Automated Sagittal Skeletal Classification of Children Based on Deep Learning.

Malocclusions are a type of cranio-maxillofacial growth and developmental deformity that occur with high incidence in children. Therefore, a simple and rapid diagnosis of malocclusions would be of great benefit to our future generation. However, the application of deep learning algorithms to the automatic detection of malocclusions in children has not been reported. Therefore, the aim of this study was to develop a deep learning-based method for automatic classification of the sagittal skeletal pattern in children and to validate its performance. This would be the first step in establishing a decision support system for early orthodontic treatment. In this study, four different state-of-the-art (SOTA) models were trained and compared by using 1613 lateral cephalograms, and the best performance model, Densenet-121, was selected was further subsequent validation. Lateral cephalograms and profile photographs were used as the input for the Densenet-121 model, respectively. The models were optimized using transfer learning and data augmentation techniques, and label distribution learning was introduced during model training to address the inevitable label ambiguity between adjacent classes. Five-fold cross-validation was conducted for a comprehensive evaluation of our method. The sensitivity, specificity, and accuracy of the CNN model based on lateral cephalometric radiographs were 83.99, 92.44, and 90.33%, respectively. The accuracy of the model with profile photographs was 83.39%. The accuracy of both CNN models was improved to 91.28 and 83.98%, respectively, while the overfitting decreased after addition of label distribution learning. Previous studies have been based on adult lateral cephalograms. Therefore, our study is novel in using deep learning network architecture with lateral cephalograms and profile photographs obtained from children in order to obtain a high-precision automatic classification of the sagittal skeletal pattern in children.

Peripheral artery disease independently associated with significantly higher risk for COVID-19 mortality: Evidence based on adjusted effect estimates.

OBJECTIVE: To investigate the influence of peripheral artery disease (PAD) on the risk of mortality among coronavirus disease 2019 (COVID-19) patients based on adjusted effect estimates. METHODS: Systematic searches were performed through electronic databases. A random-effect model was applied to calculate the pooled effect and corresponding 95% confidence interval (CI). Inconsistency index (I2) was used to evaluate the heterogeneity across studies. Sensitivity analysis, subgroup analysis, and Begg's test were all implemented. RESULTS: On the basis of 16 eligible studies with 142,832 COVID-19 patients, the meta-analysis showed that PAD significantly increased the risk for mortality among COVID-19 patients (pooled effect = 1.29, 95% CI: 1.10-1.51). The significant association was also observed in the subgroup analysis stratified by hospitalized patients, mean age ≥ 60 years, Europe and North America. Sensitivity analysis verified the robustness of our findings. Begg's test (P = 0.15) showed there was no potential publication bias. CONCLUSIONS: COVID-19 patients with PAD may have a greater risk of mortality. Clinicians and nursing staff are supposed to identify and monitor these high-risk patients in a timely manner and provide appropriate clinical treatment for them.

Heavy metals (HMs) pollution in the aquatic environment: Role of probiotics and gut microbiota in HMs remediation.

The presence of heavy metals (HMs) in aquatic ecosystems is a universal concern due to their tendency to accumulate in aquatic organisms. HMs accumulation has been found to cause toxic effects in aquatic organisms. The common HMs-induced toxicities are growth inhibition, reduced survival, oxidative stress, tissue damage, respiratory problems, and gut microbial dysbiosis. The application of dietary probiotics has been evolving as a potential approach to bind and remove HMs from the gut, which is called "Gut remediation". The toxic effects of HMs in fish, mice, and humans with the potential of probiotics in removing HMs have been discussed previously. However, the toxic effects of HMs and protective strategies of probiotics on the organisms of each trophic level have not been comprehensively reviewed yet. Thus, this review summarizes the toxic effects caused by HMs in the organisms (at each trophic level) of the aquatic food chain, with a special reference to gut microbiota. The potential of bacterial probiotics in toxicity alleviation and their protective strategies to prevent toxicities caused by HMs in them are also explained. The dietary probiotics are capable of removing HMs (50-90%) primarily from the gut of the organisms. Specifically, probiotics have been reported to reduce the absorption of HMs in the intestinal tract via the enhancement of intestinal HM sequestration, detoxification of HMs, changing the expression of metal transporter proteins, and maintaining the gut barrier function. The probiotic is recommended as a novel strategy to minimize aquaculture HMs toxicity and safe human health.

Effect of dibutyl phthalate on microalgal growth kinetics, nutrients removal, and stress enzyme activities.

The dibutyl phthalate (DPB) is an emerging plasticizer contaminant that disrupts the biological processes of primary producers, especially phytoplankton. In this study, two microalgal species (Chlorella sp. GEEL-08 and Tetradesmus dimorphus GEEL-04) were exposed to various concentrations of DBP extending from 0 to 100 mg/L. The growth kinetics, N-nitrate, and P-phosphate removal efficiency were assessed. The response enzymes such as malonaldehyde (MDA) and superoxide dismutase (SOD) were also investigated. The results revealed that the Chlorella sp. GEEL-08 at 10 mg/L concentration of DBP exhibited higher growth (0.88 OD680nm) compared to T. dimorphus GEEL-04 (0.80 OD680nm). More than 94% of N and P were removed from culture media by both microalgal species. The DBP (>50 mg/L) significantly exacerbates the growth of both microalgae species and the growth inhibition ratio was in the range of 3.6%-25.9%. The SOD activity and MDA were higher in T. dimorphus culture media than in the culture media of Chlorella sp. The results reflect the hazard and the risk of plasticizers on primary producers in the ecosystem.

Significant association between anemia and higher risk for COVID-19 mortality: A meta-analysis of adjusted effect estimates.

OBJECTIVE: This study aimed to evaluate whether there was a significant relationship between anemia and the risk for mortality among coronavirus disease 2019 (COVID-19) patients by a quantitative meta-analysis based on the adjusted effect estimates. METHODS: A systematic search was conducted in electronic databases to identify all published literature. A random-effects meta-analysis model was used to estimate the pooled effect size and 95% confidence interval (CI). Heterogeneity test, Begg's test, subgroup analysis and meta-regression were performed. RESULTS: Twenty-three articles with 573,928 COVID-19 patients were included in the quantitative meta-analysis. There was a significant association between anemia and an elevated risk of COVID-19 mortality (pooled effect size = 1.47, 95% CI [1.30-1.67]). We observed this significant association in the further subgroup analyses by age, proportion of males, sample size, study design, region and setting. Sensitivity analysis exhibited that our results were reliable. Begg's test showed that there was no publication bias. Meta-regression indicated that the tested variables might not be the source of heterogeneity. CONCLUSION: Our meta-analysis based on risk factors-adjusted effect estimates indicated that anemia was independently associated with a significantly elevated risk for mortality among COVID-19 patients.

Rapid and Ultrasensitive Detection of Methicillin-Resistant Staphylococcus aureus Based on CRISPR-Cas12a Combined With Recombinase-Aided Amplification.

Staphylococcus aureus is one of the main pathogens causing hospital and community-acquired infections, in particular, infections caused by methicillin-resistant Staphylococcus aureus (MRSA) cause a higher mortality rate than those caused by methicillin-sensitive strains, which poses a serious global public health problem. Therefore, rapid and ultrasensitive detection of patients with clinical MRSA infection and timely control of infection are essential. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) based on nucleic acid detection methods are well-known for its high specificity and sensitivity and programmability. Here, we successfully proposed a method based on CRISPR-Cas12a combined with recombinase-aided amplification (RAA) through fluorescent readout to achieve accurate identification and highly sensitive detection of MRSA in clinical samples. Results showed that the limit of detection (LoD) of the RAA-Cas12a method could reach 10 copies/µl at 60 min of reaction. Specificity tests showed that the method could distinguish MRSA from clinically common bacteria. The results of RAA-Cas12a were consistent with that of antimicrobial susceptibility tests (AST) and polymerase chain reaction (PCR) in 83 clinical samples. These results indicated that the detection method based on RAA-Cas12a has high sensitivity and specificity, and provides important value for rapid detection of MRSA.

Effects of Smad4 on the expression of caspase­3 and Bcl­2 in human gingival fibroblasts cultured on 3D PLGA scaffolds induced by compressive force.

Human gingival fibroblasts (HGFs) are the main cells that comprise gingival tissue, where they transfer mechanical signals under physiological and pathological conditions. The exact mechanism underlying gingival tissue reconstruction under compressive forces remains unclear. The present study aimed to explore the effects of Smad4, caspase­3 and Bcl­2 on the proliferation of HGFs induced by compressive force. HGFs were cultured on poly(lactide­co­glycolide) (PLGA) scaffolds under an optimal compressive force of 25 g/cm2. Cell viability was determined via Cell Counting Kit­8 assays at 0, 12, 24, 48 and 72 h. The expression levels of Smad4, caspase­3 and Bcl­2 were measured via reverse transcription­quantitative PCR and western blotting. The application of compressive force on HGFs for 24 h resulted in a significant increase in cell proliferation and Bcl­2 expression, but a significant decrease in the expression of Smad4 and caspase­3; however, inverse trends were observed by 72 h. Subsequently, a lentivirus was used to overexpress Smad4 in HGFs, which attenuated the effects of compressive force on HGF proliferation and Bcl­2 expression, but enhanced caspase­3 expression, suggesting that Smad4 may regulate compressive force­induced apoptosis in HGFs. In conclusion, these findings increased understanding regarding the mechanisms of compressive force­induced HGF proliferation and apoptosis, which may provide further insight for improving the efficacy and stability of orthodontic treatment.

JAK2-STAT5 signaling is insensitive to porcine growth hormone (pGH) in hepatocytes of neonatal pig.

Porcine growth hormone (pGH) is most important hormone which is involved in the growth and development of pig. However, a series of studies have indicated that neonatal pig is insensitive to pGH; the reason for this phenomenon is still not fully understood. In this work, we try to investigate this issue from the angle of intracellular signaling induced by pGH. In the present study, porcine hepatocytes from neonatal pig were used as a model, and confocal laser scanning microscopy (CLSM), Western blot, co-immunoprecipitation and colocalization assay were used to study pGH's signaling properties in hepatocytes of neonatal pig and explore the possible mechanism(s) for why intracellular signaling is insensitive to pGH. The results indicated that Janus kinase 2 and signal transducers and activators of transcription 5/3/1 (JAK2-STATs) signaling are not activated. We further investigated the possible mechanism(s) by which JAK2-STATs' signaling is not activated by pGH and growth hormone receptor (GHR) and found that the negative regulatory molecules of JAK2-STATs signaling may be associated with this phenomenon in the hepatocytes of neonatal pig. In addition, we also explored pGH's biology in hepatocytes from neonatal pig, it can be found that pGH/GHR could translocate into the cell nucleus, which implies that pGH/GHR may exhibit physiological roles based on their nuclear localization. We found that pGH could not trigger intracellular signaling in the hepatocytes of neonatal pigs, but not young pigs, which provides an important explanation for why the growth of neonatal pig is GH independent.

Potential Role of Integrin α5ß1/Focal Adhesion Kinase (FAK) and Actin Cytoskeleton in the Mechanotransduction and Response of Human Gingival Fibroblasts Cultured on a 3-Dimension Lactide-Co-Glycolide (3D PLGA) Scaffold.

BACKGROUND The stability of orthodontic treatment is thought to be significantly affected by the compression and retraction of gingival tissues, but the underlying molecular mechanism is not fully elucidated. The objectives of our study were to explore the effects of mechanical force on the ECM-integrin-cytoskeleton linkage response in human gingival fibroblasts (HGFs) cultured on 3-dimension (3D) lactide-co-glycolide (PLGA) biological scaffold and to further study the mechanotransduction pathways that could be involved. MATERIAL AND METHODS A compressive force of 25 g/m² was applied to the HGFs-PLGA 3D co-cultured model. Rhodamine-phalloidin staining was used to evaluate the filamentous actin (F-actin) cytoskeleton. The expression level of type I collagen (COL-1) and the activation of the integrin alpha5ß1/focal adhesion kinase (FAK) signaling pathway were determined by using real-time PCR and Western blotting analysis. The impacts of the applied force on the expression levels of FAK, phosphorylated focal adhesion kinase (p-FAK), and COL-1 were also measured in cells treated with integrin alpha5ß1 inhibitor (Ac-PHSCN-NH 2, ATN-161). RESULTS Mechanical force increased the expression of integrin alpha5ß1, FAK (p-FAK), and COL-1 in HGFs, and induced the formation of stress fibers. Blocking integrin alpha5ß1 reduced the expression of FAK (p-FAK), while the expression of COL-1 was not fully inhibited. CONCLUSIONS The integrin alpha5ß1/FAK signaling pathway and actin cytoskeleton appear to be involved in the mechanotransduction of HGFs. There could be other mechanisms involved in the promotion effect of mechanical force on collagen synthesis in addition to the integrin alpha5ß1 pathway.

Pouch wall thickness and floppy pouch complex.

BACKGROUND: Floppy pouch complex (FPC) consists of disease phenotypes in patients with ileal pouches, including pouch prolapse, afferent limb syndrome, enterocele, redundant loop, and pouch folding. Our recent study demonstrated that lower body weight, lower peripouch fat, family history of inflammatory bowel disease (IBD), female gender, and dyschezia are risk factors for FPC patients with IBD. The aims of this study were to assess the relationship between pouch wall thickness and FPC, and to investigate the association between inflamed and non-inflamed pouch wall thickness. METHODS: This case-control study included all eligible patients with FPC from our prospectively maintained, IRB-approved Pouchitis Registry from 2011 to 2017. We measured pouch wall thickness of fully distended pouches on cross-sectional abdominal and pelvic imaging. Patients with stoma and non-distended pouches were completely excluded. Risk factors for FPC were analyzed. RESULTS: A total of 140 out of 451 patients from our were found to have fully distended pouches on imaging. Of the 140 patients, 36 (25.7%) were diagnosed as having FPC. We analyzed pouch wall thickness for each subcategory of FPC as well as non-FPC conditions. The thickness of pouch wall was follows: pouch prolapse (N = 19): 1.5 mm (1.5-2.0), afferent limb syndrome (N = 12): 1.5 mm (1.1-2.0), folded pouch (N = 4): 1.5 mm (1.1-1.9), and redundant pouch (N = 2): 1.3 mm (1.0-1.3). The control group (N = 104) consisting of normal pouch, pouchitis, cuffitis, Crohn's disease of the pouch, and pouch sinus with median pouch wall thickness of 1.5 mm, 2.3 mm, 2.0 mm, 2.0 mm, and 1.5 mm, respectively. There were significant differences in pouch wall thickness between normal or non-inflamed pouch versus pouchitis versus cuffitis versus Crohn's disease of the pouch with p values of 0.01, 0.04, 0.05, and 0.049, respectively. CONCLUSION: Patients with FPC were shown to have thin pouch wall, which those with inflammatory conditions of the pouch tended to have thick pouch wall. These findings will have implications in both diagnosis and investigation of etiopathogenesis of these disorders.

Mechanical force promotes the proliferation and extracellular matrix synthesis of human gingival fibroblasts cultured on 3D PLGA scaffolds via TGF­ß expression.

Human gingival fibroblasts (HGFs) are responsible for connective tissue repair and scarring, and are exposed to mechanical forces under physiological and pathological conditions. The exact mechanisms underlying gingival tissue reconstruction under mechanical forces remain unclear. The present study aimfed to investigate the effects of mechanical forces on the proliferation and extracellular matrix synthesis in HGFs by establishing a 3­dimensional (3D) HGF culture model using poly(lactide­co­glycolide) (PLGA) scaffolds. HGFs were cultured in 3D PLGA scaffolds and a mechanical force of 0, 5, 15, 25 or 35 g/cm2 was applied to HGFs for 24 h. A mechanical force of 25 g/cm2 induced the highest proliferation rate, and thus was selected for subsequent experiments. Cell viability was determined using the MTT assay at 0, 24, 48 and 72 h. The expression levels of type I collagen (COL­1) and matrix metallopeptidase (MMP)­1 were examined by reverse transcription­quantitative polymerase chain reaction and ELISA, and transforming growth factor (TGF)­ß expression was evaluated by ELISA. The application of mechanical force on HGFs cultured on the 3D PLGA scaffolds resulted in a significant increase in cell proliferation and COL­1 expression, as well as a decrease in MMP­1 expression. A TGF­ß1 inhibitor was also applied, which attenuated the effects of mechanical force on HGF proliferation, and COL­1 and MMP­1 expression, thus suggesting that TGF­ß signaling pathways may mediate the mechanical force­induced alterations observed in HGFs. In conclusion, these findings helped to clarify the mechanisms underlying mechanical force­induced HGF proliferation and ECM synthesis, which may promote the development of targeted therapeutics to treat various diseases, including gingival atrophy caused by orthodontic treatment.

Cellular internalization and trafficking of 20 KDa human growth hormone.

Twenty kilodalton human growth hormone (20K-GH) is the second most abundant GH isoform after the twenty-two kilodalton human growth hormone (22 K-GH) isoform. 20K-GH exhibits similar but not identical physiological activities as that of 22K-GH. The cell behaviour of 22K-GH has been extensively studied, but little or no information has been reported regarding 20K-GH. Here, we focussed on the internalization of 20K-GH. We found that the internalization of 20K-GH is rapid and occurs in a time- and dose-dependent manner. 20K-GH internalization is mediated by GHR. It appears that the internalization of 20K-GH and GHR into the cytoplasm is mediated by clathrin and/or caveolin. The current study indicates that 20K-GH can internalize into the cytoplasm and suggests that the internalized 20K-GH may exhibit different functions from those of 22K-GH.

Structural insights into the gating of DNA passage by the topoisomerase II DNA-gate.

Type IIA topoisomerases (Top2s) manipulate the handedness of DNA crossovers by introducing a transient and protein-linked double-strand break in one DNA duplex, termed the DNA-gate, whose opening allows another DNA segment to be transported through to change the DNA topology. Despite the central importance of this gate-opening event to Top2 function, the DNA-gate in all reported structures of Top2-DNA complexes is in the closed state. Here we present the crystal structure of a human Top2 DNA-gate in an open conformation, which not only reveals structural characteristics of its DNA-conducting path, but also uncovers unexpected yet functionally significant conformational changes associated with gate-opening. This structure further implicates Top2's preference for a left-handed DNA braid and allows the construction of a model representing the initial entry of another DNA duplex into the DNA-gate. Steered molecular dynamics calculations suggests the Top2-catalyzed DNA passage may be achieved by a rocker-switch-type movement of the DNA-gate.

What is the Optimal Age for Students to Receive Cardiopulmonary Resuscitation Training?

OBJECTIVES: Training students has been proven to be the optimal way to deliver cardiopulmonary resuscitation (CPR) skills. However, it is somehow unknown whether or not the current recommendations appropriate for Caucasian students are also suitable for East Asian students. The purpose of this study is to explore the best age for East Asian students to receive CPR training. METHODS: Students were recruited from six schools. Students participated in a standard CPR training program provided by tutors. Each student attended a 60-minute training session with a manikin. After being trained, within one hour, the student's compression quality was assessed. RESULTS: A total of 360 students who constituted 12 continuous grades were recruited for this study. Adequate compression depth and satisfactory compression rate with correct hand position could be achieved since the age of 12. However, successful compression rate and complete release could be achieved since the younger age of six. CONCLUSIONS: Current recommendations for Caucasian students to cultivate a full-capacity CPR rescuer at the age of 12 are also appropriate for East Asian students. However, the optimal age for students to receive CPR training should be decided based on evidence and importance assessment of CPR.He D, Huang K, Yang Y, Jiang W, Yang N, Yang H. What is the optimal age for students to receive cardiopulmonary resuscitation training? Prehosp Disaster Med. 2018;33(4):394-398.

Illness perception in patients with androgenetic alopecia and alopecia areata in China.

OBJECTIVE: The aim of the present study was to provide more information on the role of illness perception in patients with androgenetic alopecia (AGA) and those with alopecia areata (AA), and to further investigate the relationship of illness perception with psychological disorders and dermatological QoL. METHODS: The study included 342 patients who were diagnosed with AGA (n=212) or AA (n=130) for the first time at our institution between October 2013 and December 2014. All patients were surveyed before clinical examination by several questionnaires including the Brief Illness Perception, Self-rating Depression Scale, Self-rating Anxiety Scale, and Dermatology Life Quality Index (DLQI). RESULTS: In the AGA patients, the illness perception and QoL were low, whereas the prevalence of clinical depression and anxiety was higher compared to the AA patients. Illness perception was associated with psychological distress and low QoL in both groups, and some illness perception dimensions were found to be significant predictors of the DLQI scores. CONCLUSION: Illness perception plays an important role in AGA and AA patients, and is associated with psychological distress and low QoL. The identification of critical components of illness perception in alopecia patients could help to understand alopecia specificities, to design consultations and interventions according to the perception, and to improve physical and mental outcomes as well as QoL in alopecia patients.

Recovery of the poisoned topoisomerase II for DNA religation: coordinated motion of the cleavage core revealed with the microsecond atomistic simulation.

Type II topoisomerases resolve topological problems of DNA double helices by passing one duplex through the reversible double-stranded break they generated on another duplex. Despite the wealth of information in the cleaving operation, molecular understanding of the enzymatic DNA ligation remains elusive. Topoisomerase poisons are widely used in anti-cancer and anti-bacterial therapy and have been employed to entrap the intermediates of topoisomerase IIß with religatable DNA substrate. We removed drug molecules from the structure and conducted molecular dynamics simulations to investigate the enzyme-mediated DNA religation. The drug-unbound intermediate displayed transitions toward the resealing-compliant configuration: closing distance between the cleaved DNA termini, B-to-A transformation of the double helix, and restoration of the metal-binding motif. By mapping the contact configurations and the correlated motions between enzyme and DNA, we identified the indispensable role of the linker preceding winged helix domain (WHD) in coordinating the movements of TOPRIM, the nucleotide-binding motifs, and the bound DNA substrate during gate closure. We observed a nearly vectorial transition in the recovery of the enzyme and identified the previously uncharacterized roles of Asn508 and Arg677 in DNA rejoining. Our findings delineate the dynamic mechanism of the DNA religation conducted by type II topoisomerases.

Reliability and validity assessment of the revised Symptom Checklist 90 for alopecia areata patients in China.

No study has tested the reliability and validity of the revised Symptom Checklist 90 (SCL-90-R) for patients with alopecia areata (AA), and few have used it to evaluate the mental health of AA patients. To assess the psychological status in Chinese AA patients using the SCL-90-R, and to evaluate its reliability and validity, the psychological status of 168 patients and 100 controls was evaluated with the Chinese-version SCL-90-R. From this study, we found that The Global Severity Index and nine subscale scores on the SCL-90-R were significantly higher in AA patients than that in the controls. Moreover, The Global Severity Index and nine subscale scores on the SCL-90-R were associated with disease duration, age of onset, sex and type of AA. In addition, the SCL-90-R presented good internal consistency (whole scale α = 0.98 and split-half coefficient = 0.95). The intercorrelations between the nine subscales and their correlations with the total scale were 0.58-0.93. Factor analysis produced 22 factors with eigenvalues more than 1.0; the first factor explained 33.88% of the variance. Only hostility and paranoid ideation merged into one factor. Taken together, our data indicated that Chinese AA patients demonstrate greater psychopathology than healthy controls. The SCL-90-R can be used to assess global psychological distress in AA patients with good reliability and validity.

Drug-induced conformational population shifts in topoisomerase-DNA ternary complexes.

Type II topoisomerases (TOP2) are enzymes that resolve the topological problems during DNA replication and transcription by transiently cleaving both strands and forming a cleavage complex with the DNA. Several prominent anti-cancer agents inhibit TOP2 by stabilizing the cleavage complex and engendering permanent DNA breakage. To discriminate drug binding modes in TOP2-α and TOP2-ß, we applied our newly developed scoring function, dubbed AutoDock4RAP, to evaluate the binding modes of VP-16, m-AMSA, and mitoxantrone to the cleavage complexes. Docking reproduced crystallographic binding mode of VP-16 in a ternary complex of TOP2-ß with root-mean-square deviation of 0.65 Å. Molecular dynamics simulation of the complex confirmed the crystallographic binding mode of VP-16 and the conformation of the residue R503. Drug-related conformational changes in R503 have been observed in ternary complexes with m-AMSA and mitoxantrone. However, the R503 rotamers in these two simulations deviate from their crystallographic conformations, indicating a relaxation dynamics from the conformations determined with the drug replacement procedure. The binding mode of VP-16 in the cleavage complex of TOP2-α was determined by the conjoint use of docking and molecular dynamics simulations, which fell within a similar binding pocket of TOP2-ß cleavage complex. Our findings may facilitate more efficient design efforts targeting TOP2-α specific drugs.

Oleosin of subcellular lipid droplets evolved in green algae.

In primitive and higher plants, intracellular storage lipid droplets (LDs) of triacylglycerols are stabilized with a surface layer of phospholipids and oleosin. In chlorophytes (green algae), a protein termed major lipid-droplet protein (MLDP) rather than oleosin on LDs was recently reported. We explored whether MLDP was present directly on algal LDs and whether algae had oleosin genes and oleosins. Immunofluorescence microscopy revealed that MLDP in the chlorophyte Chlamydomonas reinhardtii was associated with endoplasmic reticulum subdomains adjacent to but not directly on LDs. In C. reinhardtii, low levels of a transcript encoding an oleosin-like protein (oleolike) in zygotes-tetrads and a transcript encoding oleosin in vegetative cells transferred to an acetate-enriched medium were found in transcriptomes and by reverse transcription-polymerase chain reaction. The C. reinhardtii LD fraction contained minimal proteins with no detectable oleolike or oleosin. Several charophytes (advanced green algae) possessed low levels of transcripts encoding oleosin but not oleolike. In the charophyte Spirogyra grevilleana, levels of oleosin transcripts increased greatly in cells undergoing conjugation for zygote formation, and the LD fraction from these cells contained minimal proteins, two of which were oleosins identified via proteomics. Because the minimal oleolike and oleosins in algae were difficult to detect, we tested their subcellular locations in Physcomitrella patens transformed with the respective algal genes tagged with a Green Fluorescent Protein gene and localized the algal proteins on P. patens LDs. Overall, oleosin genes having weak and cell/development-specific expression were present in green algae. We present a hypothesis for the evolution of oleosins from algae to plants.

Calcium content of different compositions of gallstones and pathogenesis of calcium carbonate gallstones.

BACKGROUND/OBJECTIVES: Our aim was to investigate the calcium content of different gallstone compositions and the pathogenic mechanisms of calcium carbonate gallstones. METHODS: Between August 2001 and July 2007, gallstones from 481 patients, including 68 calcium carbonate gallstones, were analyzed for total calcium content. Gallbladder bile samples from 33 cases and six controls were analyzed for pH, carbonate anion level, free-ionized calcium concentration and saturation index for calcium carbonate. RESULTS: Total calcium content averaged 75.6 %, 11.8 %, and 4.2 % for calcium carbonate, calcium bilirubinate and cholesterol gallstones. In 29.4 % of patients, chronic and/or intermittent cystic duct obstructions were caused by polypoid lesions in the neck region and 70.6 % were caused by stones. A total of 82 % of patients had chronic low-grade inflammation of the gallbladder wall and 18.0 % had acute inflammatory exacerbations. In the bile, we found the mean pH, mean carbonate anion, free-ionized calcium concentrations, and mean saturation index for calcium carbonate to be elevated in comparison to controls. CONCLUSION: From our study, we found chronic and/or intermittent cystic duct obstructions and low-grade GB wall inflammation lead to GB epithelium hydrogen secretion dysfunction. Increased calcium ion efflux into the GB lumen combined with increased carbonate anion presence increases SI_CaCO(3) from 1 to 22.4. Thus, in an alkaline milieu with pH 7.8, calcium carbonate begins to aggregate and precipitate.