Cuproptosis/OXPHOS tendency prediction of prognosis and immune microenvironment of esophageal squamous cell carcinoma: Bioinformatics analysis and experimental validation.

BACKGROUND: Cuproptosis is a newly discovered cell death mechanism that relies on mitochondrial respiration, for which oxidative phosphorylation (OXPHOS) is an essential part. However, the detailed mechanisms of cuproptosis associated with OXPHOS in esophageal squamous cell carcinoma (ESCC) and how this correlation affects prognosis still remains unclear. METHODS: scRNA-seq data of ESCC were downloaded from SRA (Sequence Read Archive) database. "AUCell" algorithm was used to grouping epithelial cells according to cuproptosis and OXPHOS score. Cell-cell communication, Pseudo-time Trajectory and transcription factor enrichment analysis were repectively conducted by "CellChat", "monocle3" package and "pySCENIC" algorithm. Univariate and LASSO cox regression analysis were used to construct the prognostic cuproptosis-OXPHOS signature. Finally, CCK-8 assay and DCFH-DA staining assay were respectively validated the sensitive and ROS production of elesclomol. RESULTS: scRNA-seq data were analyzed to identify 10 core cell types. According to the median scores for cuproptosis and OXPHOS, malignant epithelial cells were divided into double high, double low, and mixed groups. The double high group distributed at the end of the pseudo-time trajectory and harbored HMGA1(+) as specific transcriptional regulons. Knockdown of HMGA1 partly reversed the inhibition of cell viability visualized by CCK-8 assay, while reactive oxygen species (ROS) production by elesclomol was enhanced after HMGA1 silencing. Furthermore, the immunosuppressive signal was significantly increased in the double high group detected by 'CellChat' in single-cell data and 'ssGSEA' in bulk data followed by 'CIBERSORTx' algorithm. Finally, a new cuproptosis-OXPHOS prognostic signature (CNN2, ATP6V0E1, PSMD6, CCDC25, IGFBP2, MT1E, and RPS4Y1) was constructed for the prediction of the prognosis, and a high-risk group corresponding to a more sensitive tendency to erlotinib, dasatinib, and bosutinib treatment was identified. CONCLUSIONS: Our study revealed the relationship between OXPHOS and tendency of cuproptosis in ESCC, and malignant cells with this characteristic exerted immunosuppressive signals and indicated poor prognosis. Furthermore, we constructed the regulatory network in high cuproptosis-OXPHOS ESCC and identified HMGA1 as a potential regulator molecule of cuproptosis mediated by elesclomol.

Knockdown of LRRK2 inhibits the progression of lung cancer by regulating TLR4/NF-κB pathways and NLRP3 inflammasome.

Leucine-rich repeat kinase 2 (LRRK2) plays an important role in a variety of inflammatory diseases, as well as peripheral and central immune responses. At present, there are few reports about the role of LRRK2 in lung cancer, and need to be further explored. The main purpose of this study is to explore the role and mechanism of LRRK2 in lung cancer. The results revealed that the expression of LRRK2 was increased in the tissues of lung cancer patient and lung cancer cells. Further studies found that interference with LRRK2 expression significantly induced the apoptosis, and promoted the expression of caspase-3, caspase-9, and Bax. More importantly, si-LRRK2 inhibited the expression of VEGF and P-gp, indicating inhibition of cell proliferation and drug resistance. What's more, LRRK2 regulated TLR4/NF-κB signaling pathways and NLRP3 inflammasome, and TLR4/NF-κB pathways was involved in the molecular mechanism of LRRK2 on lung cancer cells. In conclusion, this study suggested that the mechanism of si-LRRK2 inhibiting the progression of lung cancer is to regulate the TLR4/NF-κB signaling pathways and NLRP3 inflammasome.

Rapid measurement method of intracavity phase retardation based on laser frequency splitting.

We present a method for phase retardation measurement of intracavity optical elements which is based on frequency splitting caused by weak phase anisotropy of Nd: YAG. The measurement range covers 0-π and the measurement uncertainty is less than 0.0300 rad. A theoretical analysis is provided to obtain the phase retardation of intracavity optical elements by using the phase difference or frequency difference of two eigenmodes. The minimum error is 0.0036 rad by using the composite wave plate to verify various phase retardation conditions. This work provides a rapid and accurate intracavity method for measuring the phase retardation of optical elements.

QCR7 affects the virulence of Candida albicans and the uptake of multiple carbon sources present in different host niches.

Background: Candida albicans is a commensal yeast that may cause life-threatening infections. Studies have shown that the cytochrome b-c1 complex subunit 7 gene (QCR7) of C. albicans encodes a protein that forms a component of the mitochondrial electron transport chain complex III, making it an important target for studying the virulence of this yeast. However, to the best of our knowledge, the functions of QCR7 have not yet been characterized. Methods: A QCR7 knockout strain was constructed using SN152, and BALb/c mice were used as model animals to determine the role of QCR7 in the virulence of C. albicans. Subsequently, the effects of QCR7 on mitochondrial functions and use of carbon sources were investigated. Next, its mutant biofilm formation and hyphal growth maintenance were compared with those of the wild type. Furthermore, the transcriptome of the qcr7Δ/Δ mutant was compared with that of the WT strain to explore pathogenic mechanisms. Results: Defective QCR7 reduced recruitment of inflammatory cells and attenuated the virulence of C. albicans infection in vivo. Furthermore, the mutant influenced the use of multiple alternative carbon sources that exist in several host niches (GlcNAc, lactic acid, and amino acid, etc.). Moreover, it led to mitochondrial dysfunction. Furthermore, the QCR7 knockout strain showed defects in biofilm formation or the maintenance of filamentous growth. The overexpression of cell-surface-associated genes (HWP1, YWP1, XOG1, and SAP6) can restore defective virulence phenotypes and the carbon-source utilization of qcr7Δ/Δ. Conclusion: This study provides new insights into the mitochondria-based metabolism of C. albicans, accounting for its virulence and the use of variable carbon sources that promote C. albicans to colonize host niches.

A Complete Genome of Nocardia terpenica NC_YFY_NT001 and Pan-Genomic Analysis Based on Different Sources of Nocardia spp. Isolates Reveal Possibly Host-Related Virulence Factors.

Objective: We aimed to identify the possible virulence genes associated with Nocardia NC_YFY_NT001 isolated by ourselves and other Nocardia spp. Methods: The genome of Nocardia terpenica NC_YFY_NT001 was completed by using PacBio and Illumina platforms. A pan-genomic analysis was applied to selected complete Nocardia genomes. Results: Nocardia terpenica NC_YFY_NT001 can cause healthy mice death by tail intravenous injection. The genome of NT001 has one circular chromosome 8,850,000 bp and one circular plasmid 70,000 bp with ~68% GC content. The chromosome and plasmid encode 7914 and 80 proteins, respectively. Furthermore, a pan-genomic analysis showed a total of 45,825 gene clusters, then 304 core, 21,045 shell and 24,476 cloud gene clusters were classified using specific parameters. In addition, we found that catalases were more abundant in human isolates. Furthermore, we also found no significant differences in the MCE proteins between different strains from different sources. The pan-genomic analysis also showed that 67 genes could only be found in humoral isolates. ReX3 and DUF853 domain protein were found in all eight human isolates. The composition of unique genes in humoral isolate genomes indicated that the transcriptional regulators may be important when Nocardia invades the host, which allows them to survive in the new ecological system. Conclusion: In this study, we confirmed that NT001 could cause infected animal death, and identified many possible virulence factors for our future studies. This study also provides new insight for our further study on Nocardia virulence mechanisms.

Molecular Epidemiology, Antifungal Susceptibility, and Virulence Evaluation of Candida Isolates Causing Invasive Infection in a Tertiary Care Teaching Hospital.

Background: The incidence of invasive candidiasis is increasing worldwide. However, the epidemiology, antifungal susceptibility, and virulence of Candida spp. in most hospitals remain unclear. This study aimed to evaluate invasive candidiasis in a tertiary care hospital in Nanchang City, China. Methods: MALDI-TOF MS and 18S rDNA ITS sequencing were used to identify Candida strains. Randomly amplified polymorphic DNA analysis was used for molecular typing; biofilm production, caseinase, and hemolysin activities were used to evaluate virulence. The Sensititre™ YeastOne YO10 panel was used to examine antifungal susceptibility. Mutations in ERG11 and the hotspot regions of FKS1 of drug-resistant strains were sequenced to evaluate the possible mechanisms of antifungal resistance. Results: We obtained 110 Candida strains, which included 40 Candida albicans (36.36%), 37 C. parapsilosis (33.64%), 21 C. tropicalis (19.09%), 9 C. glabrata (8.18%), 2 C. rugose (1.82%), and 1 C. haemulonii (0.91%) isolates. At a limiting point of 0.80, C. albicans isolates could be grouped into five clusters, C. parapsilosis and C. tropicalis isolates into seven clusters, and C. glabrata isolates into only one cluster comprising six strains by RAPD typing. Antifungal susceptibility testing revealed that the isolates showed the greatest overall resistance against fluconazole (6.36%), followed by voriconazole (4.55%). All C. albicans and C. parapsilosis isolates exhibited 100% susceptibility to echinocandins (i.e., anidulafungin, caspofungin, and micafungin), whereas one C. glabrata strain was resistant to echinocandins. The most common amino acid substitutions noted in our study was 132aa (Y132H, Y132F) in the azole-resistant strains. No missense mutation was identified in the hotpot regions of FKS1. Comparison of the selected virulence factors detectable in a laboratory environment, such as biofilm, caseinase, and hemolysin production, revealed that most Candida isolates were caseinase and hemolysin producers with a strong activity (Pz < 0.69). Furthermore, C. parapsilosis had greater total biofilm biomass (average Abs620 = 0.712) than C. albicans (average Abs620 = 0.214, p < 0.01) or C. tropicalis (average Abs620 = 0.450, p < 0.05), although all C. glabrata strains were either low- or no-biofilm producers. The virulence level of the isolates from different specimen sources or clusters showed no obvious correlation. Interesting, 75% of the C. albicans from cluster F demonstrated azole resistance, whereas two azole-resistant C. tropicalis strains belonged to the cluster Y. Conclusion: This study provides vital information regarding the epidemiology, pathogenicity, and antifungal susceptibility of Candida spp. in patients admitted to Nanchang City Hospital.

miR-152/TNS1 axis inhibits non-small cell lung cancer progression through Akt/mTOR/RhoA pathway.

AIM: The purpose of the present study was to explore the function and mechanism of tensin 1 (TNS1) in non-small cell lung cancer (NSCLC) progression. METHODS: The expression of TNS1 in NSCLC cells and tissues was assessed by RT-PCR and Western blot. Besides, Kaplan-Meier survival analysis was recruited to explore the association between TNS1 and NSCLC. Cell growth was analyzed by MTT and flow cytometry assay, while cell metastasis was determined by wound healing and transwell assays. The targeting relationship between TNS1 and miR-152 was assessed by luciferase activity assays. And Western blot was employed to determine the expression of related proteins of Akt/mTOR/RhoA pathway. RESULTS: TNS1 level was boosted in NSCLC cells and tissues, related to the prognosis of NSCLC patients. Furthermore, it was proved that TNS1 promoted the growth and metastasis of NSCLC cells via Akt/mTOR/RhoA pathway. And miR-152 targeted TNS1 to affect the progression of NSCLC. CONCLUSION: miR-152/TNS1 axis inhibits the progression of NSCLC by Akt/mTOR/RhoA pathway.

Molecular Profiling Reveals a Common Metabolic Signature of Tissue Fibrosis.

Fibrosis, or the accumulation of extracellular matrix, is a common feature of many chronic diseases. To interrogate core molecular pathways underlying fibrosis, we cross-examine human primary cells from various tissues treated with TGF-ß, as well as kidney and liver fibrosis models. Transcriptome analyses reveal that genes involved in fatty acid oxidation are significantly perturbed. Furthermore, mitochondrial dysfunction and acylcarnitine accumulation are found in fibrotic tissues. Substantial downregulation of the PGC1α gene is evident in both in vitro and in vivo fibrosis models, suggesting a common node of metabolic signature for tissue fibrosis. In order to identify suppressors of fibrosis, we carry out a compound library phenotypic screen and identify AMPK and PPAR as highly enriched targets. We further show that pharmacological treatment of MK-8722 (AMPK activator) and MK-4074 (ACC inhibitor) reduce fibrosis in vivo. Altogether, our work demonstrate that metabolic defect is integral to TGF-ß signaling and fibrosis.

A PDM-based 128-Gb/s PAM4 fibre-FSO convergent system with OBPFs for polarisation de-multiplexing.

A polarisation-division-multiplexing (PDM)-based four-level pulse amplitude modulation (PAM4) fibre-free-space optical (FSO) convergent system with optical band-pass filters (OBPFs) for polarisation de-multiplexing is feasibly demonstrated for the first time. In a PDM scenario with PAM4 modulation, the transmission capacity of fibre-FSO convergent systems is enhanced four times with an aggregate channel capacity of 128 Gb/s (64 Gb/s PAM4/polarisation × 2 polarisations). With an OBPF, polarisation-tracking free de-multiplexing is attained by eliminating other optical carrier with orthogonal polarisation. An OBPF is a simple polarisation de-multiplexing scheme in which the polarisation-orthogonal carrier can be effectively de-multiplexed and the cross-polarisation interference can be nearly eliminated. Compared with traditional PDM-based fibre-FSO convergent systems with sophisticated polarisation-tracking mechanism and elaborate digital signal processing (DSP) approach, it reveals a noteworthy one with the advantage of simplicity. Through 25 km single-mode fibre transport and 500 m FSO link, sufficiently low bit error rate and qualified PAM4 eye diagrams are attained. This proposed polarisation-tracking free PDM-based fibre-FSO convergent system is notable because it not only incorporates the fibre backbone and optical wireless feeder, but it also simplifies the framework since complicated polarisation-tracking mechanism and DSP approach are not involved.

From Screening to Targeted Degradation: Strategies for the Discovery and Optimization of Small Molecule Ligands for PCSK9.

Proprotein convertase substilisin-like/kexin type 9 (PCSK9) is a serine protease involved in a protein-protein interaction with the low-density lipoprotein (LDL) receptor that has both human genetic and clinical validation. Blocking this protein-protein interaction prevents LDL receptor degradation and thereby decreases LDL cholesterol levels. Our pursuit of small-molecule direct binders for this difficult to drug PPI target utilized affinity selection/mass spectrometry, which identified one confirmed hit compound. An X-ray crystal structure revealed that this compound was binding in an unprecedented allosteric pocket located between the catalytic and C-terminal domain. Optimization of this initial hit, using two distinct strategies, led to compounds with high binding affinity to PCSK9. Direct target engagement was demonstrated in the cell lysate with a cellular thermal shift assay. Finally, ligand-induced protein degradation was shown with a proteasome recruiting tag attached to the high-affinity allosteric ligand for PCSK9.

A PDM-based bi-directional fibre-FSO integration with two RSOAs scheme.

A polarization-division-multiplexing (PDM)-based bi-directional fibre-free-space optical (FSO) integration with two reflective semiconductor optical amplifiers (RSOAs) scheme to efficiently wipe off the modulated data for upstream modulation is proposed and successfully demonstrated. For downstream modulation, a high-speed 128 Gb/s vestigial sideband (VSB)-four-level pulse amplitude modulation (PAM4) fibre-FSO integration is feasibly established. The transmission capacity is increased up to four times through PDM operation and VSB-PAM4 modulation. For uplink transmission, a 10 Gb/s non-return-to-zero fibre-FSO integration with two RSOAs scheme to effectually erase the downstream modulated data is practically constructed. The upstream performance exhibits noticeable enhancement by using of two RSOAs scheme to wipe off the modulated data clearly. Such illustrated PDM-based bi-directional 128 Gb/s (downstream)/10 Gb/s (upstream) fibre-FSO integration is shown to be prominent not only due to its enhancement in the convergence of fibre backhaul and optical wireless reach extender but also because of its benefit in bi-directional transmission for affording high transmission capacity with long-reach optical wireless link and improved upstream performance.

A 30 Gb/s PAM4 underwater wireless laser transmission system with optical beam reducer/expander.

We have, so far as we know, proposed and demonstrated the first 30 Gb/s four-level pulse amplitude modulation (PAM4) underwater wireless laser transmission (UWLT) system with an optical beam reducer/expander over 12.5-m piped underwater channel/2.5-m high-turbidity harbour underwater channel. In piped underwater links, the performances of PAM4 UWLT systems get better with beam reduction given a small amount of light absorbed by the piped water. In highly turbid harbour underwater links, the performances of PAM4 UWLT systems get better with beam expansion given a large amount of scattered light received by the optical receiver. The effect of high-turbidity harbour water that induces scattering angle (beam divergence) on beam diameter is analyzed and optimised to enhance the transmission performances. This proposed PAM4 UWLT system, which uses an optical beam reducer/expander, provides a practical choice for high transmission capacity and considerably develops clarity and high-turbidity scenarios. It presents promising features for affording a high-transmission-rate underwater optical wireless transmission and opening an access to accelerate wide applications of UWLT systems.

Climates on incidence of childhood type 1 diabetes mellitus in 72 countries.

We are aimed to systematically assess the worldwide trend in incidence of childhood type 1 diabetes mellitus (CT1DM) from 1965 to 2012 and to discuss whether climate affect incidence of CT1DM. We searched the relevant literatures in detail to judge the effect of different climates on incidence of CT1DM. The climates included Mediterranean, monsoon, oceanic, continental, savanna, and rainforest. According to different climates, we further researched relevant factor such as sunshine durations and latitudes. The overall incidence of CT1DM in 72 countries was 11.43 (95% CI 10.31-12.55) per 100,000 children/yr. The incidence of CT1DM in Oceanic climate [10.56 (8.69-12.42)] is highest compared with other climates; the incidence in 40°-66°34'N/S [14.71 (12.30-17.29)] is higher than other latitude groups; the incidence in sunshine durations with 3-4 hours per day [15.17 (11.14-19.20)] is highest compared with other two groups; the incidence of CT1DM from 2000 to 2012 [19.58 (14.55-24.60)] is higher than other periods; all p < 0.01. Incidence of CT1DM was increasing from 1965 to 2012, but incidence in Oceanic climate is higher than other climates. Furthermore, it is higher in centers with higher latitude and lower sunshine durations. The climates might play a key role in inducing CT1DM.

Serum TNF-α concentrations in type 2 diabetes mellitus patients and diabetic nephropathy patients: A systematic review and meta-analysis.

OBJECTIVES: The aim of this study was to investigate whether the concentrations of serum tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine, increased in type 2 diabetes mellitus (T2DM) and type 2 diabetic nephropathy (T2DN) patients. METHODS: The four databases (PubMed, CNKI, WanFang and Chinese-Cqvip) were searched from Jan 1, 1999 to October 1, 2016 for all clinical case-control studies about the serum TNF-α concentrations in T2DM and T2DN patients. All relevant data were extracted from published reports. The meta-analysis was performed to compare the changes of serum TNF-α concentrations of T2DN and T2DM patients in Eastern and Western with healthy controls. We further evaluated concentrations of serum TNF-α in T2DN patients with mincroalbuminuria or macroalbuminuria. Random-effects models were adopted to assess the pooling data among various variations. RESULTS: In total of 6 studies (744 patients and 277 healthy controls) were included in this study. Compared with healthy controls (both p<0.01), the groups of different albuminuria levels and ethnicities both showed that the serum TNF-α levels were significantly elevated in T2DN patients as well as in eastern T2DN patients (p=0.001), but not significant changed in western T2DN patients (p=0.081). The results were stable through sensitivity analysis and no significant publications bias existed in this meta-analysis. CONCLUSIONS: Serum TNF-α concentrations are obviously increased in T2DN and T2DM patients, but higher in T2DN patients, suggesting an elevated inflammatory burden in T2DN patients.

Correlation between serum interleukin-6 level and type 1 diabetes mellitus: A systematic review and meta-analysis.

OBJECTIVE: This report aimed to explore the association between the change of circulating interleukin-6 (IL-6) in patients and the development of type 1 diabetes mellitus (T1DM). METHODS: Four databases (PubMed, CNKI, WanFang and Civip) were used to search and list all clinical case-control studies about serum IL-6 level in T1DM patients between Jan 1, 2000 and Aug 31, 2016. RESULTS: A total of 20 case-control studies with 1238 T1DM patients and 742 healthy controls were included in this study. Compared to healthy controls, the serum content of IL-6 in patients with T1DM was significantly greater (overall: SMD, 1.49; 95% CI, 1.04 to 1.93; p<0.001), and notably increased in all subgroup with different age, ethnic and disease duration (all p<0.001). Furthermore, the analysis in subgroup exhibited that serum levels of IL-6 in the age greater than 20-year old (SMD, 1.64; 95% CI, 0.57-2.71; p<0.001), the diseased duration among 0-10years (SMD, 2.43; 95% CI, 1.42-3.44; p<0.001) and the sorted American group (SMD, 1.68; 95% CI, 0.85-2.51; p<0.001) were higher than those in control groups. CONCLUSIONS: Patients with T1DM were found to be linked to elevated level of serum IL-6, which the age, ethnic and disease durations in T1DM patients had no effect on the serum IL-6 levels for promoting diabetes mellitus.

Pnpla3I148M knockin mice accumulate PNPLA3 on lipid droplets and develop hepatic steatosis.

UNLABELLED: A sequence polymorphism (rs738409, I148M) in patatin-like phospholipid domain containing protein 3 (PNPLA3) is strongly associated with nonalcoholic fatty liver disease (NAFLD), but the mechanistic basis for this association remains enigmatic. Neither ablation nor overexpression of wild-type PNPLA3 affects liver fat content in mice, whereas hepatic overexpression of the human 148M transgene causes steatosis. To determine whether the 148M allele causes fat accumulation in the liver when expressed at physiological levels, we introduced a methionine codon at position 148 of the mouse Pnpla3 gene. Knockin mice had normal levels of hepatic fat on a chow diet, but when challenged with a high-sucrose diet their liver fat levels increased 2 to 3-fold compared to wild-type littermates without any associated changes in glucose homeostasis. The increased liver fat in the knockin mice was accompanied by a 40-fold increase in PNPLA3 on hepatic lipid droplets, with no increase in hepatic PNPLA3 messenger RNA (mRNA). Similar results were obtained when the catalytic dyad of PNPLA3 was inactivated by substituting the catalytic serine with alanine (S47A). CONCLUSION: These data provide the first direct evidence that physiological expression of PNPLA3 148M variant causes NAFLD, and that the accumulation of catalytically inactive PNPLA3 on the surfaces of lipid droplets is associated with the accumulation of TG in the liver.

The novel application of artificial neural network on bioelectrical impedance analysis to assess the body composition in elderly.

BACKGROUND: This study aims to improve accuracy of Bioelectrical Impedance Analysis (BIA) prediction equations for estimating fat free mass (FFM) of the elderly by using non-linear Back Propagation Artificial Neural Network (BP-ANN) model and to compare the predictive accuracy with the linear regression model by using energy dual X-ray absorptiometry (DXA) as reference method. METHODS: A total of 88 Taiwanese elderly adults were recruited in this study as subjects. Linear regression equations and BP-ANN prediction equation were developed using impedances and other anthropometrics for predicting the reference FFM measured by DXA (FFMDXA) in 36 male and 26 female Taiwanese elderly adults. The FFM estimated by BIA prediction equations using traditional linear regression model (FFMLR) and BP-ANN model (FFMANN) were compared to the FFMDXA. The measuring results of an additional 26 elderly adults were used to validate than accuracy of the predictive models. RESULTS: The results showed the significant predictors were impedance, gender, age, height and weight in developed FFMLR linear model (LR) for predicting FFM (coefficient of determination, r2 = 0.940; standard error of estimate (SEE) = 2.729 kg; root mean square error (RMSE) = 2.571kg, P < 0.001). The above predictors were set as the variables of the input layer by using five neurons in the BP-ANN model (r2 = 0.987 with a SD = 1.192 kg and relatively lower RMSE = 1.183 kg), which had greater (improved) accuracy for estimating FFM when compared with linear model. The results showed a better agreement existed between FFMANN and FFMDXA than that between FFMLR and FFMDXA. CONCLUSION: When compared the performance of developed prediction equations for estimating reference FFMDXA, the linear model has lower r2 with a larger SD in predictive results than that of BP-ANN model, which indicated ANN model is more suitable for estimating FFM.

Chronic overexpression of PNPLA3I148M in mouse liver causes hepatic steatosis.

A genetic variant in PNPLA3 (PNPLA3(I148M)), a triacylglycerol (TAG) hydrolase, is a major risk factor for nonalcoholic fatty liver disease (NAFLD); however, the mechanism underlying this association is not known. To develop an animal model of PNPLA3-induced fatty liver disease, we generated transgenic mice that overexpress similar amounts of wild-type PNPLA3 (PNPLA3(WT)) or mutant PNPLA3 (PNPLA3(I148M)) either in liver or adipose tissue. Overexpression of the transgenes in adipose tissue did not affect liver fat content. Expression of PNPLA3(I148M), but not PNPLA3(WT), in liver recapitulated the fatty liver phenotype as well as other metabolic features associated with this allele in humans. Metabolic studies provided evidence for 3 distinct alterations in hepatic TAG metabolism in PNPLA3(I148M) transgenic mice: increased formation of fatty acids and TAG, impaired hydrolysis of TAG, and relative depletion of TAG long-chain polyunsaturated fatty acids. These findings suggest that PNPLA3 plays a role in remodeling TAG in lipid droplets, as they accumulate in response to food intake, and that the increase in hepatic TAG levels associated with the I148M substitution results from multiple changes in hepatic TAG metabolism. The development of an animal model that recapitulates the metabolic phenotype of the allele in humans provides a new platform in which to elucidate the role of PNLPA3(I148M) in NAFLD.

Molecular characterization of proprotein convertase subtilisin/kexin type 9-mediated degradation of the LDLR.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that promotes degradation of cell surface LDL receptors (LDLRs) in selected cell types. Here we used genetic and pharmacological inhibitors to define the pathways involved in PCSK9-mediated LDLR degradation. Inactivating mutations in autosomal recessive hypercholesterolemia (ARH), an endocytic adaptor, blocked PCSK9-mediated LDLR degradation in lymphocytes but not in fibroblasts. Thus, ARH is not specifically required for PCSK9-mediated LDLR degradation. Knockdown of clathrin heavy chain with siRNAs prevented LDLR degradation. In contrast, prevention of ubiquitination of the LDLR cytoplasmic tail, inhibition of proteasomal activity, or disruption of proteins required for lysosomal targeting via macroautophagy (autophagy related 5 and 7) or the endosomal sorting complex required for trafficking (ESCRT) pathway (hepatocyte growth factor-regulated Tyr-kinase substrate and tumor suppressor gene 101) failed to block PCSK9-mediated LDLR degradation. These findings are consistent with a model in which the LDLR-PCSK9 complex is internalized via clathrin-mediated endocytosis and then routed to lysosomes via a mechanism that does not require ubiquitination and is distinct from the autophagy and proteosomal degradation pathways. Finally, the PCSK9-LDLR complex appears not to be transported by the canonical ESCRT pathway.