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The localization of essential oils, including flavor components, in perilla herb (Perilla frutescens var. crispa) were visually determined using matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry (MS) imaging. The surface of a perilla leaf was peeled using a cyanoacrylate adhesion compound and contained oil glands that retained their morphology and chemical properties. We imaged the three essential oils perillaldehyde, β‐caryophyllene, and rosmarinic acid (RA). Perillaldehyde was derivatized using glycine to prevent evaporation and allow its detection and imaging while localized in oil glands. β‐caryophyllene also localized in the oil glands and not in the epidermis region. RA was detected throughout the leaf, including the oil glands. Quantitative data for the three essential oils were obtained by gas chromatography‐ or liquid chromatography‐MS. The concentrations of perillaldehyde, β‐caryophyllene, and RA were 12.6 ± 0.62, 0.27 ± 0.02, and 0.16 ± 0.02 [mg/g] in the paste sample of perilla herb. Peeling using a cyanoacrylate adhesion compound, and derivatization of a target such as an aroma component have great potential for mass spectrometry imaging for multiple essential oils., Perilla herb is well known as food in the whole world. This article visually revealed the localization of fragrance compounds using derivatization imaging MS by avoidance of volatilization.

Organisms orchestrate cellular functions through transcription factor (TF) interactions with their target genes, although these regulatory relationships are largely unknown in most species. Here we report a high-throughput approach for characterizing TF-target gene interactions across species and its application to 354 TFs across 48 bacteria, generating 17,000 genome-wide binding maps. This dataset revealed themes of ancient conservation and rapid evolution of regulatory modules. We observed rewiring, where the TF sensing and regulatory role is maintained while the arrangement and identity of target genes diverges, in some cases encoding entirely new functions. We further integrated phenotypic information to define new functional regulatory modules and pathways. Finally, we identified 242 new TF DNA binding motifs, including a 70% increase of known Escherichia coli motifs and the first annotation in Pseudomonas simiae, revealing deep conservation in bacterial promoter architecture. Our method provides a versatile tool for functional characterization of genetic pathways in prokaryotes and eukaryotes.

Microbial biosynthetic gene clusters (BGCs) encoding secondary metabolites are thought to impact a plethora of biologically mediated environmental processes, yet their discovery and functional characterization in natural microbiomes remains challenging. Here we describe deep long-read sequencing and assembly of metagenomes from biological soil crusts, a group of soil communities that are rich in BGCs. Taking advantage of the unusually long assemblies produced by this approach, we recovered nearly 3,000 BGCs for analysis, including 712 full-length BGCs. Functional exploration through metatranscriptome analysis of a 3-day wetting experiment uncovered phylum-specific BGC expression upon activation from dormancy, elucidating distinct roles and complex phylogenetic and temporal dynamics in wetting processes. For example, a pronounced increase in BGC transcription occurs at night primarily in cyanobacteria, implicating BGCs in nutrient scavenging roles and niche competition. Taken together, our results demonstrate that long-read metagenomic sequencing combined with metatranscriptomic analysis provides a direct view into the functional dynamics of BGCs in environmental processes and suggests a central role of secondary metabolites in maintaining phylogenetically conserved niches within biocrusts., Marc Van Goethem et al. combine short- and long-read sequencing from biocrust samples to report nearly 3,000 biosynthetic gene clusters (BGCs) encoding microbial secondary metabolites. Their results demonstrate the advantage of integrated metatranscriptomic and long-read metagenomic sequencing in analyzing BGCs and provides insight into the role of secondary metabolites in maintaining phylogenetic niches in biocrusts.

BACKGROUND AND AIMS: This study aimed to investigate whether N-benzyl-N-methyl-2-[7,8-dihydro-7-(2-[18F]fluoroethyl)-8-oxo-2-phenyl-9H-purin-9-yl]acetamide (18F-FEDAC), a probe for translocator protein (TSPO), can visualize atherosclerotic lesions in rabbits and whether TSPO is localized in human coronary plaques. METHODS: 18F-FEDAC-PET of a rabbit model of atherosclerosis induced by a 0.5% cholesterol diet and balloon injury of the left carotid artery (n = 7) was performed eight weeks after the injury. The autoradiography intensity of 18F-FEDAC in carotid artery tissue sections was measured, and TSPO expression was evaluated immunohistochemically. TSPO expression was examined in human coronary arteries obtained from autopsy cases (n = 16), and in human coronary plaques (n = 12) aspirated from patients with acute myocardial infarction (AMI). RESULTS: 18F-FEDAC-PET visualized the atherosclerotic lesions in rabbits as high-uptake areas, and the standard uptake value was higher in injured arteries (0.574 ± 0.24) than in uninjured arteries (0.277 ± 0.13, p

We studied the time-course of stem response for six months following complete girdling in branches of Aquilaria sinensis to determine the potential role of interxylary phloem (IP) in this response. It was found that the vascular cambium, as well as its derivative secondary xylem and phloem, regenerated fully through redifferentiation of IP. We confirmed that vascular cambium regenerated within one month after girdling based on observation of new vessels, IP, and secondary phloem fibers. The time-course study showed that IPs made connections with each other, merged, and became larger through the proliferation of IPs parenchyma cells and the cleaving of secondary xylem in a narrow zone 400 to 1000 μm deep inside the girdled edge. This led to the formation of a complete circular sheath of vascular cambium, followed by the regeneration of vascular tissue. It is worth noting that the secondary xylem is regenerated always following the formation of a thick belt of wound xylem.

Background DYT-TOR1A is caused by a GAG deletion in the TOR1A gene. While it usually manifests as early-onset dystonia, its phenotype is extremely diverse, even within one family. Recent reports have revealed that some DYT-TOR1A cases have novel mutations in the TOR1A gene while others have mutations in both TOR1A and another DYT gene (THAP1 or SGCE). Our understanding of the correlation between genotype and phenotype is becoming increasingly complicated. Case presentations: Here, we report on monozygotic twins who developed dystonia in childhood. The two children had different presentations in terms of onset age and dominant disturbances, but both exhibited marked diurnal fluctuation and jerking movements of the limbs as well as levodopa/levodopa-carbidopa responsiveness. These features are commonly associated with DYT/PARK-GCH1 and DYT-SGCE, yet these twins had no mutations in the GCH1 or SGCE genes. Whole exome sequencing eventually revealed a single GAG deletion in the TOR1A gene. Conclusion Monozygotic twins whose only mutation was a GAG deletion in TOR1A exhibited DYT/PARK-GCH1-asssociated features and jerking movements reminiscent of myoclonus. This finding may expand the spectrum of phenotypes associated with DYT-TOR1A, and suggests that levodopa has potential as a treatment for DYT-TOR1A with DYT/PARK-GCH1-associated features.

Objectives Systemic inflammatory response is strongly associated with poor oncological outcome in colorectal cancer (CRC). Perioperative inflammation caused by surgical stress can lead to the development of postoperative infectious complications (PIC) as well as cancer-related inflammation. We aimed to evaluate the prognostic potential of perioperative systemic inflammation by calculating the time-dependent cumulative C-reactive protein (CRP) levels during the perioperative period. Methods We analyzed clinicopathological data from 540 patients with CRC who underwent potentially curative surgery at our institution. The time-dependent aggregated CRP level was denoted "cumulative CRP," which represents the area under the line of time (days) and the CRP levels preoperatively and on postoperative days 1, 3, and 7. Results Cumulative CRP was significantly higher in patients with CRC undergoing open surgery than in patients undergoing laparoscopic surgery. In multivariate analysis, high cumulative CRP was an independent prognostic factor for disease-free survival (DFS) and overall survival (OS) in both the laparoscopic and open surgery groups. Patients with CRC and high cumulative CRP had significantly poorer DFS and OS than those with low cumulative CRP, including those patients without PIC. Conclusions Cumulative CRP is an independent predictive marker of OS and DFS in patients with CRC who undergo curative surgery.

Polymer micromaterials in a liquid or gel phase covered with a surfactant membrane are widely used materials in pharmaceuticals, cosmetics, and foods. In particular, cell-sized micromaterials of biopolymer solutions covered with a lipid membrane have been studied as artificial cells to understand cells from a physicochemical perspective. The characteristics and phase transitions of polymers confined to a microscopic space often differ from those in bulk systems. The effect that causes this difference is referred to as the cell-size space effect (CSE), but the specific physicochemical factors remain unclear. This study introduces the analysis of CSE on molecular diffusion, nanostructure transition, and phase separation and presents their main factors, i.e., short- and long-range interactions with the membrane surface and small volume (finite element nature). This serves as a guide for determining the dominant factors of CSE. Furthermore, we also introduce other factors of CSE such as spatial closure and the relationships among space size, the characteristic length of periodicity, the structure size, and many others produced by biomolecular assemblies through the analysis of protein reaction-diffusion systems and biochemical reactions.

The spread of the COVID-19 pandemic around the world has revealed that it is urgently important to develop rapid and inexpensive assays for antibodies in general and anti-SARS-CoV-2 IgG antibody (anti-SARS-CoV-2 spike glycoprotein S1 antibody) in particular. Herein we report a method to detect the anti-SARS-CoV-2 spike antibody level by using Janus emulsions or Janus particles as biosensors. Janus emulsions are composed of two immiscible hydrocarbon and fluorocarbon oils. The hydrocarbon/water interfaces are functionalized with a secondary antibody of IgG protein and SARS-CoV-2 spike receptor binding domain (RBD), to produce two different Janus emulsions. Mixtures of these Janus droplets enable the detection of the anti-SARS-CoV-2 spike IgG antibody in an agglutination assay caused by the antibody’s binding to both the secondary antibody of IgG antibody and SARS-CoV-2 spike protein RBD. Both qualitative optical images and quantitative fluorescence spectra are able to detect the level of anti-SARS-CoV-2 spike antibody at concentrations as low as 0.2 μg/mL in 2 h. The detection results of clinical human serum samples using this agglutination assay confirm that this method is applicable to clinical samples with good sensitivity and specificity. The reported method is generalizable and can be used to detect other analytes by attaching different biomolecular recognition elements to the surface of the Janus droplets., An efficient, sensitive and less expensive method of using Janus emulsions or Janus particles for the detection of the anti-SARS-CoV-2 spike IgG antibody in human serum is described.

The human enzyme As(III) S-adenosylmethionine methyltransferase (AS3MT) catalyzes arsenic biotransformations and is considered to contribute to arsenic-related diseases. AS3MT is expressed in various tissues and cell types including liver, brain, adrenal gland, and peripheral blood mononuclear cells but not in human keratinocytes, urothelial, or brain microvascular endothelial cells. This indicates that AS3MT expression is regulated in a tissue/cell type-specific manner, but the mechanism of transcriptional regulation of expression of the AS3MT gene is not known. In this study, we define the DNA sequence of the core promoter region of the human AS3MT gene. We identify a GC box in the promoter to which the stress-related transcription factor Sp1 binds, indicating involvement of regulatory elements in AS3MT gene expression.

Apples Malus domestica, known as a rich source of triterpene acids, induced more variety and quantity of triterpene acids in response to herbivory or mechanical damage. There were 3 major induced compounds: pomaceic acid and euscaphic acid, both of which are known apple triterpene acids, and 2α,19α-dihydroxy-3-oxours-12-en-28-oic acid (named eriobotoric acid), which was first identified in apples. In this study, the 3 compounds’ induction curves after damage, varietal differences in induction amounts, and physiological roles against pest insects were further investigated. Eriobotoric acid showed clear antifeedant activity against lepidopteran insect Spodoptera litura but not against apple pests.

The method for evaluating soybean (Glycine max) antixenosis against the common cutworm (Spodoptera litura) was developed based on a dual-choice assay aided by a statistical analysis model. This model was constructed from the results of a dual-choice assay in which Enrei, a soybean cultivar susceptible to S. litura, was used as both a standard and a test leaf disc for 2nd-5th instar larvae. The statistical criterion created by this model enabled the evaluation of the presence of antixenosis. This method was applied to four soybean varieties, including Tamahomare (susceptible), Himeshirazu (resistant), IAC100 (resistant), and Peking (unknown), as well as Enrei. Subsequently, the degrees of antixenosis were also compared by F-test, followed by maximum likelihood estimation (MLE). According to the results, the antixenosis of Tamahomare, Himeshirazu, and IAC100 was statistically reevaluated and Peking exhibited a novel antixenosis, which was stronger for 3rd-5th instar larvae than for 2nd instar.

BACKGROUND Neutrophils play a major role in ischemia/reperfusion injury (IRI) in renal transplantation and acute kidney injury. However, it has been difficult to observe changes in neutrophil dynamics over time in living mice kidney. We investigate neutrophil dynamics in IRI in living mice using novel in vivo multiphoton microscope imaging techniques and characterize the renoprotective effects of a selective phosphodiesterase (PDE) 5 inhibitor, tadalafil. METHODS Wild-type (WT) and eNOS knockout (eNOS-KO) mice, a model of endothelial dysfunction, were used to establish in vivo real-time imaging in living mouse kidneys. Neutrophils were labeled green with Ly-6G monoclonal antibody, and plasma flow was labeled red with bovine serum albumin. Tadalafil was administered orally 1 h before surgery. Both kidney pedicles were reperfused after 37° warm ischemia for 45 min. RESULTS Our novel approach revealed that neutrophils were trapped in glomerulus within a few minutes after reperfusion. They gradually increased over time and Infiltrated neutrophils were observed in the tubular lumen and peritubular capillary. The neutrophils were clearly visualized rolling on peritubular capillary plexus at 3 μm/min. The administration of tadalafil significantly reduced neutrophil influx into the glomerulus in both WT and eNOS-KO mice. Reduced neutrophil infiltration in tadalafil groups, which was confirmed by flow cytometry, resulted in histopathologically decreased tubular injury. The expression of VCAM-1 and KIM-1 was partially prevented by tadalafil. CONCLUSIONS Use of a novel technique contributed to elucidation of neutrophil dynamics after reperfusion. Tadalafil has a potential for inhibiting neutrophil infiltration in renal IRI.Supplemental Visual Abstract; http://links.lww.com/TP/C223.

It is difficult to diagnose immunoglobulin heavy chain amyloidosis (AH amyloidosis) without proteomic analysis due to no useful diagnostic antibodies. The aim of this study was to develop diagnostic antibodies available to immunohistochemistry and immunoblotting. Two rabbit anti-heavy chain variable region antibodies were generated and evaluated in immunohistochemical studies performed on 11 AH amyloidosis patients and 64 patients with other systemic amyloidoses. Additionally, immunoblotting was performed using extracted amyloid protein from one patient and serum samples from two patients with AH amyloidosis. Immunohistochemical analysis generated a positive outcome in 10 of 11 AH amyloidosis patients (sensitivity 90.9%). While positive staining was also observed in 9 of 64 non-AH amyloidosis patients (specificity 85.9%), substitution of the blocking agent reversed the positive reactivity in 5 of 9 patients. Amyloid protein band was clearly detected via immunoblotting analysis, and protein bands with similar molecular weights of amyloid protein were observed in serum samples from patients with AH amyloidosis. The two antibodies may represent a powerful diagnostic tool for AH amyloidosis. In addition, our data revealed the existence of amyloidogenic variable region fragments in the serum of patients, suggesting their potential as diagnostic markers for AH amyloidosis.

Francisella halioticida, the causative agent of francisellosis of the giant abalone Haliotis gigantea, has also been isolated from Yesso scallops Mizuhopecten yessoensis, which presented with orange/pinkish lesions in the adductor muscle and experienced high mortality. However, it is not clear whether the F. halioticida isolated from the giant abalone and Yesso scallops are phenotypically and genetically identical to each other. The present study revealed that isolates from the giant abalone and Yesso scallops were phenotypically different, with slower growth in modified eugon broth and a lack of prolyl aminopeptidase and phenylalanine aminopeptidase in Yesso scallop isolates. Additionally, we found that 3 of 8 housekeeping genes were different between them. Based on these phenotypic and genetic differences, we propose that F. halioticida isolated from Yesso scallops in Japan be designated as the ‘J-scallop type’ to distinguish it from strains from abalone (‘abalone type’). Whole-genome sequencing analysis of a strain belonging to the J-scallop type showed that the overall similarity between the J-scallop and abalone type strains was estimated to be 99.84%. In accordance with a lack of prolyl aminopeptidase activity, in general, all of the J-scallop type strains examined have a 1 bp deletion in the responsible gene encoding prolyl aminopeptidase. This deletion was confirmed in all F. halioticida in diseased Yesso scallops examined, suggesting that in Japan, francisellosis of Yesso scallops is caused by a novel type of F. halioticida and not by the abalone type.

Signal transducer and activator of transcription 3 (STAT3) plays a critical role in regulating cell growth, survival, and metastasis. STAT3 signaling is constitutively activated in various types of hematologic or solid malignancies. YHO-1701 has been developed as an orally available STAT3 inhibitor. Herein, YHO-1701 in combination with molecular-targeted agents was evaluated. Additive or synergistic effects were observed in a broad spectrum of “combination treatment + cell line” pairs. Of particular interest was the synergistic effect observed when YHO-1701 was combined with imatinib or dasatinib [breakpoint cluster region-abelson (BCR-ABL) inhibitors], osimertinib [epidermal growth factor receptor (EGFR) inhibitor], crizotinib, alectinib, or ceritinib [anaplastic lymphoma kinase (ALK) inhibitors]. The results further showed a close relationship between these synergistic effects and the cellular levels of the key molecules involved in the target pathways for YHO-1701 and each combination drug. The combination of YHO-1701 with alectinib resulted in a significantly greater antitumor activity without exhibiting body weight loss in an NCI-H2228 [echinoderm microtubule–associated protein-like 4 (EML4)-ALK fusion] xenograft mouse model. Our results strongly suggest that the logical strategy in combination with the novel STAT3 inhibitor YHO-1701 and other mechanistically different targeted agents could be a promising approach in the clinical setting.

Hereditary systemic amyloidosis aside from transthyretin-related familial amyloid polyneuropathy is quite uncommon in Japan. We herein report a sporadic case of hereditary apolipoprotein A-I (apoAI) amyloidosis. The patient was a 43-year-old Japanese man who exhibited marked hepatomegaly with spleno-testicular enlargement. While he was initially thought to have primary AL amyloidosis, a proteomics analysis revealed that the amyloid was composed of variant apoAI with an E34K variant. To date, only one patient with apoAI amyloidosis has been reported in Japan. However, our study suggests that more patients may be present in Japan, and the majority may have been diagnosed with other types of amyloidosis due to its clinical similarity.

The risk of fatal arrhythmias is the major concern for using chloroquine (CQ) or hydroxychloroquine (HCQ) to treat coronavirus disease 2019 (COVID‐19), but the reported number of life‐threatening arrhythmic events or deaths is relatively small. The objective of this study was to assess the arrhythmogenic risk of these two drugs using a multiscale heart simulation, which allows testing even at high concentrations, including those that cause fatal arrhythmias. We measured the inhibitory action of CQ, HCQ, and HCQ with 30 μM azithromycin (AZ) on six ion currents (fast [INa] and late [INa,L] components of the sodium current, L‐type calcium current [ICa,L], rapid [IKr/hERG], and slow [IKs] components of delayed rectifier potassium, and inward rectifier potassium [IK1]) over a wide range of concentrations using the automated patch‐clamp system. Using the concentration–inhibition relationship that was thus obtained, we simulated the drug effects while increasing the concentration until the life‐threatening arrhythmia, torsade de pointes (TdP), was observed. The obtained threshold concentrations for TdP were 12.5, 35, and 22.5 μM for CQ, HCQ, and HCQ with AZ, respectively. Adding therapeutic concentrations of mexiletine or verapamil successfully prevented the occurrence of TdP, and verapamil was more effective. CQ, HCQ, and HCQ with AZ thresholds for TdP were larger than both antiviral concentrations that were reported by in vitro experiments and free plasma concentrations that were attained by the clinically used dosage. The current simulation data provided a safety margin to the currently used clinical dose for CQ and HCQ/AZ. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? Despite the potent in vitro antiviral effect, clinical trials have failed to show the therapeutic effects of chloroquine (CQ) and hydroxychloroquine (HCQ)/azithromycin (AZ) to treat coronavirus disease 2019. Torsadogenic potentials may limit the dosage of these drugs, but the reported incidence of fatal arrhythmias is rare. WHAT QUESTION DID THIS STUDY ADDRESS? Our objective was to assess the arrhythmogenicity of CQ and HCQ/AZ over a wide range of drug concentrations using a multiscale heart simulation. WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? Our study showed that CQ and HCQ/AZ do not induce fatal arrhythmias even at concentrations much higher than in vitro antiviral half‐maximal effective concentration (EC50) values at which QT prolongation exceeds 150 ms. We also found that estimated free plasma concentrations of CQ and HCQ/AZ achieved by currently used dosing protocols are lower than the antiviral EC50 for these drugs. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? Our simulation data provided a safety margin to the currently used clinical dose for CQ and HCQ/AZ.

The clinical significance of the platelet count × C-reactive protein level multiplier (P-CRP) in patients with locally advanced rectal cancer (LARC) undergoing neoadjuvant chemoradiotherapy followed by curative surgery has not been fully evaluated. In this retrospective study, the correlation between the P-CRP and prognosis was evaluated in 135 patients with LARC. We also performed a subgroup analysis limited to patients with pathological TNM stage III [ypN(+)] LARC. The cut-off value of the P-CRP for prognosis was set at 4.11. The high and low P-CRP groups comprised 39 (28.89%) and 96 (71.11%) patients, respectively. Among the investigated clinicopathological factors, the serum carcinoembryonic antigen level and presence of recurrence were significantly associated with the P-CRP value. In the Kaplan–Meier analysis, both overall survival (OS) and disease-free survival (DFS) were shorter in the high P-CRP group (p

During a survey of myxosporean parasites of marine fish in the coastal region of Vietnam, a species of the genus Henneguya (Myxosporea: Bivalvulida) was found in the gill of yellowfin seabream Acanthopagrus latus (Perciformes: Sparidae). White and oval cysts, measuring 145-220 μm in diameter, were detected in the gill lamellae of 4 of 15 fish examined (26.7%). Mature myxospores were elongate, with smooth valves, two similar polar capsules, and having the following dimensions: spore body length 9.9 ± 0.5 (8.9-12.5) μm, body width 6.7 ± 0.3 (6.1-7.6) μm, thickness 5.1 ± 0.2 (4.8-5.4) μm, caudal appendage length 10.0 ± 1 (8.3-11.6) μm, and total myxospore length 19.3 ± 1.4 (16.5-21.5) μm. The polar capsules were ovoid, measuring 3.2 ± 0.2 (2.8-3.9) μm long and 1.9 ± 0.2 (1.5-2.3) μm wide. Each polar capsule has a polar filament with 4-5 coils. Histological analysis revealed plasmodia in the connective tissues of the gill lamellae, but inflammation and deformation of the gills were not observed. In the phylogenetic tree reconstructed from the small subunit ribosomal DNA (SSU rDNA), sequences of the Henneguya specimens found in this study form a distinct branch. Morphological characteristics and molecular data identified a new species, namely Henneguya lata n. sp.

The soil bacterium Burkholderia gladioli GSRB05 produces the natural compound arsinothricin [2-amino-4-(hydroxymethylarsinoyl) butanoate] (AST), which has been demonstrated to be a broad-spectrum antibiotic. To identify the genes responsible for AST biosynthesis, a draft genome sequence of B. gladioli GSRB05 was constructed. Three genes, arsQML, in an arsenic resistance operon were found to be a biosynthetic gene cluster responsible for synthesis of AST and its precursor, hydroxyarsinothricin [2-amino-4-(dihydroxyarsinoyl) butanoate] (AST-OH). The arsL gene product is a noncanonical radical S-adenosylmethionine (SAM) enzyme that is predicted to transfer the 3-amino-3-carboxypropyl (ACP) group from SAM to the arsenic atom in inorganic arsenite, forming AST-OH, which is methylated by the arsM gene product, a SAM methyltransferase, to produce AST. Finally, the arsQ gene product is an efflux permease that extrudes AST from the cells, a common final step in antibiotic-producing bacteria. Elucidation of the biosynthetic gene cluster for this novel arsenic-containing antibiotic adds an important new tool for continuation of the antibiotic era. IMPORTANCE Antimicrobial resistance is an emerging global public health crisis, calling for urgent development of novel potent antibiotics. We propose that arsinothricin and related arsenic-containing compounds may be the progenitors of a new class of antibiotics to extend our antibiotic era. Here, we report identification of the biosynthetic gene cluster for arsinothricin and demonstrate that only three genes, two of which are novel, are required for the biosynthesis and transport of arsinothricin, in contrast to the phosphonate counterpart, phosphinothricin, which requires over 20 genes. Our discoveries will provide insight for the development of more effective organoarsenical antibiotics and illustrate the previously unknown complexity of the arsenic biogeochemical cycle, as well as bring new perspective to environmental arsenic biochemistry.

Cetaceans, including dolphins, serve as definitive hosts of zoonotic anisakid nematodes, which are important etiological agents for human anisakiasis and allergy-associated health risks. With limited knowledge of these zoonotic parasites from the marine environment in the Philippine waters, the stranding of a Fraser’s dolphin (Lagenodelphis hosei Fraser, 1956) off the central Philippines made it possible to identify the worm species isolated from its gut. Parasitological examinations were carried out using morphological and molecular tools. Morphologically, the SEM and LM data revealed that the specimens belong to the genus Anisakis of the Type 1 group. Molecularly, PCR-RFLP results of the ITS region generated only a single fragment pattern on all worm samples corresponding to the reported molecular keys for A. typica. Further sequence and phylogenetic analyses of both ITS rDNA and mtDNA COX2 genes confirmed the anisakid nematodes’ identity as A. typica. The molecular data obtained in this study support previous findings on the possible existence of local variants of A. typica in this region.

Toads of the genus Bufo synthesize and accumulate bufadienolides (BDs) in their parotoid glands. BDs are cardiotonic steroids that play an important role in defense against the toads' predators. Three bufonid taxa occur in mainland Japan, Bufo japonicus formosus, B. j. japonicus, and B. torrenticola. The chemical structures of BDs isolated from B. j. formosus were studied several decades ago, but there is no further information on the toxic components of Japanese toads and their metabolism. In this study, we analyzed BDs of toads from throughout Japan and compared the BD profiles by liquid chromatography/mass spectrometry (LC/MS) and hierarchical cluster analysis (HCA). We observed BDs in three taxa of Japanese toads, and identified five of the most common BDs by nuclear magnetic resonance (NMR) analyses. Of the five BDs, only bufalin was detected in all individuals. HCA of individual BD profiles divided the three taxa into five primary clusters and several subclusters. This result indicates that BD profiles differ both among and within the taxa. The clustering pattern of BDs is generally concordant with a phylogenetic tree reconstructed from the mitochondrial cytochrome b gene of Japanese toads. Our results suggest that the BDs of Japanese toads have diversified not in response to specific selective pressures, but simply due to population structuring over evolutionary time.

Defining an appropriate and efficient assessment of drug‐induced corrected QT interval (QTc) prolongation (a surrogate marker of torsades de pointes arrhythmia) remains a concern of drug developers and regulators worldwide. In use for over 15 years, the nonclinical International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) S7B and clinical ICH E14 guidances describe three core assays (S7B: in vitro hERG current & in vivo QTc studies; E14: thorough QT study) that are used to assess the potential of drugs to cause delayed ventricular repolarization. Incorporating these assays during nonclinical or human testing of novel compounds has led to a low prevalence of QTc‐prolonging drugs in clinical trials and no new drugs having been removed from the marketplace due to unexpected QTc prolongation. Despite this success, nonclinical evaluations of delayed repolarization still minimally influence ICH E14‐based strategies for assessing clinical QTc prolongation and defining proarrhythmic risk. In particular, the value of ICH S7B‐based “double‐negative” nonclinical findings (low risk for hERG block and in vivo QTc prolongation at relevant clinical exposures) is underappreciated. These nonclinical data have additional value in assessing the risk of clinical QTc prolongation when clinical evaluations are limited by heart rate changes, low drug exposures, or high‐dose safety considerations. The time has come to meaningfully merge nonclinical and clinical data to enable a more comprehensive, but flexible, clinical risk assessment strategy for QTc monitoring discussed in updated ICH E14 Questions and Answers. Implementing a fully integrated nonclinical/clinical risk assessment for compounds with double‐negative nonclinical findings in the context of a low prevalence of clinical QTc prolongation would relieve the burden of unnecessary clinical QTc studies and streamline drug development.

Fusobacterium nucleatum (Fn) is frequently found in colorectal cancers (CRCs). High loads of Fn DNA are detected in CRC tissues with microsatellite instability-high (MSI-H), or with the CpG island hypermethylation phenotype (CIMP). Fn infection is also associated with the inflammatory tumor microenvironment of CRC. A subtype of CRC exhibits inflammation-associated microsatellite alterations (IAMA), which are characterized by microsatellite instability-low (MSI-L) and/or an elevated level of microsatellite alterations at selected tetra-nucleotide repeats (EMAST). Here we describe two independent CRC cohorts in which heavy or moderate loads of Fn DNA are associated with MSI-H and L/E CRC respectively. We also show evidence that Fn produces factors that induce γ-H2AX, a hallmark of DNA double strand breaks (DSBs), in the infected cells.

This study was undertaken on three species of rabbitfish, Siganus puellus, Siganus unimaculatus, and Siganus virgatus, to clarify whether stonefish toxin-like toxins are distributed in rabbitfish. The primary structures of toxins from these three species of rabbitfish were successfully elucidated by complementary DNA cloning and found to comprise two different subunits with almost the same molecular mass, i.e., 70–80 kDa. Based on the heterodimeric structure, molecular size, and amino acid sequence features, the toxins from the three species of rabbitfish were judged to be analogues of the stonefish toxins. The amino acid sequences of the toxins from S. puellus and S. unimaculatus were very similar, and distinguishable from those of S. virgatus and S. fuscescens. Homology modeling indicated that the predicted three-dimensional structures of the rabbitfish toxins are comparable to that of stonustoxin (a toxin of the stonefish Synanceia horrida), as they are composed of four domains. It is thus assumed that the rabbitfish toxins form pores in cell membranes by essentially the same mechanism as stonustoxin, although the rabbitfish toxins have alterations of some amino acid residues that are suggested to be critical for the interaction between the two subunits of stonustoxin.

Xylan deposition and lignification processes were examined in tension wood fibers with gelatinous layers (G-layers) in Mallotus japonicus (Euphorbiaceae). The cell walls consisted of a multi-layered structure of S1 + S2 + G + n(L + G), where n indicates the number of repetitions (n = 0–3) and L indicates very thin lignified layers. The formation and lignification processes of the multi-layered structure of tension wood fibers were examined by light microscopy, ultraviolet microscopy, and transmission electron microscopy (TEM) following KMnO4 staining. The deposition of xylan was examined by immunoelectron microscopy with a monoclonal antibody (LM11). Immunolabelling of xylan appeared in lignified cell wall layers, except in the compound middle lamella (CML), i.e., the S1, S2, and L layers but not the G-layers. The density of LM11 xylan immunogold labeling in S2 layers increased during the formation of G-layers. This increase was due to the shrinkage of S2 layers during development rather than intrusive deposition of xylan through G-layers. Lignification of the CML, S1, and S2 layers proceeded during G-layer formation. The shrinkage of S2 layers occurred almost simultaneously with the lignification of the S2 layers during G-layer formation, suggesting that the S2 layers shrank with lignification.

Programmed cell death ligand-1 (PD-L1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) play a pivotal role in cancer immunotherapy. Each of these molecules has a membrane-bound receptor form (mPD-L1/mCTLA-4) and a soluble form (sPD-L1/sCTLA-4). However, these prognostic impacts in colorectal cancer (CRC) remain unclear. We immunohistochemically scored tumoral mPD-L1/mCTLA-4 expression and quantified preoperative circulating sPD-L1/sCTLA-4 levels using matched serum specimens from 131 patients with pStage I–III CRC. We also examined the association between these statuses and tumor infiltrating lymphocytes (TILs) in these patients. Elevated levels of mPD-L1, mCTLA-4, sPD-L1 and sCTLA-4 were significantly correlated with poor overall survival (OS) and disease-free survival (DFS). Co-high expression of tumoral mPD-L1 and mCTLA-4 or co-elevated levels of serum sPD-L1 and sCTLA-4 were strongly correlated with poor OS and DFS. Multivariate analysis revealed that both statuses were negative independent prognostic factors for OS [hazard ratio (HR) 3.86, 95% confidence interval (95% CI) 1.71–8.51, p = 0.001; HR 5.72, 95% CI 1.87–14.54, p = 0.004, respectively] and DFS (HR 2.53, 95% CI 1.23–4.95, p = 0.01; HR 6.88, 95% CI 2.42–17.13, p = 0.0008, respectively). Although low expression of tumoral mCTLA-4 was significantly correlated with increased CD8(+) TILs, there was no correlation in any other combination. We verified the prognostic impacts of mPD-L1, mCTLA-4, sPD-L1 and sCTLA-4 in pStage I–III CRC patients. Dual evaluation of immune checkpoint molecules in primary tissues or preoperative serum could identify a patient population with poor prognosis in these patients.

Resistance to cytotoxic T cells is frequently mediated by loss of MHC class I expression or IFNγ signaling in tumor cells, such as mutations of B2M or JAK1 genes. Natural killer (NK) cells could potentially target such resistant tumors, but suitable NK-cell–based strategies remain to be developed. We hypothesized that such tumors could be targeted by NK cells if sufficient activating signals were provided. Human tumors frequently express the MICA and MICB ligands of the activating NKG2D receptor, but proteolytic shedding of MICA/B represents an important immune evasion mechanism in many human cancers. We showed that B2M- and JAK1-deficient metastases were targeted by NK cells following treatment with a mAb that blocks MICA/B shedding. We also demonstrated that the FDA-approved HDAC inhibitor panobinostat and a MICA/B antibody acted synergistically to enhance MICA/B surface expression on tumor cells. The HDAC inhibitor enhanced MICA/B gene expression, whereas the MICA/B antibody stabilized the synthesized protein on the cell surface. The combination of panobinostat and the MICA/B antibody reduced the number of pulmonary metastases formed by a human melanoma cell line in NOD/SCID gamma mice reconstituted with human NK cells. NK-cell–mediated immunity induced by a mAb specific for MICA/B, therefore, provides an opportunity to target tumors with mutations that render them resistant to cytotoxic T cells.

We determined the requirements for dormancy break/germination and kind of dormancy in seeds of the Hawaiian lobelioids Cyanea kunthiana, Delissea rhytidoperma, Lobelia grayana, L. hypoleuca, Trematolobelia grandifolia, and T. singularis. Fresh seeds were incubated in light/dark at 15/6, 20/10, and 25/15 °C, and germination monitored at two-week intervals for 14 weeks. For each species, the mean embryo length (E): seed (S) length ratio was determined for freshly matured seeds and for seeds at the time the seed coat split but before radicle emergence (germination). The embryo in seeds of all six species incubated at 25/15 °C grew inside the seed prior to germination (42%–148% increase in E:S ratio, depending on species). Seeds of L. grayana and L. hypoleuca have morphological dormancy (MD); they germinated to 82%–98% at the three temperature regimes in 4 weeks. Seeds of the other species have nondeep simple morphophysiological dormancy (MPD) and require >4 weeks for maximum germination to occur. Our results add to the growing body of knowledge about the kind (class) of seed dormancy in Campanulaceae, which suggests that seeds of members of this family have either MD or MPD and embryos grow at warm (≥15 °C) temperatures.

Significance Listeria has been a strong foodborne pathogen for years and can lead to severe diseases. Efficient sensing methods for quick detections of Listeria are still in demand. Here we designed a biosensor that installed Listeria antibodies on the surface of hydrocarbon phase of Janus droplets. We also dissolved a pair of blocking and emissive dyes in the hydrocarbon and fluorocarbon phases of Janus droplets. With the existence of Listeria , Janus droplets will form agglutinations and tilt that could be easily tracked by fluorimeter. Our work provides a sensitive, simple, and quick method for the detection of Listeria .

Interleukin-1β (IL-1β) is an inflammatory cytokine produced by monocytes/macrophages and is closely associated with periodontal diseases. The NLRP3 inflammasome is involved in IL-1β activation through pro–IL-1β processing and pyroptotic cell death in bacterial infection. Recently, glyburide, a hypoglycemic sulfonylurea, has been reported to reduce IL-1β activation by suppressing activation of the NLRP3 inflammasome. Therefore, we evaluated the possibility of targeting the NLRP3 inflammasome pathway by glyburide to suppress periodontal pathogen-induced inflammation. THP-1 cells (a human monocyte cell line) were differentiated to macrophage-like cells by treatment with phorbol 12-myristate 13-acetate and stimulated by periodontopathic bacteria, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, or Fusobacterium nucleatum, in the presence of glyburide. IL-1β and caspase-1 expression in the cells and culture supernatants were analyzed by Western blotting and enzyme-linked immunosorbent assay, and cell death was analyzed by lactate dehydrogenase assay. Stimulation of THP-1 macrophage-like cells with every periodontopathic bacteria induced IL-1β secretion without cell death, which was suppressed by the NLRP3 inhibitor, MCC950, and caspase-1 inhibitor, z-YVAD-FMK. Glyburide treatment suppressed IL-1β expression in culture supernatants and enhanced intracellular IL-1β expression, suggesting that glyburide may have inhibited IL-1β secretion. Subsequently, a periodontitis rat model was generated by injecting periodontal bacteria into the gingiva, which was analyzed histologically. Oral administration of glyburide significantly suppressed the infiltration of inflammatory cells and the number of osteoclasts in the alveolar bone compared with the control. In addition to glyburide, glimepiride was shown to suppress the release of IL-1β from THP-1 macrophage-like cells, whereas other sulfonylureas (tolbutamide and gliclazide) or other hypoglycemic drugs belonging to the biguanide family, such as metformin, failed to suppress IL-1β release. Our results suggest that pharmacological targeting of the NLRP3 pathway may be a strategy for suppressing periodontal diseases.

Zinc metallopeptidase STE24 (ZMPSTE24) is essential for the conversion of farnesyl–prelamin A to mature lamin A, a key component of the nuclear lamina. In the absence of ZMPSTE24, farnesyl–prelamin A accumulates in the nucleus and exerts toxicity, causing a variety of disease phenotypes. By ∼4 months of age, both male and female Zmpste24(−/−) mice manifest a near-complete loss of adipose tissue, but it has never been clear whether this phenotype is a direct consequence of farnesyl–prelamin A toxicity in adipocytes. To address this question, we generated a conditional knockout Zmpste24 allele and used it to create adipocyte-specific Zmpste24–knockout mice. To boost farnesyl–prelamin A levels, we bred in the “prelamin A–only” Lmna allele. Gene expression, immunoblotting, and immunohistochemistry experiments revealed that adipose tissue in these mice had decreased Zmpste24 expression along with strikingly increased accumulation of prelamin A. In male mice, Zmpste24 deficiency in adipocytes was accompanied by modest changes in adipose stores (an 11% decrease in body weight, a 23% decrease in body fat mass, and significantly smaller gonadal and inguinal white adipose depots). No changes in adipose stores were detected in female mice, likely because prelamin A expression in adipose tissue is lower in female mice. Zmpste24 deficiency in adipocytes did not alter the number of macrophages in adipose tissue, nor did it alter plasma levels of glucose, triglycerides, or fatty acids. We conclude that ZMPSTE24 deficiency in adipocytes, and the accompanying accumulation of farnesyl–prelamin A, reduces adipose tissue stores, but only modestly and only in male mice.

Spraying a calcium carbonate suspension "White Coat" on the fruit of apples significantly suppresses the oviposition of the peach fruit moth, Carposina sasakii. In gas chromatography (GC) with an electroantennographic detector analysis, adult female antennae showed responses to three compounds that were identified as 2,2,4-trimethyl-1,3-pentanediol diisobutyrate (TXIB) and its two mono-hydrolyzed analogs, texanols (1- and 3-isobutyrates), all added as a plasticizer to the agents. An oviposition-choice test using adult moths revealed that TXIB has clear deterrent properties when applied to young apple fruits. Video recording analysis showed that female moths spent longer on self-grooming and searching around TXIB-treated fruits. In the same assay, pure calcium carbonate treatment prevented the moths from climbing up or landing on the fruits, while such was not the case with White Coat-treated fruits. TXIB, an adjuvant aimed to provide rain/wind resistance, weakened the slipperiness of the calcium carbonate coating but, coincidentally, maintained the oviposition inhibitory activity of the White Coat by its deterrent odorant.

The systemic inflammatory response is attracting increasing attention as a predictive biomarker for oncological outcome in patients with colorectal cancer. This study is aimed at verifying if the lymphocyte–C-reactive protein (CRP) ratio (LCR) could be used as a predictor of oncological outcome in patients with rectal cancer (RC) receiving preoperative chemoradiotherapy (CRT). We analyzed data for 86 patients with RC who received preoperative CRT followed by total mesorectal excision at our institution. A ratio of 6000 was used as the cut-off value for LCR for further analysis. The post-CRT LCR was significantly lower than the pre-CRT LCR in patients with RC. Although post-CRT LCR status was not significantly correlated with overall survival (OS), low pre-CRT LCR was significantly associated with shorter recurrence-free survival (RFS: p = 0.02) and OS (p = 0.017) in this population and was an independent prognostic factor for both RFS and OS (hazard ratio (HR) 3.19, 95% confidence interval (CI) 1.33–7.66, p = 0.009; HR 2.83, 95%CI 1.14–7.01, p = 0.025, respectively). Furthermore, low pre-CRT LCR was a stronger indicator of early recurrence (p = 0.001) and poor prognosis (p = 0.025) in RC patients without pathological lymph node metastasis compared with patients with pathological lymph node metastasis, and prognostic potential of pre-CRT LCR was clearly revealed especially RC patients receiving long-course CRT. Assessment of pretreatment LCR status might aid decision-making regarding postoperative treatment strategies in patients with RC receiving CRT followed by potentially curative resection.

Many pathological proteins related to neurodegenerative diseases are misfolded, aggregating to form amyloid fibrils during pathogenesis. One of the pathological proteins, alpha-synuclein (α-syn), accumulates in the brains of Parkinson disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), which are designated as synucleinopathies. Recently, structural properties of abnormal accumulated proteins are suggested to determine the disease phenotype. However, the biochemical and structural characteristics of those accumulated proteins are still poorly understood. We previously reported the sequence and seed-structure-dependent polymorphic fibrils of α-syn and the polymorphism was identified by proteinase K-resistant cores determined by mass spectrometry (MS) analysis. In this study, we applied this method to analyze α-syn aggregates of MSA and DLB. To perform MS analysis on proteinase K-resistant cores, we first performed amplification of α-syn aggregates by seeding reaction and protein misfolding cyclic amplification (PMCA) to obtain a sufficient amount of aggregates. Using SDS insoluble fraction of the disease brain, we successfully amplified enough α-syn aggregates for MS analysis. We differentiated between mouse and human α-syn aggregates by MS analysis on proteinase K-resistant cores of the aggregates before and after amplification. The results suggest that structural properties of amplified α-syn fibrils are preserved after PMCA and these methods can be applicable in the study of pathological proteins of the neurodegenerative disorders.

Enhancing crop traits that increase grain yield under elevated CO2 concentrations is an important option for increasing the future productivity of rice. Here, we compared the growth and yield of five varieties with different genetic background under Free-Air CO2 Enrichment (FACE) conditions to identify traits responsible for varietal differences in yield increase under elevated CO2. Three high-yielding and two standard rice varieties grown under FACE conditions commonly had (1) shorter growth periods, (2) higher dry matter production, (3) higher numbers of spikelets (sink capacity) and panicles; and (4) higher yield than those grown under ambient CO2. Yield enhancement by elevated CO2 (FACE/Ambient), however, differed significantly among varieties, ranging from 1.10 to 1.25. The greater response of the sink capacity, defined as the product of spikelet number and single grain mass, was the main factor involved in yield increase. Three high-yielding varieties (Momiroman, Takanari, and Hokuriku 193) had greater sink capacity than two standard varieties and the sink capacity of these varieties significantly increased under FACE condition. However, yield enhancement in elevated CO2 was lower in Hokuriku 193 than in Momiroman and Takanari. In Hokuriku 193, sink production was relatively low while dry matter production was similar to the others. Therefore, larger increase in sink production efficiency per unit of dry matter production under FACE was found to be a particularly important varietal trait, suggesting that efforts to develop varieties suited to the predicted elevated CO2 condition should focus on this and the related traits.

Diffuse alveolar hemorrhage developed in an SLE mouse model is ameliorated by TANK via the suppression of type I IFN induced by cGAS-mediated sensing of intracellular DNA., Diffuse alveolar hemorrhage (DAH) is one of the serious complications associated with systemic lupus erythematosus, an autoimmune disease whose pathogenesis involves type I IFNs and cytokines. Here, we show that TANK, a negative regulator of the NF-κB signaling via suppression of TRAF6 ubiquitination, is critical for the amelioration of fatal DAH caused by lung vascular endothelial cell death in a mouse model of systemic lupus erythematosus. The development of fatal DAH in the absence of TANK is mediated by type I IFN signaling, but not IL-6. We further uncover that STING, an adaptor essential for the signaling of cytoplasmic DNA sensor cyclic GMP-AMP (cGAMP) synthase (cGAS), plays a critical role in DAH under Tank deficiency. TANK controls cGAS-mediated cGAMP production and suppresses DNA-mediated induction of IFN-stimulated genes in macrophages by inhibiting the formation of DNA-cGAS aggregates containing ubiquitin. Collectively, TANK inhibits the cGAS-dependent recognition of cytoplasmic DNA to prevent fatal DAH in the murine lupus model.

Zebrafish models have been developed for several studies involving lipid metabolism and lipid-related diseases. In the present study, the migration of dietary docosahexaenoic acid (DHA) in whole-body zebrafish was estimated by stable-isotope tracer and matrix-assisted laser desorption/ionization mass spectrometry imaging.Administration of 1-13C-2,2-D2-labeled DHA ((+3)DHA) ethyl ester to male zebrafish was conducted to evaluate its accumulation, migration, and distribution in the body. The (+3)DHA content in the body of zebrafish after administering (+3)DHA for 10 and 15 d was significantly higher than that in the control group. (+3)DHA was observed as a constituent of phosphatidylcholine (PC) in the intestine of zebrafish that were administered (+3)DHA for 5 and 10 d. (+3)DHA-containing PC tended to accumulate in the intestines of zebrafish administered (+3)DHA for 1 d, indicating that recombination of (+3)DHA from ethyl ester to PC occurs quickly at intestine.After administration for 15 d, (+3)DHA-containing PC accumulated in the intestine, liver, and muscle of whole-body zebrafish. In contrast, (+3)DHA-containing PC was not detected in the brain.These results showed that dietary DHA is initially constructed into PC as a structural component of intestinal cell membranes and gradually migrates into peripheral tissues such as muscle., 公開日: 2022-12-02

The exposure of adult fish to warm or high temperatures is known to impair reproduction, yet the long-term reproductive impacts for treatments at early life are not well clarified. This study aimed to evaluate the effects of warm temperature (WT) during juvenile stage on gonad maturation, gamete quality, and offspring thermotolerance in rainbow trout. While the comparison of basic reproductive parameters in WT females did not reveal any kind of impairment, many WT males showed an atrophied, undeveloped gonad, or a smaller testis with lower milt volume; sperm quality parameters in WT males and deformity rates in the respective progeny were also highly affected. However, despite of such negative effects, many of the remaining progeny presented better rates of survival and growth when exposed to the same conditions as those of parental fish (WT), suggesting that thermal stress in parr stage males elicited intergenerational thermotolerance after a single generation. The present results support that prolonged warming stress during early life stages can adversely affect key reproductive aspects, but contrastingly increase offspring performance at upper thermal ranges. These findings have implications on the capacity of fish to adapt and to cope with global warming. Fil: Butzge, Arno Juliano. Universidade Estadual Paulista Julio de Mesquita Filho; Brasil Fil: Yoshinaga, Tulio Teruo. Universidade de Sao Paulo; Brasil Fil: Moreno Acosta, Omar David. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Fernandino, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Sanches, Eduardo Antônio. Universidade Estadual Paulista Julio de Mesquita Filho; Brasil Fil: Tabata, Yara Aiko. Salmonid Experimental Station At Campos Do Jordão; Brasil Fil: de Oliveira, Claudio. Universidade Estadual Paulista Julio de Mesquita Filho; Brasil Fil: Takahashi, Neuza Sumico. Centro de Pesquisa de Aquicultura; Brasil Fil: Hattori, Ricardo Shohei. Salmonid Experimental Station at Campos do Jordão; Brasil

Poly(ADP-ribose) polymerases, PARPs, transfer ADP-ribose onto target proteins from nicotinamide adenine dinucleotide (NAD+). Current mass spectrometric analytical methods require proteolysis of target proteins, limiting the study of dynamic ADP-ribosylation on contiguous proteins. Herein, we present a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) method that facilitates multisite analysis of ADP-ribosylation. We observe divergent ADP-ribosylation dynamics for the catalytic domains of PARPs 14 and 15, with PARP15 modifying more sites on itself (+3-4 ADP-ribose) than the closely related PARP14 protein (+1-2 ADP-ribose)─despite similar numbers of potential modification sites. We identify, for the first time, a minimal peptide fragment (18 amino-acids) that is preferentially modified by PARP14. Finally, we demonstrate through mutagenesis and chemical treatment with hydroxylamine that PARPs 14/15 prefer acidic residues. Our results highlight the utility of MALDI-TOF in the analysis of PARP target modifications and in elucidating the biochemical mechanism governing PARP target selection.

Chromatin modifications are key epigenetic regulatory features with roles in various cellular events, yet histone mark identification, gene wide distribution and relationship to gene expression remains understudied in green algae. Histone lysine methylation is regarded as an active chromatin mark in many organisms, and is implicated in mediating active euchromatin. We interrogated the genome wide distribution pattern of mono- and trimethylated H3K4 using Chromatin-Immunoprecipitation followed by deep-sequencing (ChIP-Seq) during key phases of the Chlamydomonas cell cycle: early G1 phase (ZT1) when cells initiate biomass accumulation, S/M phase (ZT13) when cells are undergoing DNA replication and mitosis, and late G0 phase (ZT23) when they are quiescent. Tri-methylated H3K4 was predominantly enriched at TSSs of the majority of protein coding genes (85%). The likelihood of a gene being marked by H3K4me3 correlated with it being transcribed at one or more time points during the cell cycle but not necessarily by continuous active transcription. This finding even applied to early zygotic genes whose expression may be dormant for hundreds or thousands of generations between sexual cycles; but core meiotic genes were completely missing H3K4me3 peaks at their TSS. In addition, bi-directional promoters regulating expression of replication dependent histone genes, had transient H3K4me3 peaks that were present only during S/M phase when their expression peaked. In agreement with biochemical studies, mono-methylated H3K4 was the default state for the vast majority of histones that were outside of TSS and terminator regions of genes. A small fraction of the genome which was depleted of any H3 lysine methylation was enriched for DNA cytosine methylation and the genes within these DNA methylation islands were poorly expressed. Genome wide H3K4me3 ChIP-Seq data will be a valuable resource, facilitating gene structural annotation, as exemplified by our validation of hundreds of long non-coding RNA genes.