Phaseolus vulgaris leukoagglutinating lectin (L‐PHA) reactive oligosaccharides, and sialylation of L‐PHA reactive oligosaccharides, are closely associated with a worse prognosis for diffuse large B cell lymphoma (DLBCL) patients. Sialic acid is related to cell adhesion to the extracellular matrix and metastasis of HBL‐8 Burkitt lymphoma cells in a severe combined immunodeficiency (SCID) mouse animal model. In HBL‐8 clones, differential cell surface sialylation was due to different expression levels of UDP‐GlcNAc 2‐epimerase (GNE). Knockdown of beta‐galactoside alpha‐2,6‐sialyltransferase  resulted in enhanced lymphoma cell adhesion to galectin‐1 in anaplastic large cell lymphoma cell line, H‐ALCL. A fluorinated sialic acid analogue was shown to be useful for inhibiting sialyltransferase and may provide a new glycoengineering strategy for desialylation, as well as inhibiting invasion and metastasis and inducing cell death in lymphoma cell lines. This paper discusses glycosylation and sialylation in human lymphoma, and several glycoengineering therapeutic strategies for lymphoma.Research using mouse lymphoma cell lines has resulted in many reports of glycosylation being a key regulator for the distant metastasis of mouse lymphoma cells in animal models. In contrast, there are only a few reports of experiments examining human lymphoma cell metastasis. The glycosylation pattern in human lymphoma shows that loss of  Phaseolus vulgaris leukoagglutinating lectin (L‐PHA) reactive oligosaccharides are reported to be associated with patients’ prognosis in non‐small cell lung cancer,Glycosylation plays a significant role in the cell biology of many malignant tumors. For example, the concentration of sialic acid has been reported to be elevated in the serum of patients with malignant lymphoma,et al. used the mouse lymphoma cloned cell lines Esb and Eb and reported that sialylation is closely associated with distant metastasis in a mouse animal model. The Esb cell line has sialylated glycans and showed a higher metastatic phenotype than the Eb clone, which has non‐sialylated glycans. Two different clones are available for the Burkitt lymphoma cell line HBL‐8. Abe et al. reported that the 3G3 clone of HBL‐8 has a highly sialylated cell surface and is highly metastatic whereas the 3D2 clone of HBL‐8 has a low level of cell surface sialylation and is poorly metastatic in a severe combined immunodeficiency (SCID) mouse animal model.Limax flavus, and enhanced cell growth. Resialylation by ManNAc inhibits ceramide‐induced cell death and inhibits necrosis and death due to lactate dehydrogenase (LDH) release from the cytoplasm caused by membrane permeability, suggesting that resialylation may be closely associated with enhanced HBL‐8 cell growth or the inhibition of cell death. RT‐PCR and flow cytometry using small interfering RNA (siRNA) for GNE showed that the knockdown of GNE in the 3G3 clone of HBL‐8 reduced GNE mRNA in real time and decreased cell surface sialylation of the sialic acid‐specific lectin from Maachia amurensis.Altevogt L‐PHA reactive oligosaccharides in lymphoma modulate cell adhesion to extracellular matrix(CARD11), and PI3K‐Akt. Akt signaling is bidirectional, with one activity aiding survival and the other aiding apoptosis.N‐glycans, including L‐PHA reactive oligosaccharides of IgM, regulate cell growth induced by the CD40‐CD40L system in DLCBL.In malignant lymphoma, cell adhesion to or invasion of the extracellular matrix are significantly related to lymphoma cell metastasis.In our opinion, since glycosylation appears to be closely associated with tumor cell biology in human malignant lymphoma, the modulation of cell surface glycosylation by enzymatic (neuraminidase treatment or glycosylation inhibitors) or genetic (knockdown by siRNA) approaches could be useful for lymphoma therapy. We have continued to clarify detailed mechanisms in glycobiology pertaining to lymphoma cell adhesion, invasion, and apoptosis. Neuraminidase treatment or knockdown of ST6Gal1 enhances cell adhesion to galectin‐1, ‐3, ‐8, and laminin, and inhibits lymphoma cell invasion.36 and CD30 is specifically expressed in Hodgkin or anaplastic large cell lymphoma. Taken together, galectin‐1 may be effective for treating anaplastic large cell lymphoma in combination with anti‐CD30 antibody. In other studies, complementary DNA plasmid expressing galectin‐3 was transfected into human embryonic kidney (HEK) 293 cells. Galectin‐3 was expressed on the cell surface and the cells exhibited marked aggregation. Galectin‐3 was also expressed in ALCL cells and was secreted into the microenvironment in an autocrine fashion. Therefore, galectin‐3 may act as a cell to cell adhesion molecule, and this may be related to the cohesive cell growth which is a characteristic histological finding of ALCL lymphoma. In future we will clarify whether the biological behavior of ALCL cells is regulated by galectins, and whether galectins are useful tools for ALCL therapy by inducing apoptosis.CD30 is a target for therapy of Hodgkin lymphoma.We have previously reported many biological phenomena related to the glycobiology of human lymphoma. Modulation of cell surface glycosylation by enzymatic or genetic methods may provide useful strategies for controlling lymphoma cells. However, more detailed understanding of the underlying mechanisms are required to induce effective killing of lymphoma cells by altering their glycosylation. We have already clarified the significance of glycosylation in understanding lymphoma cell biology and believe that modulating the sialylation of a cell surface is the most promising candidate strategy for altering lymphoma cell behavior, given that desialylation of the cell surface by neuraminidase treatment, or knockdown of ST6Gal1 or GNE, may lead to cell death induced by Fas or anticancer drugs. In addition, the inhibition of sialyltransferase induces the desiaylation of tumor cells.None to declare.


European Commission (EC) criteria to identify the endocrine‐disrupting properties of plant protection products (PPPs) have been developed in a regulation for application across the European Union (EU) beginning 10 November 2018 population level for non‐target organisms,” and that “adequate, reliable and representative field or monitoring data and/or results from population models shall as well be considered where available” EC .This inconsistency between regulation and guidance threatens to make it difficult to disprove the relevance of endocrine effects on nontarget wildlife vertebrate populations because of an apparent reliance on a hazard assessment approach in which any indication of an endocrine‐mediated individual adverse effect in any laboratory test is considered to indicate that a PPP is an endocrine disruptor with wildlife population relevance. This excludes any consideration of population relevance based on modeling or field data, which is clearly not the intention of the regulation.® on Endocrine‐Active Substances (Matthiessen et al. Participants at a recent SETAC Pellston WorkshopAgreement on scientifically defensible methods to assess adverse population effects of PPPs in the context of endocrine‐disrupting activity is urgently required. In an associated paper , we prop


Traumatic brain injury (TBI) and posttraumatic stress disorder (PTSD) commonly cooccur. Approaches to research and treatment of these disorders have been segregated, despite overlapping symptomology. We and others have hypothesized that comorbid TBI + PTSD generates worse symptoms than either condition alone. We present a mouse model of comorbid TBI + PTSD to further explore this condition.  A mouse model of TBI + PTSD was generated using the single prolonged stress (SPS) protocol in combination with the controlled cortical impact (CCI) protocol. This resulted in four experimental groups: control, TBI, PTSD, and TBI + PTSD. Behavioral phenotyping included gait analysis, contextual fear conditioning, acoustic startle response, and prepulse inhibition.  Mice in the TBI + PTSD group showed a significantly impaired gait compared to their counterparts with TBI alone as well as control mice. Mice in the TBI + PTSD group showed significantly impaired contextual fear recall compared to controls. Prepulse inhibition testing revealed intact acoustic startle and auditory sensory gating.  These results indicate that SPS paired with CCI in mice produces unique behavioral impairments in gait and fear recall that are not present in either condition alone. Further studies are underway to examine additional behavioral, physiological, and pathological phenotypes in this combined model of TBI + PTSD. Traumatic brain injury (TBI) is a change in neural functioning caused by an external force to the head, resulting in mild to severe injury . TBI resPosttraumatic stress disorder (PTSD) can develop after a traumatizing event and frequently results in behavioral changes that overlap in symptomatology with TBI, including cognitive dysfunction, heightened anxiety, and exaggerated startle response . CompareThe comorbidity of TBI and PTSD in the same individual may present a unique symptomology; the extent of which has yet to be fully explored and may depend on the severity of either condition. Conventional approaches to research and treatment of these disorders in the human population have been segregated, despite overlapping symptomology. Thus, the aim of this study was to characterize behavioral outcomes in a mouse model of TBI + PTSD to add to the development and characterization of this emerging field of research –10. We hMale, C57Bl/6J wild-type mice (The Jackson Laboratory), 8-9 weeks in age and weighing 23–30 g, were used for this study. Mice were housed in groups of four and were allowed to acclimate to the colony after shipment for one week prior to beginning the study. Housing was in a 12 : 12 h light : dark cycle (lights on at 0700 h) with ad libitum access to food  and water. Temperature (21.6°C) and humidity (24%) were monitored and maintained at a constant level throughout the experiment. All protocols and housing were approved by the Institutional Animal Care and Use Committee at the Portland VA and adhered to guidelines set forth by the National Institutes of Health Guide for Care and Use of Laboratory Animals.Mice were randomized into one of four groups , and 1.0 mL children's cherry flavored acetaminophen (Q-Pap) in 15 mL of water. Mice in the sham surgery condition underwent anesthesia and scalp incision, followed by application of hydrogen peroxide to the skull and immediate closure of the incision with sutures. All mice were housed individually following surgery for the remainder of the study to ensure recovery from surgery.Experimental mice were randomized to either a CCI or a sham surgery condition. In combination with SPS above, this resulted in four groups in total . For CCI, mice were weighed, anesthetized using isoflurane , and secured to a stereotaxic frame (Stoelting Company). Mice had their heads shaved and sterilized using alcohol and iodine, before topical lidocaine was applied to the shaved head. An incision was made on the right side of the prepared skin to expose the skull landmarks bregma and lambda, and 30% hydrogen peroxide was used on a cotton applicator to remove the membranes covering the skull. Using a hand drill (Fine Science Tools), a 3 mm craniotomy was made to the right of the midline, between bregma and lambda. An impactor arm (Kopf Instruments) was then used to deliver an impact at a depth of 3.0 mm and velocity of 0.5 m/s, with a dwell time of 0.1 s to the cortical surface. The scalp incision was sutured, and triple antibiotic ointment was applied to the incision site. Mice were given a 1.0 mL subcutaneous injection of saline and returned to a standard home cage on a circulating warming pad with access to food, water, nestlets, DietGel® 76A (ClearHn = 2), failure to walk at least six consecutive strides (n = 2), and for technical difficulties of dim lighting affecting usable video acquisition (n = 4 per group). Mice were then removed from the treadmill chamber and returned to their home cage. All testing occurred between 0830 and 1000 h. Gait was analyzed using DigiGait imaging software (version 14) according to manufacturer's instructions by a scorer blinded to the group to interpret metrics involving stance and other indices of gait. For the purpose of this study, we focused on measures of stance  and stability (stance width and paw placement positioning) since TBI has been found to affect stance and stature in humans . Repeated measures ANOVA with the dB level of prepulse  as the within-subject factor and treatment group as the between-subject factor revealed, as expected, a significant within-subject effect of the PPI level with increasing amplitudes of prepulses leading to increased inhibition of the startle response to the subsequent pulse  .We found that mice in the TBI + PTSD group showed a significantly impaired gait compared to mice with TBI alone and controls. Mice in the TBI + PTSD group also showed significantly impaired fear recall compared to controls. Prepulse inhibition testing revealed no overall effect of TBI, PTSD, or combined TBI + PTSD on baseline acoustic startle and sensory gating ability. Taken together, these results indicate that SPS paired with CCI in mice produces unique behavioral impairments in gait and fear recall that are not present in either condition alone.Gait is frequently examined in animal models of TBI, likely owing to damage to the motor circuits that control gait caused by CCI and other common TBI protocols. As of now, there are no standardized parameters for TBI induced by CCI and research has shown that variations in speed and/or depth of impact can influence both cognitive and motor outcomes differentially . In our In this model of TBI + PTSD, we found that stance width in the forelimbs was dramatically decreased and paw placement position, a measure that indicates the amount of ipsilateral overlap between fore and hind paws, was preferentially increased on the left side in the TBI + PTSD mice. Paw overlap may reflect the ability of mice to balance and may contribute to the instability seen in the decreased forelimb stance width in the TBI + PTSD mice. In humans, narrower stance widths tend to be unstable , suggestTBI alone was not sufficient to induce gait changes, in contrast to what others have found in other CCI mouse models of TBI , 25. In Other studies using TBI alone found effects on stance duration that ranged from being present in all paws  to only To be able to definitively understand what factors are influencing gait, future studies will look at gait testing in larger cohorts of animals and with altered parameters, such as treadmill training. This will help determine if injury from the TBI is responsible for the effect or if other conditions are driving our results.In addition to changes in gait, we sought to investigate the pathological fear and startle response characteristic of PTSD within this combined TBI + PTSD mouse model. Overgeneralization of fear responses is common in patients with PTSD, presumably owing to their inability to differentiate between dangerous and neutral contexts , 28. PatAdditionally, we found that mice with combined TBI + PTSD showed deficits in contextual fear recall 48 hours after contextual fear conditioning as evidenced by decreased freezing when first returned to the feared context. It will be important in future research to determine if this deficit in memory recall is specific to contextual fear learning as well as memory of nonaversive events. We did not find differences during the contextual fear acquisition session, which suggests that fear learning and the expression of freezing are intact in all of our groups. The fear recall deficit is specific to retrieval of the contextual fear memory 48 hours after conditioning and possibly represents impaired memory systems within the combined TBI + PTSD model.Importantly, our observed contextual fear memory impairment was found only in the combined TBI + PTSD mice and not in mice that received a TBI alone. This suggests that the injury itself is not the cause of the memory impairment. Future research will expand on these results to examine how a contextual fear memory, acquired postinjury, changes over an extended period of time using this model to further explore memory impairments in TBI or TBI + PTSD animals. Sierra-Mercado et al. (2015) also used the CCI method to induce TBI in mice, examined auditory cued fear conditioning, and found no deficits in conditioning, recall, or extinction in CCI mice. Although we used a purely contextual model of fear learning, our TBI results are similar even though our test was conducted only one-week postinjury versus two weeks in the study by Sierra-Mercado et al. Additionally, our results add to data obtained by Ojo et al. , who faiWe anticipated that mice in the PTSD groups would have an increased baseline acoustic startle response, as has been reported in patients with PTSD . HoweverGiven the phenomenology of human subjects with PTSD, we sought to measure sensory gating in our mouse model of TBI and PTSD using prepulse inhibition testing. Prepulse inhibition tests the phenomenon of sensory gating whereby the occurrence of a low intensity “prepulse” milliseconds before the onset of a high intensity, startle inducing, “pulse,” leads to a dampening of the physiological startle response to the pulse. If successful prepulse inhibition has occurred, the recorded startle response would be lower than in the absence of the prepulse. The timing of these trials is on the order of milliseconds and is not consciously detectable to the subject. Importantly, the sensory gating phenomenon and ability to test it with prepulse inhibition testing equipment are conserved across species and altered in some psychiatric conditions.In human subjects with PTSD, results are mixed regarding the ability to appropriately filter out irrelevant auditory stimuli in tests of prepulse inhibition, with some report finding and others failing to see differences in individuals with PTSD compared to control populations . Here, wHere, we describe a unique model of TBI + PTSD in laboratory mice that recapitulates some of the behavioral impairments found in the patient population with these disease states. By combining single prolonged stress with controlled cortical impact, we have developed a controlled laboratory model of TBI + PTSD. Mice with comorbid TBI + PTSD present with unique impairments in their gait profile and problems with memory recall after contextual fear conditioning without affecting acoustic startle response or their ability to gate sensory stimuli.Further studies are underway to examine additional behavioral, physiological, and pathological phenotypes in this combined model of TBI + PTSD. As we begin to develop an understanding of how TBI + PTSD symptoms manifest in humans, the use of this animal model to further explore the underlying physiological changes associated with combined TBI + PTSD will be invaluable in both the diagnosis and treatment of this patient population.


The Alive & Thrive programme scaled up infant and young child feeding interventions in Bangladesh from 2010 to 2014. In all, 8.5 million mothers benefited. Approaches – including improved counselling by frontline health workers during home visits; community mobilization; mass media campaigns reaching mothers, fathers and opinion leaders; and policy advocacy – led to rapid and significant improvements in key practices related to breastfeeding and complementary feeding.  Intervention design was based on extensive formative research and behaviour change theory and principles and was tailored to the local context. The programme focused on small, achievable actions for key audience segments identified through rigorous testing. Promotion strategies took into account underlying behavioural determinants and reached a high per cent of the priority groups through repeated contacts. Community volunteers received monetary incentives for mothers in their areas who practised recommended behaviours. Programme monitoring, midterm surveys and additional small studies to answer questions led to ongoing adjustments. Scale‐up was achieved through streamlining of tools and strategies, government branding, phased expansion through BRAC – a local non‐governmental implementing partner with an extensive community‐based platform – and nationwide mainstreaming through multiple non‐governmental organization and government programmes.Well‐designed and well‐implemented large‐scale interventions that combine interpersonal counselling, community mobilization, advocacy, mass communication and strategic use of data have great potential to improve IYCF practices rapidly.Formative research and ongoing studies are essential to tailor strategies to the local context and to the perspectives of mothers, family members, influential community members and policymakers. Continued use of data to adjust programme elements is also central to the process.Scale‐up can be facilitated through strategic selection of partners with existing community‐based platforms and through mass media, where a high proportion of the target audience can be reached through communication channels such as broadcast media.Sustaining the impacts will involve commitments from government and capacity building. The next step for capacity building would involve understanding barriers and constraints and then coming up with appropriate strategies to address them. One of the limitations we experienced was rapid transition of staff in key positions of implementing agencies, in government leadership, donors and other stakeholders. There was a need for continued advocacy, orientation and teaching related to strategic programme design, behaviour change, effective implementation and use of data. Stewartet al.  emphasiz. et al. .et al. et al. et al. et al. et al. Effective IYCF interventions have included the following: (1) time‐targeted interpersonal counselling, individually and in groups, provided by trained health care professionals or lay workers; (2) assistance to pregnant and newly delivered mothers in initiating lactation immediately after delivery and in continuing breastfeeding; (3) provision of food and related supplements for complementary feeding; (4) media and marketing approaches; and (5) legislation to support restriction of the indiscriminate marketing of breast milk substitutes and parental leave to enable mothers to breastfeed exclusively for 6 months  initiative to develop scale models for IYCF in three distinct geographies: Bangladesh, Ethiopia and Vietnam. In this paper, we document the process of scaling up an evidence‐based IYCF intervention in Bangladesh. Results of quantitative studies documenting significant changes in IYCF practices in the A&T programme area are forthcoming. Information on the A&T programmes in Ethiopia and Vietnam is available at r et al. .In Bangladesh, the programme aimed to develop a streamlined and easily scaled‐up package of activities. A&T partnered initially with the Essential Health Care (EHC) programme of BRAC, a respected national non‐governmental organization (NGO) operating large community‐based public health, education and microfinance programmes. Programme design involved four components: advocacy, interpersonal counselling and community mobilization, mass communication and the strategic use of data /Government of Bangladesh (GOB), dissemination of government‐branded materials to implementing stakeholders.Engaging journalists: Orientations and scholarships for journalists, TV talk shows and newspaper supplements.Individualized dialogue with government decision makers and donors: MOUs, task forces and sharing evidence.Advocacy to promote child nutrition and accelerate scale‐up of programmes:Mothers: Counselling provided through home visits at specific ages of the child on breastfeeding, complementary feeding and handwashing before feeding, and group community meetings of pregnant and lactating women with trained community workers.Community opinion leaders: Mobilization of support for child nutrition and IYCF through orientations, video shows, seminars and forums to reach doctors, religious leaders, fathers, local government and NGOs working at community level.Health providers: 3‐ to 5‐day in‐service training, pre‐service medical/nursing curriculum through partnerships with medical associations, mass media and print materials, messages through national and regional newspapers, wall posters for government and private clinics, job aids for government staff and materials tailored for formal and informal health practitioners.Interpersonal counselling and community mobilizationFamilies, frontline workers and opinion leaders: TV and radio spots on key topics for mothers, fathers, frontline workers and opinion leaders at all levels.Rural community members in media dark areas: Interactive community events including village theatre, community video showings and quiz shows on IYCF and handwashing topics.Mass mediaet al. et al. et al. et al. Alive & Thrive's situational analysis included a review of policies and strategy guidelines of the GOB, data on media habits, case studies and relevant research Community component launched in BRAC EHC programme in 22 sub‐districts: August to December 2010Media campaign launch and intensification: December 2010 to March 2011Campaign materials on handwashing before feeding developed : April 2011 to July 2012Community component reaches 50 sub‐districts in BRAC's EHC programme: August 2011Final field testing and revisions of training module and job aids, rebranded as GOB materials and disseminated nationally: January 2011 to November 2011Interpersonal counselling scaled up through national government providers and NGO maternal newborn and child health and DFID‐funded livelihoods programmes: August 2011 to December 2013Media dark strategy designed and tested and community events launched in targeted low reach villages: February 2012 to June 2014Media campaign developed and print materials launched for handwashing before feeding: October 2012Media campaign for doctors developed and launched: January 2012Interpersonal counselling scaled up through GOB Health and Family Welfare workers: March 2013 to mid‐2014Alive & Thrive partner International Food Policy Research Institute (IFPRI) conducted a baseline household survey in 2010 with a sample of 2000 households having children below 24 months of age. The study was carried out in 20 sub‐districts from five of six regions in Bangladesh. Questions focused on the WHO‐recommended IYCF practices; household, maternal and child characteristics; and mothers' knowledge, contacts with health workers and media habits.N = 300), video data collection on 12 caregivers of children with a mean age of 15 months, an exercise for ranking motivations for handwashing with six groups, 24 in‐depth interviews with caregivers of children with a mean age of 16 months and six focus group discussions. The household survey and qualitative studies were conducted in 50 rural villages in three purposively selected districts in different regions of the country . Results are reported in Nizame et al.  and media reach and recall. Data regarding community interventions were collected through three channels: routine service registers maintained by BRAC including indicators on community mobilization, home visits and mothers' IYCF practices; sample surveys by BRAC's independent monitoring wing; and random sample home visits conducted by A&T focusing on BRAC home visits, community mobilization, IYCF practices and media recall.From 2010 onwards, A&T hired media placement companies to track TV viewing habits, via devices embedded in homes, in a random sample of households as part of a routine industry survey that calculates media reach for advertising and marketing companies. Data were triangulated with the recall‐aided questions in the IFPRI and A&T surveys to estimate coverage and recall of the media messages.N = 150). Other research included a pilot test of communication strategies for media dark areas, tests of options for short duration training and in‐kind incentives and field trials for participatory community approaches in Sylhet and Habiganj.Alive & Thrive carried out several small studies after programme launch to examine gaps highlighted by the baseline or that emerged during monitoring or to field test evolving strategies. Studies included a survey of doctors' knowledge, attitudes and practices related to providing IYCF support to mothers and their media habits  were enlisted and the protocol of age‐specific home visits was completed. A second cadre of full‐time IYCF promoters was allocated at a ratio of one per 10 volunteers to ensure that if the volunteer could not make the scheduled visits, the IYCF promoter would fill the gap. Non‐intensive areas = Less frequent, shorter duration routine home visits were ongoing plus the national mass media campaign on IYCF. programme areas and 500 from non‐intensive areas in 20 sub‐districts.International Food Policy Research Institute conducted two midline household surveys. The 2012 survey measured WHO standard indicators for complementary feeding practices, exposure to home visits (by two types of frontline workers) and recall of TV spots broadcast by the programme. The sample included 462 households having children 6–23 months of age selected randomly from 100 villages in 10 programme sub‐districts. The 2013 survey included infants below 6 months (as well as older children) and looked at all IYCF‐recommended WHO core indicators for breastfeeding and complementary feeding, plus the coverage indicators. The sample included 500 households from intensiveImplementation partners  shared information quarterly on their community‐based and facility‐based IYCF programmes. GOB mapped the location of these stakeholders, and A&T assisted with maintaining a database on the cumulative number of health workers and trainers provided with training. Information was shared at Alliance meetings.The existence of supportive national policies on child nutrition in Bangladesh meant that investments were not needed in this area and advocacy should focus on stimulating and supporting implementation of existing policies and guidelines. A&T also saw an urgent need for harmonizing efforts of multiple implementing NGOs and donors working in different geographic areas of the country. There was a clear opportunity to form a national alliance under the GOB's IPHN to leverage a high level of interest in IYCF and strengthen sustainability. As part of scale‐up efforts, A&T developed a national training module for the government's frontline workers. Government staff were trained by national and master trainers who were in turn trained by A&T at three government training centres in Dhaka and at one NGO training site .Advocacy for, and evidence of the effectiveness of interpersonal counselling, had been strong over several years, but large‐scale coverage of support for mothers was very limited. A&T saw the need to introduce strategies going beyond government health services. BRAC was the only NGO with scaled‐up presence in the country and had a ready‐made cadre of multi‐purpose community volunteers. They were a natural partner for introduction of new strategies.Prior to 2009, promotion of breastfeeding had received far more emphasis in Bangladesh than promotion of optimal complementary feeding practices. While there was public awareness about the importance of exclusive breastfeeding for the first 6 months, practice was still not widespread. A&T therefore decided to focus on converting high awareness of breastfeeding to an emphasis on specific desirable practices and building a basic understanding about complementary feeding.Secondary evidence clearly pointed to the importance and feasibility of promoting home‐based foods. Processed and/or fortified foods for children were not feasible as a nationally scalable strategy because of limited capacity for quality control and strong opposition from highly respected and influential IYCF advocates in the country. Household food insecurity was estimated to affect a quarter of the population, and three of four households were found to have animal source foods for child feeding NIPORT .Formative research revealed that the major barrier to exclusive breastfeeding was mothers' perception of ‘insufficient milk’. A&T strategies therefore emphasized counselling on breastfeeding skills and simple remedies to prevent and overcome common difficulties . Providers were trained to support mothers in accurately self‐assessing the adequacy of their breast milk production and in understanding how to maintain adequate breast milk supply.Formative research also highlighted the importance of promoting animal source foods. Even when available in the home, these were not being given to young children.The perception of poor appetite was widespread, leading mothers to lose confidence in proper complementary feeding. A&T messages discouraged giving children junk food, which was ubiquitous, and promoted simple strategies for motivating fussy eaters: encouraging food variety and age‐appropriate texture, reducing watery and low‐nutrient density snacks foods and spending more time feeding. Most mothers were available to spend time feeding children as outside employment is low and infant care is considered the mothers' main occupation, according to rural social norms.For mothers, motivating factors for changes in IYCF practices included benefits for child's brain and physical development and safety or protection from illnesses, perception of positive child responses (‘child likes it’), convenience and satisfaction in being able to breastfeed adequately or motivate a child to eat enough quantity of complementary foods.Alive & Thrive saw the importance and feasibility of developing a single national communication strategy. Results of the formative research and reviews of the DHS showed that patterns of good vs. weak IYCF practices were similar in urban and rural areas. Specifically, timely initiation, duration of exclusive breastfeeding and diversity of complementary foods were low in both urban and rural areas. The rapid trials of improved practices (TIPs) showed that mothers in urban and rural areas had similar reasons for common practices and similar motivations for adopting improved behaviours. Mass media reach and habits initially seemed largely the same, but more TV channels were available in urban areas. A&T decided to use mass media and community mobilization on a large scale. Secondary audiences who could encourage mothers  could also be reached rapidly through mass media.The media audit provided evidence that TV should be the main channel for high reach/coverage at national scale. A loyal viewership (68% of households watching TV at least once a week), cutting across economic and education levels, and a single main language and cultural context were crucial factors. However, the audit also showed that media habits change rapidly, so media placements needed to be adjusted based on ongoing assessments. The media audit demonstrated that Bangladesh has effectively controlled mass media advertising of breast milk substitutes, but the situation needed ongoing monitoring.Alive & Thrive's baseline survey confirmed the relative importance of different IYCF practices for programme emphasis. The survey also confirmed that even poor families consumed a diverse diet, including animal source foods, as well as vegetables and fruits – so that home‐based foods could be promoted as part of the IYCF strategy.Mothers said that health professionals and family members were their primary sources of support when faced with problems with infant feeding. Food purchases were primarily the responsibility of male members of the family. This validated A&T's decision to target multiple influential audiences.Although BRAC workers were found to be present in communities in many areas, baseline coverage of home visits to mothers was not very high, and there was substantial variability across sub‐districts. Overall, about a quarter of respondents had ever been visited at home by a BRAC frontline health volunteer (Shasthya Sebika). Contact between households and Shasthya Sebika went up to over 60% in some areas. A smaller proportion of respondents had been visited by a BRAC health worker (Shasthya Kormi) at 11.3%; 12% attended a health forum facilitated by a Shasthya Kormi.BRAC's capacity to manage and organize mobilization of frontline workers on a large scale was strong, with great potential for strengthening home visits and community mobilization. BRAC decided to follow the example of their community‐based programmes  to provide performance‐based monetary incentives to volunteers rather than a fixed salary to conduct home visits (one per 250–300 households) and supported them with full‐time mentors (IYCF promoters) with one mentor per 10 volunteers. Volunteers received a cash incentive  based on each eligible mother who practised the IYCF behaviours. This motivated volunteers to counsel almost every eligible woman in their respective areas and work on problem‐solving for changing behaviours rather than only giving messages.et al. Feedback from monitoring visits revealed that mothers often complained that they were unable to feed children because of ‘poor appetite’. This did not refer to ‘sick’ children only, or frequently sick children. A&T hypothesized that one reason for low appetite might be sub‐clinical enteropathic infections from contaminated complementary foods due to infrequent washing of hands with soap before feeding children  were prioritized.Results of knowledge, attitudes and practices surveys of doctors and their media habits in 2012 showed that doctors were not providing even basic counselling about child nutrition to mothers. They did not have the knowledge, skills or motivation for improving IYCF practices. There was some evidence that they may even have been reinforcing poor practices – for example, recommending breast milk substitutes for perceived insufficient milk and encouraging ‘soft foods’ that are low in energy and nutrient density for infants under 1 year old. The survey showed that a large proportion did not believe it was their responsibility to counsel mothers on IYCF. It also showed that doctors could be reached with high coverage through print media and TV.Alive & Thrive decided IYCF counselling needed to be urgently mainstreamed within national health services. GOB leadership was important for introducing IYCF counselling during antenatal care and immunization contacts and also at sick child visits. Standards of care for maternities, training on how to initiate breastfeeding and special support for C‐section deliveries were needed. A&T provided a partnership grant to the Obstetrical and Gynecological Society of Bangladesh (OGSB) to support revisions to the pre‐service curriculum. The programme also launched a mass media campaign through inserts in newspapers for 20 months starting in October 2012.et al. The household survey on handwashing revealed that 95% of participants had soap in their homes but only 37% had soap present at the place for handwashing before food preparation  conducted in Sylhet (eastern region), Manikgonj  and Dinajpur (northern region) also showed that a significant number of communities did not view TV because of electricity shortages and exclusion from services due to various reasons, and this was contributing to low TV viewership.Meena animated films and the seven TV spots being broadcast through national TV. Each material was played, and key messages were then discussed by trained facilitators. Difficulties in practising the actions were also discussed. A small feasibility trial in 19 randomly allocated low electricity villages confirmed that even a single round of such an approach would raise message recall and knowledge of correct practices. Based on this, the ‘media dark’ strategy was mainstreamed in the A&T programme.Alive & Thrive tested a supplementary communication strategy of exposing these communities to the same TV spots through mobile video shows and interactive Q&A sessions  in order to reach a wide cross‐section of community members and opinion leaders in low‐electricity communities. The materials included A&T messages contained in et al. The 2013 midline survey showed improvements in TV exposure above 2010 and 2012 levels in both A&T programme areas  compared with non‐intensive areas] as follows: early initiation of breastfeeding from 63.5% to 91.9% (DD of 18.7), exclusive breastfeeding from 48.5% to 83.4% (DD of 25.0), minimum dietary diversity from 32.1 to 61.8 (DD of 26.5), minimum acceptable diet from 16.0 to 48.0 (DD of 24.6) and iron‐rich foods from 39.5 to 72.2 (DD of 20.9).Problems in IYCF still remained, including not feeding during illness and use of sugary drinks and foods and salty snacks.et al. The processes that were associated with improvements in practices included high levels of exposure to TV spots on IYCF in both intensive and less intensive programme areas and, in intensive areas, higher frequency of home visits by trained IYCF workers  branding of the package for broader ownership and uptake. The national IYCF communication plan framework and document developed by the IYCF Alliance with A&T and UNICEF support was also branded and distributed by the government and adopted by a range of stakeholders. A&T also made adoption convenient by providing seed money, timely technical support and free materials and training of trainers to government and NGOs.After launching the programme in 2010, A&T programme components were streamlined starting in 2012 as part of the transfer process to new implementing partners. For example, training of frontline workers was reduced from 5 to 3 days. Post‐training supervision checklists were reduced from over 25 items each for breastfeeding and complementary feeding to a single two‐sided sheet with 8–10 items for observing counselling content and technique. The number of separate community mobilization forums was reduced from 8 , other NGO workers, village elite, local government leaders and village doctors) to 3 .Programme scale and effectiveness increased over time because of monitoring and purposeful adjustment of strategies that led to new implementing partners. When the 2012 midline survey showed that mothers continued to consult doctors for IYCF difficulties, A&T increased its emphasis on reaching doctors, through both in‐service and pre‐service training. New communication channels were employed (newspaper inserts) to reach medical doctors; the national OGSB was selected as a strategic partner to lead the strengthening of pre‐service medical and nursing curricula.The formation of the National IYCF Alliance of multiple stakeholders by A&T and UNICEF under GOB/IPHN facilitated expansion of the IYCF programme beyond BRAC to new implementers. The transfer of approaches, tools and skills nationwide was carried out through this central government‐led hub. Collaboration with UNICEF  and advocacy with donors such as DFID and USAID helped lay the groundwork for sustainability. In the second year of implementation, the country's national nutrition programme (supported by multiple donors) was reorganized as the National Nutrition Service, and with A&T's inputs, IYCF became a top priority in plans for mainstreaming through government health and family planning services.et al. Scale‐up took place in several ways. Partnerships led to the geographic expansion of the A&T community model and tools within different BRAC programme divisions and outside BRAC BRAC . The govTo increase the commitment of new stakeholders to IYCF, A&T's advocacy component engaged national medical associations of paediatricians and Ob/Gyn practitioners, as well as donors and senior government officials. Journalists were invited to raise awareness by reporting on the risks of stunting and related IYCF government programmes and policies.Successful scale‐up was facilitated in Bangladesh by contextual factors. Publicity around the global Scale Up Nutrition (SUN) movement movement stimulated involvement in IYCF by new donors. Different constituencies responded for different reasons. Government historically had progressive policies  and strategies  but stagnant indicators NIPORT . GovernmThe National IYCF Alliance – comprising A&T, BRAC, UNICEF and the government as its nucleus plus over 20 other stakeholders – led to building up resources for scale and is now a formal structure of the government of Bangladesh.Challenges faced during scale‐up included (1) lack of demand due to widespread gaps in understanding among communities and health workers of complementary feeding beyond introduction at 6–8 months; (2) lack of capacity, leadership and confidence including concerns among implementing partners that mainstreaming IYCF into existing programme platforms might not work because of work overload and lack of technical focus; (3) a limited number of experienced nutrition programme implementers with the necessary practical training; (4) unfamiliarity among implementers with WHO's IYCF indicators; (5) the need for training and supervising a large number of health care providers and community volunteers in the labour‐intensive interpersonal component; (6) a substantial number of the population residing in ‘media dark’ areas; and (7) one in four households facing economic constraints. Donors were reluctant to make adequate financial commitments without firm evidence of results from the endline survey in 2014.Key to scale‐up of A&T's IYCF interventions in Bangladesh included early evidence that behaviour change interventions were working, partnership with a strong community‐based NGO, the formation of alliances with like‐minded stakeholders, availability of funds and technical support from multiple donors, feasible programme implementation options, well‐defined interventions and indicators, and streamlined processes and tools to aid implementation. Formation of synergistic partnerships for sustainability and scale – particularly the IYCF Alliance, including governmental, non‐governmental and donor organizations – ensured complementary resources to support IYCF activities.Alive & Thrive is funded by the Bill & Melinda Gates Foundation and the governments of Canada and Ireland.The authors declare that they have no conflicts of interest.TS, JB, RS and AJ contributed to writing different sections. SR, RH and KA provided concepts, interpretation and facts and figures for the document.


These are mainly based on the use of a combination of synthetic/natural scaffolds and bioactive agents, obviating the incorporation of cells. However, there are some inherent limitations associated with traditional techniques, and we hypothesized that the use of cell‐based therapies as part of comprehensive regenerative protocols may help overcome these hurdles to enhance clinical outcomes. We conducted a systematic review of human controlled clinical trials investigating the clinical and/or histological effect of the use of cell‐based therapies for alveolar bone and periodontal regeneration and explored the translational potential of the different cell‐based strategies identified in the included trials. A total of 16 studies  were included for data synthesis and qualitative analysis with meta‐analyses performed when appropriate. The results suggest a clinical benefit from the use of cell therapy. Improved outcomes were shown for alveolar ridge preservation, lateral ridge augmentation, and periodontal regeneration. However, there was insufficient evidence to identify best‐performing treatment modalities amongst the different cell‐based techniques. In light of the clinical and histological outcomes, we identify extraction socket and challenging lateral and vertical bone defects requiring bone block grafts as strong candidates for the adjuvant application of mesenchymal stem cells. Given the complexity, invasiveness, and costs associated with techniques that include “substantial manipulation” of tissues and cells, their additional clinical benefit when compared with “minimal manipulation” must be elucidated in future trials.   Cell‐based therapies have the potential to improve outcomes of regenerative treatment in the oral cavity. Extraction sockets and challenging lateral/vertical bone defects requiring bone block grafts are ideal candidates for the adjuvant application of mesenchymal stem cells. The present review showed that there is insufficient evidence to identify best‐performing treatment modalities among the different cell‐based techniques. Given the complexity, invasiveness, and costs associated with the “substantial manipulation” of tissues and cells, the clinical benefit of these techniques when compared with “minimal manipulation” must be elucidated. Further research evaluating the effectiveness of simple, fast, and economical methods for cell harvesting and processing is warranted.Outcomes in alveolar bone and periodontal regeneration are largely dependent on the biological and the material characteristics of scaffolds and the availability, recruitment and activation of cells and biomolecules in the injured area during healing. A large number of reviews have assessed the outcome of bone regeneration techniques either before or at the time of implant placement and following maxillary sinus augmentation The primary aim of this systematic review is to assess the efficacy of cell therapies on clinical and histological outcomes for alveolar bone and periodontal regeneration. We will focus on the use of these innovative approaches in human clinical studies investigating specifically ridge preservation, horizontal and vertical alveolar bone augmentation, sinus augmentation techniques, and periodontal regenerative surgeries. The results will be stratified according to the defect type but also with regard to the source of mesenchymal stem cells (MSCs) and the techniques for manipulation of tissue samples and cells. As a secondary outcome, we aim to investigate the translational potential outside a hospital/university setting of the different cell‐based strategies identified in the included controlled clinical trials.www.crd.york.ac.uk/PROSPERO).The protocol was registered on the PROSPERO database: ID CRD42019121119  evaluating the effect of surgical treatment with or without the adjunctive use of cell‐based therapies for the treatment of alveolar bone/periodontal defects assessing changes in alveolar bone dimensions/periodontal clinical measurements. Secondary outcomes were assessed only for those studies reporting data on the primary outcomes. A minimum of 6 weeks follow‐up post‐surgically for alveolar ridge preservation, 3 months follow‐up for lateral/vertical ridge, and sinus augmentation and 12 months for periodontal regeneration studies were required. Studies were stratified according to the defect model and each group was analyzed independently: (a) ridge preservation (ARP); (b) lateral and/or vertical ridge augmentation (GBR); (c) sinus augmentation (SINUS) d) periodontal regeneration (PERIO). Studies investigating the use of cell therapies to reconstruct large defects due to maxillectomies were excluded. Case reports and/or case series, retrospective clinical studies, and publications reporting the outcomes of in vitro studies or preclinical  studies only were excluded.The primary outcome was dependent on the defect model: postsurgical alterations in the alveolar bone defect size, based on direct/indirect clinical and/or radiographic measurements were considered as primary outcomes. See the Supporting Information for all primary and secondary outcomes selected according to the defect type.A highly sensitive search was conducted. Electronic databases  were searched using a string of medical subject heading and free‐text terms. OpenGrey was searched for the unpublished, gray literature. The search strategy was first designed for the MEDLINE database and then modified appropriately for the other databases (see Supporting Information). There were no language or publication date restrictions. Reference lists of all studies included for full text screening and previous reviews were searched for missing records. A manual search for the period from December 2013 to May 2019 was completed for a number of scientific journals (see Supporting Information). Further details regarding study selection, data extraction and analysis, quality assessment, and the processes followed for data synthesis can be found in the Supporting Information.A total of 5,053 potentially eligible records were returned from the electronic searches. A further 39 publications were found through the manual search of the selected journals. Following deletion of duplicates, a total of 4,741 records were available for initial title/abstract screening, after which the full‐text of 74 publications were retrieved. After assessment of the full papers, a further 56 records were excluded. Therefore, a total of 18 There was a total of 11 randomized controlled trials (RCTs) We identified cell‐based therapies according to the type of MSCs harvested and the donor tissue. Seven studies reported the use of bone marrow stem cells (BMSCs) harvested from posterior iliac crest aspiration For the ROBIN‐I assessment of nonrandomized clinical trials, five of the six studies included were considered to be at a high risk of bias. Among the RCTs assessed with the Cochrane RoB 2.0 tool, two studies showed a low risk of bias, three exhibited some methods which raised some concerns about the risk of bias, and six reported systematic errors in design, adherence or reporting which translated in a high risk of bias. Figure The reader is referred to the extensive tables in the Supporting Information for a comprehensive summary of clinical, histological, and other relevant outcome data reported in the included studies categorized according to defect type. A brief synthesis is presented below with Forest plots of all meta‐analysis carried out presented in Figures Due to significant differences in study characteristics, it was not possible to complete meta‐analysis of effects size for the studies reporting on the use of stem cells for alveolar ridge preservation; a short narrative synthesis is therefore presented.Mean diff 36.5%, p < .001) and the horizontal  dimensions 45–90 days after ridge preservation surgery. Another two RCTs reported on the use of BMSCs Mean diff 23.6% [6.02–41.09], p = .01) but the benefit of the cell‐based therapy was lost at 12 weeks  Mean diff 13.9% [−5.03–33.2], p = .09) and no difference at 12 weeks  p = .014) with the test group losing on average 13.61% ± 12.5% and the control group 31.35% ± 11.88% of the original ridge width (p = .006) p = .36) Of the four original reports included I2 = 84.9%). The combined estimate for the three studies reporting data for percentage of mineralized tissue histologically was 12.76% . The three RCTs reported no complications or adverse events.Two studies reporting radiographic horizontal bone width linear measurements outcomes 3  in favor of the stem cell groups with substantial heterogeneity  was observed. These data are in line with the outcomes of the other four studies with an adequate control groups Three CCTs and three RCTs reported data for radiographic changes in alveolar ridge dimension. However due to discrepancies between studies, only two studies Histologically, two studies I2 = 0.0%) and radiographic BF  with no observed heterogeneity and a nonsignificant improvement in PD , and REC . This positive effect is further illustrated by one of the studies reporting CAL gain as a % of the original defect, showing 83.5% (±31.7) defect resolution in cell‐grafted sites compared with 55.0% (±21.9) in control sites For studies aiming to achieve periodontal regeneration, one of the RCTs did not report clinical measurements beyond 3 months and did not report the standard deviations of the differences in radiographic bone fill at 12 months postop The low number of included studies does not come as a surprise given that only recently the application of cell‐based therapies for regeneration of alveolar bone and periodontal tissues has been reported. We focused our research question around RCTs and CCTs which could contribute clinical outcome measures and not only histology analysis. For this reason, the secondary histology outcomes reported in this review were limited due to many studies reporting only histology analyses being excluded.The CCTs included in this review were considered to be at a high risk of bias There were virtually no complications and no severe adverse events across all of the included studies in all the different defect categories, demonstrating the safety of this treatment modality. Although there were insufficient data to draw any conclusions regarding best cell‐based treatment strategies, an appraisal of the clinical applicability of the different techniques identified through the systematic search is warranted. Our assessment of the different techniques will appraise aspects which may be relevant for widespread adoption later on the translational pathway. We hope this will help identifying techniques which may be prioritized in future research.The studies The regeneration of tissues depends mainly on three key players: scaffolds, cells, and biomolecules coming together to orchestrate a series of spatial‐temporal events which result in a pattern of healing that resembles the original components, structure, and function of the lost tissues. There were significant differences between the techniques appraised in this review on the use of scaffolds and “biologics.” For instance, there was a wide range of different scaffolds which were used as “carriers” for the cells. When the studies are stratified according to defect type, it becomes apparent that the scaffolds were selected according to the biomechanical and biological requirements of the defects which were being reconstructed and not for their ability to improve cell attachment and/or behavior. For this reason, we will focus the discussion on the cell harvesting and manipulation steps.We based our “clinical applicability” appraisal on domains like those described in Roger's diffusion of innovation theory We consider the invasiveness of the harvesting procedure as one of the factors potentially affecting adoption. The techniques involving DPSCs and PdlSCs harvesting require the extraction of a tooth. This invasive procedure would only be appropriate in cases were extractions are already scheduled, limiting its applicability. The harvesting of A‐SVF requires a liposuction under general anesthesia and is therefore considered highly invasive. Bone marrow aspirates were carried out under local anesthesia and, in some cases, conscious sedation and were rated as moderately invasive. The less invasive technique was the harvesting of gingival connective tissue from intra‐oral sources. The clear advantage of this technique is that tissues are readily available. Besides their invasiveness, the need of a BMA or liposuction adds an additional layer of complexity due to the need to contact and organize specialists outside the dental office while also adding significant costs.After harvesting, the cells were either “minimally” or “substantially” modified before application in the surgical site. The rationale for cell expansion is based on the fact that only a small fraction of the cells within the donor tissues are characterized as MSCs. Billions of cells can be generated from as little as 1 ml of bone marrow aspirate following ex vivo expansion The time that lapsed from cell harvesting to application in “substantially manipulated” therapies ranged from 12 days to over 6 weeks Based on a limited number of studies, cell‐based therapies have the potential to improve outcomes of regenerative treatment for the reconstruction of alveolar bone and periodontal tissues. We identified extraction socket defects and challenging lateral and vertical bone defects requiring bone block grafts as ideal candidates for the adjuvant application of MSCs. This review showed that there is insufficient evidence to identify best‐performing treatment modalities among the different cell‐based techniques described in the included publications. This research synthesis has also highlighted a high risk of bias in most of the studies and the need for well‐designed randomized clinical controlled trials including the gold standard of treatment as a positive control. Given the complexity, invasiveness and costs associated with techniques which include “substantial manipulation” of tissues and cells, their clinical benefit when compared with “minimal manipulation” must be elucidated in future trials. Further research evaluating the clinical effect and cost‐effectiveness of simple, fast, and economical methods for cell harvesting and processing is warranted.F.M.S.: protocol design, publication screening, data abstraction, data analysis, interpretation of results, writing of the manuscript; Y.L.: protocol design, publication screening, data abstraction, data analysis, interpretation of results, reviewing the manuscript; M.O., J.B.: protocol design, data analysis, interpretation of results, review of the manuscript; W.V.G., F.D.: interpretation of results, review of the manuscript.The authors indicated no potential conflicts of interest. UCL and/or UCLH and UCLH CBRC paid salaries as part of their research/clinical roles to all authors and researchers involved in the project.Appendix S1: Supporting Information 1Click here for additional data file.


Our understanding of the diversity and community dynamics of soil fungi has increased greatly through the use of DNA‐based identification. Community characterization of metabolically active communities via RNA sequencing has previously revealed differences between ‘active’ and ‘total’ fungal communities, which may be influenced by the persistence of DNA from nonactive components. However, it is not known how fungal traits influence their prevalence in these contrasting community profiles. In this study, we coextracted DNA and RNA from soil collected from three Antarctic islands to test for differences between total and active soil fungal communities. By matching these geographically isolated fungi against a global dataset of soil fungi, we show that widely dispersed taxa are often more abundant in the total community, whilst taxa restricted to Antarctica are more likely to have higher abundance in the active community. In addition, we find that active communities have lower richness, and show a reduction in the abundance of the most dominant fungi, whilst there are consistent differences in the abundances of certain taxonomic groups between the total and active communities. These results suggest that the views of soil fungal communities offered by DNA‐ and RNA‐based characterization differ in predictable ways. Molecular techniques have substantially increased our knowledge of fungal communities in soils across the globe  region, a much shorter‐lived molecule than DNA, which is present predominantly in metabolically active tissue , and the lowest for DNA in Signy Island soil . The abundances of all taxa varied across islands and between total and active communities . In contrast, OTUs of cosmopolitan fungi were significantly more likely to have higher abundance in the total community than would be expected by chance . Bipolar fungi tended towards having higher abundances in the total community, but this difference was not significant .Endemic and cosmopolitan fungi showed differential patterns of abundance between the active and total communities Fig. . Fungal r = 0.629–0.669; a plot of one random iteration is presented in Supporting Information P = 0.0501), species evenness of RNA was consistently higher than that of DNA at each of the individual islands , and there was no effect of soil depth on the magnitude of the difference in fungal richness between total and active communities  – i.e., the difference in fungal richness between total and active communities did not change with depth.Across both active and total communities, fungal richness decreased with soil depth, from an average of 147 OTUs at 2 cm to 133 OTUs at 8 cm, but the relationship was not significant  and within each of the three islands , demonstrating that when communities were sampled from the same location, those characterized using DNA were more similar to other samples characterized using DNA than to those characterized using RNA, and vice versa. A generalized linear mixed effect model also showed that the proportion of fungi shared between active and total communities decreased across all islands with increasing soil depth . Tests within each island showed that this effect was present at Signy Island , but not at the other two islands .Adonis analyses were used to test for differences in the composition of soil fungal communities characterized using either RNA or DNA. Significant differences were detected between the composition of active and total soil fungal communities across all islands (P < 0.001).Two classes of fungi showed significantly different patterns of abundance between the total and active communities across the three islands: OTUs belonging to the Leotiomycetes and the Microbotryomycetes more often showed higher abundance in the total community than in the active community  were consistently recorded as cosmopolitan taxa (Supporting Information Table S1). By contrast, all but three of the 86 OTUs in the Chytridiomycota were found to be endemic to Antarctica (Supporting Information Table S1). The same pattern was observed for lichen‐forming fungi, with all but one of the 44 OTUs in the Verrucariales and the Lecanoromycetes being recorded as endemic to the Antarctic (Supporting Information Table S1).Abundances of fungal genera are provided in Supporting Information Table S1. Eight OTUs in the Trichocomaceae , which on artificial media produce copious conidia typically measuring < 6 μm × < 4 μm  in order to achieve long‐range dispersal Fig. , which iet al., et al., et al., et al., et al., et al., et al., et al., Across all three islands, we consistently found that the richness of the active fungal community was lower than that of the total community, in contrast with previous studies that have found no differences between these two communities  Bartl., the only two native vascular plant species that occur in Antarctica. On each island, 50 ml sterile centrifuge tubes  were used to collect soil samples by hammering them directly into the vertical walls of three pits at three depths . Thus, a total of 27 soil samples were collected from across the three islands. The three pits were a maximum of 1 km apart on each island, with an average distance of 311 m separating them. The soil, kept on ice after collection and frozen at −80 °C within 5 h, was freeze dried to preserve fungal nucleotides.Between October and November 2011, soil samples were collected from Bird Island , Signy Island  and Léonie Island  in the sub‐Antarctic, low maritime and high maritime Antarctic respectively. In order to achieve consistency between islands, soil was collected from under populations of co‐occurring et al. , using RNA PowerSoil Total RNA Isolation and DNA Elution Accessory kits . Extracted DNA was amplified in triplicate PCR reactions using the primers ITS1F and ITS4 as described by Cox et al. , with coet al.  and ITS4 et al.,  primers.The triplicate PCR products were pooled and subsequently purified using AMPure XP bead purification  and quantified using a Qubit dsDNA HS Assay  before normalization to consistent concentration. The purified and normalized PCR products were run on a single plate, on the 454 Roche Titanium FLX platform at the Liverpool Centre for Genomic Research, at the same time and under identical conditions to the DNA library.et al., et al., et al., et al., The resulting RNA sequences were pooled with the DNA sequences  against a previously described global database of 32 376 soil fungal ITS2 sequences   were used to investigate whether endemic, cosmopolitan and bipolar fungi differed between total and active communities. This test was designed to identify differential abundances greater than the expected stochastic differences between sequencing runs. Based on BLAST searches against the global database of soil fungal ITS2 sequences  and active (RNA) samples. R (Pearson's) = 0.643, n = 27. 1:1 line is shown in red.Table S1. The number of OTUs of each fungal genus, assigned as endemic, cosmopolitan or bipolar when compared to a global dataset of soil fungi. Taxonomies were assigned by running representative sequences through the BLAST algorithm against the UNITE fungal database. Bipolar fungi were defined as those occurring in Antarctica, and north of the Arctic circle (approximately 66.5° north), but not occurring at latitudes in between.Click here for additional data file.


CFSs) has become a widespread intervention targeting protection and support for displaced children in humanitarian contexts. There is a lack of evidence of impact of these interventions with respect to both short‐term outcomes and longer‐term developmental trajectories.The establishment of Child Friendly Spaces  and endline  amongst 430 CFS attenders and 161 nonattenders. Follow‐up assessments after the end of CFS programming were conducted 18 months post‐baseline (T3) with caregivers of 249 previous CFS attenders and 77 CFS nonattenders.We collected data from caregivers of Congolese refugee children residing in Rwamwanja Refugee Settlement at three timepoints. To assess short‐term impact of CFSs was associated with better maintenance of psychosocial well‐being  and greater increases in developmental assets , with significantly stronger impacts for girls. CFS interventions meeting higher programing quality criteria were associated with greater impact on both PSWB and development assets . Amongst boys, benefits of program attendance were only indicated for those attending higher‐quality CFS . At follow‐up, however, there were no discernable impacts of prior CFS attendance on any measures. Age and school attendance were the only characteristics that predicted an outcome – developmental assets – at follow‐up.In the short‐term, attendance at CFSs – particularly involving higher‐quality programming – supported children's well‐being and development. However, sustained impact beyond active CFS programming was not demonstrated. Intervention goals and strategies in humanitarian contexts need to address the challenge of connecting children to other resources to facilitate developmental progress in conditions of protracted displacement.Attendance at  Forced displacement currently impacts the lives of more than 65 million people globally UNHCR, . ChildreStrategies for addressing these challenges to protection and well‐being have become an increasing focus of research  are increasingly adopted as the presumptive intervention strategy by humanitarian agencies to promote the mental health and psychosocial well‐being (PSWB) of children in crisis contexts. They are seen as a safe environment in which children may resume some sense of normalcy  and other members of the Global Protection Cluster Child Protection Working Group, to explore the protective and restorative outcomes and impacts of CFSs in various emergency contexts  before the opening of CFS locations. For endline assessment , these same caregivers were then interviewed during the final weeks of the CFS program in their respective villages. Caregiver reports of CFS attendance in the intervening period were collected (and triangulated with CFS attendance records where these were fully available). Caregivers were interviewed again 18 months following baseline assessment, when all formal CFS programming had ceased.This longitudinal study adopted a quasi‐experimental design with randomized‐cluster sampling  and the Office of the Prime Minister of Uganda.Measurement tools were selected in collaboration with the program team to assess the main objectives of CFS intervention: (a) child protection, (b) supporting caregivers and communities in strengthening systems of support, care, and protection of children, and (c) the promotion of children's social and emotional well‐being. Measures were translated into the two local dialects by a trained translator and then – following piloting in one village – confirmed through group translation and back translation to ensure team comprehension and accuracy in the contextualization of core constructs.The Child Protection Rapid Assessment (CPRA) is an interagency designed tool that provides a series of questions to rapidly identify and prioritize protection needs of children in the context of a rapid‐onset emergency  and then conducted bivariate and multivariate linear regressions controlling for baseline outcome scores, age, gender, and school attendance. We calculated Cohen's d to estimate the effect of attending CFS at T2. We additionally used logistic regressions to explore what factors  predicted CFS attendance at T2. We further explored the effects of attending a higher‐ vs. lower‐quality CFSs in comparison with nonattendance via multivariate regressions . Multilevel multivariate modeling, using households and village clusters as levels, was also attempted; however, sample size compromised the integrity of such models (models not reported).Following data collection and cleaning, to account for potential contamination, we restricted analyses to data pertaining to children residing in the same geographic areas throughout the study period. The internal consistency of measurement scales – the PSWB and CDRA – was examined using Cronbach's alpha. The intervention group was comprised of CFS attenders, defined as children reported as attending the CFS at T2 either ‘sometimes’ or ‘always’. To estimate short‐ and longer‐term impact, we compared mean scores of outcomes for CFS attenders and nonattenders at baseline and endline and at baseline and follow‐up, respectively. We began with univariate analyses , data were collected with respect to 447 children of the original sample. We retained and analyzed data for 326 children for whom we could match records regarding age and gender across all measurement points and, for children reported as previously attending CFS, with unchanged location (enabling association with a specific CFS). 249 of these 326 (76.4%) children had previously attended CFS during their operation; 77 (23.6%) were nonattenders  or village of residence (χ2n = 591). Vulnerability was not a predictor of CFS attendance . Both children who had attended a CFS and their caregivers tended to be slightly younger than children and caregivers of those who did not attend. Children whose caregivers reported them attending school in the period between baseline and endline were less likely to have attended a CFS . Scoring on protection and well‐being measures at baseline were generally similar for intervention and comparison groups. However, reported developmental assets and knowledge of child protection resources were significantly lower – and reported barriers to accessing and using these child protection resources significantly higher – for those whose children subsequently attended CFS. On the basis of these differences – and the nonrandom allocation of attenders and nonattenders – we used multivariate regressions controlling for baseline outcome scores, age, gender, and school attendance as the basis for subsequent reported findings.Baseline and endline characteristics for the sample retained at T2 are summarized in Table D effect‐size estimates of attending CFS on key outcomes at T2. Table p < .001, Cohen's d = .231) and better maintenance of PSWB . The latter was notably in the context of generally deteriorating well‐being scores for those not attending CFS .Figure p < .001) vs. attenders of lower‐quality CFS ; for CRDA, attenders of higher‐quality CFS  vs. attenders of lower‐quality CFS . Indeed, amongst boys, benefits of program attendance were only indicated for those attending higher‐quality CFS .The attribution of these trends to CFS attendance is supported by subgroup analysis  and development assets , with no significant impact of prior CFS attendance identified for girls  or attenders of higher‐quality CFS .Regression analyses confirmed that neither baseline characteristics of gender and vulnerability, nor baseline scores on well‐being and development assets, predicted scores on these outcomes at T3. Age was found to be the sole significant predictor of a T3 outcome, with respect to development assets .The lack of short‐term impact of CFS attendance on protection concerns and caregiver stresses needs to be interpreted in the context of the wider risks presenting within the settlement , which it is challenging for sessional programming to address. Significant protection impacts of CFS have, however, been reported in other contexts  toward the support and protection of children must remain central to the design and implementation of the intervention. It is vital to provide integrated services that remain flexible to the evolving needs and priorities of children and families and are not dependent on unsustainable external programming.Finally, the observation of stronger intervention effects on PSWB and development assets for CFS programming that met high programing standards underlines the general importance of ensuring quality and fidelity in interventions implemented at scale if targeted impacts are to be secured. Planned work examining the impacts of alternative approaches to CFS curricula builds upon this finding . However, there is little evidence of their impact in either the short‐ or longer‐term.This study reports on caregivers reports of children’s wellbeing – and their concerns for their children ‐ on first arrival in the studied refugee settlement (before the commencement of CFS programming); at the end of the intervention period; and at one‐year follow‐up.Those children that attended a CFS maintained better psychosocial wellbeing and secured greater development assets after 3 to 6 months, with the strongest impacts in the better‐run CFSs.A further year later, however, there were no significant differences between children who had attended or not attended a CFS in their early months in the refugee settlement, although scoring in the latter showed greater variation.If interventions in humanitarian contexts are to substantially influence longer‐term trajectories of well‐being and adjustment they will need to be more effective in connecting children to resources in conditions of protracted displacement.Table S1. Longer‐term analysis: sample characteristics.Table S2. Multivariate regressions  indicating impact of lower and higher‐quality CFS attendance at T2 on key outcomes at endline.Table S3. Multivariate regressions  indicating impact of lower and higher‐quality CFS attendance at T2 on key outcomes at follow‐up.Click here for additional data file.


DBH) and its standard deviation (SD), sample size of trees with and without active nest, amount of nest and available trees described as dead or with a broken crown, and amount of nest and available trees that were lacking these characteristics. Further collected data included bird species nesting in the cavities and nest‐building type (nonexcavator/excavator), forest type (coniferous/deciduous/mixed), biome , and naturalness . From these data, three effect sizes were calculated that describe potential nest trees in terms of DBH, vital status , and crown status (broken/intact). These tree characteristics can be easily recognized by foresters. The results show that on average large‐diameter trees, dead trees, and trees with broken crowns were selected for nesting. The magnitude of this effect varied depending primarily on bird species and the explanatory variables forest type and naturalness. Biome had lowest influence (indicated by ΔAIC). We conclude that diameter at breast height, vitality, and crown status can be used as tree characteristics for the selection of trees that should be retained in selectively harvested forests.Many studies have dealt with the habitat requirements of cavity‐nesting birds, but there is no meta‐analysis on the subject and individual study results remain vague or contradictory. We conducted a meta‐analysis to increase the available evidence for nest‐site selection of cavity‐nesting birds. Literature was searched in Web of Science and Google Scholar and included studies that provide data on the habitat requirements of cavity‐nesting birds in temperate and boreal forests of varying naturalness. To compare nest and non‐nest‐tree characteristics, the following data were collected from the literature: diameter at breast height ( These observations illustrate another key role cavity nesters play in ecosystems and underline the neccessity for explicit evidence regarding their habitat requirements.Besides being of general interest for the conservation of avian biodiversity, cavity‐nesting birds act as keystone species on a variety of grounds. Most obviously, woodpecker cavities benefit numerous subsequent nonexcavating bird species  evaluate whether cavity‐nesting birds select for nest trees with large DBH, dead trees, and trees with broken crowns. We also aimed to elucidate the influence of (b) dominating tree species and (c) naturalness in (d) temperate and boreal forests. In conjunction to the use of meta‐analytical methods to quantify nest‐tree selection of cavity‐nesting birds, we also provide an overview of management recommendations extracted from the reviewed studies as a scientific basis to (e) guide future management decisions in the selection of trees that should be retained in temperate and boreal forests.2Studies were included in the analysis according to a priori study inclusion criteria (see Supporting information List SD) [cm], including the sample size (N) of dead and live trees with and without active nests. Most studies provided information to separate nest‐ from non‐nest trees. However, this separation was not always possible. If fewer than 25% of random trees were nest trees, then the study was included (Supporting information Appendix DBH (breast height = approx. 1.3 m above ground) and standard deviation . Weak and primary excavators were pooled in the category “excavators” following the example of previous studies  and latitude  based on the study site description and complemented by a map of the world biomes (linked in Supporting information List Forest type based on the dominating tree species (coniferous/deciduous/mixed).Naturalness . Often either forest age or past management history was provided in studies  to extract data that were only provided in figures. In case of missing data, authors were contacted. If only range was provided, the SD of the mean DBH was approximated by dividing the range of DBH values by four, equivalent to 95% of the data in a normal distribution  and North America . Fifty‐one studies contributed data to this meta‐analysis. Four studies complemented data of already included studies as they were published by the same authors for the same study area. Most studies (38 of 47) were from the temperate forest biome. Forests dominated by coniferous (25 studies) or deciduous (14 studies) tree species were commonly researched. Mixed forests were underrepresented (eight studies).Our data set contained 47 cavity‐nesting bird species native to coniferous and deciduous forests in Europe  were on average 13.3 cm thicker than available trees, which had a mean diameter of 35.6 cm. Nests were on average located at a height of 8 m (standard error: 0.5 m). Nonexcavators selected nest cavities that were 97 cm below the overall average height of all cavities in the data set. Quantitative recommendations given in the reviewed studies are shown in Table Nest trees , and by the model that included forest type for the effect sizes vitality (ΔAIC: 6) and crown status (ΔAIC: 5).Effect size estimates for the explanatory variables were only slightly different between subgroups  presumably due to favorable tree characteristics such as soft heartwood and solid sapwood combined with a tendency to remain standing after death  and red‐breasted nuthatches (Sitta canadensis) were reported to nest more frequently in dead trees than hairy woodpeckers . DBH is only one of several proxies that can be useful in forest management. Further, tree characteristics such as the crown status or vital status also have predictive power and should be considered during the selection of suitable trees for retention. Retaining such trees (instead of only focussing on trees with large DBH) might also be more realistic if economic factors are taken into consideration.Studies included in the meta‐analysis based on the study inclusion criteria (shown in Supporting information Appendix CFD and FG contributed first ideas; FG collected the data and provided the first draft manuscript; CFD and FG analyzed the data. Both authors contributed substantially to the writing and gave final approval for publication. Click here for additional data file. Click here for additional data file.


Plants are able to effectively cope with invading pathogens by activating an immune response based on the detection of invasion patterns (IPs) originating from the pathogen or released by the plant after infection. At a first level, this perception takes place at the plasma membrane through cell surface immune receptors and although the involvement of proteinaceous pattern recognition receptors (PRRs) is well established, increasing data are also pointing out the role of membrane lipids in the sensing of IPs. In this review, we discuss the evolution of various conceptual models describing plant immunity and present an overview of well‐characterized IPs from different natures and origins. We summarize the current knowledge on how they are perceived by plants at the plasma membrane, highlighting the increasingly apparent diversity of sentinel‐related systems in plants. To counter pathogen attack, plants use two distinct mechanisms: (i) constitutive defences, including pre‐existing physical and chemical barriers, and (ii) inducible defences activated after perception of the invader  proteins, harpins, rhamnolipids and lipopeptides trigger plant immunity through lipid receptors and/or potential membrane lipid‐raft structure perturbations  and summarize the current knowledge on their mode of perception at the plasma membrane through proteinaceous PRRs and/or lipid‐driven processes.Apoplastic IPs display a large diversity of biochemical nature, including (glyco)protein‐, polysaccharide‐ and lipid‐based structures  promotes hydrolytic release of the active IP from glycosylated flagellin. BGAL1 acts in immunity against pathogenic Pseudomonas syringae strains only when they carry a terminal modified viosamine in the flagellin O‐glycan. Interestingly, P. syringae pathovars are able to evade host immunity by using BGAL1‐resistant O‐glycans or by producing a BGAL1 inhibitor . Interestingly, elf18 does not trigger immunity‐related mechanisms in plants outside the Brassicaceae, unlike flg22  and sensed by the rice LRR‐RK XA21 , has also been widely studied for its ability to induce a plant immune response , which provide rigidity and structure to the bacterial cell wall in both Gram‐negative and Gram‐positive bacteria, induce innate immunity in monocots and eudicots. Muropeptides that are PGN breakdown products from et al., Phytophthora protein1 (NPP1) domain, induce plant immunity and cell death in eudicot plant species  , a glycoprotein from the cell wall of et al., et al., O‐polysaccharidic extremity. In tobacco, treatment with lipid A induces late‐phase defence responses while the oligosaccharide core induces early immune responses  and lipooligosaccharides (LOS) are major constituents of bacterial Gram‐negative cell walls and are well known for inducing immunity in several plant species (Desender N‐acetyl‐d‐glucosamine (GlcNAc) present in fungi and oomycete cell wall and arthropod exoskeleton, is a widespread IP perceived by monocot and eudicot plants. Perception of chitin and chitin‐derived oligosaccharide structures by plants depends on the acetylation and/or the polymerization degree of these compounds  proteins that can use the fungal cell wall as a substrate to release chitin and glucans  in Arabidopsis, it was recently shown that non‐branched β‐‐glucans sensing requires CERK1, the co‐receptor also involved in chitin perception ‐glucans are generally specific of fungi and oomycetes, β‐‐glucans are naturally present in plant cell walls  either modulating plasma membrane physical properties (driven by insertion between lipids and/or membrane damages) or, as demonstrated more recently, using lipid decoration as the receptor/target  lipid is known to induce TLR4‐dependent mechanisms in mammals. It has also been studied for its eliciting properties in Arabidopsis and interestingly the plant defence response induced by the molecule is independent from CERK1 (Cambiagno et al., Arabidopsis. diC14 and diC16 induce defence‐related gene expression in this plant, but diC16 leads to weaker responses. diC14 pretreatment triggers Arabidopsis resistance against P. syringae, unlike diC16 (Cambiagno et al., 14 with plant plasma membrane lipids may alter the organization, compartmentalization or composition of this membrane to somehow boost the activity of the plant defence system (Cambiagno et al., Cyclic lipopeptides are amphiphilic molecules produced by a large variety of bacteria such as et al., et al., Arabidopsis and chitin‐insensitive cerk1 mutant, demonstrating that chitosan is perceived through a CERK1‐independent pathway (Povero et al., et al., +‐ATPase, giving rise to a possible link between chitosan‐triggered plant innate immunity and its putative impact on the structural organization of lipid rafts from the plant plasma membrane (Amborabé et al., et al., Some studies have shown that the LysM domain of CERK1 has very weak binding affinity to chitosan (Iizasa Apoplastic IPs are diverse in their molecular nature: some are kingdom‐specific or even specific to species, while others are present in several kingdoms, such as chitin, which is found in fungi, bacteria and arthropods. The majority of apoplastic IPs characterized to date are perceived by plants at the plasma membrane through PRRs. Interestingly, more and more studies are also suggesting a new perception system based on direct sensing through membrane lipids without the involvement of specific proteinaceous PRRs. This sensing system monitors membrane perturbations and is driven by amphiphilic compounds or toxin‐like compounds. Interestingly, some IPs, such as NLPs, elicitins or chitosan, can be perceived through direct interaction with PRRs and/or by lipid‐mediated mechanisms. In addition, IPs known or suspected to be perceived through membrane lipids are only from microbial origin. Many studies are still required to understand how IPs are sensed by plants and it seems that a large variety of processes are involved. Further integrative investigations, including biophysical approaches and functional biology on plasma membrane lipids, are required to characterize lipid‐based IP perception and its potential relationship with PRR‐mediated mechanisms. The understanding of IP‐triggered immunity is a first step in the development of new plant breeding strategies. PRR engineering and modification of the lipid composition of the plasma membrane could impact the ability of plants to perceive IPs and are examples of biotechnologies that could be used to optimize plant resistance.


We find median inequality aversion parameters of 10.95 for Atkinson and 0.15 for Kolm. These values suggest substantial concern for health inequality among the English general public which, at current levels of quality adjusted life expectancy, implies weighting health gains to the poorest fifth of people in society six to seven times as highly as health gains to the richest fifth. © 2016 The Authors. Health Economics published by John Wiley & Sons, Ltd.Health inequality aversion parameters can be used to represent alternative value judgements about policy concern for reducing health inequality versus improving total health. In this study, we use data from an online survey of the general public in England ( When these two objectives conflict, Health‐Related Social Welfare Functions (HRSWFs) can be used to articulate the trade‐off between them. Economists have explored the properties of several SWFs, which aim to capture these trade‐offs in the form of a single inequality aversion parameter. The Atkinson Index  level of health builds on the concept of EDE income, defined as the mean level ‘which if equally distributed would give the same level of social welfare as the present distribution’  for subgroup i, H¯ is the mean level of health for the entire population and f(xi) is the proportion of the population in subgroup i. Unlike most previous studies, our data allow the elicitation of negative parameters for individual respondents representing ‘inequality seeking’ judgements and positive parameters representing ‘inequality averse’ ethical judgements.In these equations, 2.4The point where a given respondent switches from one programme to the other, or selects equally good, is interpreted to reflect the point at which the respondent is indifferent between the two programmes. The implied inequality aversion parameter for each respondent can then be established by numerically solving the EDE equations.2.5In order to represent an inequality aversion parameter for the population, we use the median response rather than the mean. This is because the inequality aversion parameter approaches infinity for Maximin responses . Figure The elicited inequality aversion parameters, and the EDE level of health they imply, are given in Table The median inequality aversion parameters were robust to sensitivity analyses using larger samples (see Appendix D for details).44.1et al., et al., et al., ε value of 28.9, which is considerably larger than our value of 10.95. There are multiple factors that could explain this difference. One may be that their study did not use a ‘slow thinking’ intervention . Thus, the corresponding parameter values generated in our study (ε = 10.95 and α = 0.15) suggest that the targeted intervention is the intervention that should be chosen in order to maximise social welfare allowing for inequality aversion. Table To illustrate how this value could be used in practice, we present an example from a distributional cost‐effectiveness study of two different ways of spending the same fixed budget for increasing uptake of a pre‐existing universal bowel cancer screening programme . Richard Cookson is supported by the NIHR . The views expressed in this publication are those of the authors and not necessarily those of the funding bodies, the NHS or the Department of Health.We obtained research ethics approval from the University of York Health Sciences Research Governance Committee, in a letter dated 23 May 2013. The main ethical issues involved informed consent and data security to ensure participant responses were kept anonymous and no personal data disclosed.Appendix A: Survey MethodsAppendix B: Response CategorisationAppendix C: Sample CharacteristicsAppendix D: Data Quality and Sensitivity AnalysisAppendix E: Categorised Inequality Aversion ParametersAppendix F: Restricting Responses due to Duration of SurveySupporting InformationClick here for additional data file.