The Acute Care of at-Risk Newborns (ACoRN) program was developed in Canada for trained health care providers for the identification and management of newborns who are at-risk and/or become unwell in the first few hours or days after birth. The ACoRN process follows an 8-step framework that enables the evaluation and management of babies irrespective of the experience or expertise of the caregiving individual or team. This study assesses the applicability of the program to Chinese pediatric practitioners.Course content and educational materials were translated from English into Chinese by bilingual neonatal practitioners. Confidence and knowledge questionnaires were developed and reviewed for face and content validity by a team of ACoRN instructors. Bilingual Chinese instructors were trained at the tertiary perinatal centre in Hangzhou Zhejiang to deliver the course at 15 level II county hospitals. Participants completed pre- and post-course confidence and knowledge questionnaires and provided feedback through post-course focus groups.216 physicians and nurses were trained. Confidence and knowledge relating to neonatal stabilization improved significantly following the courses. Participants rated course utility and function between 4.2 and 4.6/5 on all items. Pre/post measures of confidence were significantly correlated with post course knowledge. Focus group data supported the perceived value of the program and recommended course adjustments to include pre-course reading, and increased content related to simulation, communication skills, and management of respiratory illness and jaundice.ACoRN, a Canadian educational program, appears to be well received by Chinese health care providers and results in improved knowledge and confidence. International program adaptation for use by health care professionals requires structured and systematic evaluation to ensure that the program meets the needs of learners, reflects their learning styles, and can be applied in their setting. Worldwide, approximately 4 million babies die in the first 4 weeks after birth with the highest risk being on the first day -3. AlmosThere are evidence-based, cost-effective interventions for improving perinatal care in low-income countries, particularly in rural settings -8 which The Acute Care of at-Risk Newborns (ACoRN) program  was deveIn a promising trend, the People’s Republic of China reduced neonatal mortality by 70% (from 34 to 10 per thousand live births) between 1990 and 2008 . DespiteThe purpose of this study was to assess whether (1) course participants in China perceived the content and program focus useful; (2) the content could be applied to the clinical practice in China and be taught in their institutions; (3) the program increased practitioner self-confidence in managing sick infants; (4) the course improved their knowledge; and (5) the gain in knowledge could be explained by initial knowledge or confidence.Funding support from the Canadian Institutes of Health Research and the National Natural Science Foundation of China made it possible for Canadian and Chinese researchers to partner in the dissemination and assessment of the ACoRN program. The Public Health Bureau of Zhejiang Province, along with the Department of Neonatology, Hangzhou Children’s Hospital, Zhejiang University School of Medicine, are committed to the introduction of ACoRN in 15 county hospitals. Zhejiang province in eastern China has a population of 46 million, 2 large cities, 9 regional cities and 36 counties. Each county is served by a county hospital and between 10–20 village clinics. There are about 30,000 births per year in the designated area. Zhejiang has excellent administrative organization and clear lines of communication between levels of care.The study was conducted in 15 level II county hospitals in South-West Zhejiang province, which is predominantly rural and economically disadvantaged. The total annual births in these hospitals was 22400 . There were 4100  annual nursery admissions. The majority of these deliveries are in a facility. Some are transferred from a lower level facility to a higher level facility for care.There was an identified need for the ACoRN program for both physicians and nurses. While, almost 100% of the nurses and physicians have participated in an NRP course, there are few opportunities for hands on practice and no policies for continuing assessment of skills or retraining. There is a shortage of nurses in China. Often nurses in special care nurseries will have to take care of 5–6 (or even 8–10 sick babies at night). The ACoRN primary survey  enables the nurses to evaluate the baby’s condition quickly. In local hospitals, where most of the medical care is delivered by a general pediatrician with little experience and training in managing sick newborn infants and no medical consultation to support them, physicians require the training in stabilization that ACoRN offers.The ACoRN program provides a systematic approach to care, using an 8-step framework to help the practitioner systematically gather information, establish priorities and intervene appropriately. The program provides tools to be used for neonatal assessment and the knowledge and skills for clinical management of sick or ‘at-risk’ newborns . The keyACoRN is designed to be delivered in the same way that other life support courses are delivered, namely that teachers participate in a teach the teacher (TTT) program prior to delivering the course to ensure the course is delivered consistently across all offerings. TTT training focuses on (1) the primary survey for a baby who is sick or at risk leading to a prioritized problem list, (2) algorithmic sequences that address concerns in specific body systems , (3) case-based learning, (4) preparation for neonatal transport, (5) support of the baby, family, and team, and (6) interactive or simulated skill stations for chest X-ray interpretation, jaundice and admission .To ensure that the content of the program was appropriate for practitioners in this area of China, the course text was translated into Mandarin and checked for accuracy by a bilingual member of the ACoRN editorial board. Canadian faculty taught the first few county hospital workshops alongside the participants of the TTT with an expectation that groups of 10–15 (physician and nurses) would be trained in their own communities. The Canadian faculty spent a day with 4 of the trainers adapting some of the content  and delivery to ensure acceptability to the learners.To evaluate the course, a 16 item  questionnaire assessed perceptions about whether participants found the content and program useful and whether the course functioned as designed. This questionnaire also asked participants if they could apply the content in their settings and could teach it to their co-workers Table . Three kDescriptive analyses  were calculated for each of the items in the ‘course utility and function’ Table  and ‘conParticipants for focus groups were recruited with the assistance of Chinese colleagues, through their contact with the Hangzhou Children’s Hospital. All participants were informed of the purpose of the study and asked to sign a written informed consent which assured confidentiality and sought permission to audio and video tape the group discussions. The focus group questions, developed by members of the ACoRN team, asked both teachers and participants what they liked about the course, to comment on each of the course components , and to provide their perceptions about how the course could be improved. Participants were asked whether they currently take care of sick babies and whether any part of the course was challenging to learn and why. Teachers were also asked if they could follow the translation, the acceptability of the course to their learners, how the course might help learners change how they take care of sick babies, and whether any part would be challenging to teach in their hospital.The interviews were conducted by a fluent English-Chinese speaking Canadian education researcher in a small comfortable meeting room free from outside distractions. Participants sat around a table so they could see and interact with each other. The audio recordings of the interviews were translated and transcribed into English by the group facilitator. All names were changed on the transcripts to ensure anonymity. Transcripts were coded and analyzed so that feedback about the central themes and issues related to all of the core components of the course could be identified and modifications made to the course as needed.A total of 216 physicians and nurses participated in the program.The participants rated the utility and function of the course highly with a range of means from 4.23 to 4.58. Table . ParticiThe knowledge evaluations indicated that the knowledge scores for each scenario increased between the pre and the post test with a total increase from 31.47 (s.d. = 5.1) to 34.7 (s.d. = 3.5) out of 40; effect size d = 0.77. While this increase in scores is only about 10%, it is noted that the increase was greater for the three scenarios in which there were abnormal findings compared to scenario D which profiled a well term infant. For the pre knowledge measure, the Cronbach’s alpha was 0.62 and post measure Cronbach’s alpha was 0.58  to 62% of the variance between the content and applying the course. Pre measure of confidence and post measure of confidence were significantly correlated with most measures of knowledge; although, a number of these correlations were small  = 36.514, p < 0.01); the majority of the variance was accounted for by pre course knowledge  with pre comfort scores  accounting for less of the variance  was seen as good, they recommended modifications so that it would be more useful and suitable for the Chinese environment. They encouraged adding more information about the respiratory system and jaundice to the course and to the textbook. Last, learners suggested that clinical simulations would be helpful.Learners also recommended pre-course material including a clear workshop schedule and textbook so they could prepare for the course. They noted that attention also needed to be paid to post course activities to reinforce the content to prevent participants from going back to their old ways.  with the teachers providing explicit information about patient management. We presented the skill stations and demonstrated the interactive participatory method of learning. The students very quickly learnt how to conduct them and were successfully engaged in the participatory learning. We speculate that in settings where traditional didactic methods of teaching are practiced exposure to participatory methods will be acceptable and may lead to acquisition and sustainability of knowledge. The initial evaluation results of these modifications to the ACoRN course in this setting are encouraging and suggest that the program with modifications could be used in China.Participants told us that the content and focus of the course were useful. Further, participants believed they could apply what they learned in their own clinical practices. The course functioned reasonably well with teachers reporting they would be comfortable teaching the content and learners demonstrating an increase in knowledge and self- confidence. For both self-confidence and knowledge, the effect size of the increase in scores could be considered to be ‘large’. The variance in the scores at the post-test was primarily explained by the pre course knowledge scores; while the pre-comfort score did make a statistically unique contribution to the equation it added very little to the variance.By taking both a quantitative and qualitative approach to data collection, we were able to assess change and improvement in scores as well as obtaining critical information to modify the course. The focus groups provided us with additional insights about participants’ desires to prepare for the learning experience, areas which needed to be expanded, and cases which were inappropriate for learners in China.One of the interesting outcomes of this study was the preference of Chinese learners for case-based learning and simulation. Research on learning through simulation suggests that when it is well planned, it has benefits, particularly in complex situations involving teams . This isThe results of this study do speak to the generalizability of the ACoRN Primary Survey and Sequences, presumably because of the generic nature of newborn diseases. Gaps, however, were identified in learning objectives, in particular, the need to add neonatal jaundice to the list of conditions addressed.This evaluation is an initial assessment of the program. As health professions educators have noted, a comprehensive assessment of educational outcomes should include a hierarchy that considers participant numbers, satisfaction, learning, competence and performance; patient health; and community health . This stEducational and other strategies continue to be important in the challenge of improving neonatal outcomes. As a recent Cochrane review noted, it continues to be important to have skilled delivery and facility-based services for newborn care as well as community-based care . The appACoRN, a Canadian educational program, was developed for a Canadian population of health care providers. Through a collaborative effort whereby Canadian and Chinese neonatologists worked together, the ACoRN program was modified both to add content and to change the teaching strategies in order to improve the program’s utility and acceptability. While data are preliminary, the program appeared to be well received by Chinese health care providers and resulted in improved knowledge and confidence. Programs developed in one setting can be modified and successfully adapted for a different setting.University of Calgary Conjoint Health Ethics Review Board.ACoRN, Acute Care of at-Risk Newborn; HBB, Helping Babies Breathe, a program of the American Academy of Pediatrics; NRP, Neonatal Resuscitation, a program of the American Academy of Pediatrics.The authors declare that they have no competing interests.NS was PI on the grant application, oversaw all aspects of the study in Canada and China, and drafted the manuscript. JL was a co-investigator on the grant, provided critical input on instruments and data analysis, oversaw the analysis, drafted the manuscript and managed manuscript revisions. HF was research associate on the study, handled the analysis, created tables, and provided critical input into the manuscript. KA was a co-investigator on the grant, collaborated on the adaptation of the course, provided critical input on instruments and data analysis, and provided critical input into the manuscript drafts. DM was a co-investigator on the study, collaborated on the adaptation of the course, provided critical input on instruments and data analysis, and provided critical input into the manuscript drafts. XQ was a research associate on the study, conducted the focus groups, analyzed the qualitative data, and provided critical input into manuscript drafts. XM collaborated on the adaptation of the course for China, co-managed the educational programming and data collection in China, and provided critical input into manuscript drafts. LD was project lead for China, collaborated on the adaptation of the course for China, co-managed the educational programming and data collection in China, and provided critical input into manuscript drafts. SL was a co-investigator on the grant and provided critical input into manuscript drafts. All authors read and approved the final manuscript.Dr Singhal is Professor, Department of Pediatrics, Faculty of Medicine, University of Calgary, Calgary, Alberta, CanadaDr Lockyer is Professor, Department of Community Health Sciences and Senior Associate Dean--Education, Faculty of Medicine, University of Calgary, Calgary, AlbertaMs Fidler is Research Associate, Office of Continuing Medical Education and Professional Development, Faculty of Medicine, University of Calgary, Calgary, Alberta, CanadaDr Aziz is Associate Professor, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, CanadaDr McMillan is Professor Department of Pediatrics, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, CanadaDr Qiu is Assistant Professor of Paediatrics, Faculty of Medicine, University of Toronto, Toronto, Ontario CanadaDr Ma is Assistant Professor, Pediatrics, Children’s Hospital of Zhejiang University School of Medicine, Hangzhou, People’s Republic of ChinaDr Du is Professor, Pediatrics, Children’s Hospital of Zhejiang University School of Medicine, Hangzhou, People’s Republic of ChinaDr Lee is Professor, Departments of Paediatrics, Obstetrics & Gynecology, and Public Health, Faculty of Medicine, University of Toronto, Toronto, Ontario, CanadaThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6920/12/44/prepubScenario A results. Participants were asked to assess a 2 hour old full term baby from the maternity unit. The baby looks pale and has blue hands and feet. He is making a noise with every breath when he breathes out. His breathing rate is 40 per minute. His pulse rate is 120 per minute. His axillary temperature is 35.8 Celsius. He does not wake up when you examine him.Click here for fileScenario B results. Participants were asked to assess a 32 week gestation, 1.8 kg baby is admitted to the nursery at two hours of age. The baby has breathing difficulty and looks pale and blue despite receiving 100% oxygen by facemask. His breathing rate is 50 per minute and his pulse rate is 150 per minute. His axillary temperature is 36.8 Celsius.Click here for fileScenario C results. Participants were asked to assess a full term baby admitted at 4 days of age with jaundice, a fever, and a seizure. The baby is breathing easily at 40 breaths per minute with aheart rate of 120 per minute. His axillary temperature is 38 Celsius. He is no longer seizing but does not wake up when examined. The transcutaneous bilirubin level is >305 micromol/l(>18 mg/dl).Click here for fileScenario D results. Participants were asked to assess a full term baby who required intubation and suctioning for meconium-stained amniotic fluid. At 10 minutes of age, the baby appears pink and is breathing easily with a respiratory rate of 40 breaths for minute. The pulse rate is 120 beats per minute. Capillary refill time is 3 seconds. The baby is alert and active.Click here for fileQualitative data from focus groups. Direct quotes from instructors and learners are documented in this file.Click here for file


This study was aimed at characterizing the expression and activity of the mostly mitochondrial enzyme GABA transaminase (GABA-T) during DRF/FEO expression. We found that DRF promotes a sustained increase of GABA-T in the liver homogenate and mitochondrial fraction throughout the entire day-night cycle. The higher amount of GABA-T promoted by DRF was not associated to changes in GABA-T mRNA or GABA-T activity. The GABA-T activity in the mitochondrial fraction even tended to decrease during the light period. We concluded that DRF influences the daily variations of GABA-T mRNA levels, stability, and catalytic activity of GABA-T. These data suggest that the liver GABAergic system responds to a metabolic challenge such as DRF and the concomitant appearance of the FEO.Daytime restricted feeding (DRF) is an experimental protocol that influences the circadian timing system and underlies the expression of a biological clock known as the food entrained oscillator (FEO). Liver is the organ that reacts most rapidly to food restriction by adjusting the functional relationship between the molecular circadian clock and the metabolic networks.  Its specificity is not strict since it can also recognize alanine, β-alanine, aspartate, and propanoate as substrates. GABA-T is dependent on pyridoxal-phosphate (PLP) as cofactor, and its catalytic mechanism consists of two coupled half reactions in which the PLP cofactor oscillates between the pyridoxal and the pyridoxamine forms [α2 dimer with 472 residues per subunit; the two monomers are tightly intertwined, and the two PLP cofactors are located close to the subunit interface [GABA transaminase  is a catabolic enzyme that converts GABA and ne forms . This ennterface . The matGABA is a well-known inhibitory transmitter in the nervous system. However, components of the GABAergic system have also been characterized in a variety of endocrine tissues such as pancreas, testis, and liver –9. MammaDaytime restricted feeding (DRF) is an experimental protocol that influences the circadian timing system by promoting the emergence of an alternate master circadian oscillator that is independent of the suprachiasmatic nucleus (SCN) . The basTo obtain further insights into the modulation of the mitochondrial adaptations of the liver GABA-handling system during the DRF protocol/FEO expression, we analyzed the daily variations of GABA-T mRNA and protein expression and enzymatic activity. The results evidenced under the DRF protocol are as follows: (1) an increased presence of GABA-T in homogenate and mitochondrial fraction, (2) changes in the rhythmic profile of GABA-T activity, and (3) disappearance of an ultradian rhythm of GABA-T mRNA.Experiments were carried out with male Wistar rats weighing from 200 to 250 g and maintained under a 12:12 h LD cycle (lights on 08:00 h) at constant temperature (22 ± 1°C). Rats were kept in groups of 4 in transparent acrylic cages (40 × 50 × 20 cm) with free access to Purina Chow and water except during food restriction or fasting conditions. Experimental procedures were conducted in accordance with our Institutional Guide for Care and Use of Experimental Animals  and with international ethical standards . ad libitum with free access to food and water throughout the 24 h period, AL group;control animals fed withexperimental group with restricted food access (exclusively from 12:00 to 14:00 h), DRF group.The experimental protocol has been published previously . BrieflyAt the end of the 3 weeks, subgroups of animals were sacrificed at 3 h intervals over a complete 24 h cycle, starting at 08:00 h. The protocol was followed until the last day. ad libitum were maintained with free food access for 3 weeks; on the last day, food was removed at 14:00 h, and, after 21 h (~1 day) of food deprivation, they were sacrificed (at 11:00 h), F group.Animals fedA second group of rats was similarly deprived of food for 21 h, then refed for 2 h (from 12:00 to 14:00), and sacrificed at 14:00 h, F + R group.In addition, 2 more groups were included to compare the acute fasting and subsequent refeeding response in the DRF group.≈5 g) and immediately placed in an ice-cold isolation medium (1 : 10 wt/vol) containing 250 mM sucrose, 0.1% BSA (fatty acid free), and 0.5 mM EGTA (pH 7.4). The tissue was homogenized in 10 volumes of 10 mM Tris-HCl, pH 7.4, with a Teflon homogenizer (40 rpm for 10 s). Subcellular fractionation was done according to Aguilar-Delfín et al. [Rats were beheaded with a guillotine-like instrument. The liver was removed  were separated on 10% SDS-PAGE, transferred to nitrocellulose membranes, and blocked for 1 h in TBST buffer  containing 5% nonfat milk. Membranes were then washed and incubated in the presence of mouse anti-GABA-T antibody  diluted 1/10,000 in TBST overnight at 4°C. As controls, in the case of homogenates, membranes were incubated in the presence of mouse anti-tubulin antibody  diluted 1/1,000 or in the case of mitochondrial fractions in the presence of rabbit anti-VDAC1/Porin antibody  diluted 1/1,000. After washing, membranes were incubated with secondary antibodies conjugated to alkaline phosphatase . Bands were revealed using AP conjugate substrate kit . Densitometric analysis was performed using the Image Lab Software .Liver homogenates and mitochondrial fractions were obtained in RIPA buffer  and subjected to denaturing SDS-PAGE under reducing conditions. Total protein concentrations were determined by the Bradford method and equal amounts (30 μg) was reverse transcribed using SuperScript III Reverse Transcriptase, Oligo (dT) 12–18 Primer, RNaseOUT Recombinant Ribonuclease Inhibitor, and dNTP Set PCR Grade . Amplification was performed in triplicate in the CFX96TM Real-Time PCR Detection System . Primers used for qPCR amplifications were synthesized by Sigma-Aldrich Co. , and the corresponding sequences were, for GABA-T (GenBank BC081787.1), forward 5′-TTCCGGAAGCTGAGAGACAT-3′ and reverse 5′-AGTCTGAACCTCGTCCACCA-3′ and, for ribosomal protein S18 (Rps18) (GenBank BC126072.1) used as housekeeping gene, forward 5′-TTCAGCACATCCTGCGAGTA-3′ and reverse 5′-TTGGTGAGGTCAATGTCTGC-3′. Amplifications were carried out with Maxima SYBR Green qPCR Master Mix  in a 10 μL final reaction volume containing cDNA (1/100) and 0.5 μM of each of the primer pairs in SYBR Green Master Mix, according to the following protocol: activation of Taq DNA polymerase and DNA denaturation at 95°C for 10 min, followed by 40 amplification cycles consisting of 10 s at 95°C, 30 s at 62°C, and 30 s at 72°C. The PCR data were analyzed by the 2−ΔΔCT method and cycle thresholds (CT) normalized to the housekeeping gene Rps18 were used to calculate the mRNA levels of GABA-T.GABA-T gene expression was evaluated by isolating total RNA from liver tissues (20–30 mg) using the SV Total RNA Isolation System . The amount and quality of RNA were estimated spectrophotometrically at 260 and 280 nm, and a constant amount of RNA (2 P < 0.05 for both. The Student's t-test was applied to detect significant differences between DRF rats and the controls of feeding conditions (acute fasting and refeeding) both before and after food access . The Student's t-test was also used to identify significant differences (P < 0.05) between the 24 h average values of the AL and DRF groups. Rhythmic analysis was performed by Chronos-Fit  based on a partial Fourier analysis of the data [Data were classified by group and time and are displayed as mean ± standard error of the mean (SEM). Data were compared using a two-way ANOVA using Fisher's least significant difference (LSD) test for multiple and independent measures, with a factor for group (2 levels) and a factor for time (8 levels). In order to determine significant time effects for each daily sampling profile, a one-way ANOVA was performed for individual groups. The one-way ANOVA was followed by Tukey's post hoc test whereas the two-way ANOVA was followed by a Bonferroni post hoc test with the significance threshold set at All results are double plotted displayed to have a better notion of the rhythmic profiles.The amount of GABA-T protein detected by Western blot analysis in the liver homogenate and mitochondrial fraction is shown in Since GABA-T is in an important proportion a mitochondrial enzyme, the amount of this protein was also evaluated by Western blot analysis in the liver mitochondrial fraction in the AL and DRF groups (b). As observed in liver homogenate, GABA-T was significantly increased in the DRF group. However, comparing the calculated average values, the elevation was more moderate ~26%, . Again, The activity of GABA-T in AL and DRF groups is shown in In both liver homogenate and the mitochondrial fraction, the food condition controls (F and F + R groups) did not show any changes in comparison to the AL and DRF rats at 11:00 h and 14:00 h, respectively.The mRNA for GABA-T was quantified in liver by RT-qPCR in the AL and DRF groups, and the results are shown in Our data show that under a protocol of daytime restricted feeding (DRF) with the subsequent appearance of the feeding entrained oscillator (FEO) the properties of liver GABA-T are affected and highlight the plasticity of the hepatic GABAergic system under a metabolic challenge.It is well documented that GABA and its metabolizing enzymes are found outside the nervous system and play a role in the cell signaling of several organs and tissues . These tGABA-T is, but not exclusively, a mitochondrial enzyme that requires PLP for its activity. In the liver, it exists as a smaller variant because of proteolytic processing that removes 12–14 amino acids . The phaOur results indicate that liver GABA-T activity, mainly in the mitochondrial fraction, and the level of GABA-T mRNA in AL groups show daily fluctuations. Previously, our group reported that the mitochondrial yield after cellular fractionation did not change by the DRF protocol . The datThe changes in the amount and activity of GABA-T associated with the protocol of daytime restricted feeding are part of an extensive set of metabolic and physiological adaptations that occur in the liver and other organs when the daily rhythmicity is modified by a new organization of the timing system , 30. TheIt is in the liver where the influence of the FEO is most evident. The liver plays central roles in both the processing of nutrients arriving from the duodenal tract and the modulating of the hunger-satiety cycle . This faIn previous reports, we have shown that DRF/FEO expression strongly modifies the metabolic status of the liver mitochondria, promoting a more oxidized state and increasing the synthesis of ATP , 35. In  ad libitum feeding. This new interaction has the characteristics of an emergent property, since it is not observed in the ad libitum condition or in the control groups of acute fasting and acute fasting followed by refeeding. For example, the increased amount GABA-T protein promoted by the protocol of DRF/FEO expression and detected by Western blot analysis (The exact molecular mechanisms that underlie the adaptive modifications of liver physiology during expression of the FEO and in response to the DRF protocol are not known. However, an initial rationale towards the identification of this sequence of events is to postulate that synchronizing circadian physiology by limited food access has an impact on the reciprocal regulation between energetic metabolic networks and the cellular timing system , 37. Duranalysis  is uniquOne way to consider the emergence of new properties in biological systems is the concept of rheostasis , which iBiochemical properties of liver GABA-T were modified by the DRF protocol. An explanation for these findings should consider the coincidence of the FEO expression with a functional light-driven SCN. In addition, GABA-T regulation could be responding to the intense arrival of nutrients during the 2 h of food access, in the context of restricted feeding schedules. The data raise the possibility that alterations in the hepatic GABAergic system could be among the metabolic and physiological adaptions that occur in the liver during expression of the FEO. This possibility needs further exploration.


Rd7 retinas have reduced levels of the outer segment (OS) structural protein retinal degeneration slow (RDS). We test the hypothesis that increasing RDS levels would improve the Rd7 phenotype. Transgenic mice over-expressing normal mouse peripherin/RDS (NMP) in rods and cones were crossed onto the rd7 background. Disease phenotypes were assessed in NMP/rd7 eyes and compared to wild-type (WT) and rd7 eyes at postnatal day 30. NMP/rd7 retinas expressed total RDS (transgenic and endogenous) message at WT levels, and NMP protein was correctly localized to the OS. NMP/rd7 retinas have shorter OSs compared to rd7 and WT and significantly reduced number of rosettes. NMP/rd7 mice also exhibited significant deficits in scotopic ERG amplitudes compared to rd7 while photopic amplitudes remained unaffected. Protein levels of rhodopsin, RDS, and the RDS homologue ROM-1 were significantly reduced in the NMP/rd7 retinas compared to rd7. We show that correcting the levels of RDS gene expression does not improve the phenotype of the rd7 suggesting that RDS deficiency is not responsible for the defect in this model. We suggest that the specific rod defect in the NMP/rd7 is likely associated with ongoing problems in the rd7 that are related to the expression of cone genes in rod cells, a characteristic of the model.The nuclear receptor NR2E3 promotes expression of rod photoreceptor genes while repressing cone genes. Mice lacking NR2E3 ( The homeobox genes CRX and OTX regulate both rod and cone genesis. Downstream of these are NRL and NR2E3  rd7/rd7Nr2e3 here referred to as rd7 for the sake of simplicity) mutant mouse lacks NR2E3 and has been used as a model for ESCS Rd7 mice exhibit rosettes in the outer nuclear layer (ONL), slow retinal degeneration, and abnormal electroretinograms (ERG) rd7 eyes demonstrate substantial alterations in the pattern of retinal gene expression, including de-repression of many cone-specific genes The retinal degeneration 7  +/−rds mouse) results in an ADRP-like phenotype, characterized by 1) early defects in rod function and later onset defects in cone function, 2) shortened OSs with membranous whorls, and 3) slow degeneration. Similarities between some RDS mutant mouse models rd7 combined with the decreased levels of RDS expression in the rd7, have led to the hypothesis that part of the degenerative phenotype in the rd7 may be due to RDS haploinsufficiency One of the transcriptional targets of NR2E3 is the photoreceptor-specific protein retinal degeneration slow (RDS). In the +/−rds haploinsufficiency model of ADRP rd7 background and evaluated the retinal phenotype. Surprisingly, instead of mediating improvement in the rd7 phenotype, over-expression of RDS exacerbated the degenerative phenotype.To test this hypothesis, we took advantage of a transgenic mouse model we have generated to over-express wild-type RDS . This transgene is driven by the interphotoreceptor retinoid binding protein (IRBP) promoter and previous characterization has established that one NMP allele results in transgenic RDS protein levels which are ∼30% of WT levels rd7 retinas could ameliorate the degenerative phenotype, we crossed the NMP transgenic line onto the rd7 background. We generated retinal cDNA and amplified using primers that recognize both endogenous RDS and NMP  brought total RDS message levels back to WT levels . This difference can also be seen on the ultrastructural level; while most rods in the NMP/rd7 still contain nicely stacked discs, they are much shorter than WT or rd7 rods  to label cone OSs  to assess subcellular localization of a panel of OS proteins at P30 Fig. 1C,rd7 compared to WT, and were further reduced in NMP/rd7 (compared to WT and rd7). In contrast, photopic b-waves in response to white , although not in the adult ed to WT Fig. 6A.−/−Nrl model also exhibits altered retinal transcription, we next assessed the effects of the NMP transgene on the −/−Nrl. As in the WT , and both S- and M-opsin are properly localized  is similar to what we see in the −/−Nrl wherein 100% of rods are converted to cone-like cells. This suggests that the hybrid rod cells rd7 are likely the primary source of the changes seen in the RDS complexes. We have previously hypothesized that the differential role of RDS in rods vs. cones  is tied to RDS having different unknown binding partners in the two cell types. Indirect support for this hypothesis is provided here: hybrid rods of the rd7 abnormally express many cone genes One of the interesting outcomes we noticed here is that in both the rd7 is not related to a deficit in functional RDS. We observe decreases in OS protein levels, including RDS, even though we show that RDS mRNA levels are corrected in the NMP/rd7. Furthermore, neither the rd7 nor the NMP/rd7 retina exhibits major defects in RDS complex formation or mislocalization of RDS. More importantly, the ultrastructure of rd7 and NMP/rd7 OSs is very different from that seen in cases of RDS haploinsufficiency rd7 or the NMP/rd7, both of which exhibit nicely flattened and organized discs, although OSs are much shorter than WT. These observations suggest that the reported similarities between the rd7 and models of RDS haploinsufficiency Several pieces of our data suggest that the degeneration in the rd7. We observe reduction in rod function and shortening of rod OSs in the NMP/rd7 compared to rd7 and WT. Likely as a consequence of OS shortening we also observe declines in the levels of proteins expressed in rods, including RDS and ROM-1. These defects do not appear to be a consequence of altering retinal transcription per se, as we observe no functional defect in NMP/−/−Nrl mice. −/−Nrl are affected by the lack of RDS −/−Nrl is not because the photoreceptors in the −/−Nrl retina are immune to alterations in RDS expression. Thus the question arises, why is there such a severe defect in the NMP/rd7 retina while the NMP/−/−Nrl and the NMP/WT retinas exhibit no defects?It is clear, however, that expression of the NMP transgene exacerbates the rod defect in the rd7rd7. This hypothesis is supported by the observation that the cones in the rd7 are not in this hybrid state of expressing both rod and cone genes, and thus do not exhibit any defect when they are induced to express NMP. In conclusion, these data suggest that the defect in the rd7 is not due to RDS haploinsufficiency and that expression of the NMP transgene may accelerate the degeneration of hybrid photoreceptors which abnormally express both rod and cone genes.The most likely explanation is tied to the effects of NRL ablation vs. NR2E3 ablation. In the absence of NRL in mice, no rods are formed at all rd7 transgenic mice were generated by cross-breeding our NMP transgenic mice rd7 mice rd7 mice were used as controls. Animals were maintained in cyclic light .All experiments and animal maintenance were approved by the local Institutional Animal Care and Use Committee  and conformed to the guidelines on the care and use of animals adopted by the Association for Research in Vision and Ophthalmology . The NMP/Primary antibodies were used as described in each section. Several antibodies used here were generated in-house and characterized previously The methods used for tissue collection, processing, plastic-embedding, and immunogold labeling were as described previously Eyes were harvested, dissected, fixed and embedded as previously described for paraffin sectioning (6 µm) Table S1.Total RNA from frozen eyes was extracted using TRIzol (Life Technologies) as described previously 2 intensity presented to the dark-adapted dilated eyes in a BigShot ganzfeld. To evaluate photopic response, animals were light adapted for 5 minutes under a light source of 29.03 cd/m2 intensity in a BigShot ganzfeld. Cone photoreceptor function (photopic ERG) was assessed by averaging responses to 25 strobe flash stimuli at either 530 nm , 365 nm (0.79 cd-s/m2), or white light . At least 8 mice per genotype were analyzed.ERGs were performed as previously described Western blot and velocity sedimentation were performed as described previously Differences between genotypes were assessed by 1-way ANOVA with Bonferroni's post-hoc pairwise comparisons, or two-tailed Student's t-test (when only two groups were tested). P<0.05 was considered significant. Graphs are presented as mean ± SEM.Table S1Primer sequences used for qRT-PCR.(PDF)Click here for additional data file.


Implementation of lifestyle interventions in patient care is a major challenge. Understanding factors that influence implementation is a first step in programs to enhance uptake of these interventions. A lifestyle-counseling intervention, Lively Legs, delivered by trained nurses, can effectively improve the lifestyle in patients with venous leg ulcers. The aim of this study was to identify factors that hindered or facilitated implementation of this intervention in outpatient dermatology clinics and in home care.A mixed-methods multiple case study in five purposefully selected healthcare settings in the Netherlands was conducted. Measurements to identify influencing factors before and after implementation of Lively Legs included interviews, focus groups, questionnaires, and nurses’ registration. Analyses focused on qualitative data as the main data source. All data were compared across multiple cases to draw conclusions from the study as a whole.A total of 53 patients enrolled in the Lively Legs program, which was delivered by 12 trained nurses. Barriers for implementation were mainly organizational. It was difficult to effectively organize reaching and recruiting patients for the program, especially in home care. Main barriers were a lack of a standardized healthcare delivery process, insufficient nursing time, and a lack of motivated nurses to deliver the program. Facilitating factors were nurse-driven coordination of care and a standardized care process to tie Lively Legs into, as this resulted in better patient recruitment and better program implementation.This study identified a range of factors influencing the implementation of a lifestyle-counseling program, mainly related to the organization of healthcare. Using a case study method proved valuable in obtaining insight into influencing factors for implementation. This study also shed light on a more general issue, which is that leg ulcer care is often fragmented, indicating that quality improvement is needed. Physical exercise and compliance with compression therapy are key elements in the course of leg ulcer healing and recurrence. However, many patients demonstrate sedentary lifestyles and noncompliance -7. Althoi.e., patients, dermatologists, nurses, a clinical psychologist, a physiotherapist, a dietician, hospital management, nursing associations, and patient organizations) [The Lively Legs program primarily aims at increasing physical activity , leg exercises, and adherence with compression therapy in venous leg ulcer patients. Next to this, the program focuses on adequate pain management, proper hygiene of feet and legs, adequate footwear, and a healthy diet . The prozations)  and is bzations) -17.et al. (2011) reported an increase in the number of patients that performed exercises after a nurse-led intervention comparable to Lively Legs [The effectiveness of the Lively Legs program on behavior change was established in a multicenter randomized controlled trial . Resultsely Legs . The Leget al. 11 reportely Legs . Positively Legs . Thus, sely Legs -24, undeely Legs -28.A systematic and stepwise approach is recommended to effectively implement innovations in healthcare . One of et al. (2007)  in the Netherlands, the implementation of the Lively Legs program was examined . A case was defined as a region around an outpatient clinic for dermatology where the program was implemented. Starting from the outpatient clinic for dermatology, possibilities for collaboration with homecare agencies were explored in each case. Thus, sometimes one case consists of more than one healthcare setting because of collaboration with one or more homecare agencies. Each case was studied using both qualitative and quantitative methods; however, qualitative data  were the main data source to identify influencing factors for implementation  to determine patients’ current health behaviors, among other things, the level of physical activity and adherence to compression therapy. During this assessment, patients receive tailored health education, including practicing how to perform leg exercises. Also, written educational materials are given to the patient at the end of the first session. In follow-up consultations (20–30 minutes), barriers and facilitators for potential behavior change are discussed using motivational interviewing, and one or two topics for behavior change are chosen to be worked on. The frequency of sessions and choice of topics is decided upon by nurse and patient. Goal setting and home assignments are part of the intervention, as well as discussing patients’ motivation and self-efficacy towards behavior change. The individual counseling consultations take place at the outpatient clinic or at patients’ homes.The essential elements of the Lively Legs program are listed below.– Session 1 – Assessment of patients’ lifestyles – Give tailored health education related to patients’ health beliefs – Demonstrate and practice leg exercises – Give education materials to the patient – Discuss motivation and self-efficacy towards behavior change – Explore barriers and facilitators for behavior change – Goal setting on one or more lifestyle topics and home assignments– Session 2–5 – Evaluate behavior change and give feedback – Motivational interviewing – Discuss motivation and self-efficacy towards behavior change – Explore barriers and facilitators for behavior change – Goal setting on one or more lifestyle topics and home assignments– Final (Preferably six months after session 1) – Evaluate behavior change and give feedback – Summarize the course of the counseling trajectory and the achieved results – Discuss maintenance of behavior changeFive cases were purposefully selected for this study. Cases were selected to compose a heterogeneous sample in terms of geography, familiarity with the Lively Legs program, and degree of cooperation between dermatology outpatient clinic and homecare. This cooperation ranged from no cooperation at all to a joint standardized care process. An overview of the cases and the participants involved in the study is presented in the Results section Table .Potential influencing factors were identified by organizing focus group discussions and semistructured interviews with healthcare providers and managers in each of the cases. Focus groups and interviews mainly addressed two elements: identifying influencing factors and (group-based) brainstorming to identify solutions or strategies to the factors. Participants and respondents included a purposeful sample of dermatologists, dermatology nurses, homecare nurses, and managers. Additionally, all nurses who had participated in the previous effectiveness study were asked to fill in a questionnaire to classify influencing factors for implementation, as they already worked with the program and were expected to estimate potential factors based on their experience. The questionnaire was developed by the Scientific Institute for Quality of Healthcare  and was used in a range of studies on influencing factors for implementing innovation in healthcare ,41. A thAll identified influencing factors and possible implementation strategies were listed by the research team. To select implementation strategies, we used two existing classifications for change strategies in the literature to broaden the scope of strategies and thus reduce the chance of overlooking important issues ,43. The Qualitative data (primarily interviews and secondary field notes and observations) were the main data source to identify influencing factors postimplementation. Nurses, dermatologists, and managers were interviewed (IMV) to explore factors that influenced implementation. Open-ended questions that were asked during these interviews were as follows: Can you tell me about your experiences with the program? What do you think of the program? What has driven/facilitated the implementation of Lively Legs? What were/are barriers? Why is that? Are those barriers resolved, and how? What was/is the most important barrier? What is needed to resolve this? What would you do differently next time? What would you advise to others that would want to implement this program? Besides the interviews, one researcher (IMV) observed counseling sessions to get an impression of how the program was delivered.Next to this, the evaluation makes use of more structured and quantitative methods. Following the model of Hasson (2010), the following categories of potential influencing factors were assessed:– Quality of the delivery of implementation strategies (implementation fidelity): Strategy delivery was assessed through registration by the researchers and checking to what extent strategies were carried out as planned in interviews with nurses and managers.– Participant responsiveness: Responsiveness is the extent to which the Lively Legs program was appealing to patients and nurses. To measure this, patients were given a self-developed evaluation questionnaire with a return envelope at the end of the last counseling session. In the questionnaire, patients had to score how they valued the program on a scale from 1 to 10. Nurses also filled in an evaluation form to assess their satisfaction with the results achieved per patient on a scale from 1 to 5. Furthermore, the facilitators and barriers questionnaire also contains one question about how nurses valued the program in general on a scale from 1 to 5.– Organizational preconditions: In interviews with nurses and managers, it was asked if there was sufficient nursing time to deliver the program and if a consulting room was available. In the facilitators and barriers questionnaire, there was one question on how nurses valued the extent to which the organization met the preconditions for delivering the program .– Knowledge and support of colleagues: All participating nurses were asked to hand out the facilitators and barriers questionnaire with a return envelope to three colleagues at the end of the project. This questionnaire also contained one question about how well they knew what the program Lively Legs was about (colleagues) and one question about how supportive colleagues were in program delivery (nurses).In addition to our main focus on identifying influencing factors, we examined whether the implementation was perceived as successful by nurses and how many patients were enrolled in the program.– Perceived implementation success: Nurses were asked to give a score on a scale from 1 to 10 for how well the program was currently implemented. In addition, they were asked to give a score on a scale from 1 to 10 for how well the program will be implemented in a half a year’s time, reflecting their expectation of the embedding of the program.– Patient recruitment: Patient recruitment was monitored weekly by nurses who filled in the number of eligible patients and the number of recruited and participating patients using an online tool. Because on average one new patient per month participated in the previous effectiveness trial , this waData collection and measures on program adherence are described in more detail in Additional file With respect to identifying influencing factors from qualitative data, the interviews, focus groups, and field notes were transcribed and analyzed just after they were carried out (IMV). New topics and preliminary findings were incorporated and checked in subsequent interviews. Consequently, some participants were requested to answer additional questions or provide extra information. Analyses were performed by coding transcripts and field notes and categorizing codes on the same topics in matrices. The transcripts and the matrices were reviewed by a second researcher (MMH), and codes and concepts identified from the qualitative data were agreed upon.Structured data were organized in a case study database in Microsoft Excel  . Data we– Influencing factors should be apparent in at least three of the five cases (and were regarded as even stronger if more than one respondent per case identified the same factor).– This also meant that a factor was counted when it was a barrier in one case and a facilitator in the other.– Influencing factors were divided by type of setting (homecare or outpatient clinic) and by phase (pre- or postimplementation) to be able to indicate patterns.– Finally, we listed explanations with respect to influencing factors; for example, we looked at all possible reasons why patient recruitment was problematic in some cases and not in others.Method triangulation was used to enhance the credibility of the conclusions drawn from the data and was applied as follows:– The results of the monitor instrument (how many patients enrolled in the program) and the evaluation forms were discussed with respondents in qualitative interviews.– Observations and field notes were checked in qualitative interviews.– We compared the results from the barriers and facilitator questionnaire with the findings from interviews afterwards, as respondents filled in the questionnaire after the qualitative interview.Table Within the five cases, nine different healthcare organizations participated Table . In two Twelve nurses  were responsible for delivering the program. Characteristics are presented in Table The way leg ulcer care was organized before implementation of the program varied. In some cases, a regional treatment protocol resulted in agreements about the care trajectory between the general practitioner (GP), homecare, and outpatient clinic (cases 3 and 5). In other cases, this was not organized, and every subsequent step in the care process was determined depending on the situation . In addition to this, the role of the nurse varied between cases. Wound care at the outpatient clinic was nurse-led in cases 3, 4, and 5, meaning that a dermatology nurse had her own clinic hours for wound care and compression therapy. A dermatologist supervised the care and usually entered the consulting room during a consult. In other outpatient clinics (case 2), patients were seen by dermatologists, and nurses entered the consulting room during a consult to execute the treatment that the dermatologist advised. Most homecare nurses were responsible for a particular district , except for the nurse practitioner in homecare who could be consulted by all districts with respect to wound care (case 1).The number of participating patients varied between cases Table . A totalfacilitate implementation were mainly related to the program itself and to the opportunities that the program may bring about. Eight expected hindering factors were identified in the pre-implementation phase.In Table e.g., in homecare, venous leg ulcer patients are coded as either “wound care” or “compression therapy”). Furthermore, competition between healthcare organizations was identified as a potential barrier. Nurses and dermatologists stated that due to different financial incentives, organizations might have reservations about referring patients to the lifestyle program. Connected to this, reimbursement was mentioned as a barrier mainly by managers and dermatologists. Subsequently, some practical barriers were identified, for instance, how many nurses should be trained to cover rural areas, whether nurses have sufficient time to deliver the program, and how to deal with changing clinic managers during the implementation period at outpatient clinics.In almost all cases, participants stated that it is not clear what would be the best place to recruit as many patients for the program as possible. Moreover, they said that patients could not be easily grouped and identified from patient information systems due to different diagnostic and procedure codes , a text to be used to inform patients about the lifestyle program, and educational materials to inform colleagues and referrers about Lively Legs.The Lively Legs program was carried out with a moderate to good adherence to protocol (65%–90%). Detailed results on program adherence are described in Additional file Various factors influenced the implementation of the Lively Legs program. These—and their illustrations—can be found in the postimplementation column in Table Participants who reported that patient recruitment was problematic felt they were dependent on GPs or dermatologists for referrals: “Maybe if you put in more time…a lot more time. Maybe, you should just keep telephoning those GPs. (…) I think, if you can just lean on the support of GPs… Well I think that’s the biggest issue” . In addition to this, nurses in homecare and those working at an outpatient clinic without a standardized care process explained that they had no overview of eligible patients themselves. Apart from that, homecare nurses reported a low concentration of venous leg ulcer patients in their particular district, indicating that there were not enough eligible patients: “Implementation is doable in theory, but I wonder if it’s sensible, at least in homecare. If I see the limited numbers of patients we are able to recruit in homecare, I wonder: wouldn’t it be better to do this elsewhere?” . Connected to this, some participants stated that the program should rather or should also be focused on patients with venous insufficiency who do not (yet) have a wound. A dermatology nurse said, “I think from the viewpoint of prevention… the program should also be offered in homecare, because the chronic venous insufficiency problems may linger in homecare. At the outpatient clinic, we don’t see these patients that often. We see them when it’s too late” .With respect to patient recruitment, patients were specifically asked if they wanted to participate in the Lively Legs program. Nurses said that intrinsically motivated patients participated in the program and that they sometimes had difficulties in motivating other patients. “Most of them are older than 70 and sometimes even older than 80. They are hesitant to join yet another thing. Or they are afraid it will cost them money. Sometimes they only agree to join if it can be combined with their other appointments at the clinic” .From interviews it appeared that working hours of participating nurses was a factor of interest. For example, in case 2, two nurses at the outpatient clinic were working for less than 12 hours per week. Managers and nurses explained in interviews that this resulted in difficulties in coordinating the program and planning counseling sessions. Apart from time, nurses’ motivation was also considered as an influencing factor. Some of them stated that, as a nurse, it was important to be dedicated to giving lifestyle counseling. In cases 2 and 4, nurses had not specifically chosen to deliver the program. In these settings, patient recruitment rates were lower, as well as nurses’ satisfaction with the program.Setting seemed an influencing factor with respect to the content of the counseling. This was identified in interviews and in field notes of observed counseling sessions. Patients at the outpatient clinic received practical tips of a different kind than in homecare, for instance, on how to take off compression stockings in an easy way or what kind of cream to use for skin care. Also, referrals to bandage therapists were made more easily at the outpatient clinic than in homecare. In one case, a homecare nurse discovered that one of the patients did not have compression therapy or stockings: “I don’t dare to say that she should have had them. Because, well … I don’t even know if she’s had the right diagnostics. I don’t think so” .From structured data, several factors were checked to see if and how they were of importance to the implementation of Lively Legs. First, the extent to which strategies were carried out as planned varied . Training and agreements on nursing time were regarded as the most facilitating strategies. According to nurses, monitoring and feedback on patient recruitment was not a helpful strategy to implementing the program because the feedback information was of no extra value to them.Patients valued the program with a mean score of 8.4 Table . Nurses With respect to perceived implementation success, nurses scored this ranging from 4.9 to 7.0 on a scale from 1 to 10 Table . HoweverFrom the hindering factors, four were referred to before and after implementation. These were problems related to patient recruitment, competition between healthcare organizations, insufficient nursing time, and lack of support of management. The factors related to patient recruitment were mentioned most and as most hindering for program implementation. Conversely, the most facilitating factors for implementation were also connected to patient recruitment. These were the existence of a standardized care process and nurses’ own practice hours and possibilities to overview patient recruitment.The aim of this study was to identify which factors hindered and facilitated the implementation of Lively Legs in outpatient clinics for dermatology and in homecare. Factors were mainly found at the organizational level. The most difficult part of the program to implement was the organization of reaching and recruiting patients. In this study, we found that if a nurse coordinated a standardized care process with a clear treatment protocol to tie Lively Legs into, this resulted in better patient recruitment and better implementation. Other factors that facilitated implementation were sufficient nursing time and motivated nurses to deliver the program.Implementing the Lively Legs program at the outpatient clinic for dermatology had a number of advantages compared to implementation in homecare organizations. It took less time to coordinate recruitment, possibly due to a higher concentration of patients at outpatient clinics and less dependency on others for referrals. Also, barriers related to market competition were avoided. Furthermore, the outpatient clinic showed salience in giving practical advice and tips to patients. Implementation in homecare is only possible when ulcer care is well organized and when sufficient nurses are trained to counsel patients. The need for a clear treatment protocol and collaboration between outpatient clinic, homecare, and GPs emerged to be important in every case in this study.The fact that patient recruitment was difficult in some cases and that there was a prominent need for a clear treatment protocol to ensure the continuity of care in every case in this study revealed more general issues with respect to leg ulcer care. Leg ulcer care was fragmented in some cases. At those settings, it was difficult to organize patient recruitment and thus implement the Lively Legs program. Moreover, this fragmented care delivery revealed that quality improvement is needed, in particular with respect to the handover of care between GP, outpatient clinic, and homecare.A case study design was used to investigate the implementation within its real-life context and to explore influencing factors before, during, and after implementation. It appeared that participants were able to identify relevant influencing factors beforehand and that these were mostly confirmed during and after implementation. However, reimbursement was regarded as an influencing factor beforehand but was not mentioned afterwards, indicating that this factor seemed only of influence in the decision-making process. Furthermore, the context and setting purposefully varied between cases, and this appeared to be crucial to understanding the influencing factors for implementation, in particular, the influence of the organization of leg ulcer care; the extent to which collaboration between homecare, outpatient clinic, and GP was established; and the extent to which the nurse coordinated the care process. By closely looking at what happened, it was made apparent that a clear treatment protocol and continuity of care facilitates the implementation of Lively Legs. Conversely, a lack of well-organized care does not only hinder implementation of the lifestyle program but also raises questions about the quality of care for this patient group.et al. (2005) have pointed out that program reach and lack of referrals from physicians is often problematic with respect to implementing lifestyle interventions [Although similar initiatives to promote better adherence to leg ulcer treatment were reported in other countries, no implementation studies have been performed yet. Nevertheless, several factors that influenced the implementation of Lively Legs have also been reported in some studies in other patient groups. For example, Lorig ventions . Direct ventions .et al. (2009) and Griffin et al. (2009), who reported that motivational interviewing was the most challenging aspect of their protocol to implement and that continued training was needed [Program adherence was moderate to good, implying that the program can be adequately carried out in daily practice. Variation between cases was found with respect to goal setting, assessing patients’ motivation and self-efficacy, and exploring patients’ barriers and facilitators for behavior change. This indicates that motivational interviewing skills need continuing attention, for example, through repetitive training or coaching on the job. This finding is in line with Whittemore s needed ,45.The strength of using a case study design lies in the opportunity to study the implementation process in real-life settings and collect multiple types of data, enabling development of an in-depth picture . By seleHowever, it may be that some nurses in this study had insufficient insight in events or influencing factors at the management level or regional level to provide valid answers. But, the variation in answers and gained insight in implementation difficulties do not point in the direction of possible unreliable answers. Furthermore, due to low patient recruitment, some nurses may not have been able to master their skills in program delivery. Program adherence could have been affected by this. Another critical reflection concerns the fact that evaluation took place after seven months in cases 1, 2, and 3 and after four months in cases 4 and 5. Due to time constraints, it was not possible to keep both evaluation periods the same. There might be a possibility that other results would have been obtained after a longer period before evaluation, but our results from all cases point in the same direction, indicating that the findings on our main question are trustworthy. Furthermore, we would like to comment that in this study, the selected implementation strategies were mostly voluntary and on the micro-level. In hindsight, it might have been necessary to first make structural changes to the care process before implementing the program or to include structural changes as one of the implementation strategies. On the other hand, these structural changes would not have been feasible within the time and scope of this research project.In this study, we used the framework of Hasson (2010) to guide us in collecting and organizing the data. For this purpose this sufficed very well. On the one hand, the framework gives structure; on the other hand, it is still generic enough to explore how factors exactly influenced the implementation of this explicit lifestyle program. At the same time, we acknowledge that other frameworks such as these are available and could have been equally suitable.With respect to leg ulcer treatment, further research is needed on effects of process redesign to improve the continuity and quality of care in such a way that prevention is also included. Next to this, expanding lifestyle counseling to patients with chronic venous insufficiency, as an early stage of leg ulceration, should also be investigated.In summary, we conclude that the main influencing factors for implementing Lively Legs are at the organizational level. The organization of patient recruitment was most difficult. When lifestyle counseling is integrated in regular care as part of a standardized treatment protocol, it helps to guarantee that all patients can at least take part in the first step of the program. Other factors that facilitated the implementation were selecting dedicated nurses for delivering the program and facilitating them with sufficient nursing time and tools to coordinate the care process. We found that nurses were able to deliver the program adequately, in such a way that patients as well as nurses were very satisfied.This study suggested that the Lively Legs program can best be implemented at the outpatient clinic for dermatology. The specific influencing factors may be relevant more widely in programs for implementing lifestyle interventions in different healthcare settings. The value of using a case study design is that an in-depth picture is obtained of how and why implementation is possible in different settings. Furthermore, it also shed light on a more general issue, that is, that leg ulcer care was often fragmented, indicating that quality improvement is needed in particular with respect to treatment protocols and the handover of care between GP, outpatient clinic, and homecare.MW is Editor in Chief of Implementation Science. All decisions on this paper were made by another editor.IMV, MMH, and TV contributed to study design. AWE and MW provided advice on study design. IMV and MMH performed the data collection and analysis. IMV was responsible for manuscript preparation. MMH, AWE, MW, and TV provided critical comments on the manuscript. All authors read and approved the final manuscript.Program adherence Lively Legs.Click here for file


Aims. To evaluate the effect of a physical activity (PA) program on markers of endothelial dysfunction and oxidative stress in adolescents with (MetS). Methods. We carried out a cohort study of 38 adolescents with and without MetS . All participants completed a 3-month PA program. All variables of the MetS as well as markers of endothelial dysfunction and oxidative stress tests were evaluated. Results. Females with and without MetS showed significant differences for almost all components of the MetS, whereas males were significantly different in half of the components. After the PA program, components of the MetS were not different from baseline values except for HDL-C levels. Some baseline endothelial dysfunction markers were significantly different among adolescents with and without MetS; however, after the PA program, most of these markers significantly improved in subjects with and without MetS. Conclusion. PA improves the markers of endothelial dysfunction in adolescents with MetS although other changes in the components of the MetS were not observed. Perhaps the benefits of PA on all components of MetS would appear after a PA program with a longer duration.The metabolic syndrome (MetS) is a precursor of diabetes. Physical activity (PA) improves endothelial dysfunction and may benefit patients with MetS.  Metabolic syndrome (MetS) is a cluster of several atherothrombotic risk factors and is considered to be a precursor of atherothrombotic diseases and diabetes mellitus . SeveralThe prevalence of MetS in adolescents according to the Adult Treatment Panel III (ATP-III) definition varies significantly. For example, the prevalence of MetS is higher in Mexico than in the US  , 5. HoweChildren older than 10 years with overweight or obesity have a high risk for the development of dyslipidemia, arterial hypertension, abnormalities in carbohydrate metabolism, and prothrombotic and proinflammatory states . One necSeveral studies have fully demonstrated the beneficial effects of physical activity (PA) in different groups in healthy controls and in patients with several pathological states –18, inclDue to the very well-known beneficial effects of PA in adult patients with MetS, the aim of this study was to evaluate the effects of a PA program on metabolic parameters and markers of endothelial cell dysfunction and oxidative stress in adolescents with MetS.A cohort study including 1200 healthy adolescents between 14 and 16 years of age was carried out between February 2011 and May 2011. A search for MetS was performed. Finally, 575 adolescents accepted to participate  . AccordiFor this study we included 38 age- and gender-matched adolescents : 19 with MetS and 19 without MetS. Exclusion criteria included smoking and presence of infectious disease at the time of evaluation or during the 15 days prior to enrollment or taking anti-inflammatory or antihistamine drugs during the same time period. Alcohol intake during the study was highly discouraged. Pregnancy was always considered as an exclusion criterion. Both groups participated in a 3-month PA program. The program was designed to exercise 45 min/day for 5 days/week with an intermediate intensity  as monitored with a heart rate monitor. This program was designed and supervised by an international certificated instructor. Importantly, although some dietary advice was offered, no specific changes in dietary habits or meal characteristics were indicated to the study participants.Before and after the PA program, fasting blood samples (10 mL) were obtained using a vacuum system. Blood was drawn in vacuum tubes containing either sodium citrate 3.8% , heparin, EDTA, and in another tube without anticoagulant. All tubes were centrifuged for 10 min at 500 g in order to obtain platelet-poor plasma and serum. Triglycerides (TGs), high-density lipoprotein cholesterol (HDL-C), and fasting glucose measurements were performed on the same day of the collection using an enzymatic assay . We measured soluble vascular cell adhesion molecule (VCAM-1) and P-selectin . Also, plasma von Willebrand factor (vWF) was evaluated using a commercially available kit . hsCRP was assayed using a commercially available kit  in an automated equipment system . Several fibrinolytic variables were evaluated, namely, plasminogen activator inhibitor (PAI-1) , tissue plasminogen activator (tPA) , factor VIII (FVIII) , and fibrinogen . Hemoglobin was evaluated in the blood collected in EDTA tubes using an AcT Diff Hematology Analyzer . Heparinized blood was used for assays evaluating oxidative stress. Lipoperoxidation, enzymatic activity of superoxide dismutase, catalase activity, and glutathione peroxidase activity were evaluated according to previously described and validated methods –25.t-test. We then described the mean and standard deviation of the same variables in males and females after application of the PA program. Before and after results were compared using a paired t-test, significant differences were considered when P < 0.05. All statistical analyses were performed using a Statistical Package for the Social Sciences (SPSS) software .We described baseline value and values after the PA program (mean and standard deviation) of the components of the MetS as well as markers of inflammation, EC dysfunction, and oxidative stress. Differences of these variables between cases and controls were established using Student's Before entering the study, all participants and their parents were informed about the characteristics and objectives of the investigation. Signed informed consent was obtained from the parents and from each adolescent. The study was conducted according to all the principles of the Declaration of Helsinki. The protocol was approved by the ethics committee of our research center.As previously mentioned, 19 adolescents  with MetS were included and compared with a paired control group without MetS. Baseline general characteristics of the adolescents in both groups are shown in  MetS is caused by a combination of genetic factors, poor dietary habits, and sedentary lifestyle. It is a major risk factor for atherothrombotic diseases . AccordiUpon appropriate stimulation with inflammatory mediators the endothelium may become dysfunctional. Endothelial dysfunction is defined as impairment in any of the normal functions of the ECs leading to vasoconstriction, thrombosis, upregulation of CAMs, increased cytokine and chemokine secretion, and leukocyte adherence, among others. EC dysfunction is regarded as a precursor for atherogenesis, and it is associated with a high risk of atherothrombotic events , 28. TheECs have other functions in addition to the regulation of vascular tone. Today it is possible to measure plasma or serum constituents as alternative markers of endothelial function. For example, CAMs are expressed on the surface of ECs in response to endothelial dysfunction, and their circulating levels are considered as surrogate markers of dysfunction, or damage . ElevateWe attempted to determine the pattern of markers of EC dysfunction before and after a PA program in adolescents with and without MetS. We measured several markers of endothelial dysfunction which are widely used and accepted in the literature , 34–36. Our most important findings appeared after the PA program. We found significant changes in HDL-C levels in all groups; however, no changes were found for TG as previously shown . An explThe effects observed after the PA program are not that surprising. Indeed, many atherothrombotic risk factors associated with MetS can be reduced or eliminated through weight control, diet, and, most importantly, regular exercise . PA induIn this study we found a beneficial effect of PA on markers of EC dysfunction, namely, CAMs and blood coagulation markers; however, we could not demonstrate a positive consequence for other variables such as hsCRP and oxidative stress tests. We can hypothesize that this lack of effect may be due to a PA program of insufficient length to induce the desired changes. Perhaps each marker of EC dysfunction requires a specific time in order to be reversed with PA, and such timing possibly was not fully achieved in our study for all variables analyzed.Most studies on EC dysfunction have been performed in adults with specific pathological states and only a few have addressed children and adolescents . In adolWe have shown that a PA program effectively reduces the levels of several markers of EC dysfunction in adolescents with MetS, although these beneficial changes are not associated with the expected changes in the clinical components of the MetS. Perhaps these benefits on EC dysfunction markers may be increased or extended either to inflammation markers, to oxidative stress tests, or to the clinical components of MetS with a PA program beyond 3 months.


Ventilation of ARDS patients focuses on delivering adequate oxygenation and allowing elimination of CO2 while protecting the lung. The objectives were to compare Intellivent-ASV with conventional ventilation on safety and efficacy, and to compare the number of manual adjustments between the two ventilatory modalities.Intellivent-ASV has been developed to provide fully closed loop mechanical ventilation using a ventilation controller keeping EtCOA randomized, controlled study including all consecutive patients receiving mechanical ventilation for at least 48 hours. Patients were randomly ventilated either with Intellivent-ASV or conventional ventilation, with a S1 . Parameters were adjusted by the clinician in charge of the patient. Ventilatory and oxygenation parameters were recorded cycle by cycle during 48 hours and blood gases were performed every 6 hours.2/FiO2 at inclusion 136 (107 to 154). Eleven were ventilated in the conventional group and 13 in the Intellivent-ASV group. The study was stopped for one patient from the Intellivent-ASV group because of a pneumothorax not caused by ventilation. The delivered Vt was slightly higher during Intellivent-ASV (7.9 (7.5 to 8.5) vs. 7.2 (6.8 to 7.8) ml/kg, P = 0.013). The time spent by the various parameters in the suboptimal zone (safety) is the same for the two ventilation modes. The time spent in the optimal zone (efficacy) is the same for the two ventilation modes, expect for SpO2 (78% (75 to 89) with Intellivent-ASV vs. 44% (41 to 58), P = 0.001). Intellivent-ASV required less manual adjustments during 48 hours (2 (0 to 7) vs. 17 (10 to 27), P <0.001). The total number of adjustments during 48 hours, including automatic regulation, was higher in the Intellivent-ASV group (379 (274 to 493) vs. 17 (10 to 27), P <0.001).Twenty-four patients with ARDS were included, 10 female, 14 male, median age 58 (46 to 63) years, APACHE II score 22 (17 to 29), PaOOver 48 hours, Intellivent-ASV allowed safe ventilation for ARDS patients, with better oxygenation and an identical ventilation efficacy when compared with conventional ventilation, with less manual adjustments.


Pierolapithecus catalaunicus  exhibits a relatively wide and shallow thorax with moderate hand length and phalangeal curvature, dorsally-oriented metacarpophalangeal joints, and loss of ulnocarpal articulation. This evidence reveals enhanced orthograde postures without modern ape-like below-branch suspensory adaptations. Therefore, it has been proposed that natural selection enhanced vertical climbing (and not suspension per se) in Pierolapithecus catalaunicus. Although limb long bones are not available for this species, its patella (IPS 21350.37) can potentially provide insights into its knee function and thus on the complexity of its total morphological pattern. Here we provide a detailed description and morphometric analyses of IPS 21350.37, which are based on four external dimensions intended to capture the overall patellar shape. Our results reveal that the patella of Pierolapithecus is similar to that of extant great apes: proximodistally short, mediolaterally broad and anteroposteriorly thin. Previous biomechanical studies of the anthropoid knee based on the same measurements proposed that the modern great ape patella reflects a mobile knee joint while the long, narrow and thick patella of platyrrhine and especially cercopithecoid monkeys would increase the quadriceps moment arm in knee extension during walking, galloping, climbing and leaping. The patella of Pierolapithecus differs not only from that of monkeys and hylobatids, but also from that of basal hominoids , which display slightly thinner patellae than extant great apes (the previously-inferred plesiomorphic hominoid condition). If patellar shape in Pierolapithecus is related to modern great ape-like knee function, our results suggest that increased knee mobility might have originally evolved in relation to enhanced climbing capabilities in great apes .The mosaic nature of the Miocene ape postcranium hinders the reconstruction of the positional behavior and locomotion of these taxa based on isolated elements only. The fossil great ape  Pierolapithecus catalaunicus. With an estimated age of 11.9 Ma  Pierolapithecus is the oldest undisputed extinct member of the great-ape-and-human clade—i.e., the Hominidae The partial hominoid skeleton IPS 21350 from the locality of Barranc de Can Vila 1 ProconsulPierolapithecus—as in other Miocene apes—relied significantly on above-branch palmigrady with a thumb-assisted grasping during arboreal locomotion —might have been the primary target of natural selection in Pierolapithecus, since it is the only other common behavior to the hominoid crown group Pierolapithecus are not preserved (other than shaft fragments), the morphology of its preserved patella (IPS 21350.37) can potentially provide hints of its knee function, as previous studies have shown for other Miocene taxa Pierolapithecus (IPS 21350) as well as an exhaustive morphometric analysis with selected extant anthropoids and available fossil hominoids. Therefore, the aim of this study is to shed light on the patellar morphology and inferred knee function of Pierolapithecus catalaunicus.IPS 21350 comprises more than 80 bones or bone fragments, including the splanchnocranium, key regions of the wrist and ankle complexes, a clavicle, vertebrae and ribs, as well as fragmentary remains of the pelvis and an almost complete patella Pierolapithecus were compared with the sample of extant anthropoids used by Ward et al. (their Proconsul heseloni) Proconsul nyanzae) Nacholapithecus kerioi, referred to Kenyapithecus in Oreopithecus bambolii, measured by S.A. from a cast: PD  = 22.2 mm, PDAS  = 19.9 mm, AP  = 8.9 mm, ML  = 23.0 mm); and KNM-MB 24738 (Equatorius africanus) The studied specimen IPS 21350.37) is housed at the Institut Català de Paleontologia Miquel Crusafont . To compare this specimen with the patellae of other (extant and extinct) anthropoids, four variables were measured following Ward et al. l. their  and 2 [11350.37 iSPSS v 15 and PAST v 2.15.For shape comparisons, linear dimension were divided by overall patellar size, which was approximated by the geometric mean (GM) of the four original lengths. Scaling the patellar linear dimensions by the GM gives individual dimensionless Mosimann shape ratios that are independent of the remaining sample  Patellar mediolateral breadth (ML) has been found to scale with body mass (BM) in non-human hominoids The left patella IPS 21350.37  is well P. heseloni and Nacholapithecus, which are similar to hylobatids and monkeys (platyrrhines and cercopithecoids), the rest of Miocene apes, including Pierolapithecus, have patellae of intermediate size between the monkey-hylobatid group (except Papio) and African ape-human group, overlapping with the ranges of orangutans and baboons.The range of variation of the patellar size (GM), as well as the Mosimann shape variables for the different extant genera and fossil individuals, are depicted in Symphalangus is exceptionally large, its lower non-interquartile range overlapping only with the upper range of cercopithecoid monkeys, but not with that of Hylobates. As for extant great apes, only the uppermost range of Pan overlaps with that of monkeys and Hylobates. Pierolapithecus (similarly as Oreopithecus) falls within the interquartil range of all great apes and humans, while the rest of Miocene apes exhibit slightly proximodistally longer patellae, falling in an intermediate position between monkeys-Hylobates and great apes.Monkeys and hylobatids exhibit proximodistally longer patellae than extant great apes and humans . That ofCebus show proximodistally shorter articular surfaces than Pan, Pongo, hylobatids and Ateles. Gorillas display a wide range, overlapping with the interquartile ranges of the remaining great apes and all monkeys. Hylobates shows the highest values of PDAS, closely followed by Ateles. Pierolapithecus overlaps with humans, gorillas and monkeys . Oreopithecus shows one of the lowest values for this ratio, conversely to the rest of Miocene apes, whose ratios overlap with those of Hylobates, Ateles and the uppermost part of the interquartile range of great apes and Symphalangus.For PDAS, differences between genera are less clear . AlthougPierolapithecus. Oreopithecus, Nacholapithecus and Proconsul show slightly thinner patellae than great apes, overlapping with the lowest range of Pan. Equatorius is more similar to humans and cercopithecoids, although it also falls in the range of gorillas. Finally, cercopithecoids display the narrowest patellae (P. heseloni (KPS PT 4) and P. nyanzae the lowest values of ML among fossils. Equatorius, Oreopithecus, the other individual of P. heseloni (KPS PT 3) and Nacholapithecus show intermediate values for fossils, being Pierolapithecus the specimen with the broadest mediolateral length of the patella.In contrast, marked differences are observed concerning anteroposterior thickness . Hylobatpatellae , followeRegression results are given in Symphalangus exhibits the extreme condition for hominoids, being statistically different from the remaining taxa except for Hylobates . Modern humans display differences with apes and cercopithecoids (p<0.05), but not with platyrrhines  among extant and fossil taxa is explained by the two first between-group principal components bgPCs; ; Table 2Proconsul, Nacholapithecus and Oreopithecus) fall close in the bgPC1-bgPC2 morphospace, highly overlapping with Symphalangus and great apes  for bgPC1. These fossil apes occupy a central position along bgPC2, overlapping with extant apes and monkeys. BAC 122 (Oreopithecus) shows the lowest values among the above-mentioned Miocene apes, and KNM-RU 17382 (P. nyanzae) the highest. Overall, the patella of these Miocene apes is relatively thin anteroposteriorly and wide mediolaterally, in the uppermost range or just above the extant great ape range (bgPC1), and in the upper range of great apes for bgPC2 (by discounting one Pan outlier), but fully within the monkey range for the latter axis. Two fossil ape patellae depart from the others: KNM-MB 24738 (Equatorius) and IPS 21350.37 (Pierolapithecus). They show both lower bgPC1  and bgPC2 values than the remaining Miocene apes. When both bgPC axes are inspected together, to the exception of KPS PT4 , the other Proconsul, Nacholapithecus and Oreopithecus specimens fall in a unique region of the morphospace. Equatorius shows its closest affinities with modern humans, and Pierolapithecus overlaps with Pongo and Gorilla.Most fossil apes  have been previously noted on the basis of the external dimensions used herein : monkeys exhibit proximodistally taller, anteroposteriorly thicker and mediolaterally narrower patellae than great apes  and 5 [1Our results agree with a previous study Symphalangus the proximodistally highest patella) than in great apes , with no habitual and stereotyped flexion-extension of the knee joint. This positional hypothesis is compatible with the orthograde body plan inferred for Pierolapithecus on the basis of its thorax morphology Pierolapithecus, based on hand morphology  Pierolapithecus probably has no modern analog, due to its tailless condition and powerful-grasping (thumb-assisted) capabilities inferred for this and other Miocene apes Pierolapithecus also exhibits a pelvic morphology similar to Proconsul, but with a slightly more marked lateral flaring of the ilia Proconsul) a mosaic postcranial morphology, combining in the case of Pierolapithecus an orthograde body plan with above-branch palmigrady, great ape-like knee function and hip joint with increased ape-like mobility , the patellae of Pierolapithecus and Oreopithecus—the only widely accepted orthograde taxa among the analyzed fossil apes In evolutionary terms, our results suggest that cercopithecoids might display, concerning the anteroposterior dimension, the most derived patella among anthropoids  and 5. Hn humans , 3 and 5Pierolapithecus suggest that its great ape-like patellar morphology might be simply attributable to the higher range of knee motion required by orthograde vertical climbing, which would have been probably most similar to that performed by extant great apes  PierolapithecusHispanopithecus/Rudapithecus (see discussion in Hispanopithecus (if ever found) would probably resemble those of modern great apes, like in Pierolapithecus and Oreopithecus.Given that the evolution of the locomotor apparatus in apes during the Miocene apparently proceeded in a mosaic fashion e.g., , and thePierolapithecus catalaunicus reveal its closest shape affinities with those of extant great apes. The great ape-like—i.e., proximodistally short, anteroposteriorly thin and mediolaterally broad—patellar morphology displayed by Pierolapithecus is functionally related  to a wider range of movements at the knee joint Proconsul or Nacholapithecus) differ by being anteroposteriorly more compressed than that of Pierolapithecus (a morphology that has been inferred to be the plesiomorphic condition for hominoids). In turn, hylobatids display a patellar morphology intermediate between those of monkeys and great apes (an even autapomorphic to some degree). We hypothesize that, taking into account the lack of specific suspensory adaptations in the orthograde Pierolapithecus , its great ape-like patellar morphology probably evolved originally in conjunction with orthograde behaviors (other than suspension) requiring increased versatile movements of the knee joint, such as specialized vertical climbing.Morphometric analyses of the patella of Table S1Significance for the ANOVA and MANOVA multiple post hoc comparisons (Bonferroni method) for scores of bgPC 1 and 2 according to extant primates genera.(PDF)Click here for additional data file.


We present a computer program developed for estimating penetrance rates in autosomal dominant diseases by means of family kinship and phenotype information contained within the pedigrees. The program also determines the exact 95% credibility interval for the penetrance estimate. Both executable  and web versions  of the software are available. The web version enables further calculations, such as heterozygosity probabilities and assessment of offspring risks for all individuals in the pedigrees. Both programs can be accessed and down-loaded freely at the home-page address http://www.ib.usp.br/~otto/software.htm. Accurate penetrance estimates are important for determining genetic disease recurrence risks in families where incompletely penetrant Mendelian disorders are segregating or for establishing genetic map locations by linkage analysis. While crude penetrance estimates can be rapidly derived by dividing the number of observed individuals expressing a disease phenotype by a rough estimate of the probable number of carriers in a given pedigree exhibiting autosomal dominant inheritance, deriving exact maximum likelihood estimates of carrier status at an individual level is time consuming and tedious. In this paper, we describe the structure and use of a computer program designed to be user friendly and assist genetic counselors and gene mappers to make accurate penetrance estimates in all sizes and complexities of autosomal dominant pedigrees, including those containing consanguineous loops and twin pairs.A, a), with allele A dominant in relation to allele a. In the case of human autosomal dominant diseases, the homozygous condition AA is generally either unknown or very rare , so that in pedigrees with cases of autosomal dominant disease the affected individuals are almost invariably Aa heterozygotes, while normal individuals are either aa homozygotes or non-penetrant Aa heterozygotes. In this sense, the penetrance rate K is assumed to be the probability of an Aa heterozygote being affected: K = Prob(affected|Aa).The program deals with the situation of a single monogenic locus  program, developed using Active Server Pages (ASP), through the languages VBScript and Jscript. Both penetrance programs are the intellectual property of the authors, and as such, any use of or reference to the materials included in them, must contain an explicit reference to their origin. Feedback from users is welcome and will be used to improve the program and to correct unforeseen flaws.Both programs are described in figures obtained directly in real time from screen images generated by the programs themselves.First we describe PenCalc for Windows. To illustrate the operation of the program, we will use as example the hypothetical pedigree shown in File, Data Input and Help appear in the main menu of the program's opening page. The File menu accesses options for exiting the program, for saving or printing the text file generated by the program to show the likelihood function, the estimate of K  and its exact 95% credibility interval. The Data Input menu program allows the writing of a data file through the File submenu option Create  or gives information on the use of the user's Manual in pdf format.Options n Create , accessiThe input of consanguineous structures is far more complicated; we shall use the example shown in We next describe the program PenCalcWeb. Initially, the program shows graphic interfaces for entering data, similar to those for PenCalc for Windows. Because of the flexibility of the programming language, PenCalcWeb is far more intuitive to use than PenCalc for Windows. Only the final screen with results is shown in