qSanger: Quantification of Genetic Variants in Bacterial Cultures by Sanger Sequencing.

Genetic variations such as mutations and recombinations arise spontaneously in all cultured organisms. Although it is possible to identify nonneutral mutations by selection or counterselection, the identification of neutral mutations in a heterogeneous population usually requires expensive and time-consuming methods such as quantitative or droplet polymerase chain reaction and high-throughput sequencing. Neutral mutations could even become dominant under changing environmental conditions enforcing transitory selection or counterselection. We propose a novel method, which we called qSanger, to quantify DNA using amplitude ratios of aligned electropherogram peaks from mixed Sanger sequencing reads. Plasmids expressing enhanced green fluorescent protein and mCherry fluorescent markers were used to validate qSanger both in vitro and in cotransformed Escherichia coli via quantitative polymerase chain reaction and fluorescence quantifications. We show that qSanger allows the quantification of genetic variants, including single-base natural polymorphisms or de novo mutations, from mixed Sanger sequencing reads, with substantial reduction of labor and costs compared to canonical approaches.

Padina pavonica Extract Promotes In Vitro Differentiation and Functionality of Human Primary Osteoblasts.

Marine algae have gained much importance in the development of nutraceutical products due to their high content of bioactive compounds. In this work, we investigated the activity of Padina pavonica with the aim to demonstrate the pro-osteogenic ability of its extract on human primary osteoblast (HOb). Our data indicated that the acetonic extract of P. pavonica (EPP) is a safe product as it did not show any effect on osteoblast viability. At the same time, EPP showed to possess a beneficial effect on HOb functionality, triggering their differentiation and mineralization abilities. In particular EPP enhanced the expression of the earlier differentiation stage markers: a 5.4-fold increase in collagen type I alpha 1 chain (COL1A1), and a 2.3-fold increase in alkaline phosphatase (ALPL), as well as those involved in the late differentiation stage: a 3.7-fold increase in osteocalcin (BGLAP) expression and a 2.8-fold in osteoprotegerin (TNFRSF11B). These findings were corroborated by the enhancement in ALPL enzymatic activity (1.7-fold increase) and by the reduction of receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) ratio (0.6-fold decrease). Moreover, EPP demonstrated the capacity to enhance the bone nodules formation by 3.2-fold in 4 weeks treated HOb. Therefore, EPP showed a significant capability of promoting osteoblast phenotype. Given its positive effect on bone homeostasis, EPP could be used as a useful nutraceutical product that, in addition to a healthy lifestyle and diet, can be able to contrast and prevent bone diseases, especially those connected with ageing, such as osteoporosis (OP).