Eating problems at age 6 years in a whole population sample of extremely preterm children

Aim: The aim of this study was to investigate the prevalence of eating problems and their association with neurological and behavioural disabilities and growth among children born extremely preterm (EPC) at age 6 years. Method: A standard questionnaire about eating was completed by parents of 223 children (125 males [56.1%], 98 females [43.9%]) aged 6 years who were born at 25 weeks' gestation or earlier (mean 24.5wks, SD 0.7wks; mean birthweight 749.1g, SD 116.8g), and parents of 148 classmates born at term (66 males [44.6%], 82 females [55.4%]). All children underwent neurological, cognitive, and anthropometric assessment, and parents and teachers completed a behaviour scale. Results: Eating problems were more common among the EPC than the comparison group (odds ratio [OR] 3.6, 95% confidence interval [CI] 2.1–6.3), including oral motor (OR 5.2, 95% CI 2.8–9.9), hypersensitivity (OR 3.0, 95% CI 1.6–5.6), and behavioural (OR 3.8, 95% CI 1.9–7.6) problems. Group differences were reduced after adjustment for cognitive impairment, neuromotor disability, and other behaviour problems. EPC with eating problems were shorter, lighter, and had lower mid-arm circumference and lower body mass index (BMI) even after adjusting for disabilities, gestational age, birthweight, and feeding problems at 30 months. Interpretation: Eating problems are still frequent in EPC at school age. They are only partly related to other disabilities but make an additional contribution to continued growth failure and may require early recognition and intervention

A Single Amino Acid Dictates Protein Kinase R Susceptibility to Unrelated Viral Antagonists

<div><p>During millions of years of coevolution with their hosts, cytomegaloviruses (CMVs) have succeeded in adapting to overcome host-specific immune defenses, including the protein kinase R (PKR) pathway. Consequently, these adaptations may also contribute to the inability of CMVs to cross species barriers. Here, we provide evidence that the evolutionary arms race between the antiviral factor PKR and its CMV antagonist TRS1 has led to extensive differences in the species-specificity of primate CMV TRS1 proteins. Moreover, we identify a single residue in human PKR that when mutated to the amino acid present in African green monkey (Agm) PKR (F489S) is sufficient to confer resistance to HCMV_TRS1. Notably, this precise molecular determinant of PKR resistance has evolved under strong positive selection among primate PKR alleles and is positioned within the αG helix, which mediates the direct interaction of PKR with its substrate eIF2α. Remarkably, this same residue also impacts sensitivity to K3L, a poxvirus-encoded pseudosubstrate that structurally mimics eIF2α. Unlike K3L, TRS1 has no homology to eIF2α, suggesting that unrelated viral genes have convergently evolved to target this critical region of PKR. Despite its functional importance, the αG helix exhibits extraordinary plasticity, enabling adaptations that allow PKR to evade diverse viral antagonists while still maintaining its critical interaction with eIF2α.</p></div

Species-specific differences in primate CMV TRS1 PKR antagonism map to a single amino acid.

<p>(A) Schematic representation of the SEAP assay. Transfection of PKR leads to decreased activity of a co-transfected reporter construct expressing SEAP. This PKR-driven repression can be counteracted by co-transfection of a functional TRS1 antagonist, resulting in a rescue of SEAP activity. (B) The SEAP assay recapitulates species-specific differences in HuPKR antagonism by TRS1 alleles. HeLa PKR KO cells were co-transfected with a SEAP reporter plasmid along with either a control vector or HuPKR and the indicated TRS1 alleles or a vector control. At 48 h post-transfection, SEAP activity in the medium was measured (mean ± s.d., n = 2). Data are representative of three independent experiments. (C) A single amino acid change, F489S, confers resistance to HCMV_TRS1. Point mutants were generated in HuPKR to introduce the six AgmPKR-specific residues that differ between HuPKR and AgmPKR within the region spanning codons 475 to 520, shown in the alignment. The ability of the point mutants to antagonize HuPKR was evaluated as described in (B) (mean ± s.d., n = 2). Data are representative of three independent experiments.</p

Species-specific differences in primate CMV TRS1 PKR antagonism.

<p>(A) HeLa PKR KO, HeLa (human), or BSC40 (Agm) cells were mock infected or infected (MOI 0.1) with WT VacV, VacVΔE3L, or VacVΔE3L recombinants containing HCMV_TRS1, AgmCMV_TRS1, RhCMV_TRS1, or SmCMV_TRS1. At 48 hpi, viral replication was quantified by measuring β-gal activity (mean ± s.d., n = 3). Data are representative of three independent experiments. (B) His-tagged TRS1 constructs were detected in lysates of the infected HeLa PKR KO cells from (A) by western blotting. TRS1 size variation is expected based on differences in coding length.</p

The PKR pathway is activated in HuPKR F489S cells after infection with VacVΔE3L+HCMV_TRS1.

<p>HeLa PKR KO cells with stably integrated HuPKR or HuPKR F489S were induced with doxycycline to express PKR and 24 hours later mock-infected or infected at an MOI of 3 with the indicated viruses. At 6 hpi, cells were lysed and levels of PKR-P, total PKR, eIF2α-P, total eIF2α and actin were evaluated by western blotting.</p

The F489S mutation eliminates HCMV_TRS1 binding to PKR.

<p>HeLa PKR KO cells were co-transfected with WT HuPKR or HuPKR F489S and either His-tagged HCMV_TRS1, AgmCMV_TRS1, SmCMV_TRS1 or EGFP. At 48h post transfection, lysates were prepared and incubated with nickel-agarose beads. Cell lysates and bound proteins were analyzed by western blotting, probing for His-tagged TRS1 proteins and for PKR. Data are representative of three independent experiments.</p

HuPKR F489S confers resistance to VacV K3L H47R.

<p>HeLa PKR KO cells inducibly expressing the indicated PKR variants were infected and evaluated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005966#ppat.1005966.g003" target="_blank">Fig 3A</a>. (mean ± s.d., n = 3, ** p<0.005; ns, not significant). Data are representative of three independent experiments. (B) K3L was detected in lysates of the uninduced, infected empty vector cells from (A) by western blotting.</p

Position 489 of HuPKR is highly tolerant of amino acid substitutions.

<p>(A) Position 489 (dark blue) falls within the αG helix of PKR (light blue) and projects into a hydrophobic pocket of eIF2α (pale orange) composed of the side chains of Y32, E42, M44 and Y81 (dark orange). (B) Position 489 is highly variable in primates. The protein sequence alignment of the αG helix of PKR from representative primates is shown. Amino acids previously found to be evolving under positive selection among primates [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005966#ppat.1005966.ref016" target="_blank">16</a>] are indicated with arrowheads. (C) Most mutations at position 489 retain PKR activity but only a subset are inhibited by HCMV_TRS1. HuPKR 489 variants were evaluated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005966#ppat.1005966.g002" target="_blank">Fig 2B</a> (mean ± s.d., n = 2). The fold change relative to a vector control is indicated for amino acids that are most sensitive to inhibition by HCMV_TRS1 (greater than 3 fold increase). KD PKR is a mutant of PKR (K296R) that lacks kinase activity. Data are representative of at least two independent experiments.</p