Evaluation of antibacterial activity of propolis on regenerative potential of necrotic immature permanent teeth in dogs.

BACKGROUND: This study evaluated the antibacterial efficiency and ability of propolis to promote regeneration of immature permanent non-vital dogs' teeth. METHODS: Ninety six immature permanent premolars teeth in 6 mongrel dogs were divided randomly into: experimental teeth (N = 72) and control teeth (N = 24). Periapical pathosis was induced in all experimental and positive control teeth. Experimental teeth were classified according to the used intra-canal medication into: group I (N = 36), propolis paste was used and group II (N = 36), triple antibiotic paste (TAP) was used. Bacteriologic samplings were collected before and after exposure to intra-canal medicaments. After the disinfection period (3 weeks), revascularization was induced in all experimental teeth. Each group was subdivided according to the root canal orifice plug into: subgroup A (N = 18), propolis paste was used and subgroup B (N = 18), mineral trioxide aggregates (MTA) was used. Each subgroup was further subdivided according to the evaluation period into 3 subdivisions (6 teeth each): subdivision 1; after 2 weeks, subdivision 2; after one month and subdivision 3; after 2 months. Positive control group had 12 teeth with induced untreated periapical pathosis. Negative control group had 12 untouched sound teeth. All teeth were evaluated with radiography and histology. The bacteriologic and radiographic data were analyzed using repeated measures ANOVA and post-hoc Tukey tests. The histologic data were analyzed using Kruskal-Wallis test, Mann-Whitney U test with Bonferroni's adjustment and Chi-square test. The significance level was set at P ≤ .05. RESULTS: There was no significant difference in the antibacterial effectiveness between TAP and propolis groups (P > .05). In all subdivisions, there was no significant difference between the experimental groups in terms of increase in root length and dentin thickness, decrease in apical closure, new hard tissue formation, vital tissue formation inside the pulp canal and apical closure scores (P > .05). CONCLUSION: Propolis can be comparable with TAP as a disinfection treatment option in regenerative endodontic. As a root canal orifice plug after revascularization of necrotic immature permanent teeth in dogs, propolis induces a progressive increase in root length and dentin thickness and a decrease in apical diameter similar to those of MTA.

Runoff of the herbicides triclopyr and glufosinate ammonium from oil palm plantation soil.

This study focused on the residue detection of the herbicides triclopyr and glufosinate ammonium in the runoff losses from the Tasik Chini oil palm plantation area and the Tasik Chini Lake under natural rainfall conditions in the Malaysian tropical environment. Triclopyr and glufosinate ammonium are post-emergence herbicides. Both herbicides were foliar-sprayed on 0.5 ha of oil palm plantation plots, which were individualized by an uneven slope of 10-15%. Samples were collected at 1, 3, 7, 15, 30, 45, 60, 90, and 120 days after treatment. The concentrations of both herbicides quickly diminished from those in the analyzed sample by the time of collection. The highest residue levels found in the field surface leachate were 0.031 (single dosage, triclopyr), 0.041 (single dosage, glufosinate ammonium), 0.017 (double dosage, triclopyr), and 0.037 µg/kg (double dosage, glufosinate ammonium). The chromatographic peaks were observed at "0" day treatment (2 h after herbicide application). From the applied active ingredients, the triclopyr and glufosinate losses were 0.025 and 0.055%, respectively. The experimental results showed that both herbicides are less potent than other herbicides in polluting water systems because of their short persistence and strong adsorption onto soil clay particles.

Contamination of rice field water with sulfonylurea and phenoxy herbicides in the Muda Irrigation Scheme, Kedah, Malaysia.

The purpose of the present study was to investigate the potential risk of herbicide contamination (2,4-dichlorophenoxy (2,4-D), 2-methyl-4-chlorophenoxyacetic acid (MCPA), metsulfuron, bensulfuron, and pyrazosulfuron) in the rice fields of the Muda Irrigation Scheme, Kedah, Malaysia. The study included two areas with different irrigation water sources namely non-recycled (N-RCL) and recycled (RCL) water. Periodic water sampling was carried out from the drainage canals during the planting period of the wet season 2006/2007 and dry season 2007. The HPLC-UV was used to detect herbicide residues in the water samples collected from the rice fields. The results showed that the concentration of sulfonylurea herbicides such as bensulfuron and metsulfuron in the rice field was 0.55 and 0.51 µg/L, respectively. The potential risk of contamination depended on the actual dosage of each herbicide applied by farmers to their rice fields. The potential risk of water pollution by the five herbicides studied in the area with RCL water tended to be more widespread compared to the area with N-RCL water due to surface water runoff with higher levels of weedicides to the surrounding areas. During the two seasons, 50-73% of the water samples collected from the area receiving RCL water contained the five herbicides studied at concentrations of more than 0.05 µg/L, and this percentage was higher than that from the areas receiving N-RCL water (45-69%). During the wet season, the overall total mean concentration of the eight herbicides found in the samples collected from the area with RCL water (6.27 µg/L) was significantly higher (P < 0.01) than that from the area receiving N-RCL water (2.39 µg/L). Meanwhile, during the dry season, there was no significant difference (P > 0.05) in the herbicide concentrations between the areas receiving RCL (6.16 µg/L) and N-RCL water (7.43 µg/L) water.

Adsorption, desorption and mobility of cyfluthrin in three Malaysian tropical soils of different textures.

The sorption and desorption of cyfluthrin mixture isomers were determined using batch equilibration method and mobility was studied under laboratory conditions, using packed soil column. The soil types used in the study were clayey, clay loam and sandy clay loam obtained from three tomato farms in Cameron Highlands. A low Freundlich adsorption distribution coefficient K(ads(f)) for cyfluthrin was observed for clayey, clay loam and sandy clay loam soils (95.69, 21.64 and 8.99 l/kg, respectively). Results showed that cyfluthrin had high Freundlich organic matter (OM) distribution coefficient K(oc) values of 5799, 2278 and 1635 lkg(-1) for clayey, clay loam and sandy clay loam soils, respectively. These values indicate that cyfluthrin is considered immobile in Malaysian soils with different textures, based on the value of K(oc) by McCall. Adsorption of cyfluthrin was significantly (P < 0.05) affected with soil pH, fertilizer NPK, organic matter content and temperature. It was observed that approximately 95.8%, 93.8% and 91.8% of the adsorbed cyfluthrin remained sorbed after four successive rinses for clayey, clay loam and sandy clay loam soils. Soil column test showed that cyfluthrin was not detected in leachate. Cyfluthrin was detected in topsoil and its concentration decreased with depth. The downward movement of cyfluthrin in sandy clay loam soil was more than that in clay loam and clayey soils. Approximately, 80.9%, 77.8% and 67.3% cyfluthrin was observed at the depth of 0-5 cm (rainfall 350 mm) for clayey, clay loam and sandy clay loam soils respectively. Mobility of cyfluthrin showed that the percentage of cyfluthrin leached into soil was not affected by the amount of rainfall. The result clearly showed that cyfluthrin molecules were bound strongly to all the three Malaysian soil types.

Complete genome sequencing and genetic characterization of Alkhumra hemorrhagic fever virus isolated from Najran, Saudi Arabia.

BACKGROUND: Alkhumra hemorrhagic fever virus (AHFV) is a newly described flavivirus first isolated in 1994-1995 from the Alkhumra district south of Jeddah, Saudi Arabia. Subsequently, the virus was also isolated from Makkah (2001-2003) and Najran (2008-2009), Saudi Arabia. METHODS: The full-length genome of an AHFV strain isolated from patients in Najran (referred to as AHFV/997/NJ/09/SA) was PCR amplified and sequenced, and compared with the sequences of 18 other AHFV strains previously isolated from Jeddah and Makkah, dengue virus (DENV), Kyasanur forest disease virus (KFDV), Langat virus, Omsk hemorrhagic fever virus (OHFV), and tick-borne encephalitis virus (TBEV). RESULTS: The RNA of the AHFV/997/NJ/09/SA strain was found to have 10,546 nucleotides encoding for a single 3,416-amino acid polyprotein, whereas the previously reported AHFV strains were composed of 10,685-10,749 nucleotides. The AHFV/997/NJ/09/SA strain showed about 99% homology with the previously reported AHFV strains. The KFDV, Langat virus, TBEV, and OHFV isolates formed a separate cluster with a variable homology. The most important variations were observed in the core protein and NS4a gene sequences of two AHFV isolates. CONCLUSION: The variation in the number of nucleotides and phylogenetic analysis with the other AHFV isolates could have resulted from recombination of circulating virus strains.

Trypsin-dependent hemagglutination of erythrocytes of a variety of mammalian and avian species by Alkhumra hemorrhagic fever virus.

Alkhumra hemorrhagic fever virus (AHFV) is an emerging flavivirus that was discovered in 1994-1995 in Saudi Arabia. Clinical manifestations of AHFV infection include hemorrhagic fever, hepatitis, and encephalitis, with a reported mortality rate as high as 25 %. Biological characteristics of this virus have not been well defined. Agglutination of erythrocytes (hemagglutination) is a laboratory tool for studying the attachment of viruses to cellular receptors. The envelope protein contains sites for attachment to host receptors to initiate the process of infection and is thus an essential component of the virion. In the present study, we examined the ability of AHFV to agglutinate erythrocytes of 13 mammalian and avian species (human group O+, camel, cow, sheep, goat, rabbit, guinea pig, mouse, rat, chicken, duck, goose and turkey) with and without trypsin-treatment. Without trypsin treatment, AHFV failed to agglutinate erythrocytes of all examined species. Following trypsin treatment, AHFV agglutinated erythrocytes of five species, namely, goose, human group O+, rat, guinea pig, and mouse, in descending order of sensitivity. This trypsin-dependent hemagglutination test has potential for use in serological and functional studies of AHFV.

Outbreak of viral hemorrhagic fever caused by dengue virus type 3 in Al-Mukalla, Yemen.

BACKGROUND: Investigations were conducted by the authors to explore an outbreak of viral hemorrhagic fever (VHF) reported in 2010 from Al-Mukalla city, the capital of Hadramout in Yemen. METHODS: From 15-17 June 2010, the outbreak investigation period, specimens were obtained within 7 days after onset of illness of 18 acutely ill patients hospitalized with VHF and 15 household asymptomatic contacts of 6 acute cases. Additionally, 189 stored sera taken from acutely ill patients with suspected VHF hospitalized in the preceding 12 months were obtained from the Ministry of Health of Yemen. Thus, a total of 222 human specimens were collected; 207 specimens from acute cases and 15 specimens from contacts. All samples were tested with RT-PCR for dengue (DENV), Alkhumra (ALKV), Rift Valley Fever (RVFV), Yellow Fever (YFV), and Chikungunya (CHIKV) viruses. Samples were also tested for DENV IgM, IgG, and NS1-antigen. Medical records of patients were reviewed and demographic, clinical, and laboratory data was collected. RESULTS: Of 207 patients tested, 181 (87.4%) patients were confirmed to have acute dengue with positive dengue NS1-antigen (97 patients, 46.9%) and/or IgM (163 patients, 78.7%). Of the 181 patients with confirmed dengue, 100 (55.2%) patients were IgG-positive. DENV RNA was detected in 2 (1%) patients with acute symptoms; both samples were molecularly typed as DENV type 3. No other VHF viruses were detected. For the 15 contacts tested, RT-PCR tests for the five viruses were negative, one contact was dengue IgM positive, and another one was dengue IgG positive. Of the 181 confirmed dengue patients, 120 (66.3%) patients were males and the median age was 24 years. The most common manifestations included fever (100%), headache (94.5%), backache (93.4%), malaise (88.4%), arthralgia (85.1%), myalgia (82.3%), bone pain (77.9%), and leukopenia (76.2%). Two (1.1%) patients died. CONCLUSIONS: DENV-3 was confirmed to be the cause of an outbreak of VHF in Al-Mukalla. It is important to use both IgM and NS1-antigen tests to confirm acute dengue particularly under the adverse field conditions, where proper storage and transportation of specimens are missing, which substantially reduce the sensitivity of the RT-PCR for detecting DENV RNA.

Superiority of the buffy coat over serum or plasma for the detection of Alkhumra virus RNA using real time RT-PCR.

RT-PCR to detect Alkhumra virus (ALKV) RNA in plasma or serum has been the standard practice to confirm this infection in the first seven days of illness. In this study, RT-PCR detection of viral RNA from the plasma, serum, and buffy coat (BC) was compared to virus isolation. Plasma, serum, and BC were obtained from seven patients with clinically suspected ALKV infection in Najran, Saudi Arabia. Baby hamster kidney (BHK-21) and rhesus monkey kidney (LLC-MK2) cell culture monolayers were used for virus isolation. Real-time RT-PCR was used to confirm ALKV infection and to detect viral RNA directly from plasma, serum, and BC. ALKV was isolated from five of the seven patients. The virus was isolated from all three specimen types (plasma, serum, and BC) of the five confirmed patients. ALKV RNA was detected directly by RT-PCR in BC in all five (100%) culture-positive patients and in plasma or serum in only four (80%) of the five patients. Three of the five patients for whom ALKV RNA was detected in BC also had detectable viral RNA in plasma and serum. In the remaining two patients with detectable ALKV RNA in the BC, the plasma was positive but the serum was negative in one patient, whereas the serum was positive and the plasma was negative in the other patient. The use of real-time RT-PCR to detect ALKV RNA in the BC was superior to using plasma and serum and equivalent to virus isolation.

Fatal neck pain in a patient on maintenance hemodialysis.

Infectious spondylodiscitis (IS) is defined as the pathogenic invasion of the vertebrae and intervertebral disks. It is a serious condition that can lead to many complications such as chronic pain, permanent neurological deficits, and even death. Vertebral surgical procedures, invasive urinary tract manipulations, and central line-associated bloodstream infection are the primary methods by which microorganisms reach the vertebrae and intervertebral disks. Hemodialysis (HD) patients are regularly exposed to bloodstream infections due to long-term catheter utilization or repeated vascular puncturing in patients with arteriovenous fistula. Due to the high risk of blood stream infections, HD patients have a higher risk of developing IS. Despite advanced diagnostic methods, diagnosis of spondylodiscitis is often delayed due to insidious and nonspecific symptoms, allowing dissemination of the infection, which explains the high level of mortality due to spondylodiscitis in HD patients. The infectious process typically occurs in the thoracic or lumbar region, although cervical IS does occur. We herein report the case of a 67-year-old man on HD who developed infectious cervical spondylodiscitis. The diagnosis was established a few days after symptom onset, but the issue was unfortunately fatal despite a well-conducted antibiotic treatment.

Towards a European health research and innovation cloud (HRIC).

The European Union (EU) initiative on the Digital Transformation of Health and Care (Digicare) aims to provide the conditions necessary for building a secure, flexible, and decentralized digital health infrastructure. Creating a European Health Research and Innovation Cloud (HRIC) within this environment should enable data sharing and analysis for health research across the EU, in compliance with data protection legislation while preserving the full trust of the participants. Such a HRIC should learn from and build on existing data infrastructures, integrate best practices, and focus on the concrete needs of the community in terms of technologies, governance, management, regulation, and ethics requirements. Here, we describe the vision and expected benefits of digital data sharing in health research activities and present a roadmap that fosters the opportunities while answering the challenges of implementing a HRIC. For this, we put forward five specific recommendations and action points to ensure that a European HRIC: i) is built on established standards and guidelines, providing cloud technologies through an open and decentralized infrastructure; ii) is developed and certified to the highest standards of interoperability and data security that can be trusted by all stakeholders; iii) is supported by a robust ethical and legal framework that is compliant with the EU General Data Protection Regulation (GDPR); iv) establishes a proper environment for the training of new generations of data and medical scientists; and v) stimulates research and innovation in transnational collaborations through public and private initiatives and partnerships funded by the EU through Horizon 2020 and Horizon Europe.

Degradation of Triazine-2-(14)C Metsulfuron-Methyl in Soil from an Oil Palm Plantation.

Triazine-2-(14)C metsulfuron-methyl is a selective, systemic sulfonylurea herbicide. Degradation studies in soils are essential for the evaluation of the persistence of pesticides and their breakdown products. The purpose of the present study was to investigate the degradation of triazine-2-(14)C metsulfuron-methyl in soil under laboratory conditions. A High Performance Liquid Chromatograph (HPLC) equipped with an UV detector and an on-line radio-chemical detector, plus a Supelco Discovery column (250 x 4.6 mm, 5 µm), and PRP-1 column (305 x 7.0 mm, 10 µm) was used for the HPLC analysis. The radioactivity was determined by a Liquid Scintillation Counter (LSC) in scintillation fluid. The soil used was both sterilized and non-sterilized in order to observe the involvement of soil microbes. The estimated DT50 and DT90 values of metsulfuron-methyl in a non-sterile system were observed to be 13 and 44 days, whereas in sterilized soil, the DT50 and DT90 were 31 and 70 days, respectively. The principal degradation product after 60 days was CO2. The higher cumulative amount of (14)CO2 in (14)C-triazine in the non-sterilized soil compared to that in the sterile system suggests that biological degradation by soil micro-organisms significantly contributes to the dissipation of the compound. The major routes of degradation were O-demethylation, sulfonylurea bridge cleavage and the triazine "ring-opened."

HIV-1 and hepatitis B transmission in Sudan.

A seroepidemiologic survey was conducted among 773 male soldiers living in five urban locations in Sudan to study the prevalence of and risk factors for HIV-1 and hepatitis B transmission. Twenty-eight per cent of the study population were born and raised in southern Sudan, an area bordering Kenya, Zaire and Uganda, whilst 72% of the study subjects were from northern Sudan. Seventy-eight per cent of the study population had serologic evidence of past hepatitis B infection, and 13 soldiers were confirmed positive for HIV-1 antibody. All 13 HIV-positive soldiers had recently been deployed in southern Sudan. Multivariate analysis indicated an association between living in southern Sudan and both hepatitis B (odds ratio 8.2) and HIV-1 infection (odds ratio 14). Additionally, sexual relations with prostitutes (odds ratio 1.5) and medical injections for schistosomiasis (odds ratio 2.72) were independent predictors of hepatitis B markers in this military population. The findings of this study suggest that sexual promiscuity is a risk factor for hepatitis B transmission in Sudan. They also indicate one possible route for the spread of HIV-1 from central to northern Africa.

Hepatitis B and HIV in Sudan: a serosurvey for hepatitis B and human immunodeficiency virus antibodies among sexually active heterosexuals.

A serosurvey was conducted in Port Sudan and Suakin, Sudan in October and March 1987 to determine the prevalence and risk factors associated with the transmission of hepatitis B, human immunodeficiency virus type 1 (HIV-1), and syphilis among sexually active heterosexuals on the coast of Sudan. A total of 536 subjects, including 202 female prostitutes, 95 long-distance truck drivers, 103 soldiers, 72 Ethiopian refugees, and 54 Sudanese outpatients, were enrolled in the study. Seventy-eight percent (202/259) of the female study subjects were engaged in prostitution, and 57% (157/277) of the men admitted to prior sexual relations with prostitutes. Serologic markers for hepatitis B and syphilis were detected in 68% and 17% of the entire study population, respectively. In contrast, antibody to HIV-1 was detected in none of the 536 sera tested. Risk factors found to be independently predictive of hepatitis B infection by multivariate analysis included prostitution, positive serology for syphilis, and a history of anti-schistosomal therapy. The absence of HIV-1 infection among the prostitutes enrolled in this study is in marked contrast to the current AIDS epidemic in neighboring sub-Saharan countries, suggesting that HIV-1 has not been widely introduced on the coast of Sudan. The high prevalence of serologic markers to hepatitis B and syphilis, however, indicates a potential for HIV-1 in this region.

Onchocerciasis in Sudan: the Abu Hamed focus.

The current status of onchocerciasis in Abu Hamed, Northern Province, Sudan, was studied. Of 208 persons attending out-patient clinics in villages in this region, 71 were microfilariae-positive on skin snips or had palpable nodules. Microfilariae and worms in nodules were identified as Onchocerca volvulus. No microfilariae were seen in peripheral blood. Most nodules and microfilariae were found in the pelvic region, but the intensity of infection was uniformly low (av. less than 3 mf/mg). Despite this, signs of onchocercal dermatitis were common and severe, especially over the buttocks. Papular eruptions and scarring often appeared to lead to black-grey hyperpigmentation, but no cases were seen of the unilateral, hyper-reactive 'sowda' described in Arabs in Yemen. No microfilariae were detected in the eyes of any of the patients who had positive outer canthus snips. Serum retinol concentrations were normal but mildly elevated concentrations of serum IgG, IgM and IgA were detected in many patients. Immunoglobulin E values in a sample of 20 microfilariae-positive patients were markedly higher than normal, with most in the 4,000 to 15,000 U/ml range. Eosinophil levels in differential counts of peripheral blood from the 208 villagers were markedly elevated. In skin snip surveys of over 400 villagers and school pupils, sample prevalence rates of 2 to 17.5% were recorded. Simulium biting was seasonal (November to May) and peaked in March. Over-all, the results indicate that O. volvulus infection persists in the Abu Hamed region as a serious cause of skin disease in the absence of other complicating filariases.

Transaxillary first rib resection for thoracic outlet syndrome: follow-up of 9 cases.

Nine cases of Thoracic Outlet Syndrome (TOS) were treated by transaxillary 1st rib resection over a three year period. Mean follow-up was 26 +/- 4 months. Patients were recalled every 3 months post-operatively for surveillance. Parameters for evaluation included physical examination, return of patient to (full-range or partial range) pre-illness activity, nerve conduction velocities, somato-sensory evoked potentials, Duplex imaging and venography or arteriography if the latter two were indicated. Neurogenic involvement with or without vascular compression was present in 7 cases (78%) and satisfactory results of surgery were recorded in 6 patients (67%). The clinical settings and detailed results are reviewed and variable factors that might alter the outcome are analyzed in this series. Neither duration of symptoms, sex differences nor associated bony anomalies had any effect on operative results. The single factor that seemingly had a negative impact on the outcome after surgery was the patient's need to return to strenuous and repetitive arm and/or hand movements.

Nebulizer therapy with antibiotics in chronic suppurative lung disease.

Aerosolized antibiotics have been shown to be a useful modality of treatment in patients with cystic fibrosis. In this investigation we examined the utility of this treatment in patients with other chronic suppurative lung disorders. These included forty patients, thirty men and ten women with chronic airway infection (27 with bronchiectasis, 6 with chronic abscess and 7 with chronic suppurative bronchitis). Pathogenic organisms were isolated from the affected part of the lung by a fiberoptic bronchoscopy using a sterile disposable bronchial microbiology brush. Cultures from these specimens were used to determine the appropriate antibiotic. A second control group of 20 patients was treated with systemic antibiotics alone. Both systemic and aerosolized antibiotics were administered in 20 patients. A statistically significant improvement in clinical, and ventilatory functions was recorded in the first group compared to the second. Nebulized antibiotics used as adjunctive therapy in association with systemic antibiotics may offer a therapeutic advantage in chronic suppurative lung diseases.

Breast cancer in south-east Republic of Yemen.

A retrospective study of breast cancer was carried out using the treatment registry of Aden Health Office and archives of Al-Gamhoria Teaching Hospital from January 1989 to December 1996 and the records of patients registered at Aden Cancer Centre from January 1997 to December 1998. The data analysed included age, sex, residence, breast affected and type of cancer. There were 227 patients with breast cancer registered between January 1989 and December 1998; 225 were female and 2 were male. Most of the patients (81.5%) had infiltrating ductal carcinoma. About 90% of the patients had lymph node involvement. The age groups most affected were 30-39 years and 40-49 years.

Propagation and titration of Alkhumra hemorrhagic fever virus in the brains of newborn Wistar rats.

Alkhumra hemorrhagic fever virus (AHFV) is a novel flavivirus identified first in Saudi Arabia. In this study, successful propagation of AHFV in the brains of newborn Wistar rats is described and the median rat lethal dose (RLD50) is determined. AHFV-RNA-positive human sera diluted 1:10 were injected intracerebrally into 16, ≤24h old rats. Post-inoculation, the rats were observed daily for 30 days. Brains of moribund rats were tested for AHFV-RNA using RT-PCR and cultured in LLC-MK2 cells. The titer of the isolated virus was determined and expressed in median tissue culture infectious dose (TCID50). To determine the RLD50, AHFV brain suspension was 10-fold diluted serially and each dilution was inoculated in the cerebral hemispheres of 10 rats for a total of 90 rats. Three days post-inoculation, the rats developed tremor, irritability, convulsion, opisthotonus, and spastic paresis starting in the hind limbs and ascending to involve the whole body. All infected rats died within 3-7 days with histopathologically confirmed meningoencephalitis. AHFV-RNA was detected in the brains of all infected rats and the virus titer was 10(9.4) RLD50/ml. The virus titer in LLC-MK2 was 10(8.2) TCID50/ml. In conclusion, AHFV was propagated successfully to high titers in the brains of newborn Wistar rats.