Conditional and inducible transgene expression in mice through the combinatorial use of Cre-mediated recombination and tetracycline induction

Here we describe a triple transgenic mouse system, which combines the tissue specificity of any Cre-transgenic line with the inducibility of the reverse tetracycline transactivator (rtTA)/tetracycline-responsive element (tet-O)-driven transgenes. To ensure reliable rtTA expression in a broad range of cell types, we have targeted the rtTA transgene into the ROSA26 locus. The rtTA expression, however, is conditional to a Cre recombinase-mediated excision of a STOP region from the ROSA26 locus. We demonstrate the utility of this technology through the inducible expression of the vascular endothelial growth factor (VEGF-A) during embryonic development and postnatally in adult mice. Our results of adult induction recapitulate several different hepatic and immune cell pathological phenotypes associated with increased systemic VEGF-A protein levels. This system will be useful for studying genes in which temporal control of expression is necessary for the discovery of the full spectrum of functions. The presented approach abrogates the need to generate tissue-specific rtTA transgenes for tissues where well-characterized Cre lines already exist

Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes

Background & Aims: Hepatocyte-like cells (HLCs), differentiated from pluripotent stem cells by the use of soluble factors, can model human liver function and toxicity. However, at present HLC maturity and whether any deficit represents a true fetal state or aberrant differentiation is unclear and compounded by comparison to potentially deteriorated adult hepatocytes. Therefore, we generated HLCs from multiple lineages, using two different protocols, for direct comparison with fresh fetal and adult hepatocytes. Methods: Protocols were developed for robust differentiation. Multiple transcript, protein and functional analyses compared HLCs to fresh human fetal and adult hepatocytes. Results: HLCs were comparable to those of other laboratories by multiple parameters. Transcriptional changes during differentiation mimicked human embryogenesis and showed more similarity to pericentral than periportal hepatocytes. Unbiased proteomics demonstrated greater proximity to liver than 30 other human organs or tissues. However, by comparison to fresh material, HLC maturity was proven by transcript, protein and function to be fetal-like and short of the adult phenotype. The expression of 81% phase 1 enzymes in HLCs was significantly upregulated and half were statistically not different from fetal hepatocytes. HLCs secreted albumin and metabolized testosterone (CYP3A) and dextrorphan (CYP2D6) like fetal hepatocytes. In seven bespoke tests, devised by principal components analysis to distinguish fetal from adult hepatocytes, HLCs from two different source laboratories consistently demonstrated fetal characteristics. Conclusions: HLCs from different sources are broadly comparable with unbiased proteomic evidence for faithful differentiation down the liver lineage. This current phenotype mimics human fetal rather than adult hepatocytes

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Temporal dynamics of microbiota before and after host death

The habitats that animals, humans and plants provide for microbial communities are inevitably transient, changing drastically when these hosts die. Because microbes associated with living hosts are ensured prime access to the deceased host's organic matter, it is feasible that opportunistic, adaptable lifestyles are widespread among host-associated microbes. Here we investigate the temporal dynamics of microbiota by starving to death a host-the planktonic Crustacean Daphnia magna-and tracking the changes in its microbial community as it approaches death, dies and decomposes. Along with obligate host-associated microbes that vanished after the host's death and decomposers that appeared after the host's death, we also detected microbes with opportunistic lifestyles, seemingly capable of exploiting the host even before its death. We suggest that the period around host death plays an important role for host-microbiota ecology and for the evolution of hosts and their microbes

Role of VEGF and VEGF Receptors in the Glomerulus

VEGF is a potent angiogenic and endothelial cell growth factor that is key for the development of the glomerulus, the main filtration unit of the kidney. It is continued to be expressed in the mature glomerulus, with podocytes being the major site of production. VEGF binds to two receptors, VEGFR-1 and VEGFR-2, which are expressed by the adjacent endothelial cells (ECs). VEGFR-2 is the primary mediator of VEGF signaling while VEGFR-1is thought to function as a ‘decoy’ receptor, sequestering VEGF away from VEGFR-2. Gene targeting studies in mice show that VEGF loss from the podocyte results in profound defects of the ECs, consistent with a paracrine signaling loop. However, the identification of VEGF receptors on podocytes in vitro suggests an additional autocrine signaling pathway for VEGF may exist. To further study the role of VEGF in the glomerulus and to address whether a VEGF autocrine loop is functional in vivo, we generated a transgenic mouse model with inducible VEGF upregulation in the podocyte and genetically deleted VEGFR-2 and VEGFR-1 from the podocyte using the Cre-loxP system. Increased VEGF production from the podocyte leads to increased glomerular permeability and ultrastructural changes in the glomerular filtration barrier depending on the time and length of induction. Podocyte-selective deletion of VEGFR-2 did not cause glomerular disease. In contrast, VEGFR-1 loss from the podocyte led to proteinuria and glomerular defects at 6 weeks of age with extensive podocyte foot process effacement. In keeping with the model that VEGFR-1 functions as a VEGF trap, similarities were observed between the glomerular lesions of VEGFR-1 mutant mice and transgenic mice that overexpress VEGF within podocytes. Strikingly, in vitro studies also revealed an increase in podocyte cell adhesion to sVEGFR-1, suggesting additional roles for sVEGFR-1. Together, these data suggest that a tight regulation of VEGF must be maintained in the adult glomerulus. Furthermore, these findings provide the first genetic evidence that VEGF autocrine signaling loop through VEGFR-2 is dispensable in normal glomeruli. In addition, podocytes express sVEGFR-1 and is required in podocytes in vivo to maintain glomerular integrity by regulating VEGF availability and podocyte cell adhesive properties.Ph

Role of VEGF and VEGF Receptors in the Glomerulus

VEGF is a potent angiogenic and endothelial cell growth factor that is key for the development of the glomerulus, the main filtration unit of the kidney. It is continued to be expressed in the mature glomerulus, with podocytes being the major site of production. VEGF binds to two receptors, VEGFR-1 and VEGFR-2, which are expressed by the adjacent endothelial cells (ECs). VEGFR-2 is the primary mediator of VEGF signaling while VEGFR-1is thought to function as a ‘decoy’ receptor, sequestering VEGF away from VEGFR-2. Gene targeting studies in mice show that VEGF loss from the podocyte results in profound defects of the ECs, consistent with a paracrine signaling loop. However, the identification of VEGF receptors on podocytes in vitro suggests an additional autocrine signaling pathway for VEGF may exist. To further study the role of VEGF in the glomerulus and to address whether a VEGF autocrine loop is functional in vivo, we generated a transgenic mouse model with inducible VEGF upregulation in the podocyte and genetically deleted VEGFR-2 and VEGFR-1 from the podocyte using the Cre-loxP system. Increased VEGF production from the podocyte leads to increased glomerular permeability and ultrastructural changes in the glomerular filtration barrier depending on the time and length of induction. Podocyte-selective deletion of VEGFR-2 did not cause glomerular disease. In contrast, VEGFR-1 loss from the podocyte led to proteinuria and glomerular defects at 6 weeks of age with extensive podocyte foot process effacement. In keeping with the model that VEGFR-1 functions as a VEGF trap, similarities were observed between the glomerular lesions of VEGFR-1 mutant mice and transgenic mice that overexpress VEGF within podocytes. Strikingly, in vitro studies also revealed an increase in podocyte cell adhesion to sVEGFR-1, suggesting additional roles for sVEGFR-1. Together, these data suggest that a tight regulation of VEGF must be maintained in the adult glomerulus. Furthermore, these findings provide the first genetic evidence that VEGF autocrine signaling loop through VEGFR-2 is dispensable in normal glomeruli. In addition, podocytes express sVEGFR-1 and is required in podocytes in vivo to maintain glomerular integrity by regulating VEGF availability and podocyte cell adhesive properties.Ph

Ingress/Egress Incidents Involving Wheelchair Users in a Fixed-Route Public Transit Environment

The objectives of this study were to characterize the ingress and egress activities of wheeled mobility device (WhMD) passengers using fixed-route large, accessible transit vehicles (LATVs), and to examine factors associated with incidents occurring on ramps. A retrospective review of public transit video surveillance footage of WhMDseated passengers accessing fixed-route LATVs was conducted. Ingress and egress activities were characterized based on travel conditions and characteristics of the WhMD and LATV ramp. Incidents were identified based on predetermined criteria, and chi-square analysis was performed to identify WhMD and/or ingress/egress characteristics associated with incidents. Video records of 250 WhMD trips were analyzed. A total of 39 incidents occurred during ingress and 12 incidents occurred during egress. Results indicated that the frequency of incidents was significantly greater for scooter users and passengers who ascended the ramp using a rear-facing WhMD orientation. Narrow LATV ramp width was associated with the greatest number of incidents, followed by problems related to LATV door width and steep ramp slope

Ingress/Egress Incidents Involving Wheelchair Users in a Fixed-Route Public Transit Environment

The objectives of this study were to characterize the ingress and egress activities of wheeled mobility device (WhMD) passengers using fixed-route large, accessible transit vehicles (LATVs), and to examine factors associated with incidents occurring on ramps. A retrospective review of public transit video surveillance footage of WhMDseated passengers accessing fixed-route LATVs was conducted. Ingress and egress activities were characterized based on travel conditions and characteristics of the WhMD and LATV ramp. Incidents were identified based on predetermined criteria, and chi-square analysis was performed to identify WhMD and/or ingress/egress characteristics associated with incidents. Video records of 250 WhMD trips were analyzed. A total of 39 incidents occurred during ingress and 12 incidents occurred during egress. Results indicated that the frequency of incidents was significantly greater for scooter users and passengers who ascended the ramp using a rear-facing WhMD orientation. Narrow LATV ramp width was associated with the greatest number of incidents, followed by problems related to LATV door width and steep ramp slope

Automated coin segregator and counter

Banking in the Philippines today is a 3.4 trillion-peso industry. It is vital to our economy that our banks provide effective and efficient service. It is also essential that they provide total satisfaction to their patrons. One of the problems of the service of banks is the management of coins. Up to now, major banks still use manual sorting of coins or require clients to sort their coins before depositing them. This is a time-consuming task that requires personnel who could be doing something else. Automation may provide solution to this problem. Automated coin sorters and counters do exist. However, they do not satisfy the needs of the Philippine market. Existing designs classify coins by diameter and thickness. The current 5-centavo and 1-centavo coins are identical in both aspects. The only difference lies in the 5-centavo coin having a hole in the middle. On the other hand, the 10- and 5-peso coins have almost identical diameters. This leaves a technological gap to the existing coin sorters and counters being used by the Philippine banking system. The machine works by first separating the coins into five groups (1-centavo and 5-centavos, 10-centavos, 25-centavos, 1-peso, and 5-peso and 10-peso) mechanically through a sorter disc. The 10-centavo, 25-centavo, and 1-peso coins are counted by a fibre-optic sensor placed at the sorting hole of these coins. An inductive sensor detects if the 1-or 5-centavo coin has a hole at the center, and solenoids push the coins to their proper sorting destinations. A color sensor senses the color of the outer part of the edge of the 5- and 10-peso coins. Solenoids are also used to sort them. From the results of the experiments, the machine is found to have a maximum percentage error for sorting at 5.24%, and a maximum percentage error for counting at 7.14%. The machine may be inaccurate, it is still found to be within the theoretical acceptable margin of error at 10% for a sample size of 100. The machine was able to sort current circulating Philippine coins into each of the seven denominations and count the coins per denomination at an acceptable accuracy