Spatiotemporal variation and distribution characteristics of crop residue burning in China from 2001 to 2018.

In this study, we integrated a remote-sensing fire product (MOD14A1) and land-use product (MCD12Q1) to extract the number of crop-residue burning (CRB) spots and the fire radiative power (FRP) in China from 2001 to 2018. Moreover, we conducted three trend analyses and two geographic distribution analyses to quantify the interannual variations and summarize the spatial characteristics of CRB on grid (0.25° × 0.25°) and regional scales. The results indicated that CRB presents distinctive seasonal patterns with each sub-region. All trend analyses suggested that the annual number of CRB spots in China increased significantly from 2001 to 2018; the linear trend reached 2615 spots/year, the Theil-Sen slope was slightly lower at 2557 spots/year, and the Mann-Kendal τ was 0.75. By dividing the study period into two sub-periods, we found that the five sub-regions presented different trends in the first and second sub-periods; e.g., the Theil-Sen slope of eastern China in the first sub-period (2001-2009) was 1021 spots/year but was -1599 spots/year in the second period (2010-2018). This suggests that summer CRB has been effectively mitigated in eastern China since 2010. Further, the average FRP of CRB spots presented a decreasing trend from 27.5 MW/spot in 2001 to only 15.8 MW/spot in 2018; this may be attributable to more scattered CRB rather than aggregated CRB. Collectively, the fire spots, FRP, and average FRP indicated that spring, summer, and autumn CRB had dropped dramatically over previous levels by 2018 due to strict mitigation measures by local governments.

Secondary structure characterization based on amino acid composition and availability in proteins.

The importance of thorough analyses of the secondary structures in proteins as basic structural units cannot be overemphasized. Although recent computational methods have achieved reasonably high accuracy for predicting secondary structures from amino acid sequences, a simple and fundamental empirical approach to characterize the amino acid composition of secondary structures was performed mainly in 1970s, with a small number of analyzed structures. To extend this classical approach using a large number of analyzed structures, here we characterized the amino acid sequences of secondary structures (12 154 alpha-helix units, 4592 3(10)-helix units, 16 787 beta-strand units, and 30 811 "other" units), using the representative three-dimensional protein structure records (1641 protein chains) from the Protein Data Bank. We first examined the length and the amino acid compositions of secondary structures, including rank order differences and assignment relationships among amino acids. These compositional results were largely, but not entirely, consistent with the previous studies. In addition, we examined the frequency of 400 amino acid doublets and 8000 triplets in secondary structures based on their relative counts, termed the availability. We identified not only some triplets that were specific to a certain secondary structure but also so-called zero-count triplets, which did not occur in a given secondary structure at all, even though they were probabilistically predicted to occur several times. Taken together, the present study revealed essential features of secondary structures and suggests potential applications in the secondary structure prediction and the functional design of protein sequences.

Visualization of free radical reactions in an aqueous sample irradiated by 290 MeV carbon beam.

The detection of free radical reactions in a gelatin sample irradiated by a heavy-ion beam was tested using electron paramagnetic resonance (EPR) spectroscopic and MRI methods. Geometry and the amount of free radical generation in a sample are described. A reaction mixture containing glutathione and a nitroxyl radical, TEMPOL, was caked with gelatin, and then irradiated with a 290 MeV carbon beam. The amount of free radical generation in a solid sample was almost flat from the surface to the beam end, except for a small peak, the peak radioactivation profile, and then steeply decreased approaching the beam end. Total free radical reactions obtained with carbon-beam irradiation were expected to be less than one-third of X-ray irradiation, when the same dose for a deeper target was considered. Both EPR and MRI are useful tools to visualize free radical generation in samples irradiated by a heavy-ion beam. The EPR-based method is more sensitive and quantitative than the MRI-based method; however, the MRI method can achieve high spatial resolution. This study gives the rationale for a redox regulation trial using antioxidant drugs to reduce the side effects on normal tissues in carbon-beam therapy.

Potential implications of availability of short amino acid sequences in proteins: an old and new approach to protein decoding and design.

Three-dimensional structure of a protein molecule is primarily determined by its amino acid sequence, and thus the elucidation of general rules embedded in amino acid sequences is of great importance in protein science and engineering. To extract valuable information from sequences, we propose an analytical method in which a protein sequence is considered to be constructed by serial superimpositions of short amino acid sequences of n amino acid sets, especially triplets (3-aa sets). Using the comprehensive nonredundant protein database, we first examined "availability" of all possible combinatorial sets of 8,000 triplet species. Availability score was mathematically defined as an indicator for the relative "preference" or "avoidance" for a given short constituent sequence to be used in protein chain. Availability scores of real proteins were clearly biased against those of randomly generated proteins. We found many triplet species that occurred in the database more than expected or less than expected. Such bias was extended to longer sets, and we found that some species of pentats (5-aa sets) that occurred reasonably frequently in the randomly generated protein population did not occur at all in any real proteins known today. Availability score was dependent on species, potentially serving as a phylogenetic indicator. Furthermore, we suggest possibilities of various biotechnological applications of characteristic short sequences such as human-specific and pathogen-specific short sequences obtained from availability analysis. Availability score was also dependent on secondary structures, potentially serving as a structural indicator. Availability analysis on triplets may be combined with a comprehensive data collection on the varphi and psi peptide-bond angles of the amino acid at the center of each triplet, i.e., a collection of Ramachandran plots for each triplet. These triplet characters, together with other physicochemical data, will provide us with basic information between protein sequence and structure, by which structure prediction and engineering may be greatly facilitated. Availability analysis may also be useful in identifying word processing units in amino acid sequences based on an analogy to natural languages. Together with other approaches, availability analysis will elucidate general rules hidden in the primary sequences and eventually contributes to rebuilding the paradigm of protein science.

Identification and characterization of an increased glycoprotein in aging: age-associated translocation of cathepsin D.

We found that 14 N-glycosylated proteins were accumulated in the rat cerebral cortex cytosolic fraction in the aging process by a comparative study with two-dimensional gel electrophoresis and concanavalin A staining. All proteins had high mannose and/or hybrid-type N-glycans, as indicated by the fact that they were sensitive to endoglycosidase H digestion. Three of these cytosolic glycoproteins were identified as cathepsin D, a lysosomal protease, by tryptic digestion and nano liquid chromatography electrospray ionization quadrupole time of flight mass spectrometry. The increase of cytosolic cathepsin D during aging was not due to lysosomal membrane disruption, as shown by the fact that the activities of beta-hexosaminidase and beta-glucuronidase, other lysosomal enzymes, did not increase in the cytosolic fraction. Although the total amount of cathepsin D increased during aging, the amount of cathepsin D in the microsomal fraction did not change, indicating a selective increase of cytosolic cathepsin D. This phenomenon was also observed in the hippocampus, cerebellum, kidney, liver, and spleen. Based on these results, we propose that cytosolic cathepsin D is a new biomarker of aging.

Availability of short amino acid sequences in proteins.

Much attention is being paid to protein databases as an important information source for proteome research. Although used extensively for similarity searches, protein databases themselves have not fully been characterized. In a systematic attempt to reveal protein-database characters that could contribute to revealing how protein chains are constructed, frequency distributions of all possible combinatorial sets of three, four, and five amino acids ("triplets," "quartets," and "pentats"; collectively called constituent sequences) have been examined in the nonredundant (nr) protein database, demonstrating the existence of nonrandom bias in their "availability" at the population level. Nonexistent short sequences of pentats were found that showed low availability in biological proteins against their expected probabilities of occurrence. Among them, six representative ones were successfully synthesized as peptides with reasonably high yields in a conventional Fmoc method, excluding the possibility that a putative physicochemical energy barrier in forming them could be a direct cause for the low availability. They were also expressed as soluble fusion proteins in a conventional Escherichia coli BL21Star(DE3) system with reasonably high yield, again excluding a possible difficulty in their biological synthesis. Together, these results suggest that information on three-dimensional structures and functions of proteins exists in the context of connections of short constituent sequences, and that proteins are composed of evolutionarily selected constituent sequences, which are reflected in their availability differences in the database. These results may have biological implications for protein structural studies.

The proto-oncogene BCL-6 is expressed in olfactory sensory neurons.

Mammalian olfactory sensory neurons harbor a large repertoire of odorant receptors. Yet, each cell chooses a single odorant receptor gene to express, which may then serve as the molecular identification of that cell. This process of differentiation or receptor gene regulation of olfactory sensory neurons has largely been enigmatic. Here, we showed that the important proto-oncogene in B lymphocyte terminal differentiation, B-Cell Leukemia/Lymphoma 6 (BCL-6), which codes for a sequence-specific transcription repressor, is expressed in olfactory sensory neurons in the mouse. We detected BCL-6 mRNA in most, if not all, mature olfactory sensory neurons by in situ hybridization histochemistry. Western blot analysis also revealed the presence of BCL-6 protein in the olfactory epithelium, but immunohistochemical analysis using anti-BCL-6 antibodies showed that only a subset of olfactory sensory neurons highly expressed BCL-6 protein. BCL-6 mRNA was detected as early as embryonic day 14 (E14) in most olfactory sensory neurons as in adults, and at E15, BCL-6 protein was detected in most cells that were likely to be differentiating into mature olfactory sensory neurons. Artificial induction of apoptosis of olfactory sensory neurons resulted in the decrease of BCL-6 mRNA, but during the subsequent phase of regeneration and differentiation, it markedly increased. Taken together, our results suggest the possibility that BCL-6 plays an important role in terminal differentiation not only in B lymphocytes but also in olfactory sensory neurons.

Morphological comparison of pupal wing cuticle patterns in butterflies.

Butterfly wing color-patterns are determined in the prospective wing tissues during the late larval and early pupal stages. To study the cellular differentiation process of wings, morphological knowledge on pupal wings is prerequisite. Here we systematically examined morphological patterns of the pupal wing cuticular surface in a wide variety of nymphalid butterflies in relation to adult color-patterns. Several kinds of pupal wing patterns corresponding to particular adult color-pattern elements were widely observed in many species. Especially noteworthy were the pupal "focal" spots corresponding to the adult border ocelli system, which were detected in many species of Nymphalinae, Apaturinae, Argynninae, Satyrinae, and Danainae. Striped patterns on the pupal wing cuticle seen in some species of Limenitinae, Ariadnae, and Marpesiinae directly corresponded to those of the adult wings. In Vanessa cardui, eyespot-like pattern elements were tentatively produced during development in the wing tissue underneath the pupal spots and subsequently erased, suggesting a mechanism for producing novel color-patterns in the course of development and evolution. The pupal focal spots reasonably correlated with the adult eyespots in size in Precis orithya and Ypthima argus. We physically damaged the pupal focal spots and their corresponding cells underneath in these species, which abolished or inhibited the formation of the adult eyespots. Taken together, our results clarified that pupal cuticle patterns were often indicative of the adult color-patterns and apparently reflect molecular activity of organizing centers for the adult color-pattern formation at least in nymphalid butterflies.

Tungstate-induced color-pattern modifications of butterfly wings are independent of stress response and ecdysteroid effect.

Systemic injections of sodium tungstate, a protein-tyrosine phosphatase (PTPase) inhibitor, to pupae immediately after pupation have been shown to efficiently produce characteristic color-pattern modifications on the wings of many species of butterflies. Here we demonstrated that the tungstate-induced modification pattern was entirely different from other chemically-induced ones in a species of nymphalid butterfly Junonia (Precis) orithya. In this species, the systemic injections of tungstate produced characteristic expansion of black area and shrinkage of white area together with the move of parafocal elements toward the wing base. Overall, pattern boundaries became obscure. In contrast, an entirely different modification pattern, overall darkening of wings, was observed by the injections of stress-inducing chemicals, thapsigargin, ionomycin, or geldanamycin, to pupae under the rearing conditions for the adult summer form. On the ventral wings, this darkening was due to an increase of the proportion of peppered dark scales, which was reminiscent of the natural fall form of this species. Under the same rearing conditions, the injections of ecdysteroid, which is a well-known hormone being responsible for the seasonal polyphenism of nymphalid butterflies, yielded overall expansion of orange area especially around eyespots. Taken together, we conclude that the tungstate-induced modifications are clearly distinguishable from those of stress response and ecdysteroid effect. This conclusion then suggests that the putative PTPase signaling pathway that is sensitive to tungstate uniquely contributes to the wing-wide color-pattern development in butterflies.

Color-pattern modifications and speciation in butterflies of the genus Vanessa and its related genera Cynthia and Bassaris.

We have previously shown that the systemic injection of sodium tungstate, a protein-tyrosine phosphatase (PTPase) inhibitor, to pupae immediately after pupation efficiently produces characteristic color-pattern modifications on the wings of many species of butterflies including Vanessa indica and Cynthia cardui. In these species, the black spots reduced in size in response to the treatment. Similar modifications are occasionally seen in the field-caught aberrant individuals. Exceptionally, however, a C. cardui individual with enlarged black spots ("reversed" modification pattern) has been reported. Here we show that these modified patterns of V. indica and C. cardui are quite similar to the normal color-patterns of other Vanessa species. V. indica with tungstate-induced modifications resembled V. tameamea, V. samani, and Bassaris itea, whereas V. dilecta, V. atalanta, and V. dejeanii are similar to the "reversed" individual. Most features seen in the experimentally-modified V. indica were observed throughout the fore- and hindwings of V. samani. In contrast, the experimentally-induced color-patterns of C. cardui did not parallel variation of Cynthia butterflies. Since it has been proposed that a hypothetical transduction pathway with a PTPase for the scale-cell differentiation globally coordinates the wing-wide color-patterns, our findings suggest that spontaneous mutations in genes in this hypothetical pathway might have played a major role in creating new color-patterns and species in the Vanessa genus but not in the Cynthia genus. This evolutionary mechanism may probably be shared more widely in Lepidoptera, although this would not be a sole determinant for the color-pattern development and evolution.

The proto-oncogene BCL6 promotes survival of olfactory sensory neurons.

For the mammalian olfactory epithelium to continually detect odorant, neuronal survival, apoptosis, and regeneration must be coordinated. Here, we showed that the proto-oncogene BCL6, which encodes a transcriptional repressor required for lymphocyte terminal differentiation, contributes to the survival of olfactory sensory neurons (OSNs). In the olfactory epithelia of the BCL6 null mutant mice, many OSNs were positive for both OMP and GAP43. The epithelium was relatively thinner, showing many apoptotic signals. These characters were phenotypically similar to those of the wild-type mice treated with nasal lectin irrigation, which acutely induces apoptosis of OSNs. Odorant receptors were expressed normally in the epithelia of the mutant mice, and their overall expression profile based on DNA microarray analyses was roughly similar to that of the apoptosis-induced olfactory epithelia of the wild-type mice. Experimental increase of BCL6 together with green fluorescent protein in OSNs using adenovirus-mediated gene transfer made the epifluorescence last longer than the control fluorescence without exogenous BCL6 after the nasal lectin irrigation, indicating that BCL6 made the infected neurons survive longer. We conclude that BCL6 plays an active role in the survival of OSNs as an anti-apoptotic factor and confers immature OSNs enough time to fully differentiate into mature ones.

Detection of free radical reactions in an aqueous sample induced by low linear-energy-transfer irradiation.

Quantitative detection of free radical reactions induced by low linear-energy-transfer (LET) irradiation in an aqueous solution was attempted using nitroxyl radicals. The stability and reactivity of reaction mixtures containing a nitroxyl radical and a hydrogen donor, i.e., glutathione (GSH), nicotinamide adenine dinucleotide (NADH), or nicotinamide adenine dinucleotide phosphate (NADPH), were tested. X-band electron paramagnetic resonance (EPR) measurements of several reaction mixtures were performed to find a suitable preparation to quantitatively detect free radical reactions produced by low LET irradiation. The EPR signal intensity of nitroxyl radicals was decreased by low LET irradiation when a hydrogen donor coexisted in the reaction mixture. The combination of 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (4-hydroxy-TEMPO, known as TEMPOL) and GSH was most preferable among other preparations tested in this paper, because of the sensitivity and irreversible reaction. The extent of the free radical reaction, i.e., formation of reactive oxygen species, in the reaction mixture depended on the radiation energy when an identical dose was given.

Alignment-free classification of G-protein-coupled receptors using self-organizing maps.

Proteins are classified mainly on the basis of alignments of amino acid sequences. Drug discovery processes based on pharmacologically important proteins such as G-protein-coupled receptors (GPCRs) may be facilitated if more information is extracted directly from the primary sequences. Here, we investigate an alignment-free approach to protein classification using self-organizing maps (SOMs), a kind of artificial neural network, which needs only primary sequences of proteins and determines their relative locations in a two-dimensional lattice of neurons through an adaptive process. We first showed that a set of 1397 aligned samples of Class A GPCRs can be classified by our SOM program into 15 conventional categories with 99.2% accuracy. Similarly, a nonaligned raw sequence data set of 4116 samples was categorized into 15 conventional families with 97.8% accuracy in a cross-validation test. Orphan GPCRs were also classified appropriately using the result of the SOM learning. A supposedly diverse family of olfactory receptors formed the most distinctive cluster in the map, whereas amine and peptide families exhibited diffuse distributions. A feature of this kind in the map can be interpreted to reflect hierarchical family composition. Interestingly, some orphan receptors that were categorized as olfactory were somatosensory chemoreceptors. These results suggest the applicability and potential of the SOM program to classification prediction and knowledge discovery from protein sequences.

Quantitative measurements of oxidative stress in mouse skin induced by X-ray irradiation.

To find efficient methods to evaluate oxidative stress in mouse skin caused by X-ray irradiation, several markers and methodologies were examined. Hairless mice were irradiated with 50 Gy X-rays and skin homogenates or skin strips were prepared. Lipid peroxidation was measured using the skin homogenate as the level of thiobarbituric acid reactive substances. The level of lipid peroxidation increased with time after irradiation and was twice that of the control at 78 h. ESR spectra of skin strips showed a clear signal for the ascorbyl radical, which increased with time after irradiation in a manner similar to that of lipid peroxidation. To measure levels of glutathione (GSH) and its oxidized forms (GSSG) simultaneously, two HPLC methods, sample derivatization with 1-fluoro-2,4-dinitrobenzene and detection with a UV detector (method A) and no derivatization and detection with an electrochemical detector (method B), were compared and the latter was found to be better. No significant change was observed within 24 h after irradiation in the levels of GSH and GSSG measured by method B. The GSH/GSSG ratio may be a less sensitive parameter for the evaluation of acute oxidative stress caused by X-ray irradiation in the skin. Monitoring the ascorbyl radical seems to be a good way to evaluate oxidative stress in skin in vivo.

Species-specific color-pattern modifications of butterfly wings.

We have previously shown that the systemic injection of sodium tungstate, a general protein-tyrosine phosphatase (PTPase) inhibitor, efficiently produces characteristic color-pattern modifications on the wings of the Painted Lady butterfly, Vanessa cardui. By using this method in the present study, we analyzed modification patterns of six species of Japanese butterflies. Whereas in Vanessa indica the black spots on the forewings reduced in size in response to the treatment, in Lycaena phlaeas the morphologically similar black spots enlarged in size. However, the metallic blue spots on the forewings of V. indica did enlarge in size, showing different behavior even within a single wing surface. The response patterns of Ypthima argus differed markedly from those of other species in that ectopic color-pattern elements were created. Colias erate showed minor modifications that coincidentally resembled the natural color-pattern of a closely related species, Colias palaeno. Through a comprehensive literature search, we confirmed the existence of naturally occurring aberrant color patterns with close similarities to the experimentally induced phenocopies in each of the modified species. Our results point out the possibility that a hypothetical transduction pathway with a PTPase for the scale-cell differentiation globally coordinates the wing-wide color-pattern development in butterflies.

Length analyses of Drosophila odorant receptors.

Odorant receptors comprise a unique family of G-protein-coupled seven-transmembrane receptors both in mammals and insects. In the fruit fly Drosophila melanogaster, all 61 candidate odorant receptor genes have been identified based on the complete genome sequence, and their expression patterns have been examined. A given odorant receptor is expressed in the antenna or maxillary palp, or not expressed at all. Here we have applied a set of statistical analyses to the length of the extra- and intracellular loops and terminals (LTs) of Drosophila odorant receptors to examine possible inter- and intramolecular relations at the population level. We have first provided some useful statistical information such as mean length values and length histograms to depict a general nature of Drosophila odorant receptors at the population level, after focusing on discrepancy on assigning transmembrane domains between researchers. In a preferable transmembrane assignment, all extracellular LTs, especially the second extracellular loops, were relatively large in length, suggesting their functional significance. Somewhat surprisingly, principle component analysis (PCA) indicated that the maxillary palp receptors were almost as diverse as the antenna receptors despite their much smaller population size. PCA together with histograms also revealed that receptors with an abnormal length configuration tended not to be expressed, suggesting that LT length deviations are related to transcriptional silencing of odorant receptor genes. Rank transformation tests pointed out possible LTs that could have different length between differently expressed receptors at the population level. Taken together, length analyses provide us with a general picture, i.e. "length configuration," of Drosophila odorant receptors at the population level that could point out putatively important functional sites for experimental studies.

Odorant receptor expression in the mouse cerebral cortex.

Mammalian odorant receptors have been known to be involved not only in odorant detection but also in neuronal development of olfactory sensory neurons. We have examined a possibility of odorant receptor expression in nonolfactory neurons in the mouse. Mouse odorant receptors (M71, C6, and OR3), two of which were already shown to be functionally activated by odorants in heterologous systems, were detected by polymerase chain reactions (PCRs) from the cerebral cortex but not from other brain tissues. Degenerate PCR further suggested that other odorant receptors were also expressed in the mouse cerebral cortex. One of these receptors showed high sequence-match with a putative chick odorant receptor OR7 transiently expressed in the notochord during development. In situ hybridization detected signals for M71 and C6 receptors in the layer II cortical pyramidal neurons located in the occipital pole. In the M71-IRES-tauLacZ mouse, in which M71 expression was genetically marked with tauLacZ, X-gal staining signals were mostly localized in the layer II neurons in the occipital pole, being consistent with the in situ hybridization result. Fluorescent immunohistochemistry using anti-beta-galactosidase antibody further detected the tauLacZ signals in the same cells. X-gal staining began at P3, peaked at P8, and continued to adults, although signals gradually decreased. These data showed that at least a few odorant receptors are expressed not only in olfactory sensory neurons but also in pyramidal neurons in the cerebral cortex, possibly playing an important role either in chemical detection of exogenous or endogenous ligands or in a developmental process such as axon guidance and target recognition.