Anionic lipids facilitate membrane development and protochlorophyllide biosynthesis in etioplasts.

Dark-germinated angiosperm seedlings develop chloroplast precursors called etioplasts in cotyledon cells. Etioplasts develop lattice membrane structures called prolamellar bodies (PLBs), where the chlorophyll intermediate protochlorophyllide (Pchlide) forms a ternary complex with NADPH and light-dependent NADPH:Pchlide oxidoreductase (LPOR). The lipid bilayers of etioplast membranes are mainly composed of galactolipids, which play important roles in membrane-associated processes in etioplasts. Although etioplast membranes also contain 2 anionic lipids, phosphatidylglycerol (PG) and sulfoquinovosyldiacylglycerol (SQDG), their roles are unknown. To determine the roles of PG and SQDG in etioplast development, we characterized etiolated Arabidopsis (Arabidopsis thaliana) mutants deficient in PG and SQDG biosynthesis. A partial deficiency in PG biosynthesis loosened the lattice structure of PLBs and impaired the insertion of Mg2+ into protoporphyrin IX, leading to a substantial decrease in Pchlide content. Although a complete lack of SQDG biosynthesis did not notably affect PLB formation and Pchlide biosynthesis, lack of SQDG in addition to partial PG deficiency strongly impaired these processes. These results suggested that PG is required for PLB formation and Pchlide biosynthesis, whereas SQDG plays an auxiliary role in these processes. Notably, PG deficiency and lack of SQDG oppositely affected the dynamics of LPOR complexes after photoconversion, suggesting different involvements of PG and SQDG in LPOR complex organization. Our data demonstrate pleiotropic roles of anionic lipids in etioplast development.

Distribution of ticks and their possession of spotted fever group Rickettsia in Ibaraki prefecture.

INTRODUCTION: Tick-borne diseases (TBDs) are a growing threat in Japan. However, distribution of ticks and their possession of human pathogens remain poorly understood. METHODS: In the present study, we collected 3477 ticks at 6 remote, woodland sites in Ibaraki prefecture between May 23 and November 4, 2021, and investigated the distribution and the possession of spotted fever group Rickettia (SFGR). RESULTS: The collected ticks included Haemaphysalis flava (78.3 %), Haemaphysalis longicornis (9.0 %), Haemaphysalis hystricis (4.6 %), Ixodes turdus (4.3 %), Amblyomma testudinarium (2.1 %), Haemaphysalis cornigera (0.9 %), Haemaphysalis formosensis (0.9 %), Haemaphysalis megaspinosa (0.2 %), Ixodes ovatus (0.1 %), Ixodes nipponensis (0.09 %), and Ixodes columnae (0.03 %). Of 2160 DNA samples extracted from the ticks, the gltA gene and the 17-kDa antigen gene of SFGR were detected in 67 samples. Among 1682 samples from adult and nymph ticks, the positive rate of SFGR was 2.7 %. Sequence analyses of the partial 17-kDa antigen gene demonstrated that the detected SFGR were classified into 8 groups (G1 to G8). The sequences of G2, G4, G5, G6, and G7 were either identical to or differed by one base pair from those of Rickettsia asiatica, Rickettsia tamurae, Rickettsia monacensis, Rickettsia canadensis, and Rickettsia felis, respectively. CONCLUSION: The present study revealed a diverse tick fauna in Ibaraki prefecture, including detection of species commonly found in southwestern Japan. Although the prevalence of SFGR in ticks was lower than in previous studies, several SFGR causing human infection may be present.

The HKT1 Na+ transporter protects plant fertility by decreasing Na+ content in stamen filaments.

Salinity stress can greatly reduce seed production because plants are especially sensitive to salt during their reproductive stage. Here, we show that the sodium ion transporter AtHKT1;1 is specifically expressed around the phloem and xylem of the stamen in Arabidopsis thaliana to prevent a marked decrease in seed production caused by salt stress. The stamens of AtHKT1;1 mutant under salt stress overaccumulate Na+, limiting their elongation and resulting in male sterility. Specifically restricting AtHKT1;1 expression to the phloem leads to a 1.5-fold increase in the seed yield upon sodium ion stress. Expanding phloem expression of AtHKT1;1 throughout the entire plant is a promising strategy for increasing plant productivity under salinity stress.

Molecular Insights into Innate Immune Response in Captive Koala Peripheral Blood Mononuclear Cells Co-Infected with Multiple Koala Retrovirus Subtypes.

Koala retrovirus (KoRV) exists in both endogenous and exogenous forms and has appeared as a major threat to koala health and conservation. Currently, there are twelve identified KoRV subtypes: an endogenous subtype (KoRV-A) and eleven exogenous subtypes (KoRV-B to -I, KoRV-K, -L, and -M). However, information about subtype-related immune responses in koalas against multiple KoRV infections is limited. In this study, we investigated KoRV-subtype (A, B, C, D, and F)-related immunophenotypic changes, including CD4, CD8b, IFN-γ, IL-6, and IL-10 mRNA expression, in peripheral blood mononuclear cells (PBMCs) obtained from captive koalas (n = 37) infected with multiple KoRV subtypes (KoRV-A to F) reared in seven Japanese zoos. Based on KoRV subtype infection profiles, no significant difference in CD4 and CD8b mRNA expression was observed in the study populations. Based on the different KoRV subtype infections, we found that the IFN-γ mRNA expression in koala PMBCs differs insignificantly (p = 0.0534). In addition, IL-6 and IL-10 mRNA expression also did not vary significantly in koala PBMCs based on KoRV subtype differences. We also investigated the Toll-like receptors (TLRs) response, including TLR2-10, and TLR13 mRNA in koala PBMCs infected with multiple KoRV subtypes. Significant differential expression of TLR5, 7, 9, 10, and 13 mRNA was observed in the PBMCs from koalas infected with different KoRV subtypes. Therefore, based on the findings of this study, it is assumed that co-infection of multiple KoRV subtypes might modify the host innate immune response, including IFN-γ and TLRs responses. However, to have a more clear understanding regarding the effect of multiple KoRV subtypes on host cytokines and TLR response and pathogenesis, further large-scale studies including the koalas negative for KoRV and koalas infected with other KoRV subtypes (KoRV-A to -I, KoRV-K, -L and -M) are required.

Detection of SARS-CoV-2 Genome for over 100 Days after COVID-19 Onset.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is spreading globally. Generally, the viral genome becomes undetectable within a couple of weeks after infection. Herein, we report a case of long-term detection of the SARS-CoV-2 genome in the same individual for 106 days. Whole genome sequencing was performed on specimens taken at the onset of the disease and 2 months after onset, and the B.1.1.7 lineage was detected in both samples. A comparison of the full-length sequences revealed a single-base difference and no amino acid mutations. This is the first case in Japan where the virus was detected over a long period, and the full-length sequences were compared.

Development of Parallel Reaction Monitoring Mass Spectrometry Assay for the Detection of Human Norovirus Major Capsid Protein.

Human Norwalk viruses (HuNoVs), the most common etiological agents of acute gastroenteritis, are genetically diverse RNA viruses that frequently cause mass food poisoning internationally. Although nucleic acid detection methods, such as reverse transcription-quantitative polymerase chain reaction (RT-qPCR), are the gold standard for the diagnosis of norovirus infection, alternative methods are needed for the specific and sensitive viral protein detection for rapid diagnosis and surveillance. In this study, we developed a robust and high-throughput targeted proteomic assay workflow to directly detect the VP1 major capsid protein of HuNoVs. A parallel reaction monitoring (PRM) assay using a high-resolution mass spectrometer was used to detect representative peptides derived from VP1 in six different HuNoV genotypes. An optimized protocol using synthesized heavy isotope-labeled peptides as internal standards was also used to simultaneously genotype and quantify the VP1 protein in human stool specimens. This method is expected to become a new tool for studying the molecular epidemiology of HuNoV and to shed new light on targeted proteomics in clinical practice.

Subtype distribution and expression of the koala retrovirus in the Japanese zoo koala population.

We investigated the proviral copies and RNA expression in koala retrovirus (KoRV)-infected koalas. To ascertain any variation in viral load by institution, age, sex, or body condition score, we quantified KoRV proviral DNA and RNA loads in captive koalas (n = 37) reared in Japanese zoos. All koalas were positive for KoRV genes (pol, LTRs, and env of KoRV-A) in genomic DNA (gDNA), and 91.89% were positive for the pol gene in RNA. In contrast, the distribution rates of KoRV-B, KoRV-C, KoRV-D, and KoRV-F env genes in gDNA were 94.59%, 27.03%, 67.57%, and 54.05%, respectively. A wide inter-individual variation and/or a significant inter-institutional difference in proviral DNA (p < 0.0001) and RNA (p < 0.001) amounts (copies/103 koala ß-actin copies) were observed in Awaji Farm England Hill Zoo koalas, which were obtained from southern koala populations, suggesting exogenous incorporation of KoRV in these koalas. Significant (p < 0.05) age differences were noted in KoRV RNA load (p < 0.05) and median total RNA load (p < 0.001), with loads higher in younger koalas (joeys and juveniles). Thus, the current study provides the distribution of KoRV subtypes in Japanese zoo koala populations and identifies several additional risk factors (sex, age, and body condition) associated with KoRV expression.

Crucial importance of length of fatty-acyl chains bound to the sn-2 position of phosphatidylglycerol for growth and photosynthesis of Synechocystis sp. PCC 6803.

Phosphatidylglycerol (PG) in thylakoid membrane is essential for growth and photosynthesis of photosynthetic organisms. Although the sn-2 position of PG in thylakoid membrane is exclusively esterified with C16 fatty acids, the functional importance of the C16 fatty-acyl chains at the sn-2 position has not been clarified. In this study, we chemically synthesized non-metabolizable PG molecules: we introduced linoleic acid (18:2, fatty acid containing 18 carbons with 2 double bonds) and one of the saturated fatty acids with different chain length (12:0, 14:0, 16:0, 18:0 and 20:0) by ether linkage to the sn-1 and sn-2 positions, respectively. With the synthesized ether-linked PG molecules, we checked whether they could complement the growth and photosynthesis of pgsA mutant cells of Synechocystis sp. PCC 6803 to understand the importance of length of fatty chains at the sn-2 position of PG. The pgsA mutant is incapable of synthesizing PG, so it requires exogenous PG added to medium for growth. The growth rate and photosynthetic activity of mutant cells depended on the length of fatty chains: the PG molecular species binding 16:0 most effectively complemented the growth and photosynthesis of mutant cells, and other PG molecular species with fatty chains shorter or longer than 16:0 were less effective; especially, those binding 12:0 inhibited the growth and photosynthetic activity of the mutant cells. These data demonstrate that length of fatty chains bound to the sn-2 position of PG is critical for PG performance in growth and photosynthesis.

Existence of giant mitochondria-containing sheet structures lacking cristae and matrix in the etiolated cotyledon of Arabidopsis thaliana.

Mitochondria are essential organelles involved in the production and supply of energy in eukaryotic cells. Recently, the use of serial section scanning electron microscopy (S3EM) has allowed accurate three-dimensional (3D) reconstructed images of even complex organelle structures. Using this method, ultrathin sections of etiolated cotyledons were observed 4 days after germination of Arabidopsis thaliana in the dark, and giant mitochondria were found. To exclude the possibility of chemical fixation artifacts, this study confirmed the presence of giant mitochondria in high-pressure frozen samples. The 3D reconstructed giant mitochondria had a complex structure that included not only the elongated region but also the flattened shape of a disk. It contained the characteristic sheet structure, and the sheet lacked cristae and matrix but consisted of outer and inner membranes. Whether this phenomenon could be observed in living cells was investigated using the transformant with mitochondrial matrix expressing green fluorescent protein. Small globular mitochondria observed in light-treated samples were also represented in etiolated cotyledons. Although no giant mitochondria were observed in light-treated samples, they were found in the dark 3 days after germination and rapidly increased in number on the fourth day. Therefore, giant mitochondria were observed only in dark samples. These findings were supported by electron microscopy results.

Back to feedback: aberrant sensorimotor control in music performance under pressure.

Precisely timed production of dexterous actions is often destabilized in anxiogenic situations. Previous studies demonstrated that cognitive functions such as attention and working memory as well as autonomic nervous functions are susceptible to psychological stress in skillful performance while playing sports or musical instruments. However, it is not known whether the degradation of sensorimotor functions underlies such a compromise of skillful performance due to psychophysiological distress. Here, we addressed this issue through a set of behavioral experiments. After artificially delaying the timing of tone production while playing the piano, the local tempo was abnormally disrupted only under pressure. The results suggest that psychological stress degraded the temporal stability of movement control due to an abnormal increase in feedback gain. A learning experiment further demonstrated that the temporal instability of auditory-motor control under pressure was alleviated after practicing piano while ignoring delayed auditory feedback but not after practicing while compensating for the delayed feedback. Together, these findings suggest an abnormal transition from feedforward to feedback control in expert piano performance with psychological stress, which can be mitigated through specialized sensorimotor training that involves piano practice while volitionally ignoring the artificially delayed provision of auditory feedback.

Fertile Arabidopsis cyp704b1 mutant, defective in sporopollenin biosynthesis, has a normal pollen coat and lipidic organelles in the tapetum.

The exine acts as a protectant of the pollen from environmental stresses, and the pollen coat plays an important role in the attachment and recognition of the pollen to the stigma. The pollen coat is made of lipidic organelles in the tapetum. The pollen coat is necessary for fertility, as pollen coat-less mutants, such as those deficient in sterol biosynthesis, show severe male sterility. In contrast, the exine is made of sporopollenin precursors that are biosynthesized in the tapetum. Some mutants involved in sporopollenin biosynthesis lose the exine but show the fertile phenotype. One of these mutants, cyp704b1, was reported to lose not only the exine but also the pollen coat. To identify the cause of the fertile phenotype of the cyp704b1 mutant, the detailed structures of the tapetum tissue and pollen surface in the mutant were analyzed. As a result, the cyp704b1 mutant completely lost the normal exine but had high-electron-density granules localized where the exine should be present. Furthermore, normal lipidic organelles in the tapetum and pollen coat embedded between high-electron-density granules on the pollen surface were observed, unlike in a previous report, and the pollen coat was attached to the stigma. Therefore, the pollen coat is necessary for fertility, and the structure that functions like the exine, such as high-electron-density granules, on the pollen surface may play important roles in retaining the pollen coat in the cyp704b1 mutant.

Plastid Anionic Lipids Are Essential for the Development of Both Photosynthetic and Non-Photosynthetic Organs in Arabidopsis thaliana.

The lipid bilayer matrix of the thylakoid membrane of cyanobacteria and chloroplasts of plants and algae is mainly composed of uncharged galactolipids, but also contains anionic lipids sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) as major constituents. The necessity of PG for photosynthesis is evident in all photosynthetic organisms examined to date, whereas the requirement of SQDG varies with species. In plants, although PG and SQDG are also found in non-photosynthetic plastids, their importance for the growth and functions of non-photosynthetic organs remains unclear. In addition, plants synthesize another anionic lipid glucuronosyldiacylglycerol (GlcADG) during phosphorus starvation, but its role in plant cells is not elucidated yet. To understand the functional relationships among PG, SQDG, and GlcADG, we characterized several Arabidopsis thaliana mutants defective in biosynthesis of these lipids. The mutants completely lacking both PG and SQDG biosynthesis in plastids showed developmental defects of roots, hypocotyls, and embryos in addition to leaves, which suggests that these lipids are pleiotropically required for the development of both photosynthetic and non-photosynthetic organs. Furthermore, our analysis revealed that SQDG, but not GlcADG, is essential for complementing the role of PG, particularly in photosynthesis under PG-deficient conditions such as phosphorus starvation.

Study Elucidating Disinfection at Nursery Schools in Ibaraki, Japan.

Control of infectious diseases requires switching from usual hygiene such as water wiping and cleaning, to control measures including appropriate and aggressive disinfection using 70% alcohol and/or hypochlorous acid depending on the pathogen. Nevertheless, some nursery schools might not understand or select proper disinfectant methods. A survey was administered by the local government of Ibaraki prefecture, Japan in January 2018 to all 456 nursery schools in Ibaraki prefecture. The surveyed items were the number of nursery teachers, usual manual hygiene among children, usual disinfection for lavatories, diaper changing spaces, tables used by children for meals, and classroom floors when there was no vomiting in the facilities and no outbreak in surrounding area. Moreover, it asked about disinfection procedures if children vomited during a community outbreak of gastroenteritis infection. We defined proper use for usual disinfection of a lavatory or diaper changing space as chlorine-based disinfectant including hypochlorous acid or 70% alcohol according to guidelines. Overall, 403 nursery schools responded to the survey. All nursery schools implemented usual hand hygiene. Association between proper disinfection and the size of nursery schools was not significant. Moreover, association between proper disinfection and nursery schools with nurse presence was not found to be significant.

A Longitudinal Study on Enteric Virus Contamination in Bivalves along the Coast of Ibaraki Prefecture, Japan.

ABSTRACT: During the 2014 to 2018 seasons, we conducted a longitudinal study involving enteric virus surveillance in bivalves, including natural oysters and clams harvested in Ibaraki Prefecture, Japan. Some norovirus (NoV) contaminations were detected in natural oysters, whereas no enteric virus was found in clams. NoVs detected in oysters were of the genotypes GII.4 and GII.6, both of which are closely related genetically to the NoV strains prevalent in humans. We found low level of enteric virus contamination in bivalves collected along the coast of Ibaraki Prefecture. The possibility of food poisoning caused by these viruses appears low, and few cases of infectious disease have been observed in the surrounding area. The harvest timing was more related to contamination quantity than the harvest area in many enteric viruses. Our results highlight that contamination of bivalves by enteric viruses may depend upon the prevalence of human diarrhea and illness.

Factors of choking under pressure in musicians.

Under pressure, motor actions, such as those required in public speech, surgery, or musical performance, can be compromised, even when these have been well-trained. The latter is often referred to as 'choking' under pressure. Although multifaceted problems mediate such performance failure in anxiogenic situations, such as compromised motor dexterity and cognitive disruption, the fundamental set of abnormalities characterizing choking under pressure and how these abnormalities are related have not been elucidated. Here, we attempted, first, to classify behavioural, psychological, and physiological abnormalities associated with choking under pressure in musicians and, second, to identify their relationship based on datasets derived from a questionnaire with 258 pianist respondents. Explorative factor analysis demonstrated eight functional abnormalities related to the musicians' choking, such as attention to the audience, erroneous motor actions, perceptual confusion, and failure of memory recall, which however did not include exaggerated attention to the performance. This suggests distraction of attention away from skill execution, which may underlie the spoiled performance under pressure. A structural equation analysis further inferred causal relationships among them. For instance, while failure of memory recall was influenced by passive behaviours manifesting under pressure, erroneous motor actions during performance were influenced by feeling rushed and a loss of body control. In addition, some specific personal traits, such as neuroticism, public self-consciousness, and a lack of confidence, were associated with the extent to which pressure brought about these abnormalities. These findings suggest that distinct psycho-behavioural abnormalities and personal traits underlie the detrimental effects of pressure on musical performance.

Ultrastructural characterization of microlipophagy induced by the interaction of vacuoles and lipid bodies around generative and sperm cells in Arabidopsis pollen.

During pollen maturation, various organelles change their distribution and function during development as male gametophytes. We analyzed the behavior of lipid bodies and vacuoles involved in lipophagy in Arabidopsis pollen using serial section SEM and conventional TEM. At the bicellular pollen stage, lipid bodies in the vegetative cells lined up at the surface of the generative cell. Vacuoles then tightly attached, drew in, and degraded the lipid bodies and eventually occupied the space of the lipid bodies. Degradation of lipid began before transfer of the entire contents of the lipid body. At the tricellular stage, vacuoles instead of lipid bodies surrounded the sperm cells. The degradation of lipid bodies is morphologically considered microautophagy. The atg2-1 Arabidopsis mutant is deficient in one autophagy-related gene (ATG). In this mutant, the assembly of vacuoles around sperm cells was sparser than that in wild-type pollen. The deficiency of ATG2 likely prevents or slows lipid degradation, although it does not prevent contact between organelles. These results demonstrate the involvement of microlipophagy in the pollen development of Arabidopsis.

Development of Monoclonal Antibodies and Antigen-Capture ELISA for Human Parechovirus Type 3.

Human parechovirus type 3 (HPeV3) is an etiologic agent of respiratory diseases, meningitis, and sepsis-like illness in both infants and adults. Monoclonal antibodies (mAbs) can be a promising diagnostic tool for antigenic diseases such as virus infection, as they offer a high specificity toward a specific viral antigen. However, to date, there is no specific mAb available for the diagnosis of HPeV3 infection. In this study, we developed and characterized mAbs specific for HPeV3 capsid protein VP0. We used cell-free, wheat germ-synthesized viral VP0 protein for immunizing BALB/c mice to generate hybridomas. From the resultant hybridoma clones, we selected nine clones producing mAbs reactive to the HPeV3-VP0 antigen, based on enzyme-linked immunosorbent assay (ELISA). Epitope mapping showed that these mAbs recognized three distinct domains in HPeV3 VP0. Six mAbs recognized HPeV3 specifically and the other three mAbs showed cross-reactivity with other HPeVs. Using the HPeV3-specific mAbs, we then developed an ELISA for viral antigen detection that could be reliably used for laboratory diagnosis of HPeV3. This ELISA system exhibited no cross-reactivity with other related viruses. Our newly developed mAbs would, thus, provide a useful set of tools for future research and ensure HPeV3-specific diagnosis.

Volitional Control of Piloerection: Objective Evidence and Its Potential Utility in Neuroscience Research.

The volitional control of piloerection has been previously reported in a small subset of individuals. Although this ability may be useful to study the mechanism underlying piloerection, there is little existing research on this ability, neither objective evidence at a group-level, nor information about its stability under experimental constraints. The present study aimed to validate existing findings of voluntarily generated piloerection (VGP) and to examine its potential contribution to neuroscientific research based on objective evidence of this ability. In Study 1, to confirm the characteristics of VGP reported in previous studies and identify individuals with VGP capability, an online survey of VGP candidates was conducted. In Study 2, 18 VGP holders participated in a mail-based piloerection measurement experiment, and the nature of VGP was examined based on the objective data obtained by image-based analysis (GooseLab). Study 1 largely confirmed the characteristics of VGP reported in previous studies, and Study 2 demonstrated VGP at a group-level and provided information about the temporal characteristics of this ability, which supports the utility of VGP in neuroscientific research. For some participants, VGP appeared to be emotionally promoted, which suggests that VGP has some relationship with the emotional nature of involuntary piloerection. Although the studies did not tightly control the environment in which VGP was elicited, the findings nonetheless demonstrate the possible contribution of VGP to elucidating the mechanism of involuntary emotional piloerection and the neural basis of piloerection itself.

Evolutionary significance of the ring-like plastid nucleus in the primitive red alga Cyanidioschyzon merolae as revealed by drying.

Primary plastids originated from a free-living cyanobacterial ancestor and possess their own genomes-probably a few DNA copies. These genomes, which are organized in centrally located plastid nuclei (CN-type pt-nuclei), are produced from preexisting plastids by binary division. Ancestral algae with a CN-type pt-nucleus diverged and evolved into two basal eukaryotic lineages: red algae with circular (CL-type) pt-nuclei and green algae with scattered small (SN-type) pt-nuclei. Although the molecular dynamics of pt-nuclei in green algae and plants are now being analyzed, the process of the conversion of the original algae with a CN-type pt-nucleus to red algae with a CL-type one has not been studied. Here, we show that the CN-type pt-nucleus in the primitive red alga Cyanidioschyzon merolae can be changed to the CL-type by application of drying to produce slight cell swelling. This result implies that CN-type pt-nuclei are produced by compact packing of CL-type ones, which suggests that a C. merolae-like alga was the original progenitor of the red algal lineage. We also observed that the CL-type pt-nucleus has a chain-linked bead-like structure. Each bead is most likely a small unit of DNA, similar to CL-type pt-nuclei in brown algae. Our results thus suggest a C. merolae-like alga as the candidate for the secondary endosymbiont of brown algae.