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Background: Monocyte Chemotactic Protein 1-Induced Protein 1 (MCPIP1, also called Regnase-1) is a negative modulator of inflammation with tumor-suppressive properties. Mice with keratinocyte-specific deletion of the Zc3h12a gene, encoding MCPIP1, (Mcpip1 eKO mice) are more susceptible to the development of epidermal papillomas initiated by 7,12-dimethylbenz[a]-anthracene (DMBA) and promoted by 2-O-tetradecanoylphorbol-13-acetate (TPA)., Methods: The aim of this study was to investigate the MCPIP1 RNase-dependent microRNA (miRNA)‒mRNA regulatory network in chemically induced squamous cell carcinoma (SCC)-like skin papillomas. Next-generation sequencing (NGS) coupled with bioinformatic analysis was used to shortlist the MCPIP1-dependent changes in protein-coding genes and miRNAs. The expression levels of the selected miRNAs were analyzed by quantitative PCR in human keratinocytes with MCPIP1 silencing. Functional studies were performed in human keratinocytes transfected with appropriate miRNA mimics. The DIANA-microT-CDS algorithm and DIANA-TarBase v7 database were used to predict potential target genes and identify the experimentally validated targets of differentially expressed (DE) miRNAs., Results: RNA sequencing (RNA-Seq) analysis of control and Mcpip1 eKO DMBA/TPA-induced papillomas revealed transcriptome changes, with 2400 DE protein-coding genes and 33 DE miRNAs. The expression of miR-223-3p, miR-376c-3p, and miR-139-5p was confirmed to be dependent on MCPIP1 activity in both murine and human models. We showed that MCPIP1 directly regulates the expression of miR-376c-3p via direct cleavage of the corresponding precursor miRNA. The pro-proliferative activity of miR-223-3p, miR-376c-3p, and miR-139-5p was experimentally confirmed in SCC-like keratinocytes. Bioinformatic prediction of the mRNA targets of the DE-miRNAs revealed 416 genes as putative targets of the 18 upregulated miRNAs and 425 genes as putative targets of the 15 downregulated miRNAs. Further analyses revealed the murine interactions that are conserved in humans. Functional analysis indicated that during the development of cutaneous SCC, the most important pathways/processes mediated by the miRNA‒mRNA MCPIP1-dependent network are the regulation of inflammatory processes, epithelial cell proliferation, Wnt signaling, and miRNA transcription., Conclusions: Loss of MCPIP1 modulates the expression profiles of 33 miRNAs in chemically induced Mcpip1 eKO papillomas, and these changes directly affect the miRNA‒mRNA network and the modulation of pathways and processes related to carcinogenesis., (© 2024. The Author(s).)

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disease. Although it leads to muscle weakness, affected individuals predominantly die from cardiomyopathy, which remains uncurable. Accumulating evidence suggests that an overexpression of utrophin may counteract some of the pathophysiological outcomes of DMD. The aim of this study was to investigate the role of utrophin in dystrophin-deficient human cardiomyocytes (CMs) and to test whether an overexpression of utrophin, implemented via the CRISPR-deadCas9-VP64 system, can improve their phenotype. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) lacking either dystrophin (DMD) or both dystrophin and utrophin (DMD KO/UTRN (+/-) ). We carried out proteome analysis, which revealed considerable differences in the proteins related to muscle contraction, cell-cell adhesion, and extracellular matrix organization. Furthermore, we evaluated the role of utrophin in maintaining the physiological properties of DMD hiPSC-CMs using atomic force microscopy, patch-clamp, and Ca 2+ oscillation analysis. Our results showed higher values of afterhyperpolarization and altered patterns of cytosolic Ca 2+ oscillations in DMD; the latter was further disturbed in DMD KO/UTRN (+/-) hiPSC-CMs. Utrophin upregulation improved both parameters. Our findings demonstrate for the first time that utrophin maintains the physiological functions of DMD hiPSC-CMs, and that its upregulation can compensate for the loss of dystrophin., Competing Interests: The authors declare that they have no conflict of interest and have no relationships with the industry., (© 2024 The Authors.)

Aims: Duchenne muscular dystrophy (DMD)-associated cardiomyopathy is a serious life-threatening complication, the mechanisms of which have not been fully established, and therefore no effective treatment is currently available. The purpose of the study was to identify new molecular signatures of the cardiomyopathy development in DMD., Methods and Results: For modelling of DMD-associated cardiomyopathy, we prepared three pairs of isogenic control and dystrophin-deficient human induced pluripotent stem cell (hiPSC) lines. Two isogenic hiPSC lines were obtained by CRISPR/Cas9-mediated deletion of DMD exon 50 in unaffected cells generated from healthy donor and then differentiated into cardiomyocytes (hiPSC-CM). The latter were subjected to global transcriptomic and proteomic analyses followed by more in-depth investigation of selected pathway and pharmacological modulation of observed defects. Proteomic analysis indicated a decrease in the level of mitoNEET protein in dystrophin-deficient hiPSC-CM, suggesting alteration in iron metabolism. Further experiments demonstrated increased labile iron pool both in the cytoplasm and mitochondria, a decrease in ferroportin level and an increase in both ferritin and transferrin receptor in DMD hiPSC-CM. Importantly, CRISPR/Cas9-mediated correction of the mutation in the patient-derived hiPSC reversed the observed changes in iron metabolism and restored normal iron levels in cardiomyocytes. Moreover, treatment of DMD hiPSC-CM with deferoxamine (DFO, iron chelator) or pioglitazone (mitoNEET stabilizing compound) decreased the level of reactive oxygen species in DMD hiPSC-CM., Conclusion: To our knowledge, this study demonstrated for the first time impaired iron metabolism in human DMD cardiomyocytes, and potential reversal of this effect by correction of DMD mutation or pharmacological treatment. This implies that iron overload-regulating compounds may serve as novel therapeutic agents in DMD-associated cardiomyopathy., Competing Interests: Conflict of interest: None declared. This manuscript was handled by Deputy Editor Pasquale Maffia., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.)

The blackening of cut carrots causes substantial economic losses to the food industry. Blackening was not observed in carrots that had been stored underground for less than a year, but the susceptibility to blackening increased with the age of the carrots that were stored underground for longer periods. Samples of black, border, and orange tissues from processed carrot batons and slices, prepared under industry standard conditions, were analyzed to identify the molecular and metabolic mechanisms underpinning processing-induced blackening. The black tissues showed substantial molecular and metabolic rewiring and large changes in the cell wall structure, with a decreased abundance of xyloglucan, pectins (homogalacturonan, rhamnogalacturonan-I, galactan and arabinan), and higher levels of lignin and other phenolic compounds when compared to orange tissues. Metabolite profiling analysis showed that there was a major shift from primary to secondary metabolism in the black tissues, which were depleted in sugars, amino acids, and tricarboxylic acid (TCA) cycle intermediates but were rich in phenolic compounds. These findings suggest that processing triggers a release from quiescence. Transcripts encoding proteins associated with secondary metabolism were less abundant in the black tissues, but there were no increases in transcripts associated with oxidative stress responses, programmed cell death, or senescence. We conclude that restraining quiescence release alters cell wall metabolism and composition, particularly regarding pectin composition, in a manner that increases susceptibility to blackening upon processing.

The early embryo of the cockroach Blattella germanica exhibits high E93 expression. In general, E93 triggers adult morphogenesis during postembryonic development. Here we show that E93 is also crucial in early embryogenesis in the cockroach, as a significant number of E93-depleted embryos are unable to develop the germ band under maternal RNAi treatment targeting E93 . Moreover, transcriptomic analysis indicates that E93 depletion results in important gene expression changes in the early embryo, and many of the differentially expressed genes are involved in development. Then, using public databases, we gathered E93 expression data in embryo and preadult stages, finding that embryonic expression of E93 is high in hemimetabolan species (whose juveniles, or nymphs, are similar to the adult) and low in holometabolans (whose juveniles, or larvae, are different from the adult). E93 expression is also low in Thysanoptera and in Hemiptera Sternorrhyncha, hemimetabolans with postembryonic quiescent stages, as well as in Odonata, the nymph of which is very different from the adult. In ametabolans, such as the Zygentoma Thermobia domestica , E93 transcript levels are very high in the early embryo, whereas during postembryonic development they are medium and relatively constant. We propose the hypothesis that during evolution, a reduction of E93 expression in the embryo of hemimetabolans facilitated the larval development and the emergence of holometaboly. Independent decreases of E93 transcripts in the embryo of Odonata, Thysanoptera, and different groups of Hemiptera Sternorrhyncha would have allowed the development of modified juvenile stages adapted to specific ecophysiological conditions.

Miniature inverted-repeat transposable elements (MITEs) are the most abundant group of Class II mobile elements in plant genomes. Their presence in genic regions may alter gene structure and expression, providing a new source of functional diversity. Owing to their small size and lack of coding capacity, the identification of MITEs has been demanding. However, the increasing availability of reference genomes and bioinformatic tools provides better means for the genome-wide identification and analysis of MITEs and for the elucidation of their contribution to the evolution of plant genomes. We mined MITEs in the carrot reference genome DH1 using MITE-hunter and developed a curated carrot MITE repository comprising 428 families. Of the 31,025 MITE copies spanning 10.34 Mbp of the carrot genome, 54% were positioned in genic regions. Stowaways and Tourists were frequently present in the vicinity of genes, while Mutator -like MITEs were relatively more enriched in introns. hAT -like MITEs were relatively more frequently associated with transcribed regions, including untranslated regions (UTRs). Some carrot MITE families were shared with other Apiaceae species. We showed that hAT -like MITEs were involved in the formation of new splice variants of insertion-harboring genes. Thus, carrot MITEs contributed to the accretion of new diversity by altering transcripts and possibly affecting the regulation of many genes.

The AT-hook motif containing nuclear localized (AHL) gene family, controlling various developmental processes, is conserved in land plants. They comprise Plant and Prokaryote Conserved (PPC) domain and one or two AT-hook motifs. DcAHLc1 has been proposed as a candidate gene governing the formation of the carrot storage root. We identified and in-silico characterized carrot AHL proteins, performed phylogenetic analyses, investigated their expression profiles and constructed gene coexpression networks. We found 47 AHL genes in carrot and grouped them into two clades, A and B, comprising 29 and 18 genes, respectively. Within Clade-A, we distinguished three subclades, one of them grouping noncanonical AHLs differing in their structure (two PPC domains) and/or cellular localization (not nucleus). Coexpression network analysis attributed AHLs expressed in carrot roots into four of the 72 clusters, some of them showing a large number of interactions. Determination of expression profiles of AHL genes in various tissues and samples provided basis to hypothesize on their possible roles in the development of the carrot storage root. We identified a group of rapidly evolving noncanonical AHLs , possibly differing functionally from typical AHLs , as suggested by their expression profiles and their predicted cellular localization. We pointed at several AHLs likely involved in the development of the carrot storage root.

Background: Diverse groups of carrot cultivars have been developed to meet consumer demands and industry needs. Varietal groups of the cultivated carrot are defined based on the shape of roots. However, little is known about the genetic basis of root shape determination., Methods: Here, we used 307 carrot plants from 103 open-pollinated cultivars for a genome wide association study to identify genomic regions associated with the storage root morphology., Results: A 180 kb-long region on carrot chromosome 1 explained 10% of the total observed phenotypic variance in the shoulder diameter. Within that region, DcDCAF1 and DcBTAF1 genes were proposed as candidates controlling secondary growth of the carrot storage root. Their expression profiles differed between the cultivated and the wild carrots, likely indicating that their elevated expression was required for the development of edible roots. They also showed higher expression at the secondary root growth stage in cultivars producing thick roots, as compared to those developing thin roots., Conclusions: We provided evidence for a likely involvement of DcDCAF1 and/or DcBTAF1 in the development of the carrot storage root and developed a genotyping assay facilitating the identification of variants in the region on carrot chromosome 1 associated with secondary growth of the carrot root.

The carrot is the most popular root vegetable worldwide. The genetic makeup underlying the development of the edible storage root are fragmentary. Here, we report the first comparative transcriptome analysis between wild and cultivated carrot roots at multiple developmental stages. Overall, 3285, 4637, and 570 genes were differentially expressed in the cultivated carrot in comparisons made for young plants versus developing roots, young plants versus mature roots, and developing roots versus mature roots, respectively. Of those, 1916, 2645, and 475, respectively, were retained after filtering out genes showing similar profiles of expression in the wild carrot. They were assumed to be of special interest with respect to the development of the storage root. Among them, transcription factors and genes encoding proteins involved in post-translational modifications (signal transduction and ubiquitination) were mostly upregulated, while those involved in redox signaling were mostly downregulated. Also, genes encoding proteins regulating cell cycle, involved in cell divisions, development of vascular tissue, water transport, and sugar metabolism were enriched in the upregulated clusters. Genes encoding components of photosystem I and II, together with genes involved in carotenoid biosynthesis, were upregulated in the cultivated roots, as opposed to the wild roots; however, they were largely downregulated in the mature storage root, as compared with the young and developing root. The experiment produced robust resources for future investigations on the regulation of storage root formation in carrot and Apiaceae .

The prevalence of non-autonomous class II transposable elements (TEs) in plant genomes may serve as a tool for relatively rapid and low-cost development of gene-associated molecular markers. Miniature inverted-repeat transposable element (MITE) copies inserted within introns can be exploited as potential intron length polymorphism (ILP) markers. ILPs can be detected by PCR with primers anchored in exon sequences flanking the target introns. Here, we designed primers for 209 DcSto ( Daucus carota Stowaway -like) MITE insertion sites within introns along the carrot genome and validated them as candidate ILP markers in order to develop a set of markers for genotyping the carrot. As a proof of concept, 90 biallelic DcS -ILP markers were selected and used to assess genetic diversity of 27 accessions comprising wild Daucus carota and cultivated carrot of different root shape. The number of effective alleles was 1.56, mean polymorphism informative content was 0.27, while the average observed and expected heterozygosity was 0.24 and 0.34, respectively. Sixty-seven loci showed positive values of Wright's fixation index. Using Bayesian approach, two clusters comprising four wild and 23 cultivated accessions, respectively, were distinguished. Within the cultivated carrot gene pool, four subclusters representing accessions from Chantenay, Danvers, Imperator, and Paris Market types were revealed. It is the first molecular evidence for root-type associated diversity structure in western cultivated carrot. DcS -ILPs detected substantial genetic diversity among the studied accessions and, showing considerable discrimination power, may be exploited as a tool for germplasm characterization and analysis of genome relationships. The developed set of DcS -ILP markers is an easily accessible molecular marker genotyping system based on TE insertion polymorphism.

Carrot is one of the most important vegetables worldwide, owing to its capability to develop fleshy, highly nutritious storage roots. It was domesticated ca. 1,100 years ago in Central Asia. No systematic knowledge about the molecular mechanisms involved in the domestication syndrome in carrot are available, however, the ability to form a storage root is undoubtedly the essential transition from the wild Daucus carota to the cultivated carrot. Here, we expand on the results of a previous study which identified a polymorphism showing a significant signature for selection upon domestication. We mapped the region under selection to the distal portion of the long arm of carrot chromosome 2, confirmed that it had been selected, as reflected in both the lower nucleotide diversity in the cultivated gene pool, as compared to the wild (π w /π c = 7.4 vs. 1.06 for the whole genome), and the high F ST (0.52 vs. 0.12 for the whole genome). We delimited the region to ca. 37 kb in length and identified a candidate domestication syndrome gene carrying three non-synonymous single nucleotide polymorphisms and one indel systematically differentiating the wild and the cultivated accessions. This gene, DcAHLc1 , belongs to the AT-hook motif nuclear localized (AHL) family of plant regulatory genes which are involved in the regulation of organ development, including root tissue patterning. AHL genes work through direct interactions with other AHL family proteins and a range of other proteins that require intercellular protein movement. Based on QTL data on root thickening we speculate that DcAHLc1 might be involved in the development of the carrot storage root, as the localization of the gene overlapped with one of the QTLs. According to haplotype information we propose that the 'cultivated' variant of DcAHLc1 has been selected from wild Central Asian carrot populations upon domestication and it is highly predominant in the western cultivated carrot gene pool. However, some primitive eastern landraces and the derived B7262 purple inbred line still carry the 'wild' variant, reflecting a likely complexity of the genetic determination of the formation of carrot storage roots.

Metallothioneins (MT) are low-molecular weight proteins implicated in heavy metal detoxification, zinc and cooper homeostasis and cell protection against free radicals. In variety of cancers MT-overexpression was shown, but there are just a few studies on the role of MT in skin carcinogenesis. Current study was undertaken to evaluate MT and Ki-67 expression in pre-cancerous skin lesions as well as in fully developed skin cancers. 73 squamous cell carcinomas (SCC), 23 actinic keratoses (AK) and 20 normal skin samples were included in the study. In obtained paraffin sections immunohistochemical reactions were performed. MT-expression in SCC (mean 2.89 ± 1.83) was significantly higher than in AK (mean 1.69 ± 1.26)(p = 0.006) and higher than in normal skin (mean 2 ± 0.79) (p = 0.0075). The MT-expression positively correlated with Ki-67 expression (R = 0.28; p = 0.017) in SCC and in AK (R = 0.49; p = 0.018). Various clinico-pathological variables, e.g. morphology, size of lesions and the depth of neoplastic infiltration were not associated to MT-expression in both SCC and AK. The grade of histological differentiation of SCC correlated positively with Ki-67 antigen (p < 0.001) and did not correlate with MT-expression (p = 0.06). Ki-67 expression was higher in SCC and in AK than in healthy skin (p = 0,003). In SCC and in AK expression of Ki-67 antigen correlated positively with MT-expression (respectively p = 0.017 and p = 0.018). MT may serve as a good markers of proliferation in SCC and AK. MT-overexpression in SCC may suggest a potential role of MT in skin carcinogenesis.

Introduction: Psoriasis is characterized by hyperproliferation and abnormal differentiation of keratinocytes,by the presence of inflammatory cell infiltrate in both the dermis and the epidermis and by alterations of capillaries. p53 protein is an important transcription factor which plays a central role in cell cycle regulation mechanisms and cell proliferation control., Objectives: This study was performed to identify the expression and localization of p53 protein in lesional and non-lesional skin samples taken from psoriatic patients in comparison with healthy controls., Material and Methods: Sections of psoriatic lesional and non-lesional skin (n=18) were examined. A control group (n=10) of healthy volunteers with no personal and family history of psoriasis was also examined. The expression of p53 was demonstrated using the avidin-biotin complex immunoperoxidase method and the monoclonal antibody DO7. The count and localization of cells with stained nuclei was evaluated using a light microscope in 10 fields for every skin biopsy., Results: In lesional psoriatic skin the count of p53 positive cells was significantly higher than in the skin samples taken from healthy individuals (p<0.01) and non-lesional skin taken from psoriatic patients (p=0.02). No significant difference between non-lesional psoriatic skin and normal skin was observed (p=0.1). A strong positive correlation between mean count and mean per cent of p53 positive cells was found (p<0.0001). P53 positive cells were located most commonly in the basal layer of the epidermis of both healthy skin and non-lesional psoriatic skin. In lesional psoriatic skin p53 positive cells were present in all layers of the epidermis., Conclusion: P53 protein appears to be an important factor in the pathogenesis of psoriasis.

45-year-old man with the Acquired Immunodeficiency Syndrome, on highly active antiretroviral therapy (HAART) resulting in rapid decline of HIV RNA and increase of CD4 T cells count, developed multiple skin umbilicated lesions (resembling molluscum contagiosum) on his face, ears, neck and chest. Histopathology and mycological cultures of a skin biopsy revealed Cryptococcus neoformans. Antigens of Cryptococcus was also identified in blood. During treatment with amphotericin B, the skin lesions regressed. This case demonstrates that skin lesions resembling molluscum contagiosum may be caused by cryptococcal infection. It is necessary to perform skin biopsy in HIV-infected persons with skin lesions to diagnose cutaneous cryptococcosis. The open question is if skin cryptococcosis may be the immune reconstruction disease.

The activity of granulocyte pyruvate kinase and glucose-6-phospho dehydrogenase in 12 patients with severe atopic dermatitis in active stage of disease and in remission was investigated. It was found that in active stage the activity of both enzymes was not significantly different than in healthy individuals. In remission pyruvate kinase activity was significantly decreased and glucose-6-phospho dehydrogenase activity was found to be non significantly decreased.