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Background Levocarnitine deficiency has been observed in patients receiving parenteral nutrition (PN) and can cause or worsen hypertriglyceridemia. The objective was to characterize use of levocarnitine supplementation in PN and evaluate its effect on triglyceride levels in hospitalized adults. Methods This retrospective, single-center study included patients with triglyceride levels ≥175 mg/dl while receiving PN who had a subsequent reduction in lipid injectable emulsion dose. A piecewise linear regression was used to evaluate trends in triglyceride levels before and after the intervention, defined as initiation of levocarnitine in PN for the levocarnitine group, or reduction in lipid injectable emulsion alone for the control group. Results Two hundred sixty-one patients who received PN had an elevated triglyceride level and lipid injectable emulsion dose reduction, of which 97 (37.2%) received levocarnitine in PN. The median (IQR) levocarnitine dose added to PN was 8.0 (5.7-9.9) mg/kg. Triglyceride levels at 30 days post-intervention did not differ between groups (125 vs 176 mg/dl, P = .345). The addition of levocarnitine to PN was associated with a significantly greater rate of reduction in triglyceride levels pre-intervention to post-intervention compared with a reduction in lipid injectable emulsion alone (-11 vs -3 mg/dl per day; 95% CI, -15 to -2; P = .012). Conclusion In hospitalized adults with hypertriglyceridemia who had a lipid injectable emulsion dose reduction, the addition of levocarnitine in PN was not associated with a difference in triglyceride levels at 30 days; however, a greater rate of improvement in pre-intervention to post-intervention triglyceride levels was observed.

Cabotegravir/rilpivirine (CAB/RPV) was recently approved by the US Food and Drug Administration (FDA) as the first complete parenteral antiretroviral (ART) regimen for treatment of people living with HIV (PLWH). As a monthly intramuscular (IM) injection, this therapy constitutes a major departure from the traditional paradigm of oral therapy requiring (at least) daily administration that has defined HIV treatment for decades. Composed of a second-generation integrase inhibitor (INSTI) and nonnucleoside reverse transcriptase inhibitor (NNRTI), CAB/RPV has achieved high rates of sustained virologic suppression with a favorable safety profile for treatment-experienced PLWH following oral lead-in (OLI) during several clinical trials. In addition to the clinical benefits of this agent, patient-reported outcomes associated with convenience, confidentiality, and the tolerability of the injections have consistently reflected positive perceptions of CAB/RPV. The novel nature of this therapy in the field of HIV presents logistical challenges. Clinics will need to address barriers related to management of clinic workflow, procurement, reimbursement, and nonadherence. The aim of this review was to summarize the available safety, efficacy, and pharmacokinetic/pharmacodynamic (PK/PD) data of this long-acting (LA) injectable regimen as well as discuss some potential considerations for prescribing and operationalization.

The Deepwater Horizon disaster inflicted untold damage on humans and the natural environment in the U.S. Gulf Coast. Less discussed is that British Petroleum (BP) was prosecuted and found guilty of...

Congenital heart defects (CHDs) are the leading cause of death in live-born infants. Currently, patches used in the repair of CHDs are exclusively inert and non-degradable, which increases the risk of arrhythmia, follow-up surgeries, and sudden cardiac death. In this preliminary study, we sought to fabricate biodegradable scaffolds that can support cardiac regeneration in the repair of CHDs. We electrospun biodegradable scaffolds using various blends of polyurethane (PU) and polycaprolactone (PCL) with and without sacrificial poly(ethylene oxide) (PEO) particles and assessed the mechanical properties, cell infiltration levels, and inflammatory response in vitro (surface cell seeding) and in vivo (subcutaneous mouse implant). We hypothesized that a blend of the two polymers would preserve the low stiffness of PU as well as the high cell infiltration observed in PCL scaffolds. The inclusion of PU in the blends, even as low as 10%, decreased cell infiltration both in vitro and in vivo. The inclusion of sacrificial PEO increased pore sizes, reduced Young’s moduli, and reduced the inflammatory response in all scaffold types. Collectively, we have concluded that a PCL patch electrospun with sacrificial PEO particles is the most promising scaffold for further assessment as in our heart defect model.

Background: There is variation in the treatment of patients with severe alcohol withdrawal and a need for effective protocols. The purpose of this study was to evaluate the implementation of a symptom-triggered benzodiazepine protocol using the 5-item Brief Alcohol Withdrawal Scale (BAWS) for treatment of alcohol withdrawal in intensive care units (ICUs). Methods: This retrospective study included admissions to ICUs of 2 hospitals over 6 months who had an alcohol withdrawal protocol ordered and experienced severe withdrawal. Records were reviewed to collect demographic data, benzodiazepine exposure, duration of treatment, and withdrawal severity. Results: The protocol was ordered and implemented in 279 admissions; 48 (17.9%) had severe withdrawal defined as a BAWS of 6 or more. The majority of the 48 patients were from the emergency department (79.2%); mean hospital length of stay was 11.2 days and mean ICU stay 6.6 days; 31.3% required mechanical ventilation. A little more than half were treated only with the protocol (53.2%); 25.0% received additional benzodiazepines, 20.8% dexmedetomidine, 10.4% propofol, 25.0% antipsychotics and 2.0% phenobarbital. Conclusion: Among ICU patients treated for alcohol withdrawal with a symptom-triggered benzodiazepine protocol using a novel 5-item scale, most did not develop severe withdrawal, and of those who did, approximately half were treated with the protocol alone.

To explore novel coatings with potential for easy release of ice (icephobicity), a series of platinum-cured silicone coatings was prepared incorporating SYL-OFF 7210, designated MQ-R, as a nanoscale reinforcing component. These optically transparent coatings are designated according to cure temperature and MQ-R wt %, for example, Pt-PDMS(25)-20 for 25 °C cure and 20 wt % MQ-R. Surface characterization included dynamic contact angles and morphology by atomic force microscopy. Bulk characterization was accomplished with stress-strain measurements at 25 °C and dynamic mechanical analysis from -110 to 150 °C. Ice adhesion tests at -10 °C showed modulus had a dominant effect in increasing

Athletic involvement is linked to increased risk for heavy alcohol use among college students. We examined whether student-athletes from diverse racial/ethnic backgrounds differ with respect to heavy drinking and related consequences. Method: Participants were 15,135 student-athlete drinkers (50.7% female) from 170 NCAA member institutions who participated in an online study. Results: Findings from our hierarchical linear models indicated that being a male student-athlete was associated with an increased likelihood of high intensity drinking (10/8 + drinks/per sitting for males/females) for White, Asian American/Pacific Islander, and Black student-athletes, but not for Hispanic student-athletes. Additionally, being a female student-athlete was associated with higher levels of negative alcohol-related consequences across all racial/ethnic groups. Finally, at similar drink quantities, compared to being a White student-athlete, being an Asian American/Pacific Islander student-athlete was associated with higher levels of alcohol-related consequences. Conclusions: Student-athlete drinkers are not homogeneous with respect to heavy drinking and related consequences.

Human offspring of obese mothers suffer higher risk of developing type 2 diabetes due to insufficient islet insulin secretion. Preclinical maternal obesity models demonstrated the sex-differences in offspring islet function, but the mechanistic targets are unknown. We found that a higher islet Regenerating Islet Derived Protein 3-Gamma (Reg3g) is associated with the maintenance of islet function in female offspring of obese dams. Reg3g binds to EXTL3 glycosyltransferase that polymerizes heparan sulfate glycosaminoglycan (HSG). To date, the role of Reg3g mediated HSG polymerization in modulating β-cell health in offspring of obese mice is unclear. We hypothesize that the upregulation of Reg3g-mediated HSG polymerization protects offspring of obese mice from glucose intolerance and islet dysfunction. We evaluated islet GSIS, islet Reg3g gene/protein, and pancreatic HSG with SAX-HPLC in offspring born to chow-fed mice (Con) and diet-induced obese mice (MatOb) on postnatal day 21 (P21). To define the causality of Reg3g, wildtype (WT) and heterozygous Reg3g mutant (Reg3g+/-) offspring of Con and MatOb were generated. Lastly, we tested the therapeutic effect of intraperitoneal recombinant Reg3g (rReg) and heparan sulfate analogue (HS) from P14-20 in male MatOb pups. Compared to same-sex controls, P21 female MatOb pups had higher islet Reg3g gene/protein* (n=3-6), higher islet (n=3/3) and pancreatic (n=6/6) HSG*, and unchanged GSIS. Male MatOb pups had unchanged islet Reg3g and a lower pancreatic HSG* (n=8/8). Male MatOb pups also had a lower glucose tolerance* (n=9-19) and 30% decrease in GSIS* (n=5/5). The sex-difference in glucose tolerance was ablated in Reg3g+/- mice, where female MatOb Reg3g+/- pups had significantly impaired GTT. Lastly, rReg3g or HS improved glucose tolerance in male MatOb pups* (n=3-7). *p In conclusion, our findings supported the role of Reg3g as an amenable mechanistic target mediating islet dysfunction in MatOb offspring. Disclosure J. C. Jarrell: None. J. Casasnovas: Employee; Self; Thermo Fisher Scientific. L. Kua: Stock/Shareholder; Spouse/Partner; Dexcom, Inc., Thermo Fisher Scientific. Funding Riley Children’s Foundation (to L.K.); Showalter Research Trust (EPAR1143 to L.K.); Center for Diabetes and Metabolic Diseases (P30DK097512)

Offspring of obese mothers suffer higher risks of type 2 diabetes due to increased adiposity and decreased β cell function. To date, the sex-differences in offspring islet insulin secretion during early life has not been evaluated extensively, particularly prior to weaning at postnatal day 21 (P21). To determine the role of maternal obesity on offspring islet insulin secretion, C57BL/6J female dams were fed chow or western diet from 4 weeks prior to mating to induce maternal obesity. First, offspring of chow-fed and obese dams were evaluated on postnatal day 21 (P21) prior to weaning for body composition, glucose and insulin tolerance, and islet phasic insulin-secretion. Compared to same-sex controls, both male and female P21 offspring born to obese dams (MatOb) had higher body adiposity and exhibited sex-specific differences in glucose tolerance and insulin secretion. The male MatOb offspring developed the highest extent of glucose intolerance and lowest glucose-induced insulin secretion. In contrast, P21 female offspring of obese dams had unimpaired insulin secretion. Using SAX-HPLC, we found that male MatOb had a decrease in pancreatic heparan sulfate glycosaminoglycan, which is a macromolecule critical for islet health. Notably, 8-weeks-old offspring of obese dams continued to exhibit a similar pattern of sex-differences in glucose intolerance and decreased islet insulin secretion. Overall, our study suggests that maternal obesity induces sex-specific changes to pancreatic HSG in offspring and a lasting effect on offspring insulin secretion, leading to the sex-differences in glucose intolerance.

Electronic cigarettes (e-cig) are an increasingly popular alternative to traditional smoking but have been in use for too short of a period of time to fully understand health risks. Furthermore, associated health risks are difficult to evaluate because of a large range of flavoring agents and their combinations for use with e-cig. Many flavoring agents are generally regarded as safe but have limited studies for effects on lung. Vanillin, an aromatic aldehyde, is one of the most commonly used flavoring agents in e-cig. Vanillin is electrophilic and can be redox active, with chemical properties expected to interact within biologic systems. Because accumulating lung metabolomics studies have identified metabolic disruptions associated with idiopathic pulmonary fibrosis, asthma and acute respiratory distress syndrome, we used human bronchial epithelial cells (BEAS-2B) with high-resolution metabolomics analysis to determine whether these disease-associated pathways are impacted by vanillin over the range used in e-cig. A metabolome-wide association study showed that vanillin perturbed specific energy, amino acid, antioxidant and sphingolipid pathways previously associated with human disease. Analysis of a small publicly available human dataset showed associations with several of the same pathways. Because vanillin is a common and high-abundance flavorant in e-cig, these results show that vanillin has potential to be mechanistically important in lung diseases and warrants in vivo toxicity testing in the context of e-cig use.

The adoption of genetically engineered (GE) crops in agriculture has increased dramatically over the last few decades. Among the transgenic plants, those tolerant to the herbicide glyphosate are among the most common. Weed resistance to glyphosate-based herbicides (GBHs) has been on the rise, leading to increased herbicide applications. This, in turn, has led to increased glyphosate residues in feed. Although glyphosate has been considered to be generally safe to animal health, recent studies have shown that GBHs have potential to cause adverse effects in animal reproduction, including disruption of key regulatory enzymes in androgen synthesis, alteration of serum levels of estrogen and testosterone, damage to reproductive tissues and impairment of gametogenesis. This review emphasizes known effects of GBHs on reproductive health as well as the potential risk GBH residues pose to animal agriculture.

OBJECTIVES: The gut microbiome changes with age, and rapamycin treatment both delays aging and modulates the microbiome. This raises important questions concerning cause-effect relationships between microbiome, mTOR signaling and aging mechanisms. Microbiome metabolite valerobetaine (VB) disrupts mitochondrial fatty acid oxidation mechanism. Thus, we tested the hypothesis that increase in VB with aging activates mTORC1 and stimulates lung aging. METHODS: Plasma collected from 179 healthy humans (age; 21 to 75) without known disease (CHDWB, Center for Health Discover and Well Being), and lungs from mice (6 mo and 24 mo, n = 24 each) were analyzed for VB by high resolution metabolomics (HRM). Lung fibroblasts (LF) treated with VB were analyzed for activation of mechanistic target of rapamycin (mTOR) pathway by Western blot analysis. RESULTS: VB concentration in human plasma was significantly increased with age (P = 0.0004, r = 0.26). Consistently, lung VB level in old mice were significantly higher than young mice (P = 0.004). LF treated with VB stimulated phosphorylation of mTOR and ribosomal protein S6 as markers for mTORC1 activation, and rapamycin mTORC1 inhibitor substantially decreased VB-stimulated phosphorylation of S6 and mTOR. CONCLUSIONS: The results show significant association of VB with aging in human and mouse lung studies, and VB-stimulated mTORC1. The finding suggests that microbiome metabolite VB could impact aging and indicates that studies are needed to understand potential dysfunctions related to age and age-associated diseases such as pulmonary fibrosis. FUNDING SOURCES: NIH R01 ES023485 and S10 OD018006.

Background Brain tumor vasculature can be significantly compromised and leakier than that of normal brain blood vessels. Little is known if there are vascular permeability alterations in the brain adjacent to tumor (BAT). Changes in BAT permeability may also lead to increased drug permeation in the BAT, which may exert toxicity on cells of the central nervous system. Herein, we studied permeation changes in BAT using quantitative fluorescent microscopy and autoradiography, while the effect of chemotherapy within the BAT region was determined by staining for activated astrocytes. Methods Human metastatic breast cancer cells (MDA-MB-231Br) were injected into left ventricle of female NuNu mice. Metastases were allowed to grow for 28 days, after which animals were injected fluorescent tracers Texas Red (625 Da) or Texas Red dextran (3 kDa) or a chemotherapeutic agent 14C-paclitaxel. The accumulation of tracers and 14C-paclitaxel in BAT were determined by using quantitative fluorescent microscopy and autoradiography respectively. The effect of chemotherapy in BAT was determined by staining for activated astrocytes. Results The mean permeability of texas Red (625 Da) within BAT region increased 1.0 to 2.5-fold when compared to normal brain, whereas, Texas Red dextran (3 kDa) demonstrated mean permeability increase ranging from 1.0 to 1.8-fold compared to normal brain. The Kin values in the BAT for both Texas Red (625 Da) and Texas Red dextran (3 kDa) were found to be 4.32 ± 0.2 × 105 mL/s/g and 1.6 ± 1.4 × 105 mL/s/g respectively and found to be significantly higher than the normal brain. We also found that there is significant increase in accumulation of 14C-Paclitaxel in BAT compared to the normal brain. We also observed animals treated with chemotherapy (paclitaxel (10 mg/kg), erubilin (1.5 mg/kg) and docetaxel (10 mg/kg)) showed activated astrocytes in BAT. Conclusions Our data showed increased permeation of fluorescent tracers and 14C-paclitaxel in the BAT. This increased permeation lead to elevated levels of activated astrocytes in BAT region in the animals treated with chemotherapy.

A method is presented for the rapid identification of tertiary aliphatic and aromatic N-oxide functionalities in protonated analytes via ion-molecule reactions with dimethylamine (DMA) in a linear quadrupole ion trap mass spectrometer (LQIT). DMA was leaked into the trapping region of the mass spectrometer and allowed to react with protonated analytes (ionized by electrospray ionization) for 500 ms, after which all ions were detected. Protonated analytes with the tertiary N-oxide functionality react with DMA to yield exclusively product ions with m/z-values that are 45 units greater than that of the protonated analyte, corresponding to a stable DMA adduct. Collision-activated dissociation of the adduct ions suggests that formation of a hydrogen bond between DMA and the protonated analytes is responsible for the formation of the stable adduct. Hydrogen bond energies were calculated for several adducts at the B3LYP/6-31++G(d,p) level of theory (including correction for basis-set superposition error) to define the potential energy wells for the formation of [M+H+DMA]+ adduct ions. The relative enthalpies calculated for the [M+H+DMA]+ adduct ions were found to be lower than those of the products resulting from proton transfer from protonated tertiary N-oxides and one aromatic ketone, ketoprofen, to DMA. On the other hand, a protonated primary N-oxide, nitrosobenzene, exclusively reacts via proton transfer with DMA, which was calculated to be more exothermic than formation of an adduct ion. Collisional cooling was found to be crucial to the formation of an adduct ion for analytes with proton affinities lower than that of DMA. Ion-molecule reactions of protonated 4-nitro-2-picoline N-oxide (PA = 206 kcal mol−1) with DMA (PA = 222 kcal mol−1) in an FT-ICR at ultra-high vacuum with no buffer gas resulted solely in proton transfer to DMA. Finally, examination of the reactions of DMA with protonated clozapine and clozapine-4′-N-oxide demonstrated that DMA can be used to identify the N-oxide functionality in the presence of other basic functionalities. Compared to other reagents reported for the identification of tertiary N-oxide functionalities via ion-molecule reactions in a mass spectrometer, DMA is the only one volatile enough to be used in a multi-ported pulsed valve system designed for rapid introduction of several reagents, each diagnostic for a different functionality, for characterization of analytes as they elute from an HPLC.

The measurable charged-particle produced from the fast neutron interactions with the Si and C nucleuses can make a wide bandgap silicon carbide (SiC) sensor intrinsically sensitive to neutrons. The 4H-SiC Schottky detectors have been fabricated and tested at up to 500 °C, presenting only a slightly degraded energy resolution. The response spectrum of the SiC detectors were also obtained by exposing the detectors to external neutron beam irradiation and at a near-core location where gamma-ray field is intense. The fast neutron flux of these two locations are ∼ 4 . 8 × 1 0 4 cm − 2 ⋅ s − 1 and ∼ 2 . 2 × 1 0 7 cm − 2 ⋅ s − 1 , respectively. At the external beam location, a Si detector was irradiated side-by-side with SiC detector to disjoin the neutron response from Si atoms. The contribution of gamma ray, neutron scattering , and charged-particles producing reactions in the SiC was discussed. The fast neutron detection efficiencies were determined to be 6 . 43 × 1 0 − 4 for the external fast neutron beam irradiation and 6 . 13 × 1 0 − 6 for the near-core fast neutron irradiation.

Brain kappa-opioid receptors (KORs) are implicated in the pathophysiology of depressive and anxiety disorders, stimulating interest in the therapeutic potential of KOR antagonists. Research on KOR function has tended to focus on KOR-expressing neurons and pathways such as the mesocorticolimbic dopamine system. However, KORs are also expressed on non-neuronal cells including microglia, the resident immune cells in the brain. The effects of KOR antagonists on microglia are not understood despite the potential contributions of these cells to overall responsiveness to this class of drugs. Previous work in vitro suggests that KOR activation suppresses proinflammatory signaling mediated by immune cells including microglia. Here, we examined how KOR antagonism affects microglia function in vivo, together with its effects on physiological and behavioral responses to an immune challenge. Pretreatment with the prototypical KOR antagonist JDTic potentiates levels of proinflammatory cytokines (IL-1ß, IL-6) in blood following administration of lipopolysaccharide (LPS), an immune-activating agent, without triggering effects on its own. Using magnetic-activated cell sorting (MACs), we found that KOR antagonism potentiates LPS-induced cytokine expression within microglia. This effect is accompanied by potentiation of LPS-induced hyperthermia, although reductions in body weight and locomotion were not affected. Histological analyses confirm that LPS produces visible changes in microglia morphology consistent with activation, but this effect is not altered by KOR antagonism. Considering that inflammation is increasingly implicated in depressive and anxiety disorders, these findings raise the possibility that KOR antagonist actions on microglia may detract from actions on neurons that contribute to their therapeutic potential.

Intracellular delivery is a critical process in biology and medicine. During intracellular delivery, different constructs (e.g., functional macromolecules such as DNA, RNA, and protein, and various complexes) are delivered across the cell membrane and into the cytosol. Herein, we use a microfluidic post array to induce hydrodynamic conditions for cell membrane poration with microfluidic vortex shedding (µVS). µVS is then used for the intracellular delivery of mRNA to primary human pan T cells. The specific microfluidic device and experimental rig used in this study contains a 960 µm wide by 40 µm deep flow cell capable of processing more than 2E6 cells/s at volumes ranging from 100 µL to 1.5 mL. Furthermore, we demonstrate efficient enhanced green fluorescent protein (EGFP) mRNA expression (e.g., 57.4 ± 6.8% of viable, recovery cells, mean ± stdev) after mRNA delivery to human pan T cells with high cell viability (e.g., 83.7 ±0.7% of recovered cells) and high cell recovery (e.g., 96.3 ± 1.1% of processed cells), resulting in net yield of 46.3 ± 5.6% viable, recovered, and GFP expressing human pan T cells at mRNA concentrations of 80 µg/mL. We also demonstrate µVS does not alter human pan T cell growth nor activation. These results demonstrated that µVS is a rapid intracellular delivery platform with promising potential for cytosolic delivery of mRNA to human primary T cells for (1) clinical applications, where larger volumes of cells are required and demonstrated value for (2) research applications, where rapid screening and minimal reagent consumption is preferable.

Seven synthesized G-lignin oligomer model compounds (ranging in size from dimers to an octamer) with 5-5 and/or β-O-4 linkages, and three synthesized S-lignin model compounds (a dimer, trimer, and tetramer) with β-O-4 linkages, were evaporated and deprotonated using negative-ion mode ESI in a linear quadrupole ion trap/Fourier transform ion cyclotron resonance mass spectrometer. The collision-activated dissociation (CAD) fragmentation patterns (obtained in MS2 and MS3 experiments, respectively) for the negative ions were studied to develop a procedure for sequencing unknown lignin oligomers. On the basis of the observed fragmentation patterns, the measured elemental compositions of the most abundant fragment ions, and quantum chemical calculations, the most important reaction pathways and likely mechanisms were delineated. Many of these reactions occur via charge-remote fragmentation mechanisms. Deprotonated compounds with only β-O-4 linkages, or both 5-5 and β-O-4 linkages, showed major 1,2-eliminations ...

Background/Aims: Endoplasmic reticulum (ER) stress has emerged as a potential mechanism contributing to diabetes and its comorbidities. However, the importance of ER stress in diabetic vascular dysfunction is unclear. The purpose of this study was to examine the effects of the ER stress inhibitor, tauroursodeoxycholic acid (TUDCA), on arterial stiffness and endothelial dysfunction in type 2 diabetic mice. Methods: Carotid and mesenteric artery endothelial function were assessed via ex vivo pressure myography, and arterial stiffness was measured by aortic pulse wave velocity. The effects of TUDCA were examined both acutely (ex vivo) and chronically (250 mg/kg/day; i.p., 4 weeks). Results: Compared to control C57BL/6J mice, db/db (DB) mice did not display carotid artery endothelial dysfunction; however, mesenteric artery endothelial function was markedly impaired. Acute incubation and chronic administration of TUDCA improved endothelium-dependent dilation in DB mesenteric arteries, without affecting endothelium-independent dilation. Chronic TUDCA administration also reduced arterial stiffness and was associated with reductions in ER stress markers in aortic and perivascular adipose tissue. Conclusions: These results suggest that ER stress may represent a novel cause of, and therapeutic target for, diabetic vascular dysfunction.

The donor substrates for the biosynthesis of bacterial polysaccharides include UDP-Glc/Gal and UDP-GlcNAc/GalNAc. The conversion of these nucleotide sugars is catalyzed by 4-epimerases. The wbpP gene of Pseudomonas aeruginosa encodes a 4-epimerase that has a preference for UDP-GlcNAc/GalNAc as substrates. Other 4-epimerases have broad specificities or preference for UDP-Glc/Gal. We have developed coupled assays where the 4-epimerase product is used as a donor substrate for glycosyltransferases that are highly specific for the nucleotide sugar structure. We describe here a method for the study of substrate specificity of WbpP, using coupled assays employing four different glycosyltransferases. These protocols can be applied to the identification and characterization of novel 4-epimerases and to determine their substrate specificities.

Unexpected wetting behavior is reported for silicone elastomers platinum cured at 37 °C in water or saline. These conditions were prompted as a way to mimic cure under physiologically relevant conditions for VK100, a Pt-cured silicone used for vertebral augmentation. Water contact angles (CAs) were determined by the drop addition/withdrawal method. Network formation in air, water, or saline gave high advancing CAs (θA). However, compared to 74° for air cure, network formation in water (56°) or saline (46°) gave low receding CAs (θR). Thus, water drop adhesion to VK100 and a model Pt-cured silicone depends on whether network formation is carried out in water or saline (“sticky”) or in air (“slippery”). For cure in water or saline, autoxidation (Si-H ➔ Si-OH) and near-surface entrapment of cross-linking chains containing –Si-OH are proposed to account for low receding CAs. The origin of the low θR and high contact angle hysteresis (54–72°) is correlated with the theory of Johnson and Dettre by which a small area fraction of polar groups impedes retraction of a receding water drop. These results are of interest given the importance of polar interactions at interfaces that favor adhesion to bone and influence on biofouling, adhesion of proteins, and interactions with human cells.

INTRODUCTION The conventional use of a prosthetic device by amputees involves contact of the residual limb tissue with the prosthetic socket using an intermediate elastomer liner. Roll-on gel liners are applied directly to the limb, and slide into the rigid hard socket; the gels are generally silicone or plastic. Regardless of the material, or the liner system used on a residual limb, problems occur because of direct skin and socket or liner contact.1 The skin tissue of the residual limb is subject to compressive, shear, and tensile forces through weight bearing against the interface wall. In addition the skin is subject to heat/sweat issues that may be seasonal and related to the insulation properties of the interface material. A common problem encountered by the prosthetic user is socket odor, or odor emanating from socket liners.2 Proper hygiene does not guarantee the reduction of strong socket or liner odor over time. In addition, skin problems among amputees include rashes, blistering, mold/fungal infections and other skin irritations. The socket/wall interface (regardless of material composition) is subject to local skin sloughing, sweat, and skin oils in a warm/moist dark environment over hours of use providing an ideal environment for fungal and bacterial growth.3 To address this problem, our team evaluated a titanium and silicone hybrid cleaning and coating technology containing a silver fatty acid complex against the odor producing, Gram-negative, facultative anaerobe, Pseudomonas aeruginosa.4 Abstract PDF Link: https://jps.library.utoronto.ca/index.php/cpoj/article/view/32016/24435 How to cite: Jarrell J.D, Garcia D.R, Born C.T. INHIBITING PSEUDOMONAS AERUGINOSA GROWTH ASSOCIATED WITH PROSTHETIC LINERS. CANADIAN PROSTHETICS & ORTHOTICS JOURNAL, VOLUME 1, ISSUE 2, 2018; ABSTRACT, POSTER PRESENTATION AT THE AOPA’S 101ST NATIONAL ASSEMBLY, SEPT. 26-29, VANCOUVER, CANADA, 2018.DOI: https://doi.org/10.33137/cpoj.v1i2.32016

A thrombotic microangiopathy (TMA) occurs when a patient presents with microangiopathic hemolytic anemia, thrombocytopenia and organ damage. There are many causes of TMAs, but thrombotic thrombocytopenic purpura (TTP) must always be considered because of its high mortality rate. Treatment with therapeutic plasma exchange and transfusions can reduce the mortality rate but those treatments carry their own morbidity. Severe cobalamin deficiency can cause a clinical picture similar to TTP. Understanding the pathologic changes of this pseudo-TTP can allow physicians to suspect this difference early in the clinical course. In the following case, elevations in homocysteine and methylmalonic acid, as well as changes in the CBC, allowed this distinction to be identified early, minimizing potentially dangerous treatments.

Polyacenes have attracted considerable attention due to their various applications in organic optoelectronic materials. This study focuses on linear polyacenes and their electron affinity (EA) and ionization potential (IP) properties. We have employed our recent implementation of EA/IP equation of motion coupled cluster singles and doubles (EA/IP-EOMCCSD) methods which are accurate, computationally efficient and are capable of treating large systems employing reasonable basis sets size. The EA/IP results obtained for naphthalene, anthracene, tetracene, pentacene, hexacene and heptacene are in a good agreement with experiment. Comparison between quality of excitation energies obtained from IP-EOMCCSD and EE-EOMCCSD formalisms were also studied.

Background Type 2 diabetes (T2D) is associated with generalized vascular dysfunction characterized by increases in large artery stiffness, endothelial dysfunction, and vascular smooth muscle dysfunction. Sodium glucose cotransporter 2 inhibitors (SGLT2i) represent the most recently approved class of oral medications for the treatment of T2D, and have been shown to reduce cardiovascular and overall mortality. Although it is currently unclear how SGLT2i decrease cardiovascular risk, an improvement in vascular function is one potential mechanism. The aim of the current study was to examine if dapagliflozin, a widely prescribed STLT2i, improves generalized vascular dysfunction in type 2 diabetic mice. In light of several studies demonstrating a bi-directional relation between orally ingested medications and the gut microbiota, a secondary aim was to determine the effects of dapagliflozin on the gut microbiota. Methods Male diabetic mice (Db, n = 24) and control littermates (Con; n = 23) were randomized to receive either a standard diet or a standard diet containing dapagliflozin (60 mg dapagliflozin/kg diet; 0.006%) for 8 weeks. Arterial stiffness was assessed by aortic pulse wave velocity; endothelial function and vascular smooth muscle dysfunction were assessed by dilatory responses to acetylcholine and sodium nitroprusside, respectively. Results Compared to untreated diabetic mice, diabetic mice treated with dapagliflozin displayed significantly lower arterial stiffness (Db = 469 cm/s vs. Db + dapa = 435 cm/s, p

Current biomass utilization processes do not make use of lignin beyond its heat value. Here we report on a bimetallic Zn/Pd/C catalyst that converts lignin in intact lignocellulosic biomass directly into two methoxyphenol products, leaving behind the carbohydrates as a solid residue. Genetically modified poplar enhanced in syringyl (S) monomer content yields only a single product, dihydroeugenol. Lignin-derived methoxyphenols can be deoxygenated further to propylcyclohexane. The leftover carbohydrate residue is hydrolyzed by cellulases to give glucose in 95% yield, which is comparable to lignin-free cellulose (solka floc). New conversion pathways to useful fuels and chemicals are proposed based on the efficient conversion of lignin into intact hydrocarbons.

Air quality monitoring was conducted during the late construction and early occupation stages of the College of DuPage Student Resource Center (SRC) addition from April 24,1995, to July 20,1995. Chemical contaminants monitored included combustibles; cleaning solvents; and human, furniture, and carpeting effluents. Carbon dioxide, carbon monoxide, ethanol, propane, 3-pentanone, methyl cyclohexane, methyl formate, tetrahydrofuran, methyl methacrylate, and cyclohexane were used as calibration standards for continuous infrared absorption measurements. Indoor water content, outdoor relative humidity, indoor and outdoor temperatures, and indoor airborne particulate matter were measured. After most construction and indoor painting and carpeting were completed, a two-week air-out was performed using a continuous supply of fresh air, without recirculated air. This resulted in a low "case study" level of contaminants. Contaminant levels increased significantly after furniture and people move-ins and student use. Contaminant level changes were observed during typical indoor construction days, before and after a power outage-caused loss of ventilation, and in the presence of carpentry machines. A "naive" sensory panel contributed its "perception" of air quality, and anair quality survey was conducted among new building employees. No significant or consistent effects of indoor contaminants or indoor temperature upon indoor perception were noted. An inverse relationship between indoor air quality perceptions and the outdoor Temperature-Humidity Index was found.

BACKGROUND Prothrombin complex concentrates (PCCs) are drug products containing varying amounts of vitamin K-dependent coagulation factors II, VII, IX, and X. The evidence comparing 3-factor PCC (3-PCC) versus 4-factor PCC (4-PCC) for warfarin reversal is conflicting. It has been hypothesized that 3-PCC may be less effective than 4-PCC because of relatively lower factor VII content. STUDY QUESTION The primary objective of this study was to compare international normalized ratio (INR) reversal between 3-PCC and 4-factor PCC (4-PCC) in warfarin-treated patients. The secondary objectives include comparing blood product use, total reversal costs, and cost-effectiveness between the groups. STUDY DESIGN This was a retrospective cohort study conducted in 2 affiliated, academic institutions in the United States. Consecutive adult patients who received 3-PCC or 4-PCC for warfarin reversal were included. MEASURES AND OUTCOMES The primary outcome was adequate INR reversal defined as a final INR ≤1.5. Secondary outcomes were the utilization of plasma, red blood cells and platelets, reversal costs, and the cost-effectiveness ratio. RESULTS There were 89 patients who were included in the overall cohort (3-PCC = 57, 4-PCC = 32). Adequate INR reversal occurred less commonly with 3-PCC (45.6%) compared with 4-PCC (87.5%) (P < 0.001). There was no significant difference in the proportion of patients who received plasma (32% vs. 28%, P = 0.813), red blood cells (37% vs. 47%, P = 0.377), or platelets (16% vs. 28%, P = 0.180) between the 3-PCC and 4-PCC groups, respectively. The median reversal cost of 3-PCC ($3663) was lower than 4-PCC ($5105) (P = 0.001). The cost-effective ratio favored 4-PCC ($5105/87.5% = $5834) compared with 3-PCC ($3663/45.6% = $8033). CONCLUSIONS Four-PCC was more effective than 3-PCC with regard to INR reversal in patients taking warfarin, but blood product use was similar. Although 4-PCC is associated with increased reversal costs, it may be cost-effective in terms of INR reversal.

The absolute configurations of the separated enantiomers of fluralaner, a racemic animal health product used to prevent fleas and ticks, have been assigned using vibrational circular dichroism (VCD). The crystallographic structure of the active enantiomer (+)-fluralaner has previously been shown to have the (S) configuration using small molecule crystallography. We sought a faster analytical method to determine the absolute configuration of the separated enantiomers. When comparing the measured IR (infrared) and VCD spectra, it is apparent that the amide carbonyl groups appear in the IR but are nearly absent in the VCD. Computational work to calculate the VCD and IR using in vacuo models, implicit solvation, and explicitly solvated complexes has implicated conformational averaging of the carbonyl VCD intensities.

Two biosurfactants, surfactin and fatty acyl-glutamate, were produced from genetically-modified strains of Bacillus subtilis on 2% glucose and mineral salts media in shake-flasks and bioreactors. Biosurfactant synthesis ceased when the main carbohydrate source was completely depleted. Surfactin titers were ∼30-fold higher than fatty acyl-glutamate in the same medium. When bacteria were grown in large aerated bioreactors, biosurfactants mostly partitioned to the foam fraction, which was recovered. Dispersion effectiveness of surfactin and fatty acyl-glutamate was evaluated by measuring the critical micelle concentration (CMC) and dispersant-to-oil ratio (DOR). The CMC values for surfactin and fatty acyl-glutamate in double deionized distilled water were 0.015 and 0.10 g/L, respectively. However, CMC values were higher, 0.02 and 0.4 g/L for surfactin and fatty acyl-glutamate, respectively, in 12 parts per trillion (ppt) Instant Ocean ® sea salt, which has been partly attributed to saline-induced conformational changes in the solvated ionic species of the biosurfactants. The DORs for surfactin and fatty acyl-glutamate were 1:96 and 1:12, respectively, in water. In Instant Ocean ® solutions containing 12 ppt sea salt, these decreased to 1:30 and 1:4, respectively, suggesting reduction in oil dispersing efficiency of both surfactants in saline. Surfactant toxicities were assessed using the Gulf killifish, Fundulus grandis , which is common in estuarine habitats of the Gulf of Mexico. Surfactin was 10-fold more toxic than fatty acyl-glutamate. A commercial surfactant, sodium laurel sulfate, had intermediate toxicity. Raising the salinity from 5 to 25 ppt increased the toxicity of all three surfactants; however, the increase was the lowest for fatty acyl-glutamate.

Lignin is an aromatic biopolymer that may yield valuable chemicals currently obtained solely from petroleum. However, extraction of lignin by using traditional methods, such as organosolv extraction, produces very complex mixtures. Molecular level characterization of the major components is essential to be able to rationally tailor methodology for the conversion of these mixtures to transportation fuel and valuable chemicals. In this study, high performance liquid chromatography/high resolution tandem mass spectrometry (HPLC/MSn) was used to obtain molecular weight, elemental composition and structural information for the major components in an organosolv lignin sample. HPLC/MSn coupled with hydroxide-doped electrospray ionization was used to identify the structures of the major components by using a Thermo Scientific linear quadrupole ion trap-Fourier transform ion cyclotron resonance hybrid mass spectrometer (LQIT/FT-ICR). The results reported here demonstrate that the major products of organosolv extraction are low molecular weight compounds, including monomeric and dimeric lignin units, with various functionalities.

Zirconia is a transition metal oxide with current applications to orthopedic implants. It has been shown to up-regulate specific genes involved in bio-integration and injury repair. This study examines the effects of zirconia and polydimethylsiloxane (PDMS) hybrids on the proliferation and viability of human primary osteoblast and fibroblast cells. In this study, zirconia-PDMS hybrid coatings were synthesized using a modified sol gel process. The hybrid material was characterized using optical microscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, and contact angle analysis. This study demonstrates that Zr-PMDS surface materials display hydrophobic surface properties coupled with a preferential deposition of polymer near the surface. Primary osteoblast and fibroblast proliferation and viability on hybrid coated surfaces were evaluated via a rapid screening methodology using WST-1 and calcein AM assays. The cells were seed at 5,000 cells per well in 96-well plates coated with various composition of Zr-PDMS hybrids. The results showed increasing cell proliferation with increasing zirconia concentration, which peaked at 90 % v/v zirconia. Proliferation of osteoblasts and fibroblasts displayed similar trends on the hybrid material, although osteoblasts displayed a bi-phasic dose response by the calcein AM assay. The results of this current study show that Zr-PDMS may be used to influence tissue-implant integration, supporting the use of the hybrid as a promising coating for orthopedic trauma implants.

In order to evaluate the importance of molecular shape of inhibitor molecules and the charge/H-bond and hydrophobic interactions, we synthesized three types of molecules and tested them against a sialyltransferase. The first type of compounds were designed as substrate mimics in which the phosphate in CMP-Neu5NAc was replaced by a non-hydrolysable, uncharged 1,2,3-triazole moiety. The second type of compound contained a 2-deoxy-2,3-dehydro-acetylneuraminic moiety which was linked to cytidine through its carboxylic acid and amide linkers. In the third type of compound the sialyl phosphate was substituted by an aryl sulfonamide which was then linked to cytidine. Inhibition study of these cytidine conjugates against Campylobacter jejuni sialyltransferase Cst 06 showed that the first type of molecules are competitive inhibitors, whereas the other two could only inhibit the enzyme non-competitively. The results indicate that although the binding specificity may be guided by molecular shape and H-bond interaction, the charge and hydrophobic interactions contributed most to the binding affinity.

This study evaluates the potential of niobium oxide-polydimethylsiloxane (PDMS) composites for tuning cellular response of fibroblasts, a key cell type of soft tissue/implant interfaces. In this study, various hybrid coatings of niobium oxide and PDMS with different niobium oxide concentrations were synthesized and characterized using scanning electron microscopy, X-ray photoelectron spectrometry (XPS), and contact angle goniometry. The coatings were then applied to 96-well plates, on which primary fibroblasts were seeded. Fibroblast viability, proliferation, and morphology were assessed after 1, 2, and 3 days of incubation using WST-1 and calcein AM assays along with fluorescent microscopy. The results showed that the prepared coatings had distinct surface features with submicron spherical composites covered in a polymeric layer. The water contact angle measurement demonstrated that the hybrid surfaces were much more hydrophobic than the original pure niobium oxide and PDMS. The combination of surface roughness and chemistry resulted in a biphasic cellular response with maximum fibroblast density on substrate with 40 wt % of niobium oxide. The results of the current study indicate that by adjusting the concentration of niobium oxide in the coating, a desirable cell response can be achieved to improve tissue/implant interfaces.

The development of chemical methods for the direct catalytic conversion of biomass to high value organic molecules is an area of increasing interest. The plant matter component known as lignin is a polymer consisting of aromatic rings that could provide a means of obtaining aromatic materials currently derived solely from petroleum. This report describes a bimetallic Pd/C and Zn catalytic system that can perform selective hydrodeoxygenation (HDO) of monomeric lignin surrogates as well as successfully cleave the β-O-4 linkages found in dimeric lignin model complexes and synthetic lignin polymers with near quantitative conversions and yields between 80–90%. The reaction with lignin polymer was highly selective affording methoxy substituted propylphenol as the major product. These reactions were performed in a Parr reactor operating at relatively mild temperature (150 °C) and pressure (20 bar H2) using methanol as a solvent. Reaction products were characterized using high-pressure liquid chromatography coupled to a linear quadrupole ion trap mass spectrometer equipped with an electrospray ionization source using negative ion mode. Hydroxide ions were doped into the analyte solutions to encourage negative ion formation. This method ionizes all the mixture components to yield a single ion/analyte with no fragmentation. The catalyst is fully recyclable without the need for additional zinc. X-ray absorption spectroscopy (EXAFS) is consistent with Pd nanoparticles (4–5 nm) and no evidence of Pd–Zn alloy formation. A mechanistic hypothesis on the synergy between Pd and Zn is presented.

The oligosaccharyltransferase is the signature enzyme for N-linked glycosylation in all domains of life. In Archaea, this enzyme termed AglB, is responsible for transferring lipid carrier-linked glycans to select asparagine residues in a variety of target proteins including archaellins, S-layer proteins and pilins. This study investigated the ability of a variety of AglBs to compensate for the oligosaccharyltransferase activity in Methanococcus maripaludis deleted for aglB, using archaellin FlaB2 as the reporter protein since all archaellins in Mc. maripaludis are modified at multiple sites by an N-linked tetrasaccharide and this modification is required for archaellation. In the Mc. maripaludis ΔaglB strain FlaB2 runs as at a smaller apparent molecular weight in western blots and is nonarchaellated. We demonstrate that AglBs from Methanococcus voltae and Methanothermococcus thermolithotrophicus functionally replaced the oligosaccharyltransferase activity missing in the Mc. maripaludis ΔaglB strain, both returning the apparent molecular weight of FlaB2 to wildtype size and restoring archaellation. This demonstrates that AglB from Mc. voltae has a relaxed specificity for the linking sugar of the transferred glycan since while the N-linked glycan present in Mc. voltae is similar to that of Mc. maripaludis, the Mc. voltae glycan uses N-acetylglucosamine as the linking sugar. In Mc. maripaludis that role is held by N-acetylgalactosamine. This study also identifies aglB from Mtc. thermolithotrophicus for the first time by its activity. Attempts to use AglB from Methanocaldococcus jannaschii, Haloferax volcanii or Sulfolobus acidocaldarius to functionally replace the oligosaccharyltransferase activity missing in the Mc. maripaludis ΔaglB strain were unsuccessful.

The rapid and complete baseline separation of both volatile (C5 to C16 alkanes in gasoline or terpenes in plant extracts) and non-volatile (>C20 alkanes) organic compounds was achieved by combining (1) low-density fluid chromatography (LDFC) using carbon dioxide at elevated temperature (>90 °C) and low pressure (1500 psi) designed to increase the retention of the most volatile compounds and (2) high-vacuum technology (

Conversion of lignin into smaller molecules provides a promising alternate and sustainable source for the valuable chemicals currently derived from crude oil. Better understanding of the chemical composition of the resulting product mixtures is essential for the optimization of such conversion processes. However, these mixtures are complex and contain isomeric molecules with a wide variety of functionalities, which makes their characterization challenging. Tandem mass spectrometry based on ion-molecule reactions has proven to be a powerful tool in functional group identification and isomer differentiation for previously unknown compounds. This study demonstrates that the identification of the phenol functionality, the most commonly observed functionality in lignin degradation products, can be achieved via ion-molecule reactions between diethylmethoxyborane (DEMB) and the deprotonated analyte in the absence of strongly electron-withdrawing substituents in the ortho- and para-positions. Either a stable DEMB adduct or an adduct that has lost a methanol molecule (DEMB adduct-MeOH) is formed for these ions. Deprotonated phenols with an adjacent phenol or hydroxymethyl functionality or a conjugated carboxylic acid functionality can be identified based on the formation of DEMB adduct-MeOH. Deprotonated compounds not containing the phenol functionality and phenols containing an electron-withdrawing ortho- or para-substituent were found to be unreactive toward diethylmethoxyborane. Hence, certain deprotonated isomeric compounds with phenol and carboxylic acid, aldehyde, carboxylic acid ester, or nitro functionalities can be differentiated via these reactions. The above mass spectrometry method was successfully coupled with high-performance liquid chromatography for the analysis of a complex biomass degradation mixture. Graphical Abstract ᅟ.

The Clean Air Act (CAA) controls routine emissions at petroleum refineries, by creating limits and penalties for excess emissions. The CAA offers provisions for upset events, air emissions released because of unforeseen or unavoidable circumstances, if companies report the emissions and take corrective action. States enforce upset event rules and many states provide exemptions for a variety of circumstances, which may allow upset emissions to become a substantial, yet mostly unregulated source of emissions. We catalog the quantity and type of emissions generated during upset events at 18 Texas petroleum refineries from 2003 to 2008. We find that upset events occur frequently at these facilities and are collectively large in magnitude, emitting a combined total of 75 million lbs of emissions. In a select number of cases, single upset events exceeded annual emissions reported to the Toxics Release Inventory. Future research should assess the accuracy of upset event reporting and impact of upset events on environmental health.

Purpose To identify metabolites and metabolic pathways altered in neovascular age-related macular degeneration (NVAMD). Methods We performed metabolomics analysis using high-resolution C18 liquid chromatography-mass spectrometry on plasma samples from 100 NVAMD patients and 192 controls. Data for mass/charge ratio ranging from 85 to 850 were captured, and metabolic features were extracted using xMSanalyzer. Nested feature selection was used to identify metabolites that discriminated between NVAMD patients and controls. Pathway analysis was performed with Mummichog 2.0. Hierarchical clustering was used to examine the relationship between the discriminating metabolites and NVAMD patients and controls. Results Of the 10,917 metabolic features analyzed, a set of 159 was identified that distinguished NVAMD patients from controls (area under the curve of 0.83). Of these features, 39 were annotated with confidence and included multiple carnitine metabolites. Pathway analysis revealed that the carnitine shuttle pathway was significantly altered in NVAMD patients (P = 0.0001). Tandem mass spectrometry confirmed the molecular identity of five carnitine shuttle pathway acylcarnitine intermediates that were increased in NVAMD patients. Hierarchical cluster analysis revealed that 51% of the NVAMD patients had similar metabolic profiles, whereas the remaining 49% displayed greater variability in their metabolic profiles. Conclusions Multiple long-chain acylcarnitines that are part of the carnitine shuttle pathway were significantly increased in NVAMD patients compared to controls, suggesting that fatty acid metabolism may be involved in NVAMD pathophysiology. Cluster analysis suggested that clinically indistinguishable NVAMD patients can be separated into distinct subgroups based on metabolic profiles.

The acute toxicities for technical grade fluridone (Sonar™) and the commercial formulation of fluridone (Sonar®AS) were assessed for male water mites (Hydrachnidiae: Arrenurus: Megaluracarus). Signs of toxicity were evaluated by detection of locomotor dysfunction or death after exposure to concentrations of 100,000, 10,000, 1,000, and 100 μg/L of Sonar™ and 10,000, 5,000, 1,000, 100, and 10 μg/L of Sonar®AS in US EPA, moderately hard reconstituted water (MHRW). The median effective concentration (EC50) was 891 and 631 μg/L for Sonar™ at 48 and 96 h and less than 10 μg/L for Sonar®AS at 96 h. Increased duration of exposure to Sonar®AS from 48 to 96 h had a significant effect on increasing the rate of combined morbidity and mortality. At the lowest concentration of Sonar®AS tested, which is half the concentration allowed within 400 m of any functioning potable water intake for human usage, 40% of the mites were adversely affected at 48 h and 70% were affected after 96 h of exposure. This study demonstrates that Sonar®AS is 60-fold more toxic to water mites than the active ingredient alone. At currently acceptable application rates of 90-150 μg/L fluridone, the addition of ingredients classified as inert, as in Sonar®AS, result in an increased risk of adverse effects on populations of male water mites (Arrenurus: Megaluracarus) in aquatic ecosystems.

Surfactants find wide commercial use as foaming agents, emulsifiers, and dispersants. Currently, surfactants are produced from petroleum, or from seed oils such as palm or coconut oil. Due to concerns with CO(2) emissions and the need to protect rainforests, there is a growing necessity to manufacture these chemicals using sustainable resources In this report, we describe the engineering of a native nonribosomal peptide synthetase pathway (i.e., surfactin synthetase), to generate a Bacillus strain that synthesizes a highly water-soluble acyl amino acid surfactant, rather than the water insoluble lipopeptide surfactin. This novel product has a lower CMC and higher water solubility than myristoyl glutamate, a commercial surfactant. This surfactant is produced by fermentation of cellulosic carbohydrate as feedstock. This method of surfactant production provides an approach to sustainable manufacturing of new surfactants.

Many archaeal proteins undergo posttranslational modifications. S-layer proteins and flagellins have been used successfully to study a variety of these modifications, including N-linked glycosylation, signal peptide removal and lipid modification. Use of these well-characterized reporter proteins in the genetically tractable model organisms,Haloferax volcanii, Methanococcus voltaeandMethanococcus maripaludis,has allowed dissection of the pathways and characterization of many of the enzymes responsible for these modifications. Such studies have identified archaeal-specific variations in signal peptidase activity not found in the other domains of life, as well as the enzymes responsible for assembly and biosynthesis of novel N-linked glycans. In vitro assays for some of these enzymes have already been developed. N-linked glycosylation is not essential for eitherHfx. volcaniior theMethanococcusspecies, an observation that allowed researchers to analyze the role played by glycosylation in the function of both S-layers and flagellins, by generating mutants possessing these reporters with only partial attached glycans or lacking glycan altogether. In future studies, it will be possible to consider questions related to the heterogeneity associated with given modifications, such as differential or modulated glycosylation.

Glycosylation is a posttranslational modification utilized in all three domains of life. Compared to eukaryotic and bacterial systems, knowledge of the archaeal processes involved in glycosylation is limited. Recently, Methanococcus voltae flagellin proteins were found to have an N-linked trisaccharide necessary for proper flagellum assembly. Current analysis by mass spectrometry of Methanococcus maripaludis flagellin proteins also indicated the attachment of an N-glycan containing acetylated sugars. To identify genes involved in sugar biosynthesis in M. maripaludis , a putative acetyltransferase was targeted for in-frame deletion. Deletion of this gene (MMP0350) resulted in a flagellin molecular mass shift to a size comparable to that expected for underglycosylated or completely nonglycoslyated flagellins, as determined by immunoblotting. Assembled flagellar filaments were not observed by electron microscopy. Interestingly, the deletion also resulted in defective pilus anchoring. Mutant cells with a deletion of MMP0350 had very few, if any, pili attached to the cell surface compared to a nonflagellated but piliated strain. However, pili were obtained from culture supernatants of this strain, indicating that the defect was not in pilus assembly but in stable attachment to the cell surface. Complementation of MMP0350 on a plasmid restored pilus attachment, but it was unable to restore flagellation, likely because the mutant ceased to make detectable flagellin. These findings represent the first report of a biosynthetic gene involved in flagellin glycosylation in archaea. Also, it is the first gene to be associated with pili, linking flagellum and pilus structure and assembly through posttranslational modifications.

Flagellins from Clostridium botulinum were shown to be post-translationally modified with novel glycan moieties by top-down MS analysis of purified flagellin protein from strains of various toxin serotypes. Detailed analyses of flagellin from two strains of C. botulinum demonstrated that the protein is modified by a novel glycan moiety of mass 417 Da in O-linkage. Bioinformatic analysis of available C. botulinum genomes identified a flagellar glycosylation island containing homologs of genes recently identified in Campylobacter coli that have been shown to be responsible for the biosynthesis of legionaminic acid derivatives. Structural characterization of the carbohydrate moiety was completed utilizing both MS and NMR spectroscopy, and it was shown to be a novel legionaminic acid derivative, 7-acetamido-5-(N-methyl-glutam-4-yl)-amino-3,5,7,9-tetradeoxy-D-glycero-alpha-D-galacto-nonulosonic acid, (alphaLeg5GluNMe7Ac). Electron transfer dissociation MS with and without collision-activated dissociation was utilized to map seven sites of O-linked glycosylation, eliminating the need for chemical derivatization of tryptic peptides prior to analysis. Marker ions for novel glycans, as well as a unique C-terminal flagellin peptide marker ion, were identified in a top-down analysis of the intact protein. These ions have the potential for use in for rapid detection and discrimination of C. botulinum cells, indicating botulinum neurotoxin contamination. This is the first report of glycosylation of Gram-positive flagellar proteins by the 'sialic acid-like' nonulosonate sugar, legionaminic acid.

Post-translational modifications account for much of the biological diversity generated at the proteome level. Of these, glycosylation is the most prevalent. Long thought to be unique to Eukarya, it is now clear that both Bacteria and Archaea are also capable of N-glycosylation, namely the covalent linkage of oligosaccharides to select target asparagine residues. However, while the eukaryal and bacterial N-glycosylation pathways are relatively well defined, little is known of the parallel process in Archaea. Of late, however, major advances have been made in describing the process of archaeal N-glycosylation. Such efforts have shown, as is often the case in archaeal biology, that protein N-glycosylation in Archaea combines particular aspects of the eukaryal and bacterial pathways along with traits unique to this life form. For instance, while the oligosaccharides of archaeal glycoproteins include nucleotide-activated sugars formed by bacterial pathways, the lipid carrier on which such oligosaccharides are assembled is the same as used in eukaryal N-glycosylation. By contrast, transfer of assembled oligosaccharides to their protein targets shows Archaea-specific properties. Finally, addressing N-glycosylation from an archaeal perspective is providing new general insight into this event, as exemplified by the solution of the first crystal structure of an oligosaccharide transferase from an archaeal source.