Marker-free CRISPR-Cas9 based genetic engineering of the phytopathogenic fungus, Penicillium expansum.

Filamentous fungi are prolific producers of secondary metabolites (SecMets), including compounds with antibiotic properties, like penicillin, that allows the producing fungus to combat competitors in a shared niche. However, the biological function of the majority of these small complex metabolites for the producing fungi remains unclear (Macheleidt et al., 2016). In an effort to address this lack of knowledge, we have chosen to study the microbial community of moldy apples in the hope of shedding more light on the role of SecMets for the dynamics of the microbial community. Penicillium expansum is one of the prevalent fungal species in this system, and in co-culture experiments with other apple fungal pathogens, we have observed up- and downregulation of several SecMets when compared to monocultures. However, molecular genetic dissection of the observed changes is challenging, and new methodologies for targeted genetic engineering in P. expansum are needed. In the current study, we have established a CRISPR-Cas9 dependent genetic engineering toolbox for the targeted genetic manipulation of P. expansum to allow for single-step construction of marker-free strains. The method and effect of different combinations of a Cas9-sgRNA expressing plasmids and repair template substrates in the NHEJ-proficient WT strain is tested by targeted deletion of melA, encoding a PKS responsible for pigment formation, which upon deletion resulted in white mutants. Co-transformation with a linear double-stranded DNA fragment consisting of two 2 kb homology arms flanking the PKS gene proved to be the most efficient strategy with 100% confirmed deletions by diagnostic PCR. Shorter homology arms (500-1000 bp) resulted in 20-30% deletion efficiency. Furthermore, we demonstrate the application of the CRISPR-Cas9 method for targeted deletion of biosynthetic genes without a visible phenotype, insertion of a visual reporter-encoding gene (mRFP), and overexpression of biosynthetic genes. Combined, these tools will advance in enabling the deciphering of SecMet biosynthetic pathways, provide in situ insight into when and where SecMets are produced, and provide an avenue to study the role of P. expansum SecMets in shaping the microbial community development on moldy apples via marker-free targeted genetic engineering of P. expansum.

Evidence of gene-gene interaction in hidradenitis suppurativa: a nationwide registry study of Danish twins.

BACKGROUND: Hidradenitis suppurativa (HS) is a recurrent inflammatory skin disease that, apart from rare causative loss-of-function mutations, has a widely unknown genetic aetiology. OBJECTIVES: To estimate the relative importance of genetic and environmental factors underlying susceptibility to HS. METHODS: Via the Danish Twin Registry and the Danish National Patient Registry we pulled together information on zygosity with that of HS status. Cases of HS were identified by the International Classification of Diseases (ICD)-8 (705·91) and ICD-10 (L73·2). Heritability was assessed by the classic biometric model and the possibility of gene-gene interaction via the multilocus modelling approach. RESULTS: Among 100 044 registered twins, we found 170 twins (from 163 pairs) diagnosed with HS. The seven concordant pairs were all monozygotic. Monozygotic twins had a case-wise concordance rate of 28% [95% confidence interval (CI) 7-49], corresponding to a familial risk of 73 (95% CI 13-133) times that of the background population. The biometrical modelling suggested a heritability of 0·80 (95% CI 0·67-0·93), and the multilocus index estimate was 230 (95% CI 60-400). This is highly indicative of gene-gene interactions, with the possibility of up to six interacting loci. CONCLUSIONS: This twin study was substantially larger and employed a more valid phenotype than previous studies. Genetics account for the majority of HS susceptibility, and HS is most likely caused by gene-gene interactions rather than monogenetic mutations or solely additive genetic factors. New approaches aimed at assessing potential interactions at a single-nucleotide polymorphism (SNP)-SNP level should be implemented in future HS genome-wide association studies.

Field method for isolation of trichostrongyle larvae from vegetation of natural pastures of Arctic ruminants.

The extent to which wild ruminant populations are exposed to infective helminth larvae on their natural pastures is relatively undetermined. In the present study, a modified method for sampling of herbage and isolation of trichostrongyle infective third-stage larvae from natural pastures was used successfully in a muskox habitat in low-Arctic Greenland. The method, a revision of the Macro-Baermann method, is particularly aimed at fieldwork under primitive conditions.

Chronic ethanol intake and reduction of lung tumours from urethane in strain A mice.

Combinations of ethanol and urethane were added to the drinking-water of female strain A/Ph mice for 12 wk, at the end of which the animals were killed. Urethane concentrations were 0, 200, 500 and 1000 ppm and ethanol concentrations, 0, 5, 10 and 20% (v/v). All possible combinations of these urethane and ethanol concentrations were tested. Urethane induced primary lung adenomas in all treated mice in a dose-dependent manner. An average of 71 +/- 15 tumours/mouse were found, when the animals were killed, after treatment with 1000 ppm urethane for 12 wk. Ethanol alone did not alter the background incidence of tumours and produced only marginal hepatotoxicity. The tumour yields induced by urethane treatment were greatly reduced by simultaneous treatment with ethanol. The effect of ethanol was independent of urethane dose. When the concentrations of ethanol in the drinking-water were 20 and 10% the incidences of lung adenomas induced by urethane were reduced by about two-thirds and one-half, respectively. The effect of 5% ethanol, if any, was not statistically significant.

Carcinogenic potential of cooked food mutagens (IQ and MeIQ) in Wistar rats after short-term exposure.

Two potent cooked food mutagens, 2-amino-3-methylimidazo/4,5-f/quinoline (IQ) and 2-amino-3,4-dimethylimidazo/4,5-f/quinoline (MeIQ), were examined in an initiation-promotion assay in the male wistar rat. Fourteen doses of 10 mg IQ or 10 mg MeIQ/kg b.wt. were given during initiation, followed by promotion with 500 p.p.m. phenobarbital sodium (PB) in the drinking water up to week 58. A small number of tumours of Zymbal's gland were seen in all groups treated with IQ or MeIQ, irrespective of PB-treatment. Though the promotional regimen failed to produce the expected number of liver tumours, it did induce a significant amount of gamma-glutamyltranspeptidase (GGT) activity. These results suggest that even short exposures to low doses of IQ or MeIQ produce persistent procarcinogenic lesions in the rat, and that secondary factors, e.g. promoters or high cell turnover, may over time develop these lesions into cancer.

Percutaneous insertion of CSF ventriculoatrial shunts--a new technique. Technical note.

In conventional techniques concerning insertion of ventriculoatrial shunt systems, the route to the caval system is accomplished by a lateral rightsided neck dissection and isolation usually of the facial or the external jugular vein, in order to introduce the atrial catheter into the internal jugular vein and consequently the superior vena cava. A new approach for catheterization of the internal jugular vein is proposed. The technique is a combination of the well proven approach for percutaneous catheterization of the vein and a technique used in the implantation of permanent pacemaker leads. We find the method suitable for cases whenever a VA-shunt is preferred.

Sensitizing potential of 2-hydroxyethylmethacrylate.

Guinea pigs exhibited none or slight responses to sensitization with low concentrations of 2-hydroxyethylmethacrylate in the guinea pig maximization test, while 60-100% reacted to high concentrations regardless of the vehicle used for induction. Petrolatum, water, soybean oil and a mixture of oil and 2-butanone (sbomek) were used as vehicles for elicitation. The neat methacrylate was less effective than dilutions in any vehicle, petrolatum being the best. The major determinant of the frequency of response was the concentration used for intradermal induction. An increase in frequency and in duration of responsiveness after treatment with cyclophosphamide 3 days before challenge suggests that hydroxyethylmethacrylate preferentially stimulates the suppressor cell function.

Mutagenic metabolites in rat urine after dermal exposure to 1,3-diaminobenzene.

Amberlite XAD-2 purified urine extracts from rats treated dermally with 1,3-diaminobenzene were fractionated by HPLC and the metabolites were identified by GC-MS. Mutagenic activity in Salmonella typhimurium TA 98 was equally distributed between the two fractions, none of which contained the parent amine. The first fraction, eluted between 2-7 minutes, contained only a very small amount of presently unidentified metabolites, while the second fraction eluted between 14-19 minutes contained N,N'-diacetyl-1,3-diaminobenzene. Both fractions showed minor, but still significant activity even without metabolic activation. A non-mutagenic fraction, eluted between 10-12 minutes contained N-acetyl -acetyl-1,3-diaminobenzene and N,N'-diacetyl-2,4-diaminophenol.

Mutagens in rat urine after dermal application of 1,3-diaminobenzene.

The mutagenicity of urine from rats treated topically on the skin with 1,3-diaminobenzene was studied by the Salmonella/mammalian-microsome assay. Urine samples were either passed directly through micropore filters or extracts were prepared using XAD-2 resin before testing in the frameshift strain TA98. Significant mutagenic activity was found only after metabolic activation with rat-liver microsomes. The activity was higher in extracts from rats treated with a mixture of hydrogen peroxide and 1,3-diaminobenzene than from rats which were exposed to 1,3-diaminobenzene only. After fractionation of the urine by HPLC it could be demonstrated that the mutagenic activity was not due to the parent amine but related to metabolites in two of the fractions. To a lesser extent these two partially purified fractions were also mutagenic without S9 activation even though it was not possible to demonstrate this effect in unfractionated urine extracts. A third fraction containing two metabolites did not exert demonstrable mutagenic activity. The implications for the assessment of hazard to man are discussed.

Toxicological studies on malachite green: a triphenylmethane dye.

The oral LD50 for malachite green oxalate was found to be 275 mg/kg in rats while the approximate lethal dose for NMRI mice was 50 mg/kg. No systemic effects were seen after dermal application of 2,000 mg/kg. Repeated administration in the diet for 28 days to rats produced only minor changes in serum urea and aspartate aminotransferase levels. The rats at the highest dose level showed decreased weight gain and appeared clinically to have elevated motor activity. No sex differences were observed in either acute or prolonged experiments. In accord with human experience malachite green was irritating to mucous membranes, but no effects were seen on intact skin nor was it shown to be sensitizing. It was found to be a mutagen in the Salmonella/microsome test after metabolic activation but without clastogenic activity when tested at maximally tolerated levels in mice in the micronucleus test.

Cross-reaction patterns in guinea pigs sensitized to acrylic monomers.

The cross-reaction patterns of selected acrylate and methacrylate esters were investigated using the guinea pig maximization test. Methacrylates were less potent sensitizers than acrylates. Cross-sensitization was found between (meth)acrylates with closely related core structures, most extensively between hydroxyethyl and hydroxypropyl congeners. The importance of considering not only the functional group but also the core structure when assessing potential interactions was emphasized as butanedioldiglycidylether, hexamethylenediisocyanate and hexanedioldiacrylate--all with linear 6-atom cores and different functional group--cross-reacted.