Biology subjects

Kim, K.-M.

Publications and source records attributed to Kim, K.-M..

4 recordsLinked to original sources

bioRxiv2021

A new protocol for speed vernalisation of winter cereals

There are many challenges facing the development of high-yielding, nutritious crops for future environments. One limiting factor is generation time, which prolongs research and plant breeding timelines. Recent advances in speed breeding protocols have dramatically reduced generation time for many short-day and long-day species by optimising light and temperature conditions during plant growth. However, winter crops with a vernalisation requirement still require up to 6-10 weeks in low-temperature conditions before transition to reproductive development. Here, we tested a suite of environmental conditions and protocols to investigate if vernalisation can be satisfied more efficiently. We identified a vernalisation method consisting of exposing seeds at the soil surface to an extended photoperiod of 22 h day:2 h night at 10{degrees}C with transfer to speed breeding conditions that dramatically reduces generation time in both winter wheat (Triticum aestivum) and winter barley (Hordeum vulgare). Implementation of this protocol achieved up to five generations per year for winter wheat or barley, instead of the two typically obtained under standard vernalisation and plant growth conditions. The protocol has great potential to enhance training and to accelerate research, pre-breeding, and breeding outcomes focussed on winter crop improvement.

plant biology↗

bioRxiv2020

Mycobiome analysis in fungal Infected formalin-fixed and paraffin-embedded tissues for identification of pathogenic fungi: A pilot study

BackgroundFungal organisms are frequently observed in surgical pathological diagnosis. In order to more accurately identify fungi in formalin-fixed and paraffin-embedded (FFPE) tissues, it is necessary to use genomic information. The purpose of our pilot study is to identify the factors to be considered for the identification of pathogenic fungi using mycobiome analysis in FFPE tissues. MethodsWe selected 49 cases in five hospitals. In each case, FFPE tissue was cut into 50 {micro}m and DNA was extracted. Multiplex PCR with four primers (ITS1, ITS2, ITS3 and ITS4) was performed. Multiplex sequencing was performed using MinION device according to the manufacturers protocol. Sequences of each case were searched using BLASTN with an ITS database from NCBI RefSeq Targeted Loci Project with default parameter. ResultsA total of 2,526 DNA nucleotides were sequenced. We were able to identify 342 fungal nucleotides in 24 (49.0%, 24/49) cases. The median value of the detected fungal DNA per case was 3 (1Q: 1 and 3Q: 14.25). The 215 (62.87%) fungal DNA contained the entire region of ITS1 or ITS2. The remaining 127 fungal DNAs were identified as fungi using partial sequence of ITS1, ITS2, 5.8S, LSU or SSU. ConclusionIn conclusion, we have identified the possibility of finding pathogenic fungi through mycobiome analysis in fungal infected FFPE tissues using nanopore sequencing method. However, we have also found several limitations to be solved for further studies. If we develop a method to characterize pathogenic fungi in FFPE tissues in a follow-up study, we think it will help patients to use appropriate antifungal agents.

microbiology↗

bioRxiv2019

Repurposing disulfiram (Tetraethylthiuram Disulfide) as a potential drug candidate against Borrelia burgdorferi in vitro and in vivo

Lyme disease caused by the Borrelia burgdorferi (Bb or B. burgdorferi) is a most common vector-borne, multi-systemic disease in USA. Although, most Lyme disease patients can be cured with a course of antibiotic treatment, a significant percent of patient population fail to be disease-free post-treatment, necessitating the development of more effective therapeutics. We previously found several drugs including disulfiram having with good activity against B. burgdorferi. In current study, we evaluated the potential of repurposing the FDA approved disulfiram drug for its borreliacidal activity. Our in vitro results indicate disulfiram shows excellent borreliacidal activity against both the log and stationary phase B. burgdorferi. Subsequent mice studies have determined that the disulfiram eliminated B. burgdorferi completely from hearts and urinary bladder by day 28 post infection, demonstrating the practical application and efficacy of disulfiram against B. burgdorferi in vivo. Moreover, disulfiram treated mice showed reduced expression of inflammatory markers and protected against histopathology and organ damage. Furthermore, disulfiram treated mice showed significantly lower amounts of total antibody titers (IgM and IgG) at day 21 and total IgG2b at day 28 post infection. Mechanistically, cellular analysis of lymph nodes revealed a decrease in percentage of CD19+ B cells and increase in total percentage of CD3+ T cells, CD3+ CD4+ T helpers, and naive and effector memory cells in disulfiram-treated mice. Together, we demonstrate that disulfiram has the potential and could be repurposed as an effective antibiotic for treating Lyme disease in near future.

microbiology↗

bioRxiv2019

Discovery and evaluation of a novel step in the flavonoid biosynthesis pathway regulated by F3H gene using a yeast expression system

Kaempferol and quercetin are the essential plant secondary metabolites that confer huge biological functions in the plant defense system. These metabolites are produced in low quantities in plants, therefore engineering microbial factory is a favorable strategy for the production of these metabolites. In this study, biosynthetic pathways for kaempferol and quercetin were constructed in Saccharomyces cerevisiae using naringenin as a substrate. The results elucidated a novel step for the first time in kaempferol and quercetin biosynthesis directly from naringenin catalyzed by flavonol 3-hydroxylase (F3H). F3H gene from rice was cloned into pRS42K yeast episomal plasmid (YEP) vector using BamH1 and Xho1 restriction enzymes. We analyzed our target gene activity in engineered and in empty strains. The results were confirmed through TLC followed by Western blotting, nuclear magnetic resonance (NMR), and LC-MS. TLC showed positive results on comparing both compounds extracted from the engineered strain with the standard reference. Western blotting confirmed lack of Oryza sativa flavonol 3-hydroxylase (OsF3H) activity in empty strains while high OsF3H expression in engineered strains. NMR spectroscopy confirmed only quercetin, while LCMS-MS results revealed that F3H is responsible for naringenin conversion to both kaempferol and quercetin. These results concluded that rice F3H catalyzes naringenin metabolism via hydroxylation and synthesizes kaempferol and quercetin.\n\nHighlightsO_LICurrent study is a discovery of a novel step in flavonoid biosynthesis pathway of rice plant.\nC_LIO_LIIn this study F3H gene from rice plant was functionally expressed in yeast expression system.\nC_LIO_LIResults confirmed that, F3H gene is responsible for the canalization of naringenin and converted into kaempferol and quercetin.\nC_LIO_LIThe results were confirmed through, western blotting, TLC, HPLC and NMR analysis.\nC_LI

molecular biology↗