An 81-year-old immunocompetent patient with bronchiectasis and refractory Mycobacterium abscessus lung disease was treated for six months with a three-phage cocktail active against the strain. In this case study of phage to lower infectious burden, intravenous administration was safe and reduced the M. abscessus sputum load 10-fold within one month. However, after two months, M. abscessus counts increased as the patient mounted a robust IgM-and IgG-mediated neutralizing antibody response to the phages, which associated with limited therapeutic efficacy.Nontuberculous mycobacteria (NTM)-especially Mycobacterium abscessus infections represent emerging pathogens of increasing clinical importance 1 . There is an urgent need for novel treatments for NTM as outcomes are often poor, particularly with macrolide-resistant strains 2-4 . Bacteriophages offer an innovative therapeutic approach for difficult-to-treat infections 5,6 Recently, a 15-year-old lung transplant recipient with cystic

Background: Hemophilia B is caused by genetic aberrations in the F9 gene. The majority of these are non-synonymous mutations that alter the primary structure of blood coagulation Factor IX (FIX). However, a synonymous mutation c.459G>A (Val107Val) was clinically reported to result in mild hemophilia B (FIX coagulant activity 15–20% of normal). The F9 mRNA of these patients showed no skipping or retention of introns and/or change in mRNA levels, suggesting that mRNA integrity does not contribute to the origin of the disease in affected individuals. The aim of this study is to elucidate the molecular mechanisms that can explain disease manifestations in patients with this synonymous mutation. Methods: We analyze the molecular mechanisms underlying the FIX deficiency through in silico analysis and reproducing the c.459G>A (Val107Val) mutation in stable cell lines. Conformation and non-conformation sensitive antibodies, limited trypsin digestion, activity assays for FIX, interaction with other proteins, and post-translation modifications were used to evaluate the biophysical and biochemical consequences of the synonymous mutation. Results: The Val107Val synonymous mutation in F9 was found to significantly diminish FIX expression. Our results suggest that this mutation slows FIX translation and affects its conformation resulting in decreased extracellular protein level. The altered conformation did not change the specific activity of the mutated protein. Conclusions: The pathogenic basis for one synonymous mutation (Val107Val) in the F9 gene associated with hemophilia B was determined. A mechanistic understanding of these synonymous variants yields potential for guiding and developing future therapeutic treatments.

Animal cells and cell lines, such as HEK-293 cells, are commonly cultured at 37°C. These cells are often used to express recombinant proteins. Having a higher expression level or a higher protein yield is generally desirable. As we demonstrate in this study, dropping culture temperature to 33°C, but not lower, 24 hours after transient transfection in HEK-293S cells will give rise to ~1.5-fold higher expression of green fluorescent protein (GFP) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. By following the time course of the GFP-expressing cells growing at 37°C and 33°C from 24 hours after transfection (including 19 hours recovery at 37°C in the normal growth medium), we found that a mild hypothermia (i.e., 33°C) reduces the growth rate of HEK-293S cells, while increasing cellular productivity of recombinant proteins. As a result, green cells remain undivided in a longer period of time. Not surprisingly, the property of a recombinant protein expressed in the cells grown at 33°C is unaffected, as shown by the use of AMPA receptors. We further demonstrate with the use of PC12 cells that this method may be especially useful when a recombinant protein is difficult to express using a chemical-based, transient transfection method.