Advancement of microfluidic modulation spectroscopy as a highly sensitive protein characterization technique for the detection of small structural changes.

The higher order structure (HOS) of a protein is vital to its function and activity, making it a critical component in the development of protein-based therapeutics. Characterization of HOS can be performed using various biophysical techniques, but many of these methods have limitations such as low throughput, complicated workflow, narrow concentration range, and low sensitivity. Microfluidic Modulation Spectroscopy (MMS) is an emerging technology that addresses these limitations, offering high sensitivity and automated analysis for protein secondary structure. This study evaluates and compares the different well plate formats and scan modes of two MMS instruments. The newer Apollo system features a high throughput 96-well plate format and sweep scan mode that allows a 50% reduction in sample volume consumption and measurement time compared to the previous system. By measuring two proteins with drastically different secondary structures, the results demonstrated that the measurements were highly repeatable (>99% repeatability by area of overlap) regardless of the well plate formats or the scan modes. The limit of quantitation (LOQ) for determining structural impurity using the sweep scan mode was 3.2% and significantly better than that of FTIR at 23% from previous studies. This work highlighted the advancement of MMS as a highly sensitive technique to detect small changes of protein structures due to aggregation or misfolding.

OnabotulinumtoxinA significantly attenuates bladder afferent nerve firing and inhibits ATP release from the urothelium.

OBJECTIVE: To investigate the direct effect of onabotulinumtoxinA (OnaBotA) on bladder afferent nerve activity and release of ATP and acetylcholine (ACh) from the urothelium. MATERIALS AND METHODS: Bladder afferent nerve activity was recorded using an in vitro mouse preparation enabling simultaneous recordings of afferent nerve firing and intravesical pressure during bladder distension. Intraluminal and extraluminal ATP, ACh, and nitric oxide (NO) release were measured using the luciferin-luciferase and Amplex(®) Red assays (Molecular Probes, Carlsbad, CA, USA), and fluorometric assay kit, respectively. OnaBotA (2U), was applied intraluminally, during bladder distension, and its effect was monitored for 2 h after application. Whole-nerve activity was analysed to classify the single afferent units responding to physiological (low-threshold [LT] afferent <15 mmHg) and supra-physiological (high-threshold [HT] afferent >15 mmHg) distension pressures. RESULTS: Bladder distension evoked reproducible pressure-dependent increases in afferent nerve firing. After exposure to OnaBotA, both LT and HT afferent units were significantly attenuated. OnaBotA also significantly inhibited ATP release from the urothelium and increased NO release. CONCLUSION: These data indicate that OnaBotA attenuates the bladder afferent nerves involved in micturition and bladder sensation, suggesting that OnaBotA may exert its clinical effects on urinary urgency and the other symptoms of overactive bladder syndrome through its marked effect on afferent nerves.

The afferent system and its role in lower urinary tract dysfunction.

PURPOSE OF REVIEW: Lower urinary tract disorders such as overactive bladder syndrome (OABS) and interstitial cystitis/painful bladder syndrome (IC/PBS) are debilitating conditions with serious adverse effects on quality of life. Common to both OABS and IC/PBS are the sensory symptoms of urgency and frequency, implicating the afferent system in the aetiology of these disorders. Thus, understanding the role that afferent pathways play in the function of the lower urinary tract is the focus of much current research. This review aims to provide an insight into the recent advances in this field. RECENT FINDINGS: Sensory transduction in the bladder is not only mediated by direct activation of the afferents via a host of receptors and ion channels located on the afferent terminal but also may be attributed to the interplay between the urothelium and the release of urothelially derived mediators. Recent studies provide compelling evidence to support this concept and highlight the complex nature of the bladder afferent system. SUMMARY: Recent studies provide further evidence that afferent control of the bladder may be dependent on integration of excitatory and inhibitory mediators from the urothelium such as ATP and nitric oxide. A number of studies have examined the role cholinergic and adrenergic mechanisms play in bladder afferent function, and several new potential mechanisms involving the cannabinoid receptors and transient receptor potential channels have emerged as areas which warrant further investigation. A better understanding of afferent mechanisms in the bladder will hopefully lead to more effective treatments of lower urinary tract disorders.

The basolateral expression of mUT-A3 in the mouse kidney.

Facilitative UT-A urea transporters play a central role in the urinary concentrating mechanism. There are three major UT-A isoforms found in the mouse kidney: mUT-A1, mUT-A2, and mUT-A3. The major aim of this study was to identify the location and function of mUT-A3. UT-A proteins were investigated using three novel mouse UT-A-targeted antibodies: ML446, MQ2, and ML194. ML446 detected mUT-A1 and mUT-A3. ML194 detected mUT-A1 and mUT-A2. Importantly, MQ2 was found to be selective for mUT-A3. MQ2 detected a 45- to 65-kDa signal in the mouse kidney inner medulla, which was deglycosylated to a 40-kDa protein band. Immunolocalization studies showed that mUT-A3 was strongly detected in the papillary tip, mainly in the basolateral regions of inner medullary collecting duct (IMCD) cells. Immunoblotting of subcellular fractions of inner medullary protein suggested that in mouse kidney mUT-A3 was present in plasma membranes. Consistent with this, immunoelectron microscopy demonstrated that mUT-A3 was predominantly localized at the basal plasma membrane domains of the IMCD cells in mouse kidney. Heterologous expression of mUT-A3-enhanced green fluorescent protein in Madin-Darby canine kidney cells showed that the protein localized to the basolateral membrane. In conclusion, our study indicates that mUT-A3 is a basolateral membrane transporter expressed in IMCD cells.

Postexercise hypotension differs between white and black women.

BACKGROUND: Because data are lacking, we examined the acute effect of exercise on ambulatory blood pressure (BP) in premenopausal white women (n = 18) and black women (n = 15) with normal (n = 21) and high (n = 12) BP. METHODS: Women performed 40 minutes of control and moderate-intensity exercise. BP and hormones were measured before, during, and after the control and exercise periods. By means of RMANCOVA (repeated measures analysis of covarience), we tested whether BP and hormones differed with time and between ethnic, BP, and experimental groups. Multiple regression analysis was used to examine hormonal mediators of the postexercise BP response. RESULTS: Among white women with hypertension, average daytime systolic (S) and diastolic (D) BP decreased 11.0 +/- 3.3 mm Hg (-2.9, -19.1; P =.017) and 8.2 +/- 2.8 mm Hg (-1.2, -13.9; P =.000), from 142.6 +/- 5.8 mm Hg and 96.1 +/- 2.8 mm Hg, respectively, after exercise. Among black women with high BP, mean daytime SBP rose 12.5 +/- 5.2 mm Hg (-2.0, 27.1; P =.000) after exercise, from 121.8 +/- 6.1 mm Hg, whereas DBP was similar before and after exercise (81.4 +/- 4.3 mm Hg and 82.8 +/- 4.7 mm Hg, respectively). In white women without hypertension, daytime SBP and DBP were similar before and after exercise. In black women without hypertension, mean daytime SBP increased 6.3 +/- 2.6 mm Hg (0.4, 12.1; P =.000) after exercise from 103.6 +/- 1.4 mm Hg, and DBP did not change. In black women, hypertension (P = 0.000) and exercise-mediated insulin decreases (P =.005) explained 85.6% of the postexercise SBP response (P =.000). In white women, hypertension (P =.003) and baseline plasma renin (P =.049) accounted for 53.3% of the postexercise SBP response (P =.001). Exercise acutely reduced daytime BP in white women, but not in black women with high BP. CONCLUSION: Endurance exercise may adversely affect the BP of black women.