Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation.

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe N-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)-linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-acetylgalactosamine-4-epimerase (GALE) like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan-specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.

Daily emotional well-being during the COVID-19 pandemic.

The COVID-19 outbreak has become one of the largest public health crises of our time. Governments have responded by implementing self-isolation and physical distancing measures that have profoundly impacted daily life throughout the world. In this study, we aimed to investigate how people experience the activities, interactions, and settings of their lives during the pandemic. The sample (N = 604) was assessed in Ireland on the 25 March 2020, following the closure of schools and non-essential businesses. We examined within-person variance in emotional well-being and how people spend their time. We found that while most time was spent in the home (74%), time spent outdoors (8%) was associated with markedly raised positive affect and reduced negative emotions. Exercising, going for walks, gardening, pursuing hobbies, and taking care of children were the activities associated with the greatest affective benefits. Home-schooling children and obtaining information about COVID-19 were ranked lowest of all activities in terms of emotional experience. These findings highlight activities that may play a protective role in relation to well-being during the pandemic, the importance of setting limits for exposure to COVID-19-related media coverage, and the need for greater educational supports to facilitate home-schooling during this challenging period.

Why is active travel more satisfying than motorized travel? Evidence from Dublin.

Some trips are better than others, and more and more studies find that active travel (walking and cycling) is more satisfying than motorized forms of travel (using the car or public transport). Why is this the case? Using data on travel satisfaction from 4134 commutes to a large University campus in Dublin, Ireland, this paper replicates the differences in travel satisfaction between active and motorized travel. We attribute these differences in large part to the duration of the trip. Subjective trip characteristics, such as safety and convenience, also play important roles. The trip duration explains rush-hour effects as well as why people starting from less affluent and more difficult-to-reach places are less satisfied with their trips. Longer-term policy options suggested by these results include infrastructure developments and spatial development strategies. A shorter-term initiative would be to delay university schedules in the morning to avoid low travel satisfaction during the slow rush-hour period and simultaneously ease pressure on the transport network at peak times.

What is wrong with quantitative structure-property relations models based on three-dimensional descriptors?

Quantitative structure-property relations (QSPR) employing descriptors derived from the three-dimensional (3D) molecular structure are frequently applied for property prediction in various fields of research. However, there is no common understanding of the necessary degree of detail to which molecular structure has to be known for reliable descriptor evaluation, but computational methods used vary from simplified molecular mechanics up to rigorous ab initio programs. In order to quantify the yet unknown error due to this heterogeneity, widely used 3D molecular descriptors from diverse fields of application are evaluated for molecular structures computed by different computational methods. The results clearly indicate that the widespread, exclusive use of the most stable molecular conformation as well as too simplistic computational methods yield systematically erroneous descriptor values with misleading information for the inferred structure-property relations. Thus, generating an awareness and understanding of this fundamental problem is considered an important first step to make 3D QSPR a generally accepted property prediction method.

Reaction of the i-C4H5 (CH2CCHCH2) radical with O2.

The resonantly stabilized radical i-C(4)H(5) (CH(2)CCHCH(2)) is an important intermediate in the combustion of unsaturated hydrocarbons and is thought to be involved in the formation of polycyclic aromatic hydrocarbons through its reaction with acetylene (C(2)H(2)) to form benzene + H. This study uses quantum chemistry and statistical reaction rate theory to investigate the mechanism and kinetics of the i-C(4)H(5) + O(2) reaction as a function of temperature and pressure, and unlike most resonantly stabilized radicals we show that i-C(4)H(5) is consumed relatively rapidly by its reaction with molecular oxygen. O(2) addition occurs at the vinylic and allenic radical sites in i-C(4)H(5), with respective barriers of 0.9 and 4.9 kcal mol(-1). Addition to the allenic radical form produces an allenemethylperoxy radical adduct with only around 20 kcal mol(-1) excess vibrational energy. This adduct can isomerize to the ca. 14 kcal mol(-1) more stable 1,3-divinyl-2-peroxy radical via concerted and stepwise processes, both steps with barriers around 10 kcal mol(-1) below the entrance channel energy. Addition of O(2) to the vinylic radical site in i-C(4)H(5) directly forms the 1,3-divinyl-2-peroxy radical with a small barrier and around 36.8 kcal mol(-1) of excess energy. The 1,3-divinyl-2-peroxy radical isomerizes via ipso addition of the O(2) moiety followed by O atom insertion into the adjacent C-C bond. This process forms an unstable intermediate that ultimately dissociates to give the vinyl radical, formaldehyde, and CO. At higher temperatures formation of vinylacetylene + HO(2), the vinoxyl radical + ketene, and the 1,3-divinyl-2-oxyl radical + O paths have some importance. Because of the adiabatic transition states for O(2) addition, and significant reverse dissociation channels in the peroxy radical adducts, the i-C(4)H(5) + O(2) reaction proceeds to new products with rate constant of around 10(11) cm(3) mol(-1) s(-1) at typical combustion temperatures (1000-2000 K). For fuel-rich flames we show that the reaction of i-C(4)H(5) with O(2) is likely to be faster than that with C(2)H(2), bringing into question the importance of the i-C(4)H(5) + C(2)H(2) reaction in initiating ring formation in sooting flames.

Preparation and pre-vivo evaluation of no-carrier-added, carrier-added and cross-complexed [(68)Ga]-EDTMP formulations.

PURPOSE: The present study aimed to develop convenient preparation and quality control protocols for [(68)Ga]-EDTMP, a potential radiotracer for skeletal PET imaging. Furthermore, bone binding characteristics with special focus on the influence of carrier addition were evaluated. METHODS: No-carrier-added (nca), carrier-added and novel cross-complexed [(68)Ga]-EDTMP formulations were prepared using [(68)Ga]-gallium chloride and a commercial EDTMP kit. Respective bone binding characteristics were determined on the basis of an established in-vitro method using hydroxyapatite and human bone powders as binding matrices. RESULTS: Pre-vivo evaluation of nca [(68)Ga]-EDTMP yielded irreversible binding on the mineral bone phase characterised by fast binding kinetics. Generally, nca [(68)Ga]-EDTMP showed low uptake values comparable to nca [(99m)Tc]-EDTMP. Interestingly, the bone binding affinity of [(68)Ga]-EDTMP could be increased by the addition of carriers, presumably by changing the complex structure. CONCLUSIONS: This fast and reliable preparation protocol could enable small PET facilities without onsite cyclotron to perform PET bone scans. A comparison of all cross-complexed [(68)Ga]-EDTMP preparations further strengthens the recently presented "foreign carrier theory", which highlights carrier addition as a factor strongly affecting bone uptake of radiolabelled polyphosphonates. The clinical applicability of [(68)Ga]-EDTMP - particularly with respect to lesion specificity and sensitivity - should be clarified in forthcoming in-vivo studies.

Three-dimensional lattice Monte Carlo simulations of model proteins. IV. Proteins at an oil-water interface.

Lattice Monte Carlo simulations describe the adsorption of protein-like heteropolymer chains at an oil/water interface. The heteropolymers are designed sequences of 27 and 64 amino acid-type lattice sites taken from a 20-letter alphabet. We use our recently suggested energy scale to model oil and water. We investigate the effect of the oil parameters on adsorption properties of a single chain and on the aggregation of adsorbed chains while keeping the water parameters fixed to their optimum values found previously. By varying the oil parameters, we can cause a large range of adsorption behavior: from no adsorption to reversible adsorption to irreversible adsorption. We compare adsorption at a liquid/solid interface to that at a liquid/liquid interface. A liquid interface leads to stronger adsorption and denaturation than a solid interface with the same water and oil interaction parameters. We propose "optimal" oil parameters and use them to study multichain adsorption at a liquid interface.

3D-Lattice Monte Carlo simulations of model proteins. Size effects on folding thermodynamics and kinetics.

Recently, we devised an energy scale to vary systematically amino-acid residue-solvent interactions for Monte Carlo simulations of lattice-model proteins in water. For 27-mer proteins, the folding behavior varies appreciably with the choice of interaction parameters. We now perform similar simulations with 64-mers to study the size dependence of the optimal energy parameter set for representing realistic behavior typical of many real proteins (i.e. fast folding and high cooperativity for single chains). We find that 64-mers are considerably more stable and more cooperative compared to 27-mers. The optimal interfacial-interaction-energy parameter set, however, is relatively size independent.

Treatment of landfill leachate--high tech or low tech? A case study.

At the sanitary landfill of the city of Penzberg (Germany), two diverse approaches to leachate treatment were studied as parts of a three-stage treatment concept. The performance of a simple aerobic pond was compared to that of an advanced multistage treatment unit, the latter comprising a membrane biological reactor and a two-stage activated carbon filter. For 274 days of the year (75%) the pond was able to provide sufficient treatment even under cold weather conditions. For temperatures lower than 5 degrees C, a higher biomass content and temporal storage of the raw leachate (e.g. increasing hydraulic retention time) could close the gap of insufficient treatment. In contrast, the advanced treatment system could only accomplish limited treatment capabilities due to insufficient maintenance, low loading conditions and deficient coordination between the individual treatment steps. As a result, degradation rates were low and operational problems frequent. Limits for Ntot were exceeded regularly (Ntot,e = 60-70 mg/L), throughput broke down and excessive nitrite production occurred (NO2-Ne = 10 mg/L) as a result of microbial activity inside the activated carbon filters. This case study clearly suggests aerobic ponds as an appropriate solution for the treatment of landfill leachate in areas where operational independence is essential.

AAA proteases of mitochondria: quality control of membrane proteins and regulatory functions during mitochondrial biogenesis.

An ubiquitous and conserved proteolytic system regulates the stability of mitochondrial inner membrane proteins. Two AAA proteases with catalytic sites at opposite membrane surfaces form a membrane-integrated quality control system and exert crucial functions during the biogenesis of mitochondria. Their activity is modulated by another membrane-protein complex that is composed of prohibitins. Peptides generated upon proteolysis in the matrix space are transported across the inner membrane by an ATP-binding cassette transporter. The function of these conserved components is discussed in the present review.

Mba1, a novel component of the mitochondrial protein export machinery of the yeast Saccharomyces cerevisiae.

The biogenesis of mitochondria requires the integration of many proteins into the inner membrane from the matrix side. The inner membrane protein Oxa1 plays an important role in this process. We identified Mba1 as a second mitochondrial component that is required for efficient protein insertion. Like Oxa1, Mba1 specifically interacts both with mitochondrial translation products and with conservatively sorted, nuclear-encoded proteins during their integration into the inner membrane. Oxa1 and Mba1 overlap in function and substrate specificity, but both can act independently of each other. We conclude that Mba1 is part of the mitochondrial protein export machinery and represents the first component of a novel Oxa1-independent insertion pathway into the mitochondrial inner membrane.

Role of the ABC transporter Mdl1 in peptide export from mitochondria.

ATP-binding cassette (ABC) adenosine triphosphatases actively transport a wide variety of compounds across biological membranes. Here, the ABC protein Mdl1 was identified as an intracellular peptide transporter localized in the inner membrane of yeast mitochondria. Mdl1 was required for mitochondrial export of peptides with molecular masses of approximately 2100 to 600 daltons generated by proteolysis of inner-membrane proteins by the m-AAA protease in the mitochondrial matrix. Proteolysis by the i-AAA protease in the intermembrane space led to the release of similar-sized peptides independent of Mdl1. Thus, two pathways of peptide efflux from mitochondria exist that may allow communication between mitochondria and their cellular environment.

Membrane protein degradation by AAA proteases in mitochondria: extraction of substrates from either membrane surface.

Two AAA proteases, each with its catalytic site at the opposite membrane surface, mediate the ATP-dependent degradation of mitochondrial inner membrane proteins. We demonstrate here that a model substrate polypeptide containing hydrophilic domains at both sides of the membrane can be completely degraded by either of the AAA proteases, if solvent-exposed domains are in an unfolded state. A short protein tail protruding from the membrane surface is sufficient to allow the proteolytic attack of an AAA protease that facilitates domain unfolding at the opposite side. Our results provide a rationale for the membrane arrangement of AAA proteases in mitochondria and demonstrate that degradation of membrane proteins by AAA proteases involves an active extraction of transmembrane segments and transport of solvent-exposed domains across the membrane.

Chaperone-like activity of the AAA domain of the yeast Yme1 AAA protease.

The AAA domain, a conserved Walker-type ATPase module, is a feature of members of the AAA family of proteins, which are involved in many cellular processes, including vesicular transport, organelle biogenesis, microtubule rearrangement and protein degradation. The function of the AAA domain, however, has not been explained. Membrane-anchored AAA proteases of prokaryotic and eukaryotic cells comprise a subfamily of AAA proteins that have metal-dependent peptidase activity and mediate the degradation of non-assembled membrane proteins. Inactivation of an orthologue of this protease family in humans causes neurodegeneration in hereditary spastic paraplegia. Here we investigate the AAA domain of the yeast protein Yme1, a subunit of the iota-AAA protease located in the inner membrane of mitochondria. We show that Yme1 senses the folding state of solvent-exposed domains and specifically degrades unfolded membrane proteins. Substrate recognition and binding are mediated by the amino-terminal region of the AAA domain. The purified AAA domain of Yme1 binds unfolded polypeptides and suppresses their aggregation. Our results indicate that the AAA domain of Ymel has a chaperone-like activity and suggest that the AAA domains of other AAA proteins may have a similar function.

AAA proteases with catalytic sites on opposite membrane surfaces comprise a proteolytic system for the ATP-dependent degradation of inner membrane proteins in mitochondria.

The mechanism of selective protein degradation of membrane proteins in mitochondria has been studied employing a model protein that is subject to rapid proteolysis within the inner membrane. Protein degradation was mediated by two different proteases: (i) the m-AAA protease, a protease complex consisting of multiple copies of the ATP-dependent metallopeptidases Yta1Op (Afg3p) and Yta12p (Rcalp); and (ii) by Ymelp (Ytallp) that also is embedded in the inner membrane. Ymelp, highly homologous to Yta1Op and Yta12p, forms a complex of approximately 850 kDa in the inner membrane and exerts ATP-dependent metallopeptidase activity. While the m-AAA protease exposes catalytic sites to the mitochondrial matrix, Ymelp is active in the intermembrane space. The Ymelp complex was therefore termed 'i-AAA protease'. Analysis of the proteolytic fragments indicated cleavage of the model polypeptide at the inner and outer membrane surface and within the membrane-spanning domain. Thus, two AAA proteases with their catalytic sites on opposite membrane surfaces constitute a novel proteolytic system for the degradation of membrane proteins in mitochondria.

Efficacy of hepatitis B screening in a private obstetrical population.

We sought to determine whether the recent Centers for Disease Control recommendation of universal prenatal screening for hepatitis B surface antigen (HBsAg) is necessary or cost-effective in a population of private patients. During the 21 months of our study there were 17,973 deliveries at Magee-Womens Hospital, the largest-volume private obstetrics service in the United States. We screened 12,377 of these patients for HBsAg. Only 11 patients, 0.09% of those screened (5 private and 6 clinic) tested positive. We administered questionnaires regarding historical risk factors for hepatitis B to all 11 patients testing positive for HBsAg and to 100 controls who tested negative for HBsAg. All private patients and 5 of 6 clinic patients testing positive for HBsAg had identifiable risk factors for hepatitis B. In addition, historical risk factors for hepatitis B were identified in 29% of the women testing negative for HBsAg. We found historical risk factors to be excellent predictors of the presence of HBsAg in our private patients. Our data indicate that universal screening for HBsAg is not necessary in private patients.

[Differential diagnosis of endogenous psychoses in relation to a symptom catalog]. / Differenzierte Diagnostik der Endogenen Psychosen unter Anlehnung an einem Symptomenkatalog.

The paper introduces a catalog of symptoms to promote in the field of endogenous psychoses differentiated diagnosis, at the same time constituting a prognosis. Symptoms should be more minutely differentiated than is customary in psychiatric questionnaires. Manic-depressive disorders and purely phasal psychoses are distinguished solely in the clinical evaluation. Thus benign cycloid psychoses can be distinguished from malignant unsystematic schizophrenia.

Catamnesis of endogenous psychoses according to the differential diagnostic method of Karl Leonhard.

The validity of the current systems of classification of endogenous psychoses has not yet been unambiguously demonstrated using the criterion of prognosis. An alternative to these systems may be seen in the Leonhard classification of endogenous psychoses. In a catamnestic study, 93% of the initial diagnoses and prognoses made between 1969 and 1973 could be confirmed in 1986.

[Goethe, Kant, Locke, Villaume knew our technic of individual therapy]. / Goethe, Kant, Locke, Villaume kannten die Methode unserer Individualtherapie.

John Locke (1632-1704) and Villaume (1746-1825) were already familiar with the method with which we, in our "individual therapy", treat neurotic patients. The question whether conditional reflexes and the elimination of their effects play an essential part in the procedure must be denied.