Regulatory elements located in the upstream region of the Rhizobium leguminosarum rosR global regulator are essential for its transcription and mRNA stability

Rhizobium leguminosarum bv. trifolii is a soil bacterium capable of establishing a symbiotic relationship with clover (Trifolium spp.). Previously, the rosR gene, encoding a global regulatory protein involved in motility, synthesis of cell-surface components, and other cellular processes was identified and characterized in this bacterium. This gene possesses a long upstream region that contains several regulatory motifs, including inverted repeats (IRs) of different lengths. So far, the role of these motifs in the regulation of rosR transcription has not been elucidated in detail. In this study, we performed a functional analysis of these motifs using a set of transcriptional rosR-lacZ fusions that contain mutations in these regions. The levels of rosR transcription for different mutant variants were evaluated in R. leguminosarum using both quantitative real-time PCR and β-galactosidase activity assays. Moreover, the stability of wild type rosR transcripts and those with mutations in the regulatory motifs was determined using an RNA decay assay and plasmids with mutations in different IRs located in the 5′ -untranslated region of the gene. The results show that transcription of rosR undergoes complex regulation, in which several regulatory elements located in the upstream region and some regulatory proteins are engaged. These include an upstream regulatory element, an extension of the -10 element containing three nucleotides TGn (TGn-extended -10 element), several IRs, and PraR repressor related to quorum sensing.Polish National Science Centre DEC-2012/07/B/NZ1/0009

Defence response of Galleria mellonella larvae to oral and intrahemocelic infection with Pseudomonas entomophila

We report differences in the course of infection of G. mellonella larvae with P. entomophila via intrahemocelic and oral routes. Survival curves, larval morphology, histology, and induction of defence response were investigated. Larvae injected with 10 and 50 cells of P. entomophila activated a dose-dependent immune response, which was manifested by induction of immune-related genes and dose-dependent defence activity in larval hemolymph. In contrast, after the oral application of the pathogen, antimicrobial activity was detected in whole hemolymph of larvae infected with the $10^{3}$ but not $10^{5}$ dose in spite of the induction of immune response manifested as immune-relevant gene expression and defence activity of electrophoretically separated low-molecular hemolymph components. Among known proteins induced after the P. entomophila infection, we identified proline-rich peptide 1 and 2, cecropin D-like peptide, galiomycin, lysozyme, anionic peptide 1, defensin-like peptide, and a 27 kDa hemolymph protein. The expression of the lysozyme gene and the amount of protein in the hemolymph were correlated with inactivity of hemolymph in insects orally infected with a higher dose of P. entomophila, pointing to its role in the host-pathogen interaction

Methotrexate as a single agent for treating pulmonary sarcoidosis: a single centre real-life prospective study

Wstęp: Kortykosteroidoterapia jest według aktualnych wytycznych WASOG/ATS/ERS leczeniem pierwszego rzutu u chorych na przewlekłą sarkoidozę. Jest to leczenie obarczone wysokim ryzykiem wystąpienia objawów ubocznych, które pogarszają rokowanie odległe. J ednocześnie nie ma pewności, że glikokortykosteroidy (GKS) modyfikują naturalny przebieg choroby. Celem pracy była ocena skuteczności i bezpieczeństwa leczenia metotreksatem (MTX) w monoterapii chorych na przewlekłą sarkoidozę płucną.Materiał i metody: Do leczenia zakwalifikowano 50 chorych na przewlekłą sarkoidozę płuc potwierdzoną badaniem histopatologicznym, 28M i 22K, w średnim wieku 45,55 ± 8,9 roku, ze średnim czasem trwania choroby do włączenia MTX wynoszącym 12,34 ± 20,49 roku. W latach 2004–2013 zastosowano u tych chorych MTX w monoterapii, w dawkach 10 mg lub 15 mg tygodniowo. Czterdziestu jeden pacjentów było wcześniej leczonych GKS. Wszyscy mieli wykonywane badania laboratoryjne, czynnościowe oraz radiologiczne układu oddechowego przed rozpoczęciem leczenia i w trakcie monitorowania skuteczności (co 6 miesięcy) oraz bezpieczeństwa terapii (co 4–6 tygodni). Do analizy statystycznej oceniającej skuteczność leczenia włączono 49 chorych. Na podstawie retrospektywnej analizy wyników badań czynnościowych (FEV1, FVC, TLC, DLCO) wykonanych na zakończenie leczenia, w porównaniu z wynikami badań czynności płuc przed podaniem MTX (poprawa o 10% w zakresie FEV1, FVC, TLC lub o 15% DLCO), wyodrębniono chorych, u których stwierdzono obiektywną, istotną poprawę wskaźników czynnościowych płuc po leczeniu (grupa „z obiektywną poprawą po leczeniu”).Wyniki: Okres leczenia wynosił od 6 do 24 miesięcy, średnio 60,75 ± 34,1 tygodnia. W całej grupie istotną poprawę po leczeniu MTX stwierdzono dla SaO2 min (%) (p = 0,043) oraz dla DSaO2 (%) (p = 0,048) ocenianej w czasie testu 6-minutowego marszu. Istotnie lepsze efekty leczenia uzyskano w grupie otrzymującej 15 mg MTX tygodniowo oraz u chorych, którzy otrzymali sumarycznie większą dawkę MTX podczas całej kuracji. Istotną statystycznie różnicę po 6 miesiącach leczenia między grupami leczonymi 15 mg v. 10 mg tygodniowo stwierdzono dla DLCO% pred (73,27 ± 12,7 v. 63,15 ± 16,4; p = 0,03). Obiektywną poprawę po leczeniu stwierdzono u 25 pacjentów (55%). Chorzy, u których stwierdzono obiektywną poprawę po MTX, mieli wyjściowo istotnie niższe wartości TLC i FVC w porównaniu z grupą bez poprawy po MTX. Po zakończeniu leczenia jedyną istotnie statystycznie różnicę między obiema grupami obserwowano w zakresie DLCO. U 11 chorych (22%) przerwano leczenie z powodu objawów ubocznych. Najczęściej obserwowanym objawem ubocznym leczenia był wzrost wskaźników wątrobowych (10 chorych, 20%). U 4 osób stwierdzono powikłania infekcyjne. U żadnego chorego nie stwierdzono powikłań zagrażających życiu.Wnioski: Metotreksat w monoterapii może być bezpieczną i skuteczną alternatywą dla steroidów. U części chorych należy spodziewać się obiektywnej poprawy czynności płuc po leczeniu. Wskazane są dalsze badania w poszukiwaniu wskaźników prognozujących skuteczność leczenia. Introduction: The first-line therapy in chronic sarcoidosis, according to WASOG/ATS/ERS recommendations, is GCS. This therapy is associated with significant adverse effects and finally does not alter the natural history of the disease. The objective of our study was to evaluate the efficacy and safety of monotherapy with MTX, as an alternative to GCS, in progressive pulmonary sarcoidosis.Material and methods: An open prospective real-life, single-centre trial was performed on 50 patients with biopsy proven sarcoidosis, 28M and 22F, mean age 45.55 ± 8.9 years. The average duration of disease before MTX therapy was 12.34 ± 20.49 years, GCS therapy in the past was applied in 41 patients. All patients received MTX (10 mg or 15 mg weekly) between 2004 and 2013 because of chronic progressive pulmonary sarcoidosis. Therapy was planned for 24 months. Patients underwent regular clinical evaluation, pulmonary function assessment, exercise ability testing (6MWT), and chest radiography for therapy effectiveness every six months and side effects monitoring every 4–6 weeks. Forty-nine patients were included for statistical analysis of treatment efficacy. They were retrospectively allocated to “MTX responder” group if an improvement of 10% of FEV1, FVC, TLC, or 15% of DLCO from the initial value was documented for at least one parameter or “non-responders” if the patient did not meet the above-mentioned criteria.Results: Duration of treatment ranged from 6 to 24 months, mean time 60.75 ± 34.1 weeks. For the whole cohort significant improvement after MTX therapy was observed for minimal SaO2 (%) (p = 0.043) and for decrease of DSaO2 (%) (p = 0.048) in six-minute walk test. The results were significantly better for patients treated with 15 mg than for those treated with 10 mg weekly and for those who obtained a greater total amount of MTX during therapy. Significant difference of DLCO%pred was observed after six months of MTX therapy between groups treated 15 mg vs 10 mg weekly (73.27 ± 12.7% vs. 63.15 ± 16.4%, p = 0.03). Twenty-five patients (55%) met the criteria of “MTX responders” group. Patients who responded well to treatment had significantly lower TLC and FVC initial values comparing to “MTX non-responders”. After treatment the only significant difference in PFT between groups was noted for DLCO%pred. Eleven patients (22%) stopped the treatment due to adverse events of MTX, mild hepatic abnormalities were observed in ten patients (20%), and concomitant infection was found in four patients. There were no patients with a fatal outcome.Conclusions: MTX as a single agent in the treatment of sarcoidosis has proved to be a safe and effective steroid alternative. Selected patients with chronic pulmonary sarcoidosis experience definite PFT improvements after MTX treatment. There is need to search for predictors of MTX treatment effectiveness.

Thermosensitivity of the Saccharomyces cerevisiae gpp1gpp2 double deletion strain can be reduced by overexpression of genes involved in cell wall maintenance

A Saccharomyces cerevisiae strain in which the GPP1 and GPP2 genes, both encoding glycerol-3-phosphate phosphatase isoforms, are deleted, displays both osmo- and thermosensitive (ts) phenotypes. We isolated genes involved in cell wall maintenance as multicopy suppressors of the gpp1gpp2 ts phenotype. We found that the gpp1gpp2 strain is hypersensitive to cell wall stress such as treatment with β-1,3-glucanase containing cocktail Zymolyase and chitin-binding dye Calcofluor-white (CFW). Sensitivity to Zymolyase was rescued by overexpression of SSD1, while CFW sensitivity was rescued by SSD1, FLO8 and WSC3-genes isolated as multicopy suppressors of the gpp1gpp2 ts phenotype. Some of the isolated suppressor genes (SSD1, FLO8) also rescued the lytic phenotype of slt2 deletion strain. Additionally, the sensitivity to CFW was reduced when the cells were supplied with glycerol. Both growth on glycerol-based medium and overexpression of SSD1, FLO8 or WSC3 had additive suppressing effect on CFW sensitivity of the gpp1gpp2 mutant strain. We also confirmed that the internal glycerol level changed in cells exposed to cell wall perturbation. © 2007 Springer-Verlag

Different strategies for attaining immune memory

Odporność nabyta inaczej adaptacyjna (swoista) rozwinęła się w ewolucji bardzo późno, bo dopiero u kręgowców żuchwowych. Oparta jest ona na limfocytach T i B oraz syntezie różnorodnych receptorów i przeciwciał. Okazuje się jednak, że u bezkręgowców, których odporność oparta jest jedynie na mechanizmach wrodzonych, obserwuje się swego rodzaju pamięć immunologiczną. Co więcej, nawet organizmy jednokomórkowe jak bakterie czy archeony wykazują cechy "pamięci immunologicznej". W artykule opisano różne strategie "zapamiętywania" infekcji: mechanizm CRISPR/Cas u bakterii, receptory DSCAM i inne formy piętnowania układu immunologicznego owadów oraz zmienność receptorów bogatych w leucynę (LRR) u bezżuchwowców. Przedstawiono także jak doszło do nabycia możliwości syntezy różnorodnych przeciwciał oraz receptorów limfocytów. Opisane mechanizmy opierają się na włączaniu obcego materiału genetycznego do genomu gospodarza, mechanizmie konwersji genów, alternatywnego składania transkryptów oraz somatycznej rearanżacji DNA.Acquired immunity (adaptive, specific) developed late in evolution - in jawed vertebrates. It is based on T and B lymphocytes and a diversity of antibodies. It turns out, however, that in invertebrates, which posses only innate mechanisms there is a kind of immune memory. Moreover, even single-cell organisms such as bacteria or archaea exhibit features of immunological memory. This article describes the various strategies used to achieve a kind of rememmbrnace of infection: a CRISPR/Cas system in bacteria, diveristy of DSCAM receptors and other forms of immune priming in insects, leucine-rich receptors in jawless vertebrates. It also describes how it came to acquire the possibility of synthesis of various forms of antibodies and lymphocyte receptors by jawed vertebrates. The described mechanisms are based on the incorporation of foreign genetic material into host genome, the gene conversion mechanisms, alternative splicing and finally, somatic rearrangements of DNA

Insect immune system in defense of organism integrity

Owady zasiedlają wszystkie lądowe nisze ekologiczne. Ewolucyjny sukces osiągnęły między innymi dzięki sprawnie funkcjonującym mechanizmom obronnym. Układ odpornościowy tej gromady zwierząt oparty jest jedynie na mechanizmach wrodzonych. Składa się on z humoralnych i komórkowych odczynów, które uzupełniają się nawzajem w walce z infekcją. W pracy zwięźle przedstawiono aktualny stan wiedzy, dotyczący układu odpornościowego owadów i zwrócono uwagę na jego rolę w utrzymaniu homeostazy organizmu. Ponadto, na przykładzie barciaka większego Galleria mellonella omówiono modulację odpowiedzi immunologicznej przez zmiany temperatury otoczenia. Przedstawiono także aktualne informacje dotyczące zjawiska piętnowania immunologicznego owadów, ze szczególnym uwzględnieniem barciaka większego.Insects populate all ecological land niches. Their evolutionary successes have been achieved thanks to well-functioning defense mechanisms. The immune system of this group of animals is based only on innate immunity mechanisms. It consists of humoral and cellular reactions that complement each other in the fight against infection. The paper briefly summarizes the state of the art of insect immune system and highlights its role in maintaining homeostasis of the organism. In addition, the modulation of immune response by changes in ambient temperature is described taking an example of a greater wax moth Galleria mellonella. Additionally, the current information concerning priming of insect immune system is presented with special emphasis on the greater wax moth

Expression of the insect metalloproteinase inhibitor IMPI in the fat body of Galleria mellonella exposed to infection with Beauveria bassiana

The inducible metalloproteinase inhibitor (IMPI) discovered in Galleria mellonella is currently the only specific inhibitor of metalloproteinases found in animals. Its role is to inhibit the activity of metalloproteinases secreted by pathogenic organisms as virulence factors to degrade immune-relevant polypeptides of the infected host. This is a good example of an evolutionary arms race between the insect hosts and their natural pathogens. In this report, we analyze the expression of a gene encoding an inducible metalloproteinase inhibitor (IMPI) in fat bodies of the greater wax moth larvae Galleria mellonella infected with an entomopathogenic fungus Beauveria bassiana. We have used a natural infection, i.e. covering larval integument with fungal aerospores, as well as injection of fungal blastospores directly into the larval hemocel. We compare the expression of IMPI with the expression of genes encoding proteins with fungicidal activity, gallerimycin and galiomycin, whose expression reflects the stimulation of Galleria mellonella defense mechanisms. Also, gene expression is analyzed in the light of survival of animals after spore injection

A child with achondroplasia – interdisciplinary approach from the perspective of biology and pedagogy

Artykuł jest próbą całościowego i interdyscyplinarnego spojrzenia na achondroplazję – chorobę rzadką (dotyczy ok. 1 na 25 000 osób), uwarunkowaną genetycznie (spowodowana mutacją w genie kodującym receptor FGFR3). Podjęto w nim próbę opisania biologicznych przyczyn achondroplazji, poruszając te zagadnienia dotyczące biologii człowieka, które są niezbędne do zrozumienia powodów niskorosłości. Opisano również sposoby dziedziczenia tej choroby. Ponadto dokonano charakterystyki niskorosłego dziecka i jego fizycznych możliwości, co może być pomocne zarówno dla rodziców, jak i pracowników placówek wychowawczo-oświatowych. Dzieci te, ponieważ nie mają wskazań do nauczania indywidualnego, mogą dobrze funkcjonować w środowisku przedszkolnym i szkolnym. Jednakże mogą borykać się z trudnościami o podłożu społeczno-emocjonalnym, gdyż niski wzrost bywa przyczyną kłopotów z samooceną, a także może okazać się barierą w budowaniu relacji rówieśniczych. Stąd całościowe spojrzenie na dziecko dotknięte achondroplazją jest ważne, by rzetelnie zająć się wspieraniem jego rozwoju oraz edukacją.The current work is an attempt to look at the problem of achondroplasia in a holistic and thus interdisciplinary way. Achondroplasia is a genetically determined disease caused by a mutation in the gene encoding the FGFR3 receptor. We have attempted to describe the biological causes of achondroplasia, addressing those issues in human biology that are necessary to understand the reasons for short stature. We also describe the chances and mechanisms of inheriting this disease. In addition, the description of the structure of a small child and his or her physical abilities which is included in the article will be helpful for the parents and in educational institutions. The children can function well in pre-school and school environments, as there are no indications for individual teaching. However, they may struggle with social and emotional difficulties, as short stature may cause problems with self-esteem, and may also be a barrier in building peer relationships. A holistic view of a child affected by achondroplasia is important to honestly support their development and education

Activation of MAP kinase pathways in Galleria mellonella infected with Bacillus thuringiensis

We followed changes in the level of phospho-MAP kinases in the greater wax moth Galleria mellonella after infection with Bacillus thuringiensis. We observed an enhanced level of phosphorylated p38 and JNK in fat bodies of the infected larvae. In hemocytes, injection of B. thuringiensis caused the highest increase in phospho-JNK, however, all pathways were activated after aseptic injection. We report that Galleria mellonella larvae exposed to heat shock before infection showed an enhanced level of phosphorylated JNK in fat body. This finding is relevant in the light of our previous reports, which submit evidence that pre-shocked animals are more resistant to infection

The effect of cold shock on the immune response of the greater wax moth Galleria mellonella after infection with entomopathogenic bacteria Bacillus thuringiensis

Insect immune system consists of only innate mechanisms relied on cellular and humoral branches. Many defence proteins and peptides exist or appear in response to infection in insect’s hemolymph. The interaction between the infected host and the entomopathogen occurs in the conditions of external environment. In this work the greater wax moth larvae of Galleria mellonella were subjected to a temperature of 120C for a short period of time, directly before infection with entomopathogenic bacteria Bacillus thuringiensis. It appeared that the induction of the immune response was higher in cold-shocked animals than in larvae permanently reared at the optimal temperature of 28 0C. This enhanced immune response was manifested as higher antibacterial and lysozyme-type activity detected in full hemolymph, and as a higher level of peptides of molecular weight below 10 kDa having antibacterial activity. Moreover, other changes in the contents of proteins in the hemolymph were observed. These changes concerned inter alia apolipophorin III, the multifunctional protein of immune significance. Its level was higher in the hemolymph of animals pre-exposed to cold shock than in nonshocked, infected ones. Altogether our results indicate that the interdependence mechanisms occur between cold shock and the immune response