Molecular and cellular organization of odorant binding protein genes in Drosophila.

Chemosensation is important for the survival and reproduction of animals. The odorant binding proteins (OBPs) are thought to be involved in chemosensation together with chemosensory receptors. While OBPs were initially considered to deliver hydrophobic odorants to olfactory receptors in the aqueous lymph solution, recent studies suggest more complex roles in various organs. Here, we use GAL4 transgenes to systematically analyze the expression patterns of all 52 members of the Obp gene family and 3 related chemosensory protein genes in adult Drosophila, focusing on chemosensory organs such as the antenna, maxillary palp, pharynx, and labellum, and other organs such as the brain, ventral nerve cord, leg, wing, and intestine. The OBPs were observed to express in diverse organs and in multiple cell types, suggesting that these proteins can indeed carry out diverse functional roles. Also, we constructed 10 labellar-expressing Obp mutants, and obtained behavioral evidence that these OBPs may be involved in bitter sensing. The resources we constructed should be useful for future Drosophila OBP gene family research.

Correlation between urine cytology results on the day after overnight continuous saline irrigation following transurethral resection of bladder tumor and bladder tumor recurrence.

PURPOSE: To investigate the relationship between urine cytology results after overnight continuous saline irrigation (OCSI) following transurethral resection of bladder tumor (TURBT) and bladder tumor recurrence in non-muscle invasive bladder cancer (NMIBC). MATERIALS AND METHODS: A retrospective study was conducted on patients diagnosed with NMIBC between 2016 and 2020 after undergoing TURBT at our hospital. All patients received OCSI following TURBT and had urine cytology test at postoperative 1 day. Urine cytology was classified into three groups: Negative, low-grade urothelial neoplasm (LGUN)+atypical urothelial cells (AUC), and suspicious for high-grade urothelial carcinoma (SHGUC)+high-grade urothelial carcinoma (HGUC). Recurrence-free survival (RFS) in each group was compared using the Kaplan-Meier method. Univariable and multivariable Cox regression analyses were performed to evaluate independent prognostic factors. RESULTS: A total of 172 patients were included in this study. Based on urine cytology group (after OCSI), RFS did not reach the median value in the Negative group. In the LGUN+AUC group, the median RFS was 615.00 days. In the SHGUC+HGUC group, the median RFS was 377.00 days. In survival analysis, the Negative group had a longer RFS than the SHGUC+HGUC group (p=0.013). However, Cox regression analysis showed that SHGUC+HGUC was not an independent prognostic factor for recurrence. CONCLUSIONS: Urine cytology results after OCSI following TURBT in NMIBC were associated with bladder tumor recurrence. Specifically, SHGUC or HGUC in urine cytology after OCSI showed earlier recurrence than negative cases. However, further research is needed to accurately determine whether it is an independent prognostic factor.

Gramicidin, a Bactericidal Antibiotic, Is an Antiproliferative Agent for Ovarian Cancer Cells.

Background and Objectives: Gramicidin, a bactericidal antibiotic used in dermatology and ophthalmology, has recently garnered attention for its inhibitory actions against cancer cell growth. However, the effects of gramicidin on ovarian cancer cells and the underlying mechanisms are still poorly understood. We aimed to elucidate the anticancer efficacy of gramicidin against ovarian cancer cells. Materials and Methods: The anticancer effect of gramicidin was investigated through an in vitro experiment. We analyzed cell proliferation, DNA fragmentation, cell cycle arrest and apoptosis in ovarian cancer cells using WST-1 assay, terminal deoxynucleotidyl transferase dUTP nick and labeling (TUNEL), DNA agarose gel electrophoresis, flow cytometry and western blot. Results: Gramicidin treatment induces dose- and time-dependent decreases in OVCAR8, SKOV3, and A2780 ovarian cancer cell proliferation. TUNEL assay and DNA agarose gel electrophoresis showed that gramicidin caused DNA fragmentation in ovarian cancer cells. Flow cytometry demonstrated that gramicidin induced cell cycle arrest. Furthermore, we confirmed via Western blot that gramicidin triggered apoptosis in ovarian cancer cells. Conclusions: Our results strongly suggest that gramicidin exerts its inhibitory effect on cancer cell growth by triggering apoptosis. Conclusively, this study provides new insights into the previously unexplored anticancer properties of gramicidin against ovarian cancer cells.

Gentian Violet Inhibits Cell Proliferation through Induction of Apoptosis in Ovarian Cancer Cells.

Gentian violet (GV) is known to have antibacterial and antifungal effects, but recent studies have demonstrated its inhibitory effects on the growth of several types of cancer cells. Here, we investigated the anticancer efficacy of GV in ovarian cancer cells. GV significantly reduced the proliferation of OVCAR8, SKOV3, and A2780 cells. Results of transferase dUTP nick and labeling (TUNEL) assay and Western blot assay indicated that the inhibitory effect of GV on ovarian cancer cells was due to the induction of apoptosis. Moreover, GV significantly increased reactive oxygen species (ROS) and upregulated the expression of p53, PUMA, BAX, and p21, critical components for apoptosis induction, in ovarian cancer cells. Our results suggest that GV is a novel antiproliferative agent and is worthy of exploration as a potential therapeutic agent for ovarian cancer.

Radiological Assessment of the Effects of Anterior Cervical Discectomy and Fusion on Distraction of the Posterior Ligamentum Flavum in Patients with Degenerative Cervical Spines.

BACKGROUND: This study aimed to assess the effects of anterior cervical discectomy and fusion (ACDF) on distraction of the posterior ligamentum flavum (LF) by increasing the intervertebral disc height and positioning a graft in patients with degenerative cervical spine disease. METHODS: Sixty-eight patients with degenerative cervical diseases who underwent single-level ACDF were included in the analysis. The intervertebral disc height, Cobb angle, and transverse thickness of the LF were measured, and magnetic resonance imaging was performed both preoperatively and 6 weeks postoperatively on each patient. Correlation analyses were performed to evaluate the relationships between age, sex, change in intervertebral disc height, Cobb angle, and position of the intervertebral implant according to the postoperative change in LF thickness. The position of the intervertebral implant was categorized as anterior, middle, or posterior. We also evaluated radiological effects according to the implant position. RESULTS: The mean intervertebral disc height increased from 5.88 mm preoperatively to 7.49 mm postoperatively. The Cobb angle was 0.88° preoperatively and 1.43° postoperatively. Age (p = 0.551), sex (p = 0.348), position of cage (p = 0.312), pre- and postoperative intervertebral disc height (p = 0.850, p = 0.900), Cobb angle (p = 0.977, p = 0.460), and LF thickness (p = 0.060, p = 1.00) were not related to changes in postoperative LF thickness. Postoperative increase in disc height was related to Cobb angle (r = 0.351, p = 0.038). No other factors were significantly related. The position of the cage was not related with the change of Cobb angle (p = 0.91), LF thickness (p = 0.31), or disc height (p = 0.54). CONCLUSIONS: Change in the intervertebral disc height and the position of the intervertebral implant after ACDF did not affect the thickness of the LF after surgery in patients with degenerative cervical spine disease.

Spatiotemporal organization of enteroendocrine peptide expression in Drosophila.

The digestion of food and absorption of nutrients occurs in the gut. The nutritional value of food and its nutrients is detected by enteroendocrine cells, and peptide hormones produced by the enteroendocrine cells are thought to be involved in metabolic homeostasis, but the specific mechanisms are still elusive. The enteroendocrine cells are scattered over the entire gastrointestinal tract and can be classified according to the hormones they produce. We followed the changes in combinatorial expression of regulatory peptides in the enteroendocrine cells during metamorphosis from the larva to the adult fruit fly, and re-confirmed the diverse composition of enteroendocrine cell populations. Drosophila enteroendocrine cells appear to differentially regulate peptide expression spatially and temporally depending on midgut region and developmental stage. In the late pupa, Notch activity is known to determine which peptides are expressed in mature enteroendocrine cells of the posterior midgut, and we found that the loss of Notch activity in the anterior midgut results in classes of enteroendocrine cells distinct from the posterior midgut. These results suggest that enteroendocrine cells that populate the fly midgut can differentiate into distinct subtypes that express different combinations of peptides, which likely leads to functional variety depending on specific needs.

Identification and characterization of GAL4 drivers that mark distinct cell types and regions in the Drosophila adult gut.

The gastrointestinal tract in the adult Drosophila serves as a model system for exploring the mechanisms underlying digestion, absorption and excretion, stem cell plasticity, and inter-organ communication, particularly through the gut-brain axis. It is also useful for studying the cellular and adaptive responses to dietary changes, alterations in microbiota and immunity, and systematic and endocrine signals. Despite the various cell types and distinct regions in the gastrointestinal tract, few tools are available to target and manipulate the activity of each cell type and region, and their gene expression. Here, we report 353 GAL4 lines and several split-GAL4 lines that are expressed in enteric neurons (ENs), progenitors (ISCs and EBs), enterocytes (ECs), enteroendocrine cells (EEs), or/and other cell types that are yet to be identified in distinct regions of the gut. We had initially collected approximately 600 GAL4 lines that may be expressed in the gut based on RNA sequencing data, and then crossed them to UAS-GFP to perform immunohistochemistry to identify those that are expressed selectively in the gut. The cell types and regional expression patterns that are associated with the entire set of GAL4 drivers and split-GAL4 combinations are annotated online at http://kdrc.kr/index.php (K-Gut Project). This GAL4 resource can be used to target specific populations of distinct cell types in the fly gut, and therefore, should permit a more precise investigation of gut cells that regulate important biological processes.

Deep Learning-Based Decision-Tree Classifier for COVID-19 Diagnosis From Chest X-ray Imaging.

The global pandemic of coronavirus disease 2019 (COVID-19) has resulted in an increased demand for testing, diagnosis, and treatment. Reverse transcription polymerase chain reaction (RT-PCR) is the definitive test for the diagnosis of COVID-19; however, chest X-ray radiography (CXR) is a fast, effective, and affordable test that identifies the possible COVID-19-related pneumonia. This study investigates the feasibility of using a deep learning-based decision-tree classifier for detecting COVID-19 from CXR images. The proposed classifier comprises three binary decision trees, each trained by a deep learning model with convolution neural network based on the PyTorch frame. The first decision tree classifies the CXR images as normal or abnormal. The second tree identifies the abnormal images that contain signs of tuberculosis, whereas the third does the same for COVID-19. The accuracies of the first and second decision trees are 98 and 80%, respectively, whereas the average accuracy of the third decision tree is 95%. The proposed deep learning-based decision-tree classifier may be used in pre-screening patients to conduct triage and fast-track decision making before RT-PCR results are available.

[Secondary Publication] Standard Operating Procedure for Post-mortem Inspection in a Focus on Coronavirus Disease-19: the Korean Society for Legal Medicine.

Coronavirus disease 2019 (COVID-19) is a respiratory syndrome caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and emerged in Wuhan, China, in late 2019. It resulted in a worldwide pandemic, and spread through community transmission in the Republic of Korea (ROK). In the ROK, SARS-CoV-2 is categorized as a first-degree infectious disease of the legal communicable disease present. The Korean Society for Legal Medicine (KSLM) is the sole official academic association of forensic professionals in the ROK. As such, this society has played an important role in forensic medicine and science in the ROK. Therefore, KSLM suggests a standard operating procedure for the postmortem inspection in a focus on COVID-19. This article includes the background of this suggested standard operation procedure, basic principles for postmortem inspections of individuals suggested of having an infectious disease, and specific procedures according to the probability level of SARS-CoV-2 infection.

Cellular Basis of Bitter-Driven Aversive Behaviors in Drosophila Larva.

Feeding, a critical behavior for survival, consists of a complex series of behavioral steps. In Drosophila larvae, the initial steps of feeding are food choice, during which the quality of a potential food source is judged, and ingestion, during which the selected food source is ingested into the digestive tract. It remains unclear whether these steps employ different mechanisms of neural perception. Here, we provide insight into the two initial steps of feeding in Drosophila larva. We find that substrate choice and ingestion are determined by independent circuits at the cellular level. First, we took 22 candidate bitter compounds and examined their influence on choice preference and ingestion behavior. Interestingly, certain bitter tastants caused different responses in choice and ingestion, suggesting distinct mechanisms of perception. We further provide evidence that certain gustatory receptor neurons (GRNs) in the external terminal organ (TO) are involved in determining choice preference, and a pair of larval pharyngeal GRNs is involved in mediating both avoidance and suppression of ingestion. Our results show that feeding behavior is coordinated by a multistep regulatory process employing relatively independent neural elements. These findings are consistent with a model in which distinct sensory pathways act as modulatory circuits controlling distinct subprograms during feeding.

Rational Design of Carbon Nanomaterials for Electrochemical Sodium Storage and Capture.

Electrochemical sodium storage and capture are considered an attractive technology owing to the natural abundance, low cost, safety, and cleanness of sodium, and the higher efficiency of the electrochemical system compared to fossil-fuel-based counterparts. Considering that the sodium-ion chemistry often largely deviates from the lithium-based one despite the physical and chemical similarities, the architecture and chemical structure of electrode materials should be designed for highly efficient sodium storage and capture technologies. Here, the rational design in the structure and chemistry of carbon materials for sodium-ion batteries (SIBs), sodium-ion capacitors (SICs), and capacitive deionization (CDI) applications is comprehensively reviewed. Types and features of carbon materials are classified into ordered and disordered carbons as well as nanodimensional and nanoporous carbons, covering the effect of synthesis parameters on the carbon structure and chemistry. The sodium storage mechanism and performance of these carbon materials are correlated with the key structural/chemical factors, including the interlayer spacing, crystallite size, porous characteristics, micro/nanostructure, morphology, surface chemistry, heteroatom incorporation, and hybridization. Finally, perspectives on current impediment and future research directions into the development of practical SIBs, SICs, and CDI are also provided.

Ionotropic Receptor 76b Is Required for Gustatory Aversion to Excessive Na+ in Drosophila.

Avoiding ingestion of excessively salty food is essential for cation homeostasis that underlies various physiological processes in organisms. The molecular and cellular basis of the aversive salt taste, however, remains elusive. Through a behavioral reverse genetic screening, we discover that feeding suppression by Na+-rich food requires Ionotropic Receptor 76b (Ir76b) in Drosophila labellar gustatory receptor neurons (GRNs). Concentrated sodium solutions with various anions caused feeding suppression dependent on Ir76b. Feeding aversion to caffeine and high concentrations of divalent cations and sorbitol was unimpaired in Ir76b-deficient animals, indicating sensory specificity of Ir76b-dependent Na+ detection and the irrelevance of hyperosmolarity-driven mechanosensation to Ir76b-mediated feeding aversion. Ir76b-dependent Na+-sensing GRNs in both L- and s-bristles are required for repulsion as opposed to the previous report where the L-bristle GRNs direct only low-Na+ attraction. Our work extends the physiological implications of Ir76b from low-Na+ attraction to high-Na+ aversion, prompting further investigation of the physiological mechanisms that modulate two competing components of Na+-evoked gustation coded in heterogeneous Ir76b-positive GRNs.

Involvement of a Gr2a-Expressing Drosophila Pharyngeal Gustatory Receptor Neuron in Regulation of Aversion to High-Salt Foods.

Regulation of feeding is essential for animal survival. The pharyngeal sense organs can act as a second checkpoint of food quality, due to their position between external taste organs such as the labellum which initially assess food quality, and the digestive tract. Growing evidence provides support that the pharyngeal sensory neurons regulate feeding, but much is still unknown. We found that a pair of gustatory receptor neurons in the LSO, a Drosophila adult pharyngeal organ which expresses four gustatory receptors, is involved in feeding inhibition in response to high concentrations of sodium ions. RNAi experiments and mutant analysis showed that the gustatory receptor Gr2a is necessary for this process. This feeding preference determined by whether a food source is perceived as appetizing or not is influenced by nutritional conditions, such that when the animal is hungry, the need for energy dominates over how appealing the food source is. Our results provide experimental evidence that factors involved in feeding function in a context-dependent manner.

A Pair of Pharyngeal Gustatory Receptor Neurons Regulates Caffeine-Dependent Ingestion in Drosophila Larvae.

The sense of taste is an essential chemosensory modality that enables animals to identify appropriate food sources and control feeding behavior. In particular, the recognition of bitter taste prevents animals from feeding on harmful substances. Feeding is a complex behavior comprised of multiple steps, and food quality is continuously assessed. We here examined the role of pharyngeal gustatory organs in ingestion behavior. As a first step, we constructed a gustatory receptor-to-neuron map of the larval pharyngeal sense organs, and examined corresponding gustatory receptor neuron (GRN) projections in the larval brain. Out of 22 candidate bitter compounds, we found 14 bitter compounds that elicit inhibition of ingestion in a dose-dependent manner. We provide evidence that certain pharyngeal GRNs are necessary and sufficient for the ingestion response of larvae to caffeine. Additionally, we show that a specific pair of pharyngeal GRNs, DP1, responds to caffeine by calcium imaging. In this study we show that a specific pair of GRNs in the pharyngeal sense organs coordinates caffeine sensing with regulation of behavioral responses such as ingestion. Our results indicate that in Drosophila larvae, the pharyngeal GRNs have a major role in sensing food palatability to regulate ingestion behavior. The pharyngeal sense organs are prime candidates to influence ingestion due to their position in the pharynx, and they may act as first level sensors of ingested food.

Maintenance of Membrane Integrity and Permeability Depends on a Patched-Related Protein in Caenorhabditis elegans.

Membrane integrity is critical for cell survival, defects of which cause pathological symptoms such as metabolic diseases. In this study, we used ethanol sensitivity of the nematode Caenorhabditis elegans to identify genetic factors involved in membrane integrity. InC. elegans, acute exposure to a high concentration (7% v/v) of ethanol changes membrane permeability, as measured by propidium iodide staining, and causes paralysis. We used the timing of complete paralysis as an indicator for alteration of membrane integrity in our genetic screen, and identified ptr-6 as a gene that confers ethanol resistance when mutated. PTR-6 is a patched-related protein and contains a sterol sensing domain. Inhibition of two PTR-encoding genes,ptr-15 and ptr-23, and mboa-1, encoding an Acyl Co-A: cholesterol acyltransferase homolog, restored ethanol sensitivity of the ptr-6 mutant, suggesting that these ptr genes and mboa-1 are involved in the maintenance of membrane integrity and permeability. Our results suggest that C. elegans can be used as a model system to identify factors involved in metabolic diseases and to screen for therapeutic drugs.

A subset of enteroendocrine cells is activated by amino acids in the Drosophila midgut.

The intestine is involved in digestion and absorption, as well as the regulation of metabolism upon sensation of the internal intestinal environment. Enteroendocrine cells are thought to mediate these internal intestinal chemosensory functions. Using the CaLexA (calcium-dependent nuclear import of LexA) method, we examined the enteroendocrine cell populations that are activated when flies are subjected to various dietary conditions such as starvation, sugar, high fat, protein, or pathogen exposure. We find that a specific subpopulation of enteroendocrine cells in the posterior midgut which express Dh31 and tachykinin are activated by the presence of proteins and amino acids.

Behavioral Analysis of Bitter Taste Perception in Drosophila Larvae.

Insect larvae, which recognize food sources through chemosensory cues, are a major source of global agricultural loss. Gustation is an important factor that determines feeding behavior, and the gustatory receptors (Grs) act as molecular receptors that recognize diverse chemicals in gustatory receptor neurons (GRNs). The behavior of Drosophila larvae is relatively simpler than the adult fly, and a gustatory receptor-to-neuron map was established in a previous study of the major external larval head sensory organs. Here, we extensively study the bitter taste responses of larvae using 2-choice behavioral assays. First, we tested a panel of 23 candidate bitter compounds to compare the behavioral responses of larvae and adults. We define 9 bitter compounds which elicit aversive behavior in a dose-dependent manner. A functional map of the larval GRNs was constructed with the use of Gr-GAL4 lines that drive expression of UAS-tetanus toxin and UAS-VR1 in specific gustatory neurons to identify bitter tastants-GRN combinations by suppressing and activating discrete subsets of taste neurons, respectively. Our results suggest that many gustatory neurons act cooperatively in larval bitter sensing, and that these neurons have different degrees of responsiveness to different bitter compounds.

The Mosquito Repellent Citronellal Directly Potentiates Drosophila TRPA1, Facilitating Feeding Suppression.

Citronellal, a well-known plant-derived mosquito repellent, was previously reported to repel Drosophila melanogaster via olfactory pathways involving but not directly activating Transient Receptor Potential Ankyrin 1 (TRPA1). Here, we show that citronellal is a direct agonist for Drosophila and human TRPA1s (dTRPA1 and hTRPA1) as well as Anopheles gambiae TRPA1 (agTRPA1). Citronellal-induced activity is isoform-dependent for Drosophila and Anopheles gambiae TRPA1s. The recently identified dTRPA1(A) and ag-TRPA1(A) isoforms showed citronellal-provoked currents with EC50s of 1.0 B1 0.2 and 0.1 B1 0.03 mM, respectively, in Xenopus oocytes, while the sensitivities of TRPA1(B)s were much inferior to those of TRPA1(A)s. Citronellal dramatically enhanced the feeding-inhibitory effect of the TRPA1 agonist N-methylmaleimide (NMM) in Drosophila at an NMM concentration that barely repels flies. Thus, citronellal can promote feeding deterrence of fruit flies through direct action on gustatory dTRPA1, revealing the first isoform-specific function for TRPA1(A).

Isoform-specific expression of the neuropeptide orcokinin in Drosophila melanogaster.

Orcokinins are neuropeptides that have been identified in diverse arthropods. In some species, an orcokinin gene encodes two isoforms of mature orcokinin peptide through alternative mRNA splicing. The existence of two orcokinin isoforms was predicted in Drosophila melanogaster as well, but the expression pattern of both isoforms has not been characterized. Here, we use in situ hybridization, antibody staining, and enhancer fusion GAL4 transgenic flies to examine the expression patterns of the A and B forms of orcokinin, and provide evidence that they are expressed differentially in the central nervous system (CNS) and the intestinal enteroendocrine system. The orcokinin A isoform is mainly expressed in the CNS of both larvae and adults. The A form is expressed in 5 pairs of neurons in abdominal neuromeres 1-5 of the larval CNS. In the adult brain, the A form is expressed in one pair of neurons in the posteriorlateral protocerebrum, and an additional four pairs of neurons located near the basement of the accessory medulla. Orcokinin A expression is also observed in two pairs of neurons in the ventral nerve cord (VNC). The orcokinin B form is mainly expressed in intestinal enteroendocrine cells in the larva and adult, with additional expression in one unpaired neuron in the adult abdominal ganglion. Together, our results provide elucidation of the existence and differential expression of the two orcokinin isoforms in the Drosophila brain and gut, setting the stage for future functional studies of orcokinins utilizing the genetically amenable fly model.

Combined Effect of Kimchi Powder and Onion Peel Extract on Quality Characteristics of Emulsion Sausages Prepared with Irradiated Pork.

This study was conducted to investigate the effects of kimchi powder and onion peel extract on the quality characteristics of emulsion sausage manufactured with irradiated pork. The emulsion sausages were formulated with 2% kimchi powder and/or 0.05% onion peel extract. The changes in pH value of all treatments were similar, depending on storage periods. The addition of kimchi powder increased the redness and yellowness of the emulsion sausage. The addition of onion peel extract decreased the thiobarbituric acid reactive substances value of the emulsion sausages prepared with irradiated pork. The volatile basic nitrogen value of the emulsion sausage prepared with kimchi powder was the highest, whereas that of the emulsion sausage prepared with onion peel extract was the lowest. The treatment without kimchi powder or onion peel extract and the treatments prepared with onion peel extract showed lower microbial populations than the other treatment. Sensory evaluations indicated that a higher acceptability was attained when kimchi powder was added to the emulsion sausages manufactured with irradiated pork. In conclusion, our results suggest that combined use of kimchi powder and onion peel extract could improve quality characteristics and shelf stability of the emulsion sausage formulated with irradiated pork during chilled storage.