Core-shell graphene oxide-polymer hollow fibers as water filters with enhanced performance and selectivity.

Commercial hollow fiber filters for micro- and ultrafiltration are based on size exclusion and do not allow the removal of small molecules such as antibiotics. Here, we demonstrate that a graphene oxide (GO) layer can be firmly immobilized either inside or outside polyethersulfone-polyvinylpyrrolidone hollow fiber (Versatile PES®, hereafter PES) modules and that the resulting core-shell fibers inherits the microfiltration ability of the pristine PES fibers and the adsorption selectivity of GO. GO nanosheets were deposited on the fiber surface by filtration of a GO suspension through a PES cartridge (cut-off 0.1-0.2 µm), then fixed by thermal annealing at 80 °C, rendering the GO coating stably fixed and unsoluble. The filtration cut-off, retention selectivity and efficiency of the resulting inner and outer modified hollow fibers (HF-GO) were tested by performing filtration on water and bovine plasma spiked with bovine serum albumin (BSA, 66 kDa, ≈15 nm size), monodisperse polystyrene nanoparticles (52 nm and 303 nm sizes), with two quinolonic antibiotics (ciprofloxacin and ofloxacin) and rhodamine B (RhB). These tests showed that the microfiltration capability of PES was retained by HF-GO, and in addition the GO coating can capture the molecular contaminants while letting through BSA and smaller polystyrene nanoparticles. Combined XRD, molecular modelling and adsorption experiments show that the separation mechanism does not rely only on physical size exclusion, but involves intercalation of solute molecules between the GO layers.

Early palliative care and quality of life of advanced cancer patients-a multicenter randomized clinical trial.

BACKGROUND: To compare quality of life (QoL) of patients receiving early palliative care (EPC) vs. standard oncologic care (SOC). METHODS: Pragmatic, multicenter, randomized trial at five University and Community Hospital Cancer Centers in Northern Italy. Advanced non-small cell lung, gastric, pancreatic and biliary tract cancer patients diagnosed within the previous 8 weeks. In the EPC arm, visits were performed systematically by a dedicated physician/nurse palliative care (PC) team, who assessed physical and psychosocial symptoms, and enacted the necessary services. In the SOC arm, PC visits were only carried out if requested. The primary outcome was the difference in the change of QoL [Functional Assessment of Cancer Therapy-General measure (FACT-G)] from baseline to 12 weeks in the two groups. RESULTS: From November 2014 to March 2016, 281 patients were enrolled (142 EPC, 139 SOC); 218 completed FACT-G at 12 weeks. Baseline demographic and clinical characteristics were similar for the two groups. Values of FACT-G at baseline and 12 weeks were 72.3 (SD 12.6) and 70.1 (SD 15.5) for patients enrolled in the EPC arm, vs. 71.7 (SD 14.7) and 69.6 (SD 15.5) for the SOC arm, but the change scores did not differ significantly between groups. In the multivariable analysis, adjusting for QoL at baseline, two potential prospective prognostic factors were statistically significant: lung cancer (P=0.03) and interaction of living without a partner and intervention arm (P=0.01). Dying within 6 months (P<0.001) was also statistically significant. CONCLUSIONS: In this study, EPC did not improve QoL in advanced cancer patients, but our findings highlight aspects which may guide future research on EPC.

Immobilization of Perylene-3,4,9,10-Tetracarboxylic Dianhydride on Hollow Polysulfone Fibers: Primary Amine Coupling and Fluorescence Reporting.

The fluorescent dye perylene-3,4,9,10-tetracarboxylic dianhydride (PBA) was immobilized onto polysulfone hollow fibers by means of a wet coating procedure. After immobilization, PBA was able to react with primary amines through a double anhydride ring opening reaction. The in situ amine coupling was further revealed by fluorescence analysis. Both emission (534 nm →538 nm) and fluorescence lifetime changes (2.7 ns →3.3 ns) of the dye are a useful tool to detect and visualize the occurrence of the reaction. The successful implementation of amine coupling with a reporting function on polysulfone fibers holds great interest for biomedical applications.

Lysosomal lipase deficiency: molecular characterization of eleven patients with Wolman or cholesteryl ester storage disease.

Wolman Disease (WD) and cholesteryl ester storage disease (CESD) represent two distinct phenotypes of the same recessive disorder caused by the complete or partial deficiency of lysosomal acidic lipase (LAL), respectively. LAL, encoded by the LIPA gene, hydrolyzes cholesteryl esters derived from cell internalization of plasma lipoproteins. WD is a rapidly progressive and lethal disease characterized by intestinal malabsorption, hepatic and adrenal failure. CESD is characterized by hepatic fibrosis, hyperlipidemia and accelerated atherosclerosis. Aim of the study was the identification of LIPA mutations in three WD and eight CESD patients. The WD patients, all deceased before the first year of age, were homozygous for two novel mutations (c.299+1G>A and c.419G>A) or a mutation (c.796G>T) previously reported as compound heterozygosity in a CESD patient. The two mutations (c.419G>A and c.796G>T) resulting in truncated proteins (p.W140* and p.G266*) and the splicing mutation (c.229+1G>A) were associated with undetectable levels of LIPA mRNA in fibroblasts. All eight CESD patients carried the common mutation c.894G>A known to result not only in a major non-functional transcript with the skipping of exon 8 (p.S275_Q298del), but also in a minor normally spliced transcript producing 5-10% residual LAL activity. The c.894G>A mutation was found in homozygosity in four patients and, as compound heterozygosity, in association with a known (p.H295Y and p.G342R) or a novel (p.W140*) mutation in four other CESD patients. Segregation analysis performed in all patients harboring c.895G>A showed its occurrence on the same haplotype suggesting a common founder ancestor. The other WD and CESD mutations were associated with different haplotypes.

Multiple abnormally spliced ABCA1 mRNAs caused by a novel splice site mutation of ABCA1 gene in a patient with Tangier disease.

BACKGROUND: Mutations in ABCA1 gene are the cause of Tangier disease (TD) and familial high density lipoprotein (HDL) deficiency. Splice site mutations of this gene were reported infrequently. METHODS: ABCA1 gene was sequenced in a TD patient and in subjects with low HDL. The effect of intronic variants on ABCA1 pre-mRNA splicing was studied in COS-1 cells expressing a mutant minigene or in patients' cells. RESULTS: A novel mutation in intron 20 (c.2961 -2 A>C) was found in the TD patient. To assess its effect, a mutant ABCA1 minigene, containing intron 18-intron 23 region, was expressed in COS-1 cells. The mutant minigene generated three transcripts: i) in the first (459bp) 61 nucleotides of intron 20 were retained; ii) in the second (384bp) exon 20 joined to exon 21 devoid of the first 14 nucleotides; and iii) in the third (255bp) the entire exon 21 was skipped. The first two transcripts were also observed in patient's peripheral blood mononuclear cells. These mRNAs encode truncated proteins. A variant in intron 8 (c.814 -14 ins A), identified in subjects with low HDL, had no effect on ABCA1 pre-mRNA splicing. CONCLUSIONS: Functional analysis is required to establish the effect of intronic mutations on ABCA1 pre-mRNA splicing.

Functional lecithin: cholesterol acyltransferase is not required for efficient atheroprotection in humans.

BACKGROUND: Mutations in the LCAT gene cause lecithin:cholesterol acyltransferase (LCAT) deficiency, a very rare metabolic disorder with 2 hypoalphalipoproteinemia syndromes: classic familial LCAT deficiency (Online Mendelian Inheritance in Man No. 245900), characterized by complete lack of enzyme activity, and fish-eye disease (Online Mendelian Inheritance in Man No. 136120), with a partially defective enzyme. Theoretically, hypoalphalipoproteinemia cases with LCAT deficiency should be at increased cardiovascular risk because of high-density lipoprotein deficiency and defective reverse cholesterol transport. METHODS AND RESULTS: The extent of preclinical atherosclerosis was assessed in 40 carriers of LCAT gene mutations from 13 Italian families and 80 healthy controls by measuring carotid intima-media thickness (IMT). The average and maximum IMT values in the carriers were 0.07 and 0.21 mm smaller than in controls (P=0.0003 and P=0.0027), respectively. Moreover, the inheritance of a mutated LCAT genotype had a remarkable gene-dose-dependent effect in reducing carotid IMT (P=0.0003 for average IMT; P=0.001 for maximum IMT). Finally, no significant difference in carotid IMT was found between carriers of LCAT gene mutations that cause total or partial LCAT deficiency (ie, familial LCAT deficiency or fish-eye disease). CONCLUSIONS: Genetically determined low LCAT activity in Italian families is not associated with enhanced preclinical atherosclerosis despite low high-density lipoprotein cholesterol levels. This finding challenges the notion that LCAT is required for effective atheroprotection and suggests that elevating LCAT expression or activity is not a promising therapeutic strategy to reduce cardiovascular risk.

Effect of ezetimibe coadministered with statins in genotype-confirmed heterozygous FH patients.

We investigated the effect of statins and statins plus ezetimibe in 65 FH heterozygotes carrying LDLR-defective or LDLR-negative mutations as well as the effect of ezetimibe monotherapy in 50 hypercholesterolemic (HCH) patients intolerant to statins. PCSK9 and NPC1L1 genes were analysed to assess the role of genetic variants in response to therapy. In FH patients combined therapy reduced LDL-C by 57%, irrespective of the type of LDLR mutation. The additional decrease of plasma LDL-C induced by ezetimibe showed wide inter-individual variability (from -39% to -4.7%) and was negatively correlated with percent LDL-C decrease due to statin alone (r=-0.713, P<0.001). The variable response to statins was not due to PCSK9 gene variants associated with statin hyper-sensitivity. The highest response to ezetimibe was observed in a carrier of R174H substitution in NPC1L1, which had been found to be associated with high cholesterol absorption. In HCH patients, ezetimibe monotherapy induced a variable decrease of plasma LDL-C (from -47.7% to -13.4%). To investigate this variability, we sequenced NPC1L1 gene in patients with the highest and the lowest response to ezetimibe. This analysis showed a higher prevalence of the G allele of the c.816 C>G polymorphism (L272L) in hyper-responders, an observation confirmed also in FH patients hyper-responders to ezetimibe. In both FH and HCH patients, the G allele carriers tended to have a higher LDL-C reduction in response to ezetimibe. These observations suggest that in FH heterozygotes LDL-C reduction following combined therapy reflects a complex interplay between hepatic synthesis and intestinal absorption of cholesterol.

A novel loss of function mutation of PCSK9 gene in white subjects with low-plasma low-density lipoprotein cholesterol.

OBJECTIVES: The PCSK9 gene, encoding a pro-protein convertase involved in posttranslational degradation of low-density lipoprotein receptor, has emerged as a key regulator of plasma low-density lipoprotein cholesterol. In African-Americans two nonsense mutations resulting in loss of function of PCSK9 are associated with a 30% to 40% reduction of plasma low-density lipoprotein cholesterol. The aim of this study was to assess whether loss of function mutations of PCSK9 were a cause of familial hypobetalipoproteinemia and a determinant of low-plasma low-density lipoprotein cholesterol in whites. METHODS AND RESULTS: We sequenced PCSK9 gene in 18 familial hypobetalipoproteinemia subjects and in 102 hypocholesterolemic blood donors who were negative for APOB gene mutations known to cause familial hypobetalipoproteinemia. The PCSK9 gene variants found in these 2 groups were screened in 42 subjects in the lowest (<5th) percentile, 44 in the highest (>95th) percentile, and 100 with the average plasma cholesterol derived from general population. In one familial hypobetalipoproteinemia kindred and in 2 hypocholesterolemic blood donors we found a novel PCSK9 mutation in exon 1 (c.202delG) resulting in a truncated peptide (Ala68fsLeu82X). Two familial hypobetalipoproteinemia subjects and 4 hypocholesterolemic blood donors were carriers of the R46L substitution previously reported to be associated with reduced low-density lipoprotein cholesterol as well as other rare amino acid changes (T77I, V114A, A522T and P616L) not found in the other groups examined. CONCLUSIONS: We discovered a novel inactivating mutation as well as some rare nonconservative amino acid substitutions of PCSK9 in white hypocholesterolemic individuals.

Denaturing high-performance liquid chromatography in the detection of ABCA1 gene mutations in familial HDL deficiency.

Mutations in the ABCA1 gene are the cause of familial high density lipoprotein deficiency (FHD). Because these mutations are spread over the entire gene, their detection requires the sequencing of all 50 exons. The aim of this study was to validate denaturing high-performance liquid chromatography (DHPLC) in mutation detection as an alternative to systematic sequencing. Exons of the ABCA1 gene were amplified using primers employed for sequencing. Temperatures for DHPLC were deducted from a software and empirically defined for each amplicon. To assess DHPLC reliability, we tested 30 sequence variants found in FHD patients and controls. Combined DHPLC and sequencing was applied to the genotyping of new FHD patients. Most of the amplicons required from two to five temperature conditions to obtain partially denatured DNA over the entire amplicon length. Twenty-nine of the variants found by sequencing were detected by DHPLC (97% sensitivity). The detection of the last variant (in exon 40) required different primers and amplification conditions. DHPLC and sequencing analysis of new FHD patients revealed that all amplicons showing a heteroduplex DHPLC profile contained sequence variants. No variants were detected in amplicons with a homoduplex profile. DHPLC is a sensitive and reliable method for the detection of ABCA1 gene mutations.

Characterization of monoclonal antibodies against prostate specific antigen produced by genetic immunization.

Prostate specific antigen (PSA) is the most important marker for prostate cancer. Antibodies against minor variants of PSA may be useful in the development of novel diagnostic tests for prostate cancer, but it has been difficult to produce such antibodies by protein immunization. In this study, we have compared the characteristics of monoclonal antibodies (MAbs) obtained by genetic immunization with those obtained by protein immunization. The whole coding region of PSA-cDNA was cloned in a mammalian expression vector pCDNA-3. Six mice were immunized four times by intra-muscular (i.m.) injection of the PSA-pCDNA3 plasmid. The MAbs produced were characterized with respect to subclass, epitope specificity, binding to various molecular forms of PSA and affinity. After intra-muscular injection of DNA, anti-PSA antibodies were detected in the serum of all mice, but the antibody titers were markedly lower than after protein immunization. After fusion of the spleen cells from the mice, five hybridomas producing MAbs to PSA were obtained. The MAbs were of IgG1 and IgG2a isotype and they all recognized equally different forms of free PSA, namely enzymatically active, nicked and proPSA. Epitope mapping showed that these MAbs reacted with the same antigenic regions as those obtained by protein immunization. Thus, genetic immunization leads to production of anti PSA MAbs with similar characteristics to those obtained by immunizing with PSA protein. As applied in the present study, it is less efficient than protein immunization, but it is a useful technique when the antigen is not available in the quantities needed for immunization.