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.

Application of RNA interference and protein localization to investigate housekeeping and developmentally regulated genes in the emerging model protozoan Paramecium caudatum.

Unicellular eukaryotes represent tremendous evolutionary diversity. However, the molecular mechanisms underlying this diversity remain largely unexplored, partly due to a limitation of genetic tools to only a few model species. Paramecium caudatum is a well-known unicellular eukaryote with an unexpectedly large germline genome, of which only two percent is retained in the somatic genome following sexual processes, revealing extensive DNA elimination. However, further progress in understanding the molecular mechanisms governing this process is hampered by a lack of suitable genetic tools. Here, we report the successful application of gene knockdown and protein localization methods to interrogate the function of both housekeeping and developmentally regulated genes in P. caudatum. Using these methods, we achieved the expected phenotypes upon RNAi by feeding, and determined the localization of these proteins by microinjection of fusion constructs containing fluorescent protein or antibody tags. Lastly, we used these methods to reveal that P. caudatum PiggyMac, a domesticated piggyBac transposase, is essential for sexual development, and is likely to be an active transposase directly involved in DNA cleavage. The application of these methods lays the groundwork for future studies of gene function in P. caudatum and can be used to answer important biological questions in the future.

Activation of METTL3 Promotes White Adipose Tissue Beiging and Combats Obesity.

The induction of beige adipocytes in white adipose tissue (WAT), also known as WAT beiging, improves glucose and lipid metabolism. However, the regulation of WAT beiging at the posttranscriptional level remains to be studied. Here, we report that METTL3, the methyltransferase of N6-methyladenosine (m6A) mRNA modification, is induced during WAT beiging in mice. Adipose-specific depletion of the Mettl3 gene undermines WAT beiging and impairs the metabolic capability of mice fed with a high-fat diet. Mechanistically, METTL3-catalyzed m6A installation on thermogenic mRNAs, including Krüppel-like factor 9 (Klf9), prevents their degradation. Activation of the METTL3 complex by its chemical ligand methyl piperidine-3-carboxylate promotes WAT beiging, reduces body weight, and corrects metabolic disorders in diet-induced obese mice. These findings uncover a novel epitranscriptional mechanism in WAT beiging and identify METTL3 as a potential therapeutic target for obesity-associated diseases. ARTICLE HIGHLIGHTS: METTL3, the methyltransferase of N6-methyladenosine (m6A) mRNA modification, is induced during WAT beiging. Depletion of Mettl3 undermines WAT beiging and impairs thermogenesis. METTL3-mediated m6A installation promotes the stability of Krüppel-like factor 9 (Klf9). KLF9 rescues impaired beiging elicited by Mettl3 depletion. Pharmaceutical activation of the METTL3 complex by its chemical ligand methyl piperidine-3-carboxylate induces WAT beiging. Methyl piperidine-3-carboxylate corrects obesity-associated disorders. The METTL3-KLF9 pathway may serve as a potential therapeutic target for obesity-associated diseases.

Adipocyte YTH N(6)-methyladenosine RNA-binding protein 1 protects against obesity by promoting white adipose tissue beiging in male mice.

Obesity, one of the most serious public health issues, is caused by the imbalance of energy intake and energy expenditure. N(6)-methyladenosine (m6A) RNA modification has been recently identified as a key regulator of obesity, while the detailed mechanism is elusive. Here, we find that YTH RNA binding protein 1 (YTHDF1), an m6A reader, acts as an essential regulator of white adipose tissue metabolism. The expression of YTHDF1 decreases in adipose tissue of male mice fed a high-fat diet. Adipocyte-specific Ythdf1 deficiency exacerbates obesity-induced metabolic defects and inhibits beiging of inguinal white adipose tissue (iWAT) in male mice. By contrast, male mice with WAT-specific YTHDF1 overexpression are resistant to obesity and shows promotion of beiging. Mechanistically, YTHDF1 regulates the translation of diverse m6A-modified mRNAs. In particular, YTHDF1 facilitates the translation of bone morphogenetic protein 8b (Bmp8b) in an m6A-dependent manner to induce the beiging process. Here, we show that YTHDF1 may be an potential therapeutic target for the management of obesity-associated diseases.

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.

ACSL4 promotes microglia-mediated neuroinflammation by regulating lipid metabolism and VGLL4 expression.

Acyl-CoA synthetase long-chain family member 4 (ACSL4) is an important isozyme in polyunsaturated fatty acid (PUFA) metabolism. The role of ACSL4 in the lipopolysaccharide (LPS)-induced inflammation of microglia, and the effects of ACSL4-mediated inflammation on the progression of Parkinson's disease (PD) are unknown. In this study, we found that ACSL4 expression was increased after LPS stimulation. Knocking down ACSL4 in microglia decreased proinflammatory cytokine production. Mechanistically, ACSL4 reduced vestigial-like family member 4(VGLL4) expression to promote NF-κB signal transduction; and ACSL4 regulated lipid composition after LPS stimulation. In addition, knocking down ACSL4 alleviated neuroinflammation in a systemic LPS model and acute l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) model. These data revealed ACSL4 to be a novel regulator that promotes microglia-mediated neuroinflammation by regulating VGLL4 expression and lipid metabolism.

Global mortality of diabetic foot ulcer: A systematic review and meta-analysis of observational studies.

AIM: To estimate the long-term mortality and risk factors in patients with diabetic foot ulcer (DFU). METHODS: We systematically searched Medline (PubMed), Embase, Scopus, Web of Science, Cochrane Library, China Science and Technology Journal Database (CQVIP), China National Knowledge Infrastructure, the Chinese Biomedical Literature Database (SinoMed) and Wanfang Data from 1 January 2011 to 31 July 2022. All observational studies that reported long-term mortality of patients with DFU were included. Random effect models were used to pool the reconstructed participant data from Kaplan-Meier curves. The primary outcome was the long-term survival of patients with DFU. An aggregate data meta-analysis was also performed. RESULTS: We identified 34 studies, with 124 376 participants representing 16 countries, among whom there were 51 386 deaths. Of these, 27 studies with 21 171 patients were included in the Kaplan-Meier-based meta-analysis. The estimated Kaplan-Meier-based survival rates were 86.9% (95% confidence interval [CI] 82.6%-91.5%) at 1 year, 66.9% (95% CI 59.3%-75.6%) at 3 years, 50.9% (95% CI 42.0%-61.7%) at 5 years and 23.1% (95% CI 15.2%-34.9%) at 10 years. The results of the aggregate data-based meta-analysis were similar. Cardiovascular disease and infection were the most common causes of death, accounting for 46.6% (95% CI 33.5%-59.7%) and 24.8% (95% CI 16.0%-33.5%), respectively. Patients with older age (per 1 year, hazard ratio [HR] 1.054, 95% CI 1.045-1.063), peripheral artery disease (HR 1.882, 95% CI 1.592-2.225), chronic kidney disease (HR 1.535, 95% CI 1.227-1.919), end-stage renal disease (HR 3.586, 95% CI 1.333-9.643), amputation (HR 2.415, 95% CI 1.323-4.408) and history of cardiovascular disease (HR 1.449, 95% CI 1.276-1.645) had higher mortality risk. CONCLUSIONS: This meta-analysis found that the overall mortality of DFU was high, with nearly 50% mortality within 5 years. Cardiovascular disease and infection were the two leading causes of death.

Small RNA-mediated genome rearrangement pathways in ciliates.

Most eukaryotes employ a combination of transcriptional and post-transcriptional silencing mechanisms to suppress transposons, yet ciliates employ a more extreme approach. They separate germline and somatic functions into distinct nuclei, enabling the elimination of transposons from the active somatic genome through diverse small RNA-mediated genome rearrangement pathways during sexual processes.

[Experimental Study of Using Aloe Vera Gel to Treat Diabetic Chronic Cutaneous Ulcers in Bama Miniature Pigs].

Objective: To investigate the efficacy, safety, and mechanism of topical application of aloe vera gel (AVG) to treat diabetic chronic cutaneous ulcers in Bama miniature pigs. Methods: The Bama miniature pig model of diabetic chronic skin wounds was constructed and the model pigs were randomly assigned to AVG daily administration group (AVG QD), aloe vera gel every-other-day administration group (AVG QOD), and diabetic control group (DC). A non-diabetic chronic skin wounds model pig was set as the non-diabetic control group (NDC). Treatment efficacy was evaluated based on the amount of time needed for complete healing of the wounds, healing rates, granulation growth rates, and skin histopathological changes. Safety was evaluated according to whether adverse reactions were observed. In addition, the dynamic changes of the relative expression levels of miR21, miR29a, miR126, miR146a, miR155, and miR210 in wound granulation tissues were examined. Results: 1) Efficacy and safety: The amount of time needed for complete healing of the wounds was shorter in the NDC group than those of the three other groups, DC group, AVG QD group, and AVG QOD group (all P<0.05). The amount of time needed for complete healing of the wounds was shorter in the AVG QD group and AVG QOD group than that of DC group (all P<0.05). The amount of time needed for complete healing of the wounds was shorter in the AVG QOD group than that of AVG QD group (all P<0.05). No adverse reactions were detected in the whole process of AVG topical treatment. The granulation growth rate of NDC group was higher than those of DC group, AVG QD group, and AVG QOD group (all P<0.05). The wound healing rate of NDC group was higher than those of DC group, AVG QD group, and AVG QOD group (all P<0.05); the wound healing rate of AVG QOD group was higher than those of DC group and AVG QD group (all P<0.05). 2) Histopathology: The results of HE staining light microscopy showed that collagen fiber production increased, and that microvascular formation with slight inflammatory cell infiltration was observed in the dermal interstitium at the initial stage of wound healing after AVG treatment. One year of after complete healing, pathological examination results of wound healing skin showed that the epidermal keratinization was complete, that collagen was arranged neatly and orderly, and that many microvessels were found in the interstitium. The results of picric acid celestite scarlet staining showed that, after AVG treatment, type Ⅰ collagen mainly increased in the initial stage of wound healing, type Ⅲ collagen gradually increased when the wound healed completely, and the collagen was arranged neatly during the whole process. 3) The relative expression of microRNAs: The relative expression of miR21, miR126, and miR210 in NDC group, AVG QD group, and AVG QOD group were higher than that in DC group (all P<0.05). The relative expression of miR29a and miR155 in NDC group, AVG QD group, and AVG QOD group was lower than that in DC group (all P<0.05). The relative expression of miR146a in NDC group was higher than that in DC group ( P<0.05). Conclusion: AVG topical application can shorten the time needed for complete healing of diabetic chronic wounds in Bama minipigs. The wound healing speed of the alternate-day treatment group was faster than that of the daily treatment group. No adverse reactions were observed over the course of the treatment. The mechanism may be related to the up-regulation of the expressions of miR21, miR126, and miR210 and the down-regulation of miR29a and miR155 in wound granulation tissue.

Transcranial Direct-Current Stimulation Regulates MCT1-PPA-PTEN-LONP1 Signaling to Confer Neuroprotection After Rat Cerebral Ischemia-Reperfusion Injury.

Propionic acid (PPA) is a critical metabolite involved in microbial fermentation, which functions to reduce fat production, inhibit inflammation, and reduce serum cholesterol levels. The role of PPA in the context of cerebral ischemia-reperfusion (I/R) injury has yet to be clarified. Increasing evidence indicate that transcranial direct-current stimulation (tDCS) is a safe approach that confers neuroprotection in cerebral ischemia injury. Here, we show that the levels of PPA were reduced in the ischemic brain following a rat cerebral I/R injury and in the cultured rat cortical neurons after oxygen-glucose deprivation (OGD), an in vitro model of ischemic injury. We found that the decreased levels of transporter protein monocarboxylate transporter-1 (MCT1) were responsible for the OGD-induced reduction of PPA. Supplementing PPA reduced ischemia-induced neuronal death after I/R. Moreover, our results revealed that the neuroprotective effect of PPA is mediated through downregulation of phosphatase PTEN and subsequent upregulation of Lon protease 1 (LONP1). We demonstrated that direct-current stimulation (DCS) increased MCT1 expression and PPA level in OGD-insulted neurons, while tDCS decreased the brain infarct volume in the MCAO rats via increasing the levels of MCT1 expression and PPA. This study supports a potential application of tDCS in ischemic stroke.

Severe Intermittent Hypoxia Modulates the Macrophage Phenotype and Impairs Wound Healing Through Downregulation of HIF-2α.

Background: Obstructive sleep apnea is prevalent in patients with diabetic foot ulcers, while the effect of intermittent hypoxia on wound healing is unclear. The objective of this study was to investigate the effect of severe intermittent hypoxia on wound healing. Methods: C57BL/6 mice were exposed to 5 weeks of severe intermittent hypoxia or normoxia. The wound healing rate were assessed. The gene expression of CD206 and HIF-2α was tested in vivo and in vitro. Inflammatory factors in RAW264.7 macrophages were measured to investigate the effect of intermittent hypoxia on macrophage polarization. The proliferation of HUVECs and HaCaT cells was also assessed after exposure to intermittent hypoxia. Results: Severe intermittent hypoxia decreased wound healing at day 3. The expression of CD206 and HIF-2α was significantly decreased after exposure to severe intermittent hypoxia. In vitro, severe intermittent hypoxia significantly promoted M1 phenotype polarization of RAW264.7 macrophages and increased the expression of proinflammatory factors (IL-1ß and TNF-α). Severe intermittent hypoxia also decreased the proliferation of HUVECs cultured in endothelial cell medium and HaCaT cells cultured in high glucose DMEM. Conclusion: Severe intermittent hypoxia could lead to M1 but not M2 macrophage polarization through downregulation of HIF-2α, and then lead to impaired wound healing.

A novel isoform of hydroxyacyl-CoA dehydrogenase inhibits cell proliferation.

Hydroxyacyl-CoA dehydrogenase (HADH) catalyzes the third reaction of mitochondrial ß-oxidation cascade, while the regulation of its expression and function remains to be elucidated. Using the quantitative translation initiation sequencing (QTI-seq), we have identified that murine Hadh mRNA has two alternative translation start codons. We demonstrated that translation from upstream start codon encodes the mitochondrial isoform of HADH, while translation from downstream start codon produces a short isoform (HADH-S) with predominant nuclear localization. Moreover, overexpression of HADH-S inhibits the proliferation of mouse embryonic fibroblasts. Overall, our results identify a novel isoform of HADH participating in cell proliferation.

Efficacy and Safety of Pentoxifylline for Venous Leg Ulcers: An Updated Meta-Analysis.

The venous leg ulcers are debilitating, painful, and often unresponsive to advanced dressing treatments, so drugs used locally and systematically are essential adjuvant therapy-pentoxifylline (PTX) whose anti-inflammatory effects may offer a promising avenue to treat venous leg ulcers. However, the current results are controversial. To further evaluate the efficacy and safety of PTX, we performed an updated meta-analysis of randomized placebo-controlled trials of PTX in the treatment of venous leg ulcers. We systematically searched multiple electronic databases PubMed, Web of Science, Embase, the Cochrane Library, the Cochrane Central Register of Controlled Trials, China Science and Technology Journal Database, WanFang Data, China National Knowledge Infrastructure, and the Chinese Biomedical Literature Database to identify eligible studies. Randomized clinical trials of pentoxifylline versus placebo treatment in patients with venous leg ulcers were considered for inclusion. The primary outcomes included ulcer healing rate and the incidence of adverse events after treatment. The secondary outcomes were the ulcer significant improvement (the ulcer size shrank by more than 60% after treatment) rate, mean duration of complete wound healing and changes in mean ulcer size. A meta-analysis and qualitative analysis were conducted to estimate endpoints. A total of 13 randomized clinical trials, including 921 individuals, were finally included. Compared with placebo, pentoxifylline significantly improved the ulcer healing rate (RR = 1.59, 95%CI 1.22 to 2.07, P < .001) and significant improvement rate (RR = 2.36, 95%CI 1.31 to 4.24, P = .004) while increased the incidence of gastrointestinal disturbances (RR = 2.29, 95%CI 1.04 to 5.03, P = .04) at the same time. Moreover, pentoxifylline also shortened mean duration of complete wound healing (P = .007) and shrank ulcer size (P = .02). Currently available evidence suggests that pentoxifylline could help venous leg ulcers heal more quickly and effectively. However, the evidence is insufficient to prove the results due to moderate-certainty evidence. Large-scale, well-designed randomized clinical trials are warranted.

A new approach for investigating the relative contribution of basal glucose and postprandial glucose to HbA1C.

To develop an accurate method for evaluating the relative contributions of basal glucose (BG) and postprandial glucose (PPG) to glycated haemoglobin (HbA1c) in subjects with hyperglycaemia using a Continuous Glucose Monitoring System (CGMS®). The subjects were divided into the normal glucose tolerance (NGT), impaired glucose tolerance (IGT), newly-diagnosed type 2 diabetes (NDDM), and drug-treated type 2 diabetes (T2DM) groups. We evaluated the relative contributions of BG and PPG to HbA1c in patients with hyperglycaemia according to three different baseline values. Subjects (n = 490) were grouped as follows: 92 NGT, 36 IGT, 131 NDDM, and 231 T2DM. The relative contributions of PPG to HbA1c were calculated using baseline values of 6.1 mmol/L, 5.6 mmol/L, and the 24-h glucose curve of the NGT group. The relative contribution of PPG to HbA1c decreased progressively from the IGT group to the T2DM group. Compared with the 24-h glucose curve as the baseline, the relative contribution of PPG was overestimated in 9.04% and 1.76% of the subjects when 6.1 mmol/L and 5.6 mmol/L were used as baselines, respectively (P < 0.01), in T2DM patients. The 24-h glucose curve of NGT is more suitable for studying the relative contributions of BG and PPG to HbA1c and it is more precise, as it considers physiological fluctuations in NGT after meals. However, 5.6 mmol/L can be used when the 24-h glucose curve for NGT is unavailable; using 6.1 mmol/L as a baseline value may overestimate the contribution to the HbA1c. There is no unified standard for assessing the contributions of basal glucose (BG) and postprandial glucose (PPG) to HbA1c. The 24-h glucose curve of NGT is more suitable for studying the relative contributions of BG and PPG to HbA1c, as it considers physiological fluctuations in NGT after meals. However, 5.6 mmol/L can be used when the 24-h glucose curve for NGT is unavailable; using 6.1 mmol/L as a baseline value may overestimate the contribution to the HbA1c.

Extremity Gangrene Caused by HBV-Related Cryoglobulinemia Vasculitis in a Patient with Diabetes - A Case Report.

We presented a case of hepatitis B virus (HBV)-related type III cryoglobulinemia vasculitis (CryoVas) characterized by extremity gangrene in a patient with diabetes. The 60-year-old female had a 10-year history of poorly controlled type 2 diabetes mellitus. She complained of sudden onset pain and swelling of toes which quickly progressed to gangrene, with fingers becoming pain and dark violet. The patient was initially misdiagnosed as diabetic foot (DF). Although DF is one of the common chronic complications of diabetes, it rarely involves the hand. What is more, the ischemic manifestations of the extremity were not consistent with the results of the vascular examination and immune system changes. The patient had Raynaud's phenomenon, arthralgia, and extremity gangrene. Test results showed cryoglobulinemia multiple positive, polyclonal immunoglobulin with rheumatoid factor negative, lower complement 3, leukocytoclastic vasculitis, and HBV infection. HBV-related type III CryoVas was finally diagnosed, and a conservative therapy strategy was given. Six months after treatment with cyclophosphamide, corticosteroid, nucleoside/nucleotide analog therapy, local debridement, and dressing change, she recovered and kept no recurrence by following up for 30 months. To our knowledge, this is the first report of extremity gangrene caused by HBV-related CryoVas in a diabetic patient.

YTHDF1-regulated expression of TEAD1 contributes to the maintenance of intestinal stem cells.

N6-methyladenosine (m6A) mRNA modification has been defined as a crucial regulator in various biological processes. Recent studies indicated an essential role of YTHDF1, an m6A reader, in the maintenance of intestinal stem cells (ISCs), while the detailed mechanism remains to be explored. By searching our m6A sequencing, RNA sequencing, and ribosome profiling data, we identified the transcriptional enhanced associate domain 1 (TEAD1) as a direct target of YTHDF1. We confirmed the presence of m6A modifications in TEAD1 mRNA and its binding with YTHDF1. Knockdown of either m6A methyltransferase METTL3 or YTHDF1 reduced the translation of TEAD1. TEAD1 was highly expressed in ISCs, while depletion of TEAD1 inhibited proliferation and induced differentiation of organoids. Overexpression of TEAD1 reversed the impaired stemness elicited by YTHDF1 depletion. These findings identify TEAD1 as a functional target of m6A-YTHDF1 in sustaining intestinal stemness.

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.