Male- and female-specific microRNA expression patterns in a mouse model of methanol poisoning.

The aims of this study were to determine the miRNAs involved in the methanol poisoning, and identify the male- and female-specific miRNA expression patterns in mice. Methanol was applied orally at the doses of 4 g/kg and 8 g/kg to induce mild and severe methanol poisoning in Balb/c mice. miRNA expression levels were detected at 3 different time periods (30, 60, and 180 min) following methanol exposure. miRNA expression profiles were determined using the high-throughput Fluidigm BioMark real-time PCR. We observed that serum miR-206 expression in male mice and miR-6357 expression in female mice could be an indicator of methanol poisoning. miR-9-3p downregulation and miR-1187 upregulation could be important for liver tissue. miR-3106-5p and miR-133a-5p upregulations and miR-122-3p downregulation could be poison biomarkers for ocular tissue in male mice. However, miR-194-5p downregulation could be a biomarker for ocular tissue in female mice. miR-122-5p and miR-124-3p downregulations and miR-499a-5p upregulation appeared to be important for kidney tissue in male mice. miR-543 and miR-6342 upregulations could be potential candidate biomarkers for kidney tissue in female mice. Our study is the first to report that differential miRNA expressions are involved in blood and tissues in male and female mice after methanol treatment.

Evaluation of the Rho-kinase gene expression and polymorphisms in adult patients with acute appendicitis: a differential impact of gender.

OBJECTIVE: Acute appendicitis represents one of the most common causes of acute intra-abdominal emergencies worldwide. In this case-control study, we aimed to investigate associations of Rho-kinase gene expression and polymorphisms with acute appendicitis in a Turkish population. We also aimed to study the effects of gender on these parameters. METHODS: A total of 93 unrelated patients with acute appendicitis and 93 healthy controls in the Department of Emergency Medicine, Erciyes University, between June 2019 and June 2021 were included in this study. Genomic DNA was isolated from peripheral leukocytes, and the LightCycler 480 II real-time polymerase chain reaction was utilized to detect Rho-kinase1 gene rs35996865 and Rho-kinase2 gene rs2230774 (Thr431Asn) polymorphisms. Quantitative real-time polymerase chain reaction was applied to determine Rho-kinase1 and Rho-kinase2 gene expressions. RESULTS: There was a marked increase in Rho-kinase1, but not in Rho-kinase2, mRNA expression, and this increase was evident only in male patients (p=0.0008). No significant differences were found in allele and genotype frequencies for Rho-kinase1 gene rs35996865 and Rho-kinase2 gene rs2230774 polymorphisms between the patients with acute appendicitis and the control group. CONCLUSIONS: Our data imply that Rho-kinase1 (rs35996865) and Rho-kinase2 (rs2230774) gene variants are not risk factors for the development of acute appendicitis in the Turkish population. However, increased mRNA expression of the Rho-kinase1 gene in males indicated that Rho-kinase1 is involved in the pathogenesis of acute appendicitis in a gender-specific way.

Evaluation of the Rho-kinase gene expression and polymorphisms in adult patients with acute appendicitis: a differential impact of gender

SUMMARY OBJECTIVE: Acute appendicitis represents one of the most common causes of acute intra-abdominal emergencies worldwide. In this case-control study, we aimed to investigate associations of Rho-kinase gene expression and polymorphisms with acute appendicitis in a Turkish population. We also aimed to study the effects of gender on these parameters. METHODS: A total of 93 unrelated patients with acute appendicitis and 93 healthy controls in the Department of Emergency Medicine, Erciyes University, between June 2019 and June 2021 were included in this study. Genomic DNA was isolated from peripheral leukocytes, and the LightCycler 480 II real-time polymerase chain reaction was utilized to detect Rho-kinase1 gene rs35996865 and Rho-kinase2 gene rs2230774 (Thr431Asn) polymorphisms. Quantitative real-time polymerase chain reaction was applied to determine Rho-kinase1 and Rho-kinase2 gene expressions. RESULTS: There was a marked increase in Rho-kinase1, but not in Rho-kinase2, mRNA expression, and this increase was evident only in male patients (p=0.0008). No significant differences were found in allele and genotype frequencies for Rho-kinase1 gene rs35996865 and Rho-kinase2 gene rs2230774 polymorphisms between the patients with acute appendicitis and the control group. CONCLUSIONS: Our data imply that Rho-kinase1 (rs35996865) and Rho-kinase2 (rs2230774) gene variants are not risk factors for the development of acute appendicitis in the Turkish population. However, increased mRNA expression of the Rho-kinase1 gene in males indicated that Rho-kinase1 is involved in the pathogenesis of acute appendicitis in a gender-specific way.

Evaluation of the rs35996865 polymorphism of the ROCK1 gene in sepsis.

OBJECTIVE: Sepsis is a complex and serious medical condition resulting from the activation of an innate host response to infections. The etiology of sepsis is complex and can be influenced by genetic susceptibility. The purpose of the present study was to investigate a possible association of Rho-kinase 1 (ROCK1) gene polymorphism with sepsis in a Turkish population. METHODS: The study group consisted of 100 unrelated patients with sepsis and 100 healthy controls. Genomic DNA was isolated from peripheral leukocytes from EDTA-containing blood using the QIAamp DNA Blood Mini Kit. ROCK1 gene rs35996865 and rs112130712 (Lys1054Arg) polymorphisms were analyzed in genomic DNA using the LightCycler 480 II real-time polymerase chain reaction. RESULTS: There were no significant differences in allele and genotype frequencies for ROCK1 gene rs35996865 polymorphism between the patients with sepsis and control group (p>0.05). Additionally, no association was detected between the rs35996865 polymorphism and mortality in the patient group. No polymorphism was detected with ROCK1 gene rs112130712 (Lys1054Arg) in our study groups. CONCLUSIONS: Our data showed that there is no marked association between the rs35996865 polymorphism and sepsis. Therefore, these results suggest that ROCK1 gene rs35996865 polymorphism is not risk factor for the development of sepsis in the Turkish population.

Evaluation of the rs35996865 polymorphism of the ROCK1 gene in sepsis

SUMMARY OBJECTIVE: Sepsis is a complex and serious medical condition resulting from the activation of an innate host response to infections. The etiology of sepsis is complex and can be influenced by genetic susceptibility. The purpose of the present study was to investigate a possible association of Rho-kinase 1 (ROCK1) gene polymorphism with sepsis in a Turkish population. METHODS: The study group consisted of 100 unrelated patients with sepsis and 100 healthy controls. Genomic DNA was isolated from peripheral leukocytes from EDTA-containing blood using the QIAamp DNA Blood Mini Kit. ROCK1 gene rs35996865 and rs112130712 (Lys1054Arg) polymorphisms were analyzed in genomic DNA using the LightCycler 480 II real-time polymerase chain reaction. RESULTS: There were no significant differences in allele and genotype frequencies for ROCK1 gene rs35996865 polymorphism between the patients with sepsis and control group (p>0.05). Additionally, no association was detected between the rs35996865 polymorphism and mortality in the patient group. No polymorphism was detected with ROCK1 gene rs112130712 (Lys1054Arg) in our study groups. CONCLUSIONS: Our data showed that there is no marked association between the rs35996865 polymorphism and sepsis. Therefore, these results suggest that ROCK1 gene rs35996865 polymorphism is not risk factor for the development of sepsis in the Turkish population.

Variants in TNF and NOS3 (eNOS) genes associated with sepsis in adult patients.

BACKGROUND: Sepsis is a life-threatening condition caused by a dysregulated host response to infections and is a leading cause of death in hospitalized patients. The present study aimed to elucidate the possible association between sepsis and the tumor necrosis factor (TNF) gene -308G/A (rs1800629) polymorphism, as well as endothelial nitric oxide synthase (eNOS, NOS3) gene -786T/C (rs2070744), 4a/4b (27 bp-VNTR in intron 4, rs61722009) and 894G/T (Glu298Asp, rs1799983) polymorphisms. METHODS: In total, 188 septic adult cases and 188 healthy controls were enrolled. Genomic DNAs from the controls and patients were analyzed by polymerase chain reaction and restriction fragment length polymorphism methods. RESULTS: There were significant associations between the G/G genotype and G allele of the TNF -308G/A (rs1800629) polymorphism in the sepsis group (p < 0.001). The presence of the T/C genotype (p = 0.002) and C allele (p = 0.001) of the -786T/C (rs2070744) was markedly associated with an increased risk of sepsis. However, no significant associations were found with 4a/4b (27 bp-VNTR in intron 4, rs61722009) and 894G/T (Glu298Asp, rs1799983) polymorphisms. Higher 4bGC and lower 4bTT haplotype frequencies were associated with sepsis. CONCLUSIONS: Our results strongly suggest that TNF gene (-308G/A, rs1800629) and NOS3 gene -786T/C (rs2070744) polymorphisms may modify individual susceptibility to sepsis in the Turkish population.

Serum vitamin D, vitamin D binding protein levels and leukocyte vitamin D receptor gene expression in patients with ischaemic stroke.

OBJECTIVE: To investigate the possible contributions of serum 25 hydroxyvitamin D and vitamin D binding protein levels along with leukocyte vitamin D receptor gene expression in patients with ischaemic stroke. METHODS: The randomised controlled single-blind study was conducted at the Mayo Hospital, Lahore, Pakistan, from September 2015 to September 2017, and comprised patients aged 40-75 years with Arbeitsgemeinschaft für Osteosynthesefragen type A2 and A3 per trochanteric fracture. The patients randomised into two equal groups. In Group A, patients were treated by closed reduction and internal fixation with dynamic hip screw, while those in Group B were treated by closed reduction and internal fixation by proximal femoral nail. Follow-up was done at 2nd, 6th and 12th weeks, and at 6th, 9th and 12th month post-operatively. Variables evaluated were frequency of union, surgical time, approximate amount of blood loss and complications. The functional assessment was done by using Harris hip score. SPSS 20 was used for data analysis. RESULTS: Of the 90 subjects, 51 (56.6%) were cases with a mean age of 65.2±14.3 years, and 39 (43.3%) were controls with a mean age of 61.1±16.7 years. There was no difference between the groups with respect to vitamin D deficiency, serum vitamin D binding protein levels and leukocyte vitamin D receptor gene expressions (p>0.05). A negative correlation was found between 25-hydroxyvitamin D levels and the severity of ischaemic stroke (p=0.0342). CONCLUSION: There was a correlation between serum 25-hydroxyvitamin D levels and severity of ischaemic stroke as assessed by the National Institutes of Health Stroke Scale.

The detection of occult CO poisoning through noninvasive measurement of carboxyhemoglobin: A cross-sectional study.

OBJECTIVES: Carbon monoxide (CO) poisoning is one of the leading causes of preventable death in the world. Our primary objective was to identify and treat individuals who are unaware of their exposure to carbon monoxide in emergency departments (EDs). Our secondary goal was to reduce the costs of diagnosis and treatment by preventing unnecessary diagnostic testing in EDs. METHODS: In this cross-sectional study, carboxyhemoglobin (COHb) levels of patients presented with the signs of CO poisoning to the Emergency Department of Kayseri Training and Research Hospital between November 2012 and May 2013 were noninvasively measured during triage. Patients with elevated COHb levels were suspected of CO poisoning and subjected to further investigation. RESULTS: A total of 4073 patients were enrolled in the study, and 106 (2.6%) of them were diagnosed with CO poisoning. Initial evaluation revealed headache to be the most common presenting complaint in patients with occult CO poisoning. Further evaluations to determine the accuracy of noninvasive measurements showed that noninvasive pulse CO-oxymeter and arterial blood gas (ABG) measurement were compatible. CONCLUSIONS: The use of noninvasive pulse CO-oxymeter might reduce the morbidity and mortality associated with occult CO poisoning in patients presented with suspected CO poisoning in emergency settings.

Prevalence of hidden carbon monoxide poisoning in auto service workers; a prospective cohort study.

BACKGROUND: Carbon monoxide (CO) is formed as a result of the incomplete burning of hydrocarbon-containing fuels such as natural gas, coal, liquid petroleum gas, and wood. CO is a colorless, odorless, and poisonous gas that produces various acute and chronic effects in CO-exposed people. In this study, we aimed to measure CO levels in auto care repairmen with chronic CO-related illnesses using a serial, non-invasive method.A prospective cohort study. METHODS: A total of 99 people from six different auto-repair services were included in the study. Carboxyhemoglobin (COHb) levels were measured at four different times with 2-hour intervals starting at 08:00 AM. Data concerning employees' ages, working hours, smoking statuses, and types of home heating fuel were collected. A control group of 100 cases was created based on this data. The measurements were done on the control group in the morning with a Masimo Rad-57 CO-oximeter. RESULTS: The highest mean (± SD) COHb value was 7.04% ± 3.32% after the third measurement. The mean value for the control group was 1.61% ± 1.43%. A statistically significant difference between the groups was found for each value. DISCUSSION: We determined that the risk of being affected by CO is high in buildings in which the auto services were located. The effects of chronic or prolonged exposure to low amounts of CO were found to be ambiguous. However, in some studies, it was found that low-grade CO exposure could lead to coronary artery disease and some neurological complications. Therefore, it is necessary to be careful about the health of employees who have been exposed to CO. CONCLUSIONS: We concluded that there is a need for more detailed studies concerning chronic CO poisoning. Also, in workplaces in which there is high exposure to CO, proper workplace safety measures should be taken to reduce this gas's harmful effects to employees.

Role of circulating microRNAs in acute appendicitis.

BACKGROUND: Acute appendicitis (AA) is a momentous, emergency, surgical pathology that has still been investigated for both etiopathogenetic unknowns and challenges in diagnosis. Presently, there is little information about the role of microRNAs (miRNAs), which have basic biological functions in the cell, can be a marker, and are associated with various pathologies, in patients with AA. The aim of this study was to investigate the expressions of some miRNAs in AA. METHODS: Overall, 41 miRNAs were screened in 48 individuals comprising 24 patients with AA and 24 healthy controls at Erciyes University Genome and Stem Cell Center (GENKOK). The obtained data were analyzed using appropriate statistical methods. RESULTS: miR-29c-3p was found to be increased 2-fold during the first 4-6 h in AA, and this increase was revealed to be statistically significant compared with healthy individuals. Similarly, expressions of let-7b-5p, let-7i-5p, miR-30a-5p, miR-29b-3p, and miR-23a-3p also increased approximately 2-fold in AA, although not statistically significant. No significant differences were found in the screening of the remaining 35 miRNAs in patients with AA. CONCLUSION: Although there is little information about the relationship between AA and miRNAs currently, miR-29c-3p was reported to increase in the acute period of AA in this study. With the current results, it can be argued that miR-29c-3p bears the potential to be a marker in patients with AA. The present study may also be a basic research for more extensive and necessary miRNAs screening in this field.

A role of the endothelial nitric oxide system in acute renal colic caused by ureteral stone.

BACKGROUND AND AIMS: Endothelial nitric oxide synthase gene polymorphisms play a role in some pathophysiological processes. In this study, the possible effects of endothelial nitric oxide synthase gene polymorphisms on ureteral stone disease in patients who were admitted to the emergency department with severe pain due to renal colic are examined. MATERIALS AND METHODS: The study groups were designed as controls and patients. The control group was formed from the healthy volunteers who applied to the blood center next to the emergency service. The patient group comprised patients who were diagnosed with ureteral stone disease with severe pain. All of the genetic studies were based on extracted peripheral blood samples using the necessary procedures from the Genome and Stem Cell Center at Erciyes University (GENKOK). The data were analyzed with SPSS (IBM, ver 20, United Sate). RESULTS: The study group comprised 62 females and 138 males, and the control group comprised 64 females and 136 males. All of the stones that caused renal colic were found to be localized in the ureters and the ureterovesical junction. The genotypes of the intron 4 polymorphism were found to be as follows: 4a/4a in 10 people, 4b/4a in 115, and 4b/4b in 275 people. The GG genotype of the eNOS-G894T polymorphism was found in 108 patients in the study group and in117 of the healthy individuals. There was no statistically significant difference between the two groups regarding these data. CONCLUSION: Although this study is the first in the literature to examine the relationship between renal colic and endothelial nitric oxide synthase gene polymorphisms, our study demonstrated that no relation was found.

A hidden household danger: Television.

BACKGROUND: Today, at least 1 television (TV) is owned in most households, and watching TV has become a significant part of daily life. Time spent in front of the TV has increased as its popularity has grown, which may pose a danger to children. The aim of the present study was to draw attention to cases of TV tip-over-related pediatric injuries. METHODS: Cases of TV tip-over injuries of pediatric patients admitted to the Emergency Department of Kayseri Training and Research Hospital recorded between June 1, 2011 and December 31, 2013 were included. Data were prospectively collected, including age and gender of patient, part(s) of the body affected, Glasgow Coma Scale score, TV screen size and brand, and whether the patient was hospitalized in the general ward or the intensive care unit (ICU). RESULTS: There were 53 pediatric patients enrolled in the study, including 30 males (56.6%) and 23 females (43.4%). Eight patients (17%) had a fracture somewhere in the body, whereas 45 patients (83%) had no fracture. Of patients with a fracture, 3 (2 males, 1 female) were treated in the general ward and 3 (male) in the intensive care unit. One male patient died. CONCLUSION: An increasing number of individuals present to emergency services with injuries sustained by TV toppling. Cases of injury, permanent disability, and even death associated with falling TVs suggest that awareness of the danger should be increased, as it may affect children in particular.

Role of endothelial nitric oxide synthases system on acute appendicitis.

BACKGROUND: Obstruction and inflammation of the appendix lumen is the leading physiopathological process during acute appendicitis (AA). Although the relationship between inflammation and endothelial nitric oxide synthases (eNOS) has been well described, no recent data describing the relationship between inflammation during AA and polymorphism of the eNOS gene has been reported. Given the limited data available, we believed that defining the relationship between AA and eNOS would be a beneficial contribution. METHODS: A total of 201 patients admitted to the emergency department with AA and 201 healthy volunteers selected from among the relatives of patients were included. Polymorphism of the eNOS was assessed. RESULTS: Intron 4a/4a was positive in 119 participants, genotype G894T GT was positive in 71 patients with AA, and 786-1 was positive in 71 patients with AA. These results suggest that no statistically significant correlation exists between genotypes of AA patients and control subjects regarding 4a/b, G894-GT, and 786-1 eNOS polymorphisms. CONCLUSION: Though the present results suggest that no statistically significant correlation exists between AA and eNOS gene polymorphism, to claim otherwise is also impractical. We believe that the present results will lay the groundwork for future, larger studies.

Spontaneous subscapular haematoma in a patient using warfarin therapy.

Warfarin is widely used. Spontaneous bleeding is one of the complications of warfarin treatment. A 70-year-old man was admitted to the emergency room with right back pain. There was no history of trauma. He was using warfarin following a bypass graft. The patient's vital signs were stable. On physical examination, swelling at the level of the right scapula was detected. Laboratory findings showed that the international normalised ratio (INR) was ↑↑ (a very high reading) (1.47 1 month previously). There was no melena on rectal examination. A chest CT was performed to differentiate the swelling of the right scapula. Warfarin was stopped and vitamin K was administered. Fresh frozen plasma was initiated and the patient was hospitalised to the cardiovascular surgical service. The fact that use of warfarin in the elderly may increase the risk of spontaneous bleeding should be especially kept in mind.

Oral deltamethrin ingestion due in a suicide attempt.

Deltamethrin intoxication is uncommon throughout the world. The toxicity of insecticides containing pyrethroids is considered relatively lower than that of other insecticides such as compounds containing organophosphate. Acute deltamethrin poisoning due to oral ingestions is relatively rare. This report describes a case of a 32-year-old woman admitted to the emergency department (ED) with irritability, muscle cramps, discomfort, sensation of burning, loss of sensation in her feet and arms and dyspnea due to deltamethrin ingestion. Deltamethrin intoxication should be considered as a differential diagnosis in patients presented to ED with nonspecific neurological symptoms. The supportive treatment in acute phase of intoxication is critical in the management of these patients since higher doses of deltamethrin ingestion may cause severe symptoms (Tab. 2, Ref. 16). Full Text (Free, PDF) www.bmj.sk.

Protective effects of Y-27632 on acute dichlorvos poisoning in rats.

Anticholinesterase poisoning is an important health problem in developing countries, and understanding of its underlying mechanisms is essential for the effective treatment. This study is designed to examine the effects of Y-27632, a selective Rho-kinase inhibitor, on organophosphate-induced cardiac toxicity and mortality in rats. Rats were randomly divided into 4 groups: control (corn oil), dichlorvos (30 mg/kg intraperitoneally), and 1- and 10-mg/kg Y-27632 + dichlorvos groups. After 6 hours of intraperitoneal injection, venous blood and cardiac samples were obtained, biochemical or immunohistochemical analyses were performed, and the intensity of muscle fasciculation was recorded. Serum cholinesterase activities were suppressed with dichlorvos, and these reductions were inhibited with Y-27632 pretreatment. Serum creatine kinase, creatine kinase-MB activities, and myoglobin and N-terminal probrain natriuretic peptide concentrations were not markedly affected with poisoning or Y-27632. Although serum nitric oxide concentrations did not change with dichlorvos, cardiac nitric oxide levels were markedly increased with Y-27632 pretreatment. Cardiac glutathione levels also increased with 1 mg/kg Y-27632. There was no staining for apoptosis, and immunohistochemical analyses of inducible nitric oxide synthase showed no change in cardiac tissue for all of the groups. Both doses of Y-27632 abolished mortality in rats with acute dichlorvos exposure (100% survival). These results show that administration of Rho-kinase inhibitor can produce protective effects against dichlorvos intoxication in rats. These findings may provide new possibilities for the treatment of organophosphate poisoning.

Formulation of effects of atropine, pralidoxime and magnesium sulfate on cardiac tissue levels of nitric oxide, malondialdehyde and glutathione in organophosphate poisoning using artificial neural network.

Anticholinesterase poisoning is an important health problem in our country, and a complete understanding of its underlying mechanisms is essential for the emergency physician. So, this study focused on two purposes. First one was aimed to investigate the biochemical analysis to determine the tissue levels of malondialdehyde (MDA), glutathione and nitric oxide (NO). Secondly, it was planned to model and formulate the effects of some drugs on cardiac tissues levels of NO, MDA and glutathione in acute organophosphate poisoning in rats by the application of neural network based on experimental results. It has been planned to determine whether artificial neural network (ANN) is appropriate tool to analyze and formulate it. As a result, it has been considered that ANN can be effectively used to model NO, MDA and glutathione level. The performances of ANN formulation versus target experimental values are found to be quite high. It is concluded that, proposed NN models are also presented as simple explicit mathematical functions for further use by researchers.

Cardiac effects of magnesium sulfate pretreatment on acute dichlorvos-induced organophosphate poisoning: an experimental study in rats.

Although atropine and oximes are traditionally used in the management of organophosphate poisoning, investigations have been directed to finding additional therapeutic approaches. Thus, the aim of this study was to evaluate the cardiac effects of magnesium sulfate pretreatment on dichlorvos intoxication in rats. Rats were randomly divided into three groups as control, dichlorvos, and magnesium sulfate groups. After 6 h of dichlorvos or corn oil (as a vehicle) injection, venous blood samples were collected, and cardiac tissue samples were obtained. Biochemical analyses were performed to measure some parameters on serum and cardiac tissue. Immunohistochemical analyses of apoptosis and inducible nitric oxide (NO) synthase showed no change in cardiac tissue. Serum cholinesterase levels were markedly depressed with dichlorvos, and further suppressed markedly with magnesium sulfate pretreatment. Although we have demonstrated that serum NO levels in dichlorvos and magnesium sulfate groups were lower than the control group, cardiac tissue NO levels in magnesium sulfate group were higher than the other two groups. Mortality was not significantly affected with magnesium sulfate pretreatment. Uncertainty still persists on the right strategies for the treatment of organophosphate acute poisoning; however, it was concluded that our results do not suggest that magnesium sulfate therapy is beneficial in the management of acute dichlorvos-induced organophosphate poisoning, and also further studies are required.

Myocardial infarction, acute ischemic stroke, and hyperglycemia triggered by acute chlorine gas inhalation.

Chlorine is one of the most common substances involved in toxic inhalation. Until now, several accidental exposures have been reported. The damage to the respiratory tract in the immediate phase after exposure to chlorine is well defined. Death occurs particularly due to pulmonary edema with respiratory failure and circulatory collapse. On the other hand, no association with myocardial infarction, acute stroke, severe hyperglycemia, and acute chlorine inhalation has been reported in literature. In the present study, an elderly (74-year-old) and diabetic case with myocardial infarction, acute stroke, hyperglycemia, and respiratory failure associated with acute chlorine intoxication after a diagnosis of acute chlorine poisoning and treatment in the emergency department is reported and the literature is revisited. Physicians should know that in elderly patients with a systemic disease who apply with chlorine gas inhalation, more serious complications along with damage in respiratory tract might be observed.