TAp63 suppresses mammary tumorigenesis through regulation of the Hippo pathway.

Mechanisms regulating the transition of mammary epithelial cells (MECs) to mammary stem cells (MaSCs) and to tumor-initiating cells (TICs) have not been entirely elucidated. The p53 family member, p63, is critical for mammary gland development and contains transactivation domain isoforms, which have tumor-suppressive activities, and the ΔN isoforms, which act as oncogenes. In the clinic, p63 is often used as a diagnostic marker, and further analysis of the function of TAp63 in the mammary gland is critical for improved diagnosis and patient care. Loss of TAp63 in mice leads to the formation of aggressive metastatic mammary adenocarcinoma at 9-16 months of age. Here we show that TAp63 is crucial for the transition of mammary cancer cells to TICs. When TAp63 is lost, MECs express embryonic and MaSC signatures and activate the Hippo pathway. These data indicate a crucial role for TAp63 in mammary TICs and provide a mechanism for its role as a tumor- and metastasis-suppressor in breast cancer.

Tuberculous meningitis in adults: a prospective study at a tertiary referral centre in Sri Lanka.

INTRODUCTION: Central nervous system tuberculosis is the most severe form of extrapulmonary TB and it is associated with a substantial morbidity and mortality. OBJECTIVES: To describe the demographic profile, clinical features, laboratory and imaging results of a cohort of adult patients with TBM (Tuberculous meningitis). METHODS: This study encompasses a prospective analysis of all adult cases of TBM diagnosed from 1st January 2010 to 31st December 2011 in the Neurology unit 2, National Hospital of Sri Lanka. Consensus case definitions for TBM were used for clinical case classification and patients were given a definite, probable, or possible tuberculous meningitis status accordingly. RESULTS: A total of 89 patients fulfilled the established diagnostic criteria for TBM and there were 22 definitive cases, 46 probable cases and 21 possible cases. The mean age of the series was 44 years and 56 (63%) were males. TBM presented with fever in 64 (71%), general constitutional symptoms in 61 (68%), headache in 53 (59%), and diminished level of consciousness in 36 (40%) patients. CSF biochemistry revealed elevated protein in all patients. MRI brain showed meningeal enhancement in 73 (82%). Twenty four (27%) died during hospitalisation and out of 65 who survived 44 (49%) had residual sequelae at the time of discharge. CONCLUSIONS: MRI evidence and biochemical analysis of CSF are still the main supportive diagnostic modalities. TBM is a relatively common but difficult to diagnose disease, which results in significant morbidity and mortality.

Molecular and functional characteristics of ovarian surface epithelial cells transformed by KrasG12D and loss of Pten in a mouse model in vivo.

Ovarian cancer is a complex and deadly disease that remains difficult to detect at an early curable stage. Furthermore, although some oncogenic (Kras, Pten/PI3K and Trp53) pathways that are frequently mutated, deleted or amplified in ovarian cancer are known, how these pathways initiate and drive specific morphological phenotypes and tumor outcomes remain unclear. We recently generated Pten(fl/fl); Kras(G12D); Amhr2-Cre mice to disrupt the Pten gene and express a stable mutant form of Kras(G12D) in ovarian surface epithelial (OSE) cells. On the basis of histopathologic criteria, the mutant mice developed low-grade ovarian serous papillary adenocarcinomas at an early age and with 100% penetrance. This highly reproducible phenotype provides the first mouse model in which to study this ovarian cancer subtype. OSE cells isolated from ovaries of mutant mice at 5 and 10 weeks of age exhibit temporal changes in the expression of specific Mullerian epithelial marker genes, grow in soft agar and develop ectopic invasive tumors in recipient mice, indicating that the cells are transformed. Gene profiling identified specific mRNAs and microRNAs differentially expressed in purified OSE cells derived from tumors of the mutant mice compared with wild-type OSE cells. Mapping of transcripts or genes between the mouse OSE mutant data sets, the Kras signature from human cancer cell lines and the human ovarian tumor array data sets, documented significant overlap, indicating that KRAS is a key driver of OSE transformation in this context. Two key hallmarks of the mutant OSE cells in these mice are the elevated expression of the tumor-suppressor Trp53 (p53) and its microRNA target, miR-34a-c. We propose that elevated TRP53 and miR-34a-c may exert negatively regulatory effects that reduce the proliferative potential of OSE cells leading to the low-grade serous adenocarcinoma phenotype.

An outbreak of Aspergillus meningitis following spinal anaesthesia for caesarean section in Sri Lanka: a post-tsunami effect?

An outbreak of Aspergillus fumigatus meningitis occurred in 5 women following spinal anaesthesia, performed between 21 June and 17 July 2005 for caesarean section, in Colombo, Sri Lanka. The patients' median age was 27 years. Different teams in 2 maternity hospitals gave spinal anaesthesia. Mean incubation period was 11.2 days. Fever, headache and nuchal rigidity were common presentations. Remittent fever continued despite broad-spectrum intravenous antibiotics. Papilloedema, lateral rectus palsy, cerebral infarction and haemorrhage developed later. Three patients died. Cerebrospinal fluid pleocytosis with low glucose yielded negative PCR for fungi. Fungal cultures subsequently grew Aspergillus fumigatus. A post-mortem of the first patient confirmed Aspergillus meningitis, followed by treatment with amphotericin B and voriconazole, that saved the lives of others. Visual and hearing impairment in one and complete recovery in the other were observed a year after treatment. Examination of unused plastic syringes, needles, cannulae, and ampoules of anaesthetic agents confirmed that 43 syringes from three different manufactures were contaminated with Aspergillus fumigatus. The stores for drugs and devices of the Ministry of Health were examined and found to be full of tsunami donations, while regular procurements of the Ministry were kept in a poorly maintained humid warehouse. Inadequate space for tsunami donations was identified as the most plausible explanation for sub-optimal storage. Withdrawal and incineration of all unused syringes controlled the outbreak. The survival of those aggressively treated for Aspergillus meningitis suggests in hindsight that the availability of diagnostic tests and specific treatment, and early recognition of the outbreak could have saved the lives of victims who died. Early life-threatening side-effects and permanent long term sequelae of antifungal medication stress the need to be cautious with empirical treatment in immuno-competent low-risk individuals.

Novel potential ALL low-risk markers revealed by gene expression profiling with new high-throughput SSH-CCS-PCR.

The current systems of risk grouping in pediatric acute lymphoblastic leukemia (ALL) fail to predict therapeutic success in 10-35% of patients. To identify better predictive markers of clinical behavior in ALL, we have developed an integrated approach for gene expression profiling that couples suppression subtractive hybridization, concatenated cDNA sequencing, and reverse transcriptase real-time quantitative PCR. Using this approach, a total of 600 differentially expressed genes were identified between t(4;11) ALL and pre-B ALL with no determinant chromosomal translocation. The expression of 67 genes was analyzed in different cytogenetic ALL subgroups and B lymphocytes isolated from healthy donors. Three genes, BACH1, TP53BPL, and H2B/S, were consistently expressed as a significant cluster associated with the low-risk ALL subgroups. A total of 42 genes were differentially expressed in ALL vs normal B lymphocytes, with no specific association with any particular ALL subgroups. The remaining 22 genes were part of a specific expression profile associated with the hyperdiploid, t(12;21), or t(4;11) subgroups. Using an unsupervised hierarchical cluster analysis, the discriminating power of these specific expression profiles allowed the clustering of patients according to their subgroups. These genes could help to understand the difference in treatment response and become therapeutical targets to improve ALL clinical outcomes.

Neurological complications of measles.

OBJECTIVE: To study the clinical features and course of neurological complications of measles. DESIGN: Prospective study. SETTING: Neurology Unit, Teaching Hospital, Kandy. PATIENTS: 10 patients admitted with neurological complications of measles during an outbreak. MEASUREMENTS: Assessment of clinical features and the neurological investigations. RESULTS: 6 had CSF IgM antibodies confirming a recent measles infection. 7 patients had encephalitis and 3 had myelitis. Complications had appeared 5 to 14 days after appearance of the rash. All patients with encephalitis had depressed level of consciousness (Glasgow coma scale 2 to 11) with seizures, 3 patients had lateralising signs, and assisted ventilation was required in 4. Cranial CTs were either normal or showed mild cerebral oedema. CSF analysis showed either normal or mildly raised protein without a cellular reaction. EEG in all 7 revealed diffuse delta activity. During the follow up period of 1 to 12 weeks, all showed a gradual improvement, except one who succumbed to respiratory complications of assisted ventilation. All others were left with some residual disability when last seen. Three patients with myelitis had symmetrical paraparesis. CSF protein was 60 to 80 mg/ml without a cellular reaction. All three improved without residual disability. CONCLUSIONS: Measles encephalitis is a life threatening complication. All three myelitis patients recovered completely within 5 to 8 weeks.

Transcriptional regulation of the murine erythroid-specific 5-aminolevulinate synthase gene.

5-Aminolevulinate synthase (ALAS) catalyzes the first step of the heme biosynthetic pathway in mammalian cells. Separate genes encode the two isoforms: ubiquitously expressed ALAS (ALAS1) and erythroid-specific ALAS (ALAS2). Transcription of the ALAS2 gene is only activated during erythroid cell differentiation. This stimulation allows for the formation of hemoglobin-specific heme. The 5'-flanking region of the mouse ALAS2 gene was studied in order to define its erythroid-specific function in transcriptional activation. Putative binding sites for the erythroid-specific nuclear factors GATA-1, NF-E2, and EKLF were identified within the first 300bp region of the mouse ALAS2 5'-flanking region. However, this 300bp region alone did not efficiently activate transient expression in erythroid MEL and K562 cell lines. Additional DNA regulatory sequences found within 300-718bp upstream of the transcription start site were required for maximal transcriptional activation, even though these regions stimulated similar expression in the non-erythroid HeLa and NIH/3T3 cells. This suggests that cis-acting elements present in the 5'-flanking region are not responsible for maintenance of transcriptional silencing in non-erythroid cell lines and that tissue-specific regulation of ALAS2 depends on other regions of the gene or on chromatin remodeling. A putative hypoxia inducible factor 1 (HIF-1) response element was identified within the 300-718bp upstream region. Significantly, two proximal GATA-1-binding sites (-118/-113 and -98/-93) and a region located within -518 to -315bp of the mouse ALAS2 promoter were essential for transcriptional activation during chemically induced differentiation of MEL cells, implying their importance in conferring erythroid specificity to the ALAS2 transcriptional activation. This is the first study to delimit the cis-acting region responsible for activation of the ALAS2 promoter upon dimethyl-sulfoxide induction in MEL cells.

Estimated contribution of known ataxia genes in ataxia patients undergoing DNA testing.

We estimated the relative contributions of known ataxia genes (SCA1, 2, 3, 6, 7 and X25) in the patient population sent to our DNA diagnostic laboratory for diagnostic testing. Approximately 28% of these patients had an abnormal triplet repeat expansion in one of these ataxia genes (3% for SCA1, 8% for SCA2, 11% for SCA3/MJD, 2% for SCA6, 3% for SCA7, and 1.5% for X25). The lack of abnormal repeat expansions in the majority of ataxis patients tested suggests that the molecular defects associated with most ataxia cases are currently undetermined and that this population includes both familial and sporadic cases. In contrast, of the patients submitted for genetic testing for Friedrich's ataxia (FRDA), 44% (69/157) showed at least one expansion in the X25 gene, indicating that FRDA accounts for a significant proportion of the recessively inherited ataxias and appears to have a high rate of accurate clinical diagnosis. On the basis of our DNA studies, we propose a comprehensive and efficient approach for molecular analysis of ataxia patients.

Duplication of the PMP22 gene in 17p partial trisomy patients with Charcot-Marie-Tooth type-1 neuropathy.

Autosomal dominant Charcot-Marie-Tooth type-1A neuropathy (CMT1A) is a demyelinating peripheral nerve disorder that is commonly associated with a submicroscopic tandem DNA duplication of a 1.5-Mb region of 17p11.2p12 that contains the peripheral myelin gene PMP22. Clinical features of CMT1A include progressive distal muscle atrophy and weakness, foot and hand deformities, gait abnormalities, absent reflexes, and the completely penetrant electrophysiologic phenotype of symmetric reductions in motor nerve conduction velocities (NCVs). Molecular and fluorescense in situ hybridization (FISH) analyses were performed to determine the duplication status of the PMP22 gene in four patients with rare cytogenetic duplications of 17p. Neuropathologic features of CMT1A were seen in two of these four patients, in addition to the complex phenotype asociated with 17p partial trisomy. Our findings show that the CMT1A phenotype of reduced NCV is specifically associated with PMP22 gene duplications, thus providing further support for the PMP22 gene dosage mechanism for CMT1A.

Tissue-specific and allele-specific replication timing control in the imprinted human Prader-Willi syndrome region.

To examine the relationship between replication timing and differential gene transcription in tissue-specific and imprinted settings we have studied the replication timing properties of the human Prader-Willi syndrome (PWS) region on human chromosome 15q11-13. Interphase fluorescence in situ hybridization with an overlapping series of cosmid clones was used to map a PWS replication timing domain to a 500- to 650-kb region that includes the SNRPN gene. This PWS domain replicates late in lymphocytes but predominantly early in neuroblasts, with replication asynchrony observed in both tissues, and appears to colocalize with a genetically imprinted transcription domain showing prominent expression in the brain. A 5- to 30-kb deletion in the 5' region of SNRPN results in the loss of late replication control of this domain in lymphocytes when the deleted chromosome is inherited paternally. This potential allele-specific replication timing control region also appears to colocalize with a putative imprinting control region that has been shown previously to abolish the expression of three imprinted transcripts in this same region.

The human homologue of the Drosophila melanogaster flightless-I gene (flil) maps within the Smith-Magenis microdeletion critical region in 17p11.2.

The Smith-Magenis syndrome (SMS) appears to be a contiguous-gene-deletion syndrome associated with a proximal deletion of the short arm of chromosome 17 in band p11.2. The spectrum of clinical findings includes short stature, brachydactyly, developmental delay, dysmorphic features, sleep disturbances, and behavioral problems. The complex phenotypic features suggest deletion of several contiguous genes. However, to date, no protein-encoding gene has been mapped to the SMS critical region. Recently, the Drosophila melanogaster flightless-I gene, fliI, and the homologous human cDNA have been isolated. Mutations in fliI result in loss of flight ability and, when severe, cause lethality due to incomplete cellularization with subsequent abnormal gastrulation. Here, we demonstrate that the human homologue (FLI) maps within the SMS critical region. Genomic cosmids were used as probes for FISH, which localized this gene to the 17p11.2 region. Somatic-cell hybrid-panel mapping further localized this gene to the SMS critical region. Southern blot analysis of somatic-cell hybrids and/or FISH analysis of lymphoblastoid cell lines from 12 SMS patients demonstrates the deletion of one copy of FLI in all SMS patients analyzed.

Protein tyrosyl phosphatase-1B is expressed by normal human epidermis, keratinocytes, and A-431 cells and dephosphorylates substrates of the epidermal growth factor receptor.

In the epidermis tyrosine kinases such as those found in the epidermal growth factor receptor (EGF-R) phosphorylate regulatory molecules on tyrosine and play an important role in controlling epidermal growth. Phosphotyrosyl phosphatases (PTPase) that dephosphorylate EGF-R and other proteins phosphorylated on tyrosine must also play an important role in controlling epidermal growth. The presence and metabolism of one such PTPase, PTP-1B, was detected and studied in human skin using biochemical, immunochemical, and molecular biologic methods. The message for PTP-1B was expressed in human epidermis, in keratinocytes cultured from human epidermis, and in human keratinocyte cell lines. The 49-kDa but not the 37-kDa form of PTP-1B was identified in membranes prepared from these cells and tissues by immunodetection on Western blots. Nearly all of the labeled proteins identified by gel electrophoresis of an A-431 particulate fraction phosphorylated with [gamma-32P] ATP in the presence of epidermal growth factor are substrates for PTP-1B because their labeling decreased after incubation with a catalytically active and purified PTP-1B fusion protein. Immunohistochemical methods were used to show that PTP-1B was primarily localized to the basal cell layers in normal thick epidermis. The presence of PTP-1B in intact human epidermis suggests that this molecule is not an artifact limited to cultured cells but is an important molecule in the in vivo regulation of epidermal functions.

An evolutionarily conserved palindrome in the Drosophila Gld promoter directs tissue-specific expression.

A conserved palindromic sequence (Gpal) in the promoter region of the Drosophila Gld directs expression of a heterologous reporter gene in transgenic flies to the anterior spiracular glands of third instar larvae and to the ejaculatory bulb of adult males. The Gld gene is normally expressed at high levels in the anterior spiracular glands but is not expressed in the ejaculatory bulb of Drosophila melanogaster. However, Gld promoters from other Drosophila species contain the Gpal element and express glucose dehydrogenase (GLD) in the adult male ejaculatory bulb. A gene fusion composed of the D. melanogaster Gld promoter and the lacZ gene is expressed in the anterior spiracular glands of transgenic larvae. Mutations of the Gpal sequence element in this gene fusion block expression of beta-galactosidase in the anterior spiracular gland. Together these experiments demonstrate that Gpal is necessary and sufficient for tissue-specific expression in the anterior spiracular glands. Based upon the tissue distribution and function of GLD, it is speculated that expression of GLD in the anterior spiracular glands represents the ancestral state and that GLD expression in other tissues arose as a fortuitous consequence of a shared combinatorial regulatory network.