Many persons infected with Hepatitis B virus (HBV) and Hepatitis C virus (HCV) in the past are now lost to follow-up. The aim of the Northern Holland Hepatitis Retrieval Project (NHHRP) is to retrieve and re-evaluate persons previously diagnosed with HBV or HCV and bring them back into care. Chronic HBV infection was defined as two positive Hepatitis B surface antigen (HBsAg) tests within 6 months and chronic HCV infection with 2 positive HCV RNA tests by polymerase chain reaction (PCR).Data files of the local public health services and microbiology laboratory were explored to identify all registered HBV and HCV cases in the Alkmaar region, the Netherlands, for the past 15 years. Identified cases were compared with patients currently known in our hospital. Patients without follow-up in primary or hospital care were approached via their primary health care physician and invited for evaluation at our hospitalIn total, 552 cases of HBV were identified. 356 (64.5%) had no follow-up. Only 113/356 (31.7%) were eligible for retrieval and 44.2% were evaluated in our hospital resulting in a change of management in 22/50 (44%) of patients. Four hundred ninety nine cases of HCV were identified, 150/499 (30.1%) were lost to follow-up. Only 20/150 (13.3%) were eligible for retrieval and 4/20 (20%) were evaluated at our clinic. Resulting in a change of management in 3/4 (75%).Only a limited part of HBV and HCV persons lost to follow-up is eligible for retrieval, nonetheless re-evaluation of these persons will lead to a change of management in the majority of persons. Infection with hepatitis B virus (HBV) and hepatitis C virus (HCV) is present worldwide and a leading cause of liver-related morbidity and mortality. It is estimated that 257 million persons worldwide are living with chronic HBV infection and 71 million persons with chronic HCV infection [In high-endemic HBV areas, such as sub-Saharan Africa and East-Asia, perinatal transmission is the most common mode of infection resulting in chronic HBV in more than 90% of infected neonates. In low-endemic countries such as Western Europe and North-America the majority of acute HBV infections occur during adolescence or adulthood and predominantly via sexual activity or intravenous drug use. In immunocompetent adults, less than 1% of acute HBV infections will progress to chronic HBV infection . Acute HIn order to prevent long-term complications, it is important to achieve an adequate viral suppression or even elimination. Successful antiviral therapy can delay progression to cirrhosis and hepatocellular carcinoma and improve survival , 5, 6. IThe primary goal of treatment in HCV infection is to cure HCV infection. The endpoint of therapy is a sustained viral response defined as undetectable HCV RNA 12 weeks after treatment completion . Every pThe WHO set the ambitious goal to eliminate HBV and HCV as public health threat by 2030.To accomplish this goal it is important to detect all persons infected with HBV or HCV. Several screening strategies are deployed in high-risk and low-risk population to detect infected persons. However, many persons previously diagnosed with HBV or HCV are lost to follow-up in primary and/or hospital care. This is an important target group. After all, the chance of spontaneous clearance is low and therefore an indication for antiviral treatment or strict follow-up might exist.The goal of the Northern Holland Hepatitis Retrieval Project (NHHRP) is to focus on persons previously diagnosed with chronic HBV or HCV who are now lost to follow-up. We aimed to retrieve these persons and bring them back into care. To the best of our knowledge this is the first structured retrieval project within a geographic region. Furthermore, we aim to create a frame-work for other local or even nationwide retrieval programmes.Data files of the local public health services and the local microbiology laboratory were explored to identify all registered cases of chronic HBV and HCV in our region for the past 15 years. The Alkmaar region in Northern Holland covers about 300.000 inhabitants.A full-time fellow was appointed to retrieve all data and evaluate patients at our clinic from January 2016 to September 2016.Persons with a chronic HBV infection were defined as two positive Hepatitis B surface antigen (HBsAg) tests within 6 months. Chronic HCV infection was defined as two positive anti-HCV tests within 6 months confirmed with a positive HCV RNA test. Identified cases were compared with patients currently under follow-up in hospital or primary care. Persons without follow-up appointment were considered lost to follow up.Due to Dutch privacy regulations, it was not allowed to directly contact identified persons since no active medical treatment agreement existed. Therefore, we informed the registered primary health care physician of identified persons and requested to refer them for evaluation at our clinic. Hence, only persons with updated contact details  were eligible for retrieval. If persons moved to another area and no longer were registered at a primary health care physician in our region it was not possible for us to contact them. We were not allowed to use other databases, for instance municipality databases, to search for updated contact details.Persons with severe comorbidity and an estimated survival of less than 1 year were excluded. If there was no referral within 2 months, we send a reminder to the primary health care physician.Re-evaluation at our hospital included physical examination, blood examination for Alanine-aminotransferase (ALT), complete serology, HBV DNA load and genotype determination, and transient elastography of the liver. A management advice was proposed based on the outcome of evaluation in accordance to the Dutch guidelines on treatment of HBV and HCV infection .The number of persons with chronic HBV or HCV infection lost to follow-upThe number of persons lost to follow-up we were able to retrieve and responded to our invitation for evaluationThe number of persons for whom re-evaluation resulted in a change of managementThe goal of the NHHRP was to retrieve patients lost to follow-up. In order to evaluate the feasibility of such a retrieval project, we aimed to evaluate the following outcomes:Results were evaluated using descriptive statistics.The Northern Holland Hepatitis Retrieval Project was approved by the local ethics committee.Concerning patients with chronic HBV we identified 552 cases and in our region.In total, 356/552 (64.5%) HBV patients had no follow-up in primary or hospital care see Fig. . Only 12In total, 50 of the 113 (44.2%) responded to the invitation and were evaluated at our hospital. Patient characteristics and outcome of evaluation are described in Table All patients were hepatitis B e antigen (HBeAg) negative. Evaluation resulted in a change of management in 22/50 (44%) of patients. An additional indication for HCC-screening was recommended in 14/50 (28%), 5/50 (10%) had an indication for strict follow-up and 3/50 (6%) an indication to start antiviral therapy. The remaining 25/50 (50%) were advised to have a 6-12 monthly check of ALT levels and in 3/50 (6%) patients viral load was undetectable.In total, 499 cases of chronic HCV were identified in our region see Fig. .Fig. 2ReIn 150/499 (30.1%) persons, no follow-up was scheduled in primary or hospital care. Only 24/150 (16%) were eligible for retrieval and after consultation of their primary health care physician, 20/24 (83.3%) were invited for evaluation. Of the 126 persons not eligible for retrieval, in 45/126 (35.7%) their primary health care physician was unknown, 21/126 (16.7%) were asylum seekers with unknown address, 23/126 (18.3%) were imprisoned, 37/126 (29.4%) resided in another region.Patient characteristics and outcome of evaluation are described in Table Only 4/20 (20%) of invited HCV patients responded to the invitation and were evaluated at our clinic and 3/4 (75%) started antiviral therapy.The NHHRP was initiated to retrieve chronic HBV and HCV patients without follow-up in primary or specialist care. We aimed to identify these patients and invite them for re-evaluation at our clinic.The number of patients eligible for retrieval for both HBV and HCV was considerably lower than the total number of patients lost to follow-up. The major reason for this drawback was that updated contact details were lacking. About one-third of those identified to lost to follow-up moved to another region and may have received adequate follow-up and care elsewhere. Due to national privacy regulations we were not allowed to search for updated data outside our own medical records, such as municipal databases. We expect this privacy regulation to be a major limitation in future retrieval projects. However, in Iceland, chronic HCV was defined as a public health threat and therefore a nationwide elimination programme, treatment as prevention for hepatitis C in Iceland (TRAP HEP C), was launched. Unlike our approach, in the context of this TRAP HEP C programme it was allowed to check updated contact details in the municipal database and to contact the patient directly .Among the patients not eligible for retrieval we identified two important groups. The first group consisted of asylum seekers whose tests were performed in asylum seeker centers upon arrival. At the time of our retrieval project current address and legal status were unknown and therefore we were not able to retrieve and re-evaluate this group. Our numbers suggest that this group could be a significant target group for retrieval and with better information and cooperation between asylum seeker centers and hepatitis treatment centers we could offer this group a chance of control or cure of their disease.Second, prisoners tested positive during detention period are lost to follow-up after transferal to other detention centers or release to freedom without organizing follow-up in their place of residence. With close cooperation between prisons and hepatological centers this group is especially suitable for further evaluation and treatment of their chronic HBV or HCV. A large state-wide programme in Australia showed that screening and treatment of prisoners can be very successful if it is done in a structured approach . HoweverOnly 44% of chronic HBV patients eligible for retrieval was referred for re-evaluation. A lack of awareness of both patients and primary health care physician is a possible explanation. Awareness about chronic HBV and HCV can be created through education or media campaign. Increased awareness will contribute to the effect of a retrieval programme and may enhance the willingness of target groups to participate in these programmes.3 in combination with ALT greater than the upper limit of normal and/or at least moderate liver necroinflammation or fibrosis should be treated.The major change of management in chronic HBV patients was strict surveillance of the patient and to a lesser extent indication for antiviral therapy. Evaluation resulted in a major change of management in 44% of the patients. The remaining 50% had an indication for 6-12 monthly check of ALT levels and viral load. Patients were evaluated using the 2012 Dutch guideline on chronic HBV infection as standard. In 2017 the European Association of Studies of the Liver (EASL) released the updated guideline on HBV infection. This guideline sets a stricter cut-off point for indications for treatment with all patients with HBeAg-positive or –negative chronic hepatitis B, a viral load of > 2.0 • 104. These patients now may have an indication for therapy if HBV DNA levels are > 2,0 • 103 according to the updated EASL guideline [Three HBV patients started antiviral therapy based on viral loads in combination with elevated ALT levels. An additional 5 patients had an indication for strict follow-up because of elevated ALT levels or fibrosis stage F2-F3 despite viral load < 2,0 • 10uideline .Concerning HCV patients, the results of retrieval were disappointing. We observed that two-third of patients had treatment or adequate follow-up, but the remaining one-third was hard to retrieve due to above-mentioned reasons. Moreover, people who inject drugs (PWID) are an important target group as well. Because this group often has no permanent address, it is especially hard to reach them. However, treatment of PWID can be successfully in a multidisciplinary setting using strategies such as directly observed therapy and the involvement of nurse-practioners . In closDespite difficulties with reaching above mentioned target groups, the effect of retrieval of chronic HCV patients will be significant. As of October 2015 direct antiviral agents for hepatitis C are approved for reimbursement of every basic health insurance in the Netherlands. Therefore, according to the Dutch guideline on hepatitis C, every patient with HCV infection has an indication for antiviral therapy. We started antiviral therapy in 3 of 4 patients, in one patient we did not start because of limited life expectancy.A considerable larger number of HBV patients was lost to follow-up compared to HCV patients. The population of HBV patients predominantly consisted of migrants whereas the HCV population mainly consisted of PWID. It is possible that migrants have more difficulties with access to health care whilst PWID often have follow-up in addiction care and are easily referred to health care. Furthermore, in the past need for treatment and follow-up for chronic HBV infection was less strict compared to HCV patients and HBV patients therefore easily got lost to follow-up.The most time-consuming element was the complicated construction to contact patients via their primary health care physician. Our region is a low-endemic region for HBV and HCV and most practices only have one or two HBV or HCV patients. Therefore, viral hepatitis is not a priority to most primary health care physicians. If it would be possible to approach the patients directly by using updated contact details, a retrieval project could be much more effective to perform. But we expect that, due to (inter) national privacy regulations, a direct approach will be hard to implement.Our retrieval project is currently expanded to other regions in the Netherlands. The results of these projects will show us if large scale retrieval projects are worth the effort.We conclude that a retrieval project comparing datafiles of the microbiological laboratory and medical records is time-consuming and will only lead to a limited percentage of patients lost to follow-up eligible for retrieval. Nonetheless it will lead to a change of management in a significant percentage of evaluated patients. Therefore, structured retrieval programmes of chronic HBV and HCV are an important element in the accomplishment of the WHO goal to eliminate HBV and HCV as public health care threat.


Moses technology modulates the laser pulse to separate the water (vapor bubble), then deliver the remaining energy through the bubble. Proprietary laser fibers were designed for the Moses technology. Our aim was to compare stone lithotripsy with and without the Moses technology.The Lumenisn=23) and without (n=11) the use of Moses technology. Surgeons ranked the Moses technology in 23 procedures, in comparison to regular lithotripsy . Laser working time and energy use were collected from the Lumenis 120H log.We designed a questionnaire for the urologist to fill immediately after each ureteroscopy in which the Lumenis 120H was used. We compared procedures with . Three urologists ranked Moses technology as much better or better in 17 procedures. In 2 cases, it was ranked equivalent, and in 4 cases ranking was not done. Overall, laser lithotripsy with Moses technology utilized laser energy in less time to achieve a satisfying stone fragmentation rate of 95.8 mmThe new Moses laser technology demonstrated good stone fragmentation capabilities when used in everyday clinical practice. Modern renal stone surgery maximizes stone removal while minimizing invasiveness. Extracorporeal shockwave lithotripsy (ESWL) is the least invasive treatment with acceptable stone-free rates (70%–85%) and the standard treatment for small kidney stones up to 2 cm). cm.2In the last two decades advances in technology made ureteroscopic access to kidney stones possible, and, as ureteroscopy made the visualization of the target stone feasible, a compatible flexible tool to fragment the stone had to be developed.That came in the form of the pulsed holmium yttrium-aluminium-garnet (YAG) laser—a solid-state laser system that operates at a wavelength of 2140 nm. Holmium laser is absorbed by water, and therefore is absorbed superficially by tissue, allowing for cutting or ablation of strictures and tumors. The main use of the holmium YAG laser is for lithotripsy—urinary stone fragmentation.4Stone movement caused by laser shockwave  is one disadvantage of laser lithotripsy.The new Lumenis High-power Holmium YAG laser  enables the surgeon to control the pulse width, and it features a unique modulated pulse mode (Moses), designed to minimize stone retropulsion.We designed a questionnaire for the urologist to fill immediately after each ureteroscopy in which the Lumenis 120H was used. We compared procedures with and without the use of Moses technology. Over a period of four months the Lumenis 120H was evaluated in 36 procedures by five different urologists. We excluded two patients who were treated for upper tract transitional cell carcinoma; the rest of the patients had urinary stone disease.In 23 procedures the Moses technology was used, while regular lithotripsy was used in 11 procedures. We used Moses laser fibers with a diameter of 200 μm, 365 μm, and 550 μm, and regular fibers of 200 μm and 365 μm. Surgeons ranked each of 23 procedures, in comparison to regular mode . The operators were also asked about their subjective experience with the Moses technology capabilities in specific domains: stone fragmentation rate, ability to minimize stone retropulsion, fiber flexibility, and fiber durability . Laser working time and energy use were collected from the Lumenis 120H log for all 34 procedures. Stone sizes were taken from the preoperative computerized tomography (CT) scan. Stone volume and total stone burden were calculated by measuring the stones’ three dimensions in millimeters and then calculating the stone volume: length × width × height × π × 1/6.9Clinical data were collected from the patients’ medical file. The study was approved by the institutional ethical committee.The comparison groups were similar in terms of demographics and by entry clinical data. Stone locations were: the kidney in 19 patients, the ureter in 15 patients. In these procedures, a flexible ureteroscope was used in 22 cases and a semi-rigid ureteroscope in 12.The Moses technology was used in 23 procedures, by three urologists. In all 23 procedures, the lithotripsy fragmentation rate was rated either excellent (15 cases) or good (8 cases). The domain of minimizing stone retropulsion was rated excellent in 8 procedures, good in 10, average in 2, and not rated in 3 cases. Moses fiber durability was rated excellent in 19 cases and good in 4. Fiber flexibility was rated excellent in 12 procedures, good in 4, average in 2, and not rated in 5. When overall lithotripsy efficacy of the Moses technology was compared to regular lithotripsy in these procedures, in 3 procedures the surgeon rated the Moses as much better, in 15 procedures it was ranked better, and in 2 procedures equivalent. In 3 procedures a comparison of the surgeon’s impression was not available.3/min versus 58.1 mm3/min, respectively (P=0.19).The median calculated stone fragmentation rate for Moses and standard technology was 95.8 mmectively , thus suAs new technologies have been developed in the field of minimally invasive lithotripsy, we notice a paradigm shift in stone disease clinical practice. Treatment using ESWL, considered to be the gold standard for small kidney and upper ureteral stones, is pushed aside by ureteroscopy as a treatment modality for kidney and upper ureteral stones .2However successful, ureteroscopy is heavily dependent on technology. Another consideration is the cost of disposable equipment as well as reusable sensitive equipment (ureteroscope). The recent advances in digital imaging and the development of digital high-definition ureteroscopy improve the quality of imaging at the time of surgery. Advances in these technologies now allow for the first time a disposable flexible high-definition ureteroscope to be produced and competitively priced. With time and competition these technologies, now deemed too expensive in many health care systems across the globe, will become affordable.The pulsed holmium YAG laser is the powerful tool that completes the ureteroscopy, allowing the cutting or ablation of strictures and tumors and the fragmenting of urinary stones.3Stone retropulsion at time of laser lithotripsy as well as the fragmentation of stones to multiple small stones is one hurdle that laser lithotripsy technology needs to address. Another is the lack of flexibility of the laser fiber, hampering the ureteroscope’s reach of urinary stones in difficult anatomical positions.Holmium laser lithotripters allow the operator to control the pulse energy and pulse frequency. Recently, newer models of holmium lasers enable the urologist to choose different pulse durations. In both settings the same amount of energy is delivered, but retropulsion is reduced when the energy is distributed on a longer pulse.16Previous studies were focused on pulse amplitude and frequency for reducing the size of fragments. These studies demonstrated that a lower amplitude reduces retropulsion and fragment sizes.6in vitro model as well as in vivo animal model.in vitro model demonstrated pronounced reduction of retropulsion in the Moses mode during fragmentation setting  as well as dusting mode . Moses modes resulted in a significantly higher ablation volume when compared with the regular mode . In vivo assessment also supported the reduction in retropulsion when treating stones in the porcine kidney.7Elhilali et al. evaluated the Moses technology with an As the Lumenis 120H with the Moses technology is now approved for clinical use and commercially available, we sought to compare the surgeon operator experience with the Moses mode as well as the objective efficacy of this technology in fragmenting stones.The subjective clinical evaluation of the Moses technology by three experienced urologists in everyday clinical practice was excellent on all domains tested. The operators experienced less stone retropulsion when working with the Moses technology. Domains linked to laser fiber quality (durability and flexibility) were good as well.Comparing the urologist lithotripsy experience when operating with the Moses mode, nearly every time Moses mode lithotripsy was ranked better.3/min versus 58.1 mm3/min, P=0.19), probably due to the small sample size. However, this trend gives some support to the surgeons’ subjective evaluation.Objective data collected included treated stone burden volume, laser working time, and energy use, which allowed us to calculate the median stone volume fragmentation rate (volume of stone fragmented per unit time). The higher fragmentation rate with the Moses mode (95.8 mmOur study is a non-randomized trial with a small study group and multiple operators, and as such is limited in the clinical outcome analysis it can offer. We were able to draw conclusions about subjective surgeon experience and objective stone fragmentation rate; however, we do not have sufficient follow-up and sample size to compare stone-free rate at this stage. Further studies assessing Moses technology impact on stone-free rate are warranted.We conclude that in our experience in an everyday clinical practice our initial impression using Moses technology is that it allows a considerable reduction in stone retropulsion. Although not statistically significant, this study reports promising results that warrants further clinical studies in large and varied groups of patients.


Alternatively, heat-stress leads to hyperventilation in pigs, which reduces CO2 concentration and with the cinnamon treatment, CO2 diminishes even more, resulting in health improvement outcomes. Overall, a good repeatability in gas profiles is also observed. The model demonstrates the strong potential of real-time gas profiler in providing new physiological information that will impact understanding of therapeutics, presenting a highly reliable device for monitoring/diagnostics of gastrointestinal disorders.Gastroenterologists are still unable to differentiate between some of the most ordinary disorders of the gut and consequently patients are misdiagnosed. We have developed a swallowable gas sensor capsule for addressing this. The gases of the gut are the by-product of the fermentation processes during digestion, affected by the gut state and can consequently provide the needed information regarding the health of the gut. Here we present the first study on gas sensor capsules for revealing the effect of a medical supplement in an animal (pig) model. We characterise the real-time alterations of gastric-gas in response to environmental heat-stress and dietary cinnamon and use the gas profiles for understanding the bio-physiological changes. Under no heat-stress, feeding increases gastric CO Currently, gastroenterology is still mired with patients’ problems stemming from misdiagnosis123572, CH4, CO2, NOx and H2S as well as many volatile organic compounds, are mainly the result of the fermentation processes and other activities by the intestinal microbial community and may appear in the metabolic pathways9in vivo and in vitro studies have shown that there are strong links between the intestinal gases and different types of gastrointestinal disorders. For instance, H2 and CH4 are used for detecting and characterising carbohydrate malabsorption4 has been found to play an important role in the peristaltic control of the gut and its presence is closely correlated to constipation-type IBS102S and NOx have been identified as gasotransmitters that are important for the intestinal motility and secretory functions. Both of them are also considered as typical markers for IBD131415It is now widely accepted that gastrointestinal gas profiles provide key information regarding the gut’s state-of-health99in vitro fermentation of faecal samples202122Nevertheless, the accuracies and reliabilities of the current intestinal gas measurements have been of great concerned. Breath analysis is currently the most common indirect method which relies on the measurement of intestinal gases that are first absorbed onto the gut walls, then recirculated to the lung through the blood stream and eventually excreted by respiration11179Early attempt of direct and accurate intestinal gas measurements have been realised by inserting tubes into patients’ oral cavity or anus to collect gas samples24in vivo for the investigation of gut under various disease and disorder conditions. The gas capsule can also be a unique tool in assessing therapeutics and their impacts on the gut. However, such capabilities have not been shown yet.Recently, we have successfully developed a non-invasive and direct gas profiling tool called “gas sensor capsule” . The capcinnamomum verum) is chosen as a representative therapeutic agent due to its anti-inflammatory, anti-oxidant and free radical scavenging properties in the gastrointestinal tract32Here, such hypothesises are validated using an animal model influenced by a representative gut disorder and a dietary therapeutic agent. The model of gut disorder is induced by stressing pigs under cyclical heat. The heat-stress effects on the gut physiological abnormalities are well-known in the literature. When under heat-stress, the shunting of blood from the central circulation to the periphery reduces the perfusion to the viscera, particularly the gastrointestinal tract, which may result in tissue hypoxia and intestinal mucosal injury282730In this pilot study, the capability of gas sensor capsule is for the first time investigated through examining gastric gas production in pigs under heat-stress and assessing the effect of oral cinnamon on such gas profiles. The results are interpreted in the light of other complementary physiological and biochemical assessments. The repeatability of the gas profiles is also evaluated in a large group of pigs for revealing the effectiveness of this novel medical tool.2 gas profile of the thermoneutral control pig (no cinnamon in the diet) was measured by the gastric gas profiler (a modified version of gas sensor capsule with higher density) and shown in 2 level by ~21% in ~3–4 h, while there was a ~8% increase of CO2 concentration at ~3 h after the second feeding. Simultaneously, a rise of gastric temperature of up to 0.8 °C corresponding to each feeding was observed. A rapid decline of CO2 gas level was seen after reaching the maximum of the day.The real-time gastric CO2 gas pattern and production kinetics in the thermoneutral cinnamon-treated pig .The gastric COated pig  were dis−1 could be ascribed to the stretching modes of C-H, amide NH, C=C and amide CH bonds, respectively. In the pig treated with cinnamon, the normalised intensities of these Raman peaks were much lower than those of control, indicating less pepsin concentration in the gastric digesta.Addition of cinnamon in the diet significantly increased the gastric pH from ~1 to ~3.2 , with th342 pattern was changed compared to those of the thermoneutral control pig. As shown in 2 concentration maximum was observed at ~4 h after the first feeding for the heat-stress controlled pig (no cinnamon supplement) with the value comparable to that in the thermoneutral control animal. At the same time, a significant increase in the gastric temperature by ~3 °C was observed. However, the second feeding resulted in a very small increase in the CO2 concentration, which was only ~2% in comparison to ~8% in the thermoneutral condition. This was associated with a decrease of blood CO2 and carbonate concentrations compared to that under thermoneutral controlled condition, while their blood pH values were similar  relative to those in the heat-stress control pig and relatively reduced respiratory rate associated with heat-stress will tend to increase gastric CO2 content. However, cinnamon increases gastric mucosal blood flow322 exchange. This is supported by the attenuated rise in gastric temperature that might reflect enhanced radiant heat loss from the mucosa. Skin blood flow might also increase with additional radiant heat loss as suggested by the reduced skin temperature in the cinnamon-treated heat-stress pig. Interestingly, rectal temperature is not reduced and this may indicate rectal mucosal blood flow to be unaffected by cinnamon, an untested but feasible hypothesis.The observation that dietary supplementation with cinnamon appeared to reduce gastric CO2 level is found to be suppressed due to the combination of reduction of gastric acid and pepsin secretion as well as hyperventilation, providing a novel marker of the physiological disturbances that enhances information from more traditional physiological and biochemical measurements. The addition of cinnamon into the diet inhibits both the gastric acid and pepsin secretion, resulting in a significant decline in the gastric CO2 gas level and alters CO2 gas pattern. When under heat-stress, partial amelioration of disorders signified by physiological indices is observed. The CO2 gas pattern corresponding to the second feeding of the day is greatly altered as the gastric CO2 gas is rapidly depleted during that period, which can be ascribed to the possible enhanced gastric mucosal blood flow in the presence of cinnamon. In addition, the relatively normal gastric acid and pepsin secretion may reflect the potential antioxidant activity of cinnamon against the intestinal mucosal injury induced by tissue hypoxia32Hence, this pilot study has demonstrated the potential utility of the real-time gastric gas profiler to enhance understanding of the physiological processes that occur in a model of a gastric disorder and of the mechanisms by which a potential therapeutic agent acts. Thus, for the heat-stress effect, the gastric COThe strong similarity and repeatability shown when comparing the gastric gas profiles obtained from the high-density profilers and normal-density gas sensor capsules indicates that the profiles obtained using the transient non-invasive capsules are precise. This suggests that this technology can be reliably used for the interpretation of the gastric gas profiles.via the gas level, pattern and production kinetics) about disorders of the gastrointestinal tract. Such information may permit new insights that will underpin future research into pathogenesis of gastrointestinal disorders, dietary and drug effects, and clinical diagnostic applications.In conclusion, this gastric gas profiler has provided novel, precise and repeatable real-time assessment of gastrointestinal physiological processes that enhance our understanding of a representative gastric disorder induced by heat-stress and the effect of a cinnamon supplement as a dietary remedial model. Since such emerging gas sensor capsule technology can readily be designed to target other parts of the gastrointestinal tract and/or other gas molecules, it presents as a novel medical tool that is capable of providing unique information  and housed under either thermoneutral (20 °C) or heat-stress conditions (35 °C from 09:00–17:00 and 28 °C for the reminder of each 24 h period). Swine were acclimatised to diets for 14 days pre-trial and fed twice a day at around 09:00 and 15:00 over the 2-day experiments. Access to feed and water was provided by troughs and nipple drinkers. Heat and humidity were monitored by using temperature and humidity Dataloggers® placed in the thermally controlled rooms.Four female crossbred pigs (averaged at ~40 kg) were kept at four different conditions. They were fed a commercial ration  supplemented with either 0 or 1.5% The repeatability of gastric gas profiles were assessed using a group of 12 pigs, which included 4 pigs from the main tests that were administrated with high-density gastric gas profilers and 8 additional pigs that were administrated with normal-density gas sensor capsules. The additional 8 pigs were divided evenly for four different feeding and environmental conditions similar to those in the main tests. This resulted in a total of three pigs for each condition (N = 3).2 gas  and cHCO3− (concentration of bicarbonate in plasma), using an automated blood gas analyser .Before the completion of the experiment, 1 mL of blood was collected from the ear vein of each pig. The samples were then immediately assayed for acid-base indices, including pH, pCOAt the completion of the experiment, pigs were euthanized and dissected. During dissection, the gastric digesta were collected and pH values were immediately measured using a calibrated pH meter . Measurements were repeated five times for each sample and the average calculated.wt/wt solution of silver nanoparticles  at ratio of 1:1. The silver nanoparticles were used for amplifying Raman spectroscopic signals of chemicals presented in the digesta−1). The final intensity of the Raman spectrum of each sample was average of four repeated Raman scans with an exposure time of 30 s each. Background luminescent offset, noise removal and baseline adjustment of the Raman spectra were conducted using a Matlab® 10 software package.After pH measurements, gastric digesta were mixed with a 5% How to cite this article: Ou, J. Z. et al. Potential of in vivo real-time gastric gas profiling: a pilot evaluation of heat-stress and modulating dietary cinnamon effect in an animal model. Sci. Rep.6, 33387; doi: 10.1038/srep33387 (2016).


FGFR1 plays an important role in the development of the nervous system as well as the regulation of the skeletal development and bone homeostasis. Mutations in FGFR1 genes affect skull development, specifically suture and synchondrosis, resulting in craniosynostosis and facial abnormalities. We examined subjects with normal skull morphology for genetic polymorphisms that might be associated with normal craniofacial variations. Genomic DNA was obtained from 216 Japanese and 227 Korean subjects. Four FGFR1 SNPs, namely, rs881301, rs6996321, rs4647905, and rs13317, were genotyped. These SNPs were tested for association with craniofacial measurements obtained from lateral and posteroanterior cephalometries, in which principle component analysis was performed to compress the data of the craniofacial measurements. We observed that SNPs rs13317 and rs6996321 were correlated with the overall head size and midfacial development, indicating that FGFR1 SNPs played crucial roles in the normal variation of human craniofacial morphology. Subjects with the derived alleles of SNPs rs13317 and rs6996321 had a small face and a facial pattern associated with a retruded midface and relatively wide-set eyes. These facial features were similar to but were milder than those of individuals with Pfeiffer syndrome, which is caused by a dysfunctional mutation in FGFR1. The human face not only hosts the most important sensory organs but also is the main symbol of expression, appearance, communication, and identification. Facial shape is one of the most important characteristics in developing a self-image and self-esteem ,2. AssesFGFR1), which is located at 8p11.1, contains 19 exons spanning 55-kb DNA and encodes at least 9 isoforms of FGFR1 . Fifteen measurements of the cranium and nine measurements of the mandible were obtained from the cephalograms , Fig 1. FGFR1 SNPs with PC scores, with sex and population being used as covariates when they were associated with the focal PC. Sex and population were used as covariates in order to control the unexplained variation that may occur from different populations in the sample, which may have different norms of the craniofacial measurements as well as the use of male and female subjects. Single and stepwise multiple SNP models in which genotypes were indicated using the number of derived alleles were examined for each PC. In the stepwise multiple SNP model, the four SNPs were used as explanatory variables at the beginning and were narrowed down later. When two or more SNPs remained after the stepwise procedure, additive multiple SNP and haplotype models were used. In the additive multiple SNP model, the number of effective alleles was added for multiple SNPs. In the haplotype model, haplotype counts in the individual were used as explanatory variables, with the ancestral haplotype as a reference. Furthermore, the 4 SNPs were tested for their association with the ratio |Or-NA|/Or-Or, which represent the relation between the facial retrusion and facial width.Principal component analysis (PCA) was performed to improve the exploration and visualization of morphological variations by decreasing the dimensionality of obtained data. Metric data of craniofacial morphology was standardized for both the populations, and these standardized data were used to create a correlation coefficient matrix. PCA was performed by using the correlation coefficient matrix, and eigenvalues, eigenvectors and PC scores were obtained. PCA was applied separately to the cranial measurements and to the mandibular measurements . LateralCraniofacial measurements of the Japanese and Korean subjects included in the study are summarized in FGFR1 SNPs in the Japanese and Korean subjects are presented in Allele frequencies of the four Cranial PC1 represented a variation in the overall size of the face, and cranial PC3 represented a variation in midfacial protrusion. High PC3 score was associated with less protrusion of the midface and large facial width in the orbit and cheek region . The numFGFRs are responsible for craniosynostosis and chondrodysplasia syndromes [FGFR2 and FGFR3 have widely varying phenotypes. The vast majority of mutations that cause craniosynostosis syndromes are associated with the FGFR2, which cause quite variable and non-specific phenotypes. As the main role of the FGFR3 is the regulation of cell proliferation in epiphyseal plate chondrocytes, many FGFR3 mutations are characterized by short-limbed bone dysplasia of varying severity ; craniosynostosis is rare in the first two of these disorders. Although a reasonable correlation between clinical description and mutation has emerged, identification of these mutations has necessitated some reappraisal of the rather confusing clinical classification of the craniosynostosis disorders. Therefore, abnormalities resulting from mutations in FGFR2 and FGFR3 genes are a good example of a disorder that is better classified by mutation rather than phenotype [FGFR1 (P252R) can cause Pfeiffer syndrome and phenocopies several Pfeiffer syndrome mutations in FGFR2 [FGFR1 [FGFR1 gene as the number of mutations already identified in this gene indicates that FGFR1 might accumulate SNPs that do not result in a pathologic deformation, but may influence normal variation. Furthermore, the FGFR1 is associated with osteoprogenitor cell differentiation suggesting that mutation in the FGFR1 gene may affect the function of the differentiated osteobalsts in early embryonic life and hence affect the bone development. Pervious studies have identified a correlation between the SNP rs4647905 of the FGFR1 gene and some head measurements [FGFR1 SNPs rs881301, rs6996321, rs4647905, and rs13317 might have certain role in the craniofacial morphogenesis. In the present study, we analyzed four FGFR1 SNPs in Japanese and Korean subjects and observed an association of SNPs rs13317 and rs6996321 with some facial traits, as determined by performing PCA. Interestingly, SNPs rs13317 and rs6996321 were associated with a small face (denoted by PC1) and midfacial retrusion, which in turn resulted in protruded forehead and relatively wide orbit and cheek area (denoted by PC3). These facial features were similar to those of patients with Pfeiffer syndrome, which is caused by a dysfunctional mutation in FGFR1 [Because FGF ligands and their receptors play important roles in craniofacial development, especially suture and synchondrosis, variations in genes encoding these ligands and their receptors can affect their expression and functions, thus exerting various effects on craniofacial morphology. Gain-of-function missense mutations in yndromes . Allelichenotype ,29. A siin FGFR2 . Moreove2 [FGFR1 –33. We f[FGFR1 P22R can caurements ,17. Moreurements –22. Therin FGFR1 . Most fain FGFR1 . This miin FGFR1 ,34–39. Ain FGFR1 ,39.FGFR1. Functions of SNPs rs13317 and rs6996321 are unknown. Moreover, it is possible that these SNPs do not have any function other than serving as markers. Because the distance between SNPs rs13317 and rs6996321 is 52.8 kb and because their LD was weak in the analyzed populations, it is likely that effective polymorphisms exist in different LD blocks.Our regression analyses by using the stepwise multiple SNP model indicated an association of SNPs rs13317 and rs6996321 with PC1 and PC3. However, the single SNP model showed that only rs13317 was significantly associated with PC1 and that rs6996321 did not show any significant association. Similarly, the single SNP model showed that only rs13317 was significantly associated with the ratio |Or-NA|/Or-Or. These results may be because of the independent effects of at least two polymorphisms in FGFR1 variants and craniofacial measurements. Coussens and van Daal (2005) observed a significant association between rs4647905 and decreased cephalic index [FGFR1 polymorphisms with the normal variation of the craniofacial morphology. However, cephalometric measurements are associated with errors classified as ‘‘errors of projection” and ‘‘errors of identification”. Errors of projection are due to the two- dimensional (2D) head film, which causes a shadow of the three-dimensional (3D) object meaning that complete control is not possible unless the positions of landmarks are known in 3D. The impact of error of projection is mainly on the linear measurements, as the values of the angular measurements remain constant. In addition, those linear measures, which were used, are conventionally defined as lying in the midsagittal plane. In theory, that plane is held at a constant distance from both the film and the emitter by the nature of cephalostat (head holding device) design, and hence all points on it have a similar and standardizable enlargement factor. Precise repositioning of patients in any head holder is, however, very difficult, resulting in a situation in which the true anatomic midsagittal plane coincides with the nominal midsagittal plane of the x-ray-cephalostat system only rarely and by chance as slight head rotation may occur. However, it was found that differences in angular or linear measurements do not become significant unless rotation of the head in the cephalostat exceeds 10 degrees. On the other hand, the recent 3D imaging techniques like the cone beam computed tomography (CBCT) can exactly record and represent the size of the object [FGFR1 polymorphisms and the craniofacial morphology is recommended using the modern 3D imaging techniques for more accurate representation of the variations occurring in the craniofacial complex.Our results may not be in agreement with those of some previous association studies involving  Daal 200 observedic index , suggeste object –42. For FGFR1 SNPs played crucial roles in inducing craniofacial morphological variations in Japanese and Korean subjects, primarily because of their effects on the overall size of the head and midfacial development. Subjects with the derived alleles of SNPs rs13317 and rs6996321 had a small face and a facial pattern associated with a retruded midface and relatively wide-set eyes. These facial features were similar to but were milder than those of patients with Pfeiffer syndrome, which is caused by a dysfunctional mutation in FGFR1.In conclusion, we showed that S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Oral mucosa has been highlighted as a suitable source of epidermal cells due to its intrinsic characteristics such as its higher proliferation rate and its obtainability. Diabetic ulcers have a worldwide prevalence that is variable (1%-11%), meanwhile treatment of this has been proven ineffective. Tissue-engineered skin plays an important role in wound care focusing on strategies such autologous dermal-epidermal substitutes.  The aim of this study was to obtain autologous dermal-epidermal skin substitutes from oral mucosa from diabetic subjects as a first step towards a possible clinical application for cases of diabetic foot.per group). Epidermal cells were isolated and cultured using autologous fibrin to develop dermal-epidermal in vitro substitutes by the air-liquid technique with autologous human serum as a supplement media. Substitutes were immunocharacterized with collagen IV and cytokeratin 5-14 as specific markers. A Student´s t- test was performed to assess the differences between both groups. Oral mucosa was obtained from diabetic and healthy subjects (n=20  It was possible to isolate epidermal cells from the oral mucosa of diabetic and healthy subjects and develop autologous dermal-epidermal skin substitutes using autologous serum as a supplement. Differences in the expression of specific markers were observed and the cytokeratin 5-14 expression was lower in the diabetic substitutes, and the collagen IV expression was higher in the diabetic substitutes when compared with the healthy group, showing a significant difference. Cells from oral mucosa could be an alternative and less invasive source for skin substitutes and wound healing. A difference in collagen production of diabetic cells suggests diabetic substitutes could improve diabetic wound healing. More research is needed to determine the crosstalk between components of these skin substitutes and damaged tissues. Great progress has been achieved in the culturing of keratinocytes and fibroblasts; this has been especially true for keratinocytes since the first successful culture by Rheinwald and Green in 197525, to the present day, with the creation of specialized in vitro techniques24. Although current tissue-engineered skin substitutes have shown promising results for wound healing26, there are still several challenges to overcome, therefore there is a continuing search for novel approaches to wound care and treatments that could be more effective, where all epidermal cells and matrices are obtained from the same patient, with less invasive sources to obtain the biological components, and with new alternatives to harvest tissue in patients with physiological and ethical limitations due to their conditions.Advancements in the field of tissue engineering have allowed the widespread use of skin substitutes to cover wounds and to promote healing22. This tissue also has the advantage that its harvesting produces less disability, and provides better aesthetic outcomes. With these special features, it is assumed that the skin substitute obtained from oral mucosa can be produced faster. A possible disadvantage for the use of oral mucosa as an epidermal cell source could be the differentiation of the epithelial pattern of keratinocytes from oral mucosa, which differs from that of epidermal keratinocytes and skin. The skin is an example of an orthokeratinized epithelium. It displays a stratum basale, stratum spinosum, stratum granulosum and stratum corneum. Gingiva tissue also consists of an epithelium on a connective tissue matrix populated mainly with fibroblasts and endothelial cells, however, in contrast to skin, the epithelium is parakeratinized27. In this regard, Ueda, et al.26 (1995) reported in their study that a grafted sheet of keratinocytes from oral mucosa changed the epithelial pattern over a period of 4 weeks, explaining this phenomenon due to epithelial-mesenchymal interactions, suggesting that the specificity of the epithelium is dependent on the influence of the underlying mesenchymal tissue.Oral mucosa has been highlighted as a viable alternative source of epidermal cells, due to its easy preparation and suitable features, such as higher cell proliferation rates, lower terminal cell differentiation degrees and an increased biological activity compared with epidermal keratinocytes7. Foot problems are indeed a global problem and there is no area in the world that does not report the development of foot lesions as a consequence, mainly of neuropathy and peripheral vascular disease. The prevalence of active foot ulceration varies from approximately 1% in certain European and North American studies to more than 11% in reports from some African countries7. Although there have been many developments in recent years, which encourages optimism for future improvement in diabetic foot care, there is still much to be done.Worldwide, diabetic foot is a major medical, social and economic problem. It is estimated that approximately 15% of the more than 150 million people with diabetes world-wide will at some stage develop diabetic foot ulcerationThe aim of the present study was to obtain an autologous dermal-epidermal skin substitute using oral mucosa tissue from diabetic subjects with a measurable immunofluorescence characterization, as a first step to perfecting the development technique with the main focus of future possible clinical applications on the treatment of diabetic foot ulcers. Also it proposes for the first time, maintaining this substitute using autologous serum, thus avoiding the use of animal products and promoting the development of the construct.in vitro test carried out at Hospital Central “Dr. Ignacio Morones Prieto”, the Basic Sciences Laboratory, Faculty of Stomatology, Universidad Autónoma de San Luis Potosí. The study was performed in accordance with the Helsinki Declaration, and it was approved by the Faculty’s Ethical Board (CEIFE-033-012).This was an experimental 16, and 20 healthy controls donated 20 ml of peripheral blood and a sample of oral mucosa from the retromolar area. Blood was collected in labeled Vacutainer tubes, 10 ml without additives, and 10 ml with sodium citrate  and frozen at 20°C, until used. Oral mucosa samples were obtained with a 3 mm biopsy punch  previous aseptic and antiseptic controls and inferior alveolar nerve blockage (IAN). Hemorrhage was controlled by direct means for 10 minutes. Patients received instructions for wound care, and a follow-up after 7 days. None of the patients presented complications.Twenty adult subjects with diabetes mellitus type II (DM2), diagnosed according to the American Diabetics Association guidelines2 culture dishes . DMEM low glucose medium  supplemented with 10% fetal calf serum  was used as culture medium for fibroblasts. Keratinocytes were cultured using QN medium (a specific culture medium for keratinocytes). After 3 days the fetal calf serum concentration was reduced to 5%, and after a further 3 days to 1%. Blood samples were defrosted and centrifuged at 1200 rpm for 10 minutes. Serum and plasma were isolated and kept at -20°C until use. Autolougus serum was used for cell culture instead of the fetal calf serum after 7 days, while the plasma was used to develop the stroma for the substitutes. Both cell lines were incubated at 37°C in an atmosphere of 5% CO2 until the cultures reached a confluence of 80%. The culture media was changed every third day.Each oral mucosa sample was collected in 1.5 ml tubes containing phosphate buffered saline (PBS), 100 μg/ml streptomycin, 100 IU/ml penicillin, and 10 μg/ml amphotericin B  and preserved at 4°C for 12 h. Oral mucosa was macroscopically separated by region, with a scalpel, into epithelium and connective tissue. By using the explant technique, each region was grown separately in 25 cm® software .Once the cultures reached a confluence of 80%, part of the cell cultures were used for an indirect immunofluorescence assessment. In order to assure that the cells were fibroblasts and keratinocytes, the characterization of these cell cultures was performed. The specific monoclonal antibody used for fibroblasts was anti-collagen I  mainly because due to its abundance in the skin, recognized as the most abundant collagen in the epidermis, and its specificity for this cell type. For keratinocyte was anti-cytokeratins 5-14 . As secondary antibodies, Alexa fluor 488  and Alexa 594  were also used. Briefly, cells were grown on round glass cover slips (12 mm). After 12 h of incubation, the cultures were fixed for 20 minutes with neutral formalin, permeabilized with Triton X100 0.025% for 20 min at room temperature then blocked with bovine serum albumina (BSA) 1% for 45 min. The primary antibodies were incubated for 2 h at room temperature and the secondary antibodies were incubated for 2 h at room temperature and were protected from light. Between each step, phosphate buffered saline rinses were made. The samples were observed by confocal microscopy  and LASAFper 1 mL. A solution was made by mixing the resuspended cells with plasma and agarose at 3% to create fibrin agarose/gels5. 300 microliters of the solution were placed in the Transwell system  following the air-liquid technique3. Culture medium was placed covering the entire substitute. Each system was incubated at 37°C in a 5% CO2 atmosphere and with a relative humidity of 95% for 10 days , centrifuged and suspended once more in the medium and quantified by One Scepter . Cells were again suspended in DMEM low glucose medium  with 1% of human serum obtained from each subject, reaching a cell concentration of 500,000  10 days , b.per 1 mL. One hundred microliters of the cell solution were placed in the Transwell system  on the top of the already set stroma, and incubated for 10 days using the air-liquid technique. Culture medium was placed covering both compartments on the first day of settlement, on the fourth day, the culture medium was placed only in the lower compartment. Each system was incubated at 37°C in a 5% CO2 atmosphere with a 95% relative humidity for 10 further days.Cultured keratinocytes were detached from the culture dishes using TrypLE Express , centrifuged and resuspended in a QN medium with 1% human serum obtained from, and quantified by One Scepter  to reach a concentration of approximately 500,000 cells per slide). Each slide was treated for its indirect immunofluorescence assessment.Substitutes were mechanically separated from each system and placed in dishes with 1X PBS to remove excess of the medium. Subsequently they were fixed with 4% paraformaldehyde and placed in a solution of 3% sucrose as a cryoprotector. Cryosections of 6 microns thickness were made at -29°C in a cryostat . From each sample, 100 cuts were made and placed on 50 slides  are expressed on basal keratinocytes and the expression of these filaments are characteristic of complex stratified epithelia1. Keratin 14 (CK14) is a prototypic marker of dividing basal keratinocytes and helps in the maintenance of epidermal cell shape; it also provides resistance to mechanical stress. Interestingly, the CK5/CK14 pair is expressed in the basal layer of the epidermis, which contains epidermal stem cells and transient amplifying (TA) cells2. Monoclonal secondary antibodies, Alexa fluor 488 and 594  were also used. Ten different fields were chosen randomly from each slide; each slide of every substitute created to be evaluated, was chosen by the sample function of the R program, version 3.0.1. The evaluation of the slide was performed by an observer blinded to the nature of the study. A total of 20 images were obtained; observations were performed by confocal microscopy  and the LASAF® software . Images obtained were evaluated by Image J program manager  with the ROI function getting the mean of the arbitrary fluorescence units per slide, per antibody for further statistical analysis.For the characterization of these dermal-epidermal substitutes, the specific monoclonal antibodies used were anti-collagen IV and anticytokeratins 5-14 . Collagen IV was assessed in this characterization due to its role as the primary collagen found in the extracellular basal membranes separating a variety of epithelial and endothelial cells. It is a major component of the dermal-epidermal junction, where it is mostly found in the lamina densa of the basal membraneper well. The evaluation times were 0, 3, 6 and 9 days.Cell growth curves were performed (N=8). Cultures of 4 healthy subjects and 4 age-matched diabetic subjects were selected. Fibroblasts were subculture in duplicate in 6 well boxes by placing 100,000 cells 20. An analysis of descriptive statistics, obtaining the measures of a central tendency and dispersion of all the variables was then performed. For the bivariate comparative analysis we used the Student’s t-test for continuous variables based on a normal distribution calculated with plot of Fox10. For categorical variables the Chi-squared test was applied. A paired Student´s t test for time 0 versus 3, 6 and 9 days for the growing cellularity between the groups was performed. Statistical significance was considered with a p value <0.05. For the cell growth curve, statistical significance was considered with a Bonferroni correction of alpha for 3 comparisons with a p value of <0.016.Statistical analysis at 95% of confidence was performed using JMP 8 software  and R 3.1.3per group) according to the previously set criteria. Of the total of samples taken, 19 corresponded to men and 21 to women. The average age was 52.15±13.5 years. Variables studied in each group are showed in A total of 40 oral mucosa samples were collected , and time 0 vs 9 days (p=0.004); however statistically wide confidence intervals are showed, indicating greater variability in the behavior of the diabetic group compared with the healthy group , Table 2Substitutes were developed using the air-liquid technique, as previously described. The culture time for the stromal and epithelial phase was 10 days for each. Once both phases were completed, diabetic and healthy substitutes were observed with an inverted microscope at 40X, showing interesting features. An extended cellular pattern with more fibers was observed in the diabetic substitutes, whereas healthy substitutes showed a globular pattern with fewer fibers . The cliEach substitute was characterized and assessed as previously described. Anti-cytokeratin 5-14 expression showed 1.14 arbitrary fluorescence units (AFU) less in the diabetic substitutes compared with the healthy control group. Anti-collagen IV expression was 1.9 AFU higher in the diabetic substitutes when compared with the healthy group. In addition, dermo-epidermal skin substitutes were immunostained with Sytox Red  showing the nucleus and the cytoskeleton was stained with Phalloidin , showing the cell distribution throughout the thickness of the substitute .A Student’s T-test was performed as planned. All variables showed a normal distribution. There was a significant difference between the diabetic group and the healthy group for arbitrary fluorescence units of collagen IV (AFU col-IV) p value=0.02 and arbitrary fluorescence units of cytokeratin 5-14 (AFU ck 5-14) p value=0.05 .13 (2005) developed an epidermal equivalent from oral mucosa based on an acellular allogeneic dermal matrix showing good results after its clinical application26. Other studies reported the development of skin substitutes using cells obtained from biopsies of human skin, mainly the foreskins of children or from established epidermal cell lines. The main disadvantage of this approach is the allogeneic nature of the cells and the latent possibility of short-term rejection by the host. Still, wound healing was accomplished with its clinical application11.Skin tissue engineering is a therapeutic field developed to aid in the healing of injured skin, especially for wounds that have difficulty healing in the primary stage. This study showed the development of the dermo-epidermal substitutes of oral mucosa obtained from the retromolar area with a 3 mm punch biopsy from diabetic and healthy subjects, using the air-liquid technique and its assessment by immunofluorescence characterizing and quantifying its intensity in AFU, as a first step towards a possible future clinical application. Recent studies show oral mucosa to be a feasible, alternative source of epidermal cells and matrices, mainly for its intrinsic characteristics studied and described in previous studies. Iida, et al.8, and also using enzymatic techniques such as cell disruption without mentioning the time at which they achieved confluence, pointing out only the use of cells in different culture passages24. In primary cell culture techniques, cell disintegration is a strategy that can reduce culture times up to 50%, yet has the main disadvantage of the possibility of damaging tissues and cells12. It is important to point out that for the clinical application of these skin dermal-epidermal substitutes, cell culture times should be reduced, and thereby the enzymatic digestion technique is a good strategy to be used for future studies.Primary cultures of fibroblasts and keratinocytes were achieved from both populations  by the explant technique. Eighty percent of confluence was achieved in 25.3 days for diabetic subjects and for healthy subjects in 26.85 days. In similar studies, the production of primary fibroblast and keratinocyte cultures has been reported by the explant technique alone2 and O2) of the epithelial layer in the final stage. It allows for a place specific culture medium for each cell line (fibroblasts and matrices in the lower compartment and keratinocytes in the upper compartment) throughout the entire culture process20. In our study, the construct remained intact throughout the in vitro process, allowing for the manipulation of both compartments independently compared with other culture techniques and materials24. Culture times reported through the air-liquid technique range from day 1 of the stromal stage, up to 5 days, and the epithelial phase for a period of 5 to 10 days21. In this study we used 10 days in the cultures for both phases; stromal and epithelial, obtaining good results in relation to the establishment of the stromal cells as evidenced by the immunofluorescence evaluation.Regarding the development of dermo-epidermal substitutes, the air-liquid technique was an efficient cell culture technique for obtaining these skin equivalents. It provides the conditions and characteristics needed for the obtention of this kind of tissue, mainly by the gas exchange (CO15 (2014) studied the effects of neurotensin on human keratinocytes under hyperglycemic conditions and normal conditions at different functional levels, mainly NT receptors, cytokines and growth factor expression and proliferation and also migration of the epidermal cells. They observed that the neurotensin did not affect the viability of keratinocytes, however, neurotensin and the expression levels of all recipients were significantly reduced. Neurotensin treatment stimulated the expression of neurotensin receptor 2 (NTR2), whereas expression levels of neurotensin receptors 1 and 3  did not change. Keratinocyte proliferation was not affected, but the migration of keratinocytes was reduced. Their results show that hyperglycemic conditions adversely affect the endogenous expression of neurotensin and its receptors, especially in keratinocytes NTR2 with important consequences for its function. This effect of neurotensin in hyperglycemic conditions has also been well studied in other cells such as macrophages, where it was observed that this neurotransmitter runs cell migration patterns and the inflammatory response in wound healing18. Given these findings, Moura, et al.17(2014) conducted two additional studies where neurotensin was added as a component to the dermo-epidermal substitutes, specifically for the scaffold or biomaterial in which cells are suspended. They tested in mediums of collagen and chitosan observing a reduction in inflammatory infiltrates up to day 3 after implantation in rats with induced diabetes, and also an improvement in wound healing promoting cell migration and deposition of collagens I and III17. Future studies concerning the effect of NT are required in order to assess if in these neurotensin substitutes have an inhibitory proliferation role.In the assessment of the substitutes by immunofluorescence, there was a significant difference observed between both groups (p=0.002 for AFU COL-IV and p=0.05 for AFU CK5-14). The results showed less arbitrary fluorescence units for keratinocytes (CK5-14 antibody) in the substitutes generated from the oral mucosa of diabetic subjects (9.56) compared to those generated from the oral mucosa of the healthy subjects (10.47). In relation to the above, recent studies show an association of Neurotensin (NT), a neurotransmitter involved in the process of wound healing. Moura, et al.4. Our results support this statement, as it showed increased immunofluorescence for anticollagen-IV and fibroblasts of the dermal-epidermal substitutes generated from diabetic subjects (8.05) compared with healthy subjects (6.15). Berlanga-Acosta, et al.5(2010) reported that under the high glucose load imposed by diabetes, skin and skin fibroblasts were disturbed in recreated in vitro clinical models showing alterations to the normal physiology of fibroblasts as well as extracellular matrix secretion, therefore, it has been suggested that high concentrations of glucose is the major trigger of a cascade of molecular changes to skin fibroblasts. In our study, cell cultures of diabetic subjects reached an 80% confluence in a shorter time (25.3 days) than healthy subjects (26.85 days) suggesting a permanent effect of hyperglycemic conditions in cell physiology; however further study is needed.Fibrosis is a complication of chronic hyperglycemia involving the excessive proliferation of extracellular matrix (ECM) and its accumulation in various tissues and organs, the most prominent being microcirculation, kidney, heart, retina and wound healing. Chronic hyperglycemia affects the cells responsible for the production of collagen IV causing this accumulationDifferences in the collagen production of diabetic subjects’ cells could suggest that diabetic substitutes might improve the healing of diabetic foot ulcers, however more research is needed to determine the clinical impact of the differences found. Additionally these substitutes may be used to test new drugs in an environment that is closer to that of real tissues.Results of our study were measurable and quantifiable (intensity of collagen-IV and anticytokeratin 5-14 in arbitrary fluorescence units) showing numerical variations in the amount of expression of both antibodies in both populations; diabetic and healthy subjects, suggesting differences in cellular function under this clinical condition and, with possible implications in the clinical application of the substitute. Also it is necessary to emphasize that the cells not only survived through the development process of the substitute, but also they were integrated in to the dermal matrix and secreted basal membrane supported by the positive expression of the specific markers CK5-14 and COL IV.Despite the above, the significance of these findings is not yet known, further studies are needed to elucidate them. However this data may provide a basis to perfect/improve the development technique of the dermal-epidermal substitutes from the oral mucosa of diabetic patients and to answer the biological questions raised by it.in vitro tests were performed to evaluate the functionality of the developed substitute, such as migration essays. In that matter, the next step would be in vivo and in vitro essays of functionality of these substitutes developed by this technique and the characterization proposed in this study. This will lead us to the future clinical application of these diabetic skin substitutes.Within the constraints of our study, it is worth mentioning that no It is possible to develop dermal-epidermal substitutes from the isolation of the epidermal cells of oral mucosa from diabetic and healthy subjects using the air-liquid technique and its assessment by immunofluorescence characterizing and quantifying its intensity in arbitrary fluorescence units (AFU).in vivo animal studies to evaluate functionality will be performed to continue with this matter.Dermal-epidermal skin substitutes of diabetic subjects showed reduced keratinocyte immunofluorescence intensity when compared to the substitutes from healthy subjects; while for fibroblasts and COLIV, substitutes of diabetic subjects showed a greater intensity, with a higher AFU value. These findings suggest differences in cellular function under this clinical condition; however more research is needed to determine the crosstalk which occurs between the components of these skin substitutes and the damaged tissues. The following long-term goal in this research is the clinical application of these substitutes; further


Bison bison) on the island. Through the application of the immunocontraceptive vaccine porcine zona pellucida (PZP), population growth was slowed within 1 year, and halted over time. In response to lingering questions about the use of PZP to manage large, free-ranging wildlife populations, we sought to determine the reversibility of PZP by ceasing the annual application to a subset of 15 bison cows and monitoring for subsequent calf arrival, and to document changes in the timing and length of the breeding season in response to PZP by monitoring breeding behavior and assessing fecal progesterone (FP) levels for all 60 resident cows over a 13-month period. As of June 2017, no new calves had been observed on the island, suggesting that, following repeated annual treatment with PZP (3 or 4 years), bison do not resume normal reproduction for at least 4 or 5 years, and that fewer treatments would be advisable if a faster return to fertility is desired. Based on observations of bull and cow behavior, and FP levels, cows displayed estrous cycles consistently throughout the study period, indicating that bison may ovulate year-round when conception and its consequences, e.g., lactation and presence of calves, are blocked. Because there is little evidence that an extended breeding season would negatively impact the health of bulls or result in large numbers of out-of-season births on Catalina, PZP appears to be a highly effective tool for managing the population of introduced bison on the island. However, the extended period of contraception and breeding activity of both cows and bulls may make PZP less suitable in high-latitude, predator-rich environments where bison conservation remains a top priority.In late 2009, the Catalina Island Conservancy began using fertility control to replace periodic removals to manage an introduced population of American bison ( Historically, humans have altered wildlife abundance in many ways, including eliminating predators to protect themselves and their livestock, over-harvesting game species, and introducing both non-native and domestic species to new areas where they outcompete or displace native wildlife. The resulting population imbalances require informed management to prevent habitat destruction caused by overgrazing, and the extinction of native species from competition or predation . Lethal Sus scrofa domesticus) stimulates her immune system to produce antibodies against the proteins in the vaccine. These antibodies target the similar protein in the zona pellucida of her eggs, attach to sperm receptors, and thereby block fertilization  was nearly driven to extinction by overexploitation  has been responsible for managing ecological resources on the island, including the bison herd . To bettBased on its well-documented safety, efficacy and reversibility characteristics, its low cost, and the ability to deliver the vaccine remotely, PZP was first administered to cows in the Catalina herd during 2 roundups in 2009. The first (September 2009) facilitated a final shipment of 149 bison off the island and reduced the island herd to approximately 120 individuals . The secn = 43) in the population had received the vaccine during the roundups or remotely via darting. Pregnancy testing, completed during the November 2009 roundup, confirmed that 67% (29) of these cows were already pregnant when they received their 1st dose and all successfully produced calves in the spring of 2010  and domestic goats (Capra hircus) maintained high antibody titers to PZP antigens for 3 years after a single year’s treatment (primer and booster), but that antibody titers in lowland wisent (Bison bonasus) and Javan banteng (Bos javanicus), species that are more closely related to bison, returned to relatively low levels within only 9 months. Although the bison contraception program met its initial goal of halting population growth by reducing the calving rate, several key problems remained to be addressed to demonstrate that PZP could be an effective method for long-term, population-wide control of fertility in bison. First, the use of contraceptives to manage high-profile species has raised public concerns about the long-term health of treated populations, and their ability to resume reproduction successfully when the vaccine is no longer administered . ReversiA 2nd concern was that PZP would alter the reproductive behavior of bison, specifically, the timing and duration of the breeding season. Adverse changes associated with social behaviors, aggression, and movement and activity patterns have been documented in animals treated with hormone-based fertility control agents . HoweverCervus elaphus—Out-of-season breeding activity has been documented in studies of PZP application to free-ranging elk , located approximately 40 km west of Los Angeles, California, United States, has a rugged mountainous terrain, with a central ridge running its length  were present by the 1890s. Feral pigs (Sus scrofa) were initially established in the 1930s (Bos taurus) operations ceased by the 1950s (Odocoileus hemionus) were introduced to the island in 1928 and have been managed since 1998 through a private land management hunting program  weighing 60–70% more than females . The matheromone .Once a bull becomes interested in a specific cow, he will tend or guard her, remaining in close proximity, often impeding her movement and warding off other bulls until he is either permitted to copulate with her or is displaced by a rival . Bulls cImmediately following copulation, which lasts only a few seconds, the cow arches her back, voids a small volume of milky secretions from the vulva (presumably semen), and erects her tail . Cows elUnlike domestic cattle, bison were initially thought to be monestrous  or to onThe reproductive physiology of bison is similar to that of domestic cattle. The estrous cycle, as described by Photoperiod has been identified as a cue for reproduction for many wild ungulates, and plant phenology and predation have been implicated as factors contributing to birth synchrony . The bre2 rifle (Pneu-Dart). At the beginning of each darting season, the tail fin of each dart was painted red or orange using Rust-Oleum specialty fluorescent spray paint and numbered to facilitate dart recovery and tracking of vaccinations. Immediately after loading the dart a small dab of petroleum jelly was applied to the tip to prevent PZP leakage. Prepared vaccine was stored in a refrigerator and transported to the field in an ice chest. Treated cows were darted in the caudal thigh or gluteus muscle.The native PZP antigen was prepared at the Science and Conservation Center from porcine oocytes, using the modified method of Following the roundup and shipment of bison off-island in late 2009, all calving-age (≥ 2 years old) cows were treated with PZP each year from 2010 to 2012 . In 2013To assess the reversibility of PZP in bison, the vaccine was withheld from 10 cows (ages 5–11 years) beginning in 2013, and 5 additional cows (ages 8–14 years) in 2014. All but 1 of the Reversal cows were within the range of ages in which the highest fecundity in bison cows has been documented  collars  were fitted on 5 mature cows on 10–11 May 2014 to assist with locating smaller groups of bison and to assess movement patterns and resource use. Monitoring of the movements of these 5 collared cows allowed us to effectively locate and sample approximately 85–90% of the Catalina herd at all times . HoweverFor each cow, we recorded the following potential signs of breeding activity  and continued until copulation or the end of an observation period.- Tail-up partial: The cow was observed with a slightly elevated, less than horizontal, tail at the beginning of an observation period and maintained it for 1 h or more.- Tail-up high: The cow held her tail at a horizontal angle or higher, for an extended period of several hours to several days.- Copulation: The bull mounted the cow with intromission.- Fresh mounting sores (FMS): The presence of fresh  wounds on the lower back, point of hip region on either side of a cow.- Healing mounting sores (HMS): The presence of healing  wounds, presumably > 2 days old, on the lower back, in the point of hip region on either side of a cow.Heavy tending, tail-up high, and copulation behaviors have all been confirmed as reliable indicators of ovulation , and werDuring behavioral observations, cows frequently voided feces. We marked the location of newly voided feces by fixing the location of the feces in the crosshairs of a rifle scope attached to a tripod. We used a range finder to record the distance between the tripod and the target cow. When it was considered safe to approach , a fecal sample was collected to fill about three-quarters of a 30-ml plastic container , and placed in a cooler with ice packs. Each collection cup and lid was labeled with the cow’s identification code, date, and time of fecal deposition just prior to sample collection. To ensure a homogeneous sample, fecal material was mixed thoroughly on the ground prior to collection using a single-use wooden tongue depressor. Samples were transferred to a freezer at the end of an observation period and stored frozen (−10°C) until time of shipment. Fecal samples were shipped to the San Diego Zoo Institute for Conservation Research  within 6 weeks of collection for progesterone radioimmunoassay analysis.g for 10 min in a Legend XTR centrifuge . The supernatant was recovered and the pellet resuspended in 5 ml of 90% aqueous ethanol, vortexed briefly and then centrifuged. The combined supernatants were dried under a stream of filtered air and dissolved in 1 ml of 100% ethanol.Fecal samples were lyophilized for 72 h in a Flexi-Dry microprocessor manifold lyophiliter  to reduce variability in water content. Extraneous material was removed from the lyophilized feces by sifting each sample through a copper mesh screen (2 × 2 mm). A 0.1-g sample was weighed and suspended in 5 ml of 90% aqueous ethanol and boiled immediately for 20 min at 80°C. The sample was then centrifuged at 1,000 × Samples were analyzed using a monoclonal progesterone antibody produced against 4-pregnen-11-ol-3,20-dione hemisuccinate:BSA , which cg for 15 min and the supernatant poured into scintillation vials. Scintillation fluid  was added to each tube and the assay counted on a Beckman LS 6500 liquid scintillation counter  to determine the FP level.One microliter of the fecal extract was assayed in duplicate and incubated overnight at 4°C. After incubation, the competitive reaction was stopped and the bound and unbound hormone were separated by adding 0.25 ml of char-dex solution to the assay, which was then incubated for 30 min at 4°C. The assay was then centrifuged at 1,000 × We plotted FP levels and breeding behaviors for all 60 cows individually to detect errors and identify possible cycles. For behavioral purposes, a cow was considered to be in estrus if any 1 of 3 characteristic behaviors  was observed on a given day. To produce a conservative estimate, we considered observations of these behaviors to represent different estrous cycles if they were separated by at least 20 days .SD of all values and then excluded ones that exceeded the mean plus 1.5 SD. A new mean was then calculated and these steps were repeated until none of the remaining values exceeded the mean plus 1.5 SD. Using the approach of SD = 100) calculated from the other 52 cows. Data from these 8 cows were included in summaries of behavior and FP values, but were excluded from the analysis of temporal patterns in cycles across the herd.We were unable to calculate a baseline FP value using the As of 1 June 2017, no calves had been detected in the herd. This included the 9 surviving Reversal cows that had not been vaccinated since 2012  and the 5 Reversal cows that had not been vaccinated since 2013 .For both behavioral observations and FP sampling, the number of days sampled and the number of total observations and samples collected tended to increase over the course of the 13-month study period (see Supplementary Data SD1). The fewest samples and observations were in summer 2014, whereas, except for a peak in October 2014, the most observations and samples were in spring 2015.Breeding behavior and FP levels were plotted together for all 60 cows. Representative profiles of PZP-treated and Reversal cows see . Fewer tThe presence or absence of mounting sores was determined to not be a good indicator of breeding or ovulation. Although ovulation and breeding behavior were documented in all 60 cows, 20% (12) never developed sores, and sores were rare on many others, despite abundant evidence of breeding activity. Moreover, sores were often persistent and could not be used to indicate the specific timing of copulation. In many instances, sores were re-aggravated or maintained by scratching on trees or bushes, wallowing, or licking, and by mounting activities that were not associated with breeding.n = 60) were collected from each cow .FP values varied greatly within and among individuals (see Supplementary Data SD2). On average, 18.4 samples . Fewer cycles were identified using behavior than FP values and the 2 measures only occasionally overlapped , whereas Reversal cows displayed 4–11 cycles . Estrous cycles were recorded throughout the 13-month study period  of the resident cows and, as of June 2017, no calves have been born on the island. Based on the documented reversibility of PZP in wild horses , we antiIt is also possible that resumption of fertility in Reversal cows has been delayed because severe drought conditions that persisted on Catalina from 2012 to 2016 reduced the amount and quality of forage. Some of the lowest pregnancy and calving rates recorded in free-ranging bison herds were documented on Catalina Island (35% in 1975 and in 2002) and on Antelope Island, Utah (46% from 1987 to 1997), and in these instances, nutritional deficiencies were suggested as the primary cause . HoweverBecause bison were reported to have at most 2 or 3 estrous cycles in a given year, we anticipated that blocking conception with PZP would, at most, modestly extend the breeding season. Aside from a brief period of low reproductive behavior and progesterone levels in mid-April through May 2015, our results instead suggest that, like domestic cattle , bison aTwo concerns that have been raised about an extension of the breeding season in PZP-treated ungulates are the potential impacts on the condition of males as a result of an extended rut  on Catalina Island from 1 June 2014 to 30 June 2015.Supplementary Data SD2.—Range of fecal progesterone values for 60 bison (Bison bison) cows on Catalina Island between 1 June 2014 and 30 June 2015.Supplementary Data SD1Click here for additional data file.Supplementary Data SD2Click here for additional data file.