CRISPR/Cas9n-mediated ELANE promoter editing for gene therapy of severe congenital neutropenia.

Severe congenital neutropenia (CN) is an inherited pre-leukemia bone marrow failure syndrome commonly caused by autosomal-dominant ELANE mutations (ELANE-CN). ELANE-CN patients are treated with daily injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF). However, some patients do not respond to rhG-CSF, and approximately 15% of ELANE-CN patients develop myelodysplasia or acute myeloid leukemia. Here, we report the development of a curative therapy for ELANE-CN through inhibition of ELANE mRNA expression by introducing two single-strand DNA breaks at the opposing DNA strands of the ELANE promoter TATA-box using CRISPR/Cas9D10A nickases - termed MILESTONE. This editing effectively restored defective neutrophil differentiation of ELANE-CN CD34+ hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo, without affecting the functions of the edited neutrophils. CRISPResso analysis of the edited ELANE-CN CD34+ HSPCs revealed on-target efficiencies of over 90%. Simultaneously, GUIDE-seq, CAST-Seq, and rhAmpSeq indicated a safe off-target profile with no off-target sites or chromosomal translocation. Taken together, ex vivo gene editing of ELANE-CN HSPCs using MILESTONE in the setting of autologous stem cell transplantation could be a universal, safe, and efficient gene therapy approach for ELANE-CN patients.

Differential transcriptional control of hematopoiesis in congenital and cyclic neutropenia patients harboring ELANE mutations.

Mutations in the ELANE gene, encoding the neutrophil elastase (NE) protein, are responsible for most CyN cases and approximately 25 % of CN cases. In CN and in CyN, a median of 2.8 % of CD34+ cells were early CD49f+ hematopoietic stem cells (eHSC) that did not express ELANE and thus escape from the unfolded protein response (UPR) caused by mutated NE. In CyN, the CD49f+ cells respond to G-CSF with a significant upregulation of the hematopoietic stem-cell-specific transcription factors, C/EBP/, MLL1, HOXA9, MEIS1, and HLF during the ascending arm of the cycle, resulting in the differentiation of myeloid cells to mature neutrophils at the cycle peak. However, NE protein released by neutrophils at the cycle's peak caused a negative feedback loop on granulopoiesis through the proteolytic digestion of G-CSF. In contrast, in CN patients, CD49f+ cells failed to express mRNA levels of HSC-specific transcription factors mentioned above. Rescue of C/EBP//expression in CN restored granulopoiesis.

The design of functional proteins using tensorized energy calculations.

In protein design, the energy associated with a huge number of sequence-conformer perturbations has to be routinely estimated. Hence, enhancing the throughput and accuracy of these energy calculations can profoundly improve design success rates and enable tackling more complex design problems. In this work, we explore the possibility of tensorizing the energy calculations and apply them in a protein design framework. We use this framework to design enhanced proteins with anti-cancer and radio-tracing functions. Particularly, we designed multispecific binders against ligands of the epidermal growth factor receptor (EGFR), where the tested design could inhibit EGFR activity in vitro and in vivo. We also used this method to design high-affinity Cu2+ binders that were stable in serum and could be readily loaded with copper-64 radionuclide. The resulting molecules show superior functional properties for their respective applications and demonstrate the generalizable potential of the described protein design approach.

Chlorpyrifos, permethrin and cyfluthrin effect on cell survival, permeability, and tight junction in an in-vitro model of the human blood-brain barrier (BBB).

The blood-brain barrier (BBB) is a structural and functional interface between the plasma and the human brain. Predictive BBB in-vitro models like immortalized human capillary microvascular endothelial cells (HCMEC/D3) can be used to explore the BBB disruption potential of daily exposed chemicals. The present study was focused on investigating the human BBB permeation potential of one organophosphate pesticide, chlorpyrifos (CPF), and two pyrethroids, permethrin (PMT) and cyfluthrin (CFT). HCMEC/D3 cells were exposed to the chemical and the time-dependent pass across BBB along with permeation coefficient (Papp) was calculated. Transendothelial electrical resistance (TEER) was measured for the cells to check the monolayer formation and later to check the reduction in integrity after chemical exposure. Real time PCR was conducted to investigate the effect of chemicals on the expression BBB´s tight and adherens junction proteins. Calculated Papp value for three chemicals was in the following order: CPF>CFT>PMT, where CPF showed the highest permeation coefficient. TEER calculation showed that the integrity decreased after CPF exposure which was in concordance with Papp value whereas for other chemicals, no change in TEER after exposure was observed. In addition, the transwell study showed a higher efflux ratio (ER) (>2) of CFT indicating that CFT could be a substrate for active transport. For CPF and PMT, ER was less than 2, so no active transport seems to be involved. The evaluation of the mRNA expression analysis revealed a statistically significant decrease in Occludin (OCLN) gene expression for CPF, VE-Cadherin (CDH5) for PMT and Zonula Occludens (ZO1) expression for CFT. Our study showed that CPF has the highest potential for inducing cell death, higher permeation, and capability to induce BBB dysfunction than among the above-mentioned chemicals. Additionally, the results of the permeation study could be useful to build a human PBPK model using in vitro-to-in vivo extrapolation approach.

Regenerative medicine meets translational oncology: Modeling leukemic bone marrow niche.

Most studies on leukemia focus on leukemia cells as isolated objects without considering the bone marrow niche. Pal et al. have recreated the bone marrow niche using induced pluripotent stem cells (iPSCs), identifying CDH2 as a therapeutically druggable leukemia-promoting factor.1.

Occupational and residential exposures to organophosphate and pyrethroid pesticides in a rural setting.

Organophosphate (OP) and pyrethroid pesticides (PYR) are extensively used in agriculture, resulting in higher exposures among farmworkers. The present study reports the occurrence of 8 urinary OP and PYR metabolites in a sample of farmworkers and residents from Sucs (n = 87), a rural township in North West Catalonia (Spain). The aim of the present study was to examine differences in urinary pesticide metabolite concentrations between occupationally-exposed (farmworkers; n = 45) and environmentally-exposed subjects (n = 42) and to assess the relationship between pesticide's exposures and occupational activities in a real-case scenario. Six OP and two PYR metabolites have been investigated, urine samples were extracted using SPE extraction and analyzed by UPLC-MS/MS. Three OP metabolites were commonly detectable in urine, namely TCPY (metabolite of chlorpyrifos), PNP (parathion) and DEAMPY (pirimiphos). Regarding pyrethroids, the two analyzed metabolites, 3-PBA and 4F-3-PBA, were detected in a high proportion of urine samples. Differences in concentrations between both groups were statistically significant for TCPY and 4F-3-PBA (Mann-Whitney U Test for independent groups, p < 0.05). In the case of TCPY, the concentrations were higher among the farmworkers, which is consistent with their occupational activity. The small differences found in DEAMPY, PNP, 3-PBA or even the significant higher concentrations of 4F-3-PBA among rural population suggest a general exposure to these compounds, even in those who do not carry an occupational activity. Specific personal protective equipment (PPE) among farmworkers, such as the use of gloves and mask during mixing, showed a decrease in the exposure levels, although the differences were not statistically significant. However, a positive association was found between the use of a cap during mixing (for PNP and 3-PBA) and during application (only for 3-PBA). However, this piece of cloth is mainly used for sun protection, and when not cleaned after the handling of pesticides, it might represent a continuous source of exposure through dermal contact. Farmworkers using tractors with cabin had statistically significant lower concentrations of DEAMPY than those using a tractor without cabin. The previous results suggest that occupational protections should be encouraged among farmworkers and other potential workers handling with pesticides.

Effects of Dynamic Suit Orthoses on the Spatio-Temporal Gait Parameters in Children with Cerebral Palsy: A Systematic Review.

Dynamic suit orthoses (DSO) are currently used as a complementary treatment method in children with Cerebral Palsy (cwCP). The aim of this review was to assess the effects of interventions with DSO on the altered spatio-temporal gait parameters (STGPs) in cwCP. An electronic search was conducted in the Web of Science, Scopus, PEDro, Cochrane Library, MEDLINE/PubMed, and CINAHL databases up to July 2021. We included a total of 12 studies, which showed great heterogeneity in terms of design type, sample size, and intervention performed (two employed a Therasuit, three employed the Adeli suit, three employed Theratogs, one employed elastomeric tissue dynamic orthosis, one employed a full-body suit, one employed external belt orthosis, and one employed dynamic orthosis composed of trousers and T-shirt). The Cochrane collaboration's tool and the Checklist for Measuring Study Quality were used to assess the risk of bias and the methodological quality of the studies. It was variable according to the Checklist for Measuring Study Quality, and it oscillated between eight and 23. The studies of higher methodological quality showed significant post-intervention changes in walking speed (which is the most widely evaluated parameter), cadence, stride length, and step length symmetry. Although the evidence is limited, the intervention with DSO combined with a programme of training/physical therapy seems to have positive effects on the STGPs in cwCP, with the functional improvements that it entails. Despite the immediate effect after one session, a number of sessions between 18 and 60 is recommended to obtain optimum results. Future studies should measure all STGPs, and not only the main ones, such as gait speed, in order to draw more accurate conclusions on the functional improvement of gait after the use of this type of intervention.

Pilot study of exposure of the male population to organophosphate and pyrethroid pesticides in a region of high agricultural activity (Córdoba, Argentina).

Urinary metabolites of organophosphate (OP) and pyrethroid (PYR) pesticides and biomarkers of effects were studied in a population (n=40) residing in an important agricultural area of the province of Córdoba (Argentina). Detection frequencies (DF) higher than 85% were observed for the metabolites of pirimiphos (2-diethylamino-6-methylpyrimidin-4-ol -DEAMPY-, median 7.5 µg/g creatinine, DF: 100%), parathion (p-nitrophenol, 0.99 µg/g creatinine, 100%), and chlorpyrifos (3,5,6-trichloro-2-pyridinol, 0.25 µg/g creatinine, 85%). The DEAMPY concentrations doubled the levels found in other studies and were negatively associated with Er-AChE activity, suggesting the appearance of health effects already in environmental exposure levels below established acceptable daily intakes (ADIs). 3-Phenoxybenzoic acid, the metabolite of several PYR pesticides, was also found in all samples. This metabolite was also significantly negatively correlated with Er-AChE, indicating effects of pyrethroid pesticides on the acetylcholine system even at concentrations below the ADI.

A Sentence Repetition Task for Early Language Assessment in Spanish.

Sentence repetition tasks have been widely used in the last years as a diagnostic tool in developmental language disorders. However in Spanish there are few (if any) of these instruments, especially for younger children. In this context, we develop a new Sentence Repetition Task for assessing language (morphosyntactic) abilities of very young Spanish children. A list of 33 sentences of different length and complexity was created and included in the task. A total of 130 typical developing children from 2 to 4 years of age were engaged in a play situation and asked to repeat the sentences. Children's answers were scored for accuracy at sentence and word level and error analysis at the word level was undertaken. Besides a subsample of 92 children completed a non-word repetition task. First results show its adequacy to children from 2 to 4 years of age, its capacity to discriminate between different developmental levels, and its concurrent validity with the nonword repetition task.

Mother/child organophosphate and pyrethroid distributions.

The present study reports one of the few cases in which organophosphate (OP) and pyrethroid (PYR) pesticide human exposure is evaluated in family contexts by the analysis of mother/child pair samples. Urinary concentrations of 6 organic metabolites of organophosphates and 2 pyrethroids were measured in mothers and their 7-to 8-year-old children (n = 168) in a general population from the central area of Slovenia. The results were adjusted for specific gravity and creatinine. The most abundant OP metabolite in children was 4-nitrophenol (PNP) (median 0.7 ng/ml) and in mothers (0.45 ng/ml), representing parathion exposure. 3-Phenoxibenzoic acid (3-PBA) (0.26 ng/ml), the general metabolite of pyrethroids, and 3,5,6-trichloro-2-pyridinol (TCPY) (0.16 ng/ml; chlorpyriphos) were the second most abundant compounds in children and mothers, respectively. The geometric mean specific gravity adjusted concentrations of OPs and PYRs were statistically significantly higher in children than in their mothers (between 3% and 24% higher), with the exception of TCPY (26% lower). All OP and PYR metabolites found in higher concentration in children showed significant positive correlations with the metabolite concentrations found in the mothers (p < 0.05 and 0.01), involving the fact that higher maternal concentrations were associated with higher children levels. These differential mother-children distributions and significant correlations were observed for the 2 types of pesticides studied, OPs and PYRs, which have different chemical properties. This agreement is consistent with the incorporation of the pesticides because of the general activities developed in the family context, instead of pesticide-dependent specific inputs. Comparison of the estimated daily intakes with the acceptable daily intakes of all detected metabolites revealed no significant risk of adverse health effects from exposure to these pesticides.

Organophosphate metabolite concentrations in maternal urine during pregnancy.

The burden of organophosphate (OP) pesticides in pregnant women from Tarragona (n = 157), a Mediterranean area of intense agricultural activity, has been assessed from the study of hydroxylated organic metabolites in urine samples in the three trimesters of pregnancy. 2-Diethylamino-6-methylpyrimidin-4-ol (DEAMPY), a metabolite of pirimiphos, was the compound found in higher concentration, medians 0.66-2.8 µg/g creatinine. 4-Nitrophenol (PNP), a metabolite of parathion, medians 0.24-0.41 µg/g creatinine, was the second most abundant compound. 2-Isopropyl-6-methyl-4-pyrimidol (IMPY), a metabolite of diazinon, was also present but in lower concentrations. Except for DEAMPY, the concentrations found in this cohort were lower than those reported in studies from other countries. Intraclass correlation coefficients (ICCs) were calculated for the compounds found in more than the 35% of the samples, the reliability between trimesters was poor (<0.40) to fair (0.40-0.60). Statistically significant differences were observed for the creatinine adjusted concentrations of the most abundant OP metabolites in these trimesters when examined with the Wilcoxon signed rank test for paired data. In general, no association was found between urinary OP metabolites and most demographic and lifestyle predictors. However, a positive significant association was observed for women with vegetarian diet and for women of higher economic status and eventual consumption of organic food which showed higher PNP concentrations. These results suggest that higher fruit and vegetable consumption may involve higher OP pesticide ingestion but the overall association was weak.

A sentence repetition task for early language assessment in Spanish

Sentence repetition tasks have been widely used in the last years as a diagnostic tool in developmental language disorders. However in Spanish there are few (if any) of these instruments, especially for younger children. In this context, we develop a new Sentence Repetition Task for assessing language (morphosyntactic) abilities of very young Spanish children. A list of 33 sentences of different length and complexity was created and included in the task. A total of 130 typical developing children from 2 to 4 years of age were engaged in a play situation and asked to repeat the sentences. Children's answers were scored for accuracy at sentence and word level and error analysis at the word level was undertaken. Besides a subsample of 92 children completed a non-word repetition task. First results show its adequacy to children from 2 to 4 years of age, its capacity to discriminate between different developmental levels, and its concurrent validity with the nonword repetition task

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Urinary metabolites of organophosphate and pyrethroid pesticides in children from an Italian cohort (PHIME, Trieste).

Urinary metabolites of organophosphate (OP) and pyrethroid (PYR) pesticides from seven years old children of a birth cohort study (n=199; PHIME cohort of Trieste, Italy) have been measured. Six OP and two PYR metabolites have been investigated, 2-diethylamino-6-methylpyrimidin-4-ol (DEAMPY, pirimiphos metabolite) was the one found at higher concentrations, median 3.4 ng/mL specific gravity adjusted (SG adjusted), followed by 4-nitrophenol (PNP, median 1.4 ng/mL SG adjusted) and 3,5,6-trichloro-2-pyridinol (TCPY, median 0.36 ng/mL SG adjusted), parathion and chlorpyriphos metabolites, respectively. TCPY concentrations were low in comparison to other distributions of OP metabolites in children from other studies. Accordingly, the PHIME cohort showed a distinct OP metabolite distribution with high concentrations of pirimiphos and parathion. Another specific characteristic of this cohort was the high concentration of 3-phenoxybenzoic acid (3-BPA, median 0.36 ng/mL SG adjusted), a general metabolite of PYR pesticides. Evaluation of anthropometric and socio-demographic characteristics of children and families only showed a positive association between family educational level and urinary concentrations of DEAMPY metabolite (p<0.05), which could reflect distinct dietary habits depending on the educational level. Estimated daily intakes were evaluated, all studied metabolites were found within safe levels.

Drivers of maternal accumulation of organohalogen pollutants in Arctic areas (Chukotka, Russia) and 4,4'-DDT effects on the newborns.

BACKGROUND: One of the most worrying consequence of the production and use of persistent organohalogen pollutants (POPs) is the high accumulation in Arctic populations because of long-range transport. Study of the effects in these populations may illustrate human impacts that are difficult to assess in other locations with lower exposure to these compounds and more diverse pollutant influences. OBJECTIVE: We aimed to identify the main maternal characteristics influencing on the accumulation of these compounds and the effects on the newborns in a highly exposed Arctic population (Chukotka, Russia). METHODS: Organochlorine and organobromine compounds were analysed in maternal venous serum (n = 250). The study included data on residence, educational level, age, parity and body mass index (BMI) from self-reported questionnaires and measured anthropometric characteristics of newborns. RESULTS: Concentrations of ß-hexachlorocyclohexanes, hexachlorobenzene, 4,4'-DDT and polychlorobiphenyls were high when compared with those generally found in adult populations later than year 2000. The polybromodiphenyl ethers were negligible. These POP concentrations were higher than in Alaska and Arctic Norway and similar to those in Canada. The Chukotka mothers living in inland areas showed significant lower concentrations than those living in the coast (p < 0.001) except for 4,4'-DDT. The population from the Chukotsky District, a specific coastal area, showed the highest concentrations. Residence was therefore a main concentration determinant (p < 0.001) followed by maternal age, and in some cases parity and BMI (p < 0.05). 4,4'-DDT showed an association with the anthropometric characteristics of the newborns (p < 0.05). Mothers with higher 4,4'-DDT concentrations had longer gestational ages and gave birth to infants with higher weight and length. CONCLUSIONS: The maternal accumulation patterns of POPs were mainly related with residence. Most of these compounds were found in higher concentration in women living at coastal areas except 4,4'-DDE and 4,4'-DDT which were of inland origin. This last pesticide was the pollutant showing positive associations with gestational age and newborn's weight and length. To the best of our knowledge, this is the first study reporting statistically significant associations between maternal 4,4'-DDT exposure and anthropometric characteristics of the newborns.

Analysis of metabolites of organophosphate and pyrethroid pesticides in human urine from urban and agricultural populations (Catalonia and Galicia).

Isotope dilution solid phase extraction UPLC-MS/MS has been used to develop a robust and rapid methodology for the determination of eight specific metabolites of organophosphate and pyrethroid pesticides in human urine. The use of methanol:acetone (25:75v/v) affords an improvement in extraction efficiency in comparison to these individual solvents. The use of synthetic urine improves selectivity and limits of detection for the calibration straight lines. The method provides detection limits of 14-69pg/ml and 18-19pg/ml for the organophosphate and pyrethroid metabolites, respectively. Urine analyses of these metabolites in urban non-occupationally exposed individuals and farm workers shows that ingestion of these pesticides occurred in both populations. The concentrations of organophosphate pesticide metabolites in the latter were twofold than those from non-exposed populations.

Variations in serum concentrations of selected organochlorines among delivering women in Argentina. The EMASAR study.

The EMASAR study is the first study to describe the body burden of OCs in Argentinian women after delivery. In total, 698 maternal serum samples from Salta (n = 498) and Ushuaia (n = 200) were collected in 2011-2012 and analyzed for a total of 7 polychlorinated biphenyls (PCBs) and 12 pesticide-related compounds. Only 11 of the compounds had detection rates above 60% in one or both places. Compared with Ushuaian women, those from Salta exhibited higher lipid-adjusted concentrations of p,p'-DDE, p,p'-DDT, ß-HCH, and PCB 118 (p ≤ 0.003), with no differences in concentrations of PCB 153 and 138. After controlling for age, parity and heritage (born in the province or migrated there from other regions of Argentina), concentrations of p,p'-DDE, p,p'-DDT, ß-HCH and all PCBs were significantly higher in Salta natives compared with Ushuaia natives or migrants (p ≤ 0.010). No variations between native and migrated Ushuaian women were observed other than for PCB 153 (6.1 versus 8.6 µg kg-1 lipid, p = 0.022). Age was generally associated positively with the body burden of nearly all OCs and parity negatively so, with p,p'-DDD, o,p'-DDT, and o,p'-DDD residues and α-HCH in Ushuaia being the exceptions. The regional differences in OC concentrations are explained by contrasting domestic sources, historical and current uses, industrial emissions, dietary patterns and lifestyle factors, as well as long-range-transport. The relatively high PCB 118/PCB 180 ratio observed for both Argentinian communities likely reflects the use of technical mixtures with congener-specific composition. In a comprehensive comparison with other countries, the Argentinian OC concentrations were mostly in the lower range. It is concluded that a latitude effect equivalent to that operative in the Arctic region seems unlikely.

Influence of maternal and sociodemographic characteristics on the accumulation of organohalogen compounds in Argentinian women. The EMASAR study.

The occurrence of organohalogen compounds in venous serum from post-partum mothers from two Argentinian cities, Salta and Ushuaia, has been investigated (n = 698). 4,4'-DDE was the most abundant compound in these cities, with geometric means of 33 and 67ng/g lipid weight, respectively. City of residence, age and parity were the main determinants of the accumulation of these compounds. Hexachlorobenzene (HCB) was the second most abundant pollutant in Ushuaia, 8.7ng/g lipid, and ß-hexachlorocyclohexane (ß-HCH) in Salta, 7.8ng/g lipid. Decabromodiphenyl ether was higher in Ushuaia than Salta, 8.2 and 4.1ng/g lipid, respectively. The predominance of ß-HCH, 4,4'-DDE and 4,4'-DDT in Salta was related with higher use of pesticides for agricultural applications. The observed higher concentrations of 4,4'-DDE and 4,4'-DDT in the mothers from rural+semi-urban sites than in urban areas were consistent with this agricultural origin. In addition, the most volatile organochlorine compounds included in this study, HCB and α-HCH, were mainly found in Ushuaia. The concentrations of the studied organohalogen pollutants in Argentina were lower than those found in other similar studies which is consistent with the location of these cities in the southern hemisphere. Age, mainly for 4,4'-DDE and polychlorobiphenyl (PCB) congeners 138, 153 and 180, and parity, mainly for HCB, ß-HCH, 4,4'-DDT and PCB congener 118, were the second main determinants of the concentrations of these compounds. Gestational weight gain also influenced on the maternal levels of HCB, ß-HCH, 4,4'-DDT and PCB congeners 118, 138 and 153. Higher weight accumulation during pregnancy involved dilution of these persistent pollutants. Body mass index (BMI) was a statistically significant determinant for 4,4'-DDT, α-HCH and PCB congeners 153 and 180. The observed direct correspondence between higher BMI and 4,4'-DDT concentrations was in agreement with the above reported inputs related with agricultural applications. The reverse correspondence of BMI with α-HCH and the PCB congeners indicated higher dilution at higher weight increase.

Regulation of carotenogenesis in the red yeast Xanthophyllomyces dendrorhous: the role of the transcriptional co-repressor complex Cyc8-Tup1 involved in catabolic repression.

BACKGROUND: The yeast Xanthophyllomyces dendrorhous produces carotenoids of commercial interest, including astaxanthin and ß-carotene. Although carotenogenesis in this yeast and the expression profiles of the genes controlling this pathway are known, the mechanisms regulating this process remain poorly understood. Several studies have demonstrated that glucose represses carotenogenesis in X. dendrorhous, suggesting that this pathway could be regulated by catabolic repression. Catabolic repression is a highly conserved regulatory mechanism in eukaryotes and has been widely studied in Saccharomyces cerevisiae. Glucose-dependent repression is mainly observed at the transcriptional level and depends on the DNA-binding regulator Mig1, which recruits the co-repressor complex Cyc8-Tup1, which then represses the expression of target genes. In this work, we studied the regulation of carotenogenesis by catabolic repression in X. dendrorhous, focusing on the role of the co-repressor complex Cyc8-Tup1. RESULTS: The X. dendrorhous CYC8 and TUP1 genes were identified, and their functions were demonstrated by heterologous complementation in S. cerevisiae. In addition, cyc8 - and tup1 - mutant strains of X. dendrorhous were obtained, and both mutations were shown to prevent the glucose-dependent repression of carotenogenesis in X. dendrorhous, increasing the carotenoid production in both mutant strains. Furthermore, the effects of glucose on the transcript levels of genes involved in carotenogenesis differed between the mutant strains and wild-type X. dendrorhous, particularly for genes involved in the synthesis of carotenoid precursors, such as HMGR, idi and FPS. Additionally, transcriptomic analyses showed that cyc8 - and tup1 - mutations affected the expression of over 250 genes in X. dendrorhous. CONCLUSIONS: The CYC8 and TUP1 genes are functional in X. dendrorhous, and their gene products are involved in catabolic repression and carotenogenesis regulation. This study presents the first report involving the participation of Cyc8 and Tup1 in carotenogenesis regulation in yeast.

The Involvement of Mig1 from Xanthophyllomyces dendrorhous in Catabolic Repression: An Active Mechanism Contributing to the Regulation of Carotenoid Production.

The red yeast X. dendrorhous is one of the few natural sources of astaxanthin, a carotenoid used in aquaculture for salmonid fish pigmentation and in the cosmetic and pharmaceutical industries for its antioxidant properties. Genetic control of carotenogenesis is well characterized in this yeast; however, little is known about the regulation of the carotenogenesis process. Several lines of evidence have suggested that carotenogenesis is regulated by catabolic repression, and the aim of this work was to identify and functionally characterize the X. dendrorhous MIG1 gene encoding the catabolic repressor Mig1, which mediates transcriptional glucose-dependent repression in other yeasts and fungi. The identified gene encodes a protein of 863 amino acids that demonstrates the characteristic conserved features of Mig1 proteins, and binds in vitro to DNA fragments containing Mig1 boxes. Gene functionality was demonstrated by heterologous complementation in a S. cerevisiae mig1- strain; several aspects of catabolic repression were restored by the X. dendrorhous MIG1 gene. Additionally, a X. dendrorhous mig1- mutant was constructed and demonstrated a higher carotenoid content than the wild-type strain. Most important, the mig1- mutation alleviated the glucose-mediated repression of carotenogenesis in X. dendrorhous: the addition of glucose to mig1- and wild-type cultures promoted the growth of both strains, but carotenoid synthesis was observed only in the mutant strain. Transcriptomic and RT-qPCR analyses revealed that several genes were differentially expressed between X. dendrorhous mig1- and the wild-type strain when cultured with glucose as the sole carbon source. The results obtained in this study demonstrate that catabolic repression in X. dendrorhous is an active process in which the identified MIG1 gene product plays a central role in the regulation of several biological processes, including carotenogenesis.

A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast.

In the fission yeast Schizosaccharomyces pombe the prevailing approach for gene manipulations is based on homologous recombination of a PCR product that contains genomic target sequences and a selectable marker. The CRISPR/Cas9 system has recently been implemented in fission yeast, which allows for seamless genome editing without integration of a selection marker or leaving any other genomic 'scars'. The published method involves manual design of the single guide RNA (sgRNA), and digestion of a large plasmid with a problematic restriction enzyme to clone the sgRNA. To increase the efficiency of this approach, we have established and optimized a PCR-based system to clone the sgRNA without restriction enzymes into a plasmid with a dominant natMX6 (nourseothricin) selection marker. We also provide a web-tool, CRISPR4P, to support the design of the sgRNAs and the primers required for the entire process of seamless DNA deletion. Moreover, we report the preparation of G1-synchronized and cryopreserved S. pombe cells, which greatly increases the efficiency and speed for transformations, and may also facilitate standard gene manipulations. Applying this optimized CRISPR/Cas9-based approach, we have successfully deleted over 80 different non-coding RNA genes, which are generally lowly expressed, and have inserted 7 point mutations in 4 different genomic regions.