Characterizing melanins in situ and determining their 3D z-epidermal distribution is paramount for understanding physiological/pathological processes of melanin neosynthesis, transfer, degradation or ...modulation with external UV exposure or cosmetic/pharmaceutical products. Multiphoton fluorescence intensity- and lifetime-based approaches have been shown to afford melanin detection, but how can one quantify melanin in vivo in 3D from multiphoton fluorescence lifetime (FLIM) data, especially since FLIM imaging requires long image acquisition times not compatible with 3D imaging in a clinical setup? We propose an approach combining (i) multiphoton FLIM, (ii) fast image acquisition times, and (iii) a melanin detection method called Pseudo-FLIM, based on slope analysis of autofluorescence intensity decays from temporally binned data. We compare Pseudo-FLIM to FLIM bi-exponential and phasor analyses of synthetic melanin, melanocytes/keratinocytes coculture and in vivo human skin. Using parameters of global 3D epidermal melanin density and z-epidermal distribution profile, we provide first insights into the in vivo knowledge of 3D melanin modulations with constitutive pigmentation versus ethnicity, with seasonality over 1 year and with topical application of retinoic acid or retinol on human skin. Applications of Pseudo-FLIM based melanin detection encompass physiological, pathological, or environmental factors-induced pigmentation modulations up to whitening, anti-photoaging, or photoprotection products evaluation.
Two-photon imaging of endogenous fluorescence can provide physiological and metabolic information from intact tissues. However, simultaneous imaging of multiple intrinsic fluorophores, such as ...nicotinamide adenine dinucleotide(phosphate) (NAD(P)H), flavin adenine dinucleotide (FAD) and retinoids in living systems is generally hampered by sequential multi-wavelength excitation resulting in motion artifacts. Here, we report on efficient and simultaneous multicolor two-photon excitation of endogenous fluorophores with absorption spectra spanning the 750-1040 nm range, using wavelength mixing. By using two synchronized pulse trains at 760 and 1041 nm, an additional equivalent two-photon excitation wavelength at 879 nm is generated, and achieves simultaneous excitation of blue, green and red intrinsic fluorophores. This method permits an efficient simultaneous imaging of the metabolic coenzymes NADH and FAD to be implemented with perfect image co-registration, overcoming the difficulties associated with differences in absorption spectra and disparity in concentration. We demonstrate ratiometric redox imaging free of motion artifacts and simultaneous two-photon fluorescence lifetime imaging (FLIM) of NADH and FAD in living tissues. The lifetime gradients of NADH and FAD associated with different cellular metabolic and differentiation states in reconstructed human skin and in the germline of live C. Elegans are thus simultaneously measured. Finally, we present multicolor imaging of endogenous fluorophores and second harmonic generation (SHG) signals during the early stages of Zebrafish embryo development, evidencing fluorescence spectral changes associated with development.
Noninvasive approaches for early detection of bladder cancer are actively being investigated. We recently developed a urine- based molecular assay for the detection and surveillance of bladder ...neoplasms (UroSEEK). UroSEEK is designed to detect alterations in 11 genes that include most common genetic alterations in bladder cancer. In this study, we analyzed 527 cases, including 373 noninvasive and 154 invasive urothelial carcinomas of bladder from transurethral resections or cystectomies performed at four institutions (1991-2016). Two different mutational analysis assays of a representative tumor area were performed: first, a singleplex PCR assay for evaluation of the TERT promoter region (TERTSeqS) and second, a multiplex PCR assay using primers designed to amplify regions of interest of 10 (FGFR3, PIK3CA, TP53, HRAS, KRAS, ERBB2, CDKN2A, MET, MLL, and VHL) genes (UroSeqS). Overall, 92% of all bladder tumors were positive for at least one genetic alteration in the UroSEEK panel. We found TERT promoter mutations in 77% of low-grade noninvasive papillary carcinomas, with a relatively lower incidence of 65% in high-grade noninvasive papillary carcinomas and carcinomas in situ; p = 0.017. Seventy-two percent of pT1 and 63% of muscle-invasive bladder tumors harbored TERT promoter mutations with g.1295228C>T alteration being the most common in all groups. FGFR3 and PIK3CA mutations were more frequent in low-grade noninvasive papillary carcinomas compared with high-grade noninvasive papillary carcinomas and carcinomas in situ (p < 0.0001), while the opposite was true for TP53 (p < 0.0001). Significantly higher rates of TP53 and CDKN2A mutation rates (p = 0.005 and 0.035, respectively) were encountered in muscle-invasive bladder tumors compared with those of pT1 stage. The overwhelming majority of all investigated tumors showed at least one mutation among UroSEEK assay genes, confirming the comprehensive coverage of the panel and supporting its potential utility as a noninvasive urine-based assay.
At the onset of nearly every American civil rights movement there are two pivotal messages: the first is the group's claims of exclusion from the life that the privileged lead and the second is a ...demand to be included. In all cases the first step in creating sustainable change has been the recognition that society was functioning on a multi-tiered system that grants access to some but not to all. Currently there is a civil rights movement taking place that focuses on the integration of students with diverse ability levels. In the United States this movement is called Inclusion. Unfortunately, the primary focus of inclusion is on the integration of students with disabilities; thus children who represent intersectional identities are often passed over and continually left on the margins of inclusive classrooms, schools and society. This paper will focus on a subgroup of students who are currently being left behind in this movement: bilingual special education students. Due to the fact that separate policies exist to address their linguistic and academic needs bilingual students with disabilities fall into what can be considered an intersectional gap. This paper addresses how this gap came to be as well as offering recommendations for mending it.
Spent coffee is the main byproduct of the brewing process and a potential source of bioactive compounds, mainly phenolic acids easily extracted with water. Free and bound caffeoylquinic (3-CQA, ...4-CQA, 5-CQA), dicaffeoylquinic (3,4-diCQA, 3,5-diCQA, 4,5-diCQA), caffeic, ferulic, p-coumaric, sinapic, and 4-hydroxybenzoic acids were measured by HPLC, after the application of three treatments (alkaline, acid, saline) to spent coffee extracts. Around 2-fold higher content of total phenolics has been estimated in comparison to free compounds. Phenolic compounds with one or more caffeic acid molecules were approximately 54% linked to macromolecules such as melanoidins, mainly by noncovalent interactions (up to 81% of bound phenolic compounds). The rest of the quantitated phenolic acids were mainly attached to other structures by covalent bonds (62–97% of total bound compounds). Alkaline hydrolysis and saline treatment were suitable to estimate total bound and ionically bound phenolic acids, respectively, whereas acid hydrolysis is an inadequate method to quantitate coffee phenolic acids.
Racialized students are overrepresented in special- and English-learner education programs in the United States. Researchers have pointed to implicit bias in evaluation tools and evaluators as a ...cause resulting in calls for more culturally competent/relevant practices/assessments. However, this paper argues that racial overrepresentation is reflective of larger settler colonial frameworks embedded in linguistic standards that continue to drive education and language ideologies/practices globally but especially in U.S. schools. First, through an analysis of an orthoepic test used during the Parsley Massacre of 1937 on the island of Hispaniola, I present how the evaluation of accented language has been used to racialize and pathologize people. Secondly, through a comparative analysis of bilingualism in the U.S. and Canada, I show how linguistic variation is only devalued when it emerges from marginalized communities, affirming the white normative gaze as a mechanism for maintaining inequitable power structures. Finally, the paper presents how these logics are present in current manifestations of bilingual education. By indicating how racially, physically, and/or neurodivergent people are othered, this paper calls on the decolonization of applied linguistics in order to effectively address the over- and disproportionate representation of Black, Indigenous, and/or Latinx students within special- and English-learner programs.
Solar ultraviolet longwave UVA1 exposure of human skin has short-term consequences at cellular and molecular level, leading at long-term to photoaging. Following exposure, reactive oxygen species ...(ROS) are generated, inducing oxidative stress that might impair cellular metabolic activity. However, the dynamic of UVA1 impact on cellular metabolism remains unknown because of lacking adequate live imaging techniques. Here we assess the UVA1-induced metabolic stress response in reconstructed human skin with multicolor two-photon fluorescence lifetime microscopy (FLIM). Simultaneous imaging of nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD) by wavelength mixing allows quantifying cellular metabolism in function of NAD(P)
/NAD(P)H and FAD/FADH
redox ratios. After UVA1 exposure, we observe an increase of fraction of bound NAD(P)H and decrease of fraction of bound FAD indicating a metabolic switch from glycolysis to oxidative phosphorylation or oxidative stress possibly correlated to ROS generation. NAD(P)H and FAD biomarkers have unique temporal dynamic and sensitivity to skin cell types and UVA1 dose. While the FAD biomarker is UVA1 dose-dependent in keratinocytes, the NAD(P)H biomarker shows no dose dependence in keratinocytes, but is directly affected after exposure in fibroblasts, thus reflecting different skin cells sensitivities to oxidative stress. Finally, we show that a sunscreen including a UVA1 filter prevents UVA1 metabolic stress response from occurring.
Field programmable gate arrays (FPGAs) have established themselves as one of the preferred digital implementation platforms in a plethora of current industrial applications, and extensions and ...improvements are still continuously being included in the devices. This paper reviews recent advancements in FPGA technology, emphasizing the novel features that may significantly contribute to the development of more efficient digital systems for industrial applications. Special attention is paid to the design paradigm shift caused by the availability of increasingly powerful embedded (and soft) processors, which transformed FPGAs from hardware accelerators to very powerful system-on-chip (SoC) platforms. New analog resources, floating-point operators, and hard memory controllers are also described, because of the great advantages they provide to designers. Software tools are being strongly influenced by the design paradigm shift, which requires from them a much better support for software developers. Focusing mainly on this issue, recent advancements in software resources intellectual property (IP) cores and design tools are also reviewed. The impact of new FPGA features in industrial applications is analyzed in detail in three main areas, namely digital real-time simulation, advanced control techniques, and electronic instrumentation, with focus on mechatronics, robotics, and power systems design. The way digital systems are being currently designed in these areas is comprehensively reviewed, and a critical analysis of how they could significantly benefit from new FPGA features is presented.
Immunotherapy has been one of the great advances in the recent years for the treatment of advanced tumors, with nonsmall-cell lung cancer (NSCLC) being one of the cancers that has benefited most from ...this approach. Currently, the only validated companion diagnostic test for first-line immunotherapy in metastatic NSCLC patients is testing for programmed death ligand 1 (PD-L1) expression in tumor tissues. However, not all patients experience an effective response with the established selection criteria and immune checkpoint inhibitors (ICIs). Liquid biopsy offers a noninvasive opportunity to monitor disease in patients with cancer and identify those who would benefit the most from immunotherapy. This review focuses on the use of liquid biopsy in immunotherapy treatment of NSCLC patients. Circulating tumor cells (CTCs), cell-free DNA (cfDNA) and exosomes are promising tools for developing new biomarkers. We discuss the current application and future implementation of these parameters to improve therapeutic decision-making and identify the patients who will benefit most from immunotherapy.
Diet-induced obesity and food allergies increase in tandem, but a potential cause-and-effect relationship between these diseases of affluence remains to be tested.
We sought to test the role of high ...dietary fat intake, diet-induced obesity, and associated changes in gut microbial community structure on food allergy pathogenesis.
Mice were fed a high-fat diet (HFD) for 12 weeks before food allergen sensitization on an atopic dermatitis–like skin lesion, followed by intragastric allergen challenge to induce experimental food allergy. Germ-free animals were colonized with a signature HFD or lean microbiota for 8 weeks before induction of food allergy. Food-induced allergic responses were quantified by using a clinical allergy score, serum IgE levels, serum mouse mast cell protease 1 concentrations, and type 2 cytokine responses. Accumulation of intestinal mast cells was examined by using flow cytometry and chloroacetate esterase tissue staining. Changes in the gut microbial community structure were assessed by using high-throughput 16S ribosomal DNA gene sequencing.
HFD-induced obesity potentiates food-induced allergic responses associated with dysregulated intestinal effector mast cell responses, increased intestinal permeability, and gut dysbiosis. An HFD-associated microbiome was transmissible to germ-free mice, with the gut microbial community structure of recipients segregating according to the microbiota input source. Independent of an obese state, an HFD-associated gut microbiome was sufficient to confer enhanced susceptibility to food allergy.
These findings identify HFD-induced microbial alterations as risk factors for experimental food allergy and uncouple a pathogenic role of an HFD-associated microbiome from obesity. Postdieting microbiome alterations caused by overindulgence of dietary fat might increase susceptibility to food allergy.
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