Insect cell expression and purification of recombinant SARS‐COV‐2 spike proteins that demonstrate ACE2 binding

The COVID‐19 pandemic caused by SARS‐CoV‐2 infection has led to socio‐economic shutdowns and the loss of over 5 million lives worldwide. There is a need for the identification of therapeutic targets to treat COVID‐19. SARS‐CoV‐2 spike is a target of interest for the development of therapeutic targets. We developed a robust SARS‐CoV‐2 S spike expression and purification protocol from insect cells and studied four recombinant SARS‐CoV‐2 spike protein constructs based on the original SARS‐CoV‐2 sequence using a baculovirus expression system: a spike protein receptor‐binding domain that includes the SD1 domain (RBD) coupled to a fluorescent tag (S‐RBD‐eGFP), spike ectodomain coupled to a fluorescent tag (S‐Ecto‐eGFP), spike ectodomain with six proline mutations and a foldon domain (S‐Ecto‐HexaPro(+F)), and spike ectodomain with six proline mutations without the foldon domain (S‐Ecto‐HexaPro(‐F)). We tested the yield of purified protein expressed from the insect cell lines Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tni) and compared it to previous research using mammalian cell lines to determine changes in protein yield. We demonstrated quick and inexpensive production of functional glycosylated spike protein of high purity capable of recognizing and binding to the angiotensin converting enzyme 2 (ACE2) receptor. To further confirm functionality, we demonstrate binding of eGFP fused construct of the spike ectodomain (S‐Ecto‐eGFP) to surface ACE2 receptors on lung epithelial cells by flow cytometry analysis and show that it can be decreased by means of receptor manipulation (blockade or downregulation).     

His‐tagged protein purification by metal‐chelate affinity extraction with nickel‐chelate reverse micelles

Di(2‐ethylhexyl) phosphoric acid (HDEHP) was used as a transition metal ion chelator and introduced to the nonionic reverse micellar system composed of equimolar Triton X‐45 and Span 80 at a total concentration of 30 mmol/L. Ni(II) ions were chelated to the HDEHP dimers in the reverse micelles, forming a complex denoted as Ni(II)R₂. The Ni(II)‐chelate reverse micelles were characterized for the purification of recombinant hexahistidine‐tagged enhanced green fluorescent protein (EGFP) expressed in Escherichia coli. The affinity binding of EGFP to Ni(II)R₂ was proved by investigation of the forward and back extraction behaviors of purified EGFP. Then, EGFP was purified with the affinity reverse micelles. It was found that the impurities in the feedstock impeded EGFP transfer to the reverse micelles, though they were little solubilized in the organic phase. The high specificity of the chelated Ni²⁺ ions toward the histidine tag led to the production of electrophoretically pure EGFP, which was similar to that purified by immobilized metal affinity chromatography. A two‐stage purification by the metal‐chelate affinity extraction gave rise to 87% recovery of EGFP. Fluorescence spectrum analysis suggests the preservation of native protein structure after the separation process, indicating the system was promising for protein purification. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Development of purification process for dual‐function recombinant human heavy‐chain ferritin by the investigation of genetic modification impact on conformation

Ferritin is a promising drug delivery platform and has been functionalized through genetic modifications. This work has designed and expressed a dual‐functional engineered human heavy‐chain ferritin (HFn) with the inserted functional peptide PAS and RGDK to extend half‐life and improve tumor targeted drug delivery. A facile and cost‐effective two‐step purification pathway for recombinant HFn was developed. The genetic modification was found to affect HFn conformation, and therefore varied the purification performance. Heat‐acid precipitation followed by butyl fast flow hydrophobic interaction chromatography (HIC) has been developed to purify HFn and modified HFns. Nucleic acid removal reached above 99.8% for HFn and modified HFns. However, HFn purity reached above 95% and recovery yield (overall) above 90%, compared with modified HFns purity above 82% and recovery yield (overall) above 58%. It is interesting to find that the inserted functional peptides significantly changed the molecule conformation, where a putative turnover of the E‐helix with the inserted functional peptides formed a “flop” conformation, in contrast with the “flip” conformation of HFn. It could be the cause of fragile stability of modified HFns, and therefore less tolerant to heat and acid condition, observed by the lower recovery yield in heat‐acid precipitation.     

Engineering defensin α‐helix to produce high‐affinity SARS‐CoV‐2 spike protein binding ligands

The binding of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike protein to the angiotensin‐converting enzyme 2 (ACE2) receptor expressed on the host cells is a critical initial step for viral infection. This interaction is blocked through competitive inhibition by soluble ACE2 protein. Therefore, developing high‐affinity and cost‐effective ACE2 mimetic ligands that disrupt this protein–protein interaction is a promising strategy for viral diagnostics and therapy. We employed human and plant defensins, a class of small (2–5 kDa) and highly stable proteins containing solvent‐exposed alpha‐helix, conformationally constrained by two disulfide bonds. Therefore, we engineered the amino acid residues on the constrained alpha‐helix of defensins to mimic the critical residues on the ACE2 helix 1 that interact with the SARS‐CoV‐2 spike protein. The engineered proteins (h‐deface2, p‐deface2, and p‐deface2‐MUT) were soluble and purified to homogeneity with a high yield from a bacterial expression system. The proteins demonstrated exceptional thermostability (Tm 70.7°C), high‐affinity binding to the spike protein with apparent K _d values of 54.4 ± 11.3, 33.5 ± 8.2, and 14.4 ± 3.5 nM for h‐deface2, p‐deface2, and p‐deface2‐MUT, respectively, and were used in a diagnostic assay that detected SARS‐CoV‐2 neutralizing antibodies. This work addresses the challenge of developing helical ACE2 mimetics by demonstrating that defensins provide promising scaffolds to engineer alpha‐helices in a constrained form for designing of high‐affinity ligands.     

Purification and characterization of the receptor‐binding domain of SARS‐CoV‐2 spike protein from Escherichia coli

SARS‐CoV‐2 is responsible for a disruptive worldwide viral pandemic, and renders a severe respiratory disease known as COVID‐19. Spike protein of SARS‐CoV‐2 mediates viral entry into host cells by binding ACE2 through the receptor‐binding domain (RBD). RBD is an important target for development of virus inhibitors, neutralizing antibodies, and vaccines. RBD expressed in mammalian cells suffers from low expression yield and high cost. E. coli is a popular host for protein expression, which has the advantage of easy scalability with low cost. However, RBD expressed by E. coli (RBD‐1) lacks the glycosylation, and its antigenic epitopes may not be sufficiently exposed. In the present study, RBD‐1 was expressed by E. coli and purified by a Ni Sepharose Fast Flow column. RBD‐1 was structurally characterized and compared with RBD expressed by the HEK293 cells (RBD‐2). The secondary structure and tertiary structure of RBD‐1 were largely maintained without glycosylation. In particular, the major β‐sheet content of RBD‐1 was almost unaltered. RBD‐1 could strongly bind ACE2 with a dissociation constant (K_D) of 2.98 × 10^–8 M. Thus, RBD‐1 was expected to apply in the vaccine development, screening drugs and virus test kit.     

Structural basis for the stereospecific inhibition of the dual proline/hydroxyproline catabolic enzyme ALDH4A1 by trans‐4‐hydroxy‐L‐proline

Aldehyde dehydrogenase 4A1 (ALDH4A1) catalyzes the final steps of both proline and hydroxyproline catabolism. It is a dual substrate enzyme that catalyzes the NAD⁺‐dependent oxidations of L‐glutamate‐γ‐semialdehyde to L‐glutamate (proline metabolism), and 4‐hydroxy‐L‐glutamate‐γ‐semialdehyde to 4‐erythro‐hydroxy‐L‐glutamate (hydroxyproline metabolism). Here we investigated the inhibition of mouse ALDH4A1 by the six stereoisomers of proline and 4‐hydroxyproline using steady‐state kinetics and X‐ray crystallography. Trans‐4‐hydroxy‐L‐proline is the strongest of the inhibitors studied, characterized by a competitive inhibition constant of 0.7 mM, followed by L‐proline (1.9 mM). The other compounds are very weak inhibitors (approximately 10 mM or greater). Insight into the selectivity for L‐stereoisomers was obtained by solving crystal structures of ALDH4A1 complexed with trans‐4‐hydroxy‐L‐proline and trans‐4‐hydroxy‐D‐proline. The structures suggest that the 10‐fold greater preference for the L‐stereoisomer is due to a serine residue that hydrogen bonds to the amine group of trans‐4‐hydroxy‐L‐proline. In contrast, the amine group of the D‐stereoisomer lacks a direct interaction with the enzyme due to a different orientation of the pyrrolidine ring. These results suggest that hydroxyproline catabolism is subject to substrate inhibition by trans‐4‐hydroxy‐L‐proline, analogous to the known inhibition of proline catabolism by L‐proline. Also, drugs targeting the first enzyme of hydroxyproline catabolism, by elevating the level of trans‐4‐hydroxy‐L‐proline, may inadvertently impair proline catabolism by the inhibition of ALDH4A1.     

An ultra‐high affinity protein–protein interface displaying sequence‐robustness

Protein–protein interactions are crucial in biology and play roles in for example, the immune system, signaling pathways, and enzyme regulation. Ultra‐high affinity interactions (K _d <0.1 nM) occur in these systems, however, structures and energetics behind stability of ultra‐high affinity protein–protein complexes are not well understood. Regulation of the starch debranching barley limit dextrinase (LD) and its endogenous cereal type inhibitor (LDI) exemplifies an ultra‐high affinity complex (K _d of 42 pM). In this study the LD–LDI complex is investigated to unveil how robust the ultra‐high affinity is to LDI sequence variation at the protein–protein interface and whether alternative sequences can retain the ultra‐high binding affinity. The interface of LD–LDI was engineered using computational protein redesign aiming at identifying LDI variants predicted to retain ultra‐high binding affinity. These variants present a very diverse set of mutations going beyond conservative and alanine substitutions typically used to probe interfaces. Surface plasmon resonance analysis of the LDI variants revealed that high affinity of LD–LDI requires interactions of several residues at the rim of the protein interface, unlike the classical hotspot arrangement where key residues are found at the center of the interface. Notably, substitution of interface residues in LDI, including amino acids with functional groups different from the wild‐type, could occur without loss of affinity. This demonstrates that ultra‐high binding affinity can be conferred without hotspot residues, thus making complexes more robust to mutational drift in evolution. The present mutational analysis also demonstrates how energetic coupling can emerge between residues at large distances at the interface.     

Cost‐effective purification process development for chimeric hepatitis B core (HBc) virus‐like particles assisted by molecular dynamic simulation

Inserting foreign epitopes to hepatitis B core (HBc) virus‐like particles (VLPs) could influence the molecular conformation and therefore vary the purification process. In this study, a cost‐effective purification process was developed for two chimeric HBc VLPs displaying Epstein–Barr nuclear antigens 1 (EBNA1), and hepatitis C virus (HCV) core. Both chimeric VLPs were expressed in soluble form with high production yields in Escherichia coli. Molecular dynamic (MD) simulation was employed to predict the stability of chimeric VLPs. HCV core‐HBc was found to be less stable in water environment compared with EBNA1‐HBc, indicating its higher hydrophobicity. Assisting with MD simulation, ammonium sulfate precipitation was optimized to remove host cell proteins with high target protein recovery yields. Moreover, 99% DNA impurities were removed using POROS 50 HQ chromatography. In characterization measurement, we found that inserting HCV core epitope would reduce the ratio of α‐helix of HCV core‐HBc. This could be another reason on the top of its higher hydrophobicity predicted by MD simulation, causing its less stability. Tertiary structure, transmission electron microscopy, and immunogenicity results indicate that two chimeric VLPs maintained correct VLP structure ensuring its bioactivity after being processed by the developed cost‐effective purification approach.     

Selective cross‐linking of coinciding protein assemblies by in‐gel cross‐linking mass spectrometry

Cross‐linking mass spectrometry has developed into an important method to study protein structures and interactions. The in‐solution cross‐linking workflows involve time and sample consuming steps and do not provide sensible solutions for differentiating cross‐links obtained from co‐occurring protein oligomers, complexes, or conformers. Here we developed a cross‐linking workflow combining blue native PAGE with in‐gel cross‐linking mass spectrometry (IGX‐MS). This workflow circumvents steps, such as buffer exchange and cross‐linker concentration optimization. Additionally, IGX‐MS enables the parallel analysis of co‐occurring protein complexes using only small amounts of sample. Another benefit of IGX‐MS, demonstrated by experiments on GroEL and purified bovine heart mitochondria, is the substantial reduction of undesired over‐length cross‐links compared to in‐solution cross‐linking. We next used IGX‐MS to investigate the complement components C5, C6, and their hetero‐dimeric C5b6 complex. The obtained cross‐links were used to generate a refined structural model of the complement component C6, resembling C6 in its inactivated state. This finding shows that IGX‐MS can provide new insights into the initial stages of the terminal complement pathway.     

CyDisCo production of functional recombinant SARS‐CoV‐2 spike receptor binding domain

The COVID‐19 pandemic caused by SARS‐CoV‐2 has applied significant pressure on overtaxed healthcare around the world, underscoring the urgent need for rapid diagnosis and treatment. We have developed a bacterial strategy for the expression and purification of a SARS‐CoV‐2 spike protein receptor binding domain (RBD) that includes the SD1 domain. Bacterial cytoplasm is a reductive environment, which is problematic when the recombinant protein of interest requires complicated folding and/or processing. The use of the CyDisCo system (cytoplasmic disulfide bond formation in E. coli) bypasses this issue by pre‐expressing a sulfhydryl oxidase and a disulfide isomerase, allowing the recombinant protein to be correctly folded with disulfide bonds for protein integrity and functionality. We show that it is possible to quickly and inexpensively produce an active RBD in bacteria that is capable of recognizing and binding to the ACE2 (angiotensin‐converting enzyme) receptor as well as antibodies in COVID‐19 patient sera.     

Genome‐wide selective detection of Mile red‐bone goat using next‐generation sequencing technology

The ecotype population of goats (Capra hircus) was created by long‐term artificial selection and natural adaptation. Mile red‐bone goat is an indigenous breed with visible red bones, and its special bone structure has received extensive attention. This study aimed to identify genetic variants and candidate genes associated with specific bone phenotypes using next‐generation sequencing technology (NGS). The results revealed that 31,828,206 single nucleotide polymorphisms (SNPs) were obtained from 72 goats (20 Mile red‐bone goats and 52 common goats) by NGS. A total of 100 candidate genes were identified on the basis top 1% window interaction from nucleotide diversity (π), π ratio (π _A/π _B), and pairwise fixation index (F _ST). Exactly 77 known signaling pathways were enriched. Specifically, three coding genes (NMNAT2, LOC102172983, and PNLIP) were annotated in the vitamin metabolism signaling pathways, and NCF2 was annotated to the osteoclast (OC) differentiation pathway. Furthermore, 5862 reliable copy number variations (CNVs) were obtained, and 14 and 24 genes were annotated with the top 1‰ CNV based on F _ST (>0.490) and V _ST (>0.527), respectively. Several pathways related to bone development and metabolism of exogenous substances in vivo, including calcium signaling pathway, OC differentiation, and glycerophospholipid metabolism, were annotated. Specifically, six genes from 19 candidate CNVs, which were obtained by interaction of the top 1‰ CNVs with F _ST and V _ST, were annotated to mucin‐type O‐glycan biosynthesis and metabolic pathways. Briefly, the results implied that pseudopurpurin and specific genetic variants work together to contribute to the red‐bone color and specific bone structure of Mile red‐bone goat. This study is helpful to understanding the genetic basis of the unique bone phenotype of Mile red‐bone goats.     

The atomic portrait of SARS‐CoV‐2 as captured by cryo‐electron microscopy

Transmission electron microscopy has historically been indispensable for virology research, as it offers unique insight into virus function. In the past decade, as cryo‐electron microscopy (cryo‐EM) has matured and become more accessible, we have been able to peer into the structure of viruses at the atomic level and understand how they interact with the host cell, with drugs or with antibodies. Perhaps, there was no time in recent history where cryo‐EM was more needed, as SARS‐CoV‐2 has spread around the globe, causing millions of deaths and almost unquantifiable economic devastation. In this concise review, we aim to mark the most important contributions of cryo‐EM to understanding the structure and function of SARS‐CoV‐2 proteins, from surface spikes to the virus core and from virus‐receptor interactions to antibody binding.     

Predictable fold switching by the SARS‐CoV‐2 protein ORF9b

Extant fold‐switching proteins remodel their secondary structures and change their functions in response to environmental stimuli. These shapeshifting proteins regulate biological processes and are associated with a number of diseases, including tuberculosis, cancer, Alzheimer''s, and autoimmune disorders. Thus, predictive methods are needed to identify more fold‐switching proteins, especially since all naturally occurring instances have been discovered by chance. In response to this need, two high‐throughput predictive methods have recently been developed. Here we test them on ORF9b, a newly discovered fold switcher and potential therapeutic target from the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2). Promisingly, both methods correctly indicate that ORF9b switches folds. We then tested the same two methods on ORF9b1, the ORF9b homolog from SARS‐CoV‐1. Again, both methods predict that ORF9b1 switches folds, a finding consistent with experimental binding studies. Together, these results (a) demonstrate that protein fold switching can be predicted using high‐throughput computational approaches and (b) suggest that fold switching might be a general characteristic of ORF9b homologs.     

Disease‐linked TDP‐43 hyperphosphorylation suppresses TDP‐43 condensation and aggregation

Post‐translational modifications (PTMs) have emerged as key modulators of protein phase separation and have been linked to protein aggregation in neurodegenerative disorders. The major aggregating protein in amyotrophic lateral sclerosis and frontotemporal dementia, the RNA‐binding protein TAR DNA‐binding protein (TDP‐43), is hyperphosphorylated in disease on several C‐terminal serine residues, a process generally believed to promote TDP‐43 aggregation. Here, we however find that Casein kinase 1δ‐mediated TDP‐43 hyperphosphorylation or C‐terminal phosphomimetic mutations reduce TDP‐43 phase separation and aggregation, and instead render TDP‐43 condensates more liquid‐like and dynamic. Multi‐scale molecular dynamics simulations reveal reduced homotypic interactions of TDP‐43 low‐complexity domains through enhanced solvation of phosphomimetic residues. Cellular experiments show that phosphomimetic substitutions do not affect nuclear import or RNA regulatory functions of TDP‐43, but suppress accumulation of TDP‐43 in membrane‐less organelles and promote its solubility in neurons. We speculate that TDP‐43 hyperphosphorylation may be a protective cellular response to counteract TDP‐43 aggregation.     

Attenuation of SARS‐CoV‐2 replication and associated inflammation by concomitant targeting of viral and host cap 2'‐O‐ribose methyltransferases

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.     

Neutralization of SARS‐CoV‐2 by highly potent,hyperthermostable, and mutation‐tolerant nanobodies

Monoclonal anti‐SARS‐CoV‐2 immunoglobulins represent a treatment option for COVID‐19. However, their production in mammalian cells is not scalable to meet the global demand. Single‐domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor‐binding domain (RBD) of the SARS‐CoV‐2 Spike protein, we isolated 45 infection‐blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS‐CoV‐2 at 17–50 pM concentration (0.2–0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X‐ray and cryo‐EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune‐escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low‐picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such “fold‐promoting” nanobodies may allow for simplified production of vaccines and their adaptation to viral escape‐mutations.     

Cryo‐EM reveals mechanisms of angiotensin I‐converting enzyme allostery and dimerization

Hypertension (high blood pressure) is a major risk factor for cardiovascular disease, which is the leading cause of death worldwide. The somatic isoform of angiotensin I‐converting enzyme (sACE) plays a critical role in blood pressure regulation, and ACE inhibitors are thus widely used to treat hypertension and cardiovascular disease. Our current understanding of sACE structure, dynamics, function, and inhibition has been limited because truncated, minimally glycosylated forms of sACE are typically used for X‐ray crystallography and molecular dynamics simulations. Here, we report the first cryo‐EM structures of full‐length, glycosylated, soluble sACE (sACE^S1211). Both monomeric and dimeric forms of the highly flexible apo enzyme were reconstructed from a single dataset. The N‐ and C‐terminal domains of monomeric sACE^S1211 were resolved at 3.7 and 4.1 Å, respectively, while the interacting N‐terminal domains responsible for dimer formation were resolved at 3.8 Å. Mechanisms are proposed for intradomain hinging, cooperativity, and homodimerization. Furthermore, the observation that both domains were in the open conformation has implications for the design of sACE modulators.     

The pro‐inflammatory response of macrophages regulated by acid degradation products of poly(lactide‐co‐glycolide) nanoparticles

Poly(lactide‐co‐glycolide) (PLGA) shows great potentials in biomedical applications, in particular with the field of biodegradable implants and control release technologies. However, there are few systematic and detailed studies on the influence of PLGA degradation behavior on the immunogenicity. In this study, in order to develop a method for dynamically assessing the immunological response of PLGA throughout the implantation process, PLGA particles are fabricated using an o/w single‐emulsion method. The physicochemical characterizations of the prepared PLGA particles during in vitro hydrolytic degradation are investigated. Then, a series of immunological effects triggered by PLGA by‐products formed with degradation process are evaluated, including cell viability, apoptosis, polarization and inflammatory reaction. THP‐1 human cell line is set as in vitro cell model. Our results show that PLGA degradation‐induced acid environment decreases cell viability and increases cell apoptosis, which is a potential factor affecting cell function. In particular, the macrophages exhibit up‐regulations in both M1 subtype related surface markers and pro‐inflammatory cytokines with the degradation process of PLGA, which indicates the degradation products of PLGA can convert macrophages to the pro‐inflammatory (M1) polarization state. All these findings provide the mechanism of PLGA‐induced inflammation and lay the foundation for the design of next‐generation PLGA‐based biomaterials endowed with immunomodulatory functions.     

Association between IGF‐1 levels ranges and all‐cause mortality: A meta‐analysis

The association between IGF‐1 levels and mortality in humans is complex with low levels being associated with both low and high mortality. The present meta‐analysis investigates this complex relationship between IGF‐1 and all‐cause mortality in prospective cohort studies. A systematic literature search was conducted in PubMed/MEDLINE, Scopus, and Cochrane Library up to September 2019. Published studies were eligible for the meta‐analysis if they had a prospective cohort design, a hazard ratio (HR) and 95% confidence interval (CI) for two or more categories of IGF‐1 and were conducted among adults. A random‐effects model with a restricted maximum likelihood heterogeneity variance estimator was used to find combined HRs for all‐cause mortality. Nineteen studies involving 30,876 participants were included. Meta‐analysis of the 19 eligible studies showed that with respect to the low IGF‐1 category, higher IGF‐1 was not associated with increased risk of all‐cause mortality (HR = 0.84, 95% CI = 0.68–1.05). Dose–response analysis revealed a U‐shaped relation between IGF‐1 and mortality HR. Pooled results comparing low vs. middle IGF‐1 showed a significant increase of all‐cause mortality (HR = 1.33, 95% CI = 1.14–1.57), as well as comparing high vs. middle IGF‐1 categories (HR = 1.23, 95% CI = 1.06–1.44). Finally, we provide data on the association between IGF‐1 levels and the intake of proteins, carbohydrates, certain vitamins/minerals, and specific foods. Both high and low levels of IGF‐1 increase mortality risk, with a specific 120–160 ng/ml range being associated with the lowest mortality. These findings can explain the apparent controversy related to the association between IGF‐1 levels and mortality.     

Concordant geographic and genetic structure revealed by genotyping‐by‐sequencing in a New Zealand marine isopod

Population genetic structure in the marine environment can be influenced by life‐history traits such as developmental mode (biphasic, with distinct adult and larval morphology, and direct development, in which larvae resemble adults) or habitat specificity, as well as geography and selection. Developmental mode is thought to significantly influence dispersal, with direct developers expected to have much lower dispersal potential. However, this prediction can be complicated by the presence of geophysical barriers to dispersal. In this study, we use a panel of 8,020 SNPs to investigate population structure and biogeography over multiple spatial scales for a direct‐developing species, the New Zealand endemic marine isopod Isocladus armatus. Because our sampling range is intersected by two well‐known biogeographic barriers (the East Cape and the Cook Strait), our study provides an opportunity to understand how such barriers influence dispersal in direct developers. On a small spatial scale (20 km), gene flow between locations is extremely high, suggestive of an island model of migration. However, over larger spatial scales (600 km), populations exhibit a clear pattern of isolation‐by‐distance. Our results indicate that I. armatus exhibits significant migration across the hypothesized barriers and suggest that large‐scale ocean currents associated with these locations do not present a barrier to dispersal. Interestingly, we find evidence of a north‐south population genetic break occurring between Māhia and Wellington. While no known geophysical barrier is apparent in this area, it coincides with the location of a proposed border between bioregions. Analysis of loci under selection revealed that both isolation‐by‐distance and adaption may be contributing to the degree of population structure we have observed here. We conclude that developmental life history largely predicts dispersal in the intertidal isopod I. armatus. However, localized biogeographic processes can disrupt this expectation, and this may explain the potential meta‐population detected in the Auckland region.