Measuring voluntary responses in healthcare utilization during the COVID-19 pandemic: Evidence from Taiwan.

Healthcare has been one of the most affected sectors during the coronavirus disease 2019 (COVID-19) pandemic. The utilization of related services for non-COVID-19 diseases fell dramatically following the point at which the virus broke out; however, little is known about whether this observed decline in healthcare use was due to voluntary behaviors or enforced measures. This paper quantifies the spontaneous change in healthcare utilization during the pandemic. We utilize a county-by-week-level dataset from Taiwan's National Health Insurance (NHI) record, covering the entire Taiwanese population, and a difference-in-differences design. Our results indicate that even if there were no human mobility restrictions or supply-side constraints, people voluntarily reduced their demand for healthcare, due to fears of contagion, or COVID-related precautionary behaviors. We find that the number of outpatient visits (inpatient admissions) decreased by 19% (10%) during the pandemic period (February to May 2020). Furthermore, the demand response of healthcare for Influenza-like illness (ILI) was much greater and more persistent than for non-ILI, thereby suggesting that the substantial decline in accessing healthcare was induced by positive public health externality of prevention measures for COVID-19. Finally, we find that the demand for healthcare services did not get back to the pre-pandemic baseline, even when there were no local coronavirus cases for 253 consecutive days (mid-April to December 2020) in Taiwan.

Delta-9 fatty acid desaturase overexpression enhanced lipid production and oleic acid content in Rhodosporidium toruloides for preferable yeast lipid production.

The oil plants provide a sufficient source of renewable lipid production for alternative fuel and chemical supplies as an alternative to the depleting fossil source, but the environmental effect from these plants' cropping is a concern. The high oleic acid (OA; C18:1) content in plant-derived products provide advantages of multiple uses with improved oxidative stability and a wide range of applicable temperature. Here we used a promising lipid producer, the oleaginous yeast Rhodosporidium toruloides, to attempt to obtain an OA-enriched lipid. Saccharomyces cerevisiae OLE1 (ScOLE1) gene encodes Δ9 fatty acid desaturase (Δ9FAD), which is generally known to synthesize palmitoleic acid (POA; C16:1) and OA, but the functions of putative R. toruloides Δ9FAD gene are not well understood. In a complementary test, the RtΔ9FAD gene rescued the survival of an OA-deficient Scole1Δ mutant, and we introduced the RtΔ9FAD gene into R. toruloides strains for the production of OA-enriched lipid. Increasing lipid production was observed in ScOLE1 and genomic RtΔ9FAD gene-overexpressing R. toruloides strains. The ScOLE1 transformant output fivefold more OA content in total amount, with >70% of total lipid. Different enhancing effects from the protein coding sequence and genomic sequence of RtΔ9FAD genes were also observed. Overall, this study resulted in ScOLE1 and RtΔ9FAD gene overexpression in R. toruloides to obtain OA-enriched lipid as a candidate source of designed biodiesel and lipid-related chemicals.

Isolation of a thermotolerant Rhodosporidium toruloides DMKU3-TK16 mutant and its fatty acid profile at high temperature.

Oleaginous yeast Rhodosporidium toruloides DMKU3-TK16 (TK16), which was isolated from Thailand, is considered a promising lipid producer for biodiesel production. For future industrial applications of this strain, thermotolerant traits are highly desired for their potential to reduce cooling costs in a commercial fermenter. Here, by using an adaptive breeding strategy, we isolated a thermotolerant R. toruloides mutant, L1-1. The isolated L1-1 strain exhibited better growth and higher lipid production at 37°C, and it was found to have significantly higher oleic acid (C18:1) content and yield compared with the wild-type TK16 when cultivated at 37°C. This is the first study to isolate a thermotolerant strain from the oleaginous yeast R. toruloides. The information gained herein will provide new clues for engineering lipid production and for studying the thermotolerance of R. toruloides.

Molecular cloning and overexpression of DGA1, an acyl-CoA-dependent diacylglycerol acyltransferase, in the oleaginous yeast Rhodosporidiobolus fluvialis DMKU-RK253.

Triacylglycerol (TAG) is a major component of lipid storage in yeast. The acyl CoA: diacylgycerol acyltransferase (DGAT) that catalyzes the final and rate-limiting step in the production of TAG is rather interesting. Consequently, cloning and analysis of the gene-encoding TAG synthase, diacylglycerol acyltransferase gene (DGA1), of the oleaginous yeast Rhodosporidiobolus fluvialis DMKU-RK253 were undertaken. Analysis of the deduced amino acid sequence of DGA1 from R. fluvialis DMKU-RK253 (RfDGA1) showed similarity with the acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) from other organisms. The cDNA of RfDGA1 was cloned into the yeast expression vector pYES2 and heterologously overexpressed in Saccharomyces cerevisiae. One of the transformants showed a 1.6-fold increase in lipid content compared with the wild-type strain harbouring the pYES2 empty vector. Furthermore, DGA1 overexpression in R. fluvialis DMKU-RK253 resulted in a 2.5-fold increase in lipid content when compared with the wild-type strain, and no significant differences in fatty acid composition were observed between RfDGA1-overexpressed and wild-type strains. Taken together, our results supported our hypothesis that the RfDGA1 is a genetic factor that can be used for the development of a strain with improved lipid accumulation capabilities.

Development of a sufficient and effective procedure for transformation of an oleaginous yeast, Rhodosporidium toruloides DMKU3-TK16.

Rhodosporidium toruloides DMKU3-TK16 (TK16), a basidiomycetous yeast isolated in Thailand, can produce a large amount of oil corresponding to approximately 70 % of its dry cell weight. However, lack of a sufficient and efficient transformation method makes further genetic manipulation of this organism difficult. We here developed a new transformation system for R. toruloides using a lithium acetate method with the Sh ble gene as a selective marker under the control of the R. toruloides ATCC 10657 GPD1 promoter. A linear DNA fragment containing the Sh ble gene expression cassette was integrated into the genome, and its integration was confirmed by colony PCR and Southern blot. Then, we further optimized the parameters affecting the transformation efficiency, such as the amount of linear DNA, the growth phase, the incubation time in the transformation mixture, the heat shock treatment temperature, the addition of DMSO and carrier DNA, and the recovery incubation time. With the developed method, the transformation efficiency of approximately 25 transformants/µg DNA was achieved. Compared with the initial trial, transformation efficiency was enhanced 417-fold. We further demonstrated the heterologous production of EGFP in TK16 by microscopic observation and immunoblot analysis, and use the technique to disrupt the endogenous URA3 gene. The newly developed method is thus simple and time saving, making it useful for efficient introduction of an exogenous gene into R. toruloides strains. Accordingly, this new practical approach should facilitate the molecular manipulation, such as target gene introduction and deletion, of TK16 and other R. toruloides strains as a major source of biodiesel.

Internal thoracic artery encircled by an unusual phrenic nerve loop.

We report an anatomic variation of the phrenic nerve. During a routine gross anatomical dissection course at our medical university, we found an unusual loop of the left phrenic nerve around the internal thoracic artery, about 1 cm from the take-off of the left subclavian artery. The phrenic nerve is close to the internal thoracic artery and is easily injured when dissecting the internal thoracic artery for coronary artery bypass conduit. Therefore, we suggest that the anatomic relationship of the phrenic nerve and internal thoracic artery is important in preventing incidental injury of the phrenic nerve.