Large-scale annotated dataset for cochlear hair cell detection and classification.

Our sense of hearing is mediated by cochlear hair cells, of which there are two types organized in one row of inner hair cells and three rows of outer hair cells. Each cochlea contains 5-15 thousand terminally differentiated hair cells, and their survival is essential for hearing as they do not regenerate after insult. It is often desirable in hearing research to quantify the number of hair cells within cochlear samples, in both pathological conditions, and in response to treatment. Machine learning can be used to automate the quantification process but requires a vast and diverse dataset for effective training. In this study, we present a large collection of annotated cochlear hair-cell datasets, labeled with commonly used hair-cell markers and imaged using various fluorescence microscopy techniques. The collection includes samples from mouse, rat, guinea pig, pig, primate, and human cochlear tissue, from normal conditions and following in-vivo and in-vitro ototoxic drug application. The dataset includes over 107,000 hair cells which have been identified and annotated as either inner or outer hair cells. This dataset is the result of a collaborative effort from multiple laboratories and has been carefully curated to represent a variety of imaging techniques. With suggested usage parameters and a well-described annotation procedure, this collection can facilitate the development of generalizable cochlear hair-cell detection models or serve as a starting point for fine-tuning models for other analysis tasks. By providing this dataset, we aim to give other hearing research groups the opportunity to develop their own tools with which to analyze cochlear imaging data more fully, accurately, and with greater ease.

PCP Auto Count: A Novel Fiji/ImageJ plug-in for automated quantification of planar cell polarity and cell counting.

Background: During development, planes of cells give rise to complex tissues and organs. The proper functioning of these tissues is critically dependent on proper inter- and intra-cellular spatial orientation, a feature known as planar cell polarity (PCP). To study the genetic and environmental factors affecting planar cell polarity investigators must often manually measure cell orientations, which is a time-consuming endeavor. Methodology: To automate cell counting and planar cell polarity data collection we developed a Fiji/ImageJ plug-in called PCP Auto Count (PCPA). PCPA analyzes binary images and identifies "chunks" of white pixels that contain "caves" of infiltrated black pixels. Inner ear sensory epithelia including cochleae (P4) and utricles (E17.5) from mice were immunostained for ßII-spectrin and imaged on a confocal microscope. Images were preprocessed using existing Fiji functionality to enhance contrast, make binary, and reduce noise. An investigator rated PCPA cochlear angle measurements for accuracy using a 1-5 agreement scale. For utricle samples, we directly compared PCPA derived measurements against manually derived angle measurements using concordance correlation coefficients (CCC) and Bland-Altman limits of agreement. Finally, PCPA was tested against a variety of images copied from publications examining PCP in various tissues and across various species. Results: PCPA was able to recognize and count 99.81% of cochlear hair cells (n = 1,1541 hair cells) in a sample set, and was able to obtain ideally accurate planar cell polarity measurements for over 96% of hair cells. When allowing for a <10° deviation from "perfect" measurements, PCPA's accuracy increased to >98%. When manual angle measurements for E17.5 utricles were compared, PCPA's measurements fell within -9 to +10 degrees of manually obtained mean angle measures with a CCC of 0.999. Qualitative examination of example images of Drosophila ommatidia, mouse ependymal cells, and mouse radial progenitors revealed a high level of accuracy for PCPA across a variety of stains, tissue types, and species. Altogether, the data suggest that the PCPA plug-in suite is a robust and accurate tool for the automated collection of cell counts and PCP angle measurements.

Large-scale annotated dataset for cochlear hair cell detection and classification.

Our sense of hearing is mediated by cochlear hair cells, localized within the sensory epithelium called the organ of Corti. There are two types of hair cells in the cochlea, which are organized in one row of inner hair cells and three rows of outer hair cells. Each cochlea contains a few thousands of hair cells, and their survival is essential for our perception of sound because they are terminally differentiated and do not regenerate after insult. It is often desirable in hearing research to quantify the number of hair cells within cochlear samples, in both pathological conditions, and in response to treatment. However, the sheer number of cells along the cochlea makes manual quantification impractical. Machine learning can be used to overcome this challenge by automating the quantification process but requires a vast and diverse dataset for effective training. In this study, we present a large collection of annotated cochlear hair-cell datasets, labeled with commonly used hair-cell markers and imaged using various fluorescence microscopy techniques. The collection includes samples from mouse, human, pig and guinea pig cochlear tissue, from normal conditions and following in-vivo and in-vitro ototoxic drug application. The dataset includes over 90'000 hair cells, all of which have been manually identified and annotated as one of two cell types: inner hair cells and outer hair cells. This dataset is the result of a collaborative effort from multiple laboratories and has been carefully curated to represent a variety of imaging techniques. With suggested usage parameters and a well-described annotation procedure, this collection can facilitate the development of generalizable cochlear hair cell detection models or serve as a starting point for fine-tuning models for other analysis tasks. By providing this dataset, we aim to supply other groups within the hearing research community with the opportunity to develop their own tools with which to analyze cochlear imaging data more fully, accurately, and with greater ease.

Absolute Quantification of Isoflavones in the Flowers of Pueraria lobata by qHNMR.

Pueraria lobata (Willd.) Ohwi. is a widely used medicinal plant in Korea, China, and Japan. The flower of P. lobata (Puerariae Flos) contains various bioactive substances such as triterpenoidal saponins and isoflavonoids. In this study, we developed a quantitative analysis of the isoflavones of Puerariae Flos by quantitative proton nuclear magnetic resonance (qHNMR) spectroscopy using the internal calibrant (IC). From the qHNMR results, the isoflavone content was found to be 7.99% and 10.57% for the MeOH sonication extract (PLs) and the MeOH reflux extract (PLr) of Puerariae Flos, respectively. The quantified isoflavone content was validated using the conventional analytical method, high-performance liquid chromatography with ultraviolet detection (HPLC-UV). The present study shows that validated qHNMR spectroscopy is a reliable method for quantifying and standardizing the isoflavone content in Puerariae Flos.

Screening and characterization of potent poly glutamic acid producing Bacillus sp. isolated from Kinema, water and soil samples.

Microbially produced gamma poly glutamic acid (γ-PGA) is a commercially important biopolymer with many applications in foods and various other substances and are abundantly used in different parts of the world. With an aim to study the potent γ-PGA producing Bacillus species, a total of 47 different samples (Kinema, soil, and water) were randomly collected from different locations across the country, and Bacillus sp. were selectively isolated, screened, and characterized by performing physiological, biochemical, morphological, and 16S rRNA gene sequencing. The microbial production of γ-PGA was assayed with the selected isolates on the PGA medium and the metabolite obtained was recovered by ethanol precipitation method and further characterized by thin-layer chromatography (TLC). Thermotolerance (25-60 °C), pH tolerance (4-9), and NaCl tolerance (1-9%) tests were performed to optimize the bacterial growth and γ-PGA production and its viscosity were measured by Ostwald's viscometer. Out of 145 randomly selected colonies, 63 isolates were Gram-positive, rods, and endospore producers and were presumptively confirmed as genus Bacillus. Higher growth of γ-PGA producers were reported in 22 isolates and was found at optimum conditions such as temperature (30-37 °C), pH (6.5-7), incubation time (3 days), and NaCl concentration (3%) and γ-PGA thus produced was further verified by TLC with the retention factor (RF) value 0.27. The potent isolates were closely similar to Bacillus subtilis subsp. stercoris, Bacillus cereus, Bacillus paranthracis, and Bacillus licheniformis etc. Based on the findings of the study, B. licheniformis is the most potent γ-PGA producing Bacillus sp. which can further be used for the commercial production of γ-PGA. To the best of our knowledge, there is yet no published research from Nepal showing the production of the γ-PGA although microbially produced γ-PGA are the major constituents in some popular foods in particular communities of the country.

Eudesmane and Eremophilane Sesquiterpenes from the Fruits of Alpinia oxyphylla with Protective Effects against Oxidative Stress in Adipose-Derived Mesenchymal Stem Cells.

Alpinia oxyphylla Miquel (Zingiberaceae) has been reported to show antioxidant, anti-inflammatory, and neuroprotective effects. In this study, two new eudesmane sesquiterpenes, 7α-hydroperoxy eudesma-3,11-diene-2-one (1) and 7ß-hydroperoxy eudesma-3,11-diene-2-one (2), and a new eremophilane sesquiterpene, 3α-hydroxynootkatone (3), were isolated from the MeOH extract of dried fruits of A. oxyphylla along with eleven known sesquiterpenes (4-14). The structures were elucidated by the analysis of 1D/2D NMR, high-resolution electrospray ionization mass spectrometry (HRESIMS), and optical rotation data. Compounds (1-3, 5-14) were evaluated for their protective effects against tert-butyl hydroperoxide (tBHP)-induced oxidative stress in adipose-derived mesenchymal stem cells (ADMSCs). As a result, treatment with isolated compounds, especially compounds 11 and 12, effectively reverted the damage of tBHP on ADMSCs in a dose-dependent manner. In particular, 11 and 12 at 50 µM improved the viability of tBHP-toxified ADMSCs by 1.69 ± 0.05-fold and 1.61 ± 0.03-fold, respectively.

Changes in the aquatic ecotoxicological effects of Triton X-100 after UV photodegradation.

Various spray adjuvants including surfactants are widely used in agricultural pesticide formulations, and some of them may remain in soils and waters and impose more adverse effects than active pesticide ingredients on organisms. However, previous studies are more focused on the active pesticide ingredients than the adjuvants. Thus, this study investigates the changes in toxic effects of surfactants during photodegradation, which is one way of naturally degrading contaminants in natural waters. Triton X-100, a water-soluble non-ionic surfactant, was degraded using different types of UV radiation (UVA, UVB, and UVC), and the changes in the toxic effects were determined using bioluminescent bacteria and water flea. The Triton X-100 removals were negligible with UVA within 24 h, while its removal was 81% with UVB and almost complete with UVC. The NMR spectra indicated possible molecule rearrangement after photolysis. On the other hand, the toxic effects based on the mortality of Daphnia magna and the bioluminescence of Aliivibrio fischeri increased (i.e., lower EC50 values) after photodegradation, suggesting the generation of photoproducts that are likely to have higher toxic effects or higher bioavailability. Furthermore, the sensitivities of D. magna and A. fischeri for Triton X-100 and the photodegraded Triton X-100 were different. This study suggests that the changes in the chemical composition of the Triton X-100 containing water with photodegradation can lead to changes in the relative toxic effects on different aquatic organisms. Therefore, not only the management of parent compound (i.e., Triton X-100) but also the photoproducts generated from the parent compound need to be considered when managing water environment subject to photodegradation.

Suntamide A, a neuroprotective cyclic peptide from Cicadidae Periostracum.

Suntamide A (1), a new cyclic peptide, was isolated from Cicadidae Periostracum. The gross structure of 1 was elucidated by detailed analysis of HRMS and 1D/2D NMR spectra, and the absolute configuration was established by C3 Marfey's method. We extended our study to examine biological activity of 1, and found that 1 protected SH-SY5Y cells against rotenone-induced neurotoxicity. This effect of 1 seemed to be attributed to antioxidant induction and protection of mitochondria from rotenone-caused injury. Along with augmentation of the antioxidant system by 1, there was an evident activation of Nrf2, a transcription factor involved in the activation of the antioxidant system. These results indicate that 1 rescued the cells from rotenone-mediated neurotoxicity by enhancing antioxidant capacity via induction of Nrf2, suggesting that the compound could be used as a therapeutic intervention in neurodegenerative diseases such as Parkinson's disease.

Cool OtOprotective Ear Lumen (COOL) Therapy for Cisplatin-induced Hearing Loss.

HYPOTHESIS: Localized cooling of the external ear has a protective effect on the susceptibility to cisplatin-induced hearing loss. BACKGROUND: We previously demonstrated significant protection from cisplatin-induced hearing loss using cool water ear canal irrigation. However, the study was limited to a single bolus injection of cisplatin and an acute time period. Here, we examined the application of localized cooling of the ear canal with repeated doses of cisplatin, over an expanded period of time, and using two methods of cooling. METHODS: Twenty-four guinea pigs (12 male and 12 female) underwent auditory physiological testing (auditory brainstem response and distortion product otoacoustic emissions at 8-32 kHz) and pre/postadministration of cisplatin. Cisplatin (4 mg/kg i.p.) was administered in 3 weekly single injections for a total of 12 mg/kg. While anesthetized, the left ears of the guinea pigs were exposed to either cool water (22°C; ICS Water Caloric Irrigator), a cool ear bar (15°C, cooled by a Peltier device; TNM, Scion NeuroStim), or left uncooled as a sham control. The animals were tested 3 days post each dosage and 1 month post the final dose. At the end of the experiment the animals were euthanized for histological evaluation. RESULTS: We found that hearing loss was significantly reduced, and hair cell survival greatly improved, in animals that received cooling treatments compared to cisplatin-only control animals. No significant difference was observed between the two methods of cooling. CONCLUSION: Localized cooling of the ear canal during administration of cisplatin mitigated loss of auditory function and loss of hair cells.

N-Acetyldopamine derivatives from Periostracum Cicadae and their regulatory activities on Th1 and Th17 cell differentiation.

Bioassay-guided fractionation of a 90% ethanol extract of Periostracum Cicadae led to the isolation of two new N-acetyldopamine dimers (1a/1b) along with six known dimers (2a/2b, 3a/3b, and 4a/4b) and two monomers (5a/5b); compounds 2a/2b, 4a/4b and 5a/5b were newly isolated from this material. All compounds were isolated as enantiomeric mixtures and each enantiomer was successfully separated by chiral-phase HPLC. The structures including absolute configurations were confirmed by high-resolution electrospray ionization mass spectrometry (HR-ESIMS), 1D/2D nuclear magnetic resonance (NMR) spectroscopy, 1H iterative Full Spin Analysis (HiFSA), and electronic circular dichroism (ECD) spectroscopy. Subsequently, the bioactivities of these isolates were evaluated via CD4+ T cell differentiations, which are critical for immune responses and inflammation. The results revealed that compound 5b was observed to enhance the IFN-γ+ Th1 differentiation, which may have a potential for cancer immunotherapy.

Purification of Phenylpropanoids from the Scaly Bulbs of Lilium Longiflorum by CPC and Determination of Their DPP-IV Inhibitory Potentials.

The scaly bulbs of Lilium longiflorum (Liliaceae) are used as a food ingredient and a traditional medicine in East Asia. A preliminary study revealed that treatment with 100 µg/mL of the ethyl acetate fraction of this plant material inhibited dipeptidyl peptidase IV (DPP-IV) to 58.99%. Phytochemical studies were conducted to identify the active ingredient, and five compounds, namely, 1 (2.9 mg, 75.8% purity at 320 nm), 2 (12.2 mg, 97.9% purity at 320 nm), 3 (3.1 mg, 66.5% purity at 320 nm), 4 (6.8 mg, 96.9% purity at 320 nm), and 5 (6.2 mg, 90.2% purity at 320 nm) were purified from 200 mg of the ethyl acetate fraction of L. longiflorum via centrifugal partition chromatography (CPC) with a two-phase solvent system composed of chloroform/methanol/isopropanol/water (5:2:2:4, v/v/v/v) in an ascending mode. Their structures were identified as 1-O-p-coumaroyl-2-O-ß-glucopyranosylglycerol (regaloside D, 1), 3,6'-O-diferuloylsucrose (2), 1-O-p-coumaroyl-2-O-ß-glucopyranosyl-3-O-acetylglycerol (regaloside B, 3), 1-O-p-coumaroylglycerol (4), and 4-O-acetyl-3,6'-O-diferuloylsucrose (5), respectively, by 1H and 13C NMR and MS analysis. Compounds 2 and 5 exhibited DPP-IV inhibitory activities with IC50 values of 46.19 and 63.26 µM, respectively. Compounds 1, 3, and 4 did not show activities, indicating that biphenylpropanoids linked via the sugar moiety are more effective than phenylpropanoids with glycerol or glyceryl glucoside. This is the first report of simultaneous separation of five phenylpropanoids from L. longiflorum by CPC and evaluation of their DPP-IV inhibitory activities.