A high-density immunoblotting methodology for quantification of total protein levels and phosphorylation modifications

The components of many signaling pathways have been identified and there is now a need to conduct quantitative data-rich temporal experiments for systems biology and modeling approaches to better understand pathway dynamics and regulation. Here we present a modified Western blotting method that allows the rapid and reproducible quantification and analysis of hundreds of data points per day on proteins and their phosphorylation state at individual sites. The approach is of particular use where samples show a high degree of sample-to-sample variability such as primary cells from multiple donors. We present a case study on the analysis of >800 phosphorylation data points from three phosphorylation sites in three signaling proteins over multiple time points from platelets isolated from ten donors, demonstrating the technique's potential to determine kinetic and regulatory information from limited cell numbers and to investigate signaling variation within a population. We envisage the approach being of use in the analysis of many cellular processes such as signaling pathway dynamics to identify regulatory feedback loops and the investigation of potential drug/inhibitor responses, using primary cells and tissues, to generate information about how a cell's physiological state changes over time.

Dysregulation of granulosal bone morphogenetic protein receptor 1B density is associated with reduced ovarian reserve and the age-related decline in human fertility

Reproductive ageing is linked to the depletion of ovarian primordial follicles, which causes an irreversible change to ovarian cellular function and the capacity to reproduce. The current study aimed to profile the expression of bone morphogenetic protein receptor, (BMPR1B) in 53 IVF patients exhibiting different degrees of primordial follicle depletion. The granulosa cell receptor density was measured in 403 follicles via flow cytometry. A decline in BMPR1B density occurred at the time of dominant follicle selection and during the terminal stage of folliculogenesis in the 23-30 y good ovarian reserve patients. The 40+ y poor ovarian reserve patients experienced a reversal of this pattern. The results demonstrate an association between age-induced depletion of the ovarian reserve and BMPR1B receptor density at the two critical time points of dominant follicle selection and pre-ovulatory follicle maturation. Dysregulation of BMP receptor signalling may inhibit the normal steroidogenic differentiation required for maturation in older patients.

The urban cool island phenomenon in a high-rise high-density city and its mechanisms

The urban heat island (UHI) phenomenon has been studied extensively, but there are relatively fewer reports on the so-called urban cool island (UCI) phenomenon. We reveal here that the UCI phenomenon exists in Hong Kong during the day, and is associated with the UHI at night under all wind and cloud conditions. The possible mechanisms for the UCI phenomenon in such a high-rise compact city have been discovered using a lumped urban air temperature model. A new concept of urban cool island degree hours (UCIdh) to measure the UCI intensity and duration is proposed. Our analyses reveal that when anthropogenic heat is small or absent, a high-rise and high-density city experiences a significant daytime UCI effect. This is explained by an intensified heat storage capacity and the reduced solar radiation gain of urban surfaces. However, if anthropogenic heat in the urban area increases further, the UCI phenomenon still exists, yet UCIdh decrease dramatically in a high-rise compact city. In a low-rise, low-density city, the UCI phenomenon also occurs when there is no anthropogenic heat, but easily disappears when there is little anthropogenic heat, and the UHI phenomenon dominates. This probably explains why the UHI phenomenon is often observed, but the UCI phenomenon is rarely observed. The co-existence of urban heat/cool island phenomena implies reduction of the daily temperature range (DTR) in such cities, and its dependence on urban morphology also implies that urban morphology can be used to control the urban thermal environment.