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BackgroundThe Coronavirus disease 2019 (COVID-19) resulted in the disruption of Sexual and Reproductive Health Rights (SRHR) services in the Eastern and Southern Africa region. To date, studies estimating the impact of COVID-19 disruptions have mainly focused on SRHR services without estimating the economic implication.MethodWe used national service coverage data on the effectiveness of interventions from the lives saved tool (LiST), a mathematical modeling tool that estimates the effects of service coverage change in mortality. We computed years lost due to COVID-19 disruption on SRHR using life expectancy at birth, number of years of life lost due to child mortality, and life expectancy at average maternal death. We calculated the economic value of the lives saved, using the values of statistical life year for each of the countries, comparing 2019 (pre-COVID-19) to 2020 (COVID-19 era).FindingsThe total life-years lost were 1,335,663, with 1,056,174 life-years lost attributed to child mortality and 279,249 linked to maternal mortalities, with high case-fatality rates in the Democratic Republic of Congo, Burundi, and Tanzania. The findings show COVID-19 disruptions on SRHR services between 2019 and 2020 resulted in US$ 3.6 billion losses, with the highest losses in Angola (USD 777 million), South Africa (USD 539 million), and Democratic Republic of Congo (USD 361 million).ConclusionThe monetized value of disability adjusted life years can be used as evidence for advocacy, increased investment, and appropriate mitigation strategies. Countries should strengthen their health systems functionality, incorporating and transforming lessons learned from shock events.

Introduction Addressing inequities in health service coverage is a global priority, especially with the resurgence of interest in universal health coverage. However, in Africa, which has the lowest health service coverage index, there is limited information on the progress of countries in addressing inequalities related to health services. Thus, we seek to map the evidence on inequalities in health service coverage in Africa.Methods and analysis We will conduct a scoping review following the Joanna Briggs Institute Manual for Evidence Synthesis. We preregistered this protocol with the Open Science Framework on 26 July 2022 (https://osf.io/zd5bt). We will consider any empirical research that assesses inequalities in relation to services for reproductive, maternal, newborn and child health (eg, family planning), infectious diseases (eg, tuberculosis treatment) and non-communicable diseases (eg, cervical cancer screening) in Africa. We will search MEDLINE, Embase, Web of Science, CINAHL, PsycINFO and Cochrane Library from their inception onwards. We will also hand-search Google and Global Index Medicus, and screen reference lists of relevant studies. We will evaluate studies for eligibility and extract data from included studies using pre-piloted and standardised forms. We will further extract a core set of health service coverage indicators, which are disaggregated by place of residence, race/ethnicity/culture, occupation, gender, religion, education, socioeconomic status and social capital plus equity stratifiers. We will summarise data using a narrative approach involving thematic syntheses and descriptive statistics. We will report our findings according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews checklist.Ethics and dissemination Ethical approval is not required as primary data will not be collected. This work will contribute to identifying knowledge gaps in the evidence of inequalities in health service coverage in Africa, and propose strategies that could help overcome current challenges. We will disseminate our findings to knowledge users through a publication in a peer-reviewed journal and organisation of workshops.

This article is part of the Research Topic‘Health Systems Recovery in the Context of COVID-19 and Protracted Conflict.’ProblemMany countries lacked rapid and nimble data systems to track health service capacities to respond to COVID-19. They struggled to assess and monitor rapidly evolving service disruptions, health workforce capacities, health products availability, community needs and perspectives, and mitigation responses to maintain essential health services.MethodBuilding on established methodologies, the World Health Organization developed a suite of methods and tools to support countries to rapidly fill data gaps and guide decision-making during COVID-19. The tools included: (1) a national “pulse” survey on service disruptions and bottlenecks; (2) a phone-based facility survey on frontline service capacities; and (3) a phone-based community survey on demand-side challenges and health needs.UseThree national pulse surveys revealed persisting service disruptions throughout 2020–2021 (97 countries responded to all three rounds). Results guided mitigation strategies and operational plans at country level, and informed investments and delivery of essential supplies at global level. Facility and community surveys in 22 countries found similar disruptions and limited frontline service capacities at a more granular level. Findings informed key actions to improve service delivery and responsiveness from local to national levels.Lessons learnedThe rapid key informant surveys provided a low-resource way to collect action-oriented health services data to inform response and recovery from local to global levels. The approach fostered country ownership, stronger data capacities, and integration into operational planning. The surveys are being evaluated to inform integration into country data systems to bolster routine health services monitoring and serve as health services alert functions for the future.

Abstract Background The recent 2018 Declaration of Astana recognized primary health care (PHC) as a means to achieve universal health coverage (UHC) and the health-related Sustainable Development Goals (SDGs). Following this declaration, country progress on operationalization of the PHC agenda and attainment of UHC has been stalled by the new challenges posed by the COVID-19 pandemic. The pandemic has also disrupted the continuity of essential health service provision and tested the resilience of the region’s health systems. Methods In accordance with this, the WHO Regional Office for Africa convened the Fifth Health Sector Directors’ Planning and Policy Meeting across the 47 Member States of the Region. The two-day forum focused on building health system resilience to facilitate service continuity during health threats, PHC revitalization, and health systems strengthening towards UHC. Results The Regional Forum provided evidence on building resilient health systems in the WHO African Region and engaged participants in meaningful and critical discussion. It is from these discussions that four key themes emerged: (1) working multisectorally/intersectorally, (2) moving from fragmentation to integration, (3) ensuring implementation and knowledge exchange, and (4) rethinking resilience and embracing antifragility. These discussions and associated groupings by thematic areas lend themselves to recommendations for the WHO. Conclusions This paper details the proceedings and key findings on building resilient health systems, the four themes that emerged from participant deliberation, and the recommendations that have emerged from the meeting. Deliberations from the Regional Forum are critical, as they have the potential to directly inform policy and program design, given that the meeting convenes health sector technocrats, who are at the helm of policy design, action, and implementation.

BackgroundDigital Health Interventions (DHIs) refers to the utilization of digital and mobile technology to support the health system in service delivery. Over the recent years, advanced computing, genomics, and artificial intelligence are considered part of digital health. In the context of the World Health Organization (WHO) global strategy 2020–2025, digital health is defined as “the field of knowledge and practice associated with the development and use of digital technologies to improve health.” The scoping review protocol details the procedure for developing a comprehensive list of DHIs in Sub-Saharan Africa and documenting their roles in strengthening health systems.Method and AnalysisA scoping review will be done according to the Joanne Briggs institute reviewers manual and following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews (PRISMA-ScR) checklist and explanation. The protocol has been registered at the Open Science Framework (OSF) database at https://osf.io/5kzq7. The review will include DHIs conceptualized/developed/designed, adapted, piloted, deployed, scaled up, and addressing health challenges in Sub-Saharan Africa. We will retrieve data from the global DHI repository-the WHO Digital Health Atlas (DHA)- and supplement it with information from the WHO eHealth Observatory, eHealth Survey (2015), and eHealth country profiles report. Additional searches will be conducted in four (4) electronic databases: PubMed, HINARI-Reasearch4Life, Cochrane Library, and Google Scholar. The review will also include gray literature and reference lists of selected studies. Data will be organized in conceptual categories looking at digital health interventions' distinct function toward achieving health sector objectives.DiscussionSub-Saharan Africa is an emerging powerhouse in DHI innovations with rapid expansion and evolvement. The enthusiasm for digital health has experienced challenges including an escalation of short-lived digital health interventions, duplication, and minimal documentation of evidence on their impact on the health system. Efficient use of resources is important when striving toward the use digital health interventions in health systems strengthening. This can be achieved through documenting successes and lessons learnt over time.ConclusionThe review will provide the evidence to guide further investments in DHIs, avoid duplication, circumvent barriers, focus on gaps, and scale-up successful interventions.

BackgroundWhile multiple studies have documented the impacts of mobile phone use on TB health outcomes for varied settings, it is not immediately clear what the spatial patterns of TB treatment completion rates among African countries are. This paper used Exploratory Spatial Data Analysis (ESDA) techniques to explore the clustering spatial patterns of TB treatment completion rates in 53 African countries and also their relationships with mobile phone use. Using an ESDA approach to identify countries with low TB treatment completion rates and reduced mobile phone use is the first step toward addressing issues related to poor TB outcomes.MethodsTB notifications and treatment data from 2000 through 2015 that were obtained from the World Bank database were used to illustrate a descriptive epidemiology of TB treatment completion rates among African health systems. Spatial clustering patterns of TB treatment completion rates were assessed using differential local Moran's I techniques, and local spatial analytics was performed using local Moran's I tests. Relationships between TB treatment completion rates and mobile phone use were evaluated using ESDA approach.ResultSpatial autocorrelation patterns generated were consistent with Low-Low and High-Low cluster patterns, and they were significant at different p-values. Algeria and Senegal had significant clusters across the study periods, while Democratic Republic of Congo, Niger, South Africa, and Cameroon had significant clusters in at least two time-periods. ESDA identified statistically significant associations between TB treatment completion rates and mobile phone use. Countries with higher rates of mobile phone use showed higher TB treatment completion rates overall, indicating enhanced program uptake (p < 0.05).ConclusionStudy findings provide systematic evidence to inform policy regarding investments in the use of mHealth to optimize TB health outcomes. African governments should identify turnaround strategies to strengthen mHealth technologies and improve outcomes.

The health challenges in Africa underscore the importance of effectively investing in health systems. Unfortunately, there is no information on systems investments adequate for an effective functional health system. We aimed to address this by conducting a scoping review of existing evidence following the Joanna Briggs Institute Manual for Evidence Synthesis and preregistered with the Open Science Framework (https://osf.io/bvg4z). We included any empirical research describing interventions that contributed to the functionality of health systems in Africa or any low-income or lower-middle-income regions. We searched Web of Science, MEDLINE, Embase, PsycINFO, Cochrane Library, CINAHL, and ERIC from their inception, and hand-searched other relevant sources. We summarized data using a narrative approach involving thematic syntheses and descriptive statistics. We identified 554 unique reports describing 575 interventions, of which 495 reported evidence of effectiveness. Most interventions were undertaken in Africa (80.9%), covered multiple elements of health systems (median: 3), and focused on service delivery (77.4%) and health workforce (65.6%). Effective interventions contributed to improving single (35.6%) or multiple (64.4%) capacities of health systems: access to essential services (75.6%), quality of care (70.5%), demand for essential services (38.6%), or health systems resilience (13.5%). For example, telemedicine models which covered software (technologies) and hardware (health workers) elements were used as a strategy to address issues of access to essential services. We inventoried these effective interventions for improving health systems functionality in Africa. Further analyses could deepen understanding of how such interventions differ in their incorporation of evidence for potential scale across African countries.

The need for resilient health systems is recognized as important for the attainment of health outcomes, given the current shocks to health services. Resilience has been defined as the capacity to "prepare and effectively respond to crises; maintain core functions; and, informed by lessons learnt, reorganize if conditions require it". There is however a recognized dichotomy between its conceptualization in literature, and its application in practice. We propose two mutually reinforcing categories of resilience, representing resilience targeted at potentially known shocks, and the inherent health system resilience, needed to respond to unpredictable shock events. We determined capacities for each of these categories, and explored this methodological proposition by computing country-specific scores against each capacity, for the 47 Member States of the WHO African Region. We assessed face validity of the computed index, to ensure derived values were representative of the different elements of resilience, and were predictive of health outcomes, and computed bias-corrected non-parametric confidence intervals of the emergency preparedness and response (EPR) and inherent system resilience (ISR) sub-indices, as well as the overall resilience index, using 1000 bootstrap replicates. We also explored the internal consistency and scale reliability of the index, by calculating Cronbach alphas for the various proposed capacities and their corresponding attributes. We computed overall resilience to be 48.4 out of a possible 100 in the 47 assessed countries, with generally lower levels of ISR. For ISR, the capacities were weakest for transformation capacity, followed by mobilization of resources, awareness of own capacities, self-regulation and finally diversity of services respectively. This paper aims to contribute to the growing body of empirical evidence on health systems and service resilience, which is of great importance to the functionality and performance of health systems, particularly in the context of COVID-19. It provides a methodological reflection for monitoring health system resilience, revealing areas of improvement in the provision of essential health services during shock events, and builds a case for the need for mechanisms, at country level, that address both specific and non-specific shocks to the health system, ultimately for the attainment of improved health outcomes.

The move towards universal health coverage is premised on having well-functioning health systems, which can assure provision of the essential health and related services people need. Efforts to define ways to assess functionality of health systems have however varied, with many not translating into concrete policy action and influence on system development. We present an approach to provide countries with information on the functionality of their systems in a manner that will facilitate movement towards universal health coverage. We conceptualise functionality of a health system as being a construct of four capacities: access to, quality of, demand for essential services and its resilience to external shocks. We test and confirm the validity of these capacities as appropriate measures of system functionality. We thus provide results for functionality of the 47 countries of the WHO African Region based on this. The functionality of health systems ranges from 34.4 to 75.8 on a 0–100 scale. Access to essential services represents the lowest capacity in most countries of the region, specifically due to poor physical access to services. Funding levels from public and out-of-pocket sources represent the strongest predictors of system functionality, compared with other sources. By focusing on the assessment on the capacities that define system functionality, each country has concrete information on where it needs to focus, in order to improve the functionality of its health system to enable it respond to current needs including achieving universal health coverage, while responding to shocks from challenges such as the 2019 coronavirus disease. This systematic and replicable approach for assessing health system functionality can provide the guidance needed for investing in country health systems to attain universal health coverage goals.

The spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been unprecedented in its speed and effects. Interruption of its transmission to prevent widespread community transmission is critical because its effects go beyond the number of COVID-19 cases and deaths and affect the health system capacity to provide other essential services. Highlighting the implications of such a situation, the predictions presented here are derived using a Markov chain model, with the transition states and country specific probabilities derived based on currently available knowledge. A risk of exposure, and vulnerability index are used to make the probabilities country specific. The results predict a high risk of exposure in states of small size, together with Algeria, South Africa and Cameroon. Nigeria will have the largest number of infections, followed by Algeria and South Africa. Mauritania would have the fewest cases, followed by Seychelles and Eritrea. Per capita, Mauritius, Seychelles and Equatorial Guinea would have the highest proportion of their population affected, while Niger, Mauritania and Chad would have the lowest. Of the World Health Organization's 1 billion population in Africa, 22% (16%–26%) will be infected in the first year, with 37 (29 – 44) million symptomatic cases and 150 078 (82 735–189 579) deaths. There will be an estimated 4.6 (3.6–5.5) million COVID-19 hospitalisations, of which 139 521 (81 876–167 044) would be severe cases requiring oxygen, and 89 043 (52 253–106 599) critical cases requiring breathing support. The needed mitigation measures would significantly strain health system capacities, particularly for secondary and tertiary services, while many cases may pass undetected in primary care facilities due to weak diagnostic capacity and non-specific symptoms. The effect of avoiding widespread and sustained community transmission of SARS-CoV-2 is significant, and most likely outweighs any costs of preventing such a scenario. Effective containment measures should be promoted in all countries to best manage the COVID-19 pandemic.

In the April 2010 issue of this journal, Date et al. expressed concern over the slow scale-up in low-income settings of two therapies for the prevention of opportunistic infections in people living with the human immunodeficiency virus: co-trimoxazole prophylaxis and isoniazid preventive therapy. This short paper discusses the important ways in which policy analysis can be of use in understanding and explaining how and why certain evidence makes its way into policy and practice and what local factors influence this process. Key lessons about policy development are drawn from the research evidence on co-trimoxazole prophylaxis, as such lessons may prove helpful to those who seek to influence the development of national policy on isoniazid preventive therapy and other treatments. Researchers are encouraged to disseminate their findings in a manner that is clear, but they must also pay attention to how structural, institutional and political factors shape policy development and implementation. Doing so will help them to understand and address the concerns raised by Date et al. and other experts. Mainstreaming policy analysis approaches that explain how local factors shape the uptake of research evidence can provide an additional tool for researchers who feel frustrated because their research findings have not made their way into policy and practice.

NADH cytochrome b 5 oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochrome b 5 (b 5 ) and cytochrome b 5 reductase (b 5 R) domains separated by a CHORD-Sgt1 (CS) linker domain. Ncb5or redox activity depends on proper inter-domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme. While full-length human Ncb5or has proven resistant to crystallization, we have succeeded in obtaining high-resolution atomic structures of the b 5 domain and a construct containing the CS and b 5 R domains (CS/b 5 R). Ncb5or also contains an N-terminal intrinsically disordered region of 50 residues that has no homologs in other protein families in animals but features a distinctive, conserved L 34 MDWIRL 40 motif also present in reduced lateral root formation (RLF) protein in rice and increased recombination center 21 in baker's yeast, all attaching to a b 5 domain. After unsuccessful attempts at crystallizing a human Ncb5or construct comprising the N-terminal region naturally fused to the b 5 domain, we were able to obtain a high-resolution atomic structure of a recombinant rice RLF construct corresponding to residues 25-129 of human Ncb5or (52% sequence identity; 74% similarity). The structure reveals Trp 120 (corresponding to invariant Trp 37 in Ncb5or) to be part of an 11-residue α-helix (S 116 QMDWLKLTRT 126 ) packing against two of the four helices in the b 5 domain that surround heme (α2 and α5). The Trp 120 side chain forms a network of interactions with the side chains of four highly conserved residues corresponding to Tyr 85 and Tyr 88 (α2), Cys 124 (α5), and Leu 47 in Ncb5or. Circular dichroism measurements of human Ncb5or fragments further support a key role of Trp 37 in nucleating the formation of the N-terminal helix, whose location in the N/b 5 module suggests a role in regulating the function of this multi-domain redox enzyme. This study revealed for the first time an ancient origin of a helical motif in the N/b 5 module as reflected by its existence in a class of cytochrome b 5 proteins from three kingdoms among eukaryotes., (© 2023 Wiley Periodicals LLC.)

Frankia and actinorhizal plants exchange signals in the rhizosphere leading to specific mutual recognition of partners and nitrogen-fixing nodule organogenesis. Frankia soli strain NRRL B-16219, from the Elaeagnus specificity group, colonizes the root tissues of its actinorhizal host through direct intercellular penetration of root epidermis cells and cortex. Here, we studied the early proteogenomic response of strain NRRL B-16219 to treatment with root exudates from compatible Elaeagnus angustifolia, and incompatible Ceanothus thyrsiflorus and Coriaria myrtifolia, host plants grown in nitrogen depleted hydroponic medium. Next-generation proteomics was used to identify the main Frankia proteins differentially expressed in response to the root exudates. No products of the nod genes present in B-16219 were detected. Proteins specifically upregulated in presence of E. angustifolia root exudates include those connected to nitrogen fixation and assimilation (glutamate synthetase, hydrogenase and squalene synthesis), respiration (oxidative phosphorylation and citric acid cycle pathways), oxidative stress (catalase, superoxide dismutase, and peroxidase), proteolysis (proteasome, protease, and peptidase) and plant cell wall degrading proteins involved in the depolymerization of celluloses (endoglucanase, glycosyltransferase, beta-mannanases, glycoside hydrolase and glycosyl hydrolase). Proteomic data obtained in this study will help link signaling molecules/factors to their biosynthetic pathways once those factors have been fully characterized., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2021 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Ncb5or (NADH-cytochrome b 5 oxidoreductase), a cytosolic ferric reductase implicated in diabetes and neurological diseases, comprises three distinct domains, cytochrome b 5 (b 5 ) and cytochrome b 5 reductase (b 5 R) domains separated by a CHORD-Sgt1 (CS) domain, and a novel 50-residue N-terminal region. Understanding how interdomain interactions in Ncb5or facilitate the shuttling of electrons from NAD(P)H to heme, and how the process compares with the microsomal b 5 (Cyb5A) and b 5 R (Cyb5R3) system, is of interest. A high-resolution structure of the b 5 domain (PDB entry 3lf5) has previously been reported, which exhibits substantial differences in comparison to Cyb5A. The structural characterization of a construct comprising the naturally fused CS and b 5 R domains with bound FAD and NAD + (PDB entry 6mv1) or NADP + (PDB entry 6mv2) is now reported. The structures reveal that the linker between the CS and b 5 R cores is more ordered than predicted, with much of it extending the β-sandwich motif of the CS domain. This limits the flexibility between the two domains, which recognize one another via a short β-sheet motif and a network of conserved side-chain hydrogen bonds, salt bridges and cation-π interactions. Notable differences in FAD-protein interactions in Ncb5or and Cyb5R3 provide insight into the selectivity for docking of their respective b 5 redox partners. The structures also afford a structural explanation for the unusual ability of Ncb5or to utilize both NADH and NADPH, and represent the first examples of native, fully oxidized b 5 R family members in which the nicotinamide ring of NAD(P) + resides in the active site. Finally, the structures, together with sequence alignments, show that the b 5 R domain is more closely related to single-domain Cyb5R proteins from plants, fungi and some protists than to Cyb5R3 from animals.

Objective: Although past research has demonstrated a link between the quality of motivational interviewing (MI) counseling and client behavior change, this relationship has not been examined in the context of sexual risk behavior among people living with HIV/AIDS. We studied MI quality and unprotected anal/vaginal intercourse (UAVI) in the context of SafeTalk, an evidence-based secondary HIV prevention intervention., Methods: We used a structured instrument (the MISC 2.0 coding system) as well as a client-reported instrument to rate intervention sessions on aspects of MI quality. Then we correlated client-reported UAVI with specific counseling behaviors and the proportion of interactions that achieved MI quality benchmarks., Results/conclusion: Higher MISC-2.0 global ratings and a higher ratio of reflections to questions both significantly predicted fewer UAVI acts at 8-month follow-up. Analysis of client ratings, which was more exploratory, showed that clients who rated their sessions higher in counselor acceptance, client disclosure, and relevance reported higher numbers of UAVIs, whereas clients who selected higher ratings for perceived benefit were more likely to have fewer UAVI episodes., Practice Implications: Further research is needed to determine the best methods of translating information about MI quality into dissemination of effective MI interventions with people living with HIV., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

We investigate the roles of measurement time scale and the nature of the fluorophores in the FRET states measured for calmodulin, a calcium signaling protein known to undergo pronounced conformational changes. The measured FRET distributions depend markedly on the measurement time scale (nanosecond or microsecond). Comparison of FRET distributions measured by donor fluorescence decay with FRET distributions recovered from single-molecule burst measurements binned over time scales of 90 μs to 1 ms reveals conformational averaging over the intervening time regimes. We find further that, particularly in the presence of saturating Ca(2+), the nature of the measured single-molecule FRET distribution depends markedly on the identity of the FRET pair. The results suggest interchange between conformational states on time scales of hundreds of microseconds or less. Interaction with a fluorophore such as the dye Texas Red alters both the nature of the measured FRET distributions and the dynamics of conformational interchange. The results further suggest that the fluorophore may not be merely a benign reporter of protein conformations in FRET studies, but may in fact alter the conformational landscape.

The history of cheese manufacture is a "natural history" in which animals, microorganisms, and the environment interact to yield human food. Part of the fascination with cheese, both scientifically and culturally, stems from its ability to assume amazingly diverse flavors as a result of seemingly small details in preparation. In this review, we trace the roots of cheesemaking and its development by a variety of human cultures over centuries. Traditional cheesemakers observed empirically that certain environments and processes produced the best cheeses, unwittingly selecting for microorganisms with the best biochemical properties for developing desirable aromas and textures. The focus of this review is on the role of fungi in cheese ripening, with a particular emphasis on the yeast-like fungus Geotrichum candidum. Conditions that encourage the growth of problematic fungi such as Mucor and Scopulariopsis as well as Arachnida (cheese mites), and how such contaminants might be avoided, are discussed. Bethlehem cheese, a pressed, uncooked, semihard, Saint-Nectaire-type cheese manufactured in the United Sates without commercial strains of bacteria or fungi, was used as a model for the study of stable microbial succession during ripening in a natural environment. The appearance of fungi during a 60-day ripening period was documented using light and scanning electron microscopy, and it was shown to be remarkably reproducible and parallel to the course of ripening of authentic Saint-Nectaire cheese in the Auvergne region of France. Geotrichum candidum, Mucor, and Trichothecium roseum predominate the microbiotas of both cheese types. Geotrichum in particular was shown to have high diversity in different traditional cheese ripening environments, suggesting that traditional manufacturing techniques selected for particular fungi. This and other studies suggest that strain diversity arises in relation to the lore and history of the regions from which these types of cheeses arose.

In January 2011, the U.S. Food and Drug Administration published new process validation guidance for pharmaceutical processes. The new guidance debunks the long-held industry notion that three consecutive validation batches or runs are all that are required to demonstrate that a process is operating in a validated state. Instead, the new guidance now emphasizes that the level of monitoring and testing performed during process performance qualification (PPQ) studies must be sufficient to demonstrate statistical confidence both within and between batches. In some cases, three qualification runs may not be enough. Nearly two years after the guidance was first published, little has been written defining a statistical methodology for determining the number of samples and qualification runs required to satisfy Stage 2 requirements of the new guidance. This article proposes using a combination of risk assessment, control charting, and capability statistics to define the monitoring and testing scheme required to show that a pharmaceutical batch process is operating in a validated state. In this methodology, an assessment of process risk is performed through application of a process failure mode, effects, and criticality analysis (PFMECA). The output of PFMECA is used to select appropriate levels of statistical confidence and coverage which, in turn, are used in capability calculations to determine when significant Stage 2 (PPQ) milestones have been met. The achievement of Stage 2 milestones signals the release of batches for commercial distribution and the reduction of monitoring and testing to commercial production levels. Individuals, moving range, and range/sigma charts are used in conjunction with capability statistics to demonstrate that the commercial process is operating in a state of statistical control., Lay Abstract: The new process validation guidance published by the U.S. Food and Drug Administration in January of 2011 indicates that the number of process validation batches or runs required to demonstrate that a pharmaceutical process is operating in a validated state should be based on sound statistical principles. The old rule of "three consecutive batches and you're done" is no longer sufficient. The guidance, however, does not provide any specific methodology for determining the number of runs required, and little has been published to augment this shortcoming. The paper titled "Risk-based Methodology for Validation of Pharmaceutical Batch Processes" describes a statistically sound methodology for determining when a statistically valid number of validation runs has been acquired based on risk assessment and calculation of process capability.

All but a few bacterial species have an absolute need for heme, and most are able to synthesize it via a pathway that is highly conserved among all life domains. Because heme is a rich source for iron, many pathogenic bacteria have also evolved processes for sequestering heme from their hosts. The heme biosynthesis pathways are well understood at the genetic and structural biology levels. In comparison, much less is known about the heme acquisition, trafficking, and degradation processes in bacteria. Gram-positive and Gram-negative bacteria have evolved similar strategies but different tactics for importing and degrading heme, likely as a consequence of their different cellular architectures. The differences are manifested in distinct structures for molecules that perform similar functions. Consequently, the aim of this chapter is to provide an overview of the structural biology of proteins and protein-protein interactions that enable Gram-positive and Gram-negative bacteria to sequester heme from the extracellular milieu, import it to the cytosol, and degrade it to mine iron.

Filamentous actinobacteria from the genus Frankia grow by hyphal tip extension and branching. The growth kinetics and branching pattern of Frankia are not well studied, especially at the early stages of mycelial development. Here, we compare the growth of Frankia sp. strain CcI3 in liquid cultures with and without proteose peptone #3 (PP3) using time-lapse photomicrography and image analysis. Individual hyphae showed a pseudolinear increase in length at early stages of development, whereas at the mycelial level, the aggregate length of hyphae described an exponential rate before slowing. Growth based on optical density or microscopic observations was similar in medium with or without PP3. However, PP3 altered the pattern of mycelial development by increasing branching. Distances between the hyphal apex and first branches were on average shorter in PP3-containing media. The final interbranch distances were also shorter in PP3 medium indicating that hyphae tended to branch earlier and more often when supplemented with PP3 to give a more compact mycelium. Vesicle development in nitrogen-fixing cultures limited cell expansion as a result of vesicles truncating growth on new branches. The results provide some explanation for the growth kinetics of Frankia and some indication of how growth rates may be improved.

Members of the noncultured clade of Frankia enter into root nodule symbioses with actinorhizal species from the orders Cucurbitales and Rosales. We report the genome sequence of a member of this clade originally from Pakistan but obtained from root nodules of the American plant Datisca glomerata without isolation in culture.

Progress in understanding symbiotic determinants involved in the N(2)-fixing actinorhizal plant symbioses has been slow but steady. Problems persist with studying the bacterial contributions to the symbiosis using traditional microbiological techniques. However, recent years have seen the emergence of several genomes from Frankia sp. strains and the development of techniques for manipulating plant gene expression. Approaches to understanding the bacterial side of the symbiosis have employed a range of techniques that reveal the proteomes and transcriptomes from both cultured and symbiotic frankiae. The picture beginning to emerge provides some perspective on the heterogeneity of frankial populations in both conditions. In general, frankial populations in root nodules seem to maintain a rather robust metabolism that includes nitrogen fixation and substantial biosynthesis and energy-generating pathways, along with a modified ammonium assimilation program. To date, particular bacterial genes have not been implicated in root nodule formation but some hypotheses are emerging with regard to how the plant and microorganism manage to coexist. In particular, frankiae seem to present a nonpathogenic presence to the plant that may have the effect of minimizing some plant defense responses. Future studies using high-throughput approaches will likely clarify the range of bacterial responses to symbiosis that will need to be understood in light of the more rapidly advancing work on the plant host.

Background: Frankia sp. strains are actinobacteria that form N2-fixing root nodules on angiosperms. Several reference genome sequences are available enabling transcriptome studies in Frankia sp. Genomes from Frankia sp. strains differ markedly in size, a consequence proposed to be associated with a high number of indigenous transposases, more than 200 of which are found in Frankia sp. strain CcI3 used in this study. Because Frankia exhibits a high degree of cell heterogeneity as a consequence of its mycelial growth pattern, its transcriptome is likely to be quite sensitive to culture age. This study focuses on the behavior of the Frankia sp. strain CcI3 transcriptome as a function of nitrogen source and culture age., Results: To study global transcription in Frankia sp. CcI3 grown under different conditions, complete transcriptomes were determined using high throughput RNA deep sequencing. Samples varied by time (five days vs. three days) and by culture conditions (NH4+ added vs. N2 fixing). Assembly of millions of reads revealed more diversity of gene expression between five-day and three-day old cultures than between three day old cultures differing in nitrogen sources. Heat map analysis organized genes into groups that were expressed or repressed under the various conditions compared to median expression values. Twenty-one SNPs common to all three transcriptome samples were detected indicating culture heterogeneity in this slow-growing organism. Significantly higher expression of transposase ORFs was found in the five-day and N2-fixing cultures, suggesting that N starvation and culture aging provide conditions for on-going genome modification. Transposases have previously been proposed to participate in the creating the large number of gene duplication or deletion in host strains. Subsequent RT-qPCR experiments confirmed predicted elevated transposase expression levels indicated by the mRNA-seq data., Conclusions: The overall pattern of gene expression in aging cultures of CcI3 suggests significant cell heterogeneity even during normal growth on ammonia. The detection of abundant transcription of nif (nitrogen fixation) genes likely reflects the presence of anaerobic, N-depleted microsites in the growing mycelium of the culture, and the presence of significantly elevated transposase transcription during starvation indicates the continuing evolution of the Frankia sp. strain CcI3 genome, even in culture, especially under stressed conditions. These studies also sound a cautionary note when comparing the transcriptomes of Frankia grown in root nodules, where cell heterogeneity would be expected to be quite high.

Mammalian type B (mitochondrial) b(5) cytochromes exhibit greater amino acid sequence diversity than their type A (microsomal) counterparts, as exemplified by the type B proteins from human (hCYB5B) and rat (rCYB5B). The comparison of X-ray crystal structures of hCYB5B and rCYB5B reported herein reveals a striking difference in packing involving the five-strand β-sheet, which can be attributed to fully buried residue 21 in strand β4. The greater bulk of Leu21 in hCYB5B in comparison to that of Thr21 in rCYB5B results in a substantial displacement of the first two residues in β5, and consequent loss of two of the three hydrogen bonds between β5 and β4. Hydrogen bonding between the residues is instead mediated by two well-ordered, fully buried water molecules. In a 10 ns molecular dynamics simulation, one of the buried water molecules in the hCYB5B structure exchanged readily with solvent via intermediates having three water molecules sandwiched between β4 and β5. When the buried water molecules were removed prior to a second 10 ns simulation, β4 and β5 formed persistent hydrogen bonds identical to those in rCYB5B, but the Leu21 side chain was forced to adopt a rarely observed conformation. Despite the apparently greater ease of access of water to the interior of hCYB5B than of rCYB5B suggested by these observations, the two proteins exhibit virtually identical stability, dynamic, and redox properties. The results provide new insight into the factors stabilizing the cytochrome b(5) fold.

NADH cytochrome b(5) oxidoreductase (Ncb5or) is found in animals and contains three domains similar to cytochrome b(5) (b(5)), CHORD-SGT1 (CS), and cytochrome b(5) reductase (b(5)R). Ncb5or has an important function, as suggested by the diabetes and lipoatrophy phenotypes in Ncb5or null mice. To elucidate the structural and functional properties of human Ncb5or, we generated its individual b(5) and b(5)R domains (Ncb5or-b(5) and Ncb5or-b(5)R, respectively) and compared them with human microsomal b(5) (Cyb5A) and b(5)R (Cyb5R3). A 1.25 Å x-ray crystal structure of Ncb5or-b(5) reveals nearly orthogonal planes of the imidazolyl rings of heme-ligating residues His(89) and His(112), consistent with a highly anisotropic low spin EPR spectrum. Ncb5or is the first member of the cytochrome b(5) family shown to have such a heme environment. Like other b(5) family members, Ncb5or-b(5) has two helix-loop-helix motifs surrounding heme. However, Ncb5or-b(5) differs from Cyb5A with respect to location of the second heme ligand (His(112)) and of polypeptide conformation in its vicinity. Electron transfer from Ncb5or-b(5)R to Ncb5or-b(5) is much less efficient than from Cyb5R3 to Cyb5A, possibly as a consequence of weaker electrostatic interactions. The CS linkage probably obviates the need for strong interactions between b(5) and b(5)R domains in Ncb5or. Studies with a construct combining the Ncb5or CS and b(5)R domains suggest that the CS domain facilitates docking of the b(5) and b(5)R domains. Trp(114) is an invariant surface residue in all known Ncb5or orthologs but appears not to contribute to electron transfer from the b(5)R domain to the b(5) domain.

Frankia species are the most geographically widespread gram-positive plant symbionts, carrying out N(2) fixation in root nodules of trees and woody shrubs called actinorhizal plants. Taking advantage of the sequencing of three Frankia genomes, proteomics techniques were used to investigate the population of extracellular proteins (the exoproteome) from Frankia, some of which potentially mediate host-microbe interactions. Initial two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of culture supernatants indicated that cytoplasmic proteins appeared in supernatants as cells aged, likely because older hyphae lyse in this slow-growing filamentous actinomycete. Using liquid chromatography coupled to tandem mass spectrometry to identify peptides, 38 proteins were identified in the culture supernatant of Frankia sp. strain CcI3, but only three had predicted export signal peptides. In symbiotic cells, 42 signal peptide-containing proteins were detected from strain CcI3 in Casuarina cunninghamiana and Casuarina glauca root nodules, while 73 and 53 putative secreted proteins containing signal peptides were identified from Frankia strains in field-collected root nodules of Alnus incana and Elaeagnus angustifolia, respectively. Solute-binding proteins were the most commonly identified secreted proteins in symbiosis, particularly those predicted to bind branched-chain amino acids and peptides. These direct proteomics results complement a previous bioinformatics study that predicted few secreted hydrolytic enzymes in the Frankia proteome and provide direct evidence that the symbiosis succeeds partly, if not largely, because of a benign relationship.

Background: Genome analysis of three Frankia sp. strains has revealed a high number of transposable elements in two of the strains. Twelve out of the 20 major families of bacterial Insertion Sequence (IS) elements are represented in the 148 annotated transposases of Frankia strain HFPCcI3 (CcI3) comprising 3% of its total coding sequences (CDS). EAN1pec (EAN) has 183 transposase ORFs from 13 IS families comprising 2.2% of its CDS. Strain ACN14a (ACN) differs significantly from the other strains with only 33 transposase ORFs (0.5% of the total CDS) from 9 IS families., Results: Insertion sequences in the Frankia genomes were analyzed using BLAST searches, PHYML phylogenies and the IRF (Inverted Repeat Finder) algorithms. To identify putative or decaying IS elements, a PSI-TBLASTN search was performed on all three genomes, identifying 36%, 39% and 12% additional putative transposase ORFs than originally annotated in strains CcI3, EAN and ACN, respectively. The distribution of transposase ORFs in each strain was then analysed using a sliding window, revealing significant clustering of elements in regions of the EAN and CcI3 genomes. Lastly the three genomes were aligned with the MAUVE multiple genome alignment tool, revealing several Large Chromosome Rearrangement (LCR) events; many of which correlate to transposase clusters., Conclusion: Analysis of transposase ORFs in Frankia sp. revealed low inter-strain diversity of transposases, suggesting that the majority of transposase proliferation occurred without recent horizontal transfer of novel mobile elements from outside the genus. Exceptions to this include representatives from the IS3 family in strain EAN and seven IS4 transposases in all three strains that have a lower G+C content, suggesting recent horizontal transfer. The clustering of transposase ORFs near LCRs revealed a tendency for IS elements to be associated with regions of chromosome instability in the three strains. The results of this study suggest that IS elements may help drive chromosome differences in different Frankia sp. strains as they have adapted to a variety of hosts and environments.

Background: An understanding of the evolution of global transcription regulators is essential for comprehending the complex networks of cellular metabolism that have developed among related organisms. The fur gene encodes one of those regulators - the ferric uptake regulator Fur - widely distributed among bacteria and known to regulate different genes committed to varied metabolic pathways. On the other hand, members of the Actinobacteria comprise an ecologically diverse group of bacteria able to inhabit various natural environments, and for which relatively little is currently understood concerning transcriptional regulation., Results: BLAST analyses revealed the presence of more than one fur homologue in most members of the Actinobacteria whose genomes have been fully sequenced. We propose a model to explain the evolutionary history of fur within this well-known bacterial phylum: the postulated scenario includes one duplication event from a primitive regulator, which probably had a broad range of co-factors and DNA-binding sites. This duplication predated the appearance of the last common ancestor of the Actinobacteria, while six other duplications occurred later within specific groups of organisms, particularly in two genera: Frankia and Streptomyces. The resulting paralogues maintained main biochemical properties, but became specialised for regulating specific functions, coordinating different metal ions and binding to unique DNA sequences. The presence of syntenic regions surrounding the different fur orthologues supports the proposed model, as do the evolutionary distances and topology of phylogenetic trees built using both Neighbor-Joining and Maximum-Likelihood methods., Conclusion: The proposed fur evolutionary model, which includes one general duplication and two in-genus duplications followed by divergence and specialization, explains the presence and diversity of fur genes within the Actinobacteria. Although a few rare horizontal gene transfer events have been reported, the model is consistent with the view of gene duplication as a main force of microbial genomes evolution. The parallel study of Fur phylogeny across diverse organisms offers a solid base to guide functional studies and allows the comparison between response mechanisms in relation with the surrounding environment. The survey of regulators among related genomes provides a relevant tool for understanding the evolution of one of the first lines of cellular adaptability, control of DNA transcription.

Frankia are nitrogen-fixing actinomycetes that form a symbiotic association with over 200 species of woody dicotyledonous plants. Recently, three Frankia genomes were completely sequenced. In this study, the synonymous codon usage patterns of three Frankia genomes (strains CcI3, ACN14a, and EAN1pec) were determined and compared to each other and to other actinobacteria. As expected for a high G+C organism, codon usage by Frankia was highly biased, but differences were observed among the three strains. Using the codon adaptation index (CAI) as a numerical estimator of gene expression level, highly expressed genes in Frankia were predicted with ribosomal protein genes as a reference. The analysis of the predicted highly expressed genes showed that Frankia strain CcI3 had a different profile from the other two strains. Strain CcI3 had fewer predicted highly expressed genes in several COG categories including lipid transport and metabolism, secondary metabolites biosynthesis, inorganic ion transport and metabolism, and general function prediction only than Frankia strains EAN1pec and ACN14a. Interestingly, Frankia EAN1pec had more predicted highly expressed genes in transcription and signal transduction mechanisms than the other two strains. These differences were not just a reflection in total gene numbers, but also based on percentage of genes within a category. These results support the hypothesis that strain CcI3 is becoming a symbiotic specialist and the other two facultative symbiotic strains are maintaining their capacity to exist as free-living soil dwellers.

Background: Frankia sp. strains, the nitrogen-fixing facultative endosymbionts of actinorhizal plants, have long been proposed to secrete hydrolytic enzymes such as cellulases, pectinases, and proteases that may contribute to plant root penetration and formation of symbiotic root nodules. These or other secreted proteins might logically be involved in the as yet unknown molecular interactions between Frankia and their host plants. We compared the genome-based secretomes of three Frankia strains representing diverse host specificities. Signal peptide detection algorithms were used to predict the individual secretomes of each strain, and the set of secreted proteins shared among the strains, termed the core Frankia secretome. Proteins in the core secretome may be involved in the actinorhizal symbiosis., Results: The Frankia genomes have conserved Sec (general secretory) and Tat (twin arginine translocase) secretion systems. The potential secretome of each Frankia strain comprised 4-5% of the total proteome, a lower percentage than that found in the genomes of other actinobacteria, legume endosymbionts, and plant pathogens. Hydrolytic enzymes made up only a small fraction of the total number of predicted secreted proteins in each strain. Surprisingly, polysaccharide-degrading enzymes were few in number, especially in strain CcI3, with more esterolytic, lipolytic and proteolytic enzymes having signal peptides. A total of 161 orthologous proteins belong to the core Frankia secretome. Of these, 52 also lack homologs in closely related actinobacteria, and are termed "Frankia-specific." The genes encoding these conserved secreted proteins are often clustered near secretion machinery genes., Conclusion: The predicted secretomes of Frankia sp. are relatively small and include few hydrolases, which could reflect adaptation to a symbiotic lifestyle. There are no well-conserved secreted polysaccharide-degrading enzymes present in all three Frankia genomes, suggesting that plant cell wall polysaccharide degradation may not be crucial to root infection, or that this degradation varies among strains. We hypothesize that the relative lack of secreted polysaccharide-degrading enzymes in Frankia reflects a strategy used by these bacteria to avoid eliciting host defense responses. The esterases, lipases, and proteases found in the core Frankia secretome might facilitate hyphal penetration through the cell wall, release carbon sources, or modify chemical signals. The core secretome also includes extracellular solute-binding proteins and Frankia-specific hypothetical proteins that may enable the actinorhizal symbiosis.

Outer mitochondrial membrane cytochrome b5 (OM b5) is the most thermostable cytochrome b5 isoform presently known. Herein, we show that OM b5 thermal stability is substantially enhanced by swapping an apparently invariant motif in its heme-independent folding core with the corresponding motif characteristic of its less stable evolutionary relative, microsomal cytochrome b5 (Mc b5). The motif swap involved replacing two residues, Arg15 with His and Glu20 with Ser, thereby introducing a Glu11-His15-Ser20 H-bonding triad on the protein surface along with a His15/Trp22 pi-stacking interaction. The ferric and ferrous forms of the OM b5 R15H/E20S double mutant have thermal denaturation midpoints (Tm values) of approximately 93 degrees C and approximately 104 degrees C, respectively. A 15 degrees C increase in apoprotein Tm plays a key role in the holoprotein thermal stability enhancement, and is achieved by one of the most common natural mechanisms for stabilization of thermophilic versus mesophilic proteins: raising the unfolding free energy along the entire stability curve.

We report a 1.55 A X-ray crystal structure of the heme-binding domain of cytochrome b(5) from Musca domestica (house fly; HF b(5)), and compare it with previously published structures of the heme-binding domains of bovine microsomal cytochrome b(5) (bMc b(5)) and rat outer mitochondrial membrane cytochrome b(5) (rOM b(5)). The structural comparison was done in the context of amino acid sequences of all known homologues of the proteins under study. We show that insect b(5)s contain an extended hydrophobic patch at the base of the heme binding pocket, similar to the one previously shown to stabilize mammalian OM b(5)s relative to their Mc counterparts. The hydrophobic patch in insects includes a residue with a bulky hydrophobic side chain at position 71 (Met). Replacing Met71 in HF b(5) with Ser, the corresponding residue in all known mammalian Mc b(5)s, is found to substantially destabilize the holoprotein. The destabilization is a consequence of two related factors: (1) a large decrease in apoprotein stability and (2) extension of conformational disruption in the apoprotein beyond the empty heme binding pocket (core 1) and into the heme-independent folding core (core 2). Analogous changes have previously been shown to accompany replacement of Leu71 in rOM b(5) with Ser. That the stabilizing role of Met71 in HF b(5) is manifested primarily in the apo state is highlighted by the fact that its crystallographic Calpha B factor is modestly larger than that of Ser71 in bMc b(5), indicating that it slightly destabilizes local polypeptide conformation when heme is in its binding pocket. Finally, we show that the final unit of secondary structure in the cytochrome b(5) heme-binding domain, a 3(10) helix known as alpha6, differs substantially in length and packing interactions not only for different protein isoforms but also for given isoforms from different species., ((c) 2007 Wiley-Liss, Inc.)

We have recently reported that aquo and thioether complexes of the ferric cytochrome c heme peptide N-acetylmicroperoxidase-8 (FeIII-1) exhibit greater low-spin character than do the corresponding complexes of a synthetic, water-soluble, monohistidine-ligated heme peptide (FeIII-2; Cowley, A. B.; Lukat-Rodgers, G. S.; Rodgers, K. R.; Benson, D. R. Biochemistry 2004, 43, 1656-1666). Herein we report results of studies showing that weak-field ligands bearing a full (fluoride, chloride, hydroxide) or partial (phenoxide, thiocyanate) negative charge on the coordinating atom trigger dissociation of the axial His ligand in FeIII-2 but not in FeIII-1. We attribute the greater sensitivity of His ligation in FeIII-1 to weak-field anionic ligands than to weak-field neutral ligands to the following phenomena: (1) anionic ligands pull FeIII further from the mean plane of a porphyrin than do neutral ligands, which will have the effect of straining the His-Fe bond in FeIII-2, and (2) heme in FeIII-2 is likely to undergo a modest doming distortion following anion binding that will render the His-ligated side of the porphyrin concave, thereby increasing porphyrin/ligand steric interactions. We propose that ruffling of the heme in FeIII-1 is an important factor contributing to its ability to resist His dissociation by weak-field anions. First, ruffling should allow His to more closely approach the porphyrin than is possible in FeIII-2, thereby reducing bond strain following anion binding. Second, the ruffling deformation in FeIII-1, which is enforced by the double covalent heme-peptide linkage, will almost certainly prevent significant porphyrin doming.

Soil bacteria that also form mutualistic symbioses in plants encounter two major levels of selection. One occurs during adaptation to and survival in soil, and the other occurs in concert with host plant speciation and adaptation. Actinobacteria from the genus Frankia are facultative symbionts that form N(2)-fixing root nodules on diverse and globally distributed angiosperms in the "actinorhizal" symbioses. Three closely related clades of Frankia sp. strains are recognized; members of each clade infect a subset of plants from among eight angiosperm families. We sequenced the genomes from three strains; their sizes varied from 5.43 Mbp for a narrow host range strain (Frankia sp. strain HFPCcI3) to 7.50 Mbp for a medium host range strain (Frankia alni strain ACN14a) to 9.04 Mbp for a broad host range strain (Frankia sp. strain EAN1pec.) This size divergence is the largest yet reported for such closely related soil bacteria (97.8%-98.9% identity of 16S rRNA genes). The extent of gene deletion, duplication, and acquisition is in concert with the biogeographic history of the symbioses and host plant speciation. Host plant isolation favored genome contraction, whereas host plant diversification favored genome expansion. The results support the idea that major genome expansions as well as reductions can occur in facultative symbiotic soil bacteria as they respond to new environments in the context of their symbioses.