Torulaspora delbrueckii and Saccharomyces cerevisiae contain helper large-size (4.6 kb) dsRNA viruses (V-LA) required for the stable maintenance and replication of killer medium-size dsRNA viruses (V-M) which bear the genes that encode for the killer toxin. The genome of the new V-LA dsRNA from the T. delbrueckii Kbarr1 killer yeast (TdV-LAbarr1) was characterized by high-throughput sequencing (HTS). The canonical genome of TdV-LAbarr1 shares a high sequence identity and similar genome organization with its Saccharomyces counterparts. It contains all the known conserved motifs predicted to be necessary for virus translation, packaging, and replication. Similarly, the Gag-Pol amino-acid sequence of this virus contains all the features required for cap-snatching and RNA polymerase activity, as well as the expected regional variables previously found in other LA viruses. Sequence comparison showed that two main clusters (99.2–100% and 96.3–98.8% identity) include most LA viruses from Saccharomyces, with TdV-LAbarr1 being the most distant from all these viruses (61.5–62.5% identity). Viral co-evolution and cross transmission between different yeast species are discussed based on this sequence comparison. Additional 5′ and 3′ sequences were found in the TdV-LAbarr1 genome as well as in some newly sequenced V-LA genomes from S. cerevisiae. A stretch involving the 5′ extra sequence of TdV-LAbarr1 is identical to a homologous stretch close to the 5′ end of the canonical sequence of the same virus (self-identity). Our modeling suggests that these stretches can form single-strand stem loops, whose unpaired nucleotides could anneal to create an intramolecular kissing complex. Similar stem loops are also found in the 3′ extra sequence of the same virus as well as in the extra sequences of some LA viruses from S. cerevisiae. A possible origin of these extra sequences as well as their function in obviating ssRNA degradation and allowing RNA transcription and replication are discussed.Wine killer yeasts such as killer strains of  Different types of M viruses have been described, each one encoding a specific killer toxin . Each ki strands . It has  strands , which iSaccharomyces sensu stricto: S. cerevisiae, S. paradoxus, S. kudriavzevii, and S. uvarum , such as the frameshift region and encapsidation signal  have very similar genomes and a close phylogenetic relationship. Among them, S. paradoxus is considered to be the closest relative to S. cerevisiae  and S. paradoxus  in ScV-LA1-EX231 and ScV-LAlus1-EX436, and 22 nt [5′-CTCTGACAAAGGTACTTTTGTT-3′] in ScV-LA2-EX1125 [C(−)57 to T(−)36] and ScV-LAlusA-EX1160 [C(−)47 to T(−)25] 25] .S. cerevisiae viruses. Other interesting sequences were also found in ScV-LA1-EX231, ScV-LAlus4-EX229, and ScV-LAlus1-EX436: 118 nt of 100% identity with a chromosome II sequence of S. cerevisiae, 178 nt of 100% identity with S. cerevisiae 18S rRNA, and 213 nt of 93% identity with S. cerevisiae 26S rRNA, respectively  and part in the 5′-end (37 nt) [C(−)14 to A37], showed 100% identity with a homologous stretch located near the 5′-end in the canonical sequence of the same virus [C401 to A451] . This stretch is almost a palindromic sequence, and about half of this sequence (5′-CACGTAGCTTTATTAATTAATATGCTACGTG-3′) can form a stem loop (ΔG = -50 kJ/mol), a loop that contains the first four nucleotides of the canonical sequence (5′-AATT) as unpaired. These nucleotides are complementary to the unpaired nucleotides in the stem loop of the homologous stretch located downstream in the RNA sequence (5′-AAUU/3′-UUAA), and it is possible that a kissing complex could be formed by four-base-pair annealing . A similectively .S. cerevisiae 26S rRNA 2 to G(+)17] of TdV-LAbarr1 showed 100% identity with a homologous stretch located close to 3′-extra in the canonical sequence of the same virus [T4457 to G4472], and part of each can form a stem loop (ΔG = -2.5 kJ/mol). The unpaired nucleotides of these homologous RNA loops are complementary to each other (5′-AUAU/3′-UAUA). Therefore, similarly to the case for the 5′-end of the same virus, intramolecular kissing-loop interaction is possible through four-base-pair annealing . A simil26S rRNA , which cS. cerevisiae viruses. This was to be expected given that they come from different yeast species with different ecological niches—S. cerevisiae is rarely isolated from natural environments  is needed to clarify this issue.The phylogenetic relationship we found for eviously , showing viruses . However viruses , LA viruS. cerevisiae. Surprisingly, two stretches of the Gag-Pol sequences previously known as poorly conserved , regardless of which type of M killer virus was supported by these helper LA viruses in each case. However, the opposite was found when comparing the same sequence stretches of the viruses included in the S. cerevisiae cluster with the rest of the viruses. The corresponding identity percentage was the lowest found even when the viruses belonged to the same yeast species isolated at only 300 km distance , to different yeast species isolated in the same area , or to different yeast species isolated at only 120 km distance . In particular therefore, if 100% identity is found in these two variable sequence stretches of Gag-Pol, those LA viruses may be considered to come from the same yeast species isolated from the same geographical area, no matter which type of M killer virus coincides with the LA virus in the same yeast strain. Therefore, these LA-virus variable sequences could be a good genetic marker of the unknown geographical origin of a given yeast killer strain.Comparing the amino acid sequence of Gag proteins represents an interesting approach to study the co-evolution of each LA virus with each yeast species. Indeed, we found that this approach was clearly the best method to group all yeast strains of the same species, at least in the case of onserved  were thoS. cerevisiae strains were isolated, viral cross transmission between yeast strains of the same species living in the same habitat is to be expected. The lowest identity among ScV-LA viruses was found between different yeast species, even if they came from the same geographical location, as was the case for T. delbrueckii EX1180 and the S. cerevisiae-cluster strains. Therefore, contrary to what has previously been suggested for some yeast species of the Saccharomyces sensu stricto taxon  when they coincide in the same habitat, as has previously been suggested . These results indicate that there was no associated co-evolution of specific LA with specific M viruses at all. It seems that an M virus can infect new yeasts and be stably maintained inside the cell as long as a given LA virus provides it with the required helper activity.As mentioned above, an associated co-evolution of specific LA virus variants with the corresponding specific type of M virus has been suggested based on a possible role of each killer toxin selecting the LA variants that best support each specific toxin-encoding M virus. This is a plausible hypothesis to explain why LAlus4 is specifically associated with Mlus4 virus  and thatS. cerevisiae strains isolated in the same location. Therefore, the co-evolution can be hypothesized of a specific LA virus with its specific host and habitat. The sequence identity percentage of TdV-LAbarr1 with its Saccharomyces counterparts was less when comparing only Gag sequences, but greater when comparing only the RdRp domain sequences. This indicates that LA viruses may have evolved to adapt their capsid’s functioning to better ensure replication in different yeast species and habitats. On the contrary, as features of the RNA polymerase are strongly conserved, no great changes in this enzyme would be required for these viruses to replicate in unrelated yeast species.TdV-LAbarr1 is the most distant from the rest of the viruses (62–63% identity), even from those viruses from S. cerevisiae LBC-2 virus, wine grape, Saccharomycopsis fibuligera, and melon) were also found in the 5′-extra sequences. It was suggested in that work that ScV-M RNA could somehow promiscuously covalently join other host viral or cellular RNAs, as has also been suggested for poliovirus RNA  suggests that at least part of these extra sequences may have a common origin. Given that viral self-identity is frequently found between some stretches of extra and canonical sequences, the presence of extra sequences may be a collateral result of some imprecise molecular mechanism involved in the viral replication cycle—cap-snatching, for example . This cicis-regulatory elements in translational efficiency, and large portions or even almost entire sequences of rRNA may have functional significance for some neurodegenerative diseases  may play an as yet unknown role in the biology of these viruses . Beyond diseases . Moreoven events . This alT. delbrueckii Kbarr1 yeast seems very similar to that previously described for S. cerevisiae. The autonomous LA viruses from the two yeast species show high nucleotide sequence identity, especially in the most relevant functional motifs, which indicates that they are phylogenetically related. LA virus transmission among yeasts of the same species living in the same geographical location seems to be feasible, but not cross-species transmission among phylogenetically distant yeasts such as T. delbrueckii and S. cerevisiae. Co-evolution of LA and M viruses does not seem likely, although co-evolution of LA virus with a given yeast species may occur in a specific location or habitat. Extra sequences located up- and down-stream from the viral canonical genome may form interesting RNA secondary structures, which could be involved in virus maintenance by avoiding ssRNA degradation and facilitating dsRNA synthesis.The killer dsRNA virus system of The datasets generated for this study can be found in the online repositories. The names of the repository/repositories and accession number(s) can be found in the article/MR conceived the project, analyzed the data, and wrote and edited the manuscript. MR, RV, AM, and MM designed and performed the experiments. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hydrogel beads based on the husk of agarwood fruit (HAF)/sodium alginate (SA), and based on the HAF/chitosan (CS) were developed for the removal of the dyes, crystal violet (CV) and reactive blue 4 (RB4), in aqueous solutions, respectively. The effects of the initial pH (2–10) of the dye solution, the adsorbent dosage (0.5–3.5 g/L), and contact time (0–540 min) were investigated in a batch system. The dynamic adsorption behavior of CV and RB4 can be represented well by the pseudo-second-order model and pseudo-first-order model, respectively. In addition, the adsorption isotherm data can be explained by the Langmuir isotherm model. Both hydrogel beads have acceptable adsorption selectivity and reusability for the study of selective adsorption and regeneration. Based on the effectiveness, selectivity, and reusability of these hydrogel beads, they can be treated as potential adsorbents for the removal of dyes in aqueous solutions. With the rapid development of industry and population growth, natural resources cannot meet the demand of human life. The extensive use of synthetic materials and the discharge of wastewater from manufacturing industries have seriously polluted water resources and the environment. Industrial wastewater contains organic compounds and heavy metals, which are water pollutants that cause worldwide environmental problems . Synthetd-mannuronate units and α-l-guluronate units and can be extracted from seaweed [2) and hydroxyl (-OH) groups that can be chemically or physically reacted with a crosslinking agent to prepare hydrogels [Recently, hydrogel-based adsorbents synthesized from bioresources such as chitosan, sodium alginate, and starch cellulosic materials have attracted particular attention for dye removal because of their three-dimensional network structure, high availability, environmental friendliness, high swelling property, versatility as a treatment medium, and simple recoverability ,15,16. S seaweed . Chitosa seaweed . SA has ydrogels ,20. In rydrogels ,22,23,24ydrogels . Agarwooydrogels . The intydrogels ,28. TherScanning electron microscopy (SEM) was applied to study the morphologies of the hydrogel beads. −1 was attributed to the stretching vibrations of the –OH groups from pure SA and HAF molecules. The peaks at 1610 cm−1, 1410, and 1028 cm−1 are assigned to the –COO− (asymmetric), –COO− (symmetric), and C-O-C stretching vibrations, respectively [−1 (C=O) and 1238 cm−1 (C-O), indicating more incorporation of –COO− groups. All the peaks associated with –COO− shifted in the spectra of MHAF-SA after CV adsorption and indicated electrostatic interactions between the negatively charged carboxyl groups of MHAF-SA and the positively charged CV dye. −1 can be assigned to the overlapping OH and NH stretching vibrations contributed by pure CS and HAF. Other characteristic peaks at 2872, 1590, 1373, and 1027 cm−1 are attributed to C-H stretching, N-H bending, C-H bending, and C-O-C stretching vibrations, respectively. After modification by sodium hydroxide, the peaks at 3350, 1590, and 1027 cm−1 were obviously enhanced compared with those of HAF-CS. After RB4 adsorption, the peaks at 3350, 1590, and 1027 cm−1 shifted to 3372, 1567, and 1016 cm−1, respectively. In addition, a new peak appears at 1160 cm−1 after RB4 adsorption, which corresponds to the −SO3− groups of RB4 molecules [2 groups.FTIR analysis was conducted to identify the functional groups on the surface of the hydrogel beads. The FTIR spectra of the MHAF-SA hydrogel beads before and after CV adsorption are shown in ectively . After molecules . Accordipzc) of the MHAF-SA and MHAF-CS hydrogel beads. The pHpzc values of the MHAF-SA and MHAF-CS hydrogel beads were 2.50 and 7.35, respectively. Therefore, the surface of MHAF-CS was more positive than MHAF-SA due to the presence of –NH2 groups, as proved by the FTIR spectra. MHAF-SA can be used as a cation attractor in solutions with pH values above the pHpzc, and MHAF-CS can be treated as an anion attractor in solutions with pH values below the pHpzc [S%) and does not change much with increasing time. The surface of the SA hydrogel bead is hydrophobic, and water molecules do not easily enter the interior. Thus, the swelling ratio of SA is low and does not change obviously (he pHpzc . The swebviously a. The swbviously b. Althoupzc of MHAF-SA), the surface of the hydrogel bead is negatively charged and prefers to adsorb the cationic dye (CV). The surface of the adsorbent is occupied by a large amount of H+ ions under acidic conditions, which compete for the adsorption sites with CV and result in a lower adsorption capacity. Moreover, the cationic dye easily reacts with oxygenic functional groups (–COO− and –OH− groups) in a high pH dye solution [pzc of MHAF-CS), the surface of the hydrogel bead is positively charged due to the protonation of amino groups (–NH3+) and facilitates RB4 (SO3− groups) adsorption by electrostatic attraction. MHAF-CS tends to dissolve at pH 2, which reduces the adsorption capacity of RB4. In addition, the precipitation of CV was observed at pH 10. The optimum pH values for the adsorption of CV and RB4 were determined to be 9 and 3 for MHAF-SA and MHAF-CS, respectively.The pH of the solution is one of the most important influencing factors in the adsorption system and can change the surface charge of the adsorbent. The pH effect on the dye adsorption of hydrogel beads at a fixed dosage of 30 mg and an initial dye concentration of 200 mg/L is shown in RE %) of both CV and RB4 increased with the increasing dosage, while the adsorption capacity was the opposite. When the dosage of hydrogel beads was 2.5 g/L, the removal efficiencies of CV and RB4 increased insignificantly with increasing dose. Therefore, the optimum dosage for both hydrogel beads to adsorb dyes was 2.5 g/L.The dosage of the adsorbent is another important key factor that affects the adsorption system to reduce the cost of wastewater treatment and maximize the benefit of the adsorbent. As shown in The effect of contact time on the adsorption of CV and RB4 onto hydrogel beads was investigated with a dosage of 2.5 g/L and an initial dye concentration of 200 mg/L at optimum pH values and 40 °C. k1 (1/min) and k2 (g/mg·min) are the adsorption rate constants of pseudo-first-order and pseudo-second-order models, respectively; qt (mg/g) and qe (mg/g) are the adsorption capacities at time t (min) and adsorption equilibrium, respectively. To study the effectiveness of the hydrogel beads, the adsorption kinetics of CV and RB4 onto the hydrogel beads were analyzed with pseudo-first-order model ) and pseudo-second-order model ) ,36.(1)lThe parameters of the kinetic models and correlation coefficients are summarized in eC (mg/L) is the dye equilibrium concentration in aqueous solution; mq (mg/g) and eq (mg/g) are the maximum adsorption capacity and adsorption equilibrium, respectively; LK (L/mg) is the Langmuir constant; FK and n are the Freundlich isotherm constants and adsorption intensity, respectively. The plots are illustrated in The adsorption isotherm study provides details about the interaction between the dye molecules and hydrogel beads at equilibrium. Various initial CV and RB4 dye concentrations (20–600 mg/L) at constant temperature were evaluated. The linear forms of isotherm models such as the Langmuir (expressed by Equation (3))  and FreuR2), the adsorption of CV onto MHAF-SA and RB4 onto MHAF-CS correlated well with the Langmuir isotherm model, indicating that the dyes CV and RB4 were mainly adsorbed onto a homogeneous surface of hydrogel beads and had monolayer coverage with uniform adsorption energies. The maximum monolayer adsorption capacity (mq) values from the Langmuir isotherm model for CV adsorbed onto MHAF-SA and RB4 adsorbed onto MHAF-CS were compared with other adsorbents reported in the literature and presented in Based on the linear regression coefficient  over 70%, suggesting the reusability of MHAF-CS hydrogel beads. The reusability of MHAF-SA and MHAF-CS is also compared with the report in the literature, as shown in The regeneration performance of the hydrogel beads is shown in − and –OH− groups) in a high pH dye solution (see 3+) and facilitates RB4 (SO3− groups) adsorption by electrostatic attraction  fruit was obtained from the local farmer in Kaohsiung, Taiwan.Acetic acid (purity ≧ 99.83%), ethanol (purity ≧ 99.8%), ammonia hydroxide (purity ≧ 35%), and sodium hydroxide (purity ≧ 97%) were purchased from Fisher Chemical . Sodium chloride (purity > 99.5%), Congo red (CR), and crystal violet (CV) were purchased from Acros Organics . Alginic acid sodium salt powder, chitosan, reactive blue 4 (RB4), and neutral red (NR) were supplied by Sigma-Aldrich . Hydrochloric acid (purity > 37%) was obtained from Scharlau . Calcium chloride (purity ≧ 89%) was purchased from Avantor . All chemicals were analytical grade and used without further purification. Agarwood (The treatment process of agarwood fruit is shown in HAF powder (400 mg) was added to 40 mL of deionized water and stirred on a magnetic stirrer for 30 min. Then, the solution (10 mL) was mixed with 200 mg of sodium alginate powder and stirred continuously for 1 h to completely dissolve the powder. The solution was dropped into a calcium chloride solution (5 wt%) via a syringe needle at a speed of 75 mL/h. The HAF-SA hydrogel beads were formed by an ion exchange process. After washing with deionized water, the hydrogel beads were immersed in 4.4 M acetic acid solution at 30 °C for 5 h at an agitation rate of 100 rpm in a shaker with a thermostatic water bath. Then, the modified HAF-SA (MHAF-SA) hydrogel beads were filtered and washed with deionized water at least three times. Finally, the MHAF-SA hydrogel beads were obtained after drying in an oven at 50 °C for 6 h.v/v) acetic acid solution. The solution was stirred continuously at 40 °C for 2 h and then remained undisturbed for 12 h to release air bubbles. Next, HAF powder (400 mg) was added to the chitosan solution and stirred for 30 min. The solution was dropped into a 0.5 M sodium hydroxide solution via a syringe needle with a speed of 75 mL/h. The hydrogel beads were formed and stirred for 3 h. Subsequently, the HAF-CS hydrogel beads were filtered, washed, and immersed in 4.4 M ammonia solution at 30 °C for 5 h at an agitation rate of 100 rpm in a shaker with a thermostatic water bath. After filtration, washing, and drying at 50 °C for 6 h, modified HAF-CS (MHAF-CS) hydrogel beads were obtained.The method to prepare chitosan hydrogels by physical crosslinking is preferred over the chemical crosslinking method due to the prevention of potential toxicity from chemical crosslinking agents . Therefopzc) was determined using the pH-drift method [S%) was determined by the following equation [wW and dW are the weights (g) of wet and dried hydrogel beads, respectively.The morphologies of the hydrogel beads were observed by scanning electron microscopy (SEM), and the functional groups on the surface were analyzed by Fourier transform infrared (FTIR) spectroscopy. The point of zero charge (pHt method  to invest method . A certaequation :(5)S(%)q) and the removal efficiency (RE %) of RB4 or CR adsorbed onto hydrogel beads RB4 were calculated by the following equations:C0 and eC are the initial and equilibrium dye concentrations (mg/L), respectively, V (L) is the volume of the dye solution, and W (g) is the dry weight of the hydrogel beads used.The dye adsorption experiments were performed on a BT-150D shaker  with a shaking speed of 100 rpm via a batch adsorption process. Equilibrium adsorption experiments were carried out by adding 30 mg of hydrogel beads into 20 mL of different concentrations of dye solution at 30 °C. After the adsorption approached equilibrium, the content of dye adsorbed onto the hydrogel beads was analyzed at the maximum wavelength  by using a UV/VIS spectrophotometer . The pH effect was investigated by adjusting the initial pH of RB4 or CR solutions in the range of 2–10 while the initial dye concentration was fixed at 200 mg/L. The dosage effect of hydrogel beads was studied from 0.5 to 3.5 g/L with an initial dye concentration of 200 mg/L. The temperature effect on the adsorption was studied from 30 to 60 °C with an initial dye concentration of 200 mg/L. The isothermal adsorption experiments were conducted with 20 mL of dye solutions with different initial concentrations (200–600 mg/L) at a fixed temperature (30 °C). All adsorption experiments were carried out in triplicate to confirm the accuracy of this study. The adsorption capacity , CV/NR (cationic dye-cationic dye), and RB4/CV (anionic dye-cationic dye), were used to study the adsorption selectivity of MHAF-SA and MHAF-CS hydrogel beads. The optimum dosage of hydrogel beads (2.5 g/L) was added to 20 mL of dye solution (200 mg/L) at a fixed temperature. After the adsorption reached equilibrium, the residual concentration of the dye mixture was monitored by a UV/VIS spectrophotometer.To reduce the damage to the hydrogel beads during the regeneration process, the agents used in the preparation process were selected to perform dye desorption experiments. The dye-adsorbed MHAF-SA and MHAF-CS hydrogel beads were immersed in 4.4 M acetic acid solution and 4.4 M ammonia solution, respectively. After shaking for 10 min, the hydrogel beads were washed several times and then dried at 50 °C for 6 h to obtain the regenerated hydrogel beads.mq values of 232.56–370.37 and 156.25–270.27 mg/g, respectively. Both hydrogel beads have excellent adsorption selectivity. After five adsorption-desorption cycles, the hydrogel beads still have a high removal efficiency. Overall, the hydrogel beads synthesized in this study have effectiveness, selectivity, and reusability and can be applied to the removal of dyes in aqueous solutions.Husk of agarwood fruit-based hydrogel beads (MHAF-SA and MHAF-CS) were successfully synthesized in this study. The functional groups of the hydrogel beads were identified and proved by the FTIR spectra. The dynamic adsorption behavior of CV onto MHAF-SA and RB4 onto MHAF-CS can be represented well by the pseudo-second-order model and pseudo-first-order model, respectively. The adsorption isotherm data of CV onto MHAF-SA and RB4 onto MHAF-CS can be explained by the Langmuir isotherm model, with


More than 30% of the German population suffers from mild to moderate iodine deficiency causing goiter and other iodine deficiency disorders (IDDs). The economic burden of iodine deficiency is still unclear. We aimed to assess costs for prevention, monitoring and treatment of IDDs in Germany.We performed a comprehensive cost analysis.We assessed direct medical costs and direct non-medical costs for inpatient and outpatient care of IDDs and costs for productivity loss due to the absence of work in 2018. Additionally, we calculated total costs for an IDD prevention program comprising universal salt iodization (USI). We performed threshold analyses projecting how many cases of IDDs or related treatments would need to be avoided for USI to be cost-saving.Annual average costs per case in the year of diagnosis were € 211 for goiter/thyroid nodules; € 308 for hyperthyroidism; and € 274 for hypothyroidism. Average one-time costs for thyroidectomy were € 4184 and € 3118 for radioiodine therapy. Average costs for one case of spontaneous abortion were € 916. Annual costs of intellectual disability were € 14,202. In the German population, total annual costs for USI would amount to 8 million Euro. To be cost-saving, USI would need to prevent, for example, 37,900 cases of goiter/thyroid nodules.USI potentially saves costs, if a minimum amount of IDDs per year could be avoided. In order to recommend the implementation of USI, a full health-economic evaluation including a comprehensive benefit-harm assessment is needed. Iodine deficiency is one of the world’s most common nutritional health problems. It causes thyroid disorders, such as goiter, hypothyroidism and hyperthyroidism and a variety of other iodine deficiency disorders (IDDs). Worldwide, about 2 billion people suffer from iodine deficiency and its consequences . AccordiAlthough Germany is no longer a designated iodine-deficient area according to the WHO, two recent epidemiological studies in Germany revealed that about 30% of children and 29% of adults are still considered to be mildl to moderately iodine deficient , 5. AccoUniversal salt iodization (USI) programs are commonly used to prevent iodine deficiency and its consequences. In the year 1994, the WHO and UNICEF Joint Committee on Health Policy recommended the fortification of household salt with small amounts of potassium iodate as a safe and sustainable way to prevent iodine deficiency . HoweverEvidence-based approaches are needed for a systematic evaluation of long-term effectiveness and cost-effectiveness of health care interventions. Decision-analytic models can be used to inform decision-makers about the value of health care technologies including benefits, risks and costs , 10. ThiThe purpose of our study was to perform a comprehensive cost analysis to assess costs for prevention, monitoring and treatment of IDDs in Germany. Additionally we aimed to estimate how many cases of IDDs or related treatments would need to be avoided for an IDD prevention program to be cost-saving.Our analysis includes two parts. In the first part, we assessed resource utilization and costs disaggregated by different items as well as a total cost per IDD per patient. In the second part, we performed threshold analyses projecting how many cases of IDDs or related treatments would need to be avoided for an IDD prevention program to be cost-saving.In general, we followed guidelines from the Institute for Quality and Efficiency in Health Care (IQWIG)  for health economic evaluations  as well We evaluated the costs associated with the prevention, diagnosis, treatment and inpatient and outpatient care of the following IDDs from the German societal perspective: hyperthyroidism, hypothyroidism, goiter/benign thyroid nodules, intellectual disability (IQ < 70), spontaneous abortion. We considered direct medical costs  and direct non-medical costs (i.e. travel costs to the clinic). We also assessed costs for productivity losses due to the absence of work.All costs are reported in Euro (€) for the index year 2018. Resource use for medical consultations, laboratory tests and medication for the different diseases is based on guidelines of national and international expert associations, revised and adapted by clinical experts , 25, 26.Inpatient costs were based on the ‘case flat rates’  of the German Diagnosis Related Groups System (G-DRG)  with theOutpatient costs for doctor’s consultations, diagnostic procedures and laboratory tests were based on the ‘Uniform Value Scale of the Federal Association of Physicians under the Statutory Health Insurance’ (Einheitlicher Bewertungsmaßstab (EBM) der Kassenärztlichen Bundesvereinigung)  and the The costs for medications were evaluated according to the German reimbursement rules. Health insurances reimburse a fixed price for drugs of a specific category or the actual drug price, if it is lower than the fixed price . We calcIndirect costs were based on productivity loss occurring due to the incapacity for work and the individual labor costs . The Federal Ministry of Health provides average days of absence due to illness for different International Classification of Diseases (ICD) -10 diagnoses according to health insurance data. We used the official numbers of the Federal Ministry of Health for average days of incapacity to work per case per year for the different IDDs to calculate productivity loss . Labor cCosts for thyroid disorders in the year of diagnosis reflect the outpatient costs for doctor’s visits, including consultations and diagnostic tests. Additionally, costs for pharmaceuticals, scintigraphy, fine-needle biopsy, thyroidectomy, and radioiodine therapy are included for the different health conditions. We calculated costs according to different probabilities and assumptions with regard to proportions. For example, we assumed that a specific proportion of patients receive scintigraphy (19.6 percent) and fine-needle biopsy (1.4 percent) each year according to data from the literature  and expeWe calculated a weighted mean of costs for different types of thyroidectomies ‘surgery and procedure codes’ (‘Operationen- und Prozedurenschlüssel (OPS-Codes)): 5-060, 5-061, 5-062, 5-063, 5-064) based on the proportional distribution of those surgeries . We calcThe average costs per case of miscarriage include the costs for doctor’s visits, sonography, and proportionate costs for the medical and surgical management. We calculated average costs using prevalence data of the different management strategies from published literature , 43.The annual costs for intellectual disability in Germany were available in the literature  for 2011We conducted an expert interview to estimate the resource utilization of an IDD prevention program including the iodine fortification of salt. We derived costs to the German government of administering and enforcing an iodine fortification program, costs for promotion campaigns and population information (every 2 years), additional costs for iodized salt for the consumer and costs for IDD monitoring . We calculated annual average cost per person for an IDD prevention program, total costs for IDD prevention and monitoring program in Germany and cases of the different IDDs and related treatments that need to be avoided for an IDD prevention program using universal salt iodization to be cost-saving.Resource use for the different diseases, health conditions and treatments are summarized in To calculate thresholds projecting how many IDD cases or related treatments would need to be avoided for an IDD prevention program to be cost-saving, we divided the total costs for an IDD prevention program in the German population by the costs for the different health conditions and related treatments.Direct costs per case for the different IDDs ranged from 167  to € 14,202  per year. Including costs due to productivity loss, costs raged from 167  to € 17,497  per year. Costs for thyroidectomy and radioiodine therapy were € 4184 and € 3118, respectively. Direct costs for IDD diagnosis and treatment and costs including productivity loss are summarized in In the German population (around 82.17 million persons), total annual costs for implementing an IDD prevention program including iodine fortification of salt would amount to € 8,152,812 in the first year and € 8,002,812 annually in the years after implementation. Costs for an IDD prevention and monitoring program are summarized in To be cost-saving, the IDD prevention and monitoring program would need to prevent 26,000 cases of hyperthyroidism or 29,200 cases of hypothyroidism or 37,900 cases of goiter/thyroid nodules or 560 cases of intellectual disability or 8700 cases of spontaneous abortions per year. It would also be cost-saving if it could prevent 1900 thyroidectomies or 2600 radioiodine therapies per year. If an IDD prevention and monitoring could prevent a combination of different cases of IDDs, even less saved cases are needed of a single disorder or treatment for a cost-saving program.We conducted a comprehensive cost analysis to evaluate costs for prevention, monitoring and treatment of IDDs in Germany, which provides important data for further health technology assessments. We also performed threshold analyses estimating how many cases of IDDs or related treatments would need to be prevented for an IDD prevention program to be cost-saving. To our knowledge, this is the first comprehensive and detailed cost analysis of IDD prevention, monitoring and treatment in a European country, including all potentially relevant consequences of iodine deficiency. Although there are few economic evaluations of IDD prevention programs , 47, 48,A study conducted in Australia and New Zealand estimated the costs of preventing one person from having an urinary iodine concentration below 50 µg/L (100 µg/L) to be A$ 104.35 (A$ 1.81) per prevented case in Australia and NZ$ 15.30 NZ$ 1.46) per prevented case in New Zealand and concluded that the costs appear small compared with the potential benefits  . Another per prevet al. estimated the cost of thyroid disorders in Germany .Cost data collected through prospective data collection processes  might also result in more reliable cost estimations. We could not analyze the detailed costs of every single resource used in the prevention, diagnosis and treatment of the different IDDs. We used average amounts of different resources such as flat rates, mean hospital days or mean days of illness with absence from work. Costs could be over- or underestimated depending on the actual use of the different resources per case. However, resource utilization was revised and adapted by German clinical experts and compared to diagnostic and therapeutic procedures conducted in clinical trials and prospective cohort studies.We also used expert estimations for different proportions and probabilities if we could not obtain these data from any study or registry (e.g. the proportion of patients with goiter or benign thyroid nodules receiving a scintigraphy). Therefore, the cost-analysis should be updated as soon as those data are available.For the treatment and care of patients with intellectual disability, we used data collected for a cost analysis in the Netherlands and transformed them into 2018 Euro. However, treatment and care for individuals with intellectual disability in the Netherlands may differ from those in Germany. Therefore, costs might be under- or overestimated, depending on the differences in resource utilization in both countries. However, the study used a top–down approach with a health-care perspective similar to our methods.Our threshold analyses only analyzed the effectiveness of an IDD prevention program on single outcomes such as the incidence of goiter or thyroidectomies. However, we expect a simultaneous effect of an IDD prevention program on multiple diseases, treatments and outcomes. Therefore, the estimated thresholds for each single disease or treatment are just upper limits and the real thresholds for an IDD prevention program to be cost-saving should be much lower.The threshold analyses indicate that the benefits of an IDD prevention program outweigh the harms of such a program based on current literature. However, there are also studies questioning the efficacy of IDD prevention programs , 71 or eBased on our cost analysis, an IDD prevention program potentially saves costs, if a specific minimum amount of iodine deficiency-related health conditions and/or treatments per year could be avoided by the prevention program and the overall benefits of such a program would outweigh adverse outcomes caused by such a program . However, in order to recommend the implementation of an IDD prevention program, epidemiological and long-term effectiveness, as well as safety data, need to be considered in a full health economic evaluation based on a decision-analytic model. The results of our cost analysis can be included in such decision analyses to evaluate the overall long-term effectiveness, benefit-harm balance and cost-effectiveness of an IDD prevention program.The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.This work was supported by EUthyroid. The project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement number 634453.


Purpose: To assess the use of calcium sulphate (CS) beads in the management of knee and hip periprosthetic joint infections (PJI) in terms of outcomes, complications and re-infection rates.Methods: A search of NICE healthcare database advanced search (HDAS) was conducted from its year of inception to October 2019 with the keywords: “Calcium Sulphate Beads” or “Calcium Sulfate Beads” or “Antibiotics beads” or “Stimulan” and “Arthroplasty” or “Hip Replacement” or “Knee Replacement” and “Periprothetic joint infection” or “Debridement, Antibiotics and implant retention” or “Revision”. A quality assessment was performed using the NIH study Quality Assessment Tool for case series.Results: Out of relevant 74 articles, 5 articles met the inclusion criteria. Variable outcomes and success rates have been reported in most of the patients. A small number of wound discharges and heterotrophic ossification (HO) were reported, which are occasionally symptomatic. Hypercalcemia is identified as a potential risk with the use of CS beads especially with doses over 40 cc per operation. The influence of CS beads on reinfection rate is reported in 4 out of 5 articles. Due to the case-mix and heterogenicity of the patients involved and the causative microorganism reported as well as varied procedures are undertaken including one and two stages revision and Debridement, Antibiotics and implant retention (DAIR), the influence of CS beads varied from high success to poor outcome. The poor outcome is higher if the primary procedure for the management of hip and knee PJI is DAIR rather than full revision.Conclusions: The use of CS beads in the treatment of PJI is a useful technique in delivering high doses of antibiotics locally. A favourable outcome is reported when antibiotics loaded CS is used as an adjuvant to revision procedure for PJI in hip and knee arthroplasty compared to its use as an adjuvant to DAIR procedure. There has been an increase in complications when higher volumes of beads are used, especially in subcutaneous structures and in high-risk patients. Another possible theoretical and unreported complication of CS beads is accelerating the wear rate in the artificial joint due to the possibility of causing abrasion to the bearing surfaces. The current evidence is not enough to indicate the superiority of antibiotic-loaded CS beads as an adjuvant for the treatment of PJI in Hip and Knee arthroplasty. Staphylococcus aureus in 12-47%. Streptococci and Enterococci are diagnosed in around 10% while gram-negative bacteria such as Escherichia coli are less than 5% PJI is a devastating complication for patients and results in greatly increased costs of care for both healthcare providers and patients. PJI occurs in around 1-2% of primary total hip and knee arthroplasties The use of CS in orthopaedics has therefore been increasing, both as a bone void filler and as a delivery agent for antibiotics in arthroplasty, chronic osteomyelitis, open fractures and combat injuries The Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) methodology guidance was employed The search threads used resulted in 74 articles Figure .Screening the references, we identified two more relevant articles. Eventually, out of the 76 papers, 5 papers were found to meet the inclusion criteria Figure .5 articles Different outcome measures were reported in these papers All authors in these papers used Stimulan from Biocomposites either from Keele in the UK or Wilmington in North Carolina, United States. The kit includes 10 cc 20 g) of calcium sulphate hemihydrate powder, a pre-mixing solution bulb, pellet mould and spatula. The mould produces three sizes of bead . One gram of vancomycin powder is mixed with each 10 cc of calcium sulphate in the mixing bowl and 240 mg of liquid tobramycin (40 mg/ml) is added. The ingredients were mixed for 30 seconds until “doughy” and the resulting paste was applied to the moulds using the spatula and allowed to set for 10 to 15 minutes in a typical theatre temperature of 16°c to 17°c. Kallala 2015 g of calThe authors implanted the beads around the infected joint and deep tissues and avoided placing beads in subcutaneous layers Table .The radiologically confirmed resorption time of calcium sulphate beads ranged between 3 to 12 weeks as reported by Kallala 2015 Kallala 2015Flierl et al. The authors used Harwin and Brooker classifications to describe the HO. Flierl et al. McPherson et al. According to this review, authors have reported variable results. With a 48% re-infection rate, Flierl et al. 3.8% had a persistent wound discharge which was managed conservatively in most of the cases and occasionally required surgical interventions. In a report by Menon et al. We also noticed that Lum et al. The use of CS beads in the treatment of PJI is a useful technique in delivering high doses of antibiotics locally. CS beads can accommodate both heat and non-heat stable antibiotic as well as antifungal, in addition to its complete absorbability. Variable outcomes and success rates have been reported in the reviewed papers. A favourable outcome was reported when antibiotics loaded CS beads are used as an adjuvant to revision procedures compared to DAIR procedures. A small number of wound discharges, heterotrophic ossifications and hypercalcaemia cases were reported, and majority were asymptomatic. There has been an increase in complications when higher volumes of beads are used, especially in subcutaneous structures and in high-risk patients.


Failing to account for the resources required to successfully implement public health interventions can lead to an underestimation of costs and budget impact, optimistic cost-effectiveness estimates, and ultimately a disconnect between published evidence and public health decision-making.We developed a conceptual framework for assessing implementation costs. We illustrate the use of this framework with case studies involving interventions for tuberculosis and HIV/AIDS in resource-limited settings.Costs of implementing public health interventions may be conceptualized as occurring across three phases: design, initiation, and maintenance. In the design phase, activities include developing intervention components and establishing necessary infrastructure . Initiation phase activities include training, initiation of supply chains and quality assurance procedures, and installation of equipment. Implementation costs in the maintenance phase include ongoing technical support, monitoring and evaluation, and troubleshooting unexpected obstacles. Within each phase, implementation costs can be incurred at the site of delivery (“site-specific” costs) or more centrally . For interventions evaluated in the context of research studies, implementation costs should be classified as programmatic, research-related, or shared research/program costs. Purely research-related costs are often excluded from analysis of programmatic implementation.In evaluating public health interventions in resource-limited settings, accounting for implementation costs enables more realistic estimates of budget impact and cost-effectiveness and provides important insights into program feasibility, scale-up, and sustainability. Assessment of implementation costs should be planned prospectively and performed in a standardized manner to ensure generalizability. Contributions to the literatureEconomic evaluations have long been used to prioritize health interventions in resource-limited settings. However, these analyses often fail to capture the resources required to successfully implement interventions and thus may greatly underestimate the actual economic costs of the intervention.Most existing studies in the health economics literature in the context of implementation science are retrospective or ‘ex-post’ in nature; methods and examples of evaluating these costs prospectively are limited, particularly in resource limited settings.Using recent examples from the literature, we highlight the importance and potential magnitude of costs associated with implementation of public health interventions in resource constrained settings.We also provide a conceptual framework that details the types of economic resources utilized during the design, initiation, and maintenance phases of implementation and the implications of neglecting implementation costs in evaluating the cost-effectiveness of interventions in resource-limited settings.Such a framework provides greater insight into the need for implementation costing practices for more accurate economic evaluation and budget impact analysis as well as insights into program feasibility, scale up and sustainability.Economic evaluations are frequently used to inform prioritization of health interventions and resource allocation for public health . UnfortuTo illustrate, the Avahan initiative was a complex intervention to prevent HIV transmission across six Indian states that included peer outreach, clinical services, condom distribution, safe needle exchange, and community mobilization . A cost-Another illustration of the importance of implementation costing is the recently published PopART HPTN 071) trial of universal testing and treatment for HIV in South Africa and Zambia . This tr1 trial oTo provide decision-makers with an honest appraisal of the potential costs and benefits of any health intervention, it is critical to document the full range of costs required for programmatic implementation, including the costs of intervention design and local adaptation, initiation and scale-up, and maintenance/sustainability. While consideration of implementation costs is universally relevant, these costs may be proportionally more important in settings where resources are highly constrained. Here, we use the example of peripheral diagnostic testing for TB as a case study to illustrate the importance of evaluating costs throughout the implementation process and to propose a structured method for investigating these costs.An estimated 13–18% of patients diagnosed with TB in Africa and Asia are lost to follow-up before starting treatment . Novel tSimilar to many consumer products, public health interventions can be conceptualized as having a “product life cycle,” with well-defined stages—design, launch, growth, maturity, and decline—from introduction to removal from the “market” . ConsideAs illustrated in Fig. In addition to the costs incurred at different phases of implementation, costs can be incurred at the site of delivery  or more centrally . Furthermore, health interventions are often evaluated in the context of research studies or evaluation programs, necessitating a differentiation between costs required for programmatic implementation and those incurred purely for the purposes of research or evaluation (the latter being less relevant to the cost-effectiveness of the intervention itself). Importantly, some degree of monitoring and evaluation is necessary for effective implementation and should therefore be included as a programmatic cost; however, the costs incurred by research studies and other knowledge generation activities often exceed this baseline requirement and may vary depending on the type of intervention being considered. As such, it is important to prospectively monitor and categorize costs as purely research-related  versus necessary for programmatic implementation (even if those costs represent “research” activities) to facilitate subsequent analyses.Table Implementation costs incurred during the design and adaptation of health interventions may include crafting of appropriate policies and algorithms, obtaining political and administrative approvals, developing necessary infrastructure, engagement of relevant stakeholders, and pilot testing in the local context. Where these costs are large and must be incurred before scale-up is certain, they may represent a majority of the total cost of the intervention. For example, in a trial of mobile Health-facilitated home-based contact investigation for TB in Uganda, a software package was required to generate short messaging service (SMS) reminders to facilitate home-based screening, incurring large up-front costs for an initial service contract and adaptation to the Ugandan telecommunications infrastructure . While tTM TB LAM Ag assay (LF-LAM) is a simple dipstick-based diagnostic test for TB that costs less than $2 per test kit and requires minimal operator time [.As interventions are initially rolled out and scaled-up, many types of implementation costs  are incurred up-front. These fixed costs often do not vary with the level of service output. The contribution of up-front costs to the overall cost and cost-effectiveness of a program may vary considerably across implementation sites depending on each site’s operational and infrastructural capacity and implementation outcomes such as program reach  . These ctor time . Cost-eftor time . Howevertor time . After itor time . Such unAlthough implementation costs are often proportionally lower during the maintenance phase Fig. b, they sThe examples above help illustrate the importance of considering costs at each stage of the implementation process. This structured approach to costing of the implementation process suggests three priorities for improving cost-effectiveness analyses of health interventions in resource-limited settings.First, cost-effectiveness analyses should not rely entirely on budgetary information but should explicitly consider activities and resources required for successful implementation. Examples of approaches for such implementation costing include collection of routine activity logs of implementing staff, structured discussions with field personnel to enable health economists to understand the extent of resources required for major implementation activities , and ongoing documentation of specific challenges in implementation .Second, implementation cost data should be classified by resource type, key activities, phase of implementation . While retrospective estimation of implementation costs is often feasible, such estimates are often subject to recall bias and difficult to appropriately classify retroactively. Early involvement of health economists (with experience in empiric costing activities) can therefore be very useful in evaluating the costs of implementation.One final consideration is whether detailed measurement of implementation costs is justified for a particular implementation research study. In making this assessment, investigators should consider two questions. First, if the intervention is found to be effective, is implementation likely to be influenced by considerations of cost-effectiveness and/or budget impact? Second, are there sufficient uncertainties in the costs of implementing the intervention that an empiric estimation of costs is warranted (as opposed to simply using pre-existing cost estimates from the literature)? In most cases, the answer to the first question will be yes, and the answer to the second question will be no, meaning that an empiric estimation of costs is scientifically justified. When this is the case, investigators must then evaluate whether the budget exists to measure these costs and  whether other scientific questions are more pressing.In summary, we argue that the costs of implementing health interventions in resource-limited settings are often very substantial, but generally neglected in economic evaluation. We provide a framework for effectively conceptualizing and prospectively measuring these costs, which—if incorporated appropriately—can improve the linkage between published results of cost-effectiveness analyses and the realities of implementation in the field.


Using soft x-ray absorption (XAS) and resonant inelastic x-ray scattering (RIXS) spectroscopy in transmission and reflection geometries, we demonstrate that the x-ray emission from Mn 3s-2p core-to-core transition (3sPFY) seen in the RIXS maps can represent the bulk-like absorption signal with minimal self-absorption effect around the Mn L3-edge. Similar measurements were also performed on a reference LSMO film grown on the SrTiO3 substrate and the agreement between measurements substantiates the claim that the bulk electronic structures can be preserved even after the freestanding treatment process. The 3sPFY spectrum obtained from analyzing the RIXS maps offers a powerful way to probe the bulk electronic structures in thin films and heterostructures when recording the XAS spectra in the transmission mode is not available.We have successfully fabricated high quality single crystalline La The proximity of the energy scales of these interactions and their couplings to lattice, charge, spin, and orbital degrees of freedom permit the tuning of disparate ground states through a variety of perturbations to induce colossal responses, thereby offering great potentials in multifunctional applications4. Of particular interest in these manganites is the wide-bandwidth La1-xSrxMnO3 (LSMO) with x = 0.3, whose fully spin-polarized metallic ground state at ambient condition (Curie temperature Tc ~ 360 K) has been proposed as the candidate for the spintronic device applications6.Rare-earth/alkaline-earth manganese oxides R8, polymers9, or even in the freestanding form to overcome this substrate constraint13, and it will be of paramount importance to verify that these TMOs retain their bulk electronic properties after the meticulous growth and preparation processes. In this letter, we report the soft x-ray spectroscopic studies on crystalline La0.7Sr0.3MnO3 in freestanding form (LSMO-FS) and on the SrTiO3 (STO) substrate . We show that these manganites have similar bulk electronic structures and demonstrate that the Mn 3s-2p core-to-core transition (3sPFY) besides the proposed inverse partial fluorescence yield (iPFY) from oxygen emission can be used to reliably obtain such information.However, to facilitate the incorporation of these manganites into actual devices, their form factors need to be flexible and integrable into different platforms. Although manganites can be routinely grown on top of oxide substrates using techniques such as pulsed laser deposition (PLD) and molecular beam epitaxy (MBE) to attain the precision of layer-by-layer growth, these hard substrates nevertheless present challenges to certain applications that require flexibility and bendability, notably in the wearable electronics. Recently, it has been shown that some transition metal oxides (TMOs) can be fabricated on flexible supports like mica0.7Sr0.3MnO3 (100 nm) and sacrificial YBa2Cu3O7 (25 nm) layers were epitaxially deposited on the (001)-oriented SrTiO3 substrate by PLD to form the LSMO/YBCO/STO structure. After coating the top LSMO layer with the protective Poly (methyl methacrylate) (PMMA), the assembly was immersed in light hydrochloric acid to etch away the YBCO layer, releasing the PMMA/LSMO into the solution that can be transferred onto rigid supports for further process to remove the PMMA coating. The reference LSMO/STO sample was fabricated following the same PLD process. More details about the fabrication and characterizations of the as-grown and similar LSMO-FS films can be found elsewhere13.The freestanding LSMO thin film (LSMO-FS) was prepared by the method illustrated in Fig. b-axis lattice constant is ~ 3.908 ± 0.018 Å (pseudo cubic notation). The x-ray diffraction (XRD) θ-2θ scan along the (00L) direction in Fig. c-axis lattice constant estimated from the XRD curve is 3.887 ± 0.016 Å (pseudo cubic notation), in agreement with the bulk parameters14. The temperature-dependent resistance was measured using the four-terminal method, see Fig. C = 369 K)15. Figure We carried out a series of characterizations on the freestanding LSMO on the Si wafer support to validate the film quality before performing the x-ray measurements on those that are supported by Cu mesh (to permit transmission geometry measurements). The atomic force microscope (AFM) was used to examine the morphology of LSMO-FS film. The AFM image in Fig. 13, it is important to verify that they have similar electronic structures; or equivalently, the electronic structures remain minimally perturbed after the freestanding treatment process. To address this issue, we resort to the soft x-ray absorption (XAS) and resonant inelastic x-ray scattering (RIXS) spectroscopy at Mn L2,3-edge, which correspond to the on-site transitions from Mn 2p63dn→2p53dn+1. The measurements on LSMO-FS and LSMO/STO were carried out at BL 8.0.1 at the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory, using the qRIXS endstation16. A high throughput modular x-ray spectrometer placed at 140° back-scattering angle was used to record the RIXS spectra when the excitation photon energy was scanned across the Mn L2,3-edge (the compilation of these RIXS spectra is termed the RIXS map)17. During the measurements, the LSMO-FS and LSMO/STO films were placed in the normal incidence geometry with incident x-ray beam less than 10° from the sample surface normal. There was no specific orientation alignment for LSMO-FS; but for the reference LSMO/STO, the Mn–O bond direction was in the horizontal scattering plane. The photon polarization was kept in the horizontal scattering plane (π-polarization) and the beamline energy resolution was set to 0.3 eV. The combined energy resolution (beamline plus spectrometer) for RIXS measurements determined from the full width at half maximum (FWHM) of the elastic peak was 0.4 eV. The sample-to-ground drain current was used for the total electron yield (TEY) mode measurement, whereas a GaAsP photodiode was used to record the total fluorescence yield signal (TFY) at two angles: 0° for the transmission geometry  and 150° back-scattering angle for the reflection geometry . For normalization, the XAS spectra were first subtracted by a linear background from fitting their respective pre-edge region below the Mn L3 edge, and then the edge jump that is 15 eV above the Mn L2 edge was normalized to 1. To calculate the branching ratio, BR = I(L3)/(I(L2) + I(L3)) where I(L3) and I(L2) are the integrated spectral area over the Mn L3 and L2 edges, respectively, the edge jump was further removed from the XAS spectra by subtracting an arctangent function18. For the nomenclature of XAS spectra, they will be called in the following fashion: (geometry)-(mode)-XAS; for example, trans-XAS means the XAS spectrum recorded in the transmission geometry. All data shown here were recorded at 300 K.Although the transport properties and structural parameters of LSMO-FS film are consistent with the reference LSMO/STO filmL3 edge at 642 eV. From this figure, one can see that the TEY-XAS spectra of both films are very similar except around the pre-edge region (639.6 eV) where LSMO-FS exhibits a more pronounced spectral feature  than the TEY channel (on the order of few nm)19, the similarity between the ref-TFY-XAS spectra implies very similar bulk electronic structures between these films. Although the ref-TFY-XAS spectrum is more bulk sensitive, it suffers from the strong self-absorption effect at Mn L3 edge that renders the branching ratio BR much smaller than the TEY-XAS spectrum20. This distorted BR can be problematic if used to estimate the strength of local spin–orbit coupling (SOC)21. The self-absorption effect also prohibits us from claiming that these films have similar bulk electronic structures. On the other hand, the enhanced pre-edge feature in the TEY-XAS spectra is known to originate from the Mn3+ state23, and the surface sensitivity of TEY-XAS spectra indicates more excess oxygen vacancies at the surface of LSMO-FS film, presumably caused by the wet chemical etching process.Figure 3+ feature is much reduced in the trans-XAS spectrum. In addition, its BR is almost the same as the TEY-XAS spectrum. Such observation reflects the general consensus that both TEY-XAS and ref-TFY-XAS spectra only partially represent the true bulk absorption signal. It has been suggested that besides the aforementioned TEY and TFY XAS, other x-ray and photoelectron emission channels can provide complimentary information for the bulk electronic structures27. With the high throughput soft x-ray spectrometer, we are able to record the RIXS spectra with the excitation photon energy spanning across the Mn L2,3-edge in a reasonable time frame (9 min per RIXS spectrum and 0.15 eV incident photon energy step). The resulting RIXS map for LSMO-FS film is shown in Fig. The LSMO-FS film allows us to perform the XAS measurement in the transmission mode (trans-XAS), which by definition is the true bulk absorption. Interestingly, if comparing the trans-XAS spectrum with the TEY-XAS and ref-TFY-XAS spectra of LSMO-FS, one can see that the surface-related Mnp→1s , Mn 3s→2p  and Mn 3d→2p ), respectively. By integrating the emission intensity within individual energy windows, we can obtain different partial fluorescence yield (PFY) spectra as shown in Fig. 28. In this figure, we also overlay the TEY-XAS (red), trans-XAS (thin black), and ref-TFY-XAS (pink) spectra of LSMO-FS film for comparison. We see that all PFY and TFY spectra show a much weaker pre-edge feature around 639.6 eV, consistent with their bulk sensitivity with photon emission. Although these spectra exhibit subtle variation around the pre-edge region due to their varying degree of probing depth and self-absorption effect30 the biggest contrast is in their BR value , see Table dPFY still experiences a strong self-absorption effect like TFY. iPFY was previously proposed to best represent the bulk absorption; however, for the case of LSMO-FS, its BR value still deviates from the nominal value. Furthermore, the iPFY spectrum also displays poor statistics likely due to the lower cross-section and excessive oxygen vacancies in this sample. Comparison in Fig. sPFY bears the closest resemblance to the trans-XAS spectrum with the nearly identical BR, implying that it is the most direct probe for the bulk electronic structures besides trans-XAS spectrum. In addition, we also note that 3sPFY channel has a higher yield than the iPFY26 such that it has better statistics for very thin films.In this RIXS map, three emission features can be clearly identified. From left (lower emission energy) to right (higher emission energy), they correspond to the following transitions: O 2sPFY spectrum gives the best agreement with the trans-XAS spectrum. Interestingly, the 3sPFY of LSMO/STO has an even smaller pre-edge spectral weight, which is consistent with the findings from the TEY-XAS spectra in Fig. sPFY spectrum from both LSMO-FS and LSMO/STO substantiates the claim that the LSMO-FS film retains its bulk electronic structures even after the freestanding treatment process, although its surface may possess additional oxygen vacancies that warrants further fine-tuning in the treatment to reduce them.With this finding, we now look at the PFY spectra produced from the LSMO/STO RIXS map (data not shown) in Fig. sPFY technique to study the electronic structures has been explored theoretically25 and experimentally26. Miedema et al.25 pointed out that although 3sPFY spectrum can be representative to the true XAS spectrum, certain care must be paid to the experimental configuration. For example, with the capability to analyze the photon polarizations on both incident (polarization control on the X-ray source) and emission side (with polarimeter on the spectrometer) and a proper placement of spectrometer to enhance the contrast, one may detect the linear dichroism between the parallel and crossed polarization channels. This can be advantageous for studying the respective orbital contributions in the resulting 3sPFY spectrum; however, it can lead to certain error in estimating quantities such as BR. In that regard, one needs to perform the polarization averaging to correct this error. In practice, this can be accomplished by averaging the incident  and emission (not using the polarimeter) polarizations and varying the sample orientations if needed . Experimentally, Busse et al.26 showed that 3sPFY yields a closer BR value to the nominal value compared with other techniques such as 3dPFY, iPFY, and TFY in the reflection geometry; however, the 3sPFY BR value still deviates from the nominal one. This is also seen in the current study, and the discrepancy is likely due to several factors such as saturation (much weaker in the 3sPFY channel), the fixed measurement geometry, the treatment of spectral background, etc. Nevertheless, these factors do not impact the main conclusion that 3sPFY is a suitable bulk probe for electronic structures than ref-TFY-XAS or 3dTFY.The potential of using 3sPFY spectra to obtain the bulk absorption information, these maps also reveal contrasting Coster-Kronig (C-K) transitions between 3s-2p and 3d-2p channels31. The C-K transitions at 3d-2p channel have been examined in the Co2+ systems32, but to our knowledge, this is the first report at 3s-2p channel in solid state TMO. Notably, besides the contrasting self-absorption effect that complicates the measurements33, the multiplets in the 3d-2p transition are dominating over the C-K related features while the situation is reversed in the 3s-2p transition. Such distinct behavior is related to different final state configurations when the photon energy is tuned to the L2 edge to activate the C-K transition. Taking the RIXS map in Fig. d-2p channel (~ 650 eV emission energy), the diagonal and vertical features correspond to (neglecting the charge-transfer) the transitions 3d4→3d5L2→3d4 and 3d4 →3d5L2→(C-K) 3d5L3→3d4, respectively (L denotes the core–hole). On the other hand, around the 3s-2p channel (~ 570 eV emission energy), they correspond to 3d4→3d5L2→ 3s13d5 and 3d4→3d5L2→(C-K) 3d5L3→x3s13d5, respectively. Because of this difference, the integrated spectral weight over respective emission energy window can be used to gauge the degree of covalency and study the decay rates of different channels, such as 2p-3d, L3-L2 Auger, and 2p-3s transitions. In addition, the final state configuration of 3s13d5 can be used to explore the internal s-d interaction. Further studies on manganites will be reported elsewhere.Besides using the RIXS maps to produce the 30.7Sr0.3MnO3 thin film and carried out soft x-ray XAS and RIXS measurements on this LSMO-FS film and a reference LSMO/STO film. We show that the bulk electronic structures of LSMO-FS can be well preserved after the wet chemical etching process to produce the freestanding film despite having more surface oxygen vacancies. Although a similar use of 3sPFY spectrum derived from RIXS maps to study the bulk electronic structures can be found in operando chemistry with using primarily a jet system to deliver the dilute targets, our work in LSMO single crystal constitutes an extensive evaluation of the pros and cons of different TFY, PFY, and TEY mode of XAS in correlated oxides. The use of 3sPFY mode to obtain the bulk-like XAS spectrum is of particular attractive for certain class of materials. For example, heterostructural oxides grown on (or transferred to) flexible support can be used for next generation electronic devices, and understanding the electronic structures of buried layers can be the target application for this technique8. Note that due to the minimal self-absorption in 3sPFY mode relative to ref-TFY-XAS and 3dPFY, one expects that this technique will be particular useful for elements like Cr, Mn, Fe in oxides whose L absorption edge is close to O K-edge. Besides the practical aspect of this technique, we show that the improved energy resolution in RIXS spectroscopy in this work allows us to examine the spectral features associated with the Coster-Kronig transition and offers an attractive approach to determine its strength over a wide class of materials that are inaccessible to photoelectron spectroscopy.In summary, we have successfully prepared the freestanding La


Asfarviridae, Bunyavirales, Flaviviridae, Orthomyxoviridae, Reoviridae, Rhabdoviridae and Togaviridae) is covered.Arthropod-borne viruses (arboviruses) are primarily maintained in nature in transmission cycles between hematophagous arthropods and vertebrate hosts, but an increasing number of arboviruses have been isolated from or indirectly detected in the urogenital tract and sexual secretions of their vertebrate hosts, indicating that further investigation on the possibility of sexual transmission of these viruses is warranted. The most widely recognized sexually-transmitted arbovirus is Zika virus but other arboviruses, including Crimean-Congo hemorrhagic fever virus and dengue virus, might also be transmitted, albeit occasionally, by this route. This review summarizes our current understanding on the ability of arboviruses to be sexually transmitted. We discuss the sexual transmission of arboviruses between humans and between vertebrate animals, but not arthropod vectors. Every taxonomic group known to contain arboviruses ( Bunyavirales and the families Flaviviridae and Togaviridae. All other known arboviruses belong to the families Asfarviridae, Orthomyxoviridae, Reoviridae and Rhabdoviridae.Viruses that cycle between blood-feeding arthropods and susceptible vertebrate hosts are known as arthropod-borne viruses (arboviruses) . ArbovirAlthough arboviruses primarily cycle between arthropod vectors and vertebrate hosts, other modes of transmission have been recognized ,2,3. SomFlaviviridae, can be sexually transmitted between humans, with dozens of sexually acquired infections now documented [Flaviviridae and, occasionally, arboviruses from the order Bunyavirales and family Togaviridae [Another mode of non-vector-borne arbovirus transmission, one that has gained considered interest in recent years, is sexual transmission. This recent interest is largely due to the discovery that Zika virus (ZIKV), a mosquito-transmitted virus belonging to the family cumented ,5,6. Manaviridae ,12,13,14Asfivirus (family Asfarviridae) and only known DNA arbovirus [African swine fever virus (ASFV) is the sole member of the genus rbovirus . The virrbovirus . HoweverBunyaviridae, formerly divided into five genera, elevating it to the order Bunyavirales. This recently established order contains 12 families, three of which contain recognized arboviruses, and these are the Nairoviridae, Peribunyaviridae and Phenuiviridae [In 2017, the International Committee on Taxonomy of Viruses reclassified the family Nairoviridae includes Crimean-Congo hemorrhagic fever virus , a tick-borne pathogen that can cause fatal hemorrhagic fever in humans [The family n humans . Human in humans ,20,21. In humans . Three mOrthonairovirus) replicates in lamb testis cell cultures, but evidence that it disseminates to the testes of sheep is lacking [Subsequent studies have provided evidence that CCHFV disseminates to the human genital tract. A case report describes the occurrence of epididymo-orchitis in a CCHF patient in Turkey . The pat lacking .Phenuiviridae includes Heartland virus (HRTV), severe fever with thrombocytopenia syndrome virus (SFTSV) and Rift Valley fever virus (RVFV). HRTV (genus Banyangvirus) is a tick-borne virus associated with sporadic cases of severe and sometimes fatal human disease in the United States [Banyangvirus) is a tick-borne virus that causes life-threatening human disease in Asia [Phlebovirus) is a mosquito-transmitted pathogen of ruminants and humans in sub-Saharan Africa and the Arabian Peninsula [Phenuivirus RNA/antigens in the patients’ semen or testes was due to the severity of disease or suggestive of the normal potential for sexual transmission of these viruses.The family d States ,27. A pod States . Congest in Asia . SFTSV R in Asia . RVFV , Akabane virus (AKAV) and Aino virus (AINOV), three Culicoides-transmitted viruses that cause pregnancy loss and congenital defects in ruminant livestock [The family ivestock ,34. Infeivestock ,36. Otheivestock ,37,38. Sivestock ,40.AKAV could not be isolated from the semen of experimentally infected bulls . The virFlaviviridae contains four genera, including the genus Flavivirus. Most viruses belonging to this genus are recognized arboviruses, and examples include dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV) and ZIKV, all of which are mosquito-transmitted viruses of global public health significance [The family ificance ,45. Flavificance .It is well documented that ZIKV can be sexually transmitted between humans ,12,13,14The majority of sexually acquired ZIKV infections have been transmitted from male to female partners, although female-to-male and male-to-male transmission has been reported ,56,57,58The first isolation of ZIKV from semen was from a patient in Tahiti in 2013 . The virHuman germ cells are highly susceptible to ZIKV infection and replication, as determined in experiments where human testicular biopsy samples were infected ex vivo, with virus continuously produced for at least 59 days with no substantial decrease in titer ,89. ZIKVMouse models have been developed to study the sexual transmission, tissue tropisms and shedding kinetics of ZIKV ,91,92,93Several non-human primate (NHP) models have been developed to study ZIKV replication, transmission and pathogenesis ,112,113.Tupaia belangeri) after subcutaneous inoculation [Artibeus jamaicensis) [Evidence of ZIKV infection has been detected in the reproductive tracts of several non-murine, non-primate vertebrate species ,126,127.icensis) . A more icensis) . InfectiProbable sexual transmission of DENV occurred in South Korea in 2013 . The indDENV RNA was detected by quantitative RT-PCR in vaginal secretions from a female patient and the semen of a male patient for up to 18 and 37 days, respectively, after illness onset ,51. The In 2014, a woman in the United States developed neuroinvasive WNV disease, despite recalling no recent mosquito exposure . The autThere is evidence to suggest that other flaviviruses can infect the vertebrate reproductive tract and persist in sexual fluids. YFV RNA was detected in the semen of a patient at 21 days after illness onset . JEV perOrthomyxoviridae, including two that contain arboviruses. Viruses in the genus Quaranjavirus appear to cycle primarily between soft ticks and aquatic birds [Thogotovirus cycle between hard ticks and mammals [There are seven genera in the family ic birds . Exampleic birds . Viruses mammals . Example mammals .Reoviridae contains 15 genera. Viruses in three genera  replicate in both arthropod vectors and vertebrate hosts. The type species of the genus Orbivirus is bluetongue virus (BTV), a Culicoides-transmitted virus that affects ruminants [Orbivirus is epizootic hemorrhagic disease virus (EHDV). The virus cycles between Culicoides spp. midges and ruminants and often causes fatal hemorrhagic disease in white-tailed deer [The family uminants . There huminants ,146,147.uminants . Isolatiuminants ,149. Cowuminants ,150. Howuminants . In studuminants . BTV hasuminants . Ecchymouminants . Anotherled deer . EHDV RNled deer .Rhabdoviridae contain arboviruses, and these are Curiovirus, Ephemerovirus, Hapavirus, Ledantevirus, Sripuvirus, Tibrovirus and Vesiculovirus. Bovine ephemeral fever virus (BEFV) is the type species of the genus Ephemerovirus and a major pathogen of cattle and water buffalo. The virus was isolated from the semen of one of 12 experimentally infected bulls [Seven of the 20 genera in the family ed bulls . Ten heiAlphavirus (family Togaviridae) are mosquito-borne [The majority of viruses in the genus to-borne . The gento-borne . CHIKV Rto-borne . Virus iEastern equine encephalitis virus (EEEV) and Highlands J virus (HJV) are also alphaviruses. Both cycle between mosquitoes and passerines and are associated with disease in poultry ,160. EEEAnother mosquito-transmitted alphavirus is Venezuelan equine encephalitis virus (VEEV). A live-attenuated vaccine strain of VEEV replicated in the testes of golden hamsters after intratesticular, but not subcutaneous, inoculation . One of Evidence is accumulating that some arboviruses can replicate in the vertebrate reproductive tract and persist in sexual fluids, revealing the important need to investigate further the potential sexual transmission of arboviruses. It is well documented that ZIKV can be transmitted between humans through sexual contact and CCHFV and DENV are potentially transmitted, albeit probably occasionally, by this route ,21,48,49Evidence also exists that select arboviruses have the potential to be sexually transmitted between vertebrate animals. TBEV and ZIKV have been transmitted between laboratory mice by natural mating and SPONV RNA has been detected in the semen of infected mice ,96,134. The identification of sexually acquired arbovirus infections is often confounded in geographical regions where vector-borne transmission is known to occur and so care must be taken not to over-interpret data. Sexual transmission was considered the most likely route of infection for a woman with severe WNV disease, but the case occurred in the United States, a WNV endemic region . AlthougMany arboviruses have been detected, either directly or indirectly, in the vertebrate reproductive tract and in sexual fluids. Most experiments were performed using indirect virus detection assays, such as RT-PCR, but this alone is not sufficient evidence that a virus can be sexually transmitted. Virus isolation is more informative but also has its limitations because sexual transmission depends on various factors, including viral dose, the amount of infectious virus in the dose and route of inoculation. Reports of virus RNA in the reproductive tract and sexual fluids of severely ill patients should also be interpreted with care. It may not be all that surprising that viral RNA can enter normally protected sites/fluids like sperm in the testes and seminal fluid in the prostate and seminal vesicles, when tissue integrity is failing. In this situation, virus entry into these sites may not be directly suggestive of a new route of transmission but rather just a symptom of serious disease with the beginnings of organ system failure. Many non-arthropod-borne viruses have been detected in the vertebrate reproductive tract and in sexual secretions, but have never been associated with sexually acquired infections .Various considerations must be taken into account to conclude that an arbovirus can be sexually transmitted and, if it can be, whether it is a rare event or epidemiologically relevant. The importance of developing animal models to study sexual transmission cannot be overstated. Animal models can be used to determine whether viable virus occurs and persists in the male and female reproductive tracts and in their sexual fluids, as done with ZIKV. If virus is detected, it is important to determine whether the amount of virus is enough to infect another host. Different routes of inoculation should be evaluated, including the vaginal and anorectal routes. Natural mating experiments designed to determine whether infected animals can transmit the virus to uninfected animals are especially informative. This work should be performed using infected males and uninfected females, along with reciprocal experiments. One advantage of studying the transmission dynamics of zoonotic diseases and diseases of veterinary importance is that natural animal hosts can be used in the experiments, although it is important that the work is performed under strict vector-protected conditions.With the exception of ZIKV, sexual transmission appears to have negligible impact on the epidemiology of arboviruses. However, it is possible that other arboviruses could acquire the ability to be efficiently sexually transmitted  or that there are undiscovered or poorly studied arboviruses that already possess this trait. If sexual transmission is shown to occur with an arbovirus, it is important to assess its epidemiological relevance and its contribution to transmission in both endemic and non-endemic regions. Sexual and arthropod-borne transmission may act synergistically in vector endemic regions to enhance transmission and maintain endemicity while sexual transmission may not be enough to sustain virus circulation in regions where arthropod vectors are not present. It is also important to determine whether the pathology and symptomatology of sexually acquired infections differ from mosquito-acquired infections. This information will assist in the diagnosis and clinical management of sexually acquired arbovirus disease. If an arbovirus is shown to be sexually transmitted between humans and if this mode of transmission is not a rare event, health authorities should recommend protected sex in populations living in or traveling to endemic countries, especially if there is an ongoing outbreak or epidemic. If efficient arbovirus transmission is shown to occur between livestock or poultry , resulting in substantial economic losses, producers need to modify their farming and disease control practices accordingly.To conclude, many sexually acquired ZIKV infections have occurred in the last decade, and evidence suggests that another major arbovirus pathogen, DENV, could be sexually transmitted, while other arboviruses possess the ability to infect the vertebrate reproductive tract. It is therefore important that additional work be performed to investigate the importance of sexual transmission in the epidemiology, spread and control of arboviruses.