A hallmark of early IVD degeneration is a decrease in proteoglycan content. Progression will eventually lead to matrix degradation, a decrease in weight bearing capacity and loss of disc height. In the final stages of IVD degradation, fissures appear in the annular ring allowing extrusion of the NP. It is crucial to understand the interplay between mechanobiology, disc composition and metabolism to be able to provide exercise recommendations to patients with early signs of disc degeneration. This study evaluates the effect of physiological loading compared to no loading on matrix homeostasis in bovine discs with induced degeneration. Bovine discs with trypsin-induced degeneration were cultured for 14 days in a bioreactor under dynamic loading with maintained metabolic activity. Chondroadherin abundance and structure was used to confirm that a functional matrix was preserved in the chosen loading environment. No change was observed in chondroadherin integrity and a non-significant increase in abundance was detected in trypsin-treated loaded discs compared to unloaded discs. The proteoglycan concentration in loaded trypsin-treated discs was significantly higher than in unloaded disc and the newly synthesised proteoglycans were of the same size range as those found in control samples. The proteoglycan showed an even distribution throughout the NP region, similar to that of control discs. Significantly more newly synthesised type II collagen was detected in trypsin-treated loaded discs compared to unloaded discs, demonstrating that physiological load not only stimulates aggrecan production, but also that of type II collagen. Taken together, this study shows that dynamic physiological load has the ability to repair the extracellular matrix depletion typical of early disc degeneration. The intervertebral discs (IVD) of the spine function as shock absorbers and allow for flexibility and movement of the head and upper body The major components of the IVD extracellular matrix are proteoglycans and collagens.A young and healthy NP is gelatinous with a very high proteoglycan content. The collagen in the NP is mainly type II fibres forming a randomly organised network. The AF is more fibrous with lower proteoglycan content. The collagen, mainly type I, is arranged into highly organized structures forming strong concentric rings. Aggrecan is the major proteoglycan in the IVD and the many glycosaminoglycan chains attached to its core-protein are highly anionic and attract and retain water. Aggrecan is entrapped in the collagen network that limits disc swelling and a combination of the two molecules provides for resistance to compression. A number of non-collagenous molecules are also found in the extracellular matrix of the IVD. Among these are the leucin-rich-repeat (LRR) family of proteins, including chondroadherin (CHAD) A sparse cell population is located in the vast disc extracellular matrix A degeneration protocol, along with a bioreactor that facilitates organ culture of intact discs in a controlled dynamically loaded environment, has been developed to address such questions The polyclonal rabbit antibody recognising type II collagen was from Abcam  and the polyclonal rabbit antibody recognizing chondroadherin was a kind gift from Dr. Dick Heinegård, Lund University, Sweden et alBovine tails from 24- to 30-month-old steers were obtained from Boucherie B Poirier, Saint-Louis-de-Gonzague, Quebec Canada. The tails were dissected free of soft surrounding tissue, and pedicles for each segment were removed. The largest first 3–4 caudal discs were isolated with intact cartilage endplates as described by Jim The discs were rinsed in PBS supplemented with 500 U/mL U/mL penicillin, 1000 µg/mL streptomycin  and 0.25 µg/mL fungizone (Gibco), then placed in culture chambers  containing 50 mL culture medium . Half of the media volume was changed twice a week. Forty-eight isolated discs were cultured without external load applied for 48 h . DegenerThe disc were placed between porous platen covering 50% of the disc cross-sectional area. Loading was carried out on a 858 Mini Bionix . The discs were preconditioned for 5 minutes at 0.1 MPa load, the load was then increased to 0.3 MPa and the pressure profiles were immediately then recorded. A needle (1.3 mm diameter) with a pressure sensor mounted 3 mm distant from the tip was inserted in the mid plane of the disc through the annulus, until the sensor was visibly outside of the opposite annular region Metabolic activity was measured in the discs after 14 days culture in loaded and un-loaded conditions. Two 1.5 mm sections were taken in the sagittal plane through the center of the discs using a custom made cutting tool The second half of the section was thinly sliced and proteins and proteoglycans were extracted on a wet weight per volume basis using 15 volumes extraction buffer . The extracts was kept at 4°C under continuous agitation for 48 h and were then cleared by centrifugation at 16 000 g for 30 min.Sulfated glycosaminoglycans (GAGs) were quantified from tissue extracts by the modified dimethyl methylene blue (DMMB) dye-binding assay described by Mort and Roughley Proteoglycans in 10 µL aliquots of disc extracts were precipitated by the addition of 9 volumes of 95% ethanol; the pellets were washed twice in 95% ethanol, and finally lyophilized and dissolved in distilled water. The samples were reduced, alkylated and mixed with sample buffer  and boiled for 10 minutes. The proteoglycans were separated by electrophoresis in 1.2% agarose gels Proteins and proteoglycans in 40 µL aliquots of disc extracts were precipitated in 9 volumes of 95% ethanol, the pellets were washed twice in 95% ethanol, and finally lyophilized. Samples were re-dissolved in 200 µL SDS sample buffer and 20 µL of this was separated by SDS-PAGE (4–12% NOVEX gels) under reducing conditions. Separated proteins were transferred to nitrocellulose membranes and western blotting was performed using antibodies recognizing type II collagen and chondroadherin. The membranes were blocked with 3% BSA in 10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, and 0.2% Tween (blocking buffer) and then incubated with the primary antibodies at a 1∶1000 (CHAD) and 1∶5000 (type II collagen) dilution in blocking buffer containing 3% BSA followed by the secondary antibody conjugated with HRP (1∶2000 dilution) in blocking buffer containing 1% BSA. The bound antibody was visualized by chemiluminescence  using the ImageQuant LAS4000 . ImageQuant TL array analysis software was used to analyze the blots. To generate data for the graphs, western blotting was performed twice for each sample run on separate membranes; two reference extracts were included on each blot. The relative quantity of each protein was extrapolated using the reference samples and negative controls included on each blot. For the blots displayed, the 6 samples per group were combined and analysed as a pool.The NP region of the second 1.5 mm section was fixed between porous platens in 4% paraformaldehyde, lysine, periodate as described by McLean and Nakane T-test was used to compare the groups as indicated in the figures. One way ANOVA with Tukey’s multiple comparison test was used for comparing multiple groups.Proteoglycan loss is a hallmark of early disc degeneration and trypsin injection has previously been used to mimic this in bovine discs Viable and metabolically active cells are necessary for matrix homeostasis or tissue remodelling. Although trypsin-treated discs only displayed a minor drop in pressure after one sequence of static load at 0.3 MPa, 14 days of dynamic loading of the same magnitude could eventually affect cell health adversely. Trypsin-treated and control discs were cultured for 14 days unloaded or under dynamic loading as indicated in Maintained chondroadherin abundance and structure was used as to evaluate the integrity of the disc extracellular matrix. The quantity of chondroadherin extracted from the trypsin-treated loaded and unloaded discs was evaluated in comparison to reference samples of freshly isolated discs. A similar level of chondroadherin was found in the loaded samples compared to the reference . A non-sp<0.0001. No statistical difference in proteoglycan content was observed amongst loaded trypsin-treated and control discs (p>0.05). The results indicate that physiological load has the ability to restore proteoglycan content in discs with mild degeneration . In trypsin-treated discs cultured in the absence of load, proteoglycan content decreased by 30% and 60% when treated with 10 and 100 ug trypsin, respectively, compared to control discs. The proteoglycan content was decreased by 44% and 64% when unloaded trypsin-treated discs were compared to loaded discs with the same dose of trypsin treatment. The proteoglycan concentrations in loaded trypsin-treated discs were significantly higher than in unloaded discs  were loaded for each sample-pool. The size distribution was similar in the loaded controls and loaded trypsin-treated samples, whereas the proteoglycans in the unloaded trypsin-treated samples were more heterogeneous in size with more fragmented molecules . The datp<0.001). Thus physiological load not only stimulates aggrecan production, but also that of type II collagen.Restoration of a functional extracellular matrix requires new collagen synthesis in addition to proteoglycan. Newly synthesised type II collagen is not cross-linked in the tissue and is therefore extractable. Therefore, the levels of extractable type II collagen were assessed . The quaTo evaluate proteoglycan distribution within the tissue, Safranin O and fast green staining were performed on 5 µm tissue sections. Very little Safranin O (red) staining was observed in the unloaded trypsin-treated samples, whereas control discs and trypsin-treated loaded discs showed a strong Safranin O staining . The resProteoglycan depletion is the first stage of disc degeneration and may be reversible with appropriate cellular stimulus. Physiological load could be such a stimulus. Trypsin was injected into the center of bovine discs to induce proteoglycan loss Disc degeneration with back pain is a leading cause of disability and loss of workplace productivity in otherwise healthy young adults exvivo have been described in the literature. Most of them were created to induce a more severe degeneration with the formation of a cavity so allowing injectable hydrogels to be evaluated A model of early degeneration is necessary in order to study the effect of load during this phase of degeneration. A number of methods using relatively high concentrations of proteases to induce degeneration in larger discs Chondroadherin is an important indicator of matrix homeostasis, as it is fragmented in degenerate adolescent scoliotic and adult human discs possibly as a result of excessive loading The healthy adult human spine sustains a wide range of compressive loads during daily activity ranging from 0.1–0.3 MPa, up to 1 MPa and more than 2 MPa while lifting a 20 kg weight with a rounded back. A loading regime mimicking a relatively sedentary lifestyle was chosen for this study. The loading regime mimics a daily regime with 2 h light physical activity in the morning followed by 6 h of a low activity work environment, another 2 h of light physical activity in the afternoon, and finally a longer rest period during the evening and night. We compared this to a situation mimicking complete bed-rest where no cyclic dynamic load was applied. We have previously found that bovine tail discs are less adapted than human IVDs to withstand cyclic dynamic load in our bioreactor system The importance of low frequency dynamic loading for IVD homeostasis is clear from the results. In addition to a restoration of the proteoglycan content throughout the NP region newly synthesized extractable type II collagen was significantly increased in the loaded discs. These molecules represent the major components of the healthy IVD extracellular matrix, with the swelling potential of aggrecan and tensile properties of collagen together providing a healthy disc with its resistance to load. Taken together our data demonstrate that tissue homeostasis in mildly degenerate IVDs is strongly improved by physiological load. Such knowledge is important for patient advice on lifestyle following a biological repair procedure for mild disc degeneration. It is also important to stress the value of physiological activity in young individuals in maintaining healthy discs.


Stingless bees were significant resources managed by Mesoamerican peoples during pre-Columbian times and remain important in particular areas. Our study aimed at inventorying stingless bees’ species, traditional knowledge and forms of use and management of them at the municipality of Nocupetaro, Michoacán, Mexico, a region of the Balsas River Basin.We inventoried the stingless bees of the municipality of Nocupétaro, Michoacán, México, through extensive collecting of bee specimens in different vegetation types. We then conducted semi-structured interviews to local experts in order to document their knowledge and management techniques of stingless bees’ species.Lestrimelitta chamelensis Ayala, 1999). The taxa identified are all used by local people. Scaptotrigona hellwegeri Friese, 1900; Melipona fasciata Latreille, 1811; Frieseomelitta nigra Cresson, 1878 and Geotrigona acapulconis Strand, 1919 are particularly valued as food (honey), medicinal (honey and pollen), and material for handcrafts (wax). All species recorded are wild and their products are obtained through gathering. On average, local experts were able to collect 4 nests of stingless bees per year obtaining on average 6 L of honey and 4 Kg of wax but some came to collect up 10–12 hives per year (18 L of honey and 24 Kg of wax).We identified a total of eight stingless bees’ species in the study area as well as three additional unidentified taxa recognized by people through the local names. Our inventory included one new record of species for the region (Local knowledge about use, management and ecological issues on stingless bees is persistent and deep in the study area. Information about this group of bees is progressively scarcer in Mexico and significant effort should be done from ethnobiological and ecological perspectives in order to complement the national inventory of bee resources and traditional knowledge and management of them. Apidae: Meliponini) are widely distributed in tropical and subtropical regions of the world[Meliponini bees are still available, mainly in the case of the Balsas River in Michoacan.Stingless bees , and these products played an important role both economically and sacred. During xunan-cab” (Melipona beecheii) being the most important cultivated species whose honey and pollen are used in traditional medical and regionally marketed. The Náhuatl and Totonac peoples of the Sierra Norte manage the “pisil-nekmej” (Scaptotrigona mexicana) which has an important regional marketing because of its medicinal honey[Melipona fasciata and Scaptotrigona hellwegeri) have been recorded to be managed by Nahuas people in a village of Atoyac de Alavarez[At present, the actual practices of the Mesoamerican meliponiculture must be considered as a strong and persistent culture of the indigenous and peasant peoples. Presence of old management techniques have been documented to currently occur among the Maya of the Yucatán Peninsula, the Nahal honey. In thesal honey, as wellAlavarez. AccordiAlavarez, of the Although recognized as an important region in terms of stingless bees’ diversity and culture, the Balsas River Basin is currently particularly poor in information on species richness, traditional knowledge and quality of their honey. We expect to find in the study area traditional ecological knowledge (TEK), management techniques, as well as high species richness of stingless bees, and we started to document such a supposition in part of this vast area. In order to fill the void contribute to this effort, our study was directed to inventorying the species richness of stingless bees in the region of the Balsas River Basin in the state of Michoacán, documenting local knowledge and management techniques by people of communities in the municipality of Nocupétaro. We consider this relevant, in view of the conservation of stingless bees’ species while exploring the potential of alternative productive projects.lugar en el valle , and is now a mestizo municipality with a total population of 7,799 inhabitants[Our study area included the territory of communities belonging to the municipality of Nocupétaro, Michoacán, located in the Central-western region of Mexico within the Balsas River Basin Figure . Nocupétabitants. The maiabitants.We inventoried the apifauna centering our attention on stingless bees. For such a purpose we conducted extensive collecting of bee specimens through the method of “direct searching” in 16 siThe specimens collected were slaughtered in lethal chambers with ethyl-acetate for subsThe specimens collected were deposited in the Entomological Collection of the Museum of Zoology, Faculty of Sciences, UNAM, Mexico. In addition to the taxonomic identification of specimens collected, distribution (defined as the proportion of sites where species were collected) was recorded.colmeneros who are skilled in collecting stingless bees’ products. Interviews were divided into two main sections: a) local knowledge of stingless bees , and b) use and management .For documenting traditional knowledge, use forms and management techniques by local people on stingless bees, we carried out 20 semi-structured interviews to local experts in 18 villages of the Nocupétaro municipality . The interviews were conducted using the collected specimens, in order to confirm the information. These local experts are locally recognized as In addition, we used the method of “participant observation”,29 workiFrieseomelitta nigra Cresson, 1878 was the species most widely found, appearing in 12 of the 16 collecting sites (75%) and covering the entire spectrum of vegetation types where the collections were conducted  in all the vegetation types and Geotrigona acapulconis Strand, 1919 at 8 sites (50%) and also found in all the vegetation types (except in the urban area) , in the case of this species an oak forest at 1210 m. Lestrimelitta chamelensis Ayala, 1999 represents a new record for the region and was collected directly in the nest, a hollow log of cirian , in a tropical dry forest at 555 m ; these are bees also called abejas de monte (wild bees) or colmenas que no pican (stingless bees colmenas). The local experts interviewed recognized 11 different types of stingless bees which are identified based on their morphological and behavioral characteristics as summarized in Table Stingless bees are locally called abeja bermeja (S. hellwegeri) by its red color and its defensive behavior. The stingless bee called zopilota (F. nigra) is recognized by its dark color and the wing tips white and it is also characterized by its docile behavior. The colmena de tierra or prieta de tierra (G. acapulconis) is identified by its dark color and its pattern of nesting in the ground as well as being very docile. Moreover, the colmena real (M. fasciata) is identified by its colors similar to Apis mellifera, its hairiness and size. The new record bee limoncilla (L. chamelensis) which is named for its distinctive lemon scent  is distinguished by local experts by attacking other species of stingless bees and steal their honey. The abeja esculcona (P. bilneata) is known for its aerial nesting and its defensive behavior. The bee cepimilla (Trigonisca sp) is identified by local experts for its small size and its habit of perching on the sweat of the people. The bee trompetera (N. perilampiodes) nests in hollow trunks and is named this way because the entrance to its nest is shaped like a trumpet made of beeswax. Additionally local experts known and cited three stingless bees’ species that were not collected and identified: abeja pintilla, abeja prieta esculcona and abeja sapito, , abeja zopilota (F. nigra), the colmena real (M. fasciata), and the colmena de tierra (G. acapulconis) are the best known, since all of them were referred by more than 80% of the local experts interviewed (Table abeja limoncilla (L. chamelensis) is moderately known since 35% of local experts referred to this bee. The least known stingless bees were the abeja esculcona (P. bilneata), abeja trompetera (N. perilampiodes) and the abeja cepimilla (Trigonisca sp).Out of all the species locally recognized, the d Table . The abemiel (honey), cerumen (beeswax), and pasacuareta (pollen), and they do not cause any damage to people (compared with Apis mellifera and wasps). Knowledge of stingless bees is transferred from parents to children and according to the 85% of the local experts interviewed; men know and recognize differences among stingless bees’ species, as well as their behavior and habits patterns, their nesting sites and characteristics, seasons and particular techniques for extracting products. For the contrary, 75% of the local experts interviewed referred to that women know better the techniques used for processing the products, particularly those requiring more specialization such as preparing wax for manufacturing candles, but also the form of preparing pollen and honey for food and medicinal purposes.Local experts expressed a positive perception in relation to stingless bees, all of them said that these bees are “good” since they provide cerumen, for preparing candles used during the día de muertos or día de las ánimas in November 2. Beeswax extraction collaterally allowed using honey and pollen. The manufacture of beeswax candles is an activity involving specialized practices which are carried out by women. The first step of preparation of beeswax is its washing with water, then boiling the washed beeswax with water, and repeating the process. The second last cooking without water was the moment for including the candlewick. Beeswax is also used for sealing traditional bowls made of gourds (Lagenaria siceraria), and nearly 30% of local experts said that beeswax is commercialized in areas out of Nocupétaro where it is used for managing grafting in fruit trees.According to 70% of the local experts interviewed, the main motive for using stingless bees has been their wax or Crescentia alata). It is also used as antiseptic, topically put on specific infected parts of the body, particularly eyes and cutaneous injures and infections. As anti-inflammatory honey is used for muscle fatigue, sprains, injures, mauls, dabbed on affected parts. The pollen or “pasacuareta” is edible; in general people said that when “pascuareta” has yellow colour its consumption should be avoided since it cause vomit.Honey is highly valued for its medicinal and food uses. According to local experts, honey is consumed almost immediately after being extracted, alone or accompanying a hot drink. As medicine, honey of stingless bees is used for treating different illnesses. It is consumed liquid in mixture with different ingredients for treating cold, cough, bronchitis and other respiratory illnesses  (MI: 80%), colmena real (M. fasciata) (MI: 60%), abeja zopilota (F. nigra) (MI: 55%) and the colmena de tierra (G. acapulconis) (MI: 45%)  and abeja zopilota (F. nigra) had the highest preference. However, when we asked about use of beeswax, the colmena de tierra (G. acapulconis) and abeja bermeja (S. hellwegeri) has the highest preference, since according to local people this species produces the highest amount of beewax. Nearly 15% of people interviewed said that honey of the abeja zopilota (F. nigra) is a good remedy for treating biliary problems and diabetes.The use of stingless bees has focused primarily on four species that have been the most widely used, the  Figure . Althougcolmenear (looking for bee nests), and this activity was complemented by identifying nests when carrying out other activities of their daily life. Although there is a deep knowledge on stingless bees in the study area, no raising practices or meliponiculture was recorded, therefore obtaining products derived from stingless bees is by direct extraction and partial or total destruction of nests. Currently, the use of stingless bees’ products is only occasional and may be considered in disuse. The extraction and use of stingless bees’ products was popular the last century until the 60’s where 90% of local experts identified the beginning of the decline of stingless bees’ populations, and became particularly strong in early 90’s when some species disappeared. The main causes identified as factors diminishing of stingless bees according to 75% of local experts have been the increase in the abundance of Apis mellifera; extractive practices and land use change (65%); drought (50%); use of insecticides, pesticides and agrochemicals (15%), and fires associated with long periods of drought (5%). Nearly 70% of local experts interviewed considered that stingless bees will disappear relatively soon.Local experts referred that in the past there were persons who went to the forest exclusively to colmenas de tierra (ground colmenas), people excavate holes using tools like picks, shovels, drills. People carefully try to find the nest from its entrance emerging up on the ground towards the main body of the nest down underground  are all called cera (wax).A nomenclature system was found for the parts of the nests. The storing pots of honey and pollen are called  Figure . The quetejón (Nasua narica), tlacuache , skunk zorrillo (Mephitis sp), coyote (Canis latrans), armadillo (Dasypus novemcinctus), ants and the wasps locally called avispas carneras. Two persons recognized the parasitic interaction of the stingless bee limoncilla (L. chamelensis) with other stingless bee species, as well as with the euorpean bee (Apis mellifera).Nearly 30% of the local experts interviewed said that they extract only the honey storing pots and a bit of wax, leaving intact the cells hive re-covering then the nest allowing the bees the reconstruction of the nest until the following harvest season. However, they also said that generally after extracting products the hives become lost because of the exposition to predators, parasites, and other environmental factors. People identified as the main predators of bee hives the badger colmenero extracted four hives of stingless bees per year, obtaining on average 6 L of honey and 4 Kg of beewax; however, some local experts said to collect up 10 to 12 hives per year obtaining on average 18 L of honey and 24 Kg of wax, amounts that indicate that the activity and use of these resources were important for people of Nocupétaro. Even when the colmeneros extract a relatively high amount of stingless bee’s products, commercialization is restricted to wax and it occurs at low scale, as candles in small store or for grafting. Honey is collected for direct consumption of households that collect it.On average, every local expert or L. chamelensis was identified as a new record of species for the Balsas River Basin of Michoacán, and was recorded the presence of M. fasciata, which is endemic to the region and are recognized as mountain species, inhabiting zones of relatively higher elevations[L. chamelensis for the area is transcendent since its presence depends in turn on the abundance of other stingless bees’ species because it is a clepto-biological species.According to Ayala, have beevations,3. The nabeja pintilla, abeja prieta esculcona and the abeja sapito. This kind of stingless bees where mentioned by local experts but not collected and they could represent different. Additionally we can assume this because were not explored all vegetation types, particularly crops such as sesame or peanut.An important aspect derived from this study is that it is possible to suppose that the number of stingless bees’ species in the study area is higher because the registration of the Local identification of stingless bees’ species incorporates not only morphological but also ecological, behavioral, and social characteristics, as described in other studies and cultural contexts.colmeneros, who practiced a specialized activity, but mainly by the extraction process.There are recently published works that integrate exhaustively the status of meliponiculture on a global scale, which uCephalotrigona eburneiventer [Cephalotrigona oaxacana [Cephalotrigona zexmeniae [Frieseomelitta nigra [Geotrigona acapulconis [Melipona beecheii [Melipona colimana [Melipona fasciata [Melipona lupitae [Melipona solani [Melipona yucatanica [Nannotrigona perilampoides [Paratrigona guatemalensis [Partamona bilineata [Plebeia frontalis [Plebeia manantlensis [Plebeia moureana [Plebeia pulchra [Scaptotrigona mexicana [Scaptotrigona pectoralis [Scaptotrigona hellwegeri [Tetragonisca angustula [Trigona nigérrima [Trigonisca maya [Trigonisca sp.,  and L. chamelensis,  totaling 26 useful species. This represents that of the 46 species described for Mexico[Doing an exhaustive literature review, we found the report of 24 species of stingless bees that are used in Mexico in some form. These species are: z, 1948), Cephaloa, 1999), Cephalol, 1912),34, Frier, 1836), Geotrigt, 1831),22, Melie, 1811),21, Melil, 1912),23, Melin, 1878),20,34,35n, 1845),19,20,35F. nigra, S. hellwegeri, M. fasciata and G. acapulconis at the same time are most preferred species for use. This represents a universe of species with high potential for local management and technological innovation in the study area. For this purpose would be of great importance to develop ecological studies to determine the distribution and abundance of stingless bees. This information is essential to generate plans for sustainable management of this group of insects. In this sense, it is important to note that M. fasciata seems to be a rare species since we collected only two specimens in one site, and no nests were identified, and G. acapulconis nests on the ground making it difficult to use.On the other hand it is clear that there are stingless bees’ species with a higher level of importance because the local preference of their products. This differential valuation in specific cultural contexts has been described in other studies, mainly in plants. In thisWhile it is true that honey is one of the main stingless bees’ products, is also true that there is a need to establish quality criteria. Recently the demand for natural, organic and homeopathic products has increased, including those of stingless bees and particularly for the stingless bees’ honey have not yet been defined formal norms or quality standards, which iXylocopini (xicota), Bombini  and Vespidae . This is relevant since other authors[uauapu  between Purépecha culture (territory adjacent to the study area). This motivates the exploration of the relationship between society and insects in the study area, including stingless bees but also in a broader sense, including other insects.During the fieldwork we recorded general information about eight different types of Hymenoptera that people of Nocupétaro know such as  authors highlighMeliponini. Nonetheless local experts of Nocupétaro perceive and recognize that extraction rates of bee products have decreased more recently alongside the increase of vegetation clearing and use of insecticides threatening the permanence of stingless bees, local knowledge and the use of resources they provide persist in the study area. For this, it is relevant to establish the consequences that environmental problems may have for both, populations of stingless bees and local culture associated with them. In this sense ethnozoological and ethnoentomolgical studies are a key reference to direct these efforts.Bees are among the main pollinators of flowering plants throughout the world and their conservation is a current main issue of both science and society. This is a general premise for all bees’ species but it acquires particular relevance for those species that in addition to pollination represent provision ecosystem services as are several species of colmeneros to develop this activity. No forms of cultivation management was recorded in the studied area, but important experiences in other regions of Mexico offer the possibilities to encourage efforts for recovering the local techniques innovating both knowledge and management practices based on those from other areas of Mexico.The information generated suggests that conservation associated to use of bees  is still possible to be enhanced in Nocupétaro. Mainly because the resource and local knowledge remain in the study area. Also, for the interest of the Written informed consent was obtained for the publication of this report and any accompanying images.The authors declare that they have no competing interests.The main author ARG was responsible for literature review, research design, fieldwork, systematization and analysis of field data, wrote the first draft and concluded the final version of this paper. ACG main coordinator-supervisor of the research project contributed with the research design, participated in fieldwork, systematization and analysis of data and reviewed several drafts of the manuscript. ORS contributed with the research design, review of background literature and the discussion of theoretical aspects of the study. AA contributed to the review of background literature and the discussion of theoretical aspects of the study. AC contributed to designing and following progress of the research, field work and data analyses, and made substantial contributions to the manuscript. All authors have read and approved the final manuscript.


Use of integrative medicine (IM) is prevalent in children, yet availability of training opportunities is limited. The Pediatric Integrative Medicine in Residency (PIMR) program was designed to address this training gap. The PIMR program is a 100-hour online educational curriculum, modeled on the successful Integrative Medicine in Residency program in family medicine. Preliminary data on site characteristics, resident experience with and interest in IM, and residents’ self-assessments of perceived knowledge and skills in IM are presented. The embedded multimodal evaluation is described. Less than one-third of residents had IM coursework in medical school or personal experience with IM. Yet most (66%) were interested in learning IM, and 71% were interested in applying IM after graduation. Less than half of the residents endorsed pre-existing IM knowledge/skills. Average score on IM medical knowledge exam was 51%. Sites endorsed 1–8 of 11 site characteristics, with most (80%) indicating they had an IM practitioner onsite and IM trained faculty. Preliminary results indicate that the PIMR online curriculum targets identified knowledge gaps. Residents had minimal prior IM exposure, yet expressed strong interest in IM education. PIMR training site surveys identified both strengths and areas needing further development to support successful PIMR program implementation. Integrative medicine (IM) is prevention-based medicine that emphasizes the therapeutic patient-clinician relationship and uses all appropriate therapies . IM has The integrative approach is personalized and addresses nutrition, mind-body medicine, sleep, exercise, whole medical systems , environmental health, and social support.Interest in IM is significant, driven by consumer demand for care that is cost effective and better aligned with patient values ,3. A 200Training in IM occurred quickly in some specialties. A four-year combined residency and fellowship in family medicine and integrative medicine launched in 2004 . A 200-hPediatrics lacks IM training programs, despite data from the 2007 National Health Interview Survey demonstrating that nearly 12% of all children used complementary therapies, with prevalence increasing to 50% in those with chronic illnesses .Despite significant IM use in children and interest among pediatricians, only 16 of 143 pediatric academic programs surveyed reported having IM programs in 2012 [This article describes the PIMR program, a 100-hour online curriculum developed at the Arizona Center for Integrative Medicine (AzCIM) at the University of Arizona, currently being implemented at five residencies. Successful implementation will rely on a program’s capacity to adopt new curriculum, and on resident readiness to learn about IM. Therefore, data will be presented on residency site characteristics, resident experience with and interest in IM, and residents’ self-assessments of perceived IM knowledge and skills.Content development was based on guidelines from the joint ACGME and American Board of Pediatrics (ABP) “Pediatric Milestone Project” , competeThe online curriculum is modular. Self-contained units can be adapted for use in core rotations, used within electives, or distributed longitudinally. Interactivity is facilitated with case-based teaching. Onsite teaching and experiential activities tailored by the faculty site leaders round out the program. Onsite activities may include case conferences, self-care assessments, and experiential seminars. An annual faculty retreat provides faculty support and maintenance of current IM training.The PIMR program’s website is the hub of the resident learning community and includes online dialogues for questions and comments. Faculty site leaders track resident participation and course completion through an online dashboard. Faculty resources are housed on the PIMR website and include an article archive, intake forms, handouts, and patient education materials. Linked access is provided to the Natural Medicines Database.Evaluation of the PIMR program’s curriculum is embedded into the website, organized into four components: (1) medical knowledge test and self-assessment; (2) course completion rates; (3) curriculum evaluation; and, (4) assessment of resident wellbeing and wellness behaviors. Both quantitative and qualitative methods are used. The University of Arizona Institutional Review Board (IRB) approved the study, as did required pilot site IRBs.In 2012, five residency programs were selected to participate in a three-year project to implement and evaluate the PIMR program’s curriculum. Site inclusion criteria included endorsement by the Department Chair and Residency Director, and agreement to deliver the entire 100-hour curriculum to all residents over three years. Pilot sites include four university affiliated or based residencies and one community hospital residency The following measures were used to assess program capacity to implement the curriculum and resident interest and readiness to learn about IM:A. Site Capacity to Implement the PIMR Program: Pilot Site Characteristics. Within the first year of the PIMR program’s implementation, program leaders at each site completed an 11-item checklist assessing infrastructure characteristics supportive of PIMR implementation. Characteristics include an established IM culture, faculty background in IM, and site activities supporting the PIMR curriculum.B. Resident Interest in and Readiness for IM Training. Resident interest in and readiness for learning IM was assessed with three measures: (1) An eight-item post-match survey assessing prior IM/CAM  medical school coursework, personal use of IM/CAM, interest in learning IM, and interest in applying IM upon graduation. Items concerning prior IM experience were rated dichotomously (yes/no). Interest in learning and applying IM were rated on a 5-point scale; (2) A 32-item resident self-assessment questionnaire measuring confidence with IM knowledge and practice skills and ability to provide an IM approach to specific medical conditions. Items were rated on a 5-point scale from strongly disagree to strongly agree; and, (3) A 49-item medical knowledge test based on course content.Four of five sites had the two most commonly found site readiness characteristics: an IM practitioner working onsite (IM culture), and faculty with IM training and resources. Sites varied in overall capacity to implement the PIMR program, ranging from endorsing only 1 characteristic (site E) to endorsing 8 (of the possible 11) characteristics (site C). See Sample. Of the 107 incoming class of 2016 residents, 97 completed the post-match, self-assessment, and/or medical knowledge measures (91% response rate). Of these, 75% were female, 68% were Caucasian, and 5% were Hispanic. Most were US medical school graduates (77%), 7% were osteopathic medical school graduates, and 16% were foreign medical school graduates.Post-match Survey. The survey was completed prior to the July 1 start date. Less than one-third had IM/CAM coursework in medical school or personal experience with IM/CAM modalities. Sixty-six percent were interested in learning IM and 71% indicated interest in applying IM after graduation. See Self-Assessment. The highest rated knowledge/skills items concerned: patient behavior change, familiarity with diets targeting cardiovascular disease, and physical activity recommendations. The lowest rated knowledge/skills items concerned dietary supplements and whole systems medicine. The ability to apply an integrative approach to medical conditions was highest for sleep, depression, and migraines, and lowest for rheumatoid arthritis, hyperlipidemia, and Type II Diabetes. See Integrative medicine offers a powerful approach to a healthy lifestyle and can expand treatment options in children and adolescents. Use of complementary therapies is high in children, especially those living with chronic illnesses . PediatrPreliminary results indicate that the PIMR curriculum targets identified knowledge gaps. The self-assessment and medical knowledge measures confirm the need for residents to receive exposure to this information. For example, few had awareness of IM approaches to common pediatric diagnoses such as asthma, attention deficit hyperactivity disorder, or migraine headaches. Self-assessment items specific to IM, such as knowledge of dietary supplements, received lower ratings, while more conventional topics, e.g., patient readiness to change, received higher ratings. Less than one-third had prior education or personal experience in IM. While deficits in skills and knowledge in IM would be expected upon starting residency, administration of the self-assessment and medical knowledge measures annually will allow us to track growing mastery of the curriculum content and identify content areas needing refinement.Program implementation relies on the ability of residency programs to deliver IM education and to create a culture supportive of IM. Our initial site survey identified faculty background in IM and presence of affiliated IM practitioners as strengths across the sites. Further development is needed to support onsite IM educational activities. Site characteristics will be assessed annually to track evolution of the PIMR program’s implementation and to identify characteristics associated with curriculum completion. Evaluation of the IMR program in family medicine to date indicates that the best predictors of successful program implementation are requiring program completion for graduation and including a greater number of onsite IM activities .Success of the PIMR program will likely depend on residents’ openness, interest, and readiness for IM training. In our survey, two-thirds of the residents expressed an interest in IM, and almost three-fourths were interested in applying IM after graduation. These findings, coupled with the self-assessment and medical knowledge results, suggest pediatric residents are interested, yet unschooled in IM when entering residency. The PIMR program provides a flexible, online curriculum that may satisfy resident interest and fill this knowledge gap. Evaluation of the curriculum is ongoing, and content will be refined in subsequent years to address identified learning gaps and feedback by participating residents and faculty.


The earthquake nucleation process has been vigorously investigated based on geophysical observations, laboratory experiments, and theoretical studies; however, a general consensus has yet to be achieved. Here, we studied nucleation process for the 2014 Iquique, Chile Mw 8.2 megathrust earthquake located within the current North Chile seismic gap, by analyzing a long-term earthquake catalog constructed from a cross-correlation detector using continuous seismic data. Accelerations in seismicity, the amount of aseismic slip inferred from repeating earthquakes, and the background seismicity, accompanied by an increasing frequency of earthquake migrations, started around 270 days before the mainshock at locations up-dip of the largest coseismic slip patch. These signals indicate that repetitive sequences of fast and slow slip took place on the plate interface at a transition zone between fully locked and creeping portions. We interpret that these different sliding modes interacted with each other and promoted accelerated unlocking of the plate interface during the nucleation phase. Subduction of the Nazca plate beneath the South American plate, at an average rate of ~7 cm/yr, has produced a series of megathrust subduction earthquakes along the Chile–Peru Trench . The mai2145A recent deployment of modern seismic and geodetic instrumentation along the northern Chile coast has provided an excellent opportunity to explore the nucleation process of megathrust earthquakes. According to previous studies, the mainshock was preceded by intensive foreshock sequences and geodetic signals lasting at least two weeks, showing gradual unlocking of the plate boundary fault updip of the largest coseismic slip patch578910Because seismicity is one of the most powerful tools for identifying nucleation processes, it is essential to investigate the spatial–temporal evolution of earthquakes leading up to the 2014 mainshock rupture8121314161718192021Finally, we computed the time-dependent rate of background seismicity by applying the non-stationary epidemic-type aftershock sequence (ETAS) model232325We identified 11690 earthquakes, which is nearly 17 times the number of available template events listed in the USGS catalog (684 events) , showing2In the summer of 2013, an intensive seismic swarm occurred around the southern edge and updip of the largest slip patch of the mainshock rupture  and 3a. A second crisis of seismic bursts occurred at the southern and northern edges of the mainshock rupture area between January and February 2014Following the reactivation of the northern edge of the rupture zone on 15 March 2014, the final seismic burst was characterized by intense multiple earthquake migrations during the periods 15–17 March, 16–18 March, and 22–24 March 2014, which extensively developed within the entire foreshock zone8et al.From our expanded earthquake catalog  and 3, wThese migrating seismic bursts were accompanied by the transient increase in aseismic slip and the background rate of seismicity  and 3. F8111429et al.During the final 17 days before the mainshock , the continuous GPS stations located along the coast near the source region started to move trench-ward. These surface deformations resulted from the gradual unlocking of the plate interface55We calculated surface deformations produced by the repeater-inferred aseismic slip  = (0.25–2.0 Hz), (0.5–4.0 Hz), (1.0–8.0 Hz), and (2.0–16.0 Hz). The time window of each seismogram extended from 5.0 s before the P-wave arrival to 15.0 s after the S-wave arrival, which was long enough to include direct and coda phases. We selected candidates of repeating earthquake pairs as those whose cross-correlation coefficients were ≥0.95 at two or more stations in one frequency band, roughly corresponding to the corner-frequency inferred from the average magnitude of each event pair assuming a circular patch model with a constant stress drop of 3 MPaWe selected earthquake pairs with epicenter separations of ≤50 km and calculated cross-correlation coefficients using passband-filtered vertical component seismograms. We used four different frequency bands, as follows:  due to the richness of high-frequency content in the P-wave seismograms. We calculated the largest differential S–P arrival times over the seismic network, all of which had a normalized cross-correlation coefficient of ≥0.75. Assuming typical P- and S-wave velocities for subducting oceanic crust , we computed d by multiplying the largest differential S–P time by 8.5 km/s. We compared these d with the rupture radii R1 and R2 of the two earthquakes estimated from a circular patch modeld ≤ max {R1, R2}. Finally, we grouped together all pairs of repeating earthquakes that share a common earthquake, resulting in 163 groups of 408 repeating earthquakes in the magnitude range 2.0 to 4.6. In addition, we are interested in the inter-event distance The displacement of aseismic slip  and 3b w17To project the aseismic slip onto the plate interface, we first estimated the aseismic slip distribution during the final 17 days before the mainshock  by averaging the cumulative slip of different groups of repeating earthquake located within 0.15 × 0.15° grids shifted in 0.075° increments . We thenIt could be argued that the assumed stress drop assumed for the extraction of repeating earthquakes could affect the evolution of aseismic slip. To investigate the sensitivity of the assumed stress drop on repeater-inferred aseismic slip, we analyzed two circular patch models with different assumed stress drops: 1 MPa  and 10 MMc). Among the parameters  of the ETAS model, the background rate of seismicity μ and the aftershock productivity K0 are most sensitive to the sources of non-stationarity2324μ and the aftershock productivity K0 in the ETAS modelWe applied the nonstationary ETAS model23μ(t) and K0(t) should be related to the crustal stress changes caused by aseismic transient deformation and triggering effect by earthquakes, respectively. Note that any potential changes to the other parameters are absorbed by the temporal change in K0(t) in our model. We first estimated the three parameters α, c and p of the ETAS model using the earthquake data from May 2009 to June 2013, where the seismicity rate had been almost constant (stationary) (c = 9.76 × 10−5 (±0.89 × 10−5), α = 0.987 (±0.103), p = 0.866 (±0.081), respectively. Fixing these three parameters  to be constant, we inverted earthquake occurrence for the optimal solution of the time variable parameters μ(t) and K0(t) under proper smoothness constraints with the assumption that the aseismic stress and the changes in the triggering parameter both evolve smoother than the seismicity rate changes. We then applied smoothness constraints to prevent arbitrarily rough variations in μ(t) and K0(t) which were not justified by the data. Defining the roughness penalty functions, Φμ and ΦK, as the sum of squares of the gradients in the respective parameters, the penalized log-likelihood against the roughness becomesare modified to be time dependent in such a way that tionary) : the valq = (μ(t), K0(t)). ln L(q) represents the log-likelihood function of the point process, and the weight parameters wμ and wK are the weights of the smoothness constraints. Rather than using ad hoc values, we selected optimal weights for the inversion via the Akaike Bayesian Information CriterionQ as a result of the linearized functions of q.where How to cite this article: Kato, A. et al. Accelerated nucleation of the 2014 Iquique, Chile Mw 8.2 Earthquake. Sci. Rep.6, 24792; doi: 10.1038/srep24792 (2016).


The aim of this study was to design the content and accompanying materials for a school-based program—Study without Drugs—for adolescents in junior secondary schools in Suriname based on the starting points and tasks of the fourth step of the Intervention Mapping protocol (which consists of six steps). A program based on this protocol should include a combination of theory, empirical evidence, and qualitative and quantitative research.Two surveys were conducted when designing the program. In Survey I, teachers and students were asked to complete a questionnaire to determine which school year they thought would be most appropriate for implementing a drug-prevention program for adolescents (we completed a similar survey as part of previous research). An attempt was made to identify suitable culturally sensitive elements to include in the program. In Survey II, the same teachers were asked to complete a questionnaire to determine the programs’ scope, sequence, structure, and topics as well as the general didactic principles to serve as a basis for program design. After outlining the program plan, lessons, and materials, we conducted a formative pretest evaluation among teachers, students, and parents. That evaluation included measures related to the program’s attractiveness, comprehensibility, and usefulness. The resulting lessons were presented to the teachers for assessment.The drug-prevention program we developed comprises 10 activities and lasts 2–2.5 months in an actual school setting. The activities take place during Dutch, biology, physical education, art, religion, and social studies lessons. We based the structure of the lessons in the program on McGuire’s Persuasion Communication Model, which takes into account important didactic principles. Evaluations of the program materials and lesson plans by students, teachers, and parents were mostly positive.We believe that using the fourth step of the Intervention Mapping protocol to develop a drug-prevention intervention for adolescents has a produced promising, feasible program.The online version of this article (doi:10.1186/s12889-015-2374-1) contains supplementary material, which is available to authorized users. The population is approximately around 566,000 and very heterogeneous. After around 300 years of colonization—first by England and then the Netherlands—Suriname became an independent republic on November 25, 1975. On November 25, 1980 a coup d’état took place, led by Desi Bouterse. During the period 1980–87, Suriname was often associated with drug trafficking. Democracy returned to the country in 1987.This article describes the development of Study without Drugs, a drug-prevention program recently implemented in junior secondary schools throughout Suriname. Suriname is located in northeastern South America and has an area of 165,000 kmThe Ministry of Education and Community Development is responsible for education in Suriname, which is based on the Dutch education system. In 2004, the Surinamese government announced it would implement education reforms in the country. The central aim of the reforms was to introduce an 11-year basic education system, consisting of integration of pre-primary, primary, and junior secondary schools. The language of instruction is Dutch.Basic education comprises 2 years of pre-primary school (for children aged 4 and 5 years) and primary school, which lasts 6 years (for children aged 6–11). After completing this education with a final test, pupils are awarded the Certificate of Primary Education. This certificate allows pupils to enter general junior secondary education, which is divided into a general program with a nominal duration of 4 years and a vocational program with a nominal duration of 1–4 years. The vocational program consists of several facets, such as vocation-oriented education. After graduating from general junior secondary education, pupils can move on to general senior secondary education.General senior secondary education is divided into general education, with a nominal duration of either 3 years (VWO) or 2 years, and vocational education, with a nominal duration of 1–4 years. General senior secondary education diplomas grant admission to higher education (colleges and university). Admission to Anton de Kom University of Suriname is usually granted on the basis of the VWO diploma. Suriname has a binary higher education system. With four faculties and a number of research institutes, Anton de Kom University is the only university in the country. Suriname also has a number of higher professional education institutes as well as an institute offering bachelor’s programs for higher professionals and master’s and postgraduate programs . Those findings and other reports detail adolescent drug use and later dependency , 40–42, The reactions of teachers regarding the scale and construction of the program and its structure are in line with the results of studies into the factors that determine the efficacy of school-based prevention projects. Dusenbury et al.  identifiMethodologically, the composition of the sample that participated in the pretest of the developed materials may have influenced the outcome since all the participants were volunteers. The subjects also performed under time pressure: limited time was available for the pretest of the developed material. That may have led to socially desirable replies because the students, teachers, and parents may have felt compelled to complete the pretest in a timely manner.After the pretest, an interview was conducted, in which each respondent could explain their scoring. In retrospect, that may have influenced their replies for reasons of accountability. First, the presence of the interviewer may have influenced the outcome of the answers. Second, the respondents who participated in the pretest were chosen by the respective school management teams. Those respondents may thus possibly have felt obliged to give positive replies to questions. Moreover, the sample was an exploratory sample: it was just a small group of parents and teachers who were approached for the pretest. Therefore, the sample was not completely representative.Regarding the cultural sensitivity of the program content, we made an attempt during Survey I to inquire which cultural elements should be adopted in the program. However, that attempt to identify cultural elements may be considered rather superficial. In reproducible research, it is generally recommended that greater attention be paid to issues of cultural sensitivity. Furthermore, we followed one of the basic principles of IM—comprehensive involvement of stakeholders by involving students, teachers, and parents during the survey and pretesting.We learned a number of lessons while developing Study with Drugs. For example, regarding responses, it is very important to take into account the opinions of all parties involved: those opinions relate to the suitability and usefulness of the materials being developed. Furthermore, it is necessary to bear in mind that developing program content and accompanying materials is not an easy task since various aspects have to be considered, e.g., sufficient finances and knowledge of technical aspects of producing such materials as posters and brochures.Program developers need to be informed about those factors that help determine whether lessons and material may be effective, e.g., whether they incorporate the language and ideas of contemporary youth regarding such subjects as drugs. It is advisable that developers familiarize themselves with existing effective programs and materials and have a planning and design team available before embarking on an ambitious task like developing a program plan and accompanying materials. We used the fourth step of the IM (basing the development of our drug-prevention program on existing evidence and theories) and followed that protocol in implementing the pretests and initial pilot evaluations; that allowed us to design the content of the supporting material for a school-based drug-prevention program in Suriname. Further research in the form of test implementation is necessary to provide insight into the program’s effectiveness. Such test implementation would allow an experiment and control group to be compared with each other. With respect to composition of the groups, VAB-matched respondents should be used and the principles of randomized controlled trails adopted as far as possible. Both the process and effect of evaluation need to be taken into account.


Supervision of meals is an essential part of intensive eating disorder treatment. However, there is a lack of evidence regarding the most effective approach when delivering this service. This study evaluated an eating disorder day program's core components of meal supervision against set criteria, and how meal supervision was received by patients.All staff of an eating disorders day program were observed completing meal supervision by an external assessor over a three week period. An audit tool was developed using the program's established mealtime guidelines. Descriptive feedback was sought to evaluate the patients' experience.One hundred percent of meals assessed were completed within program guidelines and done so consistently by all staff. Staff commonly used time and behaviour prompts. They explained meal guidelines inconsistently, but as deemed necessary. Staff were more confident with food than fluid guidelines. Patients valued autonomy and less support as they progressed in treatment.Day program staff were compliant with the program's mealtime guidelines and were consistent when doing so. Meal supervision was regarded as a positive aspect of treatment, and patients valued meal support being tapered as they advanced.Service Initiatives stream of the 2014 ANZAED Conference.This abstract was presented in the


The EQAPOL contract was awarded to Duke University to develop and manage global proficiency testing programs for flow cytometry-, ELISpot-, and Luminex bead-based assays , as well as create a genetically diverse panel of HIV-1 viral cultures to be made available to National Institutes of Health (NIH) researchers. As a part of this contract, EQAPOL was required to operate under Good Clinical Laboratory Practices (GCLP) that are traditionally used for laboratories conducting endpoint assays for human clinical trials. EQAPOL adapted these guidelines to the management of proficiency testing programs while simultaneously incorporating aspects of ISO/IEC 17043 which are specifically designed for external proficiency management. Over the first two years of the contract, the EQAPOL Oversight Laboratories received training, developed standard operating procedures and quality management practices, implemented strict quality control procedures for equipment, reagents, and documentation, and received audits from the EQAPOL Central Quality Assurance Unit. GCLP programs, such as EQAPOL, strengthen a laboratory's ability to perform critical assays and provide quality assessments of future potential vaccines. The EQAPOL Program is comprised of a Central Management Team, Central Quality Assurance Unit (CQAU), Statistical Group, Data Management Group, Biorepository, Central Laboratory, A3R5 Neutralizing Antibody Assay Validation Program, and three EQAPOL Oversight Laboratories (EOLs) described in detail in this issue of Journal of Immunological Methods . In addition to proficiency testing, EQAPOL is also tasked with creating a diverse panel of high-titer  are required to operate under Good Clinical Laboratory Practices (GCLP), since this is a set of standards designed to facilitate uniform and consistent data generation and reporting. GCLP encompasses both quality assurance (QA) and quality control (QC) principles into its standards. QA proactively and periodically reviews the various components of the research process to assess adherence to standard operating procedures (SOPs) and policies and to determine the accuracy of research records. QC measures are continuous and carried out on all records  by the Laboratory Teams. While external laboratories, participating in the EQAPOL proficiency testing programs, are not required to operate under GCLP, many of these laboratories are already GCLP-compliant and perform clinical trial related work. It is for this reason that the program operates in GCLP compliance as it ensures the quality, integrity, and validity of the test data.GCLP was initially designed by the British Association of Research Quality Assurance (BARQA) in 2003 and later expanded upon by the NIH/NIAID/DAIDS in 2008 to provide a regulatory framework to laboratories performing endpoint assays for HIV-1 human clinical trials . The twoExternal Quality Assurance (EQA) Programs serve three purposes according to GCLP guidance: 1) provide a way for laboratories to ensure that data generated are timely, accurate, and clinically appropriate; 2) provide sponsors with assurance that data generated are of the highest quality; and 3) ensure that human specimens from clinical trials will be tested accurately and reliably . AlthougIn addition to the proficiency testing programs, EQAPOL was also charged with establishing and characterizing clade-specific HIV-1 viral culture panels representing world-wide genetic diversity. These HIV-1 viral diversity panels are created from HIV-1 positive plasma specimens received from collaborators or from currently existing viral culture supernatants.Finally, as an option exercised by the NIAID contract, EQAPOL was also charged with performing formal validation of specific immunogenicity assays to be employed as endpoint assays for HIV vaccine clinical trials. The Neutralizing Antibody Assay for HIV-1 in A3R5 cells was optimized and formally validated  under the oversight of the EQAPOL CQAU.This report describes the process by which GCLP compliance was established for the entire EQAPOL Program.Prior to implementing GCLP throughout the EQAPOL Program, the CQAU performed an overall assessment of each EQAPOL Laboratory Team. In order to effectively implement document and version control of SOPs, and manage training, equipment and calibration/maintenance records, the CQAU adopted a Quality Management System (QMS)  that allowed all of the aforementioned information to be captured and maintained electronically. The software package allows laboratory personnel to access SOPs on any computer with internal server access, which eliminates the need to have paper copies of SOPs present in the laboratories – further mitigating the risk of expired/uncontrolled procedures being used in the laboratory. The use of electronic copies also allows for better version control as paper copies may be altered while in use in the laboratory. In addition, the QMS allows the CQAU to electronically distribute SOPs to laboratory staff, maintain version control of documents, and manage SOP revision and training records.http://www.access.gpo.gov/nara/cfr/waisidx_05/21cfr11_05.html.), the QMS software utilizes an electronic signature package allowing users to enter their username and password to sign-off on training records, audit findings, and equipment maintenance records, which provides an audit trail.The QMS has also been instrumental in streamlining the audit process by allowing auditors to complete a report and present findings electronically to each EQAPOL Program Team. The use of tablet computers running QMS-specific applications allows audits to be conducted in a more efficient manner in BSL-3 containment environments where removal of paper is restricted. Laboratory managers are able to view all audit findings and address them via corrective action entries. In order to ensure 21 CFR, Part 11 compliance  . An SOP how to conduct the assay,training for the assay,proper instrument use and maintenance,reagent bridging, anddata analysis.In addition to program-specific SOPs, EQAPOL Management developed SOPs that detailed how external proficiency (EP) kits are assembled and ordered using the EQAPOL web portal. While facilitating the control of SOPs, the QMS notified users of the effective dates for new SOPs and sent reminders when documents were due for review.When conducting assays, EQAPOL laboratory operators are required to complete assay-specific checklists to document who performed each step. Assay checklists are associated to version controlled SOPs and accessible through the QMS. Checklists have been used in many industries as a way to improve quality and safety . RecentlIn order to conduct a GCLP compliant study and proficiency testing program, the Study Director needs to write a complete Study Plan outlining the scope and conduct of the study. Although EQAPOL is not fully adherent to ISO standards, the Study Plan incorporates many of ISO's concepts. EQAPOL Study Directors, Leadership, and the Statistical Group prepare program-specific Study Plans for each round of testing under the CQAU oversight. The EQAPOL Study Plans for proficiency testing program include the following:a detailed list of the program's organizational and management structure,introduction and background of the program,requirements for participation,number and type of program participants,proficiency panel contents,assay specifics,timeline for completion and upload of data,methods for data reporting,confidentiality, andmethods for statistical analysis and grading.EQAPOL Study Plans are approved and finalized by the EQAPOL Study Directors, Leadership, the CQAU Director, and the Statistician prior to the implementation of a particular EP send-out. Study Plans provide evidence that the EQAPOL EP was planned thoroughly and received Central Leadership approval prior to the conduct of the study. The Study Plans are not shared with the laboratories participating in the proficiency testing programs, as they contain information that could unblind their testing.The EQAPOL CQAU performs internal and in-process audits of each EQAPOL Laboratory Team  as required by GCLP guidelines to identify any potential gaps in compliance and/or deviations from established protocols/SOPs/Study Plans. Audits are performed by the EQAPOL CQAU with the assistance of other QA personnel not involved in the EQAPOL contract, to eliminate potential bias. Checklists are used throughout all audits and reports are prepared with any findings that need to be addressed. Laboratory personnel then respond to the audit findings with appropriate corrective actions to address the issues. The CQAU oversees the proficiency test kit assembly, reagent preparation, and shipment by the EQAPOL Central Laboratory, for at least one send-out per year for each program. As these are often the only proficiency testing materials that clinical trial immunogenicity endpoint assay laboratories receive, it is crucial that all aspects of the send-out are subject to CQAU oversight. At the conclusion of the audits, the CQAU prepares a report summarizing the kit preparation, send-out process and congruence with what is described in the Study Plan. Throughout the first series of audits, common findings existed for many of the EQAPOL Laboratory Teams. These non-conformances typically included a lack of a reagent bridging SOP, assay-specific training SOP, internal competency testing program, and SOPs for all pieces of equipment used during the conduct of the assay .In previously reported studies, performed by the CQAU, it was demonstrated that it is easier to transfer an assay from one laboratory to another when the assay had been optimized and validated prior to the transfer . This prEQAPOL developed a Biorepository to manage all of the reagents and samples used in the External Quality Assurance (EQA) Programs, virus panel development, assay development, and validation. All materials are recorded electronically in the EQAPOL Application web-portal, which captures the reagent name, lot number, expiration date, and storage location.The EQAPOL Biorepository uses an electronic, wireless temperature surveillance system for constant monitoring of all freezers, refrigerators, and incubators. Repository staff members receive alerts upon deviations from established temperature ranges for all monitored equipment. With this temperature monitoring software, the Biorepository manager and CQAU auditors are able to view and determine when out-of-range temperature excursions occurred, the operator who handled the excursion, and the corrective action taken to address the deviation. This information is stored permanently on the EQAPOL server which is backed up nightly and the back-ups are stored off-site. A Laboratory Data Management System (LDMS) is utilized for the inventory of peripheral blood mononuclear cells (PBMCs) that were processed from fresh leukapheresis blood units and all source material and viruses received/cultured as part of the EQAPOL Viral Diversity Program.In accordance with GCLP guidelines, all new lots or batches of reagents and samples are parallel tested with previous lots to document their compatibility in the assay prior to being placed into use. Each EOL defined in SOPs the critical reagents required to be parallel tested as well as pass and fail criteria for their eligibility to be placed in proficiency testing kits. Examples of such reagents include PBMCs, Fetal Bovine Serum (FBS), and Streptavidin. Reagents that did not pass parallel testing were not permitted for use in the EQA proficiency test kits.As another aspect of the EQAPOL system, a user-friendly and comprehensive web-based portal was developed to secure all of the electronic data generated from the program. The web-based portal was developed specifically for the needs of EQAPOL by SciMed Solutions and operates in compliance with the Federal Information Security Management Act of 2002 . Within The EQAPOL web portal is used to document all aspects of each EQA program from send-out of material information to data upload to EQA report retrieval. It maintains reagent and specimen inventory and location, as well as certificates of analysis for reagents. For each EQA send-out, the system records all samples/reagents sent to each site, shipment manifests, tracking information and shipment sent/receipt dates. For each EQA sendout, sites can download assay protocols and data reporting templates on the web-based system. Once a site completes an assay, the data reporting template is completed and returned using the web-based system. Sites also complete a post-assay questionnaire using the portal. As data are received from each site, the templates undergo data verification to ensure that the data are in the proper format prior to importing them into the EQAPOL database. Specific data verification instructions have been developed for each EQA program. The EQAPOL statisticians access all EQA data directly from this database ensuring data integrity throughout the system.Once the statisticians complete all data analysis, site-specific reports are generated and uploaded to the web-based system with each site having access to their site report. Sites receive both a score out of 100 and a performance rating for each EP, with points awarded for different performance criteria . These scores and performance ratings are entered into the web-based system to allow performance tracking over time. Finally, the web-based system can capture comments and files used as part of the remediation process. This comprehensive system allows for complete traceability of the EQA process.The web portal also allows investigators to place orders for viral samples that are prepared by the EQAPOL Viral Diversity Core. Additionally, the portal captures virus culture and characterization data, and provides a means for approved laboratories to view virus inventory and order products. The system maintains an electronic inventory of all specimens and reagents for EQAPOL, and all applicable compliance information  to ensure that sites only receive material which they are authorized to receive. These orders are then processed by EQAPOL Program Management and sent through an approval process.A key aspect to maintaining GCLP-compliance within the EQAPOL Program is to keep Leadership abreast of key quality issues on a regular basis. The EQAPOL CQAU meets with the EQAPOL Steering Committee on a bi-weekly basis with updates on audits, SOP development, proficiency testing kit preparation and shipment status, and other key quality matters. The Steering Committee is comprised of the Program Director and Co-Director, Study Directors, IT Personnel, Finance, Regulatory Affairs Compliance, Statisticians, CQAU and other critical program managers. In addition, the CQAU presents annual progress updates to the EQAPOL External Scientific Advisory Board and receives feedback from the Board.The EQAPOL CQAU provides archival functions for all study and proficiency testing materials. The materials are required to be kept in case a study needs to be investigated. Obsolete SOPs, study plans, raw data, and quality control documents are given to the EQAPOL CQAU and are stored in a space that has restricted access and is separated from the laboratory environment. Archived material is maintained indefinitely or until the Sponsor has requested destruction of the documents. In addition to on-site archiving, the CQAU has contracted with a third party sub-contractor to store documents off-site for extended periods of time.Implementation of GCLP into the EQAPOL Laboratory Teams began with an initial laboratory assessment, followed by GCLP training of laboratory staff, implementation of a QMS, equipment qualification, SOP document control, and audits by the CQAU. Converting EQAPOL into a GCLP-compliant operation has a number of added benefits that include increased credibility for the program and improved accuracy, integrity, and traceability of generated data. Having a CQAU in charge of monitoring the EQAPOL project has allowed for more stringent quality control measures of all processes within the program.Implementing GCLP guidelines designed for clinical trial testing into a setting for proficiency testing was ultimately a challenge for the EQAPOL CQAU. Converting all of the EQAPOL Laboratory Teams into GCLP-compliant laboratories required approximately two years and multiple hurdles were encountered along the way. One of the primary challenges for the EQAPOL Program was unifying all of the programs under one system of SOPs and document control. Since EOLs were members of different departments within Duke, not all of them followed the same system of SOPs. To address this dilemma, the QAU utilized an electronic QMS which ultimately incorporated multiple sets of pre-existing SOPs. Additionally, the CQAU did eliminate SOP redundancies by creating an overarching system of SOPs that were to govern common processes across all programs.SOP development for new GCLP assays was also a considerable hurdle because some of the laboratories lacked experience in the creation and usage of SOPs. Commitment from laboratories is typically weak at the beginning of GCLP conversion and leads to many delays in SOP finalization. The quicker a laboratory “bought-in” to the concept and importance of GCLP, the smoother the overall transition went. Throughout this two-year process, it became evident that those laboratories that had already implemented some of the key GCLP components into their operations were better prepared to become GCLP-complaint.The up-front cost of operating a laboratory under GCLP is often seen as a hurdle, yet mistakes are often more costly then preventative measures . UtiliziGCLP implementation and compliance in the EQAPOL laboratories have provided assurance that all processes are planned, performed, monitored, recorded, and reported in a reliable and consistent manner. Program participants can be assured that the kits are assembled with the utmost quality and detail and that all handling of data and statistical analysis had been thoroughly planned and executed. Ultimately, GCLP programs, such as EQAPOL, strengthen laboratory's ability to perform critical assays and provide quality assessments of potential vaccines in the future. Quality improvement within EQAPOL Laboratory Teams is an ongoing process and changes will continue to be made with future funding so that all aspects of the program can operate at the highest level of compliance possible.