Lipid distributions in the Global Diagnostics Network across five continents.

AIMS: Lipids are central in the development of cardiovascular disease, and the present study aimed to characterize variation in lipid profiles across different countries to improve understanding of cardiovascular risk and opportunities for risk-reducing interventions. METHODS AND RESULTS: This first collaborative report of the Global Diagnostics Network (GDN) evaluated lipid distributions from nine laboratory organizations providing clinical laboratory testing in 17 countries on five continents. This cross-sectional study assessed aggregated lipid results from patients aged 20-89 years, tested at GDN laboratories, from 2018 through 2020. In addition to mean levels, the World Health Organization total cholesterol risk target (<5.00 mmol/L, <193 mg/dL) and proportions in guideline-based low-density lipoprotein cholesterol (LDL-C) categories were assessed. This study of 461 888 753 lipid results found wide variation by country/region, sex, and age. In most countries, total cholesterol and LDL-C peaked at 50-59 years in females and 40-49 years in males. Sex- and age-group adjusted mean total cholesterol levels ranged from 4.58 mmol/L (177.1 mg/dL) in the Republic of Korea to 5.40 mmol/L (208.8 mg/dL) in Austria. Mean total cholesterol levels exceeded the World Health Organization target in Japan, Australia, North Macedonia, Switzerland, Germany, Slovakia, and Austria. Considering LDL-C categories, North Macedonia had the highest proportions of LDL-C results >4.91 mmol/L (>190 mg/dL) for both females (9.9%) and males (8.7%). LDL-C levels <1.55 mmol/L (<60 mg/dL) were most common among females in Canada (10.7%) and males in the UK (17.3%). CONCLUSION: With nearly a half billion lipid results, this study sheds light on the worldwide variability in lipid levels, which may reflect inter-country differences in genetics, lipid testing, lifestyle habits, and pharmacologic treatment. Despite variability, elevated atherogenic lipid levels are a common global problem, and these results can help inform national policies and health system approaches to mitigate lipid-mediated risk of cardiovascular disease.

Optimizing Eco-Friendly Degradation of Polyvinyl Chloride (PVC) Plastic Using Environmental Strains of Malassezia Species and Aspergillus fumigatus.

Worldwide, huge amounts of plastics are being introduced into the ecosystem, causing environmental pollution. Generally, plastic biodegradation in the ecosystem takes hundreds of years. Hence, the isolation of plastic-biodegrading microorganisms and finding optimum conditions for their action is crucial. The aim of the current study is to isolate plastic-biodegrading fungi and explore optimum conditions for their action. Soil samples were gathered from landfill sites; 18 isolates were able to grow on SDA. Only 10 isolates were able to the degrade polyvinyl chloride (PVC) polymer. Four isolates displayed promising depolymerase activity. Molecular identification revealed that three isolates belong to genus Aspergillus, and one isolate was Malassezia sp. Three isolates showed superior PVC-biodegrading activity (Aspergillus-2, Aspergillus-3 and Malassezia) using weight reduction analysis and SEM. Two Aspergillus strains and Malassezia showed optimum growth at 40 °C, while the last strain grew better at 30 °C. Two Aspergillus isolates grew better at pH 8-9, and the other two isolates grow better at pH 4. Maximal depolymerase activity was monitored at 50 °C, and at slightly acidic pH in most isolates, FeCl3 significantly enhanced depolymerase activity in two Aspergillus isolates. In conclusion, the isolated fungi have promising potential to degrade PVC and can contribute to the reduction of environmental pollution in eco-friendly way.

Isolated and Combined Effect of Age and Gender on Neutrophil-Lymphocyte Ratio in the Hyperglycemic Saudi Population.

Inflammation is pivotal to the pathogenesis of diabetes mellitus (DM), but pathological alterations of the neutrophil−lymphocyte ratio (NLR), an emerging inflammatory index in DM management, remains understudied. The aim of this study is to examine the relationship between NLR and glycemic control in the Saudi population. Gender, age, WBC count, and fasting blood glucose (FBG) were obtained from Al-Borg Medical Laboratories for 14,205 subjects. Means, prevalence, risk measures, and the diagnostic accuracy of elevated NLR and hyperglycemia (HG) were evaluated. Subjects with elevated NLR (>3) had significantly higher FBG (105.10 ± 0.33 vs. 114.0 ± 2.81) and NLR was significantly elevated in impaired fasting glycemia (IFG; 1.21 ± 0.01 vs. 1.25 ± 0.01) and HG (1.21 ± 0.01 vs. 1.39 ± 0.02). Elevations of NLR in HG but not in IFG persisted across all age groups except young males and elderly females. The prevalence of elevated NLR in hyperglycemic subjects was 4.12% compared to 2.16% in subjects with normal FBG. HG was more prevalent in subjects with elevated NLR (17.33% vs. 12.46%) who had a relative risk (RR) of 1.68 (95% CI = 1.38−2.06, p < 0.0001) and an odds ratio (OR) of 1.94 (95% CI = 1.48−2.56, p < 0.0001) to be hyperglycemic. Nevertheless, NLR failed to discriminate individuals with normal FBG from those with HG based on ROC curve analysis. Pathological fluctuations in NLR may serve as supportive evidence in DM management.

Molecular insights into the coding region mutations of low-density lipoprotein receptor adaptor protein 1 (LDLRAP1) linked to familial hypercholesterolemia.

BACKGROUND: Familial hypercholesterolemia (FH) is a lipid disorder caused by pathogenic mutations in LDLRAP1 gene. The present study has aimed to deepen our understanding about the pathogenicity predictions of FH causative genetic mutations, as well as their relationship to phenotype changes in LDLRAP1 protein, by utilizing multidirectional computational analysis. METHODS: FH linked LDLRAP1 mutations were mined from databases, and the prediction ability of several pathogenicity classifiers against these clinical variants, was assessed through different statistical measures. Furthermore, these mutations were 3D modelled in protein structures to assess their impact on protein phenotype changes. RESULTS: Our findings suggest that Polyphen-2, when compared with SIFT, M-CAP and CADD tools, can make better pathogenicity predictions for FH causative LDLRAP1 mutations. Through, 3D simulation and superimposition analysis of LDLRAP1 protein structures, it was found that missense mutations do not create any gross changes in the protein structure, although they could induce subtle structural changes at the level of amino acid residues. Near native molecular dynamic analysis revealed that missense mutations could induce variable degrees of fluctuation differences guiding the protein flexibility. Stability analysis showed that most missense mutations shifts the free energy equilibrium and hence they destabilize the protein. Molecular docking analysis demonstrates the molecular shifts in hydrogen and ionic bonds and Van der waals bonding properties, which further cause differences in the binding energy of LDLR-LDLRAP1 proteins. CONCLUSIONS: The diverse computational approaches used in the present study may provide a new dimension for exploring the structure-function relationship of the novel and deleterious LDLRAP1 mutations linked to FH.

Assessment of medical intern's knowledge, awareness and practice of familial hypercholesterolemia at academic institutes in Jeddah, Saudi Arabia.

BACKGROUND: Familial Hypercholesterolemia (FH) is a serious under-diagnosed disease characterized by raised low-density lipoprotein cholesterol (LDL-C) and premature coronary artery diseases (CAD). The scarcity of FH reported patients in Saudi Arabia indicates lack of FH awareness among physicians. OBJECTIVE: The goal of this research was to assess knowledge, awareness, and practice (KAP) about FH disorder among Saudi medical interns and to identify areas that need educational attention. METHODS: This cross-sectional study involved 170 Saudi medical interns (83 males and 87 females) from academic institutes in Jeddah, Saudi Arabia. The interns were asked to fill an online FH-KAP questionnaire. Total score for each separate domain measured by adding correct answers. RESULTS: Although, knowledge of FH definition (76.5%) and classical lipid profile (52.4%) were reasonable; knowledge on inheritance (43.5%), prevalence (12.4%) and CAD risks (7.1%) were poor. Knowledge score was significantly higher in female than male (7.5 ± 3 vs. 5.3 ± 2.6, P < 0.001). Regarding awareness, 54.1% were familiar with FH disorder, 50.6% with the presence of lipid clinic but only 16.5% were acquainted with guidelines. Furthermore, in the practice domain 82.9% selected statin as first line treatment and 62.9% chose routinely checking the rest of the family, while 15.3% chose ages 13-18 years to screen for hypercholesterolemia in patients with a positive family history of premature CAD. CONCLUSION: Substantial defects in FH-KAP among Saudi medical interns were found, emphasizing the importance of professional training. Extensive and constant medical education programs as early as an internship are required to close the gap in CAD prevention.

Optimization of Thymoquinone-Loaded Coconut Oil Nanostructured Lipid Carriers for the Management of Ethanol-Induced Ulcer.

In the global incidence of peptic ulcer, with the associated rates of hospitalizations and mortality are increasing, in the United States, peptic ulcer disease affects approximately 4.6 million people annually, with an estimated 10% of the US population having evidence of a duodenal ulcer. The present research aims to find a novel treatment for ethanol induced ulcer by loading thymoquinone (TQ) on a nanostructured lipid carrier (NLC), using Compritol® 888 and coconut oil. The TQ-loaded coconut oil NLC was formulated using melt emulsification combined with a sonication method using Poloxamer 188 as a surfactant. Finally, the optimization of the formulations was performed on a three-factor, three-level Box-Behnken statistical design, with 85.63% entrapment efficiency of TQ in the optimized formulation. A biphasic release pattern of the formulation was recorded in an in vitro drug release study, where the initial burst release of the drug was observed in the first 2 h, followed by a gradual release. Later, the TQ-loaded coconut oil NLC was found to protect the gastric mucous membrane more effectively (78.95% in.; p < 0.01) in an alcohol-induced ulcer model, whereas the TQ suspension showed 30.87% inhibition (p < 0.05) of the ulcerative index, when compared with the ulcer control group. The histopathological evaluations of the stomach in ulcer-induced animals demonstrated protection potential of TQ-loaded coconut oil NLC against an alcohol-induced gastric ulcer. In a nutshell, the entrapment of TQ within the NLC was found to deliver the entrapped drug more effectively when administered through an oral route to possess a gastroprotective effect.

Correction to: Optimization of Thymoquinone-Loaded Coconut Oil Nanostructured Lipid Carriers for the Management of Ethanol-Induced Ulcer.

When the article was first published, the incorrect image for Fig. 1 was inadvertently uploaded.

Molecular design, synthesis and biological characterization of novel Resveratrol derivative as potential anticancer agent targeting NF-κB.

Resveratrol (RESV), an anticancer nutraceutical compound, is known to show poor bioavailability inside the human body. Therefore, this study has designed multiple chemical analogs of RESV compound for improving its pharmacokinetic as well as its anti-cancer properties. Initially, the drug likeliness and ADME-toxicity properties of these new chemical analogs were tested with the help of diverse computational approaches. Then the best predicted RESV derivative is synthesized by the organic method, and its NF-κB mediated anti-tumor activity assessed on histiocytic lymphoma U-937 cells. The new synthetic RESV analog, i.e. (E)-3-(prop-2-yn-1-yloxy)-5-(4-(prop-2-yn-1-yloxy) styryl) phenol has shown a rapid, persistent and better dose-dependent (IC50 of 7.25 µM) decrease in the viability of U937 cells than the native (IC50 of 30 µM) RESV compound. This analog has also demonstrated its potential ability in inducing apoptosis through DNA ladder formation. At 10 µg/ml concentration, this chemical derivative has shown a better NF-κB inhibition (IC50 is 2.45) compared to the native RESV compound (IC50 is 1.95). Molecular docking analysis found that this analog exerts its anti- NF-κB activity (binding energy of -6.78 kcal/mol and Ki 10 µM) by interacting with DNA binding residues (Arg246, Lys444, and Gln606) of p50 chain NF-κB. This study presents a novel RESV analog that could further develop as a potential anti-NF-κB mediated tumor inhibitor.

Prevalence of Helicobacter pylori Infection and Diagnostic Methods in the Middle East and North Africa Region.

Background and Objectives: Helicobacter pylori (H. pylori) infection is common worldwide and may cause gastroduodenal complications, including cancer. In this review, we examine the prevalence and distribution of various H. pylori genotypes and the risk factors for H. pylori infection, particularly in the Middle East and North Africa (MENA) region. We also introduce different global screening methods and guidelines and compare them to those currently in use in the MENA region. Materials and Methods: We searched the Google Scholar, PubMed, and Saudi Digital Library (SDL) databases for clinical trials and articles published in English. The data collection was mainly focused on MENA countries. However, for H. pylori genotypes and diagnostic methods, studies conducted in other regions or reporting global practices and guidelines were also included to allow a comparison with those in the MENA region. We also included studies examining the prevalence of H. pylori infection in healthy participants. Results: H. pylori infection is highly prevalent in the MENA region, mainly because of the accumulation of risk factors in developing countries. Herein, we highlight a lack of good quality studies on the prevalence of various H. pylori genotypes in the MENA region as well as a need for standard diagnostic methods and screening guidelines. Due to the complications associated with H. pylori, we recommend routine screening for H. pylori infection in all gastroenterology patients admitted in the MENA region. Conclusion: Concerted effort will first be required to validate affordable, non-invasive, and accurate diagnostic methods and to establish local guidelines with adapted cut-off values for the interpretation of the test results.

Recently reported familial hypercholesterolemia-related mutations from cases in the Middle East and North Africa region.

PURPOSE OF REVIEW: Familial hypercholesterolemia is an inherited disorder where cases have a significantly higher risk of having premature myocardial infarction than noncases. The prevalence of this genetic disease is currently unknown in countries of the Middle East and North Africa region. Given that a high percentage of marriages are consanguineous in this region, the prevalence may be much higher than assumed. We systematically reviewed the literature to identify case-related mutations reported within the last 4 years and since our first report in 2014. RECENT FINDINGS: Mutations were reported in familial hypercholesterolemia cases from the Saudi, Iranian, Lebanese, and Syrian populations. Some of the mutations were novel and a variety of familial hypercholesterolemia genotypes were identified, such as compound heterozygotes and double heterozygotes. SUMMARY: In recent years, work has been done to identify familial hypercholesterolemia cases in various countries of the Middle East and North Africa region. With regards to the prospective familial hypercholesterolemia registry for the Middle East and North Africa region, an important goal for the near future would be to have physician specialists collaborate with primary care clinicians for the identification and optimal care of familial hypercholesterolemia cases.

Thrombin activation of protein C requires prior processing by a liver proprotein convertase.

Protein C, a secretory vitamin K-dependent anticoagulant serine protease, inactivates factors Va/VIIIa. It is exclusively synthesized in liver hepatocytes as an inactive zymogen (proprotein C). In humans, thrombin cleavage of the propeptide at PR221↓ results in activated protein C (APC; residues 222-461). However, the propeptide is also cleaved by a furin-like proprotein convertase(s) (PCs) at KKRSHLKR199↓ (underlined basic residues critical for the recognition by PCs), but the order of cleavage is unknown. Herein, we present evidence that at the surface of COS-1 cells, mouse proprotein C is first cleaved by the convertases furin, PC5/6A, and PACE4. In mice, this cleavage occurs at the equivalent site, KKRKILKR198↓, and requires the presence of Arg198 at P1 and a combination of two other basic residues at either P2 (Lys197), P6 (Arg193), or P8 (Lys191) positions. Notably, the thrombin-resistant R221A mutant is still cleaved by these PCs, revealing that convertase cleavage can precede thrombin activation. This conclusion was supported by the fact that the APC-specific activity in the medium of COS-1 cells is exclusively dependent on prior cleavage by the convertases, because both R198A and R221A lack protein C activity. Primary cultures of hepatocytes derived from wild-type or hepatocyte-specific furin, PC5/6, or complete PACE4 knock-out mice suggested that the cleavage of overexpressed proprotein C is predominantly performed by furin intracellularly and by all three proprotein convertases at the cell surface. Indeed, plasma analyses of single-proprotein convertase-knock-out mice showed that loss of the convertase furin or PC5/6 in hepatocytes results in a ∼30% decrease in APC levels, with no significant contribution from PACE4. We conclude that prior convertase cleavage of protein C in hepatocytes is critical for its thrombin activation.

The Proprotein Convertase Subtilisin/Kexin Type 9-resistant R410S Low Density Lipoprotein Receptor Mutation: A NOVEL MECHANISM CAUSING FAMILIAL HYPERCHOLESTEROLEMIA.

Familial hypercholesterolemia (FH) is characterized by severely elevated low density lipoprotein (LDL) cholesterol. Herein, we identified an FH patient presenting novel compound heterozygote mutations R410S and G592E of the LDL receptor (LDLR). The patient responded modestly to maximum rosuvastatin plus ezetimibe therapy, even in combination with a PCSK9 monoclonal antibody injection. Using cell biology and molecular dynamics simulations, we aimed to define the underlying mechanism(s) by which these LDLR mutations affect LDL metabolism and lead to hypercholesterolemia. Our data showed that the LDLR-G592E is a class 2b mutant, because it mostly failed to exit the endoplasmic reticulum and was degraded. Even though LDLR-R410S and LDLR-WT were similar in levels of cell surface and total receptor and bound equally well to LDL or extracellular PCSK9, the LDLR-R410S was resistant to exogenous PCSK9-mediated degradation in endosomes/lysosomes and showed reduced LDL internalization and degradation relative to LDLR-WT. Evidence is provided for a tighter association of LDL with LDLR-R410S at acidic pH, a reduced LDL delivery to late endosomes/lysosomes, and an increased release in the medium of the bound/internalized LDL, as compared with LDLR-WT. These data suggested that LDLR-R410S recycles loaded with its LDL-cargo. Our findings demonstrate that LDLR-R410S represents an LDLR loss-of-function through a novel class 8 FH-causing mechanism, thereby rationalizing the observed phenotype.

Protein phenotype diagnosis of autosomal dominant calmodulin mutations causing irregular heart rhythms.

The life-threatening group of irregular cardiac rhythmic disorders also known as Cardiac Arrhythmias (CA) are caused by mutations in highly conserved Calmodulin (CALM/CaM) genes. Herein, we present a multidimensional approach to diagnose changes in phenotypic, stability, and Ca2+ ion binding properties of CA-causing mutations. Mutation pathogenicity was determined by diverse computational machine learning approaches. We further modeled the mutations in 3D protein structure and analyzed residue level phenotype plasticity. We have also examined the influence of torsion angles, number of H-bonds, and free energy dynamics on the stability, near-native simulation dynamic potential of residue fluctuations in protein structures, Ca2+ ion binding potentials, of CaM mutants. Our study recomends to use M-CAP method for measuring the pathogenicity of CA causing CaM variants. Interestingly, most CA-causing variants we analyzed, exists in either third (V/H-96, S/I-98, V-103) or fourth (G/V-130, V/E/H-132, H-134, P-136, G-141, and L-142) EF-hands located in carboxyl domains of the CaM molecule. We observed that the minor structural fluctuations caused by these variants are likely tolerable owing to the highly flexible nature of calmodulin's globular domains. However, our molecular docking results supports that these variants disturb the affinity of CaM toward Ca2+ ions and corroborate previous findings from functional studies. Taken together, these computational findings can explain the molecular reasons for subtle changes in structure, flexibility, and stability aspects of mutant CaM molecule. Our comprehensive molecular scanning approach demonstrates the utility of computational methods in quick preliminary screening of CA- CaM mutations before undertaking time consuming and complicated functional laboratory assays.

Assisting the integration of social media in problem-based learning sessions in the Faculty of Medicine at King Abdulaziz University.

PURPOSE: Issues related to traditional Problem-Based Learning (PBL) at King Abdulaziz University Faculty of Medicine (KAU-FOM), including lack of student interaction between sessions and outdated instructional materials have led to the examining the use of social media. This study examines factors affecting the implementation of social media into PBL sessions Methods: Mentored social media activities were incorporated between PBL sessions to third year medical students. Ground rules were set, and students were kept on track with learning objectives and authentic references. An online survey consisting of 18 questions were administered to measure the impact of the social media model embedded between PBL sessions. RESULTS: Feedback showed major improvements in students' learning process as well as identifying areas for improvement. The highest ratings were in participation and communication, knowledge and information gathering, and cooperation and team-building. CONCLUSIONS: This paper indicates that incorporating social media could facilitate learning between PBL sessions. Furthermore, guidelines are proposed to help educators implement a social media model into their PBL sessions.

Integrating an interprofessional education initiative: Evidence from King Abdulaziz University.

PURPOSE: This paper examines current issues with interprofessional education (IPE) at King Abdulaziz University (KAU) and discusses initiatives for integrating IPE into the medical curricula at KAU. METHODS: We reviewed the current body of literature, studied reports from IPE conferences and workshops organized at KAU, and synthesized participants' feedback from the IPE programs, including an online survey. RESULTS: A total of 506 participants responded to the online survey. Respondents rated Interprofessional Collaborative Learning as the highest category of IPE, followed by Interprofessional Self-Improvement and Interprofessional Relationship. A hybrid conceptual framework is proposed, to tackle the issue of role clarification across all healthcare colleges at KAU. This proposition was found to be necessary due to the current state of the undergraduate curriculum which does not prepare students properly for professional collaboration. CONCLUSIONS: The hybrid model may narrow the gap in IPE by emphasizing professional identity while reducing autonomy. Recommendations toward IPE are presented. Challenges toward IPE reform are discussed in the context of implementation at KAU and at other medical schools in the region.

PCSK9: a key modulator of cardiovascular health.

Since the discovery of proprotein convertase subtilisin kexin 9 (PCSK9) in 2003, this PC has attracted a lot of attention from the scientific community and pharmaceutical companies. Secreted into the plasma by the liver, the proteinase K-like serine protease PCSK9 binds the low-density lipoprotein (LDL) receptor at the surface of hepatocytes, thereby preventing its recycling and enhancing its degradation in endosomes/lysosomes, resulting in reduced LDL-cholesterol clearance. Surprisingly, in a nonenzymatic fashion, PCSK9 enhances the intracellular degradation of all its target proteins. Rare gain-of-function PCSK9 variants lead to higher levels of LDL-cholesterol and increased risk of cardiovascular disease; more common loss-of-function PCSK9 variants are associated with reductions in both LDL-cholesterol and risk of cardiovascular disease. It took 9 years to elaborate powerful new PCSK9-based therapeutic approaches to reduce circulating levels of LDL-cholesterol. Presently, PCSK9 monoclonal antibodies that inhibit its function on the LDL receptor are evaluated in phase III clinical trials. This review will address the biochemical, genetic, and clinical aspects associated with PCSK9's biology and pathophysiology in cells, rodent and human, with emphasis on the clinical benefits of silencing the expression/activity of PCSK9 as a new modality in the treatment of hypercholesterolemia and associated pathologies.

A semi-automated mass spectrometric immunoassay coupled to selected reaction monitoring (MSIA-SRM) reveals novel relationships between circulating PCSK9 and metabolic phenotypes in patient cohorts.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of circulating low density lipoprotein cholesterol (LDL-C) levels. Besides its full-length mature form, multiple variants of PCSK9 have been reported such as forms that are truncated, mutated and/or with posttranslational modifications (PTMs). Previous studies have demonstrated that most of these variants affect PCSK9's function and thereby LDL-C levels. Commercial ELISA kits are available for quantification of PCSK9, but do not allow discrimination between the various forms and PTMs of the protein. To address this issue and given the complexity and wide dynamic range of the plasma proteome, we have developed a mass spectrometric immunoassay coupled to selected reaction monitoring (MSIA-SRM) for the multiplexed quantification of several forms of circulating PCSK9 in human plasma. Our MSIA-SRM assay quantifies peptides spanning the various protein domains and the S688 phosphorylation site. The assay was applied in two distinct cohorts of obese patients and healthy pregnant women stratified by their circulating LDL-C levels. Seven PCSK9 peptides were monitored in plasma samples: one in the prodomain prior to the autocleavage site at Q152, one in the catalytic domain prior to the furin cleavage site at R218, two in the catalytic domain following R218, one in the cysteine and histidine rich domain (CHRD) and the C-terminal peptide phosphorylated at S688 and unmodified. The latter was not detectable in sufficient amounts to be quantified in human plasma. All peptides were measured with high reproducibility and with LLOQ and LOD below the clinical range. The abundance of 5 of the 6 detectable PCSK9 peptides was higher in obese patients stratified with high circulating LDL-C levels as compared to those with low LDL-C (p < 0.05). The same 5 peptides showed good and statistically significant correlations with LDL-C levels (0.55 < r < 0.65; 0.0002 ⩽ p ⩽ 0.002), but not the S688 phosphorylated peptide. However, this phosphopeptide was significantly correlated with insulin resistance (r = 0.48; p = 0.04). In the pregnant women cohort, none of the peptides were associated to LDL-C levels. However, the 6 detectable PCSK9 peptides, but not PCSK9 measured by ELISA, were significantly correlated with serum triglyceride levels in this cohort. Our results also suggest that PCSK9 circulates with S688 phosphorylated at high stoichiometry. In summary, we have developed and applied a robust and sensitive MSIA-SRM assay for the absolute quantification of all PCSK9 domains and a PTM in human plasma. This assay revealed novel relationships between PCSK9 and metabolic phenotypes, as compared to classical ELISA assays.

Decreased PCSK9 expression in human hepatocellular carcinoma.

BACKGROUND: The management of hepatocellular carcinoma (HCC) is limited by the lack of adequate screening biomarkers and chemotherapy. In response, there has been much interest in tumor metabolism as a therapeutic target. PCSK9 stimulates internalization of the LDL-receptor, decreases cholesterol uptake into hepatocytes and affects liver regeneration. Thus, we investigated whether PCSK9 expression is altered in HCC, influencing its ability to harness cholesterol metabolism. METHODS: Thirty-nine patients undergoing partial hepatectomy or liver transplantation for HCC were consented for use of HCC tissue to construct a tissue microarray (TMA). The TMA was immunostained for PCSK9. Imagescope software was used to objectively determine staining, and assess for pathological and clinical correlations. PCSK9 and LDL receptor mRNA levels in flash-frozen HCC and adjacent liver tissue were determined by quantitative RT-PCR. Serum PCSK9 levels were determined by ELISA. RESULTS: By immunohistochemistry, there was significantly lower expression of PCSK9 in HCC as compared to adjacent cirrhosis (p-value < 0.0001, wilcoxon signed-rank test). Significantly greater staining of PCSK9 was present in cirrhosis compared to HCC (p value <0.0001), and positivity (percentage of positive cells) was significantly greater in cirrhosis compared to HCC (p-value < 0.0001). Conversely, significantly higher expression of LDL-R was present in HCC as compared to the adjacent cirrhosis (p-value < 0.0001). There was no significant correlation of PCSK9 staining with grade of tumor, but there were significant correlations between PCSK9 staining and stage of fibrosis, according to spearman correlation test. PCSK9 mRNA levels were relatively less abundant within HCC compared to adjacent liver tissue (p-value =0.08) and normal control tissue (p-value =0.02). In contrast, serum PCSK9 levels were significantly increased among patients with HCC compared to those with chronic liver disease without HCC (p-value =0.029). LDL receptor mRNA was consistantly greater in HCC when compared to normal control tissue (p-value = 0.06) and, in general, was significantly greater in HCC when compared to adjacent liver (p-value = 0.04). CONCLUSIONS: The decreased expression of PCSK9 and conversely increased LDL-R expression in HCC suggests that HCC modulates its local microenvironment to enable a constant energy supply. Larger-scale studies should be conducted to determine whether PCSK9 could be a therapeutic target for HCC.

Proprotein convertase subtilisin/kexin type 9 (PCSK9): lessons learned from patients with hypercholesterolemia.

BACKGROUND: Identification of the proprotein convertase subtilisin/kexin type 9 (PCSK9) as the third gene causing familial hypercholesterolemia (FH) and understanding its complex biology has led to the discovery of a novel class of therapeutic agents. CONTENT: PCSK9 undergoes autocatalytic cleavage in the endoplasmic reticulum and enters the secretory pathway. The PCSK9 gene is under the regulatory control of sterol receptor binding proteins 1 and 2. Statins increase PCSK9 and this may modulate the response to this class of medications. In plasma, PCSK9 binds to the epidermal growth factor-like domain of the LDL receptor (LDL-R) on the cell and, once incorporated in the late endosomal pathway, directs the LDL-R toward lysosomal degradation rather than recycling to the plasma membrane. Thus, gain-of-function PCSK9 mutations lead to an FH phenotype, whereas loss-of-function mutations are associated with increased LDL-R-mediated endocytosis of LDL particles and lower LDL cholesterol in plasma. Inhibition of PCSK9 is thus an attractive therapeutic target. Presently, this is achieved by using monoclonal antibodies for allosteric inhibition of the PCSK9-LDL-R interaction. Phase 2 and 3 clinical trials in patients with moderate and severe hypercholesterolemia (including FH) show that this approach is safe and highly efficacious to lower LDL-C and lipoprotein(a). SUMMARY: PCSK9 has other biological roles observed in vitro and in animal studies, including viral entry into the cell, insulin resistance, and hepatic tissue repair. Given the potential number of humans exposed to this novel class of medications, careful evaluation of clinical trial results is warranted.