Domain Organization of the Monomeric Form of the Tom70 Mitochondrial Import Receptor

Tom70 is a mitochondrial protein import receptor composed of 11 tetratricopeptide repeats (TPRs). The first three TPRs form an N-terminal domain that recruits heat shock protein family chaperones, while the eight C-terminal TPRs form a domain that receives, from the bound chaperone, mitochondrial precursor proteins destined for import. Analytical ultracentrifugation and solution small-angle X-ray scattering (SAXS) analysis characterized Tom70 as an elongated monomer. A model for the Tom70 monomer was proposed based on the alternate interpretation of the domain pairings observed in the crystal structure of the Tom70 dimer and refined against the SAXS data. In this “open” model of the Tom70 monomer, the chaperone- and precursor-binding sites are exposed and lay side by side on one face of the molecule. Fluorescence anisotropy measurements indicated that monomeric Tom70 can bind both chaperone and precursor peptides and that chaperone peptide binding does not alter the affinity of Tom70 for the precursor peptide. SAXS was unable to detect any shape change in Tom70 upon chaperone binding. However, molecular modeling indicated that chaperone binding is incompatible with Tom70 dimer formation. It is proposed that the Tom70 monomer is the functional unit mediating initial chaperone docking and precursor recognition.     

Organization of Cellular Receptors into a Nanoscale Junction during HIV-1 Adhesion

The fusion of the human immunodeficiency virus type 1 (HIV-1) with its host cell is the target for new antiretroviral therapies. Viral particles interact with the flexible plasma membrane via viral surface protein gp120 which binds its primary cellular receptor CD4 and subsequently the coreceptor CCR5. However, whether and how these receptors become organized at the adhesive junction between cell and virion are unknown. Here, stochastic modeling predicts that, regarding binding to gp120, cellular receptors CD4 and CCR5 form an organized, ring-like, nanoscale structure beneath the virion, which locally deforms the plasma membrane. This organized adhesive junction between cell and virion, which we name the viral junction, is reminiscent of the well-characterized immunological synapse, albeit at much smaller length scales. The formation of an organized viral junction under multiple physiopathologically relevant conditions may represent a novel intermediate step in productive infection.     

Global epigenomic analysis of primary human pancreatic islets provides insights into type 2 diabetes susceptibility loci

Identifying cis-regulatory elements is important to understanding how human pancreatic islets modulate gene expression in physiologic or pathophysiologic (e.g., diabetic) conditions. We conducted genome-wide analysis of DNase I hypersensitive sites, histone H3 lysine methylation modifications (K4me1, K4me3, K79me2), and CCCTC factor (CTCF) binding in human islets. This identified ～18,000 putative promoters (several hundred unannotated and islet-active). Surprisingly, active promoter modifications were absent at genes encoding islet-specific hormones, suggesting a distinct regulatory mechanism. Of 34,039 distal (nonpromoter) regulatory elements, 47% are islet unique and 22% are CTCF bound. In the 18 type 2 diabetes (T2D)-associated loci, we identified 118 putative regulatory elements and confirmed enhancer activity for 12 of 33 tested. Among six regulatory elements harboring T2D-associated variants, two exhibit significant allele-specific differences in activity. These findings present a global snapshot of the human islet epigenome and should provide functional context for noncoding variants emerging from genetic studies of T2D and other islet disorders.     

Diet‐induced Obesity in Two C57BL/6 Substrains With Intact or Mutant Nicotinamide Nucleotide Transhydrogenase (Nnt) Gene

The C57BL/6J (B6/J) male mouse represents a standard for diet‐induced obesity (DIO) and is unique in expressing a loss‐of‐function nicotinamide nucleotide transhydrogenase (Nnt) gene. This mutation was associated with a marked reduction in glucose‐stimulated insulin secretion from B6/J islets in vitro and moderately impaired glucose clearance in vivo. To assess the contribution of this Nnt mutation, we compared DIO responsiveness of Nnt‐mutant B6/J males to Nnt wild‐type C57BL/6NJ (B6/NJ) males over a 14‐week period of feeding a high‐fat (60% of calories) diet. Initial mean body weights at 6 weeks did not distinguish the substrains and both substrains were DIO‐sensitive. However, B6/J males outgained the B6/NJ males, with a significant 3 g higher mean body weight at 20 weeks accompanied by significant increases in both lean and fat mass. Mean nonfasting serum glucose over time was also significantly higher in B6/J males, as was impairment of glucose tolerance assessed at 8 and 20 weeks of age. Serum leptin, but not insulin, was significantly higher in B6/J males over time. Potential contributions of the wild‐type Nnt gene were demonstrable on a lower fat diet (10% of calories) where a significantly greater weight gain over time by B6/NJ males was correlated with a significantly higher serum insulin. In conclusion, DIO developed in response to 60% fat feeding regardless of Nnt allele status. Contribution of the B6/J‐unique Nnt mutation was most evident in response to 10% fat feeding that resulted in reduced serum insulin and weight gain compared to B6/NJ males.     

Compound heterozygosity for loss-of-function lysyl-tRNA synthetase mutations in a patient with peripheral neuropathy

Charcot-Marie-Tooth (CMT) disease comprises a genetically and clinically heterogeneous group of peripheral nerve disorders characterized by impaired distal motor and sensory function. Mutations in three genes encoding aminoacyl-tRNA synthetases (ARSs) have been implicated in CMT disease primarily associated with an axonal pathology. ARSs are ubiquitously expressed, essential enzymes responsible for charging tRNA molecules with their cognate amino acids. To further explore the role of ARSs in CMT disease, we performed a large-scale mutation screen of the 37 human ARS genes in a cohort of 355 patients with a phenotype consistent with CMT. Here we describe three variants (p.Leu133His, p.Tyr173SerfsX7, and p.Ile302Met) in the lysyl-tRNA synthetase (KARS) gene in two patients from this cohort. Functional analyses revealed that two of these mutations (p.Leu133His and p.Tyr173SerfsX7) severely affect enzyme activity. Interestingly, both functional variants were found in a single patient with CMT disease and additional neurological and non-neurological sequelae. Based on these data, KARS becomes the fourth ARS gene associated with CMT disease, indicating that this family of enzymes is specifically critical for axon function.     

Genome-wide screen identifies rs646776 near sortilin as a regulator of progranulin levels in human plasma

Recent studies suggest progranulin (GRN) is a neurotrophic factor. Loss-of-function mutations in the progranulin gene (GRN) cause frontotemporal lobar degeneration (FTLD), a progressive neurodegenerative disease affecting ∼10% of early-onset dementia patients. Using an enzyme-linked immunosorbent assay, we previously showed that GRN is detectable in human plasma and can be used to predict GRN mutation status. This study also showed a wide range in plasma GRN levels in non-GRN mutation carriers, including controls. We have now performed a genome-wide association study of 313,504 single-nucleotide polymorphisms (SNPs) in 533 control samples and identified on chromosome 1p13.3 two SNPs with genome-wide significant association with plasma GRN levels (top SNP rs646776; p = 1.7 × 10⁻³⁰). The association of rs646776 with plasma GRN levels was replicated in two independent series of 508 controls (p = 1.9 × 10⁻¹⁹) and 197 FTLD patients (p = 6.4 × 10⁻¹²). Overall, each copy of the minor C allele decreased GRN levels by ∼15%. SNP rs646776 is located near sortilin (SORT1), and the minor C allele of rs646776 was previously associated with increased SORT1 mRNA levels. Supporting these findings, overexpression of SORT1 in cultured HeLa cells dramatically reduced GRN levels in the conditioned media, whereas knockdown of SORT1 increased extracellular GRN levels. In summary, we identified significant association of a locus on chromosome 1p13.3 with plasma GRN levels through an unbiased genome-wide screening approach and implicated SORT1 as an important regulator of GRN levels. This finding opens avenues for future research into GRN biology and the pathophysiology of neurodegenerative diseases.     

Missense Mutations in the Copper Transporter Gene ATP7A Cause X-Linked Distal Hereditary Motor Neuropathy

Distal hereditary motor neuropathies comprise a clinically and genetically heterogeneous group of disorders. We recently mapped an X-linked form of this condition to chromosome Xq13.1-q21 in two large unrelated families. The region of genetic linkage included ATP7A, which encodes a copper-transporting P-type ATPase mutated in patients with Menkes disease, a severe infantile-onset neurodegenerative condition. We identified two unique ATP7A missense mutations (p.P1386S and p.T994I) in males with distal motor neuropathy in two families. These molecular alterations impact highly conserved amino acids in the carboxyl half of ATP7A and do not directly involve the copper transporter''s known critical functional domains. Studies of p.P1386S revealed normal ATP7A mRNA and protein levels, a defect in ATP7A trafficking, and partial rescue of a S. cerevisiae copper transport knockout. Although ATP7A mutations are typically associated with severe Menkes disease or its milder allelic variant, occipital horn syndrome, we demonstrate here that certain missense mutations at this locus can cause a syndrome restricted to progressive distal motor neuropathy without overt signs of systemic copper deficiency. This previously unrecognized genotype-phenotype correlation suggests an important role of the ATP7A copper transporter in motor-neuron maintenance and function.     

Bradykinin receptor‐1 activation induces inflammation and increases the permeability of human brain microvascular endothelial cells

Neuroinflammatory disorders such as Alzheimer's and Parkinson's diseases are characterised by chronic inflammation and loss of vascular integrity. Bradykinin 1 receptor (B1R) activation has been implicated in many neuroinflammatory diseases, but the contribution of B1R to inflammation and vascular breakdown is yet to be determined. As a result, the present study evaluated the effect of B1R stimulation using Des‐Arg‐9‐BK on the cytokine profile and junctional properties of human cerebral microvascular endothelial cells (hCMVECs). Results showed that stimulation of B1R receptors increased secretion of pro‐inflammatory cytokines, interleukin‐6 (IL‐6), IL‐8, intracellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1) and monocyte chemoattractant protein‐1 (MCP‐1), but decreased the expression of vascular endothelial growth factor (VEGF), a cytokine and growth factor required for maintenance of the vasculature. B1R stimulation also resulted in the loss of occludin expression at tight junctions with no change in VE‐cadherin expression. There was also a significant increase in permeability to Evans blue albumin, suggesting an increase of vascular permeability. Taken together, these results suggest that B1R activation that occurs in neuroinflammatory diseases may contribute to both the inflammation and loss of blood‐brain barrier integrity that is characteristic of these diseases.     

Natural hazard threats to pollinators and pollination

Natural hazards are naturally occurring physical events that can impact human welfare both directly and indirectly, via shocks to ecosystems and the services they provide. Animal‐mediated pollination is critical for sustaining agricultural economies and biodiversity, yet stands to lose both from present exposure to natural hazards, and future climate‐driven shifts in their distribution, frequency, and intensity. In contrast to the depth of knowledge available for anthropogenic‐related threats, our understanding of how naturally occurring extreme events impact pollinators and pollination has not yet been synthesized. We performed a systematic review and meta‐analysis to examine the potential impacts of natural hazards on pollinators and pollination in natural and cultivated systems. From a total of 117 studies (74% of which were observational), we found evidence of community and population‐level impacts to plants and pollinators from seven hazard types, including climatological (extreme heat, fire, drought), hydrological (flooding), meteorological (hurricanes), and geophysical (volcanic activity, tsunamis). Plant and pollinator response depended on the type of natural hazard and level of biological organization observed; 19% of cases reported no significant impact, whereas the majority of hazards held consistent negative impacts. However, the effects of fire were mixed, but taxa specific; meta‐analysis revealed that bee abundance and species richness tended to increase in response to fire, differing significantly from the mainly negative response of Lepidoptera. Building from this synthesis, we highlight important future directions for pollination‐focused natural hazard research, including the need to: (a) advance climate change research beyond static “mean‐level” changes by better incorporating “shock” events; (b) identify impacts at higher levels of organization, including ecological networks and co‐evolutionary history; and (c) address the notable gap in crop pollination services research—particularly in developing regions of the world. We conclude by discussing implications for safeguarding pollination services in the face of global climate change.     

Robust age estimation of southern sea otters from multiple morphometrics

Reliable age estimation is an essential tool to assess the status of wildlife populations and inform successful management. Aging methods, however, are often limited by too few data, skewed demographic representation, and by single or uncertain morphometric relationships. In this study, we synthesize age estimates in southern sea otters Enhydra lutris nereis from 761 individuals across 34 years of study, using multiple noninvasive techniques and capturing all life stages from 0 to 17 years of age. From wild, stranded, and captive individuals, we describe tooth eruptions, tooth wear, body length, nose scarring, and pelage coloration across ontogeny and fit sex‐based growth functions to the data. Dental eruption schedules provided reliable and identifiable metrics spanning 0.3–9 months. Tooth wear was the most reliable predictor of age of individuals aged 1–15 years, which when combined with total length, explained >93% of observed age. Beyond age estimation, dental attrition also indicated the maximum lifespan of adult teeth is 13?17 years, corresponding with previous estimates of life expectancy. Von Bertalanffy growth function model simulations of length at age gave consistent estimates of asymptotic lengths (male L_oo = 126.0?126.8 cm, female L_oo = 115.3?115.7 cm), biologically realistic gestation periods (t₀ = 115 days, SD = 10.2), and somatic growth (male k = 1.8, SD = 0.1; female k = 2.1, SD = 0.1). Though exploratory, we describe how field radiographic imaging of epiphyseal plate development or fusions may improve aging of immature sea otters. Together, our results highlight the value of integrating information from multiple and diverse datasets to help resolve conservation problems.