The Weight of Genetic Drift: A Pedigree-Based Evaluation of the Breton Horse Population in Brazil.

The genetic diversity of Breton horses in Brazil is a critical concern, mainly due to the small population size and low number of births per year. Given that the inbreeding was overlooked by breeders for multiple generations, we estimated the genetic diversity of this population utilizing pedigree-based measures of population diversity. A total of 1394 six-generation pedigrees representing the full population of registered Breton horses in Brazil defined a total population (TP, N = 2679), with horses born between 2000 and 2022, reproductively active and alive, as reported by the breed association, representing the reference population (RP, N = 731). Using the R package PurgeR, we estimated inbreeding coefficient (F ped ), maternal inbreeding coefficient (F da m ), paternal inbreeding coefficient (F sire ), individual reproductive values, number of equivalents to complete generations (t), and unbiased ancestral inbreeding coefficient (Fa). We established the equivalent complete generations (ECG), effective population size (Ne), total number of founders (Nf), effective number of founders (Nfe), total number of ancestors (Na), effective number of ancestors (Nae), founder genomes (Ng), and the inbreeding coefficient estimated with effective population size (Ne) and generation numbers (t) (F Ne:t ), as well as Nfe/Nae and Nfe/Ng ratios for the RP. The RP inbreeding levels have stabilized, although they are still significantly rising by generation (t), and the Nfe/Ng ratio strongly suggests genetic drift. Pedigree-based analysis demonstrates that only five stallions have sired 52.83% of the RP individuals, which along with the Nae value of 36.73 implies that the observed inbreeding can be arising from patrilines. Our results suggest that observed inbreeding is due to Popular Sire Effect, highlighting the importance of monitoring breeding schemes and genetic diversity to maintain health.

A new approach to geostatistical synthesis of historical records reveals capuchin spatial responses to climate and demographic change.

Recent proliferation of GPS technology has transformed animal movement research. Yet, time-series data from this recent technology rarely span beyond a decade, constraining longitudinal research. Long-term field sites hold valuable historic animal location records, including hand-drawn maps and semantic descriptions. Here, we introduce a generalised workflow for converting such records into reliable location data to estimate home ranges, using 30 years of sleep-site data from 11 white-faced capuchin (Cebus imitator) groups in Costa Rica. Our findings illustrate that historic sleep locations can reliably recover home range size and geometry. We showcase the opportunity our approach presents to resolve open questions that can only be addressed with very long-term data, examining how home ranges are affected by climate cycles and demographic change. We urge researchers to translate historical records into usable movement data before this knowledge is lost; it is essential to understanding how animals are responding to our changing world.

Is Protecting Older Adults from COVID-19 Ageism? A Comparative Cross-cultural Constructive Grounded Theory from the United Kingdom and Colombia.

The COVID-19 pandemic impacted people's lives all over the world, requiring health and safety measures intended to stop the virus from spreading. This study explores whether an unintended consequence of these measures is a new form of ageism. We explore, using qualitative methods, the experiences of older adults living through the pandemic in the United Kingdom and Colombia. Although there were some small differences between countries, for the most part, the experiences were similar. We found that older adults reported that they were seen as a homogenous group and experienced both benevolent and hostile ageism and a loss of autonomy as a consequence of COVID-19 protection measures. Participants from both countries expressed anger and frustration, and increased anxiety, and felt that their individuality was ignored. We recommend that policy-makers, the media, and wider society consider the impact of such health and safety measures on older adults in preparing for future pandemics and health challenges.

Urban and Rural Child Deaths from Motor Vehicle Crashes: United States, 2015-2019.

OBJECTIVE: The objective of the study was to examine child deaths in motor vehicle crashes by rurality, restraint use, and state child passenger restraint laws. STUDY DESIGN: 2015-2019 Fatality Analysis Reporting System data were analyzed to determine deaths and rates by passenger and crash characteristics. Optimal restraint use was defined using age and the type of the restraint according to child passenger safety recommendations. RESULTS: Death rates per 100 000 population were highest for non-Hispanic Black (1.96; [1.84, 2.07]) and American Indian or Alaska Native children (2.67; [2.14, 3.20]) and lowest for Asian or Pacific Islander children (0.57; [0.47, 0.67]). Death rates increased with rurality with the lowest rate (0.88; [0.84, 0.92]) in the most urban counties and the highest rate (4.47; [3.88, 5.06]) in the most rural counties. Children who were not optimally restrained had higher deaths rates than optimally restrained children (0.84; [0.81, 0.87] vs 0.44; [0.42, 0.46], respectively). The death rate was higher in counties where states only required child passenger restraint use for passengers aged ≤6 years (1.64; [1.50, 1.78]) than that in those requiring child passenger restraint use for passengers aged ≤7 or ≤8 years (1.06; [1.01, 1.12]). CONCLUSIONS: Proper restraint use and extending the ages covered by child passenger restraint laws reduce the risk for child crash deaths. Additionally, racial and geographic disparities in crash deaths were identified, especially among Black and Hispanic children in rural areas. Decision makers can consider extending the ages covered by child passenger restraint laws until at least age 9 to increase proper child restraint use and reduce crash injuries and deaths.

3D kinematic of the thoracolumbar spine in Mangalarga Marchador horses performing the marcha batida gait and being led by hand-A preliminary report.

This study aimed to provide a preliminary description of the sagittal and transverse plane kinematics of the thoracolumbar spine of Mangalarga Marchador (MM) horses performing the marcha batida gait, led in-hand. We evaluated the pattern of angular movement and the mean amplitude of six specific angles. An optoelectronic system was used for 3D kinematic analysis (19 cameras, 250 Hz). They were positioned around the horses and an acquisition volume of 16 × 4.8 × 3 meters was used. Eight retroreflective markers were fixed on the spine of the animals over thoracic vertebrae 8 (T8), 12 (T12), 15 (T15) and 18 (T18); over the lumbar vertebrae 3 (L3) and 5 (L5); over the 1st sacral vertebra (S1); and over the 1st coccygeal vertebra (CD1). Five trials, led from a halter, with three complete gait cycles were evaluated for each marcha batida horse. The 3D coordinates of the markers were filtered with a second-order, low-pass, Butterworth filter (10 Hz). Six angles: T8-T12-T15, T12-T15-T18, T12-T18-L5, T15-T18-L3, T18-L3-L5, and L3-S1-CD1 were obtained and projected in the sagittal (Flexion and Extension) and transverse (Lateral bending) planes. We calculated, for each angle to represent the spine movements, the mean and standard deviation of the range of motion (ROM, difference between the maximum and minimum values in a stride cycle). In order to describe the movement over an average stride cycle we calculated the mean curve of angle variation. The T8-T12-T15 angle presented the largest ROM in the transverse plane, while in the sagittal plane the T8-T12-T15, T12-T15-T18 and T12-T18-L5 angles presented the largest ROMs. The L3-S1-CD1 angle (lumbosacral region) presented the lowest ROM in both planes. A reduced flexion close to a neutral spine was found, predominantly during the diagonal support and in the cranial thoracic region. At the same time, the thoracolumbar region remains in an extension which is highlighted in the lumbosacral region. During the change of the support phase, the cranial thoracic region moved from a flexion to a slight extent, and the thoracolumbar region was flexed which is emphasized in the lumbosacral region. The lateral bending of the spine followed the direction of the diagonal supports. The small amplitude in the latero-lateral and dorsoventral movements of the thoracolumbar spine of MM horses during the marcha batida gait could contribute to the smooth and natural sensations experienced when riding in this gait. The lower mobility of these angles should be considered during the clinical examination of marcha batida-gaited horses.

Qualitative Analysis of Knowledge, Attitudes, and Beliefs About Pessary Use Among Spanish-Speaking Women on the US-Mexico Border.

OBJECTIVES: To explore the knowledge, attitudes, and beliefs related to pessary use in Spanish-speaking women along the US-Mexico border. METHODS: Spanish-speaking women with symptoms of vaginal bulge were recruited from the urogynecology/gynecology clinics at Texas Tech University Health Sciences Center El Paso to participate in moderated focus groups. Discussion topics included knowledge of prolapse/pessaries, pros/cons of pessaries, alternatives, and prolapse surgery. Audio-recorded group discussions were transcribed verbatim, and qualitative analysis completed by independent review using grounded theory methodology. Common themes were identified and then aggregated to form consensus concepts, agreed upon by the reviewers. RESULTS: Twenty-nine Spanish-speaking women participated in 6 focus group discussions. Approximately half of women reported little or no prior knowledge about pessaries. Three main themes were identified from analysis: knowledge/perceptions, misinformation/misconceptions, and surgery-related concerns. Concepts identified from common themes included limited knowledge of pessaries, confusing "pessary" with "mesh," willingness to try pessaries in order to avoid surgery, desire to try pessary if it was recommended by physician, limited efficacy or complications of surgery, and mesh-related concerns. Interestingly, some women reported that pessaries appear to be a treatment more often offered in the United States rather than in Mexico. CONCLUSIONS: Most participants showed a willingness to try a pessary for symptoms of pelvic organ prolapse in an effort to avoid surgery, despite expressing limited knowledge about this treatment. Physician recommendations and risks of pessary use influence their likelihood of trying a pessary. These concepts serve as focus points for effective pessary counseling to help improve education and informed decision making in this patient population.

Acute increases in brain-derived neurotrophic factor following high or moderate-intensity exercise is accompanied with better cognition performance in obese adults.

The purpose of this study was to test if different intensities of aerobic exercise could influence abdominal fat, isoforms of BDNF and executive function. Twenty obese men (30.0 ± 5.4 years old; 34.4 ± 3.5 kg/m2) were randomized to moderate-intensity continuous training (MICT, n = 10) and high-intensity intermittent training (HIIT, n = 10) three times a week for 6 weeks, with isoenergetic energetic expenditure for each exercise session (~ 300 kcal) between conditions. Abdominal fat was assessed pre- and post-intervention; executive function (Coding subtest from BETA-III non-verbal intelligence test and Stroop Color and Word Test), concentrations of mBDNF and proBDNF were assessed in response to acute exercise pre- and post-intervention. Abdominal fat did not change in either group. There was a significant increase in mBDNF immediately after acute exercise in both groups before and after intervention. proBDNF did not present changes acutely nor after 6 weeks. Executive function presented a main effect of time at pre- and post-intervention time-points Stroop Word and Stroop Color and Coding subtest presented improved performance from pre- to post-acute exercise session, in both groups. In conclusion, executive function improvements and acute exercise session-induced increases in mBDNF concentration were found from pre- to post-exercise intervention similarly between MICT and HIIT in obese men.

Recurrent Pancreatitis in Children: Past, Present, and Future.

Recurrent acute and chronic pancreatitis are increasingly recognized in childhood. Etiologies are vastly different in children compared with adults and mostly involve genetic and anatomical factors with negligible contribution of environmental risks. Pediatric acute recurrent pancreatitis (ARP) and chronic pancreatitis (CP) have significant impact on quality of life with high healthcare costs. Children with these conditions suffer from recurrent acute or chronic abdominal pain and they endure multiple emergency room visits, hospitalizations, procedures, and surgeries. Diagnostic methods are being developed; treatment options are limited. This review summarizes the most recent developments in pediatric ARP and CP. These discoveries will help physicians provide optimal care for children with these conditions.

Secondhand Smoke Exposure and Serum Trypsinogen in Cystic Fibrosis Carriers.

OBJECTIVE: The objective of this study was to determine if infants carrying 1 cystic fibrosis transmembrane receptor (CFTR) mutation demonstrate pancreatic inflammation in response to tobacco exposure. METHODS: Cystic fibrosis carrier infants aged 4 to 16 weeks were prospectively enrolled. Tobacco exposure was assessed by survey and maternal hair nicotine analysis. Serum immunoreactive trypsinogen (IRT) levels at birth and at the time of recruitment were analyzed relative to the presence or absence of tobacco exposure. The effect of the severity of the CFTR mutation carried by the infant on the tobacco-IRT relationship was also analyzed. RESULTS: Forty-eight infants completed the study. Newborn screen and follow-up IRT levels were not different between exposed infants (19 by hair analysis) and nonexposed infants (29 by hair analysis). Follow-up IRT levels were lower in infants with more severe CFTR mutations (P = 0.005). There was no difference in follow-up IRT based on CFTR mutation severity in exposed infants. Nonexposed infants with milder CFTR mutations had higher median IRT values on follow-up testing than those with more severe CFTR mutations (P < 0.05). CONCLUSIONS: The pancreas of cystic fibrosis carrier infants is affected by tobacco exposure, and those carrying less severe CFTR mutations may be more susceptible to tobacco effects.

Frequency of Poison Center Exposures for Pediatric Accidental Unsupervised Ingestions of Acetaminophen after the Introduction of Flow Restrictors.

OBJECTIVE: To assess the temporal association of flow restrictor introduction and the rate of accidental unsupervised ingestions (AUIs) of liquid acetaminophen products. STUDY DESIGN: The National Poison Data System was used to identify AUIs of single ingredient acetaminophen in patients aged <12 years reported between 2007 and 2015. Six regional poison centers obtained additional information using a structured telephone survey. RESULTS: Pediatric AUIs involving acetaminophen averaged 30 000 exposures per year between 2007 and 2012. From 2012 to 2015, after flow restrictor introduction, exposures steadily decreased at a rate of 2400 fewer exposures annually, reaching 21 877 exposures in 2015. Normalized to sales volume, exposures involving liquid acetaminophen products decreased by 40% from 2010 to 2015. Exposures involving products with flow restrictors tended to have a lower estimated ingestion per exposure, fewer exposures exceeding a 150 mg/kg acetaminophen threshold, and were associated with lower rates of hospital admissions when compared with products without restrictors. Caregivers reported improper storage and child confusion of the medicine with treats as common contributing factors to exposures. CONCLUSIONS: The introduction of flow restrictors was associated with a decrease in pediatric AUIs of liquid acetaminophen products. Decreases in the dose ingested and risk of hospital admission per exposure may also have resulted. Efforts to optimize flow restrictors and increase their use with medicines associated with high pediatric overdose risk should be encouraged.

A single session of hatha yoga improves stress reactivity and recovery after an acute psychological stress task-A counterbalanced, randomized-crossover trial in healthy individuals.

OBJECTIVES: Yoga is promoted as an anti-stress activity, however, little is known about the mechanisms through which it acts. The present study investigated the acute effects of a hatha yoga session, displayed on a video, on the response to and recovery from an acute psychological stressor. METHODS: Twenty-four healthy young adults took part in a counterbalanced, randomized-crossover trial, with a yoga and a control condition (watching TV). Participants attended the laboratory in the afternoon on two days and each session comprised a baseline, control or yoga task, stress task and recovery. Blood pressure (BP), heart rate (HR) and salivary cortisol responses were measured. State cognitive- and somatic-anxiety along with self-confidence were assessed before and after the stressor. RESULTS: Although no difference in the BP or HR responses to stress were found between conditions, systolic BP (p=0.047) and diastolic BP (p=0.018) recovery from stress were significantly accelerated and salivary cortisol reactivity was significantly lower (p=0.01) in the yoga condition. A yoga session also increased self-confidence (p=0.006) in preparation for the task and after completion. Moreover, self-confidence reported after the stress task was considered debilitative towards performance in the control condition, but remained facilitative in the yoga condition. CONCLUSION: Our results show that a single video-instructed session of hatha yoga was able to improve stress reactivity and recovery from an acute stress task in healthy individuals. These positive preliminary findings encourage further investigation in at-risk populations in which the magnitude of effects may be greater, and support the use of yoga for stress reactivity and recovery.

The Benefits of Early Oral Nutrition in Mild Acute Pancreatitis.

OBJECTIVE: To determine whether early patient-directed oral nutrition in children with mild acute pancreatitis decreases the length of hospitalization without increasing complications. STUDY DESIGN: Hospitalized patients aged 2-21 years of age who met the criteria for acute pancreatitis based on the Revised Atlanta Classification were enrolled prospectively and allowed to eat by mouth at their discretion (patient-directed nutrition [PDN]). These patients were compared with a retrospective cohort of children who were allowed to eat based on traditional practices (treatment team-directed nutrition [TTDN]). Outcomes included length of hospitalization, time nil per os (NPO), and complications within 30 days of discharge. RESULTS: The study included 30 patients in the PDN group and 92 patients in the TTDN group. Patients in the PDN group had a median length of stay of 48.5 hours (IQR 37-70 hours) compared with 93 hours (IQR 52-145 hours) in the TTDN group (P < .0001). Patients were NPO for a median of 14 hours (IQR 7-19.5 hours) in the PDN group compared with 34 hours (IQR 19.3-55 hours) in the TTDN group (P < .0001). No patients in the PDN group developed complications within 30 days of discharge. CONCLUSION: Early patient-directed oral nutrition in mild acute pancreatitis was well tolerated and resulted in decreased length of NPO status and hospitalization with no obvious complications. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov: NCT01423786.

Physiological and lipid profile response to acute exercise at different intensities in individuals with spinal cord injury.

STUDY DESIGN: Experimental and cross-sectional study. OBJECTIVE: To assess the immediate effect of exercise on heart rate (HR), oxygen uptake (VO2), pulmonary ventilation (PV), oxygen pulse (OP), glucose and lipids of wheelchair basketball players with spinal cord injury (SCI). SETTING: Center of Studies in Psychobiology and Exercise-São Paulo, Brazil. METHODS: In all, nine wheelchair basketball players with SCI and nine able-bodied controls (C) performed three exercise sessions at different intensities: ventilatory threshold 1 (VT1), 15% below VT1 and 15% above VT1 with a duration of ~24-34 min. HR, VO2, PV, OP, glucose and lipids were analyzed. RESULTS: VO2, PV and OP were significantly lower in the players with SCI compared to C during the same intensity exercise sessions. However, the individuals with SCIs demonstrated increases in HR, PV and OP at similar rates to C. Triglycerides of the SCI group were elevated 30 min after the exercise session at VT1 compared to values before the exercise session (P=0.017); this elevation was not observed in group C. For the exercise sessions 15% above VT1, only glucose (P=0.040) and low-density lipoprotein (P=0.012) 30 min after the exercise were elevated in the SCI group compared to group C. CONCLUSION: We conclude that the SCI group demonstrated increases in HR, PV and OP but not VO2 with increased intensity of exercise at similar rates as in group C.

Male Breast Cancer.

Objectives: A clinicopathologic study with an emphasis on tumor immunohistochemical profile is presented. Methods: Sixty-one cases of male invasive breast cancers were studied. Median age of the cohort was 65 years. Results: Ninety-seven percent were estrogen receptor positive+ and 10% human epidermal growth factor receptor 2 positive. The individual diagnostic marker positivity was 98% for GATA-binding protein 3, 95% for androgen receptor, 90% for progesterone receptor, 88% for deleted in pancreatic cancer 4, 75% for gross cystic disease fluid protein 15, 72% for cytokeratin 7, 55% for mammaglobin, and 15% for vimentin and Wilms tumor protein 1. Caudal type homeobox 2 protein, cytokeratin 20, Napsin A, paired box gene 8, prostate-specific antigen, thyroid transcription factor 1, and uroplakin II were negative in all cases. Survival analyses showed tumor stage, receptor status, and Nottingham prognostic index to be prognostic. The overall survival was 70%, but the breast cancer­specific survival was 92% (mean follow-up, 59 months); 33% developed second malignancy. The immunohistochemistry profile was similar to female breast cancers. Conclusions: The second malignancies in this cohort affected overall survival and suggest the possibility of other germline mutations in addition to BRCA2 in male patients with breast cancer.

Algorithm to Identify Systemic Cancer Therapy Treatment Using Structured Electronic Data.

PURPOSE: With the shift in the majority of oncology clinical care in the United States from paper records to electronic health records, researchers need efficient and validated processes to obtain accurate data about the entire treatment history of patients diagnosed with cancer. The objective of this study was to develop and validate an algorithm that is agnostic to the source of data but that can identify specific regimens in the entire course of systemic therapy treatment for patients diagnosed with breast, colorectal, or lung cancer. METHODS: A cohort of patients with incident breast, colorectal, and lung cancer were randomly distributed into six groups. The algorithm was iteratively modified, and the performance was assessed until no additional modifications could be identified in the first three groups. The performance of the algorithm was confirmed in the three groups that remained. RESULTS: The final model produced ranges of sensitivity between 97.2% and 100% for first-course systemic therapy across all cancers, with a false-positive rate of 0%. The algorithm matched the exact number of courses and the exact regimens of systemic therapy agents as captured by infusion, pharmacy, and procedure electronic medical record data for all courses of therapy 88% to 100% of the time. CONCLUSION: Use of our validated algorithm that characterizes entire courses of systemic therapy treatment in patients diagnosed with breast, colorectal, and lung cancer will allow researchers in a variety of settings to conduct comparative effectiveness studies related to the uptake, safety, outcomes, and costs associated with the use of both novel and standard regimens.

Cross-national epidemiology of panic disorder and panic attacks in the world mental health surveys.

CONTEXT: The scarcity of cross-national reports and the changes in Diagnostic and Statistical Manual version 5 (DSM-5) regarding panic disorder (PD) and panic attacks (PAs) call for new epidemiological data on PD and PAs and its subtypes in the general population. OBJECTIVE: To present representative data about the cross-national epidemiology of PD and PAs in accordance with DSM-5 definitions. DESIGN AND SETTING: Nationally representative cross-sectional surveys using the World Health Organization Composite International Diagnostic Interview version 3.0. PARTICIPANTS: Respondents (n = 142,949) from 25 high, middle, and lower-middle income countries across the world aged 18 years or older. MAIN OUTCOME MEASURES: PD and presence of single and recurrent PAs. RESULTS: Lifetime prevalence of PAs was 13.2% (SE 0.1%). Among persons that ever had a PA, the majority had recurrent PAs (66.5%; SE 0.5%), while only 12.8% fulfilled DSM-5 criteria for PD. Recurrent PAs were associated with a subsequent onset of a variety of mental disorders (OR 2.0; 95% CI 1.8-2.2) and their course (OR 1.3; 95% CI 1.2-2.4) whereas single PAs were not (OR 1.1; 95% CI 0.9-1.3 and OR 0.7; 95% CI 0.6-0.8). Cross-national lifetime prevalence estimates were 1.7% (SE 0.0%) for PD with a median age of onset of 32 (IQR 20-47). Some 80.4% of persons with lifetime PD had a lifetime comorbid mental disorder. CONCLUSIONS: We extended previous epidemiological data to a cross-national context. The presence of recurrent PAs in particular is associated with subsequent onset and course of mental disorders beyond agoraphobia and PD, and might serve as a generic risk marker for psychopathology.

Effectiveness of a healthy lifestyle intervention for low back pain and osteoarthritis of the knee: protocol and statistical analysis plan for two randomised controlled trials

ABSTRACT Background These trials are the first randomised controlled trials of telephone-based weight management and healthy lifestyle interventions for low back pain and knee osteoarthritis. This article describes the protocol and statistical analysis plan. Method These trials are parallel randomised controlled trials that investigate and compare the effect of a telephone-based weight management and healthy lifestyle intervention for improving pain intensity in overweight or obese patients with low back pain or knee osteoarthritis. The analysis plan was finalised prior to initiation of analyses. All data collected as part of the trial were reviewed, without stratification by group, and classified by baseline characteristics, process of care and trial outcomes. Trial outcomes were classified as primary and secondary outcomes. Appropriate descriptive statistics and statistical testing of between-group differences, where relevant, have been planned and described. Conclusions A protocol for standard analyses was developed for the results of two randomised controlled trials. This protocol describes the data, and the pre-determined statistical tests of relevant outcome measures. The plan demonstrates transparent and verifiable use of the data collected. This a priori protocol will be followed to ensure rigorous standards of data analysis are strictly adhered to.

Effectiveness of a healthy lifestyle intervention for low back pain and osteoarthritis of the knee: protocol and statistical analysis plan for two randomised controlled trials.

BACKGROUND: These trials are the first randomised controlled trials of telephone-based weight management and healthy lifestyle interventions for low back pain and knee osteoarthritis. This article describes the protocol and statistical analysis plan. METHOD: These trials are parallel randomised controlled trials that investigate and compare the effect of a telephone-based weight management and healthy lifestyle intervention for improving pain intensity in overweight or obese patients with low back pain or knee osteoarthritis. The analysis plan was finalised prior to initiation of analyses. All data collected as part of the trial were reviewed, without stratification by group, and classified by baseline characteristics, process of care and trial outcomes. Trial outcomes were classified as primary and secondary outcomes. Appropriate descriptive statistics and statistical testing of between-group differences, where relevant, have been planned and described. CONCLUSIONS: A protocol for standard analyses was developed for the results of two randomised controlled trials. This protocol describes the data, and the pre-determined statistical tests of relevant outcome measures. The plan demonstrates transparent and verifiable use of the data collected. This a priori protocol will be followed to ensure rigorous standards of data analysis are strictly adhered to.