Marie Claude Gingras

Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.

A disease mutation identified by whole-genome sequencing of twins with dystonia allowed optimization of treatment, resulting in clinical improvements.

Cancer cells adapt metabolically to proliferate under nutrient limitation. Here we used combined transcriptional-metabolomic network analysis to identify metabolic pathways that support glucose-independent tumor cell proliferation. We found that glucose deprivation stimulated re-wiring of the tricarboxylic acid (TCA) cycle and early steps of gluconeogenesis to promote glucose-independent cell proliferation. Glucose limitation promoted the production of phosphoenolpyruvate (PEP) from glutamine via the activity of mitochondrial PEP-carboxykinase (PCK2). Under these conditions, glutamine-derived PEP was used to fuel biosynthetic pathways normally sustained by glucose, including serine and purine biosynthesis. PCK2 expression was required to maintain tumor cell proliferation under limited-glucose conditions in vitro and tumor growth in vivo. Elevated PCK2 expression is observed in several human tumor types and enriched in tumor tissue from non-small-cell lung cancer (NSCLC) patients. Our results define a role for PCK2 in cancer cell metabolic reprogramming that promotes glucose-independent cell growth and metabolic stress resistance in human tumors.

We investigated glioblastoma multiforme (GBM) for a pattern of consistent alterations in cell adhesion molecules (CAM) expression that might distinguish tumor from normal autologous brain tissue. We used frozen section immunohistochemistry with anti-CAM and computerized image analysis to quantify staining intensity which we expressed as relative intensity units (RIU). Our results showed that normal brain tissue generally did not express alpha 1 beta 1, intercellular CAM-1 (ICAM-1), and sialylated Lewisx, slightly expressed alpha 2, alpha 4, alpha 5, alpha 6 beta 1, alpha v beta 3, lymphocyte function-associated antigen-3 (LFA-3), Lewisx, sialylated LewisLewisx, had a good expression of alpha 3 beta 1 and CD44, and strongly expressed neural CAM (NCAM). GBM expressed alpha 2, alpha 3, alpha 5, alpha 6 beta 1, alpha v beta 3, ICAM-1, LFA-3, CD44, Lewisx, sialylated Lewisx, and sialylated LewisLewisx significantly higher (2-11-fold RIU) than normal brain tissue. ICAM-1 and LFA-3 were the most distinctive markers of GBM. The small blood vessel endothelial cells of the normal brain and the GBM showed a few differences. The tumor endothelium expression of alpha 2 beta 1, alpha 4 beta 1, and LFA-3 RIU appeared twice higher than in normal endothelium and alpha 6 beta 1 showed an average of 40% RIU decrease in comparison to normal. These results show that the expression of several CAM is consistently altered in GBM and its microvasculature when compared with autologous normal brain tissue.

The Warburg effect is a tumorigenic metabolic adaptation process characterized by augmented aerobic glycolysis, which enhances cellular bioenergetics. In normal cells, energy homeostasis is controlled by AMPK; however, its role in cancer is not understood, as both AMPK-dependent tumor-promoting and -inhibiting functions were reported. Upon stress, energy levels are maintained by increased mitochondrial biogenesis and glycolysis, controlled by transcriptional coactivator PGC-1α and HIF, respectively. In normoxia, AMPK induces PGC-1α, but how HIF is activated is unclear. Germline mutations in the gene encoding the tumor suppressor folliculin (FLCN) lead to Birt-Hogg-Dubé (BHD) syndrome, which is associated with an increased cancer risk. FLCN was identified as an AMPK binding partner, and we evaluated its role with respect to AMPK-dependent energy functions. We revealed that loss of FLCN constitutively activates AMPK, resulting in PGC-1α-mediated mitochondrial biogenesis and increased ROS production. ROS induced HIF transcriptional activity and drove Warburg metabolic reprogramming, coupling AMPK-dependent mitochondrial biogenesis to HIF-dependent metabolic changes. This reprogramming stimulated cellular bioenergetics and conferred a HIF-dependent tumorigenic advantage in FLCN-negative cancer cells. Moreover, this pathway is conserved in a BHD-derived tumor. These results indicate that FLCN inhibits tumorigenesis by preventing AMPK-dependent HIF activation and the subsequent Warburg metabolic transformation.

Genitopatellar syndrome (GPS) is a skeletal dysplasia with cerebral and genital anomalies for which the molecular basis has not yet been determined. By exome sequencing, we found de novo heterozygous truncating mutations in KAT6B (lysine acetyltransferase 6B, formerly known as MYST4 and MORF) in three subjects; then by Sanger sequencing of KAT6B, we found similar mutations in three additional subjects. The mutant transcripts do not undergo nonsense-mediated decay in cells from subjects with GPS. In addition, human pathological analyses and mouse expression studies point to systemic roles of KAT6B in controlling organismal growth and development. Myst4 (the mouse orthologous gene) is expressed in mouse tissues corresponding to those affected by GPS. Phenotypic differences and similarities between GPS, the Say-Barber-Biesecker variant of Ohdo syndrome (caused by different mutations of KAT6B), and Rubinstein-Taybi syndrome (caused by mutations in other histone acetyltransferases) are discussed. Together, the data support an epigenetic dysregulation of the limb, brain, and genital developmental programs. Genitopatellar syndrome (GPS) is a skeletal dysplasia with cerebral and genital anomalies for which the molecular basis has not yet been determined. By exome sequencing, we found de novo heterozygous truncating mutations in KAT6B (lysine acetyltransferase 6B, formerly known as MYST4 and MORF) in three subjects; then by Sanger sequencing of KAT6B, we found similar mutations in three additional subjects. The mutant transcripts do not undergo nonsense-mediated decay in cells from subjects with GPS. In addition, human pathological analyses and mouse expression studies point to systemic roles of KAT6B in controlling organismal growth and development. Myst4 (the mouse orthologous gene) is expressed in mouse tissues corresponding to those affected by GPS. Phenotypic differences and similarities between GPS, the Say-Barber-Biesecker variant of Ohdo syndrome (caused by different mutations of KAT6B), and Rubinstein-Taybi syndrome (caused by mutations in other histone acetyltransferases) are discussed. Together, the data support an epigenetic dysregulation of the limb, brain, and genital developmental programs. Genitopatellar syndrome (GPS) [MIM 606170] is a rare skeletal dysplasia combining hypoplastic or absent patellae, genital anomalies, craniofacial defects, and intellectual disability among other features. It has been described in 18 subjects to date.1Abdul-Rahman O.A. La T.H. Kwan A. Schlaubitz S. Barsh G.S. Enns G.M. Hudgins L. Genitopatellar syndrome: Expanding the phenotype and excluding mutations in LMX1B and TBX4.Am. J. Med. Genet. A. 2006; 140: 1567-1572Crossref PubMed Scopus (12) Google Scholar, 2Armstrong L. Clarke J.T.R. Report of a new case of “genitopatellar” syndrome which challenges the importance of absent patellae as a defining feature.J. Med. Genet. 2002; 39: 933-934Crossref PubMed Google Scholar, 3Bergmann C. Spranger S. Javaher P. Ptok M. Genitopatellar syndrome, sensorineural hearing loss, and cleft palate.Oral Maxillofac Surg. 2011; 15: 103-106Crossref PubMed Scopus (4) Google Scholar, 4Brugha R. Kinali M. Aminu K. Bridges N. Holder S.E. Genitopatellar syndrome: A further case.Clin. Dysmorphol. 2011; 20: 163-165Crossref PubMed Scopus (6) Google Scholar, 5Cormier-Daire V. Chauvet M.L. Lyonnet S. Briard M.L. Munnich A. Le Merrer M. Genitopatellar syndrome: A new condition comprising absent patellae, scrotal hypoplasia, renal anomalies, facial dysmorphism, and mental retardation.J. Med. Genet. 2000; 37: 520-524Crossref PubMed Google Scholar, 6Goldblatt J. Wallis C. Zieff S. A syndrome of hypoplastic patellae, mental retardation, skeletal and genitourinary anomalies with normal chromosomes.Dysmorphol. Clin. Genet. 1988; 2: 91-93Google Scholar, 7Lammer E.J. Abrams L. Genitopatellar syndrome: Delineating the anomalies of female genitalia.Am. J. Med. Genet. 2002; 111: 316-318Crossref PubMed Scopus (12) Google Scholar, 8Lifchez C.A. Rhead W.J. Leuthner S.R. Lubinsky M.S. Genitopatellar syndrome: Expanding the phenotype.Am. J. Med. Genet. A. 2003; 122A: 80-83Crossref PubMed Scopus (16) Google Scholar, 9Penttinen M. Koillinen H. Niinikoski H. Mäkitie O. Hietala M. Genitopatellar syndrome in an adolescent female with severe osteoporosis and endocrine abnormalities.Am. J. Med. Genet. A. 2009; 149A: 451-455Crossref PubMed Scopus (18) Google Scholar, 10Reardon W. Genitopatellar syndrome: A recognizable phenotype.Am. J. Med. Genet. 2002; 111: 313-315Crossref PubMed Scopus (25) Google Scholar, 11Schlaubitz S. Yatsenko S.A. Smith L.D. Keller K.L. Vissers L.E. Scott D.A. Cai W.W. Reardon W. Abdul-Rahman O.A. Lammer E.J. et al.Ovotestes and XY sex reversal in a female with an interstitial 9q33.3-q34.1 deletion encompassing NR5A1 and LMX1B causing features of Genitopatellar syndrome.Am. J. Med. Genet. A. 2007; 143A: 1071-1081Crossref PubMed Scopus (39) Google Scholar In one patient, we discovered a microdeletion encompassing LMX1B (explaining the patellar anomalies) and NR5A1 (explaining the genital anomalies) though this was not found to be a recurrent molecular lesion in the other subjects.11Schlaubitz S. Yatsenko S.A. Smith L.D. Keller K.L. Vissers L.E. Scott D.A. Cai W.W. Reardon W. Abdul-Rahman O.A. Lammer E.J. et al.Ovotestes and XY sex reversal in a female with an interstitial 9q33.3-q34.1 deletion encompassing NR5A1 and LMX1B causing features of Genitopatellar syndrome.Am. J. Med. Genet. A. 2007; 143A: 1071-1081Crossref PubMed Scopus (39) Google Scholar An important phenotypic difference is that the subject with the microdeletion is not microcephalic although all other subjects are. To gain insights into the molecular cause of GPS, we recruited subjects with this disease (see Figure 1 for photos, Figure S1 [available online] for pedigrees, and Table 1 and references therein for clinical details). Families provided informed consent to our study approved by the institutional review board of the Baylor College of Medicine. Subject 3 died at 8 years of age from bowel malrotation that led to volvulus and intestinal necrosis. An autopsy showed dramatic pancreatic hyperplasia (103 g for an expected weight of 15 g) with hyperplasia of some of the islets of Langerhans. An enlarged pancreas has never been described in other subjects, even though all other subjects in this study had abdominal ultrasounds. Other significant findings included kidney hypoplasia with multiple small subcapsullar cysts, a prominent suprapubic fat pad with underdeveloped clitoris and labia minora, and an anteriorly placed anus. Skeletal features included flat temporal bones, brachydactyly, flexion deformities of the hips and knees, and markedly hypoplastic patellae. Neuropathology showed microcephaly (851 g for an expected weight of 1,273 g); mild to moderate diffuse cortical atrophy; hypoplasia of the anterior portion of the corpus callosum; generalized mild gliosis; and small perivascular psammomatous calcifications in the basal ganglia, thalamus, corpus callosum, choroid plexus, and periventricular regions. See Figures 2A–2D for histology of some relevant tissues. Psammoma bodies are calcifications frequently seen in meningiomas and other malignancies (and only rarely in benign overgrowths) and are thought to result from calcification of dead cells or an active process to inhibit cell growth.12Das D.K. Psammoma body: A product of dystrophic calcification or of a biologically active process that aims at limiting the growth and spread of tumor?.Diagn. Cytopathol. 2009; 37: 534-541Crossref PubMed Scopus (105) Google ScholarTable 1Clinical Features of Enrolled Subjects and 13 Other Subjects from the LiteratureSubjects123456LiteratureaFrom Armstrong and Clarke,2 Bergmann et al.,3 Brugha et al.,4 Cormier-Daire et al.,5 Goldblatt et al.,6 Penttinen et al.,9 and Reardon10.Total (Affected/Total)Genderfemalemalefemalemalefemalemale4 females/9 males7 females/12 malesSkeletalbOccasional findings include osteoporosis, radioulnar synostosis, radial head deformity, brachydactyly, short stature, joint laxity, dislocated patellae, undertubulation of long bones, coxa vara, camptodactyly, narrow thorax, and exostoses.Absent or hypoplastic patellae++++++1218/19Flexion deformities++++++1319/19Club feet++++++1218/19Thoracolumbar kyphosis or scoliosis++35/19Pelvic anomalies++57/19Costal anomalies+++14/19NeurologicalcOccasional findings include hypotonia, hypertonia, seizures, and subdural hemorrhage (subject 5).Microcephaly++++++1319/19Developmental delay or intellectual disability++++++10/10dTen out of thirteen children survived beyond neonatal period and were included.16/16Absent or thin corpus callosum++++++915/19Colpocephaly or ventriculomegaly+++14/19Pachygyria+23/19Subependymal periventricular nodular heterotopia+12/19Optic atrophy or cortical visual impairment+++03/19Hearing loss++13/19Anal and GenitaleOccasional findings include rectal duplication (subject 6) and underdeveloped clitoris (subject 3).Anteriorly positioned anus++13/19Anal atresia or stenosis++02/19Hypoplastic labia minora or majora+++36/6Clitoromegaly++35/6Scrotal hypoplasia+++912/12Cryptochidism+++912/12RenalfOccasional findings include fused renal ectopia, dysplastic kidneys, and hypoplastic kidneys (subject 3).Hydronephrosis++++1116/19Multicystic kidneys++46/19CardiacgOccasional findings include tortuous ascending aorta, dilated aortic arch, patent ductus arteriosus, patent foramen ovale, and stenosis of the pulmonary valve.Atrial septal defect++++37/19Ventricular septal defect+34/19FacialhOccasional findings include bulbous nose, retrognatia or micrognatia, cleft or high-arched palate, gingival hyperplasia, coarse facies, full cheeks, ear anomalies, dental anomalies, sparse hair, hypertelorism, plagiocephaly, bitemporal flattening, downslanting palpebral fissures, downturned corners of the mouth, short columella, short philtrum, tented upper lip, and midface hypoplasia.Facial dysmorphisms++++++1319/19Broad nasal bridge++79/19Prominent nasal bridge++35/19OtheriOccasional findings include hypogonadotrophic hypogonadism, respiratory distress, apnea, recurrent infections, single palmar crease, and skin laxity.Tracheo or laryngomalacia++46/19Feeding difficulties++++26/19Small bowel malrotation+12/19Hypothyroidism+23/19a From Armstrong and Clarke,2Armstrong L. Clarke J.T.R. Report of a new case of “genitopatellar” syndrome which challenges the importance of absent patellae as a defining feature.J. Med. Genet. 2002; 39: 933-934Crossref PubMed Google Scholar Bergmann et al.,3Bergmann C. Spranger S. Javaher P. Ptok M. Genitopatellar syndrome, sensorineural hearing loss, and cleft palate.Oral Maxillofac Surg. 2011; 15: 103-106Crossref PubMed Scopus (4) Google Scholar Brugha et al.,4Brugha R. Kinali M. Aminu K. Bridges N. Holder S.E. Genitopatellar syndrome: A further case.Clin. Dysmorphol. 2011; 20: 163-165Crossref PubMed Scopus (6) Google Scholar Cormier-Daire et al.,5Cormier-Daire V. Chauvet M.L. Lyonnet S. Briard M.L. Munnich A. Le Merrer M. Genitopatellar syndrome: A new condition comprising absent patellae, scrotal hypoplasia, renal anomalies, facial dysmorphism, and mental retardation.J. Med. Genet. 2000; 37: 520-524Crossref PubMed Google Scholar Goldblatt et al.,6Goldblatt J. Wallis C. Zieff S. A syndrome of hypoplastic patellae, mental retardation, skeletal and genitourinary anomalies with normal chromosomes.Dysmorphol. Clin. Genet. 1988; 2: 91-93Google Scholar Penttinen et al.,9Penttinen M. Koillinen H. Niinikoski H. Mäkitie O. Hietala M. Genitopatellar syndrome in an adolescent female with severe osteoporosis and endocrine abnormalities.Am. J. Med. Genet. A. 2009; 149A: 451-455Crossref PubMed Scopus (18) Google Scholar and Reardon10Reardon W. Genitopatellar syndrome: A recognizable phenotype.Am. J. Med. Genet. 2002; 111: 313-315Crossref PubMed Scopus (25) Google Scholar.b Occasional findings include osteoporosis, radioulnar synostosis, radial head deformity, brachydactyly, short stature, joint laxity, dislocated patellae, undertubulation of long bones, coxa vara, camptodactyly, narrow thorax, and exostoses.c Occasional findings include hypotonia, hypertonia, seizures, and subdural hemorrhage (subject 5).d Ten out of thirteen children survived beyond neonatal period and were included.e Occasional findings include rectal duplication (subject 6) and underdeveloped clitoris (subject 3).f Occasional findings include fused renal ectopia, dysplastic kidneys, and hypoplastic kidneys (subject 3).g Occasional findings include tortuous ascending aorta, dilated aortic arch, patent ductus arteriosus, patent foramen ovale, and stenosis of the pulmonary valve.h Occasional findings include bulbous nose, retrognatia or micrognatia, cleft or high-arched palate, gingival hyperplasia, coarse facies, full cheeks, ear anomalies, dental anomalies, sparse hair, hypertelorism, plagiocephaly, bitemporal flattening, downslanting palpebral fissures, downturned corners of the mouth, short columella, short philtrum, tented upper lip, and midface hypoplasia.i Occasional findings include hypogonadotrophic hypogonadism, respiratory distress, apnea, recurrent infections, single palmar crease, and skin laxity. Open table in a new tab Figure 2Hematoxylin and Eosin Images of Tissues from the Autopsy of Subject 3Show full captionTissues shown are (A) the brain (400×) where we show a large cluster of perivascular calcifications in the white matter of the cerebral cortex (arrows point to two of them); these are never present in normal tissue; (B) the pancreas (100×) showing numerous islets of Langerhans, some of which are hyperplastic (black arrows), whereas others are of normal size (red arrows); (C) the kidney (100×), where we show one large and one small subcapsular cyst (arrows), and (D) the severely hypoplastic patella (100×), where there is persistence of the cartilaginous core of the trabeculae (black arrow).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Tissues shown are (A) the brain (400×) where we show a large cluster of perivascular calcifications in the white matter of the cerebral cortex (arrows point to two of them); these are never present in normal tissue; (B) the pancreas (100×) showing numerous islets of Langerhans, some of which are hyperplastic (black arrows), whereas others are of normal size (red arrows); (C) the kidney (100×), where we show one large and one small subcapsular cyst (arrows), and (D) the severely hypoplastic patella (100×), where there is persistence of the cartilaginous core of the trabeculae (black arrow). We performed whole-exome sequencing on three subjects (subjects 2, 4, and 5). For subjects 2 and 4, exomes were captured on Nimblegen's SeqCap EZ V2.0 library and sequencing was conducted on Illumina HiSeq. Sequences were aligned to the human reference genome (hg18) with Burrows-Wheeler Aligner (BWA) (v 0.5.9)13Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (26826) Google Scholar and recalibrated with the Genome Analysis Toolkit (GATK). Both samples achieved over 91% targeted bases at 20× coverage. SNPs and insertion-deletion events were called with Samtools Pileup (version 0.1.17).14Li H. Handsaker B. Wysoker A. Fennell T. Ruan J. Homer N. Marth G. Abecasis G. Durbin R. 1000 Genome Project Data Processing SubgroupThe Sequence Alignment/Map format and SAMtools.Bioinformatics. 2009; 25: 2078-2079Crossref PubMed Scopus (31781) Google Scholar Variants were annotated with ANNOVAR15Wang K. Li M. Hakonarson H. ANNOVAR: Functional annotation of genetic variants from high-throughput sequencing data.Nucleic Acids Res. 2010; 38: e164Crossref PubMed Scopus (7927) Google Scholar and protein-impacting variants that were rare (minor allele frequency < 5%), novel, and nonsynonymous were preferentially explored. We narrowed gene candidates by comparing with known functional databases such as dbNSFP16Liu X. Jian X. Boerwinkle E. dbNSFP: A lightweight database of human nonsynonymous SNPs and their functional predictions.Hum. Mutat. 2011; 32: 894-899Crossref PubMed Scopus (513) Google Scholar and SWISS-PROT17UniProt ConsortiumThe Universal Protein Resource (UniProt) in 2010.Nucleic Acids Res. 2010; 38: D142-D148Crossref PubMed Scopus (1006) Google Scholar to narrow down the list of plausible causative variants. For subject 5, the exome was captured on the Agilent SureSelect 50 Mb oligonucleotide library. DNA was sheared by sonication to an approximately 200 bp length. Fragment ends were ligated to specific adaptors and capture was performed with the manufacturer's protocol. The captured exome was reamplified by PCR (12 cycles) then applied to a single lane of Illumina HiSeq sequencer. The Illumina reads were aligned to the reference human genome (hg19) with BWA (v. 0.5.9) and Samtools (v. 0.1.12a). Pileup and varFilter commands were used to call variants, and these were filtered to retain SNPs and insertion-deletions with Phred-like quality scores of at least 20 and 50, respectively. ANNOVAR was used to annotate nonsynonymous variants according to the type of mutation, occurrence in dbSNP, SIFT score,18Ng P.C. Henikoff S. SIFT: Predicting amino acid changes that affect protein function.Nucleic Acids Res. 2003; 31: 3812-3814Crossref PubMed Scopus (4096) Google Scholar and 1000 Genomes allele frequency.19Marth G.T. Yu F. Indap A.R. Garimella K. Gravel S. Leong W.F. Tyler-Smith C. Bainbridge M. Blackwell T. Zheng-Bradley X. et al.the 1000 Genomes ProjectThe functional spectrum of low-frequency coding variation.Genome Biol. 2011; 12: R84Crossref PubMed Scopus (160) Google Scholar As shown in Table 2, only 13 genes showed rare novel variants that were potentially pathogenic and were shared by the three subjects. Variants were visualized and compared to the exomes of 20 other subjects with unrelated conditions. When keeping only high-quality variants (e.g., removing probable false positive variants in repeat regions or variants seen in unrelated conditions), only KAT6B variants remained. All variants were frameshift insertions-deletions (Figure 3, Figures S2A–2C, available online, and Table 3; RefSeq NM_012330.2 was used for the positions). We confirmed the variants by Sanger sequencing and sequenced the complete coding sequence of the gene in the other individuals recruited in our study (see Table S1 for primers). We have thus identified nonsense mutations in two additional subjects and one of the previously identified frameshift deletions in another (Figure 3 and Figures S2D–2F). Analysis of parental samples from five subjects showed that the mutations were acquired de novo (Figure S2). All mutations lead to a loss of the highly conserved transcription activation domain (Figure 3 and Figure S3). The Exome Variant Server has public information on KAT6B for over 1,100 individuals of European descent and 900 African Americans, with an average coverage of over 85× for the coding sequences of KAT6B. No truncating mutations of KAT6B were identified in this server or in other exomes performed by Baylor College of Medicine's Human Genome Sequencing Center.Table 2Number of Variants IdentifiedSubject 2Subject 4Subject 5Shared (2 of 3)Shared (3 of 3)Total variants26540262760221490885Total variants after base quality filtering10901741115186256344Novel variants (dbSNP129/1000G)872368886839237419Genes with rare nonsynonymous variants, splice site variants, insertions or deletions variants in coding regions.4403672737613 Open table in a new tab Table 3Mutations Identified in the SubjectsSubjectMutation (DNA)Mutation (Protein)Parents TestedReference1c.3892G>Tp.Gly1298∗not availablesubject 1 in Abdul-Rahman et al.1Abdul-Rahman O.A. La T.H. Kwan A. Schlaubitz S. Barsh G.S. Enns G.M. Hudgins L. Genitopatellar syndrome: Expanding the phenotype and excluding mutations in LMX1B and TBX4.Am. J. Med. Genet. A. 2006; 140: 1567-1572Crossref PubMed Scopus (12) Google Scholar See also Schlaubitz et al.11Schlaubitz S. Yatsenko S.A. Smith L.D. Keller K.L. Vissers L.E. Scott D.A. Cai W.W. Reardon W. Abdul-Rahman O.A. Lammer E.J. et al.Ovotestes and XY sex reversal in a female with an interstitial 9q33.3-q34.1 deletion encompassing NR5A1 and LMX1B causing features of Genitopatellar syndrome.Am. J. Med. Genet. A. 2007; 143A: 1071-1081Crossref PubMed Scopus (39) Google Scholar2c.4360_4368delinsAAAAACCAAAAp.Glu1454LysfsX8de novosubject 2 in in Abdul-Rahman et al.1Abdul-Rahman O.A. La T.H. Kwan A. Schlaubitz S. Barsh G.S. Enns G.M. Hudgins L. Genitopatellar syndrome: Expanding the phenotype and excluding mutations in LMX1B and TBX4.Am. J. Med. Genet. A. 2006; 140: 1567-1572Crossref PubMed Scopus (12) Google Scholar See also Schlaubitz et al.11Schlaubitz S. Yatsenko S.A. Smith L.D. Keller K.L. Vissers L.E. Scott D.A. Cai W.W. Reardon W. Abdul-Rahman O.A. Lammer E.J. et al.Ovotestes and XY sex reversal in a female with an interstitial 9q33.3-q34.1 deletion encompassing NR5A1 and LMX1B causing features of Genitopatellar syndrome.Am. J. Med. Genet. A. 2007; 143A: 1071-1081Crossref PubMed Scopus (39) Google Scholar3c.3802G>Tp.Gly1268∗de novoLammer and Abrams7Lammer E.J. Abrams L. Genitopatellar syndrome: Delineating the anomalies of female genitalia.Am. J. Med. Genet. 2002; 111: 316-318Crossref PubMed Scopus (12) Google Scholar Schlaubitz et al.11Schlaubitz S. Yatsenko S.A. Smith L.D. Keller K.L. Vissers L.E. Scott D.A. Cai W.W. Reardon W. Abdul-Rahman O.A. Lammer E.J. et al.Ovotestes and XY sex reversal in a female with an interstitial 9q33.3-q34.1 deletion encompassing NR5A1 and LMX1B causing features of Genitopatellar syndrome.Am. J. Med. Genet. A. 2007; 143A: 1071-1081Crossref PubMed Scopus (39) Google Scholar4c.3769_3772delTCTAp.Lys1258GlyfsX13de novoLifchez et al.8Lifchez C.A. Rhead W.J. Leuthner S.R. Lubinsky M.S. Genitopatellar syndrome: Expanding the phenotype.Am. J. Med. Genet. A. 2003; 122A: 80-83Crossref PubMed Scopus (16) Google Scholar5c.3788_3789delAAp.Lys1263ArgfsX7de novothis report6c.3769_3772delTCTAp.Lys1258GlyfsX13de novothis report Open table in a new tab Lymphoblastoid cells were established by Epstein-Barr virus infection for subjects 1 through 4. One million cells were collected, and RNA was extracted with Trizol, treated with DNase I, then phenol-chloroform extracted. The first strand of cDNA was synthesized with oligo dT primers via Invitrogen's SuperScript III First-Strand synthesis kit. For RT-PCR, a 5′ nuclease assay from Integrated DNA Technologies (Coralville, IA) was designed with probes having a 5′ fluorescein amidite fluorophore, a 3′ IBFQ quencher, and an internal ZEN quencher (see Table S1). Quantitative real-time PCR was performed in an ABI 7900 HT machine with ABI's TaqMan Universal PCR Master Mix according to the manufacturer's instructions (Figure 4A ). We also amplified cDNA by using primers encompassing the last exon-exon junction and the most 5′ mutations and sequenced the products (see Table S1 for primers). These experiments demonstrate that the mutant mRNAs do not undergo nonsense-mediated decay (Figures 4A and 4B), which is consistent with localization of the premature stop codons in the last exon. To assess the expression pattern of Myst4 in organs known to be affected by GPS, we performed immunohistochemistry on mice of various developmental ages. The primary antibody used is Sigma AV38985 (1:200 dilution), the secondary is Invitrogen A-11012 (1:600 dilution). The specificity of the antibody for the mouse protein was confirmed by showing a staining pattern in the brain compatible with published RNA in situ experiments20Thomas T. Voss A.K. Chowdhury K. Gruss P. Querkopf, a MYST family histone acetyltransferase, is required for normal cerebral cortex development.Development. 2000; 127: 2537-2548PubMed Google Scholar (see Figure 5A ). Myst4 is strongly expressed in the telencephalic vesicles, trigeminal ganglion, spinal cord, dorsal root ganglia, digestive tract, pancreas liver and ribs of developing embryos (Figures 5A and 5B). After birth, it is strongly expressed in the diaphysis of the long bones, the kidney, and the patella, among other organs (Figures 5C–5K).Figure 5Myst4 Expression in Wild-Type C57BL/6 Mice Detected by ImmunohistochemistryShow full captionTissues shown are (A) and (B) whole embryos at embryonic day 15.5, (C) femur at postnatal day 1, and (D) kidney at postnatal day 1. The other tissues are at 8 weeks of age: (E) patella, (F) duodenum (which required 2.5 s of exposure time instead of 5), (G) liver, (H) pancreas, (I) spleen, (J) testis, and (K) ovary. Arrows point to the telencephalic vesicles (tv), spinal cord (sc), liver, pancreas, dorsal root ganglia (drg), trigeminal ganglion (tg), ribs, and patella (pat).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Tissues shown are (A) and (B) whole embryos at embryonic day 15.5, (C) femur at postnatal day 1, and (D) kidney at postnatal day 1. The other tissues are at 8 weeks of age: (E) patella, (F) duodenum (which required 2.5 s of exposure time instead of 5), (G) liver, (H) pancreas, (I) spleen, (J) testis, and (K) ovary. Arrows point to the telencephalic vesicles (tv), spinal cord (sc), liver, pancreas, dorsal root ganglia (drg), trigeminal ganglion (tg), ribs, and patella (pat). KAT6B has a highly conserved acetyltransferase domain21Champagne N. Bertos N.R. Pelletier N. Wang A.H. Vezmar M. Yang Y. Heng H.H. Yang X.J. Identification of a human histone acetyltransferase related to monocytic leukemia zinc finger protein.J. Biol. Chem. 1999; 274: 28528-28536Crossref PubMed Scopus (131) Google Scholar and has been shown to fuse with p300 and CBP following chromosomal translocations in acute myeloid leukemia and myelodysplastic syndrome.22Yang X.J. Ullah M. MOZ and MORF, two large MYSTic HATs in normal and cancer stem cells.Oncogene. 2007; 26: 5408-5419Crossref PubMed Scopus (111) Google Scholar KAT6B has been reported to interact with the RUNX family of transcription factors22Yang X.J. Ullah M. MOZ and MORF, two large MYSTic HATs in normal and cancer stem cells.Oncogene. 2007; 26: 5408-5419Crossref PubMed Scopus (111) Google Scholar and form a tetrameric complex with BRPFs, ING5, and EAF6.23Doyon Y. Cayrou C. Ullah M. Landry A.-J. Côté V. Selleck W. Lane W.S. Tan S. Yang X.-J. Côté J. ING tumor suppressor proteins are critical regulators of chromatin acetylation required for genome expression and perpetuation.Mol. Cell. 2006; 21: 51-64Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar, 24Ullah M. Pelletier N. Xiao L. Zhao S.P. Wang K. Degerny C. Tahmasebi S. Cayrou C. Doyon Y. Goh S.-L. et al.Molecular architecture of quartet MOZ/MORF histone acetyltransferase complexes.Mol. Cell. Biol. 2008; 28: 6828-6843Crossref PubMed Scopus (158) Google Scholar KAT6B was pulled down in a PPAR-alpha interacting cofactor complex,25Surapureddi S. Yu S. Bu H. Hashimoto T. Yeldandi A.V. Kashireddy P. Cherkaoui-Malki M. Qi C. Zhu Y.-J. Rao M.S. Reddy J.K. Identification of a transcriptionally active peroxisome proliferator-activated receptor alpha -interacting cofactor complex in rat liver and characterization of PRIC285 as a coactivator.Proc. Natl. Acad. Sci. USA. 2002; 99: 11836-11841Crossref PubMed Scopus (106) Google Scholar and a yeast two-hybrid screen identified Atrophin-1 as a binding partner of KAT6B.26Lim J. Hao T. Shaw C. Patel A.J. Szabó G. Rual J.-F. Fisk C.J. Li N. Smolyar A. Hill D.E. et al.A protein-protein interaction network for human inherited ataxias and disorders of Purkinje cell degeneration.Cell. 2006; 125: 801-814Abstract Full Text Full Text PDF PubMed Scopus (635) Google Scholar Independently to its cloning in humans, the mouse ortholog of KAT6B was identified by screening a gene-trap library for genes highly expressed in the telencephalon.20Thomas T. Voss A.K. Chowdhury K. Gruss P. Querkopf, a MYST family histone acetyltransferase, is required for normal cerebral cortex development.Development. 2000; 127: 2537-2548PubMed Google Scholar Mice carrying a hypomorphic mutation of Myst4 have short stature, an absence of fusion of the tibia and fibula, microcephaly with neurogenesis defects, early demise, and infertility.27Voss A.K. Thomas T. MYST family histone acetyltransferases take center stage in stem cells and development.Bioessays. 2009; 31: 1050-1061Crossref PubMed Scopus (86) Google Scholar A subject with a Noonan-like phenotype has recently been identified to harbor a chromosomal translocation disrupting KAT6B after exon 3, and the mRNA levels in lymphoblastoid cells were half of normal.28Kraft M. Cirstea I.C. Voss A.K. Thomas T. Goehring I. Sheikh B.N. Gordon L. Scott H. Smyth G.K. Ahmadian M.R. et al.Disruption of the histone acetyltransferase MYST4 leads to a Noonan syndrome-like phenotype and hyperactivated MAPK signaling in humans and mice.J. Clin. Invest. 2011; 121: 3479-3491Crossref PubMed Scopus (68) Google Scholar Given the phenotypic difference between the subject with a Noonan-like phenotype and subjects with GPS (Table 4), the persistent expression of a truncated protein in GPS, containing intact N-terminal domain and HAT domains but lacking the C-terminal transcriptional activation domain, most likely leads to dominant-negative or gain-of-function effects on cellular signaling. Of relevance, leukemia-associated translocations also generate similar KAT6B fragments fused to p300 and CBP.22Yang X.J. Ullah M. MOZ and MORF, two large MYSTic HATs in normal and cancer stem cells.Oncogene. 2007; 26: 5408-5419Crossref PubMed Scopus (111) Google Scholar Rubinstein-Taybi syndrome [MIM 180849] is caused by de novo mutations inactivating the histone acetyltransferase activity of the latter two enzymes, and both haploinsufficiency and dominant-negative models have been postulated for this condition.29Petrij F. Giles R.H. Dauwerse H.G. Saris J.J. Hennekam R.C. Masuno M. Tommerup N. van Ommen G.J. Goodman R.H. Peters D.J. et al.Rubinstein-Taybi syndrome caused by mutations in the transcriptional co-activator CBP.Nature. 1995; 376: 348-351Crossref PubMed Scopus (1023) Google Scholar, 30Oike Y. Hata A. Mamiya T. Kaname T. Noda Y. Suzuki M. Yasue H. Nabeshima T. Araki K. Yamamura K. Truncated CBP protein leads to classical Rubinstein-Taybi syndrome phenotypes in mice: Implications for a dominant-negative mechanism.Hum. Mol. Genet. 1999; 8: 387-396Crossref PubMed Scopus (271) Google Scholar Although the presentation is distinct from GPS, the two conditions share some facial features, as well as intellectual disability, malformations of the heart and kidneys, and undescended testes. Recently, similar de novo truncating in KAT6B mutations were identified in subjects with the Say-Barber-Biesecker variant of Ohdo syndrome [MIM 249620], which overlaps with Genitopatellar syndrome in terms of facial features and congenital heart defects.31Clayton-Smith J. O'Sullivan J. Daly S. Bhaskar S. Day R. Anderson B. Voss A.K. Thomas T. Biesecker L.G. Smith P. et al.Whole-exome-sequencing identifies mutations in histone acetyltransferase gene KAT6B in individuals with the Say-Barber-Biesecker variant of Ohdo syndrome.Am. J. Hum. Genet. 2011; 89: 675-681Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 32Day R. Beckett B. Donnai D. Fryer A. Heidenblad M. Howard P. Kerr B. Mansour S. Maye U. McKee S. et al.A clinical and genetic study of the Say/Barber/Biesecker/Young-Simpson type of Ohdo syndrome.Clin. Genet. 2008; 74: 434-444Crossref PubMed Scopus (21) Google Scholar The mutations are however usually located more distally in the C terminus, specifically in the transcriptional activation domain, and there are several clinical differences: structural brain defects, skeletal defects, anal anomalies, genital anomalies and renal defects are more severe or frequent in GPS, whereas ocular, dental, palatal, and thyroid defects are more severe or frequent in the Say-Barber-Biesecker variant of Ohdo syndrome. A comparison of the phenotypes of GPS, the Say-Barber-Biesecker variant of Ohdo syndrome, the child with a translocation involving KAT6B, and Rubinstein-Taybi syndrome is shown in Table 4.Table 4Phenotypic Differences between GPS, the Say-Barber-Biesecker Variant of Ohdo Syndrome, a Subject with a Translocation Truncating KAT6B after Exon 3, and Rubinstein-Taybi SyndromeGenitopatellar SyndromeSay-Barber-Biesecker Variant of Ohdo SyndromeSubject with Noonan-Like Phenotype and N-Terminal Truncation of KAT6BRubinstein Taybi SyndromeNeurological anomaliesDD/ID,aDD/ID is used as an abbreviation for developmental delay or intellectual disability. microcephaly in all, agenesis of the corpus callosum, colpocephalyDD/ID, microcephaly in minority, hypotonia, no structural defectsmicrocephaly, ADHD,bADHD is used as an abbreviation for Attention deficit hyperactivity disorder. IQ 75-80, no structural defectsDD/ID, seizures, no structural defectsFacial anomaliesbroad or prominent nasal bridge, bulbous nose in minority, full cheeks in minorityblepharophimosis, ptosis, broad and flat nasal bridge, bulbous nose, full cheeks, abnormal ears, small mouth, expressionless or mask-like faciesblepharophimosis, ptosis, arched eyebrows, abnormal ears, smooth philtrum, retrognatia, high-arched palatearched eyebrows, downslanting palpebral fissures, beaked nose with the columella extending below the nares, high-arched palate, mild micrognathia, grimacing smileMusculo-skeletal anomaliesabsent or hypoplastic patellae in majority, flexion contractures, club feet, costo-vertebral anomalies, pelvic anomalieslong thumbs and toes, patellar anomalies in minorityshort stature, retarded bone age, ligamentous laxityshort stature, joint hypermobility, broad thumbs and broad big toesAnal and genital anomaliesanal anomalies, hypoplastic labia, clitoromegaly, scrotal hypoplasia, cryptorchidismcryptorchydism and hypospadiasHeart anomaliescongenital heart defectscongenital heart defectscongenital heart defectsStructural eye defectsnonefrequentfrequentDental anomaliesrarefrequentCleft palaterarefrequentHearing impairmentrarefrequentThyroid abnormalitiesrarefrequentRenal anomalieshydronephrosis or cysts in a majorityvesicoureteric reflux in one individualOtherfeeding difficulties, tracheomalacia, respiratory difficulties, small bowel malrotationfeeding difficultiesfeeding difficulties, respiratory difficulties, skin anomalies (hirsutism, naevus flammeus on the forehead, and keloid formation), malignanciesa DD/ID is used as an abbreviation for developmental delay or intellectual disability.b ADHD is used as an abbreviation for Attention deficit hyperactivity disorder. Open table in a new tab We have thus identified mutations in the epigenetic regulator KAT6B in several subjects with GPS. Because this acetyltransferase is a ubiquitous transcriptional coactivator, searching for more binding partners and studying its role in skeletogenesis and development in general might lead to important insights into epigenetic dysregulation in GPS and related diseases. We thank the families for participating in this study. We thank Alyssa Tran, Stephanie Dugan, and Andrea Kwan for help enrolling subjects; Kyu Sang Joeng, Yangjin Bae, Jianning Tao, and Terry Bertin for help and advice with the experiments; and the University Center for Fetal Medicine of the University of Mississippi for help collecting clinical information. Philippe Campeau is funded in part by the Clinician-Scientist Training award of the Canadian Institutes of Health Research. Download .pdf (1.31 MB) Help with pdf files Document S1. Figures S1–S3 and Table S1 The URLs for data presented herein are as follows:1000 Genomes, http://www.1000genomes.org/ANNOVAR, http://www.openbioinformatics.org/annovar/BWA, http://bio-bwa.sourceforge.net/dbNSFP, https://sites.google.com/site/jpopgen/dbNSFPdbSNP, http://www.ncbi.nlm.nih.gov/projects/SNP/Exome Variant Server, http://evs.gs.washington.edu/EVS/GATK, http://www.broadinstitute.org/gsa/wiki/index.php/The_Genome_Analysis_ToolkitOnline Mendelian Inheritance in Man (OMIM), http://www.omim.orgPolyphen-2, http://genetics.bwh.harvard.edu/pph2/Samtools Pileup, http://samtools.sourceforge.net/Swiss-Prot, http://web.expasy.org/docs/swiss-prot_guideline.htmlSIFT, http://sift.jcvi.org/UCSC Genome Browser (hg18 and hg19), http://genome.ucsc.edu/ De Novo Mutations of the Gene Encoding the Histone Acetyltransferase KAT6B Cause Genitopatellar SyndromeSimpson et al.The American Journal of Human GeneticsJanuary 19, 2012In BriefGenitopatellar syndrome (GPS) is a rare disorder in which patellar aplasia or hypoplasia is associated with external genital anomalies and severe intellectual disability. Using an exome-sequencing approach, we identified de novo mutations of KAT6B in five individuals with GPS; a single nonsense variant and three frameshift indels, including a 4 bp deletion observed in two cases. All identified mutations are located within the terminal exon of the gene and are predicted to generate a truncated protein product lacking evolutionarily conserved domains. Full-Text PDF Open Archive

Dysregulation of AMPK signaling has been implicated in many human diseases, which emphasizes the importance of characterizing AMPK regulators. The tumor suppressor FLCN, responsible for the Birt-Hogg Dubé renal neoplasia syndrome (BHD), is an AMPK-binding partner but the genetic and functional links between FLCN and AMPK have not been established. Strikingly, the majority of naturally occurring FLCN mutations predisposing to BHD are predicted to produce truncated proteins unable to bind AMPK, pointing to the critical role of this interaction in the tumor suppression mechanism. Here, we demonstrate that FLCN is an evolutionarily conserved negative regulator of AMPK. Using Caenorhabditis elegans and mammalian cells, we show that loss of FLCN results in constitutive activation of AMPK which induces autophagy, inhibits apoptosis, improves cellular bioenergetics, and confers resistance to energy-depleting stresses including oxidative stress, heat, anoxia, and serum deprivation. We further show that AMPK activation conferred by FLCN loss is independent of the cellular energy state suggesting that FLCN controls the AMPK energy sensing ability. Together, our data suggest that FLCN is an evolutionarily conserved regulator of AMPK signaling that may act as a tumor suppressor by negatively regulating AMPK function.

A reconstructed skin made of a collagen-chitosan sponge seeded with human fibroblasts and keratinocytes and grown in vitro for 31 days was developed for the treatment of deep and extensive burns. The aim of this study was to assess whether this tissue-engineered skin could promote nerve regeneration in vivo, since recovery of sensation is a major concern for burnt patients. The human reconstructed skin was transplanted on the back of nude mice and the growth of nerve fibres within it was assessed 40, 60, 90 and 120 days after graft. Nerve growth was monitored by confocal microscopy using immunohistochemical staining of PGP 9.5 and 150 kD neurofilament, while Schwann cell migration was observed using protein S100 expression and laminin deposition. Nerve growth was first detected 60 days after transplantation and was more abundant 90 and 120 days after graft. Linear arrangements of Schwann cells were observed in the graft as early as 40 days after graft. Nerve growth was observed along these Schwann cell extensions 60 days after transplantation. We conclude that the three-dimensional architecture of the collagen-chitosan tissue-engineered skin sponge encourages nerve growth. This result provides new perspectives to increase nerve regeneration within the tissue-engineered skin by linkage of neurotrophic factors in the sponge before transplantation.

Neuron-glial interactions are important in development of the nervous system and pathogenesis of disease. Primary cell cultures prepared from nervous tissue are often used to study the properties of individual cell types and how they interact with each other. Isolation of pure populations of cells and their culture is challenging, particularly from murine spinal cord. The purpose of this study was to optimize various protocols to achieve efficient, parallel isolation and purification of primary motor neurons, microglia and astrocytes from the same mouse embryonic spinal cord sample. Following dissociation of E12 embryonic spinal cords, motor neurons were isolated at 97% purity by a single step centrifugation of the cell suspension through multiple discontinuous density gradients of NycoPrep. The residual mixed cell pellet was resuspended and cultured for 2 weeks. Mixed cultures were then shaken to release microglia, which were then harvested from the medium and subjected to another round of differential adhesion to achieve 99% purity. The astrocytes remaining in the mixed cultures were culled to 98% purity by treatment with leucine methyl ester and a subsequent vigorous shaking step to remove any remaining microglia and neurons. Furthermore, no cross contamination was observed in the glial cultures. This technique provides a simple, convenient, and reliable method of obtaining highly purified preparations of motor neurons, microglia and astrocytes from embryonic spinal cord for the study of spinal cord cell biology and motor neuron diseases.

As we enter the era of precision medicine, characterization of cancer genomes will directly influence therapeutic decisions in the clinic. Here we describe a platform enabling functionalization of rare gene mutations through their high-throughput construction, molecular barcoding and delivery to cancer models for in vivo tumour driver screens. We apply these technologies to identify oncogenic drivers of pancreatic ductal adenocarcinoma (PDAC). This approach reveals oncogenic activity for rare gene aberrations in genes including NAD Kinase (NADK), which regulates NADP(H) homeostasis and cellular redox state. We further validate mutant NADK, whose expression provides gain-of-function enzymatic activity leading to a reduction in cellular reactive oxygen species and tumorigenesis, and show that depletion of wild-type NADK in PDAC cell lines attenuates cancer cell growth in vitro and in vivo. These data indicate that annotating rare aberrations can reveal important cancer signalling pathways representing additional therapeutic targets.

Primary motor neurons are difficult to study in conventional culture systems because of their short-term survival without trophic support from glia. In addition, axonal migration on a two-dimensional Petri dish does not reflect the three-dimensional (3D) environment in vivo. A unique in vitro 3D model of motor nerve regeneration was developed to study motor neuron axonal migration and myelination. Mouse spinal cord motor neurons were seeded on a collagen sponge populated with Schwann cells and fibroblasts. This fibroblast-populated sponge was intended to mimic the connective tissue through which motor axons have to elongate in vivo. Addition of conventional neurotrophic supplements was not required for motor neuron survival but was necessary to promote deep neurite outgrowth, as assessed by immunostaining of neurofilament M. A vigorous neurite elongation was detected inside the sponge after only 14 days of neuron culture, reaching more than 850 microm. The model also allowed the maturation of motor fibers as one-third of them were positive for neurofilament H. Neurites growing in the sponge were subject to myelination when Schwann cells were present, as shown by myelin basic protein immunostaining and electron microscopy. We demonstrated in this model the spontaneous formation of numerous thick myelin sheaths surrounding motor fibers after long-term culture (28 days). Thus, this model might be a valuable tool to study the effect of various cells and/or attractive or repulsive molecules on motor neurite outgrowth in vitro and also for the study of myelination and pathogenesis of motor neuron diseases.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTGlucocorticosteroid suppression of .alpha.1-fetoprotein synthesis in developing rat liver. Evidence for selective gene repression at the transcriptional levelLuc Belanger, Monique Frain, Pierre Baril, Marie Claude Gingras, Jacek Bartkowiak, and Jose M. Sala-TrepatCite this: Biochemistry 1981, 20, 23, 6665–6672Publication Date (Print):November 1, 1981Publication History Published online1 May 2002Published inissue 1 November 1981https://doi.org/10.1021/bi00526a022RIGHTS & PERMISSIONSArticle Views28Altmetric-Citations46LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

Pancreatic cancer is a multiple genetic disorder with many mutations identified during the progression. Two mouse pancreatic cancer cell lines were established which showed different phenotype in vivo: a non-metastatic cell line, Panc02, and a highly metastatic cell line, Panc02-H7, a derivative of Panc02. In order to investigate whether the genetic mutations of key genes in pancreatic cancer such as KRAS, TP53 (p53), CDKN2A (p16), SMAD4, ZIP4, and PDX-1 contribute to the phenotypic difference of these two mouse pancreatic cancer cells, we sequenced the exonic regions of these key genes in both cell lines and in the normal syngeneic mouse pancreas and compared them with the reference mouse genome sequence. The exons of KRAS, SMAD4, CDKN2A (p16), TP53 (p53), ZIP4, and PDX-1 genes were amplified and the genotype of these genes was determined by Sanger sequencing. The sequences were analyzed with Sequencher software. A mutation in SMAD4 was identified in both cell lines. This homozygote G to T mutation in the first position of codon 174 (GAA) generated a stop codon resulting in the translation of a truncated protein. Further functional analysis indicates that different TGF-β/SMAD signaling pathways were involved in those two mouse cell lines, which may explain the phonotypic difference between the two cells. A single nucleotide polymorphism (SNP) in KRAS gene (TAT to TAC at codon 32) was also identified in the normal pancreas DNA of the syngenic mouse and in both derived tumoral Panc02 and Panc02-H7 cells. No mutation or SNP was found in CDKN2A (p16), TP53 (p53), ZIP4, and PDX-1 genes in these two cell lines. The absence of mutations in genes such as KRAS, TP53, and CDKN2A, which are considered as key genes in the development of human pancreatic cancer suggests that SMAD4 might play a central and decisive role in mouse pancreatic cancer. These results also suggest that other mechanisms are involved in the substantial phenotypic difference between these two mouse pancreatic cancer cell lines. Further studies are warranted to elucidate the molecular pathways that lead to the aggressive metastatic potential of Panc02-H7.

Mechanisms of adaptation to environmental changes in osmolarity are fundamental for cellular and organismal survival. Here we identify a novel osmotic stress resistance pathway in Caenorhabditis elegans (C. elegans), which is dependent on the metabolic master regulator 5'-AMP-activated protein kinase (AMPK) and its negative regulator Folliculin (FLCN). FLCN-1 is the nematode ortholog of the tumor suppressor FLCN, responsible for the Birt-Hogg-Dubé (BHD) tumor syndrome. We show that flcn-1 mutants exhibit increased resistance to hyperosmotic stress via constitutive AMPK-dependent accumulation of glycogen reserves. Upon hyperosmotic stress exposure, glycogen stores are rapidly degraded, leading to a significant accumulation of the organic osmolyte glycerol through transcriptional upregulation of glycerol-3-phosphate dehydrogenase enzymes (gpdh-1 and gpdh-2). Importantly, the hyperosmotic stress resistance in flcn-1 mutant and wild-type animals is strongly suppressed by loss of AMPK, glycogen synthase, glycogen phosphorylase, or simultaneous loss of gpdh-1 and gpdh-2 enzymes. Our studies show for the first time that animals normally exhibit AMPK-dependent glycogen stores, which can be utilized for rapid adaptation to either energy stress or hyperosmotic stress. Importantly, we show that glycogen accumulates in kidneys from mice lacking FLCN and in renal tumors from a BHD patient. Our findings suggest a dual role for glycogen, acting as a reservoir for energy supply and osmolyte production, and both processes might be supporting tumorigenesis.

Increasing evidence suggests the existence of polarized human T cell responses described as Th1-type (promoting cell-mediated immunity) and Th2-type (promoting humoral immunity), characterized by a dominant production of either interferon-gamma (IFN-gamma) or IL-4, respectively. Little is known about the intratumoural activation of infiltrating lymphocytes (TIL) in human gliomas. Therefore, we assessed fresh TIL at cellular and molecular levels to find out if they were activated and polarized into a type 1 or 2 immune response. Flow cytometry analysis of TIL revealed that the major subset was made of T lymphocytes. Double labelling with alpha-CD3 and adhesion/ activation markers revealed T cell subsets expressing CD49a, CD49b, CD54, and CD15, some of which were almost absent in autologous T peripheral blood lymphocytes (T-PBL). Furthermore, the proportions of T-TIL expressing CD56, CD65, or CD25 were several-fold higher than in T-PBL. Intratumoural functional activation of TIL was tested by semiquantitative assessment in relative units (RU) of lymphokine gene activation with mRNA reverse transcriptase-polymerase chain reaction (RT-PCR). All TIL populations except one significantly expressed IL-4 1 to 2 logs of RU above healthy PBL baseline. Similarly, all patients expressed granulocyte-macrophage colony-stimulating factor (GM-CSF) in a range comparable to IL-4. However, most TIL populations did not express IFN-gamma, IL-2, and tumour necrosis factor-beta (TNF-beta) at higher levels than healthy normal PBL. The increase proportion of T cells expressing activation markers and the consistent detection of significant IL-4 and GM-CSF lymphokine gene activation in TIL populations suggested a predominant type 2 intratumoural immune response that does not promote cell-mediated tumouricidal activity and may contribute to the inefficiency of the antiglioma immune response.

Abstract The isolation of autologous neuronal precursors from skin‐derived precursor cells extracted from adult human skin would be a very efficient source of neurons for the treatment of various neurodegenerative diseases. The purpose of this study was to demonstrate that these neuronal precursors were able to differentiate into mature neurons. We isolated neuronal precursors from breast skin and expanded them in vitro for over ten passages. We showed that 48% of these cells were proliferating after the first passage, while this growth rate decreased after the second passage. We demonstrated that 70% of these cells were nestin‐positive after the third passage, while only 17% were neurofilament M‐positive after 7 days of differentiation. These neuronal precursors expressed βIII tubulin, the dendritic marker MAP2 and the presynaptic marker synaptophysin after 7 days of in vitro maturation. They also expressed the postsynaptic marker PSD95 and the late neuronal markers NeuN and neurofilament H after 21 days of differentiation, demonstrating they became terminally differentiated neurons. These markers were still expressed after 50 days of culture. The generation of autologous neurons from an accessible adult human source opens many potential therapeutic applications and has a great potential for the development of experimental studies on normal human neurons. J. Cell. Physiol. 210: 498–506, 2007. © 2006 Wiley‐Liss, Inc.

Objectives: The purpose of this study was to investigate whether pancreatic and duodenal homeobox factor 1 (PDX-1) could serve as a potential molecular target for the treatment of pancreatic cancer. Methods: Cell proliferation, invasion capacity, and protein levels of cell cycle mediators were determined in human pancreatic cancer cells transfected with mouse PDX-1 (mPDX-1) alone or with mPDX-1 short hairpin RNA (shRNA) and/or human PDX-1 shRNA (huPDX-1 shRNA). Tumor cell growth and apoptosis were also evaluated in vivo in PANC-1 tumor-bearing severe combined immunodeficient mice receiving multiple treatments of intravenous liposomal huPDX-1 shRNA. Results: mPDX-1 overexpression resulted in the significant increase of cell proliferation and invasion in MIA PaCa2, but not PANC-1 cells. This effect was blocked by knocking down mPDX-1 expression with mPDX-1 shRNA. Silencing of huPDX-1 expression in PANC-1 cells inhibited cell proliferation in vitro and suppressed tumor growth in vivo which was associated with increased tumor cell apoptosis. PDX-1 overexpression resulted in dysregulation of the cell cycle with up-regulation of cyclin D, cyclin E, and Cdk2 and down-regulation of p27. Conclusions: PDX-1 regulates cell proliferation and invasion in human pancreatic cancer cells. Down-regulation of PDX-1 expression inhibits pancreatic cancer cell growth in vitro and in vivo, implying its use as a potential therapeutic target for the treatment of pancreatic cancer.

We have examined the possible role of transforming growth factor-β (TGF-β) in metastatic malignancy by analyzing the production and activation of TGF-β, and -β2 and the regulation of TGF-β-responsive genes in oncogene-transformed metastatic fibrosarcomas. All transformed lines derived from either 10T1/2; or N1H 3T3 bv either H-ras or protein-kinase encoding oncogenes produced more TGF-β than parental cells. However, onlv highlv metastatic fibrosarcomas secreted activated TGF-β at rates that were greater than parental fibroblasts. Immunohistochemical staining for TGF-β, showed widespread intra- and extracellular distribution in metastatic lung nodules and adjacent tissue. Cells isolated from tumors successfully metastasizing to the lung had TGF-β1, mRNA levels which were increased 19-fold over in ritro controls. Despite the greatly enhanced rate of secretion of activated TGF-β, metastatic cells exhibited markedly altered responses of TGF-β1, and TGF-β2., being unable to either increase collagen secretion or enhance collagen a2(l) or TGF-β1, mRNA levels. This lack of response was not due to either altered TGF-β receptor affinity or numbers. Metastatic progression was, therefore, associated with an increase in the secretion of activated TGF-β1 and a loss of the ability to deregulate TGF-β-responsive genes.

A unique tissue-engineered model of peripheral nerve regeneration was developed in vitro to study neurite outgrowth. Mouse dorsal root ganglia neurons were seeded on a collagen sponge populated with human endothelial cells and/or human fibroblasts. Addition of nerve growth factor (NGF; 10 ng/ml) was not required for sensory neurons survival but was necessary to promote neurite outgrowth, as assessed by immunostaining of the 150 kDa neurofilament. A vigorous neurite elongation was detected inside the reconstructed tissue after 14 and 31 days of neurons culture, reaching up to 770 microm from day 14. Axons were often observed closely associated with the capillary-like tubes reconstructed in the model, in a similar pattern as in the human dermis. The presence of endothelial cells induced a significant increase of the neurite elongation after 14 days of culture. The addition of human keratinocytes totally avoided the twofold decrease in the amount of neurites observed between 14 and 31 days in controls. Besides the addition of NGF, axonal growth did not necessitate B27 supplement or glial cell coculture to be promoted and stabilized for long-term culture. Thus, this model might be a valuable tool to study the effect of various cells and/or attractive or repulsive molecules on neurite outgrowth in vitro.

The use of autologous reconstructed skin appears to be a promising treatment for the permanent coverage of deep and extensive burns. However, the capability of reconstructed skin transplanted on wounds to promote recovery of sensory perception is a major concern. Our aim was to assess the effect of laminin on cutaneous nerve regeneration. We prepared collagen-chitosan sponges enriched with 0, 1, 10 or 50 μg of laminin/sponge to produce tissue-engineered reconstructed skins by culture of human fibroblasts and keratinocytes, then grafted on the back of athymic mice for 120 days. Immunohistochemical studies demonstrated that there were 7 times more neurofilament 150 kD-positive nerve fibers migrating in the graft in the samples enriched with 10 μg laminin/sponge, compared to reconstructed skin without laminin, 120 days after graft. A significant improvement in the current perception threshold of the Aβ and Aδ nerve fibers was measured using a Neurometer® in all grafts enriched with laminin. In addition, the type C nerve fibers reached an identical current perception threshold than mouse skin, in all reconstructed skins enriched or not with laminin. We conclude that the use of a tissue-engineered autologous skin graft enriched with laminin has the potential to efficiently optimize cutaneous sensory nerve regeneration in vivo.

Cardiovascular disease (CVD) is the leading cause of death in adults in the United States, yet the benefits of genetic testing are not universally accepted.We developed the "HeartCare" panel of genes associated with CVD, evaluating high-penetrance Mendelian conditions, coronary artery disease (CAD) polygenic risk, LPA gene polymorphisms, and specific pharmacogenetic (PGx) variants. We enrolled 709 individuals from cardiology clinics at Baylor College of Medicine, and samples were analyzed in a CAP/CLIA-certified laboratory. Results were returned to the ordering physician and uploaded to the electronic medical record.Notably, 32% of patients had a genetic finding with clinical management implications, even after excluding PGx results, including 9% who were molecularly diagnosed with a Mendelian condition. Among surveyed physicians, 84% reported medical management changes based on these results, including specialist referrals, cardiac tests, and medication changes. LPA polymorphisms and high polygenic risk of CAD were found in 20% and 9% of patients, respectively, leading to diet, lifestyle, and other changes. Warfarin and simvastatin pharmacogenetic variants were present in roughly half of the cohort.Our results support the use of genetic information in routine cardiovascular health management and provide a roadmap for accompanying research.

TO THE EDITOR One of the major roles of skin, beyond its barrier function, is the sense of touch. As this organ is highly exposed to physical or chemical assault leading to burns, nerve regeneration is of major importance to promote sensory recovery during the healing process. We previously showed, both in vitro and in vivo, that nerve regeneration can be markedly enhanced by incorporation of laminin or Schwann cells in tissue-engineered skin (Gingras et al., 2003bGingras M. Paradis I. Berthod F. Nerve regeneration in a collagen-chitosan tissue-engineered skin transplanted on nude mice.Biomaterials. 2003; 24: 1653-1661Crossref PubMed Scopus (137) Google Scholar; Caissie et al., 2006Caissie R. Gingras M. Champigny M.F. et al.In vivo enhancement of sensory perception recovery in a tissue-engineered skin enriched with laminin.Biomaterials. 2006; 27: 2988-2993Crossref PubMed Scopus (39) Google Scholar; Blais et al., 2009Blais M. Grenier M. Berthod F. Improvement of nerve regeneration in tissue-engineered skin enriched with schwann cells.J Invest Dermatol. 2009; 129: 2895-2900Crossref PubMed Scopus (51) Google Scholar). However, if these strategies promote efficient recuperation of temperature and pain perception through C- and A-delta nerve fiber regeneration, they fail to recover the sense of touch, whereas A-beta fibers successfully innervate the graft. Indeed, to restore the sense of touch, these A-beta fibers need to be connected to a sensory receptor, such as the Merkel touch dome, sensory corpuscles, or the hair follicle. Of all these sensory receptors, the hair follicle is the only one that can be used for tissue-engineering purposes. The hair follicle constitutes a major component in the skin nerve network and is innervated by complex nerve plexuses (Botchkarev et al., 1997Botchkarev V.A. Eichmuller S. Johansson O. et al.Hair cycle-dependent plasticity of skin and hair follicle innervation in normal murine skin.J Comp Neurol. 1997; 386: 379-395Crossref PubMed Scopus (108) Google Scholar; Peters et al., 2002Peters E.M. Botchkarev V.A. Muller-Rover S. et al.Developmental timing of hair follicle and dorsal skin innervation in mice.J Comp Neurol. 2002; 448: 28-52Crossref PubMed Scopus (63) Google Scholar; Provitera et al., 2007Provitera V. Nolano M. Pagano A. et al.Myelinated nerve endings in human skin.Muscle Nerve. 2007; 35: 767-775Crossref PubMed Scopus (110) Google Scholar; Hendrix et al., 2008Hendrix S. Picker B. Liezmann C. et al.Skin and hair follicle innervation in experimental models: a guide for the exact and reproducible evaluation of neuronal plasticity.Exp Dermatol. 2008; 17: 214-227Crossref PubMed Scopus (33) Google Scholar). It participates in the sense of touch perception of hairy skin and, to a lesser extent, glabrous skin (Hamalainen et al., 1985Hamalainen H.A. Warren S. Gardner E.P. Differential sensitivity to airpuffs on human hairy and glabrous skin.Somatosens Res. 1985; 2: 281-302Crossref PubMed Scopus (13) Google Scholar; Botchkarev et al., 1997Botchkarev V.A. Eichmuller S. Johansson O. et al.Hair cycle-dependent plasticity of skin and hair follicle innervation in normal murine skin.J Comp Neurol. 1997; 386: 379-395Crossref PubMed Scopus (108) Google Scholar; Woodbury et al., 2001Woodbury C.J. Ritter A.M. Koerber H.R. Central anatomy of individual rapidly adapting low-threshold mechanoreceptors innervating the ‘hairy’ skin of newborn mice: early maturation of hair follicle afferents.J Comp Neurol. 2001; 436: 304-323Crossref PubMed Scopus (66) Google Scholar). It has been clearly shown that hair follicles modulate skin innervation (Botchkarev et al., 1997Botchkarev V.A. Eichmuller S. Johansson O. et al.Hair cycle-dependent plasticity of skin and hair follicle innervation in normal murine skin.J Comp Neurol. 1997; 386: 379-395Crossref PubMed Scopus (108) Google Scholar; Zhang et al., 2008Zhang Y. Andl T. Yang S.H. et al.Activation of beta-catenin signaling programs embryonic epidermis to hair follicle fate.Development. 2008; 135: 2161-2172Crossref PubMed Scopus (137) Google Scholar). Thus, we hypothesized that the incorporation of hair follicles in tissue-engineered skin may promote and/or guide nerve migration, with hairs establishing active targets for nerves (Uno and Montagna, 1982Uno H. Montagna W. Reinnervation of hair follicle end organs and Meissner Corpuscles in skin grafts of Macaques.J Invest Dermatol. 1982; 78: 210-214Crossref PubMed Scopus (8) Google Scholar). In addition, it should greatly improve the recovery of the sense of touch, as hair follicles are sensory receptors (Woodbury et al., 2001Woodbury C.J. Ritter A.M. Koerber H.R. Central anatomy of individual rapidly adapting low-threshold mechanoreceptors innervating the ‘hairy’ skin of newborn mice: early maturation of hair follicle afferents.J Comp Neurol. 2001; 436: 304-323Crossref PubMed Scopus (66) Google Scholar; Hendrix et al., 2008Hendrix S. Picker B. Liezmann C. et al.Skin and hair follicle innervation in experimental models: a guide for the exact and reproducible evaluation of neuronal plasticity.Exp Dermatol. 2008; 17: 214-227Crossref PubMed Scopus (33) Google Scholar). We developed a unique model of tissue-engineered skin cultured with hair buds that grew into hairs after grafting on mice (Larouche et al., 2011Larouche D. Cuffley K. Paquet C. et al.Tissue-engineered skin preserving the potential of epithelial cells to differentiate into hair after grafting.Tissue Eng Part A. 2011; 17: 819-830Crossref PubMed Scopus (23) Google Scholar). To study the influence of hair follicles on sensory neuron axonal migration in vitro, we prepared a dermal construct made of four superimposed self-assembled fibroblast sheets. After 1 week of maturation to promote sheet merging, the construct was seeded with mouse dorsal root ganglia sensory neurons on the top. Axonal migration was promoted throughout the three-dimensional tissue by adding nerve growth factor to the culture medium for 14 days (Gingras et al., 2003aGingras M. Bergeron J. Dery J. et al.In vitro development of a tissue-engineered model of peripheral nerve regeneration to study neurite growth.FASEB J. 2003; 17: 2124-2126PubMed Google Scholar). The construct was then turned over so that the neurons were on the bottom and dissociated keratinocytes or hair buds were seeded on the top, to mimic normal cutaneous innervation and skin histology. After 1 week of culture with the construct under immersion to allow epithelial proliferation, the innervated tissue-engineered skin with hair bud-like structures (iTES-HBLSs) or with dissociated keratinocytes (iTES-Ks) was lifted up to the air–liquid interface and cultured for 2 weeks to promote epidermal differentiation. In the iTES-K, a thin epidermis was observed covering the dermal compartment (Figure 1a). In the iTES-HBLS, a thin epidermis, made of keratinocytes originating from the hair buds, was also observed. In addition, epidermal inclusions were observed in the dermal portion, mimicking hair bud-like structures. These structures were stained with anti-keratin 14 antibodies, showing their epithelial origin (Figure 1d). In addition, when the iTES-HBLS was stained with a keratin 17-specific antibody, both HBLS and epidermis expressed the marker (Figure 1f), as well as the epidermis in the iTES-K (Figure 1e), whereas keratin 17 is a marker of epidermal appendages in normal skin (Figure 2h). However, keratin 17 is known to be expressed by hyperproliferative keratinocytes in skin substitutes cultured in vitro (Smiley et al., 2006Smiley A.K. Klingenberg J.M. Boyce S.T. et al.Keratin expression in cultured skin substitutes suggests that the hyperproliferative phenotype observed in vitro is normalized after grafting.Burns. 2006; 32: 135-138Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar).Figure 2Hair buds sped up and guided nerve regeneration in the TES-HBLS after grafting in nude mice. At 1 month after grafting, almost no neurofilament M-positive nerve fiber (in green) was observed in the TES-K (e), whereas several fibers were detected, mostly in the deep dermis of the TES-HBLS (c). In the upper part of the dermis, most of the fibers were observed to be closely associated with keratin 14-positive hair follicles (in red, c; white arrows in d). The pattern of nerve fiber connection to hair follicles in the TES-HBLS was similar to that observed in normal black haired C3H/HeN mouse skin (white arrows in a) or nude mouse skin (white arrows in b). The number of nerve fibers detected in the TES-HBLS was significantly higher compared with the TES-K (f: P<0.05, n=4). In the TES-HBLS, the mature hair follicles originating from hair buds expressed keratin 17, which is a marker of epidermal appendages, and in contrast with the keratinocytes from the epidermis (g), as observed in normal skin of black C3H/HeN mice (h). Bars in a, b, c, and e: 100μm; bars in d, g, and h: 50μm.View Large Image Figure ViewerDownload (PPT) When the iTES-K was stained with antibodies against neurofilament M, numerous neurites were observed homogeneously distributed underneath the keratin 14-positive epidermis (Figure 1b). In the iTES-HBLS model, neurofilament M-positive neurites were localized preferentially around the hair bud-like inclusions and even seemed to migrate inside them (Figure 1d). As a control, no neurofilament M-positive staining was observed in a TES-HBLS (without neurons) cultured in the same conditions as the iTES-HBLS (Figure 1g). To investigate whether these hair bud-like structures can promote normal hair growth in vivo, a TES-HBLS (without neurons) was transplanted on the back of a nude mouse (with approval of the Laval University animal care committee) and compared with a control TES-K. At 1 month after transplantation, a black hair tuft was observed growing from the TES-HBLS, whereas no hair was observed in the control graft with dissociated keratinocytes (Larouche et al., 2011Larouche D. Cuffley K. Paquet C. et al.Tissue-engineered skin preserving the potential of epithelial cells to differentiate into hair after grafting.Tissue Eng Part A. 2011; 17: 819-830Crossref PubMed Scopus (23) Google Scholar). When nerve regeneration was investigated in the grafts 1 month after transplantation, almost no neurofilament M-positive fiber was observed in the graft of the TES-K (Figure 2e). In contrast, several nerve fibers were detected deep in the dermis of the TES-HBLS graft (Figure 2c). Most of the neurofilament M-positive fibers located in the upper dermis were detected in close association with the newly formed hair follicles (white arrows, Figure 2d), in a pattern similar to that of normal C3H/HeN mouse skin (Figure 2a) or nude mouse skin (Figure 2b). The total number of nerve fibers was 180 times higher in the TES-HBLS compared with the TES-K (Figure 2f, and c vs e). In the TES-HBLS, 1 month after grafting, keratin 17 expression was restricted to hair follicles (Figure 2g), as observed in normal C3H/HeN mouse skin (Figure 2h), and in contrast with its expression pattern before grafting (Figure 1e-g). Thus, we showed that hair buds induced a much faster nerve migration in the graft compared with the control with keratinocytes. In addition, the nerve fibers migrating in the TES-HBLS were most often closely associated with the hair follicles in a pattern that mimics normal hairy mouse skin. This observation suggests that the hair buds achieved an attractive effect, guiding nerves to them, both in vitro and in vivo. Thus, the incorporation of hairs in tissue-engineered skin may greatly improve the recovery of the sense of touch through the combination of rapid reinnervation and an appropriate connection of sensory nerves to a sensory receptor. This study was supported by the Canadian Institutes of Health Research (MOP-67082). We thank Dr Pierre Coulombe for providing the anti-keratin-17 antibody, and are grateful to Anne-Marie Moisan for her expert technical assistance and Todd Galbraith for manuscript revision.

Pancreatic cancer has the worst five-year survival rate of all malignancies due to its aggressive progression and resistance to therapy. Current therapies are limited to gemcitabine-based chemotherapeutics, surgery, and radiation. The current trend toward "personalized genomic medicine" has the potential to improve the treatment options for pancreatic cancer. Gene identification and genetic alterations like single nucleotide polymorphisms and mutations will allow physicians to predict the efficacy and toxicity of drugs, which could help diagnose pancreatic cancer, guide neoadjuvant or adjuvant treatment, and evaluate patients' prognosis. This article reviews the multifaceted roles of genomics and pharmacogenomics in pancreatic cancer.

Adventitia surrounds, nourish, and protect large conductance vessels. This important outer layer has long been forgotten by researchers because interest in vascular diseases has focused mainly on resistance arteries, as shown by the numerous publications on the subject. However, involvement of large vessels in the pathogenesis of vascular diseases is beginning to be recognized. Indeed, the stiffness of conductance arteries could be a precursor event of high blood pressure. Pathological changes that occur in adventitia, increased vasa vasorum permeability for example, may lead or precipitate vascular diseases. Adventitia can also be affected by luminal events like shear stress and possibly atherosclerosis that may trigger adverse responses in the adventitial tissue. These adventitial changes and interrelationships, as well as the structure, including the afferent and efferent autonomic nervous system, and functions of adventitia are the subject of the present review. There is no doubt that the medical and scientific community would greatly benefit from awareness and a better consideration of adventitia and that more studies focusing on this part of blood vessels will lead to an improved comprehension of the different diseases affecting them.

Oxycodone is a semisynthetic opioid analgesic largely prescribed for post-operative and chronic pain management. The introduction of a slow release formulation of oxycodone has led to its frequent abuse and to an increase in emergency cases related to oxycodone overdose. Until recently, oxycodone testing has been confined to gas chromatography-mass spectrometry (GC-MS) analysis because the widely used automated opiate immunoassays poorly react to this compound. We investigated the utility of a new oxycodone immunoassay as a screening procedure to eliminate inappropriate GC-MS testing of negative urine specimens. We analyzed 96 urine specimens using GC-MS and two immunoassays, CEDIA® opiates and DRI® oxycodone assays from Microgenics, on a Hitachi 917 analyzer. The GC-MS allowed us to detect codeine, hydrocodone, hydromorphone, morphine, oxycodone, and oxymorphone following enzymatic hydrolysis and derivation by acetylation. The combination of the two immunoassays gave the best performance (98% sensitivity and specificity) when considering a positive result from GC-MS for any of the opiates. Considering positive GC-MS results for oxycodone or oxymorphone only, the oxycodone immunoassay resulted in two false-positives and one false-negative (50 ng/mL cutoff). Using these immunoassays for screening before GC-MS analysis provides a reduced opiate GC-MS workload without compromising quality.

Abstract Ever larger Structural Variant (SV) catalogs highlighting the diversity within and between populations help researchers better understand the links between SVs and disease. The identification of SVs from DNA sequence data is non-trivial and requires a balance between comprehensiveness and precision. Here we present a catalog of 355,667 SVs (59.34% novel) across autosomes and the X chromosome (50bp+) from 138,134 individuals in the diverse TOPMed consortium. We describe our methodologies for SV inference resulting in high variant quality and &gt;90% allele concordance compared to long-read de-novo assemblies of well-characterized control samples. We demonstrate utility through significant associations between SVs and important various cardio-metabolic and hemotologic traits. We have identified 690 SV hotspots and deserts and those that potentially impact the regulation of medically relevant genes. This catalog characterizes SVs across multiple populations and will serve as a valuable tool to understand the impact of SV on disease development and progression.

We investigated whether cytokine genes were activated in human tumour-infiltrating mononuclear leukocytes (TIML) obtained from six lung adenocarcinomas and seven glioblastomas. TIML were extracted by mechanical disruption and isolated by double density gradient of Ficoll. We performed mRNA reverse transcription-polymerase chain reaction (RT-PCR) on these fresh (noncultured) TIML and autologous peripheral blood mononuclear leukocytes (PBML) using primers for the cytokines IL-1 beta, IL-6, IL-2, IL-4, GM-CSF, IFN-gamma and TNF-beta. In addition, we compared patients' TIML and PBML populations with healthy normal and alpha-CD3 activated PBML as an optimally activated reference population. Gel bands of RT-PCR products were quantitated in relative units (RU) as a function of their size and intensity by computerized image-analysis. Lung and brain patients' TIML showed IL-1 beta and IL-6 cytokine mRNA expressed in the average of 2-log RU but not significantly different from autologous and normal healthy PBML. IL-2, IFN-gamma and TNF-beta also did not appear expressed in the TIML at higher levels than in autologous or healthy normal PBML. However in two thirds of patients, lung TIML could be distinguished from autologous PBML by specific expression of GM-CSF and from healthy normal PBML by expression of IL-4. Similarly, most brain TIML expressed mRNA significantly above healthy normal PBML for GM-CSF and IL-4. In comparison with alpha-CD3 activated healthy PBML, our results suggest that lung and brain TIML had detectable cytokine mRNA, but they seemed poorly activated in total number of genes and amount of cytokine mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Somatostatin receptor type 5 (SSTR5) P335L is a hypofunctional, single nucleotide polymorphism of SSTR5 with implications in the diagnostics and therapy of pancreatic neuroendocrine neoplasms. The purpose of this study is to determine whether a SSTR5 P335L-specific monoclonal antibody could sufficiently differentiate pancreatic neuroendocrine neoplasms (PNENs) with different SSTR5 genotypes.Cellular proliferation rate, SSTR5 mRNA level, and SSTR5 protein level were measured by performing MTS assay, a quantitative reverse transcription polymerase chain reaction study, Western blot analysis, and immunohistochemistry, respectively. SSTR5 genotype was determined with the TaqMan SNP Genotyping assay (Applied Biosystems, Foster City, CA).We found that the SSTR5 analogue RPL-1980 inhibited cellular proliferation of CAPAN-1 cells more than that of PANC-1 cells. Only PANC-1 (TT) cells, but not CAPAN-1 (CC) cells expressed SSTR5 P335L. In 29 white patients with PNENs, 38% had a TT genotype for SSTR5 P335L, 24% had a CC genotype for WT SSTR5, and 38% hada CT genotype for both SSTR5 P335L and WT SSTR5. Immunohistochemistry using SSTR5 P335L monoclonal antibody detected immunostaining signals only from the neuroendocrine specimens with TT and CT genotypes, but not those with CC genotypes.A SSTR5 P335L monoclonal antibody that specifically recognizes SSTR5 P335L but not WT SSTR5 could differentiate PNENs with different SSTR5 genotypes, thereby providing a potential tool for the clinical diagnosis of PNEN.

Gene expression profiling may improve the understanding of the biology behind relapse in pediatric acute lymphoblastic leukemia. Using suppression subtractive hybridization (SSH), cDNA concatenated sequencing (CCS), and reverse transcriptase real-time quantitative polymerase chain reaction (RT-RQ-PCR) on high-risk patient samples with nondeterminant chromosomal translocation, the authors identified 3 genes that were significantly overexpressed in the nonrelapsed patients: the calcium/calmodulin-dependent serine protein kinase (CASK), subunit 2 of the cofactor required for SP1 transcriptional activation (CRSP2), and granzyme K (GZMK). The level of expression of these biomarkers may help identify patients with potentially good prognosis within a group otherwise at high risk of relapse.

World-wide 265,000 people are diagnosed with pancreatic cancer each year, of which 74% will die within 1 year. Most pancreatic adenocarcinomas (PDACs) are diagnosed at an advanced stage with 91% of the patients with regional metastatic disease (1). As a result, PDAC has one of the worst prognoses among all cancers with a 5-year survival of less than 5%. It is one of the few cancers still increasing in incidence and by 2030, if current trends continue and without the discovery of effective treatments, PDAC will be the second leading cause of cancer death in the US (2).

We have previously shown that SSTR5 is an important regulator of insulin expression/secretion and islet proliferation via pancreatic-duodenal homeobox 1(PDX-1) in mice. We have also identified a germline, non-synonymous single nucleotide polymorphism (SNP) of SSTR5 in human blood and pancreatic cancer specimens, which results in mutation of intracellular C-terminal proline 335 to leucine (SSTR5 P335L). The purpose of this study was to determine whether SSTR5 P335L SNP has any functional significance on PDX-1 expression and proliferation in human cell lines.

Nerves and blood vessels are closely associated. Moreover, sensory nerves were shown to determine the branching pattern of the vascular network in skin and to promote the arterial differentiation of blood vessels. We developed a unique in vitro model featuring a pre‐formed three‐dimensional neurites network on which a capillary‐like network was allowed to organize and mature. Sensory neurons and glial cells were co‐cultured with fibroblasts in a collagen‐chitosan sponge to reconstruct the neural network for 14 days and human endothelial cells were then seeded on the tissue to build a capillary‐like network for 17 additional days. Neural cells induced a 27% increase in the number of capillary‐like tubes (CLT) formed in the tissue. This effect was abolished when K252a, an inhibitor of the TrkA, B and C receptors for the NGF, BDNF and NT‐3 neurotrophins respectively was added to the culture medium. Moreover, we demonstrated that when 10 ng/ml of NGF, 0.1 ng/ml of BDNF, 15 ng/ml of NT3 and 50 ng/ml of GDNF were added to our endothelialized reconstructed connective tissue model, a major increase from 40 to 80% in the number of CLT was observed. This is the first in vitro demonstration of a direct angiogenic effect of peripheral neural cells on human endothelial cells through the release of neurotrophic fators and of the angiogenic potential of NT‐3 and GDNF, the latter belonging to an other family of neurotrophic factors.

Recent findings have established that cutaneous nerves modulate both skin homeostasis and various skin diseases, by influencing cell growth and differentiation, inflammation and wound healing. In order to study the influence of epidermis, hair follicles and capillaries on sensory neurons, and vice‐versa, we developed a tissue‐engineered model of innervated endothelialized reconstructed skin (MIERS). Mouse dorsal root ganglia neurons were seeded on a collagen sponge populated with human fibroblasts and human endothelial cells. Keratinocytes or mice newborn immature hair follicle buds were then seeded on the opposite side of the MIERS to study their influence on sensory nerves growth, and vice versa. A vigorous neurite elongation was detected inside the reconstructed dermis after 14 and 31 days of neurons culture. The presence of endothelial cells induced a significant increase of the neurite elongation after 14 days of culture. The addition of human keratinocytes totally avoided the twofold decrease in the amount of neurites observed between 14 and 31 days in controls. We have successfully developed the MIERS that allowed us to study the effects of epidermis and capillaries on nerve growth. This model will be a useful tool to study the modulation of sensory nerves on wound healing, angiogenesis, hair growth and neurogenic inflammation in the skin.