Folic acid is necessary for proliferation and differentiation of C2C12 myoblasts.

Folic acid, a water soluble B vitamin, plays an important role in cellular metabolic activities, such as functioning as a cofactor in one-carbon metabolism for DNA and RNA synthesis as well as nucleotide and amino acid biosynthesis in the body. A lack of dietary folic acid can lead to folic acid deficiency and result in several health problems, including macrocytic anemia, elevated plasma homocysteine, cardiovascular disease, birth defects, carcinogenesis, muscle weakness, and walking difficulty. However, the effect of folic acid deficiency on skeletal muscle development and its molecular mechanisms are unknown. We, therefore, investigated the effect of folic acid deficiency on myogenesis in skeletal muscle cells and found that folic acid deficiency induced proliferation inhibition and cell cycle breaking as well as cellular senescence in C2C12 myoblasts, implying that folic acid deficiency influences skeletal muscle development. Folic acid deficiency also inhibited differentiation of C2C12 myoblasts and induced deregulation of the cell cycle exit and many cell cycle regulatory genes. It inhibited expression of muscle-specific marker MyHC as well as myogenic regulatory factor (myogenin). Moreover, immunocytochemistry and Western blot analyses revealed that DNA damage was more increased in folic acid-deficient medium-treated differentiating C2C12 cells. Furthermore, we found that folic acid resupplementation reverses the effect on the cell cycle and senescence in folic acid-deficient C2C12 myoblasts but does not reverse the differentiation of C2C12 cells. Altogether, the study results suggest that folic acid is necessary for normal development of skeletal muscle cells.

Clonal evolution of multidrug resistant hepatitis B virus during entecavir rescue therapy.

BACKGROUND & AIMS: Analysing the mutation pattern of multidrug resistance (MDR) is important in the treatment of chronic hepatitis B (CHB). In this study, the evolutionary pattern of MDR mutations was investigated in patients receiving entecavir (ETV) rescue therapy. METHODS: Eight CHB patients with lamivudine (LAM)- and adefovir (ADV)-resistant mutations showing suboptimal response to ETV and to subsequent ETV-plus-ADV therapy were enrolled. The clonal evolution of the mutation pattern was investigated through direct sequencing, multiplex restriction fragment mass polymorphism (RFMP), and clonal analysis and the utility of these methods was compared. RESULTS: Among 160 clones at baseline, wild-type hepatitis B virus (HBV) was present in 62 (38.8%), LAM-resistant mutations in 92 (57.6%) and ADV-resistant mutations in 55 (34.4%). LAM-resistant mutations increased to 70.6% at the end of ETV therapy and increased to 74.4% at the 12th month of ETV-plus-ADV therapy. During the same time periods, ETV-resistant mutations were present in 46.3% and 38.8%, and ADV-resistant mutations were present in 3.1% and 9.4% respectively. When 256 nucleotides from 32 samples were examined for mutations, clonal analysis detected 93 mutations (36.3%), direct sequencing detected 36 mutations (14.1%) and RFMP detected 73 mutations (28.5%). The sensitivity (73.1%, 95% CI; 64.1-82.1%) and specificity (96.9%, 95% CI; 94.4-99.4%) of RFMP were high, showing a concordance rate of 88.3% with the results from clonal analysis. All mutations exceeding 40% of the total clones detected by clonal analysis were also detected by RFMP. CONCLUSIONS: The clonal evolution of the mutation pattern in MDR HBV showed the selection of LAM-resistant (±ETV-resistant) HBV during ETV rescue therapy, which may be the primary reason for patients' suboptimal response. Multiplex RFMP is a useful method for detecting MDR mutations in clinical practice.