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The Hedgehog (Hh) signaling pathway is fundamental to embryogenesis, tissue homeostasis, and cancer. Hh signals are transduced via an unusual mechanism: upon agonist-induced phosphorylation, the noncanonical G protein-coupled receptor SMOOTHENED (SMO) binds the catalytic subunit of protein kinase A (PKA-C) and physically blocks its enzymatic activity. By combining computational structural approaches with biochemical and functional studies, we show that SMO mimics strategies prevalent in canonical GPCR and PKA signaling complexes, despite little sequence or secondary structural homology. An intrinsically disordered region of SMO binds the PKA-C active site, resembling the PKA regulatory subunit (PKA-R) / PKA-C holoenzyme, while the SMO transmembrane domain binds a conserved PKA-C interaction hub, similar to other GPCR-effector complexes. In contrast with prevailing GPCR signal transduction models, phosphorylation of SMO promotes intramolecular electrostatic interactions that stabilize key structural elements within the SMO cytoplasmic domain, thereby remodeling it into a PKA-inhibiting conformation. Our work provides a structural mechanism for a central step in the Hh cascade and defines a paradigm for disordered GPCR domains to transmit signals intracellularly.

Streams provide an array of habitat niches that may act as environmental filters for fish communities. The tropical island of Sulawesi in Indonesia is located in the Wallacea, a region isolated by marine barriers from the Asian and Australian faunas. Primary freshwater fishes are naturally absent in the Wallacea, including Sulawesi's numerous coastal streams. Diadromous species are in contrast species-rich in the area. The knowledge available on stream fishes in the Wallacea is largely restricted to taxonomic work and studies targeting single species groups, whereas baseline data on fish ecology remain extremely scarce. Such data and a deeper understanding of stream fish ecology are, however, urgently required for purposes such as informed management. We assumed that the stream fish assemblages are dominated by recruitment from the sea and are structured by macro- and microhabitat diversity. To test this hypothesis, we quantified the occurrence of individual fishes by point abundance electrofishing at 33 streams across Sulawesi. The 4632 fishes obtained represent 58 species out of 24 families. The native fishes recorded are mainly amphidromous (34 species), euryhaline (five species), and catadromous (five species). Gobiiformes make up the vast majority of records, dominated by Oxudercidae (22 species) and Eleotridae (five species). Only two of the species recorded are endemic to Sulawesi, including a single species strictly confined to freshwaters. Ten species, making up 6% of the fishes caught, are not native to Sulawesi. The outlying mean index (OMI) and BIOENV analyses suggest that effects on the scale of macro- and microhabitat shape fish assemblage composition, ranging from pH, conductivity, and temperature to current velocity, substrate, canopy cover, and elevation. Habitat niche use of species along the first two OMI axes is complementary and fine-scaled, covering a wide range of the available habitat space. Juvenile and adult conspecifics share similar habitat niches in most of the cases. Niche breadths overlap, but niche specialization is significant in most of the species. Non-native fishes link into the assemblages at the margins of habitat space, with substantial niche overlaps to native species. The present findings show that the native fish communities in coastal streams of Sulawesi are largely composed of species depending on access to the sea, highlighting the importance of connectivity down to the estuaries and sea. The ichthyofauna shows a rich diversity in habitat use, and the availability of alternative habitats along the altitudinal gradient provides plausible filters for species establishment. Non-native fishes are locally abundant, pose substantial potential for changing communities, but are still stocked intentionally. We stress the need for incorporating the need for connectivity and maintained habitat quality into management decisions, and a critical evaluation of stocking activities., (© 2024 The Author(s). Journal of Fish Biology published by John Wiley & Sons Ltd on behalf of Fisheries Society of the British Isles.)

The landscape theory of food web architecture (LTFWA) describes relationships among body size, trophic position, mobility, and energy channels that serve to couple heterogenous habitats, which in turn promotes long-term system stability. However, empirical tests of the LTFWA are rare and support differs among terrestrial, freshwater, and marine systems. Further, it is unclear whether the theory applies in highly altered ecosystems dominated by introduced species such as the Laurentian Great Lakes. Here, we provide an empirical test of the LTFWA by relating body size, trophic position, and the coupling of different energy channels using stable isotope data from species throughout the Lake Michigan food web. We found that body size was positively related to trophic position, but for a given trophic position, organisms predominately supported by pelagic energy had smaller body sizes than organisms predominately supported by nearshore benthic energy. We also found a hump-shaped trophic relationship in the food web where there is a gradual increase in the coupling of pelagic and nearshore energy channels with larger body sizes as well as higher trophic positions. This highlights the important role of body size and connectivity among habitats in structuring food webs. However, important deviations from expectations are suggestive of how species introductions and other anthropogenic impacts can affect food web structure in large lakes. First, native top predators appear to be flexible couplers that may provide food web resilience, whereas introduced top predators may confer less stability when they specialize on a single energy pathway. Second, some smaller bodied prey fish and invertebrates, in addition to mobile predators, coupled energy from pelagic and nearshore energy channels, which suggests that some prey species may also be important integrators of energy pathways in the system. We conclude that patterns predicted by the LTFWA are present in the face of species introductions and other anthropogenic stressors to a degree, but time-series evaluations are needed to fully understand the mechanisms that promote stability., (© 2024 The Author(s). Ecology published by Wiley Periodicals LLC on behalf of The Ecological Society of America. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Background: Diabetes technology is a fundamental part of modern diabetes therapy. Its widespread usage is associated with an increasing amount of "diabetes technology waste." The aim of this study was to quantify this waste in a real-world situation in a specialized diabetes practice in Germany., Methods: Eighty patients with diabetes and insulin treatment participated and collected all of their therapy-associated waste for three months. Their attitude toward sustainability of antidiabetic therapy, waste generation, and their own waste reduction/separation behavior was surveyed., Results: In total, 23 707 pieces of therapy-associated waste were collected. They comprised 5362 test strips, 630 glucose sensors, 14 619 needles, 519 insulin cartridges, 599 pens, and 1463 pieces of aids for insulin pump therapy. Type and quantity of the collected waste depended on the type of diabetes and the respective therapy, ie, multiple daily injections, usage of glucose sensors, or pump therapy. Most participants (92%) were surprised by the amounts of waste and reported an increased awareness toward the resource consumption of their therapy (87%). The survey indicated an enhanced interest in waste separation (94%) and a demand for the reduction and recycling of devices/aids (93%)., Conclusions: Our data revealed the amount and complexity of the waste generated by modern diabetes therapy. Extrapolating these data, it can be estimated that around 1.2 billion pieces of diabetes technology waste are generated in Germany per year. The major concern of the study participants was the limited number of recycling options. A clear demand for improved sustainability of the medical products was expressed., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Recently, machine learning algorithms have identified preprocedural γ-glutamyl transferase (GGT) as a significant predictor of long-term mortality after coronary revascularization in the SYNTAX (Synergy Between PCI [Percutaneous Coronary Intervention] With Taxus and Cardiac Surgery) trial. The aim of the present study is to investigate the impact of preprocedural GGT on 10-year all-cause mortality in patients with complex coronary artery disease after revascularization., Methods and Results: The SYNTAX trial was a randomized trial comparing PCI with coronary artery bypass grafting in 1800 patients with complex coronary artery disease. The present report is a post hoc subanalysis of the SYNTAXES (Synergy Between PCI With Taxus and Cardiac Surgery Extended Survival) trial, an investigator-driven extended 10-year follow-up of the SYNTAX trial. The association between preprocedural GGT and 10-year all-cause mortality was investigated. The mean values of GGT for men and women were 43.5 (SD, 48.5) and 36.4 (SD, 46.1) U/L, respectively. In multivariable Cox regression models adjusted by traditional risk factors, GGT was an independent predictor for all-cause death at 10-year follow-up, and each SD increase in log-GGT was associated with a 1.24-fold risk of all cause death at 10-year follow-up (95% CI, 1.10-1.40). According to previously reported sex-related GGT thresholds, patients with higher GGT level had a 1.74-fold risk of all-cause death at 10-year follow-up (95% CI, 1.32-2.29) compared with patients with lower GGT level., Conclusions: Preprocedural GGT is an independent predictor of 10-year mortality after coronary revascularization in patients with complex coronary artery disease. In patients with elevated GGT, strong secondary prevention may be required after revascularization and must be studied prospectively., Registration: URL: https://clinicaltrials.gov/study/NCT03417050.

Mutations of the human leucine-rich repeat kinase 2 (LRRK2) have been associated with both, idiopathic and familial Parkinson's disease (PD). Most of these pathogenic mutations are located in the kinase domain (KD) or GTPase domain of LRRK2. In this study we describe a mechanism in which protein kinase activity can be modulated by reversible oxidation or reduction, involving a unique pair of adjacent cysteines, the "CC" motif. Among all human protein kinases, only LRRK2 contains this "CC" motif (C2024 and C2025) in the Activation Segment (AS) of the kinase domain. In an approach combining site-directed mutagenesis, biochemical analyses, cell-based assays, and Gaussian accelerated Molecular Dynamics (GaMD) simulations we could attribute a role for each of those cysteines. We employed reducing and oxidizing agents with potential clinical relevance to investigate effects on kinase activity and microtubule docking. We find that each cysteine gives a distinct contribution: the first cysteine, C2024, is essential for LRRK2 protein kinase activity, while the adjacent cysteine, C2025, contributes significantly to redox sensitivity. Implementing thiolates (R-S - ) in GaMD simulations allowed us to analyse how each of the cysteines in the "CC" motif interacts with its surrounding residues depending on its oxidation state. From our studies we conclude that oxidizing agents can downregulate kinase activity of hyperactive LRRK2 PD mutations and may provide promising tools for therapeutic strategies., (© 2024. The Author(s).)

Leucine-rich repeat protein kinase 2 (LRRK2) is a multi-domain protein encompassing two of biology's most critical molecular switches, a kinase and a GTPase, and mutations in LRRK2 are key players in the pathogenesis of Parkinson's disease (PD). The availability of multiple structures (full-length and truncated) has opened doors to explore intra-domain cross-talk in LRRK2. A helix extending from the WD40 domain and stably docking onto the kinase domain is common in all available structures. This C-terminal (Ct) helix is a hub of phosphorylation and organelle-localization motifs and thus serves as a multi-functional protein : protein interaction module. To examine its intra-domain interactions, we have recombinantly expressed a stable Ct motif (residues 2480-2527) and used peptide arrays to identify specific binding sites. We have identified a potential interaction site between the Ct helix and a loop in the CORB domain (CORB loop) using a combination of Gaussian accelerated molecular dynamics simulations and peptide arrays. This Ct-Motif contains two auto-phosphorylation sites (T2483 and T2524), and T2524 is a 14-3-3 binding site. The Ct helix, CORB loop, and the CORB-kinase linker together form a part of a dynamic 'CAP' that regulates the N-lobe of the kinase domain. We hypothesize that in inactive, full-length LRRK2, the Ct-helix will also mediate interactions with the N-terminal armadillo, ankyrin, and LRR domains (NTDs) and that binding of Rab substrates, PD mutations, or kinase inhibitors will unleash the NTDs., (© 2024 The Author(s).)

Water oxidation is a bottleneck reaction for the establishment of solar-to-fuel energy conversion systems. Earth-abundant metal-based polyoxometalates are promising heterogeneous water oxidation catalysts that can operate in a wide pH range. However, detailed structure-reactivity relationships are not yet comprehensively understood, hampering the design and synthesis of more effective polyoxometalate-based oxidation catalysts. Here we report the synthesis of an ordered, mixed-metal cobalt-iron Weakley archetype [Co II 2 (H 2 O) 2 Fe III W 2 (Co II W 9 O 34 ) 2 ] 14- (Co 2 Fe 2 -WS), which unexpectedly highlights the strong influence of the central, coordinatively saturated metal ions on the catalytic water oxidation characteristics. The resulting species exhibits catalytic turnover frequencies which are up to 4× higher than those of the corresponding archetype tetracobalt-oxo species [Co II 2 (H 2 O) 2 Co II 2 (PW 9 -WS). It is further striking that the system becomes catalytically inactive when one of the central positions is occupied by a W 34 ) 2 ] 10- (Co 4 -WS). It is further striking that the system becomes catalytically inactive when one of the central positions is occupied by a W VI W II 2 W 2 O) 2 Co II W VI (Co II W-WS). Importantly, this study demonstrates that coordinatively saturated metal ions in this central position, which at first glance appear insignificant, do not solely have a structural role but also impart a distinctive structural influence on the reactivity of the polyoxometalate. These results provide unique insights into the structure-reactivity relationships of polyoxometalates with improved catalytic performance characteristics.9 O 34 ) 2 ] 12- (Co 3 W-WS). Importantly, this study demonstrates that coordinatively saturated metal ions in this central position, which at first glance appear insignificant, do not solely have a structural role but also impart a distinctive structural influence on the reactivity of the polyoxometalate. These results provide unique insights into the structure-reactivity relationships of polyoxometalates with improved catalytic performance characteristics., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Hereditary retinal degeneration (RD) is often associated with excessive cGMP signalling in photoreceptors. Previous research has shown that inhibition of cGMP-dependent protein kinase G (PKG) can reduce photoreceptor loss in two different RD animal models. In this study, we identified a PKG inhibitor, the cGMP analogue CN238, which preserved photoreceptor viability and functionality in rd1 and rd10 mutant mice. Surprisingly, in explanted retinae, CN238 also protected retinal ganglion cells from axotomy-induced retrograde degeneration and preserved their functionality. Furthermore, kinase activity-dependent protein phosphorylation of the PKG target Kv1.6 was reduced in CN238-treated rd10 retinal explants. Ca 2+ -imaging on rd10 acute retinal explants revealed delayed retinal ganglion cell repolarization with CN238 treatment, suggesting a PKG-dependent modulation of Kv1-channels. Together, these results highlight the strong neuroprotective capacity of PKG inhibitors for both photoreceptors and retinal ganglion cells, illustrating their broad potential for the treatment of retinal diseases and possibly neurodegenerative diseases in general.

Background: The SYNTAX trial demonstrated negative impact of repeat revascularization (RR) on 5-year outcomes following PCI/CABG in patients with three-vessel(3VD) and/or left main coronary artery disease(LMCAD). We aimed to investigate the impact of RR within 5 years, on 10-year mortality in patients with 3VD and/or LMCAD after PCI/CABG., Methods: The SYNTAXES study evaluated the vital status out to 10 years of patients with 3VD and/or LMCAD. Patients were stratified by RR within 5 years and randomized treatment. The association between RR within 5 years and 10-year mortality was assessed., Results: A total of 330 out of 1800 patients (18.3%) underwent RR within 5 years. RR occurred more frequently after initial PCI than after initial CABG (25.9% vs. 13.7%, p < 0.001). Overall, 10-year mortality was comparable between patients undergoing RR and those not (28.2% vs. 26.1%, adjusted HR: 1.17, 95%CI 0.93-1.48, p = 0.187). In the PCI arm, RR was associated with a trend toward higher 10-year mortality (adjusted HR: 1.29, 95%CI 0.97-1.72, p = 0.075), while in the CABG arm, the trend was opposite (adjusted HR: 0.74, 95%CI 0.46-1.20, p = 0.219). Among patients requiring RR, those who underwent PCI as initial revascularization had a higher risk of 10-year mortality compared to initial CABG (33.5% vs. 17.6%, adjusted HR: 2.09, 95%CI 1.21-3.61, p = 0.008)., Conclusion: In the SYNTAXES study, RR within 5 years had no impact on 10-year all-cause death in the population overall. Among patients requiring any repeat procedures, 10-year mortality was higher after initial treatment with PCI than after CABG. These exploratory findings should be investigated with larger populations in future studies., Trial Registration: URL: https://www., Clinicaltrials: gov ; SYNTAXES Unique identifier: NCT03417050. URL: https://www., Clinicaltrials: gov ; SYNTAX Unique identifier: NCT00114972., (© 2023. The Author(s).)

Microtubule-Associated Serine/Threonine (MAST) kinases represent an evolutionary conserved branch of the AGC protein kinase superfamily in the kinome. Since the discovery of the founding member, MAST2, in 1993, three additional family members have been identified in mammals and found to be broadly expressed across various tissues, including the brain, heart, lung, liver, intestine and kidney. The study of MAST kinases is highly relevant for unraveling the molecular basis of a wide range of different human diseases, including breast and liver cancer, myeloma, inflammatory bowel disease, cystic fibrosis and various neuronal disorders. Despite several reports on potential substrates and binding partners of MAST kinases, the molecular mechanisms that would explain their involvement in human diseases remain rather obscure. This review will summarize data on the structure, biochemistry and cell and molecular biology of MAST kinases in the context of biomedical research as well as organismal model systems in order to provide a current profile of this field.

Mutations in LRRK2, a large multi-domain protein kinase, create risk factors for Parkinson's Disease (PD). LRRK2 has seven well-folded domains that include three N-terminal scaffold domains (NtDs) and four C-terminal domains (CtDs). In full-length inactive LRRK2 there is an additional well-folded motif, the LRR-ROC Linker, that lies between the NtDs and the CtDs. This motif, which is stabilized by hydrophobic residues in the LRR and ROC/COR-A domains, is anchored to the C-Lobe of the kinase domain. The LRR-ROC Linker becomes disordered when the NtDs are unleashed from the CtDs following activation by Rab29 or by various PD mutations. A key residue within the LRR-ROC Linker, W1295, sterically blocks access of substrate proteins. The W1295A mutant blocks cis-autophosphorylation of S1292 and reduces phosphorylation of heterologous Rab substrates. GaMD simulations show that the LRR-Linker motif, P + 1 loop and the inhibitory helix in the DYGψ motif are very stable. Finally, in full-length inactive LRRK2 ATP is bound to the kinase domain and GDP:Mg to the GTPase/ROC domain. The fundamentally different mechanisms for binding nucleotide (G-Loop vs P-Loop) are captured by these GaMD simulations. In this model, where ATP binds with low affinity (μM range) to N-Lobe capping residues, the known auto-phosphorylation sites are located in the space that is sampled by the flexible phosphates thus providing a potential mechanism for cis-autophosphorylation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

LRRK2 is a multi-domain protein with three catalytically inert N-terminal domains (NtDs) and four C-terminal domains, including a kinase and a GTPase domain. LRRK2 mutations are linked to Parkinson's Disease. Recent structures of LRRK2RCKW and a full-length inactive LRRK2 (fl-LRRK2INACT) monomer revealed that the kinase domain drives LRRK2 activation. The LRR domain and also an ordered LRR- COR linker, wrap around the C-lobe of the kinase domain and sterically block the substrate binding surface in fl-LRRK2INACT. Here we focus on the crosstalk between domains. Our biochemical studies of GTPase and kinase activities of fl-LRRK2 and LRRK2RCKW reveal how mutations influence this crosstalk differently depending on the domain borders investigated. Furthermore, we demonstrate that removing the NtDs leads to altered intramolecular regulation. To further investigate the crosstalk, we used Hydrogen-Deuterium exchange Mass Spectrometry (HDX-MS) to characterize the conformation of LRRK2RCKW   and Gaussian Accelerated Molecular Dynamics (GaMD) to create dynamic portraits of fl-LRRK2 and LRRK2RCKW. These models allowed us to investigate the dynamic changes in wild type and mutant LRRK2s. Our data show that the a3ROC helix, the Switch II motif in the ROC domain, and the LRR-ROC linker play crucial roles in mediating local and global conformational changes. We demonstrate how these regions are affected by other domains in fl-LRRK2 and LRRK2RCKW and show how unleashing of the NtDs as well as PD mutations lead to changes in conformation and dynamics of the ROC and kinase domains which ultimately impact kinase and GTPase activities. These allosteric sites are potential therapeutic targets., (Copyright 2023 The Author(s).)

Although Fischer's extraordinary career came to focus mostly on the protein phosphatases, after his co-discovery of Phosphorylase Kinase with Ed Krebs he was clearly intrigued not only by cAMP-dependent protein kinase (PKA), but also by the heat-stable, high-affinity protein kinase inhibitor (PKI). PKI is an intrinsically disordered protein that contains at its N-terminus a pseudo-substrate motif that binds synergistically and with high-affinity to the PKA catalytic (C) subunit. The sequencing and characterization of this inhibitor peptide (IP20) were validated by the structure of the PKA C-subunit solved first as a binary complex with IP20 and then as a ternary complex with ATP and two magnesium ions. A second motif, nuclear export signal (NES), was later discovered in PKI. Both motifs correspond to amphipathic helices that convey high-affinity binding. The dynamic features of full-length PKI, recently captured by NMR, confirmed that the IP20 motif becomes dynamically and sequentially ordered only in the presence of the C-subunit. The type I PKA regulatory (R) subunits also contain a pseudo-substrate ATPMg2-dependent high-affinity inhibitor sequence. PKI and PKA, especially the Cβ subunit, are highly expressed in the brain, and PKI expression is also cell cycle-dependent. In addition, PKI is now linked to several cancers. The full biological importance of PKI and PKA signaling in the brain, and their importance in cancer thus remains to be elucidated., (© 2023 International Union of Biochemistry and Molecular Biology.)

Background: The long-term prognostic impact of a composite of periprocedural major adverse events (PMAE) following revascularisation for patients with complex coronary artery disease (CAD) has not yet been established., Aims: This study aimed to assess the impact on 10-year mortality of non-fatal PMAE following percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG). Other objectives were to evaluate 1) whether PMAE affect mortality predicted by the SYNTAX score II 2020 (SSII-2020) and 2) whether optimal medical therapy (OMT) positively affects the prognosis of patients with non-fatal PMAE., Methods: The association between 10-year mortality and non-fatal PMAE occurring within 30 days of PCI or CABG in patients with three-vessel disease and/or left main disease enrolled in the SYNTAXES study was investigated., Results: The main findings are that non-fatal PMAE occurred less frequently following PCI than CABG (11.2% vs 28.2%; p<0.001) and that non-fatal PMAE were an independent predictor of all-cause mortality in the first year post-procedure, but not at 5 or 10 years, in both treatment modalities. PMAE substantially alter the individual predictions of 10-year mortality by the SSII-2020. In patients with non-fatal PMAE, OMT may provide survival benefits during the first year post-procedure as well as in the long term., Conclusions: In patients with complex CAD, non-fatal PMAE were more common following CABG than PCI, but their prognostic impact was similar, being significant in the first year and then diminishing out to 10 years. Patients with non-fatal PMAE may therefore require more careful follow-up and additional preventive treatment in the first year post-procedure.

Background: Phosphodiesterase 3A ( PDE3A ) gain-of-function mutations cause hypertension with brachydactyly (HTNB) and lead to stroke. Increased peripheral vascular resistance, rather than salt retention, is responsible. It is surprising that the few patients with HTNB examined so far did not develop cardiac hypertrophy or heart failure. We hypothesized that, in the heart, PDE3A mutations could be protective., Methods: We studied new patients. CRISPR-Cas9-engineered rat HTNB models were phenotyped by telemetric blood pressure measurements, echocardiography, microcomputed tomography, RNA-sequencing, and single nuclei RNA-sequencing. Human induced pluripotent stem cells carrying PDE3A mutations were established, differentiated to cardiomyocytes, and analyzed by Ca 2+ imaging. We used Förster resonance energy transfer and biochemical assays., Results: We identified a new PDE3A mutation in a family with HTNB. It maps to exon 13 encoding the enzyme's catalytic domain. All hitherto identified HTNB PDE3A mutations cluster in exon 4 encoding a region N-terminally from the catalytic domain of the enzyme. The mutations were recapitulated in rat models. Both exon 4 and 13 mutations led to aberrant phosphorylation, hyperactivity, and increased PDE3A enzyme self-assembly. The left ventricles of our patients with HTNB and the rat models were normal despite preexisting hypertension. A catecholamine challenge elicited cardiac hypertrophy in HTNB rats only to the level of wild-type rats and improved the contractility of the mutant hearts, compared with wild-type rats. The β-adrenergic system, phosphodiesterase activity, and cAMP levels in the mutant hearts resembled wild-type hearts, whereas phospholamban phosphorylation was decreased in the mutants. In our induced pluripotent stem cell cardiomyocyte models, the PDE3A mutations caused adaptive changes of Ca 2+ cycling. RNA-sequencing and single nuclei RNA-sequencing identified differences in mRNA expression between wild-type and mutants, affecting, among others, metabolism and protein folding., Conclusions: Although in vascular smooth muscle, PDE3A mutations cause hypertension, they confer protection against hypertension-induced cardiac damage in hearts. Nonselective PDE3A inhibition is a final, short-term option in heart failure treatment to increase cardiac cAMP and improve contractility. Our data argue that mimicking the effect of PDE3A mutations in the heart rather than nonselective PDE3 inhibition is cardioprotective in the long term. Our findings could facilitate the search for new treatments to prevent hypertension-induced cardiac damage.

The sensory ion channel transient receptor potential vanilloid 1 (TRPV1) is mainly expressed in small to medium sized dorsal root ganglion neurons, which are involved in the transfer of acute noxious thermal and chemical stimuli. The Ankyrin-rich membrane spanning protein (ARMS) interaction with TRPV1 is modulated by protein kinase A (PKA) mediating sensitization. Here, we hypothesize that PKA phosphorylation sites of ARMS are crucial for the modulation of TRPV1 function, and that the phosphorylation of ARMS is facilitated by the A-kinase anchoring protein 79 (AKAP79). We used transfected HEK293 cells, immunoprecipitation, calcium flux, and patch clamp experiments to investigate potential PKA phosphorylation sites in ARMS and in ARMS-related peptides. Additionally, experiments were done to discriminate between PKA and protein kinase D (PKD) phosphorylation. We found different interaction ratios for TRPV1 and ARMS mutants lacking PKA phosphorylation sites. The degree of TRPV1 sensitization by ARMS mutants is independent on PKA phosphorylation. AKAP79 was also involved in the TRPV1/ARMS/PKA signaling complex. These data show that ARMS is a PKA substrate via AKAP79 in the TRPV1 signaling complex and that all four proteins interact physically, regulating TRPV1 sensitization in transfected HEK293 cells. To assess the physiological and/or therapeutic significance of these findings, similar investigations need to be performed in native neurons and/or in vivo.

Objective: We sought to investigate whether long-term clinical outcomes differ following percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG) in patients with three-vessel disease (3VD) and lesions in the proximal left anterior descending artery (P-LAD)., Methods: This post-hoc analysis of the Synergy between PCI with Taxus and Cardiac Surgery (SYNTAX) Extended Survival study included patients with 3VD who were classified according to the presence or absence of lesions located in the P-LAD. Ten-year all-cause death and 5-year major adverse cardiac or cerebrovascular events (MACCE) were assessed., Results: Among 1088 patients with 3VD, 559 (51.4%) had involvement of P-LAD and their 10-year mortality was numerically higher following PCI versus CABG (28.9% vs 21.9%; HR: 1.39, 95% CI 0.99 to 1.95). Although patients without P-LAD lesions had significantly higher 10-year mortality following PCI compared with CABG, there was no evidence of a treatment-by-subgroup interaction (28.8% vs 20.2%; HR: 1.47, 95% CI 1.03 to 2.09, p interaction =0.837). The incidence of MACCE at 5 years was significantly higher with PCI than CABG, irrespective of involvement of P-LAD (with P-LAD: HR: 1.86, 95% CI 1.36 to 2.55; without P-LAD: HR: 1.54, 95% CI 1.11 to 2.12; p interaction =0.408). Individualised assessment using the SYNTAX Score II 2020 established that a quarter of patients with P-LAD lesions had significantly higher mortality with PCI than CABG, whereas in the remaining three-quarters CABG had similar mortality., Conclusions: Among patients with 3VD, the presence or absence of a P-LAD lesion was not associated with any treatment effect on long-term outcomes following PCI or CABG., Trial Registration Number: SYNTAXES: NCT03417050; SYNTAX: NCT00114972., Competing Interests: Competing interests: PWS reports personal fees from Biosensors, Micel Technologies, Sino Medical Sciences Technology, Philips/Volcano, Xeltis and HeartFlow, outside the submitted work. HH reports a grant for studying overseas from the Japanese Circulation Society and a grant from Fukuda Foundation for Medical Technology, outside the submitted work. JJP reports personal fees and non-financial support from Philips/Volcano, outside the submitted work. M-CM is CEO and shareholder of CERC, a CRO not involved in this trial, and is a minor shareholder of Electroducer. SH reports to work as employee of Medtronic, outside the submitted work. APK reports to work as employee of Medtronic, outside the submitted work. All other authors have no conflict of interest to declare., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Clinical and anatomical characteristics are often considered key factors in deciding between percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG) in patients with complex coronary artery disease (CAD) such as left-main CAD or 3-vessel disease. However, little is known about the interaction between self-reported preprocedural physical/mental health and clinical outcomes after revascularization., Methods: This subgroup analysis of the SYNTAXES trial (SYNTAX Extended Survival), which is the extended follow-up of the randomized SYNTAX trial (Synergy Between PCI With Taxus and Cardiac Surgery) comparing PCI with CABG in patients with left-main CAD or 3-vessel disease, stratified patients by terciles of Physical (PCS) or Mental Component Summary (MCS) scores derived from the preprocedural 36-Item Short Form Health Survey, with higher PCS and MCS scores representing better physical and mental health, respectively. The primary end point was all-cause death at 10 years., Results: A total of 1656 patients with preprocedural 36-Item Short Form Health Survey data were included in the present study. Both higher PCS and MCS were independently associated with lower 10-year mortality (10-point increase in PCS adjusted hazard ratio, 0.84 [95% CI, 0.73-0.97]; P =0.021; in MCS adjusted hazard ratio, 0.85 [95% CI, 0.76-0.95]; P =0.005). A significant survival benefit with CABG over PCI was observed in the highest PCS (>45.5) and MCS (>52.3) terciles with significant treatment-by-subgroup interactions (PCS P interaction =0.033, MCS P interaction =0.015). In patients with both high PCS (>45.5) and MCS (>52.3), 10-year mortality was significantly higher with PCI compared with CABG (30.5% versus 12.2%; hazard ratio, 2.87 [95% CI, 1.55-5.30]; P =0.001), whereas among those with low PCS (≤45.5) or low MCS (≤52.3), there were no significant differences in 10-year mortality between PCI and CABG, resulting in a significant treatment-by-subgroup interaction ( P interaction =0.002)., Conclusions: Among patients with left-main CAD or 3-vessel disease, patient-reported preprocedural physical and mental health status was strongly associated with long-term mortality and modified the relative treatment effects of PCI versus CABG. Patients with the best physical and mental health had better 10-year survival with CABG compared with PCI. Assessment of self-reported physical and mental health is important when selecting the optimal revascularization strategy., Registration: URL: https://www., Clinicaltrials: gov; SYNTAXES Unique identifier: NCT03417050. URL: https://www., Clinicaltrials: gov; SYNTAX Unique identifier: NCT00114972.

The Hedgehog (Hh) cascade is central to development, tissue homeostasis and cancer. A pivotal step in Hh signal transduction is the activation of glioma-associated (GLI) transcription factors by the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO). How SMO activates GLI remains unclear. Here we show that SMO uses a decoy substrate sequence to physically block the active site of the cAMP-dependent protein kinase (PKA) catalytic subunit (PKA-C) and extinguish its enzymatic activity. As a result, GLI is released from phosphorylation-induced inhibition. Using a combination of in vitro, cellular and organismal models, we demonstrate that interfering with SMO-PKA pseudosubstrate interactions prevents Hh signal transduction. The mechanism uncovered echoes one used by the Wnt cascade, revealing an unexpected similarity in how these two essential developmental and cancer pathways signal intracellularly. More broadly, our findings define a mode of GPCR-PKA communication that may be harnessed by a range of membrane receptors and kinases., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Hydropower is a threat to freshwater fishes. Despite a recent boom in dam construction, few studies have assessed their impact on mega-diverse tropical rivers. Using a before-after study design, we investigated the early impacts of the Belo Monte hydroelectric complex, the third-largest hydropower project in the world, on fishes of the Xingu River, a major clear-water tributary of the lower Amazon. We explored impacts across different river sectors (upstream, reservoir, reduced flow sector, and downstream) and spatial scales (individual sectors vs. all sectors combined) using joint species distribution models and different facets of diversity (taxonomic, functional, and phylogenetic). After 5 years of the Belo Monte operation, species richness declined ~12% in lentic and ~16% in lotic environments. Changes in abundance were of less magnitude (<4%). Effects were particularly negative for species of the families Serrasalmidae (mainly pacus), Anostomidae (headstanders), Auchenipteridae, and Pimelodidae (catfishes), whereas no taxonomic group consistently increased in richness or abundance. The reservoir and downstream sectors were the most impacted, with declines of ~24-29% in fish species richness, overall reductions in fish body size and trophic level, and a change in average body shape. Richness and abundance also declined in the reduced river flow, and changes in size, shape, and position of fins were observed. Relatively minor changes were found in the upstream sector. Variation in functional and phylogenetic diversity following river impoundment was subtle; however, across sectors, we found a reduction in functional divergence, indicating a decline in the abundance of species located near the extremities of community functional space. This may be the first sign of an environmental filtering process reducing functional diversity in the region. Greater changes in flow and habitats are expected as hydropower operations ramp up, and continued monitoring is warranted to understand the full scope and magnitude of ecological impacts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Cyclic GMP-dependent protein kinases (PKGs) are key mediators of the nitric oxide/cyclic guanosine monophosphate (cGMP) signaling pathway that regulates biological functions as diverse as smooth muscle contraction, cardiac function, and axon guidance. Understanding how cGMP differentially triggers mammalian PKG isoforms could lead to new therapeutics that inhibit or activate PKGs, complementing drugs that target nitric oxide synthases and cyclic nucleotide phosphodiesterases in this signaling axis. Alternate splicing of PRKG1 transcripts confers distinct leucine zippers, linkers, and auto-inhibitory (AI) pseudo-substrate sequences to PKG Iα and Iβ that result in isoform-specific activation properties, but the mechanism of enzyme auto-inhibition and its alleviation by cGMP is not well understood. Here, we present a crystal structure of PKG Iβ in which the AI sequence and the cyclic nucleotide-binding (CNB) domains are bound to the catalytic domain, providing a snapshot of the auto-inhibited state. Specific contacts between the PKG Iβ AI sequence and the enzyme active site help explain isoform-specific activation constants and the effects of phosphorylation in the linker. We also present a crystal structure of a PKG I CNB domain with an activating mutation linked to Thoracic Aortic Aneurysms and Dissections. Similarity of this structure to wildtype cGMP-bound domains and differences with the auto-inhibited enzyme provide a mechanistic basis for constitutive activation. We show that PKG Iβ auto-inhibition is mediated by contacts within each monomer of the native full-length dimeric protein, and using the available structural and biochemical data we develop a model for the regulation and cooperative activation of PKGs., Competing Interests: RS, JK, LQ, PH, MA, BV, GK, BS, KM, GM, DC, FH, CK No competing interests declared

Background: Hematopoietic stem cell transplantation (HSCT) represents a curative treatment for patients with severe combined immunodeficiency (SCID), a group of monogenic immune disorders with an otherwise fatal outcome., Objective: We performed a comprehensive multicenter analysis of genotype-specific HSCT outcome, including detailed analysis of immune reconstitution (IR) and the predictive value for clinical outcome., Methods: HSCT outcome was studied in 338 patients with genetically confirmed SCID who underwent transplantation in 2006-2014 and who were registered in the SCETIDE registry. In a representative subgroup of 152 patients, data on IR and long-term clinical outcome were analyzed., Results: Two-year OS was similar with matched family and unrelated donors and better than mismatched donor HSCT (P < .001). The 2-year event-free survival (EFS) was similar in matched and mismatched unrelated donor and less favorable in mismatched related donor (MMRD) HSCT (P < .001). Genetic subgroups did not differ in 2-year OS (P = .1) and EFS (P = .073). In multivariate analysis, pretransplantation infections and use of MMRDs were associated with less favorable OS and EFS. With a median follow-up of 6.2 years (range, 2.0-11.8 years), 73 of 152 patients in the IR cohort were alive and well without Ig dependency. IL-2 receptor gamma chain/Janus kinase 3/IL-7 receptor-deficient SCID, myeloablative conditioning, matched donor HSCT, and naive CD4 T lymphocytes >0.5 × 10 e3 /μL at +1 year were identified as independent predictors of favorable clinical and immunologic outcome., Conclusion: Recent advances in HSCT in SCID patients have resulted in improved OS and EFS in all genotypes and donor types. To achieve a favorable long-term outcome, treatment strategies should aim for optimal naive CD4 T lymphocyte regeneration., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Creatinine clearance (CrCl) is an independent determinant of mortality in predictive models of revascularisation outcomes for complex coronary artery disease., Aims: This study aimed to investigate the impact of preprocedural biological markers on 10-year mortality following coronary revascularisation., Methods: The SYNTAX Extended Survival (SYNTAXES) study evaluated the 10-year vital status follow-up of 1,800 patients with de novo three-vessel (3VD) and/or left main coronary artery disease (LMCAD) randomised to include percutaneous or surgical coronary revascularisation. The associations between mortality and preprocedural C-reactive protein (CRP), haemoglobin, HbA1c, CrCl, fasting triglycerides, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol were analysed., Results: Out of 1,800 patients, 460 patients died before the 10-year follow-up. CRP, HbA1c and CrCl with threshold values of ≥2 mg/L, ≥6% (42 mmol/mol) and <60 ml/min, respectively, were associated with 10-year all-cause death (adjusted hazard ratio [95% confidence interval]: 1.35 [1.01-1.82], 1.51 [1.16-1.95], and 1.46 [1.07-2.00], respectively). There was no significant interaction between the biological markers on all-cause mortality and the type of revascularisation. Preprocedural lipid markers were not significantly associated with 10-year all-cause death, but the non-use of statins was a determinant factor of worse prognosis (adjusted hazard ratio [95% confidence interval]: 1.68 [1.26-2.25])., Conclusions: Preprocedural biomarkers, such as CRP and HbA1c, are associated with long-term mortality post revascularisation, regardless of the revascularisation technique. Conventional lipidic biomarkers associated with high-risk of cardiovascular events seem to be effectively mitigated by the long-term use of statins, whereas the non-use of statins was a factor of a worse prognosis, emphasising the importance of pharmacological treatment., Trial Registration: SYNTAXES ClinicalTrials.gov: NCT03417050. SYNTAX ClinicalTrials.gov: NCT00114972.

Protein kinase A (PKA) is a holoenzyme consisting of a regulatory (R)-subunit dimer and two catalytic (C)-subunits. There are two major families of C-subunits, C α and C β , and four functionally nonredundant R-subunits (RI α , RI β , RII α , RII β ). In addition to binding to and being regulated by the R-subunits, the C-subunits are regulated by two tail regions that each wrap around the N- and C-lobes of the kinase core. Although the C-terminal (Ct-) tail is classified as an intrinsically disordered region (IDR), the N-terminal (Nt-) tail is dominated by a strong helix that is flanked by short IDRs. In contrast to the Ct-tail, which is a conserved and highly regulated feature of all PKA, PKG, and protein kinase C protein kinase group (AGC) kinases, the Nt-tail has evolved more recently and is highly variable in vertebrates. Surprisingly and in contrast to the kinase core and the Ct-tail, the entire Nt-tail is not conserved in nonmammalian PKAs. In particular, in humans, C β actually represents a large family of C-subunits that are highly variable in their Nt-tail and also expressed in a highly tissue-specific manner. Although we know so much about the C α 1-subunit, we know almost nothing about these C β isoforms wherein C β 2 is highly expressed in lymphocytes, and C β 3 and C β 4 isoforms account for ∼50% of PKA signaling in brain. Based on recent disease mutations, the C β proteins appear to be functionally important and nonredundant with the C α isoforms. Imaging in retina also supports nonredundant roles for C β as well as isoform-specific localization to mitochondria. This represents a new frontier in PKA signaling. SIGNIFICANCE STATEMENT: How tails and adjacent domains regulate each protein kinase is a fundamental challenge for the biological community. Here we highlight how the N- and C-terminal tails of PKA (Nt-tails/Ct-tails) affect the structure and regulate the function of the kinase core and show the combinatorial variations that are introduced into the Nt-tail of the C α - and C β -subunits in contrast to the Ct-tail, which is conserved across the entire AGC subfamily of protein kinases., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Mutations in the gene coding for leucine-rich repeat kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinson's disease (PD), while LRRK2 overactivation is also associated with the more common idiopathic form of PD. LRRK2 is a large multidomain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common, disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for drug discovery. Currently, drug development has mainly focused on ATP-competitive kinase inhibitors. Here, we report the identification and characterization of a variety of nanobodies that bind to different LRRK2 domains and inhibit or activate LRRK2 in cells and in in vitro. Importantly, nanobodies were identified that inhibit LRRK2 kinase activity while binding to a site that is topographically distinct from the active site and thus act through an allosteric inhibitory mechanism that does not involve binding to the ATP pocket or even to the kinase domain. Moreover, while certain nanobodies completely inhibit the LRRK2 kinase activity, we also identified nanobodies that specifically inhibit the phosphorylation of Rab protein substrates. Finally, in contrast to current type I kinase inhibitors, the studied kinase-inhibitory nanobodies did not induce LRRK2 microtubule association. These comprehensively characterized nanobodies represent versatile tools to study the LRRK2 function and mechanism and can pave the way toward novel diagnostic and therapeutic strategies for PD., Competing Interests: Competing interest statement: R.K.S., A.K., C.J.G, and W.V. are inventors on a filed patent covering findings described in this manuscript (publication number: WO/2021/170540). All other authors declare no competing interests., (Copyright © 2022 the Author(s). Published by PNAS.)

The 2 major molecular switches in biology, kinases and GTPases, are both contained in the Parkinson disease-related leucine-rich repeat kinase 2 (LRRK2). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations, we generated a comprehensive dynamic allosteric portrait of the C-terminal domains of LRRK2 (LRRK2RCKW). We identified 2 helices that shield the kinase domain and regulate LRRK2 conformation and function. One helix in COR-B (COR-B Helix) tethers the COR-B domain to the αC helix of the kinase domain and faces its activation loop, while the C-terminal helix (Ct-Helix) extends from the WD40 domain and interacts with both kinase lobes. The Ct-Helix and the N-terminus of the COR-B Helix create a "cap" that regulates the N-lobe of the kinase domain. Our analyses reveal allosteric sites for pharmacological intervention and confirm the kinase domain as the central hub for conformational control., Competing Interests: The authors have declared that no competing interests exist.

In 1984, Japanese researchers led by the biochemist Hiroyoshi Hidaka described the first synthetic protein kinase inhibitors based on an isoquinoline sulfonamide structure (Hidaka et al. Biochemistry, 1984 Oct 9; 23(21): 5036-41. doi: 10.1021/bi00316a032). These led to the first protein kinase inhibitor approved for medical use (fasudil), an inhibitor of the AGC subfamily Rho kinase. With potencies strong enough to compete against endogenous ATP, the isoquinoline compounds established the druggability of the ATP binding site. Crystal structures of their protein kinase complexes, including with cAMP-dependent protein kinase (PKA), showed interactions that, on the one hand, could mimic ATP but, on the other hand, could be optimized for high potency binding, kinase selectivity, and diversification away from adenosine. They also showed the flexibility of the glycine-rich loop, and PKA became a major prototype for crystallographic and nuclear magnetic resonance (NMR) studies of protein kinase mechanism and dynamic activity control. Since fasudil, more than 70 kinase inhibitors have been approved for clinical use, involving efforts that progressively have introduced new paradigms of data-driven drug discovery. Publicly available data alone comprise over 5000 protein kinase crystal structures and hundreds of thousands of binding data. Now, new methods, including artificial intelligence techniques and expansion of protein kinase targeting approaches, together with the expiration of patent protection for optimized inhibitor scaffolds, promise even greater advances in drug discovery. Looking back to the time of the first isoquinoline hinge binders brings the current state-of-the-art into stark contrast. Appropriately for this Perspective article, many of the milestone papers during this time were published in Biochemistry (now ACS Biochemistry).

Leucine-Rich Repeat Kinase 2 (LRRK2) is a large, multidomain protein with dual kinase and GTPase function that is commonly mutated in both familial and idiopathic Parkinson's Disease (PD). While dimerization of LRRK2 is commonly detected in PD models, it remains unclear whether inhibition of dimerization can regulate catalytic activity and pathogenesis. Here, we show constrained peptides that are cell-penetrant, bind LRRK2, and inhibit LRRK2 activation by downregulating dimerization. We further show that inhibited dimerization decreases kinase activity and inhibits ROS production and PD-linked apoptosis in primary cortical neurons. While many ATP-competitive LRRK2 inhibitors induce toxicity and mislocalization of the protein in cells, these constrained peptides were found to not affect LRRK2 localization. The ability of these peptides to inhibit pathogenic LRRK2 kinase activity suggests that disruption of dimerization may serve as a new allosteric strategy to downregulate PD-related signaling pathways.

Background: Women with Li-Fraumeni syndrome (LFS) have elevated breast cancer (BC) risk. Optimal BC treatment strategies in this population are yet unknown., Methods: BC subtypes and treatment were retrospectively investigated between December 2016 and January 2019 in a multicentre study. BC risks were evaluated according to the type of surgery., Results: Thirty-five women of our study population (35/44; 79.5%) had developed 36 breast lesions at first diagnosis at a mean age of 34 years. Those breast lesions comprised 32 invasive BCs (89%), three ductal carcinoma in situ alone (8%) and one malignant phyllodes tumour (3%). BCs were mainly high-grade (18/32), of no special type (NST; 31/32), HER2-enriched (11/32) or luminal-B-(like)-type (10/32). Affected women (n = 35) received breast-conserving surgery (BCS, n = 17) or a mastectomy (ME, n = 18) including seven women with simultaneous contralateral prophylactic mastectomy (CPM) at first diagnosis. Nineteen women suffered 20 breast or locoregional axillary lesions at second diagnosis with mean age of 36. Median time between first and second diagnosis was 57 months; median time to contra- and ipsilateral recurrence depended on surgical strategies (BCS: 46 vs. unilateral ME: 93 vs. bilateral ME > 140 months). Women with a primary treatment of solitaire therapeutic ME suffered from contralateral BC earlier compared to those with therapeutic ME and CPM (median: 93 vs. >140 months)., Conclusion: Aggressive BC subtypes occur among women with LFS. Surgical treatment, i.e. ME and CPM, may prolong time to a second BC diagnosis. Conclusion on long-term survival benefit is pending. Individual competing tumour risks and long-term outcomes need to be taken into consideration., (© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: The significant interaction between major infection and 5-year mortality after percutaneous coronary intervention (PCI) or coronary artery bypass graft (CABG) for complex coronary artery disease (CAD) was observed previously. However, the very long-term outcomes beyond 5 years remains unclear., Methods and Results: This is a subgroup analysis of the SYNTAX Extended Survival (SYNTAXES) trial, which is the extended follow-up of the randomized SYNTAX trial comparing PCI versus CABG in patients with three-vessel disease (3VD) or left-main CAD (LMCAD). Out of 1517 patients enrolled in the SYNTAX trial with available survival status from 5 to 10 years, 140 patients had experienced major infections and survived at 5 years (major infection group). From 5 to 10 years, the mortality of major infection group was 19.8% whereas the mortality of no major infection group was 15.1% (p = 0.157). After the adjustment of other clinical factors, the risk of mortality from 5 to 10 years did not significantly differ between major infection and no major infection groups (HR: 1.10; 95% CI: 0.62-1.96; p = 0.740). When stratified by the presence or absence of periprocedural major infections, defined as a major infection within 60 days after index procedure, there was also no significant difference in 10-year mortality between two groups (30.8% vs. 24.5%; p = 0.057)., Conclusions: Despite the initial association between major infections and 5 years mortality, postprocedural major infection was not evident in the 10 years follow-up, suggesting that the impact of major infection on mortality subsided over time beyond 5 years., Trial Registration: SYNTAXES ClinicalTrials.gov reference: NCT03417050 SYNTAX ClinicalTrials.gov reference: NCT00114972., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Many of the fundamental concepts of signal transduction and kinase activity are attributed to the discovery and crystallization of cAMP-dependent protein kinase, or protein kinase A. PKA is one of the best-studied kinases in human biology, with emphasis in biochemistry and biophysics, all the way to metabolism, hormone action, and gene expression regulation. It is surprising, however, that our understanding of PKA's role in disease is largely underappreciated. Although genetic mutations in the PKA holoenzyme are known to cause diseases such as Carney complex, Cushing syndrome, and acrodysostosis, the story largely stops there. With the recent explosion of genomic medicine, we can finally appreciate the broader role of the G α s-PKA pathway in disease, with contributions from aberrant functioning G proteins and G protein-coupled receptors, as well as multiple alterations in other pathway components and negative regulators. Together, these represent a broad family of diseases we term the G α s-PKA pathway signalopathies. The G α s-PKA pathway signalopathies encompass diseases caused by germline, postzygotic, and somatic mutations in the G α s-PKA pathway, with largely endocrine and neoplastic phenotypes. Here, we present a signaling-centric review of G α s-PKA-driven pathophysiology and integrate computational and structural analysis to identify mutational themes commonly exploited by the G α s-PKA pathway signalopathies. Major mutational themes include hotspot activating mutations in Gαs, encoded by GNAS, and mutations that destabilize the PKA holoenzyme. With this review, we hope to incite further study and ultimately the development of new therapeutic strategies in the treatment of a wide range of human diseases. SIGNIFICANCE STATEMENT: Little recognition is given to the causative role of G α s-PKA pathway dysregulation in disease, with effects ranging from infectious disease, endocrine syndromes, and many cancers, yet these disparate diseases can all be understood by common genetic themes and biochemical signaling connections. By highlighting these common pathogenic mechanisms and bridging multiple disciplines, important progress can be made toward therapeutic advances in treating G α s-PKA pathway-driven disease., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Objectives: We investigated the impact of total stent length (TSL) and average nominal stent diameter (ASD) on 10-year mortality after percutaneous coronary intervention (PCI) in the SYNTAXES trial., Background: TSL and ASD in patients treated with PCI are associated with major adverse cardiovascular events. However, the treatment effect of PCI with extensive and/or small stenting as compared with coronary artery bypass grafting (CABG) for complex coronary artery disease has not been fully evaluated., Methods: Impacts on mortality of extensive stenting defined as TSL >100 mm and small stenting as ASD <3 mm were analyzed in 893 PCI patients and were compared to 865 CABG patients., Results: TSL as a continuous variable was significantly associated with 10-year mortality (adjusted hazard ratio [HR], 1.05 [1.01-1.09] per 10 mm increase). PCI patients with extensive stenting had a higher 10 year mortality than CABG patients (adjusted HR, 1.97 [1.41-2.74]) or not- extensive stenting PCI (adjusted HR, 1.94 [1.36-2.77]). Although ASD did not have a significant association with 10 year mortality (adjusted HR, 0.97 [0.85-1.11] per 0.25 mm increase), PCI with small stents was associated with a higher 10 year mortality, compared to CABG (adjusted HR, 1.66 [1.23-2.26]) and PCI performed with large stents (adjusted HR, 1.74 [1.19-2.53]). Patients treated with not-extensive and large stents had similar mortality rates (24.0 versus 23.8%) as those treated with CABG., Conclusions: Extensive and small stenting were associated with higher 10 year mortality, compared with CABG. When patients have to be treated with extensive or small stenting, revascularization with CABG should be preferred., (© 2021 The Authors. Catheterization and Cardiovascular Interventions published by Wiley Periodicals LLC.)

Very-low-carbohydrate diet triggers the endogenous production of ketone bodies as alternative energy substrates. There are as yet unproven assumptions that ketone bodies positively affect human immunity. We have investigated this topic in an in vitro model using primary human T cells and in an immuno-nutritional intervention study enrolling healthy volunteers. We show that ketone bodies profoundly impact human T-cell responses. CD4 + , CD8 + , and regulatory T-cell capacity were markedly enhanced, and T memory cell formation was augmented. RNAseq and functional metabolic analyses revealed a fundamental immunometabolic reprogramming in response to ketones favoring mitochondrial oxidative metabolism. This confers superior respiratory reserve, cellular energy supply, and reactive oxygen species signaling. Our data suggest a very-low-carbohydrate diet as a clinical tool to improve human T-cell immunity. Rethinking the value of nutrition and dietary interventions in modern medicine is required., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Expression of signaling proteins in bone cells depends on their embryological mesoderm-derived (e.g. tibia) or cranial neural crest (CNC)-derived (e.g. jaw) origin. Connexin 43 (Cx43) is a gap junction protein that plays an essential role in the mode of action of bisphosphonates (BP). This study aimed to investigate Cx43 expression and the influence of BP application on mesoderm- and CNC-derived bone. Using a rat model, molar extraction and tibia osteotomy with (Group 4) or without (Group 3) previous BP application was performed. Untreated (Group 1) and animals selectively treated with BPs (Group 2) served as controls. Cx43 expression was immunohistochemically determined 12 and 16 weeks postoperatively via a labeling index. Cx43 expression in CNC-derived bone was significantly higher compared with mesodermal bone. BP application decreased Cx43 expression; however, detected expression levels were still higher in jawbone (Group 2 tibia vs jaw: 5.83 ± 5.06 vs 23.52 ± 6.42; p = 0.007). During bone healing after surgical intervention (Group 3) there were no expression differences between tibia and jawbone. BP treatment prior to surgery resulted in significantly lower Cx43 expression in CNC-derived compared with tibia bone (Group 4 tibia vs jaw: 56.84 ± 15.57 vs 16.40 ± 5.66; p < 0.01). Increased Cx43 expression in jaw compared with tibia bone is in line with their embryological origins. A significant Cx43 suppression in jawbone after BP application and surgery might contribute to the selectively altered osseous turnover and development of MRONJ in CNC-derived bone., Competing Interests: Declaration of Competing Interest The authors of this manuscript have no conflicts of interest to disclose., (Copyright © 2021 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Coronary artery bypass grafting (CABG) was introduced in the 1960s as the first procedure for direct coronary artery revascularization and rapidly became one of the most common surgical procedures worldwide, with an overall total of more than 20 million operations performed. CABG continues to be the most common cardiac surgical procedure performed and has been one of the most carefully studied therapies. Best CABG techniques, optimal bypass conduits, and appropriate patient selection have been rigorously tested in landmark clinical trials, some of which have resolved controversy and most of which have stoked further debate and trials. The evolution of CABG cannot be properly portrayed without presenting it in the context of the parallel development of percutaneous coronary intervention. In this Historical Perspective, we a provide a broad overview of the history of coronary revascularization with a focus on the foundations, evolution, best evidence, and future directions of CABG., Competing Interests: Funding Support and Author Disclosures The authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2021 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Controlled transport of surface-functionalized magnetic beads in a liquid medium is a central requirement for the handling of captured biomolecular targets in microfluidic lab-on-chip biosensors. Here, the influence of the physiological liquid medium on the transport characteristics of functionalized magnetic particles and on the functionality of the coupled protein is studied. These aspects are theoretically modeled and experimentally investigated for prototype superparamagnetic beads, surface-functionalized with green fluorescent protein immersed in buffer solution with different concentrations of a surfactant. The model reports on the tunability of the steady-state particle substrate separation distance to prevent their surface sticking via the choice of surfactant concentration. Experimental and theoretical average velocities are discussed for a ratchet-like particle motion induced by a dynamic external field superposed on a static locally varying magnetic field landscape. The developed model and experiment may serve as a basis for quantitative forecasts on the functionality of magnetic particle transport-based lab-on-chip devices.

Objectives: Minimally invasive staged segmental artery (SA) coil- and plug embolization is a new method for paraplegia prevention associated with extensive aortic procedures. Near-infrared spectroscopy of the paraspinal collateral network (cnNIRS) has emerged as a non-invasive method for spinal cord monitoring. The aim of this study was to evaluate cnNIRS to guide minimally invasive SA occlusion., Methods: In a chronic large animal experiment, 18 juvenile pigs underwent two-stage minimally invasive staged SA coil- and plug embolization for complete SA occlusion. Coil-embolization was performed either by SA main stem occlusion (characteristic of pig anatomy) or separately for the left- and right SA. Lumbar cnNIRS was recorded during and after the procedure. Neurological status was assessed up to 3 days after complete SA occlusion., Results: Mean time from SA coil embolization to minimum cnNIRS values was 11 ± 5 min with an average decrease from 101 ± 2% to 78 ± 8% of baseline (difference: -23 ± 9, P < 0.001). Lumbar cnNIRS demonstrated significant differences between left and right when SAs were occluded separately in all cases (-7 ± 4%, 1 min after first SA occlusion; P = 0.001). Permanent paraplegia occurred in 2 (11%) and any kind of neurological deficit-temporary or permanent-in 7 animals (39%). Association between lumbar cnNIRS and neurological outcome after minimally invasive staged SA coil- and plug embolization suggests positive correlation (R = 0.5, P = 0.052)., Conclusions: Lumbar cnNIRS independently reacts to unilateral SA occlusion. cnNIRS-guided SA occlusion is feasible and may become a useful adjunct facilitating adequate and complete vessel occlusion., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Aims: To evaluate the impact of chronic obstructive pulmonary disease (COPD) on 10-year all-cause death and the treatment effect of CABG versus PCI on 10-year all-cause death in patients with three-vessel disease (3VD) and/or left main coronary artery disease (LMCAD) and COPD., Methods: Patients were stratified according to COPD status and compared with regard to clinical outcomes. Ten-year all-cause death was examined according to the presence of COPD and the revascularization strategy., Results: COPD status was available for all randomized 1800 patients, of whom, 154 had COPD (8.6%) at the time of randomization. Regardless of the revascularization strategy, patients with COPD had a higher risk of 10-year all-cause death, compared with those without COPD (43.1% vs. 24.9%; hazard ratio [HR]: 2.03; 95% confidence interval [CI]: 1.56-2.64; p < 0.001). Among patients with COPD, CABG appeared to have a slightly lower risk of 10-year all-cause death compared with PCI (42.3% vs. 43.9%; HR: 0.96; 95% CI: 0.59-1.56, p = 0.858), whereas among those without COPD, CABG had a significantly lower risk of 10-year all-cause death (22.7% vs. 27.1%; HR: 0.81; 95% CI: 0.67-0.99, p = 0.041). There was no significant differential treatment effect of CABG versus PCI on 10-year all-cause death between patients with and without COPD (p interaction  = 0.544)., Conclusions: COPD was associated with a higher risk of 10-year all-cause death after revascularization for complex coronary artery disease. The presence of COPD did not significantly modify the beneficial effect of CABG versus PCI on 10-year all-cause death., Trial Registration: SYNTAX: ClinicalTrials.gov reference: NCT00114972. SYNTAX Extended Survival: ClinicalTrials.gov reference: NCT03417050.

The Plasmodium falciparum cGMP-dependent protein kinase ( Pf PKG) is required for the progression of the Plasmodium 's life cycle and is therefore a promising malaria drug target. Pf PKG includes four cGMP-binding domains (CBD-A to CBD-D). CBD-D plays a crucial role in Pf PKG regulation as it is the primary determinant for the inhibition and cGMP-dependent activation of the catalytic domain. Hence, it is critical to understand how CBD-D is allosterically regulated by cGMP. Although the apo versus holo conformational changes of CBD-D have been reported, information on the intermediates of the activation pathway is currently lacking. Here, we employed molecular dynamics simulations to model four key states along the thermodynamic cycle for the cGMP-dependent activation of the Pf PKG CBD-D domain. The simulations were compared to NMR data, and they revealed that the Pf PKG CBD-D activation pathway samples a compact intermediate in which the N- and C-terminal helices approach the central β-barrel. In addition, by comparing the cGMP-bound active and inactive states, the essential binding interactions that differentiate these states were identified. The identification of structural and dynamical features unique to the cGMP-bound inactive state provides a promising basis to design Pf PKG-selective allosteric inhibitors as a viable treatment for malaria.

High-nuclearity Mn complexes have attracted significant scientific attention due to their fascinating magnetic properties and their relevance to bioinorganic systems and catalysis. In this work, we demonstrate how the strong binding characteristics of phosphonate ligands can be coupled with sterically hindered carboxylate groups to influence the symmetry of Mn coordination clusters. We describe the structure of two high-nuclearity Mn coordination cages, {Mn 12 } and {Mn 15 }, synthesized using this approach. These cages are structurally related to the truncated tetrahedral geometry and adopt rare topological features of Archimedean and Johnson-type solids. Their structural attributes distinctively influence their magnetic properties and electrocatalytic H 2 O oxidation characteristics.

3',5'-cyclic adenosine monophosphate (cAMP) dependent protein kinase or protein kinase A (PKA) has served as a prototype for the large family of protein kinases that are crucially important for signal transduction in eukaryotic cells. The PKA catalytic subunits are encoded by the two major genes PRKACA and PRKACB, respectively. The PRKACA gene encodes two known splice variants, the ubiquitously expressed Cα1 and the sperm-specifically expressed Cα2. In contrast, the PRKACB gene encodes several splice variants expressed in a highly cell and tissue-specific manner. The Cβ proteins are called Cβ1, Cβ2, Cβ3, Cβ4 and so-called abc variants of Cβ3 and Cβ4. Whereas Cβ1 is ubiquitously expressed, Cβ2 is enriched in immune cells and the Cβ3, Cβ4 and their abc variants are solely expressed in neuronal cells. All Cα and Cβ splice variants share a kinase-conserved catalytic core and a C-terminal tail encoded by exons 2 through 10 in the PRKACA and PRKACB genes, respectively. All Cα and Cβ splice variants with the exception of Cα1 and Cβ1 are hyper-variable at the N-terminus. Here, we will discuss how the PRKACA and PRKACB genes have developed as paralogs that encode distinct and functionally non-redundant proteins. The fact that Cα and Cβ splice variant mutations are associated with numerous diseases further opens new windows for PKA-induced disease pathologies., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Background: The optimal revascularization strategy for the elderly with complex coronary artery disease remains unclear., Objectives: The goal of this study was to investigate 10-year all-cause mortality, life expectancy, 5-year major adverse cardiac or cerebrovascular events (MACCE), and 5-year quality of life (QOL) after percutaneous coronary intervention (PCI) or coronary artery bypass graft (CABG) in elderly individuals (>70 years old) with 3-vessel disease (3VD) and/or left main disease (LMD)., Methods: In the present pre-specified analysis on age of the SYNTAX Extended Survival study, 10-year all-cause death and 5-year MACCE were compared with Kaplan-Meier estimates and Cox proportional hazards models among elderly or nonelderly patients. Life expectancy was estimated by restricted mean survival time within 10 years, and QOL status according to the Seattle Angina Questionnaire up to 5 years was assessed by linear mixed-effects models., Results: Among 1,800 randomized patients, 575 patients (31.9%) were elderly. Ten-year mortality did not differ significantly between PCI and CABG in elderly (44.1% vs. 41.1%; hazard ratio [HR]: 1.08; 95% confidence interval [CI]: 0.84 to 1.40) and nonelderly patients (21.1% vs. 16.6%; HR: 1.30; 95% CI: 1.00 to 1.69; p interaction  = 0.332). Among elderly patients, 5-year MACCE was comparable between PCI and CABG (39.4% vs. 35.1%; HR: 1.18; 95% CI: 0.90 to 1.56), whereas it was significantly higher in PCI over CABG among nonelderly patients (36.3% vs. 23.0%; HR: 1.69; 95% CI: 1.36 to 2.10; p interaction  = 0.043). There were no significant difference in life expectancy (mean difference: 0.2 years in favor of CABG; 95% CI: -0.4 to 0.7) and 5-year QOL status between PCI and CABG among elderly patients., Conclusions: Elderly patients with 3VD and/or LMD had comparable 10-year all-cause death, life expectancy, 5-year MACCE, and 5-year QOL status irrespective of revascularization mode. (Synergy Between PCI With TAXUS and Cardiac Surgery: SYNTAX Extended Survival [SYNTAXES]; NCT03417050) (SYNTAX Study: TAXUS Drug-Eluting Stent Versus Coronary Artery Bypass Surgery for the Treatment of Narrowed Arteries [SYNTAX]; NCT00114972)., Competing Interests: Funding Support and Author Disclosures The SYNTAX Extended Survival study, during extension of follow-up up to 10 years, was supported by the German Foundation of Heart Research (Frankfurt am Main, Germany). The SYNTAX trial, during 0- to 5-year follow-up, was funded by Boston Scientific Corporation (Marlborough, Massachusetts). The study funders had no role in the study design, data collection, data analyses, and interpretation of the study data, nor were involved in the decision to publish the final manuscript. The principal investigators and authors had complete scientific freedom. Dr. Serruys has received personal fees from Biosensors, Micel Technologies, Sinomedical Sciences Technology, Philips/Volcano, Xeltis, and HeartFlow, outside the submitted work. Dr. Hara has received a grant for studying overseas from Japanese Circulation Society and a grant from Fukuda Foundation for Medical Technology, outside the submitted work. Dr. Piek has received personal fees and nonfinancial support from Philips/Volcano, outside the submitted work. Dr. Head has served as an employee of Medtronic, outside the submitted work. Dr. Kappetein has served as an employee of Medtronic, outside the submitted work. Dr. Cohen has received grants from Boston Scientific, during the conduct of the study; and has received grants and personal fees from Boston Scientific, Medtronic, Abbott, and Svelte, outside the submitted work. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2021 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

To explore how pathogenic mutations of the multidomain leucine-rich repeat kinase 2 (LRRK2) hijack its finely tuned activation process and drive Parkinson's disease (PD), we used a multitiered approach. Most mutations mimic Rab-mediated activation by "unleashing" kinase activity, and many, like the kinase inhibitor MLi-2, trap LRRK2 onto microtubules. Here we mimic activation by simply deleting the inhibitory N-terminal domains and then characterize conformational changes induced by MLi-2 and PD mutations. After confirming that LRRK2 RCKW retains full kinase activity, we used hydrogen-deuterium exchange mass spectrometry to capture breathing dynamics in the presence and absence of MLi-2. Solvent-accessible regions throughout the entire protein are reduced by MLi-2 binding. With molecular dynamics simulations, we created a dynamic portrait of LRRK2 RCKW and demonstrate the consequences of kinase domain mutations. Although all domains contribute to regulating kinase activity, the kinase domain, driven by the DYGψ motif, is the allosteric hub that drives LRRK2 regulation., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

We review the role of signaling pathways in regulation of the key processes of merozoite egress and red blood cell invasion by Plasmodium falciparum and, in particular, the importance of the second messengers, cAMP and Ca 2+ , and cyclic nucleotide dependent kinases. cAMP-dependent protein kinase (PKA) is comprised of cAMP-binding regulatory, and catalytic subunits. The less well conserved cAMP-binding pockets should make cAMP analogs attractive drug leads, but this approach is compromised by the poor membrane permeability of cyclic nucleotides. We discuss how the conserved nature of ATP-binding pockets makes ATP analogs inherently prone to off-target effects and how ATP analogs and genetic manipulation can be useful research tools to examine this. We suggest that targeting PKA interaction partners as well as substrates, or developing inhibitors based on PKA interaction sites or phosphorylation sites in PKA substrates, may provide viable alternative approaches for the development of anti-malarial drugs. Proximity of PKA to a substrate is necessary for substrate phosphorylation, but the P. falciparum genome encodes few recognizable A-kinase anchor proteins (AKAPs), suggesting the importance of PKA-regulatory subunit myristylation and membrane association in determining substrate preference. We also discuss how Pf14-3-3 assembles a phosphorylation-dependent signaling complex that includes PKA and calcium dependent protein kinase 1 (CDPK1) and how this complex may be critical for merozoite invasion, and a target to block parasite growth. We compare altered phosphorylation levels in intracellular and egressed merozoites to identify potential PKA substrates. Finally, as host PKA may have a critical role in supporting intracellular parasite development, we discuss its role at other stages of the life cycle, as well as in other apicomplexan infections. Throughout our review we propose possible new directions for the therapeutic exploitation of cAMP-PKA-signaling in malaria and other diseases caused by apicomplexan parasites., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lasonder, More, Singh, Haidar, Bertinetti, Kennedy, Herberg, Holder, Langsley and Chitnis.)

Metal-organic frameworks (MOFs) encompass a rapidly expanding class of materials with diverse potential applications including gas storage, molecular separation, sensing and catalysis. So-called 'rod MOFs', which comprise infinitely extended 1D secondary building units (SBUs), represent an underexplored subclass of MOF. Further, porphyrins are considered privileged ligands for MOF synthesis due to their tunable redox and photophysical properties. In this study, the Cu II complex of 5,15-bis(4-carboxyphenyl)-10,20-diphenylporphyrin (H 2 L -Cu II , where H 2 refers to the ligand's carboxyl H atoms) is used to prepare two new 2D porphyrinic rod MOFs PROD-1 and PROD-2. Single-crystal X-ray analysis reveals that these frameworks feature 1D Mn II - or Co II -based rod-like SBUs that are coordinated by labile solvent molecules and photoactive porphyrin moieties. Both materials were characterised using infrared (IR) spectroscopy, powder X-ray diffraction (PXRD) spectroscopy and thermogravimetric analysis (TGA). The structural attributes of PROD-1 and PROD-2 render them promising materials for future photocatalytic investigations.