Generation of Scalable Hepatic Micro-Tissues as a Platform for Toxicological Studies

BACKGROUND@#Currently, there is an urgent need for scalable and reliable in vitro models to assess the effects oftherapeutic entities on the human liver. Hepatoma cell lines, including Huh-7, show weakly resemblance to humanhepatocytes, limiting their significance in toxicity studies. Co-culture of hepatic cells with non-parenchymal cells, and thepresence of extracellular matrix have been shown to influence the biological behavior of hepatocytes. The aim of this studywas to generate the scalable and functional hepatic micro-tissues (HMTs). @*METHODS@#The size-controllable HMTs were generated through co-culturing of Huh-7 cells by mesenchymal stem cellsand human umbilical vein endothelial cells in a composite hydrogel of liver-derived extracellular matrix and alginate, usingan air-driven droplet generator. @*RESULTS@#The generated HMTs were functional throughout a culture period of 28 days, as assessed by monitoringglycogen storage, uptake of low-density lipoprotein and indocyanine green. The HMTs also showed increased secretionlevels of albumin, alpha-1-antitrypsin, and fibrinogen, and production of urea. Evaluating the expression of genes involvedin hepatic-specific and drug metabolism functions indicated a significant improvement in HMTs compared to two-dimensional(2D) culture of Huh-7 cells. Moreover, in drug testing assessments, HMTs showed higher sensitivity tohepatotoxins compared to 2D cultured Huh-7 cells. Furthermore, induction and inhibition potency of cytochrome P450enzymes confirmed that the HMTs can be used for in vitro drug screening. @*CONCLUSION@#Overall, we developed a simple and scalable method for generation of liver micro-tissues, using Huh-7,with improved hepatic-specific functionality, which may represent a biologically relevant platform for drug studies.

Generation of Scalable Hepatic Micro-Tissues as a Platform for Toxicological Studies

BACKGROUND@#Currently, there is an urgent need for scalable and reliable in vitro models to assess the effects oftherapeutic entities on the human liver. Hepatoma cell lines, including Huh-7, show weakly resemblance to humanhepatocytes, limiting their significance in toxicity studies. Co-culture of hepatic cells with non-parenchymal cells, and thepresence of extracellular matrix have been shown to influence the biological behavior of hepatocytes. The aim of this studywas to generate the scalable and functional hepatic micro-tissues (HMTs). @*METHODS@#The size-controllable HMTs were generated through co-culturing of Huh-7 cells by mesenchymal stem cellsand human umbilical vein endothelial cells in a composite hydrogel of liver-derived extracellular matrix and alginate, usingan air-driven droplet generator. @*RESULTS@#The generated HMTs were functional throughout a culture period of 28 days, as assessed by monitoringglycogen storage, uptake of low-density lipoprotein and indocyanine green. The HMTs also showed increased secretionlevels of albumin, alpha-1-antitrypsin, and fibrinogen, and production of urea. Evaluating the expression of genes involvedin hepatic-specific and drug metabolism functions indicated a significant improvement in HMTs compared to two-dimensional(2D) culture of Huh-7 cells. Moreover, in drug testing assessments, HMTs showed higher sensitivity tohepatotoxins compared to 2D cultured Huh-7 cells. Furthermore, induction and inhibition potency of cytochrome P450enzymes confirmed that the HMTs can be used for in vitro drug screening. @*CONCLUSION@#Overall, we developed a simple and scalable method for generation of liver micro-tissues, using Huh-7,with improved hepatic-specific functionality, which may represent a biologically relevant platform for drug studies.

Methotrexate Induced Stevens Johnson Syndrome: A Rare Case Report

Methotrexate (MTX) is a folic acid antagonist with cytotoxic and immunosuppressant activity andwith potent antirheumatic action. It is commonly a first choice Disease modifying antirheumatic drug (DMARD). There were some spurious reports of Adverse Drug Reaction (ADR) by this drug. Here we report a rare occurrence of Stevens-Johnson syndrome (SJS) / Toxic Epidermal Necrolysis (TEN) after the use of Methotrexate. Naranjo score for this adverse drug event was six, thereby making it a probable ADR. Symptomatic management of the patient was done and the offending drug was withdrawn. We are presenting this case to highlight the serious adverse reactions possible from a routinely prescribeddrug

Retrospective cost-utility and budget impact assessments of Hypericum perforatum in contrast with Fluoxetine treatment for depression in Karachi, Pakistan

In this study we have compared two different types of therapies i.e. herbal and allopathic system of therapies for Depression and studied them from the social perspectives. The Hypericum perforatum is compared with Fluoxetine [HCL] in terms of cost-utility and financial savings thereby evaluating its influence on annual expenditure of depressive patients that were randomly selected from 178 union councils of the city of Karachi, Pakistan. For both system of therapies a total of 356 patients were selected by stratified random sampling. Taking frequency of depression as '1' annually with discount rate at 3% for calculating the burden-of-illness in terms of disability-adjusted-life-years. The cost-utility and the budget-impact assessments were carried out to assess incremental-cost-effectiveness-ratio, and the budget-impact-per-onset as well as budget-impact-per-year values. In comparison with the Fluoxetine therapy, the Hypericum perforatum was found to relieve symptoms in 21.47% less cost; owing 29.23% less disability-adjusted-life-years and 21.45% less budget-impact-per-onset as well as budget-impact-per-year. The annual mean incremental-cost-effectiveness-ratio was found to be at 36.95±270.74 (less than GDP per capita threshold of Rs. 38,173.02). Hypericum perforatum provide the optimal utility with less impact on budget of a patient in comparison with the treatment of symptoms of depression with Fluoxetine.

Effects of single administration of apomorphine on memory and monoamine metabolism: a dose related study

In the present study, we have monitored dose dependent effects of apomorphine on learning and memory. Behavioral sensitization and craving, which develop upon repeated treatment with dopamine receptor agonist apomorphine, are major limitations of the therapeutic use of apomorphine in Parkinson's patients. Effects of single [intraperitoneal] injection of apomorphine at different doses [i.e., 0.5, 1.0, and 2.0 mg/ml/kg] on locomotion in a familiar environment [Skinner's box] and memory in Morris water maze were investigated. Results show significantly enhanced activity in Skinner's box in a dose dependant manner. Low dose [0.5 mg/ml/kg] of apomorphine impaired both short- as well as long-term memory while both high and moderate doses of the drug [1.0, and 2.0 mg/ml/kg] enhanced the cognitive profile in rats. However, the memory-enhancing effects of apomorphine at moderate [1.0 mg/ml/kg] dose were more pronounced as compared to high [2.0 mg/ml/kg] dose of the drug. Rats were decapitated on day 2. Whole brains of rats were collected and stored at -70 degree C. Biogenic amines [i.e., 5-Hydroxytryptamine; 5-HT and dopamine] and metabolites [i.e., Dihydroxyphenylacetic acid; DOPAC, Homovanillic acid; HVA and 5-Hydroxyindoleacetic acid; 5HIAA] were estimated by reverse phase High Performance Liquid Chromatography with electrochemical detector [HPLC-EC]. Both low [0.5mg/ml/kg] as well as moderate [1.0mg/ml/kg] dose of apomorphine increased levels of dopamine, DOPAC, HVA, 5-HT and 5-HIAA. Whereas, high [4.0 mg/kg] dose of apomorphine increased levels of dopamine, DOPAC and HVA, while decreased 5-HT and 5-HIAA levels. Results would be helpful in elucidating memory enhancing effects of apomorphine at different doses and its implication for extending therapeutics in cognitive disorders

Assessment of environmental impact on physico-chemical parameters and heavy metals content of bovine milk available in Malir District

This work designed to investigate the physico-chemical properties [pH, conductivity, salinity, TDS, DO and specific gravity] and level of essential heavy metals [Fe, Cu and Zn] and deleterious metal [Cd] were analyzed in fresh bovine milk samples available for the consumers of Malir District, Karachi. Results of most of the samples revealed that the magnitude of conductivity below the range of reference. The concentrations of metals were determined after wet digestion of samples using atomic absorption spectroscopy. The ranges of average concentrations of Fe, Cu, Zn and Cd were found as 0.262-1.104mg/l, 0.001-2.740mg/l, 2.800-5.600mg/l and 0.001-0.034mg/l in identical order. Approximately 54% samples were noticed as highly contaminated with Cd

Analgesic, anti-inflammatory and diuretic activities of Cicer arietinum L.

Analgesic, anti-inflammatory and diuretic activities of the methanol extract of two varieties of Cicer arietinum viz black or Desi and white or Kabuli were tested in the doses of 200 and 400 mg/kg. For analgesic effect of the extracts, acetic acid induced writhing, tail immersion and hot plate tests were employed in mice. The anti-inflammatory activity was carried out by carrageenan induced inflammation in rats, whereas the diuretic action was determined using metabolic cages for rats. Animals were divided into six groups [n=7]: [1] Control [2] Standard [3] MECAB 200 [4] MECAB 400 [5] MECAW 200 [6] MECAW 400. All extracts and standard drugs were administered orally. Acute oral toxicity of the extracts was also checked in mice up to 2000mg/kg dose, which showed a favorable safety. Significant analgesic and anti-inflammatory effects were observed. The results of diuretic activity were significant at 12th and 24 th hrs. Therefore, it is concluded that the methanol extracts of the seeds of Cicer arietinum have analgesic, anti-inflammatory and diuretic potential

new indole derivative decreased SALL4 gene expression in acute promyelocytic leukemia cell line [NB4]

Background: Acute myeloblastic leukemia [AML] is a clonal disorder due to bone marrow failure and uncontrolled proliferation of myeloid lineage. Acute promyelocytic leukemia [APL] is a subtype of AML. Heterocyclic compounds, such as indole, are considered as attractive candidates for cancer therapy, due to their abundance in nature and known biological activity. Sal-like protein [SALL4] is a zinc finger transcription factor involving in the multi-potency of stem cells, in the NB4 cell line. This study was aimed to evaluate the effects of basal indole and its new derivative, 2-[1-[[2, 4-Aril]imino]-2,2,2-trifluoroethyl] phenyl-1H Indole-3- carbaldehyde [TFPHC], on the expression of SALL4

Methods: Cells were cultured and treated with different concentrations [75, 150, and 300 micro g/mL] of the new indole derivative and DMSO, as a vehicle control, for 24 and 48 hours. Cell proliferation was evaluated by using Trypan blue exclusion and MTT assays. The percentage of apoptotic cells was determined by flowcytometry analysis using the Annexin V/PI apoptosis detection kit; mRNA expression of SALL4 was studied using absolute quantitative RT-PCR

Results: Our findings demonstrated the effects of new indole derivatives on SALL4 mRNA expression. Expression of SALL4 mRNA was significantly decreased at 75, 150, and 300 micro g/mL concentrations

Conclusion: SALL4 plays a role in the survival of APL cells. SALL4 expression could be suppressed by the novel indole derivative. Additionally, SALL4 gene suppression can serve as a target in APL therapy

Inhibition of diet-restriction-induced behavioral deficits by tryptophan administration in rats

Diet has a great impact on brain health and function. It plays an important role to improve and control a number of psychiatric disorders such as depression, anxiety, hyperactivity and behavioral impulsivity. Anorexia Nervosa [AN] is one of the psychiatric disorder which is associated with diet. In AN, patients show extreme dieting, weight loss, hyperactivity, depression/anxiety, self-control and behavioral impulsivity. Previous studies showed that during diet restriction, tryptophan decreases serotonin [5-hydroxytryptamine; 5-HT] metabolism in the brain due to its less availability and contributes psychiatric problems associated with AN. The present study is designed to investigate the effects of tryptophan administration on 5-HT metabolism in diet-restricted rats. Tryptophan at a dose of 50 or 100mg/kg was given orally to respective freely fed [FF] or diet restricted [DR] animals daily for five weeks. Behavioral activities were also monitored weekly. The results show significant effect [p<0.05] on behavior in activity box, open field and in light/dark transition test by tryptophan administration in diet-restricted rats. This may be associated with the increased in serum tryptophan and brain 5-HT metabolism. Therefore, it is concluded that diet-restriction-induced behavioral changes might be reverted back with the administration of tryptophan and may be helpful to improve psychological problems in AN

Repeated treatment with reserpine as a progressive animal model of depression

Treatment-resistant depression is a major health problem worldwide. Restricted validity of the existing animal models of depression along with the need for the study of progressive development of resistance to antidepressants, demands the modeling of a progressive animal model of depression. Present study was designed to test the hypothesis that the repeated administration of reserpine could serve as a progressive animal model of depression. Animals were injected with reserpine [1.0mg/kg; once a day] for three weeks. Results from the present study showed impaired locomotive effects of reserpine in Skinner's box following second as well as third week. These hypolocomotive effects were more pronounced after third week than the second week. Reserpine-induced behavioral depression was evident in the animals after 2 weeks, as assessed by using forced swim test. Depletion of 5-HT, dopamine and metabolites was also observed in the brain samples. Results from the present study suggest that repeated administration of reserpine could be serve as a progressive model of depression and could be used as a convenient and economic animal model for the face validity of anxiolytic compounds. Findings have potential implications with reference to the understanding and the management of treatment-resistant depression

Neurochemical and behavioral effects of green tea [Camellia sinensis] as observed in animals exposed to restraint stress

Clinical studies on psychiatric patients suggest that life events stress precipitates depression. The possible involvement of 5-Hydroxy tryptamine [5-HT; Serotonin] in depression and other behavioral deficits is also suggested by clinical studies. As a natural stimulant, green tea [Camellia Sinensis] diminishes stress, worry and anxiety, allowing the brain to focus and concentrate better. Previously we have reported that beneficial effects of green tea might be associated with altered levels of 5-HT, which in turn may help in coping with stress. Present study therefore deals with monitoring the behavior and neurochemical profile of single restrained stress in animals previously administered [for 5 weeks] with green tea. Activities in light dark activity box were monitored 1hr post restraint stress. Cumulative food intake values were monitored 24hr post restraint stress. 24hr after restrained stress, rats were decapitated to collect plasma and brain samples. Brain samples were kept stored at -70[degree sign]C until neurochemical analysis by HPLC-EC. Findings illustrate that although food intake was decreased in both green tea- as well as water treated rats, stress-induced anxiogenic effects were attenuated in green tea treated rats. Tone of 5-HT was also normalized in restrained animals. Results suggest beneficial effects of green tea in coping the stressful conditions/stimuli are related to altered 5-HT metabolism

Hospital Based Study of Metabolic Syndrome in Female Patients of Hypothyroidism.

Background: Hypothyroidism was found to be associated with metabolic syndrome, with females being more at risk than males. The potential contributory role of the metabolic syndrome to cardiovascular risk and its scope in subjects with hypothyroidism is the focus of this study.Methods: Forty untreated hypothyroid women and forty normal, healthy subjects were recruited for the study. Fasting blood samples were collected for lipid profile, glucose and insulin level estimation.Results: Systolic and diastolic blood pressures in hypothyroidism patients were significantly higher than values in controls. Similarly, BMI, waist circumference was higher among hypothyroidism subjects. Again, fasting plasma glucose, total cholesterol, triglycerides and LDL-cholesterol levels were higher in patients with hypothyroidism in comparison to controls, while HDL- cholesterol, insulin, HOMA-IR were higher among controls. Conclusion: Hypothyroidism is significantly associated with metabolic syndrome and its components like dysglycemia, hypertension and dyslipidemia and obesity. Increased cardiovascular and other risk factor among hypothyroidism patients need to be addressed by further studies.

A Study on the Predictors of Functional Status and the Overuse of OTC Medications among Elderly Population in a Sub-Urban Region of North Bengal.

Background: Functional status can be conceptualized as an individual’s ability to manage activities related to personal self-care and self-maintenance. Functional status assessment is fundamental aspect of geriatric examination especially in the context of over the counter (OTC) medications. This assessment helps clinicians and policymakers to design and implement interventions that help elderly to live safely and independently. The primary objective was to assess the prevalence of limitation in activities of daily living (ADL) and instrumental ADL (IADL) among elderly population. The secondary objective was to identify the use of OTC medications according to the therapeutic indications among them. Methods: A total of 200 community dwelling elderly persons residing in Sushrutanagar of North Bengal were interviewed using valid and reliable functional assessment scales namely Katz ADL and Lawton and Brody IADL. Statistical procedures for the analyses included descriptive ADL and IADL activity limitation was 15 % (30/200) and 85 % (170/200), respectively. Results: The results of logistic regression analysis revealed that advancing age (70 years and above), poor self-rated health and ailments namely musculoskeletal problems, cardiovascular diseases and cataract significantly predict functional limitation. 70% of the subjects consumed analgesics and 60% the antacids as OTC medications. Conclusion: With advancing age, various co-morbidities starts creeping up and the study subjects used the OTC medications to counteract the associated functional limitations as evident from this study. Cataract surgeries patients should be identified and operated upon so as to improve visual functioning and thus their functional ability. Further, factors affecting the use of OTC drugs in elderly subjects need further research.

Effects of sugar rich diet on brain serotonin, hyperphagia and anxiety in animal model of both genders

Lower levels of 5-hydroxytryptamine [5-HT; serotonin] in the brain elicit sugar craving, while ingestion of sugar rich diet improves mood and alleviates anxiety. Gender differences occur not only in brain serotonin metabolism but also in a serotonin mediated functional responses. The present study was therefore designed to investigate gender related differences on the effects of long term consumption of sugar rich diet on the metabolism of serotonin in the hypothalamus and whole brain which may be relevant with the hyperphagic and anxiety reducing effects of sugar rich diet. Male and female rats were fed freely on a sugar rich diet for five weeks. Hyperphagic effects were monitored by measuring total food intake and body weights changes during the intervention. Anxiolytic effects of sugar rich diet was monitored in light-dark transition test. The results show that ingestion of sugar rich diet decreased serotonin metabolism more in female than male rats. Anxiolytic effects were elicited only in male rats. Hyperphagia was comparable in both male and female rats. Finings would help in understanding the role of sugar rich diet-induced greater decreases of serotonin in sweet craving in women during stress

Protective effects of rice bran oil in haloperidol-induced tardive dyskinesia and serotonergic responses in rats

Effect of administration of Rice bran oil [RBO] was evaluated on haloperidol elicited tardive dyskinesia in rats. Albino Wistar rats treated with haloperidol in drinking water at a dose of 0.2mg/kg/day and RBO by oral tubes at a dose of 0.4 mL/day for 5 weeks. Motor coordination, VCMs and 8-hydroxy-2-[di-n-propylamino] tetraline][8-OHDPAT] _syndrome were monitored. Striatal serotonin [5-hydroxytryptamine; 5-HT] and 5-hydroxyindolacetic acid [5- HIAA] levels were determined by high performance liquid chromatography [HPLC-EC]. Rats treated with haloperidol orally at a dose of for a period of 5 weeks developed VCMs, which increased progressively as the treatment continued for 5 weeks. Motor coordination impairment started after the 1[st] week and was maximally impaired after 3 weeks and gradually returned to the 1[st] week value. Co-administration of RBO prevented haloperidol_induced VCMs as well impairment of motor coordination. The intensity of 8-OH-DPAT_induced syndrome and decreased 5-HT metabolism were greater in water + haloperidol treated animals than RBO + haloperidol treated animals. The present study suggested that involvement of free radical in the development of TD and point to RBO as a possible therapeutic option to treat this hyperkinetic motor disorder

Co-treatment with imipramine averted haloperidol-instigated tardive dyskinesia: association with serotonin in brain regions

Outcome of imipramine [IMI] treatment was scrutinized on progression of haloperidol instigated tardive dyskinesia [TD]. 0.2 mg/kg/rat dosage of haloperidol provided orally to rats for 2 weeks enhanced vacuous chewing movements that escalated when the process proceeded for 5 weeks. Following 2 weeks co-injection 5 mg/kg dosage of IMI was diminished haloperidol-instigated VCMs and fully averted following five weeks. The potency of 8-OH-DPATinstigated locomotor activity exhibited higher in saline+haloperidol treated rats while not observed in IMI+ haloperidol treated rats. 8-OH-DPAT-instigated low 5-hydroxytryptamine [5-HT; serotonin] metabolism was higher in saline+ haloperidol treated rats when compare to IMI+ haloperidol treated rats in both regions of brain [striatum and midbrain]. It is recommended that IMI possibly competent in averting TD, in cases receiving treatment to antipsychotics

Behavioral deficits in rats following acute administration of glimepiride: relationship with brain serotonin and dopamine

A considerable body of literature suggests that depression and diabetes mellitus are co-morbid. The present study was designed to test any possible behavioral deficits and/or neurochemical changes in the brain as induced by the anti-diabetic drugs. Twenty-four rats were divided into four groups: [i] saline [ii] glimepiride [2.5mg/kg]- [iii] glimepiride [5.0mg/kg]- and [iv] glimepiride [10 mg/kg] injected animals. Behavioral activities in Skinner's box, open field and elevated plus maze were monitored 20, 35 and 45 minutes post injection respectively. Animals were decapitated 60 minutes post injection to collect brain samples. Samples were kept at -70[degree]C until neurochemical analysis by HPLC-EC. Results from the present study show decreased time spent in the open arm of the elevated plus maze [p<0.05] at all the three doses. A decrease in the HVA [Homovanillic acid] levels at all three doses [p<0.01] was also observed along with decreased 5-HT [5-Hydroxytryptamine] [p<0.05 at 5.0 and l0mg/kg] and 5-HIAA [5-Hydroxyindoleacetic acid] [p<0.05 at all three doses] levels. Since a decrease in 5-HT metabolism can induce depression-like effects, the present study therefore suggests that the occurrence of depression in diabetic patients is due to the use of glimipride. Effects of long-term administration of smaller doses of glimipride are to be explored further to monitor tolerance in glimipride-induced deficits of serotonin. The finding may help to explore the cause of depression in diabetics for improving pharmacotherapy in diabetes

Immobilization-induced increases of systolic blood pressure and dysregulation of electrolyte balance in ethanol-treated rats

Clinical and experimental studies revealed that alcohol drinking and life event stresses are predisposing factors to hypertension. Intra and extra cellular levels of electrolytes may play important role in the pathogenesis and treatment of hypertension. Dietary intake of sodium, potassium, calcium and magnesium is suggested to have a role in the regulation of blood pressure. The present study was designed to monitor the effects of acute exposure to 2h immobilization stress and ethanol administration at a dose of 2.5g/kg body weight [i.p.] and combined effect of acute administration of ethanol and immobilization stress on systolic blood pressure [SBP], intraerythrocyte, serum and tissue electrolytes in rats. Results showed that acute exposure to 2h immobilization increased SBP, intraerythrocyte sodium and decreased intraerythrocyte potassium in water as well as in ethanol injected rats. The concentration of Na[+] and Ca[2+] increased while that of K[+] and Mg[2+] decreased in the heart and kidney tissue. Ethanol administration also increased Na[+] and Ca[2+] levelsand decreased K+ and Mg[2+] levels in the heart and kidney tissue. Restraint stress decreased serum levels of Na[+], K[+], Ca[2+], P, and Cl- and increased serum Mg[2+], glucose and haematocrit. Ethanol administration also decreased serum levels of Na[+], K[2+], Ca[2+], P, and Cl- and increased serum Mg[2+], glucose and haematocrit. The effects of ethanol and stress on the changes of blood and tissues electrolytes were additive and may be involved in the greater occurrence of hypertension in alcoholics. Our results suggested an important role of intra and extra cellular electrolytes in both stress and ethanol-induced hypertension. The findings may help to develop strategies for the treatment of hypertension in alcoholics

Apomorphine induced conditioned place preference and sensitization is greater in rats exposed to unpredictable chronic mild stress

CNS stimulants are the class of the drugs that may be used to get relief from depression. Apomorphine is a D1 and D2 receptor agonist with a CNS stimulatory effect used for the treatment of Parkinson's disease is also abused. Although many drugs of abuse produce tolerance and dependence. Long term use of pshycostimulants produce reverse tolerance described as sensitization. These drugs also have a number of other beneficial effects but their therapeutic use is limited because of abuse potential. Conditioned place preference [CPP] test is used to monitor the reinforcing effect of drugs of abuse. Stress is an important factor that precipitates and potentiates addictive effects of different drugs of abuse. The present study was designed to investigate the addictive effect of apomorphine [1mg/kg] in rats previously exposed to repeated unpredictable chronic mild stress for 10 days [animal model of depression]. Results from present study illustrate that unpredictable chronic mild stress potentiates the reinforcing effects of apomorphine as the number of entries and the time spent in the CPP compartment associated with drug administration is increased. Motor activity was taken as a parameter for behavioral sensitization which is induced by repeated administration of apomorphine, monitored as the number of cage crossings in light compartment of the CPP apparatus, also increased

Bizarre Case of Polydactyly with Right Mirror Foot.

Mirror foot, a form of polydactyly, is a rare congenital anomaly. In this form of congenital anomaly, there are several additional digits with accessory tarsal bones. It may be associated with fibular dimelia, tibial aplasia and tibial dysplasia. Cause of such anomaly is not known. On experimental basis it appears to involve ectopic SHH (Sonic hedgehog) signaling in the limb bud mesenchyme.