Identification of Philaster apodigitiformis in aquaculture and functional characterization of its ß-PKA gene and expression analysis of infected Poecilia reticulata.

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) is a distinctive member of the serine­threonine protein AGC kinase family and an effective kinase for cAMP signal transduction. In recent years, scuticociliate has caused a lot of losses in domestic fishery farming, therefore, we have carried out morphological and molecular biological studies. In this study, diseased guppies (Poecilia reticulata) were collected from an ornamental fish market, and scuticociliate Philaster apodigitiformis Miao et al., 2009 was isolated. In our prior transcriptome sequencing research, we discovered significant expression of the ß-PKA gene in P. apodigitiformis during its infection process, leading us to speculate its involvement in pathogenesis. A complete sequence of the ß-PKA gene was cloned, and quantified by quantitative reverse transcription-polymerase chain reaction to analyse or to evaluate the functional characteristics of the ß-PKA gene. Morphological identification and phylogenetic analysis based on small subunit rRNA sequence, infection experiments and haematoxylin­eosin staining method were also carried out, in order to study the pathological characteristics and infection mechanism of scuticociliate. The present results showed that: (1) our results revealed that ß-PKA is a crucial gene involved in P. apodigitiformis infection in guppies, and the findings provide valuable insights for future studies on scuticociliatosis; (2) we characterized a complete gene, ß-PKA, that is generally expressed in parasitic organisms during infection stage and (3) the present study indicates that PKA plays a critical role in scuticociliate when infection occurs by controlling essential steps such as cell growth, development and regulating the activity of the sensory body structures and the irritability system.

Identification of a Tetrahymena species infecting guppies, pathology, and expression of beta-tubulin during infection.

Tetrahymenosis is caused by the ciliated protozoan Tetrahymena and is responsible for serious economic losses to the aquaculture industry worldwide. However, information regarding the molecular mechanism leading to tetrahymenosis is limited. In previous transcriptome sequencing work, it was found that one of the two ß-tubulin genes in T. pyriformis was significantly expressed in infected fish, we speculated that ß-tubulin is involved in T. pyriformis infecting fish. Herein, the potential biological function of the ß-tubulin gene in Tetrahymena species when establishing infection in guppies was investigated by cloning the full-length cDNA of this T. pyriformis ß-tubulin (BTU1) gene. The full-length cDNA of T. pyriformis BTU1 gene was 1873 bp, and the ORF occupied 1134 bp, whereas 5' UTR 434 bp, and 3' UTR 305 bp whose poly (A) tail contained 12 bases. The predicted protein encoded by T. pyriformis BTU1 gene had a calculated molecular weight of 42.26 kDa and pI of 4.48. Moreover, secondary structure analysis and tertiary structure prediction of BTU1 protein were also conducted. In addition, morphology, infraciliature, phylogeny, and histopathology of T. pyriformis isolated from guppies from a fish market in Harbin were also investigated. Furthermore, qRT-PCR analysis and experimental infection assays indicated that the expression of BTU1 gene resulted in efficient cell proliferation during infection. Collectively, our data revealed that BTU1 is a key gene involved in T. pyriformis infection in guppies, and the findings discussed herein provide valuable insights for future studies on tetrahymenosis.

Impact of Age at Diagnosis of Bladder Cancer on Survival: A Surveillance, Epidemiology, and End Results-Based Study 2004-2015.

BACKGROUND: An older age contributes to the development of bladder cancer. However, the relationship between advanced age at the diagnosis and prognosis of bladder cancer has been few reported. This study aimed to determine the effect of age on survival in bladder cancer with different subgroups. METHODS: 117,275 patients with bladder cancer, identified from the Surveillance, Epidemiology, and End Results database during 2004-2015 in America, were divided into 4 age groups (≤54, 55 to 64, 65 to 74, and ≥75 years). Multivariable Cox proportional-hazards model and competing risk model were conducted according to different age groups. Heat maps were plotted to show the impact of age on survival in subgroups classified by other clinicopathological variables. Moreover, restricted cubic spline was used to model the association between age and the risk of death. RESULTS: Patients aged ≥75 years had shorter overall survival in comparison with those aged ≤54 years (hazard ratio [HR] = 5.36, 95% confidence interval [CI] = 5.13-5.59). Compared with patients aged ≤54 years, patients older than 75 years experienced a decreased rate of bladder cancer-specific survival (subdistribution HR = 2.15, 95% CI = 2.04-2.25). Heat maps also showed that older ages were associated with worse overall cumulative mortality and bladder cancer-specific cumulative mortality. Similarly, restricted cubic spline verified the impact of age on survival of bladder cancer. CONCLUSIONS: Age at diagnosis of bladder cancer was found to be a significant predictor for the worse overall survival and bladder cancer-specific survival even in an era with more effective therapies. Exploring the reasons why older age contributes to poor outcomes for bladder cancer will be the focus of future research.

Accuracy of Non-Contrast MRI for the Detection of Hepatocellular Carcinoma: A systematic review and meta-analysis.

Non-contrast MRI is used for identifying patients with hepatocellular carcinoma (HCC), especially among high-risk patients with cirrhosis or chronic viral hepatitis. The accuracy of non-contrast MRI has been investigated with varying results. We performed this meta-analysis to consolidate the evidence on the accuracy of non-contrast MRI for the detection of HCC. We conducted a systematic search in the databases of PubMed Central, SCOPUS, MEDLINE, EMBASE and Cochrane from inception till November 2020. We used the STATA software "Midas" package for meta-analysis. We included 15 studies with 3,756 patients. The pooled sensitivity and specificity of non-contrast MRI for HCC detection were 84% (95%CI, 78%-88%) and 94% (95%CI, 91%-97%). The positive likelihood ratio was 14.9 (95% CI, 9.0-24.7) and the negative one 0.17 (0.12-0.23). The overall quality of the studies was high. We found significant heterogeneity based on chi-square test results and I2 statistic > 75%. Deek's test showed the absence of publication bias. We found that non-contrast MRI has high sensitivity and specificity as a tool for detecting HCC. Studies exploring its accuracy in different ethnic populations are required to strengthen the evidence.

Further insights into the phylogeny of peniculid ciliates (Ciliophora, Oligohymenophorea) based on multigene data.

Peniculids comprise a large order of ciliated protists in Class Oligohymenophorea having many unresolved evolutionary relationships. Herein, we report 27 new sequences, including 18S rRNA, ITS1-5.8S- ITS2 rRNA, 28S rRNA and the mitochondrial cox1 genes of eight peniculids. We conducted phylogenetic analyses based on each these markers and on a four-gene concatenated data set (18S rRNA, ITS1-5.8S- ITS2 rRNA, 28S rRNA, and cox1 gene). The main findings are: 1) subclass Peniculia and family Parameciidae are monophyletic, with genus Frontonia remaining non-monophyletic; 2) Urocentrids have traditionally been regarded as a family, multi-gene analyses support the rank of Urocentrida and consistently recovers this order as sister to Peniculida, and Urocentrida and Peniculida comprise subclass Peniculia in agreement with Lynn's (2008) classification; 3) discrepancies between multiple-gene phylogenies, and conflicts with morphologic data regarding genus Frontonia necessitate expansion and revision of species diagnoses and we propose consideration of Group III of Frontonia (including F. didieri, F. ocularis, F. anatolica, F. pusilla and F. elegans) as incertae sedis in Peniculida; 4) multi-gene analyses of Parameciidae support five previously established subgenera. Paramecium buetschlii is placed in subgenus Chloroparamecium, and P. chlorelligerum into subgenus Viridoparamecium.

Taxonomy and molecular phylogeny of a new freshwater ciliate Frontonia apoacuminata sp. nov. (Protista, Ciliophora, Oligohymenophorea) from Qingdao, PR China.

The morphology and ciliature of a new freshwater ciliate, Frontonia apoacuminata sp. nov., isolated from an artificial pond in Qingdao, PR China, were investigated using live observation and silver staining methods. The main features separating F. apoacuminata sp. nov. from its congeners are as follows: a broad elliptical body that is slightly pointed at the posterior end, four ophryokineties, one or two spherical micronuclei of a 'compact' type, a dorsally positioned contractile vacuole, and peniculi 1-3 each with five kinetosome rows though the left-most two rows in peniculus 3 are extremely shortened (with only two or three kinetosomes each). Additionally, an improved diagnosis of F. acuminata is provided. Phylogenetic analyses based on the small subunit ribosomal RNA (SSU rRNA) gene show that F. apoacuminata sp. nov. clusters with F. atra, F. minuta, F. acuminata and F. terricola. These five species group with Disematostoma, Marituja and Stokesia rather than with other Frontonia species, causing polyphyly of the genus Frontonia.

Redescription of a Hymenostome Ciliate, Tetrahymena setosa (Protozoa, Ciliophora) Notes on its Molecular Phylogeny.

In recent years, Tetrahymena species have been used as model organisms for research in a wide range of fields, highlighting the need for a fuller understanding of the taxonomy of this group. It is in this context that this paper uses living observation and silver staining methods to investigate the morphology and infraciliature of one Tetrahymena species, T. setosa (Schewiakoff 1892 Verh. Naturh. Med. Ver. Heidelb., 4:544) McCoy (1975) Acta Protozool., 14:253; the senior subjective synonym of T. setifera Holz and Corliss (1956) J. Protozool., 3:112; isolated from a freshwater pond in Harbin, north-eastern China. This organism can be distinguished from other described Tetrahymena species mainly by its single caudal cilium, which is about twice the length of the somatic ciliature. While the Harbin isolate appears similar to the population described by Holz and Corliss (1956) J. Protozool., 3:112, an improved diagnosis for T. setosa is given based on the previous descriptions and the Harbin population. In summary, this species can be recognized mainly by the combination of the following characters: body in vivo approximately 40 µm × 25 µm, 21-26 somatic kineties, one to four contractile vacuole pores associated with meridians 6-11 and a single caudal cilium. The small subunit ribosomal (SSU) rRNA gene and the cox1 gene sequences of Harbin population are also characterized in order to corroborate that the isolated species branches in phylogenetic trees as a T. setosa species. The phylogenetic analysis also indicated that sequences of populations of Tetrahymena species should be published with detailed morphological identifications.

One freshwater species of the genus Cyclidium, Cyclidiumsinicum spec. nov. (Protozoa; Ciliophora), with an improved diagnosis of the genus Cyclidium.

The morphology and infraciliature of one freshwater ciliate, Cyclidium sinicum spec. nov., isolated from a farmland pond in Harbin, northeastern China, was investigated using living observation and silver staining methods. Cyclidium sinicum spec. nov. could be distinguished by the following features: body approximately 20-25×10-15 µm in vivo; buccal field about 45-50 % of body length; 11 somatic kineties; somatic kinety n terminating sub-caudally; two macronuclei and one micronucleus; M1 almost as long as M2; M2 triangle-shaped. The genus Cyclidium is re-defined as follows: body outline usually oval or elliptical, ventral side concave, dorsal side convex; single caudal cilium; contractile vacuole posterior terminal; adoral membranelles usually not separated; paroral membrane 'L'-shaped, with anterior end terminating at the level of anterior end of M1; somatic kineties longitudinally arranged and continuous. Phylogenetic trees based on the SSU rDNA sequences showed that C. sinicum spec. nov. clusters with the type species, Cyclidiumglaucoma, with full support. Cyclidium is not monophyletic with members of the clade of Cyclidium+Protocyclidium+Ancistrum+Boveria.

Molecular Phylogeny and Taxonomy of a New Freshwater Hymenostomatid from Northeastern China, with the Establishment of a New Genus Anteglaucoma gen. n. (Protista, Ciliophora, Oligohymenophorea).

The morphology, infraciliature and SSU rDNA sequence of a new freshwater hymenostomatid ciliate, Anteglaucoma harbinensis gen. nov., spec. nov., collected from a farmland pond in Harbin, China, were investigated. The new genus Anteglaucoma is characterized as follows: small to medium-sized Glaucomidae with oral apparatus in anterior one-third of cell; paroral membrane composed of almost longitudinally arranged dikinetids; three adoral membranelles nearly equal in length and arranged almost longitudinally in parallel; silverline pattern tetrahymenid. The improved diagnosis of family Glaucomidae Corliss 1971 is provided based on the previous and present work. The type species Anteglaucoma harbinensis spec. nov. is defined by having 32-35 somatic kineties; four or five postoral kineties; membranelle 1 and membranelle 2 having five or six kinetosomal rows, membranelle 3 having three kinetosomal rows; single macronuclear nodule; contractile vacuole on average 15% from posterior body end; locomotion characterized by crawling with a rather hectic jerking motion; freshwater habitat. Phylogenetic analyses show that Anteglaucoma clusters in the family Glaucomidae and groups with the genera Glaucoma. The molecular and morphological data indicate that Glaucomidae is related to the family Bromeliophryidae in the phylogenetic trees.

Breviscapine Reduces Sepsis-Induced Acute Lung Injury by Targeting CASP8 to Regulate Neutrophil Apoptosis and Inflammation.

Background: Breviscapine has been demonstrated to have beneficial effects in ameliorating acute lung injury (ALI), yet its potential therapeutic value and molecular mechanisms in sepsis-induced ALI remain unexplored. Methods: We utilized network pharmacology approach to identify the potential targets and mechanisms of breviscapine in treating sepsis-induced ALI. To construct a murine model of sepsis, we performed cecal ligation and puncture (CLP). Hematoxylin and eosin (HE) staining and enzyme-linked immunosorbent assay (ELISA) were employed to respectively determine the pathologic changes and levels of inflammatory factors. Neutrophil count and total protein level in bronchoalveolar lavage fluid (BALF) were detected by corresponding kit. Additionally, we utilized flow cytometry, immunofluorescence, Western blotting, and real-time reverse transcription PCR (qRT-PCR) to detect cell apoptosis, protein expression, and gene expression. Finally, we used ELISA kits to detect the activity of myeloperoxidase (MPO) and caspase-8 (CASP8). Results: Breviscapine was revealed to target 81 potential proteins in the treatment of sepsis-induced ALI, while CASP8 was the most important one as demonstrated by network analysis. In vivo experiments demonstrated that breviscapine effectively reduced the severity of sepsis-induced ALI and inflammation, and significantly suppressed neutrophil infiltration in the lung tissues of CLP mice and promoted neutrophil apoptosis in the peripheral blood. In vitro experiments revealed that lipopolysaccharide (LPS)-induced neutrophil apoptosis was inhibited, and the expression and activity of CASP8 were down-regulated. Breviscapine intervention markedly up-regulated the expression and activity of CASP8, consequently activating neutrophil apoptosis and inhibiting inflammatory response by activating the NF-κB signaling pathway. Conclusion: Breviscapine is remarkably effective in improving sepsis-induced ALI, and its mechanism of action may be to induce neutrophil apoptosis, inhibit inflammatory overreaction and reduce its infiltration in pulmonary tissues by up-regulating the expression and activity of CASP8.

Morphology and small-subunit rRNA gene sequences of two novel marine ciliates, Metanophrys orientalis spec. nov. and Uronemella sinensis spec. nov. (Protista, Ciliophora, Scuticociliatia), with an improved diagnosis of the genus Uronemella.

The morphology and infraciliature of two novel marine scuticociliates, Metanophrys orientalis spec. nov. and Uronemella sinensis spec. nov., collected from sandy beaches at Qingdao, China, were investigated using live observation and protargol-staining methods. Metanophrys orientalis spec. nov. is distinguished by the following characteristics: marine habitat and a slender to elongate oval body with pointed anterior end and rounded caudal end, in vivo about 25-50 µm long; buccal field about a quarter to a third of body length; nine or ten somatic kineties with dikinetids approximately in anterior half of body, monokinetids in posterior half; membranelles 1 and 2 almost equal in length and composed of two and three longitudinal rows of kinetids respectively; paroral membrane with zigzag structure extending anteriorly to middle portion of membranelle 2; contractile vacuole pore located at posterior end of somatic kinety 1. The genus Uronemella is redefined as follows: marine form with an elongate-elliptical or inverted pear-shaped body; apical plate conspicuous; buccal field about two-thirds of body length, cytostome subequatorially located; oral apparatus Uronema-like; somatic kineties comprising a mixture of dikinetids and monokinetids. Uronemella sinensis spec. nov. is recognized by having an elongate-elliptical body with truncated apical frontal plate, size in vivo about 25-35 × 15-20 µm, nine or ten somatic kineties, membranelle 1 consisting of two or three basal bodies, contractile vacuole pore at posterior end of somatic kinety 1. This study also compared the small-subunit rRNA gene sequences of these two species with other closely related species to show the sequence divergence, which ranged from 3.53 to 9.60%. Phylogenetic analyses support the contention that the genus Uronemella is monophyletic, while Metanophrys is non-monophyletic.

Protargol synthesis: an in-house protocol.

The protargol staining method has proved to be indispensable for revealing the cellular structures of a variety of protozoa, especially the flagellates and ciliates. Protargol provides permanent stains of a variety of cellular structures: nuclei, extrusomes, basal bodies, and microfilamentous constituents of cells. Together with the older silver nitrate methods, protargol impregnations have provided the basis for the detailed descriptions of nearly all ciliates to date. The performance of commercially available preparations has varied widely. Recently, suppliers have stopped stocking the effective forms of protargol resulting in a worldwide shortage. Thus, it has become necessary for protistologists to explore on-site synthesis of this critically important agent. An optimum protocol for synthesis of protargol should be rapid, relatively inexpensive, simple enough to be done by non-chemists, and achievable without specialized equipment. In this article, the authors briefly review the interesting history of protargol and describe a protocol, based on the early studies of neuroanatomists, that yields a protargol producing impregnations of ciliates comparable to those obtained with previously available commercial preparations.

Further insights into the phylogeny of facultative parasitic ciliates associated with tetrahymenosis (Ciliophora, Oligohymenophorea) based on multigene data.

Tetrahymenosis, caused by about 10 Tetrahymena species, is an emerging problem inflicting a significant economic loss on the aquaculture industry worldwide. However, in the order Tetrahymenida, there are many unresolved evolutionary relationships among taxa. Here we report 21 new sequences, including SSU-rRNA, ITS1-5.8S-ITS2 rRNA and LSU-rRNA, genes of 10 facultative parasitic Tetrahymena associated with tetrahymenosis, and conduct phylogenetic analyses based on each individual gene and a three-gene concatenated dataset. The main findings are: (1) All the parasitic and facultative parasitic species cluster in borealis group. (2) With the addition of new sequences, Tetrahymena is still divided into three groups, namely the "borealis group", the "australis group," and the "paravorax group." (3) the cluster pattern of all the newly sequenced facultative parasitic Tetrahymena species shows that members of the "borealis" group may be more susceptible to parasitism. (4) phylogeny based on concatenated genes show that T. pyriformis, T. setosa, and T. leucophrys have close relationship.

Toxicological sensitivity of protozoa to pesticides and nanomaterials: A prospect review.

Protozoa are sensitive indicators of pollutant toxicity. This review presents and discusses the toxicological studies of protozoa and the toxicological conventional test species (Daphnia magna) by pesticides and nanomaterials, particularly comparing the sensitivity of through relative tolerance analysis, Z-score, and species sensitivity index. The sensitivity of different species of protozoa varies greatly. The protozoa Paramecium sp. and Tetrahymena sp. are not sensitive species; conversely, Urostyla sp. is sensitive to dimethoate and nanomaterials Ag-NPs, respectively ZnO-NPs, and CuO-NPs, fits the use as an indicator species on these substances. The prospects to explore scientific toxicity exposure protocols, expand the protozoan species examined, and screen the sensitive species under the protocols are discussed. This prospect review advances the knowledge for including the sensitive protozoa as an indicator species in comprehensive toxicological analysis for pesticides and nanomaterials.

Morphology and molecular phylogeny of three freshwater scuticociliates, with establishments of one new genus and three new species (Ciliophora, Oligohymenophorea).

The morphology and molecular phylogeny of three freshwater scuticociliates, Variuronema elegans gen. nov., spec. nov. (type species), V. similis spec. nov. and Uronema rigidum spec. nov., collected from Qixing River Wetland, northeastern China, were investigated using live observation and silver carbonate staining methods; the phylogenetic placements were inferred from the small subunit rRNA gene. Variuronema gen. nov., assigned to the family Uronematidae, is distinguished from the other genera mainly by its membranelles 1 and 2, each consisting of four longitudinal rows of kinetids, and the paroral membrane extending anteriorly to the front part of membranelle 3. The phylogenetic analyses indicate that: (1) Variuronema spp. form an independent, fully supported clade. However, its position among other uronematids is unresolved. (2) The family Uronematidae is not monophyletic based on the SSU rRNA gene, which is in accordance with previous studies.

Taxonomy and molecular phylogeny of two new Blepharisma species (Ciliophora, Heterotrichea) from northeastern China.

The morphology and ciliary pattern of two new heterotrich ciliates, Blepharisma orientale n. sp. and B. sinicum n. sp., collected from Sanjiang Plain Wetland, northeastern China, were investigated using living observation and protargol staining. Blepharisma orientale n. sp. is characterized by the following combination of characteristics: limnetic; body in vivo 280-380 × 120-190 µm, slender to elongate, cortical granules dark pink, ventral margin somewhat sigmoid; buccal field about 55-60% of body length; 30-39 somatic kineties; 70-76 adoral membranelles, each comprising two long rows of basal bodies; paroral membrane consists of dikinetids with the posterior third obliquely arranged, both basal bodies in each dikinetid are ciliated. Blepharisma sinicum n. sp. is recognized by the combination of the following characters: limnetic; body 190-260 × 100-170 µm in size in vivo, irregularly sigmoid and slightly bilaterally flattened, dorsal margin curved; cortical granules pale pink in color; buccal field about 60-75% of body length; 25-29 somatic kineties; 53-82 adoral membranelles; paroral membrane consisting entirely of dikinetids. Phylogenetic analyses based on small subunit rRNA (SSU rRNA) gene sequence data recovered the two new species as sister taxa, both the sister group of B. penardi.

Insights into the phylogeny of the ciliate of class Colpodea based on multigene data.

In the class Colpodea, there are many unresolved evolutionary relationships among taxa. Here, we report 30 new sequences including SSU-rRNA, ITS1-5.8S- ITS2 rRNA, and the mitochondrial small subunit ribosomal RNA (mtSSU-rRNA) genes of five colpodeans, and conduct phylogenetic analyses based on each individual gene and a two-gene concatenated dataset. For the first time, multi-genes were used to analyze phylogenetic relationships in the class Colpodea. The main findings are: (1) SSU-rRNA, ITS1-5.8S- ITS2 rRNA, and mtSSU-rRNA gene sequences of C. reniformis and C. grandis are provided for the first time, and these two species group into the clade including C. inflata, C. lucida, C. cucullus, and C. henneguyi; (2) clustering pattern and morphological similarity indicate that Bresslauides discoideus has a close relation with Colpodidae spp.; (3) Emarginatophrya genus diagnosis is improved to be 'Hausmanniellidae with sharply shortened and isometric leftmost 1-4 ciliary rows' and Colpoda elliotti is transferred to Emarginatophrya; (4) the genus Colpoda is still non-monophyletic with the addition of 10 populations from five Colpoda species sequences, but there are only two Colpoda groups left based on the present work: Group I comprises C. inflata, C. lucida, C. cucullus, C. henneguyi, C. reniformis, and C. grandis, Group II comprises C. maupasi and C. ecaudata, and the presence of diagonal grooves and the way the vestibular opens might be the two key features that differentiates Colpoda species groups; (5) a close molecular relationship, and highly similar merotelokinetal mode, somatic ciliary pattern, and basic organization of the oral apparatus with P. steinii suggests Bromeliothrix metopoides should be temporarily assigned to Colpodidae.

Morphology, morphogenesis and phylogeny of Anteglaucoma orientalis n. sp. (Ciliophora, Oligohymenophorea), a freshwater hymenostomatid from northeastern China.

A new hymenostomatid ciliate, Anteglaucoma orientalis n. sp., isolated from a freshwater pond in Harbin, northeastern China, was investigated using live observation and silver staining methods. Anteglaucoma orientalis is characterized as follows: size in vivo about 50-60 × 30-35 µm; oval body shape; buccal area occupies about 25% of body length; 28-36 somatic kineties; membranelle 1 having six or seven basal body rows, membranelle 2 five to seven rows, and membranelle 3 three rows; single macronucleus with one micronucleus attached. Morphogenesis of the genus Anteglaucoma is revealed for the first time. The main events during binary fission are as follows: morphogenesis begins with proliferation of kinetosomes in the middle part of postoral kinety 1, and kinetosomes of this primordial field multiply and organize to finally form the paroral membrane and membranelles 1-3 of the opisthe; the parental apparatus in the proter does not take part in the stomatogenetic process. Phylogenetic analyses based on SSU rRNA gene sequences show that Anteglaucoma orientalis n. sp. clusters with the type species, A. harbinensis Pan et al., 2017, with full support.

Low back pain: Influence of early MR imaging or CT on treatment and outcome - a randomized controlled study protocol.

OBJECTIVE: To establish whether early use of magnetic resonance imaging (MRI) or computed tomography (CT) influences treatment and outcome of patients with low back pain. METHODS: This study will be implemented from March 2021 to March 2022 at Huzhou Traditional Chinese Medicine Hospital Affiliated to Zhejiang Chinese Medical University. The experiment was granted through the Research Ethics Committee of Huzhou Traditional Chinese Medicine Hospital Affiliated to Zhejiang Chinese Medical University (R609320987). Patients who have symptomatic lumbar spine disorders at presentation are eligible for the trial if there is clinical uncertainty about the need for imaging (MRI or CT). Patients are excluded who required immediate referral for imaging (those who had signs suggestive of serious abnormalities or disease or who required surgical intervention), who have undergone MR imaging or CT of the spine within 1 year, who do not need imaging, and who have pain of a nonspinal origin. The primary outcome measure is the Aberdeen Low Back Pain (ALBP) score. Other principal outcome measure is the Short Form 36. RESULTS: Table 1 will show the quality of life outcome measures between groups. CONCLUSION: This study may guide the policy makers to develop an evidence-based protocol to assess the effect of early use of MRI or CT in the treatment of patients with low back pain.

Taxonomy and molecular phylogeny of three freshwater scuticociliates, with descriptions of one new genus and two new species (Protista, Ciliophora, Oligohymenophorea).

Three freshwater scuticociliates, Apouronema harbinensis gen. nov. spec. nov., Cyclidium vorax spec. nov., and C. glaucomaMüller, 1773, collected from rivers in Hulan District, Harbin, northeastern China, were investigated using morphological and phylogenetic criteria. Apouronema gen. nov., assigned to the family Uronematidae, is mainly distinguished from the other genera of the family by its paroral membrane extending anteriorly to the middle of membranelle 1. Apouronema harbinensis spec. nov. is defined by body size in vivo about 45-55 × 20-25 µm, buccal field about 70-80% of cell length; 12 or 13 somatic kineties; membranelle 1 having two rows, with 16-18 basal bodies in each kinety; membranelle 2 and membranelle 3 both having two rows each; scutica X-shaped with five pairs of basal bodies. Cyclidium vorax spec. nov. is characterized by the following features: body size 35-40 × 18-20 µm in vivo; 9 or 10 somatic kineties; membranelle 1 having two longitudinal rows, much shorter than M2; M2 triangle-shaped. The phylogenetic analyses show that: (1) Apouronema clustered in the Uronematidae clade, and grouped with genera Uronemita and Uronema; (2) Cyclidium vorax spec. nov. grouped with C. glaucoma and C. sinicum, which supports the assignment of the new species to the genus Cyclidium; (3) Cyclidium remains non-monophyletic with the addition of the new sequence.