Correlates of screen time in the early years (0-5 years): A systematic review.

The majority of young children engage in high levels of screen time. To inform future interventions, knowledge on correlates of screen time is important. This review expands on previous work by focusing on the entire early childhood range, and including a broad focus regarding types of correlates and screens. A literature search (PubMed, Embase, PsycINFO, SPORTDiscus) was performed from 2000 up to October 2021. Included studies (cross-sectional and prospective) examined associations between a potential correlate and screen time (duration or frequency) in typically developing, apparently healthy children aged 0-5 years. Methodological quality was assessed by two independent researchers. Fifty-two of 6,614 studies were included. Two studies had high methodological quality. We found moderate evidence for a positive association between an electronic device in the bedroom, parental screen time, having a TV on at home, descriptive norms and screen time, and a negative association between sleep duration, household features, high value on physical activity, monitoring screen time, being in childcare, parental self-efficacy and screen time. We found no evidence for an association for child sex, body mass index, physical activity, temperament, number of siblings, being a first-born, neighborhood-related factors, socio-economic indicators, and parental marital status, physical activity, weight status, depression, wellbeing, sex, age and positive outcome expectations. The evidence for other investigated correlates was inconsistent or insufficient. Despite the evidence for moderate associations, we were unable to draw strong conclusions. More high-quality research is needed to identify correlates of screen time in early childhood.

Differential transcription of the two Saccharomyces cerevisiae genes encoding elongation factor 2.

The single polypeptide chain of elongation factor 2 (EF-2) is encoded by two Saccharomyces cerevisiae genes (EFT1 and EFT2) with unique flanking sequences. One gene is necessary and either is sufficient for cell viability. In the present work, we have analyzed the transcription of EFT1 and EFT2. Although both genes harbor multiple transcription start points, EFT1 initiates primarily at residue C at -39 and EFT2 at residue C at -37. Several candidate TATA boxes were identified in each gene. Deletion analysis employing lacZ promoter fusions demonstrated that the promoter for EFT2 is located within a 79-bp region beginning 335 nucleotides (nt) upstream from the start ATG codon. This region contains two overlapping sequences with homology to the consensus binding site for the yeast transcription factor, Rap1p/Grf1p/Tuf. In contrast, the sequences essential for the transcription of EFT1 were localized to the region between the start ATG and the stop codon of the VPS17 gene that terminates 267 nt upstream on the same strand. Analysis of promoter strengths using lacZ fusions indicated that the promoter for EFT2 is approx. 2.5-fold more active than that of EFT1. Analysis of the steady-state levels of mRNAs revealed that EFT2 contributes approx. 70% of the total EF-2 mRNA while the remaining 30% is produced by EFT1. We conclude that the difference in expression of EFT1 and EFT2 is due to the differential transcription of their promoters.

Cloning, sequencing and phylogenetic analysis of a human 5-hydroxytryptamine 1D receptor pseudogene.

A third member of the human 5HT1D gene family has been identified using a combination of homology cloning and DNA sequence analysis. This human gene is most related to the 5HT1D alpha subtype (77% shared identity) and is a pseudogene, based on the lack of an open reading frame (ORF) caused by multiple in-frame stop codons and nucleotide (nt) deletions relative to the functional 5HT1D alpha gene (encoding the 5-hydroxytryptamine 1D alpha receptor). The 5HT1D pseudogene also contained an insertion that shares 87% identity to the Alu consensus sequence. Phylogenetic analysis of the three human genes in this family reveals that although the two functional genes, 5HT1D alpha and 5HT1D beta, are detected in all mammalian species examined, the 5HT1D pseudogene is only detected in a subset of primates (catarrhines) that evolved approximately 35-45 million years (Myr) ago. Alternatively, based on the 23% divergence between the functional 5HT1D alpha gene and the 5HT1D pseudogene, we estimate that these two genes began to diverge approximately 50 Myr ago.

Cloning and pharmacological characterization of a novel human 5-hydroxytryptamine1D receptor subtype.

The canine RDC4 gene was used to isolate two distinct human serotonin receptor genes. The receptor encoded by clone RH-6 was the species homolog of RDC4 and was identical to a human serotonin 5-hydroxytryptamine1D (5-HT1D) receptor that was recently reported [Mol. Pharmacol. 40:143-148 (1991)]. The receptor encoded by RH-2 was a novel 5-HT receptor that was 61% identical to RH-6 and showed the greatest homology with the rat 5-HT1B receptor sequence (94%). The RH-2 gene contained an intronless, 1170-base pair, open reading frame that encoded a 390-amino acid protein that contained all of the hallmarks of a guanine nucleotide-binding protein-linked receptor. Heterologous expression of the RH-2 gene in Chinese hamster ovary cells led to the appearance of high affinity binding sites for 5-HT (Kd = 2.6 nM, Bmax = 2.9 pmol/mg of membrane protein), and the receptor expressed in Chinese hamster ovary cells was coupled to inhibition of adenylyl cyclase. Competition binding experiments using compounds that are selective for various 5-HT receptor subtypes showed the highest correlation with a 5-HT1D-like receptor (r = 0.89) and a low correlation with 5-HT1B-like receptors. Examples of the 5-HT1D-like pharmacology displayed by RH-2 include high affinity for the 5-HT1D-selective compound sumatriptan (Ki = 9.4 nM) and for the alpha 2-adrenergic receptor antagonist rauwolscine (Ki = 47 nM). Therefore, despite the close genetic relationship between RH-2 and the rat 5-HT1B receptor, our results indicate that the receptor encoded by RH-2 2 is best classified as a human 5-HT1D receptor subtype and defines a second member of the human 5-HT1D receptor family.

A screening procedure for the intracellular expression of native proteins by Saccharomyces cerevisiae: discrimination of diphtheria toxin-resistant mutants.

A general method is described for screening Saccharomyces cerevisiae colonies for the intracellular expression of native proteins. Colonies are replicated onto nitrocellulose membranes and yeast cell walls are removed enzymatically. The resulting spheroplasts are rapidly lysed by placing chromatography paper soaked in hypotonic buffer on the membranes. Intracellular proteins released by spheroplast lysis are bound in situ to the nitrocellulose under non-denaturing conditions and potentially can be examined using enzymatic or immunologic methods. For example, in the present study colonies were screened for the presence of elongation factor 2 (EF-2) that can be [32P]ADP-ribosylated by diphtheria toxin and [32P]NAD+. Recognition by the toxin requires the presence in EF-2 of the unique post-translationally modified histidine derivative, diphthamide. The procedure described here reliably discriminates between wild-type yeast colonies and mutant colonies that do not synthesize diphthamide. In addition to facilitating the study of diphthamide biosynthesis in yeast, the more general application of this procedure will enable the screening of colonies with assays that require native proteins.

Expression of diphtheria toxin fragment A and hormone-toxin fusion proteins in toxin-resistant yeast mutants.

Mutants of the eukaryote Saccharomyces cerevisiae, previously selected for resistance to diphtheria toxin, were investigated for their suitability as hosts for the expression of tox-related proteins. The structural gene for the toxin, encoding the fragment A catalytic domain, was modified for efficient intracellular expression in eukaryotes and placed downstream of the yeast GAL1 promoter element in a plasmid. Transformed mutant yeast grown in galactose, which induces that promoter, were viable and contained active fragment A. In contrast, sensitive, wild-type cells harboring this plasmid grew normally under repressing conditions but were killed when the GAL1 promoter was induced. Additional constructions were also prepared that included sequences encoding either the lymphocyte growth factor interleukin 2 or alpha-melanocyte-stimulating hormone along with the lipid-associating domains of fragment B and the leader peptide of the Kluyveromyces lactis killer toxin. Resistant mutant strains transformed with these plasmids efficiently expressed and secreted the expected chimeric toxins.