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Next-generation sequencing (NGS) has been advancing the progress of detection of disease-associated genetic variants and genome-wide profiling of expressed sequences over the past decade. NGS enables the analyses of multiple regions of a genome in a single reaction format and has been shown to be a cost-effective and efficient tool for root-cause analysis of disease and optimization of treatment. NGS has been leading global efforts to device personalized and precision medicine (PM) in clinical practice. The effectiveness of NGS for the aforementioned applications has been proven unequivocal for multifactorial diseases like cancer. However, definitive prediction of cancer markers for all types of diseases and for global populations still remains highly rewarding because of the diversity of cancer types and genetic variants in human. We performed exome sequencing of four samples in quest of critical genetic factor/s associated with liver cancer. By imposing knowledge-based filter chains, we have revealed a panel of genetic variants, which are unrecognized by current major genomics data repositories. Total 20 MNV-induced, 5 INDEL-induced, and 31 SNV-induced neoplasm-exclusive genes were revealed through NGS data acquisition followed by data curing with the application of quality filter chains. Liver-specific expression profile of the identified gene pool is directed to the selection of 17 genes which could be the as likely causative genetic factors for liver cancer. Further study on expression level and relevant functional significance enables us to identify and conclude the following four novel variants, viz., c.416T>C (p.Phe139Ser) in SORD, c.1048_1049delGCinsCG (p.Ala350Arg) in KRT6A, c.1159G>T (p.Gly387Cys) in SVEP1, and c.430G>C (p.Gly144Arg) in MRPL38 as a critical genetic factor for liver cancer. By applying a novel data prioritizing rationale, we explored a panel of previously unaddressed liver cancer-associated variants. These findings may have an opportunity for early prediction of neoplasm/cancer in liver and designing of relevant personalized/precision liver cancer therapeutics in clinical practice. Since NGS protocol is associated with tons of non-specific mutations due to the variation in background genetic makeup of subjects, therefore, our method of data curing could be applicable for more effective screening of global genetic variants related to disease onset, progression, and remission.

Heart development is a precisely harmonized process of cellular proliferation, migration, differentiation, and integrated morphogenetic interactions, and therefore it is extremely vulnerable to developmental defects that cause congenital heart diseases (CHD). One of the major causes of CHD has been shown to be the mutations in key cardiac channel-forming proteins namely, connexins (Cxs). Cxs are tetra-spanning transmembrane proteins that form gap junction channels and hemichannels on cellular membrane. They allow passage of small molecules or ions between adjacent cells or between cells and the extracellular environment. Studies have revealed that the spatiotemporal expression of Cxs mainly, Cx31.9, Cx40, Cx43, and Cx45 is essentially involved in early developmental events, morphogenetic transformations, maturation, and functional significance of heart. Our lab and others have shown that mutations in gap junction proteins could result in impaired trafficking, misfolding, and improper channel function of these proteins. It has also been shown that differential expressions of cardiac Cxs are associated with pathophysiological conditions of heart. Collectively, these conditions are coupled with abrogated or modified functionality of relevant channels in cardiac tissue, which are associated with many pathological situations, including CHD. Since CHD are a major cause of morbidity, therefore recovery of such kind of heart defects associated with Cxs is extremely important but remains highly challenging. In this review, we will summarize the role of Cxs in development, morphogenesis, maturation, normal function, and pathology of heart, and propose possible bioengineering techniques to recover defects in cardiac tissues related to the modified functions of Cxs.

Chimeric proteins have been used for years for various purposes ranging from biomaterials to candidate drug molecules, and from bench to bulk. Regenerative medicine needs various kinds of proteins for providing essential factors for maintaining starting cells, like induced pluripotent stem cells (iPSC), and renewal, proliferation, targeted differentiation of these cells, and as extracellular matrix for the experimental cells. However, there are several challenges associated with making functional chimeric proteins for effective application as biomaterial in this field. Fc-chimeric protein technology could be an effective solution to overcome many of them. These tailored proteins are recently becoming superior choice of biomaterials in stem cell technology and regenerative medicine due to their specific advantageous biophysical and biochemical properties over other chimeric forms of same proteins. Recent advances in recombinant protein-related science and technology also expedited the popularity of this kind of engineered protein. Over the last decade our lab has been pioneering this field, and we and others have been successfully applied Fc-chimeric proteins to overcome many critical issues in stem cell technologies targeting regenerative medicine and tissue engineering. Fc-chimeric protein-based biomaterials, specifically, E-cad-Fc have been preferentially applied for coating of cell culture plates for establishing xenogeneic-agent free monolayer stem cell culture and their maintenance, enhanced directed differentiation of stem cells to specific lineages, and non-enzymatic on-site one-step purification of target cells. Here the technology, recent discoveries, and future direction related with the E-cad-Fc-chimeric protein in connection with regenerative medicine are described.

The pannexin family of mammalian proteins, composed of Panx1, Panx2, and Panx3, has been postulated to be a new class of single-membrane channels with functional similarities to connexin gap junction proteins. In this study, immunolabeling and coimmunoprecipitation assays revealed that Panx1 can interact with Panx2 and to a lesser extent, with Panx3 in a glycosylation-dependent manner. Panx2 strongly interacts with the core and high-mannose species of Panx1 but not with Panx3. Biotinylation and dye uptake assays indicated that all three pannexins, as well as the N-glycosylation-defective mutants of Panx1 and Panx3, can traffic to the cell surface and form functional single-membrane channels. Interestingly, Panx2, which is also a glycoprotein and seems to only be glycosylated to a high-mannose form, is more abundant in intracellular compartments, except when coexpressed with Panx1, when its cell surface distribution increases by twofold. Functional assays indicated that the combination of Panx1 and Panx2 results in compromised channel function, whereas coexpressing Panx1 and Panx3 does not affect the incidence of dye uptake in 293T cells. Collectively, these results reveal that the functional state and cellular distribution of mouse pannexins are regulated by their glycosylation status and interactions among pannexin family members.

The pannexin family of mammalian proteins, composed of Panx1, Panx2, and Panx3, has been postulated to be a new class of single-membrane channels with functional similarities to connexin gap junction proteins. In this study, immunolabeling and coimmunoprecipitation assays revealed that Panx1 can interact with Panx2 and to a lesser extent, with Panx3 in a glycosylation-dependent manner. Panx2 strongly interacts with the core and high-mannose species of Panx1 but not with Panx3. Biotinylation and dye uptake assays indicated that all three pannexins, as well as the N-glycosylation-defective mutants of Panx1 and Panx3, can traffic to the cell surface and form functional single-membrane channels. Interestingly, Panx2, which is also a glycoprotein and seems to only be glycosylated to a high-mannose form, is more abundant in intracellular compartments, except when coexpressed with Panx1, when its cell surface distribution increases by twofold. Functional assays indicated that the combination of Panx1 and Panx2 results in compromised channel function, whereas coexpressing Panx1 and Panx3 does not affect the incidence of dye uptake in 293T cells. Collectively, these results reveal that the functional state and cellular distribution of mouse pannexins are regulated by their glycosylation status and interactions among pannexin family members.

A new type of urea transporter was identified by a database search and shown to be highly expressed in the renal proximal tubule cells of teleosts; proximal tubule-type urea transporters have not been describe previously. We first identified urea transporter-like sequences in the fugu genome and in an EST database of rainbow trout. Based on these pieces of sequence information, we obtained a full-length cDNA for the eel ortholog, consisting of 378 amino acid residues, and named it eUT-C. Although its sequence similarity to the known urea transporters is low (∼35%), its heterologous expression in Xenopus laevisoocytes indicated that it is a facilitative urea transporter sensitive to phloretin. Its activity is not dependent on Na+. Northern blot analysis showed that expression of eUT-C is highly restricted to the kidney, with weak expression in the stomach. In both tissues, eUT-C mRNA was strongly induced when eels were transferred from freshwater to seawater. Immunohistochemistry and in situ hybridization histochemistry revealed proximal tubule cell localization of eUT-C. Taking into account that 1) urea is mainly secreted from the gill where another type of urea transporter (eUT) has been identified and 2) fish excrete a very small volume of urine in seawater, we propose that eUT-C cloned here is a key component working in combination with the gill transporter to achieve an efficient urea excretory system in fish, namely, eUT-C reabsorbs urea from glomerular filtrate and sends it to the gill, through the circulation, for excretion.