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.

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.

Continuous interscalene block using a stimulating catheter: a review of the technique.

The management of postoperative pain after major shoulder surgery can be achieved successfully with a continuous interscalene block. This article reviews the essentials of the stimulating catheter technique for the continuous interscalene block that was described by Boezaart in 1999. The authors also describe their experience and results with the first two hundred catheters they placed.

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.

Characterization and mapping of resistance to Oidium lycopersicum in two Lycopersicon hirsutum accessions: evidence for close linkage of two Ol-genes on chromosome 6 of tomato.

The cultivated tomato is susceptible to powdery mildew (Oidium lycopersicum). Several accessions of wild species are resistant. In this study we describe (i) the genetics and mapping of resistance to O. lycopersicum in G1.1290, one of the resistant accessions in Lycopersicon hirsutum, (ii) fine mapping of Ol-1 originated from L. hirsutum G1.1560, another resistant accession of L. hirsutum, and (iii) tests of allelism for resistance in G1.1290 and G1.1560. Initially, it is demonstrated that the resistance in G1.1290 to O. lycopersicum is controlled by an incompletely dominant gene, designated Ol-3. By using an advanced breeding line (ABL) containing introgression fragment(s) from G1.1290, Ol-3 was found to be associated with several RFLP and SCAR markers on chromosome 6. By using these markers, Ol-3 was mapped between markers TG25/SCAF10 and H9A11 on chromosome 6. Secondly, after testing some F3 lines and their progenies from the cross between L. esculentum cv Moneymaker and L. hirsutum G1.1560, we provided more evidence for the map position of Ol-1 to lie between SCAF10 and H9A11, indicating that Ol-1 and Ol-3 are in the same chromosome region. Thirdly, although allelism tests could not discriminate between Ol-1 and Ol-3, (indirect) evidence suggested that these two genes are not identical. They might instead represent functional genes of a cluster of Ol-homologues.

Opposite effects of Ras or PKC activation on the expression of the SPRR2A keratinocyte terminal differentiation marker.

Epidermal growth factor (EGF) enhances the expression of the keratinocyte terminal differentiation marker SPRR2A, when added to monolayers of basal keratinocytes, induced to stratify by increasing the extracellular calcium concentration. A similar stimulation is found during suspension-induced differentiation in methylcellulose. This effect, which is observed after several hours of EGF addition, is restricted to terminally differentiating keratinocytes and is dependent on PKC signaling. EGF also transiently activates the Ras signaling pathway, with a maximum induction after 10 min (Medema et al., 1994, Mol. Cell. Biol. 14, 7078-7085). The cellular effects of activated Ras were determined by transient transfection of Ha-ras(Leu-61) into normal human keratinocytes. Activated Ras completely inhibited PKC-mediated expression of SPRR2A. This inhibition is mediated via c-Jun as it is reversed by a dominant-negative c-Jun mutant (cJunDelta6/194) and c-Jun can substitute for activated Ras. The inhibitory effect is targeted to a 150-bp minimal promoter region, which is essential and sufficient for SPRR2A expression during keratinocyte terminal differentiation. This indicates that the Ras and PKC pathways, which both can be triggered by EGF, although at different time points, have opposite effects on SPRR2A gene expression.

Structure and evolution of the human SPRR3 gene: implications for function and regulation.

SPRR3, a member of the SPRR family of cornified envelope precursor proteins, is expressed in oral and esophageal epithelia, where it is strictly linked to keratinocyte terminal differentiation. This gene is characterized by intragenic duplications that have created the characteristic proline-rich repeats in the coding sequence, an alternative noncoding exon, and a 200-bp polypyrimidine tract in the promoter region. Mutational analysis of the promoter region and transient transfection in normal human keratinocytes showed that in addition to the polypyrimidine tract, multiple regulatory elements are involved in differentiation-specific expression. These elements include a high-affinity Ets binding site bound by ESE-1, an AP-1 site (TRE) recognized by the Jun/Fos family of transcription factors, and an ATF/CRE bound by Jun/Fos and ATF factors. The repositioning of the SPRR3 Ets binding site during evolution has a major effect on the relative contribution of this site to promoter activity.

Involvement of a nuclear matrix association region in the regulation of the SPRR2A keratinocyte terminal differentiation marker.

The small proline-rich protein genes ( SPRRs ) code for precursors of the cornified cell envelope, and are specifically expressed during keratinocyte terminal differentiation. The single intron of SPRR2A enhanced the activity of the SPRR2A promoter in transient transfection assays. This enhancement was position dependent, and did not function in combination with a heterologous promoter, indicating that the intron does not contain a classical enhancer, and that the enhancement was not due to the splicing reaction per se. Mild DNAse-I digestion of nuclei showed the SPRR2 genes to be tightly associated with the nuclear matrix, in contrast to the other cornified envelope precursor genes mapping to the same chromosomal location (epidermal differentiation complex). In vitro binding studies indicated that both the proximal promoter and the intron of SPRR2A are required for optimal association of this gene with nuclear matrices. Neither nuclear matrix association nor the relative transcriptional enhancement by the intron changed during keratinocyte differentiation. Apparently, the association of the SPRR2A gene with the nuclear matrix results in a general, differentiation-independent enhancement of gene expression.

AP-1 and ets transcription factors regulate the expression of the human SPRR1A keratinocyte terminal differentiation marker.

The 173-base pair proximal promoter of SPRR1A is necessary and sufficient for regulated expression in primary keratinocytes induced to differentiate either by increasing extracellular calcium or by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Whereas calcium-induced expression depends both on an AP-1 and an Ets binding site in this region, responsiveness to TPA resides mainly (but not exclusively) on the Ets element, indicating that Ets factors are important targets for protein kinase C signaling during keratinocyte terminal differentiation. This conclusion is further substantiated by the finding that expression of ESE-1, an Ets transcription factor involved in SPRR regulation, is also induced by TPA, with kinetics similar to SPRR1A. The strict AP-1 requirement in SPRR1A for calcium-induced differentiation is not found for SPRR2A, despite the presence of an identical AP-1 consensus binding site in this gene. Binding site swapping indicates that both the nucleotides flanking the TGAGTCA core sequence and the global promoter context are essential in determining the contribution of AP-1 factors in gene expression during keratinocyte terminal differentiation. In the distal SPRR1A promoter region, a complex arrangement of positive and negative regulatory elements, which are only conditionally needed for promoter activity, are likely involved in gene-specific fine-tuning of the expression of this member of the SPRR gene family.

Regulation of single-strand RNA synthesis of alfalfa mosaic virus in non-transgenic cowpea protoplasts by the viral coat protein.

We have compared the RNA synthesis of alfalfa mosaic virus in complete (by RNAs 1, 2 and 3) and incomplete infections (by RNAs 1 and 2) of cowpea protoplasts. Both viral RNA polymerase activity and accumulation of viral RNA were measured. By annealing RNA in solution with 32P-labelled probes of plus and minus polarity followed by treatment with ribonucleases, we determined viral RNAs quantitatively in both single- and double-stranded RNA fractions. The accumulation of single-stranded RNA of positive polarity differed considerably between the two types of infection (250 ng vs. less than 1 ng per 10(5) protoplasts), although viral RNA polymerase activities as measured in vitro and the concentrations of minus RNA were similar. Since the method also measured fragmented RNA, this difference is probably not due to lack of protection of viral RNA by coat protein during incomplete infection. Synthesis of single-stranded plus RNA requires either RNA 3 itself or one of its gene products. We postulate that coat protein is the stringent regulator of alfalfa mosaic virus genomic expression.

Transitions in the coupling of transcription and nucleotide excision repair within RNA polymerase II-transcribed genes of Saccharomyces cerevisiae.

The molecular mechanism of transcription-coupled nucleotide excision repair in eukaryotes is poorly understood. The identification of the dual role of basal transcription factor TFIIH in DNA repair and transcription provided a plausible link between both processes. However, TFIIH is not part of the elongating transcription complex, suggesting that additional components are required to recruit TFIIH when RNA polymerase II (RNAPII) stalls at the site of DNA damage. Previously, we have shown that the yeast Rad26 protein is involved in transcription-coupled DNA repair. This paper describes the differential contribution of the Rad26 protein to efficient removal of UV-induced cyclobutane pyrimidine dimers (CPDs) from transcribed DNA. Two distinct regions within the transcribed strand of RNAPII-transcribed genes are identified that differ in their requirement for the RAD26 gene product. Using high-resolution repair analysis, we determined the in vivo repair kinetics of cyclobutane pyrimidine dimers positioned around the transcription initiation site of RNAPII-transcribed genes RPB2 and URA3. Although transcription-coupled repair is severely reduced in rad26 mutants, lesions positioned in a small region immediately downstream of transcription initiation are efficiently removed in the absence of Rad26. The observed transition in repair characteristics is abrupt and in excellent agreement with the region where TFIIH dissociates from RNAPII in vitro, strongly suggesting an inverse correlation between TFIIH association and Rad26 requirement. These data suggest that a transcription repair coupling factor (Rad26/CSB) is required for efficient repair only during the elongating stages of RNAPII transcription.

The integration host factor-DNA complex upstream of the early promoter of bacteriophage Mu is functionally symmetric.

Inversion of the ihf site in the promoter region of the early promoter of bacteriophage Mu did not influence the integration host factor (IHF)-mediated functions. IHF bound to this inverted site could counteract H-NS-mediated repression, directly activate transcription, and support lytic growth of bacteriophage Mu. This implies that the IHF heterodimer and its asymmetrical binding site form a functionally symmetrical complex.

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.

Transcription elongation factor S-II is not required for transcription-coupled repair in yeast.

Two different subpathways play a role in removal of UV-induced cyclobutane pyrimidine dimers (CPDs) by nucleotide excision repair (NER). The relatively slow global genome repair subpathway operates on all CPDs irrespective of their position in the DNA, whereas the transcription-coupled repair subpathway is responsible for the rapid removal of CPDs from transcribed strands. In Saccharomyces cerevisiae, the RAD26 gene is implicated in transcription-coupled repair. However, transcription-coupled repair is not completely absent in rad26 mutants, and therefore other gene products are possibly involved in this subpathway. Based on in vitro experiments with purified components, the transcription elongation factor S-II appeared to be a candidate for a function in transcription-coupled repair. To investigate a possible role of S-II in transcription-coupled repair in vivo in yeast, S-II null mutations were introduced into various genetic backgrounds differing in NER capacity. UV sensitivity was not altered by disruption of the S-II gene in a RAD+ (NER proficient) strain, or in rad26 (impaired in efficient transcription-coupled repair), rad7 (lacking global genome repair), or rad7 rad26 (lacking global genome repair, but having residual transcription-coupled repair capacity) mutants. Moreover, S-II did not influence the repair rate on the transcribed strand of the RPB2 gene, either in repair-proficient or in rad7 rad26 backgrounds. Hence, transcription-coupled repair is fully functional in yeast cells lacking the gene encoding S-II. Furthermore, S-II is not required for the Rad26-independent residual transcription-coupled repair in vivo.

Expression of the SPRR cornification genes is differentially affected by carcinogenic transformation.

The small proline rich protein (SPRR) genes constitute a family of conserved genes which are part of the human epidermal differentiation complex (EDC) on chromosome 1q21 and code for precursor proteins of the cornified cell envelope. The expression of these genes is strictly linked to keratinocyte terminal differentiation both in vivo and in vitro. Here we show that cultured cell lines derived from squamous cell carcinoma (SCC) show significantly lower levels of SPRR expression than normal human keratinocytes. However, the residual SPRR expression in SCC lines appears to be both gene and cell line specific. Expression of SPRR2 appears to correlate well with the residual ability of these cells to differentiate. However, the kinetics of SPRR2 expression, following treatment with calcium, an inducer of keratinocyte differentiation, are typical for each cell line and differ substantially from the ones found in normal cells. In most cell lines a rapid transient expression of SPRR2 contrasts with a slow induction leading to a high sustained level of expression in normal cells. This pattern of expression is typical for SPRR2 and not observed for the other SPRR genes or involucrin. Our analysis indicates that the expression of various keratinocyte terminal differentiation markers, even when involved in the same biological process (cornification), can be differentially affected by carcinogenic transformation.