Lentzea aerocolonigenes to find these optimal cultivation conditions. However, there is currently no model concept for the dependence of the strength and frequency of the bead-induced stress on the process parameters. Therefore, shake flask simulations were performed for combinations of bead size, bead concentration, bead density and shaking frequency. Contact analysis showed that the highest shear stresses were caused by bead-bottom contacts. Based on this, a newly generated characteristic parameter, the stress area ratio (SAR), was defined, which relates the bead wall shear and normal stresses to the total shear area. Comparison of the SAR with previous cultivation results revealed an optimum pattern for product concentration and mean product-to-biomass related yield coefficient. Thus, this model is a suitable tool for future optimization, comparison and scaling up of shear-sensitive microorganism cultivation. Finally, the simulation results were validated using high-speed recordings of the bead motion on the bottom of the shake flask.In biotechnological processes, filamentous microorganisms are known for their broad product spectrum and complex cellular morphology. Product formation and cellular morphology are often closely linked, requiring a well-defined level of mechanical stress to achieve high product concentrations. Macroparticles were added to shake flask cultures of the filamentous actinomycete  In many process engineering unit operations, mechanical stresses occur in addition to thermal or chemical stresses. Understanding and controlling the positive or negative effects of these stresses is essential to efficiently achieve the desired process objectives. In biotechnology, filamentous microorganisms are particularly sensitive to mechanical stress due to their complex cellular morphology . The seling time , is a meurements . In the ivations .Aspergillus glaucus. In another study, an increased number of beads also showed a negative effect on the product formation of the antibiotic natamycin by a filamentous bacterium Streptomyces gilvosporeus LK-196  according to the mill model of Streptomyces tardus sp. nov  affect the magnitude and frequency of the bead-induced shear stress. Therefore, it is challenging to compare the observed effects of beads without any characteristic quantity like the EDCF for STBs. However, there is no deep systematic understanding of the dependencies of stress magnitude and frequency on these parameters in this application area.One way to increase the mechanical stress in a ShF culture is to add beads. A large number of studies in the literature describe positive or negative effects of additional beads in the lower millimeter range . For exas LK-196 . Further sp. nov , which pL. aerocolonigenes, Lentzea aerocolonigenes and related simulations were limited to the variation of bead size at constant shaker speed and mass concentration. As a result, other process parameters such as bead number, bead density, bead size, and shaking frequency have been experimentally varied by The modeling  and simuDepending on the selected parameters, the concentration of the secondary metabolite rebeccamycin, which has antibacterial and antitumoral properties , could bL. aerocolonigenes to gain general insights into the relationship between the production of the antibiotic rebeccamycin and mechanical stress. To validate the simulation results, the movement of the beads at the bottom of the ShF was recorded using a high-speed camera. The bead velocities were determined from the high-speed camera images using particle tracking velocimetry (PTV) and compared with the simulations.In the present study, CFD-DEM simulations were performed in order to determine the dependence of bead-induced mechanical stress for previously experimentally investigated parameter combinations. The numerically results were then combined with cultivation data of filamentous The performed multiphase simulations of a 250 mL baffled ShF filled with glass or ceramic beads  and 50 mL cultivation medium are based on the CFD-DEM simulation setup established by ® (® (® (version 4) for the simulation of multiphase flows. For LIGGGHTS® and CFDEMcoupling, the academic version published by the department of particulate flow modeling  was used. The transient turbulent flow inside the shake flask was simulated with the volume-of-fluid (VOF) solver “cfdemSolverMultiphase”  according to the analytical model for stirred media mills  is definIn this study, the maximum achievable SEs for different contact types were calculated analogously to the energy equations described by The translational bead velocities, used in the validation (see chapter 3.5), were read out at 0.01 s intervals for a period of five shaker rotations and saved in a list. This list was then used to calculate a cumulative distribution of bead velocity based on the evaluated DEM data for five shaker rotations.The mean volumetric power consumption equation :Pv=ϵ∙ρmData were analyzed every 0.01 s to calculate the time averaged power consumption for the last five shaker rotations.Particle tracking velocimetry (PTV) was applied for the validation of the three-phase simulations of the ShF. In contrast to particle imaging velocimetry (PIV), a Lagrangian frame of reference is used to determine the velocity of a bead from its displacement during the elapsed time between two recorded images . For thi−1 only) were installed. A high-speed camera  mounted vertically below the center of the ShF recorded images (896 × 920 pixels) at a frame rate of 200 s−1. Optimal illumination of the ShF was crucial to minimize disturbing image artifacts caused by light refraction and reflection on the water surface. On the one hand, a square 30 cm LED panel was placed statically above the orbital shaker. On the other hand, a circular LED strip was installed at the level of the bottom of the ShF. A cardboard cylinder, covered on the inside with aluminum foil, was used for homogeneous illumination. Glass  and ceramic (0.918 mm) beads  were used in the experiments at shaking frequencies of 100, 120, and 160 min−1. In contrast to the simulations, the minimum bead size was limited to approximately 1 mm, as smaller beads could not be reliably detected in the image analysis. The transparent glass beads were difficult to identify in the images during pre-processing. Therefore, a mixture of 26% black coated marker beads and 74% transparent beads of the same size was chosen. In the case of white ceramic beads, it was not possible to use beads of a different color, so the amount of beads was reduced from 40 mL L−1 (glass) to 2.63 mL L−1 to avoid overlapping beads in the images.To track the complex motion of the liquid and the beads from below the ShF, an experimental setup was constructed . An alum® to separate the beads from the background structures of the liquid-gas phase interface. In the case of glass beads, the imported images were converted to grayscale and smoothed with a flat-field correction. In the second step, a binary image was generated using an adaptive threshold. Then, the centers of circular objects within a defined radius range were detected in order to draw a new image with only the detected beads as white circles on a black background. In the case of white ceramic beads, an adapted image pre-processing was necessary, since no black marker beads could be used. In addition to image optimization and filtering steps, a marker-controlled watershed separation was applied to separate the partially overlapping beads. In addition, the centers of the beads were mapped to generate a new image with non-overlapping circles for PTV analysis. The actual calculation of bead velocities out of the pre-processed images was performed using the open-source MATLAB® tool PTVlab (version 1.0.0.0)  . The numL. aerocolonigenes , a slight shift of the sum distribution towards lower energies can be observed (data not shown). In addition, the saddle point in the sum distribution is missing in the current simulation. Another difference is that the order of the contact type fractions has shifted. Previously, translational normal and shear contacts dominated in the lower energy range, while the maximum SEs were caused by translational shear to almost 90%. In the latest results, the rotational shear and normal contacts dominate in the lower energy range with about 30% and 25%, respectively. All other contact types have only a small share of 10% of the total SE. The highest SE is still caused by translational shear with a fraction of about 60%. The reasons for the observed differences are, on the one hand, the more precise shape description of the SF geometry by the CFD mesh and, on the other hand, the application of a new lubrication force model from Compared to the first shake flask simulation by In cultivation, shear forces can break bio-agglomerates such as pellets or even suppress their formation. In addition, individual exposed hyphae can be sheared off the pellet surface (erosion). For this reason, the velocity to burst . Further−1 and then the slope decreases. Overall, larger beads reach a higher velocities than smaller ones. However, this is not true for the two largest bead diameters. In the high shaking frequency range, the shear velocities were slightly below the values for a bead size of 1.513 mm.Shaking frequency is the driving force for the beads movement. The bead-wall shear velocity for all bead diameters increases steadily with increasing shaking frequency . It is nForces affecting the bead motion pattern must be considered to explain the observed trends in ShF. For simplicity, the beads are assumed to move with velocity rot Eq. . This raward Eq. . The ShFAs the shaking frequency increases, the centrifugal effect gradually decreases because most of the beads are centrifuged to the outer zone of the ShF. As a result, it follows that the bead velocity increases linearly with the shaking frequency.−1 to determine the dependence of bead-wall shear velocity on the bead diameter. For the shaking frequency of 100 min−1, only a small linear increase in velocity is observed. Whereas at higher shaking frequencies, the shear velocity increases progressively more strongly up to a bead size of 1.183 mm. For 120 min−1 and 140 min−1, the shear velocity starts to increase only slightly from a bead diameter of 1.183 mm, while a decrease occurs at even higher shaking frequencies and larger bead diameters. The increase in velocity can be related to the influence of centrifugal forces, which depend on bead mass and shaking frequency . In addition, the side view shows that the liquid is increasingly forced against the side walls. While at 100 min−1, the experimental distributions for different bead diameters are similar, indicating that bead size has little influence on bead velocity. At higher shaking frequencies, the distributions differ depending on bead size, especially in the lower velocity range. At the same time, the maximum velocities achieved are almost identical. A higher bead mass with larger bead diameters results in stronger centrifugal forces, so that more beads are transported to the edge of the ShF. Furthermore, a constant mass of beads was used, which means that the number of beads decreases with bead diameter to the power of three as the bead size increases. In addition, the area required per bead increases as the projection area increases with bead diameter to the power of two. Assuming a single layer of beads, the required area of all beads is the product of the number of beads and the projection area. Consequently, the area requirement increases inversely with decreasing bead diameter for a constant total bead mass concentration. This, in turn, results in more beads moving at lower velocity in the central region of the ShF for smaller bead sizes, and finally in a broader velocity distribution. In reality, smaller beads form vertical layers, which partially reduces the effect discussed before.In addition the qualitative comparison in −1. However, the experimental distributions for 100 min−1 flatten out in the upper velocity range. It is noticeable that at a shaking frequency of 120 min−1, the two smallest bead sizes have a bimodal velocity distribution, while this effect is somewhat more pronounced for smaller beads. A likely cause is that the simulation data includes velocities from all vertical bead layers. In contrast, PTV captures only a two-dimensional velocity field without the frequency of bead velocities in the vertical direction. Accordingly, this method cannot capture the frequency of bead velocities in different bead layers. Smaller bead diameters tend to have more layers of beads, so the effect described above becomes more pronounced. Somewhat larger deviations between experiment and simulation are seen at the highest speed. The main reason for the deviations might be that the beads are now arranged very densely. This increases the likelihood of incorrect mappings of particle pairs between two images in the PTV analysis. −1 are slightly more compressed in the experiment than in the simulation, so this could be another reason for the observed differences.Overall, there is good agreement between the experimental and numerical distributions for the two lowest shaking frequencies of 100 and 120 minIn addition to the glass beads, white ceramic beads were also investigated. The experimental and simulated mean and maximum velocities are close. However, the experimental distribution shows slightly lower velocities in the lower velocity range. Overall, it is shown that the presented experimental method is suitable to determine the velocities of the beads in the ShF. Moreover, the comparison between experiment and simulation shows a high degree of agreement. Accordingly, the chosen simulation approach is a suitable tool for the analysis and prediction of the stresses caused by the beads.However, biomass was not considered in the simulations and experiments. The cultivation broth becomes turbid to non-transparent after a certain cultivation time. Thus, reliable identification and tracking of the beads on the camera images becomes much more difficult due to poorer illumination conditions. Depending on the morphological state of the culture and the biomass concentration, the viscosity of the culture broth increases and the viscosity behavior often becomes non-Newtonian . The useIn the following chapter, novel model quantities for the bead-induced stress of microorganisms in ShFs are presented. The EDCF is often used to model fluid induced stress in aerated STBs on filamentous microorganisms. This concept shows that in addition to the intensity of stress, the active stress volume and SF are also decisive. However, the EDCF cannot be directly applied to the ShF due to the different types of power input and the associated stress mechanisms. In STB, filamentous microorganisms are subjected to turbulent stress due to the power input of the stirrer and aeration . CFD simrate Eq.  in the c−1 depending on the shaking frequency. Thus, the shear rate is in the range of the average shear stress that occurs in the STB  for the production of the antibiotic novobiocin in deep-well plates . It is also noticeable that the product concentrations are shifted on the y-axis with increasing shaking frequencies. This indicates that other effects related to shaking frequency may be affecting product formation.Nevertheless, an optimum curve was found for the product concentration as a function of the newly defined model parameter. Only two data points for the ceramic ball series (light green) do not fall within the optimum curve. These points correspond to the two highest ceramic bead concentrations . Furthermore, these cultivations had lower bio dry mass concentrations than the corresponding unsupplemented control. Overall, the right amount of stress seems to be crucial for optimal cultivation results. In the literature, both positive and negative effects of bead addition have been reported for different strains and product types , takes into account the ratio of bead-wall shear and normal stresses to the total shear area. Comparison of the SAR with cultivation results showed an optimum pattern for both product concentration and mean product-to-biomass related yield coefficient. Accordingly, the model is able to compare different macroparticle-enhanced cultivation conditions.In order to compare the bead induced stresses with the results of filamentous and shear sensitive To validate the simulation method, the beads on the bottom of the ShF were recorded using a high-speed camera. The images were used to determine the velocities of the beads using particle tracking velocimetry. The comparison with the simulations showed a high agreement between simulation and experiment. Thus, the chosen simulation approach is a powerful method for quantifying bead-induced stresses in ShFs.In the future, the applicability of the model to other shear sensitive (filamentous) strains should be verified. Furthermore, more investigations at high shaking frequencies are necessary to explain the strongly increased product concentrations. Overall, this study will help to more systematically find the optimal cultivation conditions for shear sensitive microorganisms at the ShF scale. For a successful model-based scale-up to a STB, further research is needed to establish a relationship between the bead and fluid induced stresses. In addition, the simulations provide data for a future simulation-based investigation of the consequences of mechanical shear and normal stress at the level of bio-pellet.


As insects such as cockroaches can endure high radiation, flourish in unsanitary circumstances, thrive on germ‐infested feed, and can even digest the organic polymer cellulose, the gut microbiota of these species likely produces enzymes contributing to their ability to digest a variety of materials. The use of cockroaches as a bio‐resource to eliminate plastic is discussed. We explore whether species such as cockroaches are a potential bio‐resource to eliminate plastic pollution and contribute to the sustainable development goals adopted by the United Nations as well as the global community to reduce and/or eliminate plastic pollution. Galleria mellonella), which were shown to biodegrade low‐density polyethylene  and are one of the hardiest insects on the planet . At present, various strategies to dispose of plastic waste are in place including landfill sites, incineration, recycling, production of fuel and degradation or biodegradation  costs, human resources, managing costs, running costs such as electricity, and other utilities associated with the collection of plastic. Depending on the volume and type of the project and the number of employees, the approximate costs associated with setting up a recycling plant can vary, often running into millions of dollars ; formal analysis ; resources ; writing – original draft . Naveed Ahmed Khan: Conceptualization ; formal analysis ; project administration ; resources ; writing – original draft .The authors declare no conflicts of interest.


Pseudomonas aeruginosa has been purified. Its viral particles have a siphoviral morphology with a head 60 nm in diameter and a noncontractile tail 184 nm long. The dsDNA genome consists of 16,449 bp, has cohesive 3′ termini, and encodes 28 putative proteins in a single strain. The peptidoglycan endopeptidase encoded by ORF 16 was found to be the lytic enzyme of this virus. The recombinant, purified enzyme was active up to 55 °C in the pH range 6–9 against all tested isolates of P. aeruginosa, but, surprisingly, also against the distant Gram-positive micrococci Arthrobacter globiformis and A. pascens. Both this virus and its endolysin are further candidates for possible treatment against P. aeruginosa and probably also other bacteria.A novel virus lytic for  Pseudomonas aeruginosa is a Gram-negative polytrophic bacterium of great importance for human and animal health. It is considered an opportunistic nosocomial pathogen commonly associated with pneumonia, skin, soft tissue, eye, urinary tract, and otitis diseases [P. aeruginosa isolates have shown resistance to almost all classes of antibiotics, making conventional therapies with combined antibiotics increasingly ineffective [P. aeruginosa, such as ceftolozane–tazobactam, ceftazidime–avibactam, doripenem, and plazomicin, have become available in recent years, but they might have some unnecessary adverse effects or be restricted solely to intravenous use [P. aeruginosa infections [diseases ,2, and idiseases . Recentlffective . New antnous use . In addifections ,7,8.P. aeruginosa as a propagation host (accessed June 2023). Thus far, as is known, many of the lytic viruses have limited host ranges and preferentially replicate in one or a few related strains [P. aeruginosa was conducted in this way without adverse effects [P. aeruginosa in medicine [GenBank lists about 170 viruses (bacteriophages) with genome sizes between 3 kbp and 309 kbp using  strains . The use effects . In recemedicine ,13. Thesmedicine ,15. Endomedicine . The endmedicine ,19, theyThe endolysin of the novel Pseudomonas virus Hadban is most likely a peptidoglycan endopeptidase. It has its counterpart in Arthrobacter phage Adaia, with 96.4% amino acid (aa) sequence identity, but no other similar genes are present in GenBank . In the presented work, the optimal reaction condition of this enzyme was evaluated.P. aeruginosa-specific phage was isolated from soil when it was enriched overnight with a culture of P. aeruginosa-isolate POCH2. Plaques that developed on a soft agar plate with the POCH2 isolate were purified in three passages, propagated, then concentrated from the bacterial lysate with 10% PEG 6000 and 4% NaCl precipitation [600 = 0.3 (approximately 108 CFU/mL) and inoculated with the phage to obtain a multiplicity of infection > 1. Adsorption was performed for 10 min at 27 °C, followed by incubation on a shaker at the same temperature. Samples were taken every 20 min, diluted, plated onto lawns of the susceptible P. aeruginosa POCH strain using the double agar overlay assay, and then evaluated after 16 h.A new pitation . The hosDNA for sequencing was extracted from purified viruses after RNase, DNase, and proteinase K treatment as described previously , then seATGACGAAGTTCAGTAGCC and 5′TTAGACCTTTACGCCCATG  with Sapphire 2× Premix , cloned into the pET100 TOPO plasmid , then transformed into E. coli TOP10 cells. Clones with verified plasmid and inserts were propagated. The plasmid was then extracted and used for transformation of E. coli BL21(DE3) cells. Protein expression was induced for 16 h at 30 °C with 0.5 mM IPTG according to the manufacturer’s instructions. The protein was extracted from the bacterial lysate using a His-Trap column , dialyzed against 50 mM phosphate buffer, then freeze-dried.The endolysin gene was amplified with the primers 5′-caccP. aeruginosa cells after chloroform permeabilization, as described by Briers et al. [620 nm. The experiment was performed three times independently. To determine the optimal temperature, the purified enzyme was incubated in 20 mmol/L phosphate buffer at 30, 40, 50, 55, 60, and 70 °C with permeabilized P. aeruginosa POCH2 cells for 60 min. To determine the optimal pH for endolysin activity, the enzyme was incubated with P. aeruginosa POCH2 cells in 20 mmol/L phosphate buffer at pH values between 4 and 10. Relative activity was determined by comparing the maximum activity of the dataset and the lytic activity of each assay [Endolysin activity was detected using a turbidity reduction assay in which 20 µg of the purified endolysin in 50 µL volume was added to 150 µL volume of s et al. , and thech assay .P. aeruginosa was obtained from the ear swab of a dog with otitis media and identified using 16S rDNA sequencing. This strain was resistant to β-lactam antibiotics, valinomycin, and novobiocin, but was sensitive to chloramphenicol, kanamycin, tetracycline, and neomycin. The bacteriophage lysing this strain was purified and the purified virions had an isometric head with a diameter of 61 nm (±2.4 nm) and a non-contractile tail with a length of 184.4 ± 8.7 nm (n = 31), reflecting siphoviral morphology  identical to those of the Arthrobacter virus Adaia and similar to those of some decurroviruses (PhagesDB). Eleven genes at the 5′ end encoded structural proteins, the product of ORF 16 was predicted to be viral endolysin, and ORF 28 contained an HNH endonuclease domain (pfam14279) similar to the endonucleases of gordoniaviruses, adaiavirus, and atraxavirus . No geneThe Arthrobacter phage Adaia with genome size 15,840 bp and G+C content 56.1%, the Arthrobacter phage Atraxa with genome size 14,927 and G+C content 58%, and decurroviruses with genome sizes ranging from 14,830 to 15,556 bp and G+C contents around 60.1% were found to be the viruses most similar to this new virus . PhylogeP. aeruginosa strains from the Czech Collection of Microorganisms , from the Collection of Phytopathogenic and Agriculturally Beneficial Bacteria , and from the German Collection of Microorganisms and Cell Cultures . The complete bacteriophage lysed all P. aeruginosa strains, but not the atypical brown color-producing strain CCM 3630 and strain DSM 22644. The extraordinarily high amino acid sequence identity of the Pseudomonas virus Hadban and Arthrobacter virus Adaia in several genes, including endolysins (96.4%), prompted us to also test the lytic properties of the new bacteriophage on two Arthrobacter species. Surprisingly, the bacteriophage lysed both Arthrobacter pascens CCM 1653 and Arthrobacter globiformis CCM 193 strains  and the host of virus Hadban is a Gram-negative P. aeruginosa . The proteins involved in virus Hadban’s ability to infect P. aeruginosa bacteria are not known. We can speculate that the tail protein(s) responsible for binding the virus to the bacterial cell wall have been altered. From this point of view, the minor tail protein (product of ORF 14) is a good candidate, because this protein differs greatly in its C-terminal part and has a 74% overall aa identity with the corresponding protein of virus Adaia, while the other structural proteins are 95% to 100% identical to the corresponding proteins  in whose N-part within amino acid positions 4–142 a domain of the M23 family metallopeptidase was found and in whose C-part within amino acid positions 180–214 a putative peptidoglycan-binding protein (pfam01471) was found. This protein is, therefore, presumably an endolysin of this virus. The putative endolysin of the related Arthrobacter virus Atraxa (ORF 15) has a similar domain structure, but it is smaller (a protein 207 aas long) and has an amino acid sequence identity of 47.3% with the endolysin of virus Hadban. Decurroviruses’ endolysins are 149 aas long and have the metallopeptidase domain only . The recte virus b. There at 50 °C c, and itat 50 °C d.P. aeruginosa per se, but also its unique ability to lyse completely different, unrelated bacteria.Endolysins generally have broader specificity than do whole phages ,32,33. T


In this paper, we use unique health record data that cover outpatient care and the associated costs to quantify the health care costs of a sizable increase in the retirement age in Germany. For the identification, we exploit a sizable cohort-specific pension reform which abolished an early retirement program for all women born after 1951. Our results show that health care costs significantly increase by about 2.9% in the age group directly affected by the increase in the retirement age (women aged 60–62). We further show that the cost increase is mainly driven by the following specialist groups: Ophthalmologists, general practitioners (GPs), neurology, orthopedics, and radiology. While the effects are significant and meaningful on the individual level, we show that the increase in health care costs is modest relative to the positive fiscal effects of the pension reform. Specifically, we estimate an aggregate increase in the health costs of about 7.7 million euro for women born in 1952 aged 60–62 which amounts to less than 2% of the overall positive fiscal effects of the pension reform. Aging populations and demographic change challenge the financial stability of public pension systems. Therefore, many countries reform their pension systems and prolong work lives to increase contributions and to reduce the number of benefit recipients. However, an increasing retirement age might have adverse effects in other areas of the welfare state, specifically for the health care system. Previous studies e.g. , 31, 37), 3731, 3This paper uses unique data that cover outpatient care and associated costs to quantify the health care costs of a sizable increase in the retirement age.We exploit a sizable cohort-specific pension reform that abolished an early retirement scheme for all women born after 1951. The reform provides a clean quasi-experimental setting as it induces a substantial discontinuity in retirement behavior for two adjacent cohorts. We use this discontinuity in a difference-in-differences (DiD) estimation. This framework accounts for cohort and seasonality effects and allows us to identify the causal effect of the pension reform on health care costs. Specifically, similar to previous studies e.g. , 36), we, we36]),Our results show that outpatient care costs significantly increase by about 2.9%  in the age group directly affected by the increase in the retirement age (60–62). Moreover, we also find expectation effects for women at the age of 59 and indirect post-employment effects for women between 63 and 65. We further show that the cost increase is mainly driven by utilization of the following specialist groups: Ophthalmologists, general practitioners (GPs), oral and maxillofacial surgery, neurology, orthopedics, and radiology. The absolute effect is largest for GPs (about 3.5 euro) and thereby contributes about 25% to the increase in the overall costs. While the effects are significant and meaningful on the individual level, we show that the increase in health care costs are modest relative to the positive fiscal effects of the pension reform. Specifically, we estimate an aggregate increase in the health costs of about 7.7 million euro for women aged 60–62 and born in 1952. The corresponding estimate of the net effects of the pension reform for the tax and transfer system including social security amounts to about 4 billion euro .Thus, from an aggregate perspective, our results of an increase in the health care costs do not provide strong evidence against an increase in the retirement age. However, the increase of costs on the individual level shows that positive fiscal effects of a longer working life can be counteracted by potential negative health consequences for individuals. Moreover, our cost estimate focuses on the public health care costs and abstracts from individual disutility or other disadvantages due to worse health as well as other societal costs such as a decrease in labor productivity or an increase in sickness absence at work. For political decisions on retirement ages, such factors also need to be taken into account.There exists a large literature on the health effects of retirement and pension reforms:Despite this sizable literature on health outcomes, there is only little evidence on the effects of pension reforms on public health care costs. Two examples are studies looking at pension reforms that delayed retirement with mixed evidence. Shai  finds ne6Our paper is structured as follows: In “In this section, we provide a brief overview on the relevant institutions of the German pension system and discuss the 1999 pension reform, which induced an exogenous increase in the early retirement age for women born after 1951. Moreover, we describe the German health care system.The German public pension system covers roughly 90% of the workforce.pension for women. The 1999 reform abolished this pathway to retirement for cohorts born after 1951. Effectively, the reform raised the early retirement age (ERA) for most women from 60 to 63, which implies an extension of the working life of three years. The eligibility criteria of the pension for women were: (i) at least 15 years of pension insurance contributions; and (ii) at least 10 years of pension insurance contributions after the age of 40. About 60% of all women born in 1951 were eligible for the old-age pension for women ) We account for correlation between observations of the same individual or individuals born in the same month, and use robust standard errors clustered by month of birth. In the subgroup analysis , we additionally adjust the standard errors for multiple hypotheses testing using the Bonferroni-correction.i was born between October 1951 and March 1952. The indicator is zero if individual i was born between October 1950 and March 1951. i was born between January and March and zero otherwise. More formally, we estimate the following equation:The distribution of health costs for the pre-reform cohort born 1951) at age 59 and 60  in the “Before we turn to the discussion of the estimation results, we present graphical evidence on the effect of the pension reform on health care costs. Figure Importantly, at the cut-off, we observe the largest jump in health care costs: the fee-adjusted costs increase by about 25 euro per person after the cut-off date which corresponds to a relative increase of about 5%. This is first evidence that the increase in the retirement age leads to a sizable increase in health care costs. In the following, we turn to the estimation results of the DiD specification to empirically assess this reform effect.We estimate the effect of the pension reform on health care costs on the intensive and extensive margin for different age groups. In Table The estimation results confirm the graphical evidence: We find that the pension reform, i.e. the shift in the retirement age from 60 to 63, increases health care costs Table . In all In Table In Fig. We provide empirical evidence for our identification strategy in “In a next step, we analyze to which specialist care utilization the overall increase in costs can be attributed. This analysis provides insights into whether the increased prevalence of certain diagnoses goes along with increased utilization of the relevant specialists. We present the results for health care costs for the 28 different medical specialists that are classified in the data. To correct for multiple hypothesis testing, we adjust the standard errors using a Bonferroni-correction. Figure Panel (a) shows the results for 59–65 year old women. The increase in the retirement age leads to a significant increase in costs for six specialist groups: Ophthalmologists, GPs, oral and maxillofacial surgery, neurology, orthopedics and radiology. The absolute effect is largest for GPs (about 3.5 euro) and thereby contributes about 25% to the increase in the overall costs. In terms of relative price increases, the effects are largest for oral and maxillofacial surgery, neurology, and radiology.Panel (b) depicts the results for women at age 59 and the bottom left (Panel (c)) and right panel (Panel (d)) for women aged 60–62 years and 63–65 years, respectively. The results for the 59 year olds suggest, that the increase in the overall costs is mainly driven by increases in the utilization of GP and neurology care. For women aged 60–62 years, we find significant increases in the costs for eight specialists: Obstetricians/gynecologists, otolaryngologist, oral and maxillofacial surgery, neurology, orthopedics, psychiatry, and radiology. In absolute terms, the effects are largest for orthopedics (2.1 euro), radiology (1.3 euro) and obstetricians/gynecologists (1.2 euro). Relatively speaking, the rise in costs for oral and maxillofacial surgery, neurology and psychiatry are largest. The costs for specialists decreases due to the reform by 0.1 euro. Similarly to the 59 year old, the increase in overall costs for the 63–65 year old seems to be driven by increased utilization of mainly two specialist groups: GPs and radiology.Overall, and across the different age groups the results show a relatively clear pattern. We find the strongest increase in the costs for neurology, psychiatry, radiology, GPs, orthopedics and oral and maxillofacial surgery. Based on the data it is not possible to directly identify the mechanism why the costs in the different categories increase. However, the evidence about the effects of the pension reform on health outcomes  allows uIn this section we put our findings into perspective and compare the additional health care costs to the overall fiscal effects of the 1999 pension reform. As shown by  the pensRelative to this sizable net effect, the additional aggregated health care costs are modest. As documented in Table In this paper, we document that an increase in the retirement age leads to a significant increase in health care costs. To identify the causal effect of the increase in the retirement age, we exploit a cohort-specific pension reform which increased the early retirement age by three years between women born in two adjacent cohorts. The analysis is based on data that include the universe of individuals insured through the German public health care system  and comprises a ten-year observation period (2009–2018). Our results show that health care costs increase overall by about 2.9% for women in the age group directly affected by the increase in the retirement age (60–62). Moreover, we find expectation effects for women at the age of 59 and indirect post-employment effects for women between 63 and 65. In addition, we show that the cost increase is mainly driven by increased utilization of the following specialist groups: ophthalmologists, general practitioners (GPs), neurology, orthopedics and radiology. The absolute effect is largest for GPs (about 3.5 euro) and thereby contributes about 25% to the increase in the overall costs.While the effects are significant and meaningful on the individual level, we show that the increase in health care costs are modest relative to the positive fiscal effects of the pension reform. Specifically, we estimate an aggregate increase in the outpatient costs of about 7.7 million for women aged 60-62 and born in 1952. Relative to the corresponding estimate of the net effects of the pension reform of about four billion euro  this traThus, from an aggregate perspective, our results of an increase in the health care costs do not provide strong evidence against an increase in the retirement age. However, the increase of costs on the individual level support the findings of previous studies focusing on individual health outcomes, that positive fiscal effects of a longer working life can be counteracted by potential negative health consequences for individuals. Our cost estimation focuses on the public health care costs and abstracts from individual disutility or other disadvantages due to worse health as well as other societal costs such as a decrease in labor productivity or an increase in sickness absence at work. For political decisions on retirement ages, such non-monetary factors also need to be taken into account.


Highly productive agriculture is essential to feed humanity, but agricultural practices often harm human health and the environment. Using a nitrogen (N) mass-balance model to account for N inputs and losses to the environment, along with empirical based models of yield response, we estimate the potential gains to society from improvements in nitrogen management that could reduce health and environmental costs from maize grown in the US Midwest. We find that the monetized health and environmental costs to society of current maize nitrogen management practices are six times larger than the profits earned by farmers. Air emissions of ammonia from application of synthetic fertilizer and manure are the largest source of pollution costs. We show that it is possible to reduce these costs by 85%  while simultaneously increasing farmer profits. These gains come from (i) managing fertilizer ammonia emissions by changing the mix of fertilizer and manure applied, (ii) improving production efficiency by reducing fertilization rates, and (iii) halting maize production on land where health and environmental costs exceed farmer profits, namely on low-productivity land and locations in which emissions are especially harmful. Reducing ammonia emissions from changing fertilizer types—in (i)—reduces health and environmental costs by 46% ($11.7 billion). Reducing fertilization rates—in (ii)—limits nitrous oxide emissions, further reducing health and environmental costs by $9.5 billion, and halting production on 16% of maize-growing land in the Midwest—in (iii)—reduces costs by an additional $0.4 billion. Agricultural practices produce food, animal feed, and biofuels, but also harm human health and the environment. In the US Midwest, current maize production results in nitrogen-related health and environmental costs that are six times larger than the profits earned by farmers. Changes in farming practices can substantially reduce these impacts while maintaining or increasing profits for farmers. These changes involve changing the form or method of application of nitrogen fertilizer, reducing fertilizer rates, and shifting production away from areas of low productivity or high environmental and health costs. These changes in farming practices, when spread over millions of hectares, can yield large societal gains. Our modeling approach could be applied to other regions and crops.Accounting for health and environmental costs in agricultural production can lead to better decisions about what crops to produce and how and where to produce them. Here, we illustrate this potential by analyzing health and environmental costs of nitrogen (N) management in maize production in the United States (US) Midwest.2O) that contributes to climate change and ozone depletion (3) and nitrogen oxide (NO) that contribute to the formation of fine particulate matter (PM2.5), which is injurious to human health (2). Pollution costs from N fertilizer application depend on local soil and climatic conditions  limiting ammonia loss to the atmosphere from N fertilizer by changing fertilization practices; (ii) lowering N fertilizer input in addition to limiting ammonia losses; and (iii) retiring land from maize production in the counties where health and environmental costs exceed farmer profits in addition to (i) and (ii). These changes are based on the current paradigm of known and adopted farming practices, and do not preclude the possibility of more impactful, transformative changes. However, the implementation of the changes at the scale we propose would require overcoming difficult obstacles including political barriers, concerns over price increases for consumers, consequences for rural farming communities that may retire farmland, and buy-in from key stakeholders.We find that the overall health and environmental costs of current maize production in the US Midwest exceeds farmer profits by a considerable margin. Annual farmer profits (averaged over 2013–22) from Midwest maize production, which does not include government payments, are $4.3 billion per year (2020$), but health and environmental pollution costs from Midwest maize production are $25.6 billion per year see Fig. .3 emissions are responsible for 65%. N2O emissions, the next largest source of pollution costs, are responsible for 29%. Of the $25.6 billion in total health and environmental pollution costs, NHsts Fig. .2.5 formed from ammonia. Farmer profits, net of government payments, also vary by county, from −$96 to $42 t−1 of maize. Profits depend on regional maize yield-response functions . In a l studies , this lo3 emission rate to 2.3% reduces pollution costs by $11.7 billion per year (46%), largely from improved air quality, with the benefits concentrated in downwind dense population centers in the Midwest, and to a lesser extent the Atlantic and New England regions. Limiting NH3 losses also leads to slightly lower farmer costs—due here to a lower price of anhydrous ammonia plus the cost of applying fertilizer—however, there may be short-term implementation costs that are not accounted for here. This single change in farming practices greatly improves net benefits across the Midwest. However, pollution costs still exceed farmer profits by $9.1 billion per year  beyond that achieved by limiting NHone Fig. . Reducinone Fig. , we calcone Fig. .3 losses and lowering N application rates convert net economic losses for many counties into net gains—the net benefits are positive for 252 of 593 counties—especially in Iowa, Minnesota, central Illinois, and southern Wisconsin , an existing program with annual outlays of ∼$3.5 billion  that payDecreased production, which results in increased prices to consumers—both in the maize market and animal protein markets that rely heavily on maize—is another potential source of conflict. The maize price rise increases farmer profits and is more reflective of the full costs of crop production by including the costs of pollution; however, agricultural price increases have a detrimental impact on consumers, especially those in low-income countries. When applying large-scale changes to agricultural systems, including agricultural commodities on which the poorest populations globally depend, the distributional impacts of food price increases (or price variability) are especially important to consider. The recent increase in the price of agricultural commodities and potential impacts on global food shortages caused by the conflict in Ukraine amply illustrates the negative consequences of a reduction in supply of key agricultural commodities . BecauseDue to data limitations, our work was based on county averages. Consideration of variation within a county could allow for greater improvements in net benefits—for example, by considering retirements only on the least-productive land within a county, opposed to an all-or-nothing decision for the whole county.Our modeling approach, seeking alternative production practices that reduce health and environmental impacts while maintaining profits for farmers, could be applied to other regions and crops. N is the major limiting nutrient for growth of many important crops  and N feThere are additional changes in production practices that could be analyzed, such as inclusion of buffer strips, tiling practices, cover crops, other field amendments, or changes in residue management. We focused on N management based on a previous analysis that indicated that nitrogen-related health and environmental costs are the largest share of pollution costs in the Midwest . In addiOur analysis highlights that it is possible to identify changes in practices that lead to net gains for both farmers and those impacted by agricultural pollution. Agriculture is a significant contributor to pollution in many countries, but it has proven difficult to address both because the source of pollution is spread among many farmers rather than concentrated at a small number of point sources, and policies that do not reward farmers economically are politically unpopular. It becomes far easier to reduce pollution when there are easy-to-adopt alternatives that are profitable for farmers. In our application, this mutual gain was possible because the proposed changes in some cases reduced production costs or resulted in an increase in the commodity price.We created a model to examine maize production and the effects of changes in N use on yields and pollution. Our analysis simulates a single year of maize production, representing averages across 2013–22. The unit of analysis is at the county level, across seven states in the US Midwest Corn Belt . The model combines several components as follows: (i) sub-state maize yield-response functions (to simulate maize production based on N amendments), (ii) N mass-balance equations to account for nitrogen additions and subtractions on farmland, and (iii) estimates of pollution costs from different forms of N releases into the environment, including location-specific estimates of air pollution impacts. In the N mass-balance equations, additions make N available for the maize plant, affecting yields and farmer profits, and subtractions are from N uptake by the maize plant and from N losses to pollution leading to health and environmental costs. The model compares farmer profits with the health and environmental costs of N pollution to characterize the current situation, and three proposed interventions in N management.The model assumes an annual N balance such that the amount of N added to the field or soil is subtracted in some form. While we only model the outcome of a single maize production year, we assume that the land is used in a two-year maize-soybean rotation. The following sections lay out the parameters and the relationships between variables used in the model.Additions of N include amendments in the forms of synthetic N fertilizer , where the proportion of N in the maize grain is Nitrogen subtractions include uptake by the maize plant in the grain and emissions into the environment. N uptake is separated into two parts as follows: N in the grain which is removed from the field, and N in the other parts of the plant which remain in the field. We define N in the grain as = 0.0115 . N in thi, we use the following functional form to approximate the relationship in the yield calculator:Maize yield-response curves, as a function of N applied, are adopted from an Iowa State N management calculator , based owhere 2013–22)  and the 2, N2O, NO, NH3, and 2, which is not harmful, is emitted as a fixed constant, The remaining N losses are as emissions which are used to estimate pollution costs. Pollution costs are estimated by applying N-loss coefficients to calculate emissions, and marginal pollution-cost coefficients to translate emissions into monetary costs. We model the emission of N in five forms as follows: N kg N/ha .2O, NO, and NH3 are emissions as a function of the quantity of 2O and NO emissions, we follow the IPCC framework  of each pollutant from units of N to units of each compound. These emissions are defined as 2O emissions contribute to global climate change and pollution costs are calculated according to an N2O adjustment . PM2.5 concentrations, when inhaled, increase the risk of adult premature mortality . The evaluation of our modeling approach, including the air-quality model InMAP (that drives many of our conclusions) is documented in several publications, including against observed pollutant concentrations  and particulate ammonium . Furthermore, InMAP reproduces WRF-Chem-derived particulate ammonium concentrations fairly well for a range of policy scenarios .Emissions of NH nitrate , which aortality , 60. Agrglobally  and in tglobally . NH3 is ammonium , which cn health . The morn health . We use n health , 64  (w (the average of the bi-weekly price from 2013–22 (Revenue is yield times the maize price 2013–22) . Costs a2013–22)  to get a 2013–22 ), and thrtilizer —$32.37 p3–22 . CoIn each scenario, we calculate, for every county, the farmer profits, pollution costs, government payments and expenditures, and net social welfare , k is a constant, and k = 6.16 × 1010, using the initial price and the global quantity of maize (average between 2013 and 2022) (where nd 2022) . We alsopgad319_Supplementary_DataClick here for additional data file.


We review the role of vitamin B12 in cats. Cobalamin is an essential part of enzymes involved in multiple metabolic reactions. In mammals, two enzymes are cobalamin-dependent: methionine synthase and methylmalonyl-CoA mutase. Cats are obligate carnivores and have an essential requirement for certain nutrients present only in animal tissues, including vitamin B12, which cannot be synthesized by cats. The serum cobalamin concentration can be measured by an automated chemiluminescence competitive binding immunoassay system but does not accurately reflect the real availability of cobalamin, because cobalamin-dependent metabolic reactions occur within cells. A lack of intracellular cobalamin leads to reduced enzyme activity and methylmalonic acid accumulation. The serum cobalamin concentration is particularly useful for detecting deficiency, as it is directly related to the real bioavailability of the vitamin for intracellular enzymatic reactions. Several diseases of the gastrointestinal tract, pancreas, and liver are associated with hypocobalaminemia in cats. Treatment with parenteral cobalamin is always recommended for cats with gastrointestinal disease and low serum cobalamin concentrations. A recognized guideline for cobalamin supplementation does not yet exist for cats, so the recommendations are mainly based on clinical experience.Cobalamin is a water-soluble molecule that has an important role in cellular metabolism, especially in DNA synthesis, methylation, and mitochondrial metabolism. Cobalamin is bound by intrinsic factor (IF) and absorbed in the ileal tract. The IF in cats is synthesized exclusively by pancreatic tissue. About 75% of the total plasma cobalamin in cats is associated with transcobalamin II, while in this species, transcobalamin I is not present. In cats, the half-life of cobalamin is 11–14 days. Diagnostic biomarkers for B12 status in cats include decreased levels of circulating total cobalamin and increased levels of methylmalonic acid. The reference interval for serum cobalamin concentrations in cats is 290–1500 ng/L, and for the serum methylmalonic acid concentration, it is 139–897 nmol/L. Therapy for hypocobalaminemia mainly depends on the underlying disease. In some cases, subcutaneous or intramuscular injection of 250 μg/cat is empirically administered. In recent years, it has been demonstrated that oral cobalamin supplementation can also be used successfully in dogs and cats as a less invasive alternative to parental administration. This review describes the current knowledge regarding B12 requirements and highlights improvements in diagnostic methods as well as the role of hypocobalaminemia in its associated diseases. Cobalamin is a water-soluble, cobalt-containing B vitamin  resultinCats are obligate carnivores; therefore, they have an essential requirement for nutrients present only in animal tissues . VitaminNeither a safe upper limit of cobalamin (the maximum concentration in the diet or an amount that has been associated with adverse effects) or reports of toxicity as a result of cats ingesting high doses are present in the literature . DietaryAfter food is ingested, cobalamin is released in the stomach by peptic digestion and bound to a gastric R-protein  to form an unattackable complex . GastricThe IF–cobalamin complex enters the enterocytes by pinocytosis after binding to specific receptors known as cubam receptors. They are receptor complexes consisting of two subunits, cubilin (CUBN) and the protein amnionless (AMN) ,25. CobaFree cobalamin is stored in the liver and kidney  and is eThe serum cobalamin concentration is measured by an automated chemiluminescence competitive binding immunoassay system ,36,37. TThe direct influence of diet on serum cobalamin concentration can depend on several factors, and age, sex, and breed play important roles. Older cats frequently show decreased absorption of this vitamin, probably related to a decreased ability to digest it due to reduced ileal and pancreatic function ,40. KittThe serum cobalamin concentration does not accurately reflect the real availability of cobalamin, because cobalamin-dependent metabolic reactions occur within cells, mainly in the cytoplasm and mitochondria . MethylmSerum and urinary MMA concentrations can be determined by gas chromatography–mass spectrometry in fresh or stored samples (−20 °C) ,35,37. TPanthera tigris) and tigers affected by pancreatic insufficiency-like syndrome. The results showed much lower B12 concentrations in both healthy and affected tigers than in domestic cats, although reference ranges for serum cobalamin concentrations in tigers have not yet been established [Acinonyx jubatus) to establish reference ranges of species-specific biomarkers of gastrointestinal disease, including cobalamin and MMA. The measured ranges were 470–618 ng/L and 365–450 nmol/L for cobalamin and MMA, respectively, showing that even between closely related species such as the domestic cat and cheetah, there can be differences in gastrointestinal biomarkers [In humans, total plasma homocysteine levels are used in the diagnosis of cobalamin deficiency. Cobalamin represents an essential cofactor for the enzyme methionine synthase, which is essential for the synthesis of methionine from homocysteine. Cobalamin deficiency blocks this reaction by promoting homocysteine accumulation. When not enough cobalamin is available, the pathway is blocked and homocysteine accumulates within the cells and plasma . In humaablished . Anotheromarkers .Cobalamin status can be affected by various diseases either directly  or indirectly . A typical example of the latter is anorexia, a condition that frequently occurs in disease states associated with hypocobalaminemia. Indeed, in cats, a low capacity for storage and the short half-life of cobalamin predispose them to develop B12 deficiency in a short time. Therefore, anorexia should always be considered as a cause of hypocobalaminemia in sick cats. The pathological conditions related to hypocobalaminemia that have a direct influence are described below.Several diseases of the gastrointestinal tract, pancreas, and liver are associated with hypocobalaminemia in cats. In this species, inflammatory bowel disease (IBD), alimentary lymphoma, pancreatitis, cholangitis/cholangiohepatitis ,30,35, aRapidly dividing cells such as enterocytes and hematopoietic cells, which are essential for DNA replication, are more susceptible to decreased serum cobalamin concentrations . Other dClinical signs closely related to cobalamin deficiency in cats have not been well characterized because hypocobalaminemia is often associated with other pathologies ; frequenIncreased serum cobalamin may be indicative of liver disease or neoplastic disease ,62. The Since cobalamin–IF complex receptors are located in the ileum, chronic gastrointestinal disease, alimentary lymphoma, and ileum resection can lead to cobalamin deficiency, as these conditions reduce ileum absorption capacity . HypocobSeveral studies have reported that 100% of cats with EPI show hypocobalaminemia ,71,72, bTriaditis in cats is described as concurrent inflammation of the pancreas, liver, and small intestine caused by bacterial infection or by immune-mediated or idiopathic mechanisms . PancreaBacteroides spp., Clostridium spp.) may reduce its bioavailability [Dysbiosis resulting from EPI, gastritis, or other gastrointestinal disorders  may contlability . The metlability . In humalability . In addilability .Feline hepatic lipidosis is a common syndrome in cats due to fatty degeneration of hepatocytes with resulting cholestasis and hepatic dysfunction . This syA correlation between hyperthyroidism and hypocobalaminemia has been described , as thisMcMichael et al. (2000) observed a decrease in the serum cobalamin concentration  in cats with cardiomyopathy and thromboembolism compared to healthy cats. That study evaluated three groups of cats: a healthy group, with mean cobalamin of 1659 ± 700 pg/mL; a cardiomyopathy group, with a mean value of 939 ± 389 pg/mL; and a cardiomyopathy–thromboembolism group, with a value of 866 ± 367 pg/mL. Low serum cobalamin concentration was also associated with left atrium enlargement. Although reduced cobalamin has been recorded in cats with cardiovascular disease, it is unclear whether it is a predisposing and causative factor or a consequence of the heart disease .Several neurological manifestations in humans, such as dementia, are associated with cobalamin deficiency , while fTherapy for hypocobalaminemia mainly depends on the underlying disease . CyanocoCobalamin supplementation (CS) can be administered either parenterally or orally , accordiTreatment with parenteral cobalamin is always recommended for cats with gastrointestinal disease and low serum cobalamin concentrations . In someIn recent years, it has been demonstrated that oral CS can be used successfully in dogs and cats as a less invasive alternative to parental administration . Daily aThe suggested nutritional requirements for cobalamin have been developed from studies conducted on growing kittens. Therefore, further studies are needed to confirm that these nutritional requirements are also adequate for adult cats and pregnant and lactating queens. Moreover, determining MMA and assessing serum cobalamin can help to better identify nutritional needs by adapting them to the intracellular availability of the nutrient. Since serum MMA evaluation can better define the cobalamin status, it should be routinely performed in the clinical setting, although wide dissemination of this approach will not be possible without validating the methodologies already present in veterinary laboratories.Due to the importance of vitamin B12 in several metabolic reactions and its role in the therapy of several diseases, vitamin status and proper supplementation should always be considered in sick cats.Recent developments in our understanding of the gut microbiota open up new therapeutic horizons. For this reason, it would be interesting to explore the relationships between modifications in gut bacterial flora and cobalamin bioavailability and the development of chronic diseases in cats. Such studies could further speed up the collection of knowledge regarding the role and function of cobalamin.


V. vinifera) collected at the vineyards of the Krasnodar Krai in Russia. Ribosomal-depleted total RNA and isolated small RNAs were used for library preparation and high-throughput sequencing. Six grapevine-infecting viruses and two viroids were validated by RT-PCR and analyzed phylogenetically. We identified the presence of grapevine leafroll-associated virus 3, grapevine Pinot gris virus, grapevine virus T, grapevine rupestris stem-pitting-associated virus, grapevine fleck virus, and grapevine rupestris vein feathering virus, as well as two viroids, grapevine yellow speckle viroid 1 and hop stunt viroid. We also studied the mycovirome of the vineyard and identified nine viruses with single-stranded positive-sense RNA genomes: alternaria arborescens mitovirus 1, botrytis cinerea mitovirus 1, botrytis cinerea mitovirus 2, botrytis cinerea mitovirus 3, botrytis cinerea mitovirus 4, sclerotinia sclerotiorum mitovirus 3, botrytis cinerea hypovirus 1, grapevine-associated narnavirus 1, and botrytis virus F. In addition, we identified botrytis cinerea hypovirus 1 satellite-like RNA and two single-stranded negative-sense RNA viruses. This is the first study of grapevine mycoviruses in Russia. The obtained result will contribute to the development of biocontrol strategies in the future.Among other pathogens, more than 80 viruses infect grapevine. The aim of this work was to study the virome diversity of grapevine viruses and mycoviruses of a vineyard using high-throughput sequencing technologies. The grapevine virome was studied in symptomatic vines of the Rkatsiteli cultivar ( Plants, like all living organisms, are associated with a large number of microorganisms throughout their entire life cycle. They correspond to both endophytic and epiphytic microorganisms, including pathogenic bacteria, phytoplasmas, fungi, oomycetes, and viruses. Plant viral diseases are infectious diseases that lead to significant economic losses in agriculture and require constant monitoring to prevent their spread to new territories ,2. ImproThe development of molecular biology tests and rapid advances in sequencing technologies, in particular high-throughput sequencing (HTS) technologies, have led to the availability of powerful new tools for identifying plant pathogens and studying the microbial community (including viruses) of investigated plants. Protocols based on HTS technologies can decipher the composition of microbial populations in any studied sample. The application of HTS technologies is particularly advanced for plant-virus detection and is currently envisioned as an additional test to evaluate the phytosanitary status of plant material in the frame of certification or quarantine analyses .Grapevine is one of the most important crops in the world; it is affected by more than 80 known viral pathogens. Since the beginning of the use of HTS in the field of grapevine virology, several new viruses, such as grapevine red-blotch-associated virus (GRBaV), grapevine Pinot gris virus (GPGV), and grapevine Syrah virus 1 (GSyV-1), have been discovered, and the pathogenicity of some of them have been demonstrated ,5,6.In addition to the detection of grapevine viruses, HTS makes it possible to survey its virome as a microecosystem comprising viruses of other organisms living on plants, such as fungi and oomycetes. The long co-evolution of pathogenic fungi and oomycetes of grape and their viruses made it possible to established both symbiotic and antagonistic relationships between them. Viral infection in the fungal hosts could be associated with a weakening of the virulence of the fungi, a reduction in the epidemics due to a decrease in the number of spores, and a slowdown in the growth rate of mycelium .Botourmiaviridae) and botrytis cinerea negative-stranded RNA virus 1  RNA unclassified ssRNA negative-strand viruses) that infect the phytopathogenic fungus Botrytis ..https://Vitis reference genome with medium-low sensitivity parameters. Small RNAs with and without grapevine reads were then assembled into contigs using Geneious (with medium-low sensitivity) and Velvet (with default parameters) assemblers. Next, blastn and tblastx were performed against the NCBI database of the reference viral and viroid genomes (refseq release date of 5 May 2022). Contigs with hits to plant viruses with an E-Value cutoff of <e−5  were selected for further analysis. Trimmed reads were mapped to the reference genomes of the identified viruses, and the percentage of coverage of the reference genome was determined.To analyze the small RNA library, we used the Geneious Prime v. 2020.0.4. Reads were trimmed using a BBDuk Trimmer, and duplicated reads were removed. Host plant reads were kept or eliminated from the trimmed deduplicated reads by mapping them to the FinchTV; Version 1.4.0; Geospiza, Inc.: Seattle, WA, USA, 2004–2012) and deposited in the GenBank  according to the manufacturer’s protocol. Then, PCR was performed with primers for the 18S rRNA gene and with primers for the grapevine viruses detected as a result of bioinformatics analysis . Primersp-value of 0.005 or less (p ≤ 0.05) were considered as positive.For phylogenetic analysis, we used the complete genomes of viruses identified by HTS data analysis, together with complete genomes downloaded from the GenBank . Multipl


Obtaining information on implementation strategy costs and local budget impacts from multiple perspectives is essential to data-driven decision-making about resource allocation for successful evidence-based intervention delivery. This mixed methods study determines the costs and priorities of deploying Enhanced Replicating Effective Programs (REP) to implement the Michigan Model for Health™, a universal school-based prevention intervention, from key shareholder perspectives.Our study included teachers in 8 high schools across 3 Michigan counties as part of a pilot cluster randomized trial. We used activity-based costing, mapping key Enhanced REP activities across implementation phases. We included multiple perspectives, including state agencies, regional education service agencies, lead organization, and implementers. We also conducted a budget impact analysis  and a scenario analysis to estimate replication and account for cost variability. We used an experimental embedded mixed methods approach, conducting semi-structured interviews and collecting field notes during the trial to expand and explain the cost data and the implications of costs across relevant perspectives.Based on trial results, we estimate costs for deploying Enhanced REP are $11,903/school, with an estimated range between $8263/school and $15,201/school. We estimate that adding four additional schools, consistent with the pilot, would cost $8659/school. Qualitative results indicated misalignment in school and teacher priorities in some cases. Implementation activities, including training and implementation facilitation with the health coordinator, were sometimes in addition to regular teaching responsibilities. The extent to which this occurred was partly due to leadership priorities  and organizational priorities .Previous research findings indicate that, from a societal perspective, universal prevention is an excellent return on investment. However, notable misalignment in cost burden and priorities exists across shareholder groups. Our results indicate significant personal time costs by teachers when engaging in implementation strategy activities that impose an opportunity cost. Additional strategies are needed to improve the alignment of costs and benefits to enhance the success and sustainability of implementation. We focus on those perspectives informed by the analysis and discuss opportunities to expand a multi-level focus and create greater alignment across perspectives.ClinicalTrials.gov NCT04752189. Registered on 12 February 2021.The online version contains supplementary material available at 10.1186/s43058-023-00511-6. Contributions to the literatureUniversal prevention in schools represents an excellent societal return on investment, but costs and resources remain a notable barrier to realizing its public health benefits.Research examining implementation costs and budget impacts from multiple perspectives aids in understanding the practical implications of deploying implementation strategies in community settings.Combining cost and qualitative data from multiple perspectives deepens our understanding of how resources and priorities are aligned—or not—that ultimately influence implementation success and sustainment.not the costs of implementation strategies required to deploy and sustain them [Economic evaluation in implementation science has received increased attention as researchers, practitioners, and shareholders recognize that costs are foundational to successful implementation and sustainment. Practically speaking, costs and resources are essential reasons why implementation and sustainment efforts fail, including within educational settings , 2. Althain them . Implemeain them , 5, 7. ASystematic examination of costs for achieving program outcomes using implementation strategies is vital for evidence-based intervention (EBI) sustainability in schools as in other settings . Organizkey determinant of which costs researchers will include in an economic evaluation [Limited research has focused on practical information on implementation costs across essential partner perspectives, including within the education sector see Fig. . While aaluation . The peraluation . Figure aluation  and infoaluation , 20. Thealuation .Fig. 1Muactual costs depend on the context in which intervention implementation happens [A mixed methods approach to economic evaluation enables a comprehensive assessment of cost and resource burdens across shareholder levels as, undoubtedly, the levels are interdependent , 22. We The current study focuses on reporting costs related to deploying implementation strategies for the Michigan Model for Health™ universal prevention curriculum as part of a group RCT. Specifically, we focus on quantifying the incremental implementation costs of deploying the Enhanced REP implementation strategy bundle. We use an experimental embedded mixed method design to obtain a more in-depth understanding of the results see Fig. , focusinWe included data from a randomized controlled trial in 8 schools across 3 Michigan counties during the 2021–2022 school year: 4 randomized to Enhanced REP  and Department of Education (MDE), the RESAs  through the health coordinators and the teachers who receive the curriculum, training, and support for MMH from the health coordinator in their region. Other levels, including districts and local school buildings, vary in intensity, investment, and active versus passive involvement in the health curriculum see Fig. . DetermiThe study initially included ten schools randomized into control and intervention sites . We had schools currently using MMH that failed to meet state standards for implementation (delivering less than 80% of the curriculum) and/or reported one or more barriers to MMH implementation from a list of 10 common barriers to participation in the study. We checked randomization at the control/intervention group level and then by the specific school representations across the number of students .We used an activity-based micro-costing approach mapping key activities of Enhanced REP across implementation phases. We adopted multi-level perspectives, including state agencies , regional education service agencies (RESA), lead organization , and implementers . We estimated the incremental implementation strategy costs during the trial. We also conducted a budget impact analysis using explicit, real-world costs to assess the economic impact of replication and considering the cost distribution across levels in the educational system. We focused on these perspectives as they are consistently those perspectives that are explicitly involved in decision-making and resource allocation for health education. We used the EPIS  framework to guide implementation phases . This apWe assigned monetary values to each activity or other input upon establishing necessary implementation resources  across phases. Most resource costs related to the Enhanced REP implementation strategy were personnel, specifically implementers, implementation support professionals, and coordinating organization time. We include costs within the education sector, including fixed  and variable costs , and time outside the education sector  when these could be reasonably estimated for a pragmatic evaluation of costs in this analysis. Consistent as described by Gold and colleagues, opportunity costs refer specifically to the value of resource inputs in their next best alternative use . OpportuTo properly micro-cost, we assessed the amount of time spent on each activity and the costs associated with each person’s time. To optimize applicability across contexts, we utilized salary range information from the Bureau of Labor Statistics (BLS) 2021 Occupational Employment and Wage Statistics report for the state of Michigan . Since tThe time horizon for the current pilot study is aligned with the experimental design, one academic year. For the Sustainment phase, we projected the recurring costs of the program over time for each additional semester, following the first semester among teachers who implemented for two semesters.We estimated the labor cost, including fringe benefits by multiplying each hourly rate by 1.34, to reflect the actual time costs . We usedOur analysis also stratified relative costs by perspective groups— the agents involved in funding and deploying the implementation efforts . The perWe conducted a scenario analysis to characterize uncertainty in the cost estimates in a broad sense by estimating the “best” and “worst” case scenarios . This apWe conducted a budget impact analysis to estimate the potential budget impact for replication. While traditional BIA may imply adopting an organization perspective, the practical application of BIA in implementation science has expanded this to include other perspectives that would benefit from receiving information about the potential budget impact from these various perspectives. A recently published budget impact tool, for example, permits users to customize the BIA by selecting the resources that each user group  would require for a delivery model for MOUD (medication for opioid use disorder) . Wagner Our interview guide was informed by the Enhanced REP components that align with EPIS constructs in the outer context , innovation factors (the MMH intervention and fit with the context), the inner context , and multi-level constructs such as barriers and facilitators and resources needed and utilized . We , including placing the costs across shareholder groups in context and providing a broader explanation of the costs and benefits of deploying Enhanced REP over a school year. For expansion, we used qualitative results to deepen our understanding of what costs are not sufficiently captured with micro-costing, such as personal time costs  and areas of alignment and misalignment across groups in costs and benefits of implementation. Thus, we focus on comprehensively understanding cost and resource considerations and expand on the quantitative findings .Two schools dropped out early in the study, leaving eight for our cost analysis and 4 for the Enhanced REP cost estimates. Of the participating schools, 6 had student populations over 1000, equally divided between control and trial groups. The smallest participating school had 213 students and the largest had 2051 students.Based on publicly available school data  Additio, particiTable Table Table From the educational system perspective, we estimate the budget impact for Enhanced REP of $34,637 to add four additional schools or $8659.17 per school. We estimate only the budget impact using the same number of schools in the pilot test. Considering other schools would require speculation on the economies (or diseconomies) of scale of the implementation. We also calculated budget impacts from each shareholder’s perspective when estimating the BIA. The university tasks were transferred to the health coordinators and state agency employees, with schools bearing no additional cost in replication. This determination was based on input from the state agency and RESA partners on what types of costs they typically incur related to implementation, although it may vary. Consistent with the current partnership structure, University employees would serve only in minor consultation roles with an estimated cost of $343.53. We anticipated that school districts would have the most significant increase in costs and time commitment. We estimated a total cost of $17,483 for the school districts, an increase of $8892 or roughly doubling the cost. State agency partners funded all the fixed costs in implementation. Thus, we estimated a decrease in their share of the overall cost in the budget impact analysis. Simultaneously, the state agency would likely contribute more labor efforts to repeated implementation. For the state agency partners, we estimated a decrease in fixed costs of $14,385 and an increase in variable labor costs of $3107, resulting in a net cost reduction of $11,278.Since the estimated budget impacts of replication in new schools are all employee time costs, we may also consider these to be changes in total labor hours. We anticipate an increase in labor provided by the state from 30.5 to 65.5 h and an increase in the district’s efforts from 229 to 351 h to cover the decrease in the university efforts. In total, we estimate a net reduction of 91 h in labor related to the budget impact of replication.Teacher interviews aided in putting costs in context with quantitative data. We report three interrelated central themes. The themes include (1) priority and resource alignment across perspectives, (2) the impact of implementation leadership, and (3) the influence of collaboration and support on the implementer  burden.This theme refers to the extent to which priorities and resources are aligned or shared across implementer, organizational leadership , and district or RESA . Qualitexisting  programs that are already there (like MMH) because we (can) create stronger and deeper ties (with students) that are going to last more than the time period of the grant or that little bit of money this (new) initiative goes for.” Another teacher stated that this cycle of continuously adopting new programs was deeply engrained, and some school environments are very “resistant to change.” Teachers reported that if they did want more curriculum-specific focus, they had to find time outside of school hours. Thus, some teachers’ time to engage with health coordinators and receive IF related to the MMH updates was primarily during personal time. Other teachers attempted to consult with health coordinators during their planning hour, but this was frequently disrupted due to other school priorities . As a result, some teachers’ capacity to engage with IF was inconsistent, and health coordinators spent notable time trying to schedule and reschedule meetings with implementers ; the misalignment created additional barriers and resulted in significant opportunity costs within and outside the education sector, for teachers and health coordinators.Related, teachers shared instances when they saw misalignment between school, teacher, RESA/implementation support professionals  priorities, and resource allocation. One teacher expressed frustration with allocating significant time and resources to the “next new thing” versus investing in evidence-based health curricula that have already been adopted. For instance, why aren’t schools asking, “what can we do to help the reduced time in curriculum prep versus standard MMH implementation. She reported she was able to spend more time on activities to enhance student engagement with the curriculum, build connections, and meet the diverse needs of students. A teacher in this scenario reported having regular meetings with the health coordinator in which she was able to obtain both relational and informational support. “ is quite helpful. She’s always given me tips additional resources that might be helpful, not just for me, but for students and their families, so we’ve been meeting every (week) to check in and share how things are going, and so it’s been very valuable having her as a resource.”In cases of alignment between the district, teacher (implementer), and school priorities and resources, the implementer reported Implementation leadership refers to specific leadership behaviors that are likely to advance EBI implementation efforts; it can include different dimensions, such as proactive  and supportive  leadership . Teachernot utilizing evidence-based health curricula. “(I) still had to teach parts of what (others in her building were) teaching because our final exam has to be a common final.” Consequently, she reported the need to modify the tailored MMH content to comply with the exam.This theme centers on the extent to which the school environment permits engagement with peers and implementation support personnel to reduce individual implementer burden and enhance the overall implementation of the curriculum. For teachers who work with other health teachers in their school building, collaborating—or not—may profoundly impact the implementer’s workload and capacity to engage in implementation strategies. One teacher reported that her district requires a common final exam despite many of her peers Collaborating with implementation support practitioners and the health coordinators aided in problem-solving delivery challenges, teachers feeling supported and confident in their approach, and enhanced efficiency with classroom time. “(Before engaging in the implementation supports) I could spend two to three hours just on one lesson (to meet the needs of students), but with the (updated) lessons…sometimes 20 to 30 min is just going through it and make sure I’m abreast of the information.” Another teacher who experienced challenges with making the IF meetings expressed that she felt she would have benefitted from greater interaction, but the personal costs of engaging in the implementation support given her context were difficult to overcome. “ was awesome…I think if anything, I didn’t have the time to give to her, she was always willing to meet, to help…” This teacher reported that there was an incentive structure to cover other classes during planning time and earning days off rather than engaging with implementation support. “(School leadership) definitely didn’t give me time to work on this curriculum, so I couldn’t take a half day to work on it or anything like that.”individuals and organizations successfully implement and sustain EBIs such as MMH [This study examines the cost of deploying implementation strategies to enhance the delivery of a universal intervention (MMH) for drug use prevention. We determine costs across multiple phases of implementation and shareholder perspectives. We also use mixed methods, using qualitative data to explain and expand on the quantitative results. Robust evidence indicates that effective implementation of universal prevention is a good return on investment from a societal perspective . Howeverh as MMH . Thus, wThe base case per school for deploying Enhanced REP was $11,903, with the bulk of the costs being personnel time costs, consistent with other implementation research , 65, 66.Considering costs by phase, costs were highest during the implementation phase, followed by preparation, exploration, and sustainment. The trial-based analysis indicates that the relative cost burden was highest for the state agency during the preparation phase, as with the coordinating body , RESA cost burden was evenly distributed during the preparation and implementation phases and, as expected, the teacher cost burden was heavily concentrated in the implementation phase. This information can help inform implementation planning and understand resource burdens over time across groups. For example, when engaging in an implementation support effort such as deploying Enhanced REP, the RESA can plan for resource allocation that will vary over time, with it being highest during preparation and implementation. Similarly, teachers can prepare for the expected time costs to ramp up during the implementation phase. These reported costs, however, may not reflect the total costs of implementation, including other costs that may not be fully accounted for in these analyses.The qualitative results aid in expanding our understanding of some of the costs not fully represented in the analyses. We found that the teacher time cost burden, in some cases, for example, is likely an underestimate. Consistent with other school-based research, teacher time costs, including those outside the education sector  that may be personal opportunity costs related to time participating in implementation activities, are common . Given tfoundational to successful intervention implementation [Our analyses also focus on generating information for decision-makers to inform resource allocation for implementation via scenario analyses and BIA. We estimated cost ranges in the scenario analysis, depending on personnel salary at the lower (10th percentile: $8264/school) and higher . While our base case analysis uses median wages for each personnel category, assuming a typical labor market, school districts may be subject to differing labor market conditions based on outside factors such as location and experience levels. The results from these analyses can support comparing multiple scenarios that reflect potential uncertainties in cost estimates in a reliable manner, given the pilot study parameters. The scenario analysis information can aid educational systems in estimating possible costs and ranges based on their staff experience levels and salary ranges. We also examined the budget impact of Enhanced REP replication in other school settings. While there are varied applications of budget impact analysis, we apply BIA pragmatically consistent with implementation research. The BIA provides information on how adopting Enhanced REP may influence an organization, system, or individual from a budgetary perspective . It aidsentation . Thus, eSchools are highly heterogeneous settings. Qualitative results indicate that who takes responsibility for various tasks may vary by district or region. For example, when implementers are asked to adopt a new or updated intervention or have a need to engage with implementation supports , the extent to which this results in personal time costs, that is, opportunity costs, and highly dependent on the building leadership. If the task is to adjust to new population needs and engage in activities to meet that need, we found that school buildings and school leadership can either (a) adjust implementers’ responsibilities and provide flexibility to meet these needs, which may be at a “cost” to the building-level priorities such as covering classes with absent teachers, or (b) require or otherwise strongly incentivize the teachers to assist with using flexible time to meet building-level needs, but with a higher personal opportunity cost. The latter relies heavily on the implementers’ inclination to meet the needs of students at a notable personal cost. Thus, our reported costs at the implementer level likely reflect an underestimate without considering sufficiently the additional costs related to implementation strategy deployment, specifically time costs outside the education sector.Collectively, our results indicate that collaboration and support within school buildings and across levels notably impact the capacity for effective implementation from the teachers’ perspective. Shared norms and expectations of the importance of MMH implementation, also called strategic implementation climate , emergedOur mixed methods results also indicate the need to enhance focus on building-level implementation costs and priorities. For example, in the school where the priorities and resources were more aligned, there were also likely other consequential costs that were not included, such as additional staffing costs to protect the health teacher’s planning and PD time. While we did account for some additional staffing costs , there were likely additional costs and priorities at the building level. While the default interested parties were included in the current analysis, our results suggest a significant gap in some contexts in costs and priorities. Future research would benefit from a more explicit focus on exploring the building-level costs and benefits in the context of implementer-level costs.While we estimated costs related to the implementation strategy within the education sector and outside  when these could be reasonably estimated for this pragmatic evaluation, this did not fully capture all costs. Teacher time costs do not fully reflect the opportunity cost of time to participate in Enhanced REP as we could not capture the full scope of all such costs. While important, these costs are also complex and challenging to capture due to the high participant burden. The costs relate to time costs potentially diverted from other work activities that can impact productivity and outside work . However, pragmatic costing of implementation studies requires a balance between accurate estimates and shareholder burden. In the current pilot study, it was not feasible to estimate all costs comprehensively at all levels. The level of accuracy of reported costs, especially the non-IF costs, may vary as some of the costs were based on recall of past events. We focused on greater precision with the IF costs within the education sector, using weekly time logs, as this was a demonstrated influential parameter in previous research .t-tests of school-level characteristics do not indicate heterogeneity between the trial and control group analytic sample based on racial demographics or socioeconomic status. Despite these limitations, our cost estimates provide a starting point for estimating Enhanced REP costs as part of a more extensive study. Finally, an essential next step in this research is calculating the potential return on investment for implementation strategy deployment using methods of comparative economic evaluation such as cost-effectiveness or cost–benefit analysis. However, estimating costs each shareholder group bears is a vital first step in successfully accounting for resources needed with implementation strategy deployment.As this was a pilot study, our number of participants may not be sufficient to estimate the Enhanced REP deployment with scale-up accurately. The scenario analysis and BIA address this limitation by providing information about costs in different scenarios and with replication. This analysis intentionally has a limited scope to benefit decision-makers and other partners most. Still, it does not reflect the aggregate societal perspective regarding costs and resources. While we estimated sustainment costs based on the pilot study’s experimental design, the sustainment costs during a full-scale trial may vary from this estimate. Generally, sustainment costs are very underdeveloped. Thus, this study aims to add to the cost of sustainment evidence base. These results may not be generalizable to other settings but can inform assessing implementation strategy cost estimates in schools. Finally, we experienced attrition at the school level, partly because we conducted the study during the COVID-19 pandemic, which may influence our estimates of Enhanced REP costs. We were, however, due to including multiple teachers over multiple semesters, and through incorporating qualitative data, able to make reasonable estimates of Enhanced REP costs despite the attrition. Also, our Educational settings are central for providing health-focused interventions for children and youth, yet many implementation and sustainment efforts fail due to limited attention to the costs and resources required. Information on implementation strategy costs and local budget impacts from multiple perspectives is essential to data-driven decision-making about resource allocation for successful EBI delivery, including within educational settings. This mixed methods study identified the costs and priorities of deploying Enhanced Replicating Effective Programs (REP) to implement the Michigan Model for Health™, a universal school-based prevention intervention, from key shareholder perspectives. Additional work is needed to enhance alignment in costs and resources across perspectives for successful implementation and achieving intervention public health impact.Additional file 1. Testing for Heterogeneity of School Randomization.