Allele-specific and multiplex PCR based tools for cost-effective and comprehensive genetic testing in Congenital Adrenal Hyperplasia.

Congenital Adrenal Hyperplasia (CAH) is an autosomal recessive disorder due to enzyme defects in adrenal steroidogenesis. Several genes code for these enzymes, out of which mutations in the CYP21A2 gene resulting in 21 hydroxylase deficiency, contribute to the most common form of CAH. However, pseudogene imposed challenges complicate genotyping CYP21A2 gene, and there is also a lack of comprehensive molecular investigations in other genetic forms of CAH in India. Here, we describe a cost-effective, highly specific, and sensitive Allele Specific PCR (ASPCR) assay designed and optimized in-house to screen eight common pathogenic mutations in the CYP21A2 gene. We have also established and utilized a multiplex PCR assay for target enrichment and Next-generation sequencing (NGS) of CYP11B1, CYP17A1, POR, and CYP19A1 genes. Following preliminary amplification of the functional gene CYP21A2, ASPCR based genotyping of eight common mutations - P30L, I2G, 8BPdel, I172N, E6CLUS (I235N, V236E, M238K) V281L, Q318X, and R356W was carried out. These results were further validated using Sanger and Next-generation sequencing. Once optimized to be specific and sensitive, the advantage of ASPCR in CYP21A2 genotyping extends to provide genetic screening for both adult and paediatric subjects and carrier testing at a low cost and less time. Furthermore, multiplex PCR coupled NGS has shown to be cost-effective and robust for parallel multigene sequencing in CAH.

Growth hormone storm following infarction of a residual growth hormone secreting pituitary macroadenoma.

BACKGROUND: Thyrotroph pituitary adenomas have been reported to be a rare cause of 'thyroid storms', causing myriad metabolic and autonomic disturbances. In this case, we describe the second reported case in literature of a 'GH storm' in an infarcted somatotroph adenoma.Case description: We describe a residual invasive somatotroph macroadenoma that underwent infarction, producing a dramatic elevation in serum GH levels. While infarction of adenomas may in some cases lead to remission, the patient went on to require re-surgery and re-radiation due to growth of the residual viable tumour.Conclusions- 'GH storms' are rare but interesting events that may occur in somatotroph adenomas. Infarction or apoplexy must be considered when managing residual adenomas.

Allele-specific PCR and Next-generation sequencing based genetic screening for Congenital Adrenal Hyperplasia in India.

Genetic screening of Congenital Adrenal Hyperplasia (CAH) is known to be challenging due to the complexities in CYP21A2 genotyping and has not been the first-tier diagnostic tool in routine clinical practice. Also, with the advent of massive parallel sequencing technology, there is a need for investigating its utility in screening extended panel of genes implicated in CAH. In this study, we have established and utilized an Allele-Specific Polymerase Chain Reaction (ASPCR) based approach for screening eight common mutations in CYP21A2 gene followed by targeted Next Generation Sequencing (NGS) of CYP21A2, CYP11B1, CYP17A1, POR, and CYP19A1 genes in 72 clinically diagnosed CAH subjects from India. Through these investigations, 88.7% of the subjects with 21 hydroxylase deficiency were positive for eight CYP21A2 mutations with ASPCR. The targeted NGS assay was sensitive to pick up all the mutations identified by ASPCR. Utilizing NGS in subjects negative for ASPCR, five study subjects were homozygous positive for other CYP21A2 variants: one with a novel c.1274G>T, three with c.1451G>C and one with c.143A>G variant. One subject was compound heterozygous for c.955C>T and c.1042G>A variants identified using ASPCR and NGS. One subject suspected for a Simple Virilizing (SV) 21 hydroxylase deficiency was positive for a CYP19A1:c.1142A>T variant. CYP11B1 variants (c.1201-1G>A, c.1200+1del, c.412C>T, c.1024C>T, c.1012dup, c.623G>A) were identified in all six subjects suspected for 11 beta-hydroxylase deficiency. The overall mutation positivity was 97.2%. Our results suggest that ASPCR followed by targeted NGS is a cost-effective and comprehensive strategy for screening common CYP21A2 mutations and the CAH panel of genes in a clinical setting.

A clinicoradiological analysis of silent corticotroph adenomas after the introduction of pituitary-specific transcription factors.

BACKGROUND: Silent corticotroph adenomas (SCAs) are a rare subtype of non-functional pituitary adenoma. While it has been suggested that they are more aggressive and recur more frequently following excision, there is limited literature on the optimum treatment strategy for these tumors, especially regarding the role of radiation therapy in incompletely resected tumors. METHOD: We assimilated data from 62 SCAs and 238 other non-functional adenomas (ONAs), defined according to the WHO 2017 criteria that incorporates transcription factor analysis. We compared their clinicoradiological characteristics, such as hormonal levels, tumor configuration, size, and invasiveness. For 52 SCAs and 205 ONAs with serial follow-up imaging, we studied outcomes for progression after subtotal resection with or without radiation therapy or recurrence after gross total resection. Kaplan Meier analysis for recurrence or progression was used to determine the need for a differential treatment strategy for SCAs compared with other non-functional adenomas specifically concerning the role of radiotherapy. RESULTS: Patients with SCAs present at a younger age than ONAs (43.9 years vs. 48.2 years, p = 0.014), with larger (14.9 cc vs. 9.7 cc, p = 0.006) and more invasive adenomas (61.2% vs. 45.8%, p = 0.021). Overall, SCAs are more likely to recur or progress (48.7 vs. 15.7%, p < 0.001) following excision than ONAs, with significantly poorer event-free survival (Log rank test p < 0.001). Early adjuvant radiotherapy provides favorable outcomes among SCAs with postoperative residual tumor, on par with ONAs. Multivariate analysis identified male gender (HR: 2.217; p = 0.017), MIB index ≥ 3% (HR: 2.116; p = 0.012), and SCA tumor pathology (HR: 3.787; p < 0.001) as factors predicting recurrence. CONCLUSIONS: Based on the results of this retrospective, single-center review of 300 non-functional adenomas, we conclude that silent corticotroph adenomas are an aggressive subtype of non-functional pituitary adenomas that are larger, more likely to be invasive, and tend to recur more frequently after a subtotal excision compared with other non-functional adenomas. A gross total resection must be attempted whenever possible and earlier adjuvant radiation is recommended when re-surgery for residual tumor is difficult.

Do proximal hip geometry, trabecular microarchitecture, and prevalent vertebral fractures differ in postmenopausal women with type 2 diabetes mellitus? A cross-sectional study from a teaching hospital in southern India.

This study from southern India showed that the trabecular microarchitecture and proximal hip geometry were significantly impaired in postmenopausal women with diabetes as compared to age and BMI matched non-diabetic controls. This is despite there being no significant difference in bone mineral density at the femoral neck and hip not between both groups. One-third of the study subjects with type 2 diabetes had prevalent vertebral fractures. Bone mineral density assessment as a standalone tool may not adequately reflect bone health in subjects with diabetes. INTRODUCTION: There is limited information with regard to bone health in Indian postmenopausal women with type 2 diabetes. We studied the bone mineral density (BMD), trabecular bone score (TBS), prevalent vertebral fractures (VF), proximal hip geometry, and bone mineral biochemistry in ambulatory postmenopausal women with and without type 2 diabetes mellitus (T2DM). METHODS: This was a cross-sectional study conducted at a tertiary care center. BMD, TBS, prevalent vertebral fractures, and hip structural analysis (HSA) were assessed using a dual-energy X-ray absorptiometry (DXA) scanner. Bone mineral biochemical profiles were also studied. RESULTS: A total of 202 ambulatory postmenopausal women known to have type 2 diabetes mellitus with mean (SD) age of 65.6 (5.2) years and 200 age and BMI matched non-diabetic controls with mean (SD) age of 64.9 (4.7) years were recruited from the local community. Although the prevalence of lumbar spine osteoporosis was significantly lower among cases (30.7%) as compared to controls (42.9%), the prevalence of degraded bone microarchitecture (TBS < 1.200) was significantly higher among cases (51%) than in controls (23.5%); P < 0.001. Prevalent vertebral fractures were not significantly different in cases and controls. The various geometric indices of the proximal hip were significantly impaired in subjects with diabetes as compared to controls. CONCLUSION: This study may highlight the utility of the trabecular bone score and hip structural analysis in subjects with diabetes, where the bone mineral density tends to be paradoxically high, and may not adequately predict fracture risk.

Familial hypercholesterolemia: The skin speaks.

Familial hypercholesterolaemia (FH) is an autosomal dominant inherited disorder of lipoprotein metabolism caused by defects in the low-density lipoprotein receptor (LDLR) gene. It is characterized by high low-density lipoprotein (LDL) cholesterol levels, premature cardiovascular disease (CVD), and tendon xanthomas. We present the case of a 26-year-old gentleman who presented with multiple nodular eruptions over the extensor aspects of upper and lower limbs and was diagnosed as FH on the basis of positive family history, typical lipid profile abnormalities, and biopsy of the nodule consistent with tendon xanthomas. The diagnosis and management of this case is deftly feasible at the primary care level.

Vitamin D assessment and precision of clinical referrals: Insights gained from a teaching hospital in southern India.

OBJECTIVE: Vitamin D deficiency is widely prevalent worldwide. This has led to a significant surge in referrals for vitamin D assessment in recent years. The cost-effectiveness and rationalization of this practice is uncertain. This study aimed to evaluate the referral pattern for vitamin D testing from a tertiary center in southern India. MATERIALS AND METHODS: This was a cross-sectional study done over a period of one year (2017). A total of 95,750 individuals, referred for vitamin D screening were included in this study. Details regarding referring departments and indications for referral were obtained from the computerized hospital information processing system (CHIPS). RESULTS: The study population exhibited a female preponderance (54.1%) with mean (SD) age of 40.3 (18.5) years. Overall, 44% were found to have vitamin D deficiency. Most of the referrals were from nephrology (15.4%), neurology (10.1%), and orthopedics (9.1%). Nevertheless, dermatology, the staff-clinic, and hematology which contributed to 3.3%, 1.7%, and 1.7% of referrals, had a higher proportion of vitamin D deficiency of 59.1%, 57.7%, and 64.6%, respectively. Although the most common indications for referral were generalized body aches (20.5%) and degenerative bone disorders (20.1%), the proportion of subjects with vitamin D deficiency referred for these indications were 46.1% and 41.6%, respectively. In contrast, chronic steroid use that accounted for 3.3% of the referrals had 59.1% of subjects who were deficient in vitamin D. CONCLUSION: To ensure a rational approach to vitamin D testing, clinicians ought to use their discretion to screen those truly at risk for vitamin D deficiency on a case to case basis and avoid indiscriminate testing of the same.

Falls in older people with diabetes: Identification of simple screening measures and explanatory risk factors.

AIMS: To identify risk factors for falls in older people with diabetes mellitus (DM) and to develop a low-cost fall risk screening tool. METHODS: Older adults with DM (n = 103; age = 61.6 + 6.0 years) were recruited from diabetic clinics. Demographic, DM specific factors, lower limb strength and sensation, cognition, fear of falling, hand reaction time, balance, mobility and gait parameters were assessed using validated methods. Falls were prospectively recorded over six months. RESULTS: Past falls and female gender were identified as significant predictors of falls: history of falls and female gender increased fall rates by 4.62 (95% CI = 2.31-9.27) and 2.40 (95% CI = 1.04-5.54) respectively. Fall rates were significantly associated with Diabetic Neuropathy scores, HbA1c level, contrast sensitivity, quadriceps strength, postural sway, tandem balance, stride length and Timed Up and Go Test times. A multi-variable fall risk tool derived using five measures, revealed that absolute risk for multiple falls increased from 0% in participants with zero or one factor to 83% in participants with all five risk factors. CONCLUSIONS: Simple screening items for fall risk in people with DM were identified, with parsimonious explanatory risk factors. These findings help guide tailored interventions for preventing falls in DM.

How safe is metformin when initiated in early pregnancy? A retrospective 5-year study of pregnant women with gestational diabetes mellitus from India.

BACKGROUND: The initiation of metformin in early pregnancy in Gestational Diabetes mellitus (GDM) remains controversial. The aim of our study was to assess the influence of Metformin on maternal and fetal outcomes when initiated within the first trimester of pregnancy in GDM. METHODS AND MATERIALS: A retrospective analysis of 540 women with diabetes complicating pregnancy (IADPSG criteria) over five years (January 2011 to May 2016) was done. The study population comprised of patients initiated on (a) metformin within the first trimester (Group A:n = 186), (b) metformin after the first trimester (Group B:n = 203) and (c) insulin at any time during their pregnancy (Group C:n = 151). The primary outcomes compared were prematurity, respiratory distress, birth trauma, 5-min APGAR score, neonatal hypoglycaemia and need for phototherapy, while secondary outcomes compared were neonatal anthropometric measurements, maternal glycemic control, maternal hypertensive complications, postpartum glucose tolerance. RESULTS: Individual and composite primary or secondary outcomes in group A were similar to Groups B and C, though numerically higher premature births were seen in Group A. There was a 1.3% overall incidence of stillbirths/IUD, while 1.11% congenital anomalies were noted of which 2.15% were in group A and 1.32% were in Group C (p = .16). CONCLUSIONS: The initiation of metformin within the first trimester of pregnancy has no significant adverse maternal or fetal outcomes. However, vigilance for premature births is recommended in women exposed to metformin in early pregnancy.

Clinico-radiological characteristics and not laboratory markers are useful in diagnosing diabetic myonecrosis in Asian Indian patients with type 2 diabetes mellitus: A 10-year experience from South India.

INTRODUCTION: Diabetic myonecrosis or muscle infarction is an unusual complication of Type 2 Diabetes, usually associated with longstanding disease. It commonly presents as an acute non-traumatic palpable swelling of the affected muscle with predilection for the quadriceps and thigh muscles, often accompanied by retinopathy and nephropathy. METHODOLOGY: A retrospective review of the medical records of patients admitted with diabetic myonecrosis under the Department of Endocrinology, Christian Medical College Vellore over a period of ten years(2006-2015) was done. Data pertaining to clinical, biochemical and radiological characteristics were obtained and treatment modalities and outcomes were recorded. RESULTS AND ANALYSIS: A total of n = 4 patients with diabetic myonecrosis and completed clinical data were included in the study. In our present series, the mean age at presentation was 45.5 years (±7.3 years), the mean duration of the diabetes was 9.0 years (±2.5 years)with an equal distribution of male and female subjects. The mean HbA1c (9.5 ± 0.6%) was suggestive of poor glycemic control at presentation with all (100%) the patients in our series having concomitant one or more microvascular complications. While laboratory parameters of elevated CPK or LDH were mostly normal, the findings of T1 hyperintense and T2 hypointense heterogenous lower limb lesions were present in all the subjects (n = 4). Conservative management with bed rest, analgesics and good glycemic control were effective in good clinical improvement over a period of 1-2 months. CONCLUSIONS: Our series of diabetic myonecrosis in Indian patients with Type 2 diabetes mellitus, elucidates the varied clinical presentations, with MRI findings rather than laboratory markers being the mainstay of diagnosis.

Clinical characteristics, beta-cell dysfunction and treatment outcomes in patients with A-ß+ Ketosis-Prone Diabetes (KPD): The first identified cohort amongst Asian Indians.

OBJECTIVE: Ketosis-prone diabetes (KPD), an atypical form of diabetes, has emerged as a heterogeneous syndrome in multiple ethnic groups. The objectives of this study were to look into the clinical characteristics of adult Asian Indian patients with recently diagnosed, antibody negative diabetes presenting with unprovoked ketoacidosis (A-ß+ KPD) and to determine the natural course of recovery of beta-cell functions on serial follow-up over one year. RESEARCH DESIGN AND METHODS: Newly diagnosed adult diabetes patients (n=11) with suspected KPD (A-ß+) were prospectively studied over a period of 1-year with serial evaluations of clinical, biochemical and beta-cell secretion characteristics. These were compared with a control group (n=23) of KPD (A+ß-) (classical Type 1A diabetes) with similar presentation. Beta-cell secretion was assessed by fasting and stimulated C-peptide values after a standard mixed meal challenge. Glycaemic control and treatment outcomes were also documented. RESULTS: In comparison to the A+ß- KPD controls, the A-ß+ KPD patients had a significantly older age, higher BMI, stronger family history of type 2 diabetes, more severe ketoacidosis and higher fasting and stimulated C-peptide level at presentation. On serial follow-up, the patients with KPD achieved complete recovery of their beta-cell function with remission from insulin-dependence within 3-4months without further recurrences of DKA. CONCLUSIONS: This is the first reported series of A-ß+ KPD from India. The phenotype of Indian A-ß+ KPD patients differs from their Western counterparts in that they are relatively younger and leaner, though the male preponderance and natural history of recovery of beta-cell dysfunction bears similarity.

Demographic details, clinical features, and nutritional characteristics of young adults with Type 1 diabetes mellitus - A South Indian tertiary center experience.

CONTEXT: Type 1 diabetes mellitus (T1DM) accounts for 5-10% of all diagnosed diabetes and the highest incidence is found in India. AIMS: The main objectives were to study the demographic, clinical, and nutritional characteristics of young adults with T1DM and its effect glycosylated hemoglobin levels. SUBJECTS AND METHODS: This cross-sectional study was conducted among young adults with T1DM (18-45 years of age) in a tertiary hospital in South India. Data were obtained from updated medical records. The dietary data were assessed from food diaries and 24 h recall method. Anthropometry was determined. RESULTS: The analysis revealed that socio-economic variables did not affect the glycosylated hemoglobin levels. The mean glycosylated hemoglobin value was 8.81 ± 2.38%. Nearly, half the patients were malnourished. The overall dietary intake was inadequate. The multivariate regression model, adjusted for confounding factors such as gender, age, and body mass index, revealed that only duration of diabetes and protein intake were significant predictors of glycosylated hemoglobin status (P < 0.005). CONCLUSION: Integrated care provided at subsidized cost has been pivotal in effective diabetes management. However, there is an urgent need to educate our patients on nutrition therapy. T1DM patients need specialized advice to ensure appropriately balanced nutrition that has a significant impact on their long-term glycemic control.

Can New Ultrasound Signs Help in Identifying Follicular Variant of Papillary Carcinoma of Thyroid? - A Pilot Study.

AIM: To describe two new ultrasound signs for thyroid nodules - "nodule in nodule" and "hypoechoic internal septae" and assess their usefulness in differentiating follicular variant of papillary thyroid carcinoma (FVPTC) from benign thyroid nodules (BTN). METHODOLOGY: Ultrasound findings of 210 patients with histopathologically proven FVPTC (68 nodules, M:F=13:47 with mean age of 39.5±11.9 years) and BTN (165 nodules, (M:F=41:109 with mean age of 44±11.3 years) were retrospectively reviewed from PACS by a single radiologist blinded to the final diagnosis. Logistic regression analysis was performed to identify the best predictors of FVPTC and their diagnostic performance was assessed. RESULTS: The "nodule in nodule" sign was seen in 80.9% of FVPTC and only 12.1% of BTN. The "hypoechoic internal septae" sign was seen in 44.1% of FVPTC and 17% of BTN. Younger patients, heterogeneous echotexture, nodule in nodule sign, thick incomplete non-uniform halo and presence of significant nodes were the best predictors of FVPTC (p<0.05). The sensitivity, specificity, positive predictive value, negative predictive value, accuracy and relative risk for FVPTC given as odds ratio (95% CI) for heterogeneous echotexture and nodule in nodule sign which were the best 2 predictors of FVPTC were 91.2%, 81.8%, 67.3%, 95.7%, 84.5%, 46.5 (18.5-117.4) and 80.9%, 87.7%, 74.3%, 91.2%, 86.2%, 32.5 (15.04-70.2), respectively. There was improvement in the specificity (91.5%) and accuracy (86.6%) when a combined criterion of heterogeneous echotexture and nodule in nodule sign was applied to predict FVPTC. CONCLUSION: The "nodule in nodule" sign is common in FVPTC and when combined with heterogeneous echotexture can differentiate FVPTC and BTN with high specificity.

Marketing, Technology, and Medicine: Recommendations on How to Incorporate Psychological Principles into New Technologies to Promote Healthy Behaviors.

Although technologies have provided new forms of entertainment and improved our work efficiency, they have also reduced our need to engage in healthy physical activities. We believe that the psychological principles that make sedentary entertainment technologies (such as television and video games) engaging can be incorporated into new technologies to make new technologies both engaging and promote healthy behaviors. This short report aims to 1) describe how technology has traditionally reduced motivation to engage in health behaviors, 2) discuss key elements that may make sedentary technology (in this case, television) engaging, and 3) provide examples of how these same elements can be incorporated into new technologies to increase engagement and promote health behaviors.

Hypertensive crisis in a patient with thyroid cancer.

Phaeochromocytomas may be discovered incidentally when patients present with hypertensive crisis during general anaesthesia. A 49-year-old man underwent thyroidectomy 25 years ago and was diagnosed to have spindle cell carcinoma of the thyroid. He presented with recent onset of hoarseness of voice and was found to have a vocal cord nodule. He developed a hypertensive crisis during surgery. He was subsequently evaluated and found to have bilateral phaeochromocytoma. Further evaluation revealed a RET proto-oncogene mutation at codon 634 consistent with multiple endocrine neoplasia (MEN)-2A.

Cushing's syndrome in a case of thymic carcinoma.

A 29-year-old gentleman presented with episodic features suggestive of Cushing's syndrome. He was evaluated and diagnosed with ectopic Adrenocorticotropic hormone (ACTH)-dependent Cushing's syndrome due to a thymic tumor. The thymic lesion was excised and histopathology confirmed thymic carcinoma with neuroendocrine differentiation, with local, perineural, and vascular invasion. The postoperative problems and further treatment options have been discussed in this case report.

Healthcare planning in north-east India: a survey on diabetes awareness, risk factors and health attitudes in a rural community.

AIMS AND OBJECTIVES: To determine the level of awareness and knowledge about diabetes in the community, to identify their risk factors for diabetes, the relationship between anthropometric measures and diabetes risk and estimate the burden of diabetes in this rural North-East Indian population with an objective to identify target areas for future healthcare planning. MATERIALS AND METHODS: A healthcare team conducted a community-based survey of the residents of Khowai district, Tripura. All household members above the age of 25 years were eligible to participate, following voluntary consent. Survey questionnaires were administered with interpreter assistance including demographic information, diabetes knowledge, family history, smoking, diet, healthcare access and the international physical activity questionnaire (IPAQ). Anthropometric measurements were taken and blood glucose testing performed. RESULTS: One hundred and forty four participants completed the survey, 66 males and 78 females with a mean age of 44.4 +/- 14.8 years Although 91% had heard about diabetes and 44% were concerned about developing it in the future, only 39% were aware of its association with overweight status and 37% knew it required long-term treatment. Nine percent were known to have pre-existing diabetes mellitus and a further 9% were newly detected to have diabetes mellitus or impaired glucose tolerance. Fifteen percent were hypertensive and 8% had a family history of diabetes. Their mean BMI was 21.2 kg/m2 and 31% were overweight, despite high levels of physical activity in 47%. For each predictor of increased risk--waist circumference (female >80 cm, male >94 cm), waist-height ratio >50% or BMI>23 kg/m2, subjects measuring above the cut-off were more likely to have abnormal glucose tolerance than those in the normal range (27% vs. 14.3% p = 0.08, 26.5% vs. 9.5% p = 0.008, 27.3% vs. 13.3% p = 0.043, respectively.) with waist-height ratio being the best predictor of an abnormal BGL: OR 3.45 CI (1.34 - 8.88). CONCLUSION: This is the first rural survey for diabetes in North-East India. This population had a low baseline knowledge and awareness about diabetes, despite significant diabetes prevalence. A greater emphasis on health education and risk factor modification for diabetes is warranted in the North-East part of the country.

Hemolymph-dependent and -independent responses in Drosophila immune tissue.

Insects possess an antimicrobial defense response that is similar to the mammalian innate immune response. The innate immune system is designed to recognize conserved components of microorganisms called pathogen-associated molecular patterns (PAMPs). How host receptors detect PAMPs and transmit the signals to mount the immune response is being elucidated. Using GFP-Dorsal, -Dif, and -Relish reporter proteins in ex vivo assays, we demonstrate that Drosophila fat bodies, a major immune tissue, have both hemolymph-dependent and -independent responses. Microbial preparations such as lipoteichoic acid (LTA) and peptidoglycan (PGN) can stimulate some responses from dissected and rinsed larval fat bodies. Therefore, at least some aspects of recognition can occur on fat body cell surfaces, bypassing the requirement of hemolymph. Our results also show that supernatants from bacterial cultures can stimulate the nuclear translocation of Dorsal in dissected fat bodies, but this stimulation is strictly hemolymph-dependent. Various biochemical assays suggest that the factors from bacterial supernatants that stimulate the hemolymph-dependent nuclear translocation are likely made up of proteins. We further show that Dorsal mutant larvae have much lower phenoloxidase activity, consistent with a more important role of Dorsal in innate immunity than previously shown.

Analysis of Ras-induced overproliferation in Drosophila hemocytes.

We use the Drosophila melanogaster larval hematopoietic system as an in vivo model for the genetic and functional genomic analysis of oncogenic cell overproliferation. Ras regulates cell proliferation and differentiation in multicellular eukaryotes. To further elucidate the role of activated Ras in cell overproliferation, we generated a collagen promoter-Gal4 strain to overexpress Ras(V12) in Drosophila hemocytes. Activated Ras causes a dramatic increase in the number of circulating larval hemocytes (blood cells), which is caused by cellular overproliferation. This phenotype is mediated by the Raf/MAPK pathway. The mutant hemocytes retain the ability to phagocytose bacteria as well as to differentiate into lamellocytes. Microarray analysis of hemocytes overexpressing Ras(V12) vs. Ras(+) identified 279 transcripts that are differentially expressed threefold or more in hemocytes expressing activated Ras. This work demonstrates that it will be feasible to combine genetic and functional genomic approaches in the Drosophila hematopoietic system to systematically identify oncogene-specific downstream targets.