Normative data for aquagenic wrinkling of palms in children 1-15 years of age: A cross-sectional study.

BACKGROUND: Aquagenic wrinkling of palms (AWP) is emerging as a screening test for cystic fibrosis (CF). There is lack of normative data for the same in our population. OBJECTIVES: To generate normative data for AWP in children 1-15 years of age and to describe the factors associated with it. METHODS: Children aged 1-15 years were enrolled after obtaining informed consent and assent of the parent and child based on age. Information regarding age, gender, anthropometry, indications for visiting hospital and drug intake were noted down. Wrinkling test was done using distilled water maintained in room temperature. Sweat chloride analysis was done using Nano duct sweat analysis system for children having AWP within 3 min. The mean/median time to wrinkle was noted and presented as centile curves after smoothing. RESULTS: The mean (SD) and median (interquartile range [IQR]) aquagenic wrinkling time in children 1-15 years of age was 4.88 (1.066) and 5 (4-5.75) minutes respectively. The mean (SD) and median (IQR) time for AWP was 4.78 (1.076) and 5 (4-5) minutes respectively for boys and 4.98 (1.048) and 5 (4-6) minutes, respectively for girls. The time taken to wrinkle was observed to increase with age. Males have earlier AWP than females. There was no association between AWP and anthropometry or sweat chloride levels. CONCLUSION: We have estimated the normative data for AWP in children 1-15 years of age which can be used for CF screening in children with typical clinical features from resource limited settings.

Modified chemical method for efficient transformation and diagnosis in Pichia pastoris.

In the present study, green fluorescence protein (GFP) was used as a candidate protein to test and optimize a robust chemical transformation procedure in P. pastoris. Towards this, it was adjudged that pretreatment of P. pastoris cells with lithium chloride (LiCl) and its optimal concentration is critical for efficient transformation. Using three different methods (M1: 100 mM LiCl, 10 min, M2: 1 M LiCl, 10 min and M3: 1 M LiCl, 1 h), it was found that concentration and incubation time for LiCl treatment significantly affects the transformation efficiency. The transformation efficiency (transformants/µg DNA) was observed to be 1.01 × 102, 5.07 × 103 and 6.52 × 103 using methods M1, M2 and M3, respectively, indicating the superiority of M3. Moreover, presence of the GFP gene in the positive transformants was confirmed using a novel colony PCR method where the colonies were treated with LiCl prior to GFP specific amplification. Also, it was established using fluorescence microscopy and western blot analysis that increasing zeocin concentration as a post transformational vector amplification (PTVA) strategy increased the fluorescence and gene expression, respectively. Further, RT-qPCR revealed that the gene copy number using methods M1, M2 and M3 were 2.97, 5.26 and 7.19, respectively, when 500 µg/ml zecocin was used for selection, thus corroborating western blot results. In conclusion, we demonstrate a cheap and robust chemical method for achieving higher transformation efficiency in P. pastoris and a simple procedure for colony-PCR based-diagnosis alleviating the need for enzymatic treatment.