Comparison of cell viability assessment and visualization of Aspergillus niger biofilm with two fluorescent probe staining methods.

Filamentous fungi are ubiquitous and frequent components of biofilms. A means to visualize them and quantify their viability is essential for understanding their development and disruption. However, quantifying filamentous fungal biofilms poses challenges because, unlike yeasts and bacteria, they are not composed of discrete cells of similar size. This research focused on filamentous fungal biofilms that are representative of those in the built environment. The objective of this study was to develop a rapid method to examine biofilm structure and quantify live (metabolically active/ membrane undamaged) and dead (inactive/ membrane damaged) cells in Aspergillus niger biofilms utilizing a fluorescent probe staining method and confocal laser scanning microscopy (CLSM). For this, we compared two commercially available probe staining kits that have been developed for bacterial and yeast systems. One method utilized the classic cell stain FUN 1 that exhibits orange-red fluorescent intravacuolar structures in metabolically active cells, while dead cells are fluoresced green. The second method utilized a combination of SYTO9 and propidium iodide (PI), and stains cells based on their membrane morphology. SYTO9 is a green fluorescent stain with the capacity to penetrate the living cell walls, and PI is a red fluorescent stain that can only penetrate dead or dying cells with damaged cell membranes. Following staining, the biofilms were imaged using CLSM and biofilm volumes and thickness were quantified using COMSTAT, a computer program that measures biofilm accumulation from digital image stacks. The results were compared to independent measurements of live-dead cell density, as well as a classic cell viability assay-XTT. The data showed that the combination of SYTO9 and PI is optimal for staining filamentous fungal biofilms.

Comparison of the capillary-channeled polymer (C-CP) fiber spin-down tip approach to traditional methods for the isolation of extracellular vesicles from human urine.

Capillary-channeled polymer fiber (C-CP) solid-phase extraction tips have demonstrated the ability to produce clean and concentrated extracellular vesicle (EV) recoveries from human urine samples in the small EV size range (< 200 nm). An organic modifier-assisted hydrophobic interaction chromatography (HIC) approach is applied in the spin-tip method under non-denaturing conditions-preserving the structure and bioactivity of the recovered vesicles. The C-CP tip method can employ either acetonitrile or glycerol as an elution modifier. The EV recoveries from the C-CP tip method (using both of these solvents) were compared to those obtained using the ultracentrifugation (UC) and polymer precipitation (exoEasy and ExoQuick) EV isolation methods for the same human urine specimen. The biophysical and quantitative characteristics of the recovered EVs using the five isolation methods were assessed based on concentration, size distribution, shape, tetraspanin surface marker protein content, and purity. In comparison to the traditionally used UC method and commercially available polymeric precipitation-based isolation kits, the C-CP tip introduces significant benefits with efficient (< 15 min processing of 12 samples here) and low-cost (< $1 per tip) EV isolations, employing sample volumes (10 µL-1 mL) and concentration (up to 4 × 1012 EVs mL-1) scales relevant for fundamental and clinical analyses. Recoveries of the target vesicles versus matrix proteins were far superior for the tip method versus the other approaches.

Comparison of RNA content from hydrophobic interaction chromatography-isolated seminal plasma exosomes from intrauterine insemination (IUI) pregnancies.

Male factors account for roughly half of infertility cases, with most male infertility diagnosed as idiopathic. Researchers predicting intrauterine insemination success rates have identified multiple prognostic factors, including semen parameters and seminal fluid composition. Seminal plasma contains extracellular exosomes, which contain RNAs and proteins involved in spermatogenesis. The contents of seminal plasma exosomes may be an indicator of overall sperm quality or fertility potential; therefore, analysis of exosomes may provide a measure for sperm viability and fertilization potential. In our study, exosomes were isolated and purified from seminal plasma obtained from IUI treatments with known pregnancy outcomes. We used a unique method to isolate the exosomes which made use of the hydrophobic interaction chromatography method. RNASeq was performed on RNAs from the purified exosomes. This analysis revealed holistic trends, including increased expression associated with RNA originating from chromosomes 1, 10, 12, 16 and 21. Overall, total RNA was significantly decreased and rRNA was significantly increased in successful IUI attempts. Furthermore, we found specific mRNAs and lincRNAs associated with positive versus negative pregnancy outcomes. Our study isolated and purified seminal plasma exosomes without ultracentrifugation, and it provides further evidence for differences in seminal plasma exosome molecular contents associated with pregnancy status.

Rapid isolation of extracellular vesicles from diverse biofluid matrices via capillary-channeled polymer fiber solid-phase extraction micropipette tips.

Extracellular vesicles (EVs) play essential roles in biological systems based on their ability to carry genetic and protein cargos, intercede in cellular communication and serve as vectors in intercellular transport. As such, EVs are species of increasing focus from the points of view of fundamental biochemistry, clinical diagnostics, and therapeutics delivery. Of particular interest are 30-200 nm EVs called exosomes, which have demonstrated high potential for use in diagnostic and targeted delivery applications. The ability to collect exosomes from patient biofluid samples would allow for comprehensive yet remote diagnoses to be performed. While several exosome isolation methods are in common use, they generally produce low recoveries, whose purities are compromised by concomitant inclusion of lipoproteins, host cell proteins, and protein aggregates. Those methods often work on lengthy timescales (multiple hours) and result in very low throughput. In this study, capillary-channeled polymer (C-CP) fiber micropipette tips were employed in a hydrophobic interaction chromatography (HIC) solid-phase extraction (SPE) workflow. Demonstrated is the isolation of exosomes from human urine, saliva, cervical mucus, serum, and goat milk matrices. This method allows for quick (<15 min) and low-cost (<$1 per tip) isolations at sample volume and time scales relevant for clinical applications. The tip isolation was evaluated using absorbance (scattering) detection, nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). Exosome purity was assessed by Bradford assay, based on the removal of free proteins. An enzyme-linked immunosorbent assay (ELISA) to the CD81 tetraspanin protein was used to confirm the presence of the known exosomal-biomarker on the vesicles.

Rapid separation of blood plasma exosomes from low-density lipoproteins via a hydrophobic interaction chromatography method on a polyester capillary-channeled polymer fiber phase.

Exosomes are membrane-bound, cell-secreted vesicles, with sizes ranging from 30 to 150 nm. Exosomes in blood plasma have become proposed targets as measurable indicators of disease conditions. Current methods for plasma-based exosome isolation are time-consuming, complex, and have high operational costs. One of the most commonly reported shortcomings of current isolation protocols is the co-extraction of lipoproteins (e.g. low-density lipoproteins, LDLs) with the target exosomes. This report describes the use of a rapid, single-operation hydrophobic interaction chromatography (HIC) procedure on a polyester (PET) capillary-channeled polymer (C-CP) fiber column, demonstrating the ability to efficiently purify exosomes. The method has previously been demonstrated for isolation of exosomes from diverse biological matrices, but questions were raised about the potential co-elution of LDLs. In the method described herein, a step-gradient procedure sequentially elutes spiked lipoproteins and blood plasma-originating exosomes in 10 min, with the LDLs excluded from the desired exosome fraction. Mass spectrometry (MS) was used to characterize an impurity in the primary LDL material, identifying the presence of exosomal material. Transmission electron microscopy (TEM) and an enzyme-linked immunosorbent assay (ELISA) were used to identify the various elution components. The method serves both as a rapid means of high purity exosome isolation as well as a screening tool for the purity of LDL samples with respect to extracellular vesicles.

MOVAS Cells: A Versatile Cell Line for Studying Vascular Smooth Muscle Cell Cholesterol Metabolism.

Cholesterol metabolism is paramount to cells. Aberrations to cholesterol metabolism affects cholesterol homeostasis, which may impact the risk of several diseases. Recent evidence has suggested that vascular smooth muscle cell (VSMC) cholesterol metabolism may play a role in atherosclerosis. However, there is scant in vitro mechanistic data involving primary VSMC that directly tests how VSMC cholesterol metabolism may impact atherosclerosis. One reason for this lack of data is due to the impracticality of gene manipulation studies in primary VSMC, as cultured primary VSMC become senescent and lose their morphology rapidly. However, there are no immortalized VSMC lines known to be suitable for studying VSMC cholesterol metabolism. The purpose of this study was to determine whether MOVAS cells, a commercially available VSMC line, are suitable to use for studying VSMC cholesterol metabolism. Using immunoblotting and immunofluorescence, we showed that MOVAS cells express ABCA1, ABCG1, and SREBP-2. We also determined that MOVAS cells efflux cholesterol to apoAI and HDL, which indicates functionality of ABCA1/ABCG1. In serum-starved MOVAS cells, SREBP-2 target gene expression was increased, confirming SREBP-2 functionality. We detected miR-33a expression in MOVAS cells and determined this microRNA can silence ABCA1 and ABCG1 via identifying conserved miR-33a binding sites within ABCA1/ABCG1 3'UTR in MOVAS cells. We showed that cholesterol-loading MOVAS cells results in this cell line to transdifferentiate into a macrophage-like cell, which also occurs when VSMC accumulate cholesterol. Our characterization of MOVAS cells sufficiently demonstrates that they are suitable to use for studying VSMC cholesterol metabolism in the context of atherosclerosis.

Rapid isolation of lentivirus particles from cell culture media via a hydrophobic interaction chromatography method on a polyester, capillary-channeled polymer fiber stationary phase.

Lentiviruses are increasingly used as gene delivery vehicles for vaccines and immunotherapies. However, the purification of clinical-grade lentivirus vectors for therapeutic use is still troublesome and limits preclinical and clinical experiments. Current purification methods such as ultracentrifugation and ultrafiltration are time consuming and do not remove all of the impurities such as cellular debris, membrane fragments, and denatured proteins from the lentiviruses. The same challenges exist in terms of their analytical characterization. Presented here is the novel demonstration of the chromatographic isolation of virus particles from culture media based on the hydrophobicity characteristics of the vesicles. A method was developed to isolate lentivirus from media using a hydrophobic interaction chromatography (HIC) method performed on a polyester, capillary-channeled polymer (PET C-CP) stationary phase and a standard liquid chromatography apparatus. The method is an extension of the approach developed in this laboratory for the isolation of extracellular vesicles (EVs). Quantitative polymerase chain reaction (qPCR) was used to verify and quantify lentiviruses in elution fractions. Load and elution mobile phase compositions were optimized to affect high efficiency and throughput. The process has been visualized via scanning electron microscopy (SEM) of the fiber surfaces following media injection, the elution of proteinaceous material, and the elution of lentiviruses. This effort has yielded a rapid (<10 min), low-cost (< $15 per column, providing multiple separations), and efficient method for the isolation/purification of lentivirus particles from cell culture media at the analytical scale.

A novel method of high-purity extracellular vesicle enrichment from microliter-scale human serum for proteomic analysis.

We have developed a rapid, low-cost, and simple separation strategy to separate extracellular vesicles (EVs) from a small amount of serum (i.e.,<100 µL) with minimal contamination by serum proteins and lipoprotein particles to meet the high purity requirement for EV proteome analysis. EVs were separated by a novel polyester capillary channel polymer (PET C-CP) fiber phase/hydrophobic interaction chromatography (HIC) method which is rapid and can process small size samples. The collected EV fractions were subjected to a post-column cleanup protocol using a centrifugal filter to perform buffer exchange and eliminate potential coeluting non-EV proteins while minimizing EV sample loss. Downstream characterization demonstrated that our current strategy can separate EVs with the anticipated exosome-like particle size distribution and high yield (∼1 × 1011 EV particles per mL of serum) in approximately 15 min. Proteome profiling of the EVs reveals that a group of genuine EV components were identified that have significantly less high-abundance blood proteins and lipoprotein particle contamination in comparison to traditional separation methods. The use of this methodology appears to address the major challenges facing EV separation for proteomics analysis. In addition, the EV post-column cleanup protocol proposed in the current work has the potential to be combined with other separation methods, such as ultracentrifugation (UC), to further purify the separated EV samples.

Solid-phase extraction of exosomes from diverse matrices via a polyester capillary-channeled polymer (C-CP) fiber stationary phase in a spin-down tip format.

Exosomes, a subset of the extracellular vesicle (EV) group of organelles, hold great potential for biomarker detection, therapeutics, disease diagnosis, and personalized medicine applications. The promise and potential of these applications are hindered by the lack of an efficient means of isolation, characterization, and quantitation. Current methods for exosome and EV isolation (including ultracentrifugation, microfiltration, and affinity-based techniques) result in impure recoveries with regard to remnant matrix species (e.g., proteins, genetic material) and are performed on clinically irrelevant time and volume scales. To address these issues, a polyethylene terephthalate (PET) capillary-channeled polymer (C-CP) fiber stationary phase is employed for the solid-phase extraction (SPE) of EVs from various matrices using a micropipette tip-based format. The hydrophobic interaction chromatography (HIC) processing and a spin-down workflow are carried out using a table-top centrifuge. Capture and subsequent elution of intact, biologically active exosomes are verified via electron microscopy and bioassays. The performance of this method was evaluated by capture and elution of exosome standards from buffer solution and three biologically relevant matrices: mock urine, reconstituted non-fat milk, and exosome-depleted fetal bovine serum (FBS). Recoveries were evaluated using UV-Vis absorbance spectrophotometry and ELISA assay. The dynamic binding capacity (50%) for the 1-cm-long (~ 5 µL bed volume) tips was determined using a commercial exosome product, yielding a value of ~ 7 × 1011 particles. The novel C-CP fiber spin-down tip approach holds promise for the isolation of exosomes and other EVs from various matrices with high throughput, low cost, and high efficiency. Graphical abstract.

Evaluation of exosome loading characteristics in their purification via a glycerol-assisted hydrophobic interaction chromatography method on a polyester, capillary-channeled polymer fiber phase.

Exosomes are membrane-secreted vesicles, with sizes ranging from 30 to 150 nm, which play key roles in intercellular communication. There is intense interest in developing methods to isolate and quantify exosomes toward clinical diagnostics, fundamental studies of intercellular processes, and use of exosomes as delivery vehicles for therapeutic agents. Current methods for exosomes isolation and quantification are time consuming and have operational high costs; few combine isolation and quantification into a singular operation unit. This report describes the use of hydrophobic interaction chromatography on a polyester capillary-channeled polymer fiber column, employing a step gradient for exosome elution, including use of glycerol as a solvent modifier. The entire procedure is completed in 8 min, while maintaining the structural integrity and biological activity of the isolated exosomes. Electron microscopy was used to verify the size and structural fidelity of single exosomes. Absorbance response curves for a commercial exosome sample were used for exosome quantification in the chromatographic separations. In order to determine the dynamic loading capacity for exosomes, different volumes of Dictyostelium discoideum cell culture milieu supernatant were loaded at different column lengths (5-30 cm) and loading flow rates (0.2-0.5 ml/min). A loading capacity of 5.4 × 1012 exosomes derived from D. discoideum milieu was obtained on a 0.8 × 300 mm column; yielding recoveries of over 80%. It is believed that this isolation and purification strategy holds many advantages toward the use of exosomes across a wide breadth of medical and biotechnology applications.

Isolation and quantitation of exosomes isolated from human plasma via hydrophobic interaction chromatography using a polyester, capillary-channeled polymer fiber phase.

Exosomes are one class of extracellular vesicles (30-150 nm diameter) that are secreted by cells. These small vesicles hold a great deal of promise in disease diagnostics, as they display the same protein biomarkers as their originating cell. On a cellular level, exosomes are attributed to playing a key role in intercellular communication, and may eventually be exploited for targeted drug delivery. In order for exosomes to become useful in disease diagnostics, and as burgeoning drug delivery platforms, they must be isolated efficiently and effectively without compromising their structure. Plasma from peripheral blood is an excellent source of exosomes, as it is easily collected and the process does not normally cause undue discomfort to the patient. Unfortunately, blood plasma content is complex, containing abundant amounts of soluble proteins and aggregates, making exosomes extremely difficult to isolate in high purity from plasma. Most current exosome isolation methods have practical challenges including being too time-consuming and labor intensive, destructive to the exosomes, or too costly for use in clinical settings. To this end, this study examines the use of poly(ethylene terephthalate) (PET) capillary-channeled polymer (C-CP) fibers in a hydrophobic interaction chromatography (HIC) protocol to isolate exosomes from a human plasma sample. Initial results demonstrate the ability to isolate exosomes with comparable yields and size distributions and on a much faster time scale when compared to traditional isolation methods, while also alleviating concomitant proteins and other impurities. As a demonstration of the potential quantitative utility of the approach, a linear response (particles injected on-column vs peak area) using a commercial exosome standard was established using a standard UV absorbance detector. Based on the calibration function, the concentration of the original human plasma sample was determined and subsequently confirmed by NTA measurement. The potential for scalable separations covering sub-milliliter spin-down solid phase extraction tips to the preparative scale is anticipated.

Isolation and quantification of human urinary exosomes by hydrophobic interaction chromatography on a polyester capillary-channeled polymer fiber stationary phase.

Exosomes are vesicles secreted by cells having a size range from 30 to 150 nm and carrying genetic materials that are important for intercellular functions, including cancer progression. Mounting evidence shows that tumor cells secrete more exosomes than normal cells. Thus, it is important to be able to efficiently isolate and quantify exosomes for potential use in clinical diagnostics, as well as to develop a deeper understanding of their role in intercellular processes. Current methods for exosome isolation and quantification are time-consuming and expensive. Few of these methods are able to combine exosome isolation and quantification into a singular operation scheme. However, a new efficient, rapid, and low-cost isolation and quantification method for exosomes in human urine samples using polyester (PET) capillary-channeled polymer (C-CP) fibers in a hydrophobic interaction chromatography (HIC) protocol has been developed. The process has been verified via scanning electron microscopy (SEM) before and after the capture of exosomes on the fiber surfaces. Sample load and elution rates were optimized to affect high resolution and throughput. Isolated exosomes were quantified based on a UV absorbance response curve created using a commercial human urine-derived exosome standard with an exosome concentration of 7.32 × 1011 mL-1. The loading capacity of a 30-cm C-CP PET column was ~ 7 × 1011 exosomes. An inter-injection washing method with PBS was developed to improve the reproducibility with a 2.9% RSD achieved for 7 complete isolation cycles. Graphical abstract.

Exposure to arsenic during embryogenesis impairs olfactory sensory neuron differentiation and function into adulthood.

Arsenic is a contaminant of food and drinking water. Epidemiological studies have reported correlations between arsenic exposure and neurodevelopmental abnormalities, such as reduced sensory functioning, while in vitro studies have shown that arsenic reduces neurogenesis and alters stem cell differentiation. The goal of this study was assess whether arsenic exposure during embryogenesis reduced olfactory stem cell function and/or numbers, and if so, whether those changes persist into adulthood. Killifish (Fundulus heteroclitus) embryos were exposed to 0, 10, 50 or 200 ppb arsenite (AsIII) until hatching, and juvenile fish were raised in clean water. At 0, 2, 4, 8, 16, 28 and 40 weeks of age, odorant response tests were performed to assess specific olfactory sensory neuron (OSN) function. Olfactory epithelia were then collected for immunohistochemical analysis of stem cell (Sox2) and proliferating cell numbers (PCNA), as well as the number and expression of ciliated (calretinin) and microvillus OSNs (Gαi3) at 0, 4, 16 and 28 weeks. Odorant tests indicated that arsenic exposure during embryogenesis increased the start time of killifish responding to pheromones, and this altered start time persisted to 40 weeks post-exposure. Response to the odorant taurocholic acid (TCA) was also reduced through week 28, while responses to amino acids were not consistently altered. Immunohistochemistry was used to determine whether changes in odorant responses were correlated to altered cell numbers in the olfactory epithelium, using markers of proliferating cells, progenitor cells, and specific OSNs. Comparisons between response to pheromones and PCNA + cells indicated that, at week 0, both parameters in exposed fish were significantly reduced from the control group. At week 28, all exposure are still significantly different than control fish, but now with higher PCNA expression coupled with reduced pheromone responses. A similar trend was seen in the comparisons between Sox2-expressing progenitor cells and response to pheromones, although Sox2 expression in the 28 week-old fish only recovers back to the level of control fish rather than being significantly higher. Comparisons between calretinin expression (ciliated OSNs) and response to TCA demonstrated that both parameters were reduced in the 200 ppb arsenic-exposed fish in at weeks 4, 16, and 28. Correlations between TCA response and the number of PCNA + cells revealed that, at 28 weeks of age, all arsenic exposure groups had reductions in response to TCA, but higher PCNA expression, similar to that seen with the pheromones. Few changes in Gαi3 (microvillus OSNs) were seen. Thus, it appears that embryonic-only exposure to arsenic has long-term reductions in proliferation and differentiation of olfactory sensory neurons, leading to persistent effects in their function.

Nucleation and Formation of a Primary Clot in Insect Blood.

Blood clotting at wound sites is critical for preventing blood loss and invasion by microorganisms in multicellular animals, especially small insects vulnerable to dehydration. The mechanistic reaction of the clot is the first step in providing scaffolding for the formation of new epithelial and cuticular tissue. The clot, therefore, requires special materials properties. We have developed and used nano-rheological magnetic rotational spectroscopy with nanorods to quantitatively study nucleation of cell aggregates that occurs within fractions of a second. Using larvae of Manduca sexta, we discovered that clot nucleation is a two-step process whereby cell aggregation is the time-limiting step followed by rigidification of the aggregate. Clot nucleation and transformation of viscous blood into a visco-elastic aggregate happens in a few minutes, which is hundreds of times faster than wound plugging and scab formation. This discovery sets a time scale for insect clotting phenomena, establishing a materials metric for the kinetics of biochemical reaction cascades. Combined with biochemical and biomolecular studies, these discoveries can help design fast-working thickeners for vertebrate blood, including human blood, based on clotting principles of insect blood.

Exosome isolation and purification via hydrophobic interaction chromatography using a polyester, capillary-channeled polymer fiber phase.

Extracellular vesicles, including microvesicles and exosomes, are lipidic membrane-derived vesicles that are secreted by most cell types. Exosomes, one class of these vesicles that are 30-100 nm in diameter, hold a great deal of promise in disease diagnostics, as they display the same protein biomarkers as their originating cell. For exosomes to become useful in disease diagnostics, and as burgeoning drug delivery platforms, they must be isolated efficiently and effectively without compromising their structure. Most current exosome isolation methods have practical problems including being too time-consuming and labor intensive, destructive to the exosomes, or too costly for use in clinical settings. To this end, this study examines the use of poly(ethylene terephthalate) (PET) capillary-channeled polymer (C-CP) fibers in a hydrophobic interaction chromatography (HIC) protocol to isolate exosomes from diverse matrices of practical concern. Initial results demonstrate the ability to isolate extracellular vesicles enriched in exosomes with comparable yields and size distributions on a much faster time scale when compared to traditional isolation methods. As a demonstration of the potential analytical utility of the approach, extracellular vesicle recoveries from cell culture milieu and a mock urine matrix are presented. The potential for scalable separations covering submilliliter spin-down columns to the preparative scale is anticipated.

Three-photon imaging using defect-induced photoluminescence in biocompatible ZnO nanoparticles.

BACKGROUND: Although optical spectroscopy promises improved lateral resolution for cancer imaging, its clinical use is seriously impeded by background fluorescence and photon attenuation even in the so-called two-photon absorption (2PA) imaging modality. An efficient strategy to meet the clinical cancer imaging needs, beyond what two-photon absorption (2PA) offers, is to use longer excitation wavelengths through three-photon absorption (3PA). A variety of fluorescent dyes and nanoparticles (NPs) have been used in 3PA imaging. However, their nonlinear 3PA coefficient is often low necessitating high excitation powers, which cause overheating, photodamage, and photo-induced toxicity. Doped wide band gap semiconductors such as Mn:ZnS NPs have previously been used for 3PA but suffer from poor 3PA coefficients. METHODS: Here, we prepared ZnO NPs with intrinsic defects with high 3PA coefficients using a polyol method. We functionalized them with peptides for selective uptake by glioblastoma U87MG cells and used breast cancer MCF-7 cells as control for 3PA studies. Uptake was measured using inductively coupled plasma-mass spectrometry. Biocompatibility studies were performed using reactive oxygen species and cell viability assays. RESULTS: We demonstrate that ZnO NPs, which have a band gap of 3.37 eV with an order of magnitude higher 3PA coefficients, can facilitate the use of longer excitation wavelengths 950-1,100 nm for bioimaging. We used the presence intrinsic defects (such as O interstitials and Zn vacancies) in ZnO NPs to induce electronic states within the band gap that can support strong visible luminescence 550-620 nm without the need for extrinsic doping. The peptide functionalization of ZnO NPs showed selective uptake by U87MG cells unlike MCF-7 cells without the integrin receptors. Furthermore, all ZnO NPs were found to be biocompatible for 3PA imaging. CONCLUSION: We show that defect-induced luminescence 550-620 nm in ZnO NPs (20 nm) due to 3PA at longer excitation (975 nm) can be used for 3PA imaging of U87MG glioblastoma cells with lower background noise.

Bovine serum albumin coated nanoparticles for in vitro activated fluorescence.

A fluorophore modified nanoparticle was developed that can only fluoresce when a specific environmental parameter interacts with the system. The model system consisted of an azide modified bovine serum albumin (azBSA) that had been covalently attached to an alkyne modified silicon phthalocyanine (alSiPc) derivative through a copper catalyzed azide/alkyne Huisgen cycloaddition (click reaction). The azBSA/alSiPc assembly was then clicked to a ca. 67 nm poly(propargyl acrylate) (PA) nanoparticle (PA/azBSA/alSiPc). The resulting particles did not exhibit any florescence when the alSiPc was excited. Incubating the particles at 37 °C for 30 min with a proteolytic enzyme (trypsin) degraded the linking BSA and resulted in the appearance of florescence that was attributed to a "free" silicon phthalocyanine. The PA/azBSA/alSiPc particles were incubated with human non-small cell lung cancer cells (A549) and the florescence of the initially quenched particles was achieved with cellular uptake.

Evidence for the Intercalation of Lipid Acyl Chains into Polypropylene Fiber Matrices.

Headgroup-functionalized lipids are being developed as ligand tethers for high selectivity separations on polypropylene capillary-channeled polymer fiber stationary phases. Surface modification is affected under ambient conditions from aqueous solution. This basic methodology has promise in many areas where robust modifications are desired on hydrophobic surfaces. In order to understand the mode of adsorption of the lipid tail to the polypropylene surface, lipids labeled with the environmentally sensitive 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) fluorophore were used, with NBD covalently attached to the headgroup (NBD-PE) or the acyl chain (acyl NBD-PE) of the lipid. When modified with the acyl NBD-PE, fluorescence imaging of the fiber at excitation wavelengths increasing from 470 to 510 nm caused a 32 nm shift in emission toward the red edge of the absorption band, indicating that the NBD molecule (and thus the lipid tail) is motionally restricted. Fluorescence imaging on fibers modified with NBD-PE or the free NBD-Cl dye molecule yields no change in the emission response. The results of these imaging studies provide evidence that the acyl chain portions of the lipids intercalate into free volume of the polypropylene fiber structure, yielding a robust means of surface modification and the potential for high ligand densities.

Responses of Hyalella azteca to acute and chronic microplastic exposures.

Limited information is available on the presence of microplastics in freshwater systems, and even less is known about the toxicological implications of the exposure of aquatic organisms to plastic particles. The present study was conducted to evaluate the effects of microplastic ingestion on the freshwater amphipod, Hyalella azteca. Hyalella azteca was exposed to fluorescent polyethylene microplastic particles and polypropylene microplastic fibers in individual 250-mL chambers to determine 10-d mortality. In acute bioassays, polypropylene microplastic fibers were significantly more toxic than polyethylene microplastic particles; 10-d lethal concentration 50% values for polyethylene microplastic particles and polypropylene microplastic fibers were 4.64 × 10(4) microplastics/mL and 71.43 microplastics/mL, respectively. A 42-d chronic bioassay using polyethylene microplastic particles was conducted to quantify effects on reproduction, growth, and egestion. Chronic exposure to polyethylene microplastic particles significantly decreased growth and reproduction at the low and intermediate exposure concentrations. During acute exposures to polyethylene microplastic particles, the egestion times did not significantly differ from the egestion of normal food materials in the control; egestion times for polypropylene microplastic fibers were significantly slower than the egestion of food materials in the control. Amphipods exposed to polypropylene microplastic fibers also had significantly less growth. The greater toxicity of microplastic fibers than microplastic particles corresponded with longer residence times for the fibers in the gut. The difference in residence time might have affected the ability to process food, resulting in an energetic effect reflected in sublethal endpoints.

Dynamic transcriptional response of Escherichia coli to inclusion body formation.

Escherichia coli is used intensively for recombinant protein production, but one key challenge with recombinant E. coli is the tendency of recombinant proteins to misfold and aggregate into insoluble inclusion bodies (IBs). IBs contain high concentrations of inactive recombinant protein that require recovery steps to salvage a functional recombinant protein. Currently, no universally effective method exists to prevent IB formation in recombinant E. coli. In this study, DNA microarrays were used to compare the E. coli gene expression response dynamics to soluble and insoluble recombinant protein production. As expected and previously reported, the classical heat-shock genes had increased expression due to IB formation, including protein folding chaperones and proteases. Gene expression levels for protein synthesis-related and energy-synthesis pathways were also increased. Many transmembrane transporter and corresponding catabolic pathways genes had decreased expression for substrates not present in the culture medium. Additionally, putative genes represented over one-third of the genes identified to have significant expression changes due to IB formation, indicating many important cellular responses to IB formation still need to be characterized. Interestingly, cells grown in 3% ethanol had significantly reduced gene expression responses due to IB formation. Taken together, these results indicate that IB formation is complex, stimulates the heat-shock response, increases protein and energy synthesis needs, and streamlines transport and catabolic processes, while ethanol diminished all of these responses.