> line (Wilcoxon test, <i>p</i><0.005). (F) Survival of <i>pll^−/−</i> and <i>w¹¹¹⁸ iso</i> flies upon FHV oral infection (10¹⁰ TCID₅₀/ml) or buffer. <i>pll^−/−</i> flies were significantly more sensitive than <i>w¹¹¹⁸ iso</i> (Cox proportional hazard mixed effect model, <i>p</i><0.001). (G) FHV RNA levels upon oral infection (10¹⁰ TCID₅₀/ml). FHV loads are significantly different between <i>pll^−/−</i> and <i>w¹¹¹⁸ iso</i> line (Wilcoxon test, <i>p</i><0.005) (in the other independent replicate the difference in medians is 20-fold and <i>p</i> = 0.05). (H) <i>pll^−/−</i> and <i>w¹¹¹⁸ iso</i> flies were systemically infected with FHV at three different concentrations (10⁶, 10⁷, 10⁸ TCID₅₀/ml). <i>pll^−/−</i> mutant flies were not more susceptible to FHV systemic infection than <i>w¹¹¹⁸ iso</i> control flies (Cox Proportional Hazards Model, <i>p</i> = 0.819, <i>p</i> = 0.709 and <i>p</i> = 0.225, respectively). For survival experiments (A, C, D, F and H) sixty 3–6 days old males of each line per treatment were used and survival was scored daily. Survival experiments for oral infections were performed thrice, yielding similar results. Survival data of all replicates was analysed together using the Cox proportional hazard mixed effect model. For viral loads experiments (B, E, G) 3–6 days old males of each line were orally infected with the virus of interest and collected 5–6 dpi for RNA extraction and RT-qPCR. Relative amount of virus was calculated using host <i>Rpl32</i> mRNA as a reference and values are relative to the median of the <i>w¹¹¹⁸ iso</i> samples. Each point represents the relative virus amount of a single fly and lines are medians of these values. All viral loads experiments were performed twice yielding similar results.</p

Lack of interaction between <i>Wolbachia</i> and other microbiota with Toll resistance to viruses.

<p>(A–C) Sixty 3–6 days old males of each line were orally infected with DCV (10¹¹ TCID₅₀/ml) or buffer (Mock), and survival was monitored daily. Survival data was fitted with a Cox proportional hazard mixed effect model. (A) <i>Wolbachia</i> protection to DCV oral infection does not require the Toll pathway. There is no interaction between <i>Wolbachia</i> and genotype (<i>p</i> = 0.67). (B–C) Survival of antibiotic treated (B) and conventionally reared (C) <i>pll^−/−</i> and <i>w¹¹¹⁸ iso</i> flies after DCV oral infection. There is no effect of antibiotic treatment in fly survival (<i>p</i> = 0.28). <i>pll^−/−</i> flies show increased mortality relative to <i>w¹¹¹⁸ iso</i> flies in both antibiotic treated or conventionally reared conditions (p<0.001 in both conditions).</p

Toll Pathway mutant flies are less resistant to DCV oral Infection.

<p>(A and B) Survival of Toll pathway mutants upon DCV oral infection (10¹¹ TCID₅₀/ml) or buffer. Male flies <i>spz^−/−</i> (<i>spz⁴</i>/<i>spz⁴</i>), <i>pll^−/−</i> (<i>pll²</i>/<i>pll²¹</i>), <i>dl^−/−</i> (<i>dl¹</i>/<i>dl¹</i>), D<i>if^−/−</i> (<i>Dif¹</i>/<i>Dif¹</i>) (A) and <i>Toll^−/−</i> (<i>Tl^rv1</i>/<i>Tl^r3</i>) (B) were compared to <i>w¹¹¹⁸ iso</i>. <i>spz^−/−</i>, <i>pll^−/− dl^−/−</i> and <i>Tl^−/−</i> flies were significantly different from <i>w¹¹¹⁸ iso</i> (Cox proportional hazard mixed effect model, <i>p</i><0.001 for all four lines). D<i>if^−/−</i> mutant flies were not significantly different from <i>w¹¹¹⁸ iso</i> (<i>p</i> = 0.331). (C) Survival of Toll pathway mutants upon mock treatment. None of the mutant lines were significantly different from <i>w¹¹¹⁸ iso</i> (Cox proportional hazard mixed effect model, <i>p</i>>0.67), except <i>dl</i> (<i>p</i> = 0.003). (D) DCV protein levels after oral infection. 3–6 days old males of <i>pll^−/−</i> and <i>w¹¹¹⁸ iso</i> lines were orally infected with DCV (10¹¹ TCID₅₀/ml), collected 1, 3 and 5 days later for protein extraction, and probed in a Western blot with anti-DCV antibody (10 flies per sample). Anti-tubulin antibody was used as a loading control. (E) DCV RNA levels upon oral infection. 3–6 day old males of <i>pll^−/−</i> and <i>w¹¹¹⁸ iso</i> lines were orally infected with DCV (10¹¹ TCID₅₀/ml) and collected 2, 5, 10 and 20 days later for RNA extraction and RT-qPCR. 10 and 20 dpi infection samples are biased since they were collected after the major peak of DCV-induced mortality and therefore most highly infected flies have presumably died. Relative amount of DCV was calculated using host <i>Rpl32</i> mRNA as a reference and values are relative to median of <i>w¹¹¹⁸ iso</i> samples at 2 dpi. Each point represents a sample (one male), and lines are medians of the samples. DCV loads are significantly different between <i>pll^−/−</i> and <i>w¹¹¹⁸ iso</i> line at 2, 5 and 20 dpi (Wilcoxon test, <i>p</i><0.001, <i>p</i><0.005, <i>p</i> = 0.25 and <i>p</i><0.05 for 2, 5, 10 and 20 dpi, respectively). (F) Survival of Toll pathway mutants upon DCV systemic infection (pricked at 10⁷ TCID₅₀/ml). None of the mutant lines were significantly different from <i>w¹¹¹⁸ iso</i> (Cox proportional hazard mixed effect model, <i>p</i>>0.1). (G) Survival of <i>pll^−/−</i> and <i>w¹¹¹⁸ iso</i> male flies to different doses of DCV systemic infection. (10⁵, 10⁶ and 10⁷ TCID₅₀/ml). <i>pll^−/−</i> flies were not significantly different from <i>w¹¹¹⁸ iso</i> (Cox proportional hazard mixed effect model, <i>p</i> = 0.840, <i>p</i> = 0.626 and <i>p</i> = 0.085, respectively). (H and I) DCV tissue tropism of <i>pll^−/−</i> flies upon oral infection, 2 dpi (H) and 5 dpi (I). Twenty adult males per condition were dissected and immunostained with an antibody against DCV and analysed as before. (A, B, C, F, G) For all survival experiments, sixty 3–6 days old males, per line or condition, were infected with DCV or buffer, and their survival was monitored daily. Survival assays for oral infections were performed thrice for <i>pll</i>, <i>spz</i>, an <i>dl</i> mutants, and twice for <i>Dif</i> and <i>Tl</i> mutants. Survival assays of systemic infection in panel F were performed twice. Survival data of all replicates were analysed together using Cox proportional hazard mixed effect models.</p