Utilization of duckweed-derived biomass for polyhydroxybutyrate production in Escherichia coli via dual enzymatic saccharification using amylase and cellulase complex.

Duckweed is a rapid-growing plant with a high starch and low lignin content. The duckweed was saccharified via dual enzymatic treatment using amylase and cellulase complex. The duckweed-derived glucose was utilized for polyhydroxybutyrate (PHB) production in engineered Escherichia coli. In addition, the low concentration supplementation of the duckweed extract promoted cell growth compared to the analytical grade glucose.

Identification and characterization of the suppressed lipid accumulation-related gene, SLA1, in the oleaginous yeast Lipomyces starkeyi.

The oleaginous yeast Lipomyces starkeyi is an attractive industrial yeast that can accumulate high amounts of intracellular lipids. Identification of genes involved in lipid accumulation contributes not only to elucidating the lipid accumulation mechanism but also to breeding industrially useful high lipid-producing strains. In this study, the suppressed lipid accumulation-related gene (SLA1) was identified as the causative gene of the sr22 mutant with decreased lipid productivity. Suppressed lipid accumulation-related gene mutation reduced gene expression in lipid biosynthesis and increased gene expression in ß-oxidation. Our results suggest that SLA1 mutation may leads to decreased lipid productivity. Suppressed lipid accumulation-related gene deletion also exhibited decreased gene expression in ß-oxidation and increased lipid accumulation, suggesting that SLA1 deletion is a useful tool to improve lipid accumulation in L. starkeyi for industrialization.

LsSpt23p is a regulator of triacylglycerol synthesis in the oleaginous yeast Lipomyces starkeyi.

The oleaginous yeast Lipomyces starkeyi has considerable potential in industrial application, since it can accumulate a large amount of triacylglycerol (TAG), which is produced from sugars under nitrogen limitation condition. However, the regulation of lipogenesis in L. starkeyi has not been investigated in depth. In this study, we compared the genome sequences of wild-type and mutants with increased TAG productivity, and identified a regulatory protein, LsSpt23p, which contributes to the regulation of TAG synthesis in L. starkeyi. L. starkeyi mutants overexpressing LsSPT23 had increased TAG productivity compared with the wild-type strain. Quantitative real-time PCR analysis showed that LsSpt23p upregulated the expression of GPD1, which encodes glycerol 3-phosphate dehydrogenase; the Kennedy pathway genes SCT1, SLC1, PAH1, DGA1, and DGA2; the citrate-mediated acyl-CoA synthesis pathway-related genes ACL1, ACL2, ACC1, FAS1, and FAS2; and OLE1, which encodes ∆9 fatty acid desaturase. Chromatin immunoprecipitation-quantitative PCR assays indicated that LsSpt23p acts as a direct regulator of SLC1 and PAH1, all the citrate-mediated acyl-CoA synthesis pathway-related genes, and OLE1. These results indicate that LsSpt23p regulates TAG synthesis. Phosphatidic acid is a common substrate of phosphatidic acid phosphohydrolase, which is used for TAG synthesis, and phosphatidate cytidylyltransferase 1 for phospholipid synthesis in the Kennedy pathway. LsSpt23p directly regulated PAH1 but did not affect the expression of CDS1, suggesting that the preferred route of carbon is the Pah1p-mediated TAG synthesis pathway under nitrogen limitation condition. The present study contributes to understanding the regulation of TAG synthesis, and will be valuable in future improvement of TAG productivity in oleaginous yeasts. KEY POINTS: LsSpt23p was identified as a positive regulator of TAG biosynthesis LsSPT23 overexpression enhanced TAG biosynthesis gene expression and TAG production LsSPT23M1108T overexpression mutant showed fivefold higher TAG production than control.

Tongue and jaw movement assessed by 3D motion capture during gum chewing.

Introduction: The tongue plays an important role in mastication, swallowing, and articulation, but it cannot be directly observed because of its location inside the oral cavity. This study aimed to clarify detailed 3D tongue movements during chewing using electromagnetic articulography (EMA). Materials and Methods: The participants were 10 healthy, young volunteers (average age 26.8 ± 2.1 years; 5 males, 5 females). Tongue and jaw movement during gum chewing was measured and recorded using EMA. Four EMA sensors were attached to the anterior, posterior, left, and right surfaces of the tongue, and one sensor was also attached to the mandibular left incisor. The tongue motion during the chewing cycle was spatially and sequentially analyzed based on the motion trajectories of the tongue and mandible. Results and Discussion: The tongue moved downward and to the masticatory side in a manner similar to the movement of the jaw. The anterior tongue marker moved downward to a greater extent than the other tongue markers. However, the tongue moved forward as the jaw moved backward. The anterior marker reached the most anterior position during the jaw-opening phase and the posterior markers reached the most anterior position during the jaw-closing phase. Just before maximum jaw-opening, all markers on the tongue reached the bottom lowest position. During the jaw-closing phase, the tongue reached the dominant farthest position in the masticatory side. All the markers reached the most posterior position during the occlusal phase. Conclusion: These findings demonstrate the sequence of tongue motion patterns during gum chewing.

Deletion of LsSNF1 enhances lipid accumulation in the oleaginous yeast Lipomyces starkeyi.

The oleaginous yeast, Lipomyces starkeyi can have diverse industrial applications due to its remarkable capacity to use various carbon sources for the biosynthesis intracellular triacylglycerides (TAGs). In L. starkeyi, TAG synthesis is enhanced through upregulation of genes involved in citrate-mediated acyl-CoA synthesis and Kennedy pathways through the transcriptional regulator LsSpt23p. High expression of LsSPT23 can considerably enhance TAG production. Altering the regulatory factors associated with lipid production can substantially augment lipid productivity. In this study, we identified and examined the L. starkeyi homolog sucrose nonfermenting 1 SNF1 (LsSNF1) of YlSNF1, which encodes a negative regulator of lipid biosynthesis in the oleaginous yeast Yarrowia lipolytica. The deletion of LsSNF1 enhanced TAG productivity in L. starkeyi, suggesting that LsSnf1p is a negative regulator in TAG production. The enhancement of TAG production following deletion of LsSNF1 can primarily be attributed to the upregulation of genes in the citrate-mediated acyl-CoA synthesis and Kennedy pathways, pivotal routes in TAG biosynthesis. The overexpression of LsSPT23 enhanced lipid productivity; strain overexpressing LsSPT23 and without LsSNF1 exhibited increased TAG production capacity per cell. LsSnf1p also has a significant role in the utilization of carbon sources, including xylose or glycerol, in L. starkeyi. Our study results elucidated the role of LsSnf1p in the negative regulation of TAG synthesis in L. starkeyi, which has not previously been reported.

Masticatory Performance Test Using a Gummy Jelly for Older People with Low Masticatory Ability.

Evaluation of masticatory ability has become more important in an aging society because decreased masticatory ability has the potential to affect the general health of older people. A new masticatory performance test, intended for older people with low masticatory ability, has been developed using gummy jelly half the size of that used in the conventional masticatory performance test. This study aimed to investigate the compatibility between the new and conventional tests and the adaptation of the new test. The new test using the 8-grade visual score with half-size gummy jelly was performed among 137 removable denture wearers (mean age 75.8 ± 9.0 years) with low masticatory performance (a score of ≤4 on a conventional test). The correlation between the scores of half-size gummy jelly (VS-H) in the new test and those of full-size gummy jelly (VS) in the conventional test was evaluated. VS-H among the groups divided by VS were also compared. A strong positive correlation was detected between VS-H and VS (rs = 0.70). In groups with VS of 0 and 1, VS-H values were widely distributed from 0 to 7. There were significant differences in VS-H between the groups with VS of 0-2 but no significant differences in VS-H between the groups with VS of 2-4. Therefore, the masticatory performance test using half-size gummy jelly is suitable for a detailed evaluation of masticatory ability in older people with low masticatory ability when their visual score of full-size gummy jelly in the conventional test is 2 or less.

Developing tongue coating status assessment using image recognition with deep learning.

PURPOSE: To build an image recognition network to evaluate tongue coating status. METHODS: Two image recognition networks were built: one for tongue detection and another for tongue coating classification. Digital tongue photographs were used to develop both networks; images from 251 (178 women, 74.7±6.6 years) and 144 older adults (83 women, 73.8±7.3 years) who volunteered to participate were used for the tongue detection network and coating classification network, respectively. The learning objective of the tongue detection network is to extract a rectangular region that includes the tongue. You-Only-Look-Once (YOLO) v2 was used as the detection network, and transfer learning was performed using ResNet-50. The accuracy was evaluated by calculating the intersection over the union. For tongue coating classification, the rectangular area including the tongue was divided into a grid of 7×7. Five experienced panelists scored the tongue coating in each area using one of five grades, and the tongue coating index (TCI) was calculated. Transfer learning for tongue coating grades was performed using ResNet-18, and the TCI was calculated. Agreement between the panelists and network for the tongue coating grades in each area and TCI was evaluated using the kappa coefficient and intraclass correlation, respectively. RESULTS: The tongue detection network recognized the tongue with a high intersection over union (0.885±0.081). The tongue coating classification network showed high agreement with tongue coating grades and TCI, with a kappa coefficient of 0.826 and an intraclass correlation coefficient of 0.807, respectively. CONCLUSIONS: Image recognition enables simple and detailed assessment of tongue coating status.

Genotyping of Mycoplasma pneumoniae strains isolated in Japan during 2019 and 2020: spread of p1 gene type 2c and 2j variant strains.

We characterized 118 Mycoplasma pneumoniae strains isolated from three areas of Japan (Saitama, Kanagawa, and Osaka) during the period of 2019 and 2020. Genotyping of the p1 gene in these strains revealed that 29 of them were type 1 lineage (29/118, 24.6%), while 89 were type 2 lineage (89/118, 75.4%), thereby indicating that type 2 lineage was dominant in this period. The most prevalent variant of type 2 lineage was type 2c (57/89, 64%), while the second-most was type 2j, a novel variant identified in this study (30/89, 33.7%). Type 2j p1 is similar to type 2 g p1, but cannot be distinguished from reference type 2 (classical type 2) using the standard polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) with HaeIII digestion. Thus, we used MboI digestion in the PCR-RFLP analysis and re-examined the data from previous genotyping studies as well. This revealed that most strains reported as classical type 2 after 2010 in our studies were actually type 2j. The revised genotyping data showed that the type 2c and 2j strains have been spreading in recent years and were the most prevalent variants in Japan during the time-period of 2019 and 2020. We also analyzed the macrolide-resistance (MR) mutations in the 118 strains. MR mutations in the 23S rRNA gene were detected in 29 of these strains (29/118, 24.6%). The MR rate of type 1 lineage (14/29, 48.3%) was still higher than that of type 2 lineage (15/89, 16.9%); however, the MR rate of type 1 lineage was lower than that found in previous reports published in the 2010s, while that of type 2 lineage strains was slightly higher. Thus, there is a need for continuous surveillance of the p1 genotype and MR rate of M. pneumoniae clinical strains, to better understand the epidemiology and variant evolution of this pathogen, although M. pneumoniae pneumonia cases have decreased significantly since the COVID-19 pandemic.

Breaking away from an endemic state of multidrug-resistant Pseudomonas aeruginosa by daily sink disinfection.

The detection rate of multidrug-resistant Pseudomonas aeruginosa in patients admitted to 2 wards and the intensive care unit decreased from 20.3% (129 of 636 isolates) to 4.2% (37 of 889 isolates) after the start of disinfection of hand washing sinks using alkyl diaminoethylglycine hydrochloride.

Citrate-Mediated Acyl-CoA Synthesis Is Required for the Promotion of Growth and Triacylglycerol Production in Oleaginous Yeast Lipomyces starkeyi.

The oleaginous yeast Lipomyces starkeyi is an excellent producer of triacylglycerol (TAG) as a feedstock for biodiesel production. To understand the regulation of TAG synthesis, we attempted to isolate mutants with decreased lipid productivity and analyze the expression of TAG synthesis-related genes in this study. A mutant with greatly decreased lipid productivity, sr22, was obtained by an effective screening method using Percoll density gradient centrifugation. The expression of citrate-mediated acyl-CoA synthesis-related genes (ACL1, ACL2, ACC1, FAS1, and FAS2) was decreased in the sr22 mutant compared with that of the wild-type strain. Together with a notion that L. starkeyi mutants with increased lipid productivities had increased gene expression, there was a correlation between the expression of these genes and TAG synthesis. To clarify the importance of citrate-mediated acyl-CoA synthesis pathway on TAG synthesis, we also constructed a strain with no ATP-citrate lyase responsible for the first reaction of citrate-mediated acyl-CoA synthesis and investigated the importance of ATP-citrate lyase on TAG synthesis. The ATP-citrate lyase was required for the promotion of cell growth and TAG synthesis in a glucose medium. This study may provide opportunities for the development of an efficient TAG synthesis for biodiesel production.

Point-of-care molecular diagnosis of Mycoplasma pneumoniae including macrolide sensitivity using quenching probe polymerase chain reaction.

OBJECTIVES: Macrolides are generally considered to be the drugs of choice for treatment of patients with Mycoplasma pneumoniae infection. However, macrolide-resistant M. pneumoniae has been emerging since about 2000. The Smart Gene® system (MIZUHO MEDY Co., Ltd., Tosu, Japan) is a novel fully automated system for detection of pathogens using the method of quantitative polymerase chain reaction (qPCR) with QProbe (QProbe PCR). The entire procedure is completed within 50 min and the size of the instrument is small (15 x 34 x 30 cm). The purpose of this study was to evaluate the usefulness of the Smart Gene® system for detection of M. pneumoniae and detection of a point mutation at domain V of the 23S rRNA gene of M. pneumoniae. MATERIALS: Pharyngeal swab samples were collected from 154 patients who were suspected of having respiratory tract infections associated with M. pneumoniae. RESULTS: Compared with the results of qPCR, the sensitivity and specificity of the Smart Gene® system were 98.7% (78/79) and 100.0% (75/75), respectively. A point mutation at domain V of the 23S rRNA gene was detected from 7 (9.0%) of 78 M. pneumoniae-positive samples by the Smart Gene® system and these results were confirmed by direct sequencing. The minimum inhibitory concentrations of clarithromycin among the 5 isolates of M. pneumoniae with a point mutation at domain V of the 23S rRNA gene were >64 µg/ml and those among the 33 isolates without a mutation in the 23S rRNA gene were <0.0625 µg/ml. CONCLUSION: The Smart Gene® system is a rapid and accurate assay for detection of the existence of M. pneumoniae and a point mutation at domain V of the 23S rRNA gene of M. pneumoniae at the same time. The Smart Gene® system is suitable for point-of-care testing in both hospital and outpatient settings.

Isolation and characterization of Lipomyces starkeyi mutants with greatly increased lipid productivity following UV irradiation.

The oleaginous yeast Lipomyces starkeyi is an intriguing lipid producer that can produce triacylglycerol (TAG), a feedstock for biodiesel production. We previously reported that the L. starkeyi mutant E15 with high levels of TAG production compared with the wild-type was efficiently obtained using Percoll density gradient centrifugation. However, considering its use for biodiesel production, it is necessary to further improve the lipid productivity of the mutant. In this study, we aimed to obtain mutants with better lipid productivity than E15, evaluate its lipid productivity, and analyze lipid synthesis-related gene expression in the wild-type and mutant strains. The mutants E15-11, E15-15, and E15-25 exhibiting higher lipid productivity than E15 were efficiently isolated from cells exposed to ultraviolet light using Percoll density gradient centrifugation. They exhibited approximately 4.5-fold higher lipid productivity than the wild-type on day 3. The obtained mutants did not exhibit significantly different fatty acid profiles than the wild-type and E15 mutant strains. E15-11, E15-15, and E15-25 exhibited higher expression of acyl-CoA synthesis- and Kennedy pathway-related genes than the wild-type and E15 mutant strains. Activation of the pentose phosphate pathway, which supplies NADPH, was also observed. These results suggested that the increased expression of acyl-CoA synthesis- and Kennedy pathway-related genes plays a vital role in lipid productivity in the oleaginous yeast L. starkeyi.

P1 gene of Mycoplasma pneumoniae isolated from 2016 to 2019 and relationship between genotyping and macrolide resistance in Hokkaido, Japan.

We characterized 515 Mycoplasma pneumoniae specimens in Hokkaido. In 2013 and 2014, the p1 gene type 1 strain, mostly macrolide-resistant, was dominant and the prevalence of macrolide resistance was over 50 %. After 2017, the p1 gene type 2 lineage, mostly macrolide-sensitive, increased and the prevalence of macrolide resistance became 31.0 % in 2017, 5.3 % in 2018 and 16.3 % in 2019.

The association between father involvement in caregiving and early childhood overweight or obesity.

BACKGROUND: Despite the increasing trend of paternal engagement in caregiving, fathers remain underrepresented in studies on parenting and childhood obesity. OBJECTIVES: This study examined the association between father involvement in caregiving and child's weight status within a longitudinal dataset from Japan. METHODS: The data were obtained from a population-based cohort study in Japan (N = 29 584). The association between father's involvement in child caregiving (age 18 months) on the weight status of the same children at age 3.5 years was examined by logistic regression and linear regression analysis. Furthermore, we checked the interaction between maternal employment & father involvement by creating four mutually exclusive groups of families. RESULTS: Children whose fathers were highly involved in caregiving were less likely to be overweight (OR = 0.90, 95% CI, 0.85-0.96). Employed mothers with high paternal involvement in caregiving were 30% less likely to have children with overweight or obesity compared to those with low paternal involvement (95% CI, 0.55-0.90). CONCLUSIONS: Father involvement in caregiving is associated with the development of overweight or obesity in early childhood, and may also modify the association between maternal employment and child's weight status. Social policies to support shared child-rearing such as expanding paid parental leave could help families to raise children with healthy weight.

A novel electroporation procedure for highly efficient transformation of Lipomyces starkeyi.

Microbial lipids produced by oleaginous microorganisms as raw materials for the production of oleochemicals and biodiesel are sustainable while avoiding competition with food products. The oleaginous yeast Lipomyces starkeyi is an excellent lipid producer with a great industrial potential that is suitable as a valuable host to improve lipid production through genetic engineering modifications. However, genetic tools, including effective transformation methods, for L. starkeyi are insufficient for improvement of lipid production and analysis of lipid production mechanisms. We previously developed a polyethylene glycol (PEG)-mediated spheroplast transformation method that significantly improved the homologous recombination efficiency of L. starkeyi strain ∆lslig4. Although other transformation methods, including lithium acetate (LiAc)-mediated transformation and Agrobacterium tumefaciens-mediated transformation, have been reported, a more efficient and convenient transformation method for L. starkeyi is desired. In this study, we developed a novel electroporation transformation method that was first applied for integration of drug-resistance gene markers into the genome of L. starkeyi strain ∆lslig4 at the 18S ribosomal DNA locus of a multiple-copy gene, which yielded approximately 60 transformants/µg of DNA. Optimization of five parameters (i.e., cell growth phase, cell density, osmotic stabilizers, pretreatment agents, and electric conditions) enhanced the efficiency of transformation to approximately 1.5 × 104 transformants/µg of DNA. As compared with those of LiAc-mediated transformation and PEG-mediated spheroplast transformation, the efficiency of the proposed transformation method was increased by about 111- and 7-fold, respectively. Additionally, the transformation efficiency of our proposed electroporation method targeting a single-copy gene locus yielded 273 transformants/µg of DNA. To our knowledge, this is the first report of a successful electroporation method to accelerate analysis of lipid production by L. starkeyi.

Pet Ownership and Children's Emotional Expression: Propensity Score-Matched Analysis of Longitudinal Data from Japan.

With many children and young adolescents reporting strong emotional bonds with their pets, the impact of pet ownership on child/adolescent health-especially on their emotional development-has garnered increasing scientific interest. We examined the association between pet ownership in toddlerhood (age 3.5 years) and poor emotional expression in later childhood (age 5.5 years) using propensity score matching within a longitudinal cohort dataset from Japan (n = 31,453). A propensity score for pet ownership was calculated by logistic models based on a comprehensive list of each child's observed characteristics, including sex, household income, parental education, mother's employment status, residential environment, number of siblings, and living arrangement. Log-binomial regression analyses using matched samples revealed that children who owned pets during the toddler years were 6% less likely to have a poor emotional expression in later childhood (prevalence ratio = 0.94, 95% confidence interval = 0.90⁻0.99) compared to those without pets. This suggests that owning pets may provide children with opportunities to control their emotions, and lead to a lower prevalence of poor emotional expression. Pet ownership in toddlerhood may contribute to the development of expression.

Self-assembly of MoO3 needles in gas current for cubic formation pathway.

Nucleation and subsequent rapid growth are enigmatic due to the unrevealed pathways. Despite the relatively simpler mechanism compared to nucleation and growth in solution, that in vapor has received little attention. The largest hindrance to unveiling this process may be observing the rapid and mesoscopic-scale phenomena. To overcome this hindrance, we combine an experimental approach with in situ spatial scanning Fourier-transform infrared spectroscopy, which reveals the nucleating and growing nanoparticles in vapor. The nanoparticles are then collected at different evolutionary stages and analyzed by ex situ transmission electron microscopy (TEM). Needle-shaped molybdenum oxide (MoO3) nanoparticles were formed within ∼0.1 s after homogeneous nucleation from a highly supersaturated vapor. Over one second, the needle particles gradually evolved into a cubic shape by fusion in a crystallographically favored orientation in a free-flying state in vapor. The similar sizes of the elongated axes of the needle and cubic structures suggest an additional growth stage, in which the needle particles become the growth units of the cubic particles. The morphology of a final crystal should reflect the formation environment of the particle because growing crystals are sensitive to the formation conditions such as temperature, concentration, and impurities. Although nucleation under very high supersaturation induces the anisotropic growth of the needle particles, this information of the initial nucleation environment is lost in the final cubic crystal. These findings enrich our understanding of pathways in the nucleation and growth of nanoparticles and provide new insights into the growth stages driven by oriented attachment.