Modification of Vancomycin Dosing in Cardiac Surgery With Cardiopulmonary Bypass.

This study aims to assess the risk factors for insufficient vancomycin concentrations for its prophylactic use in adult patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) and to modify the dosing regimen to achieve appropriate plasma concentrations. A total of 27 patients with vancomycin dosing of 1 to 1.5 g based on a weight cutoff of 67 kg were included, of which only 13 (48.15%) had vancomycin plasma concentration >15 mg/L at surgical closure. Risk factors of vancomycin concentration <15 mg/L at surgical-site closure were confirmed by multivariate logistic regression analysis, which showed that CPB duration was an independent predictor. Patients with CPB duration >4 hours had significantly lower vancomycin concentrations and lower proportion in achieving target vancomycin concentration at the end of CPB and surgical closure. For patients with CPB >4 hours, the modified dosing regimen that a second dose of 0.5 to 0.75 g added at 4 hours since the onset of CPB improved the target achievement of vancomycin concentration at surgical closure. Taken together, CPB duration >4 hours was the risk factor for insufficient vancomycin concentration at surgical closure, while our modified dosing could improve the vancomycin concentrations for its prophylactic use in patients undergoing cardiac surgery with CPB.

Dosage Strategy of Linezolid According to the Trough Concentration Target and Renal Function in Chinese Critically Ill Patients.

Background: Linezolid is associated with myelosuppression, which may cause failure in optimally treating bacterial infections. The study aimed to define the pharmacokinetic/toxicodynamic (PK/TD) threshold for critically ill patients and to identify a dosing strategy for critically ill patients with renal insufficiency. Methods: The population pharmacokinetic (PK) model was developed using the NONMEM program. Logistic regression modeling was conducted to determine the toxicodynamic (TD) threshold of linezolid-induced myelosuppression. The dosing regimen was optimized based on the Monte Carlo simulation of the final model. Results: PK analysis included 127 linezolid concentrations from 83 critically ill patients at a range of 0.25-21.61 mg/L. Creatinine clearance (CrCL) was identified as the only covariate of linezolid clearance that significantly explained interindividual variability. Thirty-four (40.97%) of the 83 patients developed linezolid-associated myelosuppression. Logistic regression analysis showed that the trough concentration (Cmin) was a significant predictor of myelosuppression in critically patients, and the threshold for Cmin in predicting myelosuppression with 50% probability was 7.8 mg/L. The Kaplan-Meier plot revealed that the overall median time from the initiation of therapy to the development of myelosuppression was 12 days. Monte Carlo simulation indicated an empirical dose reduction to 600 mg every 24 h was optimal to balance the safety and efficacy in critically ill patients with CrCL of 30-60 ml/min, 450 mg every 24 h was the alternative for patients with CrCL <30 ml/min, and 600 mg every 12 h was recommended for patients with CrCL ≥60 ml/min. Conclusion: Renal function plays a significant role in linezolid PKs for critically ill patients. A dose of 600 mg every 24 h was recommended for patients with CrCL <60 ml/min to minimize linezolid-induced myelosuppression.

Mechanism of Infantile Feire Kechuan Oral Solution against Mycoplasma pneumoniae infection of A549 cells.

BACKGROUND: Mycoplasma pneumoniae is a leading cause of community-acquired respiratory infections. Infantile Feire Kechuan Oral Solution (IFKOS) is effective for treatment of M. pneumoniae infection. The aim of this study was to explore the potential mechanism of IFKOS against M. pneumoniae infection in basal epithelial human lung adenocarcinoma A549 cells. METHODS: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the effects of IFKOS on the viability of A549 cells infected with M. pneumoniae. Optical microscopy was used to observe cell morphology and a Muse cell analyzer was used to assess apoptosis and the cell cycle phase. Enzyme-linked immunosorbent assays were employed to assess the expression levels of interleukin (IL)-4, IL-6, IL-8, IL-17, tumor necrosis factor (TNF)-α, interferon (IFN)-α, and IFN-γ. RESULTS: Under certain conditions, M. pneumoniae infection reduced the viability and inhibited the proliferation of A549 cells, promoted early apoptosis, and arrested cells in the G0/G1 phase, thus shortening the S and G2/M phases (all p < 0.05). M. pneumoniae also upregulated expression of IL-8 and TNF-α and downregulated that of IL-6 (p < 0.05), which switched the immune balance of Th1/Th2 to Th1 cells. IFKOS (5.531 mg/mL) improved the viability and proliferation of M. pneumoniae-infected A549 cells, mitigated early apoptosis, and reversed cell cycle arrest in the G0/G1 phase, thereby extending the S and G2/M phases (all, p < 0.05). IFKOS downregulated expression of IL-8 and TNF-α and upregulated that of IL-6 (p < 0.01), thereby reversing the immune imbalance of Th1/Th2. Secretion of IL-4, IL-17, IFN-α, and IFN-γ was not observed. CONCLUSION: IFKOS played a protective role in the regulation of cell viability, apoptosis, the cell cycle, and Th1/Th2 immune imbalance induced by M. pneumoniae infection and conveyed an anti-inflammatory effect in A549 cells.

Automatic spinal cord segmentation from axial-view MRI slices using CNN with grayscale regularized active contour propagation.

Accurate positioning of the responsible segment for patients with cervical spondylotic myelopathy (CSM) is clinically important not only to the surgery but also to reduce the incidence of surgical trauma and complications. Spinal cord segmentation is a crucial step in the positioning procedure. This study proposed a fully automated approach for spinal cord segmentation from 2D axial-view MRI slices of patients with CSM. The proposed method was trained and tested using clinical data from 20 CSM patients (359 images) acquired by the Peking University Third Hospital, with ground truth labeled by professional radiologists. The accuracy of the proposed method was evaluated using quantitative measures, the reliability metric as well as visual assessment. The proposed method yielded a Dice coefficient of 87.0%, Hausdorff distance of 9.7 mm, root-mean-square error of 5.9 mm. Higher conformance with ground truth was observed for the proposed method in comparison to the state-of-the-art algorithms. The results are also statistically significant with p-values calculated between state-of-the-art methods and the proposed methods.

Anaplastic lymphoma kinase tyrosine kinase inhibitor-induced hepatic failure in lung cancer patients: A study of signal mining and analysis of the FDA adverse event reporting system database.

WHAT IS KNOWN AND OBJECTIVE: Anaplastic Lymphoma Kinase Tyrosine Kinase Inhibitors (ALK TKIs) are standard first-line therapy for non-small cell lung cancer patients with ALK rearrangement. Although some cases of hepatotoxicity related to these drugs have been reported, there is still a lack of investigation on severe hepatotoxicity, such as hepatic failure, with ALK TKIs. METHODS: We evaluated ALK TKI (crizotinib, alectinib, brigatinib, ceritinib and lorlatinib)-induced hepatic failure events (AIHFEs), by using the Reporting Odds Ratio (ROR) and Bayesian Confidence Propagation Neural Network method for mining the adverse event report signals in the FDA Adverse Event Reporting System (FAERS) database from Jan 2013 to Dec 2019. RESULTS AND DISCUSSION: The AIHFEs of "Hepatic failure," "hepatitis fulminant" and "hepatic necrosis" were defined as exposure event signals caused by ALK TKIs. The RORs of "Hepatic failure" were 4.95 (2.36-10.42) in alectinib, 3.77 (1.69-8.40) in ceritinib and 2.45 (1.60-3.76) in crizotinib, respectively. The ROR of "hepatitis fulminant" was 7.86 (3.52-17.54) in crizotinib. The Information Component value of "hepatic necrosis" was 1.97 (0.15) in alectinib. In reports of exposure-event signals, the clinical outcome of eventual death was common and could occur within 3 months. In the reports of "hepatic failure," there was no significant difference in the number of reports between men and women [OR=1.86 (0.94-3.67), p = 0.09]. WHAT IS NEW AND CONCLUSIONS: By mining the adverse event report signals in the FAERS database, we found the exposure event signals of AIHFEs in ALK TKIs were "hepatic failure," "hepatitis fulminant" and "hepatic necrosis". AIHFEs were more likely to appear in the reports of ceritinib, crizotinib and alectinib.

Systematic evaluation of IgG responses to SARS-CoV-2 spike protein-derived peptides for monitoring COVID-19 patients.

Serological tests play an essential role in monitoring and combating the COVID-19 pandemic. Recombinant spike protein (S protein), especially the S1 protein, is one of the major reagents used for serological tests. However, the high cost of S protein production and possible cross-reactivity with other human coronaviruses pose unavoidable challenges. By taking advantage of a peptide microarray with full spike protein coverage, we analyzed 2,434 sera from 858 COVID-19 patients, 63 asymptomatic patients and 610 controls collected from multiple clinical centers. Based on the results, we identified several S protein-derived 12-mer peptides that have high diagnostic performance. In particular, for monitoring the IgG response, one peptide (aa 1148-1159 or S2-78) exhibited a sensitivity (95.5%, 95% CI 93.7-96.9%) and specificity (96.7%, 95% CI 94.8-98.0%) comparable to those of the S1 protein for the detection of both symptomatic and asymptomatic COVID-19 cases. Furthermore, the diagnostic performance of the S2-78 (aa 1148-1159) IgG was successfully validated by ELISA in an independent sample cohort. A panel of four peptides, S1-93 (aa 553-564), S1-97 (aa 577-588), S1-101 (aa 601-612) and S1-105 (aa 625-636), that likely will avoid potential cross-reactivity with sera from patients infected by other coronaviruses was constructed. The peptides identified in this study may be applied independently or in combination with the S1 protein for accurate, affordable, and accessible COVID-19 diagnosis.

An investigation into possible interactions among four vascular epidermal growth factor receptor-tyrosine kinase inhibitors with gefitinib.

The objective of present work is to evaluate possible interactions among four clinically-used vascular epidermal growth factor receptor (VEGFR)-tyrosine kinase inhibitors (TKIs), including apatinib, cabozantinib, sorafenib, and sunitinib, with epidermal growth factor receptor (EGFR)-TKI gefitinib. This may advance knowledge regarding possible dual-target suppression strategies for advanced NSCLC, including VEGFR-TKI plus EGFR-TKI. The in vitro metabolism study demonstrated that apatinib inhibited the formation of metabolite M537194 with moderate effect, and inhibited another metabolite formation of M523595 with strong effect, in both human and rat liver microsomes. Sorafenib, cabozantinib, and sunitinib had no significant inhibitory effect on gefitinib metabolism. The results of the in vivo pharmacokinetics study were consistent with the in vitro metabolism study: the AUC0-t, AUC0-∞ and Cmax of gefitinib increased significantly when co-administered with apatinib by 26.8, 28.7, and 19.8%, respectively. Cabozantinib, sorafenib, and sunitinib exhibited no effect on gefitinib pharmacokinetics. Molecular docking was applied to investigate the binding mode between TKIs and CYP2D6. The docking results illustrated that binding characteristics of apatinib and gefitinib with CYP2D6 were similar, which accounts for competitive mechanism of apatinib-inhibited gefitinib metabolism. In summary, apatinib inhibited the metabolism of gefitinib in vitro and in vivo that were mediated by CYP2D6 and CYP3A4. In addition, cabozantinib, sorafenib, and sunitinib expressed no interaction with gefitinib. The results of the present study may provide a basis and valuable information for the development of treatment strategies.

The precursor of PI(3,4,5)P3 alleviates aging by activating daf-18(Pten) and independent of daf-16.

Aging is characterized by the loss of homeostasis and the general decline of physiological functions, accompanied by various degenerative diseases and increased rates of mortality. Aging targeting small molecule screens have been performed many times, however, few have focused on endogenous metabolic intermediates-metabolites. Here, using C. elegans lifespan assays, we conducted a worm metabolite screen and identified an eukaryotes conserved metabolite, myo-inositol (MI), to extend lifespan, increase mobility and reduce fat content. Genetic analysis of enzymes in MI metabolic pathway suggest that MI alleviates aging through its derivative PI(4,5)P2. MI and PI(4,5)P2 are precursors of PI(3,4,5)P3, which is negatively related to longevity. The longevity effect of MI is dependent on the tumor suppressor gene, daf-18 (homologous to mouse Pten), independent of its classical pathway downstream genes, akt or daf-16. Furthermore, we found MI effects on aging and lifespan act through mitophagy regulator PTEN induced kinase-1 (pink-1) and mitophagy. MI's anti-aging effect is also conserved in mouse, indicating a conserved mechanism in mammals.

Mesocosm experiments to better understand hydrocarbon half-lives for oil and oil dispersant mixtures.

Concerns on the timing and processes associated with petroleum degradation were raised after the use of Corexit during the Deepwater Horizon oil spill. There is a lack of understanding of the removal of oil associated with flocculate materials to the sediment. Mesocosm studies employing coastal and open-ocean seawater from the Gulf of Mexico were undertaken to examine changes in oil concentration and composition with time. The water accommodated fractions (WAF) and chemically enhanced WAF (CEWAF) produced using Macondo surrogate oil and Corexit were followed over 3-4 days in controlled environmental conditions. Environmental half-lives of estimated oil equivalents (EOE), polycyclic aromatic hydrocarbons (PAH), n-alkanes (C10-C35), isoprenoids pristane and phytane, and total petroleum hydrocarbons (TPH) were determined. EOE and PAH concentrations decreased exponentially following first-order decay rate kinetics. WAF, CEWAF and DCEWAF (a 10X CEWAF dilution) treatments half-lives ranged from 0.9 to 3.2 days for EOE and 0.5 to 3.3 days for PAH, agreeing with estimates from previous mesocosm and field studies. The aliphatic half-lives for CEWAF and DECWAF treatments ranged from 0.8 to 2.0 days, but no half-life for WAF could be calculated as concentrations were below the detection limits. Biodegradation occurred in all treatments based on the temporal decrease of the nC17/pristane and nC18/phytane ratios. The heterogeneity observed in all treatments was likely due to the hydrophobicity of oil and weathering processes occurring at different rates and times. The presence of dispersant did not dramatically change the half-lives of oil. Comparing degradation of oil alone as well as with dispersant present is critical to determine the fate and transport of these materials in the ocean.

A mesocosm experiment to determine half-lives of individual hydrocarbons in simulated oil spill scenarios with and without the dispersant, Corexit.

Here, we report results from a 15-day mesocosm experiment examining changes in estimated oil equivalents (EOEs), n-alkanes (n-C10 to n-C35), polycyclic aromatic hydrocarbons (PAHs) and petroleum biomarkers. Water accommodated fractions (WAF) of oil and diluted chemically enhanced WAF (DCEWAF) were prepared and concentrations of oil residues determined on day 0, 3 and 15, respectively. Significant removals of n-alkane and PAHs were observed starting from day 3. The n-C17/pristane and n-C18/phytane ratios suggested that the n-alkane removal was due to biodegradation in the mesocosms. The ratios of C2-dibenzothiophenes/C2-phenanthrenes (D2/P2) and C3-dibenzothiophenes/C3-phenanthrenes (D3/P3) were found to be stable through the experiment. DCEWAF treatment had longer half-lives for most n-alkanes but shorter half-lives for most PAHs than the WAF treatment. Most petroleum biomarkers were stable throughout the experiment. However, depletion of TAS (tricyclic aromatic steroids) was observed on day 15 of DCEWAF treatment.

Super-Toughed PLA Blown Film with Enhanced Gas Barrier Property Available for Packaging and Agricultural Applications.

Polylactic acid (PLA) holds enormous potential as an alternative to the ubiquitous petroleum-based plastics to be used in packaging film and agricultural film. However, the poor viscoelastic behavior and its extremely low melt strength means it fails to meet the requirements in film blowing processing, which is the most efficient film processing method with the lowest costs. Also, the PLA's brittleness and insufficient gas barrier properties also seriously limit PLA's potential application as a common film material. Herein, special stereocomplex (SC) networks were introduced to improve the melt strength and film blowing stability of PLA; polyethylene glycol (PEG) was introduced to improve PLA's toughness and gas barrier properties. Compared with neat poly(l-lactide) acid (PLLA), modified PLA is stable in the film blowing process and its film elongation at break increases more than 18 times and reaches over 250%, and its O2 permeability coefficient decreased by 61%. The resulting film material also has good light transmittance, which has great potential for green packaging applications, such as disposable packaging and agricultural films.

Inter-laboratory calibration of estimated oil equivalent (EOE) concentrations of a water accommodated fraction (WAF) of oil and a chemically enhanced WAF (CEWAF).

Chemical characterization of the presence of oil in environmental samples are performed using methods of varying complexity. Extraction of samples with an organic solvent and analysis by fluorescence spectrometry has been shown to be a rapid and effective screening technique for petroleum in the environment. During experiments, rapid analysis of oil by fluorescence provides the opportunity for researchers to modify the experimental conditions in real time. Estimated Oil Equivalents (EOE) relies on the fluorescence measurement of the aromatic compounds to estimate the oil concentration. The present intercalibration study was designed to investigate whether different fluorometer instruments can reliably measure EOE and whether the results are intercomparable. Additionally, the need for extraction of oil compounds into an organic solvent was investigated. Three different fluorometers were used in three different laboratories: a Horiba Aqualog, a Turner Trilogy and a Shimadzu Spectrofluorophotometer RF-1501. Results from these different instruments showed excellent agreement for EOE determinations. A very high correlation was found between the EOE results obtained with Aqualog Horiba and Turner Trilogy (r2 = 0.9999), with no significant differences between the mean EOE results (t-test, p = 0.30), and the Aqualog Horiba and Shimadzu (r2 = 0.995) fluorometers, with no statistically difference between the EOE results obtained by the two instruments (p = 0.40).

Multiplex Cell-Free DNA Reference Materials for Quality Control of Next-Generation Sequencing-Based In Vitro Diagnostic Tests of Colorectal Cancer Tolerance.

Background: Liquid biopsies based on next-generation sequencing (NGS) assays are confronted with more opportunities and challenges. Widespread clinical implementation of NGS-based cancer in vitro diagnostic tests (IVDs) highlighted the urgency to establish reference materials (RMs) which could provide full control of the process from nucleic acid extraction to test report generation. Quality control based on cell-free DNA (cfDNA) RMs is especially important for liquid biopsies. Methods: Here, we used genomic DNA from thirteen cell lines to establish four negative cfDNA RMs (N1-N4) and four multiplex cfDNA RMs (L1-L4) at serial allelic frequencies ranging from approximately 2% to 0.1%. All the cfDNA RMs were quantified and validated via both droplet digital polymerase chain reaction (ddPCR) and NGS. These RMs were distributed to eight domestic manufacturers to collaboratively evaluate the performance of several domestic NGS-based cancer IVDs covering four major NGS platforms (NextSeq, HiSeq, Ion Proton, and BGISEQ). Results: Each multiplex RM has eleven colorectal cancer-related mutations, including six KRAS mutations (G12S, G12C, G12D, G12A, G12V, and G13D), three NRAS mutations (G12D, Q61R, and Q61K), one PIK3CA mutation (H1047R), and one BRAF mutation (V600E). Each mutation in the cfDNA RMs was quantified and validated via both ddPCR and NGS, showing the good relevance of mutant allelic frequency. These RMs were distributed to eight domestic manufacturers for collaborative evaluation. All eight manufacturers provided similar results by domestic NGS-based cancer IVDs, except for manufacturer #5. The coefficient of variation (CV) was increased with decreasing mutant allelic frequency, and poor repetition occurred when the allelic frequency was lower than 0.5%. Conclusions: These results indicated that these cfDNA RMs would be pivotal for NGS-based cancer IVDs, especially for liquid biopsies of colorectal cancer-related mutations and would guide the further development of RMs covering more onco-related mutations.

Accurate Drug Repositioning through Non-tissue-Specific Core Signatures from Cancer Transcriptomes.

Experimental large-scale screens for drug repositioning are limited by restriction to in vitro conditions and lack of applicability to real human conditions. Here, we developed an in silico screen in human in vivo conditions using a reference of single gene mutations' non-tissue-specific "core transcriptome signatures" (CSs) of 8,476 genes generated from the TCGA database. We developed the core-signature drug-to-gene (csD2G) software to scan 3,546 drug treatment profiles against the reference signatures. csD2G significantly outperformed conventional cell line-based gene perturbation signatures and existing drug-repositioning methods in both coverage and specificity. We highlight this with 3 demonstrated applications: (1) repositioned category of psychiatric drugs to inhibit the TGF-ß pathway; (2) antihypertensive calcium channel blockers predicted to activate AMPK and inhibit AKT pathways, and validated by clinical electronic medical records; and (3) 7 drugs predicted and validated to selectively target the AKT-FOXO and AMPK pathways and thus regulate worm lifespan.

Polyparasitism of Rhabditis Axei and Enterobius Vermicularis in a Child from Beijing, China.

BACKGROUND: Rhabditis (Rhabditellae) axei is a common species in soil, which has been reported repeatedly in human urine and the digestive system. Humans exposed to sewage or mistakenly polluted sewage is the cause of larvae infecting the digestive tract or via the urethra. We reported a patient infected with Rhabditis axei and Enterobius Vermicularis. The migration of the nematodes caused true signs of hematuria, diarrhea, and high eosinophilia. METHODS: Stool and urine are collected to detect parasite eggs and genotype. Specimens are sent for polymerase chain reaction (PCR)-based species identification. Amplification of the 18S ribosomal RNA gene was performed by PCR as described [1]. RESULTS: Morphological features and PCR amplification of the 18S ribosomal RNA gene confirmed Rhabditis axei and Enterobius vermicularis as the pathogen of infection. CONCLUSIONS: Herein, we presented a case that confirmed Rhabditis axei and Enterobius vermicularis infection in humans can be associated with high eosinophilia.

Quality Control of Next-generation Sequencing-based In vitro Diagnostic Test for Onco-relevant Mutations Using Multiplex Reference Materials in Plasma.

Background: Widespread clinical implementation of next-generation sequencing (NGS)-based cancer in vitro diagnostic tests (IVDs) highlighted the urgency to establish reference materials which could provide full control of the process from nucleic acid extraction to test report generation. The formalin-fixed, paraffin-embedded (FFPE) tissue and blood plasma containing circulating tumor deoxyribonucleic acid (ctDNA) were mostly used for clinically detecting onco-relevant mutations. Methods: We respectively developed multiplex FFPE and plasma reference materials covering three clinically onco-relevant mutations within the epidermal growth factor receptor (EGFR) gene at serial allelic frequencies. All reference materials were quantified and validated via droplet digital polymerase chain reaction (ddPCR), and then were distributed to eight domestic manufacturers for the collaborative evaluation of the performance of several domestic NGS-based cancer IVDs covering four major NGS platforms (NextSeq, HiSeq, Ion Proton and BGISEQ). Results: All expected mutations except one at extremely low allelic frequencies were detected, despite some differences in coefficient of variation (CV) which increased with the decrease of allelic frequency (CVs ranging from 18% to 106%). It was worth noting that the CV value seemed to correlate with a particular mutation as well. The repeatability of determination of different mutations was L858R>T790M>19del. Conclusions: The results indicated our reference materials would be pivotal for quality control of NGS-based cancer IVDs and would guide the further development of reference materials covering more onco-relevant mutations.

Effects of repeated allopurinol administration on rat cytochrome P450 activity.

Allopurinol is a popular and widely-prescribed anti-hyperuricemic agent that has been implicated in drug interactions with substrates of several cytochrome P450 (CYP) enzymes. The effect of repeated allopurinol administration (20 mg/kg, once daily for 14 days) on metabolic activity of CYP was assessed in rats. This was a randomized, double-blind, two-way crossover study with a 4-week washout period between phases. The substrates used in this study were phenacetin (CYP1A2), tolbutamide (CYP2C9), omeprazole (CYP2C19) and dextromethorphan (CYP2D6). Validated HPLC-MS/MS was used to quantify all compounds. Our study showed that allopurinol administration inhibited CYP1A2 activity, causing a significant increase in AUC (0-infinity) (P < 0.01) and t1/2 (P < 0.05) of phenacetin, and a distinct decline in CL (P < 0.01). However, there were no significant differences of another three probe drugs in plasma concentrations and the corresponding pharmacokinetic parameters between the allopurinol-treated and normal saline-treated rats. The findings in this study suggested that allopurinol could inhibit CYP1A2 but did not influence CYP2C9, CYP2C19 and CYP2D6 enzymes.

Smoothened oligomerization/higher order clustering in lipid rafts is essential for high Hedgehog activity transduction.

The Hedgehog (Hh) signaling pathway plays evolutionarily conserved roles in controlling embryonic development and tissue homeostasis, and its dysregulation has been implicated in many human diseases including congenital disorder and cancer. The Hh pathway has a unique signal reception system that includes two membrane proteins, the receptor Patched (Ptc) and the transducer Smoothened (Smo). In the Hh signaling cascade, Smo plays a critical role in controlling transduction of Hh gradient signal from the outside into the inside of cells. Although the Smo downstream signal transduction has been intensively studied, the mechanism by which Smo on the plasma membrane is regulated has not been fully understood. As a specific membrane structure of metazoan cells, lipid rafts act as a platform to regulate signal transduction by forming a nanoscale cluster through protein-protein or protein-lipid interactions. However, it remains largely unknown whether lipid rafts are also involved in the regulation of Hh signal transduction. Here, we show that Smo extracellular domain (N terminus) and transmembrane domains form oligomers/higher order clusters in response to Hh signal. Furthermore, we identify that lipid rafts on the plasma membrane are essential for high level activity of Smo during the Hh signal transduction. Finally, our observation suggests that oligomerization/higher order clustering of Smo C-terminal cytoplasmic tail (C-tail) is essential for the transduction of high level Hh signal. Collectively, our data support that in response to Hh gradient signals, Smo transduces high level Hh signal by forming oligomers/higher order clusters in the lipid rafts of cell plasma membrane.

A SNX10/V-ATPase pathway regulates ciliogenesis in vitro and in vivo.

Sorting nexins (SNXs) are phosphoinositide-binding proteins implicated in the sorting of various membrane proteins in vitro, but the in vivo functions of them remain largely unknown. We reported previously that SNX10 is a unique member of the SNX family genes in that it has vacuolation activity in cells. We investigate the biological function of SNX10 by loss-of-function assay in this study and demonstrate that SNX10 is required for the formation of primary cilia in cultured cells. In zebrafish, SNX10 is involved in ciliogenesis in the Kupffer's vesicle and essential for left-right patterning of visceral organs. Mechanistically, SNX10 interacts with V-ATPase complex and targets it to the centrosome where ciliogenesis is initiated. Like SNX10, V-ATPase regulates ciliogenesis in vitro and in vivo and does so synergistically with SNX10. We further discover that SNX10 and V-ATPase regulate the ciliary trafficking of Rab8a, which is a critical regulator of ciliary membrane extension. These results identify an SNX10/V-ATPase-regulated vesicular trafficking pathway that is crucial for ciliogenesis, and reveal that SNX10/V-ATPase, through the regulation of cilia formation in various organs, play an essential role during early embryonic development.