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Biomedical subjects

S R Chen

Publications and source records attributed to S R Chen.

At least 37 records · Page 2Linked to original sources
PubMed2000

Synergistic effect between intrathecal non-NMDA antagonist and gabapentin on allodynia induced by spinal nerve ligation in rats.

BACKGROUND: Glutamate and non-N-methyl-D-aspartate (NMDA) receptors have been implicated in the development of neuroplasticity in the spinal cord in neuropathic pain. The spinal cord has been identified as one of the sites of the analgesic action of gabapentin. In the current study, the authors determined the antiallodynic effect of intrathecal 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) in a rat model of neuropathic pain. Also tested was a hypothesis that intrathecal injection of CNQX and gabapentin produces a synergistic effect on allodynia in neuropathic rats. METHODS: Allodynia was produced in rats by ligation of the left L5 and L6 spinal nerves. Allodynia was determined by application of von Frey filaments to the left hind paw. Through an implanted intrathecal catheter, 10-100 microg gabapentin or 0.5-8 microg CNQX disodium (a water-soluble formulation of CNQX) was injected in conscious rats. Isobolographic analysis was performed comparing the interaction of intrathecal gabapentin and CNQX using the ED50 dose ratio of 15:1. RESULTS: Intrathecal treatment with gabapentin or CNQX produced a dose-dependent increase in the withdrawal threshold to mechanical stimulation. The ED50 for gabapentin and CNQX was 45.9+/-4.65 and 3.4+/-0.22 microg, respectively. Intrathecal injection of a combination of CNQX and gabapentin produced a strong synergistic antiallodynic effect in neuropathic rats. CONCLUSIONS: This study shows that intrathecal administration of CNQX exhibits an antiallodynic effect in this rat model of neuropathic pain. Furthermore, CNQX and gabapentin, when combined intrathecally, produce a potent synergistic antiallodynic effect on neuropathic pain in spinal nerve-ligated rats.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2000

Cardiac interstitial bradykinin release during ischemia is enhanced by ischemic preconditioning.

Ischemic preconditioning is known to protect the myocardium from ischemia-reperfusion injury. We examined the transmural release of bradykinin during myocardial ischemia and the influence of ischemic preconditioning on bradykinin release during subsequent myocardial ischemia. Myocardial ischemia was induced by occlusion of the left anterior descending coronary artery in anesthetized cats. Cardiac microdialysis was performed by implantation and perfusion of dialysis probes in the epicardium and endocardium. In eight animals, bradykinin release was greater in the endocardium than in the epicardium (14.4 +/- 2.8 vs. 7.3 +/- 1.7 ng/ml, P < 0.05) during 30 min of ischemia. In seven animals subjected to preconditioning, myocardial bradykinin release was potentiated significantly from 2.4 +/- 0.6 ng/ml during the control period to 23.1 +/- 2.5 ng/ml during 30 min of myocardial ischemia compared with the non-preconditioning group (from 2.7 +/- 0.6 to 13.4 +/- 1.9 ng/ml, P < 0.05, n = 6). Thus this study provides further evidence that transmural gradients of bradykinin are produced during ischemia. The results also suggest that ischemic preconditioning enhances bradykinin release in the myocardial interstitial fluid during subsequent ischemia, which is likely one of the mechanisms of cardioprotection of ischemic preconditioning.

Animals↗
PubMed1999

The RCAF complex mediates chromatin assembly during DNA replication and repair.

Chromatin assembly is a fundamental biological process that is essential for the replication and maintenance of the eukaryotic genome. In dividing cells, newly synthesized DNA is rapidly assembled into chromatin by the deposition of a tetramer of the histone proteins H3 and H4, followed by the deposition of two dimers of histones H2A and H2B to complete the nucleosome-the fundamental repeating unit of chromatin. Here we describe the identification, purification, cloning, and characterization of replication-coupling assembly factor (RCAF), a novel protein complex that facilitates the assembly of nucleosomes onto newly replicated DNA in vitro. RCAF comprises the Drosophila homologue of anti-silencing function 1 protein ASF1 and histones H3 and H4. The specific acetylation pattern of H3 and H4 in RCAF is identical to that of newly synthesized histones. Genetic analyses in Saccharomyces cerevisiae demonstrate that ASF1 is essential for normal cell cycle progression, and suggest that RCAF mediates chromatin assembly after DNA replication and the repair of double-strand DNA damage in vivo.

Amino Acid Sequence↗
PubMed1999

HEK-293 cells possess a carbachol- and thapsigargin-sensitive intracellular Ca2+ store that is responsive to stop-flow medium changes and insensitive to caffeine and ryanodine.

Because HEK-293 cells are widely used for the functional expression of channels, exchangers and transporters involved in Ca(2+) homoeostasis, the properties of intracellular Ca(2+) stores and the methods used for measuring intracellular Ca(2+) release in HEK-293 cells were evaluated. Ca(2+) imaging was used to show caffeine-, carbachol- and thapsigargin-induced Ca(2+) release in HEK-293 cells transfected with ryanodine receptor (RyR) cDNA, but only carbachol- and thapsigargin-induced Ca(2+) release in untransfected HEK-293 cells. Intracellular Ca(2+) release in untransfected HEK-293 cells was also observed if medium changes were performed by aspirating and replacing fresh medium (stop-flow), but not if medium changes were performed by a continuous over-flow procedure. Stop-flow medium-change-induced Ca(2+) release in HEK-293 cells was independent of caffeine and ryanodine, demonstrating that it did not occur through RyR channels. Consistent with these observations was the observation that the level of expression of endogenous RyR proteins was below the limits of detection by Western blotting or [(3)H]ryanodine binding. Thus the level of endogenous expression of RyR is so low in HEK-293 cells as to provide a negligible background in relation to functional analysis of recombinant RyR molecules. These results are inconsistent with those of Querfurth et al. [Querfurth, Haughey, Greenway, Yacono, Golan and Geiger (1998) Biochem. J. 334, 79-86], who reported higher levels of endogenous RyR expression in untransfected HEK-293 cells.

Caffeine↗
PubMed1999

Molecular identification of the ryanodine receptor pore-forming segment.

A sequence motif, GXRXGGGXGD, located in the putative channel-forming domain, is conserved in all known ryanodine receptors and inositol 1,4,5-trisphosphate receptors. The functional significance of this conserved region was investigated by using site-directed mutagenesis together with functional assays consisting of Ca(2+) release measurements, [(3)H]ryanodine binding, and single channel recordings in planar lipid bilayers. We report here that single point mutations introduced into this region of the mouse cardiac ryanodine receptor reduce or abolish high affinity [(3)H]ryanodine binding. Single channel analysis revealed that a single substitution of alanine for glycine 4824 within this region reduced the single channel conductance by 97%, from 798 picosiemens (pS) for the wild type channel to 22 pS. The G4824A mutant channel was modulated by Ca(2+), Mg(2+), ATP, caffeine, ruthenium red, and ryanodine. Co-expression of the wild type and G4824A mutant proteins produced single channels that have intermediate unitary conductances of 516, 256, 176, and 60 pS. These data suggest that this conserved region constitutes an essential part of the ryanodine binding site and the channel conduction pathway of the ryanodine receptor.

Amino Acid Sequence↗
PubMed1999

Role of protons in activation of cardiac sympathetic C-fibre afferents during ischaemia in cats.

1. Chest pain caused by myocardial ischaemia is mediated by cardiac sympathetic afferents. The mechanisms of activation of cardiac afferents during ischaemia remain poorly understood. Increased lactic acid production is associated closely with myocardial ischaemia. The present study examined the role of protons generated during ischaemia in activation of cardiac sympathetic C-fibre afferents. 2. Single-unit activity of cardiac afferents innervating both ventricles was recorded from the left sympathetic chain in anaesthetized cats. Epicardial tissue pH was measured within 1-1.5 mm of the surface by a pH-sensitive needle electrode. Responses of cardiac afferents to myocardial ischaemia, lactic acid, sodium lactate, acidic phosphate buffer and hypercapnia were determined. 3. Occlusion of the coronary artery for 5 min decreased epicardial tissue pH from 7.35 +/- 0.21 to 6.98 +/- 0.22 (P < 0.05). Epicardial placement of isotonic neutral phosphate buffer, but not saline, prevented the ischaemia-induced decrease in epicardial pH. This manoeuvre significantly attenuated the response of 16 afferents to 5 min of ischaemia (1.56 +/- 0.23 pre-treatment vs. 0.67 +/- 0.18 impulses s-1). Topical application of 10-100 microg ml-1 of lactic acid, but not sodium lactate, concentration-dependently stimulated 18 cardiac afferents. Inhalation with high-CO2 gas failed to activate 12 separate cardiac afferents. Furthermore, lactic acid stimulated cardiac afferents to a greater extent than acidic phosphate buffer solution, applied at a similar pH to the same afferents. 4. Collectively, this study provides important in vivo evidence that protons contribute to activation/sensitization of cardiac sympathetic C-fibre afferents during myocardial ischaemia.

Acid-Base Equilibrium↗
PubMed1999

A frame shift mutation in the DNA-binding domain of the androgen receptor gene associated with complete androgen insensitivity, persistent müllerian structures, and germ cell tumors in dysgenetic gonads.

OBJECTIVE: To describe the molecular, cytogenetic, immunohistochemical, and endocrinologic characteristics of a young 46,XY female with persistent müllerian structures and germ cell tumors in dysgenetic gonads. DESIGN: Descriptive case study. SETTING: Mackay Memorial Hospital and National Yang-Ming University, Taipei, Taiwan. PATIENT(S): A 22-year-old 46,XY female with persistent müllerian structures, a low level of serum testosterone, and no apparent adnexal masses. INTERVENTION(S): Laparoscopic removal of the dysgenetic gonads. MAIN OUTCOME MEASURE(S): Detection of an androgen receptor gene mutation by a semiautomated DNA sequencer, of the chromosomal complement by cytogenetic examination, of placental alkaline phosphatase activity by immunohistochemical analysis, and of neoplasms in dysgenetic gonads by histologic studies. RESULT(S): A unilateral gonadoblastoma and a contralateral gonadoblastoma associated with a dysgerminoma were found in the excised gonads. The tumors had a 46,XY complement. Placental alkaline phosphatase was present in the tumor cells. A frameshift mutation in the DNA-binding domain of the androgen receptor gene was detected in the patient's blood and the tumor tissues. A five-nucleotide "AGGAA" deletion at codons 608 and 609 of the androgen receptor gene resulted in a missing arginine and lysine as well as a frameshift that introduced a stop codon 12 amino acid downstream from the mutation. CONCLUSION(S): Molecular genetic analysis of the androgen receptor gene aids in the rapid diagnosis of complete androgen insensitivity irrespective of atypical clinical phenotypes and endocrinologic parameters.

Adult↗
PubMed1999

Intrathecal clonidine alleviates allodynia in neuropathic rats: interaction with spinal muscarinic and nicotinic receptors.

BACKGROUND: Intrathecally administered clonidine increases release of spinal acetylcholine, which may be related to its analgesic action in neuropathic pain. The current study determined the role of spinal muscarinic and nicotinic receptors in the antiallodynic effect of intrathecally administered clonidine in spinal nerve-ligated rats. METHODS: Allodynia was produced in rats by ligation of the left L5-L6 spinal nerves. Mechanical allodynia was determined by application of von Frey filaments to the left hindpaw. The effect of intrathecal injection of saline, two muscarinic receptor antagonists (atropine and scopolamine), and two nicotinic receptor antagonists (mecamylamine and hexamethonium) on the antiallodynic action produced by intrathecal administration of 20 microg clonidine was assessed in six groups of animals. Each group consisted of six to eight rats. RESULTS: Intrathecal injection of saline or muscarinic or nicotinic receptor antagonists did not alter the withdrawal thresholds. The antiallodynic effect produced by intrathecally administered clonidine was attenuated in a dose-dependent manner by intrathecal treatment with muscarinic and nicotinic antagonists. Although nicotinic receptor antagonists only partially attenuated the effect of clonidine, blockade of spinal muscarinic receptors almost abolished the antiallodynic effect of clonidine. CONCLUSIONS: These results demonstrate that the analgesic effect of intrathecally administered clonidine on neuropathic pain is mediated by spinal muscarinic and nicotinic receptors. Therefore, this study provides functional evidence that spinally released acetylcholine plays a role in the antiallodynic effect of intrathecally administered clonidine in neuropathic pain.

Analgesics↗
PubMed1999

Gabapentin suppresses ectopic nerve discharges and reverses allodynia in neuropathic rats.

Repetitive ectopic discharges from injured afferent nerves play an important role in initiation and maintenance of neuropathic pain. Gabapentin is effective for treatment of neuropathic pain but the sites and mechanisms of its antinociceptive actions remain uncertain. In the present study, we tested a hypothesis that therapeutic doses of gabapentin suppress ectopic afferent discharge activity generated from injured peripheral nerves. Mechanical allodynia, induced by partial ligation of the sciatic nerve in rats, was determined by application of von Frey filaments to the hindpaw. Single-unit afferent nerve activity was recorded proximal to the ligated sciatic nerve site. Intravenous gabapentin, in a range of 30 to 90 mg/kg, significantly attenuated allodynia in nerve-injured rats. Furthermore, gabapentin, in the same therapeutic dose range, dose-dependently inhibited the ectopic discharge activity of 15 injured sciatic afferent nerve fibers through an action on impulse generation. However, the conduction velocity and responses of 12 normal afferent fibers to mechanical stimulation were not affected by gabapentin. Therefore, this study provides electrophysiological evidence that gabapentin is capable of suppressing the ectopic discharge activity from injured peripheral nerves. This action may contribute, at least in part, to the antiallodynic effect of gabapentin on neuropathic pain.

Acetates↗
PubMed1998

Molecular identification of the ryanodine receptor Ca2+ sensor.

We have investigated the molecular basis for ryanodine receptor (RyR) activation by Ca2+ by using site-directed mutagenesis together with functional assays consisting of Ca2+ release measurements and single channel recordings in planar lipid bilayers. We report here that a single substitution of alanine for glutamate at position 3885 (located in the putative transmembrane sequence M2 of the type 3 RyR) reduces the Ca2+ sensitivity, as measured by single channel activation, by more than 10,000-fold, without apparent changes in channel conductance and in modulation by other ligands (e.g. ATP and ryanodine). Co-expression of the wild type and mutant RyR proteins results in the synthesis of single channels that have intermediate Ca2+ sensitivities. These results suggest that the glutamates at position 3885 of each monomer may act in a coordinated way to form the Ca2+ sensor in the tetrameric structure corresponding to RyR.

Amino Acid Sequence↗
PubMed1998

Role of spinal NO in antiallodynic effect of intrathecal clonidine in neuropathic rats.

BACKGROUND: The role of spinal nitric oxide (NO) in neuropathic pain remains uncertain. Although intrathecal clonidine causes NO release in the spinal cord, the functional role of spinal NO in clonidine-produced analgesia has not been examined. The objectives of this study were to assess the role of spinal NO in maintenance of allodynia and to determine the role of spinal NO in the antiallodynic effect of intrathecal clonidine. METHODS: Allodynia was produced in rats by tight ligation of the left L5-L6 spinal nerves. Intrathecal catheters were inserted with tips in the lumbar intrathecal space. Mechanical allodynia was determined by application of von Frey filaments to the left hindpaw. In the first series of experiments, allodynia was assessed before and after intrathecal injection of saline, L-arginine, an NO donor (SNAP), two NO synthase inhibitors (TRIM and NMMA), or an NO scavenger (PTIO). In the second series of experiments, 20 microg of clonidine was injected intrathecally 15 min after intrathecal injection of saline, TRIM, NMMA, or PTIO. RESULTS: Allodynia was not affected significantly by intrathecal injection of L-arginine, SNAP, TRIM, NMMA, or PTIO. The antiallodynic effect produced by intrathecal injection of clonidine was attenuated significantly by pretreatment with TRIM, NMMA, or PTIO. CONCLUSIONS: These results demonstrate that spinal NO neither contributes significantly to maintenance of allodynia nor produces detectable antiallodynic effect in this neuropathic pain model. Furthermore, this study provides functional evidence that spinal NO plays an important role in the antiallodynic effect of intrathecal clonidine in neuropathic pain.

Adrenergic alpha-Agonists↗
PubMed1997

Functional characterization of the recombinant type 3 Ca2+ release channel (ryanodine receptor) expressed in HEK293 cells.

To investigate the channel properties of the mammalian type 3 ryanodine receptor (RyR3), we have cloned the RyR3 cDNA from rabbit uterus by reverse transcriptase-polymerase chain reaction and expressed the cDNA in HEK293 cells. Immunoblotting studies showed that the cloned RyR3 was indistinguishable from the native mammalian RyR3 in molecular size and immunoreactivity. Ca2+ release measurements using the fluorescence Ca2+ indicator fluo 3 revealed that the cloned RyR3 functioned as a caffeine- and ryanodine-sensitive Ca2+ release channel in HEK293 cells. Functional properties of the cloned RyR3 were further characterized by using single channel recordings in lipid bilayers. The cloned RyR3 channel exhibited a K+ conductance of 777 picosiemens in 250 mM KCl and a Ca2+ conductance of 137 picosiemens in 250 mM CaCl2 and displayed a pCa2+/pK+ ratio of 6.3 and an open time constant of about 1.16 ms. The response of the cloned RyR3 to cytoplasmic Ca2+ concentrations was biphasic. The channel was activated by Ca2+ at about 100 nM and inactivated at about 10 mM. Ca2+ alone was able to activate the cloned RyR3 fully. Calmodulin activated the cloned RyR3 at low Ca2+ concentrations but inhibited the channel at high Ca2+ concentrations. The cloned RyR3 was activated by ATP, caffeine, and perchlorate, inhibited by Mg2+ and ruthenium red, and modified by ryanodine. Cyclic ADP-ribose did not seem to affect single channel activity of the cloned RyR3. The most prominent differences of the cloned RyR3 from the rabbit skeletal muscle ryanodine receptor were in the gating kinetics, extent of maximal activation by Ca2+, and sensitivity to Ca2+ inactivation. Results of the present study provide initial insights into the single channel properties of the mammalian RyR3.

Adenosine Diphosphate Ribose↗
PubMed1997

Valtrac-secured intracolonic bypass device: an experimental study.

PURPOSE: The intracolonic bypass tube has been used both experimentally and clinically to protect the anastomotic site. A newly designed intracolonic bypass, the Valtrac-secured intracolonic bypass, which consisted of a biofragmentable anastomosis ring (BAR) and was connected with a soft, thin vinyl tube, was used in the colon approximately 5 to 10 cm proximal to the anastomotic site. The distal end of the vinyl tube is passed through the colonic anastomosis to the anus to bypass the fecal stream. METHODS: Eighteen piglets were divided into three groups of six each. Group A piglets underwent colon resection and rough anastomosis with large gaps between sutures, followed by Valtrac-secured intracolonic bypass. Group B piglets underwent the same procedures, but a colonic outlet obstruction also was done with pursestring sutures tied over the anus. Group C piglets underwent colon resection and rough anastomosis, but no intracolonic bypass tube was inserted (as in the control group). RESULTS: All Group A and Group B piglets survived. Passage of the BARs occurred approximately two weeks later. As the barium enema passed through the bypass tube, it showed a patent BAR-secured tube and intact anastomosis with no leakage. In Group C, anastomotic leakage occurred in four of six piglets, three of which died. Barium enema showed leakage at the anastomotic site. CONCLUSIONS: In the animal model we used, our new intracolonic bypass device proved to be a simple, safe, reliable means of protecting the anastomotic site and, thereby, eliminated the need for a diverting colostomy. Still we need further steps to test its potential in clinical use.

Anastomosis, Surgical↗
PubMed1997

Single-channel properties of the recombinant skeletal muscle Ca2+ release channel (ryanodine receptor).

We report transient expression of a full-length cDNA encoding the Ca2+ release channel of rabbit skeletal muscle sarcoplasmic reticulum (ryanodine receptor) in HEK-293 cells. The single-channel properties of the 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate-solubilized and sucrose gradient-purified recombinant Ca2+ release channels were investigated by using single-channel recordings in planar lipid bilayers. The recombinant Ca2+ release channel exhibited a K+ conductance of 780 pS when symmetrical 250 mM KCl was used as the conducting ion and a Ca2+ conductance of 116 pS in 50 mM luminal Ca2+. Opening events of the recombinant channels were brief, with an open time constant of approximately 0.22 ms. The recombinant Ca2+ release channel was more permeable to Ca2+ than to K+, with a pCa2+/pK+ ratio of 6.8. The response of the recombinant Ca2+ release channel to various concentrations of Ca2+ was biphasic, with the channel being activated by micromolar Ca2+ and inhibited by millimolar Ca2+. The recombinant channels were activated by ATP and caffeine, inhibited by Mg2+ and ruthenium red, and modified by ryanodine. Most recombinant channels were asymmetrically blocked, conducting current unidirectionally from the luminal to the cytoplasmic side of the channel. These data demonstrate that the properties of recombinant Ca2+ release channel expressed in HEK-293 cells are very similar, if not identical, to those of the native channel.

Adenosine Triphosphate↗
PubMed1994

Asymmetrical blockade of the Ca2+ release channel (ryanodine receptor) by 12-kDa FK506 binding protein.

A soluble 12-kDa FK506 binding protein (FKBP12), the cellular receptor of the immunosuppressive drug FK506, is tightly associated with the Ca2+ release channel of rabbit skeletal muscle sarcoplasmic reticulum [Jayaraman, T., Brillantes, A. M., Timerman, A. P., Fleischer, S., Erdjument-Bromage, H., Tempst, P. & Marks, A. (1992) J. Biol. Chem. 267, 9474-9477]. We have assessed the role of excess free FKBP12 in the function of single Ca2+ release channels incorporated into planar lipid bilayers. The addition of human recombinant FKBP12 (hFKBP12) to the cytoplasmic face of the Ca2+ release channel blocked the flow of cytoplasmic to luminal current (outward current) in a concentration-dependent manner but had no significant effect on the flow of luminal to cytoplasmic current (inward current). The luminal to cytoplasmic flow of current was modulated by Ca2+, Mg2+, ATP, caffeine, and ryanodine in the presence and absence of FKBP12. An immunosuppressive drug, L-683,590, an analog of FK506, did not block or reverse the asymmetrical hFKBP12 blockade of single Ca2+ release channels in planar lipid bilayers. FKBP12 may play a role in regulation of the flow of ions into the lumen of the sarcoplasmic reticulum through the Ca2+ release channel.

Animals↗
PubMed1994

Identification of calmodulin-, Ca(2+)-, and ruthenium red-binding domains in the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum.

cDNAs encoding trpE fusion proteins containing fragments of the skeletal muscle Ca2+ release channel (ryanodine receptor) were expressed in bacteria. The fusion proteins, which covered about 90% of the linear sequence of the ryanodine receptor, were used to identify calmodulin- (CaM), Ca(2+)-, and ruthenium red-binding regions in the ryanodine receptor through the use of 125I-CaM, 45Ca2+, and ruthenium red overlay procedures. Six Ca(2+)-dependent CaM-binding domains were detected in the skeletal muscle ryanodine receptor. Strong CaM-binding domains were localized in regions 6, 11, 12, and 13, in subregions 6b, 11b, and 13b, and in short sequences 6b3, 11b1, and 13b2, lying between amino acid residues 2063 and 2091, 3611 and 3642, and 4303 and 4328. Weaker CaM-binding domains were localized in regions 4, 9, and 10 and in subregions 4b, 9b, and 10a, lying between residues 921 and 1173, 2804 and 2930, and 2961 and 3084. Most of these CaM-binding domains encompassed all or part of previously predicted CaM-binding sites. Strong 45Ca(2+)- and ruthenium red-binding sites domains were localized in the NH2- and COOH-terminal regions of the ryanodine receptor and in regions 6, 12, and 13. The 45Ca(2+- and ruthenium red-binding sites in regions 6 and 12 were localized in subregions 6b and 12b, lying between residues 1861-2094 and 3657-3776. These data together with earlier studies (Chen, S. R. W., Zhang, L., and MacLennan, D. H. (1992) J. Biol. Chem. 267, 23318-23326), show that strong CaM-, Ca(2+)-, and ruthenium red-binding domains are colocalized in the skeletal muscle ryanodine receptor.

Animals↗
PubMed1994

[Protection of common peony from scarab damages].

The key to the protection of common peony from insect pests in the field is to control the damage done by Holotrichia oblita. An experimental study has been carried out on the prevention of adult pests in the field as well as on the prevention of pest larvae before planting and throughout the growing period of common peony. As a result a technological program for this particular pest prevention and elimination is proposed.

Animals↗