Term pregnant patients have similar gastric volume to non-pregnant females: a single-centre cohort study.

BACKGROUND: The physiological changes of pregnancy can increase the risk of peri-partum pulmonary aspiration. There is limited objective information regarding gastric volumes in pregnant patients. The aim of this cohort study was to characterise prospectively the range of gastric-fluid volume in term non-labouring pregnant patients compared with a historical cohort of non-pregnant females. METHODS: Fasted non-labouring term pregnant patients scheduled for elective Caesarean delivery underwent a standardised gastric ultrasound examination. Gastric content was evaluated qualitatively (type of content), semi-quantitatively (Perlas grades), and quantitatively (volume). The antral cross-sectional area and volume were compared with those of a retrospective cohort of non-pregnant females from the same institution. Descriptive statistics were used to describe the central tendency through mean and median values. Dispersion was evaluated with standard deviation and inter-quartile range, and the higher end of the distribution as 95th percentile. RESULTS: Non-labouring pregnant (59) and non-pregnant (81) subjects were studied. The range of estimated total gastric-fluid volume (P=0.96) and volume per body weight (P=0.78) was not significantly different between cohorts. An estimated volume of 115 ml (102-143) vs 136 ml (106-149) and volume per body weight of 1.4 ml kg-1 (1.2-2.8) vs 2.0 ml kg-1 (1.5-2.7) corresponded to the 95th percentile (95% confidence interval) values in the pregnant and non-pregnant cohort, respectively. CONCLUSIONS: Baseline gastric volume of non-labouring pregnant patients at term is not significantly different from that of non-pregnant females. This information will be helpful to interpreting findings of gastric point-of-care ultrasound in obstetric patients.

When fasted is not empty: a retrospective cohort study of gastric content in fasted surgical patients†.

Background: Perioperative aspiration leads to significant morbidity and mortality. Point-of-care gastric ultrasound is an emerging tool to assess gastric content at the bedside. Methods: We performed a retrospective cohort study of baseline gastric content on fasted elective surgical patients. The primary outcome was the incidence of full stomach (solid content or >1.5 ml kg−1 of clear fluid). Secondary outcomes included: gastric volume distribution (entire cohort, each antral grade); the association between gastric fullness, fasting intervals, and co-morbidities; anaesthetic management changes and incidence of aspiration. Results: We identified 538 patients. Thirty-two patients (6.2%) presented with a full stomach. Nine of these (1.7%) had solid content and 23 (4.5%) had clear fluid >1.5 ml kg−1. An empty stomach was documented in 480 (89.8%) patients. The examination was inconclusive in the remaining 20 patients (5.0%). As expected, increasing antral grade was correlated with larger antral cross-sectional area and higher gastric volume (P<0.001). Of the 32 patients with a full stomach, only six had a documented risk factor for prolonged gastric emptying. The anaesthetic management was changed in all nine patients with solid content. No aspiration was reported. Conclusions: This retrospective cohort study suggests that a small proportion of elective surgical patients may present with a full stomach despite the recommended duration of fasting. Further research is needed to establish the clinical implications of these findings in the elective setting. At present, the clinical role of gastric ultrasound continues to be for the evaluation of gastric contents to guide management when the risk of aspiration is uncertain or unknown.

Ultrasound assessment of gastric content and volume.

Pulmonary aspiration of gastric content is a serious anaesthetic complication that can lead to significant morbidity and mortality. Aspiration risk assessment is usually based on fasting times. However, fasting guidelines do not apply to urgent or emergent situations and to patients with certain co-morbidities. Gastric content and volume assessment is a new point-of-care ultrasound application that can help determine aspiration risk. This systematic review summarizes the current literature on bedside ultrasound assessment of gastric content and volume relevant to anaesthesia practice. Seventeen articles were identified using predetermined criteria. Studies were classified into those describing the sonographic characteristics of different types of gastric content (empty, clear fluid, solid), and those describing methods for quantitative assessment of gastric volume. A possible algorithm for the clinical application of this new tool is proposed, and areas that require further research are highlighted.

Gastric point-of-care ultrasound (PoCUS) during pregnancy and the postpartum period: a systematic review.

Personalised risk assessment of the likelihood of pulmonary aspiration is recommended for pregnant women undergoing general anaesthesia and gastric point-of-care ultrasound (PoCUS) may help to achieve this. Traditionally, risk assessment is based upon adherence to fasting times, but gastric emptying may vary during pregnancy and surgery often needs to be expedited. We systematically reviewed the evidence for gastric PoCUS up to August 2018 in pregnant and postpartum women to determine whether it can identify and quantify stomach contents, provide aspiration risk assessment via qualitative or quantitative means, and determine how gastric emptying is affected by pregnancy. Twenty-two articles comprising 1050 participants were included and studies were classified by qualitative or quantitative findings. The evidence suggests that gastric PoCUS is a reliable and feasible method of imaging the stomach in pregnancy in clinical practice. Qualitative assessment via the Perlas grading system can provide rapid assessment of gastric volume states. If fluid is visible, identification of patients at high risk of pulmonary aspiration requires measurement of antral cross-sectional area. Cut-off values of 608 mm2 and 960 mm2 are recommended in the semi-recumbent and right lateral semi-recumbent positions, respectively. Validated methods to quantify stomach volumes are available, however their usefulness is currently restricted to research. Gastric PoCUS also provides evidence that gastric emptying of ingested food is delayed by term pregnancy, labour and during the early postpartum period. However, the passage of fluids through the stomach appears unaffected throughout the peripartum period.

DNA inversions in phages and bacteria.

In certain phages and bacteria, there is a recombination system that specifically promotes the inversion of a DNA fragment. These inversion events appear to act as genetic switches allowing the alternate expression of different sets of genes which in general code for surface proteins. The mechanism of inversion in one class of inversion systems (Gin/Hin) has been studied in detail. It involves the formation of a highly specific nucleoprotein complex in which not only the two recombination sites and the DNA invertase participate but also a recombinational enhancer to which the DNA-bending protein Fis is bound.

Isolation, characterization, and UV-stimulated expression of two families of genes encoding polypeptides of related structure in human epidermal keratinocytes.

By screening of a cDNA library made on mRNA isolated from UV-irradiated human epidermal keratinocytes for sequences whose relative concentration increases in the cytoplasm after irradiation, we have isolated 40 cDNA clones (T. Kartasova, B. J. C. Cornelissen, P. Belt, and P. van de Putte, Nucleic Acids Res. 15:5945-5962, 1987). Here we describe two distinct groups of cDNA clones which do not cross-hybridize to each other but nevertheless encode proteins of very similar primary structure. These polypeptides are small (8 to 10 kilodaltons) and exceptionally rich in proline, cysteine, and glutamine and have similar repeating elements not found elsewhere. The new proteins were designated sprI and sprII (small, proline rich). The presence of prolines and cysteines suggests that they may be either structural proteins with a strong secondary structure or metal-binding proteins such as metallothioneins. Southern blot and sequence analyses of the cDNAs indicate that at least the sprII group of clones represents a family of related genes. The nucleotide sequence of both groups seems to be conserved upon evolution. The level of mRNAs corresponding to the two groups of cDNAs is increased in the cytoplasm of human epidermal keratinocytes after both UV irradiation and treatment with 4-nitroquinoline 1-oxide or 12-O-tetradecanoylphorbol 13-acetate.

Differential repair of UV damage in rad mutants of Saccharomyces cerevisiae: a possible function of G2 arrest upon UV irradiation.

After UV irradiation, the transcriptionally active MAT alpha locus in Saccharomyces cerevisiae is preferentially repaired compared with the inactive HML alpha locus. The effect of rad mutations from three different epistasis groups on differential repair was investigated. Three mutants, rad9, rad16, and rad24, were impaired in the removal of UV dimers from the inactive HML alpha locus, whereas they had generally normal repair of the active MAT alpha locus. Since RAD9 is necessary for G2 arrest after UV irradiation, we propose that the G2 stage plays a role in making the dimers accessible for repair, at least in the repressed HML alpha locus.

Interdependent transcription control elements regulate the expression of the SPRR2A gene during keratinocyte terminal differentiation.

Expression of the SPRR2A gene, a member of the small proline-rich family of cornified cell envelope precursor proteins, is strictly linked to keratinocyte terminal differentiation both in vivo and in vitro. In this study, we explored the molecular mechanisms underlying this regulation in transiently transfected primary keratinocytes induced to differentiate in vitro. Deletion mapping and site-directed mutagenesis of SPRR2A promoter-chloramphenicol acetyltransferase constructs indicate that four transcription control elements are essential and sufficient for promoter activity. These elements were further characterized by electrophoretic mobility shift and identified as (i) an inverted octamer doublet, bound by the POU domain factor Oct-11 (Skn-1a/i, Epoc-1), (ii) an interferon-stimulated response element recognized by interferon regulatory factors 1 and 2, (iii) an Ets binding site partially overlapping the interferon-stimulated response element, and (iv) a TG box recognized by the Sp1 family of zinc finger transcription factors. Destruction of a single terminal differentiation element is sufficient to completely abolish transcription from the SPRR2A promoter, indicating that these transcription control elements function in concert in an interdependent manner. Apparently, integration of signals transmitted by the above-mentioned transcription factors is necessary and sufficient to promote gene expression during keratinocyte terminal differentiation.

Novel protein in human epidermal keratinocytes: regulation of expression during differentiation.

Recently, two groups of cDNA clones have been isolated from human epidermal keratinocytes; the clones correspond to genes whose expression is stimulated by exposure of the cells to UV light or treatment with 4-nitroquinoline 1-oxide or 12-O-tetradecanoylphorbol 13-acetate (T. Kartasova and P. van de Putte, Mol. Cell. Biol. 8:2195-2203, 1988). The proteins predicted by the nucleotide sequence of both groups of cDNAs are small (8 to 10 kilodaltons), are exceptionally rich in proline, glutamine, and cysteine, and contain repeating elements with a common sequence, PK PEPC. These proteins were designated sprI and sprII (small, proline rich). Here we describe the characterization of the sprIa protein, which is encoded by one of the group 1 cDNAs. The expression of this protein during keratinocyte differentiation in vitro and the distribution of the sprIa protein in some human tissues was studied by using a specific rabbit antiserum directed against a synthetic polypeptide corresponding to the 30 amino acids of the C-terminal region of the sprIa gene product. The results indicate that the expression of the sprIa protein is stimulated during keratinocyte differentiation both in vitro and in vivo.

Transfer of human genes conferring resistance to methylating mutagens, but not to UV irradiation and cross-linking agents, into Chinese hamster ovary cells.

Chinese hamster ovary cells were transfected by human DNA ligated to the bacterial gpt (xanthine-guanine-phosphoribosyltransferase) gene which was used either in its native form or after partial inactivation with methylnitrosourea. The gpt+ transfectants were screened for resistance to high doses of N-methyl-N'-nitro-N-nitrosoguanidine. Using this approach, we showed that Chinese hamster ovary cells can acquire N-methyl-N'-nitro-N-nitrosoguanidine resistance upon transfection with DNA from diploid human fibroblasts, that this resistance is transferable by secondary transfection and is specific for methylating mutagens, and that it is not caused by increased removal of O6-methylguanine, 3-methyladenine, and 7-methylguanine from DNA.

The RAD7 and RAD16 genes, which are essential for pyrimidine dimer removal from the silent mating type loci, are also required for repair of the nontranscribed strand of an active gene in Saccharomyces cerevisiae.

The rad16 mutant of Saccharomyces cerevisiae was previously shown to be impaired in removal of UV-induced pyrimidine dimers from the silent mating-type loci (D. D. Bang, R. A. Verhage, N. Goosen, J. Brouwer, and P. van de Putte, Nucleic Acids Res. 20:3925-3931, 1992). Here we show that rad7 as well as rad7 rad16 double mutants have the same repair phenotype, indicating that the RAD7 and RAD16 gene products might operate in the same nucleotide excision repair subpathway. Dimer removal from the genome overall is essentially incomplete in these mutants, leaving about 20 to 30% of the DNA unrepaired. Repair analysis of the transcribed RPB2 gene shows that the nontranscribed strand is not repaired at all in rad7 and rad16 mutants, whereas the transcribed strand is repaired in these mutants at a fast rate similar to that in RAD+ cells. When the results obtained with the RPB2 gene can be generalized, the RAD7 and RAD16 proteins not only are essential for repair of silenced regions but also function in repair of nontranscribed strands of active genes in S. cerevisiae. The phenotype of rad7 and rad16 mutants closely resembles that of human xeroderma pigmentosum complementation group C (XP-C) cells, suggesting that RAD7 and RAD16 in S. cerevisiae function in the same pathway as the XPC gene in human cells. RAD4, which on the basis of sequence homology has been proposed to be the yeast XPC counterpart, seems to be involved in repair of both inactive and active yeast DNA, challenging the hypothesis that RAD4 and XPC are functional homologs.

Double mutants of Saccharomyces cerevisiae with alterations in global genome and transcription-coupled repair.

The nucleotide excision repair (NER) pathway is thought to consist of two subpathways: transcription-coupled repair, limited to the transcribed strand of active genes, and global genome repair for nontranscribed DNA strands. Recently we cloned the RAD26 gene, the Saccharomyces cerevisiae homolog of human CSB/ERCC6, a gene involved in transcription-coupled repair and the disorder Cockayne syndrome. This paper describes the analysis of yeast double mutants selectively affected in each NER subpathway. Although rad26 disruption mutants are defective in transcription-coupled repair, they are not UV sensitive. However, double mutants of RAD26 with the global genome repair determinants RAD7 and RAD16 appeared more UV sensitive than the single rad7 or rad16 mutants but not as sensitive as completely NER-deficient mutants. These findings unmask a role of RAD26 and transcription-coupled repair in UV survival, indicate that transcription-coupled repair and global genome repair are partially overlapping, and provide evidence for a residual NER modality in the double mutants. Analysis of dimer removal from the active RPB2 gene in the rad7/16 rad26 double mutants revealed (i) a contribution of the global genome repair factors Rad7p and Rad16p to repair of the transcribed strand, confirming the partial overlap between both NER subpathways, and (ii) residual repair specifically of the transcribed strand. To investigate the transcription dependence of this repair activity, strand-specific repair of the inducible GAL7 gene was investigated. The template strand of this gene was repaired only under induced conditions, pointing to a role for transcription in the residual repair in the double mutants and suggesting that transcription-coupled repair can to some extent operate independently from Rad26p. Our findings also indicate locus heterogeneity for the dependence of transcription-coupled repair on RAD26.

Towards integrating vectors for gene therapy: expression of functional bacteriophage MuA and MuB proteins in mammalian cells.

Bacteriophage Mu has one of the best studied, most efficient and largest transposition machineries of the prokaryotic world. To harness this attractive integration machinery for use in mammalian cells, we cloned the coding sequences of the phage factors MuA and MuB in a eukaryotic expression cassette and fused them to a FLAG epitope and a SV40-derived nuclear localization signal. We demonstrate that these N-terminal extensions were sufficient to target the Mu proteins to the nucleus, while their function in Escherichia coli was not impeded. In vivo transposition in mammalian cells was analysed by co-transfection of the MuA and MuB expression vectors with a donor construct, which contained a miniMu transposon carrying a Hygromycin-resistance marker (Hyg(R)). In all co-transfections, a significant but moderate (up to 2.7-fold) increase in Hyg(R) colonies was obtained if compared with control experiments in which the MuA vector was omitted. To study whether the increased efficiency was the result of bona fide Mu transposition, integrated vector copies were cloned from 43 monoclonal and one polyclonal cell lines. However, in none of these clones, the junction between the vector and the chromosomal DNA was localized precisely at the border of the Att sites. From our data we conclude that expression of MuA and MuB increases the integration of miniMu vectors in mammalian cells, but that this increase is not the result of bona fide Mu-induced transposition.

Influence of temperature on platinum binding to DNA, cell killing, and mutation induction in Escherichia coli K-12 cells treated with cis-diamminedichloroplatinum(II).

Cell killing and specificity of mutation induction by treatment of Escherichia coli K-12 cells with cis-Pt(NH3)2Cl2 at various temperatures have been studied. Survival experiments show that the cell killing by cis-Pt(NH3)2Cl2 is enhanced with increasing temperature. This effect is explained by an increase in the amount of platinum bound to the DNA. The binding is the same in repair-proficient and in repair-deficient cells. However, the mutation induction in the lacI gene is much more strongly enhanced than could be expected from the increased platinum binding. This phenomenon is attributed to a different distribution of the induced lesions by cis-Pt(NH3)2Cl2 at various temperatures and not to an aberrant excision repair. Analysis of the induced lacI mutants revealed an increase in the percentage of nonsense mutants at higher temperature. Among the nonsense mutations, base-pair substitutions at GAG and particularly at GCG sequences are enhanced by the increasing temperature. The results are in agreement with our hypothesis that local denaturation of DNA, known to be promoted at higher temperature, is necessary for the formation of intrastrand cross-links at two guanine bases separated by a third base.

Involvement of c-JUN in the regulation of terminal differentiation genes in normal and malignant keratinocytes.

In stratifying cultures of human keratinocytes, expression of the proto-oncoprotein c-JUN and the small proline rich 2 (SPRR2) protein, a precursor of the cornified cell envelope, are inversely related. Whereas c-JUN is typically found in basal proliferating cells, SPRR2 is restricted to suprabasal differentiating layers. Malignant keratinocytes (derived from squamous cell carcinoma, SCC) have reduced sprr2 expression, consistent with their low potential to differentiate, and express c-jun at higher levels than normal keratinocytes. A direct relation between c-jun and sprr2 expression was shown in several ways: transient ectopic expression of c-jun inhibits sprr2a promoter activity in normal differentiating cells, whereas in malignant keratinocytes a dominant negative c-jun mutant restored at least partially both the low promoter activity and the expression of endogenous sprr2. These effects are mediated via a 134 bp promoter fragment which does not include the sprr2a AP-1 binding site. Interestingly, in an SCC cell line, constitutively expressing the dominant c-jun mutant, expression of the terminal differentiation marker involucrin is also strongly increased, suggesting that c-JUN is a general modulator of keratinocyte terminal differentiation rather than only affecting the expression of sprr2.

Purification of the Gin recombination protein of Escherichia coli phage Mu and its host factor.

Inversion of the G-segment of Escherichia coli phage Mu was studied in vitro. The reaction requires the Gin recombination protein, which was purified to near homogeneity from overproducing cells. Upon purification the protein lost activity, which was restored by addition of an extract from uninfected E. coli cells. The stimulatory host factor is a small heat-stable protein and was purified from E. coli cells. Full recombination required both proteins, but Gin alone promoted some recombination by itself, particularly at high concentrations. Relaxation of negative supercoils and recombination of a substrate with two recombination sites in an inverted orientation both have the same specificity for Gin and the host factor. The Gin-associated topoisomerase activity appears tightly coupled to its recombination activity.

Cytosine methylation in the EcoRI site of active and inactive herpesvirus thymidine kinase promoters.

The herpesvirus thymidine kinase (tk) gene integrated in the human cell line, 2.1-a, can be inactivated by limited de novo methylation. All these TK- clones show partial EcoRI digestion of the recognition site (cGAATTCg) in the tk promoter in contrast to complete digestion of this site in the original cell line. Studies on well-defined substrates prepared in vitro showed that methylation of one cytosine in the EcoRI recognition sequence resulted in partial and methylation of both cytosines in severe inhibition of digestion by EcoRI. This characteristic was used to determine whether no, one or both cytosines in the EcoRI site of the tk promoter were methylated in various TK- clones derived from 2.1-a and in TK+ clones re-expressing the gene after 5-azacytidine treatment. A high correlation was found between inactivity of the tk gene and methylation of only one of the two cytosines in the EcoRI recognition site. The results also show that the tk promoter can be active despite the presence of a methylated cytosine.

De novo methylation as major event in the inactivation of transfected herpesvirus thymidine kinase genes in human cells.

Spontaneous inactivation of integrated thymidine kinase genes was studied in three human cell lines, one with multiple copies and two with a single copy of a transfected shuttle plasmid containing two selectable genes: the HSV tk gene and the Eco gpt gene. Selection for gpt expression prevented the isolation of TK- mutants which are the result of plasmid loss. Under these conditions TK- clones were isolated with a frequency of 5.10(-6) both with the cell line containing 5 or 6 copies of the tk gene and with one of the two cell lines containing one copy of this gene. This inactivity of the tk gene was associated with de novo methylation as the number of HAT-resistant (TK+) clones strongly increased after growth of the TK- derivatives in the presence of the demethylating agent, 5-azacytidine. Digestion with methylation-sensitive restriction enzymes revealed two different patterns of DNA methylation in the genomic DNA of TK- variants. In the TK- derivatives of the cell line containing multiple copies of the tk gene many HpaII restriction sites in the gene copies were insensitive to digestion. These HpaII sites were, however, not methylated in TK- variants of the cell line containing one copy of the plasmid, and methylated CpGs could be detected only with EcoRI which recognizes the cGAATTCg sequence in the tk promoter region. With the other of the two single-copy TK+ cell lines no TK- mutants were obtained, suggesting that the position of a gene in the genome is an important factor in determining the frequency and the extent of de novo methylation. Additionally, we observed that remethylation is an even more efficient process of gene inactivation as TK+ clones reactivated with 5-azacytidine can become TK- again at a 100-fold higher rate than the original TK+ cell line.