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An Ugi four-component reaction was used to construct propargylamide starting materials for a subsequent domino Heck–Suzuki–Miyaura cross-coupling reaction to give derivatives of 4-benzylidene-1-oxo-3,4-dihydro-1 H -isoquinoline. [ABSTRACT FROM AUTHOR]

α-Glucosidase inhibitory activity of galbanic acid and its new amide derivatives 3a-n were investigated. Galbanic acid and compounds 3a-n showed excellent anti-α-glucosidase activity with IC 50 values ranging from 0.3 ± 0.3 μM to 416.0 ± 0.2 μM in comparison to positive control acarbose with IC 50 value of = 750.0 ± 5.6. In the kinetic study, the most potent compound 3h demonstrated a competitive mode of inhibition with K i  = 0.57 µM. The interaction of the most potent compound 3h with the α-glucosidase was further elaborated by in vitro Circular dichroism assessment and in silico molecular docking and Molecular dynamics studies. Compound 3h was also non-cytotoxic on human normal cells. In silico study on pharmacokinetics and toxicity profile of the most potent galbanic acid derivatives demonstrated that these compounds are valuable lead compounds for further study in order to achieve new anti-diabetic agents., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

We present a spatial omics approach that combines histology, mass spectrometry imaging and spatial transcriptomics to facilitate precise measurements of mRNA transcripts and low-molecular-weight metabolites across tissue regions. The workflow is compatible with commercially available Visium glass slides. We demonstrate the potential of our method using mouse and human brain samples in the context of dopamine and Parkinson's disease., (© 2023. The Author(s).)

Introduction: Pompe disease is a lysosomal storage disorder. This study aimed to validate and compare 2 fluorimetric methods for measuring α-glucosidase acid activity in dried blood spot sample (DBS), with potential applications in neonatal screening, and disease follow-up of Pompe patients among the Iranian population for the first time., Materials and Methods: The evaluation involved 3 enzyme levels and 7 parameters. The analysis included 141 Healthy individuals, 8 Pompe patients, and 10 obligate heterozygotes using reference and modified methods., Results: Both methods exhibited highly linear calibration curves. The limit of detection (LOD) and limit of quantification (LOQ) were obtained in the micromolar concentration range in 2 methods. Inter-day and intra-day precision, expressed as relative standard deviations (RSD%) were calculated. The normal ranges were determined in healthy individuals. Receiver operating characteristic (ROC) curves were analyzed, and 2 parameters, total neutral α-glucosidase (NAG)/acid α-glucosidase (GAA) and pH ratio, were identified as cut-off values with excellent accuracy, sensitivity, and specificity for evaluating Pompe disease in both methods., Conclusions: Establishing and implementing these 2 methods for the Iranian population effectively differentiated between healthy and patient individuals. Method II, with its shorter incubation time, demonstrated practicality in the clinical setting., Competing Interests: Declaration of competing interest ‘Declaration of Interest Statement: none'. The authors of this article declare that there are no conflicts of interest to disclose. We have no financial, personal, or professional interests that could influence the content or interpretation of this work. This research has been conducted in an unbiased manner, and the findings and conclusions presented are solely based on the data and analysis conducted. Azadeh Tajmir-Riahi and Farzaneh Shemirani are affiliated with the School of Chemistry, College of Science, University of Tehran, Tehran, Iran, and Shohreh Khatami and Roghieh Mirzazadeh are affiliated with the Biochemistry Department, Pasteur Institute of Iran, Tehran, Iran. The research presented in this article was not funded by any specific entity, and the authors have no other associations that could be perceived as potential conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Epidermal homeostasis is governed by a balance between keratinocyte proliferation and differentiation with contributions from cell-cell interactions, but conserved or divergent mechanisms governing this equilibrium across species and how an imbalance contributes to skin disease are largely undefined. To address these questions, human skin single-cell RNA sequencing and spatial transcriptomics data were integrated and compared with mouse skin data. Human skin cell-type annotation was improved using matched spatial transcriptomics data, highlighting the importance of spatial context in cell-type identity, and spatial transcriptomics refined cellular communication inference. In cross-species analyses, we identified a human spinous keratinocyte subpopulation that exhibited proliferative capacity and a heavy metal processing signature, which was absent in mouse and may account for species differences in epidermal thickness. This human subpopulation was expanded in psoriasis and zinc-deficiency dermatitis, attesting to disease relevance and suggesting a paradigm of subpopulation dysfunction as a hallmark of the disease. To assess additional potential subpopulation drivers of skin diseases, we performed cell-of-origin enrichment analysis within genodermatoses, nominating pathogenic cell subpopulations and their communication pathways, which highlighted multiple potential therapeutic targets. This integrated dataset is encompassed in a publicly available web resource to aid mechanistic and translational studies of normal and diseased skin., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The lung contains numerous specialized cell types with distinct roles in tissue function and integrity. To clarify the origins and mechanisms generating cell heterogeneity, we created a comprehensive topographic atlas of early human lung development. Here we report 83 cell states and several spatially resolved developmental trajectories and predict cell interactions within defined tissue niches. We integrated single-cell RNA sequencing and spatially resolved transcriptomics into a web-based, open platform for interactive exploration. We show distinct gene expression programmes, accompanying sequential events of cell differentiation and maturation of the secretory and neuroendocrine cell types in proximal epithelium. We define the origin of airway fibroblasts associated with airway smooth muscle in bronchovascular bundles and describe a trajectory of Schwann cell progenitors to intrinsic parasympathetic neurons controlling bronchoconstriction. Our atlas provides a rich resource for further research and a reference for defining deviations from homeostatic and repair mechanisms leading to pulmonary diseases., (© 2023. The Author(s).)

Spatially resolved transcriptomics has enabled precise genome-wide mRNA expression profiling within tissue sections. The performance of methods targeting the polyA tails of mRNA relies on the availability of specimens with high RNA quality. Moreover, the high cost of currently available spatial resolved transcriptomics assays requires a careful sample screening process to increase the chance of obtaining high-quality data. Indeed, the upfront analysis of RNA quality can show considerable variability due to sample handling, storage, and/or intrinsic factors. We present RNA-Rescue Spatial Transcriptomics (RRST), a workflow designed to improve mRNA recovery from fresh frozen specimens with moderate to low RNA quality. First, we provide a benchmark of RRST against the standard Visium spatial gene expression protocol on high RNA quality samples represented by mouse brain and prostate cancer samples. Then, we test the RRST protocol on tissue sections collected from five challenging tissue types, including human lung, colon, small intestine, pediatric brain tumor, and mouse bone/cartilage. In total, we analyze 52 tissue sections and demonstrate that RRST is a versatile, powerful, and reproducible protocol for fresh frozen specimens of different qualities and origins., (© 2023. The Author(s).)

Defining the transition from benign to malignant tissue is fundamental to improving early diagnosis of cancer 1 . Here we use a systematic approach to study spatial genome integrity in situ and describe previously unidentified clonal relationships. We used spatially resolved transcriptomics 2 to infer spatial copy number variations in >120,000 regions across multiple organs, in benign and malignant tissues. We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumours and in nearby benign tissue using an organ-wide approach focused on the prostate. Our results suggest a model for how genomic instability arises in histologically benign tissue that may represent early events in cancer evolution. We highlight the power of capturing the molecular and spatial continuums in a tissue context and challenge the rationale for treatment paradigms, including focal therapy., (© 2022. The Author(s).)

Medullary thyroid cancer (MTC) is a rare tumor that arises from parafollicular cells within the thyroid gland. The molecular mechanism underlying MTC has not yet been fully understood. Here, we aimed to perform plasma metabolomics profiling of MTC patients to explore the perturbation of metabolic pathways contributing to MTC tumorigenesis. Plasma samples from 20 MTC patients and 20 healthy subjects were obtained to carry out an untargeted metabolomics by gas chromatography-mass spectrometry. Multivariate and univariate analyses were employed as diagnostic tools via MetaboAnalyst and SIMCA software. A total of 76 features were structurally annotated; among them, 13 metabolites were selected to be differentially expressed in MTC patients compared to controls (P < 0.05). These metabolites were mainly associated with the biosynthesis of unsaturated fatty acids and amino acid metabolisms, mostly leucine, glutamine, and glutamate, tightly responsible for tumor cells' energy production. Moreover, according to the receiver operating characteristic curve analysis, metabolites with the area under the curve (AUC) value up to 0.90, including linoleic acid (AUC = 0.935), linolenic acid (AUC = 0.92), and leucine (AUC = 0.948) could discriminate MTC from healthy individuals. This preliminary work contributes to existing knowledge of MTC metabolism by providing evidence of a distinctive metabolic profile in MTC patients relying on the metabolomics approach., (© 2022. The Author(s).)

Current methods for spatial transcriptomics are limited by low spatial resolution. Here we introduce a method that integrates spatial gene expression data with histological image data from the same tissue section to infer higher-resolution expression maps. Using a deep generative model, our method characterizes the transcriptome of micrometer-scale anatomical features and can predict spatial gene expression from histology images alone., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Formalin-fixed paraffin embedding (FFPE) is the most widespread long-term tissue preservation approach. Here, we report a procedure to perform genome-wide spatial analysis of mRNA in FFPE-fixed tissue sections, using well-established, commercially available methods for imaging and spatial barcoding using slides spotted with barcoded oligo(dT) probes to capture the 3' end of mRNA molecules in tissue sections. We applied this method for expression profiling and cell type mapping in coronal sections from the mouse brain to demonstrate the method's capability to delineate anatomical regions from a molecular perspective. We also profiled the spatial composition of transcriptomic signatures in two ovarian carcinosarcoma samples, exemplifying the method's potential to elucidate molecular mechanisms in heterogeneous clinical samples. Finally, we demonstrate the applicability of the assay to characterize human lung and kidney organoids and a human lung biopsy specimen infected with SARS-CoV-2. We anticipate that genome-wide spatial gene expression profiling in FFPE biospecimens will be used for retrospective analysis of biobank samples, which will facilitate longitudinal studies of biological processes and biomarker discovery., Competing Interests: J.L., E.G.V., L.L., R.M., L.K., A.A., and A. Mollbrink are scientific consultants to 10x Genomics Inc, which holds IP rights to the ST technology., (© 2021 The Authors.)

Oncogene-induced senescence (OIS) is an inherent and important tumor suppressor mechanism. However, if not removed timely via immune surveillance, senescent cells also have detrimental effects. Although this has mostly been attributed to the senescence-associated secretory phenotype (SASP) of these cells, we recently proposed that "escape" from the senescent state is another unfavorable outcome. The mechanism underlying this phenomenon remains elusive. Here, we exploit genomic and functional data from a prototypical human epithelial cell model carrying an inducible CDC6 oncogene to identify an early-acquired recurrent chromosomal inversion that harbors a locus encoding the circadian transcription factor BHLHE40. This inversion alone suffices for BHLHE40 activation upon CDC6 induction and driving cell cycle re-entry of senescent cells, and malignant transformation. Ectopic overexpression of BHLHE40 prevented induction of CDC6-triggered senescence. We provide strong evidence in support of replication stress-induced genomic instability being a causative factor underlying "escape" from oncogene-induced senescence., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cutaneous leishmaniasis is caused by protozoa of the genus Leishmania and spread by sandflies. The standard therapy for this ailment is the first-line medication of pentavalent antimonial and the second drug line of pentamidine amphotericin B. All are practiced over the years and exhibit adverse toxicity effects. Herbal product-derived medicine is a promising potential source for treating parasitic diseases. Xanthatin, a xanthanolide sesquiterpene lactone, is isolated from  Xanthium strumarium  L. treats several ailments in many countries. In the present study, we investigated the leishmanicidal activity of the xanthatin by using a metabolomics-based analysis in J774 macrophages and amastigotes phases in  Leishmania major . Xanthatin was isolated and identified by NMR spectroscopy. Macrophage toxicity of xanthatin performed by MTT assay. Macrophages infected by the L. major's promastigote stationary phase, the infection rate (IR), and multiplication index (MI) were calculated. Axenic amastigotes were treated with xanthatin. Cell quenching and metabolite extraction were performed, and the metabolome profile was analyzed with NMR spectroscopy. Outliers were classified by using multivariate statistical analysis software, and relevant metabolites and pathways were worked out. The xanthatin IC 50 rate defined 0.75 µg/mL base on macrophages viability and also  in-vitro  activity of xanthatin on amastigotes showed the best leishmanicidal activity in IR and MI values of 53% and 62.5%, respectively. Xanthatin altered amino sugars and nucleotide sugars metabolism, starch and sucrose metabolism, cyanoamino acid, and galactose metabolism. Our finding revealed that the main target of xanthatin is carbon metabolism, which is an essential step for amastigotes virulence., Competing Interests: The authors have no conflict of interest to declare.

A new series of quinoxalin-1,3,4-oxadiazole (10a-l) derivatives was synthesized and evaluated against some metabolic enzymes including human carbonic anhydrase (hCA) isoenzymes I and II (carbonic anhydrases I and II), cholinesterase (acetylcholinesterase and butyrylcholinesterase), and α-glucosidase. Obtained data revealed that all the synthesized compounds were more potent as compared with the used standard inhibitors against studied target enzymes. Among the synthesized compounds, 4-fluoro derivative (10f) against hCA I, 4-chloro derivative (10i) against hCA II, 3-fluoro derivative (10e) against acetylcholinesterase and butyrylcholinesterase, and 3-bromo derivative (10k) against α-glucosidase were the most potent compounds with inhibitory activity around 1.8- to 7.37-fold better than standard inhibitors. Furthermore, docking studies of these compounds were performed at the active site of their target enzymes., (© 2021 Deutsche Pharmazeutische Gesellschaft.)

Objectives: This study aimed to evaluate the biochemical factors, genetic mutations, outcome of treatment, and clinical follow-up data of Iranian patients with tetrahydrobiopterin (BH4) deficiency from April/2016 to March/2020., Methods: Forty-seven BH4 deficiency patients were included in the study and underwent biochemical and genetic analyses. The clinical outcomes of the patients were evaluated after long-term treatment., Results: Out of the 47 (25 females and 22 males) BH4 deficiency patients enrolled in the study, 23 were Dihydropteridine reductase (DHPR) deficient patients, 23 were 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficient patients, and one was GTP-Cyclohydrolase 1 deficiency (GTPCH-1) patient. No clinical symptoms were observed in 10 of the DHPR deficient patients (before and after the treatment). Also, most patients diagnosed at an early age had a proper response to the treatment. However, drug therapy did not improve clinical symptoms in three of the patients diagnosed at the age of over 10 years. Also, 16 PTPS deficiency patients who were detected within 6 months and received treatment no clinical symptoms were presented. One of the patients was detected with GTPCH deficiency. Despite being treated with BH4, this patient suffered from a seizure, movement disorder, mental retardation, speech difficulty, and hypotonia., Conclusions: The study results showed that neonatal screening should be carried out in all patients with hyperphenylalaninemia because early diagnosis and treatment can reduce symptoms and prevent neurological impairments. Although the BH4 deficiency outcomes are highly variable, early diagnosis and treatment in the first months of life are crucial for good outcomes., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

A series of new quinazolinone-dihydropyrano[3,2-b]pyran derivatives 10A-L were synthesized by simple chemical reactions and were investigated for inhibitory activities against α-glucosidase and α-amylase. New synthesized compounds showed high α-glucosidase inhibition effects in comparison to the standard drug acarbose and were inactive against α-amylase. Among them, the most potent compound was compound 10L (IC 50 value = 40.1 ± 0.6 µM) with inhibitory activity around 18.75-fold more than acarboase (IC 50 value = 750.0 ± 12.5 µM). This compound was a competitive inhibitor into α-glucosidase. Our obtained experimental results were confirmed by docking studies. Furthermore, the cytotoxicity of the most potent compounds 10L, 10G, and 10N against normal fibroblast cells and in silico druglikeness, ADME, and toxicity prediction of these compounds were also evaluated., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A new series of N,N-dimethylbarbituric-pyridinium derivatives 7a-n was synthesized and evaluated as Helicobacter pylori urease inhibitors. All the synthesized compounds (IC 50  = 10.37 ± 1.0-77.52 ± 2.7 μM) were more potent than standard inhibitor hydroxyurea against urease (IC 50  = 100.00 ± 0.2 μM). Furthermore, comparison of IC 50 values of the synthesized compounds with the second standard inhibitor thiourea (IC 50  = 22.0 ± 0.03 µM) revealed that compounds 7a-b and 7f-h were more potent than thiourea. Molecular modeling study of the most potent compounds 7a, 7b, 7f, and 7g was also conducted. Additionally, the drug-likeness properties of the synthesized compounds, based on Lipinski rule and other filters, were evaluated., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019. Published by Elsevier Inc.)

A novel series of phthalimide-dithiocarbamate hybrids was synthesized and evaluated for in vitro inhibitory potentials against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The anti-cholinesterase results indicated that among the synthesized compounds, the compounds 7g and 7h showed the most potent anti-AChE and anti-BuChE activities, respectively. Molecular docking and dynamic studies of the compounds 7g and 7h, respectively, in the active site of AChE and BuChE revealed that these compounds as well interacted with studied cholinesterases. These compounds also possessed drug-like properties and were able to cross the BBB., (© 2019 Wiley-VHCA AG, Zurich, Switzerland.)

Current multiplexing strategies for massively parallel sequencing of genomic DNA mainly rely on library indexing in the final steps of library preparation. This procedure is costly and time-consuming, because a library must be generated separately for each sample. Furthermore, library preparation is challenging in the case of fixed samples, such as DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues. Here we describe CUTseq, a method that uses restriction enzymes and in vitro transcription to barcode and amplify genomic DNA prior to library construction. We thoroughly assess the sensitivity and reproducibility of CUTseq in both cell lines and FFPE samples, and demonstrate an application of CUTseq for multi-region DNA copy number profiling within single FFPE tumor sections, to assess intratumor genetic heterogeneity at high spatial resolution. In conclusion, CUTseq is a versatile and cost-effective method for library preparation for reduced representation genome sequencing, which can find numerous applications in research and diagnostics.

The development of new compliant resonant microsystems and the trend towards further miniaturization have recently raised the issue of the accuracy and reliability of computational tools for the estimation of fluid damping. Focusing on electrostatically actuated torsional micro-mirrors, a major dissipation contribution is linked to the constrained flow of air at comb fingers. In the case of large tilting angles of the mirror plate, within a period of oscillation the geometry of the air domain at comb-drives gets largely distorted, and the dissipation mechanism is thereby affected. In this communication, we provide an appraisal of simple analytical solutions to estimate the dissipation in the ideal case of air flow between infinite plates, at atmospheric pressure. The results of numerical simulations are also reported to assess the effect on damping of the finite size of actual geometries.

DNA fluorescence in situ hybridization (DNA FISH) is a powerful method to study chromosomal organization in single cells. At present, there is a lack of free resources of DNA FISH probes and probe design tools which can be readily applied. Here, we describe iFISH, an open-source repository currently comprising 380 DNA FISH probes targeting multiple loci on the human autosomes and chromosome X, as well as a genome-wide database of optimally designed oligonucleotides and a freely accessible web interface ( http://ifish4u.org ) that can be used to design DNA FISH probes. We individually validate 153 probes and take advantage of our probe repository to quantify the extent of intermingling between multiple heterologous chromosome pairs, showing a much higher extent of intermingling in human embryonic stem cells compared to fibroblasts. In conclusion, iFISH is a versatile and expandable resource, which can greatly facilitate the use of DNA FISH in research and diagnostics.

Functional validation of candidate genes involved in adaptation and speciation remains challenging. Here, we exemplify the utility of a method quantifying individual mRNA transcripts in revealing the molecular basis of divergence in feather pigment synthesis during early-stage speciation in crows. Using a padlock probe assay combined with rolling circle amplification, we quantified cell-type-specific gene expression in the histological context of growing feather follicles. Expression of Tyrosinase Related Protein 1 ( TYRP1 ), Solute Carrier Family 45 member 2 ( SLC45A2 ) and Hematopoietic Prostaglandin D Synthase ( HPGDS ) was melanocyte-limited and significantly reduced in follicles from hooded crow, explaining the substantially lower eumelanin content in grey versus black feathers. The central upstream Melanocyte Inducing Transcription Factor ( MITF ) only showed differential expression specific to melanocytes - a feature not captured by bulk RNA-seq. Overall, this study provides insight into the molecular basis of an evolutionary young transition in pigment synthesis, and demonstrates the power of histologically explicit, statistically substantiated single-cell gene expression quantification for functional genetic inference in natural populations., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Background: Structural variants (SVs) are known to play important roles in a variety of cancers, but their origins and functional consequences are still poorly understood. Many SVs are thought to emerge from errors in the repair processes following DNA double strand breaks (DSBs)., Results: We used experimentally quantified DSB frequencies in cell lines with matched chromatin and sequence features to derive the first quantitative genome-wide models of DSB susceptibility. These models are accurate and provide novel insights into the mutational mechanisms generating DSBs. Models trained in one cell type can be successfully applied to others, but a substantial proportion of DSBs appear to reflect cell type-specific processes. Using model predictions as a proxy for susceptibility to DSBs in tumors, many SV-enriched regions appear to be poorly explained by selectively neutral mutational bias alone. A substantial number of these regions show unexpectedly high SV breakpoint frequencies given their predicted susceptibility to mutation and are therefore credible targets of positive selection in tumors. These putatively positively selected SV hotspots are enriched for genes previously shown to be oncogenic. In contrast, several hundred regions across the genome show unexpectedly low levels of SVs, given their relatively high susceptibility to mutation. These novel coldspot regions appear to be subject to purifying selection in tumors and are enriched for active promoters and enhancers., Conclusions: We conclude that models of DSB susceptibility offer a rigorous approach to the inference of SVs putatively subject to selection in tumors.

Single-molecule RNA fluorescence in situ hybridization (smFISH) represents a promising approach to quantify the expression of clinically useful biomarkers in tumor samples. However, routine application of smFISH to formalin-fixed, paraffin-embedded (FFPE) samples is challenging due to the low signal intensity and high background noise. Here we present RollFISH, a method combining the specificity of smFISH with the signal boosting of rolling circle amplification. We apply RollFISH to quantify widely used breast cancer biomarkers in cell lines and FFPE samples. Thanks to the high signal-to-noise ratio, we can visualize selected biomarkers at low magnification (20 × ) across entire tissue sections, and thus assess their spatial heterogeneity. Lastly, we apply RollFISH to quantify HER2 mRNA in 150 samples on a single tissue microarray, achieving a sensitivity and specificity of detection of HER2-positive samples of ~90%. RollFISH is a robust method for quantifying the expression and intratumor heterogeneity of biomarkers in FFPE tissues., Competing Interests: The authors declare no competing interests.

Microscale uncertainties related to the geometry and morphology of polycrystalline silicon films, constituting the movable structures of micro electro-mechanical systems (MEMS), were investigated through a joint numerical/experimental approach. An on-chip testing device was designed and fabricated to deform a compliant polysilicon beam. In previous studies, we showed that the scattering in the input&ndash;output characteristics of the device can be properly described only if statistical features related to the morphology of the columnar polysilicon film and to the etching process adopted to release the movable structure are taken into account. In this work, a high fidelity finite element model of the device was used to feed a transitional Markov chain Monte Carlo (TMCMC) algorithm for the estimation of the unknown parameters governing the aforementioned statistical features. To reduce the computational cost of the stochastic analysis, a synergy of proper orthogonal decomposition (POD) and kriging interpolation was adopted. Results are reported for a batch of nominally identical tested devices, in terms of measurement error-affected probability distributions of the overall Young&rsquo;s modulus of the polysilicon film and of the overetch depth.

In this work, we provide a numerical/experimental investigation of the micromechanics-induced scattered response of a polysilicon on-chip MEMS testing device, whose moving structure is constituted by a slender cantilever supporting a massive perforated plate. The geometry of the cantilever was specifically designed to emphasize the micromechanical effects, in compliance with the process constraints. To assess the effects of the variability of polysilicon morphology and of geometrical imperfections on the experimentally observed nonlinear sensor response, we adopt statistical Monte Carlo analyses resting on a coupled electromechanical finite element model of the device. For each analysis, the polysilicon morphology was digitally built through a Voronoi tessellation of the moving structure, whose geometry was in turn varied by sampling out of a uniform probability density function the value of the over-etch, considered as the main source of geometrical imperfections. The comparison between the statistics of numerical and experimental results is adopted to assess the relative significance of the uncertainties linked to variations in the micro-fabrication process, and the mechanical film properties due to the polysilicon morphology., Competing Interests: The authors declare no conflict of interest.

DNA double-strand breaks (DSBs) are major DNA lesions that are constantly formed during physiological processes such as DNA replication, transcription, and recombination, or as a result of exogenous agents such as ionizing radiation, radiomimetic drugs, and genome editing nucleases. Unrepaired DSBs threaten genomic stability by leading to the formation of potentially oncogenic rearrangements such as translocations. In past few years, several methods based on next-generation sequencing (NGS) have been developed to study the genome-wide distribution of DSBs or their conversion to translocation events. We developed Breaks Labeling, Enrichment on Streptavidin, and Sequencing (BLESS), which was the first method for direct labeling of DSBs in situ followed by their genome-wide mapping at nucleotide resolution (Crosetto et al., Nat Methods 10:361-365, 2013). Recently, we have further expanded the quantitative nature, applicability, and scalability of BLESS by developing Breaks Labeling In Situ and Sequencing (BLISS) (Yan et al., Nat Commun 8:15058, 2017). Here, we first present an overview of existing methods for genome-wide localization of DSBs, and then focus on the BLESS and BLISS methods, discussing different assay design options depending on the sample type and application.

In this paper, we investigate the stochastic effects of the microstructure of polysilicon films on the overall response of microelectromechanical systems (MEMS). A device for on-chip testing has been purposely designed so as to maximize, in compliance with the production process, its sensitivity to fluctuations of the microstructural properties; as a side effect, its sensitivity to geometrical imperfections linked to the etching process has also been enhanced. A reduced-order, coupled electromechanical model of the device is developed and an identification procedure, based on a genetic algorithm, is finally adopted to tune the parameters ruling microstructural and geometrical uncertainties. Besides an initial geometrical imperfection that can be considered specimen-dependent due to its scattering, the proposed procedure has allowed identifying an average value of the effective polysilicon Young's modulus amounting to 140 GPa, and of the over-etch depth with respect to the target geometry layout amounting to O = - 0.09 μ m. The procedure has been therefore shown to be able to assess how the studied stochastic effects are linked to the scattering of the measured input⁻output transfer function of the device under standard working conditions. With a continuous trend in miniaturization induced by the mass production of MEMS, this study can provide information on how to handle the foreseen growth of such scattering., Competing Interests: The authors declare no conflict of interest.

Precisely measuring the location and frequency of DNA double-strand breaks (DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity or practicality. Here we present Breaks Labeling In Situ and Sequencing (BLISS), featuring the following: (1) direct labelling of DSBs in fixed cells or tissue sections on a solid surface; (2) low-input requirement by linear amplification of tagged DSBs by in vitro transcription; (3) quantification of DSBs through unique molecular identifiers; and (4) easy scalability and multiplexing. We apply BLISS to profile endogenous and exogenous DSBs in low-input samples of cancer cells, embryonic stem cells and liver tissue. We demonstrate the sensitivity of BLISS by assessing the genome-wide off-target activity of two CRISPR-associated RNA-guided endonucleases, Cas9 and Cpf1, observing that Cpf1 has higher specificity than Cas9. Our results establish BLISS as a versatile, sensitive and efficient method for genome-wide DSB mapping in many applications.

When the dimensions of polycrystalline structures become comparable to the average grain size, some reliability issues can be reported for the moving parts of inertial microelectromechanical systems (MEMS). Not only the overall behavior of the device turns out to be affected by a large scattering, but also the sensitivity to imperfections gets enhanced. In this work, through on-chip tests, we experimentally investigate the behavior of thin polysilicon samples using standard electrostatic actuation/sensing. The discrepancy between the target and actual responses of each sample has then been exploited to identify: (i) the overall stiffness of the film and, according to standard continuum elasticity, a morphology-based value of its Young's modulus; (ii) the relevant over-etch induced by the fabrication process. To properly account for the aforementioned stochastic features at the micro-scale, the identification procedure has been based on particle filtering. A simple analytical reduced-order model of the moving structure has been also developed to account for the nonlinearities in the electrical field, up to pull-in. Results are reported for a set of ten film samples of constant slenderness, and the effects of different actuation mechanisms on the identified micromechanical features are thoroughly discussed.

Many abnormal α-chain hemoglobins (Hbs) are caused by single nucleotide mutations in α1- or α2-goblin genes. One of these Hbs is Hb Q-Iran which is resulted from a point mutation at codon 75 of the α1-globin gene (Asp→His). The identification of Hb Q-Iran was observed in two members of a family from the Central Province of Iran. In this study, Globin chain analysis on high performance liquid chromatography (HPLC) and DNA sequencing were applied. An unusual Hb variant, like HbS on alkaline pH electrophoresis was identified from samples of a father and his son from Arak city in the Central Province of Iran. The variant was further characterized by globin chain analysis and DNA sequencing methods. Globin chain analysis revealed an unknown globin chain peak after α-globin chain peak with a different retention time from βs-globin chain, as the control in both samples. Genetic analysis led to the identification of an unknown Hb variant, Hb Q-Iran. Globin chain analysis showed the presence of an unknown globin chain, and likewise DNA sequencing revealed HbQ-Iran. In other words, Globin chain analysis procedure could preliminarily detect an unknown globin chain.

A series of 2-amino-4-(nitroalkyl)-4H-chromene-3-carbonitriles were synthesized by an efficient multicomponent reaction in aqueous media using DBU as a catalyst at room temperature. Mild condition, environment friendly procedure and excellent yields are the main advantages of this procedure. The cytotoxic activity of target compounds were evaluated against three cancer cell lines MDA-MB-231, MCF-7 and T47D in comparison with etoposide as reference drug. Generally, all compounds showed good cell growth inhibitory activity with IC(50) values less than 30 μg/mL. Their activities were comparable or more potent than standard drug etoposide. The 6-bromo- derivatives 7e and 7f showed promising cytotoxic activity with IC50 values in the range of 3.46-18.76 μg/mL, being more potent than etoposide against all tested cell lines.

A library of some new fluorescent chromenopyrimidine derivatives has been synthesized by new approaches. Water-promoted and one-pot reaction can produce new dialkylylamino)-5H-chromeno[2,3-d]pyrimidin-2-yl) phenols. These compounds can also be produced using domino reaction. Two parallel methods are compared. Novel N-alkyl-N-phenyl-5H-chromeno[2,3-d]-pyrimidin-4-amines and 4-alkoxy-5H-chromeno[2, 3-d]pyrimidines are synthesized by Lewis-acid catalyzed reactions. The fluorescence emission intensity of the four compounds from each of libraries after excitation in 290 nm is measured. Compound 2-(4,5-bis(N-methyl-N-phenylamino)-5H-chromeno[2,3-d]pyrimidin-2-yl)phenol was isolated as a byproduct. The details of an interesting exchangeable intramolecular H- bonding of two of the new compounds are reported by X-ray analysis data.

The configuration of the C=N double bond of the title compound, C(7)H(6)BrNO, is E; the non-H atoms are approximately coplanar (r.m.s. deviation = 0.038 Å). In the crystal, pairs of mol-ecules are linked by a pair of O-H⋯N hydrogen bonds about a center of inversion, generating hydrogen-bonded dimers.

Vaccines require a period of at least three months for clinical trials, hence a method that can identify elicitation of immune response a few days after the first dose is a necessity. Evolutionary variable selections are modeling approaches for proper manipulation of available data which were used to set up an animal model for classification of time dependent 1HNMR metabolomic profiles and pattern recognition of fluctuations of metabolites in two groups of male rabbits. One group of rabbits was immunized with human red blood cells and the other used as control. Blood was obtained every 48 h from each rabbit for a period of six weeks and the serum monitored for antibodies and metabolites by 1HNMR spectra. Evaluation of data was carried out using orthogonal signal correction followed by principal component analysis and partial least square. A neural network was also set up to predict immunization profiles. A distinct separation in patterns of significant metabolites was obtained between the two groups, just a few days after the first and the second dose. These metabolites were used as targets of neural networks where each sample was used as test, validation and training and their quantitative influence predicted by regression. This model could be used for prediction of immunization in rabbits a few days after the first dose with 96% accuracy. Similar animals and human vaccine trials would assist greatly in reaching early conclusions in advance of the usual two month immunization schedule; resulting in an appreciable saving of cost and time.

The title compound, C(10)H(8)N(2)OS, features a nonplanar mol-ecule [dihedral angle between the two aromatic rings = 43.6 (1)°]. Two mol-ecules are linked by N-H⋯N hydrogen bonds about a centre of inversion, giving rise to a hydrogen-bonded dimer.

The present study aimed at differentiating rare types of heterozygous beta-thalassemia (thal) with normal Hb A(2) values from alpha-thal in Iranian carriers by globin chain synthesis in addition to other hematological parameters. Our study groups consisted of 51 normal subjects, 24 heterozygous beta- thalassemic subjects with high Hb A(2), 62 alpha-thal-2 subjects, 34 alpha-thal-1 subjects, six Hb H disease thalassemic subjects, 14 silent beta-thal subjects with normal Hb A(2) values, five deltabeta-thal subjects and two subjects with an association of alpha- and deltabeta-thal (total = 198). Analysis of globin chains was performed by high performance liquid chromatography (HPLC). The results showed that the alpha/beta ratio averages were close to the ones in the published literature, but with a greater standard deviation and a wider range. Globin chain synthesis (GCS) could be valuable in differentiating between microcytosis produced by silent beta-thal (heterozygous beta-thal with a normal Hb A(2) level) and that caused by alpha-thal. Since the complex genotype/phenotype relationship can lead to diagnostic difficulties, GCS cannot be used as the only diagnostic tool for thalassemia carrier detection. Therefore, a combination of different tests for each patient is required.

The diagnosis of the different forms of thalassemia is one of the important applications of analysis of globin chains. These analyses are done by high performance liquid chromatography (HPLC) using a MONO-S cation exchange column and ether is used for washing the globin powder in the final step. The presence of peroxide impurities in ether could change the structure of the globin chains. In the chromatograms, these modified globins appear as separated peaks next to the unmodified globin peaks. In these cases, the alpha/beta ratio exceed by artifact the correct value. Our study demonstrates that diagnostic centers should ensure that the ether they use is pure.