Puccinia triticina (Pt) is one of the most damaging diseases globally. Considerable progress has been made to control leaf rust through crop protection chemicals and host plant resistance breeding in southern Africa. However, frequent changes in the pathogen population still present a major challenge to achieve durable resistance. Disease surveillance and monitoring of the pathogen have revealed the occurrence of similar races across the region, justifying the need for concerted efforts by countries in southern Africa to develop and deploy more efficient and sustainable strategies to manage the disease. Understanding the genetic variability and composition of Pt is a pre-requisite for cultivar release with appropriate resistance gene combinations for sustainable disease management. This review highlights the variability and distribution of the Pt population, and the current control strategies, challenges and future prospects of breeding wheat varieties with durable leaf rust resistance in southern Africa. The importance of regular, collaborative and efficient surveillance of the pathogen and germplasm development across southern Africa is discussed, coupled with the potential of using modern breeding technologies to produce wheat cultivars with durable resistance.Leaf or brown rust of wheat caused by  Puccinia triticina Eriks. (Pt), is widely distributed across all the major wheat-growing regions in the world (Triticum aestivum), durum wheat (Triticum turgidum var. durum), and triticale  and associated regional and global projects /Mexico and the International Centre for Agricultural Research in the Dry Areas (ICARDA)/Syria and several private multinational seed companies, each contributing to large germplasm pools. Some of the wheat genetic resources preserved in these centers are populations that were developed through dedicated pre-breeding and breeding programs targeting novel traits, including rust resistance. Wheat germplasm has been introduced across southern African borders between recipient countries, but the inability to meet seed import requirements has limited germplasm exchange. Further, policy issues on Intellectual Property ownership and benefit-sharing rendered limited germplasm exchange between African countries. These limitations may be circumvented through harmonized policy and regulations for enhanced germplasm exchange and through the use of recent advances in genetic and genomic technologies to deliver novel genetic resources with durable rust resistance.Pt races in southern Africa. Focus is placed on describing the predominant races common across the region and the deployed leaf rust resistance genes in wheat. A comprehensive summary of effective leaf rust resistance genes available in different parts of the world that could be pyramided into local cultivars through advanced genetic and genomic technologies is also provided. Perspectives on the strides taken and milestones reached through collaborative research toward finding the best strategy to achieve durable leaf rust resistance in southern Africa and significant challenges encountered in the process are also discussed. Prospects of host induced gene silencing using the RNA interference (RNAi) technology has been highlighted as an option to enhance cultivar resistance against Pt.In light of the above background, the objective of this review is to present the variability and distribution of Puccinia triticina populations throughout the world are genetically diverse in terms of their molecular architecture as well as the combinations and complexity of their virulence patterns. This is influenced by the co-evolution of the pathogen with diverse wheats and genetic recombination of Pt races emerging from various Triticum species  and the University of Free State (UFS) in South Africa, Chitedze Research Station and CIMMYT in Malawi; the Crop Breeding Institute (CPB), Plant Protection Research Institute (PPRI), and Rattray Arnold Research Station, Seed Co., Ltd., in Zimbabwe; and Zambian Agriculture Research Institute, Mount Makulu Research Station, in Zambia. Results from the surveys are shared with the wheat breeding community through the global rust monitoring system developed by CIMMYT and other partners as part of the BGRI to mitigate the threat of wheat rust disease2.To understand the variability of Lr genes in a uniform background of a highly susceptible cultivar or line facilitates research in understanding the genetics of host–parasite interactions  containing different leaf rust resistance genes/alleles are used to distinguish races by their phenotypic responses to different pathogen strains. Having NILs with single ractions . The useommittee . When 16ommittee . A numbePuccinia genera  and amplified fragment length polymorphism (AFLP) have long been successfully used in mycology and plant pathology for the differentiation of species within the a genera . Kolmer ckground . Co-domienotypes . A recenPt genotypes. A number of leaf rust races have been detected in various locations in southern Africa, including Malawi, Mozambique, South Africa, Swaziland, Zambia, and Zimbabwe  was less virulent. Lr10, Lr14a, Lr17, Lr24, and Lr26 resistance genes.Using SSR markers, race PDRS was found to be genetically different from 10 other South African races, leading to speculation that it is a foreign introduction . Terefe Pt races. Due to a sustained emergence of physiological races in the leaf rust pathogen, deploying differential host genotypes will have an added advantage to distinguishing resistance or susceptibility reactions of wheat genotypes to Pt races. When large sets of genotypes are screened for disease resistance, the phenotypic data can be associated with genomic data to identify putative and novel virulence and avirulence genes, as well as genes conferring resistance to particular races. The continuous use of race-specific resistance genes in breeding programs requires established surveillance systems of the leaf rust pathogen to scout for new races  population in the United Kingdom, which can similarly be useful for leaf rust studies in southern Africa. With the increasing availability of fully sequenced pathogen genomes, this new high throughput technology will provide new insights into the biology, population structure and pathogenesis of pathogens of interest  libraries which have contributed to gene discoveries and stage-specific expression analysis  and host plant resistance are available. However, limited studies are conducted on the biological and cultural control options, leaving the last two options being widely used in southern Africa. Timely and accurate application of fungicides is effective in reducing both the incidence and severity of leaf rust in wheat. In the past years, regular fungicide spraying on commercial wheat fields of the Western Cape Province in South Africa contributed to the leaf rust fungus becoming less prevalent. Fungicides with active ingredients such as epoxiconazole, tebuconazole, pyraclostrobin, and trifloxystrobin remain effective for protecting yields of susceptible varieties from leaf rust attack. Nevertheless, fungicide use is not economically and environmentally sustainable, and can pose health hazards to people and animals, as well as phytotoxicity to the crop  seedling resistance, which is conferred by single or major genes (often race-specific) and (2) adult plant resistance (APR) or partial or polygenic or slow rusting resistance caused by minor genes. APR is mainly race-non-specific, but it can also be race-specific and short-lived, being effective only against Pt isolates carrying the corresponding avirulence gene. Seedling resistance is expressed at all growth stages and is sometimes referred to as all-stage resistance (ASR). All-stage resistance is commonly qualitative and associated with a programed cell death defence response referred to as hypersensitive immunity. Adult plant resistance, on the other hand, is most effective in adult plants and is commonly quantitative. The mechanisms of action of seedling and APR genes, including their interaction with the pathogen avirulence (Avr) genes have been discussed in detail in previous reports (Host plant resistance against  reports . Silva eThinopyrum elongatum Zhuk. (Lr 19), Aegilops tauschii (Lr21), Agropyron elongatum (Lr24), Secale cereale L. (Lr26), Aegilops peregrina (Lr59) among others  carrying Lr24 was widely used in the 1980’s and the prevalence of new virulent races substantially increased vulnerability of the cultivar to leaf rust. Other varieties including Gouritz, Dipka, Flamink, SST101, and Zaragoza which were grown abundantly in South Africa were reportedly susceptible  resistance genes  and Lr13 (race-specific) with both genes improving resistance on the selected lines even with the presence of races possessing virulence for the Lr13 gene. Lr16, Lr23, and Lr34 in North America. The complementary effect of combining resistance genes was also tested by Lr21, Lr29, Lr32, Lr34, Lr35, Lr36, Lr37, Lr41, and Lr42 to South African cv. Palmiet and Karee through the backcrossing method. Likewise, Lr16, Lr47, Lr19, Lr41, Lr21, Lr25, and Lr29 provided durable resistance when combined with APR genes such as Lr34, Sr2, and Lr46 in Argentina. A recent study by Lr34, Lr68, and Sr2 to increase leaf rust resistance, showing the importance of using APRs as key genes to be deployed in breeding programs to attain high levels of resistance. Combinations of partial resistance genes in the CIMMYT breeding program have conferred adequate resistance to leaf rust in the field for several years  markers, the use of next generation sequencing (NGS) technologies and QTL mapping have proved crucial in identifying more APR genes and other effective race specific genes that can be combined to achieve stable and durable resistance. Several studies supporting the use of these tools, looking at their advantages and occurring gaps are available. A recent NGS based transcript analysis by Avr gene, and the interaction of slow rusting resistance genes with other resistance genes or gene loci to achieve stable and durable rust resistance across environments is still limited. It has been reported in several studies that temperature variations play a significant role in the expression of many resistance genes. A study by Lr68 was smaller than the effect of Lr34, Lr67, or Lr46 in the 2007–2008 and 2008–2009 seasons, while Lr68 showed stronger effects than Lr46 in the 2010–2011 season in the same studied environment. The two cropping seasons varied greatly in temperature as the 2010–2011 season proved to be significantly cooler than the previous seasons, suggesting that Lr68 may be expressed better at lower temperatures. Studies by Lr13. In the former study, Lr13 was expressed at 25°C, resulting in an avirulent infection type in seedling plants exposed to three isolates of Pt from Mexico, China and Chile. Pt isolates from North America have high infection types to seedlings with Lr13 regardless of temperature, but many of the same isolates have low infection types to adult plants with Lr13.As much as rust pathogens, in general, have great economic importance, limited information is available on the genes and factors required for pathogenesis and virulence. Adding to this, accurate information on the effect of the environment and full knowledge of the identity of effective resistance genes in the host, their mode of action when interacting with the Limited public funding for sustained research on both the host as well as the pathogen and its variability, created a bottleneck in breeding for durable leaf rust resistance in most southern African countries. For example, despite the contribution of the ARC in improving the performance of the agricultural sector in South Africa, public funding (through Parliamentary Grant) to the ARC has been declining in real terms over the recent years . This imLr1 . Modest progress has been made in engineering durable resistance to wheat rust, especially leaf rust which causes considerable damage to wheat production. Resistance genes are continually being characterized and mapped in wheat and its relatives with only a few durable leaf rust resistance genes mapped. Adding to this, map-based cloning in wheat has become easier with the availability of whole genome sequencing. However, limited work has been done in cloning multiple effective leaf rust resistance genes. Some of the cloned leaf rust resistance genes include Lr1 , Lr10 (Fr1 (Lr10 , Lr21 (H10 (Lr21 , Lr34 (L21 (Lr34 , and Lr634 (Lr67 . CloningPuccinia genes in plants to determine whether silencing can be delivered to the pathogen and suppress the expression of the fungal genes. Results from this study clearly showed that some P. striiformis genes can be silenced through a host-induced RNAi system. This may yet prove to be a viable technology to analyse gene functions in rust fungi and control wheat rust disease in southern Africa.The discovery of RNAi technology has provided new opportunities to explore engineering of resistance against some biotrophic pathogens in plants by inducing silencing of genes essential for pathogen virulence. RNAi is an intrinsic cellular mechanism shared by all multicellular eukaryotes with apparent roles in gene regulation and defence against viral infection . The mecThe variability and constant evolution of wheat leaf rust populations exhort huge pressure on wheat breeders and researchers in general to be constantly vigilant against the emergence of new rust races. This requires timeous monitoring and collaborative surveillance of changes in the virulence patterns among rust pathogens in each country and across regions. Durable host plant resistance to leaf rust is one of the most critical traits that breeding programs should invest in, permitting a reduction in the use of fungicides as well as promoting greater stability and sustainability of yield levels. The use of highly sophisticated and high-throughput tools such as field pathogenomics, transgenics, genome editing, and NGS to study both the host and the pathogen will assist in ultimately achieving broad spectrum and durable leaf rust resistance in wheat. This will subsequently result in a realization of large proportions of returns on global economic investments in international wheat research. A multidisciplinary approach involving pathologists, breeders, geneticists, physiologists, agronomists and bioinformaticians at different stages of research and development is necessary to develop an improved cultivar with stable and durable leaf rust resistance through host plant resistance approach. The extensive training of young researchers specifically in the field of plant–pathogen interaction and in the use of high-throughput technologies mentioned above, in collaboration with universities in Africa, will also generate immeasurable benefits in maintaining or conserving valuable genetic materials for wheat and other economically important crops and generate varieties that integrate specific public-good traits such as durable rust resistance.SF wrote the first draft of the article and finalized with contributions by KN, LM, TT, TJT, and HS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In addition to diseases shared by both sexes, there are a number of illnesses and injuries that are primarily associated with women . These hIndeed women are either not aware of such healthcare needs, or they are aware but tend to ignore these needs because of their demanding role responsibilities, workload, and other caregiving activities, or they have been prevented from seeking healthcare and from maintaining their health by limited resources and structural constraints .International Journal of Environmental Research and Public Health was launched in June 2019 with the main aim of implementing international literature data on various aspects of women’s health, such as breastfeeding, HPV, and gynecological diseases [The Special Issue “Potential Risks and Factors of Women’s Health Promotion” of diseases . InterdiInternational Journal of Environmental Research and Public Health. In particular, 22 original articles (research articles) and one review article were published online upon closure of the Special Issue.At the end of July 2020, 60 manuscripts were submitted and, after the peer review process, 23 were accepted for publication in the SI “Potential Risks and Factors of Women’s Health Promotion” of Several topics were examined in this SI, and all manuscripts that were finally published and are available online in open access form are reported in chronological publishing order in A retrospective study conducted in Vietnam from 2014 to 2016, reported an association of one of the most important maternal outcomes, intrauterine growth restriction, in pregnant women that suffer from heart diseases .Wochna and colleagues, in their quasi-experimental field trial, demonstrated the impact of an aqua fitness training program on femur strength index among 18 postmenopausal women .An observational study conducted over a decade 2007–2016) in South Korea investigated the association between gender discrimination in the workplace and pregnancy planning/childbirth experiences among working women and found that gender discrimination, especially in a low/medium income workplace context, is associated with a decreased chance of pregnancy planning/childbirth experience [–2016 in Alvarez-Villareal M. et al. conducted a qualitative phenomenological study on female patients in a Spanish hospital with chronic kidney diseases (CKD), evidencing that catheters and/or fistulas affected sexual desire and satisfaction .A cohort study conducted in the Netherlands examined the health-related quality of life (HRQoL) of 4312 women after childbirth, identifying multiple determinants of suboptimal physical and mental HRQoL, such as maternal psychopathology after childbirth .An observational study conducted in Jiangxi Province in China that analyzed the utilization of inpatient services for middle-aged and elderly rural females evidenced that hospitalization and late discharge rates were significantly lower for divorced or widowed women than married females .Cernigliaro et al. analyzed the association of individual and contextual inequalities on breastfeeding in the Sicilian region, highlighting that contextual inequalities had a significant association with the risk of not following exclusive breastfeeding in the deprived classes .Another qualitative study conducted in Spain investigated urinary incontinence (UI) as a potential risk factor for women’s health . UI was A retrospective observational study in Ecuador demonstrated that the correct use of social networks could help deaf women facing considerable challenges in accessing information on sexual and reproductive health .Antoniou et al. evidenced that domestic violence during pregnancy was more frequent in Greek women with a foreign partner . UnemploIn a cross-sectional study on 31,690 Korean adults, Won Kee et al. showed that among women, night eaters had higher odds of depression and depressive symptoms compared with non-night eaters .Middle-aged and elderly females with chronic diseases and non-labor females have a higher probability of getting hospitalized in accordance with a study conducted in a Chinese Province .A cross-sectional study conducted in Vietnam on working hours in relation to work-to-family conflict and burnout found that female employees who work overtime on a voluntary basis are at risk of developing health problems .Vo Van Ha and colleagues demonstrated in a sample of 1617 Vietnamese mothers that resuming physical activity in the early postpartum period is a fundamental weight management strategy .Barcikowska et al. reported that prostaglandins (more than other cytokines) play a major role in the pathomechanism of dysmenorrhea .A quali-quantitative study in a depopulated area of Spain demonstrated that acknowledging the socioeconomic importance of women’s work and adopting measures that facilitate the reconciliation of work and family life were essential measures to consider for women’s well-being and rural development .Rouillon et al. highlighted the importance of health education on endocrine disruptors for pregnant women, taking knowledge, attitudes, and practices into account .An observational study conducted in Hong Kong from 2011 to 2014 evidenced that active play with their preschool children may increase mothers’ physical activity levels .An analysis of intentions to pay for HPV vaccination among women of childbearing age conducted by Thi Thanh Le and colleagues showed that a large majority of women had a firm intention to vaccinate, but after being informed of the current price, the number of respondents who intended to vaccinate dropped to one-fifth . An observational study conducted in Dubai found that vasomotor symptoms, fatigue, and obesity were the main symptoms of psychological distress among menopausal women .A multicenter Italian cross-sectional survey on knowledge and attitudes regarding HPV infection and prevention among healthcare workers showed overall positive attitudes towards HPV burden and prevention tools, although knowledge was largely suboptimal .Environmental tobacco smoke exposure during pregnancy, and not air pollution, decreased birth length and is an independent risk factor for fetal growth restriction according to data reported in a longitudinal study conducted in Poland .Finally, a cross-sectional study conducted on first-grade secondary schools in Sicily that evaluated the knowledge, attitudes, and willingness to vaccinate against HPV among preadolescents demonstrated a good level of knowledge and attitudes, and that willingness to receive HPV vaccination significantly increased after vaccination counselling interventions at school .In conclusion, one of the main challenges facing future public health is the improvement of preventive measures in order to reduce the burden of acute and chronic diseases among the general female population .Women’s health promotion could be supported throughout strong multidisciplinary alliances between healthcare professionals, providing evidence-based data on preventive strategies adopted to counteract cervical cancer , the breastfeeding attitudes of healthcare workers and mothers, pregnancy outcomes (with a specific focus on women with different deprivation levels), and the impact and effectiveness of communication strategies, especially for high-risk groups of women .


Approximately 60% of Australia’s beef cattle are located in the vast rangelands of northern Australia. Despite the often low stocking densities and extensive management practices of the observed herd, animal prevalence of BVDV infection and typical rates of transmission are similar to those observed in intensively managed herds in southern Australia and elsewhere in the world. A recent large three- to four-year study of factors affecting the reproductive performance of breeding herds in this region found that where there was evidence of widespread and/or recent BVDV infection, the percentage of lactating cows that became pregnant within four months of calving was reduced by 23%, and calf wastage was increased by 9%. BVDV is now considered the second most important endemic disease affecting beef cattle in northern Australia, costing the industry an estimated AUD 50.9 million annually. Although an effective killed vaccine was released in Australia in 2003, the adoption of routine whole herd vaccination by commercial beef farmers has been slow. However, routine testing to identify persistently infected replacement breeding bulls and heifers has been more widely adopted. Bos indicus genetics, enabling them to better cope with high environmental temperatures, low quality grazing pastures, and internal and external parasitism, in particular cattle tick  and buffalo fly (Haematobia irritans exigua) infestations. Median property and paddock size [2 and 419 to 2611 ha, respectively) and stocking rates are typically moderate to low; one adult equivalent (AE) in 5–30 ha, but in some areas, this is as low as 1 AE to 150 ha. Cattle are extensively managed, typically only being mustered (brought in from the paddock and handled through the cattle yards and crush or chute) twice a year, between April to June and August to October, when a number of husbandry and management practices are performed, such as branding and weaning, pregnancy diagnosis, drafting cattle into new management groups, and identification and removal of cull cows and bulls. Notably, this is generally the only time when disease control measures, such as the vaccination of cattle, are conducted. Approximately two-thirds of cow herds in the northern dry tropical rangelands are continuously mated [Approximately 60% of Australia’s beef cattle are located in northern Australia, which includes the state of Queensland, the Northern Territory, and the northern part of the state of Western Australia . This isock size  vary conIn Australia, BVDV was first reported to have been isolated from cases of acute and chronic mucosal disease of cattle in 1964 . Severaln = 24,035) and health certification  conducted at the Elizabeth MacArthur Agricultural Institute between approximately 2004 and 2010; 1.1% and 1.53% of cattle were confirmed PI, respectively (P. Kirkland pers. comm.). The prevalence of PI cattle amongst breeding-age bulls and heifers in Queensland, where approximately half of Australia’s cattle reside, was 0.35%. This review focuses on updating the knowledge and understanding of the epidemiology and management of BVDV in northern Australia. It has been estimated  that appIn the mid-1990s, as part of a structured animal health surveillance programme conducted by the Queensland government, a sample of heifers and cows from 213 Queensland beef herds was bled, and the BVDV seroprevalence was determined using a virus neutralisation test . The oveIn a four-year 2007–2011) longitudinal study [1 longituThe overall seroprevalence was similar for heifers and cows and between years, varying between 50% and 55%. The median management group seroprevalence was also similar across regions and years. Approximately one in four and one in five heifer and cow groups, respectively, were mostly naïve , and 3 in 10 and 4 in 10 heifer and cow groups, respectively, were mostly naturally immune , 60% became infected ,11 withiThere have been few reports on transmission of BVDV in the extensively managed beef herds of northern Australia. A study of BVDV transmission on two commercial beef properties in central Queensland  found thn = 199 to 506) of recently weaned calves (3 to 7 months of age), managed in large paddocks (~4000 ha) at stocking rates of one AE per 2–8 ha, 0.5% to 1% of susceptible cattle became infected per day. These rates of transmission are similar to those observed in intensively managed beef herds. Typically in these rangeland herds, calves weaned from different breeding groups are accumulated in large holding yards over one or more weeks, and after processing  graze together in a single paddock, all of which increases the likelihood of transmission of BVDV. A serological study of heifers in herds in the Northern Territory [In a north Queensland study  of ratesIn a serological study  of BVDV In the first 20 years after its initial detection in Australia, BVDV was considered to primarily cause acute or chronic mucosal disease, with the latter being a much more common presentation. However, fundamental research conducted at the Central Veterinary Laboratory, Glenfield, in the mid-1980s  demonstrAlthough in northern Australia most mixing of cattle from different paddocks and properties typically occurs around the bi-annual mustering events, problems with maintaining fence security  means that introductions of new cattle to a breeding group can occur at any time. Based on the findings  presentep < 0.05; In these extensively managed herds, the recommended approach to determining whether BVDV infection is likely to have contributed to the observed lower than expected reproductive performance requires, firstly, accounting for the likely impact of the major factors known to affect performance , and, seIn Meat Livestock Australia’s most recent 2015) assessment of the economic cost of endemic disease in the cattle and sheep industries , BVDV wa5 assessmAustralian cattle veterinarians and farmers only began to recognise the potential impact of BVDV infection on beef herd performance in the late 1980s, with those involved in the artificial breeding industry being most interested in strategies to prevent outbreaks of infection in groups of cattle undergoing artificial insemination or embryo collection and transfer. However, in contrast to other countries, Australia has only had access to a BVDV vaccine since 2003. Therefore, initial strategies such as blood sample testing of cattle suspected of being PI  and then deliberately exposing selected unmated/non-pregnant females to confirmed PI cattle, or inoculation of these cattle with the live virus either derived from a PI animal [Due to recognised differences in strains of BVDV found in Australian cattle and national biosecurity concerns, an Australian killed vaccine was developed by Pfizer Animal Health (Zoetis) using isolates provided by Dr. Peter Kirkland’s laboratory (EMAI). A field evaluation  conductePerhaps the most common BVDV control measure being used by cattle producers in northern Australia is requiring that all purchased replacement bulls be certified as being not PI. Seedstock producers increasingly test to assess the PI status of their bulls at the time of conducting breeding soundness examinations prior to sale. The availability of a reliable antigen capture by ELISA with high sensitivity and specificity, combined with the convenience of testing either hair or ear-notch samples, has encouraged the adoption of testing to confirm that replacement breeding animals are not PI. To support the ongoing efforts of veterinarians and cattle producers to control BVDV in Australia, a technical advisory group has developed comprehensive guidelines defining the approach to investigating and managing BVDV in beef and dairy herds . Further


The present study examines the impact of traumatic life events on religious complexity in later life. We anticipated that those older adults experiencing stressors that produce significant personal vulnerability  demonstrate reduced complexity of belief and behavior . From a sample of 278 semi-structured interviews of older adults (aged 55-101 years-old.) from six New England and New York states, we analyzed 166 interviews using grounded theory . Individuals who experienced trauma related to war, close familial loss, and/or severe physical illness tended to be “true believers,” . By contrast, those who experienced less severe trauma  were less apt to describe rigid belief. Temporal proximity of trauma was not consistently associated with greater complexity of belief and behavior, in the sense that with great distance from trauma, individuals were able to “work through” their experiences of trauma, and thereby increase complexity of belief and behavior. This is consistent with findings by Harris and Leak (2015), Krause and Hayward (2012), and Wong (2013) that suggest that trauma leading to personal vulnerability leads to long-term physical, mental, behavioral, and spiritual deficits that rigid religious belief and behavior help to offset. These findings are discussed in terms of psychological theories of grief resolution, personal coping, and terror management.


Tissue engineering is an interdisciplinary field that combines principles of engineering and life sciences to obtain biomaterials capable of maintaining, improving, or substituting the function of various tissues or even an entire organ. In virtue of its high availability, biocompatibility and versatility, cellulose was considered a promising platform for such applications. The combination of cellulose with graphene or graphene derivatives leads to the obtainment of superior composites in terms of cellular attachment, growth and proliferation, integration into host tissue, and stem cell differentiation toward specific lineages. The current review provides an up-to-date summary of the status of the field of cellulose composites with graphene for tissue engineering applications. The preparation methods and the biological performance of cellulose paper, bacterial cellulose, and cellulose derivatives-based composites with graphene, graphene oxide and reduced graphene oxide were mainly discussed. The importance of the cellulose-based matrix and the contribution of graphene and graphene derivatives fillers as well as several key applications of these hybrid materials, particularly for the development of multifunctional scaffolds for cell culture, bone and neural tissue regeneration were also highlighted. In recent decades, the rapidly aging population, environmental stressors, frequent cases of traumatic injuries and chronic diseases lead to a growing interest in the revolutionary domain of tissue engineering (TE) ,2,3. TisPorous three-dimensional (3D) scaffolds are an important component of tissue engineering. These constructs are used to provide an appropriate environment for tissue and organs regeneration. Biological scaffolds  are an accessible option because they already contain a broad spectrum of signaling molecules with an important role in the processes of cellular morphogenesis and function development ,12. HoweCellulose remarks itself among the biomaterials used for scaffold production due to its high availability and renewability. Cellulose is mainly extracted from plant cell walls. The nano-scaled forms of plant cellulose—cellulose nanofibers (CNFs) and cellulose nanocrystals (CNCs), are obtained following specific mechanical and chemical treatments  ,19. In vCellulose can also be produced by certain microorganisms, in this case being called bacterial cellulose (BC). BC is considered to be the most biocompatible form of cellulose because it lacks biogenic impurities such as lignin and hemicellulose and only mild chemical treatments are required to ensure its purity . The nanPure cellulose lacks solubility in common organic solvents. This represents an issue when it comes to processing techniques where a stable polymeric solution is required to prepare the final material . Cellulose was dissolved so far only in mixtures of highly toxic solvents  ; howeverGraphene (GE) is an allotrope of carbon produced by top down  or bottom up techniques  . GE has 2SO4 in a microwave device. The material was kept at 180 °C and a pressure of 40 bar, for 2 h, under nitrogen flow. Following this treatment, black solid carbon spheres were obtained. The spheres were further dispersed in concentrated nitric acid (HNO3), ultrasonicated 30 min at 45 °C and heated at 90 °C for 30 min, under magnetic stirring to obtain nGO. The ultrasonication time is a decisive factor in obtaining materials with uniform and reproducible properties . . 112]. TIn another study conducted by Kumar et al., GO nanosheets and CNCs were employed as multifunctional crosslinking agents for polyacrylamide-sodium carboxymethyl cellulose (PAA-NaCMC) hydrogels, prepared by in situ free radical polymerization. Due to GO and CNCs hydrophilic nature and ability to provide sites for hydrogen and covalent bonding, both PAA and NaCMC had favorable molecular interactions with GO and formed strong interfacial bonds with CNCs. The synergistic effect of GO and CNC lead to an improvement of the viscoelastic mechanical properties, shape recovery behavior and self-healing ability of the interpenetrating PAA/NaCMC network. Still, further studied must be conducted in order to determinate if these hybrid hydrogels represent appropriate 3D microenvironments for tissue engineering applications .Polylactic acid is a biobased plastic obtained by bacterial fermentation processes. Its biodegradability and biocompatibility recommend it for replacing conventional petroleum-based plastics used in the biomedical field. Still, its mechanical properties need further improvement. CNCs and rGO were used as reinforcing fillers for PLA ,62. The This study suggests that rGO is not only a reinforcing agent for biopolymer matrices but can also induce an antimicrobial character, very useful for preventing infections in tissue engineering applications.Undeniably, cellulose and graphene are highly versatile materials and they can be employed in multiple ways to obtain hybrid composites with superior physico-chemical and biological features compared to cellulose or graphene alone. A summary of the cellulose/graphene composites for tissue engineering applications presented in the previous chapters is found in This review highlighted some of the recent discoveries regarding cellulose-based composites with graphene and graphene derivatives. As most of the described studies concluded, the combination of cellulose and graphene renders sustainable and cost effective composites with improved physico-chemical features and bioactivity. Cellulose and graphene can be labeled as complementary materials because the incorporation of graphene in cellulose-based matrices minimizes GE’s potential cytotoxic character and, in its turn, GE improves the biocompatibility of cellulose by providing cell binding sites and positively interfering in cellular differentiation processes and also its mechanical properties. The versatility of both cellulose and graphene is another important reason cellulose/graphene composites are needed in tissue engineering. Cellulose paper was used to develop electrodes for cellular electrical stimulation or in vivo tissue models for a better understanding of a specific tissue biology and experimental drug testing. Bacterial cellulose and cellulose derivatives were employed in the form of electrospun mats, membranes, and hydrogels as ECM-mimicking scaffolds for enhanced cell adhesion, growth and proliferation whereas cellulose nanocrystals and cellulose nanofibers were incorporated in various biopolymer matrices for a reinforcing effect. Some particularly interesting features for tissue engineering are related to GE and its derivatives ability to guide stem cells differentiation toward specific lineages  and also to the electrical conductivity and fluorescent properties that they provide to cellulose matrices, thus extending the areas of application of cellulose/graphene composites Also, a synergistic reinforcing effect was observed when both cellulose and graphene-based fillers were incorporated in various biopolymer matrices.The constant research activity in the field of cellulose/graphene composites lead so far to important discoveries in the area of tissue engineering. Even if these materials could represent promising alternatives to currently used medical techniques, the progress toward obtaining clinical products is slow. Future trends consist of the collaboration of clinicians, biomaterial scientists and engineers, with expertise in their own fields, to elaborate marketable and accessible products for therapeutic purposes.


Lameness impacts negatively on dairy cattle welfare and production. Claw trimming is a routine practice for lameness management in dairy cows. Claw trimming is often applied for the treatment of clinically lame cows especially those affected with horn lesions; however, the benefits in the area of prevention are not well understood. This issue results from a combination of paucity of data on preventive trimming and shortfalls in study designs, which limits our understanding of identifying cows that will benefit more from such intervention during lactation. Computerized claw trimming database programs have the potential of curtailing some of these issues, by providing the basis for monitoring hoof health and adjusting lameness management practices in dairy herds. This review summarizes literature findings regarding claw trimming methods, their application in lameness management, as well as associations with the welfare and production of dairy cows.Lameness resulting from claw lesions remains a pressing welfare issue in dairy cows. Claw trimming (CT) is a common practice for prevention and management of clinically lame cows. This review summarizes the results of studies that have investigated various claw trimming (CT) methods, their application in lameness management, and associations with the welfare and production of dairy cows. The papers included in this review fulfilled the following inclusion criteria: published in peer review journal or book chapter within the last 20 years (1999–2019), written in English, and focused on the application of CT for lameness management and the association with either welfare or production variables. Databases used included Google scholar, Web of Science and PubMed. A total of 748 records were assessed and 61 papers were eligible for inclusion and the main objectives and results were used to categorize the results under six topics: CT techniques, association between CT and claw overgrowth/specific claw lesions, timing and frequency of CT, association between CT and behavioral variables, association between CT and physiological parameters, and association between CT and production. The literature findings showed the existence of various CT methods with the common types including the Dutch Five-step, White Line, White Line Atlas, and Kansas techniques. There is data paucity on the efficacy of these techniques in lameness management; however, the slight procedural difference yields varying sole thicknesses and presentations which may influence their prophylactic use. Results regarding the impact of CT on welfare and production were discussed in relation to potential short and long-term benefits. Depending on the lesion type and severity level, CT may induce immediate painful sensation, stress, changes in lying down activities and reduction in milk yield, but the positive impacts were more evident at later stages of lactation following improvement in locomotion score. The majority of the reviewed studies were lacking a detailed description of CT techniques and claw health of the studied animals; thus, reducing the strength of demonstrating CT-related benefits. However, electronic recording of claw health data during every CT visit provides the basis for monitoring hoof health and could assist in curtailing some of these challenges. To elucidate CT-related benefits, certain areas requiring further research were highlighted such as ascertaining the appropriate timing for preventive CT and identifying cows that will benefit more from such intervention during lactation. Dairy cow welfare is an important aspect in milk production systems ,2. LamenCorrect claw trimming (CT) by well-educated hoof trimmers or veterinarians is an essential lameness management procedure ,9. CT isMaintenance or preventive CT is primarily conducted for the management of claw disorders such as claw overgrowth and unbalanced sole or heel . PreventAside from claw health, CT also influences the behavior, physiological parameters, and milk yield; thus, culminating in an impact on welfare and production ,23,24,25Presently, there are no data that summarize the findings and extent of the research that has been conducted regarding the application of CT for lameness management, and the association with various welfare and production indicators in dairy cows. One of the means of identifying knowledge gaps and providing direction for future research is by conducting a systematic review of the literature . The objThe basic aspects of a systematic review were applied in this review. For papers to be included, they needed to fulfil the following criteria: published in peer review journal or book chapter, written in English and published between 1999 to 2019, focused on the application of CT for lameness management, and investigated the relationship between CT and welfare  and production variables . The review was conducted from August 2019 to February 2020 and databases used for the literature search included Google scholar, Web of Science and PubMed. Additionally, the works cited in the papers obtained from the databases were evaluated for inclusion. The search terms used were “claw trimming”, “dairy cows”, “welfare”, “production”, “claw lesions”, and “lameness”. The 3 aforementioned databases yielded 748 records, whereas 61 cited papers were assessed from the original papers. The Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) checklist was used for the literature selection process . Of the 809 records, 481 were identified as duplicates and they were excluded; thus, leaving 339 records for further review. After re-evaluation, 61 papers were eligible for inclusion and the objectives and major findings were used to classify the results under six main topics: CT techniques, association between CT and claw overgrowth/specific claw lesions, timing and frequency of CT, application of computerized CT database programs, association between CT and behavioral variables, association between CT and physiological parameters, and association between CT and production.The objectives of CT are to ensure both claws of the foot bear near to equal amount of weight and to preserve their function under various management systems. Results from the literature search yielded four major CT methods which comprised of the Dutch five step , White Ln = 15) that provided a clear description of the applied CT. The White Line method proposed by Blowey [The CT method developed by Toussaint Raven  is also y Blowey  is basedThe determination of sole thickness in both methods is critical to ensure that weight bearing between the lateral and medial hind claws is equal ,30. BaseThe White Line atlas method was described by Vic Daniel and Randall White . The basThis technique was developed based on the variations in hoof structure between animals . Kansas The dorsal wall length, dorsal wall angle and heel height are claw dimensions used to determine the presence of overgrown claws ,16,29,31Authors of the Dutch and White Line methods recommended an average dorsal wall length of 75 mm for a normal Holstein Friesian cow. However, while considering the influence of age and specific claw , Nuss and Paulus  reportedClaw overgrowth is one of the main reasons for CT since it affects the weight bearing within and between the claws ,30,31,32CHDL is a general term for lesions that arise from pathologies of the horn tissues. Examples of these lesions include sole ulcer (SU), sole hemorrhage (SH), white line disease (WLD), and toe ulcers (TU). CHDL are laminitis-related lesions following inflammation that sequel to breakage of the laminar corium from the hoof wall ,37. ReceCorrective CT is carried out to relieve the pain associated with CHDL, which entails the removal of affected horn while preserving the healthy parts ,42. A moFor preventive CT, the incidence of CHDL was lower in farms conducting preventive CT compared to herds lacking the practice , while lInfectious claw lesions affecting dairy cows include digital dermatitis (DD), interdigital dermatitis, heel horn erosion (slurry heel) and interdigital phlegmon (foot rot) . AssociaTreponema-like spirochetes (causative bacteria of DD) on the surface lesions compared to the group that only received AIC [The influence of therapeutic CT on recovery of DD affected cows has been demonstrated by some authors ,56. Mansived AIC . Aside fived AIC . Such prived AIC . Neverthived AIC . This miived AIC ,57. Thesived AIC . Aside from the lesions that have been documented typically as CHDL or infectious types, other important lesions include thin soles (TS), corkscrew claws (CSC), wall fissures, scissors claws, and interdigital hyperplasia (HYP) . AmongstThe two major factors associated with occurrence of TS in dairy herds are incorrect trimming (over-trimming) and biomechanical events at the floor–claw interface ,60,61,62There is paucity of data on the association between CT and wall fissures and scissors claws, but there are few reports on HYP and CSC. Schulz et al.  reportedOverall, literature findings suggest that CT is important for the prevention and treatment of claw lesions. Despite the existence of various CT methods, there are few data on their comparative effects for lameness prevention in dairy herds . PresentEarly detection and treatment of lame cows has been advocated by several research findings ,20,48,70Preventive CT is aimed at reducing the likelihood of lameness occurrence during the high-risk period ,71,72. TCumulative incidence of lameness was lower (18%) in cows trimmed at mid-lactation compared to control group (24%) . Recent Mahendran et al.  reportedPeer-reviewed papers on the frequency of preventive CT and lameness occurrence in dairy cows are scarce, with only a few studies demonstrating lower levels of CHDL in farms where cows underwent twice preventive CT during lactation compared to farms where the practice was once per lactation ,35. ThisThe application of computerized CT database programs to record the claw health data routinely at each claw CT visit provides the basis for monitoring hoof health and adjusting lameness management practices . These dThe freedom to express normal behavior is essential in assessing the welfare of the dairy cow. Since the aim of CT is to improve claw health and well-being, the behavioral changes associated with lameness should be minimal or short-lived after the procedure. Post-CT behavioral alterations included significant increase in lying duration and reduced walking speed ,48,83 inAs shown in Corrective CT for specific claw lesions is also associated with immediate painful sensation which may affect a cow’s behavior. For instance, significantly higher leg movements and reduced lying down time (for 3 weeks) were reported in cows following corrective CT for SU and WLD ,85. ThesLameness causes pain and stress in affected animals ,88. SignClaw temperatures of the trimmed foot have also been demonstrated as a measure of physiological changes associated with CT ,92. CorrOverall, comparisons between the reviewed studies need to consider the research designs, claw health status of the animals, and applied CT methods. There are more data regarding the impact of corrective CT (treatment of claw lesions) on behavioral and physiological parameters in dairy cows . More reMilk yield, reproduction performance and production lifespan are important measures of production in dairy cows. Literature findings relating to the association between CT and milk yield can be categorized based on the short-term ,22,90,93The studies reporting short-term decrease in milk yield following CT highlight the need to clarify if such changes result from cow restraint or actual horn removal. During cow restraint, the interruption of cows’ daily activity relating to feeding and lying behavior could influence milk production. In the long term, alterations in milk yield attributed to CT need to be differentiated from the impact of the existing claw lesion. This can be better understood by conducting a similar research in non-lame cows consisting of the actual trimmed and sham trimmed groups. Another area requiring more research is the role of different CT methods (prophylactic CT) on milk yield and other production variables in non-lame cows. The two studies that considered the association between CT and reproductive parameters found no significant difference in conception rate before and after corrective CT , and betAside from the reviewed sections, other considerations for routine CT include housing design, management, and expertise of the hoof trimmer. The growth rate and wear of the claw, as well as kinetic effects differ between various stall designs with floor types being a significant factor . RecoverThe scientific literature advocates that CT is beneficial for lameness management, as well as improving the welfare and production of dairy cows. There are several studies relating to the association between CT, welfare and production measures; however, determining the actual role of CT on these areas is faced with constraints such as undetailed descriptions of the claw health of enrolled animals and applied CT techniques. Different CT methods are currently used by hoof trimmers, but the benefits of their usage under various management systems or claw wear conditions need to be ascertained. The areas requiring more research regarding preventive CT include timing, frequency, long-term protective effect and identifying the cows that will benefit more from such an intervention during lactation. Such information will be vital in educating farmers on making on-farm decisions regarding CT as a lameness management strategy.


Socio-economic growth in many low and middle-income countries has resulted in more available, though not equitably accessible, healthcare. Such growth has also increased demands from citizens for their health systems to be more responsive to their needs. This paper shares a protocol for the RESPONSE study which aims to understand, co-produce, implement and evaluate context-sensitive interventions to improve health systems responsiveness to health needs of vulnerable groups in Ghana and Vietnam.We will use a realist mixed-methods theory-driven case study design, combining quantitative  and qualitative  methods. Data will be analysed retroductively. The study will comprise three Phases. In Phase 1, we will understand actors’ expectations of responsive health systems, identify key priorities for interventions, and using evidence from a realist synthesis we will develop an initial theory and generate a baseline data. In Phase 2, we will co-produce jointly with key actors, the context-sensitive interventions to improve health systems responsiveness. The interventions will seek to improve internal (i.e. intra-system) and external (i.e. people-systems) interactions through participatory workshops. In Phase 3, we will implement and evaluate the interventions by testing and refining our initial theory through comparing the intended design to the interventions’ actual performance.The study’s key outcomes will be: (1) improved health systems responsiveness, contributing to improved health services and ultimately health outcomes in Ghana and Vietnam and (2) an empirically-grounded and theoretically-informed model of complex contexts-mechanisms-outcomes relations, together with transferable best practices for scalability and generalisability. Decision-makers across different levels will be engaged throughout. Capacity strengthening will be underpinned by in-depth understanding of capacity needs and assets of each partner team, and will aim to strengthen individual, organisational and system level capacities. Responsiveness is a key goal of any national health system, and can be defined as “…when institutions… are cognisant and respond appropriately to the universally legitimate expectations of individuals… safeguarding of rights of patients to adequate… care” 1 p.3]. In addition to being an intrinsic goal, responsiveness is also a process involving multiple interactions within health systems  and is a. In addiIn the last two decades, large parts of the world have seen significant and rapid economic growth . BetweenImproved responsiveness, while itself being a health systems goal, also contributes towards equitable improvements in population health. Responsive systems can improve service uptake, ensure adherence to treatment, and ultimately enhance patient welfare , 7. PeopImproving health systems responsiveness requires targeting internal interactions  and external interactions  . Both inVulnerability, defined as defencelessness, insecurity and exposure to risks , comprisVulnerability of pregnant women is a particularly complex and contested issue . ScholarDespite significant progress, improvements in maternal health remain highly inequitable in many LMICs. This includes Ghana and Vietnam where large disparities cut across income, ethnicity, residence and other aspects of intersectionality , 19 and Psychological wellbeing in pregnancy and postpartum is crucial to mother’s and child’s health . HoweverThis paper shares a protocol for the RESPONSE study which aims to understand and improve health systems responsiveness to health needs of vulnerable groups in Ghana and Vietnam. RESPONSE seeks to contribute to advanced understanding and improving health systems responsiveness in LMICs through multiple case studies in Ghana and Vietnam. We will co-produce with service providers and service users, implement and evaluate context-sensitive interventions to improve systems responsiveness. The focus of our study is on improving health systems responsiveness to the needs of pregnant women, particularly those with mental health problems. Pregnant women were chosen as a priority group who meets the criteria for earlier-discussed vulnerability, due to the intersection of their gender, age, ethnicity, socio-economic status, health needs and other characteristics of intersectionality.Our overall health systems research question is: In what way can health systems become more responsive to neglected health needs of vulnerable groups within the contexts of lower-middle-income countries? This study has five objectives, shown in Key study’s outcomes will be: (1) improved health systems responsiveness to neglected health needs of vulnerable groups in Ghana and Vietnam, and (2) an empirically-grounded and theoretically-informed model of complex relations between the contexts, mechanisms and outcomes of the interventions, along with transferable best practices for scalability (i.e. expansion within similar contexts) and generalisability  for future health systems strengthening.The most widely known framework for health systems responsiveness, developed by the WHO in 2000, comprises seven domains: dignity, autonomy, confidentiality, prompt attention, quality of amenities, access to support networks, and choice of service provider , 8, 24. context-sensitive , and vat powers , 29) and/private , 7). ResAdaptations of the seven domains of responsiveness have been proposed for different health areas, such as HIV/AIDS or mental health , 30, 31.In RESPONSE, we will bridge some of these knowledge gaps while also responding to calls for improved understanding of health systems responsiveness to the needs of diverse health systems actors in LMICs , 27, 28.We approach ‘responsiveness’ as a dynamic social action which is produced via relationships between different actors within contexts of particular socio-economic arrangements as they negotiate experiences of professional, citizen, consumer and patient rights and responsibilities.In our theoretical framework , health Conceptually, health systems responsiveness involves two socially-constructed interactions:internal i.e. between policymakers, managers and service providers, for example as part of prioritisation, resource allocation, target-setting, staff supervision and performance appraisalexternal i.e. between people and the system, typically during service provision [rovision , 28People’s interactions with their families and communities are conceptualised as a context which shapes their initial expectations and subsequent engagements with a health system, though these can also be interpreted as a possible third type of interactions.Improving health systems responsiveness should, therefore, target both the internal  and external  interactions and be cognisant of the wider context which shapes people’s initial expectations of, and their subsequent engagements with, their health systems.commonalities and diversity provide excellent value for this research. Key similarities are:We will conduct the study in two lower-middle-income countries: Ghana and Vietnam. Our choice of these two countries is driven by three considerations. First, their th and 109th, respectively (https://fragilestatesindex.org).Both Ghana and Vietnam are rapidly transitioning LMIC contexts with stable political climate and socio-economic growth , but witIn both countries, the most vulnerable are the poor and those have been left behind amidst major developmental gains for the rest of the country. In response, policymakers in each country are committed to the agenda of health equity and universal health coverage.Both countries have largely publicly funded national health systems, comprising four-tiered organisations with national, regional or province, district and local levels. Both health systems have a high degree of decentralization and a growing private sector.The two health systems also have differences. Vietnam’s publicly-dominated system has increasing market competition, but the Ghanaian system has large private not-for-profit sector and a more substantial share of external funding for health (13% compared with 2% in Vietnam) . With rehigh interest from policymakers in making the health systems more responsive to health needs of the most vulnerable. Preliminary discussions with key decision-makers from the Ghana Health Service and Ministries of Health in each country held during the proposal development showed that all key policymakers are committed to improving health systems responsiveness. In each country, maternal health remains a priority [The second reason which guided the choice of Ghana and Vietnam is that there is priority , 36 withpriority ) and pilwe have strong and longstanding academic collaborations between the Universities of Leeds and Ghana [Finally, we have chosen these two countries because nd Ghana , and Unind Ghana , 40. We In each country, we have purposefully selected one region (in Ghana) or province (in Vietnam) with some of the largest inequities disproportionately affecting vulnerable groups. Within each region and province, we have further selected a rural and an urban district to allow comparisons between rural-urban contexts.In Ghana, we will work in Greater Accra Region located in the Coastal zone. It is 90% urban with highest maternal mortality of 336/100,000 live births as compared with the national average of 310/100,000 live births according to the 2017 Maternal Health Survey, growing squatter urban slums and some of the most deprived rural communities. In Vietnam, we will work in Bắc Giang, a mountainous Province 50km to the east of the capital Hanoi, with large vulnerable groups and 12% of the population comprising ethnic minorities.In each country, we have selected two district health systems as our case studies, based on consultations with policymakers and their demographic and health indicators  and leadBoth districts in Ghana have a Demographic Surveillance System run by the Dodowa Health Research Centre, thus giving a useful link to an existing dataset. The Vietnam’s MOH has identified the Bắc Giang Province to pilot models of support and rehabilitation for those with mental illnesses (including pregnant women), which provides an excellent opportunity and timing for our results to inform on-going reforms.In each district, we will implement interventions in the district hospital, 2–4 public and private primary health care facilities and within respective communities. As we explain later in the paper, the interventions will aim to improve external interactions  as well as internal interactions within health systems . Simultaneously, in Ghana we will engage with Greater Accra Regional Health Directorate and Ghana Health Service, in Vietnam we will engage with Province Health Department, and national Ministry of Health. The project co-investigator Dr Ashinyo is Deputy Director in the Directorate of Clinical Care of the Ghana Health Service and therefore has direct links with national policymakers. In Vietnam, we will leverage project co-investigator Professor Bui’s strong research-policy links. This will ensure sustainability, replication and scaling up of the interventions.mixed-methods realist theory-driven case study design, utilising our expertise in realist evaluations [We will use a luations –46 and eluations . Health luations , 48. It luations , 48, knoluations , and theRealist researchers develop, test and refine middle-range theories which show causal pathways of how interventions work. Such an approach is similar to studies guided by theories of change or even initial hypotheses. Our draft initial theory, to be further developed, tested and refined is:Context-sensitive interventions for better recognition of initial expectations of key actors, if co-produced by these actors to target internal and external interactions and implemented within favourable policy contexts, will improve health systems responsiveness to neglected health needs of vulnerable groups, ultimately contributing to better health outcomes for all.This 42-months study will comprise three Phases as shown in In phase 1 (first year) we will understand actors’ expectations of responsive health systems, identify key priorities for fine-tuning interventions, develop initial theory  and generate a baseline of data . This phase will particularly address the study’s objective 1 and will include two broad parts:conducting realist synthesis using established processes , 50 to ucollecting and analysing primary data using interviews, focus group discussions and community surveys, in order to understand actors’ expectations of responsive health systems, identify issues for fine-tuning the interventions in Phase 2 and establish a baseline for the interventions.We will rigorously synthesise knowledge on systems responsiveness in LMICs using realist synthesis, a “…systematic, theory-driven interpretative technique… to help make sense of heterogeneous evidence about complex interventions applied in diverse contexts”  p.2. ThiOur realist synthesis will utilise a four-step process , which winitial screening to map theoretical landscape of health systems responsiveness,formulating initial programme theories on the basis of theorisation of health systems responsivenessrefining programme theories through purposeful screening of the literature and consultations with key stakeholders as part of the intervention co-production for improving health systems responsiveness in Ghana and Vietnamtesting programme theories through empirical evaluations of implemented interventions in Ghana and Vietnam and against the substantive theories underpinning health systems responsiveness.https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=200353).A detailed protocol for the realist synthesis is available from the International Prospective Register of Systematic Reviews, PROSPERO registration CRD42020200353  and how this shapes their initial expectations from and interactions with, health systems within local contexts how responsiveness is understood and enacted at different health systems levels  within the bureaucracies of health systems  and invowhat health systems responsiveness means within preventive and curative settings of public and private healthcare facilities. We recognise, however, that public-private distinctions are increasingly blurred, with many private (not-for-profit) facilities sharing public-sector values and principles, and public-sector facilities facing market pressures.We will collect data using: (i) reviews of policy and facility documentation including analysis of facility records of service provision and use; (ii) in-depth interviews and focus group discussions with key actors from local communities and health systems and (iii) a community survey. Data will be analysed retroductively, meaning both inductively and deductively. We will draw upon established processes for data analysis such as thematic analysis of qualitative data, and regression models for analysis of quantitative data.review of policy and facility documentation to deepen our understanding of policy, regulatory and systems environments and healthcare practices—relating all back to the conceptualisations of health systems responsiveness. We will qualitatively review two purposefully-identified types of documents: key national-level policies, plans and guidelines; and relevant local-level documents within health facilities, such as minutes of management meetings, clinical reviews, and staff management and performance appraisal records. We will adopt a semi-structured template to summarise insights on the underlying conceptualisations, values and ideas around responsiveness, and approaches to ensuring health systems responsiveness. We will also quantitatively assess patterns of provision and utilisation of maternal and mental health services. To do so, we will analyse facility records for the last 2–3 years disaggregated by key dimensions of intersectionality such as age, income and residence.We will begin with a comprehensive IDIs, we will understand what responsiveness means to different health systems actors including its importance, underlying principles, components, mechanisms and intended outcomes. We will also explore the actors’ framing of underlying fundamental issues  and practices , relating them to our continuously refined understanding of responsiveness. From our experience, 20–25 IDIs should sufficiently represent views of key actor groups in each district, with further 10–15 IDIs at the province and national levels each . This may decrease if we reach data saturation earlier, i.e. when no major new themes will be emerging from subsequent IDIs.Using the FGDs with key actors in each country. These will explore their understanding and expectations of responsive health systems, their framing of underlying fundamental issues and will understand group norms and dynamics. The FGDs will be conducted at the community, facility and province levels (1–2 at each level). Each group will comprise 6–8 participants to maximise engagement, will comprise similar participants  and separate FGDs may be conducted to reduce gender-related bias.We will also conduct 4–6 The participants for IDIs and FGDs will be identified through purposive sampling and will include: (a) patients and public across their intersectionality dimensions ; (b) health facility staff including service providers and support staff; and (c) health policymakers and managers. An initial participant list will be drawn by month 3 and we will identify further ones through snowballing. All FGDs and IDIs will be guided by a semi-structured topic guide to explore causal pathways of responsiveness . Questiocommunity survey to help us understand which attributes of responsive health systems community members value and expect most, and which drivers determine health-seeking behaviours of vulnerable groups. These attributes will be based on the WHO’s seven domains of health systems responsiveness: dignity, autonomy, confidentiality, prompt attention, quality of amenities, access to support networks, and choice of service provider [We will conduct a baseline provider , 8, 24. Sample size: To understand respondents’ views on the importance of the different attributes of responsiveness, we will treat each discrete point on our chosen responsiveness attribute “importance scale” as a binary outcome and estimate the percentage of respondents selecting that point on the given scale (accounting for the complex survey design). We estimate that for the survey in each country we will require 562 respondents (assuming a response rate of 95%) to estimate the percentage of respondents selecting each point on the scale (assuming the most conservative percentage of 50% when estimating the sample size for a binary outcome based on precision) with a margin of error of ± 6 percentage points , which we judged to be a suitable balance between precision and resources, and assuming a moderate design effect of 2 given we have no existing data . We will aim to understand the relationships between the socio-economic factors and respondents’ views on the relative importance of the different attributes of responsiveness, such as differences between men and women. In doing so, we will use appropriate multiple linear regression models that treat the “importance scale” outcomes as continuous variables . Based on this approach, the above sample size (562) would also allow us to detect differences between binary subgroups, such as men and women (assuming a maximum between-subgroup size ratio of ≤6:1), of 0.65 or greater points in their responses to any of the “importance scale” questions with 80% power .Depending on the sampling frame data that is available in each country, we will use a multi-stage clustered household survey sampling approach  to allowThe outcome of Phase 1 will be an initial theoretical model explaining how health systems responsiveness works. It will explain how different contexts shape and trigger the mechanisms through which health systems responsiveness is enacted (or not) by the different actors to produce the intended and unintended outcomes.In Phase 2 (months 13–18) we will co-produce the context-sensitive interventions to improve health systems responsiveness, addressing study objective 2. The co-production will be through 2–3 meetings in each district involving key actors . These meetings themselves can also be seen as interventions, and will therefore involve elements of capacity strengthening and knowledge uptake. The meetings will be led by district  health leadership and facilitated by researchers who will present evidence from Phase 1, document causal pathways of how the interventions are intended to work  and using participant observations following a semi-structured template reflect on the co-production processes in terms of clarity, inclusivity, transparency and effectiveness.In each country, the interventions will seek to improve two key components of responsiveness:internal interactions i.e. within and across facility, district, province and national levelsexternal interactions i.e. between the health systems and vulnerable groups.The rationale is that these two components, when combined, will ultimately contribute to improving people’s experiences across the different domains of health systems responsiveness specified in our theoretical framework. Our focus on the processes of interaction is also driven by our intention to enact systemic change rather than target specific individual domains in a discreet manner.The overall design of the interventions is shown in During the intervention co-production meetings we will fine-tune this general design , to be informed by better understanding of people’s initial expectations of responsive health systems which inform their interactions with their health systems from Phase 1.In fine-tuning the interventions, we will consolidate, adapt and extend our work in Health Workers for Change (HWFC) workshops , 64, Coninternal interactions, in each district we will conduct sets of six thematic workshops, following the HWFC approach, comprising frontline health workers and managers in the district hospital and PHC facilities. These are participatory, interactive 2-hour workshops, moderated by a skilled facilitator–usually a social scientist with experience in moderating group discussions. The workshop series stems from Paulo Freire’s work on transformational learning [To improve learning  and aimslearning . The sixExperiences of using HFWC approach in Ghana and other countries, show them as an effective platform for institutionalising a process of Continuous Quality Improvement (CQI) in healthcare facilities , 64. Eviexternal interactions, to establish group consensus during these workshops, we will use a Nominal Group Technique (NGT)–a structured, multistep, group consensus building technique which comprises 6 steps: (a) individual writing of ideas; (b) group review and feedback; and (c) discussion, clarification and evaluation of each idea (d) ranking ideas in order of their significance; (e) discussion of the preliminary vote; and (f) final individual voting on significance of each idea.To improve We will draw upon the NGT’s documented effects on consensus building, including in Ghana and in Vietnam , 72, to The interventions are intentionally designed as low-cost activities to be embedded within the current structures and processes to ensure their sustainability, replication and scaling up. Although the HFWC workshops and NGT intend to improve internal and external interactions respectively, each is also likely to bridge the external-internal boundaries. We also anticipate that cumulatively these two intervention components will raise awareness and empower health workers and communities, and consequently will provide a sustainable platform for problem analysis and solution seeking. Such a platform will contribute to systemic improvements in responsiveness as a key attribute of health systems strengthening.In Phase 3 (months 19–42) we will implement and evaluate the interventions within local contexts. The interventions will be implemented for one full year and through existing systems’ structures and processes. Such duration should help embed the interventions within annual health planning and budgeting cycles and thus ensure their integration within routine practices and longer-term sustainability.Phase 1 section for proposed details of the survey design, questionnaire topics and format, and sample size/analysis. With the baseline and follow-up community surveys we can then explore whether respondents’ views on the relative importance of different attributes of responsiveness have changed subsequent to the intervention. This will be done using appropriate multiple linear regression models that adjust for the complex survey design, and also control for likely important confounding variables.Using realist evaluation , 73 we wWe will also assess the interventions’ feasibility and acceptability by key actors and processes within and between Ghana and Vietnam, utilising the UK Medical Research Council’s framework for process evaluation of complex interventions . In doinOur intra- and cross-country comparative analyses will allow us to develop transferable best practices for other areas and LMICs which also experience similar socio-economic growth and face pressures to effectively address the needs of vulnerable groups. These best practices will be in a form of a theory-informed and empirically-grounded model of health systems responsiveness to neglected health needs of vulnerable groups, specifically addressing the study objectives 3 and 4. The model will guide a deeper understanding of how the contexts shape and trigger specific mechanisms through which health systems responsiveness works for different actors. We will also produce detailed practical guidance notes on further adaptations of this model to inform future policy and practice on improving health systems responsiveness in LMICs.Ethics approvals for this study were obtained from the University of Leeds School of Medicine Research Ethics Committee (ref: MREC19-051), Ghana Health Service Ethics Review Committee (ref GHS-ERC 012/03/20) and Hanoi University of Public Health Institutional Review Board (ref 020-149/DD-YTCC). All primary data will be collected after obtaining written informed consent (or thumb print for those who can’t write) and while preserving participants’ anonymity and confidentiality.The project will be carried out with full respect of current relevant legislation such as the General Data Protection Regulation. The methods development, data collection and analysis will consider:anonymity of all study respondents will be preserved where possible and will be ensured at all times if respondent(s) request. Unnecessary collection of personal data will be avoided. Where personal data is collected it will be coded, removed from the data for analysis and stored separately from transcripts. Only PIs and designated research personnel in each organisation will have access to the keys linking the data with the personal information.informed consent will be obtained from all study participants and in the case of refusal, alternative means of data collection will be explored confidentiality and other data protection issues, which will include secure data storage and clear access rights. Only members of teams identified by the PIs in each institution will have access. Where data (e.g. transcripts) is stored on a server it will be password-protected and only research teams will have access to the passwords. Availability of documents on the internet will be following the consent of all collaborators.specific emphasis will be placed on The project will be implemented according to established robust research governance practice standards at the University of Leeds for implementing collaborative projects. This includes ensuring: regular communication between the partners and engagement with policymakers and practitioners; quality assurance through regular peer-review both within and between the teams; appropriate mentoring and coaching support to early career researchers; and equal opportunities as part of the Leeds University’s commitment to the Athena Swan equal opportunities initiative.We will embed the research into policy and practice working with facility, district, regional and national actors, and through extending our academic collaborations into South-South research and policy partnerships. Engagements of decision-makers will facilitate implementation and scaling-up of these and similar interventions within and across Ghana and Vietnam.We will communicate research findings through combinations of:developing policy briefs addressed to national and international policymakers and practitioners and designed as short and practical documents;delivering presentations at review meetings at local, district, regional/province and national levels in Ghana and Vietnam  and relevant regional meetings;regular (e.g. quarterly) project review meetings with national, regional and district actors and continuous engagement with key decision-makers through for example sharing draft policy implications for discussion of feasibility before including them in official reports;developing newsletters and press-releases aimed at communicating the key project findings in simple ways accessible to the general public in each country and their respective regions;interviews in the national media (e.g. radio and television) as well as articles for national newspapers, communicating our findings and educating the public on effective engagements and ensuring health systems responsiveness as appropriate and feasible;creating a website where all materials will be easily accessible by (inter)national decision-makers and practitioners, with mutual links to GHS and MOH websites in Ghana and Vietnamdelivering presentations at national, regional and international conferences and publication of articles in peer-reviewed journals with specific emphasis on open access where feasibledeveloping project reports for key stakeholders, with a publishable executive summary.There is a high interest from key policymakers in this topic, some of whom are the members of the project team. We will maintain equal research-policy partnerships  and willThis paper summarised the protocol for a mixed methods realist evaluation study which aims to understand and improve health systems responsiveness to health needs of vulnerable groups in Ghana and Vietnam.A key study’s theoretical outcome will be an empirically-grounded and theory-informed model of complex contexts-mechanisms-outcomes relations, together with transferable best practices for scalability (i.e. expanding within similar contexts) and generalisability  for future health systems strengthening. The model will advance the current understanding of health systems responsiveness and will form an overall theoretical contribution from this study to the published knowledge on the subject.Decision-makers from facility, district, regional and national levels will be engaged through embedded research in policy and practice and equal research-policy partnerships . This wiindividual level we will seek to improve knowledge and skills of early-career researchers particularly in Ghana and Vietnam through mentoring support and on-the-job training (e.g. to lead academic publications). We also aim to improve individual capacities of key decision-makers to effectively set research priorities and understand and apply research results in their routine practices, through their continuous engagements in the study. At the organisational level, we will improve partners’ processes for research governance  and their ability to effectively communicate results. Systems level work will target partners’ networks including strengthening and expanding existing research-policy links in Ghana and Vietnam. During the first three months of the project, we will consolidate our in-depth knowledge of partners’ current capacities and will develop a clear, coherent and feasible capacity strengthening plan. The South-South exchange and learning will be central to our capacity strengthening approach with collaborators in Ghana and Vietnam owning and driving capacity strengthening. Senior co-Is in Ghana and Vietnam will mentor early-career researchers and create organisational environment where researchers’ enhanced expertise can be effectively applied.Capacity strengthening is a distinct project objective and is therefore an important aspect of this study. We will deploy a systematic approach to capacity strengthening which will be underpinned by our in-depth understanding of current capacity needs and assets of each partner team from previous studies , 61, 76,