Value of the revised Atlanta classification (RAC) and determinant-based classification (DBC) systems in the evaluation of acute pancreatitis

<p><b>Objective:</b> Since increasing acute pancreatitis (AP) severity is significantly associated with mortality, accurate and rapid determination of severity is crucial for effective clinical management. This study investigated the value of the revised Atlanta classification (RAC) and the determinant-based classification (DBC) systems in stratifying severity of acute pancreatitis.</p> <p><b>Methods:</b> This retrospective observational cohort study included 480 AP patients. Patient demographics and clinical characteristics were recorded. The primary outcome was mortality, and secondary outcomes were admission to intensive care unit (ICU), duration of ICU stay, and duration of hospital stay.</p> <p><b>Results:</b> Based on the RAC classification, there were 295 patients with mild AP (MAP), 146 patients with moderate-to-severe AP (MSAP), and 39 patients with severe AP (SAP). Based on the DBC classification, there were 389 patients with MAP, 41 patients with MSAP, 32 patients with SAP, and 18 patients with critical AP (CAP). ROC curve analysis showed that the DBC system had a significantly higher accuracy at predicting organ failure compared to the RAC system (<i>p</i> < .001). Multivariate regression analysis showed that age and ICU stay were independent risk factors of mortality.</p> <p><b>Conclusion:</b> The DBC system had a higher accuracy at predicting organ failure. Age and ICU stay were significantly associated with risk of death in AP patients. A classification of CAP by the DBC system should warrant close attention, and rapid implementation of effective measures to reduce mortality.</p

Characterization of Tetratricopeptide Repeat-Containing Proteins Critical for Cilia Formation and Function

<div><p>Cilia formation and function require a special set of trafficking machinery termed intraflagellar transport (IFT), consisting mainly of protein complexes IFT-A, IFT-B, BBSome, and microtubule-dependent molecular motors. <u>T</u>etra<u>t</u>ri<u>c</u>opeptide repeat-containing (TTC) proteins are widely involved in protein complex formation. Nine of them are known to serve as components of the IFT or BBSome complexes. How many TTC proteins are cilia-related and how they function, however, remain unclear. Here we show that twenty TTC genes were upregulated by at least 2-fold during the differentiation of cultured mouse tracheal epithelial cells (MTECs) into multiciliated cells. Our systematic screen in zebrafish identified four novel TTC genes, <i>ttc4</i>, <i>-9c</i>, <i>-36</i>, and <i>-39c</i>, that are critical for cilia formation and motility. Accordingly, their zebrafish morphants displayed typical ciliopathy-related phenotypes, including curved body, abnormal otolith, hydrocephalus, and defective left-right patterning. The morphants of <i>ttc4</i> and <i>ttc25</i>, a known cilia-related gene, additionally showed pronephric cyst formation. Immunoprecipitation indicated associations of TTC4, -9c, -25, -36, and -39c with components or entire complexes of IFT-A, IFT-B, or BBSome, implying their participations in IFT or IFT-related activities. Our results provide a global view for the relationship between TTC proteins and cilia.</p></div

Screen for cilia-related TTC genes in zebrafish.

<p>(A) The morphologies of zebrafish morphants at 72 hpf. Note that the morphants of <i>ttc4</i>, <i>-9c</i>, <i>-25</i>, <i>-36</i>, and <i>-39c</i> displayed severe body curving. (B) Quantification results for the curved body phenotype. Three independent experiments were performed. (C) and (D) Rescue experiments for the <i>ttc</i> morphants. The indicated MO was coinjected with either the corresponding MO-resistant mRNA or GFP mRNA into zebrafish embryos. The body curvature phenotype was assayed at 72 hpf. The numbers of embryos counted are listed over the histograms. Statistical results are from three independent experiments. Student’s <i>t</i>-test, ***P < 0.001. Error bars represent s.d.</p

The expression profiles of TTC genes upregulated during MTEC differentiation into multiciliated cells.

<p>(A) Typical differentiation progression of MTECs cultured <i>in vitro</i>. MTECs were isolated from mice of 8-week old and were induced to differentiate into multiciliated cells by culturing at an air-liquid interface (ALI) for the indicated time. Approximately 90% of MTECs were multiciliated at ALI day 5. Acetylated tubulin (Ace-tub) was used to mark cilia. ZO-1 labels the tight junctions. (B) The gene expression profiles of known cilia-related TPR-containing proteins following the MTEC differentiation. The data were from cDNA microarray analyses on MTECs samples from ALI d0 to ALI d5. The gene expression pattern of Deup1, a protein critical for centriole amplification, is also included for comparison. (C) The expression profiles of ten novel TTC genes. (D) Expression profiles of the indicated genes, based on quantitative PCR (qPCR) results. One set of typical results of two independent experiments is presented (Please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124378#pone.0124378.s002" target="_blank">S2 Fig</a> for the second set of results). The mRNA levels at ALI d0 were set as 1.</p

Associations of the indicated TTC proteins with IFT complexes and BBSome.

<p>HEK293T cell lysates containing the indicated FLAG-tagged TTC proteins were mixed respectively with mouse testis lysates and subjected to co-immunoprecipitation with anti-FLAG M2 resin. Flag-luciferase was used as negative control. Immunoblotting was then performed to detect the FLAG-tagged proteins and representative components of the IFT complexes (A) or BBSome (B).</p

Examinations on cilia in the Kupffer’s vesicle and pronephric duct.

<p>(A-C) Both cilia number and cilia length in the Kupffer’s vesicle (KV) at the 7-somite stage were reduced in the indicated <i>ttc</i> morphants. Cilia were labeled using antibody against acetylated tubulin. The quantification results were based on three independent experiments. Student’s <i>t</i>-test, ***P < 0.001. Error bars represent s.d. (D) Typical morphology of cilia in pronephric ducts at 24 hpf. (E) and (F) Cilia motility in the pronephric duct at 60 hpf. The kymographs showed trajectories of the cilia marked with red line in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124378#pone.0124378.s010" target="_blank">S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124378#pone.0124378.s011" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124378#pone.0124378.s012" target="_blank">S3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124378#pone.0124378.s013" target="_blank">S4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124378#pone.0124378.s014" target="_blank">S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124378#pone.0124378.s015" target="_blank">S6</a> Videos. Cilia beat frequencies (CBFs) were measured at 60 hpf (40 cilia from 4 morphants for each gene). Student’s <i>t</i>-test, ***P < 0.001. Error bars represent s.e.m.</p

Additional phenotypes of the zebrafish morphants of <i>ttc4</i>, <i>-9c</i>, <i>-25</i>, <i>-36</i>, and <i>-39c</i>.

<p>(A) Typical otolith morphologies at 72 hpf. The otic vesicle normally contains two otoliths as shown. Otoliths that show differences in number, size and/or position were considered as abnormal. (B) Quantification results for abnormal otoliths in the indicated morphants. (C) and (D) Hydrocephalus formation (arrows in the bright-field images and asterisks in the histochemical sections) in the indicated morphants at 72 hpf. (E) and (F) The <i>ttc25</i> and <i>ttc4</i> morphants tended to develop pronephric cysts (arrows in the bright-field images and asterisks in the histochemical sections) at 72 hpf. (G) and (H) Left-right asymmetry patterning was disturbed in the indicated <i>ttc</i> morphants at 30 hpf. The <i>cmlc2</i> probe was used to label the heart tube (arrows) in whole-mount <i>in situ</i> hybridization. All the quantification results were based on three independent experiments. Student’s <i>t</i>-test, ***P < 0.001. Error bars represent s.d.</p