The influence of 2020 coronavirus lockdown on presentation of oral and maxillofacial trauma to a central London hospital.

The novel coronavirus COVID-19 was first identified in China in December 2019. Its spread resulted in a pandemic, with the United Kingdom entering a period of national lockdown on 23 March 2020 to reduce disease burden on the National Health Service (NHS). King's College Hospital is a Major Trauma Centre serving an inner-city population of 700,000 with 120,000 patients attending the emergency department (ED) annually. We aimed to determine the effect of lockdown on OMFS trauma presentations and lessons learned from emergency service provision during a pandemic. All referrals to the oral and maxillofacial surgical (OMFS) team from ED during the first six weeks of the lockdown period - 23 March 2020 - 3 May 2020 - were compared with the same six-week period in 2019. A total of 111 referrals were made to OMFS during the first six weeks of the lockdown period in 2020 compared with 380 referrals in 2019. Of these, 50.5%, (n=192) were related to facial trauma in 2019 vs (63.1%, n=70) in 2020. Fewer patients were admitted under OMFS: 17.4% (n=35) in 2019 vs 2.9% (n=2) in 2020, and a greater number of patients were discharged from OMFS care directly from the ED: 63.2% (n=127) in 2019 vs 82.9% (n=58) in 2020. There was profound effect of the lockdown on referrals to OMFS from the ED, in number and type of diagnosis. This is potentially reflective of the increased availability of acute/emergency dental services in South-East London during the lockdown period. This gives us valuable insight for service planning in the event of further restrictions.

Activation of two different resistance mechanisms in Saccharomyces cerevisiae upon exposure to octanoic and decanoic acids.

Medium-chain fatty acids (octanoic and decanoic acids) are well known as fermentation inhibitors. During must fermentation, the toxicity of these fatty acids is enhanced by ethanol and low pH, which favors their entrance in the cell, resulting in a decrease of internal pH. We present here the characterization of the mechanisms involved in the establishment of the resistance to these fatty acids. The analysis of the transcriptome response to the exposure to octanoic and decanoic acids revealed that two partially overlapping mechanisms are activated; both responses share many genes with an oxidative stress response, but some key genes were activated differentially. The transcriptome response to octanoic acid stress can be described mainly as a weak acid response, and it involves Pdr12p as the main transporter. The phenotypic analysis of knocked-out strains confirmed the role of the Pdr12p transporter under the control of WAR1 but also revealed the involvement of the Tpo1p major facilitator superfamily proteins (MFS) transporter in octanoic acid expulsion. In contrast, the resistance to decanoic acid is composite. It also involves the transporter Tpo1p and includes the activation of several genes of the beta-oxidation pathway and ethyl ester synthesis. Indeed, the induction of FAA1 and EEB1, coding for a long-chain fatty acyl coenzyme A synthetase and an alcohol acyltransferase, respectively, suggests a detoxification pathway through the production of decanoate ethyl ester. These results are confirmed by the sensitivity of strains bearing deletions for the transcription factors encoded by PDR1, STB5, OAF1, and PIP2 genes.

SUT1 is a putative Zn[II]2Cys6-transcription factor whose upregulation enhances both sterol uptake and synthesis in aerobically growing Saccharomyces cerevisiae cells.

Budding yeast Saccharomyces cerevisiae is a facultative anaerobe whose growth upon oxygen starvation depends on its capacity to import exogenously supplied sterols, whereas the cells are not permeable to these molecules when grown aerobically. Few genes have been identified as being involved in sterol uptake. A higher SUT1 gene dosage leads to a modest, but significant, increase in sterol uptake under aerobic conditions. Based on sequence and physiological data, SUT1 is a hypoxic gene negatively regulated when the cells are grown in the presence of oxygen. We replaced the SUT1 promoter with the constitutive PMA1 gene promoter in order to enhance its transcription. We observed that sterol uptake was then comparable with that obtained with a sterol importing hem1 mutant, although the heme status of the strain was not modified in a process which still occurs when the cells are not growing. Unexpectedly, SUT1 constitutive expression led to a parallel significant increase in endogenous sterol biosynthesis. Moreover, here we present new data showing that the structurally related YPR009 gene (SUT2) is a functional homologue of SUT1, and that both gene products may represent two novel yeast regulatory proteins involved in sterol uptake.

The role of ERG20 gene (encoding yeast farnesyl diphosphate synthase) mutation in long dolichol formation. Molecular modeling of FPP synthase.

The yeast Saccharomyces cerevisiae strain LB332 bearing a mutation in the ERG20 gene encoding farnesyl diphosphate synthase (FPPS) synthesizes significantly longer dolichols than the wild type strain FL100 (14-31 and 14-19 isoprene units, respectively). The measurement of the short chain prenyl alcohols excreted into the medium shows that increased amounts of geraniol, dimethylallyl and isopentenyl alcohols but not farnesol are synthesized by the mutant strain. The wild type FPPS synthesizes farnesyl diphosphate (FPP) as the only product. The K197E substitution, as opposed to F112A/F113S in avian FPPS, does not change product specificity. Consequently, the possibility that mutated yeast FPPS synthesizes longer polyprenols is unlikely. This is supported by additional evidence such as in vitro analysis of the mutated FPPS products and molecular modeling. We suggest that formation of longer dolichols in vivo is the result of a change in the isopentenyl diphosphate/farnesyl diphosphate ratio caused by the erg20 mutation which in turn affects the activity of cis-prenyltransferase.

Sterol metabolism and ERG2 gene regulation in the yeast Saccharomyces cerevisiae.

Certain exogenously-supplied sterols, like ergost-8-enol, are efficiently converted into ergosterol in yeast. We have taken advantage of this property to study the regulation of the Delta8-Delta7-sterol isomerase-encoding ERG2 gene in an ergosterol auxotrophic mutant devoid of squalene-synthase activity. Ergosterol starvation leads to an 8-16-fold increase in ERG2 gene expression. Such an increase was also observed in wild-type cells either grown anaerobically or treated with SR31747A a sterol isomerase inhibitor. Exogenously-supplied zymosterol is entirely transformed into ergosterol, which represses ERG2 transcription. By contrast, exogenously-supplied ergosterol has little or no effect on ERG2 transcription.

The regulation of activity of main mevalonic acid pathway enzymes: farnesyl diphosphate synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, and squalene synthase in yeast Saccharomyces cerevisiae.

The co-regulation of the main mevalonic acid pathway enzymes was investigated in the yeast Saccharomyces cerevisiae. It was found that a 6-fold increase in FPPS activity compared with that of the wild-type strain FL100 did not cause significant changes in HMG-CoA reductase activity, while the amounts of synthesized dolichols and ergosterol increased by 80 and 32%, respectively. The disruption of the SQS gene in the strain grown in the presence of ergosterol repressed the activities of both FPP synthase and HMG-CoA reductase to a comparable degree, whereas in the same strain starved for ergosterol the activity of FPPS was 10-fold higher and HMG-CoA reductase activity was practically unchanged. We show that FPPS is the enzyme that regulates the flow rate of synthesized mevalonic acid pathway products independent of HMG-CoA reductase and SQS.

The Saccharomyces cerevisiae mevalonate diphosphate decarboxylase (erg19p) forms homodimers in vivo, and a single substitution in a structurally conserved region impairs dimerization.

The wild-type ERG19 gene of the yeast Saccharomyces cerevisiae encoding mevalonate diphosphate decarboxylase (MVD) and the mutated recessive erg19-34 allele leading to a decrease of sterol production and to a thermosensitive phenotype have been characterized [2]. The mutated erg19-34 allele bears a single amino acid leucine 79-to-proline (L79P) substitution. It was shown that this mutation does not affect the level of production of the enzyme. We performed a two-hybrid assay to show that the yeast Saccharomyces cerevisiae MVD forms homodimers in vivo and that the single point mutation drastically impairs the oligomerization of the protein, thereby explaining the deficiency of MVD activity observed in the temperature-sensitive strain.

Heterologous expression in Saccharomyces cerevisiae of an Arabidopsis thaliana cDNA encoding mevalonate diphosphate decarboxylase.

Sequence comparison with the mevalonate diphosphate decarboxylase (MVD) amino acid sequence of Saccharomyces cerevisiae identified an EST clone corresponding to a cDNA that may encode Arabidopsis thaliana MVD (AtMVD1). This enzyme catalyses the synthesis of isopentenyl diphosphate, the building block of sterol and isoprenoid biosynthesis, and uses mevalonate diphosphate as a substrate. Sequencing of the full-length cDNA was performed. The predicted amino acid sequence presents about 55% identity with the yeast, human and rat MVDs. The sequence of the genomic region of A. thaliana MVD was also obtained and Southern blot analysis on genomic DNA showed that A. thaliana could have at least one homologous MVD gene. In order to allow heterologous expression in S. cerevisiae, the MVD open reading frame (ORF) was then cloned under the control of the yeast PMA1 strong promoter. When expressed in yeast, the A. thaliana cDNA complemented both the thermosensitive MN19-34 strain deficient in MVD, and the lethal phenotype of an ERG19 deleted strain. However, the wild-type sterol content was not fully restored suggesting that the A. thaliana MVD activity may not be optimal in yeast. A two-hybrid assay was also performed to evaluate homodimer formation of the A. thaliana MVD and heterodimer formation between the plant and yeast heterologous enzymes.

Molecular cloning, expression analysis, and chromosomal localization of human syntaxin 8 (STX8).

We report the cloning of a cDNA encoding human syntaxin 8 (STX8), using the regulator (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) as a bait to screen a human fetal lung cDNA library by the yeast two-hybrid system. This gene was found broadly transcribed and its mRNA size is about 1.3 kb. The STX8 gene maps to chromosomal band 17p12 and it encodes a 236-amino-acid protein. Syntaxin 8 contains in its C-terminal half a coiled-coil domain found highly conserved in the t-SNARE (SNAP receptor on target membrane) superfamily of proteins, which are involved in vesicular trafficking and docking. In syntaxin 8, a C-terminal hydrophobic domain may constitute a transmembrane anchor. It was recently shown that CFTR-mediated chloride currents can be regulated by syntaxin 1A, a t-SNARE family member, through direct protein-protein interaction. This raises the possibility that syntaxin 8 may also be involved in such regulations.

Effect of squalene synthase gene disruption on synthesis of polyprenols in Saccharomyces cerevisiae.

Biosynthesis of polyprenols was investigated in a wild-type strain of Saccharomyces cerevisiae and a squalene synthase deficient strain auxotrophic for ergosterol. The quantitative data showed that disruption of squalene synthase gene caused a 6-fold increase in the synthesis of polyprenols in vitro in comparison with the wild-type strain. Microsomal preparation from the deleted strain only slightly reacted to the additional exogenous FPP, while that from the wild-type strain presented a 4-fold increase of polyprenol synthesis. Restoration of ergosterol synthesis, by introducing ERG9 functional allele into the deleted strain resulted in a significant lowering of polyprenol synthesis, indicating the immediate shift of the common substrate (FPP) to the sterol pathway. The role of squalene synthase in the regulation of polyprenol synthesis and 'flow diversion hypothesis' is discussed.

Sterol uptake in Saccharomyces cerevisiae heme auxotrophic mutants is affected by ergosterol and oleate but not by palmitoleate or by sterol esterification.

The relationship between sterol uptake and heme competence in two yeast strains impaired in heme synthesis, namely, G204 and H12-6A, was analyzed. To evaluate heme availability, a heterologous 17alpha-hydroxylase cytochrome P-450 cDNA (P-450c17) was expressed in these strains, and its activity was measured in vivo. Heme deficiency in G204 led to accumulation of squalene and lethality. The heterologous cytochrome P-450 was inactive in this strain. The leaky H12-6A strain presented a slightly modified sterol content compared to that for the wild type, and the P-450c17 recovered partial activity. By analyzing sterol transfer on nongrowing cells, it was shown that the cells were permeable toward exogenous cholesterol when they were depleted of endogenous sterols, which was the case for G204 but not for H12-6A. It was concluded that the fully blocked heme mutant (G204) replenishes its diminishing endogenous sterol levels during growth by replacement with sterol from the outside medium. Endogenous sterol biosynthesis appears to be the primary factor capable of excluding exogenous sterol. Oleate but not palmitoleate was identified as a component that reduced but did not prevent sterol transfer. Sterol transfer was only slightly affected by a lack of esterification. It is described herein how avoidance of the potential cytotoxicity of the early intermediates of the mevalonate pathway could be achieved by a secondary heme mutation in erg auxotrophs.

[Biosynthesis and transport of sterols in the yeast Saccharomyces cerevisiae]. / Biosynthèse et transport des stérols chez la levure Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae was a powerful tool in the identification of the structural genes involved in sterol biosynthesis in eucaryotes. Among 20 genes, 16 were isolated by genetic techniques using either complementation of mutants or overexpression strategy using specific inhibitors. In spite of this good knowledge concerning the genes of the pathway, little is known about the regulation of the isoprenoid/steroid biosynthetic pathway. However, the existence of two genes encoding HMG-CoA reductase in yeast genome suggests strongly that this enzyme could play a fundamental function in regulation, such as in plants and mammals. The regulation mechanisms could also involve sterol trafficking and storage. Indeed, one enzyme in the pathway, the sterol-C24-methyl transferase is localized in lipid particles that correspond to the storage form of steryl esters. Yeast cells are impermeable towards exogenous sterols in aerobiosis and become permeable in anaerobiosis when ergosterol synthesis is precluded by the absence of molecular oxygen. This phenomenon called aerobic sterol exclusion is dependent on the hem status of the cell. One gene, named SUT1 was identified that directs aerobic sterol uptake in yeast SUT1 gene and his partner SUT2 present strong features common to yeast transcription factors and could regulate the expression of genes involved in sterol uptake or intracellular trafficking.

Cloning and characterization of the Arabidopsis thaliana SQS1 gene encoding squalene synthase--involvement of the C-terminal region of the enzyme in the channeling of squalene through the sterol pathway.

Squalene synthase (SQS) catalyzes the first committed step of the sterol biosynthetic pathway. A full-length Arabidopsis thaliana SQS cDNA has been isolated by combining library screening and PCR-based approaches. Arabidopsis SQS is encoded by a small gene family of two genes (SQS1 and SQS2) which are organized in a tandem array. SQS1 and SQS2 have an identical organization with regard to intron positions and exon sizes and encode SQS isoforms showing a high level of sequence conservation (79% identity and 88% similarity). The isolated cDNA has been assigned to the SQS1 gene product, SQS1. RNA blot analysis has shown that the 1.6-kb SQS1 mRNA is detected in all plant tissues analyzed (inflorescenses, leaves, stems and roots) although the transcript is especially abundant in roots. Arabidopsis SQS1 isoform is unable to complement the SQS-defective Saccharomyces cerevisiae strain 5302, although SQS activity was detected in the microsomal fraction of the transformed yeast strain. However, a chimeric SQS resulting from the replacement of the 66 C-terminal residues of the Arabidopsis enzyme by the 111 C-terminal residues of the Schizosaccharomyces pombe enzyme was able to confer ergosterol prototrophy to strain 5302. Labeling studies using [3H]farnesyl-P2 and microsomal fractions obtained from yeast strains expressing either Arabidopsis SQS1 or chimeric Arabidopsis/S. pombe SQS derivatives indicated that the C-terminal region of the enzyme is involved in the channeling of squalene through the yeast sterol pathway.

The Saccharomyces cerevisiae mevalonate diphosphate decarboxylase is essential for viability, and a single Leu-to-Pro mutation in a conserved sequence leads to thermosensitivity.

The mevalonate diphosphate decarboxylase is an enzyme which converts mevalonate diphosphate to isopentenyl diphosphate, the building block of isoprenoids. We used the Saccharomyces cerevisiae temperature-sensitive mutant defective for mevalonate diphosphate decarboxylase previously described (C. Chambon, V. Ladeveve, M. Servouse, L. Blanchard, C. Javelot, B. Vladescu, and F. Karst, Lipids 26:633-636, 1991) to characterize the mutated allele. We showed that a single change in a conserved amino acid accounts for the temperature-sensitive phenotype of the mutant. Complementation experiments were done both in the erg19-mutated background and in a strain in which the ERG19 gene, which was shown to be an essential gene for yeast, was disrupted. Epitope tagging of the wild-type mevalonate diphosphate decarboxylase allowed us to isolate the enzyme in an active form by a versatile one-step immunoprecipitation procedure. Furthermore, during the course of this study, we observed that a high level of expression of the wild-type ERG19 gene led to a lower sterol steady-state accumulation compared to that of a wild-type strain, suggesting that this enzyme may be a key enzyme in mevalonate pathway regulation.

Polyprenol formation in the yeast Saccharomyces cerevisiae: effect of farnesyl diphosphate synthase overexpression.

Biosynthesis of polyprenols was followed in the erg mutants of Saccharomyces cerevisiae impaired in various steps of the mevalonate pathway. The end products of the enzymatic reaction carried out in vitro, in the wild type yeast and all mutants tested, were identified as dehydrodolichols (alpha-unsaturated polyprenols) whereas in vivo, yeast synthesize dolichols (alpha-saturated polyprenols) (Biochimie, 1996.78:111-112.) The strain defective in the farnesyl diphosphate (FPP) synthase, (coded by the erg20-2 gene) required the presence of exogenous FPP for synthesis of dehydrodolichols to occur in vitro. Overexpression of the ERG20 gene restored synthesis of polyprenols in vitro indicating that FPP is the allylic "starter" for cis-prenyltransferase in yeast. Overexpression of the ERG20 gene in the erg 9 mutant, defective in squalene synthase activity, not only restored synthesis of dehydrodolichols in vitro, but also increased the synthesis of dolichols in vivo, almost 10-fold in comparison with wild type yeast. On the other hand overexpression of the mutated FPP synthase, coded by the gene erg20-2 in the same genetic background, resulted in a 100-fold increase of the amount of dehydrodolichols. Interestingly, in addition to the family of typical for yeast C60-C80 compounds, dehydrodolichols of chain length up to C135 were synthesized both in vitro and in vivo.

Characterization of the Saccharomyces cerevisiae RTA1 gene involved in 7-aminocholesterol resistance.

7-aminocholesterol has been described as being a strong inhibitor of yeast and of Gram+-bacteria proliferation. In order to determine the precise molecular target of the toxicity of this compound, we searched for yeast resistance linked to gene over-expression. We named the new yeast gene that was isolated RTA1 (EMBL X84736). This gene led to strong resistance to the inhibitor. Gene sequencing revealed that RTA1 is adjacent to the NAB1 gene which is orientated in an opposite direction and localized on chromosome VII. The RTA1 gene, which encodes a putative protein with seven potential membrane-spanning segments, was shown to be a non-essential gene in yeast.

Characterization of the Saccharomyces cerevisiae FMS1 gene related to Candida albicans corticosteroid-binding protein 1.

In order to investigate ergosterol metabolism in S. cerevisiae we studied the CM8 mutant strain defective in the regulation of this pathway. A genomic multicopy library was screened to reverse the CM8 phenotype. This allowed us to characterize a new gene, FMS1, which relieves mutant phenotype by extragenic functional complementation. FMS1 may encode a 508 amino-acid protein. The predicted protein shares 35% identity with Cbp1p, a Candida albicans corticosteroid binding-protein. Fms1p also shows a weaker homology with monoamine oxidases. The construction of a FMS1 null-allele yeast strain demonstrated that this gene is not essential for yeast in normal usual laboratory culture conditions. The existence of a gene related to CBP1 of C. albicans in S. cerevisiae strongly suggests a possible function of steroid-binding proteins in yeast general physiology rather than in a process related to pathogenicity.

FEN2: a gene implicated in the catabolite repression-mediated regulation of ergosterol biosynthesis in yeast.

We have isolated and characterized a pleiotropic recessive mutation. fen2-1, that causes resistance to fenpropimorph and a low level of ergosterol in Saccharomyces cerevisiae. Ergosterol synthesis in the mutant strain was 5.5-fold slower than in the wild type; however, in vitro assays of the enzymes involved in ergosterol biosynthesis could not account for this low rate in the mutant. The mutant phenotype was expressed only in media exerting both carbon and nitrogen catabolite repression. To our knowledge, this is the first locus in yeast that reveals a concerted regulation between different pathways (carbon and nitrogen catabolite repression and/or general control of amino acid biosynthesis and ergosterol biosynthesis). The yeast gene FEN2 has been isolated and contains an open reading frame (ORF) of 512 codons. This ORF was found to be identical to YCR28C of chromosome III. A possible function of the FEN2 gene product in yeast is discussed.

Arabidopsis thaliana contains two differentially expressed farnesyl-diphosphate synthase genes.

The enzyme farnesyl-diphosphate synthase (FPS; EC 2.5.1.1/EC 2.5.1.10) catalyzes the synthesis of farnesyl diphosphate (FPP) from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This reaction is considered to be a rate-limiting step in isoprenoid biosynthesis. Southern blot analysis indicates that Arabidopsis thaliana contains at least 2 genes (FPS1 and FPS2) encoding FPS. The FPS1 and FPS2 genes have been cloned and characterized. The two genes have a very similar organization with regard to intron positions and exon sizes and share a high level of sequence similarity, not only in the coding region but also in the intronic sequences. Northern blot analysis showed that FPS1 and FPS2 have a different pattern of expression. FPS1 mRNA accumulates preferentially in roots and inflorescences, whereas FPS2 mRNA is predominantly expressed in inflorescences. The cDNA corresponding to the FPS1 gene was isolated by functional complementation of a mutant yeast strain defective in FPS activity (Delourme, D., Lacroute, F., and Karst, F. (1994) Plant Mol. Biol. 26, 1867-1873). By using a reverse transcription-polymerase chain reaction strategy we have cloned the cDNA corresponding to the FPS2 gene. Analysis of the FPS2 cDNA sequence revealed an open reading frame encoding a protein of 342 amino acid residues with a predicted molecular mass of 39,825 Da. FPS1 and FPS2 isoforms share an overall amino acid identity of 90.6%. Arabidopsis FPS2 was able to rescue the lethal phenotype of an ERG20-disrupted yeast strain. We demonstrate that FPS2 catalyzes the two successive condensations of IPP with both DMAPP and geranyl diphosphate leading to FPP. The significance of the occurrence of different FPS isoforms in plants is discussed in the context of the complex organization of the plant isoprenoid pathway.

Products of S cerevisiae cis-prenyltransferase activity in vitro.

Products of cis-prenyltransferase activity, the first committed enzyme of the dolichol biosynthetic pathway, have been characterized in Saccharomyces cerevisiae. The evidence based on the results of ion exchange, HPTLC chromatography and acid phosphatase digestion has been presented indicating that the final product of the enzyme action in vitro is free polyprenol and not polyprenol mono- or diphosphate. On the other hand, the results of HPLC analysis confirmed that in vivo yeast accumulate alpha-saturated polyprenols (dolichols). Phosphorylation of endogenous dolichols by cytidine triphosphate (CTP)-dependent kinase is demonstrated. The hypothesis is put forth that in S cerevisiae free polyprenol is the substrate for the alpha-reductase responsible for its conversion to dolichol which in turn is phosphorylated into its active form: dolichyl phosphate.