Glucagon Clearance is Decreased in Chronic Kidney Disease, but Preserved in Liver Cirrhosis.

It is not completely clear which organs are responsible for glucagon elimination in humans, and disturbances in the elimination of glucagon could contribute to the hyperglucagonemia observed in chronic liver disease and chronic kidney disease (CKD). Here, we evaluated kinetics and metabolic effects of exogenous glucagon in individuals with stage 4 CKD (n =16), individuals with Child-Pugh A-C cirrhosis (n = 16) and matched control individuals (n = 16), before, during and after a 60-minute glucagon infusion (4 ng/kg/min). Individuals with CKD exhibited a significantly lower mean metabolic clearance rate of glucagon (14.0 [95% CI 12.2;15.7] mL/kg/min) both compared to individuals with cirrhosis (19.7 [18.1;21.3] mL/kg/min, P < 0.001) and to control individuals (20.4 [18.1;22.7] mL/kg/min, P < 0.001). Glucagon half-life was significantly prolonged in the CKD group (7.5 [6.9;8.2] minutes) compared to individuals with cirrhosis (5.7 [5.2;6.3] minutes, P = 0.002) and control individuals (5.7 [5.2;6.3] minutes, P < 0.001). No difference in the effects of exogenous glucagon on plasma glucose, amino acids, or triglycerides was observed between groups. In conclusion, chronic kidney disease, but not liver cirrhosis leads to a significant reduction in glucagon clearance, supporting the kidneys as a primary site for human glucagon elimination.

Exogenous glucagon-like peptide 2 counteracts exogenous cholecystokinin-induced gallbladder contraction in healthy men.

BACKGROUND AND OBJECTIVE: Studies in humans and mice have demonstrated that the gut hormone glucagon-like peptide 2 (GLP-2) promotes gallbladder relaxation and refilling. Here, we assessed the effect of exogenous GLP-2 on gallbladder motility in the fasted state of healthy men with and without infusion of the potent gallbladder-contracting hormone cholecystokinin (CCK). METHODS: In a randomized, double-blind, placebo-controlled, crossover study, 15 male participants (mean [SD]: age 24.7 [3.6] years; body mass index 22.9 [1.6] kg/m2) underwent four experimental days receiving two infusions on each day: either CCK (0.4 pmol × kg-1 × min-1, time 0-180 min) + GLP-2 (10 pmol × kg-1 × min-1, time 30-240 min), CCK + placebo, placebo + GLP-2, or placebo + placebo, respectively. Gallbladder volume was measured at baseline and throughout the 4-hour study day using ultrasonography. RESULTS: Compared to placebo + placebo, GLP-2 + placebo did not affect gallbladder volume, but when infused in combination with CCK, GLP-2 completely abolished the strong gallbladder-contracting effect seen during CCK + placebo infusion, restoring baseline levels of gallbladder volume. CONCLUSION: Exogenous GLP-2 counteracts exogenous CCK-induced gallbladder emptying in healthy men, pointing to a possible therapeutic potential for GLP-2 as a relaxing modulator of gallbladder smooth muscle tone (e.g., as bridge to surgery in biliary colic). The effect may also explain the gallbladder-related adverse events reported for GLP-2 receptor agonists used in the treatment of short bowel syndrome.

Genetic diversity, growth and heart function of Auckland Island pigs, a potential source for organ xenotransplantation.

One of the prerequisites for successful organ xenotransplantation is a reasonable size match between the porcine organ and the recipient's organ to be replaced. Therefore, the selection of a suitable genetic background of source pigs is important. In this study, we investigated body and organ growth, cardiac function, and genetic diversity of a colony of Auckland Island pigs established at the Center for Innovative Medical Models (CiMM), LMU Munich. Male and female Auckland Island pig kidney cells (selected to be free of porcine endogenous retrovirus C) were imported from New Zealand, and founder animals were established by somatic cell nuclear transfer (SCNT). Morphologically, Auckland Island pigs have smaller body stature compared to many domestic pig breeds, rendering their organ dimensions well-suited for human transplantation. Furthermore, echocardiography assessments of Auckland Island pig hearts indicated normal structure and functioning across various age groups throughout the study. Single nucleotide polymorphism (SNP) analysis revealed higher runs of homozygosity (ROH) in Auckland Island pigs compared to other domestic pig breeds and demonstrated that the entire locus coding the swine leukocyte antigens (SLAs) was homozygous. Based on these findings, Auckland Island pigs represent a promising genetic background for organ xenotransplantation.

The bile-gut axis and metabolic consequences of cholecystectomy.

Cholelithiasis and cholecystitis affect individuals of all ages and are often treated by surgical removal of the gallbladder (cholecystectomy), which is considered a safe, low-risk procedure. Nevertheless, recent findings show that bile and its regulated storage and excretion may have important metabolic effects and that cholecystectomy is associated with several metabolic diseases postoperatively. Bile acids have long been known as emulsifiers essential to the assimilation of lipids and absorption of lipid-soluble vitamins, but more recently, they have also been reported to act as metabolic signaling agents. The nuclear receptor, farnesoid X receptor (FXR), and the G protein-coupled membrane receptor, Takeda G protein-coupled receptor 5 (TGR5), are specific to bile acids. Through activation of these receptors, bile acids control numerous metabolic functions. Cholecystectomy affects the storage and excretion of bile acids, which in turn may influence the activation of FXR and TGR5 and their effects on metabolism including processes leading to metabolic conditions such as metabolic dysfunction-associated steatotic liver disease and metabolic syndrome. Here, with the aim of elucidating mechanisms behind cholecystectomy-associated dysmetabolism, we review studies potentially linking cholecystectomy and bile acid-mediated metabolic effects and discuss possible pathophysiological mechanisms behind cholecystectomy-associated dysmetabolism.

Systemic deletion of DMD exon 51 rescues clinically severe Duchenne muscular dystrophy in a pig model lacking DMD exon 52.

Duchenne muscular dystrophy (DMD) is a fatal X-linked disease caused by mutations in the DMD gene, leading to complete absence of dystrophin and progressive degeneration of skeletal musculature and myocardium. In DMD patients and in a corresponding pig model with a deletion of DMD exon 52 (DMDΔ52), expression of an internally shortened dystrophin can be achieved by skipping of DMD exon 51 to reframe the transcript. To predict the best possible outcome of this strategy, we generated DMDΔ51-52 pigs, additionally representing a model for Becker muscular dystrophy (BMD). DMDΔ51-52 skeletal muscle and myocardium samples stained positive for dystrophin and did not show the characteristic dystrophic alterations observed in DMDΔ52 pigs. Western blot analysis confirmed the presence of dystrophin in the skeletal muscle and myocardium of DMDΔ51-52 pigs and its absence in DMDΔ52 pigs. The proteome profile of skeletal muscle, which showed a large number of abundance alterations in DMDΔ52 vs. wild-type (WT) samples, was normalized in DMDΔ51-52 samples. Cardiac function at age 3.5 mo was significantly reduced in DMDΔ52 pigs (mean left ventricular ejection fraction 58.8% vs. 70.3% in WT) but completely rescued in DMDΔ51-52 pigs (72.3%), in line with normalization of the myocardial proteome profile. Our findings indicate that ubiquitous deletion of DMD exon 51 in DMDΔ52 pigs largely rescues the rapidly progressing, severe muscular dystrophy and the reduced cardiac function of this model. Long-term follow-up studies of DMDΔ51-52 pigs will show if they develop symptoms of the milder BMD.

Experimental characterization of de novo proteins and their unevolved random-sequence counterparts.

De novo gene emergence provides a route for new proteins to be formed from previously non-coding DNA. Proteins born in this way are considered random sequences and typically assumed to lack defined structure. While it remains unclear how likely a de novo protein is to assume a soluble and stable tertiary structure, intersecting evidence from random sequence and de novo-designed proteins suggests that native-like biophysical properties are abundant in sequence space. Taking putative de novo proteins identified in human and fly, we experimentally characterize a library of these sequences to assess their solubility and structure propensity. We compare this library to a set of synthetic random proteins with no evolutionary history. Bioinformatic prediction suggests that de novo proteins may have remarkably similar distributions of biophysical properties to unevolved random sequences of a given length and amino acid composition. However, upon expression in vitro, de novo proteins exhibit moderately higher solubility which is further induced by the DnaK chaperone system. We suggest that while synthetic random sequences are a useful proxy for de novo proteins in terms of structure propensity, de novo proteins may be better integrated in the cellular system than random expectation, given their higher solubility.

Heterologous expression of naturally evolved putative de novo proteins with chaperones.

Over the past decade, evidence has accumulated that new protein-coding genes can emerge de novo from previously non-coding DNA. Most studies have focused on large scale computational predictions of de novo protein-coding genes across a wide range of organisms. In contrast, experimental data concerning the folding and function of de novo proteins are scarce. This might be due to difficulties in handling de novo proteins in vitro, as most are short and predicted to be disordered. Here, we propose a guideline for the effective expression of eukaryotic de novo proteins in Escherichia coli. We used 11 sequences from Drosophila melanogaster and 10 from Homo sapiens, that are predicted de novo proteins from former studies, for heterologous expression. The candidate de novo proteins have varying secondary structure and disorder content. Using multiple combinations of purification tags, E. coli expression strains, and chaperone systems, we were able to increase the number of solubly expressed putative de novo proteins from 30% to 62%. Our findings indicate that the best combination for expressing putative de novo proteins in E. coli is a GST-tag with T7 Express cells and co-expressed chaperones. We found that, overall, proteins with higher predicted disorder were easier to express. STATEMENT: Today, we know that proteins do not only evolve by duplication and divergence of existing proteins but also arise from previously non-coding DNA. These proteins are called de novo proteins. Their properties are still poorly understood and their experimental analysis faces major obstacles. Here, we aim to present a starting point for soluble expression of de novo proteins with the help of chaperones and thereby enable further characterization.

Inequality, role reversal and cooperation in multiple group membership settings.

We investigate the role of endowment inequality in a local and global public goods setting with multiple group membership and examine the effect of temporal role reversal on cooperation decisions. Subjects can contribute to a global public good which benefits all subjects and two local public goods which benefit only subjects of either their own group or the group of the other endowment type. Endowment inequality per-se decreases contributions of subjects with a high endowment to the global public good, but increases cooperation of subjects with a low endowment on their local public good, thereby aggravating income disparities. Exogenously induced role reversal for several periods affects cooperation behavior of subjects with a high endowment positively and induces them to contribute more to the global good. Cooperation in unequal environments thus appears to be more stable when all parties have experienced the public goods game from the disadvantageous perspective. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10683-021-09705-y.

A scalable, clinically severe pig model for Duchenne muscular dystrophy.

Large-animal models for Duchenne muscular dystrophy (DMD) are crucial for the evaluation of diagnostic procedures and treatment strategies. Pigs cloned from male cells lacking DMD exon 52 (DMDΔ52) exhibit molecular, clinical and pathological hallmarks of DMD, but die before sexual maturity and cannot be propagated by breeding. Therefore, we generated female DMD+/- carriers. A single founder animal had 11 litters with 29 DMDY/-, 34 DMD+/- as well as 36 male and 29 female wild-type offspring. Breeding with F1 and F2 DMD+/- carriers resulted in an additional 114 DMDY/- piglets. With intensive neonatal management, the majority survived for 3-4 months, providing statistically relevant cohorts for experimental studies. Pathological investigations and proteome studies of skeletal muscles and myocardium confirmed the resemblance to human disease mechanisms. Importantly, DMDY/- pigs displayed progressive myocardial fibrosis and increased expression of connexin-43, associated with significantly reduced left ventricular ejection fraction, at 3 months. Furthermore, behavioral tests provided evidence for impaired cognitive ability. Our breeding cohort of DMDΔ52 pigs and standardized tissue repositories provide important resources for studying DMD disease mechanisms and for testing novel treatment strategies.

Structural and functional characterization of a putative de novo gene in Drosophila.

Comparative genomic studies have repeatedly shown that new protein-coding genes can emerge de novo from noncoding DNA. Still unknown is how and when the structures of encoded de novo proteins emerge and evolve. Combining biochemical, genetic and evolutionary analyses, we elucidate the function and structure of goddard, a gene which appears to have evolved de novo at least 50 million years ago within the Drosophila genus. Previous studies found that goddard is required for male fertility. Here, we show that Goddard protein localizes to elongating sperm axonemes and that in its absence, elongated spermatids fail to undergo individualization. Combining modelling, NMR and circular dichroism (CD) data, we show that Goddard protein contains a large central α-helix, but is otherwise partially disordered. We find similar results for Goddard's orthologs from divergent fly species and their reconstructed ancestral sequences. Accordingly, Goddard's structure appears to have been maintained with only minor changes over millions of years.

Structure and function of naturally evolved de novo proteins.

Comparative evolutionary genomics has revealed that novel protein coding genes can emerge randomly from non-coding DNA. While most of the myriad of transcripts which continuously emerge vanish rapidly, some attain regulatory regions, become translated and survive. More surprisingly, sequence properties of de novo proteins are almost indistinguishable from randomly obtained sequences, yet de novo proteins may gain functions and integrate into eukaryotic cellular networks quite easily. We here discuss current knowledge on de novo proteins, their structures, functions and evolution. Since the existence of de novo proteins seems at odds with decade-long attempts to construct proteins with novel structures and functions from scratch, we suggest that a better understanding of de novo protein evolution may fuel new strategies for protein design.

Major Depressive Disorder Is Associated with Impaired Interoceptive Accuracy: A Systematic Review.

Interoception is the sense of the physiological condition of the entire body. Impaired interoception has been associated with aberrant activity of the insula in major depressive disorder (MDD) during heartbeat perception tasks. Despite clinical relevance, studies investigating interoceptive impairments in MDD have never been reviewed systematically according to the guidelines of the PRISMA protocol, and therefore we collated studies that assessed accuracy in detecting heartbeat sensations (interoceptive accuracy, IAc) in MDD (databases: PubMed/Medline, PsycINFO, and PsycARTICLES). Out of 389 records, six studies met the inclusion criteria. The main findings suggest that (i) moderately depressed samples exhibit the largest interoceptive deficits as compared with healthy adults. (ii) difficulties in decision making and low affect intensity are correlated with low IAc, and (iii) IAc seems to normalize in severely depressed subjects. These associations may be confounded by sex, anxiety or panic disorder, and intake of selective serotonin reuptake inhibitors. Our findings have implications for the development of interoceptive treatments that might relieve MDD-related symptoms or prevent relapse in recurrent depression by targeting the interoceptive nervous system.

Scaffold Effects on Halogen Bonding Strength.

Halogen bonds have become increasingly popular interactions in molecular design and drug discovery. One of the key features is the strong dependence of the size and magnitude of the halogen's σ-hole on the chemical environment of the ligand. The term σ-hole refers to a region of lower electronic density opposite to a covalent bond, e.g., the C-X bond. It is typically (but not always) associated with a positive electrostatic potential in close proximity to the extension of the covalent bond. Herein, we use a variety of 30 nitrogen-bearing heterocycles, halogenated systematically by chlorine, bromine, or iodine, yielding 468 different ligands that are used to exemplify scaffold effects on halogen bonding strength. As a template interaction partner, we have chosen N-methylacetamide representing the ubiquitously present protein backbone. Adduct formation energies were obtained at a MP2/TZVPP level of theory. We used the local maximum of the electrostatic potential on the molecular surface in close proximity to the σ-hole, V S,max, as a descriptor for the magnitude of the positive electrostatic potential characterizing the tuning of the σ-hole. Free optimization of the complexes gave reasonable correlations with V S,max but was found to be of limited use because considerable numbers of chlorinated and brominated ligands lost their halogen bond or showed significant secondary interactions. Thus, starting from a close to optimal geometry of the halogen bond, we used distance scans to obtain the best adduct formation energy for each complex. This approach provided superior results for all complexes exhibiting correlations with R2 > 0.96 for each individual halogen. We evaluated the dependence of V S,max from the molecular surface onto which the positive electrostatic potential is projected, altering the isodensity values from 0.001 au to 0.050 au. Interestingly, the best overall fit using a third-order polynomial function (R2 = 0.99, RMSE = 0.562 kJ/mol) with rather smooth transitions between all halogens was obtained for V S,max calculated from an isodensity surface at 0.014 au.

Small-Molecule Intervention At The Dimerization Interface Of Survivin By Novel Rigidized Scaffolds.

INTRODUCTION: Survivin is a nodal protein involved in several cellular pathways. It is a member of the IAP family and an integral component of the chromosomal passenger complex, where it binds to borealin and INCENP through its dimerization interface. By targeting survivin with a small molecule at its dimerization interface, inhibition of the proliferation of cancer cells has been suggested. With Abbott 8, a small-molecule dimerization inhibitor has been recently reported. The structure-activity relationship of this series of inhibitors implied that the middle pyridin-2(1H)-one ring did not tolerate modifications of any kind. METHODS: Based on the synthetic strategy of Abbott 8 using multicomponent reactions, we synthesized a series of small molecules bearing a novel rigidized core scaffold. This rigidization strategy was accomplished by integrating the pyridin-2(1H)-one and its 6-phenyl substituent into a tricyclic structure, linking position 5 of pyridin-2(1H)-one to the phenyl substituent by rings of different sizes. The new scaffolds were designed based on in silico molecular dynamics of survivin. RESULTS: Binding of these rigidized scaffolds to the recombinant L54M mutant of survivin was evaluated, revealing affinities in the low micromolar range. CONCLUSION: This easily accessible, new class of survivin-dimerization modulators is an interesting starting point for further lead optimization.

Giving and promising gifts: Experimental evidence on reciprocity from the field.

We test the value of unconditional non-monetary gifts as a way to improve health worker performance in a low income country health setting. We randomly assigned health workers to different gift treatments within a program that visited health workers, measured performance and encouraged them to provide high quality care for their patients. We show that unconditional non-monetary gifts improve performance by 20 percent over a six-week period, compared to the control group. We compare the impact of the unconditional gift to one in which a gift is offered conditional on meeting a performance target and show that only the unconditional gift results in a statistically significant improvement. This demonstrates that organizations can improve the performance of health workers in the medium term without using financial incentives.

[The Load of Injustice: A Longitudinal Analysis of the Impact of Subjectively Perceived Income Injustice on the Risk of Stress-Associated Diseases Based on the German Socio-Economic Panel Study]. / Die Last der Ungerechtigkeit. Eine Längsschnittanalyse auf Basis des SOEPs zum Einfluss subjektiv wahrgenommener Einkommensgerechtigkeit auf das Risiko einer stressassoziierten Erkrankung.

BACKGROUND: Income injustice is regarded as a psychosocial strain and associated with an increased risk of stress-related diseases. The physiological stress response is thereby considered as a central link. The aim of the study is to reveal the influence of subjectively perceived income injustice on stress-associated diseases, taking into consideration the load duration. METHOD: Based on the German Socio-Economic Panel Study, data on 5,657 workers in the survey years 2005-2013 were analyzed. The dependent variable reflect the doctor's diagnosed new cases of diabetes, asthma, cardiopathy, stroke, hypertension and depression in the years 2009-2013 as an index. Key predictor is the injustice perception of one's income. In order to operationalize the duration of the injustice perception, the values of the variable for the years 2005, 2007 and 2009 were accumulated. Using logit models, stratified for gender and volume of employment, factors were identified that affect the probability of stress-related diseases. RESULTS: If income was perceived as unjust for over 5 years, the odds of stress-related diseases were strongly enhanced for women (OR 1.64; 95% CI 1.17-2.30). Women working full-time seemed to be particularly affected (OR 2.43; 95% CI 1.54-3.84). Men working full-time perceiving their income as unjust also showed an increased risk for stress diseases (OR 1.43; CI 1.03-1.98). The more often income was assessed as unjust, the higher was the probability of stress-related diseases. CONCLUSIONS: Perceived income injustice seems to be a significant risk factor for stress-related diseases within a dose-response relationship with increasing duration of exposure. Findings of stress research indicate that this represents the 'allostatic load'. Gender-specific differences in stress reaction as well as in the appraisal of the stressors can be associated with gender-specific work and life conditions and therefore provide explanatory approaches for the revealed effects.

Tri- and Tetrasubstituted Pyridinylimidazoles as Covalent Inhibitors of c-Jun N-Terminal Kinase 3.

The concept of covalent inhibition of c-Jun N-terminal kinase 3 (JNK3) was successfully transferred to our well validated pyridinylimidazole scaffold varying several structural features in order to deduce crucial structure-activity relationships. Joint targeting of the hydrophobic region I and methylation of imidazole-N1 position increased the activity and reduced the number of off-targets. The most promising covalent inhibitor, the tetrasubstituted imidazole 3-acrylamido-N-(4-((4-(4-(4-fluorophenyl)-1-methyl-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-yl)amino)phenyl)benzamide (7) inhibits the JNK3 in the subnanomolar range (IC50 = 0.3 nM), shows high metabolic stability in human liver microsomes, and displays excellent selectivity in a screening against a panel of 410 kinases. Covalent bond formation to Cys-154 was confirmed by incubation of the inhibitors with wild-type JNK3 and JNK3-C154A mutant followed by mass spectrometry.

Using Surface Scans for the Evaluation of Halogen Bonds toward the Side Chains of Aspartate, Asparagine, Glutamate, and Glutamine.

Using halogen-specific Connolly type molecular surfaces, we herein invented a new type of surface-based interaction analysis employed for the study of halogen bonding toward model systems of biologically relevant carboxylates (ASP/GLU) and carboxamides (ASN/GLN). Database mining and statistical assessment of the PDB revealed that such interactions are widely underrepresented at the moment. We observed important distance-dependent adaptions of the binding modes of halobenzenes from a preferential oxygen-directed to a bifurcated interaction geometry of the carboxylate. In addition, halogen···π contacts perpendicular to the nitrogen atom of the carboxamide become increasingly important for the lighter halogens. Our analysis on a MP2/TZVPP level of theory is backed by CCSD(T)/CBS reference calculations. To put the vast interaction energies into perspective, we also performed COSMO-RS calculations of the solvation free energy. Facilitating the visualization of our results mapped onto any binding site of choice, we aim to inspire more design studies showcasing these underrepresented interactions.

Fluorescence polarization-based assays for detecting compounds binding to inactive c-Jun N-terminal kinase 3 and p38α mitogen-activated protein kinase.

Two fluorescein-labeled pyridinylimidazoles were synthesized and evaluated as probes for the binding affinity determination of potential kinase inhibitors to the c-Jun N-terminal kinase 3 (JNK3) and p38α mitogen-activated protein kinase (MAPK). Fluorescence polarization (FP)-based competition binding assays were developed for both enzymes using 1-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5-yl)-3-(4-((4-(4-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-yl)amino)phenyl)thiourea (5) as an FP probe (JNK3: Kd = 3.0 nM; p38α MAPK: Kd = 5.7 nM). The validation of the assays with known inhibitors of JNK3 and p38α MAPK revealed that both FP assays correlate very well with inhibition data received by the activity assays. This, in addition to the viability of both FP-based binding assays for the high-throughput screening procedure, makes the assays suitable as inexpensive prescreening protocols for JNK3 and p38α MAPK inhibitors.