Assessing breathing effort by barometric whole-body plethysmography and its relationship with prognosis in client-owned cats with respiratory distress.

BACKGROUND: Cats in respiratory distress have limited tolerance for manipulation, hindering clinical monitoring. Minute volume (MV) can be utilized to rate dyspnea in humans, but its relationship with respiratory distress in cats remains poorly investigated. HYPOTHESIS: Cats with respiratory distress will show higher MV per kg body weight (MV/BW) than normal cats, and the MV/BW increase will correlate with survival. ANIMALS: Fifty-two cats with respiratory distress from lung parenchymal disease, pleural space disease, lower airway obstruction (LAO), or upper airway obstruction were recruited since 2014. METHODS: This is a prospective observational study. Study cats were placed in a transparent chamber, allowing clinicians to easily observe their breathing status and record ventilation using barometric whole-body plethysmography (BWBP). Ventilatory variables of the 52 cats were compared with those of 14 historic control cats. Follow-up data, including disease category, clinical outcomes, and survival, were prospectively collected. RESULTS: Cats in respiratory distress demonstrated significantly higher MV/BW (397 mL/kg; range, 158-1240) than normal cats (269 mL/kg; range, 168-389; P < .001). Among the etiologies, cats with LAO, parenchymal, and pleural space disease exhibited higher-than-normal MV/BW trends. A cutoff value of 373 mL/kg (1.4-fold increase) indicated abnormally increased breathing efforts (sensitivity, 67%; specificity, 93%). MV/BW was independently associated with increased cardiorespiratory mortality in cats with respiratory distress (adjusted hazard ratio 1.17, 95% confidence interval [CI] 1.02-1.35; P = .03). CONCLUSIONS AND CLINICAL IMPORTANCE: Breathing efforts in cats can be noninvasively quantified using BWBP. Measurement of MV/BW could serve as a prognostic index for monitoring cats experiencing respiratory distress.

Detection of Feline Coronavirus in Bronchoalveolar Lavage Fluid from Cats with Atypical Lower Airway and Lung Disease: Suspicion of Virus-Associated Pneumonia or Pneumonitis.

The premortem understanding of the role of feline coronavirus (FeCoV) in the lungs of cats is limited as viruses are seldom inspected in the bronchoalveolar lavage (BAL) specimens of small animal patients. This study retrospectively analyzed the prevalence of FeCoV in BAL samples from cats with atypical lower airway and lung disease, as well as the clinical characteristics, diagnostic findings, and follow-up information. Of 1162 clinical samples submitted for FeCoV RT-nPCR, 25 were BAL fluid. After excluding 1 case with chronic aspiration, FeCoV was found in 3/24 (13%) BAL specimens, with 2 having immunofluorescence staining confirming the presence of FeCoV within the cytoplasm of alveolar macrophages. The cats with FeCoV in BAL fluid more often had pulmonary nodular lesions (66% vs. 19%, p = 0.14) and multinucleated cells on cytology (100% vs. 48%, p = 0.22) compared to the cats without, but these differences did not reach statistical significance due to the small sample size. Three cats showed an initial positive response to the corticosteroid treatment based on the clinical signs and radiological findings, but the long-term prognosis varied. The clinical suspicion of FeCoV-associated pneumonia or pneumonitis was raised since no other pathogens were found after extensive investigations. Further studies are warranted to investigate the interaction between FeCoV and lung responses in cats.

Identification of druggable regulators of cell secretion via a kinome-wide screen and high-throughput immunomagnetic cell sorting.

The identification of genetic regulators of cell secretions is challenging because it requires the sorting of a large number of cells according to their secretion patterns. Here we report the development and applicability of a high-throughput microfluidic method for the analysis of the secretion levels of large populations of immune cells. The method is linked with a kinome-wide loss-of-function CRISPR screen, immunomagnetically sorting the cells according to their secretion levels, and the sequencing of their genomes to identify key genetic modifiers of cell secretion. We used the method, which we validated against flow cytometry for cytokines secreted from primary mouse CD4+ (cluster of differentiation 4-positive) T cells, to discover a subgroup of highly co-expressed kinase-coding genes that regulate interferon-gamma secretion by these cells. We validated the function of the kinases identified using RNA interference, CRISPR knockouts and kinase inhibitors and confirmed the druggability of selected kinases via the administration of a kinase inhibitor in an animal model of colitis. The technique may facilitate the discovery of regulatory mechanisms for immune-cell activation and of therapeutic targets for autoimmune diseases.

Quantifiable features of a tidal breathing phenotype in dogs with severe bronchomalacia diagnosed by bronchoscopy.

Dynamic lower airway obstruction is the primary component of canine bronchomalacia, but the ventilatory function remains underinvestigated. This prospective study analyzed tidal breathing characteristics in 28 dogs, comprising 14 with severe bronchomalacia diagnosed by bronchoscopy versus 14 without respiratory disease. Spirometry was conducted in all dogs. Bronchoscopy with bronchoalveolar lavage or brush under anesthesia was performed in 14 dogs with cough and expiratory effort. Severe bronchomalacia was defined by the severity of collapse and total number of bronchi affected. Ventilatory characteristics were compared between groups. Results revealed that dogs with severe bronchomalacia had lower minute volume (218 vs 338 mL/kg, p = .039) and greater expiratory-to-inspiratory time ratio (1.55 vs 1.35, p = .01) compared to control dogs. The tidal breathing pattern of dogs with bronchomalacia was different from that of normal dogs, and the pattern differed from the concave or flat expiratory curves typical of lower airway obstruction. Compared to control dogs, dogs with severe bronchomalacia had a significantly prolonged low-flow expiratory phase (p < .001) on the flow-time plot and a more exponential shape of the expiratory curve (p < .001) on the volume-time plot. Flow-time index ExpLF/Te (>0.14) and volume-time index Vt-AUCexp (≤31%) had a high ROC-AUC (1.00, 95% confidence interval 0.88 to 1.00) in predicting severe bronchomalacia. In conclusion, the tidal breathing pattern identified here indicates abnormal and complicated ventilatory mechanics in dogs with severe bronchomalacia. The role of this pulmonary functional phenotype should be investigated for disease progression and therapeutic monitoring in canine bronchomalacia.

Constrictive bronchiolitis obliterans with a presumptive etiology of preceding feline herpesvirus infection in a cat.

BACKGROUND: Bronchiolar disorders are rarely recognized in cats. Constrictive bronchiolitis obliterans is characterized by concentric peribronchiolar fibrosis and inflammation of the bronchioles, but the underlying causes remain poorly understood in current small animal medicine. CASE PRESENTATION: A 9-year-old cat presented with paroxysmal tachypnea, infrequent cough and persistent labor breathing. Thoracic radiography showed lung hyperinflation and bronchointerstitial pattern, and pulmonary function assessment revealed flow limitation in the late-expiratory phase and poor response to short-acting bronchodilator. Dorsally distributed subpleural ground glass opacities with distinct margin and tree-in-bud opacities were observed on lung high-resolution computed tomography. The cat underwent bronchoalveolar lavage (BAL) and showed severe neutrophilic inflammation. Feline herpesvirus was the only pathogen detected in the BAL fluid. Multiple therapeutic attempts were unsuccessful and the cat died 8 weeks after the initial presentation. Necropsy revealed the infiltration of inflammatory cells, obstruction of the bronchiolar lumen, and submucosal concentric fibrosis suggesting constrictive bronchiolitis obliterans. Combining the pre- and post-mortem findings, as well as the time from symptom onset or BAL to necropsy, constrictive bronchiolitis obliterans was possibly triggered by a preceding feline herpesvirus infection in this case. CONCLUSIONS: The history of nonvaccinated status, lower airway neutrophilic inflammation, and presence of feline herpesvirus in the BAL fluid without coexistence of other pathogens led to the presumption that constrictive bronchiolitis obliterans was induced by a preceding feline herpesvirus infection in this cat. The pathological changes of bronchiolitis obliterans induced by a preceding feline herpesvirus infection could be different from that of cats with acute herpesvirus pneumonia, such as intranuclear inclusions would disappear over time and were no longer found 7-10 days after inoculation. The presence of patchy distribution of subpleural ground glass opacities on lung high-resolution computed tomography should raise the suspicion of peribronchiolar fibrosis. Clinical awareness of bronchiolar disorders as a differential diagnosis is important in cats with lung hyperinflation and labored breathing who show poor reversibility to bronchodilator.

Influence of concurrent lower respiratory tract disease on point-of-care lung ultrasound in small-breed dogs with myxomatous mitral valve disease.

BACKGROUND: Small-breed dogs commonly have concurrent myxomatous mitral valve disease (MMVD) and lower respiratory tract disease (LRTD). HYPOTHESIS: Small-breed dogs with preclinical MMVD and concurrent LRTD have more B-lines on point-of-care lung ultrasound (POC-LUS) compared to dogs without concurrent LRTD and are prone to misdiagnose as cardiogenic pulmonary edema (CPE). ANIMALS: A total of 114 small-breed dogs with preclinical MMVD. METHODS: A prospective study was conducted, in which POC-LUS was obtained and the number of B-lines was calculated by a single clinician using the Veterinary Bedside Lung Ultrasound Examination protocol. The presence/absence of LRTD was assessed by clinicians blinded to the POC-LUS results. RESULTS: Fifty and 64 dogs were in ACVIM stage B1 and B2, respectively. The presence of LRTD was prevalent in 74.6% (85/114) of small-breed dogs with preclinical MMVD. When a previously reported criterion for CPE diagnosis (≥2 sites with >3 B-lines/site) was applied, false-positive results were observed in 15.8% (18/114) of dogs with preclinical MMVD. The summated number of B-lines (3 vs. 1, P = .003), as well as the false-positive rate (20% vs 3%, P = .04), were significantly higher in dogs with LRTD compared with dogs without LRTD. Multivariable logistic regression showed the presence of abnormalities other than B-line on POC-LUS (eg, thickened pleura or consolidation) could predict false-positive results (odds ratio = 3.75, 95% confidence intervals 1.12-12.54; P = .03) after adjustment for other clinical and echocardiographic factors. CONCLUSIONS AND CLINICAL IMPORTANCE: Concurrent LRTD and abnormalities other than B-lines should be considered in the interpretation of POC-LUS in MMVD dogs.

Simultaneous formation of Bi2O2(OH)(NO3)/BiOBr ultrathin hierarchical microspheres for effectively promoting visible-light-driven photocatalytic activity in environmental remediation.

As a two-dimensional nanomaterial, bismuth oxybromide (BiOBr) have attracted tremendous interest in the area of visible-light photocatalysis since it can provide the internal electric field (IEF) through z-axis through its unique electronic band structure. However, the insufficient active sites and rapid recombination rate of charged carriers hamper the efficiency of the photocatalysis. To address these two major obstacles, an enticing strategy of constructing heterojunction was established by introducing Bi2O2(OH)(NO3) (BiON) in BiOBr with the same precursor. Through a facile one-pot hydrothermal synthesis, two Sillén-type layered photocatalysts, with intimately constructed ultrathin heterostructure, was synthesized by the co-precipitation method. In this work, the formation of Bismuth-based heterojunction for charge separation is established by the excessive bismuth nitrate, which subsequently participates with the in situ growth of ultrathin hierarchical microspheres. By attenuating the thickness of BiOBr from 20 nm to 8 nm with the aid of BiON, the photogenerated charges could migrate to the active sites through shorter charge diffusion pathway. Also, the BiOBr and BiON act as an active bridge to promote the separation of electron-hole pairs, which also brings out more active sites due to its increased specific surface area. BiON/BiOBr ultrathin hierarchical microspheres exhibited enhanced visible-light photocatalytic activity for decontaminating several types of pollutants. Besides, the activity of as-prepared BiON/BiOBr was further evaluated by inhibiting the growth of kanamycin-resistant bacteria strains. This study presents a novel strategy to incorporate the crystalline bismuth hydrate nitrate into BiOBr to form ultrathin hierarchical microspheres with high surface area for environmental remediation.

Intercalating pyrene with polypeptide as a novel self-assembly nano-carrier for colon cancer suppression in vitro and in vivo.

Giving patients right dosage is an essential concept of precision medicine. Most of nanocarriers lack of flexible drug capacity and structural stability to be customized for specific treatment, resulting in low therapeutic efficacy and unexpected side effects. Thus, a growing need emerges for fast and rigorous approaches to develop nanoparticles with properties of adjustable dosage and controllable particle size. Poly-l-Lysine is known for its enhanced bioadhesivity and pH-triggered structural swelling effect, which is utilized as the main agent to activate the multistage drug releasing. Inspired by natural bio-assembly system, we report a simple method to self-assemble Poly-l-Lysine-based nanoparticles via supramolecular recognitions of cross-linked pyrenes, which provides noncovalent force to flexibly encapsulate Doxorubincin and to construct robust nanostructures. Pyrene-modified polypeptide self-assemblies are able to adjust drug payload from 1: 10 to 2:1 (drug: polypeptide) without changing its uniform nano-spherical morphology. This nanostructure remained the as-made morphology even after experiencing the long-term (~ 10 weeks) storage at room temperature. Also, the nanoparticles displayed multi-step drug release behaviours and exhibited great in vitro and in vivo cytotoxicity towards colon cancer cells. The as-mentioned nanoparticles provide a novel perspective to compensate the clinical needs of specific drug feedings and scalable synthesis with advantages of simple-synthesis, size-adaptivity, and morphology reversibility.

An observational study of the role of indoor air pollution in pets with naturally acquired bronchial/lung disease.

BACKGROUND: Indoor air pollution (IAP) is an emerging issue for both human and veterinary patients under the concept of 'One Health'. The association between IAP and respiratory disease in companion animals has been reported. OBJECTIVES: The present study investigated the relationship between quantifiable indoor air quality and clinical characteristics of naturally acquired bronchial/lung disease in pet dogs and cats. METHODS: A total of 36 clinical cases (20 dogs and 16 cats) with naturally acquired bronchial/lung disease were prospectively recruited. Lower airway samples were collected and analysed, and clinical signs and the information from pulmonary function testing were examined. Indoor air quality was estimated by the average concentration of particles measuring ≤2.5 µm (PM2.5, µg/m3 ) and volatile organic compounds (VOC, ppm) in the animals' domestic microenvironments. RESULTS: Exposure to IAP was not found to be correlated with the severity of clinical signs, pulmonary function changes or bronchoalveolar lavage fluid cytology in cats with bronchial/lung disease. However, a hypercellular response in canine lower airways was found to be associated with poor indoor air quality, including unacceptable indoor PM2.5 levels (>35 µg/m3 ) or increases in VOC concentration (>1 ppm) in places most commonly frequented by the dogs in the home. CONCLUSIONS: Poor indoor air quality may exacerbate airway disease in pets and should not be ignored in modern society.

GFP Plasmid and Chemoreagent Conjugated with Graphene Quantum Dots as a Novel Gene Delivery Platform for Colon Cancer Inhibition In Vitro and In Vivo.

Scientists have studied intensively the gene delivery carriers for treating genetic diseases. However, there are challenges that impede the application of naked gene-based therapy at the clinical level, such as quick elimination of the circulation, lack of membrane penetrability, and poor endosome trapping. Herein, we develop graphene quantum dots (GQDs)-derivative nanocarriers and introduce polyethylenimine (PEI) to equip the system with enhanced biocompatibility and abundant functional groups for modification. In addition to carrying green fluorescent protein (GFP) as an example of gene delivery, this system covalently binds colon cancer cells targeted antibody and epidermal growth factor receptor (EGFR) to enhance cell membrane penetrability and cell uptake of nanocarriers. To achieve multistrategy cancer therapy, the anticancer drug doxorubicin (Dox) is noncovalently encapsulated to achieve pH-induced drug release at tumor sites and leaves space for further functional gene modification. This nanoparticle serves as a multifunctional gene delivery system, which facilitates improved cytotoxicity and longer-sustained inhibition capacity compared to free Dox treatments in colon cancer cells. Moreover, our GQD composites display compatible tumor suppression ability compared with the free Dox treatment group in xenograft mice experiment with significantly less toxicity. This GQD nanoplatform was demonstrated as a multifunctional gene delivery system that could contribute to treating other genetic diseases in the future.

Usefulness of Chest Ultrasonography in Predicting Diagnosis in Non-emergency Small Animal Patients With Lung Parenchymal and Pleural Disease.

Chest ultrasonography has become an indispensable tool for pulmonary specialists in human medicine, but its current use in dogs and cats is primarily for emergency. The diagnostic performances of various ultrasonographic features other than comet-tail artifacts are of limited information in veterinary literatures. Therefore, the aims of this retrospective study were to investigate ultrasonographic findings in feline and canine respiratory patients with lung parenchymal and pleural space diseases, and to assess how ultrasonographic features correspond to specific diagnoses. Sixty-five non-emergency cases with radiographically identified lung parenchymal and pleural space abnormalities were included. Medical records and ultrasound video clips were reviewed, and additional follow-up information was subsequently collected. Common findings such as comet-tail artifacts (87.7% of cases), consolidation (84.6%), and thickened/irregular pleura (69.2%) were not distinguishable for a specific diagnosis. The presence of nodular/mass-like lesion (OR = 212, p < 0.001) and consolidated lesion with heteroechogenicity (OR = 240, p < 0.001) was significantly associated with and strongly predictive of neoplasia after age, body weight and other sonographic findings were adjusted. The finding of nodular/mass-like lesion has the best diagnostic performance (AUC = 0.93) for neoplasia, with sensitivity of 91.7% and specificity of 93.6%. For predicting a diagnosis of pneumonia, although several sonographic features were found to be statistically associated with pneumonia, only a negative finding of nodular/mass-like lesion showed good diagnostic performance (AUC = 0.83, sensitivity 95.7%, specificity 71%). These findings demonstrate the value of chest ultrasonography in predicting diagnosis in non-emergency cases. The application of thoracic ultrasound in small animal respiratory patients as part of non-invasive assessment warrants further investigation.

Carboxylated carbon nanomaterials in cell cycle and apoptotic cell death regulation.

Carbon nanomaterials, include carbon nanotubes and graphene nanosheets, have drawn an increasing amount of attention because of their potential applications in daily life or in providing novel therapeutic possibilities for treating diseases. However, the overall biocompatibility, the potential toxic effects of carbon nanomaterials toward human cells, and their modulations in cellular mechanism, are not fully understood. Herein, four types of carbon nanomaterials, include long and short carbon nanotubes and graphene nanosheets, at low and high concentrations, were functionalized and dispersed in the biocompatible buffer for assessment. The surface structure, the morphology, and chemical composition of carbon nanomaterials were characterized. Also, biological assays investigating cellular viability, vitality, cell cycle, and apoptotic cell death were applied on cells co-incubated with nanomaterials, to evaluate the biocompatibility of these nanomaterials in human cells. Our data suggested that even though co-incubation of nanomaterials did not seem to affect the viability of cells notably, high concentrations (50 ug/ml) of SW could lead to unhealthy cells, and we observed dramatic G2 arrest effect mediated by p21 induction in high SW incubated cells. Other nanomaterials at high concentration may also alter cell cycle profile of the cells. In summary, our data demonstrated that these nanomaterials could regulate cell cycle and lead to apoptosis at high concentrations, and the underling molecular mechanisms have been addressed. Caution should be taken on their concentration when nanomaterials are in used in future medical applications.

Association between indoor air pollution and respiratory disease in companion dogs and cats.

BACKGROUND: Indoor air pollution (IAP) leads to important respiratory morbidity and mortality in humans. Companion dogs and cats share the same household environment with their owners and are exposed to IAP. HYPOTHESIS: Pets with respiratory disease are more commonly exposed to indoor air pollutants in their homes and to worse air quality than pets without respiratory disease. ANIMALS: Three hundred and forty-eight animals (230 dogs and 118 cats) were recruited. METHODS: Dogs and cats attending the National Taiwan University Veterinary Hospital were prospectively enrolled over a 12-month period. Questionnaires were collected from pet owners regarding the status of signs of respiratory problem of animals and air pollutants in their homes. Clinical assessment was performed by veterinarians on all animals included in the case-control study and the presence/absence of respiratory disease and diagnoses were recorded. Individual exposure to particulate matter of 2.5 µm or less (PM2.5) was estimated in the domestic microenvironment of the animals. RESULTS: Dogs with respiratory disease were more commonly exposed to incense burning than control dogs (30 versus 13%, P = .045), but household PM2.5 level was not different between dogs with and without respiratory disease [median 30.8 µg/m3 , range 10.8-214.2 versus median 38.2 µg/m3 , range 5.4-69.4, P = .57]. Signalment factors (age, body weight, and body condition score) instead of IAP factors were associated with respiratory disease in dogs using multivariable logistic regression. In contrast, household PM2.5 level was significantly higher in cats with respiratory disease than in control cats [median 38.6 µg/m3 , range 17.8-131.2 versus median 27.4 µg/m3 , range 15.4-70.0, P = .017]. Cats living in households with PM2.5 > 35 µg/m3 were more likely to have respiratory disease than those living in households with acceptable levels of PM2.5 (OR = 4.13, 95% CI 1.12-15.27, P = .03). CONCLUSIONS AND CLINICAL IMPORTANCE: The link between IAP and respiratory disease in dogs is complicated. An unacceptable level of household PM2.5 (>35 µg/m3 ) is significantly associated with respiratory disease in cats. The effect of IAP on the respiratory health of companion animals warrants further attention.

Dirofilaria immitis exposure status in client-owned cats with or without lower airway/lung-associated signs: case-control study in a canine heartworm-endemic area.

Objectives Heartworm-associated respiratory disease (HARD) is a recently recognised pathological manifestation in cats caused by Dirofilaria immitis exposure. This study aimed to estimate the percentage of cats at risk of developing HARD in a heartworm-endemic area (Taipei, Taiwan), and to test the correlation of heartworm exposure and the presence of lower airway/lung clinical signs (LA/L signs). Methods This was a prospective case-control study. The study design called for the enrolment of at least 80 cats with LA/L signs and at least 80 cats without such clinical signs in a 1 year period. The D immitis antibody seroprevalence of the two cohorts was compared. Results From February 2014 to January 2015, 187 client-owned cats were prospectively enrolled: 83 clinical cases with LA/L signs and 104 cats without such signs. Antibody seropositivity was approximately twice as frequent in cats with LA/L signs (13.3%) than in cats without signs (7.8%) (odds ratio [OR] 1.814); nevertheless, no statistically significant difference between the two cohorts ( P = 0.22) was found. We used 41 frozen samples from free-roaming cats to examine the possibility of different exposure rates to mosquito bites between client-owned cats and stray cats, finding the seroprevalence to be 7.5% in free-roaming cats - a result not statistically different to that in client-owned cats ( P = 0.60). Outdoor access was a significant risk factor for heartworm exposure in client-owned cats (OR 3.748; P = 0.03); however, living entirely indoors did not provide complete protection from exposure/infection. Conclusions and relevance Our results did not show statistically significant differences in antibody seroprevalence between cats with and without LA/L signs. LA/L signs were not always present under conditions of natural exposure. However, exposure to D immitis is not rare among client-owned cats, suggesting that heartworm prophylactics should be a part of routine care in all cats living in areas endemic for canine heartworm.

Simultaneous visual inspection for barometric whole-body plethysmography waveforms during pulmonary function testing in client-owned cats.

OBJECTIVES: For the purpose of applying a barometric whole-body plethysmography (BWBP) device as a routine clinical tool in client-owned cats, the objective of this study was to evaluate the methodological importance of simultaneous visual inspection (SVI) of graphic tracing. METHODS: To investigate the effect of SVI on the results obtained, 50 client-owned cats were included. Breath-by-breath analysis was conducted with BWBP software, and a commonly used rejection setting was chosen for automatic elimination (AE) of non-breath artefactual waveforms, according to tidal volume (TV), inspiratory and expiratory time, and the difference between inspiratory and expiratory volumes. During 10 mins of data recording, SVI for BWBP waveforms was performed to record manually time periods that were free of any artefacts. The two datasets derived from AE alone (AEA method) and AE plus SVI (SVI-AE method) were compared. The inter-observer effect on the process of SVI was evaluated on six cats. RESULTS: There were statistically significant differences (P <0.001) between the AEA and SVI-AE datasets for most BWBP parameters. Bland-Altman analysis of the parameter-enhanced pause (Penh) showed heterogeneous variances, indicating less agreement when the Penh values were large. Intra-individual coefficients of variation of Penh were significantly higher with the AEA method than with the SVI-AE method (61.1% vs 34.7%, respectively; P <0.001). Inter-observer agreement on the SVI process was excellent, and no statistically significant differences between the two observers were found for any BWBP parameters obtained by the SVI-AE method (P >0.05). CONCLUSIONS AND RELEVANCE: Visual inspection for BWBP waveforms in real time can reliably identify stable breathing signals in client-owned cats. The obtained results were significantly different when the SVI method was used in addition to AE. In the interpretation of BWBP parameters or comparison of measurements among studies, whether an SVI methodology was applied should be considered.