High-Temperature Ethanol Fermentation and Transformation with Linear DNA in the Thermotolerant Yeast Kluyveromyces marxianus DMKU3-1042▿

We demonstrate herein the ability of Kluyveromyces marxianus to be an efficient ethanol producer and host for expressing heterologous proteins as an alternative to Saccharomyces cerevisiae. Growth and ethanol production by strains of K. marxianus and S. cerevisiae were compared under the same conditions. K. marxianus DMKU3-1042 was found to be the most suitable strain for high-temperature growth and ethanol production at 45°C. This strain, but not S. cerevisiae, utilized cellobiose, xylose, xylitol, arabinose, glycerol, and lactose. To develop a K. marxianus DMKU3-1042 derivative strain suitable for genetic engineering, a uracil auxotroph was isolated and transformed with a linear DNA of the S. cerevisiae ScURA3 gene. Surprisingly, Ura+ transformants were easily obtained. By Southern blot hybridization, the linear ScURA3 DNA was found to have inserted randomly into the K. marxianus genome. Sequencing of one Lys− transformant confirmed the disruption of the KmLYS1 gene by the ScURA3 insertion. A PCR-amplified linear DNA lacking K. marxianus sequences but containing an Aspergillus α-amylase gene under the control of the ScTDH3 promoter together with an ScURA3 marker was subsequently used to transform K. marxianus DMKU3-1042 in order to obtain transformants expressing Aspergillus α-amylase. Our results demonstrate that K. marxianus DMKU3-1042 can be an alternative cost-effective bioethanol producer and a host for transformation with linear DNA by use of S. cerevisiae-based molecular genetic tools

Reliability of fetal nasal bone length measurement at 11–14 weeks of gestation

<p>Abstract</p> <p>Background</p> <p>Nasal bone assessment has been incorporated into Down syndrome screening in first trimester. Several studies have established the normal reference values for fetal nasal bone length in the first trimester, which were found to be varied by population. However, the study on reliability of nasal bone length measurement was limited with contradictory results. This study aimed to investigate the reliability of fetal nasal bone length measurement at 11–14 weeks of gestation in the Thai population.</p> <p>Methods</p> <p>A total of 111 pregnant women at 11–14 weeks of gestation attending for the routine first-trimester ultrasound examination were recruited. Each case was measured separately by two examiners. Examiner 1 performed the first measurement in all cases; any of the other 5 examiners consecutively performed the second measurement. Three independent measurements were performed by each examiner and they were blinded to the results of the others. Intraobserver and interobserver variabilities were evaluated with the intraclass correlation coefficient (ICC).</p> <p>Results</p> <p>Nasal bone measurement was successfully performed in 106/111 cases (95.5%) by at least one examiner; 89 cases were performed by two examiners. The intraobserver variability was excellent for all examiners (ICC, 0.840-0.939). The interobserver variability between different pairs of examiners varied from moderate to excellent (ICC, 0.467-0.962). The interobserver variability between examiner 1 and any other examiner was good (ICC, 0.749). The Bland-Altman plot of the interobserver differences of nasal bone length measurements between examiner 1 and any other examiner showed good agreement.</p> <p>Conclusions</p> <p>The reliability of the fetal nasal bone length measurement at 11–14 weeks of gestation was good. The nasal bone length measurement was reproducible. Ethnicity has an effect on fetal nasal bone length, but reliability of nasal bone length measurement is critical to accuracy of screening and should be audited on an ongoing basis.</p