The purpose of the present study is to determine the effect of sodium alginate’s viscosity (low and medium) and the presence of Pluronic® F-127 micelles on the swelling behavior of the prepared pure beads and those loaded with Pluronic® F-127 micelles. The Pluronic® F-127 nanomicelles have a size of 120 nm. The swelling studies were carried out at pH = 1.2 (simulated gastric fluid-SGF) for two hours and at pH = 6.8  for four more hours. The swelling of both low- and medium-viscosity alginate beads was minor at pH = 1.2, irrespective of the use of Pluronic® F-127 nanomicelles. At pH = 6.8, without Pluronic® F-127, the beads showed an enhanced swelling ratio for the first four hours, which was even higher in the medium-viscosity alginate beads. With the addition of Pluronic® F-127, the beads were dissolved in the first and second hour, in the case of the low- and medium-alginate’s viscosity, respectively. In other words, the behavior of the mixed hydrogels was the same during the swelling experiments. Therefore, the presence of Pluronic® F-127 nanomicelles and medium-viscosity sodium alginate leads to a higher swelling ratio. A model drug, acetyl salicylic acid (ASA), was also encapsulated in the mixed beads and ASA’s release studies were performed. In conclusion, the prepared systems, which are well characterized, show potential as delivery platforms for the oral delivery of active pharmaceutical ingredients and biopharmaceuticals.The anionic polymer sodium alginate, a linear copolymer of guluronic and mannuronic acids, is primarily present in brown algae. Copolymers are used in the sodium alginate preparation process to confer on the material strength and flexibility. Micelles and other polymeric nanoparticles are frequently made using the triblock copolymer Pluronic Alginates, a frequent ingredient in pharmaceutical formulations and drug delivery systems, is one such polysaccharide. Alginate is an anionic polysaccharide that is naturally found in brown seaweed. Its long linear polymer chain is composed of ked in a –4 configMultifunctional hydrogels based on alginates, chitosan, ulvan, and other polysaccharides have numerous applications in biomedical sciences, including drug delivery, wound dressing, tissue engineering, and temporary scaffolding for cells. Hydrogels and nanogels are particularly well suited for the controlled release of bioactive compounds, offering several advantages, such as high-physicochemical stability, responsiveness to external stimuli, high-loading efficiency, oral administration, and biocompatibility . Stimuli-responsive hydrogels, such as glucose-responsive, pH-responsive, and thermoresponsive hydrogels, have been utilized to achieve the controlled and sustained release of active pharmaceutical ingredients, proteins, and peptides ,3. The s2+, leading to the formation of a 3D network with a distinctive “egg-box” structure [2+ chelation were used to make nanocellulose and sodium alginate miscible, creating an interpenetrating network hydrogel via double crosslinking with considerable prolongation of the aspirin release period to around 60 h [pH-sensitive hydrogels can be prepared using polymers obtained from both natural and synthetic sources. Sodium alginate has a high capacity to chelate divalent cations such as Catructure . The algtructure . In one tructure . AccordiFurthermore, in order to advance the development of alginate beads, as delivery systems for water-soluble drugs and proteins administered orally, it is crucial to investigate the swelling behavior as the primary parameter. The swelling profiles of calcium alginate beads have been thoroughly examined and analyzed in the existing literature using media that imitate the physiological conditions of the gastrointestinal tract. At pH 1.2, which is similar to the stomach’s pH, there is a slight swelling, while at pH 6.8, which is similar to the small intestine’s pH, there is strong and extensive swelling. Alginate hydrogels are effective as carriers for the oral administration of chemotherapeutics, water-insoluble medications, and proteins/peptides because of this pH-responsive behavior. Other materials, such as Pluronics, can be employed in the preparation of hydrogels to alter their swelling properties [® F-127 in the polymeric network reduces the swelling of the hydrogel beads and this might be attributed to the hydrophobic nature of micelles leading to minimum swelling [Pluronics, a group of block copolymers with both hydrophilic and hydrophobic components, have gained widespread use in medical and pharmaceutical applications, due to their remarkable biocompatibility, safety, and low immunogenicity. Pluronic F127, also named Poloxamer 407, is an FDA-approved temperature-sensitive hydrogel that has been widely used for drug delivery purposes and wound healing applications. The temperature-dependent gelation and micellization properties of these polymers also make them ideal as matrices and carriers, allowing for the efficient transport of active pharmaceutical ingredients (APIs), particularly those that are sparingly soluble in water ,9. Micelswelling .2, or the method of bead formation and subsequent drying. For instance, a combination of drugs, theophylline, and theobromine, using CaCl2 0.27 M for the formation of beads, has been already used with the aid of a Braun Mekungen infusion pump [Similar methods have already been investigated, although disparities exist in the medicinal product integrated within the beads, the concentration of CaClion pump . Alternaion pump .The drug that was used in this study is acetylsalicylic acid (ASA). ASA or aspirin belongs to the family of salicylates and is used as an anti-inflammatory agent. Yet, nowadays it is also used as an anticoagulant drug against heart attacks and cancer. ASA is administered mainly orally and its absorption depends on many factors, such as the dosage form, the gastric emptying time, and the gastric pH. ASA is hydrolyzed to salicylic acid in the plasma and salicylate anion is metabolized in the liver. When it is delivered orally, it is absorbed both in the stomach and in the small intestine. Through passive diffusion, the non-ionized acetylsalicylic acid passes through the stomach, whilst its ionized form is absorbed ideally at pH = 2.15–4.1. However, the absorption of ASA takes place at a greater rate at the pH of the intestine . ASA worHowever, aspirin can also cause irritation and damage to the mucosa of the stomach and intestine, leading to gastrointestinal bleeding and ulcers. To mitigate this, enteric-coated aspirin has been developed, which passes through the stomach and then dissolves in the small intestine, reducing the risk of gastric irritation .® F-127, in order to evaluate their characteristics and release properties.The scope of this investigation is to compare the physicochemical behavior of low- and medium-alginate capacities as delivery platforms with pH-responsive release properties . For thiD-mannuronic and L-guluronic acid chains and calcium chloride dihydrate (CaCl2·2H2O), as well as Pluronic® F-127 (in the form of white microbeads) were purchased from Sigma-Aldrich  and used as received. The simulated gastric  and the simulated intestinal fluids  were prepared as described in the US Pharmacopeia.Low-viscosity (250 cps of 2% solution) and medium-viscosity (>2000 cps of 2% solution) alginic acid sodium salt (NaAlg) salt from brown algae, composed of ® F-127 were dissolved in purified water. The colloidal concentration of Pluronic® F-127 is 100 mg/mL, 10% w/v. The dilution took place at room temperature under mild agitation. The size, size distribution, and zeta potential  of the micelles were evaluated by dynamic and electrophoretic light-scattering techniques. Measurements were performed at a detection angle of 90° and at 25 °C in a photon correlation spectrometer  and analyzed by the CONTIN method (Horiba software 2009–2018). For each system, three sets of three light-scattering measurements were collected and the results were averaged. Specifically, 100 μL of dispersion was diluted to 3 mL of HPLC-grade water, and the zeta potential was measured at room temperature at 633 nm. The zeta-potential values were calculated from electrophoretic mobilities, μE, by using the Henry correction of the Smoluchowski equation:ε0 is the permittivity of the vacuum, rε is the relative permittivity, a is the particle radius, k is the Debye length, and n is the viscosity of HPLC-grade water. All the experiments were run in triplicate. The zeta potential of the micelles was measured at the pH of HPLC-grade water, which is 5.5 at 25 °C.The appropriate amounts of Pluronicw/v of low (LV) and medium viscosity (LV) were prepared in order to synthesize the beads. The encapsulation of Pluronic® F-127 micelles was achieved by mixing the sodium alginate solution with the Pluronic® F-127 solution in a 9:1 weight ratio. Alginate solution droplets were introduced into a 0.1 M CaCl2 solution using a syringe pump, resulting in the formation of separate alginate beads for each alginate, all with the same flow rate. The beads were then left for 30 min in the CaCl2 0.1 M solution in order to be properly formed. Last, the beads were rinsed with water to remove the excess CaCl2 and were dried for 24 h at 80 °C.The protocol used has been previously described in the literature . Sodium Thermogravimetric analysis (TGA) was performed with a TA Instruments Q500 TGA Analyzer in the temperature range of 30–900 °C at a heating rate of 10 °C/min under air atmosphere.The morphological characterization of the different alginate beads was conducted using a Phenom-World desktop scanning electron microscope (SEM)  with a tungsten filament operating at 10 kV and a charge reduction sample holder. The samples were observed after sputter coating on a Mini Sputter Coater/Glow Discharge System SC7620 .The swelling studies took place in two different dispersion media, in accordance with the reported protocols ,13.t = 0, being in the dry state after 48 h in the dryer and then in regular time intervals at the different pH solutions, pH = 1.2 (SGF medium), and then at pH = 6.8 (SIF medium). Accurately weighed amounts of beads (ranging from 0.7 to 1.2 mg) were immersed in a 25 mL SGF solution for two hours and then transferred into a 25 mL SIF solution (pH 6.8) for four more hours. At fixed time intervals, the beads were separated from the medium using a stainless-steel grid and were immediately wiped gently with paper and weighed. The %weight change of the beads with respect to time was calculated according to the following mathematical formula and is called swelling ratio sQ:sW is the weight of the beads in the swollen state and dW is the initial weight of the dry beads. This is carried out by weighing the pellets at time The dissolution studies were carried out using the dissolution bath method. The motor was set at 50 rpm and the constant temperature bath at 37 °C. Two simulated fluids of 1000 mL each were prepared to evaluate the drug release performance of the LV and MV alginate beads. Three out of the six vessels were filled with 300 mL of the SGF medium (sodium chloride buffer at pH = 1.2), while the SIF medium (pH = 6.8) was added in the remaining three . The algt1 and t2, and is expressed as the percentage of the curve at maximum dissolution, y100, at the same time period (Equation (2)) [In order to compare the dissolution profiles of the LV and MV alginate beads, graphs of % acetylsalicylic acid release vs. time were constructed. The dissolution efficiency (DE) is equal to the area under the dissolution curve between the time points, ion (2)) :(2)DE=∫ttx%, corresponds to the time that x% of the active substance has been released from the pharmaceutical formulation and therefore, the values t20%, t50%, and t90% were calculated.The sampling time, MDT) values were calculated from the following equation:ABC is the area between the drug dissolution curve and its asymptote [The mean dissolution time . If the n value is between 0.45 and 0.89, the release is non-Fickian with anomalous transport. When n is equal to 0.89, the release follows zero-order (case II) kinetics.The variables f1) and the similarity factor (f2) are used. Factors f1 and f2 presented by the Equations (5) and (6), respectively, are qualitative (model independent) approaches to compare the dissolution pairs. The f1 factor measures the average relative difference between two curves across all the time points, whereas the f2 factor is used in order to compare the cumulative curves of percentage dissolution between two samples [n, is the number of time points; tR, mean % reference drug dissolved at time t after initiation of the study; tT, mean % test drug dissolved at time t after initiation of the study. In order to compare the dissolution curves, the difference factor ( samples :(5)f1=∑tf2 value ranges from 0 to 100, with the absolute similarity having the value of 100. As the difference between the test and reference profiles increases, the f2 score decreases and can approach zero. However, due to f2’s logarithmic nature, even minor differences between the profiles can lead to a significant decrease in the f2 score. The two dissolution curves are similar when the f2 value lies between 50 and 100 and the f1 value is between 0 and 15.The t-test, and multiple comparisons were carried out using one-way ANOVA; p-values < 0.05 were considered statistically significant. All statistical analyses were performed using “Excel 2016” .Results are shown as the mean value-standard deviation (SD) of three independent measurements. Statistical analysis was performed using the Student’s ® F-127 is self-assembled into micelles with a size around 120 nm . This suggests that the surface characteristics of the prepared micelles did not change in conditions mimicking the gastrointestinal tract [Pluronicd 120 nm  with a hd 120 nm , conside nm size  with a h nm size . In both nm size . These ζal tract .® F-127 nanomicelles with and without the encapsulated ASA for a period of 30 days. Both dispersions were stored at 4 °C (in the fridge). The results from the physicochemical characterization after 30 days showed that both the nanomicellar formulations are stable . The last observation suggests that steric stabilization due to the hydrophilic chains of the triblock copolymer is responsible for keeping the micelles in dispersion state, without the presence of electrostatic repulsion.We also checked the colloidal stability of the PluronicTGA was performed in order to evaluate the thermal stability of the hydrogel beads. The TGA curve  shows thAccording to 2 and methane and water being formed [® F-127 does not award any thermal stability to the treated sodium alginate beads [Further heating showed two more degradation processes of mass loss in the range from 300 °C to 450 °C and occurring due to the decomposition of oxygen-containing groups, such as hydroxyl and carboxyl groups (COO-) releasing COg formed ,20. Abovg formed . The totte beads .Although we would expect beads with medium viscosity to lose more weight, medium- and low-viscosity beads in this TGA demonstrated similar behavior. According to the published literature, MV beads are larger in size compared to LV beads. Therefore, they can store more water inside, and thus, they should lose more mass, especially in the first stage of TGA, where the mass is lost due to the evaporation of moisture .The swelling profile of the dry beads  in SGF  sodium alginate was used, the resulting beads were larger than those prepared with a lower viscosity (LV) alginate. Additionally, the beads made with the higher viscosity alginate had a higher capacity to swell . The swe® F-127 dissolve faster at pH = 6.8 with the MV alginate beads dissolving in 30 min and the LV alginate beads in 120 min, at pH = 6.8. When the pH increases, the ionized core is enhanced by repulsive interactions that lead to the increase in the micellar size, until they dissolve [Furthermore, as demonstrated in ® F-127. This can be explained by the fact that the alginate amphiphilic micelles bear ionized carboxylic acid groups. At pH = 1.2, these groups are protonated to -COOH [2+ ions are exchanged with K+. The Ca2+ ions are binding with the carboxylic acid groups, and this leads to decreased electrostatic repulsions causing chain loosening and enhancement of the gel swelling. Intermolecular cross-linkage is being formed between Ca2+ and G Blocks, shaping a hard surface of the beads [2+ ions are gradually released to the medium [In addition, the aqueous medium also has an effect on the swelling of the beads. As shown in to -COOH . Therefohe beads ,27. Howee medium .® F-127.SEM (scanning electron microscopy) provides information about the surface morphology and microstructure of hydrogels. SEM, as shown in ® F-127 appears porous and voids or interconnected pores are evident. However, there is a difference between the low- and medium-viscosity hydrogels. The surface of the LV hydrogels is smoother in contrast to that of the MVs, which are rougher with wrinkles  and zero-order release (n = 1.0), characterized as anomalous release. On the other hand, the MV alginate beads have an n value of 0.4, which suggests a release mechanism closer to a non-Fickian or anomalous release (0 < n < 0.5) [The dissolution kinetics of the alginate beads can be understood through the Korsmeyer–Peppas model. Thus, the LV alginate beads have an n < 0.5) .In this investigation, we designed and developed alginate beads for the encapsulation and release of ASA. To the best of the authors’ knowledge, this is the first report, where a comparison study was designed to evaluate the physicochemical and release behavior of beads composed of different viscosity alginates. In


A virus concentration method is required for viral vaccine manufacturing and virus-related research. However, concentration methods, such as ultracentrifugation, often require capital investment. We report a simple and easy-to-use handheld syringe method for virus concentration using a hollow fiber (HF) filter module, which can be applicable to viruses of different sizes, without incorporating any special machines or reagents. This virus concentration method does not use pumps, which might cause shear stress for virus particles; therefore, it is useful for stress-sensitive virus particles, and virus-like particles, as well as other proteins.•The Zika virus was concentrated from 200 mL to 5 mL within 45 min using the HF filter and handheld syringe module method.•The handheld HF filter method may be applicable to stress-sensitive viruses and proteins of different sizes.•The virus concentration process should be conducted in a safety cabinet, which is preferred for virus containment.The clarified harvest of flavivirus (Zika virus) was concentrated using an HF filter module and compared with a centrifugal ultrafiltration device (CUD) for demonstration of the HF filter method. The HF filter method achieved concentration of the virus solution in less time than the CUD. The yield comparison of the recovered virus solution indicated that recovery from the developed method was comparable to using the CUD, and infectivity was maintained throughout.  Specifications tableConcentration of virus particles is a key process in both viral vaccine manufacturing and viral characterization research. Commonly used methods for virus concentration are ultracentrifugation, tangential flow filtration, and chromatographic binding and elution ,2. UltraTangential flow filtration using a hollowfiber (HF) filter module is widely used in the vaccine manufacturing field for concentration and buffer exchange because of the low shear stress of virus particles compared with other types of ultrafiltration (UF), such as flat sheet cassettes or tubular membranes 2) for detecting Escherichia coli in water quality investigations A previously described HF filter method was developed to detect microbial contamination in water Escherichia Coli)The application of the syringe-based, handheld HF filter method for virus concentration has not been previously described. We have developed and customized a syringe-based HF filter method for virus concentration, which enables a higher concentration factor than previously reported method for water quality monitoring (5-fold for 2) than the method on which this was based, was used To increase the final concentration factor using a syringe-based concentration method, an approximately 10 times larger filter area  was used as the final system rinse buffer to minimize any surfactant effects on the virus. For demonstration of the viability of this method, a 40-fold concentration was evaluated using syringe-based HF filter module concentration of a flavivirus (Zika virus). Considering the 50 nm Zika virus size, a 300 kDa nominal molecular weight cut-off (MWCO) HF filter cassette was chosen for this study The methodology and manufacturer for the CUD are described in the supplementary appendix.10-layer Nunc EasyFill Cell Factory system 50 mL Luer lock syringe 10 mL Luer lock syringe 0.22 μm filter 2 300 kDa hollow fiber (HF) filter, 115 cm100 kDa centrifugal ultrafiltration device (CUD) filter 1x phosphate-buffered saline (PBS) pH 7.4 Fetal bovine serum (FBS)Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12)Water for injection (WFI)1.1.1.Vero cells were cultured in a 10-layer cell factory system until they were confluent in the cell culture medium (DMEM/F12 + 1% FBS).1.2.The 10-layer cell factory system was rinsed with a fresh medium (DMEM/F12).1.3.Zika virus was inoculated onto the cells, and the virus was propagated for 3 days in the same cell culture medium without FBS (DMEM/F12).Cell culture and virus propagation2.2.1.A clarified virus harvest was obtained by filtering the culture supernatant through a 0.22 μm filter.Clarification3.HF module concentration of virus3.1.200 mL of clarified virus harvest was prepared as described above.3.2.A new 50 mL syringe was connected at one side of the permeate port. The other side of the permeate port was capped.3.3.One of the HF inlets was connected with a new 50 mL syringe.3.4.The other inlet was connected to a 50 mL syringe containing the clarified virus harvest A.Fig. 1O3.5.On one side of the inlet syringe clarified virus harvest was injected into the other syringe and the other syringe was moved freely B. Again,3.6.When the permeate syringe was full, the permeate in the 50 mL syringe was discarded and reconnected.3.7.The inlet syringe was re-filled several times with the clarified virus harvest until the entire volume of the prepared clarified virus harvest was filled in the HF module.3.8.One of the inlet syringes was changed to a new 10 mL syringe if the concentrated clarified virus harvest volume was less than 50 mL.3.9.This was repeated until the clarified concentrate virus harvest volume was 1–2 mL, measured with a 10 mL syringe. The permeate port was then closed with a cap when the concentration was complete.3.10.The concentrated clarified virus was harvested in a 10 mL syringe C.3.11.1x PBS was prepared in a 10 mL syringe for system rinsing and connecting to the HF filter inlet. The required 1x PBS volume was calculated so that the final pool volume was 5 mL.3.12.The 1x PBS buffer was injected several times into the other side of the HF module to rinse the HF system.3.13.The system rinse was collected into a 10 mL syringe.3.14.The concentrated clarified virus harvest and system rinse were pooled.As an example, we described the concentration of 200 mL of clarified virus harvest to a final volume of approximately 5 mL.200 mL of the clarified virus harvest containing Zika virus was concentrated 40-fold using the HF filter module method (n=3). For comparison, the same volume of clarified virus harvest was concentrated using a CUD (n=1). The HF method took less than 45 min, compared to the CUD which took approximately 1 h.50/mL). Zika virus infectivity was evaluated using TCID50/mL  than that of the sample processed by the CUD (2028 µg/mL) . As the In this study, a novel handheld HF filter syringe-based concentration method for viruses isolated from clarified harvests was evaluated. To our knowledge, this is the first description of virus concentration without any special reagents or specialist equipment. From the perspective of virus infectivity and viral RNA recovery the HF-based method was confirmed to be more effective than the CUD method. Infectivity data demonstrated that this HF filter method can concentrate viruses with a greater retention of infectious particles than the CUD method. Furthermore, the HF filter method was able to concentrate 200 mL of Zika virus to 5 mL within 45 min without any specialist equipment or reagents, whereas the CUD method took approximately 1 h. Although the maximum MWCO of CUDs is usually 100 kDa, different MWCO filter sizes are available for the HF method. One limitation to our study was that only one test was performed for 100 kDa CUD method compared with triplicate repetition for the 300 kDa HF method, such that further repetitions with same pore sized filter as used in the CUD method would be needed in future studies. Therefore, the handheld HF method is applicable to a wide variety of viruses and proteins. Furthermore, because the virus handling procedures of the HF method are completed only in a safety cabinet, it is useful in terms of virus containment. In conclusion, the handheld HF filter syringe-based method established in this study may be a useful virus concentration tool for use in virus research. However, there is a need for further investigation of the reproducibility for virus concentration, as well as evaluation of different filter sizes, and development of standardization for the HF method.Shinsuke Higuchi: Conceptualization, Methodology, Writing – original draft. Tatsuki Satou: Writing – review & editing, Supervision. Yuki Uchida: Writing – review & editing, Supervision.The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Employees of Takeda


T’Ho virus is a poorly characterized orthoflavivirus most closely related to Rocio virus and Ilheus virus, two orthoflaviviruses associated with human disease, suggesting that T’Ho virus could also be a human pathogen. The genome of T’Ho virus has been sequenced but an isolate has never been recovered, impeding its phenotypic characterization. In an attempt to generate recombinant T’Ho virus, the entire viral genome was synthesized as three overlapping DNA fragments, joined by Gibson assembly, and transfected into mosquito cells. Several cell culture passages were performed, but virus was not recovered. Subsequent experiments focused on the development of a chimeric orthoflavivirus that contains the premembrane and envelope protein genes of T’Ho virus in the genetic background of Zika virus. The chimeric virus replicated in mosquito (C6/36) and vertebrate (Vero) cells, demonstrating that the major structural glycoproteins of T’Ho virus permit entry into both cell types. The chimeric virus produced plaques in Vero cells that were significantly smaller than those produced by Zika virus. The chimeric virus can potentially be used as a surrogate diagnostic reagent in place of T’Ho virus in plaque reduction neutralization tests, allowing T’Ho virus to be considered in the differential diagnosis.The online version contains supplementary material available at 10.1186/s12985-023-02172-2. Orthoflavivirus (family Flaviviridae) is comprised of small, enveloped RNA viruses with single-stranded, positive-sense RNA genomes of ~ 11 kb. The genome contains a 5′ untranslated region (UTR) of about 100 nt., followed by a long open reading frame, and 3′ UTR of about 400–700 nt. [The genus –700 nt. , 17, 24.–700 nt. . The prM–700 nt. . The non–700 nt. .T’Ho virus was discovered in mosquitoes in Mexico in 2007 and phylogenetic data revealed that its closest known relatives are Rocio virus (ROCV) and Ilheus virus (ILHV), two orthoflaviviruses associated with human disease , 11. ROCThe genome of T’Ho virus has been sequenced in its entirety, but attempts to recover an isolate have been unsuccessful, impeding its phenotypic characterization , 11. WitThe chimeric genome was created by inserting the premembrane and envelope protein (prM-E) genes of T’Ho virus (strain T'Ho-Mex07) into the genetic background of Zika virus . Four plasmids were required for these experiments: pUC19-THOVprME, pUC19-F1, pUC19-F2 and pUC19-F3  cells using Lipofectamine™ 2000 Transfection Reagent (ThermoFisher Scientific). Transfected cells were incubated for 7 days then an aliquot of supernatant was inoculated onto new monolayers of C6/36 cells. A second passage was performed and lysates and supernatants were harvested from the final cell culture passage at 5 days post-inoculation (p.i.) and assayed for virus by Western blot and immunofluorescence assay (IFA), as previously described [Additional PCRs were performed using pUC19-F1a, pUC19-F2, and pUC19-F3 as templates and chimeric primers that amplified all of the viral sequences, but none of the pUC19 sequences, from each plasmid . Western blot analysis revealed that ZIKV/THOV(prM-E) replicates in both mosquito C6/36) and vertebrate (Vero) cells  replicates in C6/36 and Vero cells . For each virus, 40 plaques were measured per experiment, with duplicate experiments performed.We compared the sizes of plaques produced by ZIKV/THOV(prM-E) and ZIKV in Vero cells at 5 days p.i. Fig. C. The chZIKV/THOV(prM-E) could potentially be used as a diagnostic tool. Researchers performing orthoflavivirus serosurveillance in Mexico and elsewhere in the Americas could include the chimeric virus in PRNTs, allowing T’Ho virus to be considered in the differential diagnosis. Other chimeric orthoflaviviruses have been developed for use in PRNTs , 16, 25.One limitation of our study is that the diagnostic efficacy of ZIKV/THOV(prM-E) was not compared to T’Ho virus by PRNT. All of the other chimeric flaviviruses and alphaviruses mentioned earlier were compared to the parental viruses that contributed the immunogenic structural protein genes and shown to be suitable surrogates in PRNTs , 21, 25.ZIKV/THOV(prM-E) requires BSL-2 containment because it was built on the genetic background of a BSL-2 agent. T’Ho virus is not listed in the Biosafety in Microbiological and Biomedical Laboratories but its closest known relative, Rocio virus, is a BSL-3 pathogen, suggesting that T’Ho virus should be considered a BSL-3 pathogen . TherefoPsorophora ferox mosquitoes [The ability of ZIKV/THOV(prM-E) to replicate in C6/36 and Vero cells demonstrates that the major structural glycoproteins of T’Ho virus permit entry into both mosquito and vertebrate cells. However, experiments designed to determine whether T’Ho virus replicates in these cells cannot be performed unless an isolate is acquired or recombinant virus is produced. At present, the arthropod and vertebrate host ranges of T’Ho can only be inferred using information obtained for its closest known relatives. ROCV cycles in nature between birds and squitoes . The majsquitoes , 22, 33.ZIKV/THOV(prM-E) plaques were significantly smaller than ZIKV plaques. Chimeric orthoflaviviruses created from prM-E gene exchanges often produce plaques smaller than at least one parental virus , 23, 34.Attempts to generate recombinant THo virus were unsuccessful. One explanation for this outcome is the viral genome sequence deposited into the Genbank database contains sequence errors. The full genome of T’Ho virus was sequenced by unbiased high-throughput sequencing, except for the terminal ends where 5′ and 3′ rapid amplification of cDNA ends and Sanger sequencing were used . The majTo conclude, we report the construction and characterization of ZIKV/THOV(prM-E), a chimeric orthoflavivirus that contains the prM-E genes of T’Ho virus in the genetic background of ZIKV. The ability of the chimeric virus to replicate in C6/36 and Vero cells provides evidence that the major structural glycoproteins of T’Ho virus permit entry into both mosquito and vertebrate cells. ZIKV/THOV(prM-E) could provide a suitable surrogate for T’Ho virus in PRNTs. Unfortunately, the diagnostic efficiencies of ZIKV/THOV(prM-E) and T’Ho virus could not be compared because infectious T’Ho virus is not available. However, there is no other virus than can be used in place of ZIKV/THOV(prM-E) and therefore, researchers performing orthoflavivirus serosurveillance in the Americas may want to include it in their PRNTs.Additional file 1.Figure S1. Mosquito and vertebrate cells support the replication of ZIKV/THOV(prM-E). A Western blot analysis of C/36 cells inoculated with ZIKV/THOV(prM-E). These are the same images as shown in Figure 2A, except they are uncropped. Experimental details are provided in the legend for Figure 2A. B Western blot analysis of Vero cells inoculated with ZIKV/THOV(prM-E). These are the same images as shown in Figure 3A, except they are uncropped. Experimental details can provided in the legend for Figure 3A


Developing open-shell singlet (OS) diradicals with high luminescent properties and exceptional single-molecule magnetoluminescence (ML) performance is extremely challenging. Herein, we propose a concept to enhance luminescent efficiency by adjusting the donor conjugation of OS diradicals, thereby achieving a highly luminescent diradical, DR1, with outstanding stability and making it a viable option for use in the emitting layer of organic light-emitting diodes (OLEDs). More importantly, the 0.5 wt%-DR1 doped film demonstrates significant single-molecule magnetoluminescence (ML) properties. A giant ML value of 210% is achieved at a magnetic field of 7 T, showing the great potential of DR1 in magneto-optoelectronic devices. A highly efficient open-shell singlet luminescent diradical demonstrates significant magnetoluminescence properties. A giant magnetoluminescence value of 210% is achieved at a magnetic field of 7 T, showing great potential in magneto-optoelectronic devices. Despite this progress, there remains a lack of research regarding luminescent OS diradicals, hindering their potential use in optoelectronic applications. In fact, the luminescent diradicals are rare chemical species, there are only a few reports to date44. Recently, we reported the first luminescent Müller’s hydrocarbon with OS ground state45, but unfortunately, its photoluminescence quantum yield (PLQY) was found to be very low (0.4%), rendering it suitable only for conceptual exploration and hindering its practical applications in optoelectronic devices.Open-shell singlet (OS) diradicals are important building blocks for functional molecular materials50. In 2018, Kusamoto and co-workers first reported MLs in organic radical excimer species, which opened the gate to this field46. Recently, Kusamoto and co-workers elegantly designed and synthesized a spatially confined luminescent diradical and observed its single-molecule ML properties50. Nevertheless, the development of highly luminescent diradicals and the achievement of their efficient single-molecule ML properties continue to be a formidable challenge.Magnetic field effects (MFEs) on the luminescence, i.e., magnetoluminescence (ML) of radicals, hold great promise for developing novel exciton spin manipulation methodologies that are unachievable by conventional closed-shell luminescent moleculesBased on the first-principle calculations, we find that for an OS diradical, the radiative decay of singlet excitons corresponds to electronic transitions between the singly occupied molecular orbital (SOMO) and the lowest unoccupied molecular orbital (LUMO). However, due to the electron correlation, the electron density distributions of the SOMO and LUMO for α and β electrons located in the different fragments  are cross-distributed Fig. S. This maES-T) of DR1–DR4 is −0.022, −0.031, −0.008, and 0.004 kcal mol−1, respectively, indicating weak interactions between the two TTM mono-radical centers and revealing that DR1–DR3 are OS diradicals (Table t)  electronic structure to 1 for a pure diradical8. DR1–DR4 show large y0 values of >0.90, revealing excellent diradical features of the four molecules methyl (TTM) as the radical center to design four diradicals, DR1–DR4  Figs. S are conses Table . Howeveres Table , S3.2 hybridized carbon atoms C7 and C28 are the two unpaired radicals. The magnetic property of DR1 was investigated using a superconducting quantum interference device (SQUID). As shown in Fig. SχmT increases rapidly with increasing temperature from 2 to 100 K, which is typical for an open-shell singlet ground state in thermal equilibrium with a triplet state51. Fitting the χmT–T curve with the Bleany–Bowers equation52, we obtain a ΔES-T of −0.051 kcal mol−1, which is consistent with the theoretically calculated value. Moreover, DR1 exhibits a single-line electron paramagnetic resonance (EPR) signal in the Δms = ±1 region, and a signal corresponding to the Δms = ±2 transitions is also observed at 77 K -2,6-dichlorophenyl) bis methyl (TTM-1Cz)55; Fig. 47. The PL decay lifetime (τ) of DR1 (10.6 ns) is between those of TTM (5.6 ns) and TTM-1Cz (41.3 ns)  of DR1 are estimated to be 1.51 × 107 and 7.92 × 107 s−1, respectively. Notably, the kr of DR1 is higher than those of TTM (0.35 × 107 s−1) and TTM-1Cz (1.28 × 107 s−1), which is due to the higher transition oscillator strength of DR1 (Table knr of DR1 is lower than that of TTM (17.5 × 107 s−1) and higher than that of TTM-1Cz (1.13 × 107 s−1). The larger knr of DR1 than TTM-1Cz may be due to its smaller energy gap and the intramolecular spin interaction, which can accelerate the internal conversion to the ground state.To evaluate the luminescent properties of DR1, a series of photophysical studies were conducted on it and its two fragments  Fig. . Then, tR1 Table . While tτ and PLQY in various solvents with different polarities . Overall, these photophysical properties indicate that the excited state of DR1 is a hybridized local and charge-transfer (CT) state, as compared with the local excited state of TTM and CT-dominated excited state of TTM-1Cz57.To investigate the ground and excited state characteristics of DR1, we conducted measurements of UV/vis absorption, PL, es Fig. S. As the ES-T. This is in contrast to the behavior of TTM-1Cz, where the PL intensity slightly increases with temperature from 100 to 2 K  film shows identical UV/Vis absorption to that in solution while exhibiting a deep-red emission (673 nm) with a PLQY of 14.1% Fig. . We foun K Fig. S because  K Fig. S is simil4 s) is 1500 and 25 times higher than that of TTM (2.4 × 101 s) and TTM-1Cz (1.5 × 103 s), respectively . Accordingly, we fabricated solution-processed OLEDs utilizing DR1 (0.5 wt%) as the emissive dopant (see details in Supporting Information). The OLEDs exhibit deep-red emission peaking at 680 nm with the maximum external quantum efficiency (EQE)0% Fig. S.46.It is interesting to find that DR1 shows quite strong ML properties. Figure 62. Therefore, the aggregation effects decreased, leading to a decreased non-radiative recombination rate and increased lifetime for the emission of aggregated single molecules. In addition, temperature-dependent ML tests were conducted on two films, as shown in Fig. 48. Otherwise, significant MLs can be observed at high temperatures.To understand the ML properties of DR1, we carried out transient PL measurements. As can be seen from Fig. 63. As shown in Fig. t ground state and the weak thermally forbidden intersystem crossing from singlet excitons to triplet excitons, leading to the decreased PL intensity as the temperature decreases , one can expect a decreased ML. For the excimer emission, since the decay processes do not change with the magnetic field, we can conclude that the magnetic field cannot influence the transition processes of the excimers. A probable mechanism for the ML of DR1 excimer emission should come from the difference in PL efficiencies between excimers formed by triplets and those formed by singlets. Therefore, an increased number of triplet excimers can lead to an increased total excimer emission intensity. However, the significantly decreased ML of the excimer emission than that of the single-molecular emission indicates that the PL efficiency of excimers formed by singlet excitons is close to that formed by triplet excitons, which is consistent with the temperature-insensitive excimer emission intensity , and its crystallographic data (CCDC 2252807) are provided free of charge by the joint Cambridge Crystallographic Data Center and Fachinformationszentrum Karlsruhe Access Structures service.All chemical agents and solvents, unless otherwise stated, were purchased from commercial suppliers and used directly without further purification. The intermediate 2(HTTM), mono-radical TTM, and TTM-1Cz were prepared according to our previous reports1H nuclear magnetic resonance (NMR) spectra were recorded in (Methyl sulfoxide)-d6 (d6-DMSO) on a Bruker Avance-III 500 NMR spectrometer at ambient temperature. GC–MS mass spectra were recorded on a Thermo Fisher ITQ1100 mass spectrometer. MALDI-TOF mass spectra were recorded on a Bruker Autoflex speed TOF/TOF mass spectrometer with DCTB as a matrix. EPR spectra were recorded on a Bruker ELEXSYS-II E500 CW-EPR spectrometer. Thermal gravimetric analysis (TGA) curves were obtained on the Pyris1 TGA thermal analysis system at a heating rate of 20 °C min−1 in a nitrogen atmosphere. Elemental analysis was conducted by an Elementar vario MICRO cube instrument. Single crystal X-ray diffraction data of DR1 was collected using a synchrotron X-ray source at the Shanghai Synchrotron Radiation Facility. The crystal structure was determined by direct methods and further refined by the full matrix least squares method of F2 using the SHELX-97 and Olex-2. Magnetic measurements were performed on a Quantum Design 6.5 Tesla SQUID-VSM system with a temperature range of 2–300 K and an applied field of 1000 Oe. A powder sample of DR1 with a weight of 5–10 mg was sealed in a plastic capsule. The magnetic moment was measured in the temperature range of 2–300 K. After correction of diamagnetic contributions from the sample, using tabulated constants, sample holder, and paramagnetic contamination, the magnetic data were fitted with Bleaney–Bowers equation52.The t) states at the (U)B3LYP/6-31 G** level, respectively. All optimized geometries were confirmed to be local minima by vibrational analysis. The OS structures were shown to be stable structures. ΔE(OS-CS) and ΔE(OS-Tt) were calculated as the energy differences between OS structure and CS structure, Tt structure, respectively.All calculations were performed with the Gaussian 16 program package. The geometries of all compounds were optimized as open-shell (OS) singlets by the spin-unrestricted broken-symmetry (BS) approach at the UB3LYP/6-31 G** theoretical level. This approach has been shown to provide reliable geometries and energies for singlet-state diradicals. Then these compounds were optimized as closed-shell (CS) singlets and thermally excited triplet (TUltraviolet-visible (UV–Vis) and photoluminescence (PL) spectra of the radicals were recorded on a Shimadzu UV-2550 spectrophotometer and a Shimadzu 5301PC spectrophotometer. The intensity of luminescence at 654 nm (for DR1), 564 nm (for TTM), and 628 nm (for TTM-1Cz) were monitored exciting at 370 nm light . The relative PLQYs were measured using a Shimadzu UV-2550 spectrophotometer and Edinburgh fluorescence spectrometer (FLS980). The fluorescence lifetimes were measured with FLS980. The temperature-dependent PL spectra of the radicals were measured using a spectrometer (Ocean Optics QE65 Pro), and a Spectromag PT liquid helium-free superconducting magneto-optical system (Oxford Instruments NanoScience) was used to provide different temperatures with 2 ~ 300 K and magnetic fields from 0 to 7 T.Supporting Information


PLOS Biology, the virulence of a zoonotic virus in humans depends on its reservoir host. Could physiology be the key to anticipating viral threats lethality?Would you rather kiss a platypus, a hedgehog, or a llama? According to a new study in this issue of  Would you rather kiss a platypus, a hedgehog or a llama? This study explores a new study in PLOS Biology which uses evolutionary epidemiology models to show that the life-history of the reservoir mammal hosts of zoonotic viruses might be the key to predicting their virulence in humans. Zoonotic viruses are recognised as a global threat. Although some of these like MERS-CoV or Ebola virus have shaped our imagination (and nightmares), should we generally expect them to be more virulent to humans than endemic viruses? Answering this question is difficult because zoonotic viruses are, almost by definition, poorly adapted to humans, and this maladaptation can either lead to low virulence (if the virus is rapidly cleared by the immune system) or huge virulence .So far, studies on the virulence of zoonoses have focused on transmission chains taking place right after the spillover. These showed, for example, that some infection life history traits, such as virulence, govern the probability of early extinction and rapid adaptation to humans . More viPLoS Biology, Brook and colleagues [In a new study published in this issue of lleagues  tackle tAccording to virulence evolution theory, the host life history matters. For example, in simple models, increased host life span and level of constitutive immunity are predicted to, respectively, favour less and more virulent strains. Mathematical models help to formalise these predictions, some of which have been validated experimentally .Importantly, in this work, virulence is an intrinsic property of the virus. It has to be distinguished from the expressed virulence , or “letThe literature on modelling within-host dynamics is dual, with deterministic models generating either chronic or acute infections. Brook and colleagues elegantly combine the two by assuming that zoonotic viruses cause chronic infections in their reservoir host and acute infections in their novel (human) host . Using aOverall, Brook and colleagues present us with the virulence that one might expect from a zoonotic virus coming from one of 19 potential reservoir hosts. Their predictions are consistent with data from 8 orders of mammals. This may appear as limited but it is the best one can do with the current data. In orders such as Chiroptera (bats), in which the authors infer a high tolerance to immunopathology and high levels of constitutive immunity, the expected (and observed) virulence is high. The expected virulence could be even higher in Monotrema (suggesting that you definitely should not kiss a platypus), but there currently is no data on emerging viruses from this order. Besides, the authors do not model the probability of emergence, and it is also likely that monotreme zoonoses are rare (or even nonexistent).Overall, this work is a call for an improved understanding of the physiology of potential reservoir hosts, as well as virus ecology in general. This road map to detecting potentially virulent viruses in the wild also raises ethical concerns as to whether such a search should be undertaken (or at least how it should be implemented).Other questions remain open. For instance, does this theory apply to non-Mammal reservoirs or to non-vertebrate hosts? Indeed, serial passage experiments already show that for arboviruses infecting humans, transmission rounds through arthropod vectors shape their evolution . FurtherThe mathematical model also makes important assumptions that could be explored in the future. For example, the (intrinsic) growth rate of the virus is assumed to be constant in the reservoir and human hosts. Even the relative contribution of the virus replication to virulence through direct exploitation or immunopathology is assumed to be constant in both types of hosts. Overall, only the host tolerance and resistance parameters vary freely in the model. In terms of validation, could sampling be ill-balanced between all reservoir species? For example, less virulent infections could be more likely to be detected when originating from domesticated species.An exciting possibility to validate the framework could be to apply it to emerging viruses in animal hosts. For example, the importance of the adaptation to the reservoir in shaping the virulence could be tested using a mouse system as the reference instead of humans. More generally, there is a need for additional laboratory studies such that key parameters could then be measured and not inferred via proxies.R0) and the delay between contagiousness and symptoms [Finally, the lethality of a zoonotic virus may be largely unrelated to the number of casualties it may cause since our ability to control an outbreak strongly depends on other factors, especially the basic reproduction number (symptoms . For exa


Consumption of plant foods, including whole grains, vegetables, fruits, pulses, nuts, and seeds, is linked to improved health outcomes. Dietary fiber is a nutrient in plant foods that is associated with improved health outcomes, including a lower risk of chronic diseases such as cardiovascular disease, type 2 diabetes, and certain cancers. Different fibers deliver different health benefits based on their physiochemical properties  and physiological effects (fermentability). Additionally, plant foods contain more than dietary fiber and are rich sources of bioactives, which also provide health benefits. The concept of the solubility of fiber was introduced in the 1970s as a method to explain physiological effects, an idea that is no longer accepted. Dividing total dietary fiber (TDF) into insoluble dietary fiber (IDF) and soluble dietary fiber (SDF) is an analytical distinction, and recent work finds that IDF intake is linked to a wide range of health benefits beyond increased stool weight. We have focused on the IDF content of plant foods and linked the concept of IDF to the bioactives in plant foods. Ancestral humans might have consumed as much as 100 g of dietary fiber daily, which also delivered bioactives that may be more important protective compounds in disease prevention. Isolating fibers to add to human diets may be of limited usefulness unless bioactives are included in the isolated fiber supplement. Plant foods and dietary fiber have been recommended in food guidance since 300 BC . Plant fBecause the most widely accepted definition of dietary fiber is physiological, it is difficult to devise methods to measure dietary fiber and its components accurately. Historically, crude fiber was used, a method that treats food with acid and alkali to isolate the indigestible component. This method works for animal feeds, but it seriously underestimates the dietary fiber content of food, recovering only 50% to 80% of the cellulose, 10% to 50% of the lignin, and 20% of the hemicellulose  . The catThe purpose of this review is to investigate the nutritive value of insoluble dietary fiber (IDF) fractions with respect to their rich bioactive content. The mass production of some goods yields high amounts of byproducts, including fruit peels and pomaces, that are rich in insoluble fiber but also rich in bioactive compounds. Plant byproducts are a potential nutrient-rich option to address low fiber intakes with minimal risk of adverse effects. These byproducts are commonly discarded, composted, used as animal feed, or used in the beauty industry. But the volume of the byproducts is large enough to be disruptive to the environment . Using tWhat IDF sources have been examined in regard to their bioactive content?What bioactive compounds are present in IDF?Do these bioactive compounds exhibit health-promoting effects?To conduct the scoping review, three databases were used to search for relevant research: Ovid Medline, Ovid Agricola, and Scopus. These three databases were chosen to encompass a variety of fiber and bioactive research. Current research was observed in both medical and agricultural journals, hence the inclusion of Medline/Scopus and Agricola. The Institute for Food and Nutrition Sciences (IAFANS) dietary fiber database was also screened. It was ultimately excluded due to its lack of research regarding phytochemicals in fiber and its focus on glycemic control, hunger/fullness, and type II diabetes. The search string was altered to fit the requirements of the respective databases. Ovid allowed for the same search string to screen two databases simultaneously, Medline and Agricola. For Scopus, the string had to be altered slightly to adhere to their format. The search string can be found in n = 51) were screened for their eligibility by reading the full text. After full-text screening, 44 articles remained, with 7 additional articles being excluded , total flavonoid content (TFC), and antioxidant activity (AA). Not every source was evaluated with all three methods. When AA was measured, it was conducted in a variety of ways. Methods that appeared in the search results can be depicted in There were a total of sixty-four bioactive compounds detected across the insoluble fiber sources that appeared in the research. They fell into three categories: phenolic acids, flavonoids, and non-flavonoid compounds. The non-flavonoid compounds included tannins, tocopherols, tocotrienols, vitamin C, retinol equivalents, carotenoids, chlorophylls, and betalains. The majority of these were detected using high-performance liquid chromatography (HPLC). The specific compounds can be found in In addition to bioactive flavonoids and phenolics, IDF can be found specifically in certain plant foods but also in different tissues within these plants. Guava  containsOverall, the studies that tested TPC found that there was an increase with the addition of IDF fractions. Fortification of IDF into a baked good did decrease TPC in comparison to the pure fraction, but the bioactive content still remained higher than the control flours. This can be expected due to the cooking process and using a combination of the IDF fraction flour and the control flour in an effort to maintain the integrity of the baked goods. In comparing the TPC of oat milk byproduct (OMB) flour and wheat flour, OMB flour had a higher TPC . And wheTotal flavonoid content was high as well across multiple IDF sources. IDF fractions that were made from peels tended to exhibit increased levels of flavonoids. The production of flavonoids is thought to be linked to exposure to sunlight . When coThe AA assays provide a general overview of the potential bioactivity of food products. Similarly, for TPC and TFC, there were increases in AA with the IDF fractions compared to the control, typically white or wheat flour. OMB had significantly higher AA on FRAP and DPPH assays compared to wheat flour . These iThe extraction and processing of IDF fractions have significant effects on the bioactive and IDF content of the said fraction. Sieve size was one factor that yielded changes in bioactivity. When using multiple sieve sizes to smooth out dried beetroot leaves powder (DLBP), Asadi et al. found that smaller sieve sizes had lower levels of IDF. They observed the highest levels of IDF in the raw DBLP . SimilarTreatment of the IDF sources also impacted bioactivity. Lau et al. used a conventional extraction method and a supercritical fluid extraction (SFE) method to acquire carotenoids from sweet corncobs . They obExtrusion temperature was another factor that played a role in bioactivity. The purification of cactus peel powders via increased temperature processing yielded decreases in TPC . MedeiroOutside of the extraction and processing method of the fiber fraction, the maintenance of bioactivity is another consideration. Like temperature during processing, temperature during cooking alters bioactivity. After boiling lentil-fortified pasta, 30% of the phenolic content was lost . And wheThe bioactivity and high IDF content of these plant fiber sources present an opportunity for fortification. Ready-to-consume food products are largely baked goods and have a low nutritive value overall. When plant sources were added to cookies, the IDF, TPC, and TFC content of the cookies increased. This occurred with increasing fractions of DBLP . As the Other goods were fortified, one being muffins with mango pulp flour. The MPFW and dried mango pulp fiber waste (DMPFW) muffins had higher beta-carotene and lutein contents compared to the control muffins with wheat flour . OutsideThe addition of insoluble fibers was also shown to be a functional additive to products. Apple pomace (AP) was used in an effort to maintain the integrity of yogurt. Over 28 days, they concluded that 0.5% (AP) yielded a yogurt that was firmer, more consistent, and had a higher IDF than the control . Sugar bIn conclusion, the addition of IDF to food increases its bioactive content. When small amounts of IDF are added to the flour used to create a baked good, AA and TPC increase without compromising the integrity of the good. Acceptability scores were high as well. Bioactivity did decrease upon cooking, but it still remained higher than the control food, and it may be useful as a supplement for consumers.Bioactive content was influenced by multiple factors outside of extraction, processing, and maintenance. Between five different apple varieties, IDF, TPC, and AA varied, with Royal Delicious having the highest for all three measurements . Three cDifferences in harvesting time in terms of year of growth, time of year, and time in the growth process also influenced bioactive content. When wheat grain was harvested in two consecutive years, water-soluble and total arabinoxylans were not detected in the first year, but they were detected in the second . The oppThe fraction of the fiber source used influenced bioactivity as well. The soluble and insoluble components of defatted rice bran (DRB) were examined, as well as the intact fraction of DRB. The intact DRB had more bioactive compounds than its counterparts, respectively, indicating that the entire plant may be more beneficial as a nutraceutical. Insoluble DRB had a higher proportion of bound phenolics and AA activity, but the phenolics were measured to be less bio-accessible . It is uBioactive compounds have a variety of health benefits. Dietary polyphenol consumption is associated with the management of free radicals and reactive oxygen species . The IDFAlpha-amylase activity is another measure of blood glucose control. The alpha-amylase enzyme slows the metabolism of glucose by limiting its digestibility and absorption ability in the GI tract, which results in lower postprandial blood glucose levels. Blue grapes exhibited a high alpha-amylase activity that was hypothesized to be due to the high tannin content of the fruit . Wild swThis research is applicable to a wide variety of consumers. Focus groups were conducted in many of the studies that fortified a baked good. These are valuable because even if the nutritive content of the good increases, if it is not palatable, consumers will not purchase or consume it. And if it is not consumed, the benefits upon consumption are going to be lost. The range of ages that participated in acceptance testing ranged from college students to middle-/late-aged adults. The majority of the focus groups utilized hedonic scales that had the participants rank characteristics like color, aroma, texture, flavor, softness, and overall likeness on a scale from “extremely dislike” to extremely like” and utilized untrained or trained participants. There was no significant difference in acceptability scores between breads fortified with 0, 3, and 6% capuassu peel powder . And theThe addition of IDF plant sources to ready-to-eat products also presents an option for gluten-free individuals to increase their DF intake, as it is typically a nutrient of concern for that population . Fiber tPlant sources of IDF and bioactives are cost-effective and environmentally friendly, as the majority of them are byproducts of commercial food processing. While food waste can be composted, too much compost can overload an ecosystem and, in turn, have negative environmental effects. These byproducts are also cost-effective because they are already being discarded. They can also be obtained without shipping long distances because they are present in a variety of products. For example, there is high sour cherry juice production in Poland, resulting in a lot of sour cherry pomace waste . Pitaya In the future, more research needs to be conducted in this area. There is potential to increase the nutritive value of food. In considering the production of a supplement or powder to use for fortification, researchers will need to consider the fiber type, season and climate of harvest, ripeness at harvest, and more to maximize the bioactive content of their product. They will then need to investigate the degradation over time. Does the byproduct need to be used right after it is harvested? And can it maintain its integrity in a product with a long shelf life? Or will it alter the shelf life of the good it is incorporated into? These are especially important for the bioactives that are known to be susceptible to degradation by light exposure or those not soluble in liquid. They should also consider and research the effects of enzymatic pre-treatment as a method of preparing the IDF fraction for storage.Consumer acceptance is also an important aspect of this research. While it is important to successfully obtain an IDF fraction rich in bioactives, if it is not acceptable to the consumer, that will not improve health. Some customers may not try a cookie if they know tomato is in it or bread that looks pink because the addition of sour cherry pomace makes it look different ,53. But Future research needs to consider the phytic acid content of plants as well. This is particularly important in the use of rice but can be applied to other plants, including grains, seeds, legumes, and tubers. Phytic acid is a chelator that can result in mineral deficiencies, especially zinc. Leavening agents can break down phytic acid and release minerals for absorption, so the processing effects will determine if phytic acid has any negative effects in the diet. When capuassu peel powder was added to bread, total tannins and total phenolics increased, but so did the phytic acid content . The useIn conclusion, insoluble dietary fiber is rich in bioactive compounds. It has the potential to be a nutritive product in the form of a supplement or fortified into a food product. Multiple considerations must be made regarding the extraction, processing, and storage of these bioactive compounds, but the majority observed an increase in bioactive content, even with the addition of a small amount of IDF. Work on whole grains has suggested that the bioactives in whole grains may be more important than the dietary fiber in the grain. Considerations on how best to maintain the bioactive compounds in plant extracts while still maintaining the fiber content are needed to increase both dietary fiber intake and the intake of bioactives. Past thinking that soluble dietary fiber has the most physiological benefits while insoluble fiber only alters bowel function is no longer accepted. Dietary guidance must continue to support increased consumption of plant foods to increase our total dietary fiber intake to the recommended levels. The health-promoting effects of insoluble dietary fiber go beyond just dietary fiber, and future efforts to isolate fibers from plant foods need to consider bioactives and processing strategies to enhance both dietary fiber and the bioactive content of the supplement.


Marburg virus disease, caused by Marburg and Ravn orthomarburgviruses, emerges sporadically in sub-Saharan Africa and is often fatal in humans. The natural reservoir is the Egyptian rousette bat (ERB), which sheds virus in saliva, urine, and feces. Frugivorous ERBs discard test-bitten and partially eaten fruit, potentially leaving infectious virus behind that could be consumed by other susceptible animals or humans. Historically, 8 of 17 known Marburg virus disease outbreaks have been linked to human encroachment on ERB habitats, but no linkage exists for the other 9 outbreaks, raising the question of how bats and humans might intersect, leading to virus spillover. We used micro‒global positioning systems to identify nightly ERB foraging locations. ERBs from a known Marburg virus‒infected population traveled long distances to feed in cultivated fruit trees near homes. Our results show that ERB foraging behavior represents a Marburg virus spillover risk to humans and plausibly explains the origins of some past outbreaks. Orthomarburgvirus marburgense) are the causative agents of Marburg virus disease (MVD). The prototypical filovirus and close relative of Ebola virus, MARV was identified in Germany and the former Yugoslavia in 1967 when laboratory workers had MVD develop after handling infected African green monkeys  imported from Uganda (Marburg virus (MARV) and Ravn virus (RAVV) , a cave-dwelling fruit bat, as the MARV natural reservoir based on the repeated detection or isolation of MARV and RAVV directly from ERBs captured at those and other locations  of historic MARV spillover events are epidemiologically unlinked to ERB cave habitats or infected secondary hosts. Because ERBs in Africa prefer to roost in unpopulated forested areas, pinpointing the intersections between ERBs and humans is difficult. Moreover, human infection with MARV occurs through broken skin or mucous membranes such as the eyes, nose, or mouth, implying the need for direct or indirect contact with an infected bat or infectious bodily fluids.The question remains then, how do MVD outbreaks that are geographically distant from and epidemiologically unlinked to caves or mines get started? One possibility is suggested by virus stability studies showing that MARV remains viable for up to 5 days on surfaces such as wool and glass . We usedAll animal work was performed with approval of the Centers for Disease Control and Prevention Institutional Animal Care and Use Committee (protocol no. 3063AMMBATX-A2) and with permissions from the Uganda National Counsel for Science and Technology (NS 614) and the Uganda Wildlife Authority (COD96/05) and through collaborations with Uganda Wildlife Authority and Uganda Virus Research Institute. All personnel wore appropriate personal protective equipment while performing all bat handling aspects of this field study as described  for RNA extraction and downstream PCR analysis. When handling bats and performing necropsies, all personnel wore appropriate personal protective equipment that included disposable gowns, double gloves )including bite-resistant gloves if necessary), and powered air-purifying respiratory protection.As part of an ongoing zoonotic virus surveillance effort at Python Cave, an additional 50 bats were captured for destructive sampling. The bats were humanely euthanized under anesthesia and necropsied following procedures outlined in Amman et al. (https://www.thermofisher.com) from homogenized tissues by using the MagMAX Total RNA Isolation Kit (Thermo Fisher Scientific). We then tested the extracted RNA by quantitative reverse transcription PCR (qRT-PCR) targeting the MARV protein 40 gene  micro-GPS units  to an area of the bat’s dorsum just between the scapulae (https://americasacres.com). We used male bats weighing >100 g  to keep the total percentage of unit weight to bodyweight ratio <10%. We kept bats fitted with GPS units in a screenhouse for several minutes to ensure a good GPS fit and unincumbered flight before release. The GPS units were preprogramed to begin collecting data at 5-minute intervals beginning at 7:00 pm and stopping at 5:00 am the next morning for the duration of the battery life. We placed a base station capable of wireless data download (Telemetry Solutions) just inside the entrance of Python Cave so that the entire cave interior was within line-of-site. To preserve GPS battery life, we used a repeater antenna outside the cave to transmit satellite signal to the GPS units on the bats inside the cave.We attached small (scapulae  by usinghttps://www.google.com) to locate foraging sites and generate minimum convex polygons. We calculated average flight distance by using ArcGIS Pro 2.9.2  to estimate movement by converting multiple KML layers to shapefiles and using the Generate Near Table Tool (https://pro.arcgis.com) with the Geodesic method selected for the Method parameter to calculate distances between the furthest 2 GPS locations. We defined foraging sites as areas where the GPS-fitted bats remained in the area, presumably feeding, for >30 minutes. We examined GPS trajectories to locate bat visits within 0–30 m from houses, cultivated crops, or forest patches near farms or cultivated crops outside the contiguous forest preserve.We collected the base station 5–7 days after the last GPS unit was deployed and downloaded the data to the Collar software (Telemetry Solutions) where it was converted to KML (Keyhole Markup Language) file format and viewed on Google Earth Pro version 7.3.4.8642  of 50 had detectable MARV RNA identified by qRT-PCR.Of the 100 ERBs fitted with GPS units, data from 70 bats were ultimately usable. Data from the other 30 units either were never received or were not usable because the data points were so few that no discernable flight pattern could be determined. We suspect that complete GPS failures were caused by the unit being pulled off by the bat or its roost mates or by the bat not returning to that roost, preventing the wireless download of data to the base station. Other reasons might include failure of the unit, including the battery; failure of the adhesive; or predation by one of the many snakes living in the cave or some other predator, such as birds of prey, in the surrounding area. Plotting the bat GPS coordinates onto satellite imagery showed that over a maximum battery life span of 4 days (range 1.5–4 days), ERBs from Python Cave had an average flight distance of 60.18 km  and visited 6 different homes and 34 independent cultivated crop localities in the surrounding agricultural communities. In some instances, bats visited the same location on consecutive nights. In total, 68/70 bats with GPS data (97.14%) made at least 1 visit to a house, a cultivated crop, or a forest patch near a farm or home in an agricultural area outside of the forest preserve surrounding Python Cave.pm‒4:10 am) overnight on February 12–13, 2022 (Sus scrofa) was observed rooting under the mango tree (am–3:30 am) on February 13, 2022, and then foraged again at the same site for 1 hour and 20 minutes (2:40 am–4:00 am) the following night  was in agricultural or community areas outside the forest   infection  (The GPS data showed that several ERBs came close to Kitaka mine, which supports earlier published accounts that infected ERBs at Python Cave, ≈32 km from Kitaka mine, routinely travel between roosts, effectively creating a small scale metapopulation within 2 forest preserves (>100 g, which are the more actively infected cohort of the population (We report findings of visits to cultivated fruit crops near homes and farms by a known MARV natural reservoir, the ERB. Although this information has public health usefulness, the study has several limitations, including battery limitations of the GPS units, shorter time frames for recording foraging activity, and that only male bats have been fitted with GPS units to this point in the study. Investigations of female and older juvenile bats If one considers the longevity of MARV on surfaces and fruit commonly consumed by humans in Uganda and the propensity of the natural reservoir to use human cultivated fruit crops as a source of food, it is conceivable that surface contamination of fruits and other surfaces around farms and homes is the mechanism by which some previous MVD outbreaks unlinked to ERB roosts were initiated. The data we collected will be developed into a larger, evidence-based risk map of MARV spillover in Uganda. Public health messaging for at-risk communities will include the need for properly washing harvested fruits, discarding partially eaten fruits, and keeping livestock away from fallen fruit. Furthermore, this type of evidence-based risk mapping can be used as a rationale for conducting enhanced surveillance activities at local health clinics servicing at-risk communities, including active mortality rate surveillance, to ensure rapid detection of spillover events when they occur.


The last two decades have seen an increase in the incidence of inflammatory bowel disease (IBD) in many regions of the world, which has had a significant impact on both the social and economic burden of governments and healthcare systems. The aim of this study was to determine the level of hospitalization and outpatient treatment costs for children and adolescents with Crohn’s disease and ulcerative colitis, depending on age, location, and activity of the disease. Methods were a retrospective analysis of the medical documentation of 240 children with IBD, hospitalized in the Gastroenterology Ward, Department of Pediatrics Medical University of Silesia , along the three years follow up. The costs of treatment consisted of calculations of the supply of oral and intravenous drugs, calculations of the costs of laboratory tests, imaging, and consultations, as well as person-day costs. The most important results, determined with high costs of IBD treatment, are associated with younger age, high disease activity, localization in the small intestine in Crohn’s disease (CD), and inflammatory changes in the entire colon in Ulcerative Colitis (UC). During the observation, it was noticed that the shortening of the hospitalization time did not significantly affect the total costs, which remained at a stable level. In 2017, there were 6–8 million cases of inflammatory bowel disease (IBD) globally, diagnosed in over one million citizens in the United States and two and a half million people in Europe, which is a significant cost for the healthcare system. Additional social costs related to IBD concerning the professional development of patients and deteriorating quality of life of patients should also be remembered. The majority of cases are diagnosed in young people, and their prevalence is constantly increasing. Therefore, the impact of IBD on healthcare systems is increasing exponentially . The incIn Poland, the increasing competitiveness in the market of health services and contracting them by the National Health Fund has influenced the interest of the management in financing the operations of hospitals. Not only is the global knowledge of costs incurred by hospitals important, but also the individual elements of the “chain” of costs, which consequently determine the outlays for the treatments of patients. Attention was also paid to sources of information for the costs of hospital operations and their records in financial and accounting systems .Pharmacoeconomics, dealing with the economic evaluation of pharmacotherapy, is of great importance in the treatment of many diseases and is based on the study of the relationship between pharmacotherapy costs and obtained health effects, treatment efficacy, and the impact on the length and quality of life. The goal of treatment of IBD is to achieve long-term remission and prevent recurrence of the disease .Due to different clinical pictures and specificities of the disease associated with a young age, the treatment of pediatric patients with IBD is characterized by therapeutic differences. In children, a more severe course of the disease is observed with a higher percentage of complications, and a higher need for surgical intervention compared to adults, and a more extensive location of lesions. In addition, in pediatric patients, growth and puberty disorders occur .The aim of this study was to determine the level of hospitalization and outpatient treatment costs for children and adolescents with CD and UC, depending on age, location, and activity of the disease.The study performed a retrospective analysis of the medical documentation of children with IBD hospitalized in the Gastroenterology Ward, Department of Pediatrics, Medical University of Silesia in Katowice (Poland) for 2014–2016. The study included the medical records of all children hospitalized with diagnosed Crohn’s disease and children with ulcerative colitis, respectively, 106 and 134 children. In the analyzed period of time, the children included in the study did not require surgery.The diagnosis of CD and UC was based on the Porto criteria (2005) and modified Porto criteria (2014) ,9.When analyzing the costs of hospitalization of patients, the costs of treatment were taken into account, consisting of the supply of drugs , treatments, laboratory tests, imaging, and medical consultations, as well as the cost of stay (hotel night). The analysis was carried out in accordance with the settlement system of the National Health Fund.Quantitative variables referring to the costs of treating patients for the needs of statistical analyses were transcribed into qualitative variables considering the minimum, maximum, and average values. A study of the non-hospital costs among parents and carers of children with IBD was conducted during the stay of patients in the Department of Gastroenterology and during control visits at the Gastroenterological Outpatient Clinic. We asked 103 parents and caregivers to take part in the study, and 61 of them agreed to the study and completed the survey. The original questionnaire was addressed to the parents and caregivers of treated children.For statistical analysis of the collected material, parametric and non-parametric dependency tests (Pearson and R Spearman X2) and variable independence tests  were used. In addition, the correlation strength between statistically significant variables was examined using appropriate correlation coefficients. Statistical analysis of the collected material was made using the following programs: Microsoft Office 2008  and Statistica 12, with the addition of the Medical Kit.The exclusion criteria concerned children in the questionnaire survey if the parents did not agree to its conduct. In the case of statistical data, all cases in the period were examined.Before the collection of data, the consent of the Bioethical Commission of the Medical University of Silesia no. KNW/0022/KB/308/15 was obtained.Among 240 children with IBD , there were 106 patients with CD  and 134 patients  with UC. During the study period 2014–2016, there were 628 hospitalizations for IBD. There were 308 hospitalizations for CD, which included 106 patients from 8 to 18 years, with an average age of 14.7 years (100 girls and 208 boys). There were 320 hospitalizations for UC, which included 134 patients aged 1 to 18 years with a mean age of 13.2 years (144 girls and 176 boys). The average age at the time of CD diagnosis was 12.7 years and 11.2 years in UC. The duration of hospitalization of children with CD in the Gastroenterology Ward ranged from 1 to 38 days (the average was 3.9 days), and the time of hospitalization of children with UC ranged from 1 to 34 days (the average was 5 days).p < 0.04).During the three-year follow-up, there was a downward trend in the average drug costs (25%), while there was a systematic increase in costs borne by the department as part of diagnostic tests (15%). In the studied period, a systematic decrease in the total costs of hospitalization of patients with CD and in children with UC on conventional treatment was found. However, there was no significant difference. The total average costs included direct costs of tests and medicines, as well as the costs of children staying in the ward, totaling 628,57 EUR. Disease type, i.e., Crohn’s disease and UC, had a statistically significant impact on the incurred costs. Higher costs (727.40 EUR) of treatment  were observed in children treated for CD, compared to UC  depends on the child’s weight (5 mg/kg bw/dose). Depending on body weight, hospitalization costs ranged from EUR 672.78 to EUR 987.32 (one dose of biologics—EUR 450.74–782.98). Annual biological treatment ranged from EUR 5362.72 to 7458.25 .p < 0.01). The highest costs were recorded in patients aged up to 9 years, while in cases of children aged 10–14 years, the costs were 38% lower. Differences in hospitalization costs depending on age were also found among children with UC (p < 0.01)  .p < 0.01). The highest costs were found in children with CD, where the disease was located in the small intestine and ileocecal region, and in children with UC with inflammation of the entire large intestine . The highest costs were recorded among children with severe disease , we conducted a survey among 61 parents and caregivers. The studied group of children consisted of 35 boys and 26 girls aged from 3.5 to 18 years. It was found that 59% (36 children) of the surveyed children were treated for UC and 41% (25 children) for CD. The average age of the surveyed children was 14.4 years and did not differ significantly depending on the form of the disease. The duration of the disease also did not show differences due to the form of the disease, and it was approximately 3 years. In more than half (53%) of children with UC, the inflammation involved the whole large intestine. Among patients with CD, the small intestine was mainly occupied (48%). The costs incurred by parents and carers of children with IBD include dietary costs, travel to treatment costs, and the costs of drugs taken on a permanent basis and periodically. The average monthly costs borne by parents and caregivers with UC amounted to 146.98 EUR and were lower by about 20% than the costs borne by parents and carers of children with CD, worth 182.56 EUR .54.1% (33 persons) of surveyed parents thought that their incomes were insufficient and did not allow for the purchase of essential drugs and to meet other expenses associated with the child’s disease. The same percentage of parents  used the childcare allowance.The incidence of IBD continues to rise, and so does the cost. The cost of caring for IBD has almost doubled over the past two decades . The majIn 2020, Matary conducted a systematic review of the literature on the treatment of IBD in children, including only nine studies, most of which describe the direct costs of treatment in 2005–2016 . The forPrenzler, A. et al. in Germany analyzed the total costs of treating 511 adult patients with CD and determined them on the average at 3767 EUR, of which only 134 patients required hospitalization (26%). The costs of hospitalization were dependent on the disease activity and, on average, ranged from approximately 650 EUR to over 1000 EUR. Scientists from Germany showed approximately 30% higher costs of treatment in cases of severe CD form (Crohn’s Disease Activity Index > 220) while being similar in remission to mild to moderate forms. In UC, the costs of treatment of mild to moderate forms were twice as high as in remission, and the costs were three times higher in severe form. Moreover, Cohen, R. showed three times higher costs, both indirect and direct, in patients with moderate to severe UC and a higher frequency of hospitalizations ,16.A. Basi in the United Kingdom showed that patients’ hospitalizations accounted for half of all costs. Similar conclusions from the research were drawn by Longobardi, T. in Canada and Heaton, P.C. in the United States ,18,19. TKappelman et al. analyzed the direct costs of healthcare for patients with IBD in the United States, both children and adults. In the analyzed group, of almost 20,000 patients, 8% were children, both in the CD and UC group. The average cost of hospitalization for a patient with CD was 1567 USD and was 30% higher than the cost of treatment of UC, which was 1099 USD. The treatment costs were significantly higher in children and adolescents under 20 years of age, compared to adults, but did not show a difference in relation to gender. Higher costs in children were caused by a more severe course of the disease, which resulted in more frequent controls in the specialist clinic and a higher frequency of hospitalization, which was confirmed by the analysis of our research. Bickstone also compared the costs of treatment of UC in three age groups, including those under 18, stating almost twice as high costs of treatment in the group of children ,20. The Van der Valk, M. et al., during the three-year observation, also showed the relationship between activity and incurred costs. The most common locations of CD were in the small and large intestine at 50% . Unfortunately, they did not analyze the costs related to the location of the disease ,22.Gibson, P. in Australia showed double costs of treating moderate and severe UC over three months of treatment. It appears that the location of the disease is also related to the severity, and the costs can be compared to the activity of the disease .There are many studies assessing the quality of life of IBD patients, while few relate to the costs of outpatient care in chronically ill children with IBD ,25.Sin, T. analyzed the questionnaires of 67 children with CD and 83 with UC, where the mean age of patients was 14 years, and the mean duration of the disease was 3.7 years. The annual costs incurred by parents and caregivers were above 500 USD for 63.5%, above 1000 USD for 28.5%, and above 5000 USD for 5% of patients .Stark, R. conducted cost analysis among 480 adult patients with IBD, and found the average monthly cost was 1425 EUR for CD and was significantly higher than for patients with UC, which was 1015 EUR. Of the total costs of CD treatment, 64% were indirect costs related to early retirement or sick leave, and only 32% were direct costs related to treatment. In the case of UC, 54% of the total costs were indirect, and 41% were direct medical costs .Among surveyed parents, 54.1% believed that their income was insufficient and did not allow them to buy necessary drugs and meet other expenses associated with the disease of their child. In addition, the same percentage of parents (54.1%) benefit from childcare allowance. In Poland, the amount of care allowance in connection with a child’s illness is 35.34 EUR per month. However, the demonstrated costs exceed this amount by several times. Therefore, support in this respect should likely be higher.It should be noted that the strength of the work is a multifaceted collective analysis of both direct and indirect costs of IBD treatment in children, currently available in a few publications, and the limitations of the study are the small number of children suffering from the discussed diseases and the limited number of publications in the field of health economics.The chronic nature of IBD has a large impact on the costs of hospital and outpatient treatment.In the study group of children, high costs of IBD treatment were associated with high disease activity, localization in the small intestine in CD, inflammatory changes in the entire colon in UC, and younger ages of sick children.During the three-years’ time of observation, we noted a shortening time of hospitalization, decreased costs of treatment, and increased costs of diagnostics tests, in particular, in children with CD, while the total cost of hospitalization remained stable.The costs of outpatient treatment of the examined patients borne by parents and caregivers are high in both types of the disease, without the possibility of meeting all the needs related to the disease of children in half of the surveyed families, indicating the need for financial support from social services.