Protein S antibody as an adjunct therapy for hemophilia B.

ABSTRACT: Hemophilia B (HB) is caused by an inherited deficiency of plasma coagulation factor IX (FIX). Approximately 60% of pediatric patients with HB possess a severe form of FIX deficiency (<1% FIX activity). Treatment typically requires replacement therapy through the administration of FIX. However, exogenous FIX has a limited functional half-life, and the natural anticoagulant protein S (PS) inhibits activated FIX (FIXa). PS ultimately limits thrombin formation, which limits plasma coagulation. This regulation of FIXa activity by PS led us to test whether inhibiting PS would extend the functional half-life of FIX and thereby prolong FIX-based HB therapy. We assayed clotting times and thrombin generation to measure the efficacy of a PS antibody for increasing FIX activity in commercially obtained plasma and plasma from pediatric patients with HB. We included 11 pediatric patients who lacked additional comorbidities and coagulopathies. In vivo, we assessed thrombus formation in HB mice in the presence of the FIXa ± PS antibody. We found an accelerated rate of clotting in the presence of PS antibody. Similarly, the peak thrombin formed was significantly greater in the presence of the PS antibody, even in plasma from patients with severe HB. Furthermore, HB mice injected with PS antibody and FIX had a 4.5-fold higher accumulation of fibrin at the thrombus induction site compared with mice injected with FIX alone. Our findings imply that a PS antibody would be a valuable adjunct to increase the effectiveness of FIX replacement therapy in pediatric patients who have mild, moderate, and severe HB.

Comparative Outcomes of Candida auris Bloodstream Infections: A Multicenter Retrospective Case-Control Study.

BACKGROUND: This study was performed to compare clinical characteristics and outcomes between patients with bloodstream infections (BSIs) caused by Candida auris and those with BSIs caused by other Candida spp. METHODS: A multicenter retrospective case-control study was performed at 3 hospitals in Brooklyn, New York, between 2016 and 2020. The analysis included patients ≥18 years of age who had a positive blood culture for any Candida spp. and were treated empirically with an echinocandin. The primary outcome was the 30-day mortality rate. Secondary outcomes were 14-day clinical failure, 90-day mortality rate, 60-day microbiologic recurrence, and in-hospital mortality rate. RESULTS: A total of 196 patients were included in the final analysis, including 83 patients with candidemia caused by C. auris. After inverse propensity adjustment, C. auris BSI was not associated with increased 30-day (adjusted odds ratio, 1.014 [95% confidence interval, .563-1.828]); P = .96) or 90-day (0.863 [.478-1.558]; P = .62) mortality rates. A higher risk for microbiologic recurrence within 60 days of completion of antifungal therapy was observed in patients with C. auris candidemia (adjusted odds ratio, 4.461 [95% confidence interval, 1.033-19.263]; P = .045). CONCLUSIONS: C. auris BSIs are not associated with a higher mortality risk than BSIs caused by other Candida spp. The rate of microbiologic recurrence was higher in the C. auris group.

Emerging Multiorgan Klebsiella pneumoniae Invasive Syndrome Leading to Septic Shock: A Case Report and Review of the Literature.

Klebsiella pneumoniae (K. pneumoniae) is a community-acquired pathogen that typically causes pneumonia and urinary tract infections. Rarely, it can affect other organ systems such as the gastrointestinal tract, as well as the meninges, ears, eyes, and spine. We present the case of a 62-year-old male admitted with septic shock secondary to necrotizing pneumonia and multiple hepatic liver abscesses, which to the best of our knowledge, is the first reported case of multiorgan invasive K. pneumoniae infection, including the presence of a newly recognized syndrome referred as Invasive Liver Abscess Syndrome (ILAS). It is important to maintain both ILAS and K. pneumoniae invasive syndrome (KPIS) in the differential diagnosis, especially in areas of the world with a large number of tourists and immigrants, such as New York City, where the presented case took place.

Cladosporium sphaerospermum as a Rare Cause of Pneumonia.

Cladosporium sphaerospermum isa radiotrophic dematiaceous fungus that can rarely cause disease in humans such as infections of the skin, eye, upper airways, and brain. To the best of our knowledge, we present the first reported case of Cladosporium sphaerospermum-induced invasive lung infection. This case presents a 51-year-old male with a medical history significant for heavy smoking and severe alcohol abuse who was admitted for acute hypoxic respiratory failure secondary to a large exudative right pleural effusion compounded by hydropneumothorax. Despite an initial positive clinical response, appropriate medical treatment, and eradication of the infection, which was confirmed by repeat negative culture studies, the patient had a complicated hospital course. It is suspected that the patient's medical history played a role in the acquisition of the Cladosporium sphaerospermum infection as smoking and alcohol use are known risk factors for aspiration of pathogens into the pulmonary tract. We believe it is important to bring to attention this less known organism as a potential differential diagnosis for a complicated lung infection.

Genetic and Bioinformatic Strategies to Improve Diagnosis in Three Inherited Bleeding Disorders in Bogotá, Colombia.

Inherited bleeding disorders (IBDs) are the most frequent congenital diseases in the Colombian population; three of them are hemophilia A (HA), hemophilia B (HB), and von Willebrand Disease (VWD). Currently, diagnosis relies on multiple clinical laboratory assays to assign a phenotype. Due to the lack of accessibility to these tests, patients can receive an incomplete diagnosis. In these cases, genetic studies reinforce the clinical diagnosis. The present study characterized the molecular genetic basis of 11 HA, three HB, and five VWD patients by sequencing the F8, F9, or the VWF gene. Twelve variations were found in HA patients, four in HB patients, and 19 in WVD patients. From these variations a total of 25 novel variations were found. Disease-causing variations were used as positive controls for validation of the high-resolution melting (HRM) variant-scanning technique. This approach is a low-cost genetic diagnostic method proposed to be incorporated in developing countries. For the data analysis, we developed an accessible open-source code in Python that improves HRM data analysis with better sensitivity of 95% and without bias when using different HRM equipment and software. Analysis of amplicons with a length greater than 300 bp can be performed by implementing an analysis by denaturation domains.

Idiopathic Bilateral Ovarian Vein Thrombosis in a Non-Pregnant Healthy Patient: A Case Report and Review of the Literature.

Ovarian vein thrombosis (OVT) is a potentially life-threatening condition, and it is typically related to the peripartum period; however, it is also associated with pelvic inflammatory disease, recent pelvic or abdominal surgery, inflammatory bowel disease, thrombophilia, malignancy, and sepsis. Idiopathic isolated OVT is rare and is usually presented as case reports in the medical literature. In this report, we present a case of bilateral OVT in a postmenopausal female with no identifiable risk factors and normal coagulation profile workup to highlight the importance of considering it as a differential diagnosis in female patients presenting with abdominal pain. Early identification can prevent potentially life-threatening complications. Management is often conservative, and the choice of anticoagulation is based on the patient's medical conditions. In this particular scenario, the patient was managed with low molecular weight heparin (LMWH) and transitioned to direct oral anticoagulant (DOAC) before discharge.

Hypercoagulable workup in a community hospital setting: to test or not to test; that is the question.

Background: Thrombophilia tests are often ordered unnecessarily and/or inappropriately, with significant impact on healthcare costs, hospital resources, time, and potential harm to the patient. Objective: To identify the incidence of unnecessary and inappropriate thrombophilia testing in a community hospital setting. Methods: We retrospectively reviewed electronic medical records for patients who underwent inpatient thrombophilia testing at The Brooklyn Hospital Center from 1/1/2018 to 12/31/2018. The indications, and details of the tests, and associated costs were recorded. Results: A total of 62 patients were included (mean age 45.8, 67.7% female). In 57/62 (91.9%) patients, tests were ordered in the acute phase of thrombosis. At the time the tests were ordered, 29/62 (46.8%) patients were on anticoagulation. Positive results were found in 21/62 (53.2%) patients, but was repeated in only 1/21 (4.7%) patient. Results for 51/62 (82%) patients were obtained after discharge. The hematology-oncology service was consulted in 5/62 (8.1%) cases and recommended testing in only 1 (1.6%) patient. Only 1 (1.6%) patient had both an appropriate indication and appropriate testing. Costs for the 273 total tests were $26,400. Conclusion: Thrombophilia tests were often ordered inappropriately and unnecessarily. We recommend testing only for patients with inpatient status under recommendation from the hematology-oncology service.

Conjugation of Triphenylphosphonium Cation to Hydrophobic Moieties to Prepare Mitochondria-Targeting Nanocarriers.

The contribution of mitochondrial dysfunctions to diseases such as cancer, diabetes, cardiovascular, and neurodegenerative diseases has made mitochondria an attractive pharmacological target. To deliver biologically active molecules to mitochondria, however, cellular and mitochondrial barriers must be first overcome. The mitochondrial transmembrane electric potential (negative inside) is among the most commonly used strategies to deliver molecules to mitochondria as it allows the accumulation of positively charged molecules. Thus, therapeutic molecules are either covalently conjugated to lipophilic cations like triphenylphosphonium (TPP) or loaded into nanocarriers conjugated to TPP.

PEGylated-nanoliposomal clusterin for amyloidogenic light chain-induced endothelial dysfunction.

Light chain (AL) amyloidosis is a disease associated with significant morbidity and mortality arising from multi-organ injury induced by amyloidogenic light chain proteins (LC). There is no available treatment to reverse the toxicity of LC. We previously showed that chaperone glycoprotein clusterin (CLU) and nanoliposomes (NL), separately, restore human microvascular endothelial function impaired by LC. In this work, we aim to prepare PEGylated-nanoliposomal clusterin (NL-CLU) formulations that could allow combined benefit against LC while potentially enabling efficient delivery to microvascular tissue, and test efficacy on human arteriole endothelial function. NL-CLU was prepared by a conjugation reaction between the carboxylated surface of NL and the primary amines of the CLU protein. NL were made of phosphatidylcholine (PC), cholesterol (Chol) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG 2000 carboxylic acid) at 70:25:5 mol%. The protective effect of NL-CLU was tested by measuring the dilation response to acetylcholine and papaverine in human adipose arterioles exposed to LC. LC treatment significantly reduced the dilation response to acetylcholine and papaverine; co-treatment of LC with PEGylated-nanoliposomal CLU or free CLU restored the dilator response. NL-CLU is a feasible and promising approach to reverse LC-induced endothelial damage.

Effect of diabetes status and hyperglycemia on global DNA methylation and hydroxymethylation.

Type 2 diabetes mellitus (T2DM) is characterized by oxidative stress that could lead to chronic micro- and macrovascular complications. We hypothesized that some of the target organ damage is mediated by oxidative alterations in epigenetic mechanisms involving DNA methylation (5mC) and DNA hydroxymethylation (5hmC). We analyzed global DNA methylation and hydroxymethylation in peripheral blood cells in well-controlled and poorly controlled patients with T2DM and compared them with healthy controls. We also analyzed microarrays of DNA methylation and gene expression of other important tissues in the context of diabetes from the GEO database repository and then compared these results with our experimental gene expression data. DNA methylation and, more importantly, DNA hydroxymethylation levels were increased in poorly controlled patients compared to well-controlled and healthy individuals. Both 5mC and 5hmC measurements were correlated with the percentage of glycated hemoglobin, indicating a direct impact of hyperglycemia on changes over the epigenome. The analysis of methylation microarrays was concordant, and 5mC levels were increased in the peripheral blood of T2DM patients. However, the DNA methylation levels were the opposite of those in other tissues, such as the pancreas, adipose tissue and skeletal muscle. We hypothesize that a process of DNA oxidation associated with hyperglycemia may explain the DNA demethylation in which the activity of ten-eleven translocation (TET) proteins is not sufficient to complete the process. High levels of glucose lead to cellular oxidation, which triggers the process of DNA demethylation aided by TET enzymes, resulting in epigenetic dysregulation of the damaged tissues.

Mitochondria-Targeted Agents: Mitochondriotropics, Mitochondriotoxics, and Mitocans.

Mitochondria, the powerhouse of the cell, have been known for many years for their central role in the energy metabolism; however, extensive progress has been made and to date substantial evidence demonstrates that mitochondria play a critical role not only in the cell bioenergetics but also in the entire cell metabolome. Mitochondria are also involved in the intracellular redox poise, the regulation of calcium homeostasis, and the generation of reactive oxygen species (ROS), which are crucial for the control of a variety of signaling pathways. Additionally, they are essential for the mitochondrial-mediated apoptosis process. Thus, it is not surprising that disruptions of mitochondrial functions can lead or be associated with human pathologies. Because of diseases like diabetes, Alzheimer, Parkinson's, cancer, and ischemic disease are being increasingly linked to mitochondrial dysfunctions, the interest in mitochondria as a prime pharmacological target has dramatically risen over the last decades and as a consequence a large number of agents, which could potentially impact or modulate mitochondrial functions, are currently under investigation. Based on their site of action, these agents can be classified as mitochondria-targeted and non-mitochondria-targeted agents. As a result of the continuous search for new agents and the design of potential therapeutic agents to treat mitochondrial diseases, terms like mitochondriotropics, mitochondriotoxics, mitocancerotropics, and mitocans have emerged to describe those agents with high affinity to mitochondria that exert a therapeutic or deleterious effect on these organelles. In this chapter, mitochondria-targeted agents and some strategies to deliver agents to and/or into mitochondria will be reviewed.

Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile.

Despite being a conceptually appealing alternative to conventional antibiotics, a major challenge toward the successful implementation of antisense treatments for bacterial infections is the development of efficient oligonucleotide delivery systems. Cationic vesicles (bolasomes) composed of dequalinium chloride ("DQAsomes") have been used to deliver plasmid DNA across the cardiolipin-rich inner membrane of mitochondria. As cardiolipin is also a component of many bacterial membranes, we investigated the application of cationic bolasomes to bacteria as an oligonucleotide delivery system. Antisense sequences designed in silico to target the expression of essential genes of the bacterial pathogen, Clostridium difficile, were synthesized as 2'-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO). These antisense gapmers were quantitatively assessed for their ability to block mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription-translation system. Cationic bolaamphiphile compounds (dequalinium derivatives) of varying alkyl chain length were synthesized and bolasomes were prepared via probe sonication of an aqueous suspension. Bolasomes were characterized by particle size distribution, zeta potential, and binding capacities for anionic oligonucleotide. Bolasomes and antisense gapmers were combined to form antisense nanocomplexes. Anaerobic C. difficile log phase cultures were treated with serial doses of gapmer nanocomplexes or equivalent amounts of empty bolasomes for 24 hours. Antisense gapmers for four gene targets achieved nanomolar minimum inhibitory concentrations for C. difficile, with the lowest values observed for oligonucleotides targeting polymerase genes rpoB and dnaE. No inhibition of bacterial growth was observed from treatments at matched dosages of scrambled gapmer nanocomplexes or plain, oligonucleotide-free bolasomes compared to untreated control cultures. We describe the novel application of cationic bolasomes to deliver ASOs into bacteria. We also report the first successful in vitro antisense treatment to inhibit the growth of C. difficile.

Monosialoganglioside-Containing Nanoliposomes Restore Endothelial Function Impaired by AL Amyloidosis Light Chain Proteins.

BACKGROUND: Light chain amyloidosis (AL) is associated with high mortality, especially in patients with advanced cardiovascular involvement. It is caused by toxicity of misfolded light chain proteins (LC) in vascular, cardiac, and other tissues. There is no treatment to reverse LC tissue toxicity. We tested the hypothesis that nanoliposomes composed of monosialoganglioside, phosphatidylcholine, and cholesterol (GM1 ganglioside-containing nanoliposomes [NLGM1]) can protect against LC-induced human microvascular dysfunction and assess mechanisms behind the protective effect. METHODS AND RESULTS: The dilator responses of ex vivo abdominal adipose arterioles from human participants without AL to acetylcholine and papaverine were measured before and after exposure to LC (20 µg/mL) with or without NLGM1 (1:10 ratio for LC:NLGM1 mass). Human umbilical vein endothelial cells were exposed for 18 to 20 hours to vehicle, LC with or without NLGM1, or NLGM1 and compared for oxidative and nitrative stress response and cellular viability. LC impaired arteriole dilator response to acetylcholine, which was restored by co-treatment with NLGM1. LC decreased endothelial cell nitric oxide production and cell viability while increasing superoxide and peroxynitrite; these adverse effects were reversed by NLGM1. NLGM1 increased endothelial cell protein expression of antioxidant enzymes heme oxygenase 1 and NAD(P)H quinone dehydrogenase 1 and increased nuclear factor, erythroid 2 like 2 (Nrf-2) protein. Nrf-2 gene knockdown reduced antioxidant stress response and reversed the protective effects of NLGM1. CONCLUSIONS: NLGM1 protects against LC-induced human microvascular endothelial dysfunction through increased nitric oxide bioavailability and reduced oxidative and nitrative stress mediated by Nrf-2-dependent antioxidant stress response. These findings point to a potential novel therapeutic approach for light chain amyloidosis.

A pilot-scale steam autoclave system for treating municipal solid waste for recovery of renewable organic content: Operational results and energy usage.

A pilot-scale (1800 kg per batch capacity) autoclave used in this study reduces municipal solid waste to a debris contaminated pulp product that is efficiently separated into its renewable organic content and non-renewable organic content fractions using a rotary trommel screen. The renewable organic content can be recovered at nearly 90% efficiency and the trommel rejects are also much easier to sort for recovery. This study provides the evaluation of autoclave operation, including mass and energy balances for the purpose of integration into organic diversion systems. Several methods of cooking municipal solid waste were explored from indirect oil heating only, a combination of oil and direct steam during the same cooking cycle, and steam only. Gross energy requirements averaged 1290 kJ kg(-1) material in vessel, including the weight of free water and steam added during heating. On average, steam recovery can recoup 43% of the water added and 30% of the energy, supplying on average 40% of steam requirements for the next cook. Steam recycle from one vessel to the next can reduce gross energy requirements to an average of 790 kJ kg(-1).

Methanotrophic production of polyhydroxybutyrate-co-hydroxyvalerate with high hydroxyvalerate content.

Type II methanotrophic bacteria are a promising production platform for PHA biopolymers. These bacteria are known to produce pure poly-3-hydroxybutyrate homopolymer (PHB). We isolated a strain, Methylocystis sp. WRRC1, that was capable of producing a wide range of polyhydroxybutyrate-co-hydroxyvalerate copolymers (PHB-co-HV) when co-fed methane and valerate or n-pentanol. The ratio of HB to HV monomer was directly related to the concentration of valeric acid in the PHA accumulation media. We observed increased incorporation of HV and total polymer under copper-free growth conditions. The PHB-co-HV copolymers produced had decreased melting temperatures and crystallinity compared with methanotroph-produced PHB.

Nanoliposomes protect against human arteriole endothelial dysfunction induced by ß-amyloid peptide.

We tested whether nanoliposomes containing phosphatidylcholine, cholesterol and phosphatidic acid (NLPA) prevent ß-amyloid 1-42 (Aß42) fibrillation and Aß42-induced human arteriole endothelial dysfunction. NLPA abolished Aß42 fibril formation (thioflavin-T fluorescence/electron microscopy). In ex-vivo human adipose and leptomeningeal arterioles, Aß42 impaired dilator response to acetylcholine that was reversed by NLPA; this protection was abolished by L-NG-nitroarginine methyl ester. Aß42 reduced human umbilical vein endothelial cell NO production that was restored by NLPA. Nanoliposomes prevented Aß42 amyloid formation, reversed Aß42-induced human microvascular endothelial dysfunction and may be useful in Alzheimer's disease.

Nanocarrier-based antioxidant therapy: promise or delusion?

INTRODUCTION: Oxidative stress has generally been recognized as an important factor in the pathogenesis of human diseases, making antioxidant therapy a plausible strategy to either prevent or treat human disorders. Yet so far, numerous antioxidant-based clinical trials aimed at developing clinically approved protocols have been disappointing and many reasons for their failure are being discussed, including the limited bioavailability of most antioxidants. To overcome the hurdles associated with the direct administration of antioxidant molecules, a variety of nanotechnology-based drug delivery systems are being developed. All the strategies currently being explored, however, appear in our opinion to underappreciate the crucial role reactive oxygen and nitrogen species (RO/NS) play in the regulation of the metabolome, as revealed by recent progress made in redox biology. AREAS COVERED: We briefly review antioxidant-based clinical trials and discuss the functions of RO/NS as crucial intracellular messengers. We emphasize the probable existence of three distinct concentration levels of RO/NS: a physiological level reflecting their functions as messenger molecules, an elevated level crucial for activation of protective pathways and a toxic level causing oxidative damage to cellular components. EXPERT OPINION: Site-specific, multifunctional nanodrug delivery systems able to sense the actual intracellular concentrations of RO/NS and release antioxidants accordingly in order to only neutralize the pathologic excess of RO/NS need to be developed.

Countercurrent extraction of soluble sugars from almond hulls and assessment of the bioenergy potential.

Almond hulls contain considerable proportions (37% by dry weight) of water-soluble, fermentable sugars (sucrose, glucose, and fructose), which can be extracted for industrial purposes. The maximum optimal solids loading was determined to be 20% for sugar extraction, and the addition of 0.5% (w/v) pectinase aided in maintaining a sufficient free water volume for sugar recovery. A laboratory countercurrent extraction experiment utilizing a 1 h steep followed by three extraction (wash) stages produced a high-concentration (131 g/L fermentable sugar) syrup. Overall, sugar recovery efficiency was 88%. The inner stage washing efficiencies were compatible with solution equilibrium calculations, indicating that efficiency was high. The concentrated sugar syrup was fermented to ethanol at high efficiency (86% conversion), and ethanol concentrations in the broth were 7.4% (v/v). Thin stillage contained 233 g SCOD/L, which was converted to biomethane at an efficiency of 90% with a biomethane potential of 297 mL/g SCODdestroyed. Overall, results suggested that a minima of 49 gal (185 L) ethanol and 75 m(3) methane/t hulls (dry whole hull basis) are achievable.

Nanopharmaceuticals (part 2): products in the pipeline.

In part I of this review we assessed nanoscience-related definitions as applied to pharmaceuticals and we discussed all 43 currently approved drug formulations, which are widely publicized as nanopharmaceuticals or nanomedicines. In continuation, here we review the currently ongoing clinical trials within the broad field of nanomedicine. Confining the definition of nanopharmaceuticals to therapeutic formulations, in which the unique physicochemical properties expressed in the nanosize range, when man-made, play the pivotal therapeutic role, we found an apparently low number of trials, which reflects neither the massive investments made in the field of nanomedicine nor the general hype associated with the term "nano." Moreover, after an extensive search for information through clinical trials, we found only two clinical trials with materials that show unique nano-based properties, ie, properties that are displayed neither on the atomic nor on the bulk material level.

Enhanced cellular uptake and gene silencing activity of siRNA molecules mediated by chitosan-derivative nanocomplexes.

The RNA interference (RNAi) constitutes a conservative mechanism in eukaryotic cells that induces silencing of target genes. In mammalians, the RNAi is triggered by siRNA (small interfering RNA) molecules. Due to its potential in silencing specific genes, the siRNA has been considered a potential alternative for the treatment of genetic and acquired diseases. However, the siRNA therapy has been limited by its low stability and rapid degradation in presence of nucleases, low cellular uptake, and immune response activation. In order to overcome these drawbacks, we propose the synthesis and characterization of non-viral delivery systems using chitosan derivatives to obtain siRNA complexes (polyplexes). The non-viral delivery systems synthesized included PEG-g-OCs (oligochitosan) and PEG-g-Cs (chitosan medium molecular weight). Both systems allowed the formation of siRNA polyplexes, increased the stability of siRNA in the presence of nucleases, enhanced cellular internalization, and showed low toxicity in the A549 cell line. Finally, the complexes obtained with the PEG-g-OCs system showed silencing activity in a GFP model in the cell line A549 in comparison with naked siRNA.