A Novel Bacterial Nitrate Transporter Composed of Small Transmembrane Proteins.

A putative silent gene of the freshwater cyanobacterium Synechococcus elongatus strain PCC 7942, encoding a small protein with two transmembrane helices, was named nrtS, since its overexpression from an inducible promoter conferred nitrate uptake activity on the nitrate transport-less NA4 mutant of S. elongatus. Homologs of nrtS, encoding proteins of 67-118 amino acid residues, are present in a limited number of eubacteria including mostly cyanobacteria and proteobacteria, but some others, e.g. the actinobacteria of the Mycobacterium tuberculosis complex, also have the gene. When expressed in NA4, the nrtS homolog of the γ-proteobacterium Marinomonas mediterranea took up nitrate with higher affinity for the substrate as compared with the S. elongatus NrtS (Km of 0.49 mM vs. 2.5 mM). Among the 61 bacterial species carrying the nrtS homolog, the marine cyanobacterium Synechococcus sp. strain PCC 7002 is unique in having two nrtS genes (nrtS1 and nrtS2) located in tandem on the chromosome. Coexpression of the two genes in NA4 resulted in nitrate uptake with a Km (NO3-) of 0.15 mM, while expression of either of the two resulted in low-affinity nitrate uptake activity with Km values of >3 mM, indicating that NrtS1 and NrtS2 form a heteromeric transporter complex. The heteromeric transporter was shown to transport nitrite as well. A Synechococcus sp. strain PCC 7002 mutant defective in the nitrate transporter (NrtP) showed a residual activity of nitrate uptake, which was ascribed to the NrtS proteins. Blue-native PAGE and immunoblotting analysis suggested a hexameric structure for the NrtS proteins.

CyanoBase: a large-scale update on its 20th anniversary.

The first ever cyanobacterial genome sequence was determined two decades ago and CyanoBase (http://genome.microbedb.jp/cyanobase), the first database for cyanobacteria was simultaneously developed to allow this genomic information to be used more efficiently. Since then, CyanoBase has constantly been extended and has received several updates. Here, we describe a new large-scale update of the database, which coincides with its 20th anniversary. We have expanded the number of cyanobacterial genomic sequences from 39 to 376 species, which consists of 86 complete and 290 draft genomes. We have also optimized the user interface for large genomic data to include the use of semantic web technologies and JBrowse and have extended community-based reannotation resources through the re-annotation of Synechocystis sp. PCC 6803 by the cyanobacterial research community. These updates have markedly improved CyanoBase, providing cyanobacterial genome annotations as references for cyanobacterial research.

Essential Role of Acyl-ACP Synthetase in Acclimation of the Cyanobacterium Synechococcus elongatus Strain PCC 7942 to High-Light Conditions.

Most organisms capable of oxygenic photosynthesis have an aas gene encoding an acyl-acyl carrier protein synthetase (Aas), which activates free fatty acids (FFAs) via esterification to acyl carrier protein. Cyanobacterial aas mutants are often used for studies aimed at photosynthetic production of biofuels because the mutation leads to intracellular accumulation of FFAs and their secretion into the external medium, but the physiological significance of the production of FFAs and their recycling involving Aas has remained unclear. Using an aas-deficient mutant of Synechococcus elongatus strain PCC 7942, we show here that remodeling of membrane lipids is activated by high-intensity light and that the recycling of FFAs is essential for acclimation to high-light conditions. Unlike wild-type cells, the mutant cells could not increase their growth rate as the light intensity was increased from 50 to 400 µmol photons m(-2) s(-1), and the high-light-grown mutant cells accumulated FFAs and the lysolipids derived from all the four major classes of membrane lipids, revealing high-light-induced lipid deacylation. The high-light-grown mutant cells showed much lower PSII activity and Chl contents as compared with the wild-type cells or low-light-grown mutant cells. The loss of Aas accelerated photodamage of PSII but did not affect the repair process of PSII, indicating that PSII is destabilized in the mutant. Thus, Aas is essential for acclimation of the cyanobacterium to high-light conditions. The relevance of the present finding s to biofuel production using cyanobacteria is discussed.

Identification of a Cyanobacterial RND-Type Efflux System Involved in Export of Free Fatty Acids.

An RND (resistance-nodulation-division)-type transporter having the capacity to export free fatty acids (FFAs) was identified in the cyanobacterium Synechococcus elongatus strain PCC 7942 during characterization of a mutant strain engineered to produce FFAs. The basic strategy for construction of the FFA-producing mutant was a commonly used one, involving inactivation of the endogenous acyl-acyl carrier protein synthetase gene (aas) and introduction of a foreign thioesterase gene ('tesA), but a nitrate transport mutant NA3 was used as the parental strain to achieve slow, nitrate-limited growth in batch cultures. Also, a nitrogen-regulated promoter PnirA was used to drive 'tesA to maximize thioesterase expression during the nitrate-limited growth. The resulting mutant (dAS2T) was, however, incapable of growth under the conditions of nitrate limitation, presumably due to toxicity associated with FFA overproduction. Incubation of the mutant culture under the non-permissive conditions allowed for isolation of a pseudorevertant (dAS2T-pr1) capable of growth on nitrate. Genome sequence and gene expression analyses of this strain suggested that expression of an RND-type efflux system had rescued growth on nitrate. Targeted inactivation of the RND-type transporter genes in the wild-type strain resulted in loss of tolerance to exogenously added FFAs including capric, lauric, myristic, oleic and linolenic acids. Overexpression of the genes in dAS2T, on the other hand, enhanced FFA excretion and cell growth in nitrate-containing medium, verifying that the genes encode an efflux pump for FFAs. These results demonstrate the importance of the efflux system in efficient FFA production using genetically engineered cyanobacteria.

Nitrite transport activity of a novel HPP family protein conserved in cyanobacteria and chloroplasts.

Some cyanobacterial genomes encode an integral membrane protein of the HPP family, which exhibited nitrite transport activity when expressed in the nitrite transport-less NA4 mutant of the cyanobacterium Synechococcus elongatus strain PCC 7942. AT5G62720 and AT3G47980 were found to encode Arabidopsis homologs of the cyanobacterial protein. The product of AT5G62720 was localized to the chloroplast envelope membrane and was shown to confer nitrite uptake activity on the NA4 mutant when expressed with an N-terminally truncated transit peptide or as a fusion with the N-terminal region of the cyanobacterial HPP family protein. Kinetic analyses showed that the Arabidopsis protein has much higher affinity for nitrite (K(m) = 13 µM) than the cyanobacterial protein (K(m) = 150 µM). Illuminated chloroplasts isolated from the mutant lines of AT5G62720 showed much lower activity of nitrite uptake than the chloroplasts isolated from the wild-type Col-0 plants, while the chloroplasts of the mutants of AT1G68570 (AtNPF3.1), the gene previously reported to encode a plastid nitrite transporter AtNitr1, showed wild-type levels of nitrite uptake activity. AT3G47980 was expressed in roots but not in shoots. It has a putative transit peptide similar to that of AT5G62720 and its fusion with the N-terminal region of the cyanobacterial HPP protein showed low but significant activity of nitrite transport in the cyanobacterial cell. Transcription of AT5G62720 (AtNITR2;1) and AT3G47980 (AtNITR2;2) was stimulated by nitrate under the control of the NIN-like proteins, suggesting that the HPP proteins represent nitrate-inducible components of the nitrite transport system of plastids.

Effects of high CO2 on growth and metabolism of Arabidopsis seedlings during growth with a constantly limited supply of nitrogen.

Elevated CO2 has been reported to stimulate plant growth under nitrogen-sufficient conditions, but the effects of CO2 on growth in a constantly nitrogen-limited state, which is relevant to most natural habitats of plants, remain unclear. Here, we maintained Arabidopsis seedlings under such conditions by growing a mutant with reduced nitrate uptake activity on a medium containing nitrate as the sole nitrogen source. Under nitrogen-sufficient conditions (i.e. in the presence of ammonium), growth of shoots and roots of both the wild type (WT) and the mutant was increased approximately 2-fold by elevated CO2. Growth stimulation of shoots and roots by elevated CO2 was observed in the WT growing with nitrate as the sole nitrogen source, but in the mutant grown with nitrate, the high-CO2 conditions stimulated only the growth of roots. In the mutant, elevated CO2 caused well-known symptoms of nitrogen-starved plants, including decreased shoot/root ratio, reduced nitrate content and accumulation of anthocyanin, but also had an increased Chl content in the shoot, which was contradictory to the known effect of nitrogen depletion. A high-CO2-responsive change specific to the mutant was not observed in the levels of the major metabolites, although CO2 responses were observed in the WT and the mutant. These results indicated that elevated CO2 causes nitrogen limitation in the seedlings grown with a constantly limited supply of nitrogen, but the Chl content and the root biomass of the plant increase to enhance the activities of both photosynthesis and nitrogen uptake, while maintaining normal metabolism and response to high CO2.

In vitro and in vivo analyses of the role of the carboxysomal ß-type carbonic anhydrase of the cyanobacterium Synechococcus elongatus in carboxylation of ribulose-1,5-bisphosphate.

The carboxylase activities of crude carboxysome preparations obtained from the wild-type Synechococcus elongatus strain PCC 7942 strain and the mutant defective in the carboxysomal carbonic anhydrase (CA) were compared. The carboxylation reaction required high concentrations of bicarbonate and was not even saturated at 50 mM bicarbonate. With the initial concentrations of 50 mM and 25 mM for bicarbonate and ribulose-1,5-bisphosphate (RuBP), respectively, the initial rate of RuBP carboxylation by the mutant carboxysome (0.22 µmol mg(-1) protein min(-1)) was only 30 % of that observed for the wild-type carboxysomes (0.71 µmol mg(-1) protein min(-1)), indicating the importance of the presence of CA in efficient catalysis by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). While the mutant defective in the ccmLMNO genes, which lacks the carboxysome structure, could grow under aeration with 2 % (v/v) CO2 in air, the mutant defective in ccaA as well as ccmLMNO required 5 % (v/v) CO2 for growth, indicating that the cytoplasmically localized CcaA helped utilization of CO2 by the cytoplasmically localized Rubisco by counteracting the action of the CO2 hydration mechanism. The results predict that overexpression of Rubisco would hardly enhance CO2 fixation by the cyanobacterium at CO2 levels lower than 5 %, unless Rubisco is properly organized into carboxysomes.

A budding yeast CAGE dataset comprising two cell types.

The budding yeast Saccharomyces cerevisiae is an excellent model organism for studying chromatin regulation with high-resolution genome-wide analyses. Since newly generated genome-wide data are often compared with publicly available datasets, expanding our dataset repertoire will be beneficial for the field. Information on transcription start sites (TSSs) determined at base pair resolution is essential for elucidating mechanisms of transcription and related chromatin regulation, yet no datasets that cover two different cell types are available. Here, we present a CAGE (cap analysis of gene expression) dataset for a-cells and α-cells grown in defined and rich media. Cell type-specific genes were differentially expressed as expected, ensuring the reliability of the data. Some of the differentially expressed TSSs were medium-specific or detected due to unrecognized chromosome rearrangement. By comparing the CAGE data with a high-resolution nucleosome map, major TSSs were primarily found in +1 nucleosomes, with a peak approximately 30 bp from the promoter-proximal end of the nucleosome. The dataset is available at DDBJ/GEA.

Evaluation of the effects of P(II) deficiency and the toxicity of PipX on growth characteristics of the P(II)-Less mutant of the cyanobacterium Synechococcus elongatus.

Among the known functions of the P(II) protein (the glnB gene product) in the cyanobacterium Synechococcus elongatus, negative regulation of the activity of PipX, a transcriptional co-activator of the NtcA regulon, has been thought to be essential for cell viability, because all the P(II)-less mutants thus far constructed carry spontaneous mutations in pipX. PipX is thus deduced to be a toxic protein, but its toxicity has not been clearly defined because of the lack of P(II)-deficient mutants carrying wild-type pipX. In this study, we developed a method to construct a targeted P(II)-less mutant of S. elongatus without the pipX mutation and determined the contribution of PipX to the detrimental effects of P(II) deficiency. Growth defects of the mutant were severe under nitrogen-replete conditions, i.e. in the presence of ammonium, but were also apparent under nitrogen-limited conditions. Genetic analyses indicated that the growth impairment observed under the nitrogen-limited conditions is largely due to the toxicity of PipX. Some of the phenotypes observed under the nitrogen-replete conditions, including reduced pigmentation and death of most of the cells after transfer from nitrogen-limited conditions to nitrogen-replete conditions, were ascribed to the toxicity of PipX, but inactivation of pipX only partially rescued the growth defect observed in the presence of ammonium, indicating the presence of an as yet unknown P(II) function(s) required for normal growth. Effects of ammonium addition on the nitrite uptake activity of the glnB mutant revealed a new function for P(II) in regulation of the activity of the ABC-type cyanate/nitrite transporter.

Prevalence and morbidity of urethral Trichomonas vaginalis in Japanese men with or without urethritis.

OBJECTIVES: Trichomonas vaginalis is one of the pathogens causing sexually transmitted infections. This microorganism is a common pathogen among women, but its significance as a cause of morbidity among men remains uncertain. We sought to determine the prevalence and morbidity of T. vaginalis infection in Japanese men with and without urethritis. METHODS: We examined urine specimens from 215 men with urethritis and 98 men without urethritis for the presence of urethral T. vaginalis by PCR assay. RESULTS: Only four patients-one with gonococcal urethritis, one with non-gonococcal chlamydial urethritis, one with non-gonococcal non-chlamydial urethritis and one without urethritis-were positive for T. vaginalis. The prevalence of T. vaginalis was 1.4% in men with urethritis and 1.0% in men without urethritis. A possible relation between the appearance of T. vaginalis and clinical symptoms was not confirmed. CONCLUSIONS: In the present study, the incidence of urethral T. vaginalis infection appears to be rare in Japanese men with or without urethritis, and T. vaginalis may be an uncommon pathogen in male urethritis in Japan.

Helical Three Dimensional Reconstruction Using Bayesian Optimization for Cryogenic Electron Microscopy.

Three-dimensional (3D) reconstruction for cryogenic electron microscopy (cryo-EM) often falls into an ill-posed problem owing to several uncertainties in observations, including noise. To reduce excessive degree of freedom and avoid overfitting, the structural symmetry is often used as a powerful constraint. In the case of the helix, the entire 3D structure is determined by the subunit 3D structure and two helical parameters. There is no analytical method to simultaneously obtain both of the subunit structure and helical parameters. A common approach is to employ an iterative reconstruction in which the two optimizations are performed alternately. However, iterative reconstruction does not necessarily converge when a heuristic objective function is used for each optimization step. Also, the obtained 3D reconstruction highly depends on the initial guess of the 3D structure and the helical parameters. Herein, we propose a method for estimating the 3D structure and helical parameters that also performs an iterative optimization; however, the objective function for each step is derived from a single objective function to make the algorithm convergent and less sensitive to the initial guess. Finally, we evaluated the effectiveness of the proposed method by testing it on cryo-EM images, which were challenging to reconstruct using conventional methods.

Regulation of nitrate assimilation in cyanobacteria.

Nitrate assimilation by cyanobacteria is inhibited by the presence of ammonium in the growth medium. Both nitrate uptake and transcription of the nitrate assimilatory genes are regulated. The major intracellular signal for the regulation is, however, not ammonium or glutamine, but 2-oxoglutarate (2-OG), whose concentration changes according to the change in cellular C/N balance. When nitrogen is limiting growth, accumulation of 2-OG activates the transcription factor NtcA to induce transcription of the nitrate assimilation genes. Ammonium inhibits transcription by quickly depleting the 2-OG pool through its metabolism via the glutamine synthetase/glutamate synthase cycle. The P(II) protein inhibits the ABC-type nitrate transporter, and also nitrate reductase in some strains, by an unknown mechanism(s) when the cellular 2-OG level is low. Upon nitrogen limitation, 2-OG binds to P(II) to prevent the protein from inhibiting nitrate assimilation. A pathway-specific transcriptional regulator NtcB activates the nitrate assimilation genes in response to nitrite, either added to the medium or generated intracellularly by nitrate reduction. It plays an important role in selective activation of the nitrate assimilation pathway during growth under a limited supply of nitrate. P(II) was recently shown to regulate the activity of NtcA negatively by binding to PipX, a small coactivator protein of NtcA. On the basis of accumulating genome information from a variety of cyanobacteria and the molecular genetic data obtained from the representative strains, common features and group- or species-specific characteristics of the response of cyanobacteria to nitrogen is summarized and discussed in terms of ecophysiological significance.

In vitro activity of azithromycin against Mycoplasma genitalium and its efficacy in the treatment of male Mycoplasma genitalium-positive nongonococcal urethritis.

Many recent studies have shown that Mycoplasma genitalium is among the pathogens responsible for Chlamydia trachomatis-negative nongonococcal urethritis (NGU). A single 1-g dose of azithromycin (AZM) has been recommended for the treatment of NGU, including M. genitalium-positive NGU, irrespective of whether it is positive or negative for Chlamydia trachomatis. The purpose of this study was to determine the minimal inhibitory concentrations of AZM against Mycoplasma genitalium strains, and to assess its clinical efficacy against Mycoplasma genitalium-positive NGU. Seven Mycoplasma genitalium strains were obtained from the American Type Culture Collection, and susceptibility testing of seven antimicrobial agents was performed using a broth microdilution method. Thirty men with M. genitalium-positive NGU were enrolled in this study and treated with a single 1-g dose of AZM. AZM and clarithromycin (CAM) were highly active against M. genitalium strains. Fluoroquinolone activities were moderate, and of the three fluoroquinolones tested, gatifloxacin (GFLX) and sparfloxacin (SPFX) were more active than levofloxacin (LVFX). In 25 of 30 (83.3%) men treated with a single 1-g dose of AZM, M. genitalium was eradicated from first-void urine samples, as determined by polymerase chain reaction. AZM was highly active against M. genitalium, and a single 1-g dose of AZM for M. genitalium-positive NGU was tolerated in Japan. These findings may be helpful in establishing optimal treatment for M. genitalium-positive NGU.

Emergence and spread of drug resistant Neisseria gonorrhoeae.

PURPOSE: The emergence and spread of Neisseria gonorrhoeae with resistance to oral antibiotics have led to difficulty in treating gonorrhea. We review drug resistance in N. gonorrhoeae with a particular emphasis on resistance to fluoroquinolones, cefixime and azithromycin. MATERIALS AND METHODS: Literature selected from peer reviewed journals listed in MEDLINE(R)/PubMed(R) from 1943 to 2009 and from resources cited in those articles was reviewed comprehensively. RESULTS: Due to the spread of fluoroquinolone resistant N. gonorrhoeae fluoroquinolones are no longer recommended for the treatment of gonorrhea. The emergence of N. gonorrhoeae with a mosaic penicillin-binding protein 2 associated with oral cephalosporin resistance has threatened cefixime treatment for gonorrhea. Emergence of N. gonorrhoeae with high level resistance to azithromycin has also been documented. However, injectable antibiotics (sepctinomycin and ceftriaxone) retain their activity against N. gonorrhoeae. To monitor drug resistance in N. gonorrhoeae several national and international programs have become functional. CONCLUSIONS: Oral regimens for the treatment of gonorrhea are limited. At present to our knowledge ceftriaxone is the most reliable and available agent for the treatment of gonorrhea. To prevent the further emergence and international spread of drug resistance, and allow for the selection of appropriate treatments, a comprehensive global program is needed including surveillance for drug resistance in N. gonorrhoeae and collection of patient epidemiological data. Clinicians should effectively treat patients with gonorrhea, always being conscious of local trends of drug resistance in N. gonorrhoeae, and should perform culture and antimicrobial susceptibility testing in those with persistent gonorrhea after treatment.

Nitrite transport activity of the ABC-type cyanate transporter of the cyanobacterium Synechococcus elongatus.

In addition to the ATP-binding cassette (ABC)-type nitrate/nitrite-bispecific transporter, which has a high affinity for both substrates (K(m), approximately 1 microM), Synechococcus elongatus has an active nitrite transport system with an apparent K(m) (NO(2)(-)) value of 20 microM. We found that this activity depends on the cynABD genes, which encode a putative cyanate (NCO(-)) ABC-type transporter. Accordingly, nitrite transport by CynABD was competitively inhibited by NCO(-) with a K(i) value of 0.025 microM. The transporter was induced under conditions of nitrogen deficiency, and the induced cells showed a V(max) value of 11 to 13 micromol/mg of chlorophyll per h for cyanate or nitrite, which could supply approximately 30% of the amount of nitrogen required for optimum growth. Its relative specificity for the substrates and regulation at transcriptional and posttranslational levels suggested that the physiological role of the bispecific cyanate/nitrite transporter in S. elongatus is to allow nitrogen-deficient cells to assimilate low concentrations of cyanate in the medium. Its contribution to nitrite assimilation was significant in a mutant lacking the ABC-type nitrate/nitrite transporter, suggesting a possible role for CynABD in nitrite assimilation by cyanobacterial species that lack another high-affinity mechanism(s) for nitrite transport.

Mechanism of low CO2-induced activation of the cmp bicarbonate transporter operon by a LysR family protein in the cyanobacterium Synechococcus elongatus strain PCC 7942.

The cmp operon of the cyanobacterium Synechococcus elongatus strain PCC 7942, encoding the subunits of the ABC-type bicarbonate transporter, is activated under CO2-limited growth conditions in a manner dependent on CmpR, a LysR family transcription factor of CbbR subfamily. The 0.7 kb long regulatory region of the operon carried a single promoter, which responded to CO2 limitation. Using the luxAB reporter system, three cis-acting elements involved in the low-CO2 activation of transcription, each consisting of a pair of LysR recognition signatures overlapping at their ends, were identified in the regulatory region. CmpR was shown to bind to the regulatory region, yielding several DNA-protein complexes in gel shift assays. Addition of ribulose-1,5-bisphosphate (> 1 mM) or 2-phosphoglycolate (> 10 microM) enhanced the binding of CmpR in a concentration-dependent manner, promoting formation of large DNA-protein complexes. Given the involvement of O2 in adaptive responses of cyanobacteria to low-CO2 conditions, our results suggest that 2-phosphoglycolate, which is produced by oxygenation by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of ribulose-1,5-bisphosphate under CO2-limited conditions, acts as the co-inducer in the activation of the cmp operon by CmpR.

Azithromycin treatment for nongonococcal urethritis negative for Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum.

Some patients with nongonococcal urethritis (NGU) are negative for Chlamydia trachomatis, mycoplasmas, and ureaplasmas. The optimal antimicrobial chemotherapy for such NGU has not fully been clarified. We assessed the efficacy of azithromycin for treatment of nonmycoplasmal, nonureaplasmal, nonchlamydial NGU (NMNUNCNGU). Thirty-eight men whose first-pass urine was negative for Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum were treated with a single dose of 1 g azithromycin. Urethritis symptoms and polymorphonuclear leukocytes in urethral smears or in first-pass urine were assessed before and after treatment with azithromycin. Thirty-two (84.2%) of the 38 men with NMNUNCNGU showed no signs of urethral inflammation after treatment. The efficacy of this azithromycin regimen was comparable to that of the 7-day regimen of levofloxacin, gatifloxacin, minocycline, or clarithromycin reported previously. A single dose of 1 g azithromycin, which is effective not only for NGU due to specific pathogens but also for NMNUNCNGU, is an appropriate treatment for NGU.

[Non-chlamydial non-gonococcal urethritis].

Besides Chlamydia trachomatis, various microorganisms could cause non-gonococcal urethritis (NGU). Recently, Mycoplasma genitalium and Ureaplasma urealyticum (biovar 2) have been suggested to be other pathogens of NGU independent of C. trachomatis. Clinical findings of non-chlamydial NGU, including M. genitalium--or U. urealyticum-postive NGU, are not different from those of chlamydial NGU. M. genitalium and U. urealyticum (biovar 2) are susceptible to tetracyclines, macrolides, and fluoroquinolones. However, the post-treatment presence of M. genitalium in the urethra is significantly associated with persistent or recurrent urethritis. Eradication of this mycoplasma from the urethra is essential for managing M. genitalium-positive NGU. In treatment of non-chlamydial NGU, therefore, the antimicrobial agents that are active against M. genitalium should be chosen.

Bayesian inference for three-dimensional helical reconstruction using a soft-body model.

Estimation of the three-dimensional (3D) structure of a protein using cryo transmission electron microscopy (cryo-TEM) is an inverse problem, which aims to estimate the parameters of a specific physical process from observations. In general, we need to model the observation process to estimate a structure. However, the inconsistency between the model and a real observation process decreases the estimation accuracy. In cryo-TEM, the flexibility of a soft protein, including the bending of a helix, can lead to inconsistencies between the observations because of the assumption that there is a consistent 3D structure behind each observed image. In this paper, we propose a 3D reconstruction algorithm for helical structures using a parametric soft-body model that can represent continuous deformation. We performed an approximate Bayesian inference for unobservable (hidden) variables, such as the deformation parameters, projection angle, and two-dimensional origin offset (shift) of each protein in the 3D structure estimation problem. Our principled approach is not only beneficial to deal with the uncertainties in the estimation, but also beneficial to make the optimization algorithm convergent and efficient. Reconstructions with artificial molecules validated the advantage of the proposed method, particularly, when deformed helices were imaged under a low signal-to-noise ratio condition. Moreover, we confirmed that the proposed method successfully reconstructed a 3D structure from cryo-TEM images of the tobacco mosaic virus.

Virtual Adversarial Training: A Regularization Method for Supervised and Semi-Supervised Learning.

We propose a new regularization method based on virtual adversarial loss: a new measure of local smoothness of the conditional label distribution given input. Virtual adversarial loss is defined as the robustness of the conditional label distribution around each input data point against local perturbation. Unlike adversarial training, our method defines the adversarial direction without label information and is hence applicable to semi-supervised learning. Because the directions in which we smooth the model are only "virtually" adversarial, we call our method virtual adversarial training (VAT). The computational cost of VAT is relatively low. For neural networks, the approximated gradient of virtual adversarial loss can be computed with no more than two pairs of forward- and back-propagations. In our experiments, we applied VAT to supervised and semi-supervised learning tasks on multiple benchmark datasets. With a simple enhancement of the algorithm based on the entropy minimization principle, our VAT achieves state-of-the-art performance for semi-supervised learning tasks on SVHN and CIFAR-10.