Exploratory use of fluorescent SmartProbes for the rapid detection of microbial isolates causing corneal ulcer

PURPOSE:To explore the use of optical SmartProbes for the rapid evaluation of corneal scrapes from patients with suspected microbial keratitis, as a clinical alternative to Gram stain. DESIGN:Experimental study with evaluation of a diagnostic technology. METHODS:Corneal scrapes were collected from 267 patients presenting with microbial keratitis at a referral cornea clinic in South India. Corneal scrapes were flooded with SmartProbes (BAC One or BAC Two) and evaluated by fluorescence microscopy (without the need for sample washing or further processing). The SmartProbe labelled samples were scored as bacteria/fungi/none (BAC One) or gram-negative bacteria/none (BAC Two) and compared to Gram stain results. RESULTS:Compared to Gram stain, BAC One demonstrated sensitivity and specificity of 80.0 and 87.5 percent respectively, positive and negative predictive values (PPV, NPV) of 93.8 and 65.1 percent, and an accuracy of 82.2. BAC Two demonstrated sensitivity and specificity of 93.3 and 84.8 percent respectively, a NPV of 99.2 percent and an accuracy of 85.6 percent. When the corresponding culture results were compared to the Gram stain result, the sensitivity and specificity were 73.4 and 70.7 percent, the PPV and NPVs were 86.5 and 51.0 percent, and an overall accuracy of 72.6. CONCLUSIONS:Fluorescent SmartProbes offer a comparative method to Gram stain for delineating gram-positive or gram-negative bacteria or fungi within corneal scrapes. We demonstrate equivalent or higher sensitivity, specificity, PPV and NPVs, and accuracy than culture to Gram stain. Our approach has scope for point-of-care clinical application to aid in the diagnosis of microbial keratitis

A fluorogenic cyclic peptide for imaging and quantification of drug-induced apoptosis

Programmed cell death or apoptosis is a central biological process that is dysregulated in many diseases, including inflammatory conditions and cancer. The detection and quantification of apoptotic cells in vivo is hampered by the need for fixatives or washing steps for non-fluorogenic reagents, and by the low levels of free calcium in diseased tissues that restrict the use of annexins. In this manuscript, we report the rational design of a highly stable fluorogenic peptide (termed Apo-15) that selectively stains apoptotic cells in vitro and in vivo in a calcium-independent manner and under wash-free conditions. Furthermore, using a combination of chemical and biophysical methods, we identify phosphatidylserine as a molecular target of Apo-15. We demonstrate that Apo-15 can be used for the quantification and imaging of drug-induced apoptosis in preclinical mouse models, thus creating opportunities for assessing the in vivo efficacy of anti-inflammatory and anti-cancer therapeutics

Chemodivergent Manganese-Catalyzed C–H Activation: Modular Synthesis of Fluorogenic Probes

Bioorthogonal diversification of peptides is generally dependent on impractical prefunctionalization methods. Here, the authors develop a manganese(I)-catalyzed C–H fluorescent labeling with BODIPY probes, which enables the development of activatable fluorophores to image cell function

Fluorescent amino acids as versatile building blocks for chemical biology

Fluorophores have transformed the way we study biological systems, enabling non-invasive studies in cells and intact organisms, which increase our understanding of complex processes at the molecular level. Fluorescent amino acids have become an essential chemical tool because they can be used to construct fluorescent macromolecules, such as peptides and proteins, without disrupting their native biomolecular properties. Fluorescent and fluorogenic amino acids with unique photophysical properties have been designed for tracking protein–protein interactions in situ or imaging nanoscopic events in real time with high spatial resolution. In this Review, we discuss advances in the design and synthesis of fluorescent amino acids and how they have contributed to the field of chemical biology in the past 10 years. Important areas of research that we review include novel methodologies to synthesize building blocks with tunable spectral properties, their integration into peptide and protein scaffolds using site-specific genetic encoding and bioorthogonal approaches, and their application to design novel artificial proteins, as well as to investigate biological processes in cells by means of optical imaging. [Figure not available: see fulltext.]

A global research priority agenda to advance public health responses to fatty liver disease

Background & aims An estimated 38% of adults worldwide have non-alcoholic fatty liver disease (NAFLD). From individual impacts to widespread public health and economic consequences, the implications of this disease are profound. This study aimed to develop an aligned, prioritised fatty liver disease research agenda for the global health community. Methods Nine co-chairs drafted initial research priorities, subsequently reviewed by 40 core authors and debated during a three-day in-person meeting. Following a Delphi methodology, over two rounds, a large panel (R1 n = 344, R2 n = 288) reviewed the priorities, via Qualtrics XM, indicating agreement using a four-point Likert-scale and providing written feedback. The core group revised the draft priorities between rounds. In R2, panellists also ranked the priorities within six domains: epidemiology, models of care, treatment and care, education and awareness, patient and community perspectives, and leadership and public health policy. Results The consensus-built fatty liver disease research agenda encompasses 28 priorities. The mean percentage of ‘agree’ responses increased from 78.3 in R1 to 81.1 in R2. Five priorities received unanimous combined agreement (‘agree’ + ‘somewhat agree’); the remaining 23 priorities had >90% combined agreement. While all but one of the priorities exhibited at least a super-majority of agreement (>66.7% ‘agree’), 13 priorities had 90% combined agreement. Conclusions Adopting this multidisciplinary consensus-built research priorities agenda can deliver a step-change in addressing fatty liver disease, mitigating against its individual and societal harms and proactively altering its natural history through prevention, identification, treatment, and care. This agenda should catalyse the global health community’s efforts to advance and accelerate responses to this widespread and fast-growing public health threat. Impact and implications An estimated 38% of adults and 13% of children and adolescents worldwide have fatty liver disease, making it the most prevalent liver disease in history. Despite substantial scientific progress in the past three decades, the burden continues to grow, with an urgent need to advance understanding of how to prevent, manage, and treat the disease. Through a global consensus process, a multidisciplinary group agreed on 28 research priorities covering a broad range of themes, from disease burden, treatment, and health system responses to awareness and policy. The findings have relevance for clinical and non-clinical researchers as well as funders working on fatty liver disease and non-communicable diseases more broadly, setting out a prioritised, ranked research agenda for turning the tide on this fast-growing public health threat

Rare-earth/transition-metal magnetic interactions in pristine and (Ni,Fe)-doped YCo5 and GdCo5

We present an investigation into the intrinsic magnetic properties of the compounds YCo5 and GdCo5, members of the RETM5 class of permanent magnets (RE = rare earth, TM = transition metal). Focusing on Y and Gd provides direct insight into both the TM magnetization and RE-TM interactions without the complication of strong crystal field effects. We synthesize single crystals of YCo5 and GdCo5 using the optical floating zone technique and measure the magnetization from liquid helium temperatures up to 800 K. These measurements are interpreted through calculations based on a Green's function formulation of density-functional theory, treating the thermal disorder of the local magnetic moments within the coherent potential approximation. The rise in the magnetization of GdCo5 with temperature is shown to arise from a faster disordering of the Gd magnetic moments compared to the antiferromagnetically aligned Co sublattice. We use the calculations to analyze the different Curie temperatures of the compounds and also compare the molecular (Weiss) fields at the RE site with previously published neutron scattering experiments. To gain further insight into the RE-TM interactions, we perform substitutional doping on the TM site, studying the compounds RECo4.5Ni0.5, RECo4Ni, and RECo4.5Fe0.5. Both our calculations and experiments on powdered samples find an increased/decreased magnetization with Fe/Ni doping, respectively. The calculations further reveal a pronounced dependence on the location of the dopant atoms of both the Curie temperatures and the Weiss field at the RE site