Distinct Changes in Synaptic Protein Composition at Neuromuscular Junctions of Extraocular Muscles versus Limb Muscles of ALS Donors

The pathophysiology of amyotrophic lateral sclerosis (ALS) is very complex and still rather elusive but in recent years evidence of early involvement of the neuromuscular junctions (NMJs) has accumulated. We have recently reported that the human extraocular muscles (EOMs) are far less affected than limb muscles at the end-stage of ALS from the same donor. The present study aimed to compare the differences in synaptic protein composition at NMJ and in nerve fibers between EOM and limb muscles from ALS donors and controls. Neurofilament light subunit and synaptophysin decreased significantly at NMJs and in nerve fibers in limb muscles with ALS whereas they were maintained in ALS EOMs. S100B was significantly decreased at NMJs and in nerve fibers in both EOMs and limb muscles of ALS donors, but other markers confirmed the presence of terminal Schwann cells in these NMJs. p75 neurotrophin receptor was present in nerve fibers but absent at NMJs in ALS limb muscles. The EOMs were able to maintain the integrity of their NMJs to a very large extent until the end-stage of ALS, in contrast to the limb muscles. Changes in Ca²⁺ homeostasis, reflected by altered S100B distribution, might be involved in the breakdown of nerve-muscle contact at NMJs in ALS.     

Nebulin isoforms of extraocular muscle

The extraocular muscles (EOMs), which are responsible for reflexive and voluntary eye movements, have many unique biochemical, physiological, and ultrastructural features that set them apart from other skeletal muscles. For example, rodent EOMs lack M-lines and express EOM-specific myosin heavy chain (MYH13) and α-cardiac myosin heavy chain. Recent gene-expression profiling studies indicate the presence of other cardiac-specific proteins in adult EOMs. This interesting mixture of myofibrillar and cytoskeletal proteins poses the questions as to whether nebulette, as opposed to nebulin, might be expressed in EOM, and what isoforms of titin are expressed in the EOM. We have performed gel electrophoresis and immunological analyses to determine the titin and nebulin isoforms expressed in the EOM. We have found that the mass of the titin isoforms expressed in the EOM most closely resemble those found in the skeletal muscles tested, viz., the soleus and extensor digitorum longus (EDL). We also demonstrate that, although the EOM expresses cardiac isoforms of myosin, it does not express nebulette and contains a nebulin isoform with a mass consistent with that found in the prototypical fast hindlimb muscle EDL. This work was supported by grants from NIH-NHLB HL073089 to C.L.M. and NEI/NIH EY12998 to F.H.A.     

Extraocular muscle degeneration and regeneration after exposure of rats to incandescent radiant energy.

Exposure of albino rats to incandescent radiant energy for a short period of time in an elevated environmental temperature (39 degrees C) causes degenerative changes in the extraocular muscles. The muscle fibres regenerate and the muscles reorganize if the animals are returned to room lighting and temperature. Extraocular muscles (EOMs) were damaged first near their insertion on the eyeball. All EOMs of both eyes were affected, but the degeneration did not extend the entire length of the muscle. The peripheral fibres of each muscle were damaged before the more central fibres. Mitochondria were swollen and often contained dense bodies. Numerous vesicular profiles, possibly from the sarcotubular system, were present. Myofibrils of the more severely damaged fibres lacked typical Z-disk structures, and I-bands had disappeared by 24 h after the exposure period, a degenerative pattern which seems to be unique for this method of EOM damage. EOM degeneration appeared to be dependent on the interaction between thermal and radiant energy on the orbital contents. However, EOMs were only rarely and very slightly affected when rats were exposed to elevated temperature in the absence of incandescent radiant energy. When an opaque, black, ocular occluder was placed over one eye and the contralateral eye was left unoccluded, EOMs and retinas of occluded eyes were undamaged, while those tissues were severely damaged in unoccluded eyes. Therefore, the most critical single variable in inducing EOM degeneration appears to be exposure to radiant energy.     

Vestibulo-ocular pathways modulate extraocular muscle myosin expression patterns

The genetic and epigenetic influences that establish and maintain the unique phenotype of the extraocular muscles (EOMs) are poorly understood. The vestibulo-ocular reflex (VOR) represents an important input into the EOMs, as it stabilizes eye position relative to the environment and provides a platform for function of all other eye movement systems. A role for vestibular cues in shaping EOM maturation was assessed in these studies using the ototoxic nitrile compound 3',3'-iminodipropionitrile (IDPN) to eliminate the receptor hair cells that drive the vestibulo-ocular reflex. Intraperitoneal injections of IDPN were followed by a 2-week survival period, after which myosin heavy chain (MyHC) analysis of the EOMs was performed. When IDPN was administered to juvenile rats, the proportion of eye muscle fibers expressing developmental and fast myosins was increased, while EOM-specific MyHC mRNA levels were downregulated. By contrast, IDPN treatment in adult rats affected only the proportion of fibers expressing developmental MyHC isoforms, leaving the EOM-specific MyHC mRNA unaltered. These data provide evidence that the VOR modulates EOM-specific MyHC expression in development. The lack of significant changes in EOM-specific MyHC expression in adult EOM following IDPN administration suggests that there may be a critical period during development when alterations in vestibular activity have significant and permanent consequences for the eye muscles.     

Muscle spindle autogenetic inhibition in the extraocular muscles of lamb

The role of extraocular muscle (EOM) proprioceptors on eye motility has been investigated in lambs on "encéphale isolé", by evaluating the tension of EOMs at various lengths and velocities of stretch before and after proprioceptive blocks. The EOM tension, in the absence of proprioceptive input, was higher than in normal conditions. Such an effect occurred at lengthening values greater than 3 mm of stretch from resting muscle length, corresponding to 18 degrees of eye deviation and was dependent on the velocity of the stretch, being more effective at high velocity. The muscle receptors responsible for this effect was determined by comparing the sensitivity to vibratory stimulation of spindles and tendon organs to the amount of inhibition provoked by the same stimulation on an EOM electromyographic activity. The tension inhibition appeared to be correlated to muscle spindle activation. Thus, the presence of muscle spindles can determine a reduction of the tension within the stretched muscles. This result suggests that the EOM length and velocity signals operate moment to moment reduction on the stiffness of the muscle which antagonizes eye displacement, thus facilitating the ocular movements.     

The development of extraocular muscle calcium homeostasis parallels visuomotor system maturation

Extraocular muscle is modulated by unique genetic and epigenetic factors to produce an atypical phenotype. As a prelude to regulation studies, we characterized the development of cation homeostasis in the predominately fast-twitch extraocular muscles. By atomic absorption spectroscopy, total muscle calcium content declined from birth to postnatal day 27 and, thereafter, stabilized at a low level in limb but increased dramatically in extraocular muscle (to 40x limb values). By ELISA, the slow isoform of sarcoplasmic reticulum Ca2+-ATPase predominated in neonatal eye muscle, but subsequently was largely replaced by the fast isoform. This replacement in eye muscle was completed later than in limb. Residual, slow Ca2+-ATPase likely resides in an unusual slow tonic fiber type characteristic of eye muscle. Maturation of the definitive extraocular muscle Ca2+-ATPase pattern paralleled myofiber Ca2+ and sarcoplasmic reticulum content. These data show that, like myosin heavy chain expression patterns, the development of cation homeostatic mechanisms in extraocular muscle parallels landmarks in the maturation of vision and eye movement control systems. Findings suggest that cation homeostasis in extraocular muscle may be susceptible to perturbations of the developing visual sensory system, as we have previously shown for myosin.     

The visual system of zebrafish and its use to model human ocular diseases

Free swimming zebrafish larvae depend mainly on their sense of vision to evade predation and to catch prey. Hence, there is strong selective pressure on the fast maturation of visual function and indeed the visual system already supports a number of visually driven behaviors in the newly hatched larvae.The ability to exploit the genetic and embryonic accessibility of the zebrafish in combination with a behavioral assessment of visual system function has made the zebrafish a popular model to study vision and its diseases.Here, we review the anatomy, physiology, and development of the zebrafish eye as the basis to relate the contributions of the zebrafish to our understanding of human ocular diseases.     

Localization and somatotopy of sensory cells innervating the extraocular muscles of lamb, pig and cat. Histochemical and electrophysiological investigation

The localization of sensory cells innervating the extraocular muscles (EOMs) was studied in the lamb, pig and cat in which horseradish peroxidase (HRP) was injected into each EOM. Electrophysiological techniques were also used to search for EOM stretch sensitive units in the semilunar ganglion. In lamb and pig labeling was observed in the semilunar ganglion only, while in cat labeled neurons were present in both the semilunar ganglion and mesencephalic trigeminal nucleus. In the semilunar ganglion of all these species a clear somatotopic organization of EOM afferents was observed. The histochemical somatotopic pattern of EOM afferents in the semilunar ganglion of lamb and pig was substantially in agreement with the electrophysiological arrangement. The responses recorded to EOM stretch in the semilunar ganglion of the pig were characterized by a low threshold and a slow adaptation as previously found in the lamb; on the contrary, in the semilunar ganglion of the cat only a few units were found, which showed high stretch threshold and quick adaptation.     

Microcirculation of the extra-ocular muscles of rats. A scanning electron-microscopic corrosion cast study.

Specific researches, employing corrosion casts, were performed on different skeletal muscles, but not on extra-ocular muscles (EOMs). The microvascular bed of EOMs was studied by the corrosion cast technique in the rat. Two histologically and physiologically different layers were present in the EOMs. On the whole, the capillary pattern of EOMs was less dense than in the other skeletal muscles. The EOM orbital layer turned out to have a higher number of transverse anastomoses and tuning-fork divisions than the global layer had. These different microcirculatory patterns can be related with the physiological function and the anatomical situation of the EOMs.     

Co-expression of multiple myosin heavy chain genes, in addition to a tissue-specific one, in extraocular musculature

We have investigated the developmental transitions of myosin heavy chain (MHC) gene expression in the rat extraocular musculature (EOM) at the mRNA level using S1-nuclease mapping techniques and at the protein level by polypeptide mapping and immunochemistry. We have isolated a genomic clone, designated lambda 10B3, corresponding to an MHC gene which is expressed in the EOM fibers (recti and oblique muscles) of the adult rat but not in hind limb muscles. Using cDNA and genomic probes for MHC genes expressed in skeletal (embryonic, neonatal, fast oxidative, fast glycolytic, and slow/cardiac beta-MHC), cardiac (alpha-MHC), and EOM (lambda 10B3) muscles, we demonstrate the concomitant expression at the mRNA level of at least six different MHC genes in adult EOM. Protein and immunochemical analyses confirm the presence of at least four different MHC types in EOM. Immunocytochemistry demonstrates that different myosin isozymes tend to segregate into individual myofibers, although some fibers seem to contain more than one MHC type. The results also show that the EOM fibers exhibit multiple patterns of MHC gene regulation. One set of fibers undergoes a sequence of isoform transitions similar to the one described for limb skeletal muscles, whereas other EOM myofiber populations arrest the MHC transition at the embryonic, neonatal/adult, or adult EOM-specific stage. Thus, the MHC gene family is not under the control of a strict developmental clock, but the individual genes can modify their expression by tissue-specific and/or environmental factors.     

Nonmuscle myosin IIB, a sarcomeric component in the extraocular muscles

Extraocular muscles (EOMs) are categorized as skeletal muscles; however, emerging evidence indicates that their gene expression profile, metabolic characteristics and functional properties are significantly different from the prototypical members of this muscle class. Gene expression profiling of developing and adult EOM suggest that many myofilament and cytoskeletal proteins have unique expression patterns in EOMs, including the maintained expression of embryonic and fetal isoforms of myosin heavy chains (MyHC), the presence of a unique EOM specific MyHC and mixtures of both cardiac and skeletal muscle isoforms of thick and thin filament accessory proteins. We demonstrate that nonmuscle myosin IIB (nmMyH IIB) is a sarcomeric component in ∼ 20% of the global layer fibers in adult rat EOMs. Comparisons of the myofibrillar distribution of nmMyHC IIB with sarcomeric MyHCs indicate that nmMyH IIB co-exists with slow MyHC isoforms. In longitudinal sections of adult rat EOM, nmMyHC IIB appears to be restricted to the A-bands. Although nmMyHC IIB has been previously identified as a component of skeletal and cardiac sarcomeres at the level of the Z-line, the novel distribution of this protein within the A band in EOMs is further evidence of both the EOMs complexity and unconventional phenotype.     

Nitric oxide synthase expression and effects of nitric oxide modulation on contractility of rat extraocular muscle.

Extraocular muscles (EOMs) are specialized skeletal muscles that are constantly active, generate low levels of force for cross sectional area, have rapid contractile speeds, and are highly fatigue resistant. The neuronal isoform of nitric oxide synthase (nNOS) is concentrated at the sarcolemma of fast-twitch muscles fibers, and nitric oxide (NO) modulates contractility. This study evaluated nNOS expression in EOM and the effect of NO modulation on lateral rectus muscle's contractility. nNOS activity was highest in EOM compared with diaphragm, extensor digitorum longus, and soleus. Neuronal NOS was concentrated to the sarcolemma of orbital and global singly innervated fibers, but not evident in the multi-innervated fibers. The NG-nitro-L-arginine methyl ester (L-NAME, a NOS inhibitor), increased submaximal tetanic and peak twitch forces. The NO donors S-nitroso-N-acetylcysteine (SNAC) and spermineNONOate reduced submaximal tetanic and peak twitch forces. The effect of NO on the contractile force of lateral rectus muscle is greater than previously observed on other skeletal muscle. NO appears more important in modulating contraction of EOM compared with other skeletal muscles, which could be important for the EOM's specialized role in generation of eye movements.     

Shared resistance to aging and ALS in neuromuscular junctions of specific muscles

Normal aging and neurodegenerative diseases both lead to structural and functional alterations in synapses. Comparison of synapses that are generally similar but respond differently to insults could provide the basis for discovering mechanisms that underlie susceptibility or resistance to damage. Here, we analyzed skeletal neuromuscular junctions (NMJs) in 16 mouse muscles to seek such differences. We find that muscles respond in one of three ways to aging. In some, including most limb and trunk muscles, age-related alterations to NMJs are progressive and extensive during the second postnatal year. NMJs in other muscles, such as extraocular muscles, are strikingly resistant to change. A third set of muscles, including several muscles of facial expression and the external anal sphinter, succumb to aging but not until the third postnatal year. We asked whether susceptible and resistant muscles differed in rostrocaudal or proximodistal position, source of innervation, motor unit size, or fiber type composition. Of these factors, muscle innervation by brainstem motor neurons correlated best with resistance to age-related decline. Finally, we compared synaptic alterations in normally aging muscles to those in a mouse model of amyotrophic lateral sclerosis (ALS). Patterns of resistance and susceptibility were strikingly correlated in the two conditions. Moreover, damage to NMJs in aged muscles correlated with altered expression and distribution of CRMP4a and TDP-43, which are both altered in motor neurons affected by ALS. Together, these results reveal novel structural, regional and molecular parallels between aging and ALS.     

The Development of Vision in the Zebrafish (Danio rerio)

We studied the development and maturation of the visual system by determining when zebrafish begin to see and to move their eyes. This information was correlated with the time courses of the development of the retina, the retinofugal projection, the retinal image, and the extraocular muscles, to obtain an integrated picture of early visual development. Two visual behaviors were monitored over 48–96 hr postfertilization (hpf). The startle response (body twitch) was evoked by an abrupt decrease in light intensity. The optokinetic response (tracking eye movements) was evoked by rotation of a striped drum. Visually evoked startle developed over 68–79 hpf, more than 20 hr after the onset of a touch-evoked startle. It was not seen in eyeless fish, excluding a role for nonretinal light senses. Tracking eye movements developed over 73–80 hpf. They were always in the direction of drum rotation, even when the fish had been light deprived from blastula stage, ruling out a “trial and error” period of learning to track the drum. The image formed by the ocular lens was examined in intact fish made transparent by suppressing the formation of melanin. The eye was initially far sighted and gradually improved, so that by 72 hpf the image plane coincided with the photoreceptor layer. The extraocular muscles assumed their adult configuration between 66 and 72 hpf. Thus, the retinal image and functional extraocular muscles appeared nearly simultaneously with the onset of tracking eye movements and probably represent the last events in the construction of this behavior.     

Palisade endings and proprioception in extraocular muscles: a comparison with skeletal muscles

This article describes current views on motor and sensory control of extraocular muscles (EOMs) based on anatomical data. The special morphology of EOMs, including their motor innervation, is described in comparison to classical skeletal limb and trunk muscles. The presence of proprioceptive organs is reviewed with emphasis on the palisade endings (PEs), which are unique to EOMs, but the function of which is still debated. In consideration of the current new anatomical data about the location of cell bodies of PEs, a hypothesis on the function of PEs in EOMs and the multiply innervated muscle fibres they are attached to is put forward.     

Presynaptic to postsynaptic relationships of the neuromuscular junction are held constant across age and muscle fiber type

The neuromuscular junction (NMJ) displays considerable morphological plasticity as a result of differences in activity level, as well as aging. This is true of both presynaptic and postsynaptic components of the NMJ. Yet, despite these variations in NMJ structure, proper presynaptic to postsynaptic coupling must be maintained in order for effective cell‐to‐cell communication to occur. Here, we examined the NMJs of muscles with different activity profiles (soleus and EDL), on both slow‐ and fast‐twitch fibers in those muscles, and among young adult and aged animals. We used immunofluorescent techniques to stain nerve terminal branching, presynaptic vesicles, postsynaptic receptors, as well as fast/slow myosin heavy chain. Confocal microscopy was used to capture images of NMJs for later quantitative analysis. Data were subjected to a two‐way ANOVA (main effects for myofiber type and age), and in the event of a significant (p < 0.05) F ratio, a post hoc analysis was performed to identify pairwise differences. Results showed that the NMJs of different myofiber types routinely displayed differences in presynaptic and postsynaptic morphology (although the effect on NMJ size was reversed in the soleus and the EDL), but presynaptic to postsynaptic relationships were tightly maintained. Moreover, the ratio of presynaptic vesicles relative to nerve terminal branch length also was similar despite differences in muscles, their fiber type, and age. Thus, in the face of considerable overall structural differences of the NMJ, presynaptic to postsynaptic coupling remains constant, as does the relationship between presynaptic vesicles and the nerve terminal branches that support them. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 744–753, 2013     

Extraocular Muscle Characteristics Related to Myasthenia Gravis Susceptibility

Background

The pathogenesis of extraocular muscle (EOM) weakness in myasthenia gravis might involve a mechanism specific to the EOM. The aim of this study was to investigate characteristics of the EOM related to its susceptibility to myasthenia gravis.

Methods

Female F344 rats and female Sprague-Dawley rats were assigned to experimental and control groups. The experimental group received injection with Ringer solution containing monoclonal antibody against the acetylcholine receptor (AChR), mAb35 (0.25 mg/kg), to induce experimental autoimmune myasthenia gravis, and the control group received injection with Ringer solution alone. Three muscles were analyzed: EOM, diaphragm, and tibialis anterior. Tissues were examined by light microscopy, fluorescence histochemistry, and transmission electron microscopy. Western blot analysis was used to assess marker expression and ELISA analysis was used to quantify creatine kinase levels. Microarray assay was conducted to detect differentially expressed genes.

Results

In the experimental group, the EOM showed a simpler neuromuscular junction (NMJ) structure compared to the other muscles; the NMJ had fewer synaptic folds, showed a lesser amount of AChR, and the endplate was wider compared to the other muscles. Results of microarray assay showed differential expression of 54 genes in the EOM between the experimental and control groups.

Conclusion

Various EOM characteristics appear to be related to the increased susceptibility of the EOM and the mechanism of EOM weakness in myasthenia gravis.     

Control of human eye movements: II. a model for the extraocular plant mechanism

Control of eye movements is essential in accomplishing visual or perceptive tasks. The brain and central nervous system process retinal information and send nervous signals to the extraocular muscles, which exert forces that cause the eye to move. A model for the human extraocular plant, which consists of the nervous input signals, the extraocular muscles, the orbit and the globe, is proposed. The derivation is based on anatomical and physiological data as well as experiments concerned with a variety of eye movements under normal and abnormal conditions. The nervous activity controlling eye movements was estimated from electromyography and single unit studies of the extraocular nuclei. The equations describing muscle properties were discussed in a previous paper by the authors; these results were incorporated into the present model. The characteristics of the isolated globe and its visco-elastic interaction with the orbit were computed from length- tension curves and isotonic experiments. Simulations using the resulting representation accurately depicted the isotonic experiments on the isolated globe and on the total extraocular plant, the isometric forces during three different types of eye movements, and the weighted globe experiment. A future paper will show that the model accurately simulates normal eye movements of different types and amplitudes.     

A continuum of myofibers in adult rabbit extraocular muscle: force, shortening velocity, and patterns of myosin heavy chain colocalization

Extraocular muscle (EOM) myofibers do not fit the traditional fiber typing classifications normally used in noncranial skeletal muscle, in part, due to the complexity of their individual myofibers. With single skinned myofibers isolated from rectus muscles of normal adult rabbits, force and shortening velocity were determined for 220 fibers. Each fiber was examined for myosin heavy chain (MyHC) isoform composition by densitometric analysis of electrophoresis gels. Rectus muscle serial sections were examined for coexpression of eight MyHC isoforms. A continuum was seen in single myofiber shortening velocities as well as force generation, both in absolute force (g) and specific tension (kN/m(2)). Shortening velocity correlated with MyHCIIB, IIA, and I content, the more abundant MyHC isoforms expressed within individual myofibers. Importantly, single fibers with similar or identical shortening velocities expressed significantly different ratios of MyHC isoforms. The vast majority of myofibers in both the orbital and global layers expressed more than one MyHC isoform, with up to six isoforms in single fiber segments. MyHC expression varied significantly and unpredictably along the length of single myofibers. Thus EOM myofibers represent a continuum in their histological and physiological characteristics. This continuum would facilitate fine motor control of eye position, speed, and direction of movement in all positions of gaze and with all types of eye movements-from slow vergence movements to fast saccades. To fully understand how the brain controls eye position and movements, it is critical that this significant EOM myofiber heterogeneity be integrated into hypotheses of oculomotor control.     

In vitro genotoxicity of PAH mixtures and organic extract from urban air particles part II: human cell lines

Principal aims of this study were at first, to find a relevant human derived cell line to investigate the genotoxic potential of PAH-containing complex mixtures and second, to use this cell system for the analysis of DNA adduct forming activity of organic compounds bound onto PM10 particles. Particles were collected by high volume air samplers during summer and winter periods in three European cities (Prague, Kosice, and Sofia), representing different levels of air pollution. The genotoxic potential of extractable organic matter (EOM) was compared with the genotoxic potential of individual carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) as well as their artificial mixtures. Metabolically competent human hepatoma HepG2 cells, confluent cultures of human diploid lung fibroblasts (HEL), and the human monocytic leukemia cell line THP-1 were used as models. DNA adducts were analyzed by ³²P-postlabeling. The total DNA adduct levels induced in HepG2 cells after exposure to EOMs were higher than in HEL cells treated under the same conditions (15–190 versus 2–15 adducts/10⁸ nucleotides, in HepG2 and HEL cells, respectively). THP-1 cells exhibited the lowest DNA adduct forming activity induced by EOMs (1.5–3.7 adducts/10⁸ nucleotides). A direct correlation between total DNA adduct levels and c-PAH content in EOM was found for all EOMs in HepG2 cells incubated with 50 μg EOM/ml (R = 0.88; p = 0.0192). This correlation was even slightly stronger when B[a]P content in EOMs and B[a]P-like adduct spots were analyzed (R = 0.90; p = 0.016). As THP-1 cells possess a limited metabolic capacity for most c-PAHs to form DNA reactive intermediates and are also more susceptible to toxic effects of PAHs and various EOM components, this cell line seemed to be an inappropriate system for genotoxicity studies of PAH-containing complex mixtures. The seasonal variability of genotoxic potential of extracts was stronger than variability among the three localities studied. In HepG2 cells, the highest DNA adduct levels were induced by EOM collected in Prague in the winter period, followed by Sofia and Kosice. However, in the summer sampling period, the order was quite opposite: Kosice > Sofia > Prague. When the EOM content per m³ of air was taken into consideration in order to compare real exposures of humans to genotoxic compounds in all three localities, extracts from respirable dust particles collected in Sofia exhibited the highest genotoxicity regardless of the sampling period. The results indicate that most of DNA adducts detected in cells incubated with EOMs have their origin in low concentrations of c-PAHs representing 0.03–0.17% of EOM total mass. Finally, our results suggest that HepG2 cells have a metabolic capacity for PAHs similar to human hepatocytes and represent therefore the best in vitro model for investigating the genotoxic potential of complex mixtures containing PAHs among the three cell lines tested in this study.