Biology subjects

Yang, T.-C.

Publications and source records attributed to Yang, T.-C..

5 recordsLinked to original sources

bioRxiv2024

Single-cell RNA sequencing of iPSC-derived brain organoids reveals Treponema pallidum infection inhibiting neurodevelopment

Congenital syphilis is a vertically transmitted bacterial infection caused by Treponema pallidum, often causing multidomain neurodevelopmental disabilities. However, little is known about the pathogenesis of this disease. Brain organoids platform derived from the induced pluripotent stem cell (iPSC) is exposed to T. pallidum infection for modelling congenital neurodevelopmental impairment. Single-cell RNA sequencing is used for identifying the subpopulations of differentially expressed genes and cellular heterogeneity and reconstructing differentiation trajectories following T. pallidum infection. The results reveal that T. pallidum infection influences the formation of neural rosette structures, reduces the cell number of the neural progenitor cell subcluster 1B (subNPC1B) and hindbrain neurons, and affects the neurodevelopment of the brain organoid. Moreover, it is speculated that T. pallidum inhibits the hindbrain neuron cell number through the suppression of subNPC1B subgroup in the organoids and inhibits transcription factor 3 activity in the subNPC1B-hindbrain neuronal axis. This is the first report on the inhibited effects of T. pallidum on the neurodevelopment of the iPSC-derived brain organoid model. T. pallidum could inhibit the differentiation of subNPC1B in brain organoids, thereby reducing the differentiation from subNPC1B to hindbrain neurons, and ultimately affecting the development and maturation of hindbrain neurons.

neuroscience↗

bioRxiv2023

A Convection-Free Apparatus for Low Force and High Efficiency Centrifugation

Centrifugation is a common technique for material separation. Most biological materials are separated under a relatively strong centrifugal force. Despite several reports highlighted the impact from centrifugal force on the separated materials, low force centrifugation typically results in low efficiency of separation and may not be achievable from the traditional centrifugation apparatus. Here, we present a novel apparatus for separating materials under low centrifugal force with high efficiency. Compare to the traditional apparatus, the design of this apparatus minimizes the convection during centrifugation. Using homogeneous polystyrene particles, we confirmed that the centrifugation process is essentially convection-free. Separation of human blood cells indicated that, under a fixed centrifugal force, less than half of the centrifugation time is required compared to the traditional apparatus. The isolated human peripheral blood mononuclear cells have higher viability and can afford multiple centrifugations without impacting cell viability. Additionally, this new apparatus has a higher resolution to separate materials by size and is sensitive to detect size heterogeneity. This apparatus provides a new opportunity for efficient centrifugation under low centrifugal force and for a systematic evaluation of the impact from centrifugal force on the quality of separated materials.

bioengineering↗

bioRxiv2019

Characterization of the novel clinical isolate X-4 containing a new tp0548 sequence-type

BackgroundA novel tp0548 sequence-type was identified in one clinical isolate (X-4) from a patient diagnosed with primary syphilis in Xiamen, China. To precisely define and characterize this new clinical isolate, we performed further genome-scale molecular analysis. Methodology/Principal findingsThe alignment of all published tp0548 genotypes revealed that this new genotype had a unique nucleotide substitution G->T at position 167, and the letter "ao" was assigned to the genotype. Phylogenetic analysis showed that the "ao" genotype belonged to the SS14-like clade of Treponema pallidum (TPA) strains. The genome of the X-4 isolate was then sequenced and analyzed, and the result of a multi-locus sequence analysis using a set of nine chromosomal loci showed that the X-4 isolate was clustered with a monophyletic group of TPA strains, which clearly identified the isolate as a TPA strain. Whole-genome phylogenetic analysis was subsequently conducted to corroborate the TPA strain classification of the X-4 isolate. And the isolate was genetically related to the SS14 strain, with 42 single nucleotide variations and 12 insertions/deletions. In addition, high intrastrain heterogeneity in the length of the poly G/C tracts was found in the TPAChi_0347 locus, which indicated that this gene was most likely involved in phase variation events. The first investigation of the length heterogeneity of the poly A/T tracts showed the variability of the ploy A/T was lower, and all the observed intrastrain variations fell within coding regions. Conclusions/SignificanceThe study demonstrated the X-4 isolate was a TPA isolate containing a novel tp0548 sequence-type. The identification of intrastrain genetic heterogeneity at poly G/C tracts and poly A/T tracts of the isolate could provide a snapshot of the genes that potentially involved in genotype-phenotype variations. These findings provide an unequivocal characterization for better understanding the molecular variation of this emerging isolate. Author summaryThree subspecies of Treponema pallidum (pallidum, pertenue, and endemicum) are increasingly showing overlap in terms of transmission and clinical manifestations. We recently identified a novel tp0548 genotype in the X-4 isolate, which was obtained from an adult male with genital lesions. The novel genotype contained a unique nucleotide substitution G->T at position 167 and belonged to the SS14-like clade of TPA strains, as determined by phylogenetic analysis. We conducted an in-depth exploration of the genome of the X-4 isolate using the pooled segment genome sequencing method followed by Illumina sequencing. Multi-locus sequence analysis of nine chromosomal loci demonstrated that the X-4 isolate was clustered within a monophyletic group of TPA strains, which identified the isolate as a TPA strain. Whole-genome phylogenetic analysis subsequently corroborated the TPA strain classification of the X-4 isolate and revealed that the isolate was very closely related to the SS14 strain, with 42 single nucleotide variations and 12 insertions/deletions. In addition, characterization of the intrastrain heterogeneity in the lengths of homopolymeric tracts in the X-4 isolate showed that the heterogeneity of the poly G/C tracts was greater than that of the poly A/T tracts, and high poly G/C tract diversity was observed in the TPAChi_0347 locus.

genetics↗

bioRxiv2019

Insights into the mysterious genetic variation profile of tprK inTreponema pallidum under the development of natural human syphilis infection

Although the variations of the tprK gene in Treponema pallidum were considered to play a critical role in the pathogenesis of syphilis, how actual variable characteristics of tprK in the course of natural human infection enabling the pathogens survive has thus far remained unclear. Here, we performed NGS to investigate tprK of T. pallidum directly from primary and secondary syphilis samples. Compared with diversity in tprK of the strains from primary syphilis samples, there were more mixture variants found within seven V regions of the tprK gene among the strains from secondary syphilis samples, and the frequencies of predominant sequences within V regions of tprK were generally decreased (less than 80%) with the proportion of minor variants in 10-60% increasing. Noteworthy, the variations within V regions of tprK always obeyed a strict 3 bp changing pattern. And tprK in the strains from the two-stage samples kept some stable amino acid sequences within V regions. Particularly, the amino acid sequences IASDGGAIKH and IASEDGSAGNLKH in V1 not only presented a high proportion of inter-population sharing, but also presented a relatively high frequency (above 80%) in the populations. Besides, tprK always demonstrated remarkable variability in V6 at both the intra- and inter-strain levels regardless of the course. These findings unveiled that the different profile of tprK in T. pallidum directly from primary and secondary syphilis samples, indicating that throughout the development of syphilis T. pallidum constantly varies its domain tprK gene to obtain the best adaptation to the host. While this changing was always subjected a strict gene conversion mechanism to keep an abnormal TprK. The highly stable peptides found in V1 would probably be promising potential vaccine components. And the highly heterogenetic regions (e.g. V6) could provide insight into the mysterious role of tprK in immune evasion. Author summaryAlthough the variations of the tprK gene in Treponema pallidum were considered to play a critical role in the pathogenesis of syphilis, how actual variable characteristics of tprK in the course of natural human infection enabling the pathogens survive has thus far remained unclear. Here, we performed next-generation sequencing, a more sensitive and reliable approach, to investigate tprK of Treponema pallidum directly from primary and secondary syphilis patients, revealing that the profile of tprK in T. pallidum from the two-stage samples was different. Within the strains from secondary syphilis patients, more mixture variants within seven V regions of tprK were found, the frequencies of their predominant sequences were generally decreased with the proportion of minor variants in 10-60% was increased. And the variations within V regions of tprK always obeyed a strict 3 bp changing pattern. Noteworthy, the amino acid sequences IASDGGAIKH and IASEDGSAGNLKH in V1 presented a high proportion of inter-population sharing and presented a relatively high frequency in the populations. And V6 region always demonstrated remarkable variability at intra- and inter-patient levels regardless of the course. These findings provide insights into the mysterious role of TprK in immune evasion and for further exploring the potential vaccine components.

microbiology↗

bioRxiv2018

Profile of the tprK gene in primary syphilis patients based on next-generation sequencing

BackgroundThe highly variable tprK gene of Treponema pallidum has been acknowledged to be the cause of persistent infection. Previous studies mainly focused on the heterogeneity in tprK in propagated strains using a clone-based Sanger approach. Few studies have investigated tprK directly from clinical samples using deep sequencing.\n\nMethods/Principal findingsWe conducted a comprehensive analysis of 14 primary syphilis clinical isolates of T. pallidum via next-generation sequencing to gain better insight into the profile of tprK in primary syphilis patients. Our results based on primary syphilis clinical samples showed that there was a mixture of distinct sequences within each V region of tprK. Except for the predominant sequence for each region as previously reported using the clone-based Sanger approach, there were many minor variants of all strains that were mainly observed at a frequency of 1-5%. Interestingly, the identified distinct sequences within the regions were variable in length and differed only by 3 bp or multiples of 3 bp. In addition, amino acid sequence consistency within each region was found between the 14 strains. Among the regions, the sequence IASDGGAIKH in V1 and the sequence DVGHKKENAANVNGTVGA in V4 showed a high stability of inter-strain redundancy.\n\nConclusionsThe seven V regions of the tprK gene in primary syphilis infection demonstrated high diversity; they generally contained a high proportion sequence and numerous low-frequency minor variants, most of which are far below the detection limit of Sanger sequencing. The rampant variation in each region was regulated by a strict gene conversion mechanism that maintained the length difference to 3 bp or multiples of 3 bp. The highly stable sequence of inter-strain redundancy may indicate that the sequences play a critical role in T. pallidum virulence. These highly stable peptides are also likely to be potential targets for vaccine development.\n\nAuthor summaryVariations in tprK have been acknowledged to be the major contributors to persistent Treponema pallidum infections. Previous studies were based on the clone-based Sanger approach, and most of them were performed in propagated strains using rabbits, which could not reflect the actual heterogeneous characteristics of tprK in vivo. In the present study, we employed next-generation sequencing (NGS) to explore the profile of tprK directly from 14 patients with primary syphilis. Our results showed a mixture of distinct sequences within each V region of tprK in these clinical samples. First, the length of identified distinct sequences within the region was variable, which differed by only 3 bp or multiples of 3 bp. Then, among the mixtures, a predominant sequence was usually observed for each region, and the remaining minor variants were mainly observed at a frequency of 1-5%. In addition, there was a scenario of amino acid sequence consistency within the regions between the 14 primary syphilis strains. The identification of the profile of tprK in the context of human primary syphilis infection contributes to further exploration of the pathogenesis of syphilis.

microbiology↗