Integrative taxonomy clarifies the evolution of a cryptic primate clade.

Global biodiversity is under accelerating threats, and species are succumbing to extinction before being described. Madagascar's biota represents an extreme example of this scenario, with the added complication that much of its endemic biodiversity is cryptic. Here we illustrate best practices for clarifying cryptic diversification processes by presenting an integrative framework that leverages multiple lines of evidence and taxon-informed cut-offs for species delimitation, while placing special emphasis on identifying patterns of isolation by distance. We systematically apply this framework to an entire taxonomically controversial primate clade, the mouse lemurs (genus Microcebus, family Cheirogaleidae). We demonstrate that species diversity has been overestimated primarily due to the interpretation of geographic variation as speciation, potentially biasing inference of the underlying processes of evolutionary diversification. Following a revised classification, we find that crypsis within the genus is best explained by a model of morphological stasis imposed by stabilizing selection and a neutral process of niche diversification. Finally, by clarifying species limits and defining evolutionarily significant units, we provide new conservation priorities, bridging fundamental and applied objectives in a generalizable framework.

Biosynthesis of the corallorazines, a widespread class of antibiotic cyclic lipodipeptides.

Corallorazines are cyclic lipodipeptide natural products produced by the myxobacterium Corallococcus coralloides B035. To decipher the basis of corallorazine biosynthesis, the corallorazine nonribosomal peptide synthetase (NRPS) biosynthetic gene cluster crz was identified and analyzed in detail. Here, we present a model of corallorazine biosynthesis, supported by bioinformatic analyses and in vitro investigations on the bimodular NRPS synthesizing the corallorazine core. Corallorazine biosynthesis shows several distinct features, such as the presence of a dehydrating condensation domain, and a unique split adenylation domain on two open reading frames. Using an alternative fatty acyl starter unit, the first steps of corallorazine biosynthesis were characterized in vitro, supporting our biosynthetic model. The dehydrating condensation domain was bioinformatically analyzed in detail and compared to other modifying C domains, revealing unreported specific sequence motives for this domain subfamily. Using global bioinformatics analyses, we show that the crz gene cluster family is widespread among bacteria and encodes notable chemical diversity. Corallorazine A displays moderate antimicrobial activity against selected Gram-positive and Gram-negative bacteria. Mode of action studies comprising whole cell analysis and in vitro test systems revealed that corallorazine A inhibits bacterial transcription by targeting the DNA-dependent RNA polymerase.

Structural response of G protein binding to the cyclodepsipeptide inhibitor FR900359 probed by NMR spectroscopy.

The cyclodepsipeptide FR900359 (FR) and its analogs are able to selectively inhibit the class of Gq proteins by blocking GDP/GTP exchange. The inhibitor binding site of Gq has been characterized by X-ray crystallography, and various binding and functional studies have determined binding kinetics and mode of inhibition. Here we investigate isotope-labeled FR bound to the membrane-anchored G protein heterotrimer by solid-state nuclear magnetic resonance (ssNMR) and in solution by liquid-state NMR. The resulting data allowed us to identify regions of the inhibitor which show especially pronounced effects upon binding and revealed a generally rigid binding mode in the cis conformation under native-like conditions. The inclusion of the membrane environment allowed us to show a deep penetration of FR into the lipid bilayer illustrating a possible access mode of FR into the cell. Dynamic nuclear polarization (DNP)-enhanced ssNMR was used to observe the structural response of specific segments of the Gα subunit to inhibitor binding. This revealed rigidification of the switch I binding site and an allosteric response in the α5 helix as well as suppression of structural changes induced by nucleotide exchange due to inhibition by FR. Our NMR studies of the FR-G protein complex conducted directly within a native membrane environment provide important insights into the inhibitors access via the lipid membrane, binding mode, and structural allosteric effects.

Pharmacological Gq inhibition induces strong pulmonary vasorelaxation and reverses pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a life-threatening disease with limited survival. Herein, we propose the pharmacological inhibition of Gq proteins as a novel concept to counteract pulmonary vasoconstriction and proliferation/migration of pulmonary artery smooth muscle cells (PASMCs) in PAH. We demonstrate that the specific pan-Gq inhibitor FR900359 (FR) induced a strong vasorelaxation in large and small pulmonary arteries in mouse, pig, and human subjects ex vivo. Vasorelaxation by FR proved at least as potent as the currently used triple therapy. We also provide in vivo evidence that local pulmonary application of FR prevented right ventricular systolic pressure increase in healthy mice as well as in mice suffering from hypoxia (Hx)-induced pulmonary hypertension (PH). In addition, we demonstrate that chronic application of FR prevented and also reversed Sugen (Su)Hx-induced PH in mice. We also demonstrate that Gq inhibition reduces proliferation and migration of PASMCs in vitro. Thus, our work illustrates a dominant role of Gq proteins for pulmonary vasoconstriction as well as remodeling and proposes direct Gq inhibition as a powerful pharmacological strategy in PH.

GRK specificity and Gßγ dependency determines the potential of a GPCR for arrestin-biased agonism.

G protein-coupled receptors (GPCRs) are mainly regulated by GPCR kinase (GRK) phosphorylation and subsequent ß-arrestin recruitment. The ubiquitously expressed GRKs are classified into cytosolic GRK2/3 and membrane-tethered GRK5/6 subfamilies. GRK2/3 interact with activated G protein ßγ-subunits to translocate to the membrane. Yet, this need was not linked as a factor for bias, influencing the effectiveness of ß-arrestin-biased agonist creation. Using multiple approaches such as GRK2/3 mutants unable to interact with Gßγ, membrane-tethered GRKs and G protein inhibitors in GRK2/3/5/6 knockout cells, we show that G protein activation will precede GRK2/3-mediated ß-arrestin2 recruitment to activated receptors. This was independent of the source of free Gßγ and observable for Gs-, Gi- and Gq-coupled GPCRs. Thus, ß-arrestin interaction for GRK2/3-regulated receptors is inseparably connected with G protein activation. We outline a theoretical framework of how GRK dependence on free Gßγ can determine a GPCR's potential for biased agonism. Due to this inherent cellular mechanism for GRK2/3 recruitment and receptor phosphorylation, we anticipate generation of ß-arrestin-biased ligands to be mechanistically challenging for the subgroup of GPCRs exclusively regulated by GRK2/3, but achievable for GRK5/6-regulated receptors, that do not demand liberated Gßγ. Accordingly, GRK specificity of any GPCR is foundational for developing arrestin-biased ligands.

Adjuvant Gemcitabine Versus Neoadjuvant/Adjuvant FOLFIRINOX in Resectable Pancreatic Cancer: The Randomized Multicenter Phase II NEPAFOX Trial.

BACKGROUND: Although addition of adjuvant chemotherapy is the current standard, the prognosis of pancreatic cancers still remains poor. The NEPAFOX trial evaluated perioperative treatment with FOLFIRINOX in resectable pancreatic cancer. PATIENTS AND METHODS: This multicenter phase II trial randomized patients with resectable or borderline resectable pancreatic cancer without metastases into arm (A,) upfront surgery plus adjuvant gemcitabine, or arm (B,) perioperative FOLFIRINOX. The primary endpoint was overall survival (OS). RESULTS: Owing to poor accrual, recruitment was prematurely stopped after randomization of 40 of the planned 126 patients (A: 21, B: 19). Overall, approximately three-quarters were classified as primarily resectable (A: 16, B: 15), and the remaining patients were classified as borderline resectable (A: 5, B: 4). Of the 12 evaluable patients, 3 achieved partial response under neoadjuvant FOLFIRINOX. Of the 21 patients in arm A and 19 patients in arm B, 17 and 7 underwent curative surgery, and R0-resection was achieved in 77% and 71%, respectively. Perioperative morbidity occurred in 72% in arm A and 46% in arm B, whereas non-surgical toxicity was comparable in both arms. Median RFS/PFS was almost doubled in arm B (14.1 months) compared with arm A (8.4 months) in the population with surgical resection, whereas median OS was comparable between both arms. CONCLUSIONS: Although the analysis was only descriptive owing to small patient numbers, no safety issues regarding surgical complications were observed in the perioperative FOLFIRINOX arm. Thus, considering the small number of patients, perioperative treatment approach appears feasible and potentially effective in well-selected cohorts of patients. In pancreatic cancer, patient selection before initiation of neoadjuvant therapy appears to be critical.

Long-term outcome of permanent tracheostomy management in two brachycephalic dogs using a commercial and a three-dimensional-printed silicone stent.

OBJECTIVE: To report the long-term outcome of utilization of a silicone stent to support the management of a permanent tracheostomy. STUDY DESIGN: Short case series. ANIMALS: Two client-owned brachycephalic dogs. METHODS: Two brachycephalic dogs with stage III laryngeal collapse underwent permanent tracheostomy. After the tracheostomy had healed, a silicone stent was inserted to support the stoma and facilitate home care. One dog wore a commercially available silicone stent for the follow-up period of 2 years. For the dog in Case 2, a 3D-printed, medical-grade silicone stent with an increased length was designed, as the dog had developed skin sores from the commercial device. RESULTS: Both dogs tolerated the silicone stent well. Stent care was managed by the owners without need for assistance. They reported that the silicone stent facilitated cleaning of the stoma surroundings and that they felt an increased confidence in airway patency, as the device prevented the tracheal stoma from collapsing. In Case 1, tracheoscopy 1 year after first stent insertion revealed minimal visible changes to the tracheal stoma. In Case 2, the 3D printed silicone stent led to a remission of skin sores and the dog wore the device comfortably until succumbing to an unrelated disease 13 months later. CONCLUSION: The insertion of a silicone stent is a simple and cost-effective method to improve home care of dogs with permanent tracheostomy. Larger dogs, as in Case 2, may benefit from custom-designed 3D-printed stents.

Economic activity and suicides: Causal evidence from macroeconomic shocks in England and Wales.

The relationship between economic activity and suicides has been the subject of much scrutiny, but the focus in the extant literature has been almost exclusively on estimating associations rather than causal effects. In this paper, using data from England and Wales between January 1, 1997 and December 31, 2017, we propose a plausible set of assumptions to estimate the causal impacts of well-known macroeconomic variables on the daily suicide rate. Our identification strategy relies on scheduled macroeconomic announcements and professional economic forecasts. An important advantage of using these variables to model suicide rates is that they can efficiently capture the elements of 'surprise or shock' via the observed difference between how the economy actually performed and how it was expected to perform. Provided that professional forecasts are unbiased and efficient, the estimated 'surprises or shocks' are 'as good as random', and therefore are exogenous. We employ time series regressions and present robust evidence that these exogenous macroeconomic shocks affect the suicide rate. Overall, our results are consistent with economic theory that shocks that reduce estimated permanent income, and therefore expected lifetime utility, can propel suicide rates. Specifically, at the population level, negative shocks to consumer confidence and house prices accelerate the suicide rate. However, there is evidence of behavioural heterogeneity between sexes, states of the economy, and levels of public trust in government. Negative shocks to the retail price index (RPI) raise the suicide rate for males. Negative shocks to the growth rate in gross domestic product (GDP) raise the population suicide rate when the economy is doing poorly. When public trust in government is low, increases in the unemployment rate increase the suicide rate for females.

Coronary Artery Surgery: Past, Present, and Future.

Coronary artery bypass grafting (CABG) is the most commonly performed and studied major cardiac operation worldwide. An understanding of the evolution of CABG, including the early days of cardiac surgery, the first bypass operation, continuous improvements in techniques, and streamlining of the operation, is important to inform current trends and future innovations. This article will examine how CABG evolved (from techniques to conduits), describe current trends in the field, and explore what lies on the horizon for the future of CABG.

Morphological variability or inter-observer bias? A methodological toolkit to improve data quality of multi-researcher datasets for the analysis of morphological variation.

OBJECTIVES: The investigation of morphological variation in animals is widely used in taxonomy, ecology, and evolution. Using large datasets for meta-analyses has dramatically increased, raising concerns about dataset compatibilities and biases introduced by contributions of multiple researchers. MATERIALS AND METHODS: We compiled morphological data on 13 variables for 3073 individual mouse lemurs (Cheirogaleidae, Microcebus spp.) from 25 taxa and 153 different sampling locations, measured by 48 different researchers. We introduced and applied a filtering pipeline and quantified improvements in data quality (Shapiro-Francia statistic, skewness, and excess kurtosis). The filtered dataset was then used to test for genus-wide sexual size dimorphism and the applicability of Rensch's, Allen's, and Bergmann's rules. RESULTS: Our pipeline reduced inter-observer bias (i.e., increased normality of data distributions). Inter-observer reliability of measurements was notably variable, highlighting the need to reduce data collection biases. Although subtle, we found a consistent pattern of sexual size dimorphism across Microcebus, with females being the larger (but not heavier) sex. Sexual size dimorphism was isometric, providing no support for Rensch's rule. Variations in tail length but not in ear size were consistent with the predictions of Allen's rule. Body mass and length followed a pattern contrary to predictions of Bergmann's rule. DISCUSSION: We highlighted the usefulness of large multi-researcher datasets for testing ecological hypotheses after correcting for inter-observer biases. Using genus-wide tests, we outlined generalizable patterns of morphological variability across all mouse lemurs. This new methodological toolkit aims to facilitate future large-scale morphological comparisons for a wide range of taxa and applications.

Coronary artery bypass grafting in left ventricular dysfunction: when and how.

PURPOSE OF REVIEW: The surgical management of patients undergoing coronary artery bypass grafting (CABG) with low ejection fraction presents unique challenges that require meticulous attention to details and good surgical technique and judgement. This review details the latest evidence and best practices in the care of such patients. RECENT FINDINGS: CABG in patients with low ejection fraction carries a significant risk of perioperative mortality and morbidity related to the development of postcardiotomy shock. Preoperative optimization with pharmacological or mechanical support is required, especially in patients with cardiogenic shock. Rapid and complete revascularization is what CABG surgeons aim to achieve. Multiple arterial revascularization should be reserved to selected patients. Off-pump CABG, on-pump breathing heart CABG, and new cardioplegic solutions remain of uncertain benefit compared with traditional CABG. SUMMARY: Tremendous advancements in CABG allowed surgeons to offer revascularization to patients with severe left ventricular dysfunction and multivessel disease with acceptable risk. Despite that, there is a lack of comprehensive and robust studies particularly on long-term outcomes. Individualized patient assessment and a heart team approach should be used to determine the optimal surgical strategy for each patient.

GPER1 deficiency causes sex-specific dysregulation of hippocampal plasticity and cognitive function.

Estrogens regulate synaptic properties and influence hippocampus-related learning and memory via estrogen receptors, which include the G-protein-coupled estrogen receptor 1 (GPER1). Studying mice, in which the GPER1 gene is dysfunctional (GPER1-KO), we here provide evidence for sex-specific roles of GPER1 in these processes. GPER1-KO males showed reduced anxiety in the elevated plus maze, whereas the fear response ('freezing') was specifically increased in GPER1-KO females in a contextual fear conditioning paradigm. In the Morris water maze, spatial learning and memory consolidation was impaired by GPER1 deficiency in both sexes. Notably, in the females, spatial learning deficits and the fear response were more pronounced if mice were in a stage of the estrous cycle, in which E2 serum levels are high (proestrus) or rising (diestrus). On the physiological level, excitability at Schaffer collateral synapses in CA1 increased in GPER1-deficient males and in proestrus/diestrus ('E2 high') females, concordant with an increased hippocampal expression of the AMPA-receptor subunit GluA1 in GPER1-KO males and females as compared to wildtype males. Further changes included an augmented early long-term potentiation (E-LTP) maintenance specifically in GPER1-KO females and an increased hippocampal expression of spinophilin in metestrus/estrus ('E2 low') GPER1-KO females. Our findings suggest modulatory and sex-specific functions of GPER1 in the hippocampal network, which reduce rather than increase neuronal excitability. Dysregulation of these functions may underlie sex-specific cognitive deficits or mood disorders.

The Bacterial Gq Signal Transduction Inhibitor FR900359 Impairs Soil-Associated Nematodes.

The cyclic depsipeptide FR900359 (FR) is derived from the soil bacterium Chromobacterium vaccinii and known to bind Gq proteins of mammals and insects, thereby abolishing the signal transduction of their Gq protein-coupled receptors, a process that leads to severe physiological consequences. Due to their highly conserved structure, Gq family of proteins are a superior ecological target for FR producing organisms, resulting in a defense towards a broad range of harmful organisms. Here, we focus on the question whether bacteria like C. vaccinii are important factors in soil in that their secondary metabolites impair, e.g., plant harming organisms like nematodes. We prove that the Gq inhibitor FR is produced under soil-like conditions. Furthermore, FR inhibits heterologously expressed Gαq proteins of the nematodes Caenorhabditis elegans and Heterodera schachtii in the micromolar range. Additionally, in vivo experiments with C. elegans and the plant parasitic cyst nematode H. schachtii demonstrated that FR reduces locomotion of C. elegans and H. schachtii. Finally, egg-laying of C. elegans and hatching of juvenile stage 2 of H. schachtii from its cysts is inhibited by FR, suggesting that FR might reduce nematode dispersion and proliferation. This study supports the idea that C. vaccinii and its excreted metabolome in the soil might contribute to an ecological equilibrium, maintaining and establishing the successful growth of plants.

Effects of Forest Fragmentation on Connectivity and Genetic Diversity in an Endemic and an Invasive Rodent in Northwestern Madagascar.

Habitat loss and fragmentation are of concern to conservation biologists worldwide. However, not all organisms are affected equally by these processes; thus, it is important to study the effects of living in fragmented habitats on species that differ in lifestyle and habitat requirements. In this study, we examined the dispersal and connectivity patterns of rodents, one endemic (Eliurus myoxinus) and one invasive (Rattus rattus), in two landscapes containing forest fragments and adjacent continuous forest patches in northwestern Madagascar. We generated genetic (RADseq) data for 66 E. myoxinus and 81 R. rattus individuals to evaluate differences in genetic diversity as well as inbreeding and connectivity in two landscapes. We found higher levels of inbreeding and lower levels of genetic diversity in E. myoxinus compared with R. rattus. We observed related dyads both within and between habitat patches and positive spatial autocorrelation at lower distance classes for both species, with a stronger pattern of spatial autocorrelation in R. rattus. Across each site, we identified contrasting migration rates for each species, but these did not correspond to habitat-matrix dichotomies. The relatively low genetic diversity in the endemic E. myoxinus suggests ecological constraints that require further investigation.

Effects of 24-hour oral estradiol-valerate administration on hormone levels in men and pre-menopausal women.

In order to translate the findings from the vast animal literature on the effect of 17ß-estradiol (E2) on brain and behavior to humans, a placebo-controlled pharmacological enhancement of E2 levels for at least 24 h is necessary. However, an exogenous increase in E2 for such a prolonged period might affect the endogenous secretion of other (neuroactive) hormones. Such effects would be of relevance for the interpretation of the effects of this pharmacological regimen on cognition and its neural correlates as well as be of basic scientific interest. We therefore administered a double dose of 12 mg of estradiol-valerate (E2V) to men and of 8 mg to naturally cycling women in their low-hormone phase, and assessed the concentration of two steroids critical to hormone regulation: follicle stimulating hormone (FSH) and luteinizing hormone (LH). We also assessed any changes in concentration of the neuroactive hormones progesterone (P4), testosterone (TST), dihydrotestosterone (DHT) and immune-like growth factor 1 (IGF-1). This regimen resulted in similar E2 levels in both sexes (saliva and serum). FSH and LH levels in both sexes were down-regulated to the same degree. P4 concentration decreased in both sexes only in serum but not saliva. TST and DHT levels dropped only in men whereas sex-hormone binding globulin was not affected. Finally, the concentration of IGF-1 decreased in both sexes. Based on previous studies on the effects of these neuroactive hormones, only the degree of downregulation of TST and DHT levels in men might have an impact on brain and behavior, which should be considered when interpreting the effects of the presented E2V regimes.

Adenylation Domain-Guided Recruitment of Trans-Acting Nonheme Monooxygenases in Nonribosomal Peptide Biosynthesis.

Nonheme diiron monooxygenases (NHDMs) interact with nonribosomal peptide synthetase (NRPS) assembly lines to install ß-hydroxylations at thiolation-domain-bound amino acids during nonribosomal peptide biosynthesis. The high potential of this enzyme family to diversify the products of engineered assembly lines is disproportionate to the currently small knowledge about their structures and mechanisms of substrate recognition. Here, we report the crystal structure of FrsH, the NHDM which catalyzes the ß-hydroxylation of l-leucines during biosynthesis of the depsipeptide G protein inhibitor FR900359. Using biophysical approaches, we provide evidence that FrsH interacts with the cognate monomodular NRPS FrsA. By AlphaFold modeling and mutational studies, we detect and examine structural features within the assembly line crucial to recruit FrsH for leucine ß-hydroxylation. These are, in contrast to cytochrome-dependent NRPS ß-hydroxylases, not located on the thiolation domain, but on the adenylation domain. FrsH can be functionally substituted by homologous enzymes from biosyntheses of the cell-wall-targeting antibiotics lysobactin and hypeptin, indicating that these features are generally applicable to members of the family of trans-acting NHDMs. These insights give important directions for the construction of artificial assembly lines to yield bioactive and chemically complex peptide products.

Intracaval Leiomyoma with Intracardiac Extension.

The objective of this study was to explain step by step how to achieve a complete resection of an intravascular leiomyoma. A 48-year-old woman was referred to our institution with progressive dyspnea on exertion, lightheadedness, and previous history of total abdominal hysterectomy and bilateral salpingo-oophorectomy for a uterine leiomyoma echocardiography, computed tomography, and magnetic resonance imaging of the heart and abdomen/pelvis were performed and an intracaval mass with extension into the right heart and pulmonary artery was identified. After multidisciplinary review, a single-stage sternotomy-laparotomy procedure on cardiopulmonary bypass (with beating heart, mild hypothermia, and no deep hypothermic circulatory arrest) ensured complete resection of a giant intravenous leiomyoma (IVL). Multidisciplinary approach, multimodality imaging, and single-stage sternotomy-laparotomy procedure on cardiopulmonary bypass (with heart beating and mild hypothermia) ensure complete resection of IVL.