Signy Chow

The requirement for bone-marrow aspirates for genomic profiling of multiple myeloma poses an obstacle to enrolment and retention of patients in clinical trials. We evaluated whether circulating cell-free DNA (cfDNA) analysis is comparable to molecular profiling of myeloma using bone-marrow tumour cells. We report here a hybrid-capture-based Liquid Biopsy Sequencing (LB-Seq) method used to sequence all protein-coding exons of KRAS, NRAS, BRAF, EGFR and PIK3CA in 64 cfDNA specimens from 53 myeloma patients to >20,000 × median coverage. This method includes a variant filtering algorithm that enables detection of tumour-derived fragments present in cfDNA at allele frequencies as low as 0.25% (median 3.2%, range 0.25-46%). Using LB-Seq analysis of 48 cfDNA specimens with matched bone-marrow data, we detect 49/51 likely somatic mutations, with subclonal hierarchies reflecting tumour profiling (96% concordance), and four additional mutations likely missed by bone-marrow testing (>98% specificity). Overall, LB-Seq is a high fidelity adjunct to genetic profiling of bone-marrow in multiple myeloma.

NHE1 (Na+/H+ exchanger isoform 1) is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammalian cells. Proline residues within transmembrane segments have unusual properties, acting as helix breakers and increasing flexibility of membrane segments, since they lack an amide hydrogen. We examined the importance of three conserved proline residues in TM IV (transmembrane segment IV) of NHE1. Pro167 and Pro168 were mutated to Gly, Ala or Cys, and Pro178 was mutated to Ala. Pro168 and Pro178 mutant proteins were expressed at levels similar to wild-type NHE1 and were targeted to the plasma membrane. However, the mutants P167G (Pro167-->Gly), P167A and P167C were expressed at lower levels compared with wild-type NHE1, and a significant portion of P167G and P167C were retained intracellularly, possibly indicating induced changes in the structure of TM IV. P167G, P167C, P168A and P168C mutations abolished NHE activity, and P167A and P168G mutations caused markedly decreased activity. In contrast, the activity of the P178A mutant was not significantly different from that of wild-type NHE1. The results indicate that both Pro167 and Pro168 in TM IV of NHE1 are required for normal NHE activity. In addition, mutation of Pro167 affects the expression and membrane targeting of the exchanger. Thus both Pro167 and Pro168 are strictly required for NHE function and may play critical roles in the structure of TM IV of the NHE.

Linoleic acid absorption was studied using everted rat jejunal sacs. At low concentrations (42-1260 microM), the relationship between linoleic acid concentration and its absorption rate fitted best to a rectangular hyperbola. At high concentrations (2.5-4.2 mM) the relationship between the two parameters was linear. The separate additions of 2,4-dinitrophenol, cyanide, or azide, or decrease in the incubation temperature from 37 to 20 degrees C did not change the absorption rate of linoleic acid. Absorption rate of linoleic acid at low concentrations increased as the hydrogen ion and taurocholate concentrations were increased or as the unstirred water layer thickness was decreased. Linoleic acid absorption rate was decreased after the additions of lecithin, oleic, linolenic, and arachidonic acids or the substitution of taurocholate with the nonionic surfactant Pluronic F 68. These observations indicate that a concentration-dependent, dual mechanism of transport is operative in linoleic acid absorption. Facilitated diffusion is the predominant mechanism of absorption at low concentrations, while at high concentrations, simple diffusion is predominant. At low concentrations, the absorption rate of linoleic acid is influenced by the pH, surfactant type and concentration, the simultaneous presence of other polyunsaturated fatty acids, and the thickness of the unstirred water layer.

The final diagnosis of 158 patients who had a cerebrospinal fluid (CSF) lactate concentration greater than 2 mmol/l was ascertained. The conditions included seizures, inflammatory changes, and proven metabolic disorders. For the diagnosis of congenital lactic acidoses, CSF lactate should ideally be measured in a seizure free patient after any acute illness.