Efficient temporally-controlled targeted mutagenesis in smooth muscle cells of the adult mouse

To generate temporally-controlled targeted somatic mutations selectively and efficiently in smooth muscles, we have established a transgenic SMA-Cre-ER(T2) mouse line in which the expression of the Tamoxifen-dependent Cre-ER(T2) recombinase is under the control of a large genomic DNA segment of the mouse smooth muscle alpha actin (SMA) gene, contained in a Bacterial artificial chromosome (Bac). In this transgenic mouse line, Cre-ER(T2)-mediated recombination of LoxP-flanked target DNA is strictly Tamoxifen-dependent, and efficient in both vascular and visceral smooth muscle cells. Moreover, with the exception of few cardiomyocytes, LoxP-flanked DNA excision is restricted to smooth muscle cells. Thus, SMA-Cre-ER(T2) mice should be of great value to analyze gene function in smooth muscles, and to establish new animal models of human smooth muscle disorders.     

Working on a chain: E3s ganging up for ubiquitylation

Substrate specificity in ubiquitylation is conferred by ubiquitin ligases (E3s). Now, several ways that E3s can interact to mediate ubiquitylation are illustrated for Ubr1 (a RING finger E3) and Ufd4 (a HECT domain E3), in Saccharomyces cerevisiae. These interactions and the related concept of E4 activity are discussed.     

<Emphasis Type="Italic">ScreenMill</Emphasis>: A freely available software suite for growth measurement,analysis and visualization of high-throughput screen data

Background  

Many high-throughput genomic experiments, such as Synthetic Genetic Array and yeast two-hybrid, use colony growth on solid media as a screen metric. These experiments routinely generate over 100,000 data points, making data analysis a time consuming and painstaking process. Here we describe ScreenMill, a new software suite that automates image analysis and simplifies data review and analysis for high-throughput biological experiments.     

SWI/SNF complexes containing Brahma or Brahma-related gene 1 play distinct roles in smooth muscle development

SWI/SNF ATP-dependent chromatin-remodeling complexes containing either Brahma-related gene 1 (Brg1) or Brahma (Brm) play important roles in mammalian development. In this study we examined the roles of Brg1 and Brm in smooth muscle development, in vivo, through generation and analysis of mice harboring a smooth muscle-specific knockout of Brg1 on wild-type and Brm null backgrounds. Knockout of Brg1 from smooth muscle in Brg1(flox/flox) mice expressing Cre recombinase under the control of the smooth muscle myosin heavy-chain promoter resulted in cardiopulmonary defects, including patent ductus arteriosus, in 30 to 40% of the mice. Surviving knockout mice exhibited decreased expression of smooth muscle-specific contractile proteins in the gastrointestinal tract, impaired contractility, shortened intestines, disorganized smooth muscle cells, and an increase in apoptosis of intestinal smooth muscle cells. Although Brm knockout mice had normal intestinal structure and function, knockout of Brg1 on a Brm null background exacerbated the effects of knockout of Brg1 alone, resulting in an increase in neonatal lethality. These data show that Brg1 and Brm play critical roles in regulating development of smooth muscle and that Brg1 has specific functions within vascular and gastrointestinal smooth muscle that cannot be performed by Brm.     

RING-type E3 ligases: Master manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination

RING finger domain and RING finger-like ubiquitin ligases (E3s), such as U-box proteins, constitute the vast majority of known E3s. RING-type E3s function together with ubiquitin-conjugating enzymes (E2s) to mediate ubiquitination and are implicated in numerous cellular processes. In part because of their importance in human physiology and disease, these proteins and their cellular functions represent an intense area of study. Here we review recent advances in RING-type E3 recognition of substrates, their cellular regulation, and their varied architecture. Additionally, recent structural insights into RING-type E3 function, with a focus on important interactions with E2s and ubiquitin, are reviewed. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

The Relation of Hepcidin to Iron Disorders,Inflammation and Hemoglobin in Chronic Kidney Disease

The metabolism of hepcidin is profoundly modified in chronic kidney disease (CKD). We investigated its relation to iron disorders, inflammation and hemoglobin (Hb) level in 199 non-dialyzed, non-transplanted patients with CKD stages 1–5. All had their glomerular filtration rate measured by ⁵¹Cr-EDTA renal clearance (mGFR), as well as measurements of iron markers including hepcidin and of erythropoietin (EPO). Hepcidin varied from 0.2 to 193 ng/mL. The median increased from 23.3 ng/mL [8.8–28.7] to 36.1 ng/mL [14.1–92.3] when mGFR decreased from ≥60 to <15 mL/min/1.73 m² (p = 0.02). Patients with absolute iron deficiency (transferrin saturation (TSAT) <20% and ferritin <40 ng/mL) had the lowest hepcidin levels (5.0 ng/mL [0.7–11.7]), and those with a normal iron profile (TSAT ≥20% and ferritin ≥40), the highest (34.5 ng/mL [23.7–51.6]). In multivariate analysis, absolute iron deficiency was associated with lower hepcidin values, and inflammation combined with a normal or functional iron profile with higher values, independent of other determinants of hepcidin concentration, including EPO, mGFR, and albuminemia. The hepcidin level, although it rose overall when mGFR declined, collapsed in patients with absolute iron deficiency. There was a significant interaction with iron status in the association between Hb and hepcidin. Except in absolute iron deficiency, hepcidin’s negative association with Hb level indicates that it is not down-regulated in CKD anemia.     

Decrease in Urinary Creatinine Excretion in Early Stage Chronic Kidney Disease

Background

Little is known about muscle mass loss in early stage chronic kidney disease (CKD). We used 24-hour urinary creatinine excretion rate to assess determinants of muscle mass and its evolution with kidney function decline. We also described the range of urinary creatinine concentration in this population.

Methods

We included 1072 men and 537 women with non-dialysis CKD stages 1 to 5, all of them with repeated measurements of glomerular filtration rate (mGFR) by ⁵¹Cr-EDTA renal clearance and several nutritional markers. In those with stage 1 to 4 at baseline, we used a mixed model to study factors associated with urinary creatinine excretion rate and its change over time.

Results

Baseline mean urinary creatinine excretion decreased from 15.3±3.1 to 12.1±3.3 mmol/24 h (0.20±0.03 to 0.15±0.04 mmol/kg/24 h) in men, with mGFR falling from ≥60 to <15 mL/min/1.73 m², and from 9.6±1.9 to 7.6±2.5 (0.16±0.03 to 0.12±0.03) in women. In addition to mGFR, an older age, diabetes, and lower levels of body mass index, proteinuria, and protein intake assessed by urinary urea were associated with lower mean urinary creatinine excretion at baseline. Mean annual decline in mGFR was 1.53±0.12 mL/min/1.73 m² per year and that of urinary creatinine excretion rate, 0.28±0.02 mmol/24 h per year. Patients with fast annual decline in mGFR of 5 mL/min/1.73 m² had a decrease in urinary creatinine excretion more than twice as big as in those with stable mGFR, independent of changes in urinary urea as well as of other determinants of low muscle mass.

Conclusions

Decrease in 24-hour urinary creatinine excretion rate may appear early in CKD patients, and is greater the more mGFR declines independent of lowering protein intake assessed by 24-hour urinary urea. Normalizing urine analytes for creatininuria may overestimate their concentration in patients with reduced kidney function and low muscle mass.     

Should We Use Standard Survival Models or the Illness-Death Model for Interval-Censored Data to Investigate Risk Factors of Chronic Kidney Disease Progression?

Background

In studies investigating risk factors of chronic kidney disease (CKD) progression, one may be interested in estimating factors effects on both a fall of glomerular filtration rate (GFR) below a specific level (i.e., a CKD stage) and death. Such studies have to account for the fact that GFR is measured at intermittent visit only, which implies that progression to the stage of interest is unknown for patients who die before being observed at that stage. Our objective was to compare the results of an illness-death model that handles this uncertainty, with frequently used survival models.

Methods

This study included 1,519 patients from the NephroTest cohort with CKD stages 1–4 at baseline (69% males, 59±15 years, median protein/creatinine ratio [PCR] 27.4 mg/mmol) and subsequent annual measures of GFR (follow-up time 4.3±2.7 years). Each model was used to estimate the effects of sex, age, PCR, and GFR at baseline on the hazards of progression to CKD stage 5 (GFR<15 mL/min/1.73 m², n = 282 observed) and death (n = 168).

Results

For progression to stage 5, there were only minor differences between results from the different models. The differences between results were higher for the hazard of death before or after progression. Our results also suggest that previous findings on the effect of age on end-stage renal disease are more likely due to a strong impact of age on death than to an effect on progression. The probabilities of progression were systematically under-estimated with the survival model as compared with the illness-death model.

Conclusions

This study illustrates the advantages of the illness-death model for accurately estimating the effects of risk factors on the hazard of progression and death, and probabilities of progression. It avoids the need to choose arbitrary time-to-event and time-to-censoring, while accounting for both interval censoring and competition by death, using a single analytical model.     

A Framework to Optimize Biodiversity Restoration Efforts Based on Habitat Amount and Landscape Connectivity

The effectiveness of ecological restoration actions toward biodiversity conservation depends on both local and landscape constraints. Extensive information on local constraints is already available, but few studies consider the landscape context when planning restoration actions. We propose a multiscale framework based on the landscape attributes of habitat amount and connectivity to infer landscape resilience and to set priority areas for restoration. Landscapes with intermediate habitat amount and where connectivity remains sufficiently high to favor recolonization were considered to be intermediately resilient, with high possibilities of restoration effectiveness and thus were designated as priority areas for restoration actions. The proposed method consists of three steps: (1) quantifying habitat amount and connectivity; (2) using landscape ecology theory to identify intermediate resilience landscapes based on habitat amount, percolation theory, and landscape connectivity; and (3) ranking landscapes according to their importance as corridors or bottlenecks for biological flows on a broader scale, based on a graph theory approach. We present a case study for the Brazilian Atlantic Forest (approximately 150 million hectares) in order to demonstrate the proposed method. For the Atlantic Forest, landscapes that present high restoration effectiveness represent only 10% of the region, but contain approximately 15 million hectares that could be targeted for restoration actions (an area similar to today's remaining forest extent). The proposed method represents a practical way to both plan restoration actions and optimize biodiversity conservation efforts by focusing on landscapes that would result in greater conservation benefits .