Modular Fluorescent Cholesterol Naphthalimide Probes And Their Application For Cholesterol Trafficking Studies In Cells.

Development of fluorescent cholesterol analogs to better understand subcellular cholesterol trafficking is of great interest for cell biology and medicine. Our approach utilizes a bifunctional 1,8-naphthalimide scaffold with a push-pull character, modified on one side with a head group and a linker on the other side connecting it to cholesterol via an ester bond. Through structure-function studies, we've explored how different substituents-linkers and head groups-affect the ability of these fluorescent cholesterol naphthalimide analogs (CNDs) to mimic natural cholesterol behavior at both molecular and cellular levels. We categorized the resulting analogs into three groups: neutral, charged, and those featuring a hydroxyl group. Each compound was assessed for its solvatochromic behavior in organic solvents and model membranes. Extensive all-atom molecular dynamics simulations helped us examine how these analogs perform in model membranes compared to cholesterol. Additionally, we investigated the partitioning of these fluorescent probes in phase-separated giant unilamellar vesicles. We evaluated the uptake and distribution of these probes within mouse fibroblast cells and astrocytes, for their subcellular distributions in lysosomes and compared that to BODIPY-cholesterol, a well-regarded fluorescent cholesterol analog. The internalization efficiency of the fluorescent probes varies in different cell types and is affected mainly by the head groups. Our results demonstrate that the modular design significantly simplifies the creation of fluorescent cholesterol probes bearing distinct spectral, biophysical, and cellular targeting features, which makes it a valuable toolkit for the investigation of subcellular distribution and trafficking of cholesterol and its derivatives.

Simple Rescue of Opaque Tissue Previously Cleared by iDISCO.

Recent advancements in tissue-clearing techniques and volumetric imaging have greatly facilitated visualization and quantification of biomolecules, organelles, and cells in intact organs or even entire organisms. Generally, there are two types of clearing methods: hydrophobic and hydrophilic (i.e., clearing with organic or aqueous solvents, respectively). The popular iDISCO approach and its modifications are hydrophobic methods that involve dehydration, delipidation, decolorization (optional), decalcification (optional), and refractive-index (RI) matching steps. Cleared samples are often stored for a relatively long period of time and imaged repeatedly. However, cleared tissues can become opaque over time, which prevents accurate reimaging. We reasoned that the resurgent haziness is likely due to rehydration, residual lipids, and uneven RI deep inside those tissue samples. For rescue, we have developed a simple procedure based on iDISCO. Beginning with a methanol dehydration, samples are delipidated using dichloromethane, followed by RI matching with dibenzyl ether (DBE). This simple method effectively re-clears mouse brains that have turned opaque during months of storage, allowing the user to effectively image immunolabeled samples over longer periods of time. Key features • This simple protocol rescues previously cleared tissue that has turned opaque. • The method does not cause detectable loss of immunofluorescence from previously stained samples. Graphical overview.

Trivalent soluble TNF Receptor, a potent TNF-α antagonist for the treatment collagen-induced arthritis.

Tumor necrosis factor is a major pro-inflammatory cytokine which triggers various physiological consequences by binding to and trimerizing its receptors, and has been the single most sought-after drug target for intervening autoimmune diseases such as rheumatoid arthritis and psoriasis. However, current TNF-α blockers, including soluble receptor-Fc fusion and therapeutic antibodies, are all dimeric in structure, whereas their target TNF-α itself is homotrimeric in nature. Here we describe the development of a trivalent soluble TNF receptor and show that it is a more potent than the dimeric TNF receptor decoys in inhibiting TNF-α signaling both in vitro and in vivo. The process involves gene fusion between a soluble receptor TNFRII with a ligand binding domain and a trimerization tag from the C-propeptide of human collagen (Trimer-Tag), which is capable of self-assembly into a covalently linked trimer. We show that the homotrimeric soluble TNF receptor (TNFRII-Trimer) produced with such method is more potent in ligand binding kinetics and cell based bioassays, as well as more efficacious in attenuating collagen-induced arthritis (CIA) in a mouse model than its dimeric TNFRII-Fc counterpart. Thus, this work demonstrates the proof of concept of Trimer-Tag and provides a new platform for rational designs of next generation biologic drugs.

Consolidating CCDs from multiple data sources: a modular approach.

BACKGROUND: Healthcare providers sometimes receive multiple continuity of care documents (CCDs) for a single patient encompassing the patient's various encounters and medical history recorded in different information systems. It is cumbersome for providers to explore different pages of CCDs to find specific data which can be duplicated or even conflicted. This study describes initial steps toward a modular system that integrates and de-duplicates multiple CCDs into one consolidated document for viewing or processing patient-level data. MATERIALS AND METHODS: The authors developed a prototype system to consolidate and de-duplicate CCDs. The system is engineered to be scalable, extensible, and open source. Using a corpus of 150 de-identified CCDs synthetically generated from a single data source with a common vocabulary to represent 50 unique patients, the authors tested the system's performance and output. Performance was measured based on document throughput and reduction in file size and volume of data. The authors further compared the output of the system with manual consolidation and de-duplication. Testing across multiple vendor systems or implementations was not performed. RESULTS: All of the input CCDs was successfully consolidated, and no data were lost. De-duplication significantly reduced the number of entries in different sections (49% in Problems, 60.6% in Medications, and 79% in Allergies) and reduced the size of the documents (57.5%) as well as the number of lines in each document (58%). The system executed at a rate of approximately 0.009-0.03 s per rule depending on the complexity of the rule. DISCUSSION AND CONCLUSION: Given increasing adoption and use of health information exchange (HIE) to share data and information across the care continuum, duplication of information is inevitable. A novel system designed to support automated consolidation and de-duplication of information across clinical documents as they are exchanged shows promise. Future work is needed to expand the capabilities of the system and further test it using heterogeneous vocabularies across multiple HIE scenarios.

Effect of plasmid replication deregulation via inc mutations on E. coli proteome & simple flux model analysis.

When the replication of a plasmid based on sucrose selection is deregulated via the inc1 and inc2 mutations, high copy numbers (7,000 or greater) are attained while the growth rate on minimal medium is negligibly affected. Adaptions were assumed to be required in order to sustain the growth rate. Proteomics indicated that indeed a number of adaptations occurred that included increased expression of ribosomal proteins and 2-oxoglutarate dehydrogenase. The operating space prescribed by a basic flux model that maintained phenotypic traits (e.g. growth, byproducts, etc.) within typical bounds of resolution was consistent with the flux implications of the proteomic changes.

High-Level Production of Plasmid DNA by Escherichia coli DH5α ΩsacB by Introducing inc Mutations.

For small-copy-number pUC-type plasmids, the inc1 and inc2 mutations, which deregulate replication, were previously found to increase the plasmid copy number 6- to 7-fold. Because plasmids can exert a growth burden, it was not clear if further amplification of copy number would occur due to inc mutations when the starting point for plasmid copy number was orders of magnitude higher. To investigate further the effects of the inc mutations and the possible limits of plasmid synthesis, the parent plasmid pNTC8485 was used as a starting point. It lacks an antibiotic resistance gene and has a copy number of ~1,200 per chromosome. During early stationary-phase growth in LB broth at 37°C, inc2 mutants of pNTC8485 exhibited a copy number of ~7,000 per chromosome. In minimal medium at late log growth, the copy number was found to be significantly increased, to approximately 15,000. In an attempt to further increase the plasmid titer (plasmid mass/culture volume), enzymatic hydrolysis of the selection agent, sucrose, at late log growth extended growth and tripled the total plasmid amount such that an approximately 80-fold gain in total plasmid was obtained compared to the value for typical pUC-type vectors. Finally, when grown in minimal medium, no detectable impact on the exponential growth rate or the fidelity of genomic or plasmid DNA replication was found in cells with deregulated plasmid replication. The use of inc mutations and the sucrose degradation method presents a simplified way for attaining high titers of plasmid DNA for various applications.

Use of flux pre-analysis to enable ¹³C tracer studies in pyruvate kinase-deficient Escherichia coli.

Pyruvate kinase-deficient Escherichia coli (PB25) is a low by-product-producing yet fast-growing mutant that has been shown to have technological potential. Determining the flux limits through finding the extreme point flux sets was previously reported to identify alternate metabolite trafficking scenarios. Previously, the extreme point flux sets were used to design tracer experiments; however, variation in extracellular measurements was not considered, and reaction reversibility was assumed to be low to moderate. In this study, we examined the utility of limiting the fluxes and predetermining the trafficking scenarios in PB25, including confirmation of quasi-linearity between extreme points to ensure sensitivity is maintained. The effects of variation in extracellular measurements and reaction reversibilities were also examined. Tightened flux limits reduced the nonlinearity between label distribution and fluxes. For low to moderate reversibility, contrast was also preserved. However, for highly reversible phosphoglucoisomerase activity, information from common analytes could lead to a flux solution that is biased towards one extreme point. Based on the PB25 model, some suggestions are provided for how predetermining flux limits and trafficking scenarios could enable flux identification in larger network problems.

Linear dynamic range for signal detection in fluorescent differential display.

We have determined the linear dynamic range in signal detection by Fluorescent Differential Display (FDD) using conditionally induced mRNA expression of the p53 tumor-suppressor gene as a control. By serial spiking of p53-induced RNA into that of non-induced RNA, we were able to quantitatively measure up to 100-fold change in p53 mRNA expression level. The linear dynamic range of signal detection per mRNA message was determined to be from 1000 up to 20,000 in fluorescence signal, in which the signals for the majority of mRNAs reside. Thus, FDD can be used to accurately quantify differences in mRNA expression among eukaryotic cells.

Proof-reading signal accuracy of gene expression by binary differential display.

Differential display (DD) is commonly used for identifying differentially expressed genes. However, each cDNA species identified by DD must be verified so a "real difference" can be differentiated from false positives. Although Northern blot analysis is the gold standard it is labor intensive, time-consuming and requires a significant amount of RNA. To speed up and streamline the confirmation process, we developed a new strategy: binary differential display (BDD) based on the binding kinetics of the arbitrary primers in DD. After determining a cDNA sequence of interest from a DD screen, two more 13mer primers derived from the original arbitrary primer used can be designed to target a corresponding cDNA sequence of interest: one with perfect 5'-base matches and the other with additional mismatches at the 5'-base to the corresponding mRNA being confirmed. A separate reverse transcription and FDD are then performed with the same RNA samples being compared. BDD can quickly and accurately determine if a cDNA sequence identified by DD corresponds to a truly differentially expressed gene. In addition, the method is especially useful when more than one cDNA sequence was recovered from a DD band where the masking effect of false-positives can be clearly resolved. Given its simplicity and limited RNA sample required, BDD can be used as a general strategy for rapid confirmation of differentially expressed genes discovered by DD.

Automated fluorescent differential display for cancer gene profiling.

Since its invention in 1992, differential display (DD) has become the most commonly used technique for identifying differentially expressed genes because of its many advantages over competing technologies such as DNA microarray, serial analysis of gene expression (SAGE), and subtractive hybridization. A large number of these publications have been in the field of cancer, specifically on p53 target genes. Despite the great impact of the method on biomedical research, there had been a lack of automation of DD technology to increase its throughput and accuracy for systematic gene expression analysis. Many previous DD work has taken a "shotgun" approach of identifying one gene at a time, with a limited number of polymerase chain reactions (PCRs) set up manually, giving DD a low-tech and low-throughput image. We have optimized the DD process with a platform that incorporates fluorescent digital readout, automated liquid handling, and large-format gels capable of running entire 96-well plates. The resulting streamlined fluorescent DD (FDD) technology offers an unprecedented accuracy, sensitivity, and throughput in comprehensive and quantitative analysis of gene expression. These major improvements will allow researchers to find differentially expressed genes of interest, both known and novel, quickly and easily.

Orexin-induced feeding requires NMDA receptor activation in the perifornical region of the lateral hypothalamus.

Food intake is stimulated following administration of orexin-A into the perifornical region of the lateral hypothalamus (LH/PFA). Orexin neurons originating in the LH/PFA interact with a number of hypothalamic systems known to influence food intake, including glutamatergic neurons. Glutamatergic systems in the LH/PFA were demonstrated to initiate feeding through N-methyl-d-aspartic acid (NMDA) receptors. Male Sprague-Dawley rats fitted with brain guide cannulas to the LH/PFA were used in two experiments. In the first experiment, a combination microdialysis/microinjection probe was used to deliver artificial cerebrospinal fluid (aCSF) or 500 pmol of orexin-A into the LH/PFA. Orexin-A increased interstitial glutamate to 143 +/- 12% of baseline (P < 0.05), which remained elevated over the 120-min collection period. In the second experiment, the NMDA receptor antagonist d-2-amino-5-phosphonopentanoic acid (d-AP5; 10 nmol) was administered before orexin-A. The orexin-induced increase in food intake (from 1.1 +/- 0.4 to 3.2 +/- 0.5 g, P < 0.05) during the first hour was absent in rats receiving d-AP5 + orexin-A (1.2 +/- 0.5 g). There was no effect of d-AP5 alone on food intake. These data support glutamatergic systems in the LH/PFA mediating the feeding response to orexin-A through NMDA receptors.

A protocol for differential display of mRNA expression using either fluorescent or radioactive labeling.

Since its invention in the early 1990s, differential display (DD) has become one of the most commonly used techniques for identifying differentially expressed genes at the mRNA level. Unlike other genomic approaches, such as DNA microarrays, DD systematically detects changes in mRNA profiles among multiple samples being compared without the need of any prior knowledge of genomic information of the living organism being studied. Here, we present an optimized DD protocol with a fluorescent digital readout as well as traditional radioactive labeling. The resulting streamlined fluorescent DD process offers an unprecedented accuracy, sensitivity and throughput in comprehensive and quantitative analysis of eukaryotic gene expression. Results usually can be obtained within days using a limited number of primer combinations, but a comprehensive DD screen may take weeks or months to accomplish, depending on gene coverage required and the number of differentially expressed genes present within a biological system being compared.

Estradiol-induced conditioned place preference may require actions at estrogen receptors in the nucleus accumbens.

Intrinsic rewarding effects of estradiol (E(2)) may underlie some of the sex differences that emerge postpuberty for the prevalence of drug use and behavioral responses to drugs, but the effects and mechanisms of E(2) for reward have not been well characterized. Conditioned place preference (CPP), as measured by the time spent on the nonpreferred/drug-associated side of the chamber, was utilized as a functional assay to investigate the effects and mechanisms of E(2) in the nucleus accumbens for reward. To determine whether intracellular estrogen receptors (ERs) are important for E(2)-induced CPP, rats were administered E(2) (10 microg; subcutaneously (s.c.)), which produced CPP in each experiment, and/or ER blockers, such as tamoxifen (Experiment 1), ICI 182,780 (Experiment 2), or antisense oligonucleotides targeted to ERs (Experiment 3). Experiment 1: E(2) significantly increased the time spent on the originally nonpreferred side of the chamber. Coadministration of tamoxifen (10 mg/kg; s.c.) attenuated effects of E(2) to produce a CPP, but tamoxifen alone, increased time spent on the nonpreferred side. Experiment 2: coadministration of ICI 182,780 (10 microg/microl) to the nucleus accumbens attenuated effects of E(2) to enhance CPP and did not produce a CPP when administered alone. Experiment 3: coadministration of s.c. E(2) with ER antisense oligonucleotides to the nucleus accumbens significantly decreased time spent on the nonpreferred side and expression of ERs in the nucleus accumbens compared to scrambled antisense oligonucleotides or saline vehicle administration. Thus, E(2)'s rewarding effects may involve actions at ERs in the nucleus accumbens.

Automation of fluorescent differential display with digital readout.

Since its invention in 1992, differential display (DD) has become the most commonly used technique for identifying differentially expressed genes because of its many advantages over competing technologies such as DNA microarray, serial analysis of gene expression (SAGE), and subtractive hybridization. Despite the great impact of the method on biomedical research, there has been a lack of automation of DD technology to increase its throughput and accuracy for systematic gene expression analysis. Most of previous DD work has taken a "shot-gun" approach of identifying one gene at a time, with a limited number of polymerase chain reaction (PCR) reactions set up manually, giving DD a low-tech and low-throughput image. We have optimized the DD process with a new platform that incorporates fluorescent digital readout, automated liquid handling, and large-format gels capable of running entire 96-well plates. The resulting streamlined fluorescent DD (FDD) technology offers an unprecedented accuracy, sensitivity, and throughput in comprehensive and quantitative analysis of gene expression. These major improvements will allow researchers to find differentially expressed genes of interest, both known and novel, quickly and easily.