Search

Abstract Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of all cancers. To improve PDAC therapy, we establish screening systems based on organoid and co-culture technologies and find a payload of antibody–drug conjugate (ADC), a bromodomain and extra-terminal (BET) protein degrader named EBET. We select CEACAM6/CD66c as an ADC target and developed an antibody, #84.7, with minimal reactivity to CEACAM6-expressing normal cells. EBET-conjugated #84.7 (84-EBET) has lethal effects on various PDAC organoids and bystander efficacy on CEACAM6-negative PDAC cells and cancer-associated fibroblasts. In mouse studies, a single injection of 84-EBET induces marked tumor regression in various PDAC-patient-derived xenografts, with a decrease in the inflammatory phenotype of stromal cells and without significant body weight loss. Combination with standard chemotherapy or PD-1 antibody induces more profound and sustained regression without toxicity enhancement. Our preclinical evidence demonstrates potential efficacy by delivering BET protein degrader to PDAC and its microenvironment via CEACAM6-targeted ADC.

Summary: Mating behavior is an essential fitness trait. We used the inbred, sequenced lines of the Drosophila Genetic Reference Panel (DGRP) to gain insights into the evolution of mating success and to evaluate the overlap in genetic architecture of mating behavior between the sexes. We found significant genetic variation for mating success when DGRP males and females from the same line were mated together, and when DGRP males and females were mated to an unrelated strain of the opposite sex. The mating success of DGRP males and females was not correlated when they were paired with the unrelated strain, suggesting independent genetic architecture of mating success in males and females that was confirmed by genome-wide association analyses. However, the mating success between pairs of the same or different DGRP lines was predicted accurately by the respective female and male mating success with the unrelated line.

Corynebacterium ulcerans is a closely related bacterium to the diphtheria bacterium C. diphtheriae, and some C. ulcerans strains produce toxins that are similar to diphtheria toxin. C. ulcerans is widely distributed in the environment and is considered one of the most harmful pathogens to livestock and wildlife. Infection with C. ulcerans can cause respiratory or nonrespiratory symptoms in patients. Recently, the microorganism has been increasingly recognized as an emerging zoonotic agent of diphtheria-like illness in Japan. To clarify the overall clinical characteristics, treatment-related factors, and outcomes of C. ulcerans infection, we analyzed 34 cases of C. ulcerans that occurred in Japan during 2001–2020. During 2010–2020, the incidence rate of C. ulcerans infection increased markedly, and the overall mortality rate was 5.9%. It is recommended that adults be vaccinated with diphtheria toxoid vaccine to prevent the spread of this infection.

ABSTRACTClostridium tetani is the etiological agent of the disease tetanus, resulting in various symptoms, including neck tension, tongue-twisting, a decline in walking ability, and death in severe cases. To date, publicly available genome sequences of C. tetani are limited. C. tetani was isolated from soil in Kumamoto Prefecture, Japan, between 2020 and 2022. Subsequently, bacteriological, biochemical, genetic, and immunological tests were conducted. Among the 46 soil sampling sites, 151 C. tetani strains were isolated from 33 sites, with a 71.7% positivity rate. Comparative genomic analysis subdivided clade 1 into clades 1-1, 1-2, and 1-3. The enzyme-linked immunosorbent assay revealed that partial clade 1-3 strains produced over sevenfold more tetanus neurotoxin (TeNT) than the reference strain (first isolate of C. tetani KHSU-154301-001). RNA-Seq analysis supported this elevated tetanus toxin tetX gene expression. Additionally, tetX-negative strains were isolated from the clade, indicating that TeNT may not be essential for ensuring the survival of their offspring without its representative toxin-causing tetanus. Five strains acquired the tetracycline resistance gene tet(M) in the tetX plasmid, resulting in significantly reduced susceptibility to tetracycline. In conclusion, although we investigated C. tetani isolates from a specific region of the Kumamoto Prefecture, we demonstrated that these isolates exhibit variable genomic features, toxin productivity, and substantial acquired antimicrobial resistance. These findings indicate that multiple C. tetani lineages have successfully adapted to the soil in Japan, as evidenced by the additional genome information and toxin productivity assays in this study.IMPORTANCEC. tetani is a spore-forming, anaerobic bacterium that produces a toxin causing muscle stiffness and paralysis. Tetanus is preventable with the toxoid vaccine, but it remains a significant public health threat in regions with low vaccine coverage. However, there are relatively few isolates and limited genomic information available worldwide. In Japan, about 100 cases are reported each year, but there have been no nationwide surveys of isolates, and no genomic information from Japanese isolates has been published. In our study, we analyzed the genomes of 151 strains from a limited survey of soil in Kumamoto, Japan. Our findings revealed a high degree of genetic diversity, and we also identified a subset of strains that produced significantly more toxin, which provides new insights into the pathogenesis of tetanus. Our findings lay the foundation for future studies to investigate the distribution and evolution of C. tetani in Japan and neighboring countries.

Some calderas are geometrically complex that may be related not to a single eruption, magma body, or structure. In order to reveal their forming processes, multidisciplinary methods should be applied. Akan volcano has E-W elongated and irregular-shaped caldera (24 × 13 km), implying a complex mechanism of formation. Akan caldera results from successive explosive eruptions from 1.4 to 0.1 Ma. On the basis of duration of dormancy and petrological features (mainly whole-rock major element compositions) of juvenile materials, these eruptions have been grouped into 17 eruptive groups (Ak1–Ak17), each of which consists of a single or a sequential phase. In order to investigate the processes of caldera formation, we focus on the younger eruptive groups (Ak1–Ak7: 0.8 to 0.2 Ma) that have relatively large magnitudes (>10 km3) and likely control the present caldera shape. We performed component analysis of lithic fragments from pyroclastic fallout deposits, whole-rock trace element analysis of juveniles, and gravitational survey of the caldera. We grouped Ak1–Ak7 into three types, namely, type A (Ak1, Ak2), type B (Ak3–Ak5), and type C (Ak6, Ak7), based on the lithic componentry, most of which are accessary and accidental fragments from vent and conduit areas. The characteristic lithic component in each type is as follows: altered rock in type A, aphyric dacite in type B, and pyroxene andesite in type C. These data suggest that explosive eruptions of each type are derived from distinct sources. The whole-rock composition of juvenile materials of each type also shows distinct trends on Harker diagrams. These three chemical trends are nearly parallel, suggesting that three different magma systems were active. This is consistent with the lithic componentry showing three spatially distinct vent sources. The geological and petrological evidence is supported by a Bouguer anomaly map. Akan caldera is characterized by three circular closed minima, indicative of three depressed segments that correspond to the source regions, each of which possibly discharged the three types of magma. Caldera-forming eruptions of Akan caldera occurred from at least three distinct sources with distinct magma systems. In conclusion, Akan caldera is a composite caldera, and its shape reflects the distribution of multiple source regions. The case study of Akan caldera shows a possible time-space evolutionary pathway for a caldera complex where several smaller calderas are nested.

Spontaneous grooming behavior is a component of insect fitness. We quantified spontaneous grooming behavior in 201 sequenced lines of the Drosophila melanogaster Genetic Reference Panel and observed significant genetic variation in spontaneous grooming, with broad-sense heritabilities of 0.25 and 0.24 in females and males, respectively. Although grooming behavior is highly correlated between males and females, we observed significant sex by genotype interactions, indicating that the genetic basis of spontaneous grooming is partially distinct in the two sexes. We performed genome-wide association analyses of grooming behavior, and mapped 107 molecular polymorphisms associated with spontaneous grooming behavior, of which 73 were in or near 70 genes and 34 were over 1 kilobase from the nearest gene. The candidate genes were associated with a wide variety of gene ontology terms, and several of the candidate genes were significantly enriched in a genetic interaction network. We performed functional assessments of 29 candidate genes using RNA interference, and found that 11 affected spontaneous grooming behavior. The genes associated with natural variation in Drosophila grooming are involved with glutamate metabolism (Gdh) and transport (Eaat); interact genetically with (CCKLR-17D1) or are in the same gene family as (PGRP-LA) genes previously implicated in grooming behavior; are involved in the development of the nervous system and other tissues; or regulate the Notch and Epidermal growth factor receptor signaling pathways. Several DGRP lines exhibited extreme grooming behavior. Excessive grooming behavior can serve as a model for repetitive behaviors diagnostic of several human neuropsychiatric diseases.

Drosophila suzukii (D. suzukii) (Matsumura, 1931; Diptera: Drosophilidae), also known as spotted wing Drosophila, is a worldwide pest of fruits with soft skins such as blueberries and cherries. Originally from Asia, D. suzukii is now present in the Americas and Europe and has become a significant economic pest. Growers largely rely on insecticides for the control of D. suzukii. Genetic strategies offer a species-specific environmentally friendly way for suppression of D. suzukii populations. We previously developed a transgenic strain of D. suzukii that produced only males on a diet that did not contain tetracycline. The strain carried a single copy of the FL19 construct on chromosome 3. Repeated releases of an excess of FL19 males led to suppression of D. suzukii populations in laboratory cage trials. Females died as a consequence of overexpression of the tetracycline transactivator (tTA) and tTA-activated expression of the head involution defective proapoptotic gene. The aim of this study was to generate additional male-only strains that carried two copies of the FL19 transgene through crossing the original line with a piggyBac jumpstarter strain. Males that carried either two chromosome 3 or a singleX-linked transgene were identified through stronger expression of the red fluorescent protein marker gene. The brighter fluorescence of the X-linked lines was likely due to dosage compensation of the red fluorescent protein gene. In total, four X-linked lines and eleven lines with two copies on chromosome 3 were obtained, of which five were further examined. All but one of the strains produced only males on a diet without tetracycline. When crossed with wild type virgin females, all of the five two copy autosomal strains examined produced only males. However, the single copy X-linked lines did not show dominant female lethality. Five of the autosomal lines were further evaluated for productivity (egg to adult) and male competition. Based on these results, the most promising lines have been selected for future population suppression experiments with strains from different geographical locations.

Introduction:Rhabdophis snakes, which include 27 species, are rear-fanged venomous snakes that are widely distributed from India to East Asia and Russia. Severe envenomation by R. tigrinus (Yamakagashi snake) in Japan and R. subminiatus in Southeast Asia has been reported. The epidemiology of R. tigrinus bites, such as geographical features, the incidence, and changes in the number of bites over time have not been comprehensively examined. Hence, we intended to clarify the epidemiological features of R. tigrinus bites through a careful review of scientific data over the last 50 years in Japan.Methods: Patient records of R. tigrinus bites between 1971 and 2020 at the Japan Snake Institute were examined retrospectively. The following were ascertained: patient characteristics, clinical symptoms, laboratory data, treatment-related factors, and hospital mortality. These variables were compared in the antivenom and the without-antivenom groups.Results: Over the 50-year study period, 43 R. tigrinus bites, including five fatal cases, were encountered. Severe cases of R. tigrinus bites have been treated with antivenom since 1985; however, fatalities occurred in 2006 and 2020. R. tigrinus bite cases have been well-distributed in the western part of Japan since 2000. The mortality rate in the antivenom group was significantly lower in the patient group that was not administered the antivenom (0 vs. 23.8%, p = 0.048).Conclusion: This study clarified the epidemiology of R. tigrinus bites in Japan over a 50-year period. Almost all severe cases of R. tigrinus bites have been treated with the antivenom in the current situation, and fatalities occurred in cases not treated with the antivenom. It is important to diagnose R. tigrinus bites in the early phase of the clinical course. The antivenom, the definitive treatment for R. tigrinus bites, is an unapproved drug. Hence, approval needs to be obtained for the drug.

We conducted molecular typing of a Corynebacterium ulcerans isolate from a woman who died in Japan in 2016. Genomic DNA modification might have affected the isolate’s ribotyping profile. Multilocus sequence typing results (sequence type 337) were more accurate. Whole-genome sequencing had greater ability to discriminate lineages at high resolution.

Background Infections caused by Corynebacterium ulcerans, a zoonotic pathogen, have been reported worldwide. This microorganism is known to produce the diphtheria toxin and cause diphtheria‐like illness. Case presentation A 63‐year‐old woman with a history of diabetes and hypertension developed cold and flu‐like symptoms, which gradually progressed into respiratory distress. Therefore, the patient was intubated for dyspnea with pseudomembrane formation. A toxin‐producing strain of C. ulcerans was identified, also detected in the patient’s domestic cats. Multilocus sequence typing confirmed all strains, including the patient’s isolate, as ST337. Conclusion Multilocus sequence typing revealed zoonotic transmission of C. ulcerans from domestic cats to a human.

Yamakagashi (Rhabdophis tigrinus) inhabits Japan widely, and incidents involving its bites occur every year. Its bite causes disseminated intravascular coagulation when the amount of infused venom is high, and it can be fatal if treatment with Yamakagashi antivenom is delayed. Although Yamakagashi antivenom is used for treating Yamakagashi bites, it is an unapproved drug and its capacity for storage is limited. Hence, it is difficult to administer to patients promptly. As a therapeutic agent for this bite, we investigated the application of recombinant thrombomodulin alpha, a commercially available disseminated intravascular coagulation therapeutic agent. Its therapeutic effect on Yamakagashi venom was confirmed in a coagulation system of human plasma using in vitro Yamakagashi venom as well as a rat experimental model of disseminated intravascular coagulation using in vivo Yamakagashi venom. The administration of recombinant thrombomodulin alpha induced an effect that prolonged the blood coagulation time of Yamakagashi venom in vitro, and the drug was administered in vivo within 0.5 h after the administration of Yamakagashi venom to save rats. Blood coagulation markers such as platelet count, prothrombin time, fibrinogen concentration, and D-dimer levels recovered to normal values in rats. Therefore, recombinant thrombomodulin alpha may be used as a therapeutic agent for Yamakagashi bites.

Corynebacterium ulcerans infection was recently recognized as a zoonosis. We present 2 cases of severe pneumonia complicated by diffuse pseudomembrane formation on the bronchus caused by C. ulcerans–producing diphtheria toxin. Our purpose is to alert medical professionals to the virulence of Corynebacterium species other than C. diphtheriae.

Many pathogenic bacteria, including Escherichia coli and Vibrio cholerae, can become viable but nonculturable (VBNC) following exposure to specific stress conditions. Corynebacterium diphtheriae, a known human pathogen causing diphtheria, has not previously been shown to enter the VBNC state. Here, we report that C. diphtheriae can become VBNC when exposed to low temperatures. Morphological differences in culturable and VBNC C. diphtheriae were examined using scanning electron microscopy. Culturable cells presented with a typical rod-shape, whereas VBNC cells showed a distorted shape with an expanded center. Cells could be transitioned from VBNC to culturable following treatment with catalase. This was further evaluated via RNA sequence-based transcriptomic analysis and reverse-transcription quantitative PCR of culturable, VBNC, and resuscitated VBNC cells following catalase treatment. As expected, many genes showed different behavior by resuscitation. The expression of both the diphtheria toxin and the repressor of diphtheria toxin genes remained largely unchanged under all four conditions (culturable, VBNC, VBNC after the addition of catalase, and resuscitated cells). This is the first study to demonstrate that C. diphtheriae can enter a VBNC state and that it can be rescued from this state via the addition of catalase. This study helps to expand our general understanding of VBNC, the pathogenicity of VBNC C. diphtheriae, and its environmental survival strategy.

Disseminated intravascular coagulation, a severe clinical condition caused by an underlying disease, involves a markedly continuous and widespread activation of coagulation in the circulating blood and the formation of numerous microvascular thrombi. A snakebite, including that of the Yamakagashi (Rhabdophis tigrinus), demonstrates this clinical condition. Thus, an animal model using Yamakagashi venom was constructed. Yamakagashi venom was administered to rats, and its lethality and the changes in blood coagulation factors were detected after venom injection. When 300 μg venom was intramuscularly administered to 12-week-old rats, (1) they exhibited hematuria with plasma hemolysis and died within 48 h; (2) Thrombocytopenia in the blood was observed in the rats; (3) irreversible prolongation of prothrombin time in the plasma to the measurement limit occurred; (4) fibrinogen concentration in the plasma irreversibly decreased below the measurement limit; and (5) A transient increase in the plasma concentration of D-dimer was observed. In this model, a fixed amount of Rhabdophis tigrinus venom injection resulted in the clinical symptom similar to the human pathology with snakebite. The use of the rat model is very effective in validating the therapeutic effect of human disseminated intravascular coagulation condition due to snakebite.

Aim Redback spiders are rapidly becoming a nationwide problem in Japan. The domestic production of antivenom for redback spider bites has been started because of extremely low supply. The purpose of this study was to investigate the ability of emergency physicians to recognize redback spider bites and to examine the demand for antivenom and identify the ideal choice for storage. Methods Questionnaires examining the ability to recognize redback spider bites, the demand for antivenom, and overall level of preparedness were sent to 271 emergency medical service centers in Japan in March 2015. Results Completed questionnaires were returned by 156 medical institutions (58%). Only 25% of the institutes correctly answered regarding the recognition of redback spider bites. A demand for the new, domestically produced antivenom was reported by 55% of the institutes. Antivenom demand was high and increased with the ability to recognize redback spider bites (0 correct answers, n = 24 [42.9%]; 1–2 answers, n = 32 [55.2%]; three answers, n = 28 [71.8%]; P = 0.02). The storage of antivenom within their prefectures or regions was the best option for initial supply planning. Approximately 90% of the emergency centers showed an ability to use the antivenom safely. Conclusion The recognition of redback spider bites was low; however, the demand for antivenom was high. The storage of antivenom within their own prefectures or regions was considered to be the best option for initial supply planning. Emergency medical service centers are also good candidates for storage and safe use of antivenom.

In 2008 in Japan, 15/60 captive Japanese macaques died. Clostridium tetani was isolated from 1 monkey, and 11 had tetanus-specific symptoms. We conclude the outbreak resulted from severe environmental C. tetani contamination. Similar outbreaks could be prevented by vaccinating all monkeys, disinfecting housing areas/play equipment, replacing highly C. tetani–contaminated soil, and conducting epidemiologic surveys.

Rhabdophis lateralis, a colubrid snake distributed throughout the continent of Asia, has recently undergone taxonomic revisions. Previously, Rhabdophis lateralis was classified as a subspecies of R. tigrinus (Yamakagashi) until 2012, when several genetic differences were discovered which classified this snake as its own species. To elucidate the toxicity of venom from this poorly studied colubrid, various biological activities were compared between the venom from the two snake species. The components of their venom were compared by the elution profiles of reversed-phase HPLC and SDS-PAGE, and gel filtrated fractions were tested for effects on blood coagulation. Proteolytic activities of these fractions were also assayed by using synthetic substrates, fibrinogen, and matrix proteins. Similar to the R. tigrinus venom, the higher molecular weight fraction of R. lateralis venom contained a prothrombin activator. Both prothrombin time (PT) and activated partial thromboplastin time (APTT) of human plasma were shortened by the addition of R. lateralis and R. tigrinus venom. The thrombin formation was estimated by the uses of SDS-PAGE and chromogenic substrates. These venom fractions also possessed very specific proteinase activity on human fibrinogen, but the substrates for matrix metalloproteinase, such as collagen and laminin, were not hydrolyzed. However, there were some notable differences in reactivity to synthetic substrates for matrix metalloproteinase, and R. tigrinus venom possessed relatively higher activity. Our chemical investigation indicates that the components included in both venoms resemble each other closely. However, the ratio of components and proteolytic activity of some ingredients are slightly different, indicating differences between two closely-related snakes.

Understanding the genetic and environmental factors that affect variation in life span and senescence is of major interest for human health and evolutionary biology. Multiple mechanisms affect longevity, many of which are conserved across species, but the genetic networks underlying each mechanism and cross-talk between networks are unknown. We report the results of a screen for mutations affecting Drosophila life span. One third of the 1,332 homozygous P-element insertion lines assessed had quantitative effects on life span; mutations reducing life span were twice as common as mutations increasing life span. We confirmed 58 mutations with increased longevity, only one of which is in a gene previously associated with life span. The effects of the mutations increasing life span were highly sex-specific, with a trend towards opposite effects in males and females. Mutations in the same gene were associated with both increased and decreased life span, depending on the location and orientation of the P-element insertion, and genetic background. We observed substantial--and sex-specific--epistasis among a sample of ten mutations with increased life span. All mutations increasing life span had at least one deleterious pleiotropic effect on stress resistance or general health, with different patterns of pleiotropy for males and females. Whole-genome transcript profiles of seven of the mutant lines and the wild type revealed 4,488 differentially expressed transcripts, 553 of which were common to four or more of the mutant lines, which include genes previously associated with life span and novel genes implicated by this study. Therefore longevity has a large mutational target size; genes affecting life span have variable allelic effects; alleles affecting life span exhibit antagonistic pleiotropy and form epistatic networks; and sex-specific mutational effects are ubiquitous. Comparison of transcript profiles of long-lived mutations and the control line reveals a transcriptional signature of increased life span.

Glaucoma is the world's second leading cause of bilateral blindness with progressive loss of vision due to retinal ganglion cell death. Myocilin has been associated with congenital glaucoma and 2-4% of primary open angle glaucoma (POAG) cases, but the pathogenic mechanisms remain largely unknown. Among several hypotheses, activation of the unfolded protein response (UPR) has emerged as a possible disease mechanism.We used a transgenic Drosophila model to analyze whole-genome transcriptional profiles in flies that express human wild-type or mutant MYOC in their eyes. The transgenic flies display ocular fluid discharge, reflecting ocular hypertension, and a progressive decline in their behavioral responses to light. Transcriptional analysis shows that genes associated with the UPR, ubiquitination, and proteolysis, as well as metabolism of reactive oxygen species and photoreceptor activity undergo altered transcriptional regulation. Following up on the results from these transcriptional analyses, we used immunoblots to demonstrate the formation of MYOC aggregates and showed that the formation of such aggregates leads to induction of the UPR, as evident from activation of the fluorescent UPR marker, xbp1-EGFP. CONCLUSIONS / SIGNIFICANCE: Our results show that aggregation of MYOC in the endoplasmic reticulum activates the UPR, an evolutionarily conserved stress pathway that culminates in apoptosis. We infer from the Drosophila model that MYOC-associated ocular hypertension in the human eye may result from aggregation of MYOC and induction of the UPR in trabecular meshwork cells. This process could occur at a late age with wild-type MYOC, but might be accelerated by MYOC mutants to account for juvenile onset glaucoma.

Corynebacterium ulcerans is a closely related bacterium to the diphtheria bacterium C. diphtheriae, and some C. ulcerans strains produce toxins that are similar to diphtheria toxin. C. ulcerans is widely distributed in the environment and is considered one of the most harmful pathogens to livestock and wildlife. Infection with C. ulcerans can cause respiratory or nonrespiratory symptoms in patients. Recently, the microorganism has been increasingly recognized as an emerging zoonotic agent of diphtheria-like illness in Japan. To clarify the overall clinical characteristics, treatment-related factors, and outcomes of C. ulcerans infection, we analyzed 34 cases of C. ulcerans that occurred in Japan during 2001-2020. During 2010-2020, the incidence rate of C. ulcerans infection increased markedly, and the overall mortality rate was 5.9%. It is recommended that adults be vaccinated with diphtheria toxoid vaccine to prevent the spread of this infection. [ABSTRACT FROM AUTHOR]

Genetic-based methods offer environmentally friendly species-specific approaches for control of insect pests. One method, CRISPR homing gene drive that target genes essential for development, could provide very efficient and cost-effective control. While significant progress has been made in developing homing gene drives for mosquito disease vectors, little progress has been made with agricultural insect pests. Here, we report the development and evaluation of split homing drives that target the doublesex (dsx) gene in Drosophila suzukii, an invasive pest of soft-skinned fruits. The drive component, consisting of dsx single guide RNA and DsRed genes, was introduced into the female-specific exon of dsx, which is essential for function in females but not males. However, in most strains, hemizygous females were sterile and produced the male dsx transcript. With a modified homing drive that included an optimal splice acceptor site, hemizygous females from each of the four independent lines were fertile. High transmission rates of the DsRed gene (94 to 99%) were observed with a line that expressed Cas9 with two nuclear localization sequences from the D. suzukii nanos promoter. Mutant alleles of dsx with small in-frame deletions near the Cas9 cut site were not functional and thus would not provide resistance to drive. Finally, mathematical modeling showed that the strains could be used for suppression of lab cage populations of D. suzukii with repeated releases at relatively low release ratios (1:4). Our results indicate that the split CRISPR homing gene drive strains could potentially provide an effective means for control of D. suzukii populations. [ABSTRACT FROM AUTHOR]