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.

Successful Retrieval of a Broken Aspiration Needle Penetrated into the Right Pulmonary Artery: A Case Report with Experience Sharing.

INTRODUCTION: Endobronchial ultrasound-guided transbronchial needle aspiration is increasingly used as a minimally invasive procedure in clinical settings. It is generally regarded as a safe procedure with high diagnostic accuracy. However, a complication involving a needle fracture that penetrated a nearby artery has not been reported during this procedure. CASE PRESENTATION: A male patient, 58 years of age, underwent endobronchial ultrasound-guided transbronchial needle aspiration for a mediastinal lymph node biopsy at a local hospital. The aspiration needle fractured and penetrated from the right middle segmental bronchus into the right pulmonary artery. The patient was then transferred to our hospital. After conducting repeated chest imaging examinations to confirm the presence of the foreign body and holding multidisciplinary team consultations, we first inserted a deflated balloon catheter near the puncture site in the right middle segmental bronchus. Following the needle retrieval through a flexible bronchoscope, the balloon catheter was inflated to ensure local hemostasis. Follow-up evaluations revealed no further complications for this patient. CONCLUSION: Intragenic vascular injury can occur during endobronchial ultrasound-guided transbronchial needle aspiration. Careful pre-procedure preparations should be planned to minimize complications. In patients experiencing complications due to needle penetration, consultation and coordination with a multidisciplinary team are essential to ensure the safe retrieval of the broken needle.

Comparative analysis of single-cell genome sequencing techniques toward the characterization of germline and somatic genomes in ciliated protists.

Ciliated protists contain both germline micronucleus (MIC) and somatic macronucleus (MAC) in a single cytoplasm. Programmed genome rearrangements occur in ciliates during sexual processes, and the extent of rearrangements varies dramatically among species, which lead to significant differences in genomic architectures. However, genomic sequences remain largely unknown for most ciliates due to the difficulty in culturing and in separating the germline from the somatic genome in a single cell. Single-cell whole genome amplification (WGA) has emerged as a powerful technology to characterize the genomic heterogeneity at the single-cell level. In this study, we compared two single-cell WGA, multiple displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC) in characterizing the germline and somatic genomes in ciliates with different genomic architectures. Our results showed that: 1) MALBAC exhibits strong amplification bias towards MAC genome while MDA shows bias towards MIC genome of ciliates with extensively fragmented MAC genome; 2) both MDA and MALBAC could amplify MAC genome more efficiently in ciliates with moderately fragmented MAC genome. Moreover, we found that more sample replicates could help to obtain more genomic data. Our work provides a reference for selecting the appropriate method to characterize germline and somatic genomes of ciliates.

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.

Key determinants of misdiagnosis of tracheobronchial tuberculosis among senile patients in contemporary clinical practice: A retrospective analysis.

BACKGROUND: Tracheobronchial tuberculosis (TBTB) is a common subtype of pulmonary tuberculosis. Concomitant diseases often obscure the diagnosis of senile TBTB. AIM: To characterize senile patients with TBTB and to identify the potential causes of misdiagnosis. METHODS: One hundred twenty patients with senile TBTB who were admitted to the Anhui Chest hospital between May 2017 and May 2019 were retrospectively analyzed. Patients were classified as diagnosed group (n = 58) and misdiagnosed group (n = 62). Clinical manifestations, laboratory results, radiographic data, and endoscopic findings were compared between the two groups. RESULTS: Patients in the misdiagnosed group were most commonly diagnosed as pulmonary tuberculosis (non-TBTB, 29/62, 46.8%), general pneumonia (9/62, 14.5%), chronic obstructive pulmonary disease (8/62, 12.9%), and tracheobronchial carcinoma (7/62, 11.3%). The time elapsed between disease onset and confirmation of diagnosis was significantly longer in the misdiagnosed group [median (first quartile, third quartile): 6.32 (4.94, 16.02) mo vs 3.73 (2.37, 8.52) mo]. The misdiagnosed group had lower proportion of patients who underwent bronchoscopy [33.87% (21/62) vs 87.93% (51/58)], chest computed tomography (CT) scan [69.35% (43/62) vs 98.28% (57/58)], and those who showed CT signs of tuberculosis [27.91% (12/62) vs 50% (29/58)] as compared to that in the diagnosed group (P < 0.05). There were no significant between-group differences with respect to age, gender, occupation, clinical manifestations, or prevalence of comorbid chronic diseases (P > 0.05). CONCLUSION: Insufficient or inaccurate radiographic or bronchoscopic assessment was the predominant cause of delayed diagnosis of TBTB. Increased implementation and better interpretation of CT scan and early implementation of bronchoscopy can help reduce misdiagnosis of senile TBTB.

A proper triage for detecting women with high-risk human papillomavirus genotypes other than HPV16/18.

OBJECTIVE: To evaluate the effect of human papillomavirus (HPV) L1 capsid protein detection in cervical exfoliated cells as a proper triage for women with high-risk human papillomavirus (hrHPV) genotypes other than HPV 16/18. STUDY DESIGN: From January 2013 to June 2015, a total of 513 women aged 30-65 years infected with non-16/18 hrHPV were enrolled into the study. Primary HPV testing, HPV 16/18 genotyping and Papanicolaou (Pap) test were performed in all eligible women. HPV L1 capsid protein was detected by immunocytochemistry in cervical exfoliated cells. All hrHPV positive women underwent colposcopy and further biopsy if indicated. Relationships between HPV L1 capsid protein expression and histologic diagnosis, as well as cytology were analyzed. RESULTS: The positive expression rate of HPV L1 capsid protein in CIN2+ group was significantly lower than that in CIN1 or better group (16.3% vs. 66.7%, P=0.000). Compared with the Pap test, HPV L1 detection achieved higher sensitivity (83.7% vs. 51.2%, P=0.000), higher negative predictive value (NPV) (95.3% vs. 88.6%, P=0.002), and significant lower specificity (66.7% vs.76.1%, P=0.002) while identifying CIN2+. Compared with the Pap test, HPV L1 detection achieved higher sensitivity (89.7% vs. 51.7%, P=0.008), higher NPV (99.0% vs. 96.2%, P=0.043), and significant lower specificity (61.2% vs.73.8%, P=0.000) while identifying CIN3+. CONCLUSION: Because of its higher sensitivity and NPV than that of Pap test, HPV L1 capsid protein detection in cervical exfoliated cells reduces the missed identification of high-grade cervical intraepithelial neoplasia (CIN) in women with hrHPV genotypes other than HPV 16/18. HPV L1 capsid protein detection could be a potential triage for women with non-16/18 hrHPV infection.

[Clinical significance of HPV L1 capsid protein detection in cervical exfoliated cells in high-risk HPV positive women].

OBJECTIVE: To explore the clinical significance of human papillomavirus L1 capsid protein detection in cervical exfoliated cells in high-risk HPV positive women. METHODS: From November 2012 to June 2013, 386 high-risk HPV positive (detected by hybrid capture II) cases were enrolled as eligible women from Huzhou Maternity & Child Care Hospital and Women's Hospital, School of Medicine, Zhejiang University. All eligible women underwent liquid-based cytology (ThinPrep) followed by colposcopy. Biopsies were taken if indicated. Cervical exfoliated cells were collected for HPV L1 capsid protein detection by immunocytochemistry. Expression of HPV L1 capsid protein in groups with different histological diagnosis were compared, and the role of HPV L1 capsid protein detection in cervical exfoliated cells in cervical lesions screening was accessed. RESULTS: Total 386 enrolled eligible women were finally diagnosed histologically as follwed: 162 normal cervix, 94 low-grade squamous intraepithelial lesion (LSIL), 128 high-grade squamous intraepithelial lesion (HSIL) and 2 squamous cervical cancer (SCC). The positive expression rate of HPV L1 in HSIL+ (HSIL or worse) group was significantly lower than that in LSIL- (LSIL or better) group (19.2% vs 66.4%, P=0.000). While identifying HSIL+ in HPV positive cases and compared with cytology, HPV L1 detection resulted in significant higher sensitivity (80.77% vs 50.77%, P=0.000) and negative predictive value (NPV; 87.18% vs 76.47%, P=0.004), significant lower specificity (66.41% vs 81.25%, P=0.000), and comparable positive predictive value (PPV; 54.97% vs 57.89%, P=0.619). To identify HSIL+ in HPV-positive/cytology-negative women, the sensitivity, specificity, PPV, and NPV of HPV L1 detection were 87.50%, 61.54%, 41.18%, and 94.12% respectively, while 80.00%, 86.36%, 80.00% and 86.36% respectively in HPV-positive/atypical squamous cell of undetermined significance (ASCUS) women. CONCLUSIONS: HPV L1 capsid detection in cervical exfoliated cells have a role in cervical lesions screening in high-risk HPV positive women, and may be a promising triage for high-risk HPV-positive/cytology-negative or ASCUS women.