A natural variation-based screen in mouse cells reveals USF2 as a regulator of the DNA damage response and cellular senescence.

Cellular senescence is a program of cell cycle arrest, apoptosis resistance, and cytokine release induced by stress exposure in metazoan cells. Landmark studies in laboratory mice have characterized a number of master senescence regulators, including p16INK4a, p21, NF-κB, p53, and C/EBPß. To discover other molecular players in senescence, we developed a screening approach to harness the evolutionary divergence between mouse species. We found that primary cells from the Mediterranean mouse Mus spretus, when treated with DNA damage to induce senescence, produced less cytokine and had less-active lysosomes than cells from laboratory Mus musculus. We used allele-specific expression profiling to catalog senescence-dependent cis-regulatory variation between the species at thousands of genes. We then tested for correlation between these expression changes and interspecies sequence variants in the binding sites of transcription factors. Among the emergent candidate senescence regulators, we chose a little-studied cell cycle factor, upstream stimulatory factor 2 (USF2), for molecular validation. In acute irradiation experiments, cells lacking USF2 had compromised DNA damage repair and response. Longer-term senescent cultures without USF2 mounted an exaggerated senescence regulatory program-shutting down cell cycle and DNA repair pathways, and turning up cytokine expression, more avidly than wild-type. We interpret these findings under a model of pro-repair, anti-senescence regulatory function by USF2. Our study affords new insights into the mechanisms by which cells commit to senescence, and serves as a validated proof of concept for natural variation-based regulator screens.

A risk-associated Active transcriptome phenotype expressed by histologically normal human breast tissue and linked to a pro-tumorigenic adipocyte population.

BACKGROUND: Previous studies have identified and validated a risk-associated Active transcriptome phenotype commonly expressed in the cancer-adjacent and histologically normal epithelium, stroma, and adipose containing peritumor microenvironment of clinically established invasive breast cancers, conferring a 2.5- to 3-fold later risk of dying from recurrent breast cancer. Expression of this Active transcriptome phenotype has not yet been evaluated in normal breast tissue samples unassociated with any benign or malignant lesions; however, it has been associated with increased peritumor adipocyte composition. METHODS: Detailed histologic and transcriptomic (RNAseq) analyses were performed on normal breast biopsy samples from 151 healthy, parous, non-obese (mean BMI = 29.60 ± 7.92) women, ages 27-66 who donated core breast biopsy samples to the Komen Tissue Bank, and whose average breast cancer risk estimate (Gail score) at the time of biopsy (1.27 ± 1.34) would not qualify them for endocrine prevention therapy. RESULTS: Full genome RNA sequencing (RNAseq) identified 52% (78/151) of these normal breast samples as expressing the Active breast phenotype. While Active signature genes were found to be most variably expressed in mammary adipocytes, donors with the Active phenotype had no difference in BMI but significantly higher Gail scores (1.46 vs. 1.18; p = 0.007). Active breast samples possessed 1.6-fold more (~ 80%) adipocyte nuclei, larger cross-sectional adipocyte areas (p < 0.01), and 0.5-fold fewer stromal and epithelial cell nuclei (p < 1e-6). Infrequent low-level expression of cancer gene hotspot mutations was detected but not enriched in the Active breast samples. Active samples were enriched in gene sets associated with adipogenesis and fat metabolism (FDR q ≤ 10%), higher signature scores for cAMP-dependent lipolysis known to drive breast cancer progression, white adipose tissue browning (Wilcoxon p < 0.01), and genes associated with adipocyte activation (leptin, adiponectin) and remodeling (CAV1, BNIP3), adipokine growth factors (IGF-1, FGF2), and pro-inflammatory fat signaling (IKBKG, CCL13). CONCLUSIONS: The risk-associated Active transcriptome phenotype first identified in cancer-adjacent breast tissues also occurs commonly in healthy women without breast disease who do not qualify for breast cancer chemoprevention, and independently of breast expressed cancer-associated mutations. The risk-associated Active phenotype appears driven by a pro-tumorigenic adipocyte microenvironment that can predate breast cancer development.

Somatic mosaicism for a lethal TRPV4 mutation results in non-lethal metatropic dysplasia.

Dominant mutations in TRPV4, which encodes the Transient Receptor Potential Cation Channel Subfamily V Member 4 calcium channel, result in a series of musculoskeletal disorders that include a set of peripheral neuropathies and a broad phenotypic spectrum of skeletal dysplasias. The skeletal phenotypes range from brachyolmia, in which there is scoliosis with mild short stature, through perinatal lethal metatropic dysplasia. We describe a case with phenotypic findings consistent with metatropic dysplasia, but in whom no TRPV4 mutation was detected by Sanger sequence analysis. Exome sequence analysis identified a known lethal metatropic dysplasia mutation, TRPV4L618P , which was present at lower frequency than would be expected for a heterozygous change. The affected individual was shown to be a somatic mosaic for the mutation, providing an explanation for the milder than expected phenotype. The data illustrate that high-throughput sequencing of genomic DNA can facilitate detection of mosaicism with higher sensitivity than Sanger sequence analysis and identify a new genetic mechanism for metatropic dysplasia. © 2016 Wiley Periodicals, Inc.

Concentration gradient generator using a convective-diffusive balance.

Since biomolecular concentration gradients are an important factor in various biological phenomena, many concentration gradient generators utilizing microfluidic techniques have been devised in order to establish stable in vitro biomolecular concentration gradients, each with their own strengths. However, all the fundamental issues, simplicity in apparatus, dynamic controllability, and convection-free conditions, have not been addressed simultaneously. In this paper, we describe a novel way of establishing concentration gradients using a convective-diffusive balance in a counterflow configuration. With the help of this method, stable, reproducible concentration gradient profiles can be formed in under a minute and the shape of the profiles are easily predicted with a simple mathematical formula.

Molecular epidemiological study for tick-borne disease (Ehrlichia and Anaplasma spp.) surveillance at selected U.S. military training sites/installations in Korea.

Vector-borne diseases are a potential public health threat to U.S. Forces Korea (USFK). Ehrlichia and Anaplasma spp., transmitted by ticks, are only two of several diseases that may affect military readiness and operations. Rodents were collected at selected U.S. military installations and training sites in the Republic of Korea. DNA was extracted from spleen tissues and assayed by PCR methods for Ehrlichia and Anaplasma species. From rodents and mustelids collected during 1999 and 2000, a total of 196 Apodemus agrarius (striped field mouse), 2 Mustela sibirica (weasel), and 1 Cricetulus triton nestor (Korean greater long-tailed hamster) were assayed for Ehrlichia and Anaplasma species-specific DNA fragments. Rodent surveillance indicated a very high prevalence of Ehrlichia and Anaplasma spp. at selected training sites. Ehrlichia/Anaplasma DNA were identified from spleen tissue from 157 Apodemus agrarius, 1 Mustela sibirica, and 1 Cricetulus riton nestor. Species-specific DNA fragments of E. canis (45), E. ewingii (16), A. phagocytophila (5), and A. platys (62) were amplified by PCR techniques. Seventy-one striped field mice had single infections, while 24 had mixed infections of 2 (17 specimens), 3 (7 specimens), or 4 (1 specimen) pathogens. The striped field mouse plays a role as a reservoir for latent infections of various Ehrlichia or Anaplasma species.