Kurzfassung: The theoretical description of the forces that shape ecological communities focuses around two classes of models. In niche theory, deterministic interactions between species, individuals, and the environment are considered the dominant factor, whereas in neutral theory, stochastic forces, such as demographic noise, speciation, and immigration, are dominant. Species abundance distributions predicted by the two classes of theory are difficult to distinguish empirically, making it problematic to deduce ecological dynamics from typical measures of diversity and community structure. Here, we show that the fusion of species abundance data with genome-derived measures of evolutionary distance can provide a clear indication of ecological dynamics, capable of quantifying the relative roles played by niche and neutral forces. We apply this technique to six gastrointestinal microbiomes drawn from three different domesticated vertebrates, using high-resolution surveys of microbial species abundance obtained from carefully curated deep 16S rRNA hypervariable tag sequencing data. Although the species abundance patterns are seemingly well fit by the neutral theory of metacommunity assembly, we show that this theory cannot account for the evolutionary patterns in the genomic data; moreover, our analyses strongly suggest that these microbiomes have, in fact, been assembled through processes that involve a significant nonneutral (niche) contribution. Our results demonstrate that high-resolution genomics can remove the ambiguities of process inference inherent in classic ecological measures and permits quantification of the forces shaping complex microbial communities.

Kurzfassung: Abnormal processing of the amyloid precursor protein (APP) and β-amyloid (Aβ) plaque accumulation are defining features of Alzheimer disease (AD), a genetically complex neurodegenerative disease that is characterized by progressive synapse loss and neuronal cell death. Aβ induces synaptic dysfunction in part by altering the endocytosis and trafficking of AMPA and NMDA receptors. Reelin is a neuromodulator that increases glutamatergic neurotransmission by signaling through the postsynaptic ApoE receptors Apoer2 and Vldlr and thereby potently enhances synaptic plasticity. Here we show that Reelin can prevent the suppression of long-term potentiation and NMDA receptors, which is induced by levels of Aβ comparable to those present in an AD-afflicted brain. This reversal is dependent upon the activation of Src family tyrosine kinases. At high concentrations of Aβ peptides, Reelin can no longer overcome the Aβ induced functional suppression and this coincides with a complete blockade of the Reelin-dependent phosphorylation of NR2 subunits. We propose a model in which Aβ, Reelin, and ApoE receptors modulate neurotransmission and thus synaptic stability as opposing regulators of synaptic gain control.

Kurzfassung: De novo mutations (DNMs), or mutations that appear in an individual despite not being seen in their parents, are an important source of genetic variation whose impact is relevant to studies of human evolution, genetics, and disease. Utilizing high-coverage whole-genome sequencing data as part of the Trans-Omics for Precision Medicine (TOPMed) Program, we called 93,325 single-nucleotide DNMs across 1,465 trios from an array of diverse human populations, and used them to directly estimate and analyze DNM counts, rates, and spectra. We find a significant positive correlation between local recombination rate and local DNM rate, and that DNM rate explains a substantial portion (8.98 to 34.92%, depending on the model) of the genome-wide variation in population-level genetic variation from 41K unrelated TOPMed samples. Genome-wide heterozygosity does correlate with DNM rate, but only explains 〈 1% of variation. While we are underpowered to see small differences, we do not find significant differences in DNM rate between individuals of European, African, and Latino ancestry, nor across ancestrally distinct segments within admixed individuals. However, we did find significantly fewer DNMs in Amish individuals, even when compared with other Europeans, and even after accounting for parental age and sequencing center. Specifically, we found significant reductions in the number of C→A and T→C mutations in the Amish, which seem to underpin their overall reduction in DNMs. Finally, we calculated near-zero estimates of narrow sense heritability ( h 2 ), which suggest that variation in DNM rate is significantly shaped by nonadditive genetic effects and the environment.

Kurzfassung: The gap between chronological age (CA) and biological brain age, as estimated from magnetic resonance images (MRIs), reflects how individual patterns of neuroanatomic aging deviate from their typical trajectories. MRI-derived brain age (BA) estimates are often obtained using deep learning models that may perform relatively poorly on new data or that lack neuroanatomic interpretability. This study introduces a convolutional neural network (CNN) to estimate BA after training on the MRIs of 4,681 cognitively normal (CN) participants and testing on 1,170 CN participants from an independent sample. BA estimation errors are notably lower than those of previous studies. At both individual and cohort levels, the CNN provides detailed anatomic maps of brain aging patterns that reveal sex dimorphisms and neurocognitive trajectories in adults with mild cognitive impairment (MCI, N  = 351) and Alzheimer’s disease (AD, N  = 359). In individuals with MCI (54% of whom were diagnosed with dementia within 10.9 y from MRI acquisition), BA is significantly better than CA in capturing dementia symptom severity, functional disability, and executive function. Profiles of sex dimorphism and lateralization in brain aging also map onto patterns of neuroanatomic change that reflect cognitive decline. Significant associations between BA and neurocognitive measures suggest that the proposed framework can map, systematically, the relationship between aging-related neuroanatomy changes in CN individuals and in participants with MCI or AD. Early identification of such neuroanatomy changes can help to screen individuals according to their AD risk.

Kurzfassung: A common challenge in drug design pertains to finding chemical modifications to a ligand that increases its affinity to the target protein. An underutilized advance is the increase in structural biology throughput, which has progressed from an artisanal endeavor to a monthly throughput of hundreds of different ligands against a protein in modern synchrotrons. However, the missing piece is a framework that turns high-throughput crystallography data into predictive models for ligand design. Here, we designed a simple machine learning approach that predicts protein–ligand affinity from experimental structures of diverse ligands against a single protein paired with biochemical measurements. Our key insight is using physics-based energy descriptors to represent protein–ligand complexes and a learning-to-rank approach that infers the relevant differences between binding modes. We ran a high-throughput crystallography campaign against the SARS-CoV-2 main protease (M Pro ), obtaining parallel measurements of over 200 protein–ligand complexes and their binding activities. This allows us to design one-step library syntheses which improved the potency of two distinct micromolar hits by over 10-fold, arriving at a noncovalent and nonpeptidomimetic inhibitor with 120 nM antiviral efficacy. Crucially, our approach successfully extends ligands to unexplored regions of the binding pocket, executing large and fruitful moves in chemical space with simple chemistry.

Kurzfassung: Most genetic studies consider autism spectrum disorder (ASD) and developmental disorder (DD) separately despite overwhelming comorbidity and shared genetic etiology. Here, we analyzed de novo variants (DNVs) from 15,560 ASD (6,557 from SPARK) and 31,052 DD trios independently and also combined as broader neurodevelopmental disorders (NDDs) using three models. We identify 615 NDD candidate genes (false discovery rate [FDR] 〈 0.05) supported by ≥1 models, including 138 reaching Bonferroni exome-wide significance ( P 〈 3.64e–7) in all models. The genes group into five functional networks associating with different brain developmental lineages based on single-cell nuclei transcriptomic data. We find no evidence for ASD-specific genes in contrast to 18 genes significantly enriched for DD. There are 53 genes that show mutational bias, including enrichments for missense ( n = 41) or truncating ( n = 12) DNVs. We also find 10 genes with evidence of male- or female-bias enrichment, including 4 X chromosome genes with significant female burden ( DDX3X , MECP2 , WDR45 , and HDAC8) . This large-scale integrative analysis identifies candidates and functional subsets of NDD genes.

Kurzfassung: Hemispheric asymmetry is a cardinal feature of human brain organization. Altered brain asymmetry has also been linked to some cognitive and neuropsychiatric disorders. Here, the ENIGMA (Enhancing NeuroImaging Genetics through Meta-Analysis) Consortium presents the largest-ever analysis of cerebral cortical asymmetry and its variability across individuals. Cortical thickness and surface area were assessed in MRI scans of 17,141 healthy individuals from 99 datasets worldwide. Results revealed widespread asymmetries at both hemispheric and regional levels, with a generally thicker cortex but smaller surface area in the left hemisphere relative to the right. Regionally, asymmetries of cortical thickness and/or surface area were found in the inferior frontal gyrus, transverse temporal gyrus, parahippocampal gyrus, and entorhinal cortex. These regions are involved in lateralized functions, including language and visuospatial processing. In addition to population-level asymmetries, variability in brain asymmetry was related to sex, age, and intracranial volume. Interestingly, we did not find significant associations between asymmetries and handedness. Finally, with two independent pedigree datasets ( n = 1,443 and 1,113, respectively), we found several asymmetries showing significant, replicable heritability. The structural asymmetries identified and their variabilities and heritability provide a reference resource for future studies on the genetic basis of brain asymmetry and altered laterality in cognitive, neurological, and psychiatric disorders.

Kurzfassung: Since the identification of the FGF23 ADHR mutations, reconciling our current knowledge of phosphate handling with the development of ADHR in patients has been difficult. Under normal circumstances, the expression of FGF23 is reduced strongly by hypophosphatemia; therefore, as serum phosphate levels decrease, serum FGF23 concentrations should normalize through transcriptional repression of the FGF23 gene. The fact that ADHR patients cannot suppress FGF23 suggests that a stimulus outside the traditional calcium–phosphate endocrine axis, namely, iron deficiency, can stimulate Fgf23 production strongly despite frank hypophosphatemia, as we observed in the ADHR mice. These findings could lead to novel diagnostic monitoring and therapeutic approaches for ADHR and for common disorders of impaired phosphate metabolism, such as chronic kidney disease-mineral bone disorder. This work supported the following concepts: ( i ) iron deficiency can induce ADHR biochemical and skeletal phenotypes in a model animal in vivo; ( ii ) the 176 RXXR 179 SPC site is critical for the normal control of circulating Fgf23 at a secondary regulatory step; and ( iii ) Fgf23 production is downstream of (i.e., responds to or is controlled by) transcriptional pathways associated with iron-deficiency sensing. Our findings show that ADHR, unlike other diseases with elevated FGF23, is a syndrome that can be caused by gene–environment interactions, whereby the combined presence of an ADHR mutation and altered iron status are capable of producing the disease phenotype. To isolate the responses of bone cells to iron deficiency, we treated a cell line derived from bone tissue (the UMR-106 osteoblastic cell line) with the therapeutic iron chelator deferoxamine (DFO) to reduce total iron content. DFO treatment resulted in a significant increase in Fgf23 mRNA and elevated intracellular signaling; both these effects were blocked by inhibitors. Furthermore, the hypoxia-induced factor (HIF) transcription factor HIF1α, known to respond to reductions in cellular iron, was stabilized with DFO and with a specific HIF activator, l -mimosine ( l -MIM). In parallel, l -MIM treatment also significantly stimulated Fgf23 mRNA expression, implicating an increase in Fgf23 mRNA transcription by HIF1α as the molecular basis for elevated Fgf23 during iron deficiency ( Fig. P1 ). To test the mechanisms for late-onset ADHR, serum Fgf23 was measured in the treated mice by using serum assays that measured either intact, bioactive Fgf23 or intact and C-terminal Fgf23 protein. Of significance, both the wild-type and ADHR mice receiving the low-iron diet had markedly elevated C-terminal Fgf23; however, the wild-type mice on the low-iron diet displayed normal levels of intact Fgf23, indicating that these animals were capable of regulating phosphate homeostasis through active proteolysis of the Fgf23 protein. In contrast, the ADHR mice on the low-iron diet had elevated intact and C-terminal Fgf23, demonstrating that intact Fgf23 cleavage was impaired by the Fgf23 ADHR R176Q mutation, resulting in hypophosphatemic bone disease. To test the potential sources for the elevated serum Fgf23, we next examined Fgf23 expression in the bones of the mice receiving the two different diets. Wild-type and ADHR mice on the low-iron diet showed marked increases in Fgf23 mRNA and protein levels at 8 and 12 wk compared with the respective control mice (summarized in Fig. P1 ). The ADHR spectrum contains two subgroups of affected individuals: One subgroup consists of patients who present during childhood with phosphate wasting, rickets, and lower limb deformity; the second subgroup consists of those who are unaffected as children but present clinically later, during adolescence or adulthood ( 1 ). To test the role of iron deficiency in the regulation of Fgf23 and ADHR onset in vivo (i.e., in the organism itself), wild-type and R176Q-Fgf23 knock-in (ADHR) mice were placed on normal (control) or low-iron diets for 8–12 wk. The low-iron diet decreased serum iron levels in both wild-type and ADHR mice and caused similar degrees of iron-deficiency anemia in both groups compared with controls. Interestingly, the low-iron diet resulted in significant hypophosphatemia and bone osteomalacia (improper mineralization) only in the ADHR mice receiving the low-iron diet but not in the other groups. Further, alkaline phosphatase (a marker for hypophosphatemic bone disease) was elevated only in the ADHR mice on the low-iron diet, and no changes in serum calcium or parathyroid hormone, which promotes increases in serum calcium, were observed. These skeletal and biochemical phenotypes parallel those of patients with late-onset ADHR. Our goal was to test iron deficiency as the cause of late-onset ADHR and to determine the interaction of genes, proteins, and molecules that contribute to this delayed disease phenotype. Here, we demonstrate that iron deficiency substantially increases Fgf23 mRNA levels in the bone of both wild-type mice (controls) and mice genetically altered to carry an Fgf23 mutation that causes ADHR in humans (these mice have been designated “ADHR R176Q-Fgf23 knock-in” mice). We showed that increased expression of Fgf23 mRNA likely occurs through the activation of key pathways involved in sensing iron. Importantly, wild-type mice proteolytically cleaved excess Fgf23 protein into smaller pieces to maintain normal phosphate metabolism. In contrast, the proteolysis in R176Q-Fgf23 knock-in mice was compromised, resulting in elevated levels of intact serum Fgf23 and hypophosphatemic bone disease, consistent with the late-onset ADHR phenotype. Thus, our work supports the concept that iron deficiency controls Fgf23 and phosphate metabolism and that ADHR is caused by gene–environment interactions. The study of disorders caused by single gene defects that alter how phosphate is processed by the body can provide critical insights into normal phosphate metabolism. Autosomal dominant hypophosphatemic rickets (ADHR), a condition that can result in bone fracture, is characterized by low levels of phosphate and vitamin D in the blood serum ( 1 ). ADHR is caused by missense mutations ( 2 ) in the gene encoding fibroblast growth factor-23 (FGF23), a hormone produced by the bones that helps maintain normal phosphate levels (i.e., phosphate homeostasis) by causing the kidney to decrease reabsorption of phosphate so that less phosphate enters the bloodstream. The missense mutations that cause ADHR effectively replace arginine (R) residues at positions 176 or 179 within the amino acid sequence of FGF23 with glutamine (Q) or tryptophan (W) residues. The mutated region of the protein, known as the 176 RXXR 179 /S 180 subtilisin-like proprotein convertase (SPC) site, normally helps stabilize the intact protein ( 2 – 4 ). ADHR is unique among the disorders involving FGF23, because individuals with this disease carry gain-of-function FGF23 mutations that can manifest as disease states ranging from mild or no symptoms to late-onset of hypophosphatemic bone disease. Delayed onset often occurs in physiological states associated with iron deficiency, including puberty and pregnancy.

Kurzfassung: Integrins are involved in the binding and internalization of both enveloped and nonenveloped viruses. By using 3 distinct cell systems—CHO cells lacking expression of α 5 β 1 -integrin, HeLa cells treated with siRNA to α 5 -integrin, and mouse β 1 -integrin knockout fibroblasts, we show that α 5 β 1 -integrin is required for efficient infection by pseudovirions bearing the ebolavirus glycoprotein (GP). These integrins are necessary for viral entry but not for binding or internalization. Given the need for endosomal cathepsins B and L (CatB and CatL) to prime GPs for fusion, we investigated the status of CatB and CatL in integrin-positive and integrin-negative cell lines. α 5 β 1 -Integrin-deficient cells lacked the double-chain (DC) forms of CatB and CatL, and this correlated with decreased CatL activity in integrin-negative CHO cells. These data indicate that α 5 β 1 -integrin-negative cells may be refractory to infection by GP pseudovirions because they lack the necessary priming machinery (the double-chain forms of CatB and CatL). In support of this model, we show that GP pseudovirions that have been preprimed in vitro to generate the 19-kDa form of GP overcome the requirement for α 5 β 1 -integrin for infection. These results provide further support for the requirement for endosomal cathepsins for ebolavirus infection, identify the DC forms of these cathepsins as previously unrecognized factors that contribute to cell tropism of this virus, and reveal a previously undescribed role for integrins during viral entry as regulators of endosomal cathepsins, which are required to prime the entry proteins of ebolavirus and other pathogenic viruses.

Kurzfassung: Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G protein-coupled receptors, which are enzymatically cleaved to expose a truncated extracellular N terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease α-thrombin, is expressed in various tissues (e.g., platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. We have discovered a series of potent peptide-mimetic antagonists of PAR-1, exemplified by RWJ-56110. Spatial relationships between important functional groups of the PAR-1 agonist peptide epitope SFLLRN were employed to design and synthesize candidate ligands with appropriate groups attached to a rigid molecular scaffold. Prototype RWJ-53052 was identified and optimized via solid-phase parallel synthesis of chemical libraries. RWJ-56110 emerged as a potent, selective PAR-1 antagonist, devoid of PAR-1 agonist and thrombin inhibitory activity. It binds to PAR-1, interferes with PAR-1 calcium mobilization and cellular function (platelet aggregation; cell proliferation), and has no effect on PAR-2, PAR-3, or PAR-4. By flow cytometry, RWJ-56110 was confirmed as a direct inhibitor of PAR-1 activation and internalization, without affecting N-terminal cleavage. At high concentrations of α-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, albeit not in human platelets; whereas, at high concentrations of SFLLRN-NH 2 , RWJ-56110 blocked activation responses in both cell types. Thus, thrombin activates human platelets independently of PAR-1, i.e., through PAR-4, which we confirmed by PCR analysis. Selective PAR-1 antagonists, such as RWJ-56110, should serve as useful tools to study PARs and may have therapeutic potential for treating thrombosis and restenosis.