Early infant male circumcision (EIMC) conducted by nurse-midwives using the AccuCirc device was safe and less costly per procedure than when conducted by doctors: for nurse-midwives, US$38.87 in vertical programs and US$33.72 in integrated programs; for doctors, US$49.77 in vertical programs. Early infant male circumcision (EIMC) conducted by nurse-midwives using the AccuCirc device was safe and less costly per procedure than when conducted by doctors: for nurse-midwives, US$38.87 in vertical programs and US$33.72 in integrated programs; for doctors, US$49.77 in vertical programs. The 14 countries that are scaling up voluntary male medical circumcision (VMMC) for HIV prevention are also considering early infant male circumcision (EIMC) to ensure longer-term reductions in HIV incidence. The cost of implementing EIMC is an important factor in scale-up decisions. We conducted a comparative cost analysis of EIMC performed by nurse-midwives and doctors using the AccuCirc device in Zimbabwe.Between August 2013 and July 2014, nurse-midwives performed EIMC on 500 male infants using AccuCirc in a field trial. We analyzed the overall unit cost and identified key cost drivers of EIMC performed by nurse-midwives and compared these with costing data previously collected during a randomized noninferiority comparison trial of 2 devices (AccuCirc and the Mogen clamp) in which doctors performed EIMC. We assessed direct costs  and indirect costs . We performed one-way sensitivity analyses to assess cost changes when we varied key component costs.The unit costs of EIMC performed by nurse-midwives and doctors in vertical programs were US$38.87 and US$49.77, respectively. Key cost drivers of EIMC were consumable supplies, personnel costs, and the device price. In this cost analysis, major cost drivers that explained the differences between EIMC performed by nurse-midwives and doctors were personnel and training costs, both of which were lower for nurse-midwives.EIMC unit costs were lower when performed by nurse-midwives compared with doctors. To minimize costs, countries planning to scale up EIMC should consider using nurse-midwives, who are in greater supply than doctors and are the main providers at the primary health care level, where most infants are born. For countries planning to roll out EIMC, using nurse-midwives to perform the procedure instead of doctors may be a solution. Nurse-midwives are able to provide comparably high-quality, affordable primary care.In relation to substituting nurse-midwives for doctors for medical male circumcision in general and EIMC in particular, there is a dearth of information on quality, outcomes, and costs. This situation is exacerbated by the fact that in some sub-Saharan African countries, including Zimbabwe, ministry of health guidelines preclude nurse-midwives from performing EIMC with devices that are currently in use, such as the Gomco clamp, Plastibell, and the Mogen clamp.AccuCirc (introduced in 2008) requires less surgical skill than the Mogen clamp and can thus be more easily used by nurse–midwives.In this study, we report results from a cost analysis of EIMC performed by nurse-midwives and compare them with costs estimated in a previous cost analysis of EIMC performed by doctors using the AccuCirc and the Mogen clamp in the same study setting in Zimbabwe.In this cost analysis, we sought to estimate the unit costs of EIMC performed by nurse-midwives in comparison with doctor-performed EIMC, and identify the key cost contributors. We collected cost data for nurse-midwives as part of a field trial (n = 500) which assessed the safety, acceptability, and feasibility of EIMC by nurse-midwives using the AccuCirc device.Costing Guidelines for HIV Prevention Strategies from the Joint United Nations Programme on HIV/AIDS (UNAIDS).Results from our companion paper suggest equivalence in safety profiles of EIMC performed by nurse-midwives in comparison with those for doctors.We accessed costs of supplies from invoices and receipts sourced from the respective research partners’ procurement and accounts departments. Nurse-midwives routinely recorded actual resource-use data  for each EIMC procedure on a supplies charge sheet in each infant’s binder. We captured and analyzed direct and indirect cost data using the Decision Makers’ Program Planning Tool (DMPPT), a Microsoft Excel-based model. The United States Agency for International Development (USAID) Health Policy Initiative and UNAIDS developed DMPPT to enable policy makers to analyze costs and impacts of different options for scaling up male circumcision services.We estimated the total cost of each consumable and nonconsumable drug and supply for 1 EIMC procedure using data on the number of units of each drug or supply needed per procedure and the corresponding unit cost of that drug or supply. The AccuCirc device was obtained for a unit price of US$10 from the international supplier, Clinical Innovations. Since AccuCirc is a single-use device, US$10 was the per-procedure device cost.,A local master trainer (doctor) provided theoretical and practical team training over the course of 5 days for 4 nurse-midwives. Personnel training costs included costs of the time spent by both the master trainer and the nurse-midwives, training material costs, and meal costs. We amortized training costs over a 3.6-year period in line with turnover rates of health care workers in Zimbabwe.To determine personnel unit costs, we conducted a time and motion study alongside the procedure to capture the time spent by nurse-midwives performing each task for all 500 infants involved in the study, as had been done in the earlier study involving doctors who performed EIMC.To determine personnel unit costs, we conducted a time and motion study alongside the procedure to capture the time spent by nurse-midwives performing each task for all 500 infants involved in the study.A nurse-midwife salary comprised basic pay plus housing and transport allowances and totaled US$13,596 per year. In the earlier comparative trial, a doctor's salary comprising basic pay and benefits was US$31,800 per year. We derived the total unit cost contribution of personnel to each procedure by summing costs of time for each step of the EIMC procedure for each nurse-midwife. We calculated personnel costs by multiplying the total length of time spent by nurse-midwives performing EIMC by their salary per hour. To estimate the hourly salary rate of a nurse-midwife, we divided the annual salary  by the total annual number of hours worked (assuming a 40-hour week × 48 weeks per year). Our primary analysis used research personnel salaries. However, as EIMC rollout is likely to be integrated into routine primary health care services, salary costs are expected to be lower and based on MoHCC salary scales. In sensitivity analysis, we analyzed personnel costs using MoHCC staff salaries, which are lower, in order to assess the cost impacts of different salary scales.To estimate waste management costs, we tracked the number of burn bins (sharps tin containers) used throughout the field trial. We multiplied the total number of burn bins (n = 90) sent to the clinic incinerators by the cost of incineration based on quotes from private service providers (US$10). To determine the contribution to unit cost, we divided the total waste management cost by the number of EIMCs performed in the field trial (n = 500).Because EIMC is likely to be rolled out using existing public health facilities, we excluded costs of buildings. For durable equipment costs, we divided the purchase price of each capital good by an appropriate amortization period to derive an annualized depreciation value. We then summed the annualized depreciation values and divided the total by the estimated annual number of EIMC procedures  to estimate the unit cost contribution of durable equipment. We charged 100% of the capital cost to EIMC, as capital equipment was exclusively used by EIMC.The services of support staff (clinic clerk and caretaker) were shared across other clinic services, such as maternity services, antenatal and postnatal services, HIV testing and counseling, pharmacy, outreach, outpatient, and family health services. We therefore apportioned a conservative estimate of 10% of the salaries of both types of personnel as support personnel costs for EIMC. We then divided the summed annual salary costs (including benefits) by the estimated annual number of EIMC procedures  to derive the unit cost contribution of support personnel.-In one-way sensitivity analysis, we assessed the extent to which the main cost-differentiating components influenced our results by varying each of them, one at a time.The Medical Research Council of Zimbabwe, the University College London Research Ethics Committee, and the London School of Hygiene and Tropical Medicine Research Ethics Committee approved the EIMC comparative trial and the field study.The unit cost per EIMC procedure by nurse-midwives in a vertical setting was US$38.87 compared with US$49.77 by doctors.Costs that were identical for nurse-midwives and doctors included consumable supplies (US$13.48) and nonconsumable supplies (US$0.27), both of which we assumed would remain inflexible even in an integrated setting because they would be dedicated to services offered exclusively in an EIMC department. Other identical costs were the AccuCirc device (US$10.00), waste management (US$1.80), capital equipment (US$0.08), and support personnel (US$1.16).In secondary analyses, we also compared costs of an EIMC procedure when provided by nurse-midwives in a vertical setting with a procedure provided in a public health facility or clinic (integrated setting). As personnel in a public health facility are employed by the civil service, we also analyzed the impact of using public-sector salaries in the secondary cost analysis. We assumed zero capital equipment costs since setting up EIMC service provision within an existing public health facility would not require new capital equipment. In an integrated public health setting, we found that the unit cost of EIMC was US$33.72 . Device,In one-way sensitivity analysis , we asseWe found the largest cost difference between vertical and integrated EIMC in personnel costs  . TraininThe largest cost difference between vertical and integrated EIMC by nurse-midwives was found in personnel costs (US$10.57 vs. US$6.53).Substituting nurse-midwives for doctors presents considerable opportunities for minimizing costs in a future EIMC rollout, without impacting safety. Our results show that in a vertical EIMC program it would cost substantially less to perform an EIMC procedure in Zimbabwe using nurse-midwives compared with doctors. Further opportunities for cost savings exist if EIMC were rolled out using an integrated model because civil service medical personnel costs are lower. In our analysis, personnel costs for EIMC performed by nurse-midwives fall between US$10.57 in a vertical program and US$6.53 in an integrated program . A combination of these factors, therefore, makes it more feasible for MoHCC policy makers to meet the goal of rolling out the EIMC procedure more widely and at a lower cost.This cost analysis of EIMC performed by nurse-midwives and doctors is, to the best of our knowledge, the first analysis based on actual costs of providing EIMC using nurse-midwives in sub-Saharan Africa. For other contexts, actual EIMC costs may differ from those described in this analysis. In a hypothetical modeling study conducted in Rwanda, EIMC was estimated to cost US$15 (direct costs only) using the Mogen clamp.This study has at least 3 limitations that influence whether the costs reported here would be the same as those in a scaled-up program. First, EIMC procedures were performed in an urban setting, and thus the results may be different for rural settings. Second, we based this cost analysis on a pilot implementation among a sample of 500 infants. To facilitate our analysis, we assumed that each team of nurse-midwives would perform 1,440 EIMC procedures each year. While this number was based on conservative assumptions, lower demand for EIMC may lead to lower capacity utilization resulting in higher unit costs of EIMC. Finally, our analyses did not include costs of demand creation due to the short duration (6 months) of the field study and the small sample size. Including realistic estimates of demand creation will be essential for fully costing a scaled-up EIMC program.,The potential impact of VMMC in reducing the number of new HIV infections has led some countries in sub-Saharan Africa to consider rolling out EIMC in order to achieve higher prevalence of male circumcision in the medium to long term.Our study highlights that an integrated EIMC program is cost-minimizing compared with a vertical program and helps to determine the overall resources needed to implement a scaled-up EIMC program.


Functional results of index pollicisation are usually assessed by the clinical score of Percival. This score is based on elementary hand movements and does not reflect the function of the neo thumb in daily life activities. The aim of this study was to develop a new video-assisted scoring system based on daily life activities to assess index pollicisation functional outcomes.Twenty-two consecutive children, operated between 1998 and 2012, were examined with a mean of 77 months after surgery. The mean age at surgery was 34 months. Post-operative results were evaluated by a new video-assisted 14-point scoring system consisting of seven basic tasks that are frequently used in daily activities. The series of tasks was performed both on the request of the examiner and in real-life conditions with the use of a hidden camera. Each video recording was examined by three different examiners. Each examiner rated the video recordings three times, with an interval of one week between examinations. Inter- and intra-observer agreements were calculated.p = 0.045). The correlation between the video-assisted scoring system and the Percival score was poor.Inter- and intra-observer agreements were excellent both on request (κ = 0.87 [0.84–0.97] for inter-observer agreement and 0.92 [0.82–0.98] for intra-observer agreement) and on hidden camera (κ = 0.83 [0.78–0.91] for inter-observer agreement and 0.89 [0.83–0.96] for intra-observer agreement). The results were significantly better on request than on hidden camera (The video-assisted scoring system is a reliable tool to assess index pollicisation functional outcomes. The scoring system on hidden camera is more representative of the neo thumb use in daily life complex movements.Level IV. Pollicisation is a challenging surgical technique, which consists in the transposition of the index into the thumb position. This procedure is used for thumb congenital hypoplasia or aplasia reconstruction, and aims to create a neo pinch in order to restore opposition and improve daily life activities function –4. SinceAfter institutional review board approval, 22 patients operated for index pollicisation between 1998 and 2012 were included. According to Blauth’s classification, indications were type IV hypoplasia in 12 cases, type IIIb hypoplasia in two cases and type V aplasia in eight cases . The defThe video-assisted evaluation consisted of seven basic tasks that are frequently used in daily activities: writing, drinking, eating, combing hair, moving an object from one point to another, moving an object from one hand to the other and, finally, one complex gesture  to assess two-hand coordination. The patients were filmed in two different conditions: (a) on request of the examiner and (b) in real-life conditions with the use of a hidden camera. During the test in real-life conditions, the child was sitting in a playing room and ignored that he was being filmed. He/she was allocated to different stands by his/her parents. On the first table, various colouring books with markers were disposed for the assessment of writing. On the second table, the parents role played a dinner party to assess drinking and eating and moving objects. Finally, on the third table, dressing and styling accessories were arranged for the evaluation of the remaining activities. The surgeon stood behind and filmed the hand movements using a hidden camera. Each condition (on request and on hidden camera) took 15–30 min.Each video recording was analysed by three different examiners: an orthopaedic surgeon, who did not perform the surgery, a paediatrician and a physiotherapist. Each examiner analysed the video recordings three times, with an interval of one week between examinations. Video recordings were presented to the examiners in a random order. Each activity was scored 0–2 points , leading to a total score of 14 points  . The results were excellent, good, fair and poor in 2, 12, 5 and 3 patients, respectively.r = 0.41 when the video-assisted system was performed on request and r = 0.36 when it was performed on hidden camera .In conclusion, our study introduces a new reliable video-assisted scoring system method for the assessment of index pollicisation functional outcomes and highlights the specific importance of the hidden camera analysis, which is more representative of the neo thumb use in daily life activities.


To present the co-creation of a whiteboard animation video, an enhanced e-storytelling technique for relaying traditional knowledge interview results as narratives.We present a design for translating interview results into a script and accompanying series of figures, followed by technical steps to create a whiteboard animation product.Our project used content analysis and researcher triangulation, followed by a collaborative process to develop an animated video to disseminate research findings. A 13-minute long whiteboard animation video was produced from a research study about changing environments in northern Canadian communities and was distributed to local people. Three challenging issues in the video creation process including communication issues, technical difficulties and contextual debate were resolved among the supporting agencies and researchers.Dissemination of findings is a crucial step in the research process. Whiteboard animation video products may be a viable and culturally-appropriate form of relaying research results back to Indigenous communities in a storytelling format. Oral storytelling traditions in Indigenous communities contribute in several distinct ways to the maintenance of traditional knowledge, educational and social development of the people, culture and advocacy for change –4. StorySlave Watershed Environmental Effects Program (SWEEP), a monitoring program, the team sought to devise a tool that advocates for potential and perceived health and environmental issues arising from these changes using culturally relevant means that transcend technological and demographic boundaries. Digital storytelling, primarily by the combination of Photovoice, PowerPoint slides and narration, has been utilized to engage Indigenous communities in climate-health and eco-health research  are concerned about the health of the Slave River and Delta (SRD). Industrial activities upstream in the Peace-Athabasca and Slave River watersheds are impacting water quantity and quality in the SRD. In response to community concerns about the health of the ecosystem and to facilitate community-based monitoring in the SRD, the Slave River and Delta Partnership (SRDP) was created in 2010 by the Government of the NWT Environment and Natural Resources (GNWT-ENR) Lands and Water Division, as part of the NWT Water Stewardship Strategy. The SRDP is a collaboration of agencies and organizations working and living in the SRD basin. Partners include 5 First Nations and Métis organizations, 1 territorial government agency, 4 federal government agencies, and Aurora College and Aurora Research Institute. This project design is one part of an ongoing research program working collaboratively with the SRDP seeking to identify, prioritize, measure and report on indicators of environmental health in the SDR communities. The project received research ethics and community approval (University of Saskatchewan REB 13-165 and Aurora Research Institute License No. 15383).The digital storytelling and whiteboard animation design emerged from stakeholder feedback indicating a desire to have results from traditional knowledge interviews relayed back to the community in a non-textual format, and a format that would endear youths in the community. We began with interviewing 11 Elders and local people for their ethnographical accounts of the changes in the river and delta across their lifetimes see . The focThe creation of the video project was a single, exploratory test of a method to disseminate interview findings in a digital storytelling format. In the SWEEP, we accepted our role as “marginal” tools, under the direction of community members, but the video was not decolonizing, or participatory action research . The larThe 11 interviews  took place between April and November 2014 in 2 locations. The first location was Fort Smith, NWT  and, the second location was Fort Resolution, NWT . The interviews were audio-recorded with permission and ranged from 45 minutes to over 3 hours. Participants were shown several pictures and maps of the SRD region ranging from the 1940s up to 2015. Participants could point out places on the map that were important to them while they were telling their stories, if desired.The interview transcripts underwent content analysis , and emeA single, encompassing narrative account of a “lifetime” in the region was developed using data from all 11 interviews across 5 broad themes: where the participants came from (early life experiences), development and disease, changing foods, changing waters and changing spiritual understandings of the world. The narrative account was massaged into a script. At times, the script contained quoted text from the interviews with participant permission. A sequence of images was then created alongside the script. Symbols were used to represent activities, places or events in the history of the region; for example, 2 sets of footprints leading to silhouettes of 2 boys carrying fishing rods was used to depict a narrative sequence where a participant remembered going fishing with his brothers as a young boy; a sketch of the outline of a dam and sequence of the reservoir behind filling with water over time was used to represent the construction and filling of the W. C. Bennett Dam and Williston Reservoir upstream in British Columbia, during the late 1960s and 1970s; and a sequence of falling water droplets containing medicine wheel elements was used to depict a traditional knowledge system of the region's connection to the waters of the river and delta .A high-quality camera capturing 5 images a second was set up in front of a large whiteboard. A fourth-year Fine Arts student with Indigenous ancestry was recruited to draw key images on the whiteboard. The student prepared by examining different images online, consulting with relatives and creating a set of drawings on paper to scaffold the animation sequencing. Images drawn to visually depict the verbal descriptions within the stories were shared with our Indigenous partners to confirm the accuracy of the drawings, for example, the illustration of a trapper's cabin or the flooding of the delta. Once finalized, the student carried out the drawing on the large whiteboard in 2 separate sequences as it was a lengthy and physically challenging process of drawing with her arm held up . The camThe technical team at the university's media and production unit zoomed the images to focus on the artist's hand and forearm as she drew the pictures. Once the zoom level and sequencing were established, camera steadiness was resolved and the 8 hour long sequence of images was sped up to look like it was a continuous 13-minute video of an arm drawing the chosen images. The narration was edited to fit the timing of particular images, and music and soundscaping were added. Some special effects were used to ensure the sequence and timing were visually appealing.www.youtube.com/watch?v=XHjmcdNwVpEDraft segments from the video were shared with Indigenous partners and individual research scientists involved in the larger program to confirm the accuracy of the narrative. Corrections to species names, locations and ice dynamics were made as a result. These segments were also shown to supporting agencies for feedback during several conference calls. Editing was completed at the request of partners and the final whiteboard animation video was gifted to the community during two showings in May 2015. Community members decided to allow broadcast of the video more widely  after an arranged public viewing. The video can be viewed at The process of completing the video was not tension-free. We faced 3 main challenges: there were communication difficulties, technical issues to overcome and contextual debate (see below).Communication difficulties arose when working with interview participants to finalize symbols and ensure accuracy. It was difficult for interview participants to understand how the images were going to represent parts of the story without the full animation sequence with the narrative included to show them. Doing so would have required more time and resources in the production stage. Participants also lived remotely and were engaged in traditional livelihoods during the production stage, thus not always able to verify that edits captured their desired changes. Culturally-important symbols such as the medicine wheel needed to be included in sensitive and appropriate ways, but while government agency representatives were cautious about using the medicine wheel, when asked, interviewees and community members welcomed the familiarity of the graphic. The accuracy of maps was also important to the community members who contributed hand-drawn pictures of what the channels looked like, as well as historic photographs they owned. Time pressures and a shortage of funding were also of concern. While the artist needed time and space to draw freely and creatively, and she had prepared by sharing various ideas with the researchers and technical team, the actual capturing of the drawing was a time-intensive part of the process. These constraints made deciding on the content of the final video challenging. We found that using the table to build understanding of the compiled storyline was helpful. To better communicate the inception and goal of the video, researchers and funders suggested including an introduction and conclusion that depicted the larger research program and ongoing efforts.Technical issues were also faced; because of the need for the still images to be captured at a high rate to make the animation sequence look realistic, and because of memory limitations of devices, the actual drawing was captured in segments that needed to be merged in the final product. The camera “bounce” due to the frequency of picture taking also needed repairing which was completed with “coding” designed by the technical producer. Photographic images shared by research team members from the larger research program were included in the introduction and closing of the video which meant extra production time was necessary.The third challenge involved contextual debate. The researchers at times felt tension between including the messaging from supporting government agencies and research centres, and the themes that emerged from the participants’ interviews. One particularly contentious issue arose during the sharing of draft segments of the video. Government agency partners flagged a segment depicting perceptions of safety and contamination of country foods. The sequence used the medicine wheel to show the relationships between certain harvested food items and importance to traditional lifestyles. The sequence included the erasing of animals as their perceived toxicity is described , while an image of a skull and crossbones is drawn inside the medicine wheel. The picture is controversial because the messaging from the Government of NWT and the perception of the local people is that country foods are on the whole, healthier choices than unhealthy choices in the grocery stores or community food programs , is not enough . Within Our product differs from other knowledge mobilization digital storytelling products in three key ways; first, we created the video with Indigenous participants providing feedback during the production stages. Research scientists, representatives from supporting agencies and funding partners contributed to the reviewing of draft videos. Second, the narrative was a compilation of 11 different interview participants’ stories and traditional knowledge. Where many digital storytelling outputs are short pieces including individual perspectives on a topic (e.g. 15–17), and photographic libraries, our video tells the story of Indigenous groups struggling against dramatic environmental and social changes in their region. We also used symbolic hand-drawn images which were “animated” through digital technology to tell the story. The inclusion of university students in our practice meant the knowledge was mobilized further afield through their networks, and their own portfolios were bolstered.Agencies seeking to co-produce knowledge with communities and researchers could benefit from these types of projects because of the ability to condense large amounts of data into accessible formats for northern communities and Indigenous people. Our First Nations and Métis partners indicated they enjoyed the video and validated its local relevance and ability to reach people. They believed it would be an effective educational tool for use in schools to identify culturally-important historical events, and notable places in their region. Copies of the video have been placed in the local schools for use as an educational tool and it is streaming live in the Health Clinic of one of our partnering communities.There are important lessons learned from the transdisciplinary collaboration in this project. Our research design and technical production needed to allow for more consultation with both the supporting agencies, and the local people so that the balance between advocacy and information sharing could be established early on. The debates that emerged could have been eased with earlier agreement on particular themes emerging from the interviews. We also needed to ensure that everyone agreed on the list of images to be drawn for the artist so that there would be less post-production editing.Arts-based approaches make visible peoples’ experiences that are often left unarticulated or hidden . This pr


Fms-related tyrosine kinase 3 (FLT3) gene (FLT3-ITD) are associated with poor prognosis in patients with acute myeloid leukemia (AML). Due to the development of drug resistance, few FLT3-ITD inhibitors are effective against FLT3-ITD+ AML. In this study, we show that FLT3-ITD activates a novel pathway involving p21Cdkn1a (p21) and pre-B cell leukemia transcription factor 1 (Pbx1) that attenuates FLT3-ITD cell proliferation and is involved in the development of drug resistance. FLT3-ITD up-regulated p21 expression in both mouse bone marrow c-kit+-Sca-1+-Lin- (KSL) cells and Ba/F3 cells. The loss of p21 expression enhanced growth factor-independent proliferation and sensitivity to cytarabine as a consequence of concomitantly enriching the S+G2/M phase population and significantly increasing the expression of Pbx1, but not Evi-1, in FLT3-ITD+ cells. This enhanced cell proliferation following the loss of p21 was partially abrogated when Pbx1 expression was silenced in FLT3-ITD+ primary bone marrow colony-forming cells and Ba/F3 cells. When FLT3-ITD was antagonized with AC220, a selective inhibitor of FLT3-ITD, p21 expression was decreased coincident with Pbx1 mRNA up-regulation and a rapid decline in the number of viable FLT3-ITD+ Ba/F3 cells; however, the cells eventually became refractory to AC220. Overexpressing p21 in FLT3-ITD+ Ba/F3 cells delayed the emergence of cells that were refractory to AC220, whereas p21 silencing accelerated their development. These data indicate that FLT3-ITD is capable of inhibiting FLT3-ITD+ cell proliferation through the p21/Pbx1 axis and that treatments that antagonize FLT3-ITD contribute to the subsequent development of cells that are refractory to a FLT3-ITD inhibitor by disrupting p21 expression.Internal tandem duplication (ITD) mutations in the  Cdkn1a (p21) was originally identified as a mediator of p53-induced growth arrest . Th. Thfx/fx+ AML [+ AML. The capacity of the FLT3-ITD-mediated up-regulation of p21 expression to inhibit the proliferation of FLT3-ITD+ HPCs suggests that FLT3-ITD signaling attenuates the proliferation and cell cycle progression of FLT3-ITD+ HPCs. However, the p21 down-regulation in FLT3-ITD+ cells triggered by inhibitors of FLT3-ITD, p53, PI3 kinase, and PKA suggests that the sustained inhibition of FLT3-ITD or other molecular pathways downstream of FLT3-ITD may block endogenous anti-growth signaling, possibly contributing to the recurrence of FLT3-ITD+ AML. To determine whether inhibiting p21 expression in FLT3-ITD+ cells by antagonizing FLT3-ITD is associated with the proliferation and/or emergence of FLT3-ITD+ cells that are refractory to a FLT3-ITD inhibitor, the association between p21 expression and cell proliferation was investigated in FLT3-ITD+ cells treated with a FLT3-ITD inhibitor. FLT3-ITD+ Ba/F3 cells were cultured in the absence of cytokines with 2 or 5 nM AC220, a selective inhibitor of FLT3-ITD, for 24 hours, and FLT3 phosphorylation was inhibited . Pbx1 itself was classified in 7 of the functional groups , the quantitative RT-PCR analysis confirmed that Pbx1 was up-regulated by 2.1 ± 0.5-fold in FLT3-ITD+ p21-/- KSL cells compared to FLT3-ITD+ p21+/+ KSL cells , whereas Pbx1 expression in p21-/- KSL cells in S phase was elevated by 6.1 ± 1.1-fold  compared to the p21+/+ cells  compared to FLT3-ITD- AML cells  [p21 expression  was inversely correlated with Pbx1 expression, but not with Evi-1 expression, in FLT3-ITD+ AML subjects, particularly those with the FAB M1 subtype  [Although the loss of p21 did not significantly affect or Evi-1  and FLT3/+ cells . Becauseov/geo/) . These rov/geo/) .+ HPCs induced by the loss of p21 was accompanied by the up-regulation of Pbx1 expression, we next examined whether Pbx1 mediates the increased proliferation induced by p21 deletion. FLT3-ITD+ p21-/- bone marrow cells transduced with a control shRNA showed a 1.9 ± 0.3-fold increase in the number of growth factor-independent CFCs compared to p21+/+ cells . Similarly, transduction of the Pbx1 shRNAs partially but significantly decreased the Pbx1 mRNA levels in the FLT3-ITD+ Ba/F3 cells containing the control shRNA, along with those harboring the p21 shRNA  uncovered a potential non-coding sequence in the 5’ region of the mouse Pbx1 gene. A transcription factor-binding analysis using up to 2.0 kb of the identified 5’ non-coding sequence revealed candidate binding sequences for c-Myc, C/EBP-α and estrogen receptor-α, which are known to be directly regulated by p21 [Our data also indicate that the enhanced proliferation of FLT3-ITDopoiesis . Pbx1 reopoiesis ,29. The  of Pbx1 , p21 deld by p21 ,17,18. T+ cell proliferation by modulating the p21/Pbx1 axis. Although the FLT3-ITD-mediated increase in p21 expression increases resistance to Ara-C by reducing cell cycle progression, disruption of p21 expression using an FLT3-ITD inhibitor accelerates the development of refractory FLT3-ITD+ cells. This result suggests that the deregulated expression of p21 and/or Pbx1 by FLT3-ITD likely contributes to the refractory phenotype of FLT3-ITD+ AML cells. P21 and/or PBX1 may represent additional therapeutic targets for patients with FLT3-ITD+ AML, particularly those who are refractory to FLT3-ITD inhibitors.Our data demonstrate that FLT3-ITD attenuates FLT3-ITDS1 Table+ KSL cells but not the freshly isolated KSL cells are listed. The functional classifications of these genes based on Gene Ontology biological processes and molecular functions and KEGG pathways are also listed.The genes that were exclusively up- and down-regulated by p21 deletion in the FLT3-ITD(XLSX)Click here for additional data file.


The objective of this study was to compare the 2 leading human acellular dermal matrices in breast reconstruction with implants. This retrospective study draws on the experience of 2 expert surgeons with a history of long-standing use of the Alloderm-RTU  product who switched to the DermACELL acellular dermal matrix  product. The consecutive nature of these data over this change allowed comparison between the 2 products without the confounding effects of patient selection or change in technique. The postoperative complications of seroma, infection, implant loss, and unplanned return to the operating room were studied, and no statistical differences were noted between these 2 products. The overall complications rates were low, with implant loss and infection less than 2% in 249 cases. Recommendations are for continued use of acellular dermal matrix in breast reconstruction and product selection based on price and availability. Sinai Health System. After obtaining IRB approval at each of the authors’ institutions, a retrospective review of prospectively maintained databases of long-term ADM users (M.R.Z. and C.A.S.) was conducted to obtain a consecutive series of ADM-based breast reconstructions that spanned their use of Alloderm-RTU and DermACELL. Both had been long-term users of Alloderm and switched to DermACELL use during this study. The techniques used in reconstructing breasts did not differ between products. This allowed direct comparison of the 2 products in the hands of experienced surgeons without patient selection bias. Minimum follow-up for patients in this study was 6 months and ranged from 6 months to 2 years. Patient characteristics included age, indication for mastectomy , immediate versus delayed reconstruction, and history of radiation or chemotherapy. The primary outcomes of complications evaluated included seroma, hematoma, capsule formation requiring surgery within 6 months, infection, and implant removal. Treatment of subsequent infections was recorded as treated with oral antibiotics or intravenous antibiotics.There were a total of 140 patients included in this retrospective review. These represent a consecutive series for both surgeons during their transition from Alloderm-RTU (70 patients) to DermACELL (70 patients). There was no statistical difference between the groups regarding age, indication (cancer vs prophylactic mastectomy), or the incidence of radiation or chemotherapy. The number of implants is not equal due to differences in these groups between the number of unilateral and bilateral cases see .The number of complications noted in this study was small , with onN = 249), the Fisher exact test P value (.7452) indicated that the association was not statistically significant at the .05 α level. Thus, we concluded that there was no relationship between ADM type and infection.Statistical analysis of the data was performed using the Fisher exact test. First, differences between Alloderm-RTU and DermACELL from the standpoint of infection were explored. Using the data set of all expanders and implants (N = 12), the Fisher exact test P value (.5692) was greater than the significance at the .05 α level. Thus, we concluded that there was no relationship between ADM type and need for surgery in the early postoperative period. All 3 implant losses involved skin necrosis as the inciting factor and were not believed to be related to ADM type.Next, the relationship between ADM type and need for surgery in the early postoperative period (first 6 months) was investigated. When using only the subset data of patients who had surgery (P = .0045) and a lack of association between chemotherapy and infection (P = 1.0).Other significant findings in review of the data showed an association between the infections and radiotherapy , has clinically been equally effective in our hands in breast reconstruction and prompted our inquiry as to which product has the lowest complication rate. DermACELL is also a human product, produced by LifeNet Health , a 501(c)3 federally designated organ procurement agency, tissue bank, and medical device manufacturer. LifeNet Health has more than 32 years of experience supplying tissue allografts for a variety of surgical applications and is certified by the American Association of Tissue Banks. Ironically, LifeNet Health was once the donated tissue supplier for the older Alloderm RTM. The similarities between the products stop there, as each is processed differently to create the end matrix. DermACELL undergoes a proprietary Matracell processing that experimentally has demonstrated less residual DNA and better tissue ingrowth than Alloderm.−6 usually required for implantable medical devices.−3 is acceptable but leaves the risk of contamination in the manufacturing process at one in a thousand compared with DermACELL's risk of one in a million.Today's reconstructive surgeon faces many choices in ADMs for breast reconstruction. These can be divided by their source into human products, bovine or porcine products, or synthetic products. To differing degree, all can be effective but the best ADM in terms of effectiveness with the least complications has yet to be determined. Human products have been favored in the United States, whereas porcine products have been favored elsewhere. The market leader, Alloderm, a human product, created the market and has the most literature supporting its use. This literature, though, has been based on its older, freeze-dried, aseptically processed product that has recently been changed to a ready-to-use product that is terminally sterilized with a Sterility Assurance Level (SAL) of 10Meta-analyses of ADM in breast reconstruction have shown, when compared with the standard of muscle coverage without ADM, the incidence of infection increases as much as 4-fold.This study retrospectively reviewed the consecutive data of 2 experienced ADM users who had used Alloderm for more than a decade and had extensive experience with the newer Alloderm-RTU before utilizing DermACELL in this study. The consecutive nature of the data, spanning both products with no change in technique between products, has provided an opportunity to compare these products directly while controlling for surgeon expertise and selection bias. The data show that both products were equally effective for use in implant-based reconstruction.Both Alloderm-RTU and DermACELL had low rates of infection and showed excellent incorporation in all cases except for the 3 cases of explantation. The need for surgery in the first 6 months was higher with Alloderm-RTU, but this did not reach statistical significance .This study has demonstrated that both Alloderm-RTU and DermACELL are effective in breast reconstruction with expanders or implants. Another important finding revealed in this review is that in the hands of experienced surgeons, rates of seroma and infection, the 2 complications most related to ADM type, can be lower than those seen with non-ADM reconstructions.An important implication of this study, given the equal effectiveness of these products with a comparable risk profile, is the potential cost savings to our health system. DermACELL is 15% to 35% less expensive in the US market for commonly used sizes. Centers such as ours that use DermACELL have realized thousands and thousands of dollars in savings over the study period.In conclusion, the paradigm shift that was created by the use of ADMs has changed the way that we practice implant-based reconstruction. This study has shown that there is no clinical difference between Alloderm-RTU and DermACELL and that there is no statistical difference between these ADMs in infection rate, implant loss, or need for corrective surgery within 6 months of placement. With all else being equal, based on generally accepted surgical principles, a sterile product will always be preferable to an aseptic product. With equal clinical performance between Alloderm-RTU and DermACELL, value-based care would dictate that the decision on which product to use will likely be made on nonclinical factors, such as availability and price.


There is increasing public and scientific concern regarding the long-term behavioural effects of video game use in children, but currently little consensus as to the nature of any such relationships. We investigated the relationship between video game use in children, degree of violence in games, and measures of depression and a 6-level banded measure of conduct disorder. Data from the Avon Longitudinal Study of Parents and Children were used. A 3-level measure of game use at age 8/9 years was developed, taking into account degree of violence based on game genre. Associations with conduct disorder and depression, measured at age 15, were investigated using ordinal logistic regression, adjusted for a number of potential confounders. Shoot-em-up games were associated with conduct disorder bands, and with a binary measure of conduct disorder, although the strength of evidence for these associations was weak. A sensitivity analysis comparing those who play competitive games to those who play shoot-em-ups found weak evidence supporting the hypothesis that it is violence rather than competitiveness that is associated with conduct disorder. However this analysis was underpowered, and we cannot rule out the possibility that increasing levels of competition in games may be just as likely to account for the observed associations as violent content. Overall game exposure as indicated by number of games in a household was not related to conduct disorder, nor was any association found between shoot-em-up video game use and depression. Despite frequent expressions of concern in the media, the behavioural and psychological effects of prolonged video game playing are not well understood. Although it has been claimed recently that there is a consensus among video game researchers , the eviOne issue is the lack of consistent and robust methodologies across different studies ; in partAside from differences in the range and type of potential confounding factors that are adjusted for across such studies, others have made the more general observation that it is difficult to isolate the violent content of video games from other features. For example, after systematically analysing games for levels of violence (and matching for factors such as level of difficulty and pace of action), Adachi and Willoughby  found exOne theoretical framework that has been developed in an attempt to explain some of these findings is the Catalyst Model . In thisIt remains unclear how adolescent social behaviour and mental health is affected by video game use. Many studies tend to look at the effects of games generally, while in reality there are a wide range of differences in game style, content and play that may confer differing, even opposite effects on behaviour. We were therefore interested in whether the level of violent content in games is associated with negative behaviour outcomes. We investigated the association between video game use at age 8/9 years, and subsequent measures of conduct disorder and depression at age 15 years, taking into account a wide range of potential confounding factors. We also attempted to assess any relationship between the degree of violence in the games played, and any behavioural outcomes.st April 1991 to 31st December 1992. The cohort has been described in detail previously [The Avon Longitudinal Study of Parents and Children (ALSPAC) is a prospective, population-based birth cohort study that recruited 14,541 pregnant women living in Avon, UK, with expected delivery dates between 1eviously . The curhttp://www.bris.ac.uk/alspac/researchers/data-access/data-dictionary/).Ethics approval for the study was obtained from the ALSPAC Ethics and Law Committee and the Local Research Ethics Committees. Please note that the study website contains details of all the data available through a fully-searchable data dictionary ”, “role-playing ”, “puzzles ”, “strategy ”, “flight simulator”, “platform ”, “other ”.Responses were used to derive a 3-level measure intended to capture whether or not participants played video games and, if they did, the degree of violence in the games played: 0) does not play video games; 1) plays puzzle games; 2) plays “shoot-‘em-up” games. If participants played games in more than one category, they were categorised upwards. Number of video games was also assessed: participants were asked “How many computer games do you have at home?” and could respond 0, 1–2, 3–4, 5–9 or 10 or more. A further binary measure was created of those who used only racing or sports games (competitive games), and those who reported using only shoot-em-up games (violent games).Conduct disorder and depression were assessed at age 15 via the DAWBA semi-structured interview. Conduct disorder was assessed via parental report and depression via child self-report. From participants’ responses, DAWBA “bands” can be created, corresponding to ordered categorical measures of likelihood of conduct disorder and depression. These are created using computer algorithms, and have been validated in samples of children in the United Kingdom. The bands contain 6 levels, and are based on the probability of disorder, ranging from very unlikely to probable. As well as the ordered categorical variables, the top two levels in each category can be used to create binary “disorder” variables . The DAWPotential confounders, for which there was a theoretical or empirical basis for considering that they might influence any observed association between video game use and subsequent mental health, were: a) pre-birth confounders, including family history of mental health problems ; maternal education ; ancestry ; religiosity ; family structure during pregnancy ; and offspring sex; and, b) childhood confounders, including IQ at age 8 ; whetheWe assessed the relationship between video game use and DAWBA ordered categorical bands of conduct disorder and depression using ordinal logistic regression, before and after adjustment for confounders. We excluded pre-existing conduct problems or depression measured at baseline, in order to assess incidence of the outcome, and adjusted for variation in baseline conduct disorder or depression in those who remained. We confirmed the proportional odds/parallel regression assumption had not been violated using the Brant test . The relWe also conducted multiple imputation of 100 datasets in order to investigate and account for potential bias introduced due to attrition. Over 50 auxiliary variables were used to make the missing-at-random assumption plausible, including measures related to pre-birth and childhood factors. The main analyses were repeated using imputed exposure and confounder data . These imputed datasets also accounted for pre-existing conduct problems or depression, as appropriate.Of 14,701 children in the initial full cohort, 5,400 completed at least some of the DAWBA at age 15. From this sample, 2,453 provided video game usage data at age 8/9. The final sample available for analysis consisted of 1,815 children for whom data on the above confounders were available. Of these, a total of 26 participants met the criteria for conduct disorder case status, which increased slightly by video game exposure category . A total of 22 participants met the criteria for depression case status, which decreased slightly by video game exposure category . The characteristics of all participants, grouped by video game exposure category, are presented in Ordered regression of conduct disorder band (6 levels) at age 15 and genre of game played  at age 8/9 years indicated weak evidence of an association in the unadjusted model  which was not altered substantially in the partially adjusted  or fully adjusted  models . That isnumber of games played at age 8/9 years (which was not stratified by genre) and conduct disorder band at age 15 in the unadjusted, partially adjusted or fully adjusted models. These results are presented in There did not appear to be any association between the A further sensitivity analysis was conducted whereby those selectively playing shoot-em-up games were compared with those selectively playing competitive racing or sport games. Despite larger effect sizes, there was only weak evidence that shoot-em-ups were associated with an increased risk of conduct disorder in comparison to competitive games . The effIn order to test the specificity of any associations with mental health outcomes, we also looked at whether there was an association between video game use and depression. Ordered regression of genre of game played at age 8/9 years on depression band (6 levels) at age 15 indicated some evidence of a negative association in the unadjusted model , but after partial adjustment results attenuated to the null  .The analyses of the multiply imputed datasets were of a smaller magnitude than the complete case findings for conduct disorder outcome, but were not markedly different. Findings for depression were very similar to the complete case .Our results indicate that playing video games that are more likely to include violent content  in childhood is weakly associated with an increased risk of conduct disorder in late adolescence. There was also weak evidence that individuals who selectively play shoot-em-ups differed in risk to those who selectively play competitive games. However, the absolute risk of developing conduct disorder is small, and the modest effect sizes we observed should be interpreted in this context. Overall game exposure, as indicated by number of games in a household, was not related to conduct disorder, nor was any association found between video game use and depression.While our results are broadly in line with findings suggesting that violent game content is associated with increased aggressive tendencies, the associations we observe  are modest, and do not seem to be consistent with claims that the effects of playing violent video games on aggressive behaviour are of a sizeable magnitude. For example, some have claimed  that theIt is worth considering alternative explanations for our findings. One possibility is unmeasured confounding; for example, Ferguson and colleagues  show thaOverall, our data are consistent with the Catalyst Model. Although there is a weak association between playing shoot-em-ups at a young age and subsequent negative behaviours, the effect is small and the statistical evidence for these associations is weak. Moreover, there was only very weak evidence that individuals who selectively played shoot-em-up games  differed in risk from those individuals who selectively played competitive games, suggesting that violent video game content alone may not be a sufficient indicator of risk for later aggressive behaviour. Furthermore, given that in the present study we do not consider the impact of media exposure aside from video games, an assumption that any association between violent game use and aggression is causal  is inappIt is also important to consider the nature of the questions asked in the study. Children aged 8 or 9 years old may not know what a ‘shoot-em -up’ game is, so may have not correctly classified games that they were playing. Participants were given exemplar games for some categories, but not all. Nevertheless, given that there was no ‘first/third person shooter’ category, it is reasonable to assume that any games in this genre may have been included in the ‘shoot-em-up’ category. Although video games carry age classifications that should restrict access to inappropriate games, these games may nevertheless be present in the home due to the presence of older siblings, or parents that play them. However, if it is the case that children aged 8–9 are not playing extremely violent games, one possible interpretation of our results is that they may actually underestimate the association of playing shoot-em-ups with later aggressive behavioural outcomes. It would be interesting to explore this association at a later age , when children have more potential access to other types of games that may contain more violent content.We attempted to differentiate between types of video game played, and one difference between the game categorisations is the potential amount of violent content within each genre–shoot-em-ups are more likely to contain violence than puzzle games or sports games. Nevertheless, our categorisation of violence may not fully reflect the true nature of the violence in these games. This is a general limitation of all current video game research, and is important because violent content and play styles can vary considerably between games that are broadly categorised as ‘violent’. Moreover, the time point at which participants took part in our study means any games they reported playing are now over 15 years old, which should be taken into consideration when using these results in the context of the potential effects of more recent games. As such, our categories may not adequately represent levels of violence in modern games and do not take into account the amount of time played . Therefore our data may not be representative of gaming today. Moreover, modern video games blur traditional genre boundaries in increasingly complex ways–for example, puzzle games (ostensibly a ‘non-violent’ category) now range from simple numerical tasks to more graphic crime scene investigations. Given the nature of the data, we were unable to assess the specific games that individuals played. Therefore, there is still a need to look at how the specific content within individual games may impact upon behaviour. Given that there are differences in the literature in outcomes depending on the tested game content, we believe that it is essential that future research pays closer attention to both the specific content within video games, and also the context in which they are played, and moves away from a generalised discussion of ‘video game’ use.We also attempted to determine whether factors other than violent game content may be associated with aggressive behaviours. Previous studies ,29 have In conclusion, our data show a weak association between playing shoot-em-up games at an early age, and an increased risk of conduct disorder in late adolescence. Additionally, our data show weak evidence that those who selectively played shoot-em-ups differed in risk to those who selectively played competitive games; however, our analysis was not sufficiently powered to be able to answer with any certainty whether or not the level of competition present in games accounts for the associations we observed. Larger studies, ideally with richer data on video game use, are required needed to address this question. It is also important to note that most of our sample played shoot-em-up games when they were aged 8, and importantly, only very few met the criteria for conduct disorder case status at age 15. Therefore, the association between violent game use and conduct disorder in fact appears to be weak. We observed no association between violent game use and subsequent depression in adolescence, a result that supports earlier studies . Further


Clostridium absonum (CA 7α-HSDH) complexed with taurochenodeoxycholic acid (TCDCA) and NADP+ by X-ray diffraction, which, as a tetramer, possesses the typical α/β folding pattern. The four subunits of an asymmetric unit lie in the fact that there are the stable hydrophobic interactions between Q-axis-related subunits. Significantly, we captured an active state of the NADP+, confirming that nicotinamide moiety of NADP+ act as electron carrier in the dehydrogenation. On the basis of crystal structure analysis, site-directed mutagenesis and MD simulation, furthermore, we find that the guanidinium of Arg38 can form the stable cation-π interaction with the adenine ring of NADP+, and the cation-π interaction and hydrogen bonds between Arg38 and NADP+ have a significant anchor effect on the cofactor binding to CA 7α-HSDH.7α-hydroxysteroid dehydrogenase (7α-HSDH) can catalyse the oxidation of C7 α-OH of the steroid nucleus in the bile acid metabolism. In the paper we determined the crystal structure of 7α-HSDH from  In mammals, the primary bile acids, including cholic acid (CA) and chenodeoxycholic acid (CDCA), are synthesised from cholesterol in hepatocyte, and then intestinal bacteria convert primary bile acids to secondary bile acids such as deoxycholic acid (DA), lithocholic acid (LCA) and ursodesoxycholic acid (UDCA). Bile acids/salts and their conjugations with glycine or taurineEscherichia coliBrucella Melitensis have been determined, and the characteristics of folding pattern in both the two 7α-HSDHs indicate that they belong to the short-chain dehydrogenases/reductases (SDRs), whose members are dimeric or tetrameric and generally share low sequence identity possessing a single-domain globular Rossmann-related fold13144Clostridium absonum 7α-HSDH (CA 7α-HSDH) has no significant change after incubation at 25 °C for 108 hBacteroides fragilis 7α-HSDH (BF 7α-HSDH) retains 90% of activity after heat treatment for 1 h at 65 °C7α-hydroxysteroid dehydrogenases (7α-HSDH) is responsible for reversibly catalysing the oxidation of C7 α-oriented hydroxyl of the steroid nucleus in the bile acid metabolism and has been detected in numerous genera of bacteria34568β-strand for NAD(H)-utilising21β strand indicates that the enzyme is NAD(H)-preferred and this residue forms hydrogen bonds to the 2′- and 3′-hydroxyl group of the adenine ribose moiety23β strand, and the other is positioned just before the sequence ‘GIG’ of the cofactor binding sequence motif mentioned above19All of the dehydrogenases require coenzyme such as NAD(H) or NADP(H) which acts as electron carriers for catalysing the redox reaction, and NADP(H) is much more efficient than NAD(H) in bile acids synthesis process. Therefore, the cofactor specific dehydrogenases attracted a lot of attention. The first crystal structure of NADP(H)-dependent SDR member  was determined in 1996 and this studyCA 7α-HSDH complexed with NADP+ and taurochenodeoxycholic acid (TCDCA) at resolution 2.0 Å. Site-directed mutageneses were performed to investigate the function of the residues involved in cofactor specificity. Furthermore, molecular dynamics (MD) simulation was carried out to study the interactions between 7α-HSDH and NADP(H)/TCDCA.To unravel the NADP(H) recognition in 7α-HSDH, we determined the crystal structure of CA 7α-HSDH mainly existed as dimers. However, seemingly a small proportion (arrows) possesses the association pattern of tetramer  see  and massamer see . To veri SEC see . The tet SEC see  and all CA 7α-HSDH complexed with NADP+ and TCDCA belonged to the P212121 space group and was determined at resolution of 2.0 Å by the molecular displacement method and refined to crystallographic R-factor of 0.182 (Rfree = 0.222). For the current model, the backbone dihedral angles of 97.7% of the residues are in the most favored region of the Ramachandran plot, and all the non-glycine residues were in the allowed region. More details of data collection and refinement can be found in The three-dimensional structure of CA 7α-HSDH crystal structure includes four subunits (An asymmetric unit of subunits . There iBrucella Melitensis 7α-HSDH with no ligand (PDB code: 3GAF) at resolution 2.20 Å and Escherichia coli 7α-HSDH . Structure of 1AHH contains ligand NAD+ at resolution 2.30 Å, while both 1FMC and 1AHI have the ligands NADH and 7-oxo-GCDCA at resolution 1.80 Å and 2.30 Å, respectively. In spite of the low sequence identity (only 15–30%), SDRs generally display the well-conserved Rossmann fold. As one of the SDRs, with the similar structure to the two 7α-HSDHs above, CA 7α-HSDH possesses the typical Rossmann-fold that each monomer consists of seven parallel β-strands as a core (β-sheet core) and α-helices located between β-strands at the surface of the globular molecules. The three-dimensional structures and folding topologies of CA 7α-HSDH and EC 7α-HSDH are shown as CA 7α-HSDH has longer C-terminal segment including an extra α-helix (αI) which involves Gln255-Tyr256-Ser257-Glu258-Tyr259. Differing from that of EC 7α-HSDH, such C-terminal structure of CA 7α-HSDH does not directly surround the substrate-binding pocket but stretch outward. We have previously proved that only 19.1% of catalytic efficiency (kcat/Km) remained after truncation of 2 amino acids at C-terminal end of CA 7α-HSDHCA 7α-HSDH.To date, four three-dimensional structures of 7α-HSDHs have been deposited in PDB database. They are CA 7α-HSDH are shown in et al.P, Q and R axes, respectively. Along Q-axis, the interactions between subunit A and C (B and D) are mediated by four α-helices, αD (residues 107–135) and αE (residues 156–176), which form αD-αD and αE-αE interfaces. As shown in Q-axis related interactions exist in the crystal structure of EC 7α-HSDHP-axis related subunits  mainly result from αH (residues 220–231) and βG (residues 242–245). P-axis. In the aqueous solution, salvation effect could weaken the interaction between P-axis related subunits, resulting in that a multitude of tetramers dissociated into the dimmers. This well explains the dynamic equilibrium between dimers and tetramers of CA 7α-HSDH in the aqueous solution mentioned above.The four subunits in an asymmetric unit of residues 7–135 andStreptomyces exfoliatus (PDB code: 2HSD)In an asymmetric unit, the polar interactions between the two subunits in the diagonal position  involves the C-terminal as shown in CA 7α-HSDH at 25 °CIn addition, we found many salt bridges exist in the surface of the monomer such as Asp49-Arg20, Asp85-Arg83, Asp152-Arg155, Asp202-Arg205. The thermostability of +, electron density of the nicotinamide moiety of NADP+ was rather weak . +/TCDCA and the distances (Å) between relative atoms in CA 7α-HSDH. The residue Thr145 forms hydrogen bond with C7-OH of TCDCA while the residue Lys162 forms hydrogen bonds with 2′- and 3′-hydroxyl group of the nicotinamide ribose moiety in NADP+. The phenolic hydroxyl of Tyr158 form hydrogen bonds with C7-OH of TCDCA and 2′-hydroxyl group of the nicotinamide ribose of NADP+. To observe difference of active-site spatial arrangement between CA 7α-HSDH and EC 7α-HSDH, we superposed the two catalytic triads  of SDRs, and the tyrosine is the most conserved residue which exists in all members of SDRs12c triads . Althoug+ in CA 7α-HSDH are similar to that of other NADP(H)-dependent SDRs such as human estrogenic 17β-HDSHCA 7α-HSDH and its cofactor NADP+ mainly involving Ser13, Thr15, Arg16, Ile18, Arg38, Asn63, Asn90, Tyr158, Lys162, Ile191, Thr193, Arg194, Ala195. Thr15, Arg16, Arg38 and Arg194 formed a positive charge-rich environment surrounding the 2′-phosphate of adenine ribose and these residues determined the cofactor specificity in CA 7α-HSDH. According to the theory mentioned above, Arg16 and Arg38 are the determinant residues for cofactor specificity. In previous study, we performed the mutation analysis to verified the function of Arg38 of CA 7α-HSDH in the cofactor recognition and found that the R38D mutant did not display enzyme activity with either NADP+ or NAD+ CA 7α-HSDH were prepared as follows: A37D, R16G, R16G/A37D, R16G/R38D, A37D/R38I, A37D/R38Y, A37D/R38V, R16G/A37D/R38I and R16G/A37D/R38V. Interestingly, the results of enzyme assay indicate that only the mutant R16G has enzyme activity with NADP+ and no activity with NAD+. No activity of the other eight mutants was detected with either NADP+ or NAD+. Such results show the alteration of cofactor specificity in CA 7α-HSDH is more complex and implementation of the residue with negative charges (aspartic acid) at position 37 or 38 could not form proper space orientation for NAD(H) recognition. From a structural point of view, the three arginines in the position 16, 38 and 194 form a positive environment surrounded the 2′-phosphate of adenine ribose of NADP+ in CA 7α-HSDH, but not more than two arginines were at the corresponding position in the other 10 SDRs listed as On account of the conserved hydrogen bonding of cofactor onto protein main chain of SDRs+. Especially, the nitrogen atom in guanidinium of Arg38 is close (~4 Å) to the adenine ring of NADP+ forming the cation-π stacking. Cation-π interactions are ubiquitous in biological systems, which play central roles in molecular recognition, stabilisation of protein structures and nucleic acid structures, and their biological functions. Cheng et al.4+ and benzene by the density function calculation, finding that the energy difference between the lowest unoccupied molecular orbital (LUMO) of NH4+ and the highest occupied molecular orbital (HOMO) of benzene is more than that between LUMO of benzene and HOMO of NH4+. So the electrons preferentially move from the benzene to NH4+ combined with the analysis of net charge of atom. We speculate that the cation-π interactions might lead to the electrons of the adenine ring of NADP+ moving to the NH2+ of Arg38.Furthermore, the amino groups in Arg16, Arg38 and Arg194 form strong hydrogen bonds with the oxygen atoms in the 2′-phosphate of adenine ribose of NADP+ binding, the mutants, R16G and R194G, were prepared and the enzyme activities were determined. +. The kcat and Km of R16G mutant increase by more than four times and five times compared with those of WT, respectively, while the catalytic efficiency (kcat/Km) of R16G mutant decrease by 17.26%. The increase in Km indicates that affinity of R16G mutant toward NADP+ become weak, while the cofactor NADP(H) dissociated more easily from the binding site resulting in the increase in kcat. Moreover, similar change of Km value also occurred in the R194G mutants. Hence, the formation of the electrostatic environment which mainly involves the 3 arginines  mediates the cofactor specificity and recognition in right orientation.To investigate the effects of this electrostatic environment on NADP+ (WT-ternary), R16G-TCDCA-NADP+ (R16G-ternary), R38D-TCDCA-NADP+ (R38D-ternary), during 5ns MD simulations the RMSD of the backbone achieved stability quickly and kept fluctuation range from 1.0 to 1.5Å. Based on MD simulations, Molecular Mechanics Poisson Boltzmann Surface Area (MM/PBSA) calculations were preformed to evaluate the binding energies between enzymes and ligands in the three systems mentioned above. The plasticity of WT-ternary, R16G-ternary and R38D-ternary was evaluated through root mean square fluctuations (RMSF)  simulations were performed to investigate the dynamics of ternary complexes of wild type, mutant R16G and R38D. The conformational stability was verified by root mean square deviation (RMSD) of the main chain of protein compared with the initial structure of protein see . For terMSF) see , and the+ in the WT-ternary structure remain stable and the donor-acceptor distances of these H-bonds remain mostly between 2.7 Å and 3.1 Å during 5 ns dynamics  may remarkably change the NADP(H) binding pattern that nicotinamide moiety deviates the right position for interactions with substrate-enzyme. iii) Although the side-chain removal of Arg16 changed the electrostatic surroundings constructed by Arg16–Arg38–Arg194, the hydrogen bonds between 2′-phosphate of adenine ribose and Arg38 remained (CA 7α-HSDH. Such MD simulation result corresponded to that of mutagenesis experiments mentioned above. iv) The anchor effect of Arg38 though cation-π interactions with adenine moiety may play an important role in the cofactor binding to CA 7α-HSDH. Similar interactions could be observed in some of SDRs, such as Sus scrofa carbonyl reductase/20 beta-hydroxysteroid dehydrogenase and Mus musculus Sepiapterin reductase  The correct binding position of nicotinamide moiety which directly involved in the oxidation-reduction reaction of NAD(P)(H) is the key determinant of the activity of R16G, and the correct spatial orientation ensure that the catalytic sites could form the proper interaction with substrate and cofactor. ii) The rotation of the pyrophosphate moiety of NADP(H) indicates that the flexibility of the pyrophosphate bridge may allow the shaking of nicotinamide moiety for electron transfer. However, molecular modification of remained  in the btase see .CA 7α-HSDH including four subunits. CA 7α-HSDH, as a member of SDRs, possesses the typical α/β folding pattern and the very similar topological structure to EC 7α-HSDH. Nevertheless, differing from EC 7α-HSDH, CA 7α-HSDH is NADP(H)-dependent and has a more complex C-terminal structure. The tetramer of CA 7α-HSDH lies in the fact that there are the stable hydrophobic interactions between subunit A and C (B and D) and the hydrogen bonds between subunit A and D (B and C). Especially, the latter fixes the four subunits of CA 7α-HSDH, but in the aqueous solution the hydrogen bonds between subunit A and D (B and C) could be weaken by salvation effect, resulting in the dissociation of the tetramer to the dimer. We further find that the residue Thr145 performs hydrogen abstraction through the hydrogen bond with C7-OH of TCDCA, while the serine executes the same function in the other SDRs. So the active residues related to the dehydrogenation catalysis involve in Thr-Lys-Tyr combination in CA 7α-HSDH, differing from the Ser-Lys-Tyr combination in the other SDRs.We showed the crystal structure of + with the rather weak electron density of the nicotinamide moiety, which confirmed that nicotinamide moiety of NADP+ act as electron carrier in the dehydrogenation reaction. In the cofactor-binding site, the three arginines at the position 16, 38 and 194 form a positive environment surrounded the 2′-phosphate of adenine ribose of NADP+ in CA 7α-HSDH. The residues Arg16, Arg38 and Arg194 can form strong hydrogen bonds with the oxygen atoms in the 2′-phosphate of adenine ribose of NADP+. Especially, the guanidinium of Arg38 with positive charge can form the cation-π interaction with the adenine ring of NADP+. On the basis of crystal structure analysis, site-directed mutagenesis and MD simulation, we find that the cation-π interaction and hydrogen bonds between Arg38 and NADP+ have a significant anchor effect on the cofactor binding to CA 7α-HSDH. So we strongly believe that the residue Arg38 play an important role in the dehydrogenation reaction mediated by CA 7α-HSDH.Significantly, we captured an active state of the coenzyme NADPCA 7α-HSDH gene was cloned into plasmid pGEX-6p-1 and the GST-fused enzyme was heterologously expressed in E. coli BL21 (DE3) using the previously reported methodCA 7α-HSDH in aqueous solution was analysed through size exclusion chromatography (SEC) with standard protein  molecular weight 669, 440, 158, 75, 44, 29 and 13.7 kDa.+ and TCDCA in protein solution are 1.12 mM, 5.6 mM and 5.6 mM, respectively. Commercial crystallisation kits from Hampton Research were used for crystal screening. Initial crystals of complex were obtained in 0.1 M HEPES (pH 7.5) buffer containing 25% PEG-3350.Crystal screening using a crystallisation robot was performed at 293 K by sitting-drop vapour-diffusion method. 200 nL protein solution was mixed with 200 nL reservoir solution and equilibrated against 30 μL reservoir solution. The concentrations of enzyme, NADP+ and R-AXIS IV of RIGAKU. The structure of EC 7α-HSDH (PDB code: 1FMC) was used as model for solving the CA 7α-HSDH structure by molecular replacement techniques. The software iMosflmCA 7α-HSDH has been deposited into the PDB (PDB code: 5EPO).The crystal was transferred to the cryo-protection solution . Immediately, we mounted the crystal onto the goniometer which was surrounded by nitrogen at 100 K and all of the data collection works by using FR-ECA 7α-HSDH crystal, Initial structure of subunit A, NADP+ and TCDCA were extracted for molecular dynamic simulation. Initial coordinates of Arg16Gly mutant was prepared by virtual mutation and structurally minimised using SYBYL program . Wild-type ternary complex (WT-TCDCA-NADP+) or mutant ternary (R16G-TCDCA-NADP+) was immersed in TIP3 water box. Na+ ions were added to neutralise negative charges of the system, then, topology and coordinate files were created by LEaP module. Three rounds of minimisation were carried out: (1) 4,000-step minimisation of solvent and ions; (2) 5,000-step minimisation of solution and side chains; (3) 10,000-step minimisation of whole system. Combination of steepest descent and conjugate gradient method was applied in these minimisations. Temperature of system increased from 0 K to 300 K within 50 ps MD simulation. Then, another 50 ps MD simulation in NPT ensemble and 5 ns final MD simulation were performed at constant temperature (300 K). The Amber Molecular Dynamics Package (AMBER12) was used for all the MD simulationIn the asymmetric unit (4 subunits) of CA 7α-HSDH and the mutants were determined by the previously reported methods18Enzyme assay and kinetic analysis of wild-type How to cite this article: Lou, D. et al. The three-dimensional structure of Clostridium absonum 7α-hydroxysteroid dehydrogenase: new insights into the conserved arginines for NADP(H) recognition. Sci. Rep.6, 22885; doi: 10.1038/srep22885 (2016).