Rapid biomechanical imaging at low irradiation level via dual line-scanning Brillouin microscopy.

Brillouin microscopy is a technique for mechanical characterization of biological material without contact at high three-dimensional resolution. Here, we introduce dual line-scanning Brillouin microscopy (dLSBM), which improves acquisition speed and reduces irradiation dose by more than one order of magnitude with selective illumination and single-shot analysis of hundreds of points along the incident beam axis. Using tumor spheroids, we demonstrate the ability to capture the sample response to rapid mechanical perturbations as well as the spatially resolved evolution of the mechanical properties in growing spheroids.

HMGB2 Loss upon Senescence Entry Disrupts Genomic Organization and Induces CTCF Clustering across Cell Types.

Processes like cellular senescence are characterized by complex events giving rise to heterogeneous cell populations. However, the early molecular events driving this cascade remain elusive. We hypothesized that senescence entry is triggered by an early disruption of the cells' three-dimensional (3D) genome organization. To test this, we combined Hi-C, single-cell and population transcriptomics, imaging, and in silico modeling of three distinct cells types entering senescence. Genes involved in DNA conformation maintenance are suppressed upon senescence entry across all cell types. We show that nuclear depletion of the abundant HMGB2 protein occurs early on the path to senescence and coincides with the dramatic spatial clustering of CTCF. Knocking down HMGB2 suffices for senescence-induced CTCF clustering and for loop reshuffling, while ectopically expressing HMGB2 rescues these effects. Our data suggest that HMGB2-mediated genomic reorganization constitutes a primer for the ensuing senescent program.

Extracellular vesicles and PD-L1 suppress macrophages, inducing therapy resistance in TP53-deficient B-cell malignancies.

Genetic alterations in the DNA damage response (DDR) pathway are a frequent mechanism of resistance to chemoimmunotherapy (CIT) in B-cell malignancies. We have previously shown that the synergy of CIT relies on secretory crosstalk elicited by chemotherapy between the tumor cells and macrophages. Here, we show that loss of multiple different members of the DDR pathway inhibits macrophage phagocytic capacity in vitro and in vivo. Particularly, loss of TP53 led to decreased phagocytic capacity ex vivo across multiple B-cell malignancies. We demonstrate via in vivo cyclophosphamide treatment using the Eµ-TCL1 mouse model that loss of macrophage phagocytic capacity in Tp53-deleted leukemia is driven by a significant downregulation of a phagocytic transcriptomic signature using small conditional RNA sequencing. By analyzing the tumor B-cell proteome, we identified a TP53-specific upregulation of proteins associated with extracellular vesicles (EVs). We abrogated EV biogenesis in tumor B-cells via clustered regularly interspaced short palindromic repeats (CRISPR)-knockout (KO) of RAB27A and confirmed that the EVs from TP53-deleted lymphoma cells were responsible for the reduced phagocytic capacity and the in vivo CIT resistance. Furthermore, we observed that TP53 loss led to an upregulation of both PD-L1 cell surface expression and secretion of EVs by lymphoma cells. Disruption of EV bound PD-L1 by anti-PD-L1 antibodies or PD-L1 CRISPR-KO improved macrophage phagocytic capacity and in vivo therapy response. Thus, we demonstrate enhanced EV release and increased PD-L1 expression in TP53-deficient B-cell lymphomas as novel mechanisms of macrophage function alteration in CIT resistance. This study indicates the use of checkpoint inhibition in the combination treatment of B-cell malignancies with TP53 loss.

Medicinal chemistry insights in neuronal nitric oxide synthase inhibitors containing nitrogen heterocyclic compounds: a mini review.

Many scientific reports over the last two decades have focused on the discovery and development of novel nNOS inhibitors. The structural identity of isoforms, bioavailability, pharmacokinetic, and safety profile issues remain major obstacles in the discovery of more potent and selective nNOS inhibitors. This review aims to provide an in-depth overview of the molecular interaction patterns between nNOS active site and inhibitors containing structurally diverse nitrogen heterocyclic compounds and highlight the structural properties needed to develop selective nNOS inhibitors. Previously published data allowed the usage of the structure-driven approach in the designing of selective nNOS inhibitors, which relies on the specific structural features required to achieve isoform-selectivity towards nNOS. The incorporation of chiral pyrrolidine ring, two aminopyridine heads, or a specific amino tail group, along with the inhibitor's capacity to adopt the curled conformation in the nNOS environment significantly strengthens the molecular interaction between the inhibitor and nNOS residues by forming specific electrostatic interactions and non-bonded contacts that are vital for isoform selectivity. Additional structure-activity relationship investigations are necessary to elucidate more structural characteristics that will ultimately resolve the exact structural basis required for isoform-selective inhibition of nNOS.

Potentilla tormentilla Extract Loaded Gel: Formulation, In Vivo and In Silico Evaluation of Anti-Inflammatory Properties.

The objective of the study was to develop a novel topical gel by mixing Potentilla tormentilla ethanolic extract, thermosensitive poloxamer 407, and carbomer 940 and evaluating its stability and rheological behavior. The irritation potential of the gel was evaluated in accordance with the Organization for Economic Cooperation and Development Guidelines 404. The potential anti-inflammatory effects of the developed gel were evaluated in vivo in rats using the carrageenan-induced paw edema test. Moreover, the in silico binding affinity for chlorogenic and ellagic acid, as dominant components in the extract, against cyclooxygenase (COX) 1 and 2 was also determined. Our findings suggest that the gel containing Potentilla tormentilla extract remained stable throughout the observation period, exhibited pseudoplastic behavior, and caused no irritation in rats, thus being considered safe for topical treatment. Additionally, the developed gel showed the capability to reduce rat paw edema, which highlights significant anti-inflammatory potential. In silico analysis revealed that chlorogenic and ellagic acid exhibited a reduced binding affinity against COX-1 but had a similar inhibitory effect on COX-2 as flurbiprofen, which was confirmed by molecular dynamics results. The study proposes the possible application of Potentilla tormentilla ethanolic extract gel for the alleviation of localized inflammatory diseases; however, future clinical evaluation is required.

Active Akt signaling triggers CLL toward Richter transformation via overactivation of Notch1.

Richter's transformation (RT) is an aggressive lymphoma that occurs upon progression from chronic lymphocytic leukemia (CLL). Transformation has been associated with genetic aberrations in the CLL phase involving TP53, CDKN2A, MYC, and NOTCH1; however, a significant proportion of RT cases lack CLL phase-associated events. Here, we report that high levels of AKT phosphorylation occur both in high-risk CLL patients harboring TP53 and NOTCH1 mutations as well as in patients with RT. Genetic overactivation of Akt in the murine Eµ-TCL1 CLL mouse model resulted in CLL transformation to RT with significantly reduced survival and an aggressive lymphoma phenotype. In the absence of recurrent mutations, we identified a profile of genomic aberrations intermediate between CLL and diffuse large B-cell lymphoma. Multiomics assessment by phosphoproteomic/proteomic and single-cell transcriptomic profiles of this Akt-induced murine RT revealed an S100 protein-defined subcluster of highly aggressive lymphoma cells that developed from CLL cells, through activation of Notch via Notch ligand expressed by T cells. Constitutively active Notch1 similarly induced RT of murine CLL. We identify Akt activation as an initiator of CLL transformation toward aggressive lymphoma by inducing Notch signaling between RT cells and microenvironmental T cells.

A novel rapamycin cream formulation improves facial angiofibromas associated with tuberous sclerosis complex: a double-blind randomized placebo-controlled trial.

BACKGROUND: Facial angiofibromas (FAs) are a major feature of tuberous sclerosis complex (TSC). Topical rapamycin can successfully treat FAs. A new stabilized cream formulation that protects rapamycin from oxidation has been developed in 0.5% and 1% concentrations. OBJECTIVES: To assess the efficacy and safety of a novel, stabilized topical rapamycin cream formulation. METHODS: This multicentre double-blind randomized placebo-controlled dose-response phase II/III study with a parallel design included participants aged 6-65 years with FAs of mild or moderate severity according to the Investigator's Global Assessment (IGA) scale. Participants were randomized to one of three treatment arms: topical rapamycin 0.5%, topical rapamycin 1% or placebo. Treatment was applied once daily for 26 weeks. Safety and efficacy measures were assessed at days 14, 56, 98, 140 and 182. The primary endpoint was the percentage of participants achieving IGA scores of 'clear' or 'almost clear' after 26 weeks of treatment. Secondary measures included Facial Angiofibroma Severity Index (FASI) and participant- and clinician-reported percentage-based improvement. Safety measures included the incidence of treatment-emergent adverse events and blood rapamycin concentration changes over time. RESULTS: Participants (n = 107) were randomized to receive either rapamycin 1% (n = 33), rapamycin 0.5% (n = 36) or placebo (n = 38). All treated participants were included in the final analysis. The percentage of participants with a two-grade IGA improvement was greater in the rapamycin 0.5% treatment group (11%) and rapamycin 1% group (9%) than in the placebo group (5%). However, this was not statistically significant [rapamycin 0.5%: odds ratio (OR) 1.71, 95% confidence interval (CI) 0.36-8.18 (P = 0.50); rapamycin 1%: OR 1.68, 95% CI 0.33-8.40 (P = 0.53)]. There was a statistically significant difference in the proportion of participants treated with rapamycin cream that achieved at least a one-grade improvement in IGA [rapamycin 0.5%: 56% (OR 4.73, 95% CI 1.59-14.10; P = 0.005); rapamycin 1%: 61% (OR 5.14, 95% CI 1.70-15.57; P = 0.004); placebo: 24%]. Skin adverse reactions were more common in patients following rapamycin application (64%) vs. placebo (29%). CONCLUSIONS: Both rapamycin cream formulations (0.5% and 1%) were well tolerated, and either strength could lead to clinical benefit in the treatment of FA.

Native and endemic Iranian Nepeta spp.: powerful antimicrobial agents.

Pathogenic microorganisms are more or less successfully treated by synthetic chemical compounds, whose residues often cause serious health problems. Plant specialized metabolites with antimicrobial properties have for a long time been the focus of both medicine and pharmacology. This study was conducted to evaluate the in vitro antimicrobial activity of methanol extracts of selected endemic and native Iranian Nepeta species against some of the most important pathogenic bacteria and fungi. The results indicated that N. kotschyi leaf extract was the most efficient against the tested bacteria, with Pseudomonas aeruginosa being the most sensitive and fungal species were more susceptible to the extracts than bacterial strains. Nepeta spp. extracts showed a strong antifungal activity against micromycetes, except for quite resistant Aspergillus niger. Antibacterial MIC values (mg.mL-1) ranged from 0.01 (N. kotschyi) to 0.20 (N. crassifolia), while antifungal MIC values ranged from 0.02 (N. crassifolia, N. kotschyi, N. menthoides, and N. cataria) to 0.13 (N. crassifolia and N. menthoides). When compared to positive controls, in most cases the extracts performed much better. The recorded antimicrobial activity candidates the selected 4 endemic and native Iranian Nepeta spp. as prospective and promising antimicrobial agents to be used in both pharmacology and biotechnology.

Serum Zinc Concentration in Patients with Alopecia Areata.

Alopecia areata is an autoimmune non-scarring disease in which the exact mechanism that induces loss of immune privilege is unknown. Zinc is important for DNA stability and repair mechanisms that are essential in maintaining normal hair growth. Zinc deficiency has been investigated as an important factor in many autoimmune diseases, and may have a possible role in the aetiopathogenesis of alopecia areata. This study included 32 patients with severe forms of alopecia areata, and 32 age- and sex-matched healthy controls. When comparing serum zinc levels in these 2 groups, statistically significantly lower zinc concentrations were found in the alopecia areata group (p = 0.017). Detected zinc deficiency was statistically more prevalent in patients with alopecia areata (p = 0.011). Evaluating patients with alopecia areata, a statistically significant negative correlation between serum zinc levels and severity of the disease was found (ρ = 0.006). The results indicate that zinc serum assessment is necessary in patients with alopecia areata. Low serum zinc levels were found to correlate with severity of alopecia areata. Given that most severe forms of alopecia areata are frequently most treatment-resistant, additional randomized control trials examining zinc supplementation are necessary to investigate its potential role in the restoration of hair follicles.

Multimodal microscale mechanical mapping of cancer cells in complex microenvironments.

The mechanical phenotype of the cell is critical for survival following deformations due to confinement and fluid flow. One idea is that cancer cells are plastic and adopt different mechanical phenotypes under different geometries that aid in their survival. Thus, an attractive goal is to disrupt cancer cells' ability to adopt multiple mechanical states. To begin to address this question, we aimed to quantify the diversity of these mechanical states using in vitro biomimetics to mimic in vivo two-dimensional (2D) and 3D extracellular matrix environments. Here, we used two modalities Brillouin microscopy (∼GHz) and broadband frequency (7-15 kHz) optical tweezer microrheology to measure microscale cell mechanics. We measured the response of intracellular mechanics of cancer cells cultured in 2D and 3D environments where we modified substrate stiffness, dimensionality (2D versus 3D), and presence of fibrillar topography. We determined that there was good agreement between two modalities despite the difference in timescale of the two measurements. These findings on cell mechanical phenotype in different environments confirm a correlation between modalities that employ different mechanisms at different temporal scales (Hz-kHz versus GHz). We also determined that observed heterogeneity in cell shape is more closely linked to the cells' mechanical state. Moreover, individual cells in multicellular spheroids exhibit a lower degree of mechanical heterogeneity when compared with single cells cultured in monodisperse 3D cultures. The observed decreased heterogeneity among cells in spheroids suggested that there is mechanical cooperativity between cells that make up a single spheroid.

HMGB1 coordinates SASP-related chromatin folding and RNA homeostasis on the path to senescence.

Spatial organization and gene expression of mammalian chromosomes are maintained and regulated in conjunction with cell cycle progression. This is perturbed once cells enter senescence and the highly abundant HMGB1 protein is depleted from nuclei to act as an extracellular proinflammatory stimulus. Despite its physiological importance, we know little about the positioning of HMGB1 on chromatin and its nuclear roles. To address this, we mapped HMGB1 binding genome-wide in two primary cell lines. We integrated ChIP-seq and Hi-C with graph theory to uncover clustering of HMGB1-marked topological domains that harbor genes involved in paracrine senescence. Using simplified Cross-Linking and Immuno-Precipitation and functional tests, we show that HMGB1 is also a bona fide RNA-binding protein (RBP) binding hundreds of mRNAs. It presents an interactome rich in RBPs implicated in senescence regulation. The mRNAs of many of these RBPs are directly bound by HMGB1 and regulate availability of SASP-relevant transcripts. Our findings reveal a broader than hitherto assumed role for HMGB1 in coordinating chromatin folding and RNA homeostasis as part of a regulatory loop controlling cell-autonomous and paracrine senescence.

New targeted approaches for epigenetic age predictions.

BACKGROUND: Age-associated DNA methylation changes provide a promising biomarker for the aging process. While genome-wide DNA methylation profiles enable robust age-predictors by integration of many age-associated CG dinucleotides (CpGs), there are various alternative approaches for targeted measurements at specific CpGs that better support standardized and cost-effective high-throughput analysis. RESULTS: In this study, we utilized 4647 Illumina BeadChip profiles of blood to select CpG sites that facilitate reliable age-predictions based on pyrosequencing. We demonstrate that the precision of DNA methylation measurements can be further increased with droplet digital PCR (ddPCR). In comparison, bisulfite barcoded amplicon sequencing (BBA-seq) gave slightly lower correlation between chronological age and DNA methylation at individual CpGs, while the age-predictions were overall relatively accurate. Furthermore, BBA-seq data revealed that the correlation of methylation levels with age at neighboring CpG sites follows a bell-shaped curve, often associated with a CTCF binding site. We demonstrate that within individual BBA-seq reads the DNA methylation at neighboring CpGs is not coherently modified, but reveals a stochastic pattern. Based on this, we have developed a new approach for epigenetic age predictions based on the binary sequel of methylated and non-methylated sites in individual reads, which reflects heterogeneity in epigenetic aging within a sample. CONCLUSION: Targeted DNA methylation analysis at few age-associated CpGs by pyrosequencing, BBA-seq, and particularly ddPCR enables high precision of epigenetic age-predictions. Furthermore, we demonstrate that the stochastic evolution of age-associated DNA methylation patterns in BBA-seq data enables epigenetic clocks for individual DNA strands.

Epigenetic Clocks Are Not Accelerated in COVID-19 Patients.

Age is a major risk factor for severe outcome of the 2019 coronavirus disease (COVID-19). In this study, we followed the hypothesis that particularly patients with accelerated epigenetic age are affected by severe outcomes of COVID-19. We investigated various DNA methylation datasets of blood samples with epigenetic aging signatures and performed targeted bisulfite amplicon sequencing. Overall, epigenetic clocks closely correlated with the chronological age of patients, either with or without acute respiratory distress syndrome. Furthermore, lymphocytes did not reveal significantly accelerated telomere attrition. Thus, these biomarkers cannot reliably predict higher risk for severe COVID-19 infection in elderly patients.

Nuclear Mechanics within Intact Cells Is Regulated by Cytoskeletal Network and Internal Nanostructures.

The mechanical properties of the cellular nucleus are extensively studied as they play a critical role in important processes, such as cell migration, gene transcription, and stem cell differentiation. While the mechanical properties of the isolated nucleus have been tested, there is a lack of measurements about the mechanical behavior of the nucleus within intact cells and specifically about the interplay of internal nuclear components with the intracellular microenvironment, because current testing methods are based on contact and only allow studying the nucleus after isolation from a cell or disruption of cytoskeleton. Here, all-optical Brillouin microscopy and 3D chemomechanical modeling are used to investigate the regulation of nuclear mechanics in physiological conditions. It is observed that the nuclear modulus can be modulated by epigenetic regulation targeting internal nuclear nanostructures such as lamin A/C and chromatin. It is also found that nuclear modulus is strongly regulated by cytoskeletal behavior through a robust mechanism conserved in different culturing conditions. Given the active role of cytoskeletal modulation in nearly all cell functions, this work will enable to reveal highly relevant mechanisms of nuclear mechanical regulations in physiological and pathological conditions.

Poikilodermatous mycosis fungoides with CD30-positive large cell transformation successfully treated by brentuximab vedotin.

We present a patient with a 33-year history of poikilodermatous mycosis fungoides (MF) who subsequently developed CD30-positive large cell transformation. After 6 years of conventional MF treatment, side effects of therapy and/or concomitant diseases prevented the previously applied treatment modalities. The CD30-directed antibody-cytotoxic drug conjugate (brentuximab vedotin) was introduced and followed by quick and excellent therapeutic response.

Annular lichenoid dermatitis of youth-Recurrent case of rare skin disease treated with cyclosporine.

Annular lichenoid dermatitis of youth (ALDY), first described in 2003, represents an uncommon entity whose etiopathogenesis is still debated. Futhermore, the optimal treatment for ALDY is yet to be established. We report a 9-year-old girl who presented with annular and oval erythematous lesions mostly on her trunk, with several lesions on the neck, groin, flanks, and upper extremities. The lesions had histological and immunohistochemical features characteristic for ALDY. Treatment with H1-antihistamines, topical corticosteroid, and UVB therapy was unsuccessful, while systemic treatment with cyclosporine induced complete remission.

Initial validation of the epidermolysis bullosa-specific module of the Infants and Toddlers Dermatology Quality of Life questionnaire.

Children with epidermolysis bullosa (EB) experienced the highest quality of life impact among several skin conditions and have problems which had not been reported by parents of children with other skin diseases. The EB-specific module of the Infants and Toddlers Dermatology Quality of Life (InToDermQoL) questionnaire was recently developed to measure the impact of disease-specific aspects in children from birth to the age of 4 years. The aim of this study was initial validation of the InToDermQoL-EB questionnaire. Parents of 44 children with EB from seven countries completed the InToDermQoL-EB questionnaire. Cronbach's alpha was .86, .89 and .91 for three age-specific versions. Differences between severity levels were all significant except for that between moderate and severe level in the version for 3- to 4-year-old children. All items of the three versions of the InToDermQoL-EB showed very high levels of relevance except "problems with defecation" in children younger than 1 year and "rejection by other children" in 3- to 4-year-old children. The three versions of the InToDermQoL-EB instrument showed good internal consistency and discriminated well between different severity levels. All InToDermQoL-EB items were confirmed as being of high relevance and the questionnaire may be used in practice and clinical trials.

GARLIC: a bioinformatic toolkit for aetiologically connecting diseases and cell type-specific regulatory maps.

Genome-wide association studies (GWAS) have emerged as a powerful tool to uncover the genetic basis of human common diseases, which often show a complex, polygenic and multi-factorial aetiology. These studies have revealed that 70-90% of all single nucleotide polymorphisms (SNPs) associated with common complex diseases do not occur within genes (i.e. they are non-coding), making the discovery of disease-causative genetic variants and the elucidation of the underlying pathological mechanisms far from straightforward. Based on emerging evidences suggesting that disease-associated SNPs are frequently found within cell type-specific regulatory sequences, here we present GARLIC (GWAS-based Prediction Toolkit for Connecting Diseases and Cell Types), a user-friendly, multi-purpose software with an associated database and online viewer that, using global maps of cis-regulatory elements, can aetiologically connect human diseases with relevant cell types. Additionally, GARLIC can be used to retrieve potential disease-causative genetic variants overlapping regulatory sequences of interest. Overall, GARLIC can satisfy several important needs within the field of medical genetics, thus potentially assisting in the ultimate goal of uncovering the elusive and complex genetic basis of common human disorders.

Imputation of orofacial clefting data identifies novel risk loci and sheds light on the genetic background of cleft lip ± cleft palate and cleft palate only.

Nonsyndromic cleft lip with or without cleft palate (nsCL/P) is among the most common human birth defects with multifactorial etiology. Here, we present results from a genome-wide imputation study of nsCL/P in which, after adding replication cohort data, four novel risk loci for nsCL/P are identified (at chromosomal regions 2p21, 14q22, 15q24 and 19p13). On a systematic level, we show that the association signals within this high-density dataset are enriched in functionally-relevant genomic regions that are active in both human neural crest cells (hNCC) and mouse embryonic craniofacial tissue. This enrichment is also detectable in hNCC regions primed for later activity. Using GCTA analyses, we suggest that 30% of the estimated variance in risk for nsCL/P in the European population can be attributed to common variants, with 25.5% contributed to by the 24 risk loci known to date. For each of these, we identify credible SNPs using a Bayesian refinement approach, with two loci harbouring only one probable causal variant. Finally, we demonstrate that there is no polygenic component of nsCL/P detectable that is shared with nonsyndromic cleft palate only (nsCPO). Our data suggest that, while common variants are strongly contributing to risk for nsCL/P, they do not seem to be involved in nsCPO which might be more often caused by rare deleterious variants. Our study generates novel insights into both nsCL/P and nsCPO etiology and provides a systematic framework for research into craniofacial development and malformation.