Differential role of EGF and BFGF in human GBM-TIC proliferation: relationship to EGFR-tyrosine kinase inhibitor sensibility.

Glioblastoma multiforme (GBM) is among the most devastating human tumors being rapidly fatal despite aggressive surgery, radiation and chemotherapies. It is characterized by extensive dissemination of tumor cells within the brain that hinders complete surgical resection. GBM tumor initiating-cells (TICs) are a rare subpopulation of cells responsible for tumor development, growth, invasiveness and recurrence after chemotherapy. TICs from human GBM can be selected in vitro using the same conditions permissive for the growth of normal neural cells, of which share some features including marker expression, self-renewal capacity, long-term proliferation, and ability to differentiate into neuronal and glial cells. EGFR overexpression and its constitutive activation is one of the most important signaling alteration identified in GBM, and its pharmacological targeting represents an attractive therapeutic goal. We previously demonstrated that human GBM TICs have different sensitivity to the EGFR kinase inhibitors erlotinib and gefitinib, depending on the differential modulation of downstream signaling cascades. In this work we investigated the mechanisms of resistance to erlotinib in two human GBM TIC cultures, analyzing EGF and bFGF individual contribution to proliferation, clonogenicity, and migration. We demonstrated the presence of a small cell subpopulation whose proliferation is supported by EGF and a larger one mainly dependent on bFGF. Thus, insensitivity to EGFR kinase inhibitors as far as TIC proliferation results from a predominant FGFR activation that hides the inhibitory effects induced on EGFR signaling. Conversely, EGF and bFGF induced cell migration with similar efficacy. In addition, unlike neural stem/progenitors cells, the removal of chondroitin sulphate proteoglycans from cell surface was unable to discern EGF- and bFGF-dependent subpopulations in GBM TICs.

Point mutations and a large intragenic deletion in SPG11 in complicated spastic paraplegia without thin corpus callosum.

BACKGROUND: Hereditary spastic paraplegia (HSP) with thin corpus callosum (HSP-TCC) is a frequent subtype of complicated HSP clinically characterised by slowly progressive spastic paraparesis with cognitive impairment and thin corpus callosum (TCC). SPG11, the gene associated with the major locus involved, encodes spatacsin, a protein of unknown function. METHODS: Different types of mutations were identified in patients with the complex form of HSP (cHSP) including TCC. We screened a series of 45 index patients with different types of cHSP with (n = 10) and without (n = 35) TCC. RESULTS: Ten mutations, of which five are novel, were detected in seven patients. Of importance, three out of seven mutated patients present with cHSP without TCC. Among the novel mutations identified, we characterised a large intragenic rearrangement deleting 2.6 kb of the SPG11 gene. The rearrangement is due to non-allelic homologous recombination between Alu sequences flanking the breakpoints. CONCLUSIONS: These findings expand the mutation spectrum of SPG11 and suggest that SPG11 mutations may occur more frequently in familial than sporadic forms of cHSP without TCC. This helps to define further clinical and molecular criteria for a correct diagnosis of the SPG11 related form of cHSP. In addition, the intragenic deletion detected here, and the mechanism involved, both provide clues to address the issue of SPG11 missing mutant alleles previously reported.

In vivo functional analysis of the Ras exchange factor son of sevenless.

The Son of sevenless (Sos) protein functions as a guanine nucleotide transfer factor for Ras and interacts with the receptor tyrosine kinase Sevenless through the protein Drk, a homolog of mammalian Grb2. In vivo structure-function analysis revealed that the amino terminus of Sos was essential for its function in flies. A molecule lacking the amino terminus was a potent dominant negative. In contrast, a Sos fragment lacking the Drk binding sites was functional and its activity was dependent on the presence of the Sevenless receptor. Furthermore, membrane localization of Sos was independent of Drk. A possible role for Drk as an activator of Sos is discussed and a Drk-independent interaction between Sos and Sevenless is proposed that is likely mediated by the pleckstrin homology domain within the amino terminus.

Interactions of Drosophila Cbl with epidermal growth factor receptors and role of Cbl in R7 photoreceptor cell development.

The human proto-oncogene product c-Cbl and a similar protein in Caenorhabditis elegans (Sli-1) contain a proline-rich COOH-terminal region that binds Src homology 3 (SH3) domains of proteins such as the adapter Grb2. Cb1-Grb2 complexes can be recruited to tyrosine-phosphorylated epidermal growth factor (EGF) receptors through the SH2 domain of Grb2. Here we identify by molecular cloning a Drosophila cDNA encoding a protein (Drosophila Cbl [D-Cbl]) that shows high sequence similarity to the N-terminal region of human c-Cbl but lacks proline-rich sequences and fails to bind Grb2. Nonetheless, in COS-1 cells, expression of hemagglutinin epitope-tagged D-Cbl results in its coimmunoprecipitation with EGF receptors in response to EGF. EGF also caused tyrosine phosphorylation of D-Cbl in such cells, but no association of phosphatidylinositol 3-kinase was detected in assays using anti-p85 antibody. A point mutation in D-Cbl (G305E) that suppresses the negative regulation of LET-23 by the Cbl homolog Sli-1 in C. elegans prevented tyrosine phosphorylation of D-Cbl as well as binding to the liganded EGF receptor in COS-1 cells. Colocalization of EGF receptors with both endogenous c-Cbl or expressed D-Cbl in endosomes of EGF-treated COS-1 cells is also demonstrated by immunofluorescence microscopy. In lysates of adult transgenic Drosophila melanogaster, GST-DCbl binds to the tyrosine-phosphorylated 150-kDa torso-DER chimeric receptor. Expression of D-Cbl directed by the sevenless enhancer in intact Drosophila compromises severely the development of the R7 photoreceptor neuron. These data suggest that despite the lack of Grb2 binding sites, D-Cbl functions as a negative regulator of receptor tyrosine kinase signaling in the Drosophila eye by a mechanism that involves its association with EGF receptors or other tyrosine kinases.

Unit Cost Analysis of PET-CT at an Apex Public Sector Health Care Institute in India.

CONTEXT: PET/CT scan service is one of the capital intensive and revenue-generating centres of a tertiary care hospital. The cost associated with the provisioning of PET services is dependent upon the unit costs of the resources consumed. AIMS: The study aims to determine the cost of providing PET/CT Scan services in a hospital. METHODS AND MATERIAL: This descriptive and observational study was conducted in the Department of Nuclear Medicine at a tertiary apex teaching hospital in New Delhi, India in the year 2014-15. Traditional costing methodology was used for calculating the unit cost of PET/CT scan service. The cost was calculated under two heads that is capital and operating cost. Annualized cost of capital assets was calculated using methodology prescribed by WHO and operating costs was taken on an actual basis. RESULTS: Average number of PET/CT scan performed in a day is 30. The annual cost of providing PET/CT scan services was calculated to be 65,311,719 Indian Rupees (INR) (US$ 1,020,496), while the unit cost of PET scan was calculated to be 9625.92 INR (US$ 150). 3/4th cost was spent on machinery and equipment (75.3%) followed by healthcare personnel (11.37%), electricity (5%), consumables and supplies (4%) engineering maintenance (3.24%), building, furniture and HVAC capital cost (0.76%), and manifold cost (0.05%). Of the total cost, 76% was capital cost while the remaining was operating cost. CONCLUSIONS: Total cost for establishing PET/CT scan facility with cyclotron and chemistry module and PET/CT scan without cyclotron and chemistry module was calculated to be INR 610,873,517 (US$9944899) and 226,745,158 (US$3542893), respectively. (US$ 1=INR 64).

Enhancing neonatal survival: what can we do today?

OBJECTIVE: Neonatal deaths account for 44% of the world's under-5 child mortality. Over half of all neonatal deaths globally occur in preterm babies. Therefore, improving care of a preterm baby is particularly important to reduce under-5 mortality. The objective of this study was to spell out components of care of preterm/low birth weight babies at first level health facility and at first referral unit (FRU) in low resource settings. STUDY DESIGN: We have analyzed weight-wise survivals at two hospitals attached to medical colleges, J.J. Hospital, Mumbai and General Hospital, Talegaon, and at Rural Hospital, Dahanu. There were three-tier interventions: (i) warmth+ feeding and antibiotics, (ii) improved care at birth plus increased oxygen availability and (iii) use of dopamine. J.J. Hospital went through all these stages one after another; General Hospital had all three going simultaneously. The Rural Hospital had a 1+2. RESULTS: During 1978 to 1984, J.J. Hospital saved 50 to 55% very low birth weight (VLBW) babies by providing warmth, feeding and antibiotics. This percentage increased to 56 to 58%, when adequate oxygen and good care at birth was available (1984 to 1989). For babies in the moderately low birth weight category (MLBW), 1500 to 2000 g at birth, the corresponding figures were 56 to 58% and 84 to 86%. The same interventions led to statistically significant decline in MLBW and VLBW categories at General Hospital, Talegaon (2010 to 2013). The Rural Hospital, Dahanu (1987 to 1992) achieved better survival rates in VLBW (61.5%) and MLBW (92.5%) categories with identical interventions and less staff. CONCLUSION: On the basis of our results, we suggest that in resource-limited settings, the first level health facility may be able to look after short-stay babies that weigh more than 1500 g and that have no respiratory distress. The FRU may look after MLBW babies, with or without respiratory distress, and VLBW babies without respiratory distress by giving special care.

Emx2 in adult neural precursor cells.

Emx2 is a vertebrate homeobox gene involved in the control of the central nervous system development. In the formation of cerebral cortex, Emx2 expression is restricted mainly to the germinal ventricular zone fading away in the first postmitotic neurons. This expression pattern, the severe impairment of cortex organization and the size in mutant mice suggest a role of Emx2 in the control of proliferation and migration of neural precursor cells. The observed persistence of Emx2 expression in adult neurogenic areas in vivo is here confirmed at later stages. We also find that Emx2 is expressed at high levels in adult neural stem cells (ANSCs) in vitro and is down modulated upon differentiation. Overexpression of Emx2 gene in ANSCs has an anti-proliferative effect but it does not influence a particular differentiation pathway. Our results suggest that Emx2 may act promoting an asymmetric mode of cell division thereby increasing the size of a transit amplifying population.

The retroviral transduction of HOXC4 into human CD34(+) cells induces an in vitro expansion of clonogenic and early progenitors.

OBJECTIVE: +HOX genes are expressed in the hematopoietic system and increasing data point to their involvement in the control of proliferation and/or differentiation. Genes belonging to the C cluster are preferentially expressed in developing and differentiated lymphoid lineages. However, recent studies demonstrated, by RT-PCR, that the HOXC4 gene is also actively transcribed in the most undifferentiated hematopoietic cells (CD34(+)38(low)) and in more mature myeloid and erythroid progenitors. We evaluated the expression of HOXC4 protein on human CD34(+) cells and the in vitro effect of its overexpression on proliferation and differentiation. MATERIALS AND METHODS: We assessed the expression of HOXC4 on human CD34(+) cells using a polyclonal antibody raised against the C-terminal portion of the protein expressed using the baculovirus system. Overexpression of HOXC4 in human CD34(+) cells was obtained by retroviral gene transfer; its effect on clonogenic (CFU-GM, BFU-E, and CFU-GEMM) and early progenitors (LTC-IC) was evaluated. RESULTS: The HOXC4 protein is indeed expressed in human CD34(+) cells, and its overexpression in human CD34(+) cells increases the proliferation potential of clonogenic and early progenitors. CFU-GM showed a median threefold expansion (range: 1.1-19.4; p < 0.002) compared with control transduced with the vector alone. The increment of BFU-E was higher (median ninefold, range 2.5-35; p < 0. 0009) and erythroid colonies presented a larger size with normal morphology. An even more marked effect was observed on LTC-IC (median 13, onefold; range 4.1-102.1, p < 0.0001). CONCLUSION: We demonstrate that HOXC4 is expressed in CD34(+) cells and that its overexpression induces an in vitro expansion of committed as well as very early hematopoietic progenitors. The most striking effect was obtained on LTC-IC with an expansion of 13.1-fold. The enforced expression of HOXC4 induced a significant increase (p < 0.009) in the number of erythroid colonies compared with CFU-GM, although without perturbing, at least in vitro, the maturation program of the cells. On the other hand, the effect of the gene overexpression did not induce any skewing in the colony types derived from the myeloid lineage.

Endocrine, metabolic, and clinical effects of gestrinone in women with endometriosis.

The effect of oral gestrinone, 2.5 mg twice weekly for 6 months, was studied in 11 women with mild or moderate endometriosis laparoscopically confirmed. The mean laparoscopic score decreased from 17.18 to 9.09 (P greater than 0.005). Painful symptoms were relieved in all patients within 2 months from start of therapy. Gonadotropins, prolactin (PRL) 17 beta-estradiol (17 beta-E2), estrone (E1), progesterone (P), androstenedione (A), and dehydroepiandrosterone sulfate (DHEA-S) remained in the follicular phase range. Total testosterone (TT) and sex hormone-binding globulin (SHBG) decreased, whereas free testosterone (FT) slightly increased. Metabolic studies showed a decrease of total triglycerides, very low-density lipoprotein (VLDL) triglycerides, and high-density lipoprotein (HDL) and VLDL cholesterol, parallel to the decrease of associated apoproteins. Low-density lipoprotein cholesterol and apoprotein B increased during therapy. The results suggest that gestrinone possesses antiestrogenic, androgenic, and progestigenic effects at therapeutic dosages both by acting on central and peripheral steroid receptors. For its efficacy and good tolerance, gestrinone may be considered an option for treating endometriosis.

Food security among preschool children.

A cross-sectional study of preschool children from 450 families from a residential colony of 'D' class hospital employees was undertaken to study food security & associated variables. Food security was established from (a) 24 hours recall method with 1 day weighment and (b) monthly food purchase inventory for cereals and pulses. Relationship between food secure status and variables of interest was studied from Chi-square value and odds ratio. Only 42.6% households and 54% preschool children from these households were calorically secure. Insecurity was the highest in 48-59 months age group. Per capital income, increasing birth order, family size, household size, less years of schooling of the mother, less than 4 meals per day and pulse insufficiency at home were associated with food insecurity. Per capita income ensures food availability at home. Family size and household size probably ensure distribution. Mother's education, frequent feeds more than four, ensure that it reaches the preschool children.

Anganwadi worker's participation in rural newborn care.

Anganwadi worker was involved in rural newborn care as a link between a dai and a health worker. She was trained to ensure that, (i) borderline LBW/preterm baby was kept warm at home and (ii) a very small baby was referred to hospital. The training was conducted during routine monthly meetings and cost of equipping each anganwadi worked out to be Rs 110. Newborn survival, infant survivals and overall MCH performance improved. Thus, newborn care formed an ideal entry point into MCH activities.

PIP: The Rural Neonatal Care Program was initiated in January 1988 in Ganjad Primary Health Center, Dahanu, India, with the training of dais in care of the newborn. Later in the year training was conducted for anganwadi workers, who would follow-up newborn care until the child was 6 years of age. The anganwadi worker was a link between the dai and health worker and the health center. Home visits were made on the day of birth. The infant was measured and an health assessment made. If the infant was preterm or low birth weight and with a foot length of between 6.5 and 7 cm, repeat home visits were made to assess the breast feeding progress, the infant activity level, and general signs of health, as reflected in warm and pink feet. Training was conducted at monthly meetings at the block level, at the primary health care level, and during field visits to show how to care for hypothermic babies. A training manual and record book were developed and used. Footprints were made, and referrals were made to the hospital for those with feet under 6.5 to 7 cm. General observations were that birth registration improved, and infants were more easily identified for immunization. Newborn referral improved, and infant mortality declined. There was an important role for the anganwadi worker in providing continuity of care, identification of referrals, and training in the home for how to keep a baby warm. The number of referrals averaged 1-2 per month and 1-2 with special home attention. The training of the anganwadi worker in newborn care was included in the normal course of training and cost about Rs. 110 per worker.

Rural neonatal care: Dahanu experience.

The Rural Neonatal Care Project, started by the Government of Maharashtra in the Ganjad Primary Health Centre, Dahanu block in Maharashtra, had the TBA as the sheet anchor for delivery of neonatal care. Maintenance of "warm chain" and resuscitation of an asphyxiated baby were recognized as the most important interventions besides detection of a very low birth weight/preterm baby and safe transportation of such a baby. Foot length measurement from foot print was used as a surrogate to birth weight as an indicator for referral. Neonatal and perinatal mortality rates dropped appreciably over 3 years and the antenatal registration went up by 30%. The cost of this programme is affordable and the programme itself was acceptable to the community and the TBAs because of its simplicity.

Training of traditional birth attendants in newborn care.

PIP: Appropriate training of traditional birth attendants (TBAs) can both increase the proportion of births attended by trained persons and enhance linkages between rural communities and modern health services. Described is a TBA training program in newborn care developed by the Rural Neonatal Care Project in Maharashtra State, India. To improve attendance, a sub-center was established for TBAs from the northern part of the primary health care area. In addition, an effort was made to train women who assist the TBAs (usually a close relative). Two training sessions per month were held for 6 months, then the frequency was reduced to once a month. TBAs received 5 rupees for attending a training session and an additional 5 rupees for each registered birth. The training, delivered by lady health visitors and auxiliary nurse midwives, used photographs and dolls to communicate information about keeping the newborn warm, resuscitating a depressed baby, identifying very small infants, and safely transporting at-risk infants to the primary health care center. Also addressed were immunization, management of diarrhea, and referral of acute respiratory infection cases. Program evaluation highlighted the importance of brief, task-oriented sessions that use demonstrations, case histories, oral questioning, and reviews of material presented in earlier sessions. Since TBAs have extensive experience in deliveries in village conditions, they should be regarded by trainers as equal partners.^ieng

Orogastric versus nasogastric feeding of newborn babies.

Oxygen saturations were compared, 10 min before and 10, 20 and 30 min after orogastric and nasogastric feeds, in 10 stable newborns. The mean saturations were significantly lower with mere passage of nasogastric tube and continued to be so during feeds. There was no difficulty in securing the orogastric tube and no baby aspirated milk.

Estrogen receptor ß activation impairs mitochondrial oxidative metabolism and affects malignant mesothelioma cell growth in vitro and in vivo.

Estrogen receptor (ER)-ß has been shown to possess a tumor suppressive effect, and is a potential target for cancer therapy. Using gene-expression meta-analysis of human malignant pleural mesothelioma, we identified an ESR2 (ERß coding gene) signature. High ESR2 expression was strongly associated with low succinate dehydrogenase B (SDHB) (which encodes a mitochondrial respiratory chain complex II subunit) expression. We demonstrate that SDHB loss induced ESR2 expression, and that activated ERß, by over-expression or by selective agonist stimulation, negatively affected oxidative phosphorylation compromising mitochondrial complex II and IV activity. This resulted in reduced mitochondrial ATP production, increased glycolysis dependence and impaired cell proliferation. The observed in vitro effects were phenocopied in vivo using a selective ERß agonist in a mesothelioma mouse model. On the whole, our data highlight an unforeseen interaction between ERß-mediated tumor suppression and energy metabolism that may be exploited to improve on the therapy for clinical management of malignant mesothelioma.

Liver transplantation without abdominal drainage.

This observational cohort compared 70 consecutive liver transplantations (OLT) with no intra-abdominal drain and 70 control subjects C with an intra-abdominal drain who were operated immediately prior to them. We sought to assess the impact of abdominal drainage on the diagnosis and prevention of early postoperative complications of hemoperitoneum, reinterventions, biliary leaks or percutaneous drainage. We assessed variables related to the recipient (age, indication, pretransplant ascites, body mass index, Model for End-stage Liver Disease score, and rejection episodes, to the donor (age, steatosis and, ischemia time) as well as intra- and postoperative factors (surgery time, blood product use, and coagulopathy). The endpoint was defined as the need for a reintervention, postoperative paracentesis, appearance/drainage of collections, as well as lengths of hospital and intensive care unit (ICU) stays. Postoperative ICU and in-hospital stay were similar between the groups (3.6 versus 3.7 days and 12 versus 14 days respectively). Six patients in the drainage group were reoperated due to hemoperitoneum, whereas it was one in the cohort without drainage. Three patients presented a biliary fistula, two in the group without drainage, and one in the drainage group. One patient in the drainage group required percutaneous drainage of an intra-abdominal collection. The need for postoperative paracentesis was greater among the group without drainage (30% versus 6%; P < .008) and among those with a preoperative ascites > 1000 mL (38%). Patients with drainage displayed a greater incidence of perihepatic hematomas upon ultrasound (50% versus 22%, P < .008) and required more postoperative blood products, especially plasma (P < .01). In conclusion, OLT without intra- abdominal drainage is safe and does not increase morbidity. It seems likely that drainage may be responsible for intra-abdominal hematomas and greater consumption of blood products.