Vertical Ex Vivo Dermoscopy in Assessment of Malignant Skin Lesions.

The role of vertical ex vivo dermoscopy relevant to clinical diagnosis has not been investigated yet. Study objectives were defining, describing, and determining the importance of the structures visible using vertical ex vivo dermoscopy in the diagnosis of malignant skin lesions, as well as determining their accuracy in the assessment of tumor margins. A prospective, descriptive study was conducted in two University centers. Digital images of completely excised skin lesions, fixed in formalin, before histopathological diagnosis were used for analysis. BCCs had the most diverse dermoscopic presentation on the vertical section, while SCCs showed a similar presentation in most cases. Vertical dermoscopy of thin melanomas was almost identical, unlike nodular melanomas. Thickness accuracy assessed by dermatologist was 0.753 for BCC, 0.810 for SCC, and 0.800 for melanomas, whereas assessment by pathologist was 0.654, 0.752, and 0.833, respectively. The accuracy of tumor width assessment was 0.819 for BCCs, 0.867 for SCCs and 1.000 for melanoma as estimated by a Dermatologist. Interobserver agreement was 0.71 for BCC, 0.799 for SCC and 0.832 for melanomas. Vertical ex vivo dermoscopy may contribute to the distinction between BCCs, SCCs, and melanomas. Moreover, regardless of the doctor's specialty, it enables a good assessment of the tumor's margins.

Influence of male body mass index on semen analysis parameters and in vitro fertilization outcomes.

Concurrent global increase of prevalence of obesity and male fertility implies link between overweight and obesity with male subfertility. This hypothesis is supported by numerous population-based epidemiological studies. Increase in body mass index (BMI) is associated with poor sperm quality in fertile, and more noticeable in infertile men. Nevertheless, some studies disprove damaging effect of BMI on semen quality. To examine the influence of men's BMI in infertile couples undergoing in vitro fertilization (IVF) on semen analysis parameters and IVF outcomes. Study encompassed all couples who underwent IVF at Gynecology and Obstetrics Clinic Narodni Front in Belgrade during 2018 and 2019. Exclusion criteria were azoospermia, conditions and diseases that could affect the semen analysis parameters (diabetes, malignant diseases treated with radiation and/or chemotherapy, trauma or surgery of the genital organs, mumps or undescended testicles in childhood). Evaluated semen analysis parameters included semen ejaculate volume, sperm pH, sperm count, sperm motility, and sperm morphology. IVF outcomes comprised total number of embryos, number and percentage of obtained good-quality embryos and clinical pregnancy rates. Based on BMI value, participants were divided into a group of underweight (Group 1), normally weight (Group 2), overweight (Group 3), and obese men (Group 4). After applying inclusion and exclusion criteria, 411 men (couples) were included in the analysis. The largest number of men were overweight, while the smallest belonged to the group of underweight participants. There are no significant differences in the semen analysis parameters between study groups. Correlation analysis shown weak and insignificant correlation between BMI and semen analysis parameters. The number and proportion of good quality embryos is significantly lower in overweight and obese study groups compared to normal weight and underweight groups (2.89, 2.91, 2.42, and 2.36, respectively, P = .041). The differences in other IVF outcomes: total number of embryos (3.61, 3.74, 3.21, and 3.37, respectively) and clinical pregnancy rates (41.26%, 43.09%, 42.78%, and 39.95%, respectively) between study groups were not significant (P > .05). BMI does not significantly affect semen analysis parameters, but a higher BMI is associated with a lower number and proportion of good quality embryos in IVF outcomes.

TULIPs decorate the three-dimensional genome of PFA ependymoma.

Posterior fossa group A (PFA) ependymoma is a lethal brain cancer diagnosed in infants and young children. The lack of driver events in the PFA linear genome led us to search its 3D genome for characteristic features. Here, we reconstructed 3D genomes from diverse childhood tumor types and uncovered a global topology in PFA that is highly reminiscent of stem and progenitor cells in a variety of human tissues. A remarkable feature exclusively present in PFA are type B ultra long-range interactions in PFAs (TULIPs), regions separated by great distances along the linear genome that interact with each other in the 3D nuclear space with surprising strength. TULIPs occur in all PFA samples and recur at predictable genomic coordinates, and their formation is induced by expression of EZHIP. The universality of TULIPs across PFA samples suggests a conservation of molecular principles that could be exploited therapeutically.

Maize miRNAs and their putative target genes involved in chilling stress response in 5-day old seedlings.

BACKGROUND: In the context of early sowing of maize as a promising adaptation strategy that could significantly reduce the negative effects of climate change, an in-depth understanding of mechanisms underlying plant response to low-temperature stress is demanded. Although microRNAs (miRNAs) have been recognized as key regulators of plant stress response, research on their role in chilling tolerance of maize during early seedling stages is scarce. Therefore, it is of great significance to explore chilling-responsive miRNAs, reveal their expression patterns and associated target genes, as well as to examine the possible functions of the conserved and novel miRNAs. In this study, the role of miRNAs was examined in 5d-old maize seedlings of one tolerant and one sensitive inbred line exposed to chilling (10/8 °C) stress for 6 h and 24 h, by applying high throughput sequencing. RESULTS: A total of 145 annotated known miRNAs belonging to 30 families and 876 potentially novel miRNAs were identified. Differential expression (DE) analysis between control and stress conditions identified 98 common miRNAs for both genotypes at one time point and eight miRNAs at both time points. Target prediction and enrichment analysis showed that the DE zma-miR396, zma-miR156, zma-miR319, and zma-miR159 miRNAs modulate growth and development. Furthermore, it was found that several other DE miRNAs were involved in abiotic stress response: antioxidative mechanisms (zma-miR398), signal transduction (zma-miR156, zma-miR167, zma-miR169) and regulation of water content (zma-miR164, zma-miR394, zma-miR396). The results underline the zma-miRNAs involvement in the modulation of their target genes expression as an important aspect of the plant's survival strategy and acclimation to chilling stress conditions. CONCLUSIONS: To our understanding, this is the first study on miRNAs in 5-d old seedlings' response to chilling stress, providing data on the role of known and novel miRNAs post-transcriptional regulation of expressed genes and contributing a possible platform for further network and functional analysis.

Effects of Different Therapeutic Approaches on Redox Balance in Psoriatic Patients.

Given that oxidative stress represents an important etiological factor in the pathogenesis of psoriasis, the aim of this study was to assess the effects of different therapeutic approaches, methotrexate, secukinumab, and ustekinumab on systemic oxidative stress biomarkers in psoriatic patients. This study involved 78 psoriatic patients, divided into the group treated with methotrexate (23 patients), secukinumab (28 patients), and ustekinumab (27 patients), and 15 healthy controls. Oxidative stress biomarkers (index of lipid peroxidation measured as TBARS, nitrites (NO2-), superoxide anion radical (O2-), and hydrogen peroxide (H2O2)) and antioxidative defense system (superoxide dismutase (SOD) activity, catalase (CAT) activity, and reduced glutathione (GSH)) were determined spectrophotometrically from the blood before the initiation of therapy in 16th, 28th, and 52nd week. O2- and SOD showed the most prominent changes comparing the psoriatic patients and healthy controls. CAT activity was significantly lower in psoriatic patients, and methotrexate induced a further decline in CAT activity. Ustekinumab induced a significant increase in GSH level after 52 weeks of treatment, while methotrexate reduced GSH. All applied therapeutic options induced a reduction in PASI, BSA, DLQI, and EARP. Biological drugs exert more pronounced antioxidant effects compared to methotrexate, which is most clearly observed in the values of O2- and SOD.

Glioblastoma evolution and heterogeneity from a 3D whole-tumor perspective.

Treatment failure for the lethal brain tumor glioblastoma (GBM) is attributed to intratumoral heterogeneity and tumor evolution. We utilized 3D neuronavigation during surgical resection to acquire samples representing the whole tumor mapped by 3D spatial coordinates. Integrative tissue and single-cell analysis revealed sources of genomic, epigenomic, and microenvironmental intratumoral heterogeneity and their spatial patterning. By distinguishing tumor-wide molecular features from those with regional specificity, we inferred GBM evolutionary trajectories from neurodevelopmental lineage origins and initiating events such as chromothripsis to emergence of genetic subclones and spatially restricted activation of differential tumor and microenvironmental programs in the core, periphery, and contrast-enhancing regions. Our work depicts GBM evolution and heterogeneity from a 3D whole-tumor perspective, highlights potential therapeutic targets that might circumvent heterogeneity-related failures, and establishes an interactive platform enabling 360° visualization and analysis of 3D spatial patterns for user-selected genes, programs, and other features across whole GBM tumors.

The Influence of Treatment Modalities on the Quality of Life of Psoriasis Patients.

Background: We aimed to identify the quality of life (QoL) of patients with psoriasis, to determine the possible differences depending on the therapeutic modalities (biologic, conventional treatment and phototherapy), and to examine other variables that could affect the success of the treatment. Methods: This research was a non-experimental, quantitative, observational study that included 183 psoriasis patients. The study was conducted from November 2021 to December 2022 at the University Clinical Center Kragujevac, Serbia. The following instruments were used: Dermatology Life Quality Index (DLQI), Psoriasis Area and Severity Index (PASI), as well as a general questionnaire that contained a set of questions which referred to sociodemographic data. Results: There was a statistically significant difference in the average values of the DLQI score concerning the application of different therapeutic modalities (P<0.001). Biologic treatment was the modality with the lowest impairments in the QoL domain (average value of DLQI score 10.6±7.3), followed by patients on conventional treatment (average value of DLQI score 12.9±7.9), and the highest levels of impaired QoL were in patients who received phototherapy (average value of the DLQI score 13.7±9.3). Conclusion: Patients on biological therapy at all four time points individually (baseline, 4, 12 and 16 weeks) had the lowest average values of the DLQI score, i.e. the best QoL compared to subjects who received other therapy.

First report of Aspergillus welwitschiae causing maize ear rot in Serbia.

In recent years, countries in Southeast Europe are facing climate changes characterized by extreme hot weather, which contribute to the increased frequency of Aspergillus species. Because of these changes, Aspergillus parasiticus was isolated, for the first time, from maize grain in Serbia (Nikolic et al, 2018). The presence of black powdery mycelia on maize ears indicated occurrence of species of the genus Aspergillus section Nigri, which led to the need for detailed identification of these fungi. Disease incidence ranged from 10 and 15% in August 2013. Maize ears with black powdery symptoms were collected from field in Zemun Polje, Serbia. Symptomatic kernels were surface sterilized with 1% sodium hypochlorite solution for 3 min, rinsed three times with sterilized water, then incubated at 25°C in the dark for 7 days on potato dextrose agar (PDA). Twenty isolates were identified as genus Aspergillus section Nigri. Monospore cultures formed black cottony colonies with a yellowish border on PDA. The average colony diameter was 50 mm. In order to reliably identify, isolates were transferred to Malt Extract agar (MEA) and Czapek Yeast Autolysate agar (CYA) (Samson et al, 2014). On CYA fungal colonies consisted of a white mycelium, covered by a layer of black conidiophores. On MEA fungal colonies were dense, black, with yellowish border. The reverse side was colorless to pale yellow, with a yellow ring in the middle. The average size of conidia was 4.3 µm. The conidia were globose to sub-globose, smooth to roughened, which coincides with previous research (Silva et al, 2020). Given that the fungi Aspergillus niger and Aspergillus welwitschiae are morphologically indistinguishable (Susca et al, 2016), species level identification was completed by analysis of a partial sequence of the internal transcribed spacer (ITS) region (ITS1/ITS4 primers) and calmodulin gene (CMD5/CMD6 primers) (Samson et al., 2014). The sequences were compared with the sequences of A. welwitschiae strains registered in the GenBank database based on nucleotide similarity, and results showed 99,64 and 100% similarity with ITS (OL711714) and calmodulin (KX894585), respectively. The sequence was deposited in GenBank with accession numbers OQ456471 (ITS) and OQ426518 (calmodulin). We also confirmed the presence of this species with specific primers (AWEL1/AWEL2) designed by Susca et al. 2020. Pathogenicity test was performed in Zemun Polje on the same maize hybrid from which the fungal species was isolated. Using artificial inoculations by the injecting conidial suspension into the silk channel, three days after 50% of plants reached the silking stage. Twenty ears were inoculated with each isolate, in four replicates (Reid et al, 1996). Inoculum was prepared from 7-day-old colonies on PDA, and 2 ml of a conidial suspension (1×106 spores/ml) was used. Control plants were inoculated with sterile water. All inoculated ears showed symptoms, similar to those from field infections. Control ears were symptomless. The fungus was reisolated and was morphologically identical to the original isolates, thus completing Koch's postulates. Based on molecular, morphological and pathogenic properties, the isolates were identified as A. welwitschiae. This is the first report of A. welwitschiae as the causal agent of black maize ear rot not only in Serbia, but also in the other countries of the Western Balkans. Given that the fungus A. welwitschiae synthesizes both ochratoxin A (OTA) (Battilani et al, 2006) and fumonisin (FB) (Frisvad et al, 2011), further studies should be focused on assessment its aggressiveness and toxicological profile.

macroH2A2 antagonizes epigenetic programs of stemness in glioblastoma.

Self-renewal is a crucial property of glioblastoma cells that is enabled by the choreographed functions of chromatin regulators and transcription factors. Identifying targetable epigenetic mechanisms of self-renewal could therefore represent an important step toward developing effective treatments for this universally lethal cancer. Here we uncover an epigenetic axis of self-renewal mediated by the histone variant macroH2A2. With omics and functional assays deploying patient-derived in vitro and in vivo models, we show that macroH2A2 shapes chromatin accessibility at enhancer elements to antagonize transcriptional programs of self-renewal. macroH2A2 also sensitizes cells to small molecule-mediated cell death via activation of a viral mimicry response. Consistent with these results, our analyses of clinical cohorts indicate that high transcriptional levels of this histone variant are associated with better prognosis of high-grade glioma patients. Our results reveal a targetable epigenetic mechanism of self-renewal controlled by macroH2A2 and suggest additional treatment approaches for glioblastoma patients.

Single-Cell Spatial Analysis Identifies Regulators of Brain Tumor-Initiating Cells.

Glioblastomas (GBM) are aggressive brain tumors with extensive intratumoral heterogeneity that contributes to treatment resistance. Spatial characterization of GBMs could provide insights into the role of the brain tumor microenvironment in regulating intratumoral heterogeneity. Here, we performed spatial transcriptomic and single-cell analyses of the mouse and human GBM microenvironment to dissect the impact of distinct anatomical regions of brains on GBM. In a syngeneic GBM mouse model, spatial transcriptomics revealed that numerous extracellular matrix (ECM) molecules, including biglycan, were elevated in areas infiltrated with brain tumor-initiating cells (BTIC). Single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin using sequencing showed that ECM molecules were differentially expressed by GBM cells based on their differentiation and cellular programming phenotypes. Exogeneous biglycan or overexpression of biglycan resulted in a higher proliferation rate of BTICs, which was associated mechanistically with low-density lipoprotein receptor-related protein 6 (LRP6) binding and activation of the Wnt/ß-catenin pathway. Biglycan-overexpressing BTICs developed into larger tumors and displayed mesenchymal phenotypes when implanted intracranially in mice. This study points to the spatial heterogeneity of ECM molecules in GBM and suggests that the biglycan-LRP6 axis could be a therapeutic target to curb tumor growth. SIGNIFICANCE: Characterization of the spatial heterogeneity of glioblastoma identifies regulators of brain tumor-initiating cells and tumor growth that could serve as candidates for therapeutic interventions to improve the prognosis of patients.

Roles of Chromatin Remodelling and Molecular Heterogeneity in Therapy Resistance in Glioblastoma.

Cancer stem cells (CSCs) represent a therapy-resistant reservoir in glioblastoma (GBM). It is now becoming clear that epigenetic and chromatin remodelling programs link the stemlike behaviour of CSCs to their treatment resistance. New evidence indicates that the epigenome of GBM cells is shaped by intrinsic and extrinsic factors, including their genetic makeup, their interactions and communication with other neoplastic and non-neoplastic cells, including immune cells, and their metabolic niche. In this review, we explore how all these factors contribute to epigenomic heterogeneity in a tumour and the selection of therapy-resistant cells. Lastly, we discuss current and emerging experimental platforms aimed at precisely understanding the epigenetic mechanisms of therapy resistance that ultimately lead to tumour relapse. Given the growing arsenal of drugs that target epigenetic enzymes, our review addresses promising preclinical and clinical applications of epidrugs to treat GBM, and possible mechanisms of resistance that need to be overcome.

Psoriasis between Autoimmunity and Oxidative Stress: Changes Induced by Different Therapeutic Approaches.

Psoriasis is defined as chronic, immune-mediated disease. Regardless of the development of new therapeutic approaches, the precise etiology of psoriasis remains unknown and speculative. The aim of this review was to systematize the results of previous research on the role of oxidative stress and aberrant immune response in the pathogenesis of psoriasis, as well as the impact of certain therapeutic modalities on the oxidative status in patients with psoriasis. Complex immune pathways of both the innate and adaptive immune systems appear to be major pathomechanisms in the development of psoriasis. Oxidative stress represents another important contributor to the pathophysiology of disease, and the redox imbalance in psoriasis has been reported in skin cells and, systemically, in plasma and blood cells, and more recently, also in saliva. Current immune model of psoriasis begins with activation of immune system in susceptible person by some environmental factor and loss of immune tolerance to psoriasis autoantigens. Increased production of IL-17 appears to be the most prominent role in psoriasis pathogenesis, while IL-23 is recognized as master regulator in psoriasis having a specific role in cross bridging the production of IL-17 by innate and acquired immunity. Other proinflammatory cytokines, including IFN-γ, TNF-α, IL-1ß, IL-6, IL-22, IL-26, IL-29, or IL-36, have also been reported to play important roles in the development of psoriasis. Oxidative stress can promote inflammation through several signaling pathways. The most noticeable and most powerful antioxidative effects exert various biologics compared to more convenient therapeutic modalities, such as methotrexate or phototherapy. The complex interaction of redox, immune, and inflammatory signaling pathways should be focused on further researches tackling the pathophysiology of psoriasis, while antioxidative supplementation could be the solution in some refractory cases of the disease.

Hi-C detects genomic structural variants in peripheral blood of pediatric leukemia patients.

B-cell acute lymphoblastic leukemia (B-ALL) is often driven by chromosome translocations that result in recurrent and well-studied gene fusions. Currently, fluorescent in situ hybridization probes are used to detect candidate translocations in bone marrow samples from B-ALL patients. Recently Hi-C, a sequencing-based technique originally designed to reconstruct the three-dimensional architecture of the nuclear genome, was shown to effectively recognize structural variants. Here, we demonstrate that Hi-C can be used as a genome-wide assay to detect translocations and other structural variants of potential clinical interest. Structural variants were identified in both bone marrow and peripheral blood samples, including an ETV6-RUNX1 translocation present in one pediatric B-ALL patient. Our report provides proof of principle that Hi-C could be an effective strategy to globally detect driver structural variants in B-ALL peripheral blood specimens, reducing the need for invasive bone marrow biopsies and candidate-based clinical tests.

Intronic elements associated with insomnia and restless legs syndrome exhibit cell-type-specific epigenetic features contributing to MEIS1 regulation.

A highly evolutionarily conserved myeloid ecotropic viral integration site 1 (MEIS1) intronic region is strongly associated with restless legs syndrome (RLS) and insomnia. To understand its regulatory function, we dissected the region by analyzing chromatin accessibility, enhancer-promoter contacts, DNA methylation and expression quantitative trait locus (eQTLs) in different human neural cell types and tissues. We observed specific activity with respect to cell type and developmental maturation, indicating a prominent role for distinct highly conserved intronic elements in forebrain inhibitory neuron differentiation. Two elements were hypomethylated in neural cells with higher MEIS1 expression, suggesting a role of enhancer demethylation in gene regulation. MEIS1 eQTLs showed a striking modular chromosomal distribution, with forebrain eQTLs clustering in intron 8/9. Clustered regularly interspersed short palindromic repeats interference targeting of individual elements in this region attenuated MEIS1 expression, revealing a complex regulatory interplay of distinct elements. In summary, we found that MEIS1 regulation is organized in a modular pattern. Disease-associated intronic regulatory elements control MEIS1 expression with cell type and maturation stage specificity, particularly in the inhibitory neuron lineage. The precise spatiotemporal activity of these elements likely contributes to the pathogenesis of insomnia and RLS.

Toxigenic Species Aspergillus parasiticus Originating from Maize Kernels Grown in Serbia.

In Serbia, aspergillus ear rot caused by the disease pathogen Aspergillus parasiticus (A. parasiticus) was first detected in 2012 under both field and storage conditions. Global climate shifts, primarily warming, favour the contamination of maize with aflatoxins in temperate climates, including Serbia. A five-year study (2012-2016) comprising of 46 A. parasiticus strains isolated from maize kernels was performed to observe the morphological, molecular, pathogenic, and toxigenic traits of this pathogen. The HPLC method was applied to evaluate mycotoxin concentrations in this causal agent. The A. parasiticus isolates synthesised mainly aflatoxin AFB1 (84.78%). The percentage of isolates synthesising aflatoxin AFG1 (15.22%) was considerably lower. Furthermore, the concentration of AFG1 was higher than that of AFB1 in eight isolates. The polyphase approach, used to characterise isolates, showed that they were A. parasiticus species. This identification was verified by the multiplex RLFP-PCR detection method with the use of restriction enzymes. These results form an excellent baseline for further studies with the aim of application in the production, processing, and storage of cereal grains and seeds, and in technological processes to ensure the safe production of food and feed.

Copy-scAT: Deconvoluting single-cell chromatin accessibility of genetic subclones in cancer.

Single-cell epigenomic assays have tremendous potential to illuminate mechanisms of transcriptional control in functionally diverse cancer cell populations. However, application of these techniques to clinical tumor specimens has been hampered by the current inability to distinguish malignant from nonmalignant cells, which potently confounds data analysis and interpretation. Here, we describe Copy-scAT, an R package that uses single-cell epigenomic data to infer copy number variants (CNVs) that define cancer cells. Copy-scAT enables studies of subclonal chromatin dynamics in complex tumors like glioblastoma. By deploying Copy-scAT, we uncovered potent influences of genetics on chromatin accessibility profiles in individual subclones. Consequently, some genetic subclones were predisposed to acquire stem-like or more differentiated molecular phenotypes, reminiscent of developmental paradigms. Copy-scAT is ideal for studies of the relationships between genetics and epigenetics in malignancies with high levels of intratumoral heterogeneity and to investigate how cancer cells interface with their microenvironment.

Extracellular signal-regulated kinase mediates chromatin rewiring and lineage transformation in lung cancer.

Lineage transformation between lung cancer subtypes is a poorly understood phenomenon associated with resistance to treatment and poor patient outcomes. Here, we aimed to model this transition to define underlying biological mechanisms and identify potential avenues for therapeutic intervention. Small cell lung cancer (SCLC) is neuroendocrine in identity and, in contrast to non-SCLC (NSCLC), rarely contains mutations that drive the MAPK pathway. Likewise, NSCLCs that transform to SCLC concomitantly with development of therapy resistance downregulate MAPK signaling, suggesting an inverse relationship between pathway activation and lineage state. To test this, we activated MAPK in SCLC through conditional expression of mutant KRAS or EGFR, which revealed suppression of the neuroendocrine differentiation program via ERK. We found that ERK induces the expression of ETS factors that mediate transformation into a NSCLC-like state. ATAC-seq demonstrated ERK-driven changes in chromatin accessibility at putative regulatory regions and global chromatin rewiring at neuroendocrine and ETS transcriptional targets. Further, ERK-mediated induction of ETS factors as well as suppression of neuroendocrine differentiation were dependent on histone acetyltransferase activities of CBP/p300. Overall, we describe how the ERK-CBP/p300-ETS axis promotes a lineage shift between neuroendocrine and non-neuroendocrine lung cancer phenotypes and provide rationale for the disruption of this program during transformation-driven resistance to targeted therapy.