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Secreted chemokines are critical mediators of cellular communication that elicit intracellular signaling by binding membrane-bound receptors. Here we demonstrate the development and use of a sensitive real-time approach to quantify secretion and receptor binding of native chemokines in live cells to better understand their molecular interactions and function. [https://www.selleckchem.com/products/ag-1478-tyrphostin-ag-1478.html AG-1478] CRISPR/Cas9 genome editing was used to tag the chemokine CXCL12 with the nanoluciferase fragment HiBiT. CXCL12 secretion was subsequently monitored and quantified by luminescence output. Binding of tagged CXCL12 to either chemokine receptors or membrane glycosaminoglycans could be monitored due to the steric constraints of nanoluciferase complementation. Furthermore, binding of native CXCL12-HiBiT to AlexaFluor488-tagged CXCR4 chemokine receptors could also be distinguished from glycosaminoglycan binding and pharmacologically analyzed using BRET. These live cell approaches combine the sensitivity of nanoluciferase with CRISPR/Cas9 genome editing to detect, quantify, and monitor binding of low levels of native secreted proteins in real time.Hypoxia is one of the critical stressors encountered by various cells of the human body under diverse pathophysiologic conditions including cancer and has profound impacts on several metabolic and physiologic processes. Hypoxia prompts internal ribosome entry site (IRES)-mediated translation of key genes, such as VEGF, that are vital for tumor progression. Here, we describe that hypoxia remarkably upregulates RNA Polymerase I activity. We discovered that in hypoxia, rRNA shows a different methylation pattern compared to normoxia. Heterogeneity in ribosomes due to the diversity of ribosomal RNA and protein composition has been postulated to generate "specialized ribosomes" that differentially regulate translation. We find that in hypoxia, a sub-set of differentially methylated ribosomes recognizes the VEGF-C IRES, suggesting that ribosomal heterogeneity allows for altered ribosomal functions in hypoxia.Circadian rhythms regulate adaptive alterations in mammalian physiology and are maximally entrained by the short wavelength blue spectrum; cataracts block the transmission of light, particularly blue light. Cataract surgery is performed with two types of intraocular lenses (IOL) (1) conventional IOL that transmit the entire visible spectrum and (2) blue-light-filtering (BF) IOL that block the short wavelength blue spectrum. We hypothesized that the transmission properties of IOL are associated with long-term survival. This retrospective cohort study of a 15-hospital healthcare system identified 9,108 participants who underwent bilateral cataract surgery; 3,087 were implanted with conventional IOL and 6,021 received BF-IOL. Multivariable Cox proportional hazards models that included several a priori determined subgroup and sensitivity analyses yielded estimates supporting that conventional IOL compared with BF-IOL may be associated with significantly reduced risk of long-term death. Confirming these differences and identifying any potential causal mechanisms await the conduct of appropriately controlled prospective translational trials.Implant devices have = proven a successful treatment modality in reconstructive surgeries. However, increasing rates of peri-implant diseases demand further examination of their pathogenesis. Polymicrobial biofilm formation on titanium surfaces has been considered the main risk factor for inflammatory processes on tissues surrounding implant devices, which often lead to implant failure. To overcome microbial accumulation on titanium surfaces biofilm targeting strategies have been developed to modify the surface and incorporate antimicrobial coatings. Because antibiotics are widely used to treat polymicrobial infections, these agents have recently started to be incorporated on titanium surface. This review discusses the biofilm formation on titanium dental implants and key factors to be considered in therapeutic and preventative strategies. Moreover, a systematic review was conducted on coatings developed for titanium surfaces using different antibiotics. This review will also shed light on potential alternative strategies aiming to reduce microbial loads and control polymicrobial infection on implanted devices.The genomes of naturally competent Pasteurellaceae and Neisseriaceae have many short uptake sequences (USS), which allow them to distinguish self-DNA from foreign DNA. To fully characterize this preference we developed genome-wide maps of DNA uptake using both a sequence-based computational model and genomic DNA that had been sequenced after uptake by and recovery from competent Haemophilus influenzae cells. When DNA fragments were shorter than the average USS spacing of ∼1,000 bp, sharp peaks of uptake were centered at USS and separated by valleys with 1000-fold lower uptake. Long DNA fragments (1.5-17 kb) gave much less variation, with 90% of positions having uptake within 2-fold of the mean. All detectable uptake biases arose from sequences that fit the USS uptake motif. Simulated competition predicted that, in its respiratory tract environment, H. influenzae will efficiently take up its own DNA even when human DNA is present in 100-fold excess.To end the decade-long, obstinately stagnant number of new leprosy cases, there is an urgent need for field-applicable diagnostic tools that detect infection with Mycobacterium leprae, leprosy's etiologic agent. Since immunity against M. leprae is characterized by humoral and cellular markers, we developed a lateral flow test measuring multiple host proteins based on six previously identified biomarkers for various leprosy phenotypes. This multi-biomarker test (MBT) demonstrated feasibility of quantitative detection of six host serum proteins simultaneously, jointly allowing discrimination of patients with multibacillary and paucibacillary leprosy from control individuals in high and low leprosy endemic areas. Pilot testing of fingerstick blood showed similar MBT performance in point-of-care (POC) settings as observed for plasma and serum. Thus, this newly developed prototype MBT measures six biomarkers covering immunity against M. leprae across the leprosy spectrum. The MBT thereby provides the basis for immunodiagnostic POC tests for leprosy with potential for other (infectious) diseases as well.