Cyclic AMP (cAMP) phosphodiesterase-4 (PDE4) enzymes degrade cAMP and underpin the compartmentalization of cAMP signaling through their targeting to particular protein complexes and intracellular ...locales. We describe the discovery and characterization of a small-molecule compound that allosterically activates PDE4 long isoforms. This PDE4-specific activator displays reversible, noncompetitive kinetics of activation (increased V
max with unchanged K
m), phenocopies the ability of protein kinase A (PKA) to activate PDE4 long isoforms endogenously, and requires a dimeric enzyme assembly, as adopted by long, but not by short (monomeric), PDE4 isoforms. Abnormally elevated levels of cAMP provide a critical driver of the underpinning molecular pathology of autosomal dominant polycystic kidney disease (ADPKD) by promoting cyst formation that, ultimately, culminates in renal failure. Using both animal and human cell models of ADPKD, including ADPKD patient-derived primary cell cultures, we demonstrate that treatment with the prototypical PDE4 activator compound lowers intracellular cAMP levels, restrains cAMP-mediated signaling events, and profoundly inhibits cyst formation. PDE4 activator compounds thus have potential as therapeutics for treating disease driven by elevated cAMP signaling as well as providing a tool for evaluating the action of long PDE4 isoforms in regulating cAMP-mediated cellular processes.
Antibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and possibly transmission. An anticipated correlate of such countermeasures is the level of neutralizing antibodies ...against the SARS-CoV-2 spike protein, which engages with host ACE2 receptor for entry. Using an infectious molecular clone of vesicular stomatitis virus (VSV) expressing eGFP as a marker of infection, we replaced the glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and developed a high-throughput-imaging-based neutralization assay at biosafety level 2. We also developed a focus-reduction neutralization test with a clinical isolate of SARS-CoV-2 at biosafety level 3. Comparing the neutralizing activities of various antibodies and ACE2-Fc soluble decoy protein in both assays revealed a high degree of concordance. These assays will help define correlates of protection for antibody-based countermeasures and vaccines against SARS-CoV-2. Additionally, replication-competent VSV-eGFP-SARS-CoV-2 provides a tool for testing inhibitors of SARS-CoV-2 mediated entry under reduced biosafety containment.
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•Vesicular stomatitis virus encoding the SARS-CoV-2 spike replicates to high titers•Virus propagation is enhanced by a truncation in the cytoplasmic tail of the spike•Neutralization can be assessed by BSL2 and BSL3 high-throughput assays•SARS-CoV-2- and VSV-SARS-CoV-2-based neutralization assays correlate
Case, Rothlauf et al. generate a replication-competent vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike and compare the neutralizing activity of antibodies with VSV-SARS-CoV-2 to fully infectious SARS-CoV-2. They show that VSV-SARS-CoV-2 is a useful BSL2 surrogate virus, as neutralization profiles strongly correlate with focus-reduction neutralization tests using SARS-CoV-2.
The primary cilium constitutes an organelle that orchestrates signal transduction independently from the cell body. Dysregulation of this intricate molecular architecture leads to severe human ...diseases, commonly referred to as ciliopathies. However, the molecular underpinnings how ciliary signaling orchestrates a specific cellular output remain elusive. By combining spatially resolved optogenetics with RNA sequencing and imaging, we reveal a novel cAMP signalosome that is functionally distinct from the cytoplasm. We identify the genes and pathways targeted by the ciliary cAMP signalosome and shed light on the underlying mechanisms and downstream signaling. We reveal that chronic stimulation of the ciliary cAMP signalosome transforms kidney epithelia from tubules into cysts. Counteracting this chronic cAMP elevation in the cilium by small molecules targeting activation of phosphodiesterase‐4 long isoforms inhibits cyst growth. Thereby, we identify a novel concept of how the primary cilium controls cellular functions and maintains tissue integrity in a specific and spatially distinct manner and reveal novel molecular components that might be involved in the development of one of the most common genetic diseases, polycystic kidney disease.
Synopsis
This study reveals a cAMP signalosome in primary cilia that evokes a distinct gene expression program via PKA‐dependent CREB phosphorylation in the cilium. Chronic stimulation of this pathway remodels renal epithelial cells and drives cystogenesis.
The ciliary signalosome relies on an increase in ciliary cAMP level and subsequent PKA‐dependent phosphorylation of CREB in the cilium.
Chronic stimulation of this signalosome remodels renal epithelial cells from tubules into cysts.
Phosphodiesterase 4 (PDE4) long isoforms maintain the ciliary cAMP compartment.
Activation of PDE4 long isoforms counteracts chronic ciliary cAMP signaling and inhibits cyst growth.
This study reveals a cAMP signalosome in primary cilia that evokes a distinct gene expression program via PKA‐dependent CREB phosphorylation in the cilium. Chronic stimulation of this pathway remodels renal epithelial cells and drives cystogenesis.
Summary
Accurate diagnosis of rare inherited anaemias is challenging, requiring a series of complex and expensive laboratory tests. Targeted next‐generation‐sequencing (NGS) has been used to ...investigate these disorders, but the selection of genes on individual panels has been narrow and the validation strategies used have fallen short of the standards required for clinical use. Clinical‐grade validation of negative results requires the test to distinguish between lack of adequate sequencing reads at the locations of known mutations and a real absence of mutations. To achieve a clinically‐reliable diagnostic test and minimize false‐negative results we developed an open‐source tool (CoverMi) to accurately determine base‐coverage and the ‘discoverability’ of known mutations for every sample. We validated our 33‐gene panel using Sanger sequencing and microarray. Our panel demonstrated 100% specificity and 99·7% sensitivity. We then analysed 57 clinical samples: molecular diagnoses were made in 22/57 (38·6%), corresponding to 32 mutations of which 16 were new. In all cases, accurate molecular diagnosis had a positive impact on clinical management. Using a validated NGS‐based platform for routine molecular diagnosis of previously undiagnosed congenital anaemias is feasible in a clinical diagnostic setting, improves precise diagnosis and enhances management and counselling of the patient and their family.
Abstract
DNA sequence data from mitochondrial genomes and c. 1000 nuclear exons were analysed for a complete taxon sampling of manta and devilrays (Mobulidae) to estimate a current molecular ...phylogeny for the family. The resulting inferences were combined with morphological information to adopt an integrated approach to resolving the taxonomic arrangement of the family. The members of the genus Manta were found to consistently nest within the Mobula species and consequently the genus Manta is placed into the synonymy of Mobula. Mobula eregoodootenkee, M. japanica and M. rochebrunei were each found to be junior synonyms of M. kuhlii, M. mobular and M. hypostoma, respectively. The mitochondrial and nuclear tree topologies were in agreement except for the placement of M. tarapacana which was basal to all other mobulids in the nuclear exon analysis, but as the sister group to the M. alfredi–M. birostris–M. mobular clade in the mitochondrial genome analysis. Results from this study are used to a revise the taxonomy for the family Mobulidae. A single genus is now recognized (where there were previously two) and eight nominal species (where there were previously 11).
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Major malformations of the human eye, including microphthalmia and anophthalmia, are examples of phenotypes that recur in families yet often show no clear Mendelian inheritance pattern. Defining loci ...by mapping is therefore rarely feasible. Using a candidate-gene approach, we have identified heterozygous coding-region changes in the homeobox gene
OTX2 in eight families with ocular malformations. The expression pattern of
OTX2 in human embryos is consistent with the eye phenotypes observed in the patients, which range from bilateral anophthalmia to retinal defects resembling Leber congenital amaurosis and pigmentary retinopathy. Magnetic resonance imaging scans revealed defects of the optic nerve, optic chiasm, and, in some cases, brain. In two families, the mutations appear to have occurred de novo in severely affected offspring, and, in two other families, the mutations have been inherited from a gonosomal mosaic parent. Data from these four families support a simple model in which
OTX2 heterozygous loss-of-function mutations cause ocular malformations. Four additional families display complex inheritance patterns, suggesting that
OTX2 mutations alone may not lead to consistent phenotypes. The high incidence of mosaicism and the reduced penetrance have implications for genetic counseling.
cAMP-specific PDE (phosphodiesterase) 4 isoforms underpin compartmentalized cAMP signalling in mammalian cells through targeting to specific signalling complexes. Their importance is apparent as PDE4 ...selective inhibitors exert profound anti-inflammatory effects and act as cognitive enhancers. The p38 MAPK (mitogen-activated protein kinase) signalling cascade is a key signal transduction pathway involved in the control of cellular immune, inflammatory and stress responses. In the present study, we show that PDE4A5 is phosphorylated at Ser147, within the regulatory UCR1 (ultraconserved region 1) domain conserved among PDE4 long isoforms, by MK2 (MAPK-activated protein kinase 2, also called MAPKAPK2). Phosphorylation by MK2, although not altering PDE4A5 activity, markedly attenuates PDE4A5 activation through phosphorylation by protein kinase A. This modification confers the amplification of intracellular cAMP accumulation in response to adenylate cyclase activation by attenuating a major desensitization system to cAMP. Such reprogramming of cAMP accumulation is recapitulated in wild-type primary macrophages, but not MK2/3-null macrophages. Phosphorylation by MK2 also triggers a conformational change in PDE4A5 that attenuates PDE4A5 interaction with proteins whose binding involves UCR2, such as DISC1 (disrupted in schizophrenia 1) and AIP (aryl hydrocarbon receptor-interacting protein), but not the UCR2-independent interacting scaffold protein β-arrestin. Long PDE4 isoforms thus provide a novel node for cross-talk between the cAMP and p38 MAPK signalling systems at the level of MK2.
Background. The urinary tract is the most common source for Escherichia coli bacteremia. Mortality from E. coli urinary-source bacteremia is higher than that from urinary tract infection. ...Predisposing factors for urinary-source E. coli bacteremia are poorly characterized. Methods. In order to identify urinary-source bacteremia risk factors, we conducted a 12-month prospective cohort study of adult inpatients with E. coli bacteriuria that were tested for bacteremia within ±1 day of the bacteriuria. Patients with bacteremia were compared with those without bacteremia. Bacterial isolates from urine were screened for 16 putative virulence genes using high-throughput dot-blot hybridization. Results. Twenty-four of 156 subjects (15%) had E. coli bacteremia. Bacteremic patients were more likely to have benign prostatic hyperplasia (56% vs 19%; P = .04), a history of urogenital surgery (63% vs 28%; P = .001), and presentation with hesitancy/retention (21% vs 4%; P = .002), fever (63% vs 38%; P = .02), and pyelonephritis (67% vs 41%; P = .02). The genes kpsMT (group II capsule) (17 71% vs 62 47%; P = .03) and prf (P-fimbriae family) (13 54% vs 40 30%; P = .02) were more frequent in the urinary strains from bacteremic patients. Symptoms of hesitancy/retention (odds ratio OR, 7.8; 95% confidence interval CI, 1.6-37), history of a urogenital procedure (OR, 5.4; 95% CI, 2-14.7), and presence of kpsMT (OR, 2.9; 95% CI, 1-8.2) independently predicted bacteremia. Conclusions. Bacteremia secondary to E. coli bacteriuria was frequent (15%) in those tested for it. Urinary stasis, surgical disruption of urogenital tissues, and a bacterial capsule characteristic contribute to systemic invasion by uropathogenic E. coli.
This paper is a summary of key presentations from a workshop in Iceland on May 3-4, 2023 arranged by Aarhus University and with participation of the below-mentioned scientists. Below you will find ...the key messages from the presentations made by: Professor Jan Vandenbroucke, Department of Clinical Epidemiology, Aarhus University, Emeritus Professor, Leiden University; Honorary Professor, London School of Hygiene & Tropical Medicine, UKProfessor, Chair Henrik Toft Sørensen, Department of Clinical Epidemiology, Aarhus University and Aarhus University Hospital, DenmarkProfessor David H. Rehkopf, Director, the Stanford Center for Population Health Sciences, Stanford University, CA., USProfessor Jaimie Gradus, Department of Epidemiology, School of Public Health, Boston University, Boston, Massachusetts, USProfessor Johan Mackenbach, Emeritus Professor, Department of Public Health, Erasmus University Rotterdam, HollandProfessor, Chair M Maria Glymour, Department of Epidemiology, Boston University School of Public Health, Boston University, Boston, Massachusetts, USProfessor, Dean Sandro Galea, School of Public Health, Boston University, Boston, Massachusetts, USProfessor Victor W. Henderson, Departments of Epidemiology & Population Health and of Neurology & Neurological Sciences, Stanford University, Stanford, CA, US; Department of Clinical Epidemiology, Aarhus University, Aarhus, DK.
Manual therapy and exercise have not previously been compared with a home exercise program for patients with osteoarthritis (OA) of the knee. The purpose of this study was to compare outcomes between ...a home-based physical therapy program and a clinically based physical therapy program.
One hundred thirty-four subjects with OA of the knee were randomly assigned to a clinic treatment group (n=66; 61% female, 39% male; mean age +/-SD=64+/-10 years) or a home exercise group (n=68, 71% female, 29% male; mean age +/-SD=62+/-9 years).
Subjects in the clinic treatment group received supervised exercise, individualized manual therapy, and a home exercise program over a 4-week period. Subjects in the home exercise group received the same home exercise program initially, reinforced at a clinic visit 2 weeks later. Measured outcomes were the distance walked in 6 minutes and the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC).
Both groups showed clinically and statistically significant improvements in 6-minute walk distances and WOMAC scores at 4 weeks; improvements were still evident in both groups at 8 weeks. By 4 weeks, WOMAC scores had improved by 52% in the clinic treatment group and by 26% in the home exercise group. Average 6-minute walk distances had improved about 10% in both groups. At 1 year, both groups were substantially and about equally improved over baseline measurements. Subjects in the clinic treatment group were less likely to be taking medications for their arthritis and were more satisfied with the overall outcome of their rehabilitative treatment compared with subjects in the home exercise group.
Although both groups improved by 1 month, subjects in the clinic treatment group achieved about twice as much improvement in WOMAC scores than subjects who performed similar unsupervised exercises at home. Equivalent maintenance of improvements at 1 year was presumably due to both groups continuing the identical home exercise program. The results indicate that a home exercise program for patients with OA of the knee provides important benefit. Adding a small number of additional clinical visits for the application of manual therapy and supervised exercise adds greater symptomatic relief.
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DOBA, FSPLJ, IZUM, KILJ, NUK, OILJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK, VSZLJ
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