Generation of a recombinant version of a biologically active cell-permeant human HAND2 transcription factor from E. coli.

Transcription factor HAND2 has a significant role in vascularization, angiogenesis, and cardiac neural crest development. It is one of the key cardiac factors crucial for the enhanced derivation of functional and mature myocytes from non-myocyte cells. Here, we report the generation of the recombinant human HAND2 fusion protein from the heterologous system. First, we cloned the full-length human HAND2 gene (only protein-coding sequence) after codon optimization along with the fusion tags (for cell penetration, nuclear translocation, and affinity purification) into the expression vector. We then transformed and expressed it in Escherichia coli strain, BL21(DE3). Next, the effect (in terms of expression) of tagging fusion tags with this recombinant protein at two different terminals was also investigated. Using affinity chromatography, we established the one-step homogeneous purification of recombinant human HAND2 fusion protein; and through circular dichroism spectroscopy, we established that this purified protein had retained its secondary structure. We then showed that this purified human protein could transduce the human cells and translocate to its nucleus. The generated recombinant HAND2 fusion protein showed angiogenic potential in the ex vivo chicken embryo model. Following transduction in MEF2C overexpressing cardiomyoblast cells, this purified recombinant protein synergistically activated the α-MHC promoter and induced GFP expression in the α-MHC-eGFP reporter assay. Prospectively, the purified bioactive recombinant HAND2 protein can potentially be a safe and effective molecular tool in the direct cardiac reprogramming process and other biological applications.

Type IVa choledochal cyst: Results following haemihepatectomy and mucosectomy of intrahepatic cyst.

Choledochal cyst is a rare condition characterised by congenital dilatation of the biliary tree. Commonly seen in the oriental countries, patients usually present with a varying combination of abdominal pain, jaundice, lump or cholangitis. Untreated patients or incomplete removal of the cysts usually leads to portal hypertension and cholangiocarcinoma. Almost one-third of the cases have intrahepatic cyst, making complete cyst excision often impossible. We are reporting a 9-year-old girl with type IVa choledochal cyst presented to us with recurrent pain abdomen and intermittent jaundice for 1 year. Excision of extrahepatic cyst, left hepatectomy, mucosectomy of the residual cyst wall of right lobe of the liver and a wide bilioenteric anastomosis was done. Patients followed up with an magnetic resonance cholangiopancreatography (MRCP) 2 months later showed shrinkage of the residual cyst and good bile drainage. Mucosectomy of intrahepatic cyst may prevent recurrent cholangitis, calculus formation and cholangiocarcinoma in the long run.