Decryption of the survival "black box": gene family expansion promotes the encystment in ciliated protists.

BACKGROUND: Encystment is an important survival strategy extensively employed by microbial organisms to survive unfavorable conditions. Single-celled ciliated protists (ciliates) are popular model eukaryotes for studying encystment, whereby these cells degenerate their ciliary structures and develop cyst walls, then reverse the process under more favorable conditions. However, to date, the evolutionary basis and mechanism for encystment in ciliates is largely unknown. With the rapid development of high-throughput sequencing technologies, genome sequencing and comparative genomics of ciliates have become effective methods to provide insights into above questions. RESULTS: Here, we profiled the MAC genome of Pseudourostyla cristata, a model hypotrich ciliate for encystment studies. Like other hypotrich MAC genomes, the P. cristata MAC genome is extremely fragmented with a single gene on most chromosomes, and encodes introns that are generally small and lack a conserved branch point for pre-mRNA splicing. Gene family expansion analyses indicate that multiple gene families involved in the encystment are expanded during the evolution of P. cristata. Furthermore, genomic comparisons with other five representative hypotrichs indicate that gene families of phosphorelay sensor kinase, which play a role in the two-component signal transduction system that is related to encystment, show significant expansion among all six hypotrichs. Additionally, cyst wall-related chitin synthase genes have experienced structural changes that increase them from single-exon to multi-exon genes during evolution. These genomic features potentially promote the encystment in hypotrichs and enhance their ability to survive in adverse environments during evolution. CONCLUSIONS: We systematically investigated the genomic structure of hypotrichs and key evolutionary phenomenon, gene family expansion, for encystment promotion in ciliates. In summary, our results provided insights into the evolutionary mechanism of encystment in ciliates.

Towards Multi-Objective Object Push-Grasp Policy Based on Maximum Entropy Deep Reinforcement Learning under Sparse Rewards.

In unstructured environments, robots need to deal with a wide variety of objects with diverse shapes, and often, the instances of these objects are unknown. Traditional methods rely on training with large-scale labeled data, but in environments with continuous and high-dimensional state spaces, the data become sparse, leading to weak generalization ability of the trained models when transferred to real-world applications. To address this challenge, we present an innovative maximum entropy Deep Q-Network (ME-DQN), which leverages an attention mechanism. The framework solves complex and sparse reward tasks through probabilistic reasoning while eliminating the trouble of adjusting hyper-parameters. This approach aims to merge the robust feature extraction capabilities of Fully Convolutional Networks (FCNs) with the efficient feature selection of the attention mechanism across diverse task scenarios. By integrating an advantage function with the reasoning and decision-making of deep reinforcement learning, ME-DQN propels the frontier of robotic grasping and expands the boundaries of intelligent perception and grasping decision-making in unstructured environments. Our simulations demonstrate a remarkable grasping success rate of 91.6%, while maintaining excellent generalization performance in the real world.

Three closely-related subclasses Phacodiniidia Small & Lynn, 1985, Protohypotrichia Shi et al., 1999, and Euplotia Jankowski, 1979 (Protista, Ciliophora): A new contribution to their phylogeny with reconsiderations on the evolutionary hypotheses.

The huge variety of species and worldwide distribution of ciliated protists in class Spirotrichea continue to make it one of the most complicated and confused groups in Ciliophora, despite significant research interest in the unique molecular genetics of these organisms. In this study, the morphological and molecular information were integrated, and it is inferred from a new perspective for the evolutionary relationship among Phacodiniidia, Protohypotrichia, Hypotrichia and Euplotia. Our results indicate that Kiitricha and Caryotricha, two members in Protohypotrichia, may represent two parallel branches of evolution; Euplotidae and Aspidiscidae represent the most recently diverged taxa within Euplotida, followed by Certesiidae, Gastrocirrhidae, and Uronychidae. Further, representative morphological characters (e.g. fronto-ventral-transverse cirral anlagen, undulating membranes, marginal cirri and caudal cirri) were stochastically mapped on phylogenies to speculate evolutionary path and morphological characters of the evolutionary transition node groups were assumed.

Insights into the phylogeny of the family Deviatidae (Protozoa, Ciliophora, Hypotrichia) based on multi-gene, morphological and ontogenetic information, with the establishment of a new species Deviata multilineae n. sp.

Hitherto, the phylogeny of ciliated protists, an important group of model organisms in many fields, has been mainly based on a single marker gene (SSU rDNA, nuclear small subunit ribosomal RNA gene). However, there is increasing evidence showing this is insufficient to provide robust phylogenies and has resulted in confusing systematics in many ciliates groups. Among these, the phylogenies within family Deviatidae (Spirotrichea, Hypotrichia) are ambiguous due to the dependence on SSU rDNA and undersampling. Here, we provide eight new sequences and conduct phylogenetic analyses based on both multi-gene and single-gene to clarify evolutionary relationships among all deviatids for which gene sequence are available. The results reveal that: (1) the monophyly of Deviatidae is well-supported by both single-gene and concatenated data; (2) the presence of fine cirri and relatively wide spacing of these cirri within all rows are plesiomorphies of Deviatidae; (3) Pseudosincirra longicirrata is closely related to Deviata rositae, which is supported by their shared possession of dorsomarginal kineties; (4) phylogenetic analyses and approximately unbiased test based on multi-gene support a close relationship among taxa lacking dorsomarginal kineties (D. parabacilliformis, D. multilineae nov. spec., D. abbrevescens, D. brasiliensis and Perisincirra paucicirrata); (5) Deviatidae shows a close relationship with Dorsomarginalia and Strongylidium-Hemiamphisiella-Pseudouroleptus assemblage, suggesting the presence/absence of dorsomarginal kineties is phylogenetically informative in this family and presence of them may be a plesiomorphy. Based on the morphological, morphogenetic and phylogenetic data, the evolutionary relationships within Deviatidae are hypothesized, and a new ciliate, Deviata multilineae nov. spec., collected from China, is investigated.

The hidden genomic diversity of ciliated protists revealed by single-cell genome sequencing.

BACKGROUND: Ciliated protists are a widely distributed, morphologically diverse, and genetically heterogeneous group of unicellular organisms, usually known for containing two types of nuclei: a transcribed polyploid macronucleus involved in gene expression and a silent diploid micronucleus responsible for transmission of genetic material during sexual reproduction and generation of the macronucleus. Although studies in a few species of culturable ciliated protists have revealed the highly dynamic nature of replicative and recombination events relating the micronucleus to the macronucleus, the broader understanding of the genomic diversity of ciliated protists, as well as their phylogenetic relationships and metabolic potential, has been hampered by the inability to culture numerous other species under laboratory conditions, as well as the presence of symbiotic bacteria and microalgae which provide a challenge for current sequencing technologies. Here, we optimized single-cell sequencing methods and associated data analyses, to effectively remove contamination by commensal bacteria, and generated high-quality genomes for a number of Euplotia species. RESULTS: We obtained eight high-quality Euplotia genomes by using single-cell genome sequencing techniques. The genomes have high genomic completeness, with sizes between 68 and 125 M and gene numbers between 14K and 25K. Through comparative genomic analysis, we found that there are a large number of gene expansion events in Euplotia genomes, and these expansions are closely related to the phenotypic evolution and specific environmental adaptations of individual species. We further found four distinct subgroups in the genus Euplotes, which exhibited considerable genetic distance and relative lack of conserved genomic syntenies. Comparative genomic analyses of Uronychia and its relatives revealed significant gene expansion associated with the ciliary movement machinery, which may be related to the unique and strong swimming ability. CONCLUSIONS: We employed single-cell genomics to obtain eight ciliate genomes, characterized the underestimated genomic diversity of Euplotia, and determined the divergence time of representative species in this subclass for the first time. We also further investigated the extensive duplication events associated with speciation and environmental adaptation. This study provides a unique and valuable resource for understanding the evolutionary history and genetic diversity of ciliates.

Evaluation of Cell Viability with a Single Fluorescent Probe Based on Two Kinds of Fluorescence Signal Modes.

Accurate evaluation of cell viability is important for dosage tests of anticancer drugs, pathology, and numerous biological experiments. However, due to the serious insufficieny of fluorescent probes, which can distinguish various cell states, the study of cell viability is immensely limited. To resolve this issue, we design and synthesize a new probe ACD-E to monitor cell viability with two kinds of fluorescence signal modes, the first single fluorescent probe that can distinguish three different cell states and furnish accurate information in biological experiments. ACD-E can discriminate live and dead cells in a dual-color mode by evaluating cell mitochondrial esterase activity and can also discriminate live and early necrosis cells by determining mitochondrial viscosity in a "turn-on" mode in the near-infrared region. Significantly, the novel ACD-E can also distinguish cell viability in vivo. This work establishes a robust strategy for monitoring multiple cell states using a single fluorescent probe.

Large-scale phylogenomic analysis provides new insights into the phylogeny of the class Oligohymenophorea (Protista, Ciliophora) with establishment of a new subclass Urocentria nov. subcl.

The class Oligohymenophorea is one of the most diverse assemblage of ciliated protists, which are particularly important in fundamental biological studies including understanding the evolutionary relationships among the lineages. Phylogenetic relationships within the class remain largely elusive, especially within the subclass Peniculia, which contains the long-standing problematic taxa Urocentrum and Paranassula. In the present study, we sequenced the genomes and/or transcriptomes of six non-culturable oligohymenophoreans using single-cell sequencing techniques. Phylogenomic analysis was performed based on expanded taxon sampling of 85 taxa, including 157 nuclear genes encoding 36,953 amino acids. The results indicate that: (1) urocentrids form an independent branch that is sister to the clade formed by Scuticociliatia and Hymenostomatia, which, together with the morphological data, supports the establishment of a new subclass, Urocentria n. subcl., within Oligohymenophorea; (2) phylogenomic analysis and ortholog comparison reveal a close relationship between Paranassula and peniculines, providing corroborative evidence for removing Paranassula from Nassulida and elevating it as an order, Paranassulida, within the subclass Peniculia; (3) based on the phylogenomic analyses and morphological data, we hypothesize that Peritrichia is the earliest diverging clade within Oligohymenophorea while Scuticociliatia and Hymenostomatia share the most common ancestor, followed successively by Urocentria and Peniculia. In addition, stop codon analyses indicate that oligohymenophoreans widely use UGA as the stop codon, while UAR are reassigned to glutamate (peritrichs) or glutamine (others), supporting the evolutionary hypothesis.

A New GNSS Interference Detection Method Based on Rearranged Wavelet-Hough Transform.

Since radio frequency interference (RFI) seriously degrades the performance of a global navigation satellite system (GNSS) receiver, interference detection becomes very important for GNSS receivers. In this paper, a novel rearranged wavelet-Hough transform (RWHT) method is proposed in GNSS interference detection, which is obtained by the combination of rearranged wavelet transform and Hough transform (HT). The proposed RWHT method is tested for detecting sweep interference and continuous wave (CW) interference, the major types of GNSS interfering signals generated by a GNSS jammer in a controlled test bench experiment. The performance of the proposed RWHT method is compared with the conventional techniques such as Wigner-Ville distribution (WVD) and Wigner-Hough transform (WHT). The analysis results show that the proposed RWHT method reduces the influence of cross-item problem and improves the energy aggregation property in GNSS interference detection. When compared with the WHT approach, this proposed RWHT method presents about 90.3% and 30.8% performance improvement in the initial frequency and chirp rate estimation of the GNSS sweep interfering signal, respectively. These results can be further considered to be the proof of the validity and effectiveness of the developed GNSS interference detection method using RWHT.

A Novel GNSS Interference Detection Method Based on Smoothed Pseudo-Wigner-Hough Transform.

This paper develops novel Global Navigation Satellite System (GNSS) interference detection methods based on the Hough transform. These methods are realized by incorporating the Hough transform into three Time-Frequency distributions: Wigner-Ville distribution, pseudo -Wigner-Ville distribution and smoothed pseudo-Wigner-Ville distribution. This process results in the corresponding Wigner-Hough transform, pseudo-Wigner-Hough transform and smoothed pseudo-Wigner-Hough transform, which are used in GNSS interference detection to search for local Hough-transformed energy peak in a small limited area within the parameter space. The developed GNSS interference detection methods incorporate a novel concept of zero Hough-transformed energy distribution percentage to analyze the properties of energy concentration and cross-term suppression. The methods are tested with real GPS L1-C/A data collected in the presence of sweep interference. The test results show that the developed methods can deal with the cross-term problem with improved interference detection performance. In particular, the GNSS interference detection performance obtained with the smoothed pseudo-Wigner-Hough transform method is at least double that of the Wigner-Hough transform-based approach; the smoothed pseudo-Wigner-Hough transform-based GNSS interference detection method is improved at least 20% over the pseudo-Wigner-Hough transform-based technique in terms of the zero Hough-transformed energy percentage criteria. Therefore, the proposed smoothed pseudo-Wigner-Hough transform-based method is recommended in the interference detection for GNSS receivers, particularly in challenging electromagnetic environments.

Characterization and Comparative Analyses of Mitochondrial Genomes in Single-Celled Eukaryotes to Shed Light on the Diversity and Evolution of Linear Molecular Architecture.

Determination and comparisons of complete mitochondrial genomes (mitogenomes) are important to understand the origin and evolution of mitochondria. Mitogenomes of unicellular protists are particularly informative in this regard because they are gene-rich and display high structural diversity. Ciliates are a highly diverse assemblage of protists and their mitogenomes (linear structure with high A+T content in general) were amongst the first from protists to be characterized and have provided important insights into mitogenome evolution. Here, we report novel mitogenome sequences from three representatives (Strombidium sp., Strombidium cf. sulcatum, and Halteria grandinella) in two dominant ciliate lineages. Comparative and phylogenetic analyses of newly sequenced and previously published ciliate mitogenomes were performed and revealed a number of important insights. We found that the mitogenomes of these three species are linear molecules capped with telomeric repeats that differ greatly among known species. The genomes studied here are highly syntenic, but larger in size and more gene-rich than those of other groups. They also all share an AT-rich tandem repeat region which may serve as the replication origin and modulate initiation of bidirectional transcription. More generally we identified a split version of ccmf, a cytochrome c maturation-related gene that might be a derived character uniting taxa in the subclasses Hypotrichia and Euplotia. Finally, our mitogenome comparisons and phylogenetic analyses support to reclassify Halteria grandinella from the subclass Oligotrichia to the subclass Hypotrichia. These results add to the growing literature on the unique features of ciliate mitogenomes, shedding light on the diversity and evolution of their linear molecular architecture.

Two new scuticociliates from southern China: Uronema apomarinum sp. nov. and Homalogastra parasetosa sp. nov., with improved diagnoses of the genus Homalogastra and its type species Homalogastra setosa (Ciliophora, Oligohymenophorea).

The morphology of two new scuticociliates, Uronema apomarinum sp. nov. and Homalogastra parasetosa sp. nov., isolated from a mangrove wetland in Shenzhen, PR China, was studied using live observation and the protargol impregnation method. Uronema apomarinum is characterized by a body size of about 20-35×10-15 µm in vivo, a partly two-rowed membranelle 1, and 12 or 13 somatic kineties. Homalogastra parasetosa is distinguished by a membranelle 1 comprising two longitudinal rows of basal bodies. Three Homalogastra setosa populations are suggested as subjective synonyms of the new species. Improved diagnoses of the genus Homalogastra Kahl, 1926 and its type species Homalogastra setosa Kahl, 1926 are provided. Results of phylogenetic analyses based on 18S rRNA gene and ITS1-5.8S-ITS2 region sequences indicate that U. apomarinum is most closely related to U. marinum, while the closest relative of H. parasetosa is H. setosa.

Further analyses on the phylogeny of the subclass Scuticociliatia (Protozoa, Ciliophora) based on both nuclear and mitochondrial data.

So far, the phylogenetic studies on ciliated protists have mainly based on single locus, the nuclear ribosomal DNA (rDNA). In order to avoid the limitations of single gene/genome trees and to add more data to systematic analyses, information from mitochondrial DNA sequence has been increasingly used in different lineages of ciliates. The systematic relationships in the subclass Scuticociliatia are extremely confused and largely unresolved based on nuclear genes. In the present study, we have characterized 72 new sequences, including 40 mitochondrial cytochrome oxidase c subunit I (COI) sequences, 29 mitochondrial small subunit ribosomal DNA (mtSSU-rDNA) sequences and three nuclear small subunit ribosomal DNA (nSSU-rDNA) sequences from 47 isolates of 44 morphospecies. Phylogenetic analyses based on single gene as well as concatenated data were performed and revealed: (1) compared to mtSSU-rDNA, COI gene reveals more consistent relationships with those of nSSU-rDNA; (2) the secondary structures of mtSSU-rRNA V4 region are predicted and compared in scuticociliates, which can contribute to discrimination of closely related species; (3) neither nuclear nor mitochondrial data support the monophyly of the order Loxocephalida, which may represent some divergent and intermediate lineages between the subclass Scuticociliatia and Hymenostomatia; (4) the assignments of thigmotrichids to the order Pleuronematida and the confused taxon Sulcigera comosa to the genus Histiobalantium are confirmed by mitochondrial genes; (5) both nuclear and mitochondrial data reveal that the species in the family Peniculistomatidae always group in the genus Pleuronema, suggesting that peniculistomatids are more likely evolved from Pleuronema-like ancestors; (6) mitochondrial genes support the monophyly of the order Philasterida, but the relationships among families of the order Philasterida remain controversial due to the discrepancies between their morphological and molecular data.

Long non-coding RNA FTH1P3 activates paclitaxel resistance in breast cancer through miR-206/ABCB1.

Emerging evidence has indicated the important function of long non-coding RNAs (lncRNAs) in tumour chemotherapy resistance. However, the underlying mechanism is still ambiguous. In this study, we investigate the physiopathologic role of lncRNA ferritin heavy chain 1 pseudogene 3 (FTH1P3) on the paclitaxel (PTX) resistance in breast cancer. Results showed that lncRNA FTH1P3 was up-regulated in paclitaxel-resistant breast cancer tissue and cells (MCF-7/PTX and MDA-MB-231/PTX cells) compared with paclitaxel-sensitive tissue and parental cell lines (MCF-7, MDA-MB-231). Gain- and loss-of-function experiments revealed that FTH1P3 silencing decreased the 50% inhibitory concentration (IC50) value of paclitaxel and induced cell cycle arrest at G2/M phase, while FTH1P3-enhanced expression exerted the opposite effects. In vivo, xenograft mice assay showed that FTH1P3 silencing suppressed the tumour growth of paclitaxel-resistant breast cancer cells and ABCB1 protein expression. Bioinformatics tools and luciferase reporter assay validated that FTH1P3 promoted ABCB1 protein expression through targeting miR-206, acting as a miRNA "sponge." In summary, our results reveal the potential regulatory mechanism of FTH1P3 on breast cancer paclitaxel resistance through miR-206/ABCB1, providing a novel insight for the breast cancer chemoresistance.

An improved indirect ELISA for specific detection of antibodies against classical swine fever virus based on structurally designed E2 protein expressed in suspension mammalian cells.

Classical swine fever (CSF), which is caused by classical swine fever virus (CSFV), is a highly contagious disease of pigs. CSFV is genetically and serologically related to bovine viral diarrhea virus (BVDV), a ruminant pestivirus. However, currently available ELISAs based on the full-length E2 protein of CSFV cannot discriminate anti-CSFV from anti-BVDV antibodies. In this study, a truncated CSFV E2 protein (amino acids 690 to 879) covering antigenic domains B/C/D/A (E2B/C/D/A) was designed based on homologous modeling according to the crystal structure of the BVDV E2 protein. The E2B/C/D/A protein was expressed in CHO cells adapted to serum-free suspension culture, and an indirect ELISA (iELISA) was established based on the recombinant protein. No serological cross-reaction was observed for anti-BVDV sera in the iELISA. When testing 282 swine serum samples, the iELISA displayed a high sensitivity (119/127, 93.7%) and specificity (143/155, 92.3%), with an agreement of 92.9% (262/282) and 92.2% (260/282) with virus neutralization test and the IDEXX CSFV blocking ELISA, respectively. Taken together, the newly developed iELISA is highly specific and sensitive and able to differentiate anti-CSFV from anti-BVDV antibodies.

Disentangling sources of variation in SSU rDNA sequences from single cell analyses of ciliates: impact of copy number variation and experimental error.

Small subunit ribosomal DNA (SSU rDNA) is widely used for phylogenetic inference, barcoding and other taxonomy-based analyses. Recent studies indicate that SSU rDNA of ciliates may have a high level of sequence variation within a single cell, which impacts the interpretation of rDNA-based surveys. However, sequence variation can come from a variety of sources including experimental errors, especially the mutations generated by DNA polymerase in PCR. In the present study, we explore the impact of four DNA polymerases on sequence variation and find that low-fidelity polymerases exaggerate the estimates of single-cell sequence variation. Therefore, using a polymerase with high fidelity is essential for surveys of sequence variation. Another source of variation results from errors during amplification of SSU rDNA within the polyploidy somatic macronuclei of ciliates. To investigate further the impact of SSU rDNA copy number variation, we use a high-fidelity polymerase to examine the intra-individual SSU rDNA polymorphism in ciliates with varying levels of macronuclear amplification: Halteria grandinella, Blepharisma americanum and Strombidium stylifer We estimate the rDNA copy numbers of these three species by single-cell quantitative PCR. The results indicate that: (i) sequence variation of SSU rDNA within a single cell is authentic in ciliates, but the level of intra-individual SSU rDNA polymorphism varies greatly among species; (ii) rDNA copy numbers vary greatly among species, even those within the same class; (iii) the average rDNA copy number of Halteria grandinella is about 567 893 (s.d. = 165 481), which is the highest record of rDNA copy number in ciliates to date; and (iv) based on our data and the records from previous studies, it is not always true in ciliates that rDNA copy numbers are positively correlated with cell or genome size.

Further consideration on the phylogeny of the Ciliophora: Analyses using both mitochondrial and nuclear data with focus on the extremely confused class Phyllopharyngea.

Most ciliate phylogenetic analyses have largely relied on the nuclear small subunit ribosome DNA (nSSU-rDNA) locus. However, single locus or multi-loci from the same genome or chromosome may not be sufficient enough to elucidate phylogenetic relationships among ciliate taxa. Therefore, in addition to nSSU-rDNA, the mitochondrial small subunit ribosome DNA (mtSSU-rDNA) was applied in this study. We expanded the taxon sampling especially within the class Phyllopharyngea. Phylogenetic analyses based on nSSU-rDNA and mtSSU-rDNA, independently, as well as concatenated were performed and revealed the following: (1) mtSSU-rDNA is more variable than nSSU-rDNA, and is better at elucidating relationships at lower levels, e.g. intra-/inter-specific or generic relationships; (2) the validity of the two genera Mirodysteria and Spirodysteria is challenged based on their similar morphology with Dysteria and the analyses from both mtSSU-rDNA and nSSU-rDNA; (3) Brooklynella is confirmed to be an intermediate taxon between Dysteriidae and Hartmannulidae, and may represent a distinct family; (4) Trithigmostoma should remain in Chilodonellidae; (5) the separation of Paraspathidium from Litostomatea is supported and it groups with prostomateans and plagiopyleans. In summary, results from mtSSU-rDNA corroborated those of nSSU-rDNA for highly supported clades, and the mtSSU-rDNA tree with its secondary structure gave topologies that could be explained by the morphology; therefore it can be useful in some cases towards better resolution of robust phylogenies.

The diagnostic value of bronchial provocation testing combined with fractional exhaled nitric oxide (FeNO) in children with chest tightness-variant asthma (CTVA).

BACKGROUND: Chest tightness-variant asthma (CTVA) is a novel atypical asthma characterized by chest tightness as the sole or primary symptom. OBJECTIVES: To investigate the value of bronchial provocation testing combined with fractional exhaled nitric oxide (FeNO) in the diagnosis of CTVA in children. METHODS: This study included 95 children aged 6-14 years with chest tightness as the sole symptom, with a duration of symptoms exceeding 4 weeks. All subjects underwent FeNO measurement, pulmonary function testing, and bronchial provocation testing using the Astograph method. Subjects with positive bronchial provocation testing were classified as the CTVA group, while those with negative results served as the non-CTVA control group. RESULTS: The lung function of children in both groups was normal. The FeNO level in the CTVA group was (22.35 ± 9.91) ppb, significantly higher than the control group (14.85 ± 5.63) ppb, with a statistically significant difference (P < 0.05). The value of FeNO in diagnosing CTVA was analyzed using an ROC curve, with an area under the curve of 0.073 (P < 0.05). The optimal cutoff point for diagnosing CTVA using FeNO was determined to be 18.5 ppb, with a sensitivity of 60.3 % and specificity of 77.8 %. There was a negative correlation between FeNO and Dmin as well as PD15 (P = 0.006). CONCLUSION: FeNO can serve as an adjunctive diagnostic tool for CTVA, with the optimal cutoff point for diagnosing CTVA being 18.5 ppb. However, FeNO is not a specific diagnostic marker for CTVA and should be used in conjunction with bronchial provocation testing to enhance its diagnostic value.

Morphology, morphogenesis and molecular phylogeny of a new soil ciliate, Lamtostyla paravitiphila nov. spec. (Ciliophora, Hypotrichia).

The morphology and morphogenesis of Lamtostyla paravitiphila nov. spec., a novel soil hypotrichous ciliate collected from eastern China, were investigated based on live observations and protargol-stained specimens. The new species is morphologically characterized as follows: seven to twelve macronuclear nodules, cortical granules absent, 19-26 adoral membranelles, three or four frontoventral cirri, the amphisiellid median cirral row extends to about mid-body and composed of 12-18 cirri, two or three transverse cirri, 27-39 left and 30-41 right marginal cirri, three almost bipolar dorsal kineties. Morphogenetically, it is characterized by the initial formation of six frontal-ventral-transverse cirral anlagen as primary primordia. Notably, the amphisiellid median cirral row and the posterior frontoventral cirrus (or cirri) contribute to the development of the frontal-ventral-transverse cirral anlagen, while the buccal cirrus may not participate in this process. Phylogenetic analyses based on small subunit ribosomal DNA sequence data indicate that the Lamtostyla species with available molecular data do not form a monophyletic group.

From germline genome to highly fragmented somatic genome: genome-wide DNA rearrangement during the sexual process in ciliated protists.

Genomes are incredibly dynamic within diverse eukaryotes and programmed genome rearrangements (PGR) play important roles in generating genomic diversity. However, genomes and chromosomes in metazoans are usually large in size which prevents our understanding of the origin and evolution of PGR. To expand our knowledge of genomic diversity and the evolutionary origin of complex genome rearrangements, we focus on ciliated protists (ciliates). Ciliates are single-celled eukaryotes with highly fragmented somatic chromosomes and massively scrambled germline genomes. PGR in ciliates occurs extensively by removing massive amounts of repetitive and selfish DNA elements found in the silent germline genome during development of the somatic genome. We report the partial germline genomes of two spirotrich ciliate species, namely Strombidium cf. sulcatum and Halteria grandinella, along with the most compact and highly fragmented somatic genome for S. cf. sulcatum. We provide the first insights into the genome rearrangements of these two species and compare these features with those of other ciliates. Our analyses reveal: (1) DNA sequence loss through evolution and during PGR in S. cf. sulcatum has combined to produce the most compact and efficient nanochromosomes observed to date; (2) the compact, transcriptome-like somatic genome in both species results from extensive removal of a relatively large number of shorter germline-specific DNA sequences; (3) long chromosome breakage site motifs are duplicated and retained in the somatic genome, revealing a complex model of chromosome fragmentation in spirotrichs; (4) gene scrambling and alternative processing are found throughout the core spirotrichs, offering unique opportunities to increase genetic diversity and regulation in this group. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00213-x.

HLA-DPB1 and DPA1 ~ DPB1 linkage mismatch affects the survival of recipients receiving HLA-14/14 matched unrelated donor HSCT.

To analyse the effect of HLA-DPA1 and HLA-DPB1 allelic mismatches on the outcomes of unrelated donor haematopoietic stem cell transplantation (URD-HSCT), we collected 258 recipients with haematological disease who underwent HLA-10/10 matched URD-HSCT. HLA-A, -B, -C, -DRB1, -DQB1, -DRB3/4/5, -DQA1, -DPA1 and -DPB1 typing was performed for the donors and recipients using next-generation sequencing (NGS) technology. After excluding 8 cases with DQA1 or DRB3/4/5 mismatches, we included 250 cases with HLA-14/14 matching for further analysis. Our results showed that the proportion of matched DPA1 and DPB1 alleles was only 10.4% (26/250). The remaining 89.6% of donors and recipients demonstrated DPA1 or DPB1 mismatch. In the DPA1 matched and DPB1 mismatched group, accounting for 18.8% (47/250) of the cohort, DPB1*02:01/DPB1*03:01 allelic mismatches were associated with decreased 2-year OS and increased NRM. DPB1*02:02/DPB1*05:01 and DPB1*02:01/DPB1*05:01 mismatches showed no impact on outcomes. Moreover, the specific allelic mismatches observed were consistent with the DPB1 T-cell epitope (TCE) classification as permissive and non-permissive. We innovatively established an analysis method for DPA1 ~ DPB1 linkage mismatch for cases with both DPA1 and DPB1 mismatched, accounting for 70% (175/250) of the total. DPA1*02:02 ~ DPB1*05:01/DPA1*02:01 ~ DPB1*17:01 linkage mismatches were associated with lower 2-year OS, especially among AML/MDS recipients. DPA1*02:02 ~ DPB1*05:01/DPA1*01:03 ~ DPB1*02:01 linkage mismatches showed no impact on outcomes. In conclusion, applying the DPA1 ~ DPB1 linkage mismatch analysis approach can identify different types of mismatches affecting transplant outcomes and provide valuable insight for selecting optimal donors for AML/MDS and ALL recipients.