Isolation and reconstruction of cardiac mitochondria from SBEM images using a deep learning-based method

Mitochondrial morphological defects are a common feature of diseased cardiac myocytes. However, quantitative assessment of mitochondrial morphology is limited by the time-consuming manual segmentation of electron micrograph (EM) images. To advance understanding of the relation between morphological defects and dysfunction, an efficient morphological reconstruction method is desired to enable isolation and reconstruction of mitochondria from EM images. We propose a new method for isolating and reconstructing single mitochondria from serial block-face scanning EM (SBEM) images. CDeep3M, a cloud-based deep learning network for EM images, was used to segment mitochondrial interior volumes and boundaries. Post-processing was performed using both the predicted interior volume and exterior boundary to isolate and reconstruct individual mitochondria. Series of SBEM images from two separate cardiac myocytes were processed. The highest F1-score was 95% using 50 training datasets, greater than that for previously reported automated methods and comparable to manual segmentations. Accuracy of separation of individual mitochondria was 80% on a pixel basis. A total of 2315 mitochondria in the two series of SBEM images were evaluated with a mean volume of 0.78 µm³. The volume distribution was very broad and skewed; the most frequent mitochondria were 0.04–0.06 µm³, but mitochondria larger than 2.0 µm³ accounted for more than 10% of the total number. The average short-axis length was 0.47 µm. Primarily longitudinal mitochondria (0–30 degrees) were dominant (54%). This new automated segmentation and separation method can help quantitate mitochondrial morphology and improve understanding of myocyte structure–function relationships.     

Seasonal development of bacterioplankton in two forest lakes in central Sweden

Bacteria were counted with acridine orange epifluorescence technique in two humic lakes during 3 years. Less than 1% of the cells were found attached to detritus aggregates. 73% of the total number and 48% of the total volume were smaller than 1 µm. The mean cell volume ranged from 0.10 to 0.35 µm³ with the highest cell volumes occurring during early summer contemporarily with the growth of the bacterial biomass and probably indicating favourable growth conditions. The mean density of bacteria in oligotrophic brown-water lakes is higher than in oligotrophic clear-water lakes. The development of bacterial biomass showed a regular and seasonally dependent pattern with maxima during early summer and autumn. The importance of different factors for the regulation of bacterial biomass is discussed. Three different approaches were used to estimate bacterial production. These resulted in an average production rate of 15–60 µg C · l^?1 · d^?1 during the growing season. It was concluded that allochthonous sources comprised a significant part of the energy supply to the bacteria in the two humic lakes.     

Long-term acrylamide intoxication induces atrophy of dorsal root ganglion A-cells and of myelinated sensory axons

We have examined the effects of acrylamide on primary sensory nerve cell bodies and their myelinated axons in chronic acrylamide intoxication. The numbers and sizes of dorsal root ganglion cell bodies (L5) and myelinated nerve fibers were estimated with sterelogical techniques in severely disabled rats which had been treated with 33.3 mg/kg acrylamide twice a week for 7.5 weeks. There was no loss of dorsal root ganglion cells or myelinated nerve fibers in the roots, the sciatic nerve, sural nerve, and a tibial nerve branch. The mean perikaryal volume of A-cells was reduced by 20% (2P < 0.001) from 50000 μm³ in controls (CV = 0.13) to 40000 μm³ (0.12), whereas B-cell volume was unchanged. All size-frequency distribution curves of myelinated axon area of peripheral nerves and sensory roots were shifted to the left towards smaller values in rats exposed to acrylamide. In the L5 sensory root 3 mm from the ganglion, there was a significant reduction of mean cross sectional area of myelinated axons by 14% (2P < 0.05) from 7.6 μm² (0.11) in controls to 6.5 μm² (0.13) in intoxicated rats. The mean cross sectional area of myelinated sural nerve axons was reduced by 22% (2P < 0.001) from 8.6 μm² (0.08) in controls to 6.7 μm² (0.17) in intoxicated rats. We conclude that chronic intoxication with acrylamide leads to selective atrophy of type A dorsal root ganglion cell bodies and simultaneous atrophy along their peripheral axons, whereas neuronal B-cell bodies and motor axons are spared. It is suggested that the neuronal atrophy might well represent a defect of neurofilament synthesis and transport.     

Risk of adult street vendor exposure to traffic-related air pollution in Bangkok,Thailand

Traffic-related air pollution (TRAP), including particulate matter (PM) in respirable coarse and fine size fractions (PM10 and PM2.5), is known to have exposure effects on human health and environment. Real-time PM10 and PM2.5 concentrations were collected from the study locations in Bangkok, Thailand, using TSI AM510 particle counters. Temperature and % relative humidity (%RH) were also collected. Data were compared to data from the closest station of the Pollution Control Department (PCD), Thailand. Real-time mean concentration varied from 86 to 1107 µg/m³ (PM10) and varied from 25 to 664 µg/m³ (PM2.5). In addition, real-time mean PM10 (223.1 µg/m³) was nearly four times greater than that measured by the PCD station, 60 µg/m³. Temperature and %RH from real-time air monitoring and PCD station were comparable. In each study location (five locations, two in morning and afternoon/evening), there were significant positive correlations between PM10 and PM2.5 concentrations and significant negative correlations between temperature and RH%. Results suggested that outdoor TRAP via measured real-time PM concentrations were more realistic exposure concentration estimates among street vendors as related to respiratory and other symptoms than data obtained from PCD station. Nevertheless, PM10 as measured by the PCD station might be a reasonable surrogate for estimated outdoor PM2.5 exposure.     

Blood platelets of the cheetah (Acinonyx jubatus)

The blood platelet count and platelet morphology of 22 adult cheetahs was investigated. The platelet counts of the animals displayed a normal distribution, with a mean count of 344×10⁹/l and a mean platelet volume of 11 fl. Morphological and ultrastructural features of the cheetah platelets revealed the typical platelet morphology of anuclear cells, with granules scattered throughout the cytoplasm. The characteristic surface canalicular system and microtubules were present. True cross‐sections of the platelets had a mean area of 2.146 µm², circumference of 6.805 µm, and mean minimum and maximum projections of 1.000 µm and 2.933 µm, respectively. Zoo Biol 23:263–271, 2004. © 2004 Wiley‐Liss, Inc.     

Volume and Shape Changes of Human Erythrocytes Induced by Electrical Fields

The effects of electrical fields on the shape and volume of normal and abnormal red blood cells (RBC) are discussed. If an electrical field of 2-3 kV/cm is applied to the suspension of RBC, normal biconcave erythrocytes transform into spherocytes and their mean volume will increase from the usual volume of 90 µm³ to 160-170/µm³ in several minutes. There are two possible explanations for this phenomenon. The one is the membrane perforation by the potential induced by electrical field and subsequent influx of water. The other Is the mechanical surface traction caused by applied fields. The second mechanism will be discussed mainly in this paper.     

Evaluation of current densities and total contact currents in occupational exposure at 400 kV substations and power lines

This investigation studied the current densities in the neck and total contact currents in occupational exposure at 400 kV substations and power lines. Eight voluntary workers simulated their normal work tasks using the helmet–mask measuring system. In all, 151 work tasks with induced current measurements were made. Work situations were: tasks in 400 kV substations, tasks in 400–110 kV towers and the cutting of vegetation under 400 kV power lines. The average current density in the neck was estimated from the current induced in the helmet. The calculated maximum average current densities in the neck varied from 1.5 to 6.4 mA/m² and the maximum total contact currents from 66.8 to 458.4 µA. The study shows that the maximum average current densities and the total contact currents (caused by electric field) in occupational exposure at 400 kV substations and power lines does not exceed the limit and action values (10 mA/m² and 1 mA) of the new EU‐directive 2004/40/EC (live‐line bare‐hand works excluded). Bioelectromagnetics 30:231–240, 2009. © 2009 Wiley‐Liss, Inc.     

Investigation of expression and activity levels of primary rat hepatocyte detoxication genes under various flow rates and cell densities in microfluidic biochips

We investigated the behavior of primary rat hepatocytes in biochips using a microfluidic platform (the integrated dynamic cell culture microchip). We studied the effects of cell inoculation densities (0.2–0.5 × 10⁶ cells/biochip) and perfusion flow rates (10, 25, and 40 µL/min) during 72 h of perfusion. No effects were observed on hepatocyte morphology, but the levels of mRNA and CYP1A2 activity were found to be dependent on the initial cell densities and flow rates. The dataset made it possible to extract a best estimated range of parameters in which the rat hepatocytes appeared the most functional in the biochips. Namely, at 0.25 × 10⁶ inoculated cells cultivated at 25 µL/min for 72 h, we demonstrated better induction of the expression of all the genes analyzed in comparison with other cell densities and flow rates. More precisely, when primary rat hepatocytes were cultivated at these conditions, the time‐lapse analysis demonstrated an over expression of CYP3A1, CYP2B1, ABCC1b and ABCC2 in the biochips when compared to the postextraction levels. Furthermore, the AHR, CYP1A2, GSTA2, SULT1A1, and UGT1A6 levels remained higher than 50% of the postextraction values whereas values of HNF4α, CEBP, and PXR remained higher than 20% during the duration of the culture process. Nevertheless, an important reduction in mRNA levels was found for the xenosensors CAR and FXR, and the related CYP (CYP2E1, CYP7A1, CYP3A2, and CYP2D2). CYP1A2 functionality was illustrated by 700 ± 100 pmol/h/10⁶ cells resorufin production. This study highlighted the functionality in optimized conditions of primary rat hepatocytes in parallelized microfluidic cultures and their potential for drug screening applications. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:401–410, 2014     

The corpus allatum,neurosecretion and photoperiodically controlled polymorphism in an aphid

Corpus allatum volumes were measured in adult vetch aphids, M. viciae, reared in long or short days. Initially, gland volumes were similar (23,228 ± 1311 (SE) and 21,413 ± 1208 μm³ respectively) but in short days the volumes increased with age to 32,283 ± 1917 (SE) μm³ on day 20 while in long days they decreased to 18,390 ± 1193 μm³. Cauterization of the anterior protocerebrum, in the region of the Group-I neurosecretory cells, of long-day insects or transfer to short days at the imaginal moult resulted in a change in the morph of the progeny from vivipara to ovipara some 3 weeks later. Concomitant with this switch to ovipara production was an increased corpus allatum volume by day 20 (30,494 ± 3032 μm³ after successful cautery and 32,568 ± 1595 μm³ after transfer to short days). Sham cautery has no effect on progeny morph or gland size. These results, together with those concerning the delay between treatment and progeny switch-over, are discussed in relation to the hormonal control of photoperiodically determined polymorphism.     

A comparative ultrastructural study of the trigeminal ganglion in the rat and chicken

The neurons of the trigeminal ganglia of the rat and chicken were characterized by means of light microscopic, electron microscopic, and histochemical methods. Light microscopy disclosed four types of neurons, based on the characteristics of Nissl granules: (1) large neurons with diffusely distributed and very fine granules, (2) neurons containing coarse and sparsely distributed Nissl granules, (3) neurons containing dense Nissl granules of varying size, and (4) small neurons with granules concentrated peripherally. Electron microscopy allowed further definition of these four types of neurons by the length and arrangement of flattened cisterns of granular endoplasmic reticulum (gER) and the number of neurofilaments. Type 1 cells were largest, with a mean nuclear area of 139.8 ± 28.3 μm². Type 4 cells were smallest, with a mean nuclear area of 74.6 ± 20.9 μm². The mean nuclear areas of type 2 and 3 cells were intermediate to those of the type 1 and 4 cells. Type 3 and 4 neurons lacked neurofilaments. Four forms of Golgi apparatus were found: (1) large bent grains forming a network throughout the soma, (2) dispersed fine granular deposits, (3) fine or small granules, and (4) coarse bent deposits arranged confluently in the perinuclear zone. In some rat neurons, the concentration of acid phosphatase reaction products suggested a high enzymatic activity, whereas the chicken ganglion cells showed no such concentration. These findings are discussed and compared with the classifications of previous studies.     

SPARCL1-containing neurons in the human brainstem and sensory ganglion

Secreted protein, acidic and rich in cysteine-like 1 (SPARCL1) is a member of the osteonectin family of proteins. In this study, immunohistochemistry for SPARCL1 was performed to obtain its distribution in the human brainstem, cervical spinal cord, and sensory ganglion. SPARCL1-immunoreactivity was detected in neuronal cell bodies including perikarya and proximal dendrites, and the neuropil. The motor nuclei of the IIIrd, Vth, VIth, VIIth, IXth, Xth, XIth, and XIIth cranial nerves and spinal nerves contained many SPARCL1-immunoreactive (-IR) neurons with medium-sized to large cell bodies. Small and medium-sized SPARCL1-IR neurons were distributed in sensory nuclei of the Vth, VIIth, VIIIth, IXth, and Xth cranial nerves. In the medulla oblongata, the dorsal column nuclei also had small to medium-sized SPARCL1-IR neurons. In addition, SPARCL1-IR neurons were detected in the nucleus of the trapezoid body and pontine nucleus within the pons and the arcuate nucleus in the medulla oblongata. In the cervical spinal cord, the ventral horn contained some SPARCL1-IR neurons with large cell bodies. These findings suggest that SPARCL1-containing neurons function to relay and regulate motor and sensory signals in the human brainstem. In the dorsal root (DRG) and trigeminal ganglia (TG), primary sensory neurons contained SPARCL1-immunoreactivity. The proportion of SPARCL1-IR neurons in the TG (mean?±?SD, 39.9?±?2.4%) was higher than in the DRG (30.6?±?2.1%). SPARCL1-IR neurons were mostly medium-sized to large (mean?±?SD, 1494.5?±?708.3?μm²; range, 320.4–4353.4?μm²) in the DRG, whereas such neurons were of various cell body sizes in the TG (mean?±?SD, 1291.2?±?532.8?μm²; range, 209.3–4326.4?μm²). There appears to be a SPARCL1-containing sensory pathway in the ganglion and brainstem of the spinal and trigeminal nervous systems.     

Chemical speciation and health risks of airborne heavy metals around an industrial community in Nigeria

Abstract

Quantification of PM_2.5 (particulate matter <2.5?µm) bound heavy metals and their potential health risks were carried out around a cement manufacturing company in Ewekoro, Nigeria. The PM_2.5 samples were collected using Environtech gravimetric sampler. A four-staged sequential extraction procedure was used to fractionate PM_2.5 bound chromium (Cr), lead (Pb), aluminum (Al), copper (Cu), and silver (Ag), and further analyzed using inductively coupled plasma mass spectrometry. Chemical speciation results reveal bioavailable levels of Pb (4.05?µg/m³), Cr (10.75?µg/m³), Al (16.47?µg/m³), Cu (4.38E-01?µg/m³), and Ag (1.22E-02?µg/m³) in the airborne particulates. Pb and Cr levels exceeded the World Health Organization allowable limit of 0.5 and 2.5E-05?µg/m³, respectively. The labile phases showed strong indication of the presence of Cr and Cu metal. Excess cancer risks exposure for adults, outdoor workers and children were higher than the acceptable risk target level of 1E-06. Non-carcinogenic health risk estimated using hazard quotients (HQs) and hazard indices (HIs) showed ingestion route within the safe level of HI <1 implying no adverse effect while inhalation route exceeded the safe level for all receptors. Enforcement of pollution control by authorized agencies, and screening of greenbelts as sinks for air pollutants is strongly recommended.     

Estimation of amoeba cell volume from nuclear diameter and its application to studies in protozoan ecology

To facilitate the estimation of cell volume in uninucleate, naked amoebae (gymnamoebae) the relationship, log cell volume (µm³) = 0.882 + 3.117log nuclear diameter (µm³), is presented. This links mean cell volume to mean nuclear diameter and provides a useful tool for protozoan ecologists interested in estimating the biovolume of amoebae in laboratory or field samples. While it is virtually impossible to measure rigid axes from which volume can be calculated in these amorphous cells, it is relatively easy to measure the diameter of the nucleus in living or fixed material. This relationship has shown that most uninucleate amoebae surveyed have volumes ranging between only 188 µm³ and 2860 µm³; this range reflects the volumes of the majority of amoebae in the field. These small volumes are unexpected since many amoebae have locomotive forms greater than 20 µm in length giving the impression that their cell volumes should be correspondingly large. This is not the case, however, because most amoebae are extremely flat when viewed in profile. The small cell volume of most amoeba species has ecological implications when numerical data is transformed to biovolume and biomass units.     

Age-related changes in sensory neurons containing calcitonin gene-related peptide under conditions of afferentation deficit in rats

Morphological features of calcitonin gene-related peptide (CGRP)-immunoreactive neurons were studied in the sensory ganglia of the vagus and thoracic nerves in 3-, 10-, 20-, 30-, 60-, 90-, and 180-day-old rats under conditions of chemically-induced deafferentation. We found that, in rats, CGRP-containing neurons appeared in both ganglia immediately after they were born and their number decreased with aging. Most of CGRP-immunoreactive neurons were small in size, i.e., up to 600 ??m². Administration of capsaicin modified age-related changes in the number of CGRP-immunopositive neurons. In the thoracic nerve ganglion, the mean square of these cells and their number substantially decreased, whereas, in the vagus nerve ganglion, positive cells were not observed.     

Spiny versus stubby: 3D reconstruction of human myenteric (type I) neurons

We have compared the three-dimensional (3D) morphology of stubby and spiny neurons derived from the human small intestine. After immunohistochemical triple staining for leu-enkephalin (ENK), vasoactive intestinal peptide (VIP) and neurofilament (NF), neurons were selected and scanned based on their immunoreactivity, whether ENK (stubby) or VIP (spiny). For the 3D reconstruction, we focused on confocal data pre-processing with intensity drop correction, non-blind deconvolution, an additional compression procedure in z-direction, and optimizing segmentation reliability. 3D Slicer software enabled a semi-automated segmentation based on an objective threshold (interrater and intrarater reliability, both 0.99). We found that most dendrites of stubby neurons emerged only from the somal circumference, whereas in spiny neurons, they also emerged from the luminal somal surface. In most neurons, the nucleus was positioned abluminally in its soma. The volumes of spiny neurons were significantly larger than those of stubby neurons (total mean of stubbies 806 ± 128 μm³, of spinies 2,316 ± 545 μm³), and spiny neurons had more dendrites (26.3 vs. 11.3). The ratios of somal versus dendritic volumes were 1:1.2 in spiny and 1:0.3 in stubby neurons. In conclusion, 3D reconstruction revealed new differences between stubby and spiny neurons and allowed estimations of volumetric data of these neuron populations.     

An examination of the method of sizing the spermatozoa of the domestic fowl and duck with an electronic counter

Although many estimations have been made electronically of mammalian sperm volume, detailed investigations have not been reported for avian spermatozoa with an electronic counter. In the present study, sizing of spermatozoa of fowls and Muscovy and Pekin drakes was examined using a Coulter counter (model ZB). In our preliminary work on fowl sperm volumes, we found mono- or di-morphic distribution displays that were modified depending on the combination of amplification (AMP) and aperture current (APC). Therefore, methodology to estimate the fowl and drake sperm volume was examined. Dilution of semen had no effect on the dimorphic distribution pattern of the sperm volume. Density-gradient centrifugation did not separate two kind of particles in the semen in either continuous or discontinuous Percoll gradients; therefore, we varied settings of AMP and APC, and found that the most suitable settings for measuring sperm volumes of these birds are 1 for AMP and 8 for APC. With these settings, mean volumes of spermatozoa were 5.1 μm³ for fowls, 5.7 μm³ for Muscovy drakes, and 5.6 μm³ for Pekin drakes.     

Home range,movements, and density of the central stoneroller,Campostoma anomalum,in a small Ohio stream

Synopsis A population of central stonerollers, Campostoma anomalum, in Harker's Run, Butler County, Ohio U.S.A., was examined during autumn, 1980, to determine the species' movements, density, and home range size. During the study period, 170 fish were marked by fin clips and released into the specific pool or riffle where they were captured within the 187 m study section. Eighty percent of the recaptured, marked fish (20) were recaptured in their initial capture area (specific pool or riffle). No fish moved more than 135 m from its original capture area. Density estimates of C. anomalum for each pool and riffle within the study section ranged from 0.10–3.91 fish m⁻² (mean = 1.25 fish m⁻²). Home range size was estimated to be 35.2 ± 14.1 m (mean ± 95% CI) of stream length. Campostoma anomalum in Harker's Run occurred at densities similar to those reported for the species in other streams, and their home ranges in this system were of similar size to those of other small, stream-dwelling fishes.     

Automatic Determination of Bacterioplankton Biomass by Image Analysis

Image analysis was applied to epifluorescense microscopy of acridine orange-stained plankton samples. A program was developed for discrimination and binary segmentation of digitized video images, taken by an ultrasensitive video camera mounted on the microscope. Cell volumes were estimated from area and perimeter of the objects in the binary image. The program was tested on fluorescent latex beads of known diameters. Biovolumes measured by image analysis were compared with directly determined carbon biomasses in batch cultures of estuarine and freshwater bacterioplankton. This calibration revealed an empirical conversion factor from biovolume to biomass of 0.35 pg of C μm⁻³ (± 0.03 95% confidence limit). The deviation of this value from the normally used conversion factors of 0.086 to 0.121 pg of C μm⁻³ is discussed. The described system was capable of measuring 250 cells within 10 min, providing estimates of cell number, mean cell volume, and biovolume with a precision of 5%.     

Production estimates of the benthic invertebrate community in a small Danish stream

Quantitative samples were used to investigate density, biomass and annual production of the benthic invertebrate fauna in a small Danish stream. Forty-eight taxa were found and the total invertebrate densities varied from 3 810 m^?2 in July to 20 040 m^?2 in December. The total mean annual biomass of the invertebrate fauna was 6.1 g ash-free dry wt m^?2. The annual production of the invertebrates was estimated from their mean annual biomass and their annual P/B ratio. Production of the primary consumers (herbivores and detritivores) was 21.4 g ash-free dry wt m^?2 y^?1 and of secondary consumers (carnivores) 1.1 g m^?2 y^?1. The amount of invertebrate production available to the trout population and the importance of the species as food for trout are discussed.     

Quantitative investigations of spinal motoneurons and their synaptic structures in a teleost: A morphometrical analysis with special reference to axosomatic synapses

The ultrastructural and morphometrical synaptology of the spinal motoneurons in Carassius auratus (goldfish) was analyzed based on profiles of 23 photomontages of large motoneurons and 25 putative small motoneurons with a semiautomatic digitizing system connected with a computer. In the 23 large motoneurons (mean circumference, 142.45 ± 39.76 μm) the total linear perikaryal circumference was 3,276.28 μm, of which 1,548.9 μm (46.1 ± 13.9%) was covered by terminal boutons. In contrast, a total linear perikaryal circumference of 1,375.24 μm (55.01 ± 14.34 μm) of the 25 putative small motoneurons was covered with terminal boutons only occupying a length of 287.45 μm (19.8 ± 11.9%). There were a total of 1,045 boutons on the large motoneurons and 204 on the small ones. The distribution of S- and F-type boutons may reflect excitatory and inhibitory synapses. The relative number of S-type boutons (58%) was larger than that of F-type boutons (42%) in the large neurons and showed similar values (S-type, 57%; F type, 43%) in the small ones. This is in contrast to mammalian spinal motoneurons in which F-type boutons are more prevalent than the S-type. The total numbers of the axosomatic boutons in large and small neurons were estimated, based on geometrical assumptions and found to differ substantially (840 vs. 59). This indicates large quantitative differences in the total number of synaptic inputs with the large motoneurons having a greater more axo-somatic bouton density. No large M- and C-type boutons occur on the spinal motoneurons of goldfish, suggesting a synaptic organization which is simple compared to that of terrestrial vertebrates. © 1993 Wiley-Liss, Inc.