scholarly journals Novel small RNAs expressed by Bartonella bacilliformis under multiple conditions reveal potential mechanisms for persistence in the sand fly vector and human host

2020 ◽  
Vol 14 (11) ◽  
pp. e0008671
Author(s):  
Shaun Wachter ◽  
Linda D. Hicks ◽  
Rahul Raghavan ◽  
Michael F. Minnick
Keyword(s):  
Life Cycle ◽  
Northern Blot Analysis ◽  
Sand Fly ◽  
Bartonella Bacilliformis ◽  
Phlebotomine Sand Flies ◽  
Non Coding Rnas ◽  
Srp Rna ◽  
Multiple Conditions

Bartonella bacilliformis, the etiological agent of Carrión’s disease, is a Gram-negative, facultative intracellular alphaproteobacterium. Carrión’s disease is an emerging but neglected tropical illness endemic to Peru, Colombia, and Ecuador. B. bacilliformis is spread between humans through the bite of female phlebotomine sand flies. As a result, the pathogen encounters significant and repeated environmental shifts during its life cycle, including changes in pH and temperature. In most bacteria, small non-coding RNAs (sRNAs) serve as effectors that may post-transcriptionally regulate the stress response to such changes. However, sRNAs have not been characterized in B. bacilliformis, to date. We therefore performed total RNA-sequencing analyses on B. bacilliformis grown in vitro then shifted to one of ten distinct conditions that simulate various environments encountered by the pathogen during its life cycle. From this, we identified 160 sRNAs significantly expressed under at least one of the conditions tested. sRNAs included the highly-conserved tmRNA, 6S RNA, RNase P RNA component, SRP RNA component, ffH leader RNA, and the alphaproteobacterial sRNAs αr45 and speF leader RNA. In addition, 153 other potential sRNAs of unknown function were discovered. Northern blot analysis was used to confirm the expression of eight novel sRNAs. We also characterized a Bartonella bacilliformis group I intron (BbgpI) that disrupts an un-annotated tRNACCUArg gene and determined that the intron splices in vivo and self-splices in vitro. Furthermore, we demonstrated the molecular targeting of Bartonella bacilliformis small RNA 9 (BbsR9) to transcripts of the ftsH, nuoF, and gcvT genes, in vitro.

scholarly journals Novel small RNAs expressed by Bartonella bacilliformis under multiple conditions reveal potential mechanisms for persistence in the sand fly vector and human host

2020 ◽  
Author(s):  
Shaun Wachter ◽  
Linda D. Hicks ◽  
Rahul Raghavan ◽  
Michael F. Minnick
Keyword(s):  
Life Cycle ◽  
Rna Sequencing ◽  
Vascular Endothelial Cells ◽  
Sand Fly ◽  
Sand Flies ◽  
Bartonella Bacilliformis ◽  
Phlebotomine Sand Flies ◽  
Group I

AbstractBartonella bacilliformis, the etiological agent of Carrión’s disease, is a Gram-negative, facultative intracellular alphaproteobacterium. Carrión’s disease is an emerging but neglected tropical illness endemic to Peru, Colombia, and Ecuador. B. bacilliformis is spread between humans through the bite of female phlebotomine sand flies. As a result, the pathogen encounters significant and repeated environmental shifts during its life cycle, including changes in pH and temperature. In most bacteria, small non-coding RNAs (sRNAs) serve as effectors that may post-transcriptionally regulate the stress response to such changes. However, sRNAs have not been characterized in B. bacilliformis, to date. We therefore performed total RNA-sequencing analyses on B. bacilliformis grown in vitro then shifted to one of ten distinct conditions that simulate various environments encountered by the pathogen during its life cycle. From this, we identified 160 sRNAs significantly expressed under at least one of the conditions tested. sRNAs included the highly-conserved tmRNA, 6S RNA, RNase P RNA component, SRP RNA component, ffH leader RNA, and the alphaproteobacterial sRNAs αr45 and speF leader RNA. In addition, 153 other potential sRNAs of unknown function were discovered. Northern blot analysis was used to confirm the expression of eight novel sRNAs. We also characterized a Bartonellabacilliformisgroup I intron (BbgpI) that disrupts an un-annotated tRNACCUArg gene and determined that the intron splices in vivo and self-splices in vitro. Furthermore, we demonstrated the molecular targeting of Bartonellabacilliformissmall RNA 9(BbsR9) to transcripts of the ftsH, nuoF, and gcvT genes, in vitro.Author summaryB. bacilliformis is a bacterial pathogen that is transmitted between humans by phlebotomine sand flies. Bacteria often express sRNAs to fine-tune the production of proteins involved in a wide array of biological processes. We cultured B. bacilliformis in vitro under standard conditions then shifted the pathogen for a period of time to ten distinct environments, including multiple temperatures, pH levels, and infections of human blood and human vascular endothelial cells. After RNA-sequencing, a manual transcriptome search identified 160 putative sRNAs, including seven highly-conserved sRNAs and 153 novel potential sRNAs. We then characterized two of the novel sRNAs, BbgpI and BbsR9. BbgpI is a group I intron (ribozyme) that self-splices and disrupts an unannotated gene coding for a transfer RNA (tRNACCUArg). BbsR9 is an intergenic sRNA expressed under conditions that simulate the sand fly. We found that BbsR9 targets transcripts of the ftsH, nuoF, and gcvT genes. Furthermore, we determined the specific sRNA-mRNA interactions responsible for BbsR9 binding to its target mRNAs through in vitro mutagenesis and binding assays.

scholarly journals A sand fly salivary protein acts as a neutrophil chemoattractant

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Anderson B. Guimaraes-Costa ◽  
John P. Shannon ◽  
Ingrid Waclawiak ◽  
Jullyanna Oliveira ◽  
Claudio Meneses ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Calcium Influx ◽  
Parasite Burden ◽  
Salivary Protein ◽  
Sand Fly ◽  
Human Neutrophils ◽  
Chemotactic Protein ◽  
The Impact

AbstractApart from bacterial formyl peptides or viral chemokine mimicry, a non-vertebrate or insect protein that directly attracts mammalian innate cells such as neutrophils has not been molecularly characterized. Here, we show that members of sand fly yellow salivary proteins induce in vitro chemotaxis of mouse, canine and human neutrophils in transwell migration or EZ-TAXIScan assays. We demonstrate murine neutrophil recruitment in vivo using flow cytometry and two-photon intravital microscopy in Lysozyme-M-eGFP transgenic mice. We establish that the structure of this ~ 45 kDa neutrophil chemotactic protein does not resemble that of known chemokines. This chemoattractant acts through a G-protein-coupled receptor and is dependent on calcium influx. Of significance, this chemoattractant protein enhances lesion pathology (P < 0.0001) and increases parasite burden (P < 0.001) in mice upon co-injection with Leishmania parasites, underlining the impact of the sand fly salivary yellow proteins on disease outcome. These findings show that some arthropod vector-derived factors, such as this chemotactic salivary protein, activate rather than inhibit the host innate immune response, and that pathogens take advantage of these inflammatory responses to establish in the host.

scholarly journals Considerations when investigating lncRNA function in vivo

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Andrew R Bassett ◽  
Asifa Akhtar ◽  
Denise P Barlow ◽  
Adrian P Bird ◽  
Neil Brockdorff ◽  
...  
Keyword(s):  
Normal Cell ◽  
Regulatory Elements ◽  
Genomic Locus ◽  
Cellular Processes ◽  
Cell Processes ◽  
Non Coding Rnas ◽  
Per Se ◽  
Dna Regulatory Elements

Although a small number of the vast array of animal long non-coding RNAs (lncRNAs) have known effects on cellular processes examined in vitro, the extent of their contributions to normal cell processes throughout development, differentiation and disease for the most part remains less clear. Phenotypes arising from deletion of an entire genomic locus cannot be unequivocally attributed either to the loss of the lncRNA per se or to the associated loss of other overlapping DNA regulatory elements. The distinction between cis- or trans-effects is also often problematic. We discuss the advantages and challenges associated with the current techniques for studying the in vivo function of lncRNAs in the light of different models of lncRNA molecular mechanism, and reflect on the design of experiments to mutate lncRNA loci. These considerations should assist in the further investigation of these transcriptional products of the genome.

scholarly journals In vitro life cycle of pentamidine-resistant amastigotes: stability of the chemoresistant phenotypes is dependent on the level of resistance induced.

1997 ◽  
Vol 41 (9) ◽  
pp. 1898-1903 ◽  
Author(s):  
D Sereno ◽  
J L Lemesre
Keyword(s):  
Life Cycle ◽  
Culture Conditions ◽  
Cross Resistance ◽  
Diminazene Aceturate ◽  
Drug Pressure ◽  
Mouse Macrophages ◽  
Wild Type Clone ◽  
Different Levels

Using a continuous drug pressure protocol, we induced pentamidine resistance in an active and dividing population of amastigote forms of Leishmania mexicana. We selected in vitro two clones with different levels of resistance to pentamidine, with clone LmPENT5 being resistant to 5 microM pentamidine, while clone LmPENT20 was resistant to 20 microM pentamidine. Resistance indexes (50% inhibitory concentration [IC50] after drug presure/IC50 before drug pressure) of 2 (LmPENT5) and 6 (LmPENT20) were determined after drug selection. Both resistant clones expressed significant cross-resistance to diminazene aceturate and primaquine. Pentamidine resistance was not reversed by verapamil, a calcium channel blocker known to reverse multidrug resistance (A. J. Bitonti, et al., Science 242:1301-1303, 1988; A. R. C. Safa et al., J. Biol. Chem. 262:7884-7888, 1987). No difference in the in vitro infectivity for resident mouse macrophages was observed between the wild-type clone (clone LmWT) and pentamidine-resistant clones. During in vitro infectivity experiments, when the life cycle was performed starting from the intramacrophagic amastigote stage, the drug resistance of the resulting LmPENT20 amastigotes was preserved even if the intermediate promastigote stage could not be considered resistant to 20 microM pentamidine. In the same way, when a complete developmental sequence of L. mexicana was achieved axenically by manipulation of appropriate culture conditions, the resulting axenically grown LmPENT20 amastigotes remained pentamidine resistant, whereas LmPENT5 amastigotes lost their ability to resist pentamidine, with IC50s and index of resistance values close to those for the LmWT clone. These results strongly indicate that the level of pentamidine tolerated by resistant amastigotes after the life cycle was dependent on the induced level of resistance. This fact could be significant in the in vivo transmission of drug-resistant parasites by Phlebotominae. Particular attention should be given to the finding that the emergence of parasite resistance is a potential risk of the use of inadequate doses as therapy in humans.

scholarly journals Organoids and Bioengineered Intestinal Models: Potential Solutions to the Cryptosporidium Culturing Dilemma

2020 ◽  
Vol 8 (5) ◽  
pp. 715 ◽  
Author(s):  
Samantha Gunasekera ◽  
Alireza Zahedi ◽  
Mark O’Dea ◽  
Brendon King ◽  
Paul Monis ◽  
...  
Keyword(s):  
Life Cycle ◽  
Host Cells ◽  
Protozoan Parasites ◽  
Future Directions ◽  
In Vivo Models ◽  
Severe Diarrhea ◽  
Transformed Cell Lines ◽  
Gnotobiotic Piglets

Cryptosporidium is a major cause of severe diarrhea-related disease in children in developing countries, but currently no vaccine or effective treatment exists for those who are most at risk of serious illness. This is partly due to the lack of in vitro culturing methods that are able to support the entire Cryptosporidium life cycle, which has led to research in Cryptosporidium biology lagging behind other protozoan parasites. In vivo models such as gnotobiotic piglets are complex, and standard in vitro culturing methods in transformed cell lines, such as HCT-8 cells, have not been able to fully support fertilization occurring in vitro. Additionally, the Cryptosporidium life cycle has also been reported to occur in the absence of host cells. Recently developed bioengineered intestinal models, however, have shown more promising results and are able to reproduce a whole cycle of infectivity in one model system. This review evaluates the recent advances in Cryptosporidium culturing techniques and proposes future directions for research that may build upon these successes.

scholarly journals miRNA Clusters with Down-Regulated Expression in Human Colorectal Cancer and Their Regulation

2020 ◽  
Vol 21 (13) ◽  
pp. 4633 ◽  
Author(s):  
Paulína Pidíkova ◽  
Richard Reis ◽  
Iveta Herichova
Keyword(s):  
Colorectal Cancer ◽  
Transcriptional Control ◽  
In Vitro Models ◽  
Mirna Regulation ◽  
Regulated Expression ◽  
Mirna Clusters ◽  
Position Regulation ◽  
Non Coding Rnas

Regulation of microRNA (miRNA) expression has been extensively studied with respect to colorectal cancer (CRC), since CRC is one of the leading causes of cancer mortality worldwide. Transcriptional control of miRNAs creating clusters can be, to some extent, estimated from cluster position on a chromosome. Levels of miRNAs are also controlled by miRNAs “sponging” by long non-coding RNAs (ncRNAs). Both types of miRNA regulation strongly influence their function. We focused on clusters of miRNAs found to be down-regulated in CRC, containing miR-1, let-7, miR-15, miR-16, miR-99, miR-100, miR-125, miR-133, miR-143, miR-145, miR-192, miR-194, miR-195, miR-206, miR-215, miR-302, miR-367 and miR-497 and analysed their genome position, regulation and functions. Only evidence provided with the use of CRC in vivo and/or in vitro models was taken into consideration. Comprehensive research revealed that down-regulated miRNA clusters in CRC are mostly located in a gene intron and, in a majority of cases, miRNA clusters possess cluster-specific transcriptional regulation. For all selected clusters, regulation mediated by long ncRNA was experimentally demonstrated in CRC, at least in one cluster member. Oncostatic functions were predominantly linked with the reviewed miRNAs, and their high expression was usually associated with better survival. These findings implicate the potential of down-regulated clusters in CRC to become promising multi-targets for therapeutic manipulation.

In vitro infection of megakaryocytes and their precursors by human cytomegalovirus

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 487-493 ◽  
Author(s):  
Kirsten Crapnell ◽  
Esmail D. Zanjani ◽  
Aniruddho Chaudhuri ◽  
Joao L. Ascensao ◽  
Stephen St. Jeor ◽  
...  
Keyword(s):  
Life Cycle ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Allogeneic Bone Marrow Transplantation ◽  
Laboratory Strain ◽  
Allogeneic Bone ◽  
Cd34 Cells ◽  
Hcmv Disease

Apart from congenital human cytomegalovirus (HCMV) infection, manifest HCMV disease occurs primarily in immunocompromised patients. In allogeneic bone marrow transplantation, HCMV is frequently associated with graft failure and cytopenias involving all hematopoietic lineages, but thrombocytopenia is the most commonly reported hematologic complication. The authors hypothesized that megakaryocytes (MK) may be a specific target for HCMV. Although the susceptibility of immature hematopoietic progenitors cells to HCMV has been established, a productive viral life cycle has only been linked to myelomonocytic maturation. The authors investigated whether HCMV can also infect MK and impair their function. They demonstrated that HCMV did not affect the thrombopoietin (TPO)-driven proliferation of CD34+ cells until MK maturation occurred. MK challenged with HCMV showed a 50% more rapid loss of viability than mock-infected cells. MK and their early precursors were clearly shown to be susceptible to HCMV in vitro, as evidenced by the presence of HCMV in magnetic column-purified CD42+ MK and 2-color fluorescent staining with antibodies directed against CD42a and HCMV pp65 antigen. These findings were confirmed by the infection of MK with a laboratory strain of HCMV containing the β-galactosidase (β-gal) gene. Using chromogenic β-gal substrates, HCMV was detected during MK differentiation of infected CD34+ cells and after infection of fully differentiated MK. Production of infectious virus was observed in cultures infected MK, suggesting that HCMV can complete its life cycle. These results demonstrate that MK are susceptible to HCMV infection and that direct infection of these cells in vivo may contribute to the thrombocytopenia observed in patients infected with HCMV.

scholarly journals The TWIST1-centered competing endogenous RNA network promotes proliferation, invasion, and migration of lung adenocarcinoma

Oncogenesis ◽  
2019 ◽  
Vol 8 (11) ◽  
Author(s):  
Wenjie Xia ◽  
Qixing Mao ◽  
Bing Chen ◽  
Lin Wang ◽  
Weidong Ma ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Migration And Invasion ◽  
Circular Rnas ◽  
Messenger Rnas ◽  
Competing Endogenous Rna ◽  
Invasion And Migration ◽  
Non Coding Rnas ◽  
And Migration

Abstract The proposed competing endogenous RNA (ceRNA) mechanism suggested that diverse RNA species, including protein-coding messenger RNAs and non-coding RNAs such as long non-coding RNAs, pseudogenes and circular RNAs could communicate with each other by competing for binding to shared microRNAs. The ceRNA network (ceRNET) is involved in tumor progression and has become a hot research topic in recent years. To date, more attention has been paid to the role of non-coding RNAs in ceRNA crosstalk. However, coding transcripts are more abundant and powerful than non-coding RNAs and make up the majority of miRNA targets. In this study, we constructed a mRNA-mRNA related ceRNET of lung adenocarcinoma (LUAD) and identified the highlighted TWIST1-centered ceRNET, which recruits SLC12A5 and ZFHX4 as its ceRNAs. We found that TWIST1/SLC12A5/ZFHX4 are all upregulated in LUAD and are associated with poorer prognosis. SLC12A5 and ZFHX4 facilitated proliferation, migration, and invasion in vivo and in vitro, and their effects were reversed by miR-194–3p and miR-514a-3p, respectively. We further verified that SLC12A5 and ZFHX4 affected the function of TWIST1 by acting as ceRNAs. In summary, we constructed a mRNA-mRNA related ceRNET for LUAD and highlighted the well-known oncogene TWIST1. Then we verified that SLC12A5 and ZFHX4 exert their oncogenic function by regulating TWIST1 expression through a ceRNA mechanism.

scholarly journals Non-Coding RNAs as Mediators of Epigenetic Changes in Malignancies

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3657
Author(s):  
Subhasree Kumar ◽  
Edward A. Gonzalez ◽  
Pranela Rameshwar ◽  
Jean-Pierre Etchegaray
Keyword(s):  
Cellular Proliferation ◽  
Circular Rnas ◽  
Cancer Type ◽  
Epigenetic Alterations ◽  
Gene Expressions ◽  
Aberrant Expression ◽  
In Vivo Experiments ◽  
Non Coding Rnas

Non-coding RNAs (ncRNAs) are untranslated RNA molecules that regulate gene expressions. NcRNAs include small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), circular RNAs (cRNAs) and piwi-interacting RNAs (piRNAs). This review focuses on two types of ncRNAs: microRNAs (miRNAs) or short interfering RNAs (siRNAs) and long non-coding RNAs (lncRNAs). We highlight the mechanisms by which miRNAs and lncRNAs impact the epigenome in the context of cancer. Both miRNAs and lncRNAs have the ability to interact with numerous epigenetic modifiers and transcription factors to influence gene expression. The aberrant expression of these ncRNAs is associated with the development and progression of tumors. The primary reason for their deregulated expression can be attributed to epigenetic alterations. Epigenetic alterations can cause the misregulation of ncRNAs. The experimental evidence indicated that most abnormally expressed ncRNAs impact cellular proliferation and apoptotic pathways, and such changes are cancer-dependent. In vitro and in vivo experiments show that, depending on the cancer type, either the upregulation or downregulation of ncRNAs can prevent the proliferation and progression of cancer. Therefore, a better understanding on how ncRNAs impact tumorigenesis could serve to develop new therapeutic treatments. Here, we review the involvement of ncRNAs in cancer epigenetics and highlight their use in clinical therapy.

scholarly journals LncRNA DQ786243 contributes to proliferation and metastasis of colorectal cancer both in vitro and in vivo

2016 ◽  
Vol 36 (3) ◽  
Author(s):  
Longci Sun ◽  
Hanbing Xue ◽  
Chunhui Jiang ◽  
Hong Zhou ◽  
Lei Gu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cancer Progression ◽  
Cell Cycle Progression ◽  
Migration And Invasion ◽  
Cycle Progression ◽  
Proliferation And Metastasis ◽  
Non Coding Rnas ◽  
Colorectal Cancer Progression

This article aims to find the key long non-coding RNAs (LncRNAs) associated with colorectal cancer (CRC) and to study its biological functions in colorectal cancer progression. Our study has shown that upregulated LncRNA DQ786243 can regulate cell proliferation, cell cycle progression, cell apoptosis, migration, and invasion in CRC cells. Xenograft experiments confirmed that the growth of xenograft tumors formed by CRC cells was suppressed after silencing LncRNA DQ786243 expression. In conclusion, our study suggests that LncRNA DQ786243 is an oncogene that promotes tumor progression and leads us to propose that LncRNAs may serve as key regulatory hubs in CRC progression.

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