Current Articles
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2023,
5(4):
431-434.
doi: 10.1007/s42995-023-00208-8
Abstract:
Abstract:
The ascidian Styela clava is an ecologically important species that is distributed along coastal regions worldwide. It has a long history as a model animal for evolutionary and developmental biology research owing to its phylogenetic position between vertebrates and invertebrates, and its classical mosaic expression patterns. However, the standard developmental atlas and protocols and tools for molecular manipulation of this organism are inadequate. In this study, we established a standard developmental table and provided a web-based digital image resource for S. clava embryogenesis at each developmental stage from fertilized eggs to hatching larvae by utilizing confocal laser microscopy and 3D reconstruction images. It takes around 10 h for fertilized eggs to develop into swimming larvae and 20–30 min to complete the tail regression processes at the metamorphic stage. We observed that the notochord cells in S. clava embryos did not produce an extracellular lumen like Ciona robusta, but showed polarized elongation behaviors, providing us an ideal comparative model to study tissue morphogenesis. In addition, we established a chemical-washing procedure to remove the chorion easily from the fertilized eggs. Based on the dechorionation technique, we further realized transgenic manipulation by electroporation and successfully applied tissue-specific fluorescent labeling in S. clava embryos. Our work provides a standard imaging atlas and powerful genetic tools for investigating embryogenesis and evolution using S. clava as a model organism.
The ascidian Styela clava is an ecologically important species that is distributed along coastal regions worldwide. It has a long history as a model animal for evolutionary and developmental biology research owing to its phylogenetic position between vertebrates and invertebrates, and its classical mosaic expression patterns. However, the standard developmental atlas and protocols and tools for molecular manipulation of this organism are inadequate. In this study, we established a standard developmental table and provided a web-based digital image resource for S. clava embryogenesis at each developmental stage from fertilized eggs to hatching larvae by utilizing confocal laser microscopy and 3D reconstruction images. It takes around 10 h for fertilized eggs to develop into swimming larvae and 20–30 min to complete the tail regression processes at the metamorphic stage. We observed that the notochord cells in S. clava embryos did not produce an extracellular lumen like Ciona robusta, but showed polarized elongation behaviors, providing us an ideal comparative model to study tissue morphogenesis. In addition, we established a chemical-washing procedure to remove the chorion easily from the fertilized eggs. Based on the dechorionation technique, we further realized transgenic manipulation by electroporation and successfully applied tissue-specific fluorescent labeling in S. clava embryos. Our work provides a standard imaging atlas and powerful genetic tools for investigating embryogenesis and evolution using S. clava as a model organism.
Abstract:
The D-quadrant organizer sets up the dorsal–ventral (DV) axis and regulates mesodermal development of spiralians. Studies have revealed an important role of mitogen-activated protein kinase (MAPK) signaling in organizer function, but the related molecules have not been fully revealed. The association between fibroblast growth factor receptor (FGFR) and MAPK signaling in regulating organizer specification has been established in the annelid Owenia fusiformis. Now, comparable studies in other spiralian phyla are required to decipher whether this organizer-inducing function of FGFR is prevalent in Spiralia. Here, we indicate that treatment with the FGFR inhibitor SU5402 resulted in deficiency of organizer specification in the mollusk Lottia peitaihoensis. Subsequently, the bone morphogenetic protein (BMP) signaling gradient and DV patterning were disrupted, suggesting the roles of FGFR in regulating organizer function. Changes in multiple aspects of organizer function (the morphology of vegetal blastomeres, BMP signaling gradient, expression of DV patterning markers, etc.) indicate that these developmental functions have different sensitivities to FGFR/MAPK signaling. Our results reveal a functional role of FGFR in organizer specification as well as DV patterning of Lottia embryos, which expands our knowledge of spiralian organizers.
The D-quadrant organizer sets up the dorsal–ventral (DV) axis and regulates mesodermal development of spiralians. Studies have revealed an important role of mitogen-activated protein kinase (MAPK) signaling in organizer function, but the related molecules have not been fully revealed. The association between fibroblast growth factor receptor (FGFR) and MAPK signaling in regulating organizer specification has been established in the annelid Owenia fusiformis. Now, comparable studies in other spiralian phyla are required to decipher whether this organizer-inducing function of FGFR is prevalent in Spiralia. Here, we indicate that treatment with the FGFR inhibitor SU5402 resulted in deficiency of organizer specification in the mollusk Lottia peitaihoensis. Subsequently, the bone morphogenetic protein (BMP) signaling gradient and DV patterning were disrupted, suggesting the roles of FGFR in regulating organizer function. Changes in multiple aspects of organizer function (the morphology of vegetal blastomeres, BMP signaling gradient, expression of DV patterning markers, etc.) indicate that these developmental functions have different sensitivities to FGFR/MAPK signaling. Our results reveal a functional role of FGFR in organizer specification as well as DV patterning of Lottia embryos, which expands our knowledge of spiralian organizers.
Abstract:
Many marine invertebrate phyla are characterized by indirect development. These animals transit from planktonic larvae to benthic spats via settlement and metamorphosis, which contributes to their adaption to the marine environment. Studying the biological process of metamorphosis is, thus, key to understanding the origin and evolution of indirect development. Although numerous studies have been conducted on the relationship between metamorphosis and the marine environment, microorganisms, and neurohormones, little is known about gene regulation network (GRN) dynamics during metamorphosis. Metamorphosis-competent pediveligers of the Pacific oyster Crassostrea gigas were assayed in this study. By assaying gene expression patterns and open chromatin region changes of different samples of larvae and spats, the dynamics of molecular regulation during metamorphosis were examined. The results indicated significantly different gene regulation networks before, during and post-metamorphosis. Genes encoding membrane-integrated receptors and those related to the remodeling of the nervous system were upregulated before the initiation of metamorphosis. Massive biogenesis, e.g., of various enzymes and structural proteins, occurred during metamorphosis as inferred from the comprehensive upregulation of the protein synthesis system post epinephrine stimulation. Hierarchical downstream gene networks were then stimulated. Some transcription factors, including homeobox, basic helix–loop–helix and nuclear receptors, showed different temporal response patterns, suggesting a complex GRN during the transition stage. Nuclear receptors, as well as their retinoid X receptor partner, may participate in the GRN controlling oyster metamorphosis, indicating an ancient role of the nuclear receptor regulation system in animal metamorphosis.
Many marine invertebrate phyla are characterized by indirect development. These animals transit from planktonic larvae to benthic spats via settlement and metamorphosis, which contributes to their adaption to the marine environment. Studying the biological process of metamorphosis is, thus, key to understanding the origin and evolution of indirect development. Although numerous studies have been conducted on the relationship between metamorphosis and the marine environment, microorganisms, and neurohormones, little is known about gene regulation network (GRN) dynamics during metamorphosis. Metamorphosis-competent pediveligers of the Pacific oyster Crassostrea gigas were assayed in this study. By assaying gene expression patterns and open chromatin region changes of different samples of larvae and spats, the dynamics of molecular regulation during metamorphosis were examined. The results indicated significantly different gene regulation networks before, during and post-metamorphosis. Genes encoding membrane-integrated receptors and those related to the remodeling of the nervous system were upregulated before the initiation of metamorphosis. Massive biogenesis, e.g., of various enzymes and structural proteins, occurred during metamorphosis as inferred from the comprehensive upregulation of the protein synthesis system post epinephrine stimulation. Hierarchical downstream gene networks were then stimulated. Some transcription factors, including homeobox, basic helix–loop–helix and nuclear receptors, showed different temporal response patterns, suggesting a complex GRN during the transition stage. Nuclear receptors, as well as their retinoid X receptor partner, may participate in the GRN controlling oyster metamorphosis, indicating an ancient role of the nuclear receptor regulation system in animal metamorphosis.
Abstract:
The evolution of a two-chambered heart, with an atrium and a ventricle, has improved heart function in both deuterostomes (vertebrates) and some protostomes (invertebrates). Although studies have examined the unique structure and function of these two chambers, molecular comparisons are few and limited to vertebrates. Here, we focus on the two-chambered protostome heart of the mollusks, offering data that may provide a better understanding of heart evolution. Specifically, we asked if the atrium and ventricle differ at the molecular level in the mollusk heart. To do so, we examined two very different species, the giant African land snail (Lissachatina fulica) and the relatively small, aquatic yesso scallop (Mizuhopecten yessoensis), with the assumption that if they exhibited commonality these similarities would likely reflect those across the phylum. We found that, although the hearts of these two species differed histologically, their cardiac gene function enrichments were similar, as revealed by transcriptomic analysis. Furthermore, the atrium and ventricle in each species had distinct gene function clusters, suggesting an evolutionary differentiation of cardiac chambers in mollusks. Finally, to explore the relationship between vertebrate and invertebrate two-chambered hearts, we compared our transcriptomic data with published data from the zebrafish, a well-studied vertebrate model with a two-chambered heart. Our analysis indicated a functional similarity of ventricular genes between the mollusks and the zebrafish, suggesting that the ventricle was differentiated to achieve the same functions in invertebrates and vertebrates. As the first such study on protostomes, our findings offered initial insights into how the two-chambered heart arose, including a possible understanding of its occurrence in both protostomes and deuterostomes.
The evolution of a two-chambered heart, with an atrium and a ventricle, has improved heart function in both deuterostomes (vertebrates) and some protostomes (invertebrates). Although studies have examined the unique structure and function of these two chambers, molecular comparisons are few and limited to vertebrates. Here, we focus on the two-chambered protostome heart of the mollusks, offering data that may provide a better understanding of heart evolution. Specifically, we asked if the atrium and ventricle differ at the molecular level in the mollusk heart. To do so, we examined two very different species, the giant African land snail (Lissachatina fulica) and the relatively small, aquatic yesso scallop (Mizuhopecten yessoensis), with the assumption that if they exhibited commonality these similarities would likely reflect those across the phylum. We found that, although the hearts of these two species differed histologically, their cardiac gene function enrichments were similar, as revealed by transcriptomic analysis. Furthermore, the atrium and ventricle in each species had distinct gene function clusters, suggesting an evolutionary differentiation of cardiac chambers in mollusks. Finally, to explore the relationship between vertebrate and invertebrate two-chambered hearts, we compared our transcriptomic data with published data from the zebrafish, a well-studied vertebrate model with a two-chambered heart. Our analysis indicated a functional similarity of ventricular genes between the mollusks and the zebrafish, suggesting that the ventricle was differentiated to achieve the same functions in invertebrates and vertebrates. As the first such study on protostomes, our findings offered initial insights into how the two-chambered heart arose, including a possible understanding of its occurrence in both protostomes and deuterostomes.
Abstract:
The Gli transcription factors are the primary mediators of Hedgehog (Hh) signaling. Vertebrate genomes contain multiple Gli paralogues with different functions downstream of Hh signal receipt, in part explaining the complexity of cellular responses to Hh that allow concentration-dependent target gene activation. Amphioxus is a chordate that split from the vertebrate lineage early in the evolution of chordates, before the genome duplications that occurred in early vertebrate evolution. It has a single Gli gene whose transcripts can be alternately spliced to yield two protein isoforms called GliS and GliL. We generated two knockout mutations in amphioxus Gli, one that affects the whole gene and a second that only affects GliL. Both knockouts showed major morphological and molecular defects in the development of left–right asymmetry, a phenotype that is similar but not identical to that previously found in Hh mutants. Hh signaling also patterns the amphioxus neural tube. Here, however, knockout of GliL showed no identifiable phenotype, while knockout of the full gene showed only small changes to the expression of one gene family, Olig. Other genes that were prominently affected by Hh knockout were not altered in expression in either knockout. Reasons for the differences between Hh and Gli knockouts in the pharynx and neural tube are discussed in the context of the likely different functions of amphioxus Gli isoforms.
The Gli transcription factors are the primary mediators of Hedgehog (Hh) signaling. Vertebrate genomes contain multiple Gli paralogues with different functions downstream of Hh signal receipt, in part explaining the complexity of cellular responses to Hh that allow concentration-dependent target gene activation. Amphioxus is a chordate that split from the vertebrate lineage early in the evolution of chordates, before the genome duplications that occurred in early vertebrate evolution. It has a single Gli gene whose transcripts can be alternately spliced to yield two protein isoforms called GliS and GliL. We generated two knockout mutations in amphioxus Gli, one that affects the whole gene and a second that only affects GliL. Both knockouts showed major morphological and molecular defects in the development of left–right asymmetry, a phenotype that is similar but not identical to that previously found in Hh mutants. Hh signaling also patterns the amphioxus neural tube. Here, however, knockout of GliL showed no identifiable phenotype, while knockout of the full gene showed only small changes to the expression of one gene family, Olig. Other genes that were prominently affected by Hh knockout were not altered in expression in either knockout. Reasons for the differences between Hh and Gli knockouts in the pharynx and neural tube are discussed in the context of the likely different functions of amphioxus Gli isoforms.
Abstract:
Vertebrates have developed various modes of reproduction, some of which are found in Teleosts. Over 300 species of the Syngnathidae (seahorses, pipefishes and seadragons) exhibit male pregnancies; the males have specialized brood pouches that provide immune protection, nourishment, and oxygen regulation. Chemokines play a vital role at the mammalian maternal–fetal interface; however, their functions in fish reproduction are unclear. This study revealed the evolutionary traits and potential functions of chemokine genes in 22 oviparous, ovoviviparous, and viviparous fish species through comparative genomic analyses. Our results showed that chemokine gene copy numbers and evolutionary rates vary among species with different modes of reproduction. Syngnathidae lost cxcl13 and cxcr5, which are involved in key receptor–ligand pairs for lymphoid organ development. Notably, Syngnathidae have site-specific mutations in cxcl12b and ccl44, suggesting immune function during gestation. Moreover, transcriptome analysis revealed that chemokine gene expression varies among Syngnathidae species with different types of brood pouches, suggesting adaptive variations in chemokine functions among seahorses and their relatives. Furthermore, challenge experiments on seahorse brood pouches revealed a joint immune function of chemokine genes during male pregnancy. This study provides insights into the evolutionary diversity of chemokine genes associated with different reproductive modes in fish.
Vertebrates have developed various modes of reproduction, some of which are found in Teleosts. Over 300 species of the Syngnathidae (seahorses, pipefishes and seadragons) exhibit male pregnancies; the males have specialized brood pouches that provide immune protection, nourishment, and oxygen regulation. Chemokines play a vital role at the mammalian maternal–fetal interface; however, their functions in fish reproduction are unclear. This study revealed the evolutionary traits and potential functions of chemokine genes in 22 oviparous, ovoviviparous, and viviparous fish species through comparative genomic analyses. Our results showed that chemokine gene copy numbers and evolutionary rates vary among species with different modes of reproduction. Syngnathidae lost cxcl13 and cxcr5, which are involved in key receptor–ligand pairs for lymphoid organ development. Notably, Syngnathidae have site-specific mutations in cxcl12b and ccl44, suggesting immune function during gestation. Moreover, transcriptome analysis revealed that chemokine gene expression varies among Syngnathidae species with different types of brood pouches, suggesting adaptive variations in chemokine functions among seahorses and their relatives. Furthermore, challenge experiments on seahorse brood pouches revealed a joint immune function of chemokine genes during male pregnancy. This study provides insights into the evolutionary diversity of chemokine genes associated with different reproductive modes in fish.
Abstract:
Psychrophilic microalgae successfully survive in the extreme and highly variable polar ecosystems, which represent the energy base of most food webs and play a fundamental role in nutrient cycling. The success of microalgae is rooted in their adaptive evolution. Revealing how they have evolved to thrive in extreme polar environments will help us better understand the origin of life in polar ecosystems. We isolated a psychrophilic unicellular green alga, Microglena sp. YARC, from Antarctic sea ice which has a huge genome. Therefore, we predicted that gene replication may play an important role in its polar adaptive evolution. We found that its protein-coding gene number significantly increased and the duplication time was dated between 37 and 48 million years ago, which is consistent with the formation of the circumpolar Southern Ocean. Most duplicated paralogous genes were enriched in pathways related to photosynthesis, DNA repair, and fatty acid metabolism. Moreover, there were a total of 657 Microglena-specific families, including collagen-like proteins. The divergence in the expression patterns of the duplicated and species-specific genes reflects sub- and neo-functionalization during stress acclimation. Overall, key findings from this study provide new information on how gene duplication and their functional novelty contributed to polar algae adaptation to the highly variable polar environmental conditions.
Psychrophilic microalgae successfully survive in the extreme and highly variable polar ecosystems, which represent the energy base of most food webs and play a fundamental role in nutrient cycling. The success of microalgae is rooted in their adaptive evolution. Revealing how they have evolved to thrive in extreme polar environments will help us better understand the origin of life in polar ecosystems. We isolated a psychrophilic unicellular green alga, Microglena sp. YARC, from Antarctic sea ice which has a huge genome. Therefore, we predicted that gene replication may play an important role in its polar adaptive evolution. We found that its protein-coding gene number significantly increased and the duplication time was dated between 37 and 48 million years ago, which is consistent with the formation of the circumpolar Southern Ocean. Most duplicated paralogous genes were enriched in pathways related to photosynthesis, DNA repair, and fatty acid metabolism. Moreover, there were a total of 657 Microglena-specific families, including collagen-like proteins. The divergence in the expression patterns of the duplicated and species-specific genes reflects sub- and neo-functionalization during stress acclimation. Overall, key findings from this study provide new information on how gene duplication and their functional novelty contributed to polar algae adaptation to the highly variable polar environmental conditions.
Abstract:
Mollusca is the second-largest animal phylum with over 100, 000 extant species representing eight classes. Across 1000 extant species in the class Polyplacophora, chitons have a relatively constrained morphology but with some notable deviations. Several genera possess "shell eyes", i.e., true eyes with a lens and retina that are embedded within the dorsal shells. The phylogeny of the major chiton clades is mostly well established, in a set of superfamily-level and higher level taxa supported by various approaches, including morphological studies, multiple gene markers, mitogenome-phylogeny, and phylotranscriptomic approaches. However, one critical lineage has remained unclear, namely Schizochiton which was controversially suggested as being the potential independent origin of chiton shell eyes. Here, with the draft genome sequencing of Schizochiton incisus (superfamily Schizochitonoidea) plus assemblies of transcriptome data from other polyplacophorans, we present phylogenetic reconstructions using both mitochondrial genomes and phylogenomic approaches with multiple methods. We found that phylogenetic trees from mitogenomic data are inconsistent, reflecting larger scale confounding factors in molluscan mitogenomes. However, a consistent and robust topology was generated with protein-coding genes using different models and methods. Our results support Schizochitonoidea as the sister group to other Chitonoidea in Chitonina, in agreement with the established classification. Combined with evidence from fossils, our phylogenetic results suggest that the earliest origin of shell eyes is in Schizochitonoidea, and that these structures were also gained secondarily in other genera in Chitonoidea. Our results have generated a holistic review of the internal relationship within Polyplacophora, and a better understanding of the evolution of Polyplacophora.
Mollusca is the second-largest animal phylum with over 100, 000 extant species representing eight classes. Across 1000 extant species in the class Polyplacophora, chitons have a relatively constrained morphology but with some notable deviations. Several genera possess "shell eyes", i.e., true eyes with a lens and retina that are embedded within the dorsal shells. The phylogeny of the major chiton clades is mostly well established, in a set of superfamily-level and higher level taxa supported by various approaches, including morphological studies, multiple gene markers, mitogenome-phylogeny, and phylotranscriptomic approaches. However, one critical lineage has remained unclear, namely Schizochiton which was controversially suggested as being the potential independent origin of chiton shell eyes. Here, with the draft genome sequencing of Schizochiton incisus (superfamily Schizochitonoidea) plus assemblies of transcriptome data from other polyplacophorans, we present phylogenetic reconstructions using both mitochondrial genomes and phylogenomic approaches with multiple methods. We found that phylogenetic trees from mitogenomic data are inconsistent, reflecting larger scale confounding factors in molluscan mitogenomes. However, a consistent and robust topology was generated with protein-coding genes using different models and methods. Our results support Schizochitonoidea as the sister group to other Chitonoidea in Chitonina, in agreement with the established classification. Combined with evidence from fossils, our phylogenetic results suggest that the earliest origin of shell eyes is in Schizochitonoidea, and that these structures were also gained secondarily in other genera in Chitonoidea. Our results have generated a holistic review of the internal relationship within Polyplacophora, and a better understanding of the evolution of Polyplacophora.
Abstract:
Antibiotic-resistant bacteria severely threaten human health. Besides spontaneous mutations generated by endogenous factors, the resistance might also originate from mutations induced by certain antibiotics, such as the fluoroquinolones. Such antibiotics increase the genome-wide mutation rate by introducing replication errors from the SOS response pathway or decreasing the efficiency of the DNA repair systems. However, the relative contributions of these molecular mechanisms remain unclear, hindering understanding of the generation of resistant pathogens. Here, using newly-accumulated mutations of wild-type and SOS-uninducible Escherichia coli strains, as well as those of the strains deficient for the mismatch repair (MMR) and the oxidative damage repair pathways, we find that the SOS response is the major mutagenesis contributor in mutation elevation, responsible for ~ 30–50% of the total base-pair substitution (BPS) mutation-rate elevation upon treatment with sublethal levels of norfloxacin (0 ~ 50 ng/mL). We further estimate the significance of the effects on other mutational features of these mechanisms (i.e., transversions, structural variations, and mutation spectrum) in E. coli using linear models. The SOS response plays a positive role in all three mutational features (mutation rates of BPSs, transversions, structural variations) and affects the mutational spectrum. The repair systems significantly reduce the BPS mutation rate and the transversion rate, regardless of whether antibiotics are present, while significantly increasing the structural variation rate in E. coli. Our results quantitatively disentangle the contributions of the SOS response and DNA repair systems in antibiotic-induced mutagenesis.
Antibiotic-resistant bacteria severely threaten human health. Besides spontaneous mutations generated by endogenous factors, the resistance might also originate from mutations induced by certain antibiotics, such as the fluoroquinolones. Such antibiotics increase the genome-wide mutation rate by introducing replication errors from the SOS response pathway or decreasing the efficiency of the DNA repair systems. However, the relative contributions of these molecular mechanisms remain unclear, hindering understanding of the generation of resistant pathogens. Here, using newly-accumulated mutations of wild-type and SOS-uninducible Escherichia coli strains, as well as those of the strains deficient for the mismatch repair (MMR) and the oxidative damage repair pathways, we find that the SOS response is the major mutagenesis contributor in mutation elevation, responsible for ~ 30–50% of the total base-pair substitution (BPS) mutation-rate elevation upon treatment with sublethal levels of norfloxacin (0 ~ 50 ng/mL). We further estimate the significance of the effects on other mutational features of these mechanisms (i.e., transversions, structural variations, and mutation spectrum) in E. coli using linear models. The SOS response plays a positive role in all three mutational features (mutation rates of BPSs, transversions, structural variations) and affects the mutational spectrum. The repair systems significantly reduce the BPS mutation rate and the transversion rate, regardless of whether antibiotics are present, while significantly increasing the structural variation rate in E. coli. Our results quantitatively disentangle the contributions of the SOS response and DNA repair systems in antibiotic-induced mutagenesis.
2023,
5(4):
551-563.
doi: 10.1007/s42995-023-00191-0
Abstract:
Currently, new species of freshwater fish trypanosomes, which are economically important parasites, are being described based on subjectively selected features, i.e., their cell morphology and the host species. We have performed detailed phylogenetic and haplotype diversity analyses of all 18S rRNA genes available for freshwater fish trypanosomes, including the newly obtained sequences of Trypanosoma carassii and Trypanosoma danilewskyi. Based on a sequence similarity of 99.5%, we divide these trypanosomes into 15 operational taxonomic units, and propose three nominal scenarios for distinguishing T. carassii and other aquatic trypanosomes. We find evidences for the existence of a low number of freshwater fish trypanosomes, with T. carassii having the widest geographic and host ranges. Our analyses support the existence of an umbrella complex composed of T. carassii and two sister species.
Currently, new species of freshwater fish trypanosomes, which are economically important parasites, are being described based on subjectively selected features, i.e., their cell morphology and the host species. We have performed detailed phylogenetic and haplotype diversity analyses of all 18S rRNA genes available for freshwater fish trypanosomes, including the newly obtained sequences of Trypanosoma carassii and Trypanosoma danilewskyi. Based on a sequence similarity of 99.5%, we divide these trypanosomes into 15 operational taxonomic units, and propose three nominal scenarios for distinguishing T. carassii and other aquatic trypanosomes. We find evidences for the existence of a low number of freshwater fish trypanosomes, with T. carassii having the widest geographic and host ranges. Our analyses support the existence of an umbrella complex composed of T. carassii and two sister species.
Abstract:
High-throughput sequencing of amplicons has been widely used to precisely and efficiently identify species compositions and analyze community structures, greatly promoting biological studies involving large amounts of complex samples, especially those involving environmental and pathogen-monitoring ones. Commercial library preparation kits for amplicon sequencing, which generally require multiple steps, including adapter ligation and indexing, are expensive and time-consuming, especially for applications at a large scale. To overcome these limitations, a "one-step PCR approach" has been previously proposed for constructions of amplicon libraries using long fusion primers. However, efficient amplifications of target genes and accurate demultiplexing of pooled sequencing data remain to be addressed. To tackle these, we present an integrative protocol for one-step PCR amplicon library construction (OSPALC). High-quality reads have been generated by this approach to reliably identify species compositions of mock bacterial communities and environmental samples. With this protocol, the amplicon library is constructed through one regular PCR with long primers, and the total cost per DNA/cDNA sample decreases to just 7% of the typical cost via the multi-step PCR approach. Empirically tested primers and optimized PCR conditions to construct OSPALC libraries for 16S rDNA V4 regions are demonstrated as a case study. Tools to design primers targeting at any genomic regions are also presented. In principle, OSPALC can be readily applied to construct amplicon libraries of any target genes using DNA or RNA samples, and will facilitate research in numerous fields.
High-throughput sequencing of amplicons has been widely used to precisely and efficiently identify species compositions and analyze community structures, greatly promoting biological studies involving large amounts of complex samples, especially those involving environmental and pathogen-monitoring ones. Commercial library preparation kits for amplicon sequencing, which generally require multiple steps, including adapter ligation and indexing, are expensive and time-consuming, especially for applications at a large scale. To overcome these limitations, a "one-step PCR approach" has been previously proposed for constructions of amplicon libraries using long fusion primers. However, efficient amplifications of target genes and accurate demultiplexing of pooled sequencing data remain to be addressed. To tackle these, we present an integrative protocol for one-step PCR amplicon library construction (OSPALC). High-quality reads have been generated by this approach to reliably identify species compositions of mock bacterial communities and environmental samples. With this protocol, the amplicon library is constructed through one regular PCR with long primers, and the total cost per DNA/cDNA sample decreases to just 7% of the typical cost via the multi-step PCR approach. Empirically tested primers and optimized PCR conditions to construct OSPALC libraries for 16S rDNA V4 regions are demonstrated as a case study. Tools to design primers targeting at any genomic regions are also presented. In principle, OSPALC can be readily applied to construct amplicon libraries of any target genes using DNA or RNA samples, and will facilitate research in numerous fields.
Abstract:
Exogenous RNA poses a continuous threat to genome stability and integrity across various organisms. Accumulating evidence reveals complex mechanisms underlying the cellular response to exogenous RNA, including endo-lysosomal degradation, RNA-dependent repression and innate immune clearance. Across a variety of mechanisms, the natural anti-sense RNA-dependent defensive strategy has been utilized both as a powerful gene manipulation tool and gene therapy strategy named RNA-interference (RNAi). To optimize the efficiency of RNAi silencing, a comprehensive understanding of the whole life cycle of exogenous RNA, from cellular entry to its decay, is vital. In this paper, we review recent progress in comprehending the recognition and elimination of foreign RNA by cells, focusing on cellular entrance, intracellular transportation, and immune-inflammatory responses. By leveraging these insights, we highlight the potential implications of these insights for advancing RNA interference efficiency, underscore the need for future studies to elucidate the pathways and fates of various exogenous RNA forms, and provide foundational information for more efficient RNA delivery methods in both genetic manipulation and therapy in different organisms.
Exogenous RNA poses a continuous threat to genome stability and integrity across various organisms. Accumulating evidence reveals complex mechanisms underlying the cellular response to exogenous RNA, including endo-lysosomal degradation, RNA-dependent repression and innate immune clearance. Across a variety of mechanisms, the natural anti-sense RNA-dependent defensive strategy has been utilized both as a powerful gene manipulation tool and gene therapy strategy named RNA-interference (RNAi). To optimize the efficiency of RNAi silencing, a comprehensive understanding of the whole life cycle of exogenous RNA, from cellular entry to its decay, is vital. In this paper, we review recent progress in comprehending the recognition and elimination of foreign RNA by cells, focusing on cellular entrance, intracellular transportation, and immune-inflammatory responses. By leveraging these insights, we highlight the potential implications of these insights for advancing RNA interference efficiency, underscore the need for future studies to elucidate the pathways and fates of various exogenous RNA forms, and provide foundational information for more efficient RNA delivery methods in both genetic manipulation and therapy in different organisms.
Abstract:
Cell–cell adhesion is at the center of structure and dynamics of epithelial tissue. E-cadherin–catenin complexes mediate Ca2+-dependent trans-homodimerization and constitute the kernel of adherens junctions. Beyond the basic function of cell–cell adhesion, recent progress sheds light the dynamics and interwind interactions of individual E-cadherin–catenin complex with E-cadherin superclusters, contractile actomyosin and mechanics of the cortex and adhesion. The nanoscale architecture of E-cadherin complexes together with cis-interactions and interactions with cortical actomyosin adjust to junctional tension and mechano-transduction by reinforcement or weakening of specific features of the interactions. Although post-translational modifications such as phosphorylation and glycosylation have been implicated, their role for specific aspects of in E-cadherin function has remained unclear. Here, we provide an overview of the E-cadherin complex in epithelial cell and tissue morphogenesis focusing on nanoscale architectures by super-resolution approaches and post-translational modifications from recent, in particular in vivo, studies. Furthermore, we review the computational modelling in E-cadherin complexes and highlight how computational modelling has contributed to a deeper understanding of the E-cadherin complexes.
Cell–cell adhesion is at the center of structure and dynamics of epithelial tissue. E-cadherin–catenin complexes mediate Ca2+-dependent trans-homodimerization and constitute the kernel of adherens junctions. Beyond the basic function of cell–cell adhesion, recent progress sheds light the dynamics and interwind interactions of individual E-cadherin–catenin complex with E-cadherin superclusters, contractile actomyosin and mechanics of the cortex and adhesion. The nanoscale architecture of E-cadherin complexes together with cis-interactions and interactions with cortical actomyosin adjust to junctional tension and mechano-transduction by reinforcement or weakening of specific features of the interactions. Although post-translational modifications such as phosphorylation and glycosylation have been implicated, their role for specific aspects of in E-cadherin function has remained unclear. Here, we provide an overview of the E-cadherin complex in epithelial cell and tissue morphogenesis focusing on nanoscale architectures by super-resolution approaches and post-translational modifications from recent, in particular in vivo, studies. Furthermore, we review the computational modelling in E-cadherin complexes and highlight how computational modelling has contributed to a deeper understanding of the E-cadherin complexes.
