A proof of concept: Clinical anti-aging efficacy and safety of Lactiplantibacillus plantarum LB244R® applied topically in a double-blinded placebo-controlled study.

BACKGROUND: With the increasing age of the westernized population, there is also increasing economic and aesthetic interest in reducing the signs of skin aging. Additionally, the physical aspect of aging can be displeasing and have detrimental effects psychologically in individuals. Probiotics have shown potential as anti-aging agents, albeit proper studies are needed to confirm their potential. AIMS: Proving that Lactiplantibacillus plantarum LB244R® could alleviate aging signs relative to its placebo vehicle. PATIENTS/METHODS: In total, 46 subjects were randomly assigned either the ointment with live bacteria, L. plantarum LB244R® or its vehicle ointment, and had to use the assigned ointment twice daily for 56 days. On Day 0, Day 28, and Day 56 subepidermal low echogenic band (SLEB) thickness, dermal density, skin firmness and elasticity, skin hydration, transepidermal water loss (TEWL), skin pH, collagen fiber visualization using confocal microscopy, Crow's feet, spot score, skin smoothness, and complexion radiance were assessed by dermatologists. RESULTS: All parameters except TEWL improved relative to their baseline (D0) for the active group. L. plantarum LB244R® improved SLEB thickness, dermal density, skin elasticity, skin hydration, and Crow's feet wrinkle score relative to the placebo vehicle ointment. CONCLUSION: The study demonstrates an anti-aging effect of L. plantarum LB244R® for topical skin use in the first double-blinded, vehicle-ointment placebo-controlled clinical study.

Topical Lactiplantibacillus plantarum LB244R® ointment alleviates skin aging: An exploratory trial.

BACKGROUND: The skin is of vital importance for health and well-being. As people age, the skin undergoes visual and morphological changes such as wrinkling, loss of elasticity, increased pigmentation, and decreased cell turnover. This is not only visually unappealing to many but can also pose health issues. AIM: In this study, a probiotic ointment (PO) containing live lactic acid bacteria (LAB) (Lactiplantibacillus plantarum LB244R®) was investigated for its ability to alleviate symptoms of skin aging in an exploratory clinical trial. METHODS: The PO was applied twice daily for 56 days by 21 subjects. Anti-aging efficacy was evaluated by skin ultrasonography, skin biomechanical properties, skin hydration, and clinical evaluations at day 0, 28, and 56. RESULTS: Sub-epidermal low echogenic band thickness decreased (0.261 ± 0.069 mm to 0.247 ± 0.055 mm) after 56 days. Dermal density increased (324.689 ± 57.506 pixel/mm2 to 367.831 ± 75.790 pixel/mm2 ). Skin hydration increased (34.1 ± 6.9 to 51.3 ± 10.0 AU). Additionally, skin firmness increased, as shown by decreasing values (0.264 ± 0.038 to 0.228 ± 0.037 mm). Skin elasticity increased (0.578 ± 0.045 to 0.618 ± 0.044). Trans-epidermal water loss decreased (9.1 ± 2.0 g/h/m2 to 8.5 ± 1.3). All clinical evaluations, Crow's feet, spot score, smoothness score, and complexion radiance, were improved. CONCLUSION: The PO improved all measured parameters with statistical significance after 56 days of application, clearly demonstrating the potential of the PO as an anti-aging agent and reaffirming the potential of topical probiotic LAB. Future studies need to elucidate the mode of action of anti-aging effects by probiotics, but at present time, this study paves the way for the use of probiotic LAB topically to alleviate aging of the skin.

Targeted Screening of Lactic Acid Bacteria With Antibacterial Activity Toward Staphylococcus aureus Clonal Complex Type 1 Associated With Atopic Dermatitis.

Atopic dermatitis (AD) is a common inflammatory skin disease characterized by an epidermal barrier impairment, as well as a Th2/Th22-skewed immune response, both favoring skin colonization with Staphylococcus aureus. Colonization is strongly related to severity of the disease, and a reduction of S. aureus has been found to alleviate symptoms. Lactic acid bacteria (LAB) produce antimicrobial compounds such as organic acids and bacteriocins and are widely used as probiotics. The aim of this study was to isolate LAB and screen for antibacterial effect specifically toward S. aureus clonal complex type 1. A total of 680 LAB were isolated from fermented vegetables and swab samples from healthy volunteers (vaginal, stool and skin). Screening for antibacterial activity toward S. aureus, narrowed the field of isolates down to four LAB strains with high antibacterial activity. The activity varied according to the specific LAB strain and the origin of the strain. The results suggested different modes of action, including co-aggregation, expression of bacteriocins and production of specific organic acids. However, the ability to acidify the surroundings appeared as the main effect behind inhibition of S. aureus. Broth microdilution assays showed a significant reduction of S. aureus growth when using down to 10% cell free supernatant (CFS). Our results underline the use of specific living LAB or their CFS as potential future treatment strategies to reduce S. aureus colonization of AD skin.

Supplementation of a lacto-fermented rapeseed-seaweed blend promotes gut microbial- and gut immune-modulation in weaner piglets.

BACKGROUND: The direct use of medical zinc oxide in feed will be abandoned after 2022 in Europe, leaving an urgent need for substitutes to prevent post-weaning disorders. RESULTS: This study investigated the effect of using rapeseed-seaweed blend (rapeseed meal added two brown macroalgae species Ascophylum nodosum and Saccharina latissima) fermented by lactobacilli (FRS) as feed ingredients in piglet weaning. From d 28 of life to d 85, the piglets were fed one of three different feeding regimens (n = 230 each) with inclusion of 0%, 2.5% and 5% FRS. In this period, no significant difference of piglet performance was found among the three groups. From a subset of piglets (n = 10 from each treatment), blood samples for hematology, biochemistry and immunoglobulin analysis, colon digesta for microbiome analysis, and jejunum and colon tissues for histopathological analyses were collected. The piglets fed with 2.5% FRS manifested alleviated intraepithelial and stromal lymphocytes infiltration in the gut, enhanced colon mucosa barrier relative to the 0% FRS group. The colon microbiota composition was determined using V3 and V1-V8 region 16S rRNA gene amplicon sequencing by Illumina NextSeq and Oxford Nanopore MinION, respectively. The two amplicon sequencing strategies showed high consistency between the detected bacteria. Both sequencing strategies indicated that inclusion of FRS reshaped the colon microbiome of weaned piglets with increased Shannon diversity. Prevotella stercorea was verified by both methods to be more abundant in the piglets supplied with FRS feed, and its abundance was positively correlated with colonic mucosa thickness but negatively correlated with blood concentrations of leucocytes and IgG. CONCLUSIONS: FRS supplementation relieved the gut lymphocyte infiltration of the weaned piglets, improved the colon mucosa barrier with altered microbiota composition. Increasing the dietary inclusion of FRS from 2.5% to 5% did not lead to further improvements.

Effects of Increasing Doses of Lactobacillus Pre-Fermented Rapeseed Product with or without Inclusion of Macroalgae Product on Weaner Piglet Performance and Intestinal Development.

This study evaluated the effects of increasing doses of pre-fermented rapeseed meal (FRM) without or with inclusion of the brown macroalgae Ascophyllum nodosum (AN) on weaner piglets' performance and gut development. Ten days pre-weaning, standardized litters were randomly assigned to one of nine isoenergetic and isoproteic diets comprising (on DM basis): no supplement (negative control, NC), 2500 ppm ZnO (positive control, PC), 8, 10, 12, 15 or 25% FRM, and 10% FRM plus 0.6 or 1.0% AN. Fifty piglets receiving the same pre-weaning diets were weaned at 28 days of age and transferred to one pen, where they continued on the pre-weaning diet until day 92. At 41 days, six piglets per treatment were sacrificed for blood and intestinal samplings. The average daily gain was at least sustained at any dose of FRM (increased at 8% FRM, 28-41 days) from 18-41 days similar to PC but unaffected by inclusion of AN. The percentage of piglets that completed the experiment was increased by FRM compared to NC, despite detection of diarrhea symptoms. FRM showed quadratic dose-response effects on colon and mid-jejunum crypts depth, and enterocyte and mid-jejunum villus heights with optimum development at 8% or 10% FRM, respectively, but this was abolished when AN was also added. In conclusion, FRM sustained piglet growth performance and intestinal development similar to ZnO with an optimum inclusion level of 8-10% of dietary DM.

Impact of Dietary Supplementation of Lactic Acid Bacteria Fermented Rapeseed with or without Macroalgae on Performance and Health of Piglets Following Omission of Medicinal Zinc from Weaner Diets.

The feeding of medicinal zinc oxide (ZnO) to weaner piglets will be phased out after 2022 in Europe, leaving pig producers without options to manage post-weaning disorders. This study assessed whether rapeseed meal, fermented alone (FRM) or co-fermented with a single (Ascophylum nodosum; FRMA), or two (A. nodossum and Saccharina latissima; FRMAS) brown macroalagae species, could improve weaner piglet performance and stimulate intestinal development as well as maturation of gut microbiota in the absence of in-feed zinc. Weaned piglets (n = 1240) were fed, during 28-85 days of age, a basal diet with no additives (negative control; NC), 2500 ppm in-feed ZnO (positive control; PC), FRM, FRMA or FRMAS. Piglets fed FRM and FRMA had a similar or numerically improved, respectively, production performance compared to PC piglets. Jejunal villus development was stimulated over NC in PC, FRM and FRMAS (gender-specific). FRM enhanced colon mucosal development and reduced signs of intestinal inflammation. All fermented feeds and PC induced similar changes in the composition and diversity of colon microbiota compared to NC. In conclusion, piglet performance, intestinal development and health indicators were sustained or numerically improved when in-feed zinc was replaced by FRM.

Viable acrosome-intact human spermatozoa in the ejaculate as a marker of semen quality and fertility status.

STUDY QUESTION: Is it possible, in an unbiased and clinical relevant way, to determine the number of viable acrosome-intact human spermatozoa in ejaculates and to use this as a measure of fertility chances? SUMMARY ANSWER: Image cytometry enables easy and unbiased quantification of viable acrosome-intact spermatozoa and it correlates with semen quality and fertility status. WHAT IS KNOWN ALREADY: The presence of the acrosome and its ability to respond to physiological inducers (e.g. progesterone) in the female reproductive tract at the appropriate time and place is required for fertilization. However, the available assays are labor intensive and therefore not used clinically. STUDY DESIGN, SIZE, DURATION: Washed semen samples and capacitated swim-up fractions from volunteers were used to develop the assay. Subsequently washed ejaculates from patients in fertility treatment (n = 156), proven fertile men (n = 54) and volunteers (n = 10) were assessed to evaluate the number of acrosome-intact spermatozoa in the ejaculate (acrosomal status) and compared to other semen parameters, fertility status, fertility treatments and pregnancy rates. PARTICIPANTS/MATERIALS, SETTING, METHODS: Image cytometry was used to assess the fluorescence intensity of Pisum sativum agglutinin and Propidium iodide. MAIN RESULTS AND THE ROLE OF CHANCE: The assay was validated by inducing the acrosome reaction in swim-up-purified and capacitated spermatozoa with progesterone and ionomycin, and in repeated acrosomal status measurements of washed ejaculates a small coefficient of variation (3.7%) was observed. Men with poor semen quality had fewer viable acrosome-intact spermatozoa in the ejaculate (P = 0.0012; median 32.6% vs. 49.3%). A large proportion (44%) of normozoospermic men from infertile couples had less than the observed median fraction (46%) of viable acrosome-intact spermatozoa in the ejaculate. Furthermore, the total number of viable acrosome-intact spermatozoa was significantly lower among men with male factor infertility compared to fertile men (median 35 vs. 97 mill, P = 1 × 10-7). Men from couples going through one or more ICSI cycles had significant fewer viable acrosome-intact spermatozoa than men from couples who only underwent IUI (P = 0.002; 44.4% vs. 62.0%) and the fraction of viable acrosome-intact spermatozoa appeared better than classical semen parameters in classifying whether or not couples needed ICSI. A positive, although non-significant, tendency toward ongoing pregnancy with an increasing number of viable acrosome-intact spermatozoa was observed (P = 0.2). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Even larger cohorts of infertile couples are needed to substantiate the clinical application of the assay in regard to estimation of fertility potential of an individual. WIDER IMPLICATIONS OF THE FINDINGS: The presented assay makes it possible to measure the number of acrosome competent spermatozoa in an ejaculate in a standardized manner and hence may serve as a new biomarker for male fertility. Few spermatozoa in an ejaculate are acrosome competent and it might be a valuable measure when evaluating male reproductive function. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from the Innovation Fund Denmark. M.G. and S.K. work at ChemoMetec, which produces the image cytometer used in the study, M.G. hold shares in the company. The other authors have no conflict of interest.

Human ß-defensin-2 production from S. cerevisiae using the repressible MET17 promoter.

BACKGROUND: Baker's yeast Saccharomyces cerevisiae is a proven host for the commercial production of recombinant biopharmaceutical proteins. For the manufacture of heterologous proteins with activities deleterious to the host it can be desirable to minimise production during the growth phase and induce production late in the exponential phase. Protein expression by regulated promoter systems offers the possibility of improving productivity in this way by separating the recombinant protein production phase from the yeast growth phase. Commonly used inducible promoters do not always offer convenient solutions for industrial scale biopharmaceutical production with engineered yeast systems. RESULTS: Here we show improved secretion of the antimicrobial protein, human ß-defensin-2, (hBD2), using the S. cerevisiae MET17 promoter by repressing expression during the growth phase. In shake flask culture, a higher final concentration of human ß-defensin-2 was obtained using the repressible MET17 promoter system than when using the strong constitutive promoter from proteinase B (PRB1) in a yeast strain developed for high-level commercial production of recombinant proteins. Furthermore, this was achieved in under half the time using the MET17 promoter compared to the PRB1 promoter. Cell density, plasmid copy-number, transcript level and protein concentration in the culture supernatant were used to study the effects of different initial methionine concentrations in the culture media for the production of human ß-defensin-2 secreted from S. cerevisiae. CONCLUSIONS: The repressible S. cerevisiae MET17 promoter was more efficient than a strong constitutive promoter for the production of human ß-defensin-2 from S. cerevisiae in small-scale culture and offers advantages for the commercial production of this and other heterologous proteins which are deleterious to the host organism. Furthermore, the MET17 promoter activity can be modulated by methionine alone, which has a safety profile applicable to biopharmaceutical manufacturing.

Isolation of a novel protein, P12-from adult Drosophila melanogaster that inhibits deoxyribonucleoside and protein kinase activities and activates 3'-5'- exonuclease activity.

We have previously found that Drosophila melanogaster only has one deoxyribonucleoside kinase, Dm-dNK, however, capable to phosphorylate all four natural deoxyribonucleosides. Dm-dNK was originally isolated from an embryonic cell line. We wanted to study the expression of Dm-dNK during development from embryonic cells to adult flies and found declining Dm-dNK activity during development and no activity in adult flies. Surprisingly, the extract from adult flies exhibited a strong inhibitory effect on deoxyribonucloside kinase activity. The dNK-inhibitor was precipitable with ammonium sulfate, and was purified to a high degree by gel-filtration as indicated by LC-MS/MS analysis. Since the inhibitor eluted from G-200 gel-filtration with a size of 10-13 kDa, we named it P12. We tested the purified fraction for specificity towards various enzymes and found that both mammalian and bacterial dNKs were inhibited, whereas there was no effect on hexokinase and pyruvate kinases and acidic phosphatase. However, when tested against cyclin B-dependent kinase, we found a strong inhibitory effect. Both with human Cdk1/CycB and S. pombe Cdc2/B-type cyclin the purified fraction from Superdex 200 that inhibited Dm-dNK, also inhibited the two protein kinases to the same degree. Furthermore, testing P12 in a DNA polymerase based assay we found that the 3'-5'-exonuclease part of the DNA polymerase (Klenow polymerase) was activated.

Generation of a double transgenic humanized neonatal Fc receptor (FcRn)/albumin mouse to study the pharmacokinetics of albumin-linked drugs.

Human serum albumin (HSA) is a natural carrier protein possessing multiple ligand binding sites with a plasma half-life ~19days, facilitated by interaction with the human neonatal Fc receptor (FcRn), that promotes it as a highly attractive drug delivery technology. A lack of adequate rodent models, however, is a major challenge in the preclinical development of albumin-linked therapeutics. This work describes the first double transgenic mouse model bearing both human FcRn and HSA genes (hFcRn(+/+), hAlb(+/+)) under the control of an endogenous promoter. Human FcRn was shown by immunohistochemical and qPCR analysis to be ubiquitously expressed in the major organs. Physiological levels of HSA were detected in the blood that exhibited similar FcRn binding kinetics to recombinant or human serum-derived HSA. The circulatory half-life (t1/2) was shown to be dependent on FcRn binding affinity that increased from low affinity (t1/2 29h), to wild type (t1/2 50h), to high affinity (t1/2 80h) variants, that validates the application of the model for optimizing the pharmacokinetics of drug carriers who's circulatory half-life is dependent in some manner upon interaction with endogenous FcRn. This study presents a novel mouse model that better mimics the human physiological conditions and, thus, has potential wide applications in the development of albumin-linked drugs or conventional drugs whose action is influenced by reversible binding to endogenous HSA.

An IPTG-inducible derivative of the fission yeast nmt promoter.

We here describe an IPTG-inducible system that reveals that the lac repressor alone can function as a potent transmodulator to regulate gene expression in the fission yeast, Schizosaccharomyces pombe. This expression system is a derivative of the Sz. pombe nmt promoter, which normally is strongly repressed by thiamine. With appropriate positioning of a lac operator site (lacO) downstream of the TATA-box, we show that gene expression from a chimeric nmt::lacO promoter can be regulated by the lac repressor up to two orders of magnitude in response to IPTG. The chimeric nmt::lacO promoter is rapidly induced and when GFP is used as a reporter; almost full induction is achieved 40 min after the addition of IPTG. Like the wild-type nmt promoter, the chimeric nmt::lacO is repressed by thiamine. This allows expression in a short and defined window, e.g. the S-phase of a synchronized cell population, by first adding IPTG to turn on expression, followed by addition of thiamine to switch off expression.

Extending serum half-life of albumin by engineering neonatal Fc receptor (FcRn) binding.

A major challenge for the therapeutic use of many peptides and proteins is their short circulatory half-life. Albumin has an extended serum half-life of 3 weeks because of its size and FcRn-mediated recycling that prevents intracellular degradation, properties shared with IgG antibodies. Engineering the strictly pH-dependent IgG-FcRn interaction is known to extend IgG half-life. However, this principle has not been extensively explored for albumin. We have engineered human albumin by introducing single point mutations in the C-terminal end that generated a panel of variants with greatly improved affinities for FcRn. One variant (K573P) with 12-fold improved affinity showed extended serum half-life in normal mice, mice transgenic for human FcRn, and cynomolgus monkeys. Importantly, favorable binding to FcRn was maintained when a single-chain fragment variable antibody was genetically fused to either the N- or the C-terminal end. The engineered albumin variants may be attractive for improving the serum half-life of biopharmaceuticals.

A versatile selection system for folding competent proteins using genetic complementation in a eukaryotic host.

Recombinant expression of native or modified eukaryotic proteins is pivotal for structural and functional studies and for industrial and pharmaceutical production of proteins. However, it is often impeded by the lack of proper folding. Here, we present a stringent and broadly applicable eukaryotic in vivo selection system for folded proteins. It is based on genetic complementation of the Schizosaccharomyces pombe growth marker gene invertase fused C-terminally to a protein library. The fusion proteins are directed to the secretion system, utilizing the ability of the eukaryotic protein quality-control systems to retain misfolded proteins in the ER and redirect them for cytosolic degradation, thereby only allowing folded proteins to reach the cell surface. Accordingly, the folding potential of the tested protein determines the ability of autotrophic colony growth. This system was successfully demonstrated using a complex insertion mutant library of TNF-alpha, from which different folding competent mutant proteins were uncovered.

Cdk phosphorylation of the Ste11 transcription factor constrains differentiation-specific transcription to G1.

Eukaryotic cells normally differentiate from G(1); here we investigate the mechanism preventing expression of differentiation-specific genes outside G(1). In fission yeast, induction of the transcription factor Ste11 triggers sexual differentiation. We find that Ste11 is only active in G(1) when Cdk activity is low. In the remaining part of the cell cycle, Ste11 becomes Cdk-phosphorylated at Thr 82 (T82), which inhibits its DNA-binding activity. Since the ste11 gene is autoregulated and the Ste11 protein is highly unstable, this Cdk switch rapidly extinguishes Ste11 activity when cells enter S phase. When we mutated T82 to aspartic acid, mimicking constant phosphorylation, cells no longer underwent differentiation. Conversely, changing T82 to alanine rendered Ste11-controlled transcription constitutive through the cell cycle, and allowed mating from S phase with increased frequency. Thus, Cdk phosphorylation mediates periodic expression of Ste11 and its target genes, and we suggest this to be part of the mechanism restricting differentiation to G(1).

Genomewide identification of pheromone-targeted transcription in fission yeast.

BACKGROUND: Fission yeast cells undergo sexual differentiation in response to nitrogen starvation. In this process haploid M and P cells first mate to form diploid zygotes, which then enter meiosis and sporulate. Prior to mating, M and P cells communicate with diffusible mating pheromones that activate a signal transduction pathway in the opposite cell type. The pheromone signalling orchestrates mating and is also required for entry into meiosis. RESULTS: Here we use DNA microarrays to identify genes that are induced by M-factor in P cells and by P-factor in M-cells. The use of a cyr1 genetic background allowed us to study pheromone signalling independently of nitrogen starvation. We identified a total of 163 genes that were consistently induced more than two-fold by pheromone stimulation. Gene disruption experiments demonstrated the involvement of newly discovered pheromone-induced genes in the differentiation process. We have mapped Gene Ontology (GO) categories specifically associated with pheromone induction. A direct comparison of the M- and P-factor induced expression pattern allowed us to identify cell-type specific transcripts, including three new M-specific genes and one new P-specific gene. CONCLUSION: We found that the pheromone response was very similar in M and P cells. Surprisingly, pheromone control extended to genes fulfilling their function well beyond the point of entry into meiosis, including numerous genes required for meiotic recombination. Our results suggest that the Ste11 transcription factor is responsible for the majority of pheromone-induced transcription. Finally, most cell-type specific genes now appear to be identified in fission yeast.

Alternative protein secretion: the Mam1 ABC transporter supports secretion of M-factor linked GFP in fission yeast.

To examine whether the fission yeast Mam1 ABC transporter can be used for secretion of heterologous proteins, thereby bypassing the classical secretion pathway, we have analyzed chimeric forms of the M-factor precursor. It was demonstrated that GFP can be exported when fused to both the amino-terminal prosequence from mfm1 and a CaaX motif. This secretion was dependent on the Mam1 transporter and not the classical secretion pathway. The secretion efficiency of GFP, however, was relatively low and most of the reporter protein was trapped in the vacuolar membranes. Our findings suggest that the Mam1 ABC protein is a promiscuous peptide transporter that can accommodate globular proteins of a relatively large size. Furthermore, our results help in defining the sequences required for processing and secretion of natural M-factor.

Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus.

Animals and higher plants express endogenous peptide antibiotics called defensins. These small cysteine-rich peptides are active against bacteria, fungi and viruses. Here we describe plectasin-the first defensin to be isolated from a fungus, the saprophytic ascomycete Pseudoplectania nigrella. Plectasin has primary, secondary and tertiary structures that closely resemble those of defensins found in spiders, scorpions, dragonflies and mussels. Recombinant plectasin was produced at a very high, and commercially viable, yield and purity. In vitro, the recombinant peptide was especially active against Streptococcus pneumoniae, including strains resistant to conventional antibiotics. Plectasin showed extremely low toxicity in mice, and cured them of experimental peritonitis and pneumonia caused by S. pneumoniae as efficaciously as vancomycin and penicillin. These findings identify fungi as a novel source of antimicrobial defensins, and show the therapeutic potential of plectasin. They also suggest that the defensins of insects, molluscs and fungi arose from a common ancestral gene.

Comparison of different signal peptides for secretion of heterologous proteins in fission yeast.

In the fission yeast Schizosaccharomyces pombe, there are relatively few signal peptides available and most reports of their activity have not been comparative. Using sequence information from the S. pombe genome database we have identified three putative signal peptides, designated Cpy, Amy and Dpp, and compared their ability to support secretion of green fluorescent protein (GFP). In the comparison we also included the two well-described secretion signals derived from the precursors of, respectively, the Saccharomyces cerevisiae alpha-factor and the S. pombe P-factor. The capability of the tested signal peptides to direct secretion of GFP varied greatly. The alpha-factor signal did not confer secretion to GFP and all the produced GFP was trapped intracellular. In contrast, the Cpy signal peptide supported efficient secretion of GFP with yields approximating 10 mg/L. We also found that the use of an attenuated version of the S. cerevisiae URA3 marker substantially increases vector copy number and expression yield in fission yeast.

Constitutive activation of the fission yeast pheromone-responsive pathway induces ectopic meiosis and reveals ste11 as a mitogen-activated protein kinase target.

In the fission yeast Schizosaccharomyces pombe, meiosis normally takes place in diploid zygotes resulting from conjugation of haploid cells. In the present study, we report that the expression of a constitutively activated version of the pheromone-responsive mitogen-activated protein kinase kinase kinase (MAP3K) Byr2 can induce ectopic meiosis directly in haploid cells. We find that the Ste11 transcription factor becomes constitutively expressed in these cells and that the expression of pheromone-responsive genes no longer depends on nitrogen starvation. Epistasis analysis revealed that these conditions bypassed the requirement for the meiotic activator Mei3. Since Mei3 is normally needed for inactivation of the meiosis-repressing protein kinase Pat1, this finding suggests that the strong Byr2 signal causes inactivation of Pat1 by an alternative mechanism. Consistent with this possibility, we found that haploid meiosis was dramatically reduced when Ste11 was mutated to mimic phosphorylation by Pat1. The mutation of two putative MAPK sites in Ste11 also dramatically reduced the level of haploid meiosis, suggesting that Ste11 is a direct target of Spk1. Supporting this, we show that Spk1 can interact physically with Ste11 and also phosphorylate the transcription factor in vitro. Finally, we demonstrate that ste11 is required for pheromone-induced G1 arrest. Interestingly, when we mutated Ste11 in the sites for Pat1 and Spk1 phosphorylation simultaneously, the cells could still arrest in G1 in response to pheromone, suggesting the existence of yet a third bifurcation of the signaling pathway.