Trimerization profile of type IV collagen COL4A5 exon deletion in X-linked Alport syndrome.

BACKGROUND: Alport syndrome (AS) is a genetic kidney disease caused by a mutation in type IV collagen α3, α4, and α5, which are normally secreted as heterotrimer α345(IV). Nonsense mutation in these genes causes severe AS phenotype. We previously revealed that the exon-skipping approach to remove a nonsense mutation in α5(IV) ameliorated the AS pathology. However, the effect of removing an exon on trimerization is unknown. Here, we assessed the impact of exon deletion on trimerization to evaluate their possible therapeutic applicability and to predict the severity of mutations associated with exon-skipping. METHODS: We produced exon deletion constructs (ΔExon), nonsense, and missense mutants by mutagenesis and evaluated their trimer formation and secretion activities using a nanoluciferase-based assay that we previously developed. RESULTS: Exon-skipping had differential effects on the trimer secretion of α345(IV). Some ΔExons could form and secrete α345(IV) trimers and had higher activity compared with nonsense mutants. Other ΔExons had low secretion activity, especially for those with exon deletion near the C-terminal end although the intracellular trimerization was normal. No difference was noted in the secretion of missense mutants and their ΔExon counterpart. CONCLUSION: Exon skipping is advantageous for nonsense mutants in AS with severe phenotypes and early onset of renal failure but applications may be limited to ΔExons capable of normal trimerization and secretion. This study provides information on α5(IV) exon-skipping for possible therapeutic application and the prediction of the trimer behavior associated with exon-skipping in Alport syndrome.

Diet choice: The two-factor host acceptance system of silkworm larvae.

Many herbivorous insects are mono- or oligophagous, having evolved to select a limited range of host plants. They specifically identify host-plant leaves using their keen sense of taste. Plant secondary metabolites and sugars are thought to be key chemical cues that enable insects to identify host plants and evaluate their quality as food. However, the neuronal and behavioral mechanisms of host-plant recognition are poorly understood. Here, we report a two-factor host acceptance system in larvae of the silkworm Bombyx mori, a specialist on several mulberry species. The first step is controlled by a chemosensory organ, the maxillary palp (MP). During palpation at the leaf edge, the MP detects trace amounts of leaf-surface compounds, which enables host-plant recognition without biting. Chemosensory neurons in the MP are tuned with ultrahigh sensitivity (thresholds of attomolar to femtomolar) to chlorogenic acid (CGA), quercetin glycosides, and ß-sitosterol (ßsito). Only if these 3 compounds are detected does the larva make a test bite, which is evaluated in the second step. Low-sensitivity neurons in another chemosensory organ, the maxillary galea (MG), mainly detect sucrose in the leaf sap exuded by test biting, allowing larvae to accept the leaf and proceed to persistent biting (feeding). The two-factor host acceptance system reported here may commonly underlie stereotyped feeding behavior in many phytophagous insects and determine their feeding habits.

The roles of enteroendocrine cell distribution and gustatory receptor expression in regulating peptide hormone secretion in the midgut of Bombyx mori larvae.

To regulate physiological homeostasis and behavior in Bombyx mori, more than 20 peptide hormones in the midgut of larvae are secreted upon detection of food substances at the lumen. Although it is logical to assume that the timings of peptide hormone secretions are regulated, little is known about the mechanisms. In this study, the distributions of enteroendocrine cells (EECs) producing five peptide hormones and EECs expressing gustatory receptors (Grs), as candidate receptors for luminal food substances and nutrients, were examined via immunostaining in B. mori larvae. Three patterns of peptide hormone distribution were observed. Tachykinin (Tk)- and K5-producing EECs were located throughout the midgut; myosuppressin-producing EECs were located in the middle-to-posterior midgut; and allatostatin C- and CCHamide-2-producing EECs were located in the anterior-to-middle midgut. BmGr4 was expressed in some Tk-producing EECs in the anterior midgut, where food and its digestive products arrived 5 min after feeding began. Enzyme-linked immunosorbent assay (ELISA) revealed secretion of Tk starting approximately 5 min after feeding began, suggesting that food sensing by BmGr4 may regulate Tk secretion. BmGr6 was expressed in a few Tk-producing EECs in the middle-to-posterior midgut, although its significance was unclear. BmGr6 was also expressed in many myosuppressin-producing EECs in the middle midgut, where food and its digestive products arrived 60 min after feeding began. ELISA revealed secretion of myosuppressin starting approximately 60 min after feeding began, suggesting that food sensing by BmGr6 may regulate myosuppressin secretion. Finally, BmGr9 was expressed in many BmK5-producing EECs throughout the midgut, suggesting that BmGr9 may function as a sensor for the secretion of BmK5.

Spätzle1 is a constituent of the extracellular signaling pathway that promotes nodule formation, a cell-mediated immune response, in caterpillar hemolymph.

The existence of an extracellular signaling pathway that mediates nodule formation, a cell-mediated immune response, has been reported in Bombyx mori larvae. In this pathway, C-type lectins and the hemolymph serine proteinase BmHP-8 function in pathogen associated molecular pattern (PAMPs) recognition and signaling transduction. However, which molecule elicits the cellular response at the end of the pathway is unknown. In this study, the Toll ligand Bombyx mori Spätzel1 was shown to be involved in the pathway by applying anit-Spätzel1 antiserum in an in vitro nodule-like aggregate formation assay and an in vivo nodule formation assay.

Mechanisms and roles of the first stage of nodule formation in lepidopteran insects.

Nodule formation is a process of cellular immunity in insects and other arthropods with open circulatory systems. Based on histological observations, nodule formation occurs in 2 stages. The first stage occurs immediately after microbial inoculation and includes aggregate formation by granulocytes. The second stage occurs approximately 2-6 h later and involves the attachment of plasmatocytes to melanized aggregates produced during the first stage. The first stage response is thought to play a major role in the rapid capture of invading microorganisms. However, little is known regarding how granulocytes in the hemolymph form aggregates, or how the first stage of the immunological response protects against invading microorganisms. Since the late 1990s, our understanding of the molecules and immune pathways that contribute to nodule formation has improved. The first stage of nodule formation involves a hemocyte-induced response that is triggered by pathogen-associated molecular pattern (PAMP) recognition proteins in the hemolymph regulated by a serine proteinase cascade and cytokine (Spätzle) and Toll signaling pathways. Hemocyte agglutination proceeds through stepwise release of biogenic amine, 5-HT, and eicosanoids that act downstream of the Toll pathway. The first stage of nodule formation is closely linked to melanization and antimicrobial peptide (AMP) production, which is critical for insect humoral immunity. Nodule formation in response to artificial inoculation with millions of microorganisms has long been studied. It has recently been suggested that this system is the original natural immune system, and enables insects to respond to a single invading microorganism in the hemocoel.

Transition Probability Test for an RO-Based Generator and the Relevance between the Randomness and the Number of ROs.

A ring oscillator is a well-known circuit used for generating random numbers, and interested readers can find many research results concerning the evaluation of the randomness with a packaged test suit. However, the authors think there is room for evaluating the unpredictability of a sequence from another viewpoint. In this paper, the authors focus on Wold's RO-based generator and propose a statistical test to numerically evaluate the randomness of the RO-based generator. The test adopts the state transition probabilities in a Markov process and is designed to check the uniformity of the probabilities based on hypothesis testing. As a result, it is found that the RO-based generator yields a biased output from the viewpoint of the transition probability if the number of ROs is small. More precisely, the transitions 01→01 and 11→11 happen frequently when the number l of ROs is less than or equal to 10. In this sense, l>10 is recommended for use in any application, though a packaged test suit is passed. Thus, the authors believe that the proposed test contributes to evaluating the unpredictability of a sequence when used together with available statistical test suits, such as NIST SP800-22.

Seroprevalence of antibodies to SARS-CoV-2 in healthcare workers in a nonepidemic region, Japan: A hospital-based study on May, 2020.

The polymerase chain reaction (PCR) testing rate is low in our local area and the true rate of coronavirus disease 2019 (COVID-19) infection may include many asymptomatic individuals. We conducted a serosurveillance using antibody testing in an area where official report of COVID-19 infection is not done yet. Blood samples were obtained from 1404 healthcare workers (41 ± 11 years) in our hospital on May 29-31, 2020. First, the potential infection frequency was confirmed using two quantitative antibody tests. In addition, the usefulness of rapid antibody kit testing for COVID-19 serosurveillance was examined. A COVID-19-indected case was defined as showing positive results in both quantitative tests. None of 1404 samples had positive results from the two quantitative tests. The false positive rates were 0.36% and 0.07%, whereas those in rapid antibody kits were 3.3% and 3.0%. In conclusion, as of May, 2020, potential spread mainly by asymptomatic individuals infected with COVID-19 was not found in our local area where there was no official report of COVID-19, even if the PCR testing rate was low. Rapid antibody kits might not be useful due to the high false positive rate in an area with a low incidence of COVID-19 infected individuals.

Consideration for Affects of an XOR in a Random Number Generator Using Ring Oscillators.

A cloud service to offer entropy has been paid much attention to. As one of the entropy sources, a physical random number generator is used as a true random number generator, relying on its irreproducibility. This paper focuses on a physical random number generator using a field-programmable gate array as an entropy source by employing ring oscillator circuits as a representative true random number generator. This paper investigates the effects of an XOR gate in the oscillation circuit by observing the output signal period. It aims to reveal the relationship between inputs and the output through the XOR gate in the target generator. The authors conduct two experiments to consider the relevance. It is confirmed that combining two ring oscillators with an XOR gate increases the complexity of the output cycle. In addition, verification using state transitions showed that the probability of the state transitions was evenly distributed by increasing the number of ring oscillator circuits.

Extracellular loop structures in silkworm ABCC transporters determine their specificities for Bacillus thuringiensis Cry toxins.

Bacillus thuringiensis Cry toxins are insecticidal proteins used widely for pest control. They are lethal to a restricted range of insects via specific interactions with insect receptors such as the ABC transporter subfamily members C2 (ABCC2) and C3 (ABCC3). However, it is still unclear how these different receptors contribute to insect susceptibility to Cry1A toxins. Here, we investigated the differences between the silkworm (Bombyx mori) ABCC2 (BmABCC2_S) and ABCC3 (BmABCC3) receptors in mediating Cry toxicity. Compared with BmABCC2_S, BmABCC3 exhibited 80- and 267-fold lower binding affinities to Cry1Aa and Cry1Ab, respectively, and these decreased affinities correlated well with the lower receptor activities of BmABCC3 for these Cry1A toxins. To identify the amino acid residues responsible for these differences, we constructed BmABCC3 variants containing a partial amino acid replacement with extracellular loops (ECLs) from BmABCC2_S. Replacing three amino acids from ECL 1 or 3 increased BmABCC3 activity toward Cry1Aa and enabled its activity toward Cry1Ab. Meanwhile, BmABCC2_S and BmABCC3 exhibited no receptor activities for Cry1Ca, Cry1Da, and Cry3Bb, correlating with markedly lower binding affinities for these Cry toxins. ABCC2 from a Cry1Ab-resistant B. mori strain (BmABCC2_R), which has a tyrosine insertion in ECL 2, displayed 93-fold lower binding affinity to Cry1Ab compared with BmABCC2_S but maintained high binding affinity to Cry1Aa. These results indicate that the Cry toxin-binding affinities of ABCC transporters are largely linked to the level of Cry susceptibility of ABCC-expressing cells and that the ABCC ECL structures determine the specificities to Cry toxins.

DAY-LENGTH-DEPENDENT DELAYED-GREENING1, the Arabidopsis Homolog of the Cyanobacterial H+-Extrusion Protein, Is Essential for Chloroplast pH Regulation and Optimization of Non-Photochemical Quenching.

Plants convert solar energy into chemical energy through photosynthesis, which supports almost all life activities on earth. Because the intensity and quality of sunlight can change dramatically throughout the day, various regulatory mechanisms help plants adjust their photosynthetic output accordingly, including the regulation of light energy accumulation to prevent the generation of damaging reactive oxygen species. Non-photochemical quenching (NPQ) is a regulatory mechanism that dissipates excess light energy, but how it is regulated is not fully elucidated. In this study, we report a new NPQ-regulatory protein named Day-Length-dependent Delayed-Greening1 (DLDG1). The Arabidopsis DLDG1 associates with the chloroplast envelope membrane, and the dldg1 mutant had a large NPQ value compared with wild type. The mutant also had a pale-green phenotype in developing leaves but only under continuous light; this phenotype was not observed when dldg1 was cultured in the dark for ≥8 h/d. DLDG1 is a homolog of the plasma membrane-localizing cyanobacterial proton-extrusion-protein A that is required for light-induced H+ extrusion and also shows similarity in its amino-acid sequence to that of Ycf10 encoded in the plastid genome. Arabidopsis DLDG1 enhances the growth-retardation phenotype of the Escherichia coli K+/H+ antiporter mutant, and the everted membrane vesicles of the E. coli expressing DLDG1 show the K+/H+ antiport activity. Our findings suggest that DLDG1 functionally interacts with Ycf10 to control H+ homeostasis in chloroplasts, which is important for the light-acclimation response, by optimizing the extent of NPQ.

Genetic characterization of a flap1 null mutation in Arabidopsis npq4 and pgr5 plants suggests that the regulatory role of FLAP1 involves the control of proton homeostasis in chloroplasts.

Precise control of the proton concentration gradient across thylakoid membranes (ΔpH) is essential for photosynthesis and its regulation because the gradient contributes to the generation of the proton motive force used for ATP synthesis and also for the fast and reversible induction of non-photochemical quenching (NPQ) to avoid photoinhibition and photodamage. However, the regulatory mechanism(s) controlling ΔpH in response to fluctuating light has not been fully elucidated. We previously described a new NPQ-regulatory chloroplastic protein, Fluctuating-Light-Acclimation Protein1 (FLAP1), which is important for plant growth and modulation of ΔpH under fluctuating light conditions. For this report, we further characterized FLAP1 activity by individually crossing an Arabidopsis flap1 mutant with npq4 and pgr5 plants; npq4 is defective in PsbS-dependent NPQ, and pgr5 is defective in induction of steady-state proton motive force (pmf) and energy-dependent quenching (qE). Both npq4 and npq4 flap1 exhibited similar NPQ kinetics and other photosynthetic parameters under constant or fluctuating actinic light. Conversely, pgr5 flap1 had recovered NPQ, photosystem II quantum yield and growth under fluctuating light, each of which was impaired in pgr5. Together with other data, we propose that FLAP1 activity controls proton homeostasis under steady-state photosynthesis to manipulate luminal acidification levels appropriately to balance photoprotection and photochemical processes.

Significance of PGR5-dependent cyclic electron flow for optimizing the rate of ATP synthesis and consumption in Arabidopsis chloroplasts.

The proton motive force (PMF) across the chloroplast thylakoid membrane that is generated by electron transport during photosynthesis is the driving force for ATP synthesis in plants. The PMF mainly arises from the oxidation of water in photosystem II and from electron transfer within the cytochrome b6f complex. There are two electron transfer pathways related to PMF formation: linear electron flow and cyclic electron flow. Proton gradient regulation 5 (PGR5) is a major component of the cyclic electron flow pathway, and the Arabidopsis pgr5 mutant shows a substantial reduction in the PMF. How the PGR5-dependent cyclic electron flow contributes to ATP synthesis has not, however, been fully delineated. In this study, we monitored in vivo ATP levels in Arabidopsis chloroplasts in real time using a genetically encoded bioluminescence-based ATP indicator, Nano-lantern(ATP1). The increase in ATP in the chloroplast stroma of pgr5 leaves upon illumination with actinic light was significantly slower than in wild type, and the decrease in ATP levels when this illumination stopped was significantly faster in pgr5 leaves than in wild type. These results indicated that PGR5-dependent cyclic electron flow around photosystem I helps to sustain the rate of ATP synthesis, which is important for growth under fluctuating light conditions.

Insect taste receptors relevant to host identification by recognition of secondary metabolite patterns of non-host plants.

The taste sensing system is crucial for food recognition in insects and other animals. It is commonly believed that insect gustatory receptors (Grs) expressed in gustatory organs are indispensable for host plant selection. Many behavioral studies have shown that mono- or oligo-phagous lepidopteran insects use the balance between feeding attractants and feeding deterrents in host plants and that these are sensed by taste organs for host plant recognition. However, the molecular mechanism underlying taste recognition, especially of feeding deterrents, remains to be elucidated. To better understand this mechanism, we studied orphan Grs, including Bombyx mori Gr (BmGr) 16, BmGr18, and BmGr53, from the mono-phagous insect, Bombyx mori. Using Calcium imaging in mammalian cells, we first confirmed in lepidoptera insects that three of the putative bitter Grs widely responded to structurally different feeding deterrents. Although the phylogenetic distance of these Grs was considerable, they responded to partially overlapping deterrents of plant secondary metabolites. These findings suggest that not only these three Grs but also most of the Grs that have been assigned to putative bitter Grs are feeding-deterrent receptors that play a role in host plant recognition.

Differential expression of a fructose receptor gene in honey bee workers according to age and behavioral role.

Honey bee (Apis mellifera) workers contribute to the maintenance of colonies in various ways. The primary functions of workers are divided into two types depending on age: young workers (nurses) primarily engage in such behaviors as cleaning and food handling within the hive, whereas older workers (foragers) acquire floral nutrients beyond the colony. Concomitant with this age-dependent change in activity, physiological changes occur in the tissues and organs of workers. Nurses supply younger larvae with honey containing high levels of glucose and supply older larvae with honey containing high levels of fructose. Given that nurses must determine both the concentration and type of sugar used in honey, gustatory receptors (Gr) expressed in the chemosensory organs likely play a role in distinguishing between sugars. Glucose is recognized by Gr1 in honey bees (AmGr1); however, it remains unclear which Gr are responsible for fructose recognition. This study aimed to identify fructose receptors in honey bees and reported that AmGr3, when transiently expressed in Xenopus oocytes, responded only to fructose, and to no other sugars. We analyzed expression levels of AmGr3 to identify which tissues and organs of workers are involved in fructose recognition and determined that expression of AmGr3 was particularly high in the antennae and legs of nurses. Our results suggest that nurses use their antennae and legs to recognize fructose, and that AmGr3 functions as an accurate nutrient sensor used to maintain food quality in honey bee hives.

Identification of functional enolase genes of the silkworm Bombyx mori from public databases with a combination of dry and wet bench processes.

BACKGROUND: Various insect species have been added to genomic databases over the years. Thus, researchers can easily obtain online genomic information on invertebrates and insects. However, many incorrectly annotated genes are included in these databases, which can prevent the correct interpretation of subsequent functional analyses. To address this problem, we used a combination of dry and wet bench processes to select functional genes from public databases. RESULTS: Enolase is an important glycolytic enzyme in all organisms. We used a combination of dry and wet bench processes to identify functional enolases in the silkworm Bombyx mori (BmEno). First, we detected five annotated enolases from public databases using a Hidden Markov Model (HMM) search, and then through cDNA cloning, Northern blotting, and RNA-seq analysis, we revealed three functional enolases in B. mori: BmEno1, BmEno2, and BmEnoC. BmEno1 contained a conserved key amino acid residue for metal binding and substrate binding in other species. However, BmEno2 and BmEnoC showed a change in this key amino acid. Phylogenetic analysis showed that BmEno2 and BmEnoC were distinct from BmEno1 and other enolases, and were distributed only in lepidopteran clusters. BmEno1 was expressed in all of the tissues used in our study. In contrast, BmEno2 was mainly expressed in the testis with some expression in the ovary and suboesophageal ganglion. BmEnoC was weakly expressed in the testis. Quantitative RT-PCR showed that the mRNA expression of BmEno2 and BmEnoC correlated with testis development; thus, BmEno2 and BmEnoC may be related to lepidopteran-specific spermiogenesis. CONCLUSIONS: We identified and characterized three functional enolases from public databases with a combination of dry and wet bench processes in the silkworm B. mori. In addition, we determined that BmEno2 and BmEnoC had species-specific functions. Our strategy could be helpful for the detection of minor genes and functional genes in non-model organisms from public databases.

FLUCTUATING-LIGHT-ACCLIMATION PROTEIN1, Conserved in Oxygenic Phototrophs, Regulates H+ Homeostasis and Non-Photochemical Quenching in Chloroplasts.

Plants have mechanisms allowing them to acclimate to intense light conditions, which involves the dissipation of excess light energy. These mechanisms allow plants to perform photosynthesis efficiently and, therefore, must be accurately and precisely controlled. However, how plants dissipate excess light energy has yet to be fully elucidated. Herein we report the identification of a gene, which we named Fluctuating-Light-Acclimation Protein1 (FLAP1), that is conserved in oxygenic phototrophs. We show that Arabidopsis FLAP1 is associated with chloroplast thylakoid and envelope membranes and that the flap1 mutant shows delayed non-photochemical quenching (NPQ) relaxation during induction of photosynthesis at moderate light intensity. Under fluctuating light conditions, NPQ levels in the flap1 mutant were higher than those in the wild type during the high light period, and the mutant exhibited a pale-green phenotype. These findings suggest that FLAP1 is involved in NPQ control, which is important for an acclimation response to fluctuating light.

Relative contributions of PGR5- and NDH-dependent photosystem I cyclic electron flow in the generation of a proton gradient in Arabidopsis chloroplasts.

MAIN CONCLUSION: Respective contributions of PGR5- and NDH-dependent cyclic electron flows around photosystem I for generating the proton gradient across the thylakoid membrane are ~30 and ~5%. The proton concentration gradient across the thylakoid membrane (ΔpH) produced by photosynthetic electron transport is the driving force of ATP synthesis and non-photochemical quenching. Two types of electron transfer contribute to ΔpH formation: linear electron flow (LEF) and cyclic electron flow (CEF, divided into PGR5- and NDH-dependent pathways). However, the respective contributions of LEF and CEF to ΔpH formation are largely unknown. We employed fluorescence quenching analysis with the pH indicator 9-aminoacridine to directly monitor ΔpH formation in isolated chloroplasts of Arabidopsis mutants lacking PGR5- and/or NDH-dependent CEF. The results indicate that ΔpH formation is mostly due to LEF, with the contributions of PGR5- and NDH-dependent CEF estimated as only ~30 and ~5%, respectively.

The domain II loops of Bacillus thuringiensis Cry1Aa form an overlapping interaction site for two Bombyx mori larvae functional receptors, ABC transporter C2 and cadherin-like receptor.

Information about the receptor-interaction region of Cry toxins, insecticidal proteins produced by Bacillus thuringiensis, is needed to elucidate the mode of action of Cry toxins and improve their toxicity through protein engineering. We analyzed the interaction sites on Cry1Aa with ABC transporter C2 (ABCC2), one of the most important Cry1A toxin receptors. A competitive binding assay revealed that the Bombyx mori ABCC2 (BmABCC2) Cry1A binding site was the same as the BtR175 binding site, suggesting that the loop region of Cry1Aa domain II is a binding site. Next, we constructed several domain II loop mutant toxins and tested their binding affinity in an SPR analysis, and also performed a cell swelling assay to evaluate receptor-mediated cytotoxicity. Our results indicate that the loop regions required for BtR175 and BmABCC2 binding and the regions important for cytotoxicity partially overlap. Our results also suggest that receptor binding is necessary but not sufficient for cytotoxicity. This is the first report showing the region of interaction between ABCC2 and Cry1Aa and the cytotoxicity-relevant properties of the Cry1Aa domain II loop region.

Male-specific pulmonary hemorrhage and cytokine gene expression in golden hamster in early-phase Leptospira interrogans serovar Hebdomadis infection.

Leptospirosis causes severe clinical signs more frequently in men than in women, but the mechanism underlying the gender differences in leptospirosis remains unclear. In this study, petechial hemorrhage was observed in male but not in female hamster lung tissues infected with Leptospira interrogans serovar Hebdomadis at 120 h pi, demonstrating that male hamsters were more susceptible to the development of a severe disease upon Leptospira infection. No leptospiral DNA was detected in the lung tissues at 120 h pi when pulmonary hemorrhage was observed, indicating that pulmonary hemorrhage is attributable to the immune reactions of the host rather than from the direct effect of leptospires. The upregulation of nitric oxide synthase genes in the hamsters without pulmonary hemorrhage, inos and enos in female hamsters at 96 h pi and enos in male animals without hemorrhage at 120 h pi, may suggest that nitric oxide has a suppressive effect on leptospirosis-associated pulmonary hemorrhage.

FRET sensor-based quantification of intracellular trehalose in mammalian cells.

Trehalose acts as a stress protectant and an autophagy inducer in mammalian cells. The molecular mechanisms of action remain obscure because intracellular trehalose at micromolar level is difficult to quantitate. Here, we show a novel trehalose monitoring technology based on FRET. FLIP-suc90µ∆1Venus sensor expressed in mammalian cells enables to quickly and non-destructively detect an infinitesimal amount of intracellular trehalose.