LC3-associated phagocytosis - The highway to hell for phagocytosed microbes.

Phagocytes ingest, kill and degrade invading microbes in a process called phagocytosis. LC3-associated phagocytosis (LAP) combines the molecular machinery of phagocytosis with that of autophagy, the cellular pathway for ingestion of cytoplasmic components, resulting in the eponymous association of 'microtubule-associated proteins 1 A/1B light chain 3' (LC3) with the phagosomal membrane. The LC3-decorated phagosomes, or LAPosomes, show enhanced fusion with lysosomes resulting in enhanced killing and degradation of contained pathogens. Thus, LAP is a particularly microbicidal pathway. In this review, we discuss the molecular mechanisms involved in induction and execution of LAP and its crucial role in antimicrobial immunity against bacteria, fungi and parasites. As LAP has only recently been defined, we also point out the key open questions that remain to be answered.

Acid sphingomyelinase is a key regulator of cytotoxic granule secretion by primary T lymphocytes.

Granule-mediated cytotoxicity is the main effector mechanism of cytotoxic CD8+ T cells. We report that CD8+ T cells from acid sphingomyelinase (ASMase)-deficient (ASMase-KO) mice are defective in exocytosis of cytolytic effector molecules; this defect resulted in attenuated cytotoxic activity of ASMase-KO CD8+ T cells and delayed elimination of lymphocytic choriomeningitis virus from ASMase-KO mice. Cytolytic granules of ASMase-KO and wild-type CD8+ T cells were equally loaded with granzymes and perforin, and correctly directed to the immunological synapse. In wild-type CD8+ T cells, secretory granules underwent shrinkage by 82% after fusion with the plasma membrane. In ASMase-KO CD8+ T cells, the contraction of secretory granules was markedly impaired. Thus, ASMase is required for contraction of secretory granules and expulsion of cytotoxic effector molecules.

Au-Cavitands: Size governed arene-alkyne cycloisomerization.

With an inwardly directed reactive center and a well-defined binding pocket, Au(I) functionalized resorcin[4]arene cavitands have been shown to catalyze molecular transformations. The reactivity profiles that emerge differ from other Au(I) catalysts. The added constraint of a binding pocket gives rise to the possibility that the substrates might have to fit into the resorcinarene pocket; our hypothesis is that substrates that match the available space have different reaction outcomes than those that do not. Herein we report on the intramolecular cyclization of alkyne-aromatic substrates with variable alkynes and aromatic composition. We see that scaffold size most drastically dictates reactivity, especially when the substrate's features are particularly designed. The results of these experiments add to the veritable goldmine of information about the selectivity in catalysis that cavitands offer.

Autophagy determines efficiency of liver-directed gene therapy with adeno-associated viral vectors.

Use of adeno-associated viral (AAV) vectors for liver-directed gene therapy has shown considerable success, particularly in patients with severe hemophilia B. However, the high vector doses required to reach therapeutic levels of transgene expression caused liver inflammation in some patients that selectively destroyed transduced hepatocytes. We hypothesized that such detrimental immune responses can be avoided by enhancing the efficacy of AAV vectors in hepatocytes. Because autophagy is a key liver response to environmental stresses, we characterized the impact of hepatic autophagy on AAV infection. We found that AAV induced mammalian target of rapamycin (mTOR)-dependent autophagy in human hepatocytes. This cell response was critically required for efficient transduction because under conditions of impaired autophagy (pharmacological inhibition, small interfering RNA knockdown of autophagic proteins, or suppression by food intake), recombinant AAV-mediated transgene expression was markedly reduced, both in vitro and in vivo. Taking advantage of this dependence, we employed pharmacological inducers of autophagy to increase the level of autophagy. This resulted in greatly improved transduction efficiency of AAV vectors in human and mouse hepatocytes independent of the transgene, driving promoter, or AAV serotype and was subsequently confirmed in vivo. Specifically, short-term treatment with a single dose of torin 1 significantly increased vector-mediated hepatic expression of erythropoietin in C57BL/6 mice. Similarly, coadministration of rapamycin with AAV vectors resulted in markedly enhanced expression of human acid-α-glucosidase in nonhuman primates. CONCLUSION: We identified autophagy as a pivotal cell response determining the efficiency of AAVs intracellular processing in hepatocytes and thus the outcome of liver-directed gene therapy using AAV vectors and showed in a proof-of-principle study how this virus-host interaction can be employed to enhance efficacy of this vector system. (Hepatology 2017;66:252-265).

Preparation of Diverse BODIPY Diesters.

The synthesis and properties of a variety of substituted BODIPY diesters is presented. We find that certain substitution patterns afford appreciable yields of the target compounds and that electronic effects result in predicable differential fluorescent behavior. Challenges to further water solubilize these dyes and/or provide new points of attachment for biological tagging remain, these strategies are discussed.

Calixarene-mediated liquid membrane transport of choline conjugates 3: The effect of handle variation on neurotransmitter transport.

Upper rim phosphonic acid functionalized calix[4]arene affects selective transport of multiple molecular payloads through a liquid membrane. The secret is in the attachment of a receptor-complementary handle to the payload. We find that the trimethylammonium ethylene group present in choline is one of several general handles for the transport of drug and drug-like species. Herein we compare the effect of handle variation against the transport of serotonin and dopamine. We find that several ionizable amine termini handles are sufficient for transport and identify two ideal candidates. Their performance is significantly enhanced in HEPES buffered solutions. This inquiry completes a series of 3 studies aimed at optimization of this strategy. In completion a new approach towards synthetic receptor mediated selective small molecule transport has emerged; future work in vesicular and cellular systems will follow.

Oxidatively Locked [Co2L3]6+ Cylinders Derived from Bis(bidentate) 2-Pyridyl-1,2,3-triazole "Click" Ligands: Synthesis, Stability, and Antimicrobial Studies.

A small family of [Co2(Lpytrz)3]6+ cylinders was synthesised from bis(bidentate) 2-pyridyl-1,2,3-triazole "click" ligands (Lpytrz) through an "assembly-followed-by-oxidation" method. The cylinders were characterised using ¹H, 13C, and DOSY NMR, IR, and UV-Vis spectroscopies, along with electrospray ionisation mass spectrometry (ESMS). Stability studies were conducted in dimethyl sulfoxide (DMSO) and D2O. In contrast to similar, previously studied, [Fe2(Lpytrz)3]4+ helicates the more kinetically inert [Co2(Lpytrz)3]6+ systems proved stable (over a period of days) when exposed to DMSO and were even more stable in D2O. The triply stranded [Co2(Lpytrz)3]6+ systems and the corresponding "free" ligands were tested for antimicrobial activity in vitro against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) microorganisms. Agar-based disk diffusion and Mueller-Hinton broth micro-dilution assays showed that the [Co2(Lpytrz)3]6+ cylinders were not active against either strain of bacteria. It is presumed that a high charge of the [Co2(Lpytrz)3]6+ cylinders is preventing them from crossing the bacterial cell membranes, rendering the compounds biologically inactive.

Riboflavin (vitamin B2 ) deficiency impairs NADPH oxidase 2 (Nox2) priming and defense against Listeria monocytogenes.

Riboflavin, also known as vitamin B2 , is converted by riboflavin kinase (RFK) into flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are essential cofactors of dehydrogenases, reductases, and oxidases including the phagocytic NADPH oxidase 2 (Nox2). Riboflavin deficiency is common in young adults and elderly individuals, who are at the coincidental risk for listeriosis. To address the impact of acute riboflavin deficiency on host defense against Listeria monocytogenes (L.m.), we generated conditional RFK knockout (KO) strains of mice. Phagocyte-specific RFK KO impaired the capability of phagocytes to control intracellular L.m., which corresponded to a greater susceptibility of mice to in vivo challenge with L.m. The oxidative burst of RFK-deficient phagocytes in response to L.m. infection was significantly reduced. Mechanistically, TNF-induced priming of Nox2, which is needed for oxidative burst, was defective in RFK-deficient phagocytes. Lack of riboflavin in wild-type macrophages for only 6 h shut down TNF-induced, RFK-mediated de novo FMN/FAD generation, which was accompanied by diminished ROS production and impaired anti-listerial activity. Vice versa, ROS production by riboflavin-deprived macrophages was rapidly restored by riboflavin supplementation. Our results suggest that acute riboflavin deficiency immediately impairs priming of Nox2, which is of crucial relevance for an effective phagocytic immune response in vivo.

Irgm1 (LRG-47), a regulator of cell-autonomous immunity, does not localize to mycobacterial or listerial phagosomes in IFN-γ-induced mouse cells.

The IFN-inducible protein Irgm1 (LRG-47) belongs to the family of immunity-related GTPases that function in cell-autonomous resistance against intracellular pathogens in mice. Irgm1 deficiency is associated with a severe immunodeficiency syndrome. The protein has been variously interpreted as a direct effector molecule on bacterial phagosomes or on other organelles or as an inducer of autophagy. In this study, we re-examined one of these claims, namely that Irgm1 targets mycobacterial and listerial phagosomes. We found no colocalization of endogenous Irgm1, using two immunofluorescent staining techniques, either in fibroblasts or in macrophages. We demonstrated the predicted existence of two protein isoforms of Irgm1 derived from differential splicing and described immunological reagents for their detection. Both Irgm1 isoforms localize to the Golgi apparatus and weakly to mitochondria; however, only the long Irgm1 isoforms can be detected on endolysosomal membranes. Together with the previous observation that the general immunodeficiency phenotype of Irgm1(-/-) mice is reversed in Irgm1/Irgm3 double-deficient mice, our results argue against a direct effector function of Irgm1 at the bacterial phagosome. We discuss these findings in the context of evidence that Irgm1 functions as a negative regulator of other members of the immunity-related GTPase protein family.

Riboflavin kinase couples TNF receptor 1 to NADPH oxidase.

Reactive oxygen species (ROS) produced by NADPH oxidase function as defence and signalling molecules related to innate immunity and various cellular responses. The activation of NADPH oxidase in response to plasma membrane receptor activation depends on the phosphorylation of cytoplasmic oxidase subunits, their translocation to membranes and the assembly of all NADPH oxidase components. Tumour necrosis factor (TNF) is a prominent stimulus of ROS production, but the molecular mechanisms by which TNF activates NADPH oxidase are poorly understood. Here we identify riboflavin kinase (RFK, formerly known as flavokinase) as a previously unrecognized TNF-receptor-1 (TNFR1)-binding protein that physically and functionally couples TNFR1 to NADPH oxidase. In mouse and human cells, RFK binds to both the TNFR1-death domain and to p22(phox), the common subunit of NADPH oxidase isoforms. RFK-mediated bridging of TNFR1 and p22(phox) is a prerequisite for TNF-induced but not for Toll-like-receptor-induced ROS production. Exogenous flavin mononucleotide or FAD was able to substitute fully for TNF stimulation of NADPH oxidase in RFK-deficient cells. RFK is rate-limiting in the synthesis of FAD, an essential prosthetic group of NADPH oxidase. The results suggest that TNF, through the activation of RFK, enhances the incorporation of FAD in NADPH oxidase enzymes, a critical step for the assembly and activation of NADPH oxidase.

Calixarene-Mediated Liquid Membrane Transport of Choline Conjugates 2: Transport of Drug-Choline Conjugates and Neurotransmitters.

Lower rim carboxylic acid calix[n]arenes and upper rim phosphonic acid functionalized calix[4]arenes effect selective transport of distinct molecular payloads through a liquid membrane. The secret to this success lies in the attachment of a receptor-complementary handle. We find that the trimethylammonium ethylene group present in choline is a general handle for the transport of drug and drug-like species. Furthermore, neurotransmitters possessing ionizable amine termini are also transported. Some limitations to this strategy have been uncovered as payloads become increasingly lipophilic. These developments reveal new approaches to synthetic receptor-mediated selective small molecule transport in vesicular and cellular systems.

Pb, Sr and Ba calix[6]arene hexacarboxylic acid octahedral complexation: a dramatic effect of dealkylation.

Calix[6]arene hexacarboxylic acid binds instantly and with low symmetry to Pb, Sr and Ba. Later a highly symmetric up-down alternating conformation emerges. The solution structures are identical to their p-tert-butylcalix[6]arene hexacarboxylic acid counterparts. With either receptor an octahedral cage is formed around the metal. The transformation from low to high symmetry however proceeds at significantly faster rates for the de-t-butylated host.

Calixarene-Mediated Liquid-Membrane Transport of Choline Conjugates.

A series of supramolecular calixarenes efficiently transport distinct molecular species through a liquid membrane when attached to a receptor-complementary choline handle. Calix-[6]arene hexacarboxylic acid was highly effective at transporting different target molecules against a pH gradient. Both carboxylic- and phosphonic-acid-functionalized calix[4]arenes effect transport without requiring a pH or ion gradient. NMR binding studies, two-phase solvent extraction, and three-phase transport experiments reveal the necessary and subtle parameters to effect the transport of molecules attached to a choline "handle". On the other hand, rescorin[4]arene cavitands, which have similar guest recognition profiles, did not transport guest molecules. These developments reveal new approaches towards attempting synthetic-receptor-mediated selective small-molecule transport in vesicular and cellular systems.

Electricity consumption of anesthesia workstations and potential emission savings by avoiding standby.

BACKGROUND: Anesthesiology has a relevant carbon footprint, mainly due to volatile anesthetics (scope 1 emissions). Additionally, energy used in the operating theater (scope 2 emissions) contributes to anesthesia-related greenhouse gas (GHG) emissions. OBJECTIVES: Optimizing the electricity use of medical devices might reduce both GHG emissions and costs might hold potential to reduce anaesthesia-related GHG-emissions and costs. We analyzed the electricity consumption of six different anesthesia workstations, calculated their GHG emissions and electricity costs and investigated the potential to reduce emissions and cost by using the devices in a more efficient way. METHODS: Power consumption (active power in watt , W) was measured with the devices off, in standby mode, or fully on with the measuring instrument SecuLife ST. Devices studied were: Dräger Primus, Löwenstein Medical LeonPlus, Getinge Flow C, Getinge Flow E, GE Carestation 750 and GE Aisys. Calculations of GHG emissions were made with different emission factors, ranging from very low (0.09 kg CO2-equivalent/kWh) to very high (0.660 kg CO2-equivalent/kWh). Calculations of electricity cost were made assuming a price of 0.25 € per kWh. RESULTS: Power consumption during operation varied from 58 W (GE CareStation 750) to 136 W (Dräger Primus). In standby, the devices consumed between 88% and 93% of the electricity needed during use. The annual electricity consumption to run 96 devices in a large clinical department ranges between 45 and 105 Megawatt-hours (MWh) when the devices are left in standby during off hours. If 80% of the devices are switched off during off hours, between 20 and 46 MWh can be saved per year in a single institution. At the average emission factor of our hospital, this electricity saving corresponds to a reduction of GHG emissions between 8.5 and 19.8 tons CO2-equivalent. At the assumed prices, a cost reduction between 5000 € and 11,600 € could be achieved by this intervention. CONCLUSION: The power consumption varies considerably between the different types of anesthesia workstations. All devices exhibit a high electricity consumption in standby mode. Avoiding standby mode during off hours can save energy and thus GHG emissions and cost. The reductions in GHG emissions and electricity cost that can be achieved with this intervention in a large anesthesiology department are modest. Compared with GHG emissions generated by volatile anesthetics, particularly desflurane, optimization of electricity consumption of anesthesia workstations holds a much smaller potential to reduce the carbon footprint of anesthesia; however, as switching off anesthesia workstations overnight is relatively effortless, this behavioral change should be encouraged from both an ecological and economical point of view.

Selective cavitand-mediated endocytosis of targeted imaging agents into live cells.

A water-soluble synthetic receptor molecule is capable of selective, controlled endocytosis of a specifically tagged target molecule in different types of living human cells. The presence of suitable choline-derived binding handles is essential for the molecular recognition and transport process, allowing selective guest transport and imaging of cancer cells.

Dragon PolyA Spotter: predictor of poly(A) motifs within human genomic DNA sequences.

MOTIVATION: Recognition of poly(A) signals in mRNA is relatively straightforward due to the presence of easily recognizable polyadenylic acid tail. However, the task of identifying poly(A) motifs in the primary genomic DNA sequence that correspond to poly(A) signals in mRNA is a far more challenging problem. Recognition of poly(A) signals is important for better gene annotation and understanding of the gene regulation mechanisms. In this work, we present one such poly(A) motif prediction method based on properties of human genomic DNA sequence surrounding a poly(A) motif. These properties include thermodynamic, physico-chemical and statistical characteristics. For predictions, we developed Artificial Neural Network and Random Forest models. These models are trained to recognize 12 most common poly(A) motifs in human DNA. Our predictors are available as a free web-based tool accessible at http://cbrc.kaust.edu.sa/dps. Compared with other reported predictors, our models achieve higher sensitivity and specificity and furthermore provide a consistent level of accuracy for 12 poly(A) motif variants. CONTACT: vladimir.bajic@kaust.edu.sa SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Linking watershed nutrient loading to estuary water quality with generalized additive models.

Evaluating estuary water quality responses to reductions (or increases) in nutrient loading attributed to on the ground management actions can be challenging due to the strong influence of environmental drivers on nutrient loads and non-linear relationships. This study applied generalized additive models to calculate watershed nutrient loads and assess responses in estuary water quality to seasonally-adjusted freshwater inflow and flow-adjusted nutrient loads in Lavaca Bay, Texas. Lavaca Bay is a secondary embayment on the Texas coast displaying early potential for eutrophication and water quality degradation. Use of flow-adjusted nutrient loads allowed the study to evaluate the response in water quality to changes in nutrient loads driven by anthropogenic sources. Cross-validation indicated that, despite data constraints, semiparametric models performed well at nutrient load prediction. Based on these models, delivered annual nutrient loads varied substantially from year to year. In contrast, minimal changes in flow-normalized loads indicate that nutrient loadings were driven by natural variation in precipitation and runoff as opposed to changes in management of nonpoint sources. Models indicated no evidence of long-term changes in dissolved oxygen or chlorophyll-a within Lavaca Bay. However, site specific long-term increases in both organic and inorganic nitrogen are concerning for their potential to fuel eutrophication. Further analysis found freshwater inflow had strong influences on nutrient and chlorophyll-a concentrations but there was no evidence that changes in watershed nutrient loading explained additional variation in dissolved oxygen and limited evidence that watershed nutrient loadings explained chlorophyll-a concentrations. In addition to providing a baseline assessment of watershed nutrient loading and water quality responses in the Lavaca Bay watershed, this study provides methodological support for the use of semiparametric models in load regression models and estuary assessments.

Let's talk about PFAS: Inconsistent public awareness about PFAS and its sources in the United States.

The presence of per- and polyfluoroalkyl substances (PFAS) in U.S. drinking water has recently garnered significant attention from the media, federal government, and public health professionals. While concerns for PFAS exposure continue to mount, the general public's awareness and knowledge of the contaminant has remained unknown. This exploratory study sought to fill this data gap by administering a nationwide survey in which the awareness of PFAS and community contamination, awareness of PFAS containing products and intentions to change product use, and awareness and concern about PFAS in drinking water were assessed. The results indicated that almost half the respondents had never heard of PFAS and do not know what it is (45.1%). Additionally, 31.6% responded that they had heard of PFAS but do not know what it is. A large portion of respondents (97.4%) also responded that they did not believe their drinking water had been impacted by PFAS. Demographic association did not influence knowledge of PFAS or levels of concern with PFAS in drinking water. The strongest predictor of PFAS awareness was awareness due to known community exposure. The respondents aware of community exposure were more likely to have knowledge of PFAS sources, change their use of items with potential PFAS contamination, and answer that their drinking water sources were also contaminated with PFAS. Based on the received responses, PFAS information and health risks need to be better communicated to the public to help increase awareness. These efforts should also be coordinated between government agencies, utilities, the research community, and other responsible entities to bolster their effectiveness.

Synthesis and Host-Guest Chemistry of Chiral Spirobifluorene-Based Macrocycles Soluble in Basic Aqueous Solution.

Synthesis and host-guest chemistry of water-soluble (pH 12.5) chiral spirobifluorene-based macrocycles 2-[n] were carried out. Cationic guests, such as quaternary ammonium salts, were accommodated well in the hosts. Cp2Co+ was especially strongly bound in 2-[4] (Ka of up to 3.0 × 105 M-1). Enantioselective recognition with (l)-carnitine was also achieved.

Strategies for responding to RAC requests electronically.

Providers that would like to respond to complex RAC reviews electronically should consider three strategies: Invest in an EHR software package or a high-powered scanner that can quickly scan large amounts of paper. Implement an audit software platform that will allow providers to manage the entire audit process in one place. Use a CONNECT-compatible gateway capable of accessing the Nationwide Health Information Network (the network on which the electronic submission of medical documentation program runs).