An automatic glucose monitoring system based on periplasmic binding proteins for online bioprocess monitoring.

Glucose is one of the most vital nutrients in all living organisms, so its monitoring is critical in healthcare and bioprocessing. Enzymatic sensors are more popular as a technology solution to meet the requirement. However, periplasmic binding proteins have been investigated extensively for their high sensitivity, enabling microdialysis sampling to replace existing complex and expensive glucose monitoring solutions based on enzymatic sensors. The binding proteins are used as optical biosensors by introducing an environment-sensitive fluorophore to the protein. The biosensor's construction, characterization, and potential application are well studied, but a complete glucose monitoring system based on it is yet to be reported. This work documents the development of the first glucose sensor prototype based on glucose binding protein (GBP) for automatic and continuous glucose measurements. The development includes immobilizing the protein into reusable chips and a low-cost solution for non-invasive glucose sampling in bioprocesses using microdialysis sampling technique. A program was written in LabVIEW to accompany the prototype for the complete automation of measurement. The sampling technique allowed glucose measurements of a few micromolar to 260 mM glucose levels. A thorough analysis of the sampling mode and the device's performance was conducted. The reported measurement accuracy was 81.78%, with an RSD of 1.83%. The prototype was also used in online glucose monitoring of E. coli cell culture. The mode of glucose sensing can be expanded to the measurement of other analytes by switching the binding proteins.

Bioburden detection on surface and water samples in a rapid, ultra-sensitive and high-throughput manner.

Bioburden detection is crucial for food, water, and biopharmaceutical applications as it can directly impact public health. The objective of this study is to develop and validate an assay and protocol for detecting bioburden on solid surfaces, as well as in water, with high sensitivity and accuracy in a rapid manner. Henceforth, a resazurin-based assay optimized for detecting bioburden has been integrated with a previously developed portable multichannel fluorometer. The microbes were isolated from solid surfaces in different laboratory settings by swabbing technique, and stream water was collected for contamination analysis. Based on the results, the assay and protocol can successfully detect bioburden as low as 20 CFU/cm2 and 10 CFU/mL present in both surface and water samples, respectively.

A novel, low-cost microfluidic device with an integrated filter for rapid, ultrasensitive, and high-throughput bioburden detection.

Rapid and accurate bioburden detection has become increasingly necessary for food, health, pharmaceutical and environmental applications. To detect bioburden accurately, and in a highly sensitive manner, we have fabricated a novel microfluidic device with an integrated filter to trap the cells. Bioburden is detected on the filter paper in situ using the redox reaction of fluorescent label resorufin and a portable multichannel fluorometer is used for fluorescence measurement. The microfluidic device was fabricated in a facile, low-cost, and rapid way with microwave-induced thermally assisted bonding. To characterize the bonding quality of the microfluidic cassettes, different tests were performed, and the filter paper material and size were optimized. Primary Bacillus subtilis culture bacterial samples were filtered through the device to validate and investigate the performance parameters. Our results show that a limit of detection (LOD) of 0.037 CFU/mL can be achieved through this microfluidic device whereas the LOD in a normal microfluidic cassette in the fluorometer and the golden standard spectrophotometer are 0.378 and 0.128 CFU/mL respectively. The results depict that three to ten times LOD improvement is possible through this microfluidic cassette and more sensitive detection is possible depending on the volume filtered within a rapid 3 min. This novel microfluidic device along with the fluorometer can be used as a rapid portable tool for highly sensitive, accurate and high-throughput bacterial detection for different applications.

The RNA-binding protein and stress granule component ATAXIN-2 is expressed in mouse and human tissues associated with glaucoma pathogenesis.

Polyglutamine repeat expansions in the Ataxin-2 (ATXN2) gene were first implicated in Spinocerebellar Ataxia Type 2, a disease associated with degeneration of motor neurons and Purkinje cells. Recent studies linked single nucleotide polymorphisms in the gene to elevated intraocular pressure in primary open angle glaucoma (POAG); yet, the localization of ATXN2 across glaucoma-relevant tissues of the vertebrate eye has not been thoroughly examined. This study characterizes ATXN2 expression in the mouse and human retina, and anterior eye, using an antibody validated in ATXN2-/- retinas. ATXN2-ir was localized to cytosolic sub compartments in retinal ganglion cell (RGC) somata and proximal dendrites in addition to GABAergic, glycinergic, and cholinergic amacrine cells in the inner plexiform layer (IPL) and displaced amacrine cells. Human, but not mouse retinas showed modest immunolabeling of bipolar cells. ATXN2 immunofluorescence was prominent in the trabecular meshwork and pigmented and nonpigmented cells of the ciliary body, with analyses of primary human trabecular meshwork cells confirming the finding. The expression of ATXN2 in key POAG-relevant ocular tissues supports the potential role in autophagy and stress granule formation in response to ocular hypertension.

Piezo1 channels mediate trabecular meshwork mechanotransduction and promote aqueous fluid outflow.

KEY POINTS: Trabecular meshwork (TM) is a highly mechanosensitive tissue in the eye that regulates intraocular pressure through the control of aqueous humour drainage. Its dysfunction underlies the progression of glaucoma but neither the mechanisms through which TM cells sense pressure nor their role in aqueous humour outflow are understood at the molecular level. We identified the Piezo1 channel as a key TM transducer of tensile stretch, shear flow and pressure. Its activation resulted in intracellular signals that altered organization of the cytoskeleton and cell-extracellular matrix contacts and modulated the trabecular component of aqueous outflow whereas another channel, TRPV4, mediated a delayed mechanoresponse. This study helps elucidate basic mechanotransduction properties that may contribute to intraocular pressure regulation in the vertebrate eye. ABSTRACT: Chronic elevations in intraocular pressure (IOP) can cause blindness by compromising the function of trabecular meshwork (TM) cells in the anterior eye, but how these cells sense and transduce pressure stimuli is poorly understood. Here, we demonstrate functional expression of two mechanically activated channels in human TM cells. Pressure-induced cell stretch evoked a rapid increase in transmembrane current that was inhibited by antagonists of the mechanogated channel Piezo1, Ruthenium Red and GsMTx4, and attenuated in Piezo1-deficient cells. The majority of TM cells exhibited a delayed stretch-activated current that was mediated independently of Piezo1 by TRPV4 (transient receptor potential cation channel, subfamily V, member 4) channels. Piezo1 functions as the principal TM transducer of physiological levels of shear stress, with both shear and the Piezo1 agonist Yoda1 increasing the number of focal cell-matrix contacts. Analysis of TM-dependent fluid drainage from the anterior eye showed significant inhibition by GsMTx4. Collectively, these results suggest that TM mechanosensitivity utilizes kinetically, regulatory and functionally distinct pressure transducers to inform the cells about force-sensing contexts. Piezo1-dependent control of shear flow sensing, calcium homeostasis, cytoskeletal dynamics and pressure-dependent outflow suggests potential for a novel therapeutic target in treating glaucoma.

Abnormalities of vascular histology and collagen fiber configuration in patients with advanced chronic kidney disease.

INTRODUCTION:: Several histologic features have been identified in the upper-extremity arteries and veins of patients with advanced chronic kidney disease, which may affect arteriovenous fistula maturation. However, it is unclear whether these chronic kidney disease vascular features are abnormal. METHODS:: We obtained upper-extremity arterial and venous specimens from 125 advanced chronic kidney disease patients undergoing arteriovenous fistula creation and from 15 control subjects. We quantified medial fibrosis, micro-calcification, and intimal hyperplasia with appropriate histology stains. We characterized medial collagen fiber configuration in second-harmonic-generation microscopy images for the fiber anisotropy index and the dominant fiber direction. RESULTS:: The advanced chronic kidney disease patients were significantly younger than control subjects (53 ± 14 years vs 76 ± 11 years, p < 0.001). After controlling for age, the chronic kidney disease patients had greater arterial medial fibrosis (69% ± 14% vs 51% ± 10%, p < 0.001) and greater arterial micro-calcification (3.03% ± 5.17% vs 0.01% ± 0.03%, p = 0.02), but less arterial intimal thickness (30 ± 25 µm vs 63 ± 25 µm, p < 0.001), as compared to control subjects. The anisotropy index of medial collagen fibers was lower in both arteries (0.24 ± 0.10 vs 0.44 ± 0.04, p < 0.001) and veins (0.28 ± 0.09 vs 0.53 ± 0.10, p < 0.001) in chronic kidney disease patients, indicating that orientation of the fibers was more disordered. The dominant direction of medial collagen fibers in chronic kidney disease patients was greater in the arteries (49.3° ± 23.6° vs 4.0° ± 2.0°, p < 0.001) and the veins (30.0° ± 19.6° vs 3.9° ± 2.1°, p < 0.001), indicating that the fibers in general were aligned more perpendicular to the lumen. CONCLUSION:: Advanced chronic kidney disease is associated with several abnormalities in vascular histology and collagen fiber configuration. Future research is needed to investigate whether these abnormalities affect the maturation outcomes of arteriovenous fistulas.

Fibrotic Venous Remodeling and Nonmaturation of Arteriovenous Fistulas.

The frequency of primary failure in arteriovenous fistulas (AVFs) remains unacceptably high. This lack of improvement is due in part to a poor understanding of the pathobiology underlying AVF nonmaturation. This observational study quantified the progression of three vascular features, medial fibrosis, intimal hyperplasia (IH), and collagen fiber organization, during early AVF remodeling and evaluated the associations thereof with AVF nonmaturation. We obtained venous samples from patients undergoing two-stage upper-arm AVF surgeries at a single center, including intraoperative veins at the first-stage access creation surgery and AVFs at the second-stage transposition procedure. Paired venous samples from both stages were used to evaluate change in these vascular features after anastomosis. Anatomic nonmaturation (AVF diameter never ≥6 mm) occurred in 39 of 161 (24%) patients. Neither preexisting fibrosis nor IH predicted AVF outcomes. Postoperative medial fibrosis associated with nonmaturation (odds ratio [OR], 1.55; 95% confidence interval [95% CI], 1.05 to 2.30; P=0.03, per 10% absolute increase in fibrosis), whereas postoperative IH only associated with failure in those individuals with medial fibrosis over the population's median value (OR, 2.63; 95% CI, 1.07 to 6.46; P=0.04, per increase of 1 in the intima/media ratio). Analysis of postoperative medial collagen organization revealed that circumferential alignment of fibers around the lumen associated with AVF nonmaturation (OR, 1.38; 95% CI, 1.03 to 1.84; P=0.03, per 10° increase in angle). This study demonstrates that excessive fibrotic remodeling of the vein after AVF creation is an important risk factor for nonmaturation and that high medial fibrosis determines the stenotic potential of IH.