β-Arrestin2 Regulates Lysophosphatidic Acid-Induced Human Breast Tumor Cell Migration and Invasion via Rap1 and IQGAP1

β-arrestins play critical roles in chemotaxis and cytoskeletal reorganization downstream of several receptor types, including G protein-coupled receptors (GPCRs), which are targets for greater than 50% of all pharmaceuticals. Among them, receptors for lysophosphatidic acid (LPA), namely LPA₁ are overexpressed in breast cancer and promote metastatic spread. We have recently reported that β-arrestin2 regulates LPA₁-mediated breast cancer cell migration and invasion, although the underlying molecular mechanisms are not clearly understood. We show here that LPA induces activity of the small G protein, Rap1 in breast cancer cells in a β-arrestin2-dependent manner, but fails to activate Rap1 in non-malignant mammary epithelial cells. We found that Rap1A mRNA levels are higher in human breast tumors compared to healthy patient samples and Rap1A is robustly expressed in human ductal carcinoma in situ and invasive tumors, in contrast to the normal mammary ducts. Rap1A protein expression is also higher in aggressive breast cancer cells (MDA-MB-231 and Hs578t) relative to the weakly invasive MCF-7 cells or non-malignant MCF10A mammary cells. Depletion of Rap1A expression significantly impaired LPA-stimulated migration of breast cancer cells and invasiveness in three-dimensional Matrigel cultures. Furthermore, we found that β-arrestin2 associates with the actin binding protein IQGAP1 in breast cancer cells, and is necessary for the recruitment of IQGAP1 to the leading edge of migratory cells. Depletion of IQGAP1 blocked LPA-stimulated breast cancer cell invasion. Finally, we have identified that LPA enhances the binding of endogenous Rap1A to β-arrestin2, and also stimulates Rap1A and IQGAP1 to associate with LPA₁. Thus our data establish novel roles for Rap1A and IQGAP1 as critical regulators of LPA-induced breast cancer cell migration and invasion.     

Synthesis and structure-activity relationship of N-alkyl Gly-boro-Pro inhibitors of DPP4, FAP, and DPP7

The structure-activity relationship of various N-alkyl Gly-boro-Pro derivatives against three dipeptidyl peptidases (DPPs) was studied. In a series of N-cycloalkyl analogs, DPP4 and fibroblast activation protein-alpha (FAP) optimally preferred N-cycloheptyl whereas DPP7 tolerated even larger cycloalkyl rings. Gly alpha-carbon derivatization of N-cyclohexyl or N-(2-adamantyl) Gly-boro-Pro resulted in a significant decrease in potency against all the three DPPs.     

Maternal Complications and Women's Behavior in Seeking Care from Skilled Providers in North Gondar,Ethiopia

Background

Maternal complications are morbidities suffered during pregnancy through the postpartum period of 42 days. In Ethiopia, little is known about women''s experience of complications and their care-seeking behavior. This study attempted to assess experiences related to obstetric complication and seeking assistance from a skilled provider among women who gave birth in the last 12 months preceding the study.

Methods

This study was a cross-sectional survey of women who gave birth within one year preceding the study regardless of their delivery place. The study was carried out in six selected districts in North Gondar Zone, Amhara Region. Data was collected house-to-house in 12 selected clusters (kebeles) using a pretested Amharic questionnaire. During the survey, 1,668 women were interviewed. Data entry was done using Epi Info version 3.5.3 and was exported to SPSS for analysis. Logistic regression was applied to control confounders.

Results

Out of the total sample, 476 women (28.5%, 95% CI: 26.4%, 30.7%) reported some kind of complication. The most common complications reported were; excessive bleeding and prolonged labor that occurred mostly at the time of delivery and postpartum period. Out of the total women who faced complications, 248 (52.1%, 95% CI: 47.6%, 56.6%) sought assistance from a skilled provider. Inability to judge the severity of morbidities, distance/transport problems, lack of money/cost considerations and use of traditional options at home were the major reasons for not seeking care from skilled providers. Belonging to a wealthier quintile, getting antenatal care from a skilled provider and agreement of a woman in planning for possible complications were significantly associated with seeking assistance from a skilled provider.

Conclusion

Nearly half of the women who faced complications did not use skilled providers at the time of obstetric complications. Cognitive, geographic, economic and cultural barriers were involved in not using skilled maternal care.     

Childhood Diarrhea Exhibits Spatiotemporal Variation in Northwest Ethiopia: A SaTScan Spatial Statistical Analysis

Background

Childhood diarrhea continues to be a public health problem in developing countries, including Ethiopia. Detecting clusters and trends of childhood diarrhea is important to designing effective interventions. Therefore, this study aimed to investigate spatiotemporal clustering and seasonal variability of childhood diarrhea in northwest Ethiopia.

Methods

Retrospective record review of childhood diarrhea was conducted using quarterly reported data to the district health office for the seven years period beginning July 1, 2007. Thirty three districts were included and geo-coded in this study. Spatial, temporal and space-time scan spatial statistics were employed to identify clusters of childhood diarrhea. Smoothing using a moving average was applied to visualize the trends and seasonal pattern of childhood diarrhea. Statistical analyses were performed using Excel^® and SaTScan programs. The maps were plotted using ArcGIS 10.0.

Results

Childhood diarrhea in northwest Ethiopia exhibits statistical evidence of spatial, temporal, and spatiotemporal clustering, with seasonal patterns and decreasing temporal trends observed in the study area. A most likely purely spatial cluster was found in the East Gojjam administrative zone of Gozamin district (LLR = 7123.89, p <0.001). The most likely spatiotemporal cluster was detected in all districts of East Gojjam zone and a few districts of the West Gojjam zone (LLR = 24929.90, p<0.001), appearing from July 1, 2009 to June 30, 2011. One high risk period from July 1, 2008 to June 30, 2010 (LLR = 9655.86, p = 0.001) was observed in all districts. Peak childhood diarrhea cases showed a seasonal trend, occurring more frequently from January to March and April to June.

Conclusion

Childhood diarrhea did not occur at random. It has spatiotemporal variation and seasonal patterns with a decreasing temporal trend. Accounting for the spatiotemporal variation identified in the study areas is advised for the prevention and control of diarrhea.     

Aggregation behavior of ibuprofen, cholic acid and dodecylphosphocholine micelles

Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used to treat chronic pain and inflammation. However, prolonged use of NSAIDs has been known to result in Gastrointestinal (GI) ulceration/bleeding, with a bile-mediated mechanism underlying their toxicity to the lower gut. Bile acids (BAs) and phosphatidylcholines (PCs), the major components of bile, form mixed micelles to reduce the membrane disruptive actions of monomeric BAs and simple BA micelles. NSAIDs are suspected to alter the BA/PC balance in the bile, but the molecular interactions of NSAID-BA or NSAID-BA-PC remain undetermined. In this work, we used a series of all-atom molecular dynamics simulations of cholic acid (CA), ibuprofen (IBU) and dodecylphosphocholine (DPC) mixtures to study the spontaneous aggregation of CA and IBU as well as their adsorption on a DPC micelle. We found that the size of CA-IBU mixed micelles varies with their molar ratio in a non-linear manner, and that micelles of different sizes adopt similar shapes but differ in composition and internal interactions. These observations are supported by NMR chemical shift changes, NMR ROESY crosspeaks between IBU and CA, and dynamic light scattering experiments. Smaller CA-IBU aggregates were formed in the presence of a DPC micelle due to the segregation of CA and IBU away from each other by the DPC micelle. While the larger CA-IBU aggregates arising from higher IBU concentrations might be responsible for NSAID-induced intestinal toxicity, the absence of larger CA-IBU aggregates in the presence of DPC micelles may explain the observed attenuation of NSAID toxicity by PCs.     

Calves' sex ratio in naturally and artificially bred cattle in central Ethiopia

A study was undertaken with the objective to identify some intrinsic (genotype of the cow, estrus time and parity) and extrinsic factors (service type, service time and estrus seasons) that affect calf sex ratio in naturally and artificially bred cattle in the central highlands of Ethiopia. A total of 4657 calving events were extracted from the long-term dairy cattle genetic improvement experiment at Holetta Agricultural Research Center. Factors that affect the logit of the probability of a female calf being born were obtained by using PROC GENMODE in Statistical Analysis System. Moreover, multivariate analysis was performed using PROC LOGISTIC procedure using forward selection procedure. Accordingly, genotype of the cow, parity, estrus season, and service type had considerable influences on calf sex ratio. However, estrus time and service time did not affect calf sex ratio (χ² = 0.83 and 0.79, respectively). In Ethiopia, smallholder dairy farmers often complain that artificial insemination (AI) skewed to producing more male calves. However, our study showed that AI did not alter female-to-male calf sex ratio. On the contrary, natural mating increases the probability of female calves born (odds ratio 1.38) over AI. Heifer/cows that showed estrus and bred during the harsh seasons of the years produced more female calves than those that bred during the good seasons of the year. This strongly agreed with Trivers and Willard sex allocation theory.     

Genome of a virulent bacteriophage Lb338-1 that lyses the probiotic Lactobacillus paracasei cheese strain

There is a lack of fundamental knowledge about the influence of bacteriophage on probiotic bacteria and other commensals in the gut. Here, we present the isolation and morphological and genetic characterization of a virulent narrow-host-range bacteriophage, φLb338-1. This phage was isolated from fresh sewage and was shown to infect the probiotic cheese strain Lactobacillus paracasei NFBC 338. Electron microscopy studies revealed that φLb338-1 is a member of the Myoviridae family, with an isometric head, a medium-sized contractile tail, and a complex base plate. Genome sequencing revealed a 142-kb genome with 199 open reading frames. Putative functions could be assigned to 22% of the open reading frames; these had significant homology to genes found in the broad-host-range SPO1-like group of phages which includes the Enterococcus faecalis phage φEF24C, Listeria phage A511, and Lactobacillus plantarum phage LP65. Interestingly, no significant genomic similarity was observed between the phage and the probiotic host strain. Future studies will determine if the presence of bacteriophage φLb338-1 or others in the human or animal gut plays an antagonistic role against the probiotic effect of beneficial bacteria.     

Landscape genetic structure of <Emphasis Type="Italic">Olea europaea</Emphasis> subsp. <Emphasis Type="Italic">cuspidata</Emphasis> in Ethiopian highland forest fragments

Understanding patterns of genetic diversity at the landscape scale will enhance conservation and management of natural populations. Here we analyzed the genetic diversity, population connectivity, and spatial genetic structure among subpopulations and age groups of Olea europaea subsp. cuspidata, a cornerstone species of the Afromontane highlands. The study was conducted at the landscape level within a radius of approximately 4 km, as well as on a fine scale (intensive study plot) of less than 300 m radius. In total 542 samples from four natural subpopulations in northwestern Ethiopia were analyzed using ten nuclear microsatellite markers. Inbreeding was higher in smaller populations. No genetic difference was detected among cohorts of different tree sizes in the intensive studied plot. Average population differentiation was low but significant (F _ST ?=?0.016). Landscape genetic analysis inferred two groups: the most distant subpopulation WE located less than 4 kms from the other three subpopulations formed a separate group. Sixty-four percent of the total migrants were shared among the three latter subpopulations, which are spatially clustered. Immigrants were non-randomly distributed inside of the intensive study plot. Significant spatial genetic structure (SGS) was found both at the landscape scale and in the intensive study plot, and adults showed stronger SGS than young trees. An indirect estimate of 220 m as mean gene dispersal distance was obtained. We conclude that even under fragmentation migration is not disrupted in wild olive trees and that large protected populations at church forests are very important to conserve genetic resources. However, the higher level of inbreeding and evidence for population bottlenecks in the small populations, as well as the persisting heavy pressure on most remaining populations, warrants quick action to maintain genetic diversity of wild olive in the Ethiopian highlands.     

3- and 19-oximes of 16alpha,17alpha-cyclohexanoprogesterone derivatives: synthesis and interactions with progesterone receptor and other proteins

Series of 3- and 19-oximes of 16alpha,17alpha-cyclohexanoprogesterone derivatives (pregna-d'-pentaranes) have been synthesized with the aim of probing the surfaces of progesterone receptor's and two other protein ligand binding pockets neighboring to 3- and 19-positions of steroid core. The same derivatives were also studied as possible intermediates for attachment to matrixes. The data on affinity constants suggest the presence of hydrophobic cavities with hydrophilic necks in the progesterone receptor and serum pentaranophylin near C19 of bound ligand and the lack of such a cavity in uterine pentaranophylin. Any of 3-oxime substitutions were found to significantly diminish the ligand affinity for the progesterone receptor. It was also found that some of these modifications, in the Z-configuration particularly, might increase the affinity for serum and uterine pentaranophylins. The latter finding suggests the presence of large cavities near C3 of bound ligand in these proteins and interchangeability between 3-keto and 3-oxime groups in ligand-protein interactions.     

A novel switch region regulates H-ras membrane orientation and signal output

The plasma membrane nanoscale distribution of H-ras is regulated by guanine nucleotide binding. To explore the structural basis of H-ras membrane organization, we combined molecular dynamic simulations and medium-throughput FRET measurements on live cells. We extracted a set of FRET values, termed a FRET vector, to describe the lateral segregation and orientation of H-ras with respect to a large set of nanodomain markers. We show that mutation of basic residues in helix alpha4 or the hypervariable region (HVR) selectively alter the FRET vectors of GTP- or GDP-loaded H-ras, demonstrating a critical role for these residues in stabilizing GTP- or GDP-H-ras interactions with the plasma membrane. By a similar analysis, we find that the beta2-beta3 loop and helix alpha5 are involved in a novel conformational switch that operates through helix alpha4 and the HVR to reorient the H-ras G-domain with respect to the plasma membrane. Perturbation of these switch elements enhances MAPK activation by stabilizing GTP-H-ras in a more productive signalling conformation. The results illustrate how the plasma membrane spatially constrains signalling conformations by acting as a semi-neutral interaction partner.