Purification of fatty acid methyl esters by high-performance liquid chromatography

The determination by gas chromatography (GC) of fatty acid methyl esters (FAMEs) prepared from complex biological samples is subject to interference from cholesterol. During sample injection on the GC system of FAMEs prepared from tissues that contain cholesterol, we observed a major contaminant that co-eluted with docosahexaenoic acid (DHA, 22:6n-3). To address this problem, FAMEs were purified on an amino-phase high-performance liquid chromatography (HPLC) column using a hexane–isopropanol gradient. The HPLC retention times for both the FAME fraction and cholesterol were stable and reproducible when the amino column was used for sample purification. The purified extracts were analyzed by GC without artifacts or impurity peaks after 50 analytical runs. The method described here will be useful for measurement of 22:6n-3 and other fatty acids important for studies of nutrition or pathology.     

Alpha Satellite Insertion Close to an Ancestral Centromeric Region

Human centromeres are mainly composed of alpha satellite DNA hierarchically organized as higher-order repeats (HORs). Alpha satellite dynamics is shown by sequence homogenization in centromeric arrays and by its transfer to other centromeric locations, for example, during the maturation of new centromeres. We identified during prenatal aneuploidy diagnosis by fluorescent in situ hybridization a de novo insertion of alpha satellite DNA from the centromere of chromosome 18 (D18Z1) into cytoband 15q26. Although bound by CENP-B, this locus did not acquire centromeric functionality as demonstrated by the lack of constriction and the absence of CENP-A binding. The insertion was associated with a 2.8-kbp deletion and likely occurred in the paternal germline. The site was enriched in long terminal repeats and located ∼10 Mbp from the location where a centromere was ancestrally seeded and became inactive in the common ancestor of humans and apes 20–25 million years ago. Long-read mapping to the T2T-CHM13 human genome assembly revealed that the insertion derives from a specific region of chromosome 18 centromeric 12-mer HOR array in which the monomer size follows a regular pattern. The rearrangement did not directly disrupt any gene or predicted regulatory element and did not alter the methylation status of the surrounding region, consistent with the absence of phenotypic consequences in the carrier. This case demonstrates a likely rare but new class of structural variation that we name “alpha satellite insertion.” It also expands our knowledge on alphoid DNA dynamics and conveys the possibility that alphoid arrays can relocate near vestigial centromeric sites.     

Variation in growth rate,carbon assimilation,and photosynthetic efficiency in response to nitrogen source and concentration in phytoplankton isolated from upper San Francisco Bay

Six species of phytoplankton recently isolated from upper San Francisco Bay were tested for their sensitivity to growth inhibition by ammonium (NH₄⁺), and for differences in growth rates according to inorganic nitrogen (N) growth source. The quantum yield of photosystem II (F_v/F_m) was a sensitive indicator of NH₄⁺ toxicity, manifested by a suppression of F_v/F_m in a dose‐dependent manner. Two chlorophytes were the least sensitive to NH₄⁺ inhibition, at concentrations of >3,000 μmoles NH₄⁺ · L^?1, followed by two estuarine diatoms that were sensitive at concentrations >1,000 μmoles NH₄⁺ · L^?1, followed lastly by two freshwater diatoms that were sensitive at concentrations between 200 and 500 μmoles NH₄⁺ · L^?1. At non‐inhibiting concentrations of NH₄⁺, the freshwater diatom species grew fastest, followed by the estuarine diatoms, while the chlorophytes grew slowest. Variations in growth rates with N source did not follow taxonomic divisions. Of the two chlorophytes, one grew significantly faster on nitrate (NO₃^?), whereas the other grew significantly faster on NH₄⁺. All four diatoms tested grew faster on NH₄⁺ compared with NO₃^?. We showed that in cases where growth rates were faster on NH₄⁺ than they were on NO₃^?, the difference was not larger for chlorophytes compared with diatoms. This holds true for comparisons across a number of culture investigations suggesting that diatoms as a group will not be at a competitive disadvantage under natural conditions when NH₄⁺ dominates the total N pool and they will also not have a growth advantage when NO₃^? is dominant, as long as N concentrations are sufficient.     

Secreted frizzled-related protein 4 regulates two Wnt7a signaling pathways and inhibits proliferation in endometrial cancer cells

In the endometrium, hormonal effects on epithelial cells are often elicited through stromal hormone receptors via unknown paracrine mechanisms. Several lines of evidence support the hypothesis that Wnts participate in stromal-epithelial cell communication. Wnt7a is expressed in the luminal epithelium, whereas the extracellular modulator of Wnt signaling, secreted frizzled-related protein 4 (SFRP4), is localized to the stroma. Studies have reported that SFRP4 expression is significantly decreased in endometrial carcinoma and that both SFRP4 and Wnt7a genes are differentially regulated in response to estrogenic stimuli. Aberrant Wnt7a signaling irrevocably causes organ defects and infertility and contributes to the onset of disease. However, specific frizzled receptors (Fzd) that bind Wnt7a and the particular signal transduction pathway each Wnt7a-Fzd pair activates have not been identified. Additionally, the function of SFRP4 in the endometrium has not been addressed. We show here that Wnt7a coimmunoprecipitates with Fzd5, Fzd10, and SFRP4 in Ishikawa cells. Wnt7a binding to Fzd5 was shown to activate beta-catenin/canonical Wnt signaling and increase cellular proliferation. Conversely, Wnt7a signaling mediated by Fzd10 induced a noncanonical c-Jun NH2-terminal kinase-responsive pathway. SFRP4 suppresses activation of Wnt7a signaling in both an autocrine and paracrine manner. Stable overexpression of SFRP4 and treatment with recombinant SFRP4 protein inhibited endometrial cancer cell growth in vitro. These findings support a mechanism by which the nature of the Wnt7a signal in the endometrium is dependent on the Fzd repertoire of the cell and can be regulated by SFRP4.     

Design and synthesis of orally bioavailable serum and glucocorticoid-regulated kinase 1 (SGK1) inhibitors

The lead serum and glucocorticoid-related kinase 1 (SGK1) inhibitors 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid (1) and {4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid (2) suffer from low DNAUC values in rat, due in part to formation and excretion of glucuronic acid conjugates. These PK/glucuronidation issues were addressed either by incorporating a substituent on the 3-phenyl ring ortho to the key carboxylate functionality of 1 or by substituting on the group in between the carboxylate and phenyl ring of 2. Three of these analogs have been identified as having good SGK1 inhibition potency and have DNAUC values suitable for in vivo testing.     

Using Inquiry and Tree-Thinking to “March Through the Animal Phyla”: Teaching Introductory Comparative Biology in an Evolutionary Context

Biodiversity was originally taught in our Introductory Organismal Biology course at Michigan State University (LB144; freshman/sophomore majors) by rote memorization of isolated facts about organisms. When we moved to an inquiry-based laboratory framework to improve pedagogy, an unfortunate and unforeseen result was the loss of much of our study of biodiversity. In this paper, we describe the restructuring of LB144 to restore the study of biodiversity and organismal groups while retaining the benefits of an inquiry-based approach. The curricular intervention was accomplished through the creation and implementation of a four-week Comparative Biology laboratory stream. During this stream, student research teams recorded and organized observations that they made on a range of organisms and analyzed their data in a phylogenetic framework. During the stream, our students worked through a set of exercises designed to help them learn how to read, interpret, and manipulate phylogenetic trees. We placed particular emphasis on the concept that phylogenetic trees are hypotheses of relationship that can be tested with scientific data. This incorporation of phylogenies and phylogenetic analysis, or “tree-thinking,” into our students’ work provided an explicit synthetic evolutionary framework for their comparative biodiversity studies. End-of-stream products included a team phylogenetic analysis exercise and an individual comparative biology oral presentation.     

Escherichia coli O157:H7 Strains That Persist in Feedlot Cattle Are Genetically Related and Demonstrate an Enhanced Ability To Adhere to Intestinal Epithelial Cells

A longitudinal study was conducted to investigate the nature of Escherichia coli O157:H7 colonization of feedlot cattle over the final 100 to 110 days of finishing. Rectal fecal grab samples were collected from an initial sample population of 788 steers every 20 to 22 days and microbiologically analyzed to detect E. coli O157:H7. The identities of presumptive colonies were confirmed using a multiplex PCR assay that screened for gene fragments unique to E. coli O157:H7 (rfbE and fliC_h7) and other key virulence genes (eae, stx₁, and stx₂). Animals were classified as having persistent shedding (PS), transient shedding (TS), or nonshedding (NS) status if they consecutively shed the same E. coli O157:H7 genotype (based on the multiplex PCR profile), exhibited variable E. coli O157 shedding, or never shed morphologically typical E. coli O157, respectively. Overall, 1.0% and 1.4% of steers were classified as PS and NS animals, respectively. Characterization of 132 E. coli O157:H7 isolates from PS and TS animals by pulsed-field gel electrophoresis (PFGE) typing yielded 32 unique PFGE types. One predominant PFGE type accounted for 53% of all isolates characterized and persisted in cattle throughout the study. Isolates belonging to this predominant and persistent PFGE type demonstrated an enhanced (P < 0.0001) ability to adhere to Caco-2 human intestinal epithelial cells compared to isolates belonging to less common PFGE types but exhibited equal virulence expression. Interestingly, the attachment efficacy decreased as the genetic divergence from the predominant and persistent subtype increased. Our data support the hypothesis that certain E. coli O157:H7 strains persist in feedlot cattle, which may be partially explained by an enhanced ability to colonize the intestinal epithelium.Escherichia coli serotype O157:H7 was first linked to human illness in the early 1980s, when it was determined to cause severe abdominal pain with initially watery diarrhea that progressed to grossly bloody diarrhea accompanied by little or no fever (42). Initially, E. coli O157:H7 can cause nonbloody diarrhea through attachment to, and subsequent destruction of, intestinal microvilli (24). In addition to microvillus damage, serious health complications can arise due to the ability of E. coli O157:H7 to produce Shiga toxins (Stx1 and Stx2). Shiga toxins are very potent cytotoxins that are absorbed into the intestinal microvasculature and initiate apoptosis of vascular epithelium, resulting in hemorrhagic colitis (41). Persistent uptake of these toxins may lead to more severe manifestations of disease, such as hemolytic-uremic syndrome, which may ultimately result in kidney failure (24). Most recent estimates have identified E. coli O157:H7 as the cause of at least 70,000 cases of food-borne illness annually in the United States, and in 4% of cases life-threatening hemolytic-uremic syndrome develops (37). Epidemiological studies have implicated the consumption of meat, dairy products, produce, and water contaminated by animal feces, as well as person-to-person contact and direct contact with farm animals or their environment, as routes of E. coli O157:H7 transmission leading to human illness (36).It is generally accepted that cattle and other animals are the major reservoir of E. coli O157:H7, but it is still not clear if animals are colonized for prolonged periods with E. coli O157:H7 or if they transiently shed this organism following repeated exposure to it through ingestion of contaminated feedstuffs or water or through exposure to other contaminated environmental sources. Based on results of numerous epidemiological studies (4, 6, 21, 30, 32), the prevalence of E. coli O157:H7 in feedlot cattle is highly variable and can range from less than 1% to 80%. Several other studies (7, 8, 23) have found evidence of persistent E. coli O157:H7 colonization in individual cattle, supporting the hypothesis that at least some animals are susceptible to persistent E. coli O157:H7 colonization. Multiple experimental inoculation studies (15, 23, 39, 46) showed that E. coli O157:H7 persists in the bovine gastrointestinal (GI) tract for at least 14 days up to 140 days postinfection. Studies have implicated the lower GI tract and specifically the recto-anal junction (RAJ) as the major location of E. coli O157:H7 colonization and proliferation (9, 12, 23, 39); however, this organism also can be found throughout the bovine GI tract (7, 8, 31, 40, 54).It stands to reason that if the E. coli O157:H7 prevalence in cattle presented for harvest were reduced, there would be a decrease in the probability of beef product contamination, if good manufacturing procedures were used. Although there is consensus concerning the importance of preharvest pathogen mitigation and its role in minimizing entry of E. coli O157:H7 into harvest facilities, there is disagreement about the significance of “supershedders” (animals that excrete large quantities of a pathogen for various amounts of time) for E. coli O157:H7 transmission dynamics at the preharvest level (12, 34, 35, 39). Utilizing statistical modeling, researchers have estimated that, on average, the prevalence of “supershedders” in a population is 4% and that these animals excrete 50 times more E. coli O157:H7 than other animals colonized by this organism (34). Additionally, the same researchers suggested that approximately 80% of E. coli O157:H7 transmission is generated by a few “supershedders” (35).Research by our group discovered a unique association between E. coli O157:H7 prevalence in pen floor fecal pats and carcass contamination by this pathogen (57). When the prevalence in fecal pats from a pen floor exceeded 20%, carcasses of animals from the pen had E. coli O157:H7 prevalence values of 14.3, 2.9, and 0.7% before evisceration, after evisceration, and after final intervention, respectively. However, when the prevalence in pen floor fecal pats was less than 20%, the preeviscerated carcass prevalence value was 6.3%, and there was no detectable E. coli O157:H7 contamination of carcass samples after evisceration and after final intervention (57). Thus, we hypothesize that animals which persistently excrete normal levels of E. coli O157:H7 over prolonged periods (persistent shedders [PS]) rather than animals that periodically shed abnormally high levels (supershedders) are the most significant source of E. coli O157:H7 contamination in the food continuum. Although previous studies suggested that cattle may be persistently colonized by E. coli O157:H7 and shed this organism in their feces for prolonged periods, molecular subtyping data are required to further investigate whether cattle are persistently colonized by the same strain (i.e., molecular subtype) or if they are repeatedly exposed to different strains through contaminated feedstuffs, water, or other environmental sources. Thus, the objectives of this study were to determine if naturally colonized feedlot cattle persistently shed E. coli O157:H7, using combined cultural microbiological analyses, molecular subtyping approaches, and in vitro virulence phenotype assays to probe the factors (agent, host, environment, or a combination of these factors) that contribute to the complex ecology of E. coli O157:H7 persistence at the preharvest level.     

Identification of EMS-Induced Mutations in Drosophila melanogaster by Whole-Genome Sequencing

Next-generation methods for rapid whole-genome sequencing enable the identification of single-base-pair mutations in Drosophila by comparing a chromosome bearing a new mutation to the unmutagenized sequence. To validate this approach, we sought to identify the molecular lesion responsible for a recessive EMS-induced mutation affecting egg shell morphology by using Illumina next-generation sequencing. After obtaining sufficient sequence from larvae that were homozygous for either wild-type or mutant chromosomes, we obtained high-quality reads for base pairs composing ~70% of the third chromosome of both DNA samples. We verified 103 single-base-pair changes between the two chromosomes. Nine changes were nonsynonymous mutations and two were nonsense mutations. One nonsense mutation was in a gene, encore, whose mutations produce an egg shell phenotype also observed in progeny of homozygous mutant mothers. Complementation analysis revealed that the chromosome carried a new functional allele of encore, demonstrating that one round of next-generation sequencing can identify the causative lesion for a phenotype of interest. This new method of whole-genome sequencing represents great promise for mutant mapping in flies, potentially replacing conventional methods.