Refining the Galleria mellonella Model by Using Stress Marker Genes to Assess Clostridioides difficile Infection and Recuperation during Phage Therapy

The Galleria mellonella is an effective model for probing Clostridioides difficile interactions with phages. Despite valuable insights from this model, the larvae are not easily amenable to assessing detailed clinical responses to either bacteria or phages. Here, larval survival, colonisation and toxin levels were compared to expression profiles of 17 G. mellonella stress genes to monitor Clostridiodes difficile infection (CDI), and recuperation during phage therapy. The larvae were infected with a ribotype 014/020 isolate and treated with an optimised phage cocktail. Larvae treated prophylactically with phages and the phage-control larval group were protected, showing the highest survival, and low C. difficile colonisation and toxin rates, compared to co-infection, remedial and bacterial-control larval groups. Expression of growth (9) and reproduction (2) genes were enhanced within prophylaxis and phage-control larval groups compared to the co-infection, remedial and bacterial control groups. In contrast, expression of infection (2), humoral (1) and cellular (3) immunity genes declined in the prophylactic and phage-control groups but increased in the co-infection, remedial and bacterial control larvae. The molecular markers augment the survival, colonisation and toxin data and allow detailed monitoring of CDI and recovery. This data support the use of stress marker genes as tools to analyse clinical symptoms in this model.

Download Full-text

Impact of Phage CDHS-1 on the Transcription, Physiology and Pathogenicity of a Clostridioides difficile Ribotype 027 Strain, R20291

Viruses ◽

10.3390/v13112262 ◽

2021 ◽

Vol 13 (11) ◽

pp. 2262

Author(s):

Janet Y. Nale ◽

Thekra Sideeq Al-Tayawi ◽

Shaun Heaphy ◽

Martha R. J. Clokie

Keyword(s):

Galleria Mellonella ◽

Expression Profiles ◽

Burst Size ◽

Late Phase ◽

Phage Infection ◽

Marker Genes ◽

Bacterial Gene ◽

Bacterial Physiology ◽

Ribotype 027 ◽

Clostridioides Difficile

All known Clostridioides difficile phages encode integrases rendering them potentially able to lyse or lysogenise bacterial strains. Here, we observed the infection of the siphovirus, CDHS-1 on a ribotype 027 strain, R20291 and determined the phage and bacterial gene expression profiles, and impacts of phage infection on bacterial physiology and pathogenicity. Using RNA-seq and RT-qPCR we analysed transcriptomic changes during early, mid-log and late phases of phage replication at an MOI of 10. The phage has a 20 min latent period, takes 80 min to lyse cells and a burst size of ~37. All phage genes are highly expressed during at least one time point. The Cro/C1-transcriptional regulator, ssDNA binding protein and helicase are expressed early, the holin is expressed during the mid-log phase and structural proteins are expressed from mid-log to late phase. Most bacterial genes, particularly the metabolism and toxin production/regulatory genes, were downregulated from early phage replication. Phage-resistant strains and lysogens showed reduced virulence during Galleria mellonella colonization as ascertained by the larval survival and expression of growth (10), reproduction (2) and infection (2) marker genes. These data suggest that phage infection both reduces colonization and negatively impacts bacterial pathogenicity, providing encouraging data to support the development of this phage for therapy to treat C. difficile infection.

Download Full-text

Antibiotics Act with vB_AbaP_AGC01 Phage against Acinetobacter baumannii in Human Heat-Inactivated Plasma Blood and Galleria mellonella Models

International Journal of Molecular Sciences ◽

10.3390/ijms21124390 ◽

2020 ◽

Vol 21 (12) ◽

pp. 4390

Author(s):

Bartłomiej Grygorcewicz ◽

Marta Roszak ◽

Piotr Golec ◽

Daria Śleboda-Taront ◽

Natalia Łubowska ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Phage Therapy ◽

Galleria Mellonella ◽

Ex Vivo ◽

Bacterial Species ◽

Larval Survival ◽

Broad Host Range ◽

Phenotypic Analysis ◽

In Vivo Models

Increasing multidrug resistance has led to renewed interest in phage-based therapy. A combination of the bacteriophages and antibiotics presents a promising approach enhancing the phage therapy effectiveness. First, phage candidates for therapy should be deeply characterized. Here we characterize the bacteriophage vB_AbaP_AGC01 that poses antibacterial activity against clinical Acinetobacter baumannii strains. Moreover, besides genomic and phenotypic analysis our study aims to analyze phage–antibiotic combination effectiveness with the use of ex vivo and in vivo models. The phage AGC01 efficiently adsorbs to A. baumannii cells and possesses a bacteriolytic lifecycle resulting in high production of progeny phages (317 ± 20 PFU × cell−1). The broad host range (50.27%, 93 out of 185 strains) against A. baumannii isolates and the inability of AGC01 to infect other bacterial species show its high specificity. Genomic analysis revealed a high similarity of the AGC01 genome sequence with that of the Friunavirus genus from a subfamily of Autographivirinae. The AGC01 is able to significantly reduce the A. baumannii cell count in a human heat-inactivated plasma blood model (HIP-B), both alone and in combination with antibiotics (gentamicin (GEN), ciprofloxacin (CIP), and meropenem (MER)). The synergistic action was observed when a combination of phage treatment with CIP or MER was used. The antimicrobial activity of AGC01 and phage-antibiotic combinations was confirmed using an in vivo larva model. This study shows the greatest increase in survival of G. mellonella larvae when the combination of phage (MOI = 1) and MER was used, which increased larval survival from 35% to 77%. Hence, AGC01 represents a novel candidate for phage therapy. Additionally, our study suggests that phages and antibiotics can act synergistically for greater antimicrobial effect when used as combination therapy.

Download Full-text

Trichostatin A-Assisted Epigenomic Modulation Affects the Expression Profiles of Not Only Recombinant Human α1,2-Fucosyltransferase and α-Galactosidase A Enzymes But Also Galα1→3Gal Epitopes in Porcine Bi-Transgenic Adult Cutaneous Fibroblast Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22031386 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1386

Author(s):

Jerzy Wiater ◽

Marcin Samiec ◽

Maria Skrzyszowska ◽

Daniel Lipiński

Keyword(s):

Genetic Resource ◽

Ex Vivo ◽

Trichostatin A ◽

Expression Profiles ◽

Fibroblast Cells ◽

Control Groups ◽

Transgenic Pigs ◽

Cell Tissue ◽

Donor Cells ◽

Lower Expression

This study was conducted to explore whether trichostatin A-assisted epigenomic modulation (TSA-EM) can affect the expression of not only recombinant human α1,2-fucosyltransferase (rhα1,2-FT) and α-galactosidase A (rhα-Gal A) immune system enzymes but also Galα1→3Gal epitopes in ex vivo proliferating adult cutaneous fibroblast cells (ACFCs) derived from hFUT2×hGLA bi-transgenic pigs that had been produced for the needs of future xenotransplantation efforts. The ACFC lines were treated with 50 nM TSA for 24 h and then the expression profiles of rhα1,2-FT and rhα-Gal A enzymes were analyzed by Western blot and immunofluorescence. The expression profiles of the Galα1→3Gal epitope were determined by lectin blotting and lectin fluorescence. The ACFCs derived from non-transgenic (nTG) pigs were served as the negative (TSA−) and positive (TSA+) control groups. For both hFUT2×hGLA and nTG samples, the expression levels of α1,2-FT and α-Gal A proteins in TSA+ cells were more than twofold higher in comparison to TSA− cells. Moreover, a much lower expression of the Galα1→3Gal epitopes was shown in TSA− hFUT2×hGLA cells as compared to the TSA− nTG group. Interestingly, the levels of Galα1→3Gal expression in TSA-treated hFUT2×hGLA and nTG ACFCs were significantly higher than those noticed for their TSA-untreated counterparts. Summing up, ex vivo protection of effectively selected bi-transgenic ACFC lines, in which TSA-dependent epigenetic transformation triggered the enhancements in reprogrammability and subsequent expression of hFUT2 and hGLA transgenes and their corresponding transcripts, allows for cryopreservation of nuclear donor cells, nuclear-transferred female gametes, and resultant porcine cloned embryos. The latter can be used as a cryogenically conserved genetic resource of biological materials suitable for generation of bi-transgenic cloned offspring in pigs that is targeted at biomedical research in the field of cell/tissue xenotransplantation.

Download Full-text

Galleria mellonella Larvae as an Infection Model to Investigate sRNA-Mediated Pathogenesis in Staphylococcus aureus

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.631710 ◽

2021 ◽

Vol 11 ◽

Author(s):

Guillaume Ménard ◽

Astrid Rouillon ◽

Gevorg Ghukasyan ◽

Mathieu Emily ◽

Brice Felden ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Galleria Mellonella ◽

Inflammatory Responses ◽

Expression Profiles ◽

Bacterial Pathogens ◽

Infection Model ◽

Animal Testing ◽

Easy Method ◽

Larval Infection ◽

Over Time

Small regulatory RNAs (sRNAs) are key players in bacterial regulatory networks. Monitoring their expression inside living colonized or infected organisms is essential for identifying sRNA functions, but few studies have looked at sRNA expression during host infection with bacterial pathogens. Insufficient in vivo studies monitoring sRNA expression attest to the difficulties in collecting such data, we therefore developed a non-mammalian infection model using larval Galleria mellonella to analyze the roles of Staphylococcus aureus sRNAs during larval infection and to quickly determine possible sRNA involvement in staphylococcal virulence before proceeding to more complicated animal testing. We began by using the model to test infected larvae for immunohistochemical evidence of infection as well as host inflammatory responses over time. To monitor sRNA expression during infection, total RNAs were extracted from the larvae and invading bacteria at different time points. The expression profiles of the tested sRNAs were distinct and they fluctuated over time, with expression of both sprD and sprC increased during infection and associated with mortality, while rnaIII expression remained barely detectable over time. A strong correlation was observed between sprD expression and the mortality. To confirm these results, we used sRNA-knockout mutants to investigate sRNA involvement in Staphylococcus aureus pathogenesis, finding that the decrease in death rates is delayed when either sprD or sprC was lacking. These results demonstrate the relevance of this G. mellonella model for investigating the role of sRNAs as transcriptional regulators involved in staphylococcal virulence. This insect model provides a fast and easy method for monitoring sRNA (and mRNA) participation in S. aureus pathogenesis, and can also be used for other human bacterial pathogens.

Download Full-text

Oxidative stress and outcome of antioxidant supplementation in patients with polycystic ovarian syndrome (PCOS)

International Journal of Reproduction Contraception Obstetrics and Gynecology ◽

10.18203/2320-1770.ijrcog20181892 ◽

2018 ◽

Vol 7 (5) ◽

pp. 1667 ◽

Cited By ~ 5

Author(s):

Abubakar A. Panti ◽

Constance E. Shehu ◽

Yusuf Saidu ◽

Karima A. Tunau ◽

Emmanuel I. Nwobodo ◽

...

Keyword(s):

Oxidative Stress ◽

Antioxidant Enzymes ◽

Serum Levels ◽

Clinical Pregnancy ◽

Oxidative Stress Marker ◽

Randomised Control Trial ◽

Antioxidant Supplementation ◽

Control Groups ◽

Stress Marker ◽

And Control

Background: PCOS is a condition with significant decrease in antioxidant with an increased risk of oxidative stress. Antioxidant supplementation has been shown to improve insulin sensitivity in PCOS and may improve outcome of management of PCOS. Objectives of this study were to determine the oxidative stress level of PCOS patients, to assess the effect of antioxidant supplementation on the outcome of management of PCOS and to compare with a control group.Methods: The study was a single blind randomised control trial involving 200 patients with PCOS. The study was conducted at Usmanu Danfodiyo University Teaching Hospital Sokoto. They were randomly divided into intervention and control groups and base line serum levels of oxidative stress marker, antioxidant enzymes, vitamins and minerals were determined. Antioxidant supplementation and placebo were given to the intervention and control groups respectively. All the patients had ovulation induction with clomiphene citrate and were followed up for 6 months. Outcome measures were clinical pregnancy or menstrual regularisation. Level of significance was <0.05.Results: There was statistical significance in the serum levels of oxidative stress marker, antioxidant enzymes, vitamins and minerals between the two groups (post intervention). Glutathione peroxidase (p = 0.001), superoxide dismutase (p = 0.0001), catalase (p= 0.0369), melondialdehyde (p= 0.007), Vitamin A, Vitamin C, Vitamin E (p = 0.0001), zinc and copper (p = 0.0001). The clinical pregnancy outcomes were 22 (22%) versus 2 (2%); live births 18 (18%) versus 2 (2%) and menstrual regularisation 48 (48%) versus 46 (46%) in the intervention and control groups respectively.Conclusions: Antioxidant supplementation in this study significantly affected pregnancy rate in patients with PCOS. Larger studies are suggested to revisit the conclusion of the Cochrane review that antioxidants supplementation had no significant role in female infertility.

Download Full-text

Molecular Classification of Multiple Myeloma: A Distinct Transcriptional Profile Characterizes Patients Expressing CCND1 and Negative for 14q32 Translocations

Journal of Clinical Oncology ◽

10.1200/jco.2005.01.3870 ◽

2005 ◽

Vol 23 (29) ◽

pp. 7296-7306 ◽

Cited By ~ 90

Author(s):

Luca Agnelli ◽

Silvio Bicciato ◽

Michela Mattioli ◽

Sonia Fabris ◽

Daniela Intini ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Expression Profiles ◽

Quantitative Polymerase Chain Reaction ◽

Gene Expression Pattern ◽

Marker Genes ◽

Published Data ◽

Cyclin D ◽

Chromosome 13Q ◽

13Q Deletion

Purpose The deregulation of CCND1, CCND2 and CCND3 genes represents a common event in multiple myeloma (MM). A recently proposed classification grouped MM patients into five classes on the basis of their cyclin D expression profiles and the presence of the main translocations involving the immunoglobulin heavy chain locus (IGH) at 14q32. In this study, we provide a molecular characterization of the identified translocations/cyclins (TC) groups. Materials and Methods The gene expression profiles of purified plasma cells from 50 MM cases were used to stratify the samples into the five TC classes and identify their transcriptional fingerprints. The cyclin D expression data were validated by means of real-time quantitative polymerase chain reaction analysis; fluorescence in situ hybridization was used to investigate the cyclin D loci arrangements, and to detect the main IGH translocations and the chromosome 13q deletion. Results Class-prediction analysis identified 112 probe sets as characterizing the TC1, TC2, TC4 and TC5 groups, whereas the TC3 samples showed heterogeneous phenotypes and no marker genes. The TC2 group, which showed extra copies of the CCND1 locus and no IGH translocations or the chromosome 13q deletion, was characterized by the overexpression of genes involved in protein biosynthesis at the translational level. A meta-analysis of published data sets validated the identified gene expression signatures. Conclusion Our data contribute to the understanding of the molecular and biologic features of distinct MM subtypes. The identification of a distinctive gene expression pattern in TC2 patients may improve risk stratification and indicate novel therapeutic targets.

Download Full-text

Spatial sampling approach to unravel the impact of soil texture and root genotype on maize root gene expression profiles

10.5194/egusphere-egu2020-9707 ◽

2020 ◽

Author(s):

Minh Ganther ◽

Marie-Lara Bouffaud ◽

Lucie Gebauer ◽

François Buscot ◽

Doris Vetterlein ◽

...

Keyword(s):

Gene Expression ◽

Soil Texture ◽

Expression Profiles ◽

Soil Column ◽

Column Experiment ◽

Spatial Sampling ◽

Marker Genes ◽

Sampling Depth ◽

Plant Growth Hormones ◽

The Impact

The complex interactions between plant roots and soil microbes enable a range of beneficial functions such as nutrient acquisition, defense against pathogens and production of plant growth hormones. The role of soil type and plant genotype in shaping rhizosphere communities has been explored in the past, but often without spatial context. The spatial resolution of rhizosphere processes enables us to observe pattern formation in the rhizosphere and investigate how spatial soil organization is shaped through soil&#8211;plant&#8211;microbiome interactions.We applied spatial sampling in a standardized soil column experiment with two maize genotypes (wildtype vs. roothairless3) and two different soil textures (loam vs. sand) in order to investigate how in particular functions of the maize roots relating to nutrient/water uptake, immunity/defense, stress and exudation are affected. RNA sequencing and differential gene expression analysis were used to dissect impact of soil texture, root genotype and sampling depth. Our results indicate that variance in gene expression is predominantly explained by soil texture as well as sampling depth, whereas genotype appears to play a less pronounced role at the analyzed depths. Gene Ontology enrichment analysis of differentially expressed genes between soil textures revealed several functional categories and pathways relating to phytohormone-mediated signaling, cell growth, secondary metabolism, and water homeostasis. Community analysis of rhizosphere derived ACC deaminase active (acdS gene including) plant beneficial bacteria, which suppress the phytohormone ethylene production, suggests that soil texture and column depth are the major factors that affect acdS community composition.From the comprehensive gene expression analyses we aim to identify maize marker genes from the relevant core functional groups. These marker genes will be potentially useful for future experiments; such as field plot experiments for investigation of later-emerging plant properties.This research was conducted within the research program &#8220;Rhizosphere Spatiotemporal Organisation &#8211; a Key to Rhizosphere Functions&#8221; of the German Science Foundation (TA 290/5-1).

Download Full-text

Inhibition of Elastase or SDF-1/CXCR4 Axis Promotes Differentiation of Human AML Cells.

Blood ◽

10.1182/blood.v108.11.1891.1891 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1891-1891

Author(s):

Sigal Tavor ◽

Jasmine Jacob-Hirsch ◽

Manny Eisenbach ◽

Sigi Kay ◽

Shoshana Baron ◽

...

Keyword(s):

Gene Expression ◽

Cell Line ◽

Expression Profiles ◽

Expression Patterns ◽

Critical Role ◽

Marker Genes ◽

Granulocytic Differentiation ◽

Elastase Inhibitor ◽

Granule Proteins ◽

Cxcr4 Axis

Abstract Elastase, along with other azurophil granule proteins like proteinase 3 regulates normal and leukemic granulopoiesis in an un-defined mechanism. We have recently showed that human acute myeloid leukemic (AML) cells constitutively express and secrete stromal derived factor 1 (SDF-1) dependent cell surface elastase, which regulates their migration and proliferation. To elucidate the molecular events and genes regulated by elastase and SDF-1/CXCR4 axis in AML cells, we examined gene expression of U937 AML cell line treated with neutralizing anti-CXCR4 Abs or elastase inhibitor (EI) compared to untreated cells, using DNA microarray technology. Unsupervised hierarchical clustering analysis showed very similar gene expression profiles of EI and anti CXCR4 Abs treated cells as compared to control. 230 of 8400 genes interrogated were repressed, and 164 were induced after culturing AML cells in the presence of EI or anti CXCR4 Abs at different time points as compared to untreated cells. Inhibition of elastase or CXCR4 was accompanied by down regulation of the transcripts of primary granule proteins. Functional classification of elastase or SDF-1/CXCR4 axis regulated genes revealed downregulation of HOXA9, HOXA10, ETS2, as well as other transcription factors that are over expressed in AML and are important for the development of leukemia. Whereas, transcriptional factors and regulators known to be induced during myeloid differentiation like C/EBPε, ID1, RUNX3 and HHEX were up-regulated in treated cells. Expression patterns of apoptosis genes indicated decline in death control by the p53 dependent pathway and a more prominent control by mitochondrial mediated apoptotic pathway like bcl2 related genes. In addition, receptors for interleukins, growth factors (G-CSFR and GM-CSF), complement component (C1QR1) were upregulated in the treated cells. In contrast, FLT-3, a growth factor receptor stimulating growth of early progenitor cells and AML blasts, was down regulated in AML cell treated with EI or anti CXCR4 Abs. These data were confirmed by real time PCR for selected marker genes of granulocytic differentiation. Interestingly, many of the differentially expressed genes were common to the transcriptional program of normal terminal granulocytic differentiation (Theilgaard-Monch & Borregarrd 2005. Blood 105:1785) suggesting that inhibition of elastase may induce differentiation in AML cells. Thus we further analyzed the effect of elastase inhibition on AML cell differentiation and growth. Treatment of HL60 AML cell line with EI triggered a proliferative arrest, apoptosis and mimicked terminal granulocytic differentiation, including morphologic changes, increased CD11b expression, and the ability to produce oxidative bursts. In summary, our study showed that inhibition of elastase or SDF-1/CXCR4 axis in AML cells affects similar pathways related to differentiation and malignant transformation, implying a critical role for those molecules in regulating leukemic development. Repression of elastase decreases proliferation and induces differentiation of AML cells, suggesting a potential new therapeutic approach for AML.

Download Full-text

Molecular characteristics of papillary thyroid carcinomas without BRAF mutation or RET/PTC rearrangement: relationship with clinico-pathological features

Endocrine Related Cancer ◽

10.1677/erc-08-0081 ◽

2009 ◽

Vol 16 (2) ◽

pp. 467-481 ◽

Cited By ~ 15

Author(s):

Stéphanie Durand ◽

Carole Ferraro-Peyret ◽

Mireille Joufre ◽

Annie Chave ◽

Françoise Borson-Chazot ◽

...

Keyword(s):

Gene Expression ◽

Normal Tissue ◽

Expression Profiles ◽

Strong Association ◽

The Other ◽

Marker Genes ◽

Molecular Characteristics ◽

Papillary Thyroid ◽

Thyroid Carcinomas ◽

Gene Alteration

About 60–70% of papillary thyroid carcinomas (PTC) present a BRAFT1799A gene mutation or a rearrangement of RET gene (RET/PTC). In this study, we examined whether PTC without BRAFT1799A mutation and without RET/PTC rearrangement named PTC-ga(−) were distinguishable from PTC-ga(+) (with one or the other gene alteration) on the basis of gene expression characteristics. We analyzed the mutational state of 116 PTC and we compared gene expression profiles of PTC-ga(+) and PTC-ga(−) from data of a 200 gene macroarray and quantitative PCR. Seventy five PTC were PTC-ga(+) and 41 were PTC-ga(−). Unsupervised analyses of macroarray data by hierarchical clustering led to a complete segregation of PTC-ga(+) and PTC-ga(−). In a series of 42 genes previously recognized as PTC ‘marker’ genes, 22 were found to be expressed at a comparable level in PTC-ga(−) and normal tissue. Thyroid-specific genes, TPO, TG, DIO1, and DIO2 were under-expressed in PTC-ga(+) but expressed at a normal level in PTC-ga(−). A few genes including DUOX1 and DUOX2 were selectively dys-regulated in PTC-ga(−). Tumor grade of PTC-ga(−) was lower than that of PTC-ga(+). There was a strong association between the mutational state and histiotype of PTC; 81% of PTC follicular variants were corresponded to PTC-ga(−), whereas 84% of PTC of classical form were PTC-ga(+). In conclusion, we show that PTC without BRAFT1799A mutation or RET/PTC rearrangement, mainly corresponding to follicular variants, maintain a thyroid differentiation expression level close to that of normal tissue and should be of better prognosis than PTC with one or the other gene alteration.

Download Full-text