No improvement in mortality among critically ill patients with carbapenems as initial empirical therapy and more detection of multi-drug resistant pathogens associated with longer use: a post hoc analysis of a prospective cohort study.

Whether empirical therapy with carbapenems positively affects the outcomes of critically ill patients with bacterial infections remains unclear. This study aimed to investigate whether the use of carbapenems as the initial antimicrobial administration reduces mortality and whether the duration of carbapenem use affects the detection of multidrug-resistant (MDR) pathogens. This was a post hoc analysis of data acquired from Japanese participating sites from a multicenter, prospective observational study [Determinants of Antimicrobial Use and De-escalation in Critical Care (DIANA study)]. A total of 268 adult patients with clinically suspected or confirmed bacterial infections from 31 Japanese intensive care units (ICUs) were analyzed. The patients were divided into two groups: patients who were administered carbapenems as initial antimicrobials (initial carbapenem group, n = 99) and those who were not administered carbapenems (initial non-carbapenem group, n = 169). The primary outcomes were mortality at day 28 and detection of MDR pathogens. Multivariate logistic regression analysis revealed that mortality at day 28 did not differ between the two groups [18 (18%) vs 27 (16%), respectively; odds ratio: 1.25 (95% confidence interval (CI): 0.59-2.65), P = 0.564]. The subdistribution hazard ratio for detecting MDR pathogens on day 28 per additional day of carbapenem use is 1.08 (95% CI: 1.05-1.13, P < 0.001 using the Fine-Gray model with death regarded as a competing event). In conclusion, in-hospital mortality was similar between the groups, and a longer duration of carbapenem use as the initial antimicrobial therapy resulted in a higher risk of detection of new MDR pathogens.IMPORTANCEWe found no statistical difference in mortality with the empirical use of carbapenems as initial antimicrobial therapy among critically ill patients with bacterial infections. Our study revealed a lower proportion of inappropriate initial antimicrobial administrations than those reported in previous studies. This result suggests the importance of appropriate risk assessment for the involvement of multidrug-resistant (MDR) pathogens and the selection of suitable antibiotics based on risk. To the best of our knowledge, this study is the first to demonstrate that a longer duration of carbapenem use as initial therapy is associated with a higher risk of subsequent detection of MDR pathogens. This finding underscores the importance of efforts to minimize the duration of carbapenem use as initial antimicrobial therapy when it is necessary.

Redefining awn development in rice through the breeding history of Japanese awn reduction.

The study challenges the conventional understanding of awn loss as a domestication syndrome, showing instead that many awned varieties continued to be widely grown in Japan until the early twentieth century and that selection for awn reduction was active at that time, demonstrating that awn loss is not a domestication syndrome but "a trait that emerged during crop improvement". Although selection for awnless mutants was carried out independently using different types of awned cultivars in the early twentieth century in Japan, awn loss was caused by the mutation in OsEPFL1. This suggests that a single mutant haplotype of OsEPFL1 was conserved in the genomes of different cultivars and subsequently selected within each line to meet the demand for awnless varieties. The study also conducts phylogenetic analyses of EPFL1 in 48 grass plants, revealing its unique involvement in awn formation in rice while potentially playing a different role in the domestication of other grass plants. Finally, an attempt is made to isolate an awn-forming gene that has not been identified from the awned rice cultivar "Omachi", which is still cultivated in Japan. The results presented in this paper provide a new perspective on domestication against the conventional understanding of awn development, shedding light on its potential as a useful organ for breeding to mitigate environmental stress.

α-Synuclein pathology in Drosophila melanogaster is exacerbated by haploinsufficiency of Rop: connecting STXBP1 encephalopathy with α-synucleinopathies.

Syntaxin-binding protein 1 (STXBP1) is a presynaptic protein that plays important roles in synaptic vesicle docking and fusion. STXBP1 haploinsufficiency causes STXBP1 encephalopathy (STXBP1-E), which encompasses neurological disturbances including epilepsy, neurodevelopmental disorders, and movement disorders. Most patients with STXBP1-E present with regression and movement disorders in adulthood, highlighting the importance of a deeper understanding of the neurodegenerative aspects of STXBP1-E. An in vitro study proposed an interesting new role of STXBP1 as a molecular chaperone for α-Synuclein (αSyn), a key molecule in the pathogenesis of neurodegenerative disorders. However, no studies have shown αSyn pathology in model organisms or patients with STXBP1-E. In this study, we used Drosophila models to examine the effects of STXBP1 haploinsufficiency on αSyn-induced neurotoxicity in vivo. We demonstrated that haploinsufficiency of Ras opposite (Rop), the Drosophila ortholog of STXBP1, exacerbates compound eye degeneration, locomotor dysfunction, and dopaminergic neurodegeneration in αSyn-expressing flies. This phenotypic aggravation was associated with a significant increase in detergent-insoluble αSyn levels in the head. Furthermore, we tested whether trehalose, which has neuroprotective effects in various models of neurodegenerative disorders, mitigates αSyn-induced neurotoxicity exacerbated by Rop haploinsufficiency. In flies expressing αSyn and carrying a heterozygous Rop null variant, trehalose supplementation effectively alleviates neuronal phenotypes, accompanied by a decrease in detergent-insoluble αSyn in the head. In conclusion, this study revealed that Rop haploinsufficiency exacerbates αSyn-induced neurotoxicity by altering the αSyn aggregation propensity. This study not only contributes to understanding the mechanisms of neurodegeneration in STXBP1-E patients, but also provides new insights into the pathogenesis of α-synucleinopathies.

Low mutation rate of spontaneous mutants enables detection of causative genes by comparing whole genome sequences.

In the early 1900s, mutation breeding to select varieties with desirable traits using spontaneous mutation was actively conducted around the world, including Japan. In rice, the number of fixed mutations per generation was estimated to be 1.38-2.25. Although this low mutation rate was a major problem for breeding in those days, in the modern era with the development of next-generation sequencing (NGS) technology, it was conversely considered to be an advantage for efficient gene identification. In this paper, we proposed an in silico approach using NGS to compare the whole genome sequence of a spontaneous mutant with that of a closely related strain with a nearly identical genome, to find polymorphisms that differ between them, and to identify the causal gene by predicting the functional variation of the gene caused by the polymorphism. Using this approach, we found four causal genes for the dwarf mutation, the round shape grain mutation and the awnless mutation. Three of these genes were the same as those previously reported, but one was a novel gene involved in awn formation. The novel gene was isolated from Bozu-Aikoku, a mutant of Aikoku with the awnless trait, in which nine polymorphisms were predicted to alter gene function by their whole-genome comparison. Based on the information on gene function and tissue-specific expression patterns of these candidate genes, Os03g0115700/LOC_Os03g02460, annotated as a short-chain dehydrogenase/reductase SDR family protein, is most likely to be involved in the awnless mutation. Indeed, complementation tests by transformation showed that it is involved in awn formation. Thus, this method is an effective way to accelerate genome breeding of various crop species by enabling the identification of useful genes that can be used for crop breeding with minimal effort for NGS analysis.

Locomotor Assays in Drosophila Larvae and Adult Flies.

Brain defects often lead to motor dysfunctions in humans. Drosophila melanogaster has been one of the most useful organisms in the study of neuronal biology due to its similarities with humans and has contributed to a more detailed understanding of the effects of genetic dysfunctions in the brain on behavior. We herein present modified protocols for the crawling assay with larvae and the climbing assay with adult flies that are simple to perform as well as a series of commands for ImageJ to automatically analyze data for the crawling assay.

Progress to Clarify How NOTCH3 Mutations Lead to CADASIL, a Hereditary Cerebral Small Vessel Disease.

Notch signaling is conserved in C. elegans, Drosophila, and mammals. Among the four NOTCH genes in humans, NOTCH1, NOTCH2, and NOTCH3 are known to cause monogenic hereditary disorders. Most NOTCH-related disorders are congenital and caused by a gain or loss of Notch signaling activity. In contrast, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) caused by NOTCH3 is adult-onset and considered to be caused by accumulation of the mutant NOTCH3 extracellular domain (N3ECD) and, possibly, by an impairment in Notch signaling. Pathophysiological processes following mutant N3ECD accumulation have been intensively investigated; however, the process leading to N3ECD accumulation and its association with canonical NOTCH3 signaling remain unknown. We reviewed the progress in clarifying the pathophysiological process involving mutant NOTCH3.

Knockdown of Chronophage in the nervous system mimics features of neurodevelopmental disorders caused by BCL11A/B variants.

Neurodevelopmental disorders (NDD) are a group of disorders that include intellectual disability. Although several genes have been implicated in NDD, the molecular mechanisms underlying its pathogenesis remain unclear. Therefore, it is important to develop novel models to analyze the functions of NDD-causing genes in vivo. Recently, rare pathogenic variants of the B-cell lymphoma/leukemia11A/B (BCL11A/B) gene have been identified in several patients with NDD. Drosophila carries the Chronophage (Cph) gene, which has been predicted to be a homolog of BCL11A/B based on the conservation of the amino acid sequence. In the present study, we investigated whether nervous system-specific knockdown of Cph mimics NDD phenotypes in Drosophila. Nervous system-specific knockdown of Cph induced learning and locomotor defects in larvae and epilepsy-like behaviors in adults. The number of synaptic branches was also elevated in the larval neuromuscular junction without a corresponding increase in the number of boutons. Furthermore, the expression levels of putative target genes that are Drosophila homologs of the mammalian BCL11 target genes were decreased in Cph knockdown flies. These results suggest that Cph knockdown flies are a promising model for investigating the pathology of NDD-induced BCL11A/B dysfunction.

Factors associated with improvement in impaired consciousness during the acute phase of cerebral infarction: a prospective observational study.

[Purpose] In this study, we investigated factors that contribute to improvement in impaired consciousness following cerebral infarction. [Participants and Methods] This prospective observational study included 186 patients with cerebral infarction. We investigated 21 variables including the rehabilitation status to determine factors that contribute to improvement in impaired consciousness. [Results] Improvement in impaired consciousness was correlated with age, delirium, the Japan Coma Scale score at initiation of rehabilitation, worsening, cerebral edema, and standing practice. [Conclusion] We conclude that the aforementioned factors may serve as predictors of possible improvement and that standing practice may contribute to improvement in impaired consciousness.

Integrated genome-wide differentiation and association analyses identify causal genes underlying breeding-selected grain quality traits in japonica rice.

Improving grain quality is a primary objective in contemporary rice breeding. Japanese modern rice breeding has developed two different types of rice, eating and sake-brewing rice, with different grain characteristics, indicating the selection of variant gene alleles during the breeding process. Given the critical importance of promptly and efficiently identifying genes selected in past breeding for future molecular breeding, we conducted genome scans for divergence, genome-wide association studies, and map-based cloning. Consequently, we successfully identified two genes, OsMnS and OsWOX9D, both contributing to rice grain traits. OsMnS encodes a mannan synthase that increases the white core frequency in the endosperm, a desirable trait for sake brewing but decreases the grain appearance quality. OsWOX9D encodes a grass-specific homeobox-containing transcription factor, which enhances grain width for better sake brewing. Furthermore, haplotype analysis revealed that their defective alleles were selected in East Asia, but not Europe, during modern improvement. In addition, our analyses indicate that a reduction in grain mannan content during African rice domestication may also be caused a defective OsMnS allele due to breeding selection. This study not only reveals the delicate balance between grain appearance quality and nutrition in rice but also provides a new strategy for isolating causal genes underlying complex traits, based on the concept of "breeding-assisted genomics" in plants.

Global Trends in Highly Cited Studies in COVID-19 Research.

Importance: Since the onset of the COVID-19 outbreak, an extremely high number of studies have been published worldwide, with variable quality. Research trends of highly cited papers may enable identification of influential research, providing insights for new research ideas; it is therefore important to investigate trends and focus on more influential publications in COVID-19-related studies. Objective: To examine research trends of highly cited studies by conducting a bibliometric analysis of highly cited studies in the previous 2 months about COVID-19. Design, Setting, and Participants: In this cross-sectional study, Essential Science Indicators (ESI) and Web of Science (WOS) Core Collection were used to find studies with a focus on COVID-19 that were identified as highly cited studies from Clarivate Analytics. Highly cited studies were extracted from the ESI database bimonthly between January 2020 and December 2022. Bibliographic details were extracted from WOS and combined with ESI data using unique accession numbers. The number of highly cited studies was counted based on the fractional counting method. Data were analyzed from January through July 2023. Main Outcomes and Measures: The number of publications by research field, country, and institutional affiliation. Results: The number of published COVID-19-related highly cited studies was 14 studies in January to February 2020, peaked at 1292 studies in November to December 2021, and showed a downward trend thereafter, reaching 649 studies in November to December 2022. China had the highest number of highly cited studies per 2-month period until July to August 2020 (138.3 studies vs 103.7 studies for the US, the second highest country), and the US had the greatest number of highly cited studies afterward (159.9 studies vs 157.6 studies for China in September to October 2020). Subsequently, the number of highly cited studies per 2-month period published by China declined (decreasing from 179.7 studies in November to December 2020 to 40.7 studies in September to October 2022), and the UK produced the second largest number of such studies in May to June 2021 (171.3 studies). Similarly, the top 5 institutional affiliations in May to June 2020 by highly cited studies per 2-month period were from China (Huazhong University: 14.7 studies; University of Hong Kong: 6.8 studies; Wuhan University: 4.8 studies; Zhejiang University: 4.5 studies; Fudan University: 4.5 studies), while in November to December 2022, the top 5 institutions were in the US and UK (Harvard University: 15.0 studies; University College London: 11.0 studies; University of Oxford: 10.2 studies; University of London: 9.9 studies; Imperial College London: 5.8 studies). Conclusions and Relevance: This study found that the total number of highly cited studies related to COVID-19 peaked at the end of 2021 and showed a downward trend until the end of 2022, while the origin of these studies shifted from China to the US and UK.

The BCL-2 family protein BCL-RAMBO interacts and cooperates with GRP75 to promote its apoptosis signaling pathway.

The BCL-2 family protein BCL-RAMBO, also known as BCL2-like 13, anchors at the outer mitochondrial membrane and regulates apoptosis, mitochondrial fragmentation, and mitophagy. However, the mechanisms underlying the proapoptotic role of BCL-RAMBO remain unclear. In the present study, we demonstrated that BCL-RAMBO interacted with glucose-regulated protein 75 (GRP75), also known as heat shock protein family A member 9, and mortalin using co-immunoprecipitation and glutathione S-transferase-based pull-down assays. BCL-RAMBO interacted with GRP75 via its No BCL-2 homology domain. The interaction between BCL-RAMBO and GRP75 was confirmed by genetic interactions in Drosophila because a rough eye phenotype caused by the ectopic expression of BCL-RAMBO was partially suppressed by mutations in Hsc70-5, a mammalian GRP75 ortholog. In human embryonic kidney 293T cells, the co-expression of BCL-RAMBO and GRP75 facilitated an elevation in executioner caspase activity and poly (ADP-ribose) polymerase 1 (PARP-1) cleavage. In contrast, the knockdown of GRP75 suppressed elevated executioner caspase activity and PARP-1 cleavage in BCL-RAMBO-transfected cells. The mitochondrial release of cytochrome c induced by BCL-RAMBO was also attenuated by the knockdown of GRP75. These results indicate that GRP75 interacts with BCL-RAMBO and plays a crucial role in the BCL-RAMBO-dependent apoptosis signaling pathway.

Leukaemic cells expressing ETV6::FRK are sensitive to dasatinib in vivo.

ETV6::Fyn-related kinase (FRK), which is a Src family tyrosine-kinase-related fusion gene and firstly identified in our patient with paediatric high risk B cell precursor acute lymphoblastic leukaemia (B-ALL), has no evidence of efficacy of tyrosine kinase inhibitor in vivo. We performed functional analysis of ETV6::FRK to establish molecular targeting therapy and determined that dasatinib could abrogate proliferation activity of ETV6::FRK through the repression of FRK-STAT3/STAT5 pathway in vitro and significantly extended the survival time of the xenografted mice in vivo (p < 0.01). Our data support the potential of dasatinib as a therapeutic option for patients with B-ALL harboring FRK rearrangements.

Lipin knockdown in pan-neuron of Drosophila induces reduction of lifespan, deficient locomotive behavior, and abnormal morphology of motor neuron.

The Lipin family is evolutionarily conserved among insects and mammals, and its crucial roles in lipid synthesis and homeostatic control of energy balance have been well documented. This study investigated the function of Lipin in neuronal function and neurodegeneration. The GAL4/UAS system was used to knock down Lipin in the nervous system of Drosophila and investigate its behavioral and cellular phenotypes. The neuromuscular junction (NMJ) morphology was detected by immunostaining. Moreover, triacylglycerol and ATP levels were analyzed by using assay Kit. This study found that Lipin is localized almost in the cytoplasm of neurons in the brain lobe and ventral nerve cord, which are part of the central nervous system (CNS) of Drosophila melanogaster. Lipin knockdown larvae exhibit decreased locomotor activity, aberrant morphology of motor nerve terminals at NMJs, and reduced number and size of lipid droplets in the CNS. Furthermore, neuron-specific knockdown of Lipin leads to locomotor defects and a shortened lifespan, accompanied by a reduction in ATP levels in the adult stage. These results indicate that Lipin plays a crucial role in the CNS of Drosophila.

A Novel Drosophila-based Drug Repurposing Platform Identified Fingolimod As a Potential Therapeutic for TDP-43 Proteinopathy.

Pathogenic changes to TAR DNA-binding protein 43 (TDP-43) leading to alteration of its homeostasis are a common feature shared by several progressive neurodegenerative diseases for which there is no effective therapy. Here, we developed Drosophila lines expressing either wild type TDP-43 (WT) or that carrying an Amyotrophic Lateral Sclerosis /Frontotemporal Lobar Degeneration-associating G384C mutation that recapitulate several aspects of the TDP-43 pathology. To identify potential therapeutics for TDP-43-related diseases, we implemented a drug repurposing strategy that involved three consecutive steps. Firstly, we evaluated the improvement of eclosion rate, followed by the assessment of locomotive functions at early and late developmental stages. Through this approach, we successfully identified fingolimod, as a promising candidate for modulating TDP-43 toxicity. Fingolimod exhibited several beneficial effects in both WT and mutant models of TDP-43 pathology, including post-transcriptional reduction of TDP-43 levels, rescue of pupal lethality, and improvement of locomotor dysfunctions. These findings provide compelling evidence for the therapeutic potential of fingolimod in addressing TDP-43 pathology, thereby strengthening the rationale for further investigation and consideration of clinical trials. Furthermore, our study demonstrates the utility of our Drosophila-based screening pipeline in identifying novel therapeutics for TDP-43-related diseases. These findings encourage further scale-up screening endeavors using this platform to discover additional compounds with therapeutic potential for TDP-43 pathology.

Stable High-Capacity Elemental Sulfur Cathodes with Simple Process for Lithium Sulfur Batteries.

Lithium sulfur batteries are suitable for drones due to their high gravimetric energy density (2600 Wh/kg of sulfur). However, on the cathode side, high specific capacity with high sulfur loading (high areal capacity) is challenging due to the poor conductivity of sulfur. Shuttling of Li-sulfide species between the sulfur cathode and lithium anode also limits specific capacity. Sulfur-carbon composite active materials with encapsulated sulfur address both issues but require expensive processing and have low sulfur content with limited areal capacity. Proper encapsulation of sulfur in carbonaceous structures along with active additives in solution may largely mitigate shuttling, resulting in cells with improved energy density at relatively low cost. Here, composite current collectors, selected binders, and carbonaceous matrices impregnated with an active mass were used to award stable sulfur cathodes with high areal specific capacity. All three components are necessary to reach a high sulfur loading of 3.8 mg/cm2 with a specific/areal capacity of 805 mAh/g/2.2 mAh/cm2. Good adhesion between the carbon-coated Al foil current collectors and the composite sulfur impregnated carbon matrices is mandatory for stable electrodes. Swelling of the binders influenced cycling retention as electroconductivity dominated the cycling performance of the Li-S cells comprising cathodes with high sulfur loading. Composite electrodes based on carbonaceous matrices in which sulfur is impregnated at high specific loading and non-swelling binders that maintain the integrated structure of the composite electrodes are important for strong performance. This basic design can be mass produced and optimized to yield practical devices.

The Nup98::Nsd1 fusion gene induces CD123 expression in 32D cells.

The NUP98::NSD1 fusion gene is associated with extremely poor prognosis in patients with acute myeloid leukemia (AML). NUP98::NSD1 induces self-renewal and blocks differentiation of hematopoietic stem cells, leading to development of leukemia. Despite its association with poor prognosis, targeted therapy for NUP98::NSD1-positive AML is lacking, as the details of NUP98::NSD1 function are unknown. Here, we generated 32D cells (a murine interleukin-3 (IL-3)-dependent myeloid progenitor cell line) expressing mouse Nup98::Nsd1 to explore the function of NUP98::NSD1 in AML, including comprehensive gene expression analysis. We identified two properties of Nup98::Nsd1 + 32D cells in vitro. First, Nup98::Nsd1 promoted blocking of AML cell differentiation, consistent with a previous report. Second, Nup98::Nsd1 increased dependence on IL-3 for cell proliferation, due to overexpression of the alpha subunit of the IL-3 receptor (IL3-RA, also known as CD123). Consistent with our in vitro data, IL3-RA was also upregulated in samples from patients with NUP98::NSD1-positive AML. These results highlight CD123 as a potential new therapeutic target in NUP98::NSD1-positive AML.

In ovo chorioallantoic membrane assay as a xenograft model for pediatric rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common highly malignant pediatric soft tissue sarcoma. While recent multidisciplinary treatments have improved the 5­year survival rate of low/intermediate­risk patients to 70­90%, there are various complications that arise due to treatment­related toxicities. Immunodeficient mice­derived xenograft models have been widely used in cancer drug research; however, these models have some limitations, including i) they are time­consuming and expensive, ii) their use needs to be approved by animal experimental ethics committees, and iii) the inability to visualize where tumor cells or tissues were engrafted. The present study performed a chorioallantoic membrane (CAM) assay in fertilized chicken eggs, which is time­saving, simple, and easy to standardize and handle because of the high vascularization and the immature immune system of the fertilized eggs. The present study aimed to examine the usability of the CAM assay as a novel therapeutic model for the development of precision medicine for pediatric cancer. A protocol was developed for constructing cell line­derived xenograft (CDX) models using a CAM assay by transplanting RMS cells on the CAM. It was then examined as to whether these CDX models could be used as therapeutic drug evaluation models using vincristine (VCR) and human RMS cell lines. After grafting and culturing the RMS cell suspension on the CAM, three­dimensional proliferation over time was observed visually and by comparing volumes. VCR reduced the size of the RMS tumor on the CAM in a dose­dependent manner. Currently, treatment strategies based on patient­specific oncogenic backgrounds have not been adequately developed in the field of pediatric cancer. The establishment of a CDX model with the CAM assay may lead to the advancement of precision medicine and help formulate novel therapeutic strategies for intractable pediatric cancer.