| 1. |
- Bygdell, Joakim, et al.
(författare)
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Protein expression in tension wood formation monitored at high tissue resolution in Populus
- 2017
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Ingår i: Journal of Experimental Botany. - : Oxford University Press. - 0022-0957 .- 1460-2431. ; 68:13, s. 3405-3417
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Tidskriftsartikel (refereegranskat)abstract
- Tension wood (TW) is a specialized tissue with contractile properties that is formed by the vascular cambium in response to gravitational stimuli. We quantitatively analysed the proteomes of Populus tremula cambium and its xylem cell derivatives in stems forming normal wood (NW) and TW to reveal the mechanisms underlying TW formation. Phloem-, cambium-, and wood-forming tissues were sampled by tangential cryosectioning and pooled into nine independent samples. The proteomes of TW and NW samples were similar in the phloem and cambium samples, but diverged early during xylogenesis, demonstrating that reprogramming is an integral part of TW formation. For example, 14-3-3, reactive oxygen species, ribosomal and ATPase complex proteins were found to be up-regulated at early stages of xylem differentiation during TW formation. At later stages of xylem differentiation, proteins involved in the biosynthesis of cellulose and enzymes involved in the biosynthesis of rhamnogalacturonan-I, rhamnogalacturonan-II, arabinogalactan-II and fasciclin-like arabinogalactan proteins were up-regulated in TW. Surprisingly, two isoforms of exostosin family proteins with putative xylan xylosyl transferase function and several lignin biosynthesis proteins were also up-regulated, even though xylan and lignin are known to be less abundant in TW than in NW. These data provided new insight into the processes behind TW formation.
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| 2. |
- Kaur, S., et al.
(författare)
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Biochemical and proteomic analysis reveals oxidative stress tolerance strategies of Scenedesmus abundans against allelochemicals released by Microcystis aeruginosa
- 2019
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Ingår i: Algal Research. - : Elsevier B.V.. - 2211-9264. ; 41
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Tidskriftsartikel (refereegranskat)abstract
- We studied the possible survival strategies of a green alga, Scenedesmus abundans, against allelochemicals secreted by Microcystis aeruginosa. We exposed the monoculture of S. abundans to a cell free-filtrate (allelochemicals)of M. aeruginosa at the start of our experiment and measured the growth behaviour, morphological changes and oxidative stress markers. The results suggest that exposure to allelochemicals induced oxidative stress in S. abundans, which had significantly reduced the growth of green alga with certain morphological changes. However, after seven days, S. abundans found ways to reduce oxidative stress by recovering its morphology and growth close to that of control. To understand possible survival strategies of test alga, we measured biochemical as well as protein level changes in S. abundans. Biochemical response of the green alga clearly showed that as a response to allelochemicals, enzymatic and non-enzymatic antioxidants were induced. Proteomic analysis showed that exposure to allelochemicals induced accumulation of 13 proteins on the 2-DE gel of S. abundans, which falls in three functional categories, i.e., (i)energy metabolism (photosynthesis, carbon fixation and respiration), (ii)ROS scavenging enzymes and molecular chaperones, and (iii)amino acid and protein biosynthesis. After chronic oxidative stress, these proteins presumably retained glycolysis, pentose phosphate pathway and turnover rate of the Calvin-Benson cycle. Moreover, these proteins assisted in the adequate detoxification of ROS and played an important role in the damage removal and repair of oxidized proteins, lipids and nucleic acids. Therefore, our study anticipates that S. abundans embraces biochemical and proteomic reprogramming to thrives against allelochemicals released by M. aeruginosa.
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| 3. |
- Srivastava, Akanksha, et al.
(författare)
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Acute cadmium toxicity and post-stress recovery : Insights into coordinated and integrated response/recovery strategies of Anabaena sp. PCC 7120
- 2021
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Ingår i: Journal of Hazardous Materials. - : Elsevier BV. - 0304-3894 .- 1873-3336. ; 411
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Tidskriftsartikel (refereegranskat)abstract
- Cyanobacteria, the first photoautotrophs have remarkable adaptive capabilities against most abiotic stresses, including Cd. A model cyanobacterium, Anabaena sp. PCC 7120 has been commonly used to understand cyanobacterial plasticity under different environmental stresses. However, very few studies have focused on the acute Cd toxicity. In this context, Anabaena was subjected to 100 ?M Cd for 48 h (acute Cd stress, ACdS) and then transferred into the fresh medium for post-stress recovery (PSR). We further investigated the dynamics of morpho-ultrastructure, physiology, cytosolic proteome, thylakoidal complexes, chelators, and transporters after ACdS, as well as during early (ER), mid (MR), and late (LR) phases of PSR. The findings revealed that ACdS induced intracellular Cd accumulation and ROS production, altered morpho-ultrastructure, reduced photosynthetic pigments, and affected the structural organization of PSII, which subsequently hindered photosynthetic efficiency. Anabaena responded to ACdS and recovered during PSR by reprogramming the expression pattern of proteins/genes involved in cellular defense and repair; CO2 access, Calvin-Benson cycle, glycolysis, and pentose phosphate pathway; protein biosynthesis, folding, and degradation; regulatory functions; PSI-based cyclic electron flow; Cd chelation; and efflux. These modulations occurred in an integrated and coordinated manner that facilitated Anabaena to detoxify Cd and repair ACdS-induced cellular damage.
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| 4. |
- Srivastava, Akanksha, et al.
(författare)
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Cd-induced cytosolic proteome changes in the cyanobacterium Anabaena sp. PCC7120 are mediated by LexA as one of the regulatory proteins
- 2023
-
Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1570-9639 .- 1878-1454. ; 1871:3, s. 140902-
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Tidskriftsartikel (refereegranskat)abstract
- LexA, a well-characterized transcriptional repressor of SOS genes in heterotrophic bacteria, has been shown to regulate diverse genes in cyanobacteria. An earlier study showed that LexA overexpression in a cyanobacterium, Anabaena sp. PCC7120 reduces its tolerance to Cd stress. This was later shown to be due to modulation of photosynthetic redox poising by LexA under Cd stress. However, due to the global regulatory nature of LexA and the prior prediction of AnLexA-box in a few heavy metal-responsive genes, we speculated that LexA has a broad role in Cd tolerance, with regulation over a variety of Cd stress-responsive genes in addition to photosynthetic genes. Thus, to further expand the knowledge on the regulatory role of LexA in Cd stress tolerance, a cytosolic proteome profiling of Anabaena constitutively overexpressing LexA upon Cd stress was performed. The proteomic study revealed 25 differentially accumulated proteins (DAPs) in response to the combined effect of LexA over -expression and Cd stress, and the other 11 DAPs exclusively in response to either LexA overexpression or Cd stress. The 36 identified proteins were related with a variety of functions, including photosynthesis, C -meta-bolism, antioxidants, protein turnover, post-transcriptional modifications, and a few unknown and hypothetical proteins. The regulation of LexA on corresponding genes, and six previously reported Cd efflux transporters, was further validated by the presence of AnLexA-boxes, transcript, and/or promoter analyses. In a nutshell, this study identifies the regulation of Anabaena LexA on several Cd stress-responsive genes of various functions, hence expanding the regulatory role of LexA under Cd stress.
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| 5. |
- Srivastava, A., et al.
(författare)
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Gamma (γ)-radiation stress response of the cyanobacterium Anabaena sp. PCC7120 : Regulatory role of LexA and photophysiological changes
- 2023
-
Ingår i: Plant Science. - : Elsevier BV. - 0168-9452 .- 1873-2259. ; 326, s. 111529-
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Tidskriftsartikel (refereegranskat)abstract
- High radioresistance of the cyanobacterium, Anabaena sp. PCC7120 has been attributed to efficient DNA repair, protein recycling, and oxidative stress management. However, the regulatory network involved in these batteries of responses remains unexplored. In the present study, the role of a global regulator, LexA in modulating gamma (γ)-radiation stress response of Anabaena was investigated. Comparison of the cytosolic proteome profiles upon γ-radiation in recombinant Anabaena strains, AnpAM (vector-control) and AnlexA+ (LexA-overexpressing), revealed 41 differentially accumulated proteins, corresponding to 29 distinct proteins. LexA was found to be involved in the regulation of 27 of the corresponding genes based on the presence of AnLexA-Box, EMSA, and/or qRT-PCR studies. The majority of the regulated genes were found to be involved in C-assimilation either through photosynthesis or C-catabolism and oxidative stress alleviation. Photosynthesis, measured in terms of PSII photophysiological parameters and thylakoid membrane proteome was found to be affected by γ-radiation in both AnpAM and AnlexA+ cells, with LexA affecting them even under control growth conditions. Thus, LexA functioned as one of the transcriptional regulators involved in modulating γ-radiation stress response in Anabaena. This study could pave the way for a deeper understanding of the regulation of γ-radiation-responsive genes in cyanobacteria at large.
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| 6. |
- Srivastava, Akanksha, et al.
(författare)
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Physiological and thylakoid proteome analyses of Anabaena sp. PCC 7120 for monitoring the photosynthetic responses under cadmium stress
- 2021
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Ingår i: Algal Research. - : Elsevier BV. - 2211-9264. ; 54
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Tidskriftsartikel (refereegranskat)abstract
- Photosynthetic organisms are highly susceptible to cadmium (Cd) as it interferes with the structural and functional aspects of the photosynthesis. As a major group of photosynthetic prokaryotes, cyanobacteria are also affected by Cd. The inhibitory effects of Cd on cyanobacterial photosynthesis have been studied from an early stage, but the mechanism of Cd toxicity is still unclear. Therefore, we investigated the photosynthetic responses of Anabaena sp. PCC 7120 under Cd stress (LC50) at physiological and thylakoid proteome levels via chlorophyll a fluorescence measurements and blue native (BN)-SDS PAGE, respectively. The findings revealed that Cd exposure triggered the intracellular ROS production and negatively affected the photosynthetic performance. With Cd exposure, effective photochemical quantum yield of PSII (Y(II)) and photochemical quenching efficiency (qP and qL) were significantly reduced, whereas the non-regulated energy dissipation (Y(NO)) was increased. Fast fluorescence kinetic measurements showed that Cd exposure resulted in the inactivation of PSII reaction centres, which interfered with the transfer of energy from antenna complexes to reaction centre, decreased the ability to convert excitation energy into electron transport, increased thermal dissipation, and thus decreased linear electron flow efficiency. Conversely, the efficiency of electron transfer from intermediate carriers to final PSI acceptors was increased, which might be related to the induction of PSI-based cyclic electron flow. This assumption was confirmed by an enhanced post-illumination fluorescence transient. Consistent with these observations, BN-SDS PAGE showed that Cd exposure significantly decreased PSII subunit proteins accumulation, but slightly increased PSI proteins accumulation. Additionally, only minor reductions in the accumulation of ATP synthase/NDH-1 and Cytochrome b6f subunit proteins were observed. The transcript levels of most of the selected subunit proteins were in accordance with BN-SDS PAGE. Collectively, our experiments show that a shift to PSI-based cyclic electron flow was a protective strategy of Anabaena sp. PCC 7120 against Cd-induced PSII photo-damage.
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| 7. |
- Srivastava, Akanksha, et al.
(författare)
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Regulatory role of LexA in modulating photosynthetic redox poise and cadmium stress tolerance in the cyanobacterium, Anabaena sp. PCC7120
- 2022
-
Ingår i: Environmental and Experimental Botany. - : Elsevier BV. - 0098-8472 .- 1873-7307. ; 195
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Tidskriftsartikel (refereegranskat)abstract
- Strategies developed by organisms to overcome disruption in redox poise of photosynthetic electron transport chain (pETC) are important for its survival under abiotic stress. The process needs to be tightly regulated for optimal functioning. While the redox poising processes are well known in cyanobacteria, understanding of their regulatory network is lacking. Since LexA is one of the known global regulators of stress response in the cyanobacterium Anabaena sp. PCC7120, its role in pETC redox poising was investigated using cadmium (Cd) as an abiotic stressor to disrupt photosynthesis. Assessment of the photosynthetic responses of recombinant Anabaena strains, AnlexA+ (LexA-overexpressing) and AnpAM (vector control), under unstressed and Cd-stressed conditions using transmission electron microscopy (TEM) and chlorophyll a fluorescence, indicated that some pETC redox poising responses, including PSII photodamage, energy dissipation, PSI photoprotection, and NDHmediated cyclic electron flow were decreased in AnlexA+ under unstressed conditions. Disturbance in pETC redox poise during Cd stress observed in Anabaena was accentuated upon overexpression of LexA. The decreased photodamage of PSII and increased photoinhibition of PSI in AnlexA+ in the presence or absence of Cd stress, correlated well with the changes in pETC complexes observed in blue native (BN)-PAGE and the regulation of over 70 of the 90 pETC component genes by LexA demonstrated through transcript, electromobility shift assay (EMSA), and bioinformatics studies. In a nutshell, LexA has been identified as one of the regulators involved in the streamlining of pETC redox poising responses under normal growth and during abiotic stress through transcriptional regulation of some of the redox-controlled pETC component genes.
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| 8. |
- Srivastava, Vaibhav, et al.
(författare)
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Alternative splicing studies of the reactive oxygen species gene network in Populus reveal two isoforms of high-isoelectric-point superoxide dismutase
- 2009
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Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 149:4, s. 1848-1859
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Tidskriftsartikel (refereegranskat)abstract
- Recent evidence has shown that alternative splicing (AS) is widely involved in the regulation of gene expression, substantially extending the diversity of numerous proteins. In this study, a subset of expressed sequence tags representing members of the reactive oxygen species gene network was selected from the PopulusDB database to investigate AS mechanisms in Populus. Examples of all known types of AS were detected, but intron retention was the most common. Interestingly, the closest Arabidopsis (Arabidopsis thaliana) homologs of half of the AS genes identified in Populus are not reportedly alternatively spliced. Two genes encoding the protein of most interest in our study (high-isoelectric-point superoxide dismutase [hipI-SOD]) have been found in black cottonwood (Populus trichocarpa), designated PthipI-SODC1 and PthipI-SODC2. Analysis of the expressed sequence tag libraries has indicated the presence of two transcripts of PthipI-SODC1 (hipI-SODC1b and hipI-SODC1s). Alignment of these sequences with the PthipI-SODC1 gene showed that hipI-SODC1b was 69 bp longer than hipI-SODC1s due to an AS event involving the use of an alternative donor splice site in the sixth intron. Transcript analysis showed that the splice variant hipI-SODC1b was differentially expressed, being clearly expressed in cambial and xylem, but not phloem, regions. In addition, immunolocalization and mass spectrometric data confirmed the presence of hipI-SOD proteins in vascular tissue. The functionalities of the spliced gene products were assessed by expressing recombinant hipI-SOD proteins and in vitro SOD activity assays.
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| 9. |
- Srivastava, Vaibhav, et al.
(författare)
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Downregulation of high-isoelectric-point extracellular superoxide dismutase mediates alterations in the metabolism of reactive oxygen species and developmental disturbances in hybrid aspen
- 2007
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Ingår i: The Plant Journal. - : Blackwell Publishing. - 0960-7412 .- 1365-313X. ; 49:1, s. 135-148
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Tidskriftsartikel (refereegranskat)abstract
- Transgenic hybrid aspen (Populus tremula L. x P. tremuloides Michx.) plants expressing a high-isoelectric-point superoxide dismutase (hipI-SOD) gene in antisense orientation were generated to investigate its function. Immunolocalization studies showed the enzyme to be localized extracellularly, in the secondary cell wall of xylem vessels and phloem fibers. The antisense lines of hipI-SOD exhibited a distinct phenotype; growth rate was reduced, stems were thinner and leaves smaller than in wild-type (WT) plants. The abundance of hipI-SOD was reduced in the bark and xylem of plants from these antisense lines. The vascular tissue of transgenic lines became lignified earlier than in WT plants and also showed an increased accumulation of reactive oxygen species (ROS). Xylem fibers and vessels were shorter and thinner in the transgenic lines than in WT plants. The total phenolic content was enhanced in the antisense lines. Furthermore, microarray analysis indicated that several enzymes involved in cell signaling, lignin biosynthesis and stress responses were upregulated in apical vascular tissues of transgenic plants. The upregulation of selected genes involved in lignin biosynthesis was also verified by real-time PCR. The results suggest that, in the transgenic plants, a premature transition into maturation occurs and the process is discussed in terms of the effects of increased accumulation of ROS due to reduced expression of hipI-SOD during development and differentiation.
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| 10. |
- Yadav, Sandhya, et al.
(författare)
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Comparison and optimization of protein extraction and two-dimensional gel electrophoresis protocols for liverworts
- 2020
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Ingår i: BMC Research Notes. - : Springer Nature. - 1756-0500. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Objective Liverworts possess historical adaptive strategies for abiotic stresses because they were the first plants that shifted from water to land. Proteomics is a state-of-the-art technique that can capture snapshots of events occurring at the protein level in many organisms. Herein, we highlight the comparison and optimization of an effective protein extraction and precipitation protocol for two-dimensional gel electrophoresis (2-DE) of liverworts. Results We compared three different protein extraction methods, i.e.,1.5 M Tris-HCl (pH 8.8), 50 mM Tris-HCl (pH 7.5), and polyvinylpolypyrrolidone (PVPP) extraction, followed by three precipitation methods, i.e., 80% ethanol, 80% acetone, and 20% tricholoroacetic acid (TCA)-acetone, in a liverwort Dumortiera hirsuta. Among these methods, 50 mM Tris-HCl (pH 7.5) extraction, followed by 20% TCA-acetone precipitation, appeared to be more suitable for 2-DE. Furthermore, we performed modifications during protein washing, re-solubilization in rehydration buffer and isoelectric focusing (IEF). The modifications provided us better results in terms of protein yield, resolution, spot numbers, and intensities for 2-DE gels of D. hirsuta and other two liverworts, i.e., Marchantia paleacea and Plagiochasma appendiculatum. Furthermore, we randomly selected spots from the 2-DE gel of D. hirsuta and identified using mass spectrometry, which confirms the applicability of this protocol for liverworts proteomics.
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| 11. |
- Bergemalm, Daniel, et al.
(författare)
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Superoxide dismutase-1 and other proteins in inclusions from transgenic amyotrophic lateral sclerosis model mice
- 2010
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Ingår i: Journal of Neurochemistry. - : Wiley. - 0022-3042 .- 1471-4159. ; 114:2, s. 408-418
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Tidskriftsartikel (refereegranskat)abstract
- Mutant superoxide dismutase-1 (SOD1) causes amyotrophic lateral sclerosis (ALS) through a cytotoxic mechanism of unknown nature. A hallmark in ALS patients and transgenic mouse models carrying human SOD1 (hSOD1) mutations are hSOD1-immunoreactive inclusions in spinal cord ventral horns. The hSOD1 inclusions may block essential cellular functions or cause toxicity through sequestering of other proteins. Inclusions from four different transgenic mouse models were examined after density gradient ultracentrifugation. The inclusions are complex structures with heterogeneous densities and are disrupted by detergents. The aggregated hSOD1 was mainly composed of subunits that lacked the native stabilizing intra-subunit disulfide bond. A proportion of subunits formed hSOD1 oligomers or was bound to other proteins through disulfide bonds. Dense inclusions could be isolated and the protein composition was analyzed using proteomic techniques. Mutant hSOD1 accounted for half of the protein. Ten other proteins were identified. Two were cytoplasmic chaperones, four were cytoskeletal proteins, and 4 were proteins that normally reside in the endoplasmic reticulum (ER). The presence of ER proteins in inclusions containing the primarily cytosolic hSOD1 further supports the notion that ER stress is involved in ALS.
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| 12. |
- Bergemalm, Daniel, 1977-, et al.
(författare)
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Superoxide dismutase-1 and other proteins in inclusions from transgenic amyotrophic lateral sclerosis model mice
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Mutant superoxide dismutase-1 (SOD1) causes amyotrophic lateral sclerosis (ALS) through a cytotoxic mechanism of unknown nature. A hallmark in ALS patients and transgenic mouse models carrying human SOD1 (hSOD1) mutations are hSOD1-immunoreactive inclusions in spinal cord ventral horns. The hSOD1 inclusions may block essential cellular functions or cause toxicity through sequestering of other proteins. Inclusions from 4 different transgenic mouse models were examined after density gradient ultracentrifugation. The inclusions are complex structures with heterogeneous densities and are disrupted by detergents. The aggregated hSOD1 was mainly composed of subunits that lacked the native stabilizing intrasubunit disulfide bond. A proportion of subunits formed hSOD1 oligomers or was bound to other proteins through disulfide bonds. Dense inclusions could be isolated and the protein composition was analyzed using proteomic techniques. Mutant hSOD1 accounted for half of the protein. Ten other proteins were identified. Two were cytoplasmic chaperones, 4 were cytoskeletal proteins, and 4 were proteins that normally reside in the endoplasmic reticulum (ER). The presence of ER proteins in inclusions containing the primarily cytosolic hSOD1 further supports the notion that ER stress is involved in ALS.
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| 13. |
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| 14. |
- Brown, Christian, et al.
(författare)
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Structural and functional characterization of the microtubule interacting and trafficking domains of two oomycete chitin synthases
- 2016
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 283:16, s. 3072-3088
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Tidskriftsartikel (refereegranskat)abstract
- Chitin synthases (Chs) are responsible for the synthesis of chitin, a key structural cell wall polysaccharide in many organisms. They are essential for growth in certain oomycete species, some of which are pathogenic to diverse higher organisms. Recently, a Microtubule Interacting and Trafficking (MIT) domain, which is not found in any fungal Chs, has been identified in some oomycete Chs proteins. Based on experimental data relating to the binding specificity of other eukaryotic MIT domains, there was speculation that this domain may be involved in the intracellular trafficking of Chs proteins. However, there is currently no evidence for this or any other function for the MIT domain in these enzymes. To attempt to elucidate their function, MIT domains from two Chs enzymes from the oomycete Saprolegnia monoica were cloned, expressed and characterized. Both were shown to interact strongly with the plasma membrane component phosphatidic acid, and to have additional putative interactions with proteins thought to be involved in protein transport and localization. Aiding our understanding of these data, the structure of the first MIT domain from a carbohydrate-active enzyme (MIT1) was solved by NMR, and a model structure of a second MIT domain (MIT2) was built by homology modelling. Our results suggest a potential function for these MIT domains in the intracellular transport and/or regulation of Chs enzymes in the oomycetes.
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| 15. |
- Bylesjö, Max, et al.
(författare)
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Integrated analysis of transcript, protein and metabolite data to study lignin biosynthesis in hybrid aspen
- 2009
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 8:1, s. 199-210
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Tidskriftsartikel (refereegranskat)abstract
- Tree biotechnology will soon reach a mature state where it will influence the overall supply of fiber, energy and wood products. We are now ready to make the transition from identifying candidate genes, controlling important biological processes, to discovering the detailed molecular function of these genes on a broader, more holistic, systems biology level. In this paper, a strategy is outlined for informative data generation and integrated modeling of systematic changes in transcript, protein and metabolite profiles measured from hybrid aspen samples. The aim is to study characteristics of common changes in relation to genotype-specific perturbations affecting the lignin biosynthesis and growth. We show that a considerable part of the systematic effects in the system can be tracked across all platforms and that the approach has a high potential value in functional characterization of candidate genes.
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| 16. |
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| 17. |
- Dahlin, Paul, et al.
(författare)
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Comparative analysis of sterol acquisition in the oomycetes Saprolegnia parasitica and Phytophthora infestans
- 2017
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Ingår i: PLOS ONE. - : PUBLIC LIBRARY SCIENCE. - 1932-6203. ; 12:2
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Tidskriftsartikel (refereegranskat)abstract
- The oomycete class includes pathogens of animals and plants which are responsible for some of the most significant global losses in agriculture and aquaculture. There is a need to replace traditional chemical means of controlling oomycete growth with more targeted approaches, and the inhibition of sterol synthesis is one promising area. To better direct these efforts, we have studied sterol acquisition in two model organisms: the sterol-autotrophic Saprolegnia parasitica, and the sterol-heterotrophic Phytophthora infestans. We first present a comprehensive reconstruction of a likely sterol synthesis pathway for S. parasitica, causative agent of the disease saprolegniasis in fish. This pathway shows multiple potential routes of sterol synthesis, and draws on several avenues of new evidence: bioinformatic mining for genes with sterol-related functions, expression analysis of these genes, and analysis of the sterol profiles in mycelium grown in different media. Additionally, we explore the extent to which P. infestans, which causes the late blight in potato, can modify exogenously provided sterols. We consider whether the two very different approaches to sterol acquisition taken by these pathogens represent any specific survival advantages or potential drug targets.
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| 18. |
- Dahlin, Paul, et al.
(författare)
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Comparative analysis of sterol acquisition in the oomycetes Saprolegnia parasitica and Phytophthora infestans
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The oomycete class includes pathogens of animals and plants which are responsible for some of the most significant global losses in agriculture and aquaculture. There is a need to replace traditional chemical means of controlling oomycete growth with more targeted approaches, and the inhibition of sterol synthesis is one promising area. To better direct these efforts, we have studied sterol acquisition in two model organisms: the sterol-autotrophic Saprolegnia parasitica, and the sterol-heterotrophic Phytophthora infestans. We first present a comprehensive reconstruction of a likely sterol synthesis pathway for S. parasitica, causative agent of the disease saprolegniasis in fish. This pathway shows multiple potential routes of sterol synthesis, and draws on several avenues of new evidence: bioinformatic mining for genes with sterol-related functions, expression analysis of these genes, and analysis of the sterol profiles in mycelium grown in different media. Additionally, we explore the extent to which P. infestans, which causes the late blight in potato, can modify exogenously provided sterols. We consider whether the two very different approaches to sterol acquisition taken by these pathogens represent any specific survival advantages or potential drug targets.
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| 19. |
- Dahlin, Paul, et al.
(författare)
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The Oxidosqualene Cyclase from the Oomycete Saprolegnia parasitica Synthesizes Lanosterol as a Single Product
- 2016
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Ingår i: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- The first committed step of sterol biosynthesis is the cyclisation of 2,3-oxidosqualene to form either lanosterol (LA) or cycloartenol (CA). This is catalyzed by an oxidosqualene cyclase (OSC). LA and CA are subsequently converted into various sterols by a series of enzyme reactions. The specificity of the OSC therefore determines the final composition of the end sterols of an organism. Despite the functional importance of OSCs, the determinants of their specificity are not well understood. In sterol-synthesizing oomycetes, recent bioinformatics, and metabolite analysis suggest that LA is produced. However, this catalytic activity has never been experimentally demonstrated. Here, we show that the OSC of the oomycete Saprolegnia parasitica, a severe pathogen of salmonid fish, has an uncommon sequence in a conserved motif important for specificity. We present phylogenetic analysis revealing that this sequence is common to sterol-synthesizing oomycetes, as well as some plants, and hypothesize as to the evolutionary origin of some microbial sequences. We also demonstrate for the first time that a recombinant form of the OSC from S. parasitica produces LA exclusively. Our data pave the way for a detailed structural characterization of the protein and the possible development of specific inhibitors of oomycete OSCs for disease control in aquaculture.
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| 20. |
- de Oliveira, Andressa Souza, et al.
(författare)
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Antifungal activity of sustainable histone deacetylase inhibitors against planktonic cells and biofilms of Candida spp. and Cryptococcusneoformans
- 2023
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Ingår i: Medical Mycology. - : Oxford University Press (OUP). - 1369-3786 .- 1460-2709. ; 61:8
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Tidskriftsartikel (refereegranskat)abstract
- The limited therapeutic options for fungal infections and the increased incidence of fungal strains resistant to antifungal drugs, especially Candida spp., require the development of new antifungal drugs and strategies. Histone deacetylase inhibitors (HDACi), like vorinostat, have been studied in cancer treatment and have antifungal effects, acting alone or synergistically with classical antifungals. Here we investigated the antifungal activity of two novel sustainable HDACi (LDT compounds) based on vorinostat structure. Molecular docking simulation studies reveal that LDT compounds can bind to Class-I HDACs of Candida albicans, C. tropicalis, and Cryptococcus neoformans, which showed similar binding mode to vorinostat. LDT compounds showed moderate activity when tested alone against fungi but act synergistically with antifungal azoles against Candida spp. They reduced biofilm formation by more than 50% in C. albicans (4 µg/mL), with the main action in fungal filamentation. Cytotoxicity of the LDT compounds against RAW264.7 cells was evaluated and LDT536 demonstrated cytotoxicity only at the concentration of 200 µmol/L, while LDT537 showed IC50 values of 29.12 µmol/L. Our data indicated that these sustainable and inexpensive HDACi have potential antifungal and antibiofilm activities, with better results than vorinostat, although further studies are necessary to better understand the mechanism against fungal cells.
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| 21. |
- Dimitroff, George, et al.
(författare)
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(1,3;1,4)-beta-Glucan Biosynthesis by the CSLF6 Enzyme : Position and Flexibility of Catalytic Residues Influence Product Fine Structure
- 2016
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 55:13, s. 2054-2061
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Tidskriftsartikel (refereegranskat)abstract
- Cellulose synthase-like F6 (CslF6) genes encode polysaccharide synthases responsible for (1,3;1,4)-beta-glucan biosynthesis in cereal grains. However, it is not clear how both (1,3)- and (1,4) -linkages are incorporated into a single polysaccharide chain and how the frequency and arrangement of the two linkage types that define the fine structure of the polysaccharide are controlled. Through transient expression in Nicotiana benthamiana leaves, two CSLF6 orthologs from different cereal species were shown to mediate the synthesis of (1,3;1,4)-beta-glucans with very different fine structures. Chimeric cDNA constructs with interchanged sections of the barley and sorghum CslF6 genes were developed to identify regions of the synthase enzyme responsible for these differences. A single amino acid residue upstream of the TED motif in the catalytic region was shown to dramatically change the fine structure of the polysaccharide produced. The structural basis of this effect can be rationalized by reference to a homology model of the enzyme and appears to be related to the position and flexibility of the TED motif in the active site of the enzyme. The region and amino acid residue identified provide opportunities to manipulate the solubility of (1,3;1,4)-beta-glucan in grains and vegetative tissues of the grasses and, in particular, to enhance the solubility of dietary fibers that are beneficial to human health.
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| 22. |
- Dong, Yiran, et al.
(författare)
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Physiology, Metabolism, and Fossilization of Hot-Spring Filamentous Microbial Mats
- 2019
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Ingår i: Astrobiology. - : MARY ANN LIEBERT, INC. - 1531-1074 .- 1557-8070. ; 19:12
-
Tidskriftsartikel (refereegranskat)abstract
- The evolutionarily ancient Aquificales bacterium Sulfurihydrogenibium spp. dominates filamentous microbial mat communities in shallow, fast-flowing, and dysoxic hot-spring drainage systems around the world. In the present study, field observations of these fettuccini-like microbial mats at Mammoth Hot Springs in Yellowstone National Park are integrated with geology, geochemistry, hydrology, microscopy, and multi-omic molecular biology analyses. Strategic sampling of living filamentous mats along with the hot-spring CaCO3 (travertine) in which they are actively being entombed and fossilized has permitted the first direct linkage of Sulfurihydrogenibium spp. physiology and metabolism with the formation of distinct travertine streamer microbial biomarkers. Results indicate that, during chemoautotrophy and CO2 carbon fixation, the 87-98% Sulfurihydrogenibium-dominated mats utilize chaperons to facilitate enzyme stability and function. High-abundance transcripts and proteins for type IV pili and extracellular polymeric substances (EPSs) are consistent with their strong mucus-rich filaments tens of centimeters long that withstand hydrodynamic shear as they become encrusted by more than 5mm of travertine per day. Their primary energy source is the oxidation of reduced sulfur (e.g., sulfide, sulfur, or thiosulfate) and the simultaneous uptake of extremely low concentrations of dissolved O-2 facilitated by bd-type cytochromes. The formation of elevated travertine ridges permits the Sulfurihydrogenibium-dominated mats to create a shallow platform from which to access low levels of dissolved oxygen at the virtual exclusion of other microorganisms. These ridged travertine streamer microbial biomarkers are well preserved and create a robust fossil record of microbial physiological and metabolic activities in modern and ancient hot-spring ecosystems.
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| 23. |
- Ekström, Jens-Ola, et al.
(författare)
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Drosophila Nora virus capsid proteins differ from those of other picorna-like viruses
- 2011
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Ingår i: Virus Research. - Amsterdam : Elsevier. - 0168-1702 .- 1872-7492. ; 160:1-2, s. 51-58
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Tidskriftsartikel (refereegranskat)abstract
- The recently discovered Nora virus from Drosophila melanogaster is a single-stranded RNA virus. Its published genomic sequence encodes a typical picorna-like cassette of replicative enzymes, but no capsid proteins similar to those in other picorna-like viruses. We have now done additional sequencing at the termini of the viral genome, extending it by 455 nucleotides at the 5' end, but no more coding sequence was found. The completeness of the final 12,333-nucleotide sequence was verified by the production of infectious virus from the cloned genome. To identify the capsid proteins, we purified Nora virus particles and analyzed their proteins by mass spectrometry. Our results show that the capsid is built from three major proteins, VP4A, B and C, encoded in the fourth open reading frame of the viral genome. The viral particles also contain traces of a protein from the third open reading frame, VP3. VP4A and B are not closely related to other picorna-like virus capsid proteins in sequence, but may form similar jelly roll folds. VP4C differs from the others and is predicted to have an essentially α-helical conformation. In a related virus, identified from EST database sequences from Nasonia parasitoid wasps, VP4C is encoded in a separate open reading frame, separated from VP4A and B by a frame-shift. This opens a possibility that VP4C is produced in non-equimolar quantities. Altogether, our results suggest that the Nora virus capsid has a different protein organization compared to the order Picornavirales.
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| 24. |
- Ford, Kristina L., et al.
(författare)
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Comparative "Golgi" Proteome Study of Lolium multiflorum and Populus trichocarpa
- 2016
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Ingår i: PROTEOMES. - : MDPI AG. - 2227-7382. ; 4:3
-
Tidskriftsartikel (refereegranskat)abstract
- The Golgi apparatus (GA) is a crucial organelle in the biosynthesis of non-cellulosic polysaccharides, glycoproteins and proteoglycans that are primarily destined for secretion to the cell surface (plasma membrane, cell wall and apoplast). Only a small proportion of the proteins involved in these processes have been identified in plants, with the majority of their functions still unknown. The availability of a GA proteome would greatly assist plant biochemists, cell and molecular biologists in determining the precise function of the cell wall-related proteins. There has been some progress towards defining the GA proteome in the model plant system Arabidopsis thaliana, yet in commercially important species, such as either the cereals or woody species there has been relatively less progress. In this study, we applied discontinuous sucrose gradient centrifugation to partially enrich GA from suspension cell cultures (SCCs) and combined this with stable isotope labelling (iTRAQ) to determine protein sub-cellular locations. Results from a representative grass species, Italian ryegrass (Lolium multiflorum) and a dicot species, black cottonwood (Populus trichocarpa) are compared. The results confirm that membrane fractionation approaches that provide effective GA-enriched fractions for proteomic analyses in Arabidopsis are much less effective in the species examined here and highlight the complexity of the GA, both within and between species.
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| 25. |
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| 26. |
- Gallo, Valentina, et al.
(författare)
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Proteomic Analysis Identifies Markers of Exposure to Cadmium Sulphide Quantum Dots (CdS QDs)
- 2020
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Ingår i: Nanomaterials. - : MDPI. - 2079-4991. ; 10:6
-
Tidskriftsartikel (refereegranskat)abstract
- The use of cadmium sulphide quantum dot (CdS QD)-enabled products has become increasingly widespread. The prospect of their release in the environment is raising concerns. Here we have used the yeast modelSaccharomyces cerevisiaeto determine the potential impact of CdS QD nanoparticles on living organisms. Proteomic analyses and cell viability assays performed after 9 h exposure revealed expression of proteins involved in oxidative stress and reduced lethality, respectively, whereas oxidative stress declined, and lethality increased after 24 h incubation in the presence of CdS QDs. Quantitative proteomics using the iTRAQ approach (isobaric tags for relative and absolute quantitation) revealed that key proteins involved in essential biological pathways were differentially regulated over the time course of the experiment. At 9 h, most of the glycolytic functions increased, and the abundance of the number of heat shock proteins increased. This contrasts with the situation at 24 h where glycolytic functions, some heat shock proteins as well as oxidative phosphorylation and ATP synthesis were down-regulated. It can be concluded from our data that cell exposure to CdS QDs provokes a metabolic shift from respiration to fermentation, comparable to the situation reported in some cancer cell lines.
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| 27. |
- Gupta, Nitish Kumar, et al.
(författare)
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Singular Phase Characteristics of Electromagnetic Absorbers and a Framework for Low-RCS Target Detection
- 2023
-
Ingår i: IEEE Antennas and Wireless Propagation Letters. - 1536-1225 .- 1548-5757. ; 22:1, s. 134-138
-
Tidskriftsartikel (refereegranskat)abstract
- We conceptualize, numerically validate, and experimentally demonstrate a scheme for distant line-of-sight detection of low-observable targets, such as those covered by metamaterial absorbers, by solely relying on their nontrivial phase characteristics. Relying upon the temporal coupled-mode theory (TCMT), first, we put forward a generalized description of open resonators where we identify the presence of a spectral inflection point in the phase response function as the signature of interest. Thereafter, a realistic phase response function has been modeled and analyzed by incorporating the propagation aspects as well as band-limited Gaussian noise. Change of curvature information owing to the resonant absorption is then ascertained from this involute phase response by developing an algorithm. For pragmatic scrutiny of our proposition, we design a singly resonant (at 12 GHz) wideband resistive sheet-based metamaterial absorber providing − 10 dB absorption across the X- and Ku-bands and employ its de-embedded scattering parameters to confirm the existence of inflection point around 12 GHz. As a final validation, the metamaterial absorber has been fabricated using ITO/PET sheets, and its phase characteristics have been measured in a pseudomonostatic configuration. The information regarding the presence of resonant absorption has been then ascertained by the algorithm extracted phase, thereby validating our proposition.
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| 28. |
- Kaesdorf, Benjamin T., et al.
(författare)
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Mucin-Inspired Lubrication on Hydrophobic Surfaces
- 2017
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Ingår i: Biomacromolecules. - : AMER CHEMICAL SOC. - 1525-7797 .- 1526-4602. ; 18:8, s. 2454-2462
-
Tidskriftsartikel (refereegranskat)abstract
- In the human body, high-molecular-weight glycoproteins called mucins play a key role in protecting epithelial surfaces against pathogenic attack, controlling the passage of molecules toward the tissue and enabling boundary lubrication with very low friction coefficients. However, neither the molecular mechanisms nor the chemical motifs of those biomacromolecules involved in these fundamental processes are fully understood. Thus, identifying the key features that render biomacromolecules such as mucins outstanding boundary lubricants could set the stage for creating versatile artificial superlubricants. We here demonstrate the importance of the hydrophobic terminal peptide domains of porcine gastric mucin (MUCSAC) and human salivary mucin (MUCSB) in the processes of adsorbing to and lubricating a hydrophobic PDMS surface. Tryptic digestion of those mucins results in removal of those terminal domains, which is accompanied by a loss of lubricity as well as surface adsorption. We show that this loss can in part be compensated by attaching hydrophobic phenyl groups to the glycosylated central part of the mucin macromolecule. Furthermore, we demonstrate that the simple biopolysaccharide dextran can be functionalized with hydrophobic groups which confers efficient surface adsorption and good lubricity on PDMS to the polysaccharide.
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| 29. |
- Kao, Mu-Rong, et al.
(författare)
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Substrate Specificities of Variants of Barley (1,3)- and (1,3;1,4)-β-d-Glucanases Resulting from Mutagenesis and Segment Hybridization
- 2024
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 63:9, s. 1194-1205
-
Tidskriftsartikel (refereegranskat)abstract
- Barley (1,3;1,4)-β-d-glucanase is believed to have evolved from an ancestral monocotyledon (1,3)-β-d-glucanase, enabling the hydrolysis of (1,3;1,4)-β-d-glucans in the cell walls of leaves and germinating grains. In the present study, we investigated the substrate specificities of variants of the barley enzymes (1,3;1,4)-β-d-glucan endohydrolase [(1,3;1,4)-β-d-glucanase] isoenzyme EII (HvEII) and (1,3)-β-d-glucan endohydrolase [(1,3)-β-d-glucanase] isoenzyme GII (HvGII) obtained by protein segment hybridization and site-directed mutagenesis. Using protein segment hybridization, we obtained three variants of HvEII in which the substrate specificity was that of a (1,3)-β-d-glucanase and one variant that hydrolyzed both (1,3)-β-d-glucans and (1,3;1,4)-β-d-glucans; the wild-type enzyme hydrolyzed only (1,3;1,4)-β-d-glucans. Using substitutions of specific amino acid residues, we obtained one variant of HvEII that hydrolyzed both substrates. However, neither protein segment hybridization nor substitutions of specific amino acid residues gave variants of HvGII that could hydrolyze (1,3;1,4)-β-d-glucans; the wild-type enzyme hydrolyzed only (1,3)-β-d-glucans. Other HvEII and HvGII variants showed changes in specific activity and their ability to degrade the (1,3;1,4)-β-d-glucans or (1,3)-β-d-glucans to larger oligosaccharides. We also used molecular dynamics simulations to identify amino-acid residues or structural regions of wild-type HvEII and HvGII that interact with (1,3;1,4)-β-d-glucans and (1,3)-β-d-glucans, respectively, and may be responsible for the substrate specificities of the two enzymes.
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| 30. |
- Kootala, Sujit, et al.
(författare)
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Reinforcing Mucus Barrier Properties with Low Molar Mass Chitosans
- 2018
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Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 19:3, s. 872-882
-
Tidskriftsartikel (refereegranskat)abstract
- The mucus gel covers the wet epithelia that forms the inner lining of the body. It constitutes our first line of defense protecting the body from infections and other deleterious molecules. Failure of the mucus barrier can lead to the inflammation of the mucosa such as in inflammatory bowel diseases. Unfortunately, there are no effective strategies that reinforce the mucus barrier properties to recover or enhance its ability to protect the epithelium. Herein, we describe a mucus engineering approach that addresses this issue where we physically cross-link the mucus gel with low molar mass chitosan variants to reinforce its barrier functions. We tested the effect of these chitosans on mucus using in-lab purified porcine gastric mucins, which mimic the native properties of mucus, and on mucus-secreting HT29-MTX epithelial cell cultures. We found that the lowest molar mass chitosan variant (degree of polymerization of 8) diffuses deep into the mucus gels while physically cross-linking the mucin polymers, whereas the higher molar mass chitosan variants (degree of polymerization of 52 and 100) interact only superficially. The complexation resulted in a tighter mucin polymer mesh that slowed the diffusion of dextran polymers and of the cholera toxin B subunit protein through the mucus gels. These results uncover a new use for low molar mass mucoadhesive polymers such as chitosans as noncytotoxic mucosal barrier enhancers that could be valuable in the prevention and treatment of mucosal diseases.
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| 31. |
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| 32. |
- Kumar, Rajender, et al.
(författare)
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Biochemical characterization and molecular insights into substrate recognition of pectin methylesterase from Phytophthora infestans
- 2022
-
Ingår i: Computational and Structural Biotechnology Journal. - : Elsevier BV. - 2001-0370. ; 20, s. 6023-6032
-
Tidskriftsartikel (refereegranskat)abstract
- Pectin methylesterases (PMEs) are a class of carbohydrate-active enzymes that act on the O6-methyl ester groups of the homogalacturonan component of pectins, resulting in de-esterification of the sub-strate polymers and formation of pectate and methanol. PMEs occur in higher plants and microorgan-isms, including fungi, oomycetes, bacteria, and archaea. Microbial PMEs play a crucial role in pathogens' invasion of plant tissues. Here, we have determined the structural and functional properties of Pi-PME, a PME from the oomycete plant pathogen Phytophthora infestans. This enzyme exhibits maxi-mum activity at alkaline pH (8.5) and is active over a wide temperature range (25-50 degrees C). In silico deter-mination of the structure of Pi-PME reveals that the protein consists essentially of three parallel 8-sheets interconnected by loops that adopt an overall 8-helix organization. The loop regions in the vicinity of the active site are extended compared to plant and fungal PMEs, but they are shorter than the corresponding bacterial and insect regions. Molecular dynamic simulations revealed that Pi-PME interacts most strongly with partially de-methylated homogalacturonans, suggesting that it preferentially uses this type of sub-strates. The results are compared and discussed with other known PMEs from different organisms, high-lighting the specific features of Pi-PME.
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| 33. |
- Kumar, Rajender, et al.
(författare)
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Improved Binding Affinity of Omicron's Spike Protein for the Human Angiotensin-Converting Enzyme 2 Receptor Is the Key behind Its Increased Virulence
- 2022
-
Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 23:6
-
Tidskriftsartikel (refereegranskat)abstract
- The new variant of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), Omicron, has been quickly spreading in many countries worldwide. Compared to the original virus, Omicron is characterized by several mutations in its genomic region, including the spike protein's receptor-binding domain (RBD). We have computationally investigated the interaction between the RBD of both the wild type and Omicron variant of SARS-CoV-2 with the human angiotensin-converting enzyme 2 (hACE2) receptor using molecular dynamics and molecular mechanics-generalized Born surface area (MM-GBSA)-based binding free energy calculations. The mode of the interaction between Omicron's RBD with the hACE2 receptor is similar to the original SARS-CoV-2 RBD except for a few key differences. The binding free energy difference shows that the spike protein of Omicron has an increased affinity for the hACE2 receptor. The mutated residues in the RBD showed strong interactions with a few amino acid residues of hACE2. More specifically, strong electrostatic interactions (salt bridges) and hydrogen bonding were observed between R493 and R498 residues of the Omicron RBD with D30/E35 and D38 residues of the hACE2, respectively. Other mutated amino acids in the Omicron RBD, e.g., S496 and H505, also exhibited hydrogen bonding with the hACE2 receptor. A pi-stacking interaction was also observed between tyrosine residues (RBD-Tyr501: hACE2-Tyr41) in the complex, which contributes majorly to the binding free energies and suggests that this is one of the key interactions stabilizing the formation of the complex. The resulting structural insights into the RBD:hACE2 complex, the binding mode information within it, and residue-wise contributions to the free energy provide insight into the increased transmissibility of Omicron and pave the way to design and optimize novel antiviral agents.
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| 34. |
- Kumar, Rajender, et al.
(författare)
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Sequence, structure and functionality of pectin methylesterases and their use in sustainable carbohydrate bioproducts : A review
- 2023
-
Ingår i: International Journal of Biological Macromolecules. - : Elsevier BV. - 0141-8130 .- 1879-0003. ; 244
-
Tidskriftsartikel (refereegranskat)abstract
- Pectin methylesterases (PMEs) are enzymes that play a critical role in modifying pectins, a class of complex polysaccharides in plant cell walls. These enzymes catalyze the removal of methyl ester groups from pectins, resulting in a change in the degree of esterification and consequently, the physicochemical properties of the polymers. PMEs are found in various plant tissues and organs, and their activity is tightly regulated in response to developmental and environmental factors. In addition to the biochemical modification of pectins, PMEs have been implicated in various biological processes, including fruit ripening, defense against pathogens, and cell wall remodelling. This review presents updated information on PMEs, including their sources, sequences and structural diversity, biochemical properties and function in plant development. The article also explores the mechanism of PME action and the factors influencing enzyme activity. In addition, the review highlights the potential applications of PMEs in various industrial sectors related to biomass exploitation, food, and textile industries, with a focus on development of bioproducts based on eco-friendly and efficient industrial processes.
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| 35. |
- Kumar, Sanjiv, et al.
(författare)
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Identification of Growth Inhibitors of the Fish Pathogen Saprolegnia parasitica Using in silico Subtractive Proteomics, Computational Modeling, and Biochemical Validation
- 2020
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Ingår i: Frontiers in Microbiology. - : FRONTIERS MEDIA SA. - 1664-302X. ; 11
-
Tidskriftsartikel (refereegranskat)abstract
- Many Stramenopile species belonging to oomycetes from the genus Saprolegnia infect fish, amphibians, and crustaceans in aquaculture farms and natural ecosystems. Saprolegnia parasitica is one of the most severe fish pathogens, responsible for high losses in the aquaculture industry worldwide. Most of the molecules reported to date for the control of Saprolegnia infections either are inefficient or have negative impacts on the health of the fish hosts or the environment resulting in substantial economic losses. Until now, the whole proteome of S. parasitica has not been explored for a systematic screening of novel inhibitors against the pathogen. The present study was designed to develop a consensus computational framework for the identification of potential target proteins and their inhibitors and subsequent experimental validation of selected compounds. Comparative analysis between the proteomes of Saprolegnia, humans and fish species identified proteins that are specific and essential for the survival of the pathogen. The DrugBank database was exploited to select food and drug administration (FDA)-approved inhibitors whose high binding affinity to their respective protein targets was confirmed by computational modeling. At least six of the identified compounds significantly inhibited the growth of S. parasitica in vitro. Triclosan was found to be most effective with a minimum inhibitory concentration (MIC100) of 4 mu g/ml. Optical microscopy showed that the inhibitors affect the morphology of hyphal cells, with hyper-branching being commonly observed. The inhibitory effects of the compounds identified in this study on Saprolegnia's mycelial growth indicate that they are potentially usable for disease control against this class of oomycete pathogens. Similar approaches can be easily adopted for the identification of potential inhibitors against other plant and animal pathogenic oomycete infections.
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| 36. |
- Kumari, Poonam, et al.
(författare)
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Plants and endophytes interaction : a “secret wedlock” for sustainable biosynthesis of pharmaceutically important secondary metabolites
- 2023
-
Ingår i: Microbial Cell Factories. - : BioMed Central Ltd. - 1475-2859. ; 22:1
-
Forskningsöversikt (refereegranskat)abstract
- Many plants possess immense pharmacological properties because of the presence of various therapeutic bioactive secondary metabolites that are of great importance in many pharmaceutical industries. Therefore, to strike a balance between meeting industry demands and conserving natural habitats, medicinal plants are being cultivated on a large scale. However, to enhance the yield and simultaneously manage the various pest infestations, agrochemicals are being routinely used that have a detrimental impact on the whole ecosystem, ranging from biodiversity loss to water pollution, soil degradation, nutrient imbalance and enormous health hazards to both consumers and agricultural workers. To address the challenges, biological eco-friendly alternatives are being looked upon with high hopes where endophytes pitch in as key players due to their tight association with the host plants. The intricate interplay between plants and endophytic microorganisms has emerged as a captivating subject of scientific investigation, with profound implications for the sustainable biosynthesis of pharmaceutically important secondary metabolites. This review delves into the hidden world of the "secret wedlock" between plants and endophytes, elucidating their multifaceted interactions that underpin the synthesis of bioactive compounds with medicinal significance in their plant hosts. Here, we briefly review endophytic diversity association with medicinal plants and highlight the potential role of core endomicrobiome. We also propose that successful implementation of in situ microbiome manipulation through high-end techniques can pave the way towards a more sustainable and pharmaceutically enriched future.
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| 37. |
- Leijon, Felicia, et al.
(författare)
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Proteomic Analysis of Plasmodesmata From Populus Cell Suspension Cultures in Relation With Callose Biosynthesis.
- 2018
-
Ingår i: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 9
-
Tidskriftsartikel (refereegranskat)abstract
- Plasmodesmata are channels that link adjacent cells in plant tissues through which molecular exchanges take place. They are involved in multiple processes vital to plant cells, such as responses to hormonal signaling or environmental challenges including osmotic stress, wounding and pathogen attack. Despite the importance of plasmodesmata, their proteome is not well-defined. Here, we have isolated fractions enriched in plasmodesmata from cell suspension cultures of Populus trichocarpa and identified 201 proteins that are enriched in these fractions, thereby providing further insight on the multiple functions of plasmodesmata. Proteomics analysis revealed an enrichment of proteins specifically involved in responses to stress, transport, metabolism and signal transduction. Consistent with the role of callose deposition and turnover in the closure and aperture of the plasmodesmata and our proteomic analysis, we demonstrate the enrichment of callose synthase activity in the plasmodesmata represented by several gene products. A new form of calcium-independent callose synthase activity was detected, in addition to the typical calcium-dependent enzyme activity, suggesting a role of calcium in the regulation of plasmodesmata through two forms of callose synthase activities. Our report provides the first proteomic investigation of the plasmodesmata from a tree species and the direct biochemical evidence for the occurrence of several forms of active callose synthases in these structures. Data are available via ProteomeXchange with identifier PXD010692.
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| 38. |
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| 39. |
- Li, He, D.Sc, 1985-, et al.
(författare)
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Family 92 carbohydrate-binding modules specific for β-1,6-glucans increase the thermostability of a bacterial chitinase
- 2023
-
Ingår i: Biochimie. - : Elsevier BV. - 0300-9084 .- 1638-6183. ; 212, s. 153-160
-
Tidskriftsartikel (refereegranskat)abstract
- In biomass-processing industries there is a need for enzymes that can withstand high temperatures. Extensive research efforts have been dedicated to finding new thermostable enzymes as well as developing new means of stabilising existing enzymes. The attachment of a stable non-catalytic domain to an enzyme can, in some instances, protect a biocatalyst from thermal denaturation. Carbohydrate-binding modules (CBMs) are non-catalytic domains typically found appended to biomass-degrading or modifying enzymes, such as glycoside hydrolases (GHs). Most often, CBMs interact with the same polysaccharide as their enzyme partners, leading to an enhanced reaction rate via the promotion of enzyme-substrate interactions. Contradictory to this general concept, we show an example of a chitin-degrading enzyme from GH family 18 that is appended to two CBM domains from family 92, both of which bind preferentially to the non-substrate polysaccharide β-1,6-glucan. During chitin hydrolysis, the CBMs do not contribute to enzyme-substrate interactions but instead confer a 10–15 °C increase in enzyme thermal stability. We propose that CBM92 domains may have a natural enzyme stabilisation role in some cases, which may be relevant to enzyme design for high-temperature applications in biorefinery.
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| 40. |
- Li, Jing, et al.
(författare)
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Production of Structurally Defined Chito-Oligosaccharides with a Single N-Acetylation at Their Reducing End Using a Newly Discovered Chitinase from Paenibacillus pabuli
- 2021
-
Ingår i: Journal of Agricultural and Food Chemistry. - : American Chemical Society. - 0021-8561 .- 1520-5118. ; 69:11, s. 3371-3379
-
Tidskriftsartikel (refereegranskat)abstract
- Partially acetylated chito-oligosaccharides (paCOSs) are bioactive compounds with potential medical applications. Their biological activities are largely dependent on their structural properties, in particular their degree of polymerization (DP) and the position of the acetyl groups along the glycan chain. The production of structurally defined paCOSs in a purified form is highly desirable to better understand the structure/bioactivity relationship of these oligosaccharides. Here, we describe a newly discovered chitinase from Paenibacillus pabuli (PpChi) and demonstrate by mass spectrometry that it essentially produces paCOSs with a DP of three and four that carry a single N-acetylation at their reducing end. We propose that this specific composition of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) residues, as in GlcN(n)GlcNAc1, is due to a subsite specificity toward GlcN residues at the −2, −3, and −4 positions of the partially acetylated chitosan substrates. In addition, the enzyme is stable, as evidenced by its long shelf life, and active over a large temperature range, which is of high interest for potential use in industrial processes. It exhibits a kcatof 67.2 s–1 on partially acetylated chitosan substrates. When PpChi was used in combination with a recently discovered fungal auxilary activity (AA11) oxidase, a sixfold increase in the release of oligosaccharides from the lobster shell was measured. PpChi represents an attractive biocatalyst for the green production of highly valuable paCOSs with a well-defined structure and the expansion of the relatively small library of chito-oligosaccharides currently available.
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| 41. |
- Malm, Erik, 1981-, et al.
(författare)
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APP : An Automated Proteomics Pipeline for the analysis of mass spectrometry data based on multiple open access tools
- 2014
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Ingår i: BMC Bioinformatics. - : Springer Science and Business Media LLC. - 1471-2105. ; 15:1
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Mass spectrometry analyses of complex protein samples yield large amounts of data and specific expertise is needed for data analysis, in addition to a dedicated computer infrastructure. Furthermore, the identification of proteins and their specific properties require the use of multiple independent bioinformatics tools and several database search algorithms to process the same datasets. In order to facilitate and increase the speed of data analysis, there is a need for an integrated platform that would allow a comprehensive profiling of thousands of peptides and proteins in a single process through the simultaneous exploitation of multiple complementary algorithms. Results: We have established a new proteomics pipeline designated as APP that fulfills these objectives using a complete series of tools freely available from open sources. APP automates the processing of proteomics tasks such as peptide identification, validation and quantitation from LC-MS/MS data and allows easy integration of many separate proteomics tools. Distributed processing is at the core of APP, allowing the processing of very large datasets using any combination of Windows/Linux physical or virtual computing resources. Conclusions: APP provides distributed computing nodes that are simple to set up, greatly relieving the need for separate IT competence when handling large datasets. The modular nature of APP allows complex workflows to be managed and distributed, speeding up throughput and setup. Additionally, APP logs execution information on all executed tasks and generated results, simplifying information management and validation.
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| 42. |
- Marczynski, Matthias, et al.
(författare)
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Structural Alterations of Mucins Are Associated with Losses in Functionality
- 2021
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Ingår i: Biomacromolecules. - : AMER CHEMICAL SOC. - 1525-7797 .- 1526-4602. ; 22:4, s. 1600-1613
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Tidskriftsartikel (refereegranskat)abstract
- Commercial mucin glycoproteins are routinely used as a model to investigate the broad range of important functions mucins fulfill in our bodies, including lubrication, protection against hostile germs, and the accommodation of a healthy microbiome. Moreover, purified mucins are increasingly selected as building blocks for multifunctional materials, i.e., as components of hydrogels or coatings. By performing a detailed side-by-side comparison of commercially available and lab-purified variants of porcine gastric mucins, we decipher key molecular motifs that are crucial for mucin functionality. As two main structural features, we identify the hydrophobic termini and the hydrophilic glycosylation pattern of the mucin glycoprotein; moreover, we describe how alterations in those structural motifs affect the different properties of mucins-on both microscopic and macroscopic levels. This study provides a detailed understanding of how distinct functionalities of gastric mucins are established, and it highlights the need for high-quality mucins-for both basic research and the development of mucin-based medical products.
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| 43. |
- Meghwanshi, Gautam Kumar, et al.
(författare)
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Archaeal lipolytic enzymes : Current developments and further prospects
- 2022
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Ingår i: Biotechnology Advances. - : Elsevier BV. - 0734-9750 .- 1873-1899. ; 61
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Forskningsöversikt (refereegranskat)abstract
- Lipolytic enzymes include triacylglycerol lipases (EC 3.1.1.3) and esterases (EC 3.1.1.1) that catalyze the cleavage and formation of ester bonds. They are potential industrial biocatalysts because of their broad range of activities on natural and synthetic substrates, high stability in organic solvents, thermal stability, stability in highly acidic and alkaline pH conditions and enantio-, regio-and chemo-selectivity. They also have varied ap-plications in different sectors, among which industrial biotechnology, the production of cleaning agents, and pharmaceuticals are the most important ones. Identifying extremophilic lipolytic enzymes is of paramount in-terest and is a growing field in academic and industrial research. This review is focused on the current knowledge and future avenues of investigation on lipolytic enzymes sourced from the underexploited archaeal domain. Archaea is a potential source for novel extremophilic enzymes, which have high demand in the industries. The archaeal lipases and esterases are clustered into different families based on their similarity/dissimilarity at the genetic level and protein structures. The updated information on characterized and putative lipase sequences has also been presented in this paper. Common structural scaffolds of archaeal lipases have been deduced and dis-cussed in this review. However, huge diversity at the level of their genetic sequences has yet to be correlated with the structure-function relationship. Based on their biochemical properties, possible applications and future prospective of archaeal lipolytic enzymes have also been proposed.
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| 44. |
- Mishra, A., et al.
(författare)
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Progress in paper-based analytical devices for climate neutral biosensing
- 2022
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Ingår i: Biosensors and Bioelectronics: X. - : Elsevier BV. - 2590-1370. ; 11, s. 100166-
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Tidskriftsartikel (refereegranskat)abstract
- Disposable diagnostics is ramping up the development of green practices for healthcare. Paper is an excellent natural substrate for emerging low-cost, ecologically acceptable analytical devices. Flexibility, three-dimensional fibre structure, mechanical characteristics, ease of manufacturing and customization are the key advantages of cellulose as preferable materials for green analytical devices. This article overviews the advances in paper-based optical and electroanalytical devices, including manufacturing, validation of functions, detection methodology, and applications. The renewable resources hold the potential to accomplish the biodegradable analytical techniques due to their climate neutral waste management. The paper-based electrodes have demonstrated excellent performance for diagnostics while being eco-friendly at the same time. Therefore, the forthcoming sustainable diagnostics approaches require integrating paper-based analytical devices (PADs) built out of disposable electrodes. The diverse green materials and methods facilitate climate neural point-of-care products for the net-zero market. Numerous researches have been conducted in recent years to develop innovative technologies capable of analysing clinical samples quickly, accurately, simply, and sensitively, with the primary objective of offering competent tools for illness diagnosis and treatment efficacy follow-up. The micro-PADs show excellent abilities for quick onsite diagnostics within μ-level detection range. The sensitivity, stability, and reliability of analytical devices are the essential factors enabling larger-scale production and commercialization. The projected fabrication and analytical methods in the direction of paper-based devices could be nicely and reliably utilized for understanding and adaptation of climate neutral diagnostic technologies in healthcare sector. This detailed article discusses the sustainable framework that integrates green bioelectronics and sensor technologies adhering to diverse functionality for health and the environment. The paper-based digital technology (PDigiT) could be employed as a simple, cost-effective, and efficient net-zero technology for real-time monitoring of outbreaks and bio-surveillance.
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| 45. |
- Natarajan Arul, Murugan, et al.
(författare)
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Computational investigation of the increased virulence and pathogenesis of SARS-CoV-2 lineage B.1.1.7
- 2022
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Ingår i: Physical Chemistry, Chemical Physics - PCCP. - : Royal Society of Chemistry (RSC). - 1463-9076 .- 1463-9084. ; 24:34, s. 20371-20380
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Tidskriftsartikel (refereegranskat)abstract
- New variants of SARS-CoV-2 are being reported worldwide. The World Health Organization has reported Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529) as the variants of concern. There are speculations that the variants might evade the host immune responses induced by currently available vaccines and develop resistance to drugs under consideration. The first step of viral infection in COVID-19 occurs through the interaction of the spike protein's receptor-binding domain (RBD) with the peptidase domain of the human ACE-2 (hACE-2) receptor. This study aims to get a molecular-level understanding of the mechanism behind the increased infection rate in the alpha variant. We have computationally studied the spike protein interaction in both the wild-type and B.1.1.7 variant with the hACE-2 receptor using molecular dynamics and MM-GBSA based binding free energy calculations. The binding free energy difference shows that the mutant variant of the spike protein has increased binding affinity for the hACE-2 receptor (i.e. Delta G(N501Y,A570D) is in the range -7.2 to -7.6 kcal mol(-1)) and the results were validated using Density functional theory. We demonstrate that with the use of state-of-the-art computational approaches, we can, in advance, predict the virulent nature of variants of SARS-CoV-2 and alert the world healthcare system.
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| 46. |
- Natarajan Arul, Murugan, et al.
(författare)
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Searching for target-specific and multi-targeting organics for Covid-19 in the Drugbank database with a double scoring approach
- 2020
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Ingår i: Scientific Reports. - : Nature Research. - 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- The current outbreak of Covid-19 infection due to SARS-CoV-2, a virus from the coronavirus family, has become a major threat to human healthcare. The virus has already infected more than 44 M people and the number of deaths reported has reached more than 1.1 M which may be attributed to lack of medicine. The traditional drug discovery approach involves many years of rigorous research and development and demands for a huge investment which cannot be adopted for the ongoing pandemic infection. Rather we need a swift and cost-effective approach to inhibit and control the viral infection. With the help of computational screening approaches and by choosing appropriate chemical space, it is possible to identify lead drug-like compounds for Covid-19. In this study, we have used the Drugbank database to screen compounds against the most important viral targets namely 3C-like protease (3CLpro), papain-like protease (PLpro), RNA-dependent RNA polymerase (RdRp) and the spike (S) protein. These targets play a major role in the replication/transcription and host cell recognition, therefore, are vital for the viral reproduction and spread of infection. As the structure based computational screening approaches are more reliable, we used the crystal structures for 3C-like main protease and spike protein. For the remaining targets, we used the structures based on homology modeling. Further, we employed two scoring methods based on binding free energies implemented in AutoDock Vina and molecular mechanics-generalized Born surface area approach. Based on these results, we propose drug cocktails active against the three viral targets namely 3CLpro, PLpro and RdRp. Interestingly, one of the identified compounds in this study i.e. Baloxavir marboxil has been under clinical trial for the treatment of Covid-19 infection. In addition, we have identified a few compounds such as Phthalocyanine, Tadalafil, Lonafarnib, Nilotinib, Dihydroergotamine, R-428 which can bind to all three targets simultaneously and can serve as multi-targeting drugs. Our study also included calculation of binding energies for various compounds currently under drug trials. Among these compounds, it is found that Remdesivir binds to targets, 3CLpro and RdRp with high binding affinity. Moreover, Baricitinib and Umifenovir were found to have superior target-specific binding while Darunavir is found to be a potential multi-targeting drug. As far as we know this is the first study where the compounds from the Drugbank database are screened against four vital targets of SARS-CoV-2 and illustrates that the computational screening using a double scoring approach can yield potential drug-like compounds against Covid-19 infection.
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| 47. |
- Pang, Zhili, et al.
(författare)
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Analysis of a cellulose synthase catalytic subunit from the oomycete pathogen of crops Phytophthora capsici
- 2020
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Ingår i: Cellulose. - : Springer Science and Business Media B.V.. - 0969-0239 .- 1572-882X. ; 27:15, s. 8551-8565
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Tidskriftsartikel (refereegranskat)abstract
- Phytophthora capsici Leonian is an important oomycete pathogen of crop vegetables, causing significant economic losses each year. Its cell wall, rich in cellulose, is vital for cellular integrity and for interactions with the host organisms. Predicted cellulose synthase (CesA) proteins are expected to catalyze the polymerization of cellulose, but this has not been biochemically demonstrated in an oomycete. Here, we present the properties of the four newly identified CesA proteins from P. capsici and compare their domain organization with that of CesAs from other lineages. Using a newly constructed glucosyltransferase-deficient variant of Saccharomyces cerevisiae with low residual background activity, we have achieved successful heterologous expression and biochemical characterization of a CesA protein from P. capsici (PcCesA1). Our results demonstrate that the individual PcCesA1 enzyme produces cellobiose as the major reaction product. Co-immunoprecipitation studies and activity assays revealed that several PcCesA proteins interact together to form a complex whose multiproteic nature is most likely required for cellulose microfibril formation. In addition to providing important insights into cellulose synthesis in the oomycetes, our data may assist the longer term identification of cell wall biosynthesis inhibitors to control infection by pathogenic oomycetes.
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| 48. |
- Pang, Zhili, et al.
(författare)
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Quantitative proteomics links metabolic pathways to specific developmental stages of the plant-pathogenic oomycete Phytophthora capsici
- 2016
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Ingår i: Molecular plant pathology. - : John Wiley & Sons. - 1464-6722 .- 1364-3703.
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Tidskriftsartikel (refereegranskat)abstract
- The oomycete Phytophthora capsici is a plant pathogen responsible for important losses to vegetable production worldwide. Its asexual reproduction plays an important role in the rapid propagation and spread of the disease in the field. A global proteomics study was conducted to compare two key asexual life stages of P. capsici, i.e. the mycelium and cysts, to identify stage-specific biochemical processes. A total of 1200 proteins was identified using qualitative and quantitative proteomics. The transcript abundance of some of the enriched proteins was also analysed by quantitative real-time polymerase chain reaction. Seventy-three proteins exhibited different levels of abundance between the mycelium and cysts. The proteins enriched in the mycelium are mainly associated with glycolysis, the tricarboxylic acid (or citric acid) cycle and the pentose phosphate pathway, providing the energy required for the biosynthesis of cellular building blocks and hyphal growth. In contrast, the proteins that are predominant in cysts are essentially involved in fatty acid degradation, suggesting that the early infection stage of the pathogen relies primarily on fatty acid degradation for energy production. The data provide a better understanding of P. capsici biology and suggest potential metabolic targets at the two different developmental stages for disease control.
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| 49. |
- Pavankumar, Asalapuram R., et al.
(författare)
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Dimerization of a flocculent protein from Moringa oleifera : experimental evidence and in silico interpretation
- 2014
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Ingår i: Journal of Biomolecular Structure and Dynamics. - : Informa UK Limited. - 0739-1102 .- 1538-0254. ; 32:3, s. 406-415
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Tidskriftsartikel (refereegranskat)abstract
- Many proteins exist in dimeric and other oligomeric forms to gain stability and functional advantages. In this study, the dimerization property of a coagulant protein (MO2.1) from Moringa oleifera seeds was addressed through laboratory experiments, protein-protein docking studies and binding free energy calculations. The structure of MO2.1 was predicted by homology modelling, while binding free energy and residues-distance profile analyses provided insight into the energetics and structural factors for dimer formation. Since the coagulation activities of the monomeric and dimeric forms of MO2.1 were comparable, it was concluded that oligomerization does not affect the biological activity of the protein.
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| 50. |
- Pham, Trang A.T., et al.
(författare)
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Analysis of cell wall synthesis and metabolism during early germination of Blumeria graminis f. sp. hordei conidial cells induced in vitro
- 2019
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Ingår i: The Cell Surface. - : Elsevier BV. - 2468-2330. ; 5, s. 100030-
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Tidskriftsartikel (refereegranskat)abstract
- As an obligate biotroph, Blumeria graminis f. sp. hordei (Bgh) cannot be grown in an axenic culture, and instead must be cultivated on its host species, Hordeum vulgare (barley). In this study an in vitro system utilizing n-hexacosanal, a constituent of the barley cuticle and known inducer of Bgh germination, was used to cultivate Bgh and differentiate conidia up to the appressorial germ tube stage for analysis. Transcriptomic and proteomic profiling of the appressorial germ tube stage revealed that there was a significant shift towards energy and protein production during the pre-penetrative phase of development, with an up-regulation of enzymes associated with cellular respiration and protein synthesis, modification and transport. Glycosidic linkage analysis of the cell wall polysaccharides demonstrated that during appressorial development an increase in 1,3- and 1,4-linked glucosyl residues and xylosyl residues was detected along with a significant decrease in galactosyl residues. The use of this in vitro cultivation method demonstrates that it is possible to analyse the pre-penetrative processes of Bgh development in the absence of a plant host.
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