| 1. |
- Guo, Enen, et al.
(author)
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A Pacifastacus leniusculus serine protease interacts with WSSV
- 2017
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In: Fish and Shellfish Immunology. - : Elsevier BV. - 1050-4648 .- 1095-9947. ; 68, s. 211-219
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Journal article (peer-reviewed)abstract
- Serine proteases are involved in many critical physiological processes including virus spread and replication. In the present study, we identified a new clip-domain serine protease (PIcSP) in the crayfish Pacifastacus leniusculus hemocytes, which can interact with the White Spot Syndrome Virus (WSSV) envelope protein VP28. It was characterized by a classic clip domain with six strictly conserved Cys residues, and contained the conserved His-Asp-Ser (H-D-S)motif in the catalytic domain. Furthermore, signal peptide prediction revealed that it has a 16-residue secretion signal peptide. Tissue distribution showed that it was mainly located in P. leniusculus hemocytes, and its expression was increased in hemocytes upon WSSV challenge. In vitro knock down of PIcSP decreased both the expression of VP28 and the WSSV copy number in hematopoietic stem (HPT) cells. Accordingly, these data suggest that the new serine protease may be of importance for WSSV infection into hematopoietic cells.
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| 2. |
- He, Bing, et al.
(author)
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Single-cell RNA sequencing reveals the mesangial identity and species diversity of glomerular cell transcriptomes
- 2021
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In: Nature Communications. - : Springer Nature. - 2041-1723. ; 12:1
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Journal article (peer-reviewed)abstract
- Molecular characterization of the individual cell types in human kidney as well as model organisms are critical in defining organ function and understanding translational aspects of biomedical research. Previous studies have uncovered gene expression profiles of several kidney glomerular cell types, however, important cells, including mesangial (MCs) and glomerular parietal epithelial cells (PECs), are missing or incompletely described, and a systematic comparison between mouse and human kidney is lacking. To this end, we use Smart-seq2 to profile 4332 individual glomerulus-associated cells isolated from human living donor renal biopsies and mouse kidney. The analysis reveals genetic programs for all four glomerular cell types (podocytes, glomerular endothelial cells, MCs and PECs) as well as rare glomerulus-associated macula densa cells. Importantly, we detect heterogeneity in glomerulus-associated Pdgfrb-expressing cells, including bona fide intraglomerular MCs with the functionally active phagocytic molecular machinery, as well as a unique mural cell type located in the central stalk region of the glomerulus tuft. Furthermore, we observe remarkable species differences in the individual gene expression profiles of defined glomerular cell types that highlight translational challenges in the field and provide a guide to design translational studies. The molecular identity of renal glomerular cells is poorly characterized and rodent glomerulopathy models translate poorly to humans. Here, the authors show molecular signatures of glomerulus-associated cells using single cell RNA sequencing and highlight differences between mouse and human cells.
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| 3. |
- Korkut, Gül Gizem, et al.
(author)
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Expression of an active and inactive clip-domain Serine Protease Isolated from Signal Crayfish in an insect cell line
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Other publication (other academic/artistic)abstract
- In this study, we aimed to elucidate the cleavage specificity and endogenous substrate of a clip-domain serine protease from the crayfish Pacifastacus leniusculus (PlcSP). This serine protease can bind to white spot syndrome virus (WSSV) and is important for the entry of WSSV into the hematopoietic tissue (HPT). In order to understand its intrinsic role in the serine protease cascade, we wanted to use substrate phage display method to clarify cleavage specificity of PlcSP. Firstly, we aimed to make recombinant active and inactive forms of this PlcSP using human embryonic kidney cell line (HEK293) however PlcSP appeared to be toxic for mammalian cells and we were not able to express this crustacean protein. However, we were able to express these recombinantPlcSP both in their zymogenic and active forms in Sf9 insect cell line.
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| 4. |
- Korkut, Gül Gizem, 1986-
(author)
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Interaction between crayfish and some microorganisms; Effect of temperature
- 2018
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Doctoral thesis (other academic/artistic)abstract
- Innate immunity, which constitutes the first line of defense in vertebrates, is the only immune system that invertebrates rely on to protect themselves from pathogens. The invertebrate immune system is composed of cellular and humoral components. Cellular immunity is phagocytosis, opsonization and encapsulation. The humoral part is mainly composed of the events taking place upon secretion of granules and the enzymes within that lead to the lysis of the pathogen by antimicrobial peptides (AMPs) and the melanization cascade. The Prophenoloxidase (proPO) activating system is an important pathway that is stored in the granules of semigranular and granular hemocytes (blood cells). These cells will degranulate and release the proPO system when activated upon pathogen recognition. This cascade results in the melanization reaction and to trap and eliminate pathogens. White spot syndrome virus (WSSV) is a deadly pathogen mainly targeting crustaceans and causing huge economic losses since its first emergence in 1992 in Taiwan. It is known that WSSV disables the immune system of the host by interfering with the proPO cascade. Temperature is a restricting factor for the WSSV infections however it is not known if its affects are on host immunity or on the virus itself.With the aim of elucidating WSSV infection, we studied the virus entry mechanisms. By crosslinking WSSV with the hemocytes we showed that a new clip-domain serine protease (PlcSP) plays an important role during the WSSV infection in crayfish by means of interacting with WSSV envelope protein VP28. Moreover, we have shown that the viral entry is inhibited at cold temperatures due to temperature’s inhibitory effect on PlcSP expression. We also showed that by slowing down of the host’s metabolism hence proliferation in host tissue either by low temperature or cell cycle inhibitors, we could inhibit WSSV replication once it has entered the host cell. We tested if the temperature effects host or pathogen, or both, we investigated the mortalities, phagocytosis, bacterial clearance, total hemocyte counts, degranulation and melanization rate of crayfish under a cold and warm temperature by using two strains of gram-negative bacteria and LPS. It is apparent that the cellular immunity is more effective at low temperature while the humoral immunity can become overactivated and toxic for the host at higher temperature. Furthermore, we aimed to study the cleavage specificity for PlcSP since it is predicted to be secreted from hemocytes and takes part in the serine protease cascade during melanization reaction.
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| 5. |
- Korkut, Gül Gizem, et al.
(author)
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The effect of temperature on bacteria-host interactions in the freshwater crayfish, Pacifastacus leniusculus
- 2018
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In: Journal of Invertebrate Pathology. - : Elsevier BV. - 0022-2011 .- 1096-0805. ; 157, s. 67-73
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Journal article (peer-reviewed)abstract
- Water temperature is known to affect many aspects of aquatic life including immune responses and susceptibility to diseases. In this context, we studied the effect of temperature on the defense system of the freshwater crayfish Pacifastacus leniusculus. Animals were challenged with two pathogenic Gram-negative bacteria, Aeromonas hydrophila and Pseudomonas gessardii, as well as the bacterial cell wall component lipopolysaccharide (LPS) at two different temperatures, cold (6 °C) and room temperature (22 °C). The immune responses were compared by means of differences in mortality, phagocytosis, bacterial clearance, and the melanization reaction of the hemolymph at these two temperatures. We observed that crayfish survival was higher at cold temperature. The mortality rate was zero at 6 °C following A. hydrophila or LPS injections. Furthermore, the bacteria were completely cleared from crayfish after they had been held at 6 °C for more than 9 days. We also observed a strong melanization reaction of hemolymph at 22 °C when stimulated with LPS, as well as with bacteria. Taken together, our results suggest that the cellular immunity is more effective at low temperature in this cold-adapted animal and pathogens are efficiently removed from the body by mean of phagocytosis.
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| 6. |
- Korkut, Gül Gizem, et al.
(author)
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The effect of temperature on White Spot Disease progression in a crustacean, Pacifastacus leniusculus
- 2018
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In: Developmental and Comparative Immunology. - : Elsevier BV. - 0145-305X .- 1879-0089. ; 89, s. 7-13
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Journal article (peer-reviewed)abstract
- The effects of temperature on the progression of White Spot Disease (WSD) have been studied in the freshwater crayfish Pacifastacus leniusculus. In this study, we aimed to understand the reason for previously observed low mortalities with white spot syndrome virus (WSSV) infected crayfish at low temperatures. The susceptibility of freshwater crayfish to WSSV was studied at different temperatures. The mortality rate at 6°C was zero, meanwhile the animals kept at 22°C developed WSD symptoms and died in a few days after WSSV injections, however upon transfer of animals from 6°C to 22°C the mortality reached 100% indicating that the virus is not cleared. Moreover, the VP28 expression at 6°C was significantly lower compared to animals kept at 22°C. We injected animals with demecolcine, an inhibitor that arrests the cell cycle in metaphase, and observed a delayed mortality. Furthermore, the VP28 expression was found to be lower in these animals receiving both injections with WSSV and demecolcine since cell proliferation was inhibited by demecolcine. We quantified WSSV copy numbers and found that virus entry was blocked at 6°C, but not in demecolcine treatments. We supported this result by quantifying the expression of a clip domain serine protease (PlcSP) which plays an important role for WSSV binding, and we found that the PlcSP expression was inhibited at 6°C. Therefore, our hypothesis is that the WSSV needs proliferating cells to replicate, and an optimum temperature to enter the host hematopoietic stem cells successfully.
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| 7. |
- Korkut, Gül Gizem, et al.
(author)
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The effect of temperature on Pacifastacus leniusculus immunity
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Journal article (peer-reviewed)abstract
- Global climate change is an upcoming threat to marine as well as freshwater invertebrates andthe temperature around the globe is expected to rise with up to 4°C in the next decade. Thewater temperature is known to affect many aspects of aquatic life including immuneresponses and susceptibility to diseases. In this context, we studied the effect of temperatureon the defense system of the freshwater crayfish Pacifastacus leniusculus. Animals werechallenged with two pathogenic Gram-negative bacteria Aeromonas hydrophila andPseudomonas gessardii, as well as the bacterial cell wall component lipopolysaccharide(LPS) at two different temperatures, one cold and one room temperature. The immuneresponses were compared by means of differences in mortality, phagocytosis, bacterialclearance, and the melanization reaction of the hemolymph at these two temperatures. Weobserved that crayfish survived infections better at cold temperatures. The mortality rate waszero at 6°C following A. hydrophila or LPS injections. Furthermore, the bacteria werecompletely cleared from crayfish after they had been kept at this low temperature for morethan 9 days. We also observed a strong melanization reaction of hemolymph at 22°C whenstimulated with LPS, as well as with bacteria. Taken together, our results suggest that thecellular immunity is more effective at low temperature in this cold-adapted animal andpathogens are efficiently removed from the body by means of phagocytosis.
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| 8. |
- Yang, Hairu, et al.
(author)
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JAK/STAT signaling in Drosophila muscles controls the cellular immune response against parasitoid infection
- 2015
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In: EMBO Reports. - : EMBO. - 1469-221X .- 1469-3178. ; 16:12, s. 1664-1672
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Journal article (peer-reviewed)abstract
- The role of JAK/STAT signaling in the cellular immune response of Drosophila is not well understood. Here, we show that parasitoid wasp infection activates JAK/STAT signaling in somatic muscles of the Drosophila larva, triggered by secretion of the cytokines Upd2 and Upd3 from circulating hemocytes. Deletion of upd2 or upd3, but not the related os (upd1) gene, reduced the cellular immune response, and suppression of the JAK/STAT pathway in muscle cells reduced the encapsulation of wasp eggs and the number of circulating lamellocyte effector cells. These results suggest that JAK/STAT signaling in muscles participates in a systemic immune defense against wasp infection.
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