VasQ U.S. pivotal study demonstrates the safety and effectiveness of an external vascular support for arteriovenous fistula creation.

OBJECTIVE: Arteriovenous fistula (AVF) creation is a commonly performed vascular operation that reports 6-month functional success rates as low as 50%. Recently, a nitinol external vascular support device, VasQ, has shown potential in studies outside the United States (U.S.) to improve AVF outcomes when implanted at creation. Here, the pivotal study results of this novel technology in treating patients in the U.S. are described. METHODS: VasQ was implanted in 144 patients at 16 centers across the U.S. who were referred for creation of a new AVF and consented for enrollment in a 2-year, prospective, multicenter, single-arm, open-label study. Brachiocephalic (n = 129) and radiocephalic (n = 15) AVFs were analyzed. The primary endpoint was primary patency at 6 months compared against a performance goal of 55% derived from a systematic literature search. Safety endpoints included device-related events, ischemic steal, infection, aneurysm, and seroma at up to 6 months. Minimum arterial size was 2.0 mm; target veins were required to measure 2.5 to 6 mm. Key exclusion criteria were patients <18 or >80 years, those with known ipsilateral central venous occlusion, target cannulation zone venous depth greater than 8 mm, and New York Heart Association class 3 or 4. RESULTS: Patients were 61% male, 53% White, 35% African American, and 14% Hispanic. Mean age was 60 years, and median body mass index was 30.4. Of the patients, 69% were diabetic, 66% were on dialysis at the time of creation, and 70% had a prior access surgery. At 6 months, steal was observed in 2.1%, infection in 0.7%, and no aneurysms or seromas were seen. Primary patency at 6 months was 66% (P < .021 vs performance goal). Physiological maturation was achieved in 92.4% of patients. Successful two-needle cannulation for patients that entered the study on dialysis was achieved in 88% of VasQ AVFs at a median of 56 days. Pre-dialysis patients who initiated dialysis during the study achieved two-needle cannulation in 81.6% VasQ AVFs. Interventions were required at a rate of 1.07 per patient year over the entire study period. Two-year cumulative patency was 76.6% (95% confidence interval, 67.9%-83.4%) with no statistical difference between patients requiring interventions and those that did not. No patency differences were observed between brachiocephalic and radiocephalic AVFs. CONCLUSIONS: The U.S. pivotal study results demonstrated improved AVF outcomes and an excellent safety profile with VasQ use relative to traditional AVFs. Under the conditions of this trial, VasQ shows great promise in expeditiously and efficiently enhancing AVF functional success.

Prospective multicenter study of a novel endovascular venous anastomotic procedure and device for implantation of an arteriovenous graft for hemodialysis.

INTRODUCTION: The traditional sutured venous anastomosis used during arteriovenous graft implantation is associated with a high incidence of subsequent stenosis that is attributed to neointimal hyperplasia. Hyperplasia results from multiple factors, including hemodynamic abnormalities and vessel trauma during implantation. A novel anastomotic connector device was designed to provide an alternative, less traumatic, endovascular venous anastomosis that may ameliorate the clinical challenges associated with a sutured anastomosis. A prospective single-arm multicenter study was performed to evaluate safety and effectiveness of graft implantation using the study device. METHODS: Patients requiring graft creation and who met the study criteria were enrolled between February 2018 and July 2021 and observed for 6 months. Collected data included baseline characteristics, graft patency and use for hemodialysis, graft interventions, and adverse events. The primary study endpoint was cumulative graft patency, compared to a pre-specified Performance Goal of 75%. Secondary endpoints included primary unassisted patency and serious adverse events, defined as the occurrence of death, graft infection, emergent surgery, significant bleeding, and pseudoaneurysm. RESULTS: A total of 158 patients were enrolled from 10 study sites, among which 144 subjects were evaluable at 6 months and 14 were censored with partial follow-up observation. Three patients died and the graft was abandoned in 12. The primary endpoint was met (p-value < 0.001). By Kaplan Meier survival analysis, cumulative patency was 92.08% with a lower 95% Confidence Bound of 86.98%. Primary unassisted patency was 60.21% with a lower 95% Confidence Bound of 50.84%. Graft infections occurred in six patients, all unrelated to the study device. There were no reports of emergent surgery, significant bleeding or pseudoaneurysm. CONCLUSION: These results demonstrate that the study device can be used for successful endovascular anastomosis of a vein to a graft for hemodialysis, with acceptable cumulative patency and safety profile at 6 months. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02532621.

Maturing secretory granules: Where secretory and endocytic pathways converge.

Secretory granules (SGs) are specialized organelles responsible for the storage and regulated release of various biologically active molecules from the endocrine and exocrine systems. Thus, proper SG biogenesis is critical to normal animal physiology. Biogenesis of SGs starts at the trans-Golgi network (TGN), where immature SGs (iSGs) bud off and undergo maturation before fusing with the plasma membrane (PM). How iSGs mature is unclear, but emerging studies have suggested an important role for the endocytic pathway. The requirement for endocytic machinery in SG maturation blurs the line between SGs and another class of secretory organelles called lysosome-related organelles (LROs). Therefore, it is important to re-evaluate the differences and similarities between SGs and LROs.

Serum trace metal association with response to erythropoiesis stimulating agents in incident and prevalent hemodialysis patients.

Alterations in hemodialysis patients' serum trace metals have been documented. Early studies addressing associations levels of serum trace metals with erythropoietic responses and/or hematocrit generated mixed results. These studies were conducted prior to current approaches for erythropoiesis stimulating agent (ESA) drug dosing guidelines or without consideration of inflammation markers (e.g. hepcidin) important for regulation of iron availability. This study sought to determine if the serum trace metal concentrations of incident or chronic hemodialysis patients associated with the observed ESA response variability and with consideration to ESA dose response, hepcidin, and high sensitivity C-reactive protein levels. Inductively-coupled plasma-mass spectrometry was used to measure 14 serum trace metals in 29 incident and 79 prevalent dialysis patients recruited prospectively. We compared these data to three measures of ESA dose response, sex, and dialysis incidence versus dialysis prevalence. Hemoglobin was negatively associated with ESA dose and cadmium while positively associated with antimony, arsenic and lead. ESA dose was negatively associated with achieved hemoglobin and vanadium while positively associated with arsenic. ESA response was positively associated with arsenic. Vanadium, nickel, cadmium, and tin were increased in prevalent patients. Manganese was increased in incident patients. Vanadium, nickel, and arsenic increased with time on dialysis while manganese decreased. Changes in vanadium and manganese were largest and appeared to have some effect on anemia. Incident and prevalent patients' chromium and antimony levels exceeded established accepted upper limits of normal.

An optimized QF-binary expression system for use in zebrafish.

The zebrafish model organism has been of exceptional utility for the study of vertebrate development and disease through the application of tissue-specific labelling and overexpression of genes carrying patient-derived mutations. However, there remains a need for a binary expression system that is both non-toxic and not silenced over animal generations by DNA methylation. The Q binary expression system derived from the fungus Neurospora crassa is ideal, because the consensus binding site for the QF transcription factor lacks CpG dinucleotides, precluding silencing by CpG-meditated methylation. To optimize this system for zebrafish, we systematically tested several variants of the QF transcription factor: QF full length; QF2, which lacks the middle domain; QF2w, which is an attenuated version of QF2; and chimeric QFGal4. We found that full length QF and QF2 were strongly toxic to zebrafish embryos, QF2w was mildly toxic, and QFGal4 was well tolerated, when injected as RNA or expressed ubiquitously from stable transgenes. In addition, QFGal4 robustly activated a Tg(QUAS:GFPNLS) reporter transgene. To increase the utility of this system, we also modified the QF effector sequence termed QUAS, which consists of five copies of the QF binding site. Specifically, we decreased both the CpG dinucleotide content, as well as the repetitiveness of QUAS, to reduce the risk of transgene silencing via CpG methylation. Moreover, these modifications to QUAS removed leaky QF-independent neural expression that we detected in the original QUAS sequence. To demonstrate the utility of our QF optimizations, we show how the Q-system can be used for lineage tracing using a Cre-dependent Tg(ubi:QFGal4-switch) transgene. We also demonstrate that QFGal4 can be used in transient injections to tag and label endogenous genes by knocking in QFGal4 into sox2 and ubiquitin C genes.

An early endosome-derived retrograde trafficking pathway promotes secretory granule maturation.

Regulated secretion is a fundamental cellular process in which biologically active molecules stored in long-lasting secretory granules (SGs) are secreted in response to external stimuli. Many studies have described mechanisms responsible for biogenesis and secretion of SGs, but how SGs mature remains poorly understood. In a genetic screen, we discovered a large number of endolysosomal trafficking genes required for proper SG maturation, indicating that maturation of SGs might occur in a manner similar to lysosome-related organelles (LROs). CD63, a tetraspanin known to decorate LROs, also decorates SG membranes and facilitates SG maturation. Moreover, CD63-mediated SG maturation requires type II phosphatidylinositol 4 kinase (PI4KII)-dependent early endosomal sorting and accumulation of phosphatidylinositol 4-phosphate (PI4P) on SG membranes. In addition, the PI4P effector Past1 is needed for formation of stable PI4KII-containing endosomal tubules associated with this process. Our results reveal that maturation of post-Golgi-derived SGs requires trafficking via the endosomal system, similar to mechanisms employed by LROs.

Human PAH is characterized by a pattern of lipid-related insulin resistance.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a deadly disease of the small pulmonary vasculature with an increased prevalence of insulin resistance (IR). Insulin regulates both glucose and lipid homeostasis. We sought to quantify glucose- and lipid-related IR in human PAH, testing the hypothesis that lipoprotein indices are more sensitive indices of IR in PAH. METHODS: Oral glucose tolerance testing in PAH patients and triglyceride-matched (TG-matched) controls and proteomic, metabolomics, and lipoprotein analyses were performed in PAH and controls. Results were validated in an external cohort and in explanted human PAH lungs. RESULTS: PAH patients were similarly glucose intolerant or IR by glucose homeostasis metrics compared with control patients when matched for the metabolic syndrome. Using the insulin-sensitive lipoprotein index, TG/HDL ratio, PAH patients were more commonly IR than controls. Proteomic and metabolomic analysis demonstrated separation between PAH and controls, driven by differences in lipid species. We observed a significant increase in long-chain acylcarnitines, phosphatidylcholines, insulin metabolism-related proteins, and in oxidized LDL receptor 1 (OLR1) in PAH plasma in both a discovery and validation cohort. PAH patients had higher lipoprotein axis-related IR and lipoprotein-based inflammation scores compared with controls. PAH patient lung tissue showed enhanced OLR1 immunostaining within plexiform lesions and oxidized LDL accumulation within macrophages. CONCLUSIONS: IR in PAH is characterized by alterations in lipid and lipoprotein homeostasis axes, manifest by elevated TG/HDL ratio, and elevated circulating medium- and long-chain acylcarnitines and lipoproteins. Oxidized LDL and its receptor OLR1 may play a role in a proinflammatory phenotype in PAH. FUNDING: NIH DK096994, HL060906, UL1 RR024975-01, UL1 TR000445-06, DK020593, P01 HL108800-01A1, and UL1 TR002243; American Heart Association 13FTF16070002.

CB1 cannabinoid receptor-phosphorylated fourth intracellular loop structure-function relationships.

A peptide comprising the juxtamembrane C-terminal intracellular loop 4 (IL4) of the CB1 cannabinoid receptor possesses three Serine residues (Ser402, Ser411 and Ser415). Here we report the effect of Ser phosphorylation on the CB1 IL4 peptide conformation and cellular signaling functions using nuclear magnetic resonance spectroscopy, circular dichroism, G protein activation and cAMP production. Circular dichroism studies indicated that phosphorylation at various Ser residues induced helical structure in different environments. NMR data indicates that helical content varies in the order of IL4pSer411 > IL4pSer415 > IL4 > IL4pSer402. The efficacy of phosphorylated IL4 peptides in activating Go and Gi3 ([35S]GTPγS binding) and inhibiting cAMP accumulation in N18TG2 cells were correlated with helicity changes. Treatment of cells with bradykinin, which activates PKC, augmented CB1-mediated inhibition of cAMP accumulation, and this was reversed by a PKC inhibitor, suggesting that phosphorylation of serine might be a physiologically relevant modification in vivo. We conclude that phosphorylation-dependent alterations of helicity of CB1 IL4 peptides can increase efficacy of G protein signaling.

Type II phosphatidylinositol 4-kinase regulates nerve terminal growth and synaptic vesicle recycling.

Type II phosphatidylinositol 4-kinase (PI4KII) is thought to be associated with synaptic vesicles (SVs) and to be responsible for the majority of PI4K activity in the nervous system. However, the function of PI4KII at the synapse is unknown. We characterized the synaptic phenotypes of a Drosophila melanogaster PI4KII null mutant. We found increased nerve terminal growth in PI4KII null mutants indicating that PI4KII restrains nerve terminal growth. Evoked neurotransmitter release elicited in response to low frequency stimulation and spontaneous neurotransmitter release were not altered in PI4KII null mutants. However, PI4KII null mutants displayed reduced FM1-43 uptake in response to stimulation by high K+ saline, indicating impaired SV endocytosis. PI4KII null mutants did not display any defects in FM1-43 unloading, consistent with normal SV exocytosis. Thus, PI4KII is required for SV endocytosis but dispensable for SV exocytosis. Overall, our data show that PI4KII regulates both nerve terminal growth and SV recycling.

The phosphoinositide phosphatase Sac1 regulates cell shape and microtubule stability in the developing Drosophila eye.

Epithelial patterning in the developing Drosophila melanogaster eye requires the Neph1 homolog Roughest (Rst), an immunoglobulin family cell surface adhesion molecule expressed in interommatidial cells (IOCs). Here, using a novel temperature-sensitive (ts) allele, we show that the phosphoinositide phosphatase Sac1 is also required for IOC patterning. Sac1ts mutants have rough eyes and retinal patterning defects that resemble rst mutants. Sac1ts retinas exhibit elevated levels of phosphatidylinositol 4-phosphate (PI4P), consistent with the role of Sac1 as a PI4P phosphatase. Indeed, genetic rescue and interaction experiments reveal that restriction of PI4P levels by Sac1 is crucial for normal eye development. Rst is delivered to the cell surface in Sac1ts mutants. However, Sac1ts mutant IOCs exhibit severe defects in microtubule organization, associated with accumulation of Rst and the exocyst subunit Sec8 in enlarged intracellular vesicles upon cold fixation ex vivo Together, our data reveal a novel requirement for Sac1 in promoting microtubule stability and suggest that Rst trafficking occurs in a microtubule- and exocyst-dependent manner.

Caged Naloxone: Synthesis, Characterization, and Stability of 3- O-(4,5-Dimethoxy-2-nitrophenyl)carboxymethyl Naloxone (CNV-NLX).

The photolabile analogue of the broad-spectrum opioid antagonist naloxone, 3- O-(4,5-dimethoxy-2-nitrophenyl)carboxymethyl naloxone (also referred to as "caged naloxone", 3- O-(α-carboxy-6-nitroveratryl)naloxone, CNV-NLX), has been found to be a valuable biochemical probe. While the synthesis of CNV-NLX is simple, its characterization is complicated by the fact that it is produced as a mixture of α R,5 R,9 R,13 S,14 S and α S,5 R,9 R,13 S,14 S diastereomers. Using long-range and heteronuclear NMR correlations, the 1H NMR and 13C NMR resonances of both diastereomers have been fully assigned, confirming the structures. Monitoring of solutions of CNV-NLX in saline buffer, in methanol, and in DMSO has shown CNV-NLX to be stable for over a week under fluorescent laboratory lights at room temperature. Exposure of such solutions to λ 365 nm from a hand-held UV lamp led to the formation of naloxone and CNV-related breakdown products.

Metabolites as biomarkers of adverse reactions following vaccination: A pilot study using nuclear magnetic resonance metabolomics.

An Adverse Event Following Immunization (AEFI) is an adverse reaction to a vaccination that goes above and beyond the usual side effects associated with vaccinations. One serious AEFI related to the smallpox vaccine is myopericarditis. Metabolomics involves the study of the low molecular weight metabolite profile of cells, tissues, and biological fluids, and provides a functional readout of the phenotype. Metabolomics may help identify a particular metabolic signature in serum of subjects who are predisposed to developing AEFIs. The goal of this study was to identify metabolic markers that may predict the development of adverse events following smallpox vaccination. Serum samples were collected from military personnel prior to and following receipt of smallpox vaccine. The study population included five subjects who were clinically diagnosed with myopericarditis, 30 subjects with asymptomatic elevation of troponins, and 31 subjects with systemic symptoms following immunization, and 34 subjects with no AEFI, serving as controls. Two-hundred pre- and post-smallpox vaccination sera were analyzed by untargeted metabolomics using 1H nuclear magnetic resonance (NMR) spectroscopy. Baseline (pre-) and post-vaccination samples from individuals who experienced clinically verified myocarditis or asymptomatic elevation of troponins were more metabolically distinguishable pre- and post-vaccination compared to individuals who only experienced systemic symptoms, or controls. Metabolomics profiles pre- and post-receipt of vaccine differed substantially when an AEFI resulted. This study is the first to describe pre- and post-vaccination metabolic profiles of subjects who developed an adverse event following immunization. The study demonstrates the promise of metabolites for determining mechanisms associated with subjects who develop AEFI and the potential to develop predictive biomarkers.

Prospective multicenter study with a 1-year analysis of a new vascular graft used for early cannulation in patients undergoing hemodialysis.

OBJECTIVE: More than 85% of patients with end-stage renal disease start dialysis through a tunneled dialysis catheter (TDC) for long periods while their arteriovenous fistula or vascular access graft (arteriovenous graft [AVG]) matures. Because TDCs are associated with a high risk of complications, including death and infection, use of an AVG that can be cannulated safely immediately after implantation may reduce morbidity in these patients by allowing earlier TDC removal. We report a prospective multicenter study of a new early-cannulation AVG (Gore ACUSEAL Vascular Graft; W. L. Gore & Associates, Flagstaff, Ariz). METHODS: Patients requiring creation of a prosthetic vascular access for hemodialysis were enrolled between July 2010 and February 2012 and observed for 12 months. Data were collected on the patients' baseline characteristics; location, position, loss of patency, and revisions of prior AVGs; dialysis sessions using the AVG; and major adverse events related to graft implantation or cannulation. Cumulative and primary unassisted graft patency rates were calculated. A subgroup analysis compared outcomes in patients in whom the AVG was first cannulated within 72 hours after implantation with outcomes in patients in whom the initial cannulation was performed >21 days postoperatively. RESULTS: The population of this study was formed by 138 patients who received an ACUSEAL graft. During follow-up, 17 patients died and the AVG was abandoned in 27. The median value for follow-up was 360 days for all patients (variance 15,387). The overall mean time to initial cannulation was 15 days, with 54 grafts (40%) first cannulated within 72 hours after graft implantation and 33 grafts first cannulated >21 days afterward. The reason for late cannulation in some patients was dependent on the implanting surgeon's decision and the surgeon's personal experience with early cannulating grafts. The 1-year overall cumulative patency rate was 79% (95% confidence interval, 71%-85%); the primary unassisted patency rate was 35% (95% confidence interval, 27%-44%). Adverse events included 6 hematomas (two of which were related to cannulation and occurred 107 and 169 days, respectively, after AVG implantation), 15 graft infections, and 15 cases of steal syndrome requiring intervention. Patients in the early- and later-cannulation groups had similar characteristics and no significant differences in rates of cumulative or primary unassisted patency or adverse events. CONCLUSIONS: This study demonstrated that the new, early-cannulation AVG graft can be cannulated soon after implantation without a significant difference in patency and complication rates compared with rates associated with standard cannulation of expanded polytetrafluoroethylene grafts in the literature. This new AVG may allow early removal or avoidance of TDC use in patients undergoing hemodialysis, potentially reducing or eliminating the number of days of catheter-dependent dialysis, but further studies will be needed to demonstrate this potential.

PI4KIIIα is required for cortical integrity and cell polarity during Drosophila oogenesis.

Phosphoinositides regulate myriad cellular processes, acting as potent signaling molecules in conserved signaling pathways and as organelle gatekeepers that recruit effector proteins to membranes. Phosphoinositide-generating enzymes have been studied extensively in yeast and cultured cells, yet their roles in animal development are not well understood. Here, we analyze Drosophila melanogaster phosphatidylinositol 4-kinase IIIα (PI4KIIIα) during oogenesis. We demonstrate that PI4KIIIα is required for production of plasma membrane PtdIns4P and PtdIns(4,5)P2 and is crucial for actin organization, membrane trafficking and cell polarity. Female germ cells mutant for PI4KIIIα exhibit defects in cortical integrity associated with failure to recruit the cytoskeletal-membrane crosslinker Moesin and the exocyst subunit Sec5. These effects reflect a unique requirement for PI4KIIIα, as egg chambers from flies mutant for either of the other Drosophila PI4Ks, fwd or PI4KII, show Golgi but not plasma membrane phenotypes. Thus, PI4KIIIα is a vital regulator of a functionally distinct pool of PtdIns4P that is essential for PtdIns(4,5)P2-dependent processes in Drosophila development.

Characterization of the isomeric configuration and impurities of (Z)-endoxifen by 2D NMR, high resolution LC⬜MS, and quantitative HPLC analysis.

(Z)-Endoxifen (4-hydroxy-N-desmethyltamoxifen), an active metabolite generated via actions of CYP3A4/5 and CYP2D6, is a more potent selective estrogen receptor modulator (SERM) than tamoxifen. In the MCF-7 human mammary tumor xenograft model with female athymic mice, (Z)-endoxifen, at an oral dose of 4⬜8 mg/kg, significantly inhibits tumor growth. (Z)-Endoxifen's potential as an alternative therapeutic agent independent of CYP2D6 activities, which can vary widely in ER+ breast cancer patients, is being actively evaluated. This paper describes confirmation of the configuration of the active (Z)-isomer through 2D NMR experiments, including NOE (ROESY) to establish spatial proton⬜proton correlations, and identification of the major impurity as the (E)-isomer in endoxifen drug substance by HPLC/HRMS (HPLC/MS-TOF). Stability of NMR solutions was confirmed by HPLC/UV analysis. For pre-clinical studies, a reverse-phase HPLC⬜UV method, with methanol/water mobile phases containing 10 mM ammonium formate at pH 4.3, was developed and validated for the accurate quantitation and impurity profiling of drug substance and drug product. Validation included demonstration of linearity, method precision, accuracy, and specificity in the presence of impurities, excipients (for the drug product), and degradation products. Ruggedness and reproducibility of the method were confirmed by collaborative studies between two independent laboratories. The method is being applied for quality control of the API and oral drug product. Kinetic parameters of Z- to E-isomerization were also delineated in drug substance and in aqueous formulation, showing conversion at temperatures above 25 °C.

Indigenous and immigrant populations' use and experience of community pharmacies in New Zealand.

We sought to identify what services indigenous (Maori) and immigrant populations use pharmacies for, and how long pharmacy staff spend interacting with them, as longer interactions are associated with better quality care. We review literature on counseling in pharmacy, and interaction length as an indicator of counseling quality. 1,086 interactions were discretely observed in 36 pharmacies in 5 cities around New Zealand. Maori or Pacific people, along with men, were more likely to treat pharmacies as prescription 'depots', being less likely to buy over-the-counter or pharmacist only medicines (ORs: 0.25-0.72). However, the influence of demographic factors on interaction length was small (|B|s < 7.7 s). The weak effect of ethnicity on interaction length suggests that pharmacies are providing advice of relatively consistent quality to different population groups. Possible barriers to use of pharmacies for primary healthcare, including over-the-counter medicines in Maori and Pacific people are discussed.