Generation and comparative analysis of approximately 3.3 Mb of mouse genomic sequence orthologous to the region of human chromosome 7q11.23 implicated in Williams syndrome.

Williams syndrome is a complex developmental disorder that results from the heterozygous deletion of a approximately 1.6-Mb segment of human chromosome 7q11.23. These deletions are mediated by large (approximately 300 kb) duplicated blocks of DNA of near-identical sequence. Previously, we showed that the orthologous region of the mouse genome is devoid of such duplicated segments. Here, we extend our studies to include the generation of approximately 3.3 Mb of genomic sequence from the mouse Williams syndrome region, of which just over 1.4 Mb is finished to high accuracy. Comparative analyses of the mouse and human sequences within and immediately flanking the interval commonly deleted in Williams syndrome have facilitated the identification of nine previously unreported genes, provided detailed sequence-based information regarding 30 genes residing in the region, and revealed a number of potentially interesting conserved noncoding sequences. Finally, to facilitate comparative sequence analysis, we implemented several enhancements to the program, including the addition of links from annotated features within a generated percent-identity plot to specific records in public databases. Taken together, the results reported here provide an important comparative sequence resource that should catalyze additional studies of Williams syndrome, including those that aim to characterize genes within the commonly deleted interval and to develop mouse models of the disorder.     

Heat-Induced Hyperactivation

Purpose: The objectives of this study were (1) to determine the sperm hyperactivation and related kinematic parameters at 40°C after using four sperm wash procedures and (2) to correlate the heat-induced hyperactivation data with cases of clinical pregnancies from either artificial insemination or standard in vitro fertilization (IVF). Methods: Semen samples (n = 51) were collected by ejaculation, and semen analyses were carried out to determine the pretreatment data. Sperm kinematic measurements were performed using the Hamilton Thorn HTM-C computer-aided sperm analyzer. Hyperactivation was determined using the sort module on the HTM-C. Membrane integrity was assessed using the hypoosmotic sperm swelling procedure. Sperm morphology and acrosomal status were also determined using the Spermac stain. Each semen specimen was divided and processed through either the swim-up wash, the 1-h test-yolk buffer (TYB) wash, the 1 mg/ml pentoxifylline stimulant procedure, or the two-layer 90:47% gradient colloidal solution procedure. The washed sperm were incubated at 25 or at 40° C for 4 hr. After incubation, kinematic parameters were assessed for the posttreatment data. Semen specimens were obtained on different occasions for artificial insemination or standard IVF. Data from intracytoplasmic sperm injection cases were not included to avoid confounding factors. Live births and/or pregnancies with fetal heartbeat examined by ultrasound were considered clinical pregnancies. Results: Heat-induced hyperactive motility was significantly higher in sperm of the male partner of pregnant (n = 7) patients compared with nonpregnant (n = 44) patients (mean ± SE, 10.0 ± 3.3 versus 5.5 ± 0.8%) after TYB processing fallowed by 4 hr of incubation at 40°C. This was also observed after colloid (Percoll) processing (11.6 ± 4.6 versus 5.8 ± 0.8%). There were no differences in hyperactivation after 4 hr at 23°C between pregnant and nonpregnant cases. Parameters such as count, volume, motility, viability, and acrosomal status were not different for the groups. However, the percentage of sperm with normal morphology (WHO classification) was twice as high in the pregnant group versus the nonpregnant group. Conclusions: Heat-induced hyperactivation was associated with fertile sperm and was predictive of pregnancy obtained after artificial insemination or IVF. The association was evident only after TYB or Percoll sperm processing. The study could not confirm the finding of significant decreases in motility after heat treatment of sperm derived from infertile males. The mechanism for heat-induced hyperactivation did not involve membrane integrity or the sperm acrosome, although an involvement of heat shock proteins was postulated. Interestingly, there were no pregnancies when sperm did not exhibit heat-induced hyperactivation.     

Solving the puzzle of Alzheimer disease

ABSTRACT: Managing patients with dementia and Alzheimer disease can be a challenge. Often, families and caregivers ask clinicians about the latest treatments. This article summarizes the latest evidence-based practice related to pharmacologic and nonpharmacologic management of patients with Alzheimer disease.     

Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells

The development of regenerative therapies for cartilage injury has been greatly aided by recent advances in stem cell biology. Induced pluripotent stem cells (iPSCs) have the potential to provide an abundant cell source for tissue engineering, as well as generating patient-matched in vitro models to study genetic and environmental factors in cartilage repair and osteoarthritis. However, both cell therapy and modeling approaches require a purified and uniformly differentiated cell population to predictably recapitulate the physiological characteristics of cartilage. Here, iPSCs derived from adult mouse fibroblasts were chondrogenically differentiated and purified by type II collagen (Col2)-driven green fluorescent protein (GFP) expression. Col2 and aggrecan gene expression levels were significantly up-regulated in GFP+ cells compared with GFP− cells and decreased with monolayer expansion. An in vitro cartilage defect model was used to demonstrate integrative repair by GFP+ cells seeded in agarose, supporting their potential use in cartilage therapies. In chondrogenic pellet culture, cells synthesized cartilage-specific matrix as indicated by high levels of glycosaminoglycans and type II collagen and low levels of type I and type X collagen. The feasibility of cell expansion after initial differentiation was illustrated by homogenous matrix deposition in pellets from twice-passaged GFP+ cells. Finally, atomic force microscopy analysis showed increased microscale elastic moduli associated with collagen alignment at the periphery of pellets, mimicking zonal variation in native cartilage. This study demonstrates the potential use of iPSCs for cartilage defect repair and for creating tissue models of cartilage that can be matched to specific genetic backgrounds.     

Comprehensive evaluation of ESR2 variation and ovarian cancer risk.

Studies indicate that estrogen receptor beta, encoded by the ESR2 gene on chromosome 14q, may play a role in ovarian carcinogenesis. Using the genetic structure data generated by the Breast and Prostate Cohort Consortium (BPC3), we have comprehensively characterized the role of haplotype diversity in ESR2 and risk of ovarian cancer. Five haplotypes with a frequency of > or =5% were observed in White subjects and five haplotype tagging SNPs (htSNP) were selected to capture the locus diversity with a minimum R(h)(2) of 0.81. The htSNPs were genotyped in 574 White controls, 417 White invasive ovarian cancer cases, and 123 White borderline ovarian cancer cases from case-control studies carried out in Los Angeles County from 1994 through 2004. No statistically significant association was observed between the five htSNPs and related haplotypes and risk of ovarian cancer overall. Haplotype D was associated with a nonstatistically significant increased risk of invasive ovarian cancer overall (odds ratio, 1.38; 95% confidence interval, 0.93-2.02; P = 0.11) relative to the most common haplotype and a statistically significant increased risk of invasive clear cell ovarian cancer (odds ratio, 3.88; 95% confidence interval, 1.28-11.73; P = 0.016). Haplotype D was also reported by the BPC3 to be associated with increased risk of breast cancer. This haplotype warrants further investigation to rule out any effect with invasive ovarian cancer risk.     

Cyclooxygenase (COX)-2 and granulosa cell apoptosis in vitro

Purpose : C-myc was studied in cyclooxygenase (COX)-2 associated granulosa cell apoptosis. Methods : Granulosa cells (N = 5 cases) were incubated for 24 h in either 1 or 50 M COX-2 inhibitor, 1 or 50 M COX-1/COX-2 inhibitor, negative or positive controls. Single primer polymerase chain reaction of c-myc exon 1 were performed. Bisbenzimide-stained control single-stranded (ssDNA) were hybridized to SYBR Gold-stained ssDNA and fluorescent images analyzed. Results : C-myc was disrupted by the high-dose COX-2 inhibitor. Cell viability decreased with COX-1 and COX-2 inhibition. However, cell viability was similar for the positive control and at low-dose COX-2 inhibition. Conclusions : Inhibition of both COX-1 and COX-2 initiated apoptosis without disrupting c-myc suggesting a protective effect on c-myc. The low dosage of the COX-2 inhibitor did not disrupt c-myc and cell viability. C-myc sensitization was not part of apoptosis.     

Fat discrimination: a phenotype with potential implications for studying fat intake behaviors and obesity

Variations in fat preference and intake across humans are poorly understood in part because of difficulties in studying this behavior. The objective of this study was to develop a simple procedure to assess fat discrimination, the ability to accurately perceive differences in the fat content of foods, and assess the associations between this phenotype and fat ingestive behaviors and adiposity. African-American adults (n=317) were tested for fat discrimination using 7 forced choice same/different tests with Italian salad dressings that ranged in fat-by-weight content from 5 to 55%. Performance on this procedure was determined by tallying the number of trials in which a participant correctly identified the pair of samples as "same" or "different" across all test pairs (ranging from 1 to 7). Individuals who received the lowest scores on this task (≤3 out of 7 correct) were classified as fat non-discriminators (n=33) and those who received the highest scores (7 out of 7 correct) were classified as fat discriminators (n=59). These 2 groups were compared for the primary outcome variables: reported food intake, preferences, and adiposity. After adjusting for BMI, sex, age, and dietary restraint and disinhibition, fat non-discriminators reported greater consumption of both added fats and reduced fat foods (p<0.05 for both). Fat non-discriminators also had greater abdominal adiposity compared to fat discriminators (p<0.05). Test-retest scores performed in a subset of participants (n=40) showed moderate reliability of the fat discrimination test (rho=0.53; p<0.01). If these results are replicated, fat discrimination may serve as clinical research tool to identify participants who are at risk for obesity and other chronic diseases due to increased fat intake.