Unifying community-wide whole-brain imaging datasets enables robust automated neuron identification and reveals determinants of neuron positioning in C. elegans.

We develop a data harmonization approach for C. elegans volumetric microscopy data, still or video, consisting of a standardized format, data pre-processing techniques, and a set of human-in-the-loop machine learning based analysis software tools. We unify a diverse collection of 118 whole-brain neural activity imaging datasets from 5 labs, storing these and accompanying tools in an online repository called WormID (wormid.org). We use this repository to generate a statistical atlas that, for the first time, enables accurate automated cellular identification that generalizes across labs, approaching human performance in some cases. We mine this repository to identify factors that influence the developmental positioning of neurons. To facilitate communal use of this repository, we created open-source software, code, web-based tools, and tutorials to explore and curate datasets for contribution to the scientific community. This repository provides a growing resource for experimentalists, theorists, and toolmakers to investigate neuroanatomical organization and neural activity across diverse experimental paradigms, develop and benchmark algorithms for automated neuron detection, segmentation, cell identification, tracking, and activity extraction, and inform models of neurobiological development and function.

Automated cell annotation in multi-cell images using an improved CRF_ID algorithm.

Cell identification is an important yet difficult process in data analysis of biological images. Previously, we developed an automated cell identification method called CRF_ID and demonstrated its high performance in C. elegans whole-brain images (Chaudhary et al, 2021). However, because the method was optimized for whole-brain imaging, comparable performance could not be guaranteed for application in commonly used C. elegans multi-cell images that display a subpopulation of cells. Here, we present an advance CRF_ID 2.0 that expands the generalizability of the method to multi-cell imaging beyond whole-brain imaging. To illustrate the application of the advance, we show the characterization of CRF_ID 2.0 in multi-cell imaging and cell-specific gene expression analysis in C. elegans. This work demonstrates that high accuracy automated cell annotation in multi-cell imaging can expedite cell identification and reduce its subjectivity in C. elegans and potentially other biological images of various origins.

National Trends in Rotavirus Enteritis among Infants in South Korea, 2010-2021: A Nationwide Cohort.

Rotavirus causes a gastrointestinal tract infection that primarily affects young children. Due to the COVID-19 pandemic, individuals infected with the virus were subjected to quarantine measures, with strong emphases on personal hygiene and social distancing. The present study aimed to evaluate the characteristics of rotaviruses and compare the prevalence of rotavirus infection before and during the COVID-19 pandemic. This nationwide representative study was conducted using data acquired from the National Health Insurance Service between 2010 and 2021. We analyzed the data of patients younger than 12 months old who were diagnosed with rotavirus enteritis between January 2010 and December 2021. During the study period, a total of 34,487 infants younger than 12 months were diagnosed with rotavirus enteritis in South Korea. During the two-year COVID-19 pandemic (2020-2021), the rate of decline was significant (5843 cases in 2010 and 1125 in 2019), and by 2021, the total number of patients was almost negligible, as there are only 18 cases in 2021. A significant increase in the ratio of low birth weight (LBW) infants of inpatient department was observed from 2010 to 2021 (4.86% in 2010; 7.77% in 2019; and 23.08% in 2021), indicating that LBW infants are more vulnerable than infants born with normal weight. Average medical expenses related to rotavirus infections also declined significantly from 3,789,443,998 per year (pre-pandemic) to 808,353,795 per year (pandemic). Overall, personal hygiene and social distancing may play important roles in reducing rotavirus infections. However, further studies are needed to determine whether this decreasing trend persists after quarantine and whether the social life of individuals resumes.

An expanded GCaMP reporter toolkit for functional imaging in Caenorhabditis elegans.

In living organisms, changes in calcium flux are integral to many different cellular functions and are especially critical for the activity of neurons and myocytes. Genetically encoded calcium indicators (GECIs) have been popular tools for reporting changes in calcium levels in vivo. In particular, GCaMPs, derived from GFP, are the most widely used GECIs and have become an invaluable toolkit for neurophysiological studies. Recently, new variants of GCaMP, which offer a greater variety of temporal dynamics and improved brightness, have been developed. However, these variants are not readily available to the Caenorhabditis elegans research community. This work reports a set of GCaMP6 and jGCaMP7 reporters optimized for C. elegans studies. Our toolkit provides reporters with improved dynamic range, varied kinetics, and targeted subcellular localizations. Besides optimized routine uses, this set of reporters is also well suited for studies requiring fast imaging speeds and low magnification or low-cost platforms.

Bacteriology and trends of antimicrobial resistance of Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus in otitis media: A retrospective data analysis.

OBJECTIVES: Otitis media (OM) has a high prevalence worldwide and the treatment is crucial because hearing loss in children can lead to growth disorders such as language development disorders. The aim of this study is to analyse the changes in bacterial strains and the trends of antibiotic susceptibility in otitis media with effusion (OME), chronic otitis media (COM) and cholesteatomatous otitis media (Chole OM). DESIGN: This retrospective study involved 2926 patients diagnosed with OME, COM, or Chole OM between January 2000 and December 2020. The clinical data were collected and analysed through chart review from May 2021 to July 2021. SETTING: Two tertiary medical centres. PARTICIPANTS: The 2926 OM patients. MAIN OUTCOMES AND MEASURES: An otorrhea sample was collected on the first day of their hospital visit. Middle ear fluid samples for bacterial culture and antibiotics susceptibility test were collected from patients during middle ear surgery, including ventilation tube insertion. In each type of OM, the distribution of bacterial strains in the 2000s and the 2010s was compared. In addition, changes in the detection rate of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PA) and trends in their antibiotic susceptibility over the last 10 years were analysed. RESULTS: The most frequently detected bacterial strains in OME, COM and Chole OM over the study period were coagulase-negative Staphylococcus (CNS) (29.6%), MRSA (24.1%), and PA (20.1%). Compared to the 2000s, the proportion of non-typable Haemophilus influenzae in OME and MRSA in COM increased in the 2010s (27.4%-31.6% and 1.5%-29.5%, respectively). In total three types of OM, although there was no significant trend of change in detection rates of MRSA, PA, and multidrug resistant-P. aeruginosa (MDR-PA) during the last 10 years, resistance to the Quinolone class of MRSA and PA tended to increase (P < .05). CONCLUSIONS: The composition of bacterial strains in each types of OM has changed over the past 20 years. Additionally, the antibiotic resistance of MRSA and PA has increased in the last decade. Therefore, when using empirical antibiotics in necessary situations, it is necessary to change to an appropriate antibiotic through a bacterial culture test and antimicrobial susceptibility test.

An expanded GCaMP reporter toolkit for functional imaging in C. elegans.

In living organisms, changes in calcium flux are integral to many different cellular functions and are especially critical for the activity of neurons and myocytes. Genetically encoded calcium indicators (GECIs) have been popular tools for reporting changes in calcium levels in vivo . In particular, GCaMP, derived from GFP, are the most widely used GECIs and have become an invaluable toolkit for neurophysiological studies. Recently, new variants of GCaMP, which offer a greater variety of temporal dynamics and improved brightness, have been developed. However, these variants are not readily available to the Caenorhabditis elegans research community. This work reports a set of GCaMP6 and jGCaMP7 reporters optimized for C. elegans studies. Our toolkit provides reporters with improved dynamic range, varied kinetics, and targeted subcellular localizations. Besides optimized routine uses, this set of reporters are also well-suited for studies requiring fast imaging speeds and low magnification or low-cost platforms.

Hearing Aid Effects and Satisfaction in Patients with Tinnitus.

This study aimed to evaluate the effectiveness of and satisfaction with hearing aids as a treatment option for tinnitus with hearing loss. METHODS: This retrospective study used the tinnitus handicap inventory (THI), the satisfaction with amplification in daily life (SADL) questionnaire, and a medical chart review. A total of 116 patients treated between August 2018 and December 2020 were included. All patients with tinnitus and hearing loss underwent the same counseling sessions. Sixty patients chose to have hearing aids fitted (aided group), whereas 56 patients chose not to (non-aided group). Both the groups had similar audiometric configurations, durations of tinnitus, and ages. Structured interviews were performed, with various measures evaluated using the visual analog scale (VAS) and the THI questionnaire, before and six months after fitting the hearing aids. The SADL questionnaire was administered 6 months after fitting the hearing aids. RESULTS: The patients' THI scores reduced 6 months after the counseling, but the improvement in the THI scores was only significant in the group that received hearing aids. There were significant differences between the VAS scores of the two groups, and the changes in the VAS scores in the groups were statistically different. Subjective satisfaction with a hearing aid increased with improvements to tinnitus-related discomfort. CONCLUSION: The study's results indicated that patients with hearing loss and tinnitus can be treated with hearing aids and counseling.

Precipitation-based colorimetric multiplex immunoassay in hydrogel particles.

Despite a growing demand for more accessible diagnostic technologies, current methods struggle to simultaneously detect multiple analytes with acceptable sensitivity and portability. Colorimetric assays have been widely used due to their simplicity of signal readout, but the lack of multiplexibility has been a perpetual constraint. Meanwhile, particle-based assays offer multiplex detection by assigning an identity code to each analyte, but they often require lab-based equipment unsuitable for portable diagnostics. Here, by merging the two approaches, this paper reports a colorimetric multiplex immunoassay based on hydrogel microparticles that achieves the best of both worlds. The low-cost portable multiplex assay demonstrates sensitivities as high as and dynamic ranges greater than the lab-based enzyme-linked immunosorbent assay (ELISA). These critical advances are made possible by local precipitation and amplification of insoluble colour dyes inside the hydrogel networks. For the first time, enzymatic accumulation of colour dyes in hydrogel particles is reported and the kinetics of colour development is characterized in this work. By taking advantage of the colour signals in the visible spectrum, the hydrogel microparticles were imaged and analysed using low-cost portable devices. The colorimetric multiplex immunoassay was used to successfully detect three target biomarkers of preeclampsia and validated clinically using healthy and patient-derived plasma samples.

Elimination of Unreacted Acrylate Double Bonds in the Polymer Networks of Microparticles Synthesized via Flow Lithography.

Flow lithography (FL), a versatile technique used to synthesize anisotropic multifunctional microparticles, has attracted substantial interest, given that the resulting particles with complex geometries and multilayered biochemical functionalities can be used in a wide variety of applications. However, after this process, there are double bonds remaining from the cross-linkable groups of monomers. The unreacted cross-linkable groups can affect the particles' biochemical properties. Here, we verify that the microparticles produced by FL contain a significant number of unreacted acrylate double bonds (UADBs), which could cause irreversible biochemical changes in the particle and pernicious effects to biological systems. We also confirm that the particles contain a considerable number of UADBs, regardless of the various synthetic (lithographic) conditions that can be used in a typical FL process. We present an effective way to eliminate a substantial amount of UADBs after synthesis by linking biochemically inert poly(ethylene glycol) based on click chemistry. We verify that eliminating UADBs by using this click chemistry approach can efficiently resolve problems, such as the occurrence of random reactions and the cytotoxicity of UADBs.

Degassed micromolding lithography for rapid fabrication of anisotropic hydrogel microparticles with high-resolution and high uniformity.

Replica molding techniques, which are used to synthesize microparticles inside anisotropic micromolds, have been developed to enable the mass production of hydrogel particles. However, these techniques are limited in their ability to synthesize only a narrow range of particle compositions and shapes because of the difficulty in loading precursors into the micromolds as well as the low particle homogeneity due to the uneven evaporation of the precursors. Herein, we describe a simple yet powerful technique, called degassed micromolding lithography, which can load precursors within 1 min regardless of the wettability. This technique is based on the gas-solubility of a degassed micromold that acts as a suction pump to completely fill the mold by drawing precursor liquids in. The semi-closed system within the micromold prevents the uneven evaporation of the precursor, which is essential for the production of homogeneous particles. Furthermore, controlled uniformity of the hydrogel microparticles (C.V. < 2%) can be achieved by engineering the design of the micromold array.

Linker-free antibody conjugation for sensitive hydrogel microparticle-based multiplex immunoassay.

Multiplex immunoassay, or the simultaneous detection of multiple proteins in a single sample, is expected to enable a new level of protein analysis across diverse disciplines, such as medical diagnostics and biomarker discovery. A bead-based assay using graphically encoded hydrogel microparticles synthesized using stop flow lithography has been a promising platform because of its high multiplex capacity and its superior sensitivity and dynamic range compared to the enzyme-linked immunosorbent assay (ELISA). The functionalization of these particles has been dependent on the use of a heterobifunctional linker to conjugate the capture antibodies on the hydrogel. However, the linker chemistry, which is based on linking the primary amine groups of antibodies with acrylate functional groups on the hydrogel monomer, is vulnerable to hydrolysis in aqueous conditions and can potentially damage the antigen binding region of the antibody. In this work, we introduce a new antibody conjugation method that avoids the use of the linker and further enhances the sensitivity of hydrogel microparticle-based immunoassays. Disulfide bonds in antibodies are reduced to liberate free thiols, which can directly bond with the double bonds remaining in the hydrogel after particle synthesis. We characterize the optimal reduction of antibodies for producing the highest detection signal and demonstrate an average two-fold improvement in sensitivity compared to the linker-dependent antibody conjugation method. Lastly, we validate the accuracy and specificity of the multiplex assays with particles conjugated with antibodies using the linker-free method.

Post-synthesis functionalized hydrogel microparticles for high performance microRNA detection.

Encoded hydrogel microparticles, synthesized by Stop Flow Lithography (SFL), have shown great potential for microRNA assays for their capability to provide high multiplexing capacity and solution-like hybridization kinetics. However, due to the low conversion of copolymerization during particle synthesis, current hydrogel microparticles can only utilize ∼10% of the input probes that functionalize the particles for miRNA assay. Here, we present a novel method of functionalizing hydrogel microparticles after particle synthesis by utilizing unconverted double bonds remaining inside the hydrogel particles to maximize functional probe incorporation and increase the performance of miRNA assay. This allows covalent bonding of functional probes to the hydrogel network after particle synthesis. Because of the abundance of the unconverted double bonds and accessibility of all probes, the probe density increases about 8.2 times compared to that of particles functionalized during the synthesis. This results lead to an enhanced miRNA assay performance that improves the limit of detection from 4.9 amol to 1.5 amol. In addition, higher specificity and shorter assay time are achieved compared to the previous method. We also demonstrate a potential application of our particles by performing multiplexed miRNA detections in human plasma samples.

Structural Basis of Karrikin and Non-natural Strigolactone Perception in Physcomitrella patens.

In plants, strigolactones are perceived by the dual receptor-hydrolase DWARF14 (D14). D14 belongs to the superfamily of α/ß hydrolases and is structurally similar to the karrikin receptor KARRIKIN INSENSITIVE 2 (KAI2). The moss Physcomitrella patens is an ideal model system for studying this receptor family, because it includes 11 highly related family members with unknown ligand specificity. We present the crystal structures of three Physcomitrella D14/KAI2-like proteins and describe a loop-based mechanism that leads to a permanent widening of the hydrophobic substrate gorge. We have identified protein clades that specifically perceive the karrikin KAR1 and the non-natural strigolactone isomer (-)-5-deoxystrigol in a highly stereoselective manner.

Multiplexed immunoassay using post-synthesis functionalized hydrogel microparticles.

In response to a growing demand for simultaneous detection of multiple proteins in a single sample, multiplex immunoassay platforms have emerged at the forefront of proteomic analysis. In particular, detections using graphically encoded hydrogel microparticles synthesized via flow lithography have received attention for integrating a hydrogel, a substrate that can provide enhanced kinetics and high loading capacity, into the bead-based multiplex platform. Currently, the method of microparticle functionalization involves copolymerization of antibodies with the gel during particle synthesis. However, its practical operation is too precarious to be adopted because antibodies are susceptible to aggregation due to incompatibility with hydrophobic photoinitiators used in the photo-induced gel polymerization. In this work, we present a multiplex immunoassay platform that uses encoded hydrogel microparticles that are functionalized after particle synthesis by conjugating antibodies with remnant active groups readily available in the hydrogels. The method not only precludes antibody aggregation but also augments the loading density of the antibodies, which translates into enhanced detection performance. In addition to multiplexing, our platform demonstrates high sensitivity, a broad assay range, and a fast detection rate that outperform the enzyme linked immunosorbent assay (ELISA).

Effects of CYP2C9 genetic polymorphisms on the pharmacokinetics of zafirlukast.

Zafirlukast, a cysteinyl leukotriene receptor antagonist, is indicated for the treatment of patients with mild to moderate asthma. Zafirlukast is metabolized mainly by CYP3A4 and CYP2C9. We investigated the effects of the major CYP2C9 variant alleles in Asian populations, CYP2C9*3 and CYP2C9*13, on the pharmacokinetics of zafirlukast in healthy Korean subjects. A single 20-mg oral dose of zafirlukast was given to 23 Korean male subjects divided into two genotype groups according to CYP2C9 genotypes, CYP2C9EM (n = 11; CYP2C9*1/*1) and CYP2C9IM (n = 12; 9 and 3 carriers of CYP2C9*1/*3 and *1/*13, respectively). Zafirlukast concentrations were determined using a validated HPLC-MS/MS analytical method in plasma samples collected after the drug intake. Compared with the CYP2C9EM group, the Cmax and AUCinf of zafirlukast in the CYP2C9IM group were 1.44- and 1.70-fold higher, respectively (p < 0.01 and p < 0.0001). The CL/F of zafirlukast was 42.8 % lower in the CYP2C9IM group compared with the CYP2C9EM group (p < 0.001). Slightly higher Cmax and AUC, and lower CL/F of zafirlukast were observed in subjects with the CYP2C9*1/*13 genotype compared with the CYP2C9*1/*3 genotype subjects. CYP2C9*3 and CYP2C9*13 alleles significantly affected the plasma concentrations of zafirlukast.

Genome-wide identification of haploinsufficiency in fission yeast.

Abnormal phenotypes resulting from haploinsufficiency (HI) are due to the loss of one allele. Recent studies in budding yeast have shown that HI originates from insufficient protein levels or from a stoichiometric imbalance between subunits of protein complexes. In humans, however, HI often involves transcription factors. Therefore, the species differences in HI and the molecular mechanisms of species-specific HI remain under investigation. In this study, HI in fission yeast was systematically surveyed. HI in fission yeast affected genes related to signaling and to basic cellular processes, as observed in budding yeast. These results suggest that there are species differences in HI and that the HI that occurs in fission yeast is intermediate to and HI in budding yeast and humans.

Cerulenin-mediated apoptosis is involved in adenine metabolic pathway.

Cerulenin, a fatty acid synthase (FAS) inhibitor, induces apoptosis of variety of tumor cells. To elucidate mode of action by cerulenin, we employed the proteomics approach using Schizosaccharomyces pombe. The differential protein expression profile of S. pombe revealed that cerulenin modulated the expressions of proteins involved in stresses and metabolism, including both ade10 and adk1 proteins. The nutrient supplementation assay demonstrated that cerulenin affected enzymatic steps transferring a phosphoribosyl group. This result suggests that cerulenin accumulates AMP and p-ribosyl-s-amino-imidazole carboxamide (AICAR) and reduces other necessary nucleotides, which induces feedback inhibition of enzymes and the transcriptional regulation of related genes in de novo and salvage adenine metabolic pathway. Furthermore, the deregulation of adenine nucleotide synthesis may interfere ribonucleotide reductase and cause defects in cell cycle progression and chromosome segregation. In conclusion, cerulenin induces apoptosis through deregulation of adenine nucleotide biosynthesis resulting in nuclear division defects in S. pombe.