Methionine: An Indispensable Amino Acid in Cellular Metabolism and Health of Atlantic Salmon.

Methionine is an indispensable amino acid with an important role as the main methyl donor in cellular metabolism for both fish and mammals. Metabolization of methionine to the methyl donor S-adenosylmethionine (SAM) has consequence for polyamine, carnitine, phospholipid, and creatine synthesis as well as epigenetic modifications such as DNA- and histone tail methylation. Methionine can also be converted to cysteine and contributes as a precursor for taurine and glutathione synthesis. Moreover, methionine is the start codon for every protein being synthetized and thereby serves an important role in initiating translation. Modern salmon feed is dominated by plant ingredients containing less taurine, carnitine, and creatine than animal-based ingredients. This shift results in competition for SAM due to an increasing need to endogenously synthesize associated metabolites. The availability of methionine has profound implications for various metabolic pathways including allosteric regulation. This necessitates a higher nutritional need to meet the requirement as a methyl donor, surpassing the quantities for protein synthesis and growth. This comprehensive review provides an overview of the key metabolic pathways in which methionine plays a central role as methyl donor and unfolds the implications for methylation capacity, metabolism, and overall health particularly emphasizing the development of fatty liver, oxidation, and inflammation when methionine abundance is insufficient focusing on nutrition for Atlantic salmon (Salmo salar).

Effectiveness of isolation policies in schools: evidence from a mathematical model of influenza and COVID-19.

BACKGROUND: Non-pharmaceutical interventions such as social distancing, school closures and travel restrictions are often implemented to control outbreaks of infectious diseases. For influenza in schools, the Center of Disease Control (CDC) recommends that febrile students remain isolated at home until they have been fever-free for at least one day and a related policy is recommended for SARS-CoV-2 (COVID-19). Other authors proposed using a school week of four or fewer days of in-person instruction for all students to reduce transmission. However, there is limited evidence supporting the effectiveness of these interventions. METHODS: We introduced a mathematical model of school outbreaks that considers both intervention methods. Our model accounts for the school structure and schedule, as well as the time-progression of fever symptoms and viral shedding. The model was validated on outbreaks of seasonal and pandemic influenza and COVID-19 in schools. It was then used to estimate the outbreak curves and the proportion of the population infected (attack rate) under the proposed interventions. RESULTS: For influenza, the CDC-recommended one day of post-fever isolation can reduce the attack rate by a median (interquartile range) of 29 (13-59)%. With 2 days of post-fever isolation the attack rate could be reduced by 70 (55-85)%. Alternatively, shortening the school week to 4 and 3 days reduces the attack rate by 73 (64-88)% and 93 (91-97)%, respectively. For COVID-19, application of post-fever isolation policy was found to be less effective and reduced the attack rate by 10 (5-17)% for a 2-day isolation policy and by 14 (5-26)% for 14 days. A 4-day school week would reduce the median attack rate in a COVID-19 outbreak by 57 (52-64)%, while a 3-day school week would reduce it by 81 (79-83)%. In both infections, shortening the school week significantly reduced the duration of outbreaks. CONCLUSIONS: Shortening the school week could be an important tool for controlling influenza and COVID-19 in schools and similar settings. Additionally, the CDC-recommended post-fever isolation policy for influenza could be enhanced by requiring two days of isolation instead of one.

Effectiveness of Isolation Policies in Schools: Evidence from a Mathematical Model of Influenza and COVID-19.

BACKGROUND: Non-pharmaceutical interventions such as social distancing, school closures and travel restrictions are often implemented to control outbreaks of infectious diseases. For influenza in schools, the Center of Disease Control (CDC) recommends that febrile students remain isolated at home until they have been fever-free for at least one day and a related policy is recommended for SARS-CoV2 (COVID-19). Other authors proposed using a school week of four or fewer days of in-person instruction for all students to reduce transmission. However, there is limited evidence supporting the effectiveness of these interventions. METHODS: We introduced a mathematical model of school outbreaks that considers both intervention methods. Our model accounts for the school structure and schedule, as well as the time-progression of fever symptoms and viral shedding. The model was validated on outbreaks of seasonal and pandemic influenza and COVID-19 in schools. It was then used to estimate the outbreak curves and the proportion of the population infected (attack rate) under the proposed interventions. RESULTS: For influenza, the CDC-recommended one day of post-fever isolation can reduce the attack rate by a median (interquartile range) of 29 (13 - 59)%. With two days of post-fever isolation the attack rate could be reduced by 70 (55 - 85)%. Alternatively, shortening the school week to four and three days reduces the attack rate by 73 (64 - 88)% and 93 (91 - 97)%, respectively. For COVID-19, application of post-fever isolation policy was found to be less effective and reduced the attack rate by 10 (5 - 17)% for a two-day isolation policy and by 14 (5 - 26)% for 14 days. A four-day school week would reduce the median attack rate in a COVID-19 outbreak by 57 (52 - 64)%, while a three-day school week would reduce it by 81 (79 - 83)%. In both infections, shortening the school week significantly reduced the duration of outbreaks. CONCLUSIONS: Shortening the school week could be an important tool for controlling influenza and COVID-19 in schools and similar settings. Additionally, the CDC-recommended post-fever isolation policy for influenza could be enhanced by requiring two days of isolation instead of one.

[Indications and limitations of fresh frozen sections in the pulmonary apparatus]. / Indikation und Grenzen des intraoperativen Schnellschnitts im Organbereich Lunge.

Recommendations for the diagnosis of lung tumors almost limit the use of fresh frozen sections to the evaluation of resection margins. In pathology pretherapeutic methods for assessment of clinically suspected lung cancer are favored over intraoperative frozen section diagnosis. For the interdisciplinary management of uncertain lung findings diagnostic methods, such as cytopathology and examination of biopsy material are available. The use of rapid on-site evaluation (ROSE) in cytopathology is limited due to the lack of necessary personnel. Diagnosis of unclear pulmonary lesions or distinction of metastases from primary lung tumors by intraoperative frozen sections is therefore limited to exceptional cases that were not resolved by preoperative biopsies. Such rare cases require a common consensus strategy between thoracic surgeons and pathologists in a preoperative tumor board.

The ribosomal DNA of the Zygomycete Mucor miehei.

The ribosomal DNA-from the Zygomycete Mucor miehei has been characterised. The complete rDNA unit was cloned by heterologous PCR using primers whose sequence matched conserved regions of the rDNA from related fungal species. The sequence of the overlapping PCR products revealed the existence of a repeated unit of 9574 bp. The genes encoding the different rRNA species were identified by their homology to the corresponding sequences from other fungi. We estimate that the rDNA unit is present in the genome of M. miehei in about 100 copies. This estimation was made by comparing the intensity of its hybridisation signal in a Southern blot with that of the mmp gene coding for aspartyl protease, which was assumed to be contained in single copy. The size and structure of the M. miehei rDNA unit was similar to that of other fungi. The genes encoding the 25S, 18S and 5.8S RNAs are closely linked within the repeated unit which also contains the 5S gene. This latter gene appears to be transcribed in the opposite direction. The 25S, 18S and 5.8S genes showed 70-80% homology to the corresponding genes from other fungi, whereas the degree of homology for the 5S gene was much lower. The highest homology (about 80%) corresponded to the few available sequences from other Mucor species. Homology to genes from other Zygomycota was no higher than that observed for genes from the Ascomycota or Basidiomycota fungi.

Transformation of Escherichia coli with DNA from Saccharomyces cerevisiae cell lysates.

We developed a system to monitor the transfer of heterologous DNA from a genetically manipulated strain of Saccharomyces cerevisiae to Escherichia coli. This system is based on a yeast strain that carries multiple integrated copies of a pUC-derived plasmid. The bacterial sequences are maintained in the yeast genome by selectable markers for lactose utilization. Lysates of the yeast strain were used to transform E. coli. Transfer of DNA was measured by determining the number of ampicillin-resistant E. coli clones. Our results show that transmission of the Amp(r) gene to E. coli by genetic transformation, caused by DNA released from the yeast, occurs at a very low frequency (about 50 transformants per microg of DNA) under optimal conditions (a highly competent host strain and a highly efficient transformation procedure). These results suggest that under natural conditions, spontaneous transmission of chromosomal genes from genetically modified organisms is likely to be rare.

Construction of a lactose-assimilating strain of baker's yeast.

A recombinant strain of baker's yeast has been constructed which can assimilate lactose efficiently. This strain has been designed to allow its propagation in whey, the byproduct resulting from cheese-making. The ability to metabolize lactose is conferred by the functional expression of two genes from Kluyveromyces lactis, LAC12 and LAC4, which encode a lactose permease and a beta-galactosidase, respectively. To make the recombinant strain more acceptable for its use in bread-making, the genetic transformation of the host baker's yeast was carried out with linear fragments of DNA of defined sequence, carrying as the only heterologous material the coding regions of the two K. lactis genes. Growth of the new strain on cheese whey affected neither the quality of bread nor the yeast gassing power. The significance of the newly developed strain is two-fold: it affords a cheap alternative to the procedure generally used for the propagation of baker's yeast, and it offers a profitable use for cheese whey.

Highly efficient assimilation of lactose by a metabolically engineered strain of Saccharomyces cerevisiae.

A diploid strain of Saccharomyces cerevisiae able to metabolize lactose with high efficiency has been obtained. Haploid strains of Saccharomyces able to grow on lactose were constructed by cotransformation with two genes of Kluyveromyces lactis required for the utilization of the sugar, LAC4 and LAC12, encoding beta-galactosidase and lactose permease respectively. Both genes were placed under the control of a galactose-inducible promoter and targeted to the rDNA encoding region (RDN1 locus) of the Saccharomyces genome. Lac+ transformants were selected on medium with lactose as the only carbon source. These transformants were mitotically stable, they maintained the Lac+ phenotype after growing in non-selective medium for more than 60 generations, but their growth was slow. We found that this lack of vigour was caused by their genetic background and not by a deficient expression of the heterologous genes. Therefore, their performance could be improved by crossing with a wild-type strain. Among the offspring of the crosses, two strains of opposite mating type were selected and mated to obtain a fast-growing Lac+ diploid. This diploid strain showed the typical fermentative behaviour of S. cerevisiae when it was grown in aerated liquid medium with glucose. In lactose medium, it exhibited a respiro-fermentative metabolism similar to that of K. lactis, with low ethanol production and high biomass yield.

Structural and functional architecture of the yeast cell-cycle transcription factor swi6.

The structural and functional organisation of Swi6, a transcriptional regulator of the budding yeast cell cycle has been analysed by a combination of biochemical, biophysical and genetic methods. Limited proteolysis indicates the presence of a approximately 15 kDa N-terminal domain which is dispensable for Swi6 activity in vivo and which is separated from the rest of the molecule by an extended linker of at least 43 residues. Within the central region, a 141 residue segment that is capable of transcriptional activation encompasses a structural domain of approximately 85 residues. In turn, this is tightly associated with an adjacent 28 kDa domain containing at least four ankyrin-repeat (ANK) motifs. A second protease sensitive region connects the ANK domain to the remaining 30 kDa C-terminal portion of Swi6 which contains a second transcriptional activator and sequences required for heteromerisation with Swi4 or Mbp1. Transactivation by the activating regions of Swi6 is antagonised when either are combined with the central ankyrin repeat motifs. Hydrodynamic measurements indicate that an N-terminal 62 kDa fragment comprising the first three domains is monomeric in solution and exhibits an unusually high frictional coefficient consistent with the extended, multi-domain structure suggested by proteolytic analysis.

Induced expression of bacterial beta-glucosidase activity in Saccharomyces.

The bglA gene which encodes a beta-glucosidase from Bacillus polymyxa, has been expressed in Saccharomyces cerevisiae under control of the yeast CYC-GAL promoter. Strains have been constructed which carry the gene in different locations: in a multicopy plasmid, a single integration at the URA3 locus, or multiple integrations at the RDN1 locus. Integrative transformation at RDN1 yielded genetically stable clones with a high level of beta-glucosidase activity. Coordinated overexpression of the GAL4 inducer protein further increased the level of enzyme activity, although eventually caused the lysis of the cultures. Diploid, triploid and tetraploid strains derived from the transformants with multiple integrations were constructed and expression of beta-glucosidase activity in different conditions of growth was assayed. While per-cell activity increased with ploidy, specific activity was about the same in strains of equivalent genotype regardless of ploidy. Genetically stable and regulated expression in Saccharomyces of beta-glucosidase activity is interesting for the development of strains able to ferment beta-glycosidic sugars (i.e. cellobiose and lactose). From another point of view, the bglA product proved to be a convenient reporter enzyme to monitor heterologous gene expression.

Pollination ecology and patch-dependent reproductive success of the rare perennial Gentiana pneumonanthe L.

The reproductive behaviour of Gentiana pneumonanthe L., a rare plant species in The Netherlands, was studied in a relatively large wet heathland population during summer 1989. The species co-occurred with the grass Molinia caerulea and co-flowered with Erica tetralix and Calluna vulgaris. The flowering period lasted from July to October, peaking in late August to late September. Flowers are protandrous. The species appeared to be self-compatible, but spontaneous self-pollination was strongly limited. Hand-crossing and hand-selfing resulted in the same amount of seed set as in natural pollination. Reproductive success was dramatically reduced late in the season. Pollination was achieved sternotribically by the species Bombus pascuorum, which visited the flowers for nectar. In the study area, three patch types were distinguished: co-dominated by Erica, by Calluna and Erica, and by Molinia, respectively. Frequency of visits to Gentiana was highest in the Erica-patch. However, this did not result in a higher seed set. In the Molinia-patch seed set was reduced. However, in the Molinia-patch the mean number of ovules was greater than in the others (as high as in plants raised indoors and in cross-pollinated plants). Fruits from the Calluna-patch had less ovules than those from the Erica-patch. We conclude that, in this remnant population, Gentiana is not pollination-limited. However, it is likely that in the Molinia-patch geitonogamy is frequent, which may lead to inbreeding depression. The greater number of ovules observed in Molinia-patches may reflect an excess of nutrients available there, relative to the Calluna-patch where there may be reduced water availability.

Self-diploidization in Saccharomyces cerevisiae kar2 heterokaryons.

Zygotes isolated by micromanipulation from crosses of Saccharomyces cerevisiae strains, one of which carries a kar mutation, give rise most frequently to cytoductant colonies showing the nuclear constitution of either one of the two haploid parental strains. In crosses of kar2-1 strains to wild-type, about 10% of the cytoductants of both mating types are homozygous autodiploids. There is evidence indicating that self-diploidization occurs by fusion between sibling nuclei in the heterokaryotic zygote. Here we describe this phenomenon and propose to take advantage of it for the construction of genotypically-defined diploids able to mate, and of polyploid strains, which are useful tools in genetic and cytological studies.

Construction of a Saccharomyces cerevisiae strain able to ferment cellobiose.

The bglA gene, encoding a beta-glucosidase from Bacillus polymyxa, has been expressed in Saccharomyces cerevisiae under control of the CYC-GAL promoter inducible by galactose. The expression of bglA-encoded activity in the strain used as a host was not sufficient to allow its growth with cellobiose as a carbon source. However, a recessive mutation in a gene designated cem1 has been obtained which, combined with the expression of beta-glucosidase activity, allows the growth of S. cerevisiae on cellobiose. The expression of the blgA gene in a cem1 strain confers on S. cerevisiae the capability for an efficient fermentation of cellobiose, as detected by the formation of CO2.