Snacking practices from infancy to adolescence: parental perspectives from longitudinal lived experience research in England.

Consumption of snacks and ultra-processed foods (UPF) high in fat, salt and sugar (HFSS) is associated with rising rates of obesity and growing socioeconomic disparities in nutrition. While infancy, childhood and adolescence are critical periods for development of dietary preferences, there remains a dearth of research exploring factors that underpin snacking behaviour over this time. This review aims to address this gap by drawing from qualitative lived experience research, with 122 families of different socioeconomic position (SEP), to explore how the (i) home food environment, (ii) food environment and (iii) social value and meanings of food shape parental provision of snacks. This review shows that snacking holds important meanings in everyday family life, with infants integrated into existing snacking practices from an early age. Price promotions, low-cost and long shelf-lives all make UPF and HFSS snacks an appealing option for many low-SEP parents; while children's requests and preferences for HFSS snacks present a challenge across SEP. However, higher-SEP parents can ensure fresh fruits are always available as an alternative snack, while fruit is described as a financially risky expenditure for low-SEP families. The present findings also indicate that retailers and producers are increasingly promoting 'healthier' snacks through product packaging and marketing, such as 'meets one of your five a day', despite these products displaying similar nutritional profiles to traditional UPF and HFSS snacks. We outline a series of policy recommendations, including extending Healthy Start Vouchers and the Fruit and Vegetable Scheme in schools and action to address misleading product marketing and packaging.

Nivolumab induced pulmonary sarcoid-like granulomas with a concurrent pleural schwannoma: A pathologic-radiologic correlation of a rare condition mimicking metastatic melanoma.

Nivolumab is an anti-PD-1 antibody. The mechanism of action of nivolumab is inhibition of binding between PD-1 and PD-1 ligand. This causes activation of antigen-specific T cells that were previously unresponsive to cancer cells. This unique mechanism of action attributes the widespread use of nivolumab for the treatment of a variety of neoplastic conditions. On the other hand, this mode of action is associated with adverse effects as well. Schwannoma, also called neurilemmoma, is a benign peripheral nerve sheath tumor. Pleural schwannomas are very rare and very few cases have been reported in the medical English literature so far. Herein, we report a very rare case of concurrent presence of Nivolumab induced pulmonary sarcoid-like granulomas along with primary benign pleural schwannoma in a 49-year-old male. He was diagnosed with malignant melanoma of the right upper arm for which he underwent surgery and was receiving adjuvant chemotherapy. He developed pneumonitis during chemotherapy, and on imaging multiple reticular and nodular interstitial infiltrates were seen along with an incidental pleural mass with a high suspicion for metastasis. Wedge biopsy of the interstitial infiltrates was done and they were found to be pulmonary granulomas related to the nivolumab therapy he was receiving. The patient underwent excision of the pleural mass which showed histopathological and immunohistochemical features of schwannoma. The two conditions are unrelated and rarely encountered simultaneously. The radiologic and pathologic correlation along with differential diagnosis of these conditions are discussed.

Extra-framework cation release from heulandite-type rich tuffs on exchange with NH(4)(+).

The outgoing cations of Greek heulandite-rich tuff samples (heulandite type-III, 91wt.%, mica 4wt.%, feldspar 5wt. %, CEC 2.22meq/g) were analysed upon exchange with ammonium acetate using atomic absorption spectrometry (AAS). The kinetic curves of each cation were investigated over a total time of contact of 720h with sampling at frequent intervals. The materials were examined by powder X-ray diffraction, SEM-EDS, and AAS. The sorption ability was measured using the ammonium acetate saturation method. It was found that Ca(2+) presents an unexpected extra-framework release and a surprisingly high degree of exchange (90%). The exchange of Mg (57%) is also worthy of note whereas the behavior of K(+) showed an expected rapid initial release. The behavior of Na(+) must be similar. However, its lower concentration in the zeolitic material minimizes its overall significance somewhat. On the other hand, Ca(2+) and Mg(2+) release is kinetically much slower, compared to that of alkali metal ions, and this phenomenon indicates that different exchange energies are needed till final equilibrium.

Use of autologous blood as part of the perfusate for cardiopulmonary bypass: a priming technique.

In an attempt to replace the oncotic and protein coating capabilities of serum albumin in the perfusate, we established a priming protocol that used autologous blood as part of the perfusate solution. Prior to March 1, 1999, our standard priming protocol was 1650 ml of crystalloid with 250 ml of 5% serum albumin and 5,000 units of heparin. After removing albumin from our prime, our standard protocol was altered to include 40 ml of the patient's autologous blood in 1,800 ml of crystalloid and 10,000 units of heparin. To determine the intraoperative effects of using albumin/crystalloid primes (Group A), autologous blood/crystalloid primes (Group B) and crystalloid primes (Group C), a total of 178 patients were sequentially evaluated. Intraoperative parameters evaluated were total protein (TP), colloid osmotic pressure (COP), platelets (Plts) and fluid requirements during cardiopulmonary bypass (CPB). During an overlapping 12-month period of time, 1,092 consecutive cardiac surgical cases using CPB (584 albumin prime; 508 autologous blood prime) were evaluated for clinical outcomes in terms of mortality and length of hospitalization. In addition, over a period of 15 months, 1,458 patients in both the autologous blood/crystalloid group and the crystalloid only group were evaluated for the incidence of high-pressure excursions (HPE) after going on bypass. Comparative reviews of TP, COP and Plts demonstrated no significant difference 10 min after the start of bypass between Groups A and B. However, in Group C, there was a statistically significant increase in the intraoperative fluid requirements during CPB, compared to both of the other groups. There was no significant difference in the incidence of HPE, with an occurrence of 1.04% in the crystalloid only group and 1.11% in the autologous blood/crystalloid group. Autologous blood perfusates were identical to albumin perfusates in their platelet protection and reduction of fluid shifts during the intraoperative period.

Ribosomal protein S4 is a transcription factor with properties remarkably similar to NusA, a protein involved in both non-ribosomal and ribosomal RNA antitermination.

Escherichia coli ribosomal RNA (rRNA) operons contain antitermination motifs necessary for forming terminator-resistant transcription complexes. In preliminary work, we isolated 'antiterminating' transcription complexes and identified four new proteins potentially involved in rRNA transcription antitermination: ribosomal (r-) proteins S4, L3, L4 and L13. We show here that these r-proteins and Nus factors lead to an 11-fold increase in terminator read-through in in vitro transcription reactions. A significant portion of the effect was a result of r-protein S4. We show that S4 acted as a general antitermination factor, with properties very similar to NusA. It retarded termination and increased read-through at Rho-dependent terminators, even in the absence of the rRNA antiterminator motif. High concentrations of NusG showed reduced antitermination by S4. Like rrn antitermination, S4 selectively antiterminated at Rho-dependent terminators. Lastly, S4 tightly bound RNA polymerase in vivo. Our results suggest that, like NusA, S4 is a general transcription antitermination factor that associates with RNA polymerase during normal transcription and is also involved in rRNA operon antitermination. A model for key r-proteins playing a regulatory role in rRNA synthesis is presented.

DNA shuffling method for generating highly recombined genes and evolved enzymes.

We introduce a method of in vitro recombination or "DNA shuffling" to generate libraries of evolved enzymes. The approach relies on the ordering, trimming, and joining of randomly cleaved parental DNA fragments annealed to a transient polynucleotide scaffold. We generated chimeric libraries averaging 14.0 crossovers per gene, a several-fold higher level of recombination than observed for other methods. We also observed an unprecedented four crossovers per gene in regions of 10 or fewer bases of sequence identity. These properties allow generation of chimeras unavailable by other methods. We detected no unshuffled parental clones or duplicated "sibling" chimeras, and relatively few inactive clones. We demonstrated the method by molecular breeding of a monooxygenase for increased rate and extent of biodesulfurization on complex substrates, as well as for 20-fold faster conversion of a nonnatural substrate. This method represents a conceptually distinct and improved alternative to sexual PCR for gene family shuffling.

Mutagenesis of the peptidyltransferase center of 23S rRNA: the invariant U2449 is dispensable.

U2449 is one of many invariant residues in the central loop of domain V of 23S rRNA, a region that constitutes part of the peptidyltransferase center of the ribosome. In Escherichia coli, this U is post-transcriptionally modified to dihydrouridine (D) and is the only D modification found in E.coli rRNAs. To analyze the role of this base and its modification in ribosomal function, all three base substitutions were constructed on a plasmid copy of the rrnB operon and assayed for their ability to support cell growth in a strain of E.coli lacking chromosomal rrn operons. Both purine substitution mutations were not viable. However, growth and antibiotic sensitivity of cells expressing only the mutant D2449C rRNA was indistinguishable from wild type. We conclude that while a pyrimidine is required at position 2449 for proper ribosomal function, the D modification is dispensable.

Proteins shared by the transcription and translation machines.

It is becoming increasingly clear that the complex machines involved in transcription and translation, the two major activities leading to gene expression, communicate directly with one another by sharing proteins. For some proteins, such as ribosomal proteins S10 and L4, there is strong evidence of their participation in both processes, and much is known about their role in both activities. The exact roles and interactions of other proteins, such as Nus factors B and G, in both transcription and translation remain a mystery. Although there are not, at present, many examples of such shared proteins, the importance of understanding their behavior and intimate involvement with two major cellular machines is beginning to be appreciated. Studies related to the dual activities of these proteins and searches for more examples of proteins shared between the transcription and translation machines should lead to a better understanding of the communication between these two activities and the purposes it serves.

The feedback response of Escherichia coli rRNA synthesis is not identical to the mechanism of growth rate-dependent control.

Growth rate-independent rrn P1 promoter mutants were tested for their ability to respond to changes in rrn gene dosage. Most were found to be normal for the feedback response. In addition, cellular levels of the initiating nucleoside triphosphates remained unchanged when the rrn gene dosage was altered. These results suggest that the feedback response cannot be the mechanism for growth rate-dependent control of rRNA synthesis and that the relationship between these two processes may be more complicated than is currently understood.

In vivo selection of functional ribosomes with variations in the rRNA-binding site of Escherichia coli ribosomal protein S8: evolutionary implications.

The highly conserved nature of rRNA sequences throughout evolution allows these molecules to be used to build philogenic trees of different species. It is unknown whether the stability of specific interactions and structural features of rRNA reflects an optimal adaptation to a functional task or an evolutionary trap. In the work reported here, we have applied an in vivo selection strategy to demonstrate that unnatural sequences do work as a functional replacement of the highly conserved binding site of ribosomal protein S8. However, growth competition experiments performed between Escherichia coli isolates containing natural and unnatural S8-binding sites showed that the fate of each isolate depended on the growth condition. In exponentially growing cells, one unnatural variant was found to be equivalent to wild type in competition experiments performed in rich media. In culture conditions leading to slow growth, however, cells containing the wild-type sequence were the ultimate winner of the competition, emphasizing that the wild-type sequence is, in fact, the most fit solution for the S8-binding site.

Major rearrangements in the 70S ribosomal 3D structure caused by a conformational switch in 16S ribosomal RNA.

Dynamic changes in secondary structure of the 16S rRNA during the decoding of mRNA are visualized by three-dimensional cryo-electron microscopy of the 70S ribosome. Thermodynamically unstable base pairing of the 912-910 (CUC) nucleotides of the 16S RNA with two adjacent complementary regions at nucleotides 885-887 (GGG) and 888-890 (GAG) was stabilized in either of the two states by point mutations at positions 912 (C912G) and 885 (G885U). A wave of rearrangements can be traced arising from the switch in the three base pairs and involving functionally important regions in both subunits of the ribosome. This significantly affects the topography of the A-site tRNA-binding region on the 30S subunit and thereby explains changes in tRNA affinity for the ribosome and fidelity of decoding mRNA.

Isolation of kasugamycin resistant mutants in the 16 S ribosomal RNA of Escherichia coli.

Three ribosomal RNA mutations conferring resistance to the antibiotic kasugamycin were isolated using a strain of Escherichia coli in which all of the rRNA is transcribed from a plasmid-encoded rrn operon. The mutations, A794G, G926A, and A1519C, mapped to universally conserved sites in the 16 S RNA gene. Site-directed mutagenesis studies showed that virtually all mutations at these three sites conferred kasugamycin resistance and had very slight effects on cell growth. It has been known for many years that the absence of post-transcriptional modification at A1519 and the adjacent A1518 in strains lacking a functional KsgA methylase produces a kasugamycin resistance phenotype. Mutations at A1519 conferred kasugamycin resistance and had minor effects on cell growth, whereas mutations at 1518 did not confer resistance and increased the doubling time of the cells dramatically. Expression of mutations at A1518/A1519 in a methylase deficient ksgA(-)strain had divergent effects on the phenotype of the rRNA mutants, suggesting that the base identity at either position does not affect methylation at the adjacent site. Residues A794 and G926 are protected from chemical modification by kasugamycin and tRNA, and have been implicated in the initiation of protein synthesis. Despite the universal conservation and functional importance of these residues, the results presented here show that the identity of the bases is not critical for ribosomal function.

Enhancement of translation by the downstream box does not involve base pairing of mRNA with the penultimate stem sequence of 16S rRNA.

The downstream box (DB) is a sequence element that enhances translation of several bacterial and phage mRNAs. It has been proposed that the DB enhances translation by base pairing transiently to bases 1469-1483 of 16S rRNA, the so-called anti-DB, during the initiation phase of translation. We have tested this model of enhancer action by constructing mutations in the anti-DB that alter its mRNA base-pairing potential and examining expression of a variety of DB-containing mRNAs in strains expressing the mutant anti-DB 16S rRNA. We found that the rRNA mutant was viable and that expression of all tested DB-containing mRNAs was completely unaffected by radical alterations in the proposed anti-DB. These findings lead us to conclude that enhancement of translation by the DB does not involve mRNA-rRNA base pairing.

Increased rrn gene dosage causes intermittent transcription of rRNA in Escherichia coli.

When the number of rRNA (rrn) operons in an Escherichia coli cells is increased by adding an rrn operon on a multicopy plasmid, the rate of rRNA expression per operon is reduced to maintain a constant concentration of rRNA in the cell. We have used electron microscopy to examine rRNA transcription in cells containing a multicopy plasmid carrying rrnB. We found that there were fewer RNA polymerase molecules transcribing the rrn genes, as predicted from previous gene dosage studies. Furthermore, RNA polymerase molecules were arranged in irregularly spaced groups along the operon. No apparent pause or transcription termination sites that would account for the irregular spacing of the groups of polymerase molecules were observed. We also found that the overall transcription elongation rate was unchanged when the rrn gene dosage was increased. Our data suggest that when rrn gene dosage is increased, initiation events, or promoter-proximal elongation events, are interrupted at irregular time intervals.

Construction and initial characterization of Escherichia coli strains with few or no intact chromosomal rRNA operons.

The Escherichia coli genome carries seven rRNA (rrn) operons, each containing three rRNA genes. The presence of multiple operons has been an obstacle to many studies of rRNA because the effect of mutations in one operon is diluted by the six remaining wild-type copies. To create a tool useful for manipulating rRNA, we sequentially inactivated from one to all seven of these operons with deletions spanning the 16S and 23S rRNA genes. In the final strain, carrying no intact rRNA operon on the chromosome, rRNA molecules were expressed from a multicopy plasmid containing a single rRNA operon (prrn). Characterization of these rrn deletion strains revealed that deletion of two operons was required to observe a reduction in the growth rate and rRNA/protein ratio. When the number of deletions was extended from three to six, the decrease in the growth rate was slightly more than the decrease in the rRNA/protein ratio, suggesting that ribosome efficiency was reduced. This reduction was most pronounced in the Delta7 prrn strain, in which the growth rate, unlike the rRNA/protein ratio, was not completely restored to wild-type levels by a cloned rRNA operon. The decreases in growth rate and rRNA/protein ratio were surprisingly moderate in the rrn deletion strains; the presence of even a single operon on the chromosome was able to produce as much as 56% of wild-type levels of rRNA. We discuss possible applications of these strains in rRNA studies.

Antiterminator-dependent modulation of transcription elongation rates by NusB and NusG.

Ribosomal RNA is transcribed about twice as fast as messenger RNA in vivo, and this increased transcription rate requires the rrn boxA antitermination system. Because several Nus factors have been implicated in rrn antitermination, we have examined the role of NusB, NusE and NusG in controlling the rate of rrn boxA-mediated transcript elongation. In vivo RNA polymerase transcription rates were determined by measuring the rate of appearance of lacZ transcript using a plasmid that contained an inducible T7 promoter fused to the rrn boxA sequence followed by the lacZ gene. This plasmid was introduced into Escherichia coli mutant strains that can be conditionally depleted of NusG, or that carry a deficient nusB gene or a nusE mutation. We found that, in addition to the rrn boxA antiterminator sequence, both NusG and NusB were required to maintain the high transcription rate. The nusE mutation used in this study may be specific for lambda antitermination, as it did not influence the boxA-mediated increase in transcription rate.

An Escherichia coli strain with all chromosomal rRNA operons inactivated: complete exchange of rRNA genes between bacteria.

Current global phylogenies are built predominantly on rRNA sequences. However, an experimental system for studying the evolution of rRNA is not readily available, mainly because the rRNA genes are highly repeated in most experimental organisms. We have constructed an Escherichia coli strain in which all seven chromosomal rRNA operons are inactivated by deletions spanning the 16S and 23S coding regions. A single E. coli rRNA operon carried by a multicopy plasmid supplies 16S and 23S rRNA to the cell. By using this strain we have succeeded in creating microorganisms that contain only a foreign rRNA operon derived from either Salmonella typhimurium or Proteus vulgaris, microorganisms that have diverged from E. coli about 120-350 million years ago. We also were able to replace the E. coli rRNA operon with an E. coli/yeast hybrid one in which the GTPase center of E. coli 23S rRNA had been substituted by the corresponding domain from Saccharomyces cerevisiae. These results suggest that, contrary to common belief, coevolution of rRNA with many other components in the translational machinery may not completely preclude the horizontal transfer of rRNA genes.

A flavin reductase stimulates DszA and DszC proteins of Rhodococcus erythropolis IGTS8 in vitro.

Rhodococcus erythropolis IGTS8 is a gram positive bacterium, which can catabolize dibenzothiophene to 2-hydroxybiphenyl and inorganic sulfur without the cleavage of carbon-carbon bonds. Three structural genes, dszA, dszB, and dszC, have been cloned and shown to be necessary for this phenotype. Here, we demonstrate that a FMN:NADPH oxidoreductase from Vibrio harveyi complements activities of purified DszA and DszC proteins. Furthermore, we propose that DszA and DszC are oxygenase units that do not use NAD(P)H directly, but instead use FMNH2 from a FMN:NADPH oxidoreductase for oxygenation.