Unraveling the interaction of co-encapsulated Saccharomyces cerevisiae and Metarhizium brunneum in calcium alginate-based attract-and-kill beads.

BACKGROUND: Attract-and-kill (AK) beads are biological, microbial insecticides developed as an alternative to synthetic soil insecticides. For wireworm control, beads are based on calcium alginate/starch co-encapsulating the carbon dioxide (CO2) producing yeast Saccharomyces cerevisiae H205 as the attract component, and the entomopathogenic fungus Metarhizium brunneum CB15-III as the kill component. However, the physicochemical processes inside beads during co-cultivation are still unclear. Here we reveal for the first time the spatiotemporal conditions of oxygen and pH inside AK beads measured with microelectrodes and describe the impact of S. cerevisiae on CO2 and conidia formation. RESULTS: Measurements revealed a steep oxygen gradient already 2 days after co-encapsulation, with an internal hypoxic zone. Encapsulating either S. cerevisiae or M. brunneum already decreased the average pH from 5.5 to 4.7 and 4.6, respectively. However, on day 3, co-cultivation lead to temporal strong acidification of beads down to pH 3.6 which followed the maximum CO2 productivity and coincided with the maximum conidiation rate. Decreasing the yeast load decreased the total CO2 productivity to half, and the conidial production by 93%, while specific productivities normalized to 1% yeast load increased eight-fold and three-fold, respectively, with day 3 being an exception. CONCLUSION: Our findings indicate a general beneficial interaction between M. brunneum and S. cerevisiae, but also suggest competition for resources. These findings will contribute to develop innovative co-formulations with maximum efficiency to save application rates and costs. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Polyvinyl alcohol coating releasing fungal blastospores improves kill effect of attract-and-kill beads.

Polyvinyl alcohol (PVA) is a biodegradable, water-soluble polymer with excellent film forming properties, commonly studied or used as tablet coating, food packaging or controlled release fertilizers. Attract-and-kill (AK) beads are sustainable, microbial alternatives to synthetic soil insecticides, whose onset of lethal effect largely depend on how fast the encapsulated entomopathogenic fungus forms virulent conidia. Therefore, the objective of this study was to develop a water-soluble coating accelerating the kill effect of AK beads by immediately releasing virulent Metarhizium brunneum CB15-III blastospores. We assessed three PVA types (PVA 4-88, 8-88, 10-98) which differed in their degree of hydrolysis or molecular weight for their ability to release viable blastospores from thin films after drying at 60-40 °C, and examined how polyethylene glycol and soy-lecithin impact the blastospore survival. Finally, we evaluated the effectiveness of coated AK beads in a bioassay against Tenebrio molitor larvae. The blastospore release rate quadrupled within the first 5 min with decreasing molecular weight and degree of hydrolysis, with PVA 4-88 releasing 79 ± 19% blastospores. Polyethylene glycol and soy-lecithin significantly increased the blastospore survival to 18-28% for all three PVA types. Coated beads exhibited a uniform, 22.4 ± 7.3 µm thin coating layer, with embedded blastospores, as confirmed by scanning electron microscopy. The blastospore coating increased the mortality rate of T. molitor larvae over uncoated AK beads, decreasing the median lethal time from 10 to 6 days. Consequently, the blastospore coating accelerated the kill effect of regular AK beads. These findings pave the way to enhanced pest control efficacy from coated systems such as beads or seeds.

Long-Term Antibody Response to SARS-CoV-2 in Children.

Almost 2 years into the pandemic and with vaccination of children significantly lagging behind adults, long-term pediatric humoral immune responses to SARS-CoV-2 are understudied. The C19.CHILD Hamburg (COVID-19 Child Health Investigation of Latent Disease) Study is a prospective cohort study designed to identify and follow up children and their household contacts infected in the early 2020 first wave of SARS-CoV-2. We screened 6113 children < 18 years by nasopharyngeal swab-PCR in a low-incidence setting after general lockdown, from May 11 to June 30, 2020. A total of 4657 participants underwent antibody testing. Positive tests were followed up by repeated PCR and serological testing of all household contacts over 6 months. In total, the study identified 67 seropositive children (1.44%); the median time after infection at first presentation was 83 days post-symptom onset (PSO). Follow-up of household contacts showed less than 100% seroprevalence in most families, with higher seroprevalence in families with adult index cases compared to pediatric index cases (OR 1.79, P = 0.047). Most importantly, children showed sustained seroconversion up to 9 months PSO, and serum antibody concentrations persistently surpassed adult levels (ratio serum IgG spike children vs. adults 90 days PSO 1.75, P < 0.001; 180 days 1.38, P = 0.01; 270 days 1.54, P = 0.001). In a low-incidence setting, SARS-CoV-2 infection and humoral immune response present distinct patterns in children including higher antibody levels, and lower seroprevalence in families with pediatric index cases. Children show long-term SARS-CoV-2 antibody responses. These findings are relevant to novel variants with increased disease burden in children, as well as for the planning of age-appropriate vaccination strategies.

Specific CD4+ T Cell Responses to Ancestral SARS-CoV-2 in Children Increase With Age and Show Cross-Reactivity to Beta Variant.

SARS-CoV-2 is still a major burden for global health despite effective vaccines. With the reduction of social distancing measures, infection rates are increasing in children, while data on the pediatric immune response to SARS-CoV-2 infection is still lacking. Although the typical disease course in children has been mild, emerging variants may present new challenges in this age group. Peripheral blood mononuclear cells (PBMC) from 51 convalescent children, 24 seronegative siblings from early 2020, and 51 unexposed controls were stimulated with SARS-CoV-2-derived peptide MegaPools from the ancestral and beta variants. Flow cytometric determination of activation-induced markers and secreted cytokines were used to quantify the CD4+ T cell response. The average time after infection was over 80 days. CD4+ T cell responses were detected in 61% of convalescent children and were markedly reduced in preschool children. Cross-reactive T cells for the SARS-CoV-2 beta variant were identified in 45% of cases after infection with an ancestral SARS-CoV-2 variant. The CD4+ T cell response was accompanied most predominantly by IFN-γ and Granzyme B secretion. An antiviral CD4+ T cell response was present in children after ancestral SARS-CoV-2 infection, which was reduced in the youngest age group. We detected significant cross-reactivity of CD4+ T cell responses to the more recently evolved immune-escaping beta variant. Our findings have epidemiologic relevance for children regarding novel viral variants of concern and vaccination efforts.

De novo FZR1 loss-of-function variants cause developmental and epileptic encephalopathies.

FZR1, which encodes the Cdh1 subunit of the anaphase-promoting complex, plays an important role in neurodevelopment by regulating the cell cycle and by its multiple post-mitotic functions in neurons. In this study, evaluation of 250 unrelated patients with developmental and epileptic encephalopathies and a connection on GeneMatcher led to the identification of three de novo missense variants in FZR1. Whole-exome sequencing in 39 patient-parent trios and subsequent targeted sequencing in an additional cohort of 211 patients was performed to identify novel genes involved in developmental and epileptic encephalopathy. Functional studies in Drosophila were performed using three different mutant alleles of the Drosophila homologue of FZR1 fzr. All three individuals carrying de novo variants in FZR1 had childhood-onset generalized epilepsy, intellectual disability, mild ataxia and normal head circumference. Two individuals were diagnosed with the developmental and epileptic encephalopathy subtype myoclonic atonic epilepsy. We provide genetic-association testing using two independent statistical tests to support FZR1 association with developmental and epileptic encephalopathies. Further, we provide functional evidence that the missense variants are loss-of-function alleles using Drosophila neurodevelopment assays. Using three fly mutant alleles of the Drosophila homologue fzr and overexpression studies, we show that patient variants can affect proper neurodevelopment. With the recent report of a patient with neonatal-onset with microcephaly who also carries a de novo FZR1 missense variant, our study consolidates the relationship between FZR1 and developmental and epileptic encephalopathy and expands the associated phenotype. We conclude that heterozygous loss-of-function of FZR1 leads to developmental and epileptic encephalopathies associated with a spectrum of neonatal to childhood-onset seizure types, developmental delay and mild ataxia. Microcephaly can be present but is not an essential feature of FZR1-encephalopathy. In summary, our approach of targeted sequencing using novel gene candidates and functional testing in Drosophila will help solve undiagnosed myoclonic atonic epilepsy or developmental and epileptic encephalopathy cases.

Formalin-casein enhances water absorbency of calcium alginate beads and activity of encapsulated Metarhizium brunneum and Saccharomyces cerevisiae.

The control of root-feeding wireworms has become more challenging as synthetic soil insecticides have been progressively phased out due to environmental risk concerns. Innovative microbial control alternatives such as the so-called attract-and-kill strategy depend on the rapid and successful development of dried encapsulated microorganisms, which is initiated by rehydration. Casein is a functional additive that is already used in food or pharmaceutical industry due to its water binding capacity. Cross-linked forms such as formalin-casein (FC), exhibit altered network structures. To determine whether FC influences the rehydration of alginate beads in order to increase the efficacy of an attract-and-kill formulation for wireworm pest control, we incorporated either casein or FC in different alginate/starch formulations. We investigated the porous properties of alginate/starch beads and subsequently evaluated the activities of the encapsulated entomopathogenic fungus Metarhizium brunneum and the CO2 producing yeast Saccharomyces cerevisiae. Adding caseins altered the porous structure of beads. FC decreased the bead density from (1.0197 ± 0.0008) g/mL to (1.0144 ± 0.0008) g/mL and the pore diameter by 31%. In contrast to casein, FC enhanced the water absorbency of alginate/starch beads by 40%. Furthermore, incorporating FC quadrupled the spore density on beads containing M. brunneum and S. cerevisiae, and simultaneous venting increased the spore density even by a factor of 18. Moreover, FC increased the total CO2 produced by M. brunneum and S. cerevisiae by 29%. Thus, our findings suggest that rehydration is enhanced by larger capillaries, resulting in an increased water absorption capacity. Our data further suggest that gas exchange is improved by FC. Therefore, our results indicate that FC enhances the fungal activity of both fungi M. brunneum and S. cerevisiae, presumably leading to an enhanced attract-and-kill efficacy for pest control.

Excessive Seizure Clusters in an Otherwise Well-Controlled Epilepsy as a Possible Hallmark of Untreated Vitamin B6-Responsive Epilepsy due to a Homozygous PLPBP Missense Variant.

Recently, mutations in the PLPBP gene were described as a novel cause for vitamin B6-responsive epilepsy. We report the outcome in case of a male adolescent with a novel homozygous missense variant in PLPBP who was never treated with pyridoxine until the age of 16 years. He presented with only mild cognitive impairment and an early-onset, well-controlled epilepsy. In our patient, excessive seizure clusters and anxiety states occurred intermittently, suggesting that the combination might be a hallmark in untreated patients. Thus, mutations in PLPBP should be addressed even in adolescent patients with only mild learning disabilities and relatively good seizure control over the years.

Kinetics of lipid bilayer permeation of a series of ionisable drugs and their correlation with human transporter-independent intestinal permeability.

For low molecular weight drugs, lipid bilayer permeation is considered the major route for in vivo cell barrier passage. We recently introduced a fluorescence assay with liposomes to determine permeation kinetics of ionisable compounds across the lipid bilayer by monitoring drug-induced pH changes inside the liposomes. Here, we determined the permeability coefficients (PFLipP, FLipP for "Fluorescence Liposomal Permeability") across 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers of 35 ionisable drugs at pH6.0 and compared them to available in vivo human jejunal permeability (Peff) data. PFLipP values were furthermore compared with published Caco-2 cell permeability coefficients (PCaco-2), permeability coefficients determined with the parallel artificial membrane permeability assay (PAMPA) and with log D (pH6.0). The log PFLipP, corrected for predicted para-cellular diffusion, and log PCaco-2 correlated best with log Peff, with similar adjusted R2 (0.75 and 0.74, n=12). Our results suggest that transporter-independent intestinal drug absorption is predictable from liposomal permeability.

When barriers ignore the "rule-of-five".

Why are a few drugs with properties beyond the rule of 5 (bRo5) absorbed across the intestinal mucosa while most other bRo5 compounds are not? Are such exceptional bRo5 compounds exclusively taken up by carrier-mediated transport or are they able to permeate the lipid bilayer (passive lipoidal diffusion)? Our experimental data with liposomes indicate that tetracycline, which violates one rule of the Ro5, and rifampicin, violating three of the rules, significantly permeate a phospholipid bilayer with kinetics similar to labetalol and metoprolol, respectively. Published data from experimental work and molecular dynamics simulations suggest that the formation of intramolecular H-bonds and the possibility to adopt an elongated shape besides the presence of a significant fraction of net neutral species facilitate lipid bilayer permeation. As an alternative to lipid bilayer permeation, carrier proteins can be targeted to improve absorption, with the potential drawbacks of drug-drug interactions and non-linear pharmacokinetics.

The growing caseload of chronic life-long conditions calls for a move towards full self-management in low-income countries.

BACKGROUND: The growing caseload caused by patients with chronic life-long conditions leads to increased needs for health care providers and rising costs of health services, resulting in a heavy burden on health systems, populations and individuals. The professionalised health care for chronic patients common in high income countries is very labour-intensive and expensive. Moreover, the outcomes are often poor. In low-income countries, the scarce resources and the lack of quality and continuity of health care result in high health care expenditure and very poor health outcomes. The current proposals to improve care for chronic patients in low-income countries are still very much provider-centred.The aim of this paper is to show that present provider-centred models of chronic care are not adequate and to propose 'full self-management' as an alternative for low-income countries, facilitated by expert patient networks and smart phone technology. DISCUSSION: People with chronic life-long conditions need to 'rebalance' their life in order to combine the needs related to their chronic condition with other elements of their life. They have a crucial role in the management of their condition and the opportunity to gain knowledge and expertise in their condition and its management. Therefore, people with chronic life-long conditions should be empowered so that they become the centre of management of their condition. In full self-management, patients become the hub of management of their own care and take full responsibility for their condition, supported by peers, professionals and information and communication tools.We will elaborate on two current trends that can enhance the capacity for self-management and coping: the emergence of peer support and expert-patient networks and the development and distribution of smart phone technology both drastically expand the possibilities for full self-management. CONCLUSION: Present provider-centred models of care for people with chronic life-long conditions are not adequate and we propose 'full self-management' as an alternative for low-income countries, supported by expert networks and smart phone technology.

Keeping health staff healthy: evaluation of a workplace initiative to reduce morbidity and mortality from HIV/AIDS in Malawi.

BACKGROUND: In Malawi, the dramatic shortage of human resources for health is negatively impacted by HIV-related morbidity and mortality among health workers and their relatives. Many staff find it difficult to access HIV care through regular channels due to fear of stigma and discrimination. In 2006, two workplace initiatives were implemented in Thyolo District: a clinic at the district hospital dedicated to all district health staff and their first-degree relatives, providing medical services, including HIV care; and a support group for HIV-positive staff. METHODS: Using routine programme data, we evaluated the following outcomes up to the end of 2009: uptake and outcome of HIV testing and counselling among health staff and their dependents; uptake and outcomes of antiretroviral therapy (ART) among health staff; and membership and activities of the support group. In addition, we included information from staff interviews and a job satisfaction survey to describe health workers' opinions of the initiatives. RESULTS: Almost two-thirds (91 of 144, 63%) of health workers and their dependents undergoing HIV testing and counselling at the staff clinic tested HIV positive. Sixty-four health workers had accessed ART through the staff clinic, approximately the number of health workers estimated to be in need of ART. Of these, 60 had joined the support group. Cumulative ART outcomes were satisfactory, with more than 90% alive on treatment as of June 2009 (the end of the study observation period). The availability, confidentiality and quality of care in the staff clinic were considered adequate by beneficiaries. CONCLUSIONS: Staff clinic and support group services successfully provided care and support to HIV-positive health workers. Similar initiatives should be considered in other settings with a high HIV prevalence.

Providing universal access to antiretroviral therapy in Thyolo, Malawi through task shifting and decentralization of HIV/AIDS care.

OBJECTIVE: To describe how district-wide access to HIV/AIDS care was achieved and maintained in Thyolo District, Malawi. METHOD: In mid-2003, the Ministry of Health and Médecins Sans Frontières developed a model of care for Thyolo district (population 587, 455) based on decentralization of care to health centres and community sites and task shifting. RESULTS: After delegating HIV testing and counseling to lay counsellors, uptake of testing increased from 1300 tests per month in 2003 to 6500 in 2009. Shifting responsibility for antiretroviral therapy (ART) initiations to non-physician clinicians almost doubled ART enrollment, with a majority of initiations performed in peripheral health centres. By the end 2009, 23, 261 people had initiated ART of whom 11, 042 received ART care at health-centre level. By the end of 2007, the universal access targets were achieved, with nearly 9000 patients alive and on ART. The average annual cost for achieving these targets was € 2.6 per inhabitant/year. CONCLUSION: The Thyolo programme has demonstrated the feasibility of district-wide access to ART in a setting with limited resources for health. Expansion and decentralization of HIV/AIDS service-capacity to the primary care level, combined with task shifting, resulted in increased access to HIV services with good programme outcomes despite staff shortages.

Human resource aspects of antiretroviral treatment delivery models: current practices and recommendations.

UNLABELLED: PURPOSE OF VIEW: To illustrate and critically assess what is currently being published on the human resources for health dimension of antiretroviral therapy (ART) delivery models. RECENT FINDINGS: The use of human resources for health can have an effect on two crucial aspects of successful ART programmes, namely the scale-up capacity and the long-term retention in care. Task shifting as the delegation of tasks from higher qualified to lower qualified cadres has become a widespread practice in ART delivery models in low-income countries in recent years. It is increasingly shown to effectively reduce the workload for scarce medical doctors without compromising the quality of care. At the same time, it becomes clear that task shifting can only be successful when accompanied by intensive training, supervision and support from existing health system structures. SUMMARY: Although a number of recent publications have focussed on task shifting in ART delivery models, there is a lack of accessible information on the link between task shifting and patient outcomes. Current ART delivery models do not focus sufficiently on retention in care as arguably one of the most important issues for the long-term success of ART programmes. There is a need for context-specific re-designing of current ART delivery models in order to increase access to ART and improve long-term retention.

Community health workers for ART in sub-Saharan Africa: learning from experience--capitalizing on new opportunities.

Low-income countries with high HIV/AIDS burdens in sub-Saharan Africa must deal with severe shortages of qualified human resources for health. This situation has triggered the renewed interest in community health workers, as they may play an important role in scaling-up antiretroviral treatment for HIV/AIDS by taking over a number of tasks from the professional health workers. Currently, a wide variety of community health workers are active in many antiretroviral treatment delivery sites. This article investigates whether present community health worker programmes for antiretroviral treatment are taking into account the lessons learnt from past experiences with community health worker programmes in primary health care and to what extent they are seizing the new antiretroviral treatment-specific opportunities. Based on a desk review of multi-purpose community health worker programmes for primary health care and of recent experiences with antiretroviral treatment-related community health workers, we developed an analytic framework of 10 criteria: eight conditions for successful large-scale antiretroviral treatment-related community health worker programmes and two antiretroviral treatment-specific opportunities. Our appraisal of six community health worker programmes, which we identified during field work in Ethiopia, Malawi and Uganda in 2007, shows that while some lessons from the past have been learnt, others are not being sufficiently considered and antiretroviral treatment-specific opportunities are not being sufficiently seized.In particular, all programmes have learnt the lesson that without adequate remuneration, community health workers cannot be retained in the long term. Yet we contend that the apparently insufficient attention to issues such as quality supervision and continuous training will lead to decreasing quality of the programmes over time. The life experience of people living with HIV/AIDS is still a relatively neglected asset, even though it may give antiretroviral treatment-related community health worker programmes better chances of success than their predecessors and may be crucially important for adherence and retention in large-scale antiretroviral treatment programmes. Community health workers as a community-based extension of health services are essential for antiretroviral treatment scale-up and comprehensive primary health care. The renewed attention to community health workers is thus very welcome, but the scale-up of community health worker programmes runs a high risk of neglecting the necessary quality criteria if it is not aligned with broader health systems strengthening. To achieve universal access to antiretroviral treatment, this is of paramount importance and should receive urgent attention.

Effect of gastric banding on aminoterminal pro-brain natriuretic peptide in the morbidly obese.

OBJECTIVE: Aminoterminal pro-brain natriuretic peptide (NT-proBNP), like brain natriuretic peptide, might have diagnostic utility in detecting left ventricular hypertrophy and/or left ventricular dysfunction. The aim of the study was to investigate the relationship between morbid obesity and NT-proBNP and the effect of weight reduction on this parameter. RESEARCH METHODS AND PROCEDURES: A total of 34 morbidly obese patients underwent laparoscopic adjustable gastric banding (LAGB). NT-proBNP levels were measured before and 12 months after the surgery. RESULTS: Metabolic features and systolic and diastolic blood pressure were significantly decreased (p < 0.00001 for both) after a cumulative weight loss of 19.55 kg 1 year after LAGB. NT-proBNP concentration was significantly higher in morbidly obese patients before LAGB than in normal-weight control subjects (341.15 +/- 127.78 fmol/mL vs. 161.68 +/- 75.78 fmol/mL; p < 0.00001). After bariatric surgery, NT-proBNP concentration decreased significantly from 341.15 +/- 127.78 fmol/mL to 204.87 +/- 59.84 fmol/mL (p < 0.00001) and remained statistically significantly elevated (204.88 +/- 59.84 fmol/mL vs. 161.68 +/- 75.78 fmol/mL; p = 0.04) compared with normal-weight subjects. DISCUSSION: This investigation demonstrates higher levels of NT-proBNP in morbidly obese subjects and a significant decrease during weight loss after laparoscopic adjustable gastric banding. In obesity, NT-proBNP might be useful as a routine screening method for identifying left ventricular hypertrophy and/or left ventricular dysfunction.