Ashley Zehnder

Control of somatic tissue differentiation by the long non-coding RNA TINCR

Several of the thousands of human long non-coding RNAs (lncRNAs) have been functionally characterized; however, potential roles for lncRNAs in somatic tissue differentiation remain poorly understood. Here we show that a 3.7-kilobase lncRNA, terminal differentiation-induced ncRNA (TINCR), controls human epidermal differentiation by a post-transcriptional mechanism. TINCR is required for high messenger RNA abundance of key differentiation genes, many of which are mutated in human skin diseases, including FLG, LOR, ALOXE3, ALOX12B, ABCA12, CASP14 and ELOVL3. TINCR-deficient epidermis lacked terminal differentiation ultrastructure, including keratohyalin granules and intact lamellar bodies. Genome-scale RNA interactome analysis revealed that TINCR interacts with a range of differentiation mRNAs. TINCR–mRNA interaction occurs through a 25-nucleotide ‘TINCR box’ motif that is strongly enriched in interacting mRNAs and required for TINCR binding. A high-throughput screen to analyse TINCR binding capacity to approximately 9,400 human recombinant proteins revealed direct binding of TINCR RNA to the staufen1 (STAU1) protein. STAU1-deficient tissue recapitulated the impaired differentiation seen with TINCR depletion. Loss of UPF1 and UPF2, both of which are required for STAU1-mediated RNA decay, however, did not have differentiation effects. Instead, the TINCR–STAU1 complex seems to mediate stabilization of differentiation mRNAs, such as KRT80. These data identify TINCR as a key lncRNA required for somatic tissue differentiation, which occurs through lncRNA binding to differentiation mRNAs to ensure their expression.

Suppression of progenitor differentiation requires the long noncoding RNA ANCR

Long noncoding RNAs (lncRNAs) regulate diverse processes, yet a potential role for lncRNAs in maintaining the undifferentiated state in somatic tissue progenitor cells remains uncharacterized. We used transcriptome sequencing and tiling arrays to compare lncRNA expression in epidermal progenitor populations versus differentiating cells. We identified ANCR (anti-differentiation ncRNA) as an 855-base-pair lncRNA down-regulated during differentiation. Depleting ANCR in progenitor-containing populations, without any other stimuli, led to rapid differentiation gene induction. In epidermis, ANCR loss abolished the normal exclusion of differentiation from the progenitor-containing compartment. The ANCR lncRNA is thus required to enforce the undifferentiated cell state within epidermis.

IQGAP1 scaffold-kinase interaction blockade selectively targets RAS-MAP kinase-driven tumors.

Upregulation of the ERK1 and ERK2 (ERK1/2) MAP kinase (MAPK) cascade occurs in >30% of cancers, often through mutational activation of receptor tyrosine kinases or other upstream genes, including KRAS and BRAF. Efforts to target endogenous MAPKs are challenged by the fact that these kinases are required for viability in mammals. Additionally, the effectiveness of new inhibitors of mutant BRAF has been diminished by acquired tumor resistance through selection for BRAF-independent mechanisms of ERK1/2 induction. Furthermore, recently identified ERK1/2-inducing mutations in MEK1 and MEK2 (MEK1/2) MAPK genes in melanoma confer resistance to emerging therapeutic MEK inhibitors, underscoring the challenges facing direct kinase inhibition in cancer. MAPK scaffolds, such as IQ motif–containing GTPase activating protein 1 (IQGAP1), assemble pathway kinases to affect signal transmission, and disrupting scaffold function therefore offers an orthogonal approach to MAPK cascade inhibition. Consistent with this, we found a requirement for IQGAP1 in RAS-driven tumorigenesis in mouse and human tissue. In addition, the ERK1/2-binding IQGAP1 WW domain peptide disrupted IQGAP1-ERK1/2 interactions, inhibited RAS- and RAF-driven tumorigenesis, bypassed acquired resistance to the BRAF inhibitor vemurafenib (PLX-4032) and acted as a systemically deliverable therapeutic to significantly increase the lifespan of tumor-bearing mice. Scaffold-kinase interaction blockade acts by a mechanism distinct from direct kinase inhibition and may be a strategy to target overactive oncogenic kinase cascades in cancer.

Genomic analysis of mycosis fungoides and Sézary syndrome identifies recurrent alterations in TNFR2

Mycosis fungoides and Sézary syndrome comprise the majority of cutaneous T cell lymphomas (CTCLs), disorders notable for their clinical heterogeneity that can present in skin or peripheral blood. Effective treatment options for CTCL are limited, and the genetic basis of these T cell lymphomas remains incompletely characterized. Here we report recurrent point mutations and genomic gains of TNFRSF1B, encoding the tumor necrosis factor receptor TNFR2, in 18% of patients with mycosis fungoides and Sézary syndrome. Expression of the recurrent TNFR2 Thr377Ile mutant in T cells leads to enhanced non-canonical NF-κB signaling that is sensitive to the proteasome inhibitor bortezomib. Using an integrative genomic approach, we additionally discovered a recurrent CTLA4-CD28 fusion, as well as mutations in downstream signaling mediators of these receptors.

Enhancer-targeted genome editing selectively blocks innate resistance to oncokinase inhibition

Thousands of putative enhancers are characterized in the human genome, yet few have been shown to have a functional role in cancer progression. Inhibiting oncokinases, such as EGFR, ALK, ERBB2, and BRAF, is a mainstay of current cancer therapy but is hindered by innate drug resistance mediated by up-regulation of the HGF receptor, MET. The mechanisms mediating such genomic responses to targeted therapy are unknown. Here, we identify lineage-specific enhancers at the MET locus for multiple common tumor types, including a melanoma lineage-specific enhancer 63 kb downstream from the MET TSS. This enhancer displays inducible chromatin looping with the MET promoter to up-regulate MET expression upon BRAF inhibition. Epigenomic analysis demonstrated that the melanocyte-specific transcription factor, MITF, mediates this enhancer function. Targeted genomic deletion (<7 bp) of the MITF motif within the MET enhancer suppressed inducible chromatin looping and innate drug resistance, while maintaining MITF-dependent, inhibitor-induced melanoma cell differentiation. Epigenomic analysis can thus guide functional disruption of regulatory DNA to decouple pro- and anti-oncogenic functions of a dominant transcription factor and block innate resistance to oncokinase therapy.

Evaluation of indirect blood pressure monitoring in awake and anesthetized red-tailed hawks (Buteo jamaicensis): effects of cuff size, cuff placement, and monitoring equipment.

OBJECTIVE To compare Doppler and oscillometric methods of indirect arterial blood pressure (IBP) with direct arterial measurements in anesthetized and awake red-tailed hawks. STUDY DESIGN Prospective, randomized, blinded study. ANIMALS Six, sex unknown, adult red-tailed hawks. METHODS Birds were anesthetized and IBP measurements were obtained by oscillometry (IBP-O) and Doppler (IBP-D) on the pectoral and pelvic limbs using three cuffs of different width based on limb circumference: cuff 1 (20-30% of circumference), cuff 2 (30-40%), and cuff 3 (40-50%). Direct arterial pressure measurements were obtained from the contralateral superficial ulnar artery. Indirect blood pressure measurements were compared to direct systolic arterial pressure (SAP) and mean arterial pressure (MAP) during normotension and induced states of hypotension and hypertension. Measurements were also obtained in awake, restrained birds. Three-way anova, linear regression and Bland-Altman analyses were used to evaluate the IBP-D data. Results are reported as mean bias (95% confidence intervals). RESULTS The IBP-O monitor reported errors during 54% of the measurements. Indirect blood pressure Doppler measurements were most accurate with cuff 3 and were comparable to MAP with a bias of 2 (-9, 13 mmHg). However, this cuff consistently underestimated SAP with a bias of 33 (19, 48 mmHg). Variability in the readings within and among birds was high. There was no significant difference between sites of cuff placement. Awake birds had SAP, MAP and diastolic arterial pressure that were 56, 43, and 38 mmHg higher than anesthetized birds. CONCLUSIONS AND CLINICAL RELEVANCE Indirect blood pressure (oscillometric) measurements were unreliable in red-tailed hawks. Indirect blood pressure (Doppler) measurements were closer to MAP measurements than SAP measurements. There was slightly better agreement with the use of cuff 3 on either the pectoral or pelvic limbs. Awake, restrained birds have significantly higher arterial pressures than those under sevoflurane anesthesia.

Burkholderia pseudomallei isolates in 2 pet iguanas, California, USA.

Burkholderia pseudomallei, the causative agent of melioidosis, was isolated from abscesses of 2 pet green iguanas in California, USA. The international trade in iguanas may contribute to importation of this pathogen into countries where it is not endemic and put persons exposed to these animals at risk for infection.

Disseminated Lymphoma of Presumptive T-cell Origin in a Great Horned Owl (Bubo virginianus)

ABSTRACT A geriatric male great horned owl (Bubo virginianus) that was a resident at a raptor center was presented for examination because of stridor and weight loss. Results of physical examination, diagnostic imaging, and biopsy were consistent with disseminated lymphoma involving the oropharynx, neck region (including thyroid and parathyroid glands), keel, spleen, and liver. Attempts to treat the owl with chlorambucil failed, and the owl was euthanatized 5 months later. Neoplastic cells from this owl were immunoreactive to CD-3 antibody, suggesting the lymphoma was of T-cell origin.

Genetic Mapping and Biochemical Basis of Yellow Feather Pigmentation in Budgerigars

Parrot feathers contain red, orange, and yellow polyene pigments called psittacofulvins. Budgerigars are parrots that have been extensively bred for plumage traits during the last century, but the underlying genes are unknown. Here we use genome-wide association mapping and gene-expression analysis to map the Mendelian blue locus, which abolishes yellow pigmentation in the budgerigar. We find that the blue trait maps to a single amino acid substitution (R644W) in an uncharacterized polyketide synthase (MuPKS). When we expressed MuPKS heterologously in yeast, yellow pigments accumulated. Mass spectrometry confirmed that these yellow pigments match those found in feathers. The R644W substitution abolished MuPKS activity. Furthermore, gene-expression data from feathers of different bird species suggest that parrots acquired their colors through regulatory changes that drive high expression of MuPKS in feather epithelia. Our data also help formulate biochemical models that may explain natural color variation in parrots. VIDEO ABSTRACT.

Calculation of body surface area via computed tomography–guided modeling in domestic rabbits (Oryctolagus cuniculus)

OBJECTIVE To optimize the use of CT-guided modeling for the calculation of body surface area (BSA) in domestic rabbits (Oryctolagus cuniculus). Animals-12 domestic rabbits. PROCEDURES Adult rabbits (body weight, 1 to > 4 kg) that were client-owned animals undergoing CT for disease diagnosis or deceased laboratory animals donated from other research projects were scanned with a CT scanner. Images were transferred to a radiation therapy planning software program. Image slices were captured as contiguous slices at 100 kVp and 100 mA and processed to 0.1-cm-thick sections. The length of each contoured slice was summed to calculate a final BSA measurement. Nonlinear regression analysis was then used to derive an equation for the calculation of BSA in rabbits. RESULTS The constant calculated by use of this method was 9.9 (range, 9.59 to 10). The R(2) for the goodness of fit was 0.9332. The equation that best described BSA as a function of body weight for domestic rabbits with this method was as follows: BSA = (9.9 × [body weight {in grams}](2/3))/10,000. CONCLUSIONS AND CLINICAL RELEVANCE The BSA calculated via the CT-guided method yielded results similar to those obtained with equations for other similarly sized mammals and verified the use of such equations for rabbits. Additionally, this technique can be used for species that lack equations for the accurate calculation of BSA.